a s the application notebookfiles.alfresco.mjh.group/alfresco_images/pharma/2019/01/... ·...

SUPPLEMENT TO

THE

APPLICATION NOTEBOOK

September 2014

www.chromatographyonline.com

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ES494689_LCGCAN0914_cv1.pgs 08.29.2014 00:54 ADV blackyellowmagentacyan

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ES495749_LCGCAN0914_CV2_FP.pgs 08.29.2014 20:06 ADV blackyellowmagentacyan

THE APPLICATION NOTEBOOK – SEPTEMBER 2014 3

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ES494703_LCGCAN0914_003.pgs 08.29.2014 00:57 ADV blackyellowmagentacyan

THE APPLICATION

NOTEBOOK

Medical/Biological

8 Utilization of CESI Technology for

Comprehensive Characterization of Biologics

Rajeswari Lakshmanan, PhD, AB Sciex

10 Analysis of Oligopeptide Hormones at Low

and High pH on EternityXT Columns

Fredrik Limé, Robert Fredriksson, and Cecilia Mazza, Akzo Nobel

11 New Solution for Oligonucleotides

Separation Using Scherzo SW-C18 Column

Piotr Macech, Robert Puryear, and Itaru Yazawa, Imtakt USA

12 Characterization of Amyloid Fibrils

Formed by the Cell Death Regulator Bcl-2

Malvern Instruments Ltd.

13 Determination of Methadone and Metabolite

(EDDP) in Urine Samples Using Solid Phase

Extraction and GC–MS Analysis

Jeffery Hackett, UCT, LLC

14 Analysis of Monoclonal Antibody Aggregates

by SEC Using MS-Friendly Mobile Phases

Justin Steve and Atis Chakrabarti, PhD, Tosoh Bioscience LLC

Environmental

16 Keeping Water Safe: Detecting Pharmaceutical

and Personal Care Products in Water Using

Liquid Chromatography–Mass Spectrometry

Joe Anacleto, Zicheng Yang, Helen (Qingyu) Sun, and Kefei Wang, Bruker Daltonics

4 THE APPLICATION NOTEBOOK – SEPTEMBER 2014

TABLE OF CONTENTS


18 Fast Organochlorine Pesticides GC-Micro-ECD

Analysis Using Either Helium or Hydrogen Carrier Gas

Jason Thomas, Chris English, Jack Cochran, and

Gary Stidsen, Restek Corporation

20 Automated Multi-Cartridge Solid Phase Extraction of

Phosphorus Containing Pesticides in Drinking Water

FMS

Food and Beverage

21 Arsenic Speciation on Hamilton PRP-X100

Hamilton Company

22 Determination of Deoxynivalenol in Shredded

Wheat Cereal Using Automated Solid Phase

Extraction with Immunoaffinity Cartridges

Toni Hofhine*, Elizabeth K. Krantz†, Pamela Doolittle, PhD†,

and Cheri A. Barta, PhD†, *Horizon Technology, Inc.

and †University of Wisconsin-Madison

23 Detection of Low-Level Sulfur Compounds in Spearmint

Oil Using the Pulsed Flame Photometric Detector (PFPD)

Gary Engelhart and Cynthia Elmore, OI Analytical

24 Fast GC–MS-MS Analysis of Multicomponent

Pesticide Residues (360) in Food Matrix

Hendrik J. Schulte*, Hans-Ulrich Baier*, Stéphane Moreau*,

Robert H. Clifford†, and Laura Chambers†, *Shimadzu

Europa GmbH and †Shimadzu Scientific Instruments

25 A New HILIC Column for Saccharide Analysis

Melissa Turcotte* and Naoya Nakajima†,

*Showa Denko America, Inc. and †Showa Denko K.K.

26 Determination of Carbendazim in Orange Juice

Using an Automated QuEChERS Solution

Tyler Trent, Teledyne Tekmar

Industrial

27 AFFF-MALS-RI for Determining the Mass and Size

Distributions of Amylose and Amylopectins in Starch

Wyatt Technology


TABLE OF CONTENTS


Pharmaceutical

28 Use of Poroshell HPH-C18 Columns

at Elevated pH as a Tool for Method Development

William Long, Agilent Technologies, Inc.

31 HPLC UV Determination of Very High and Very

Low Concentrations of Compounds in One Run

A.G. Huesgen-Gratzfeld, Agilent Technologies

32 Separation of Apo-Transferrin and

Bovine Serum Albumin (BSA ) Proteins

Janusz Zukowski, Diamond Analytics

33 Advanced Analytical SEC Measurements of

Anthrolysin Molecular Weight and Structure


34 Amino Acid Analysis According to

European Pharmacopoeia 8.0

Pickering Laboratories

36 Screening of Drugs of Abuse Using

the Velox 360™ Paper Spray System

Joseph H. Kennedy and Justin M. Wiseman, Prosolia Inc.

37 Separation of Statistic MMA-MAA

Copolymers Using Gradient SEC

PSS Polymer Standards Service

38 Improved USP Chlorhexidine Gluconate Assay on YMC-

Triart C18 and YMC Meteoric Core HPLC Packing Materials

Jeffrey A. Kakaley, YMC America, Inc.

39 Fast Methods for Structurally Similar

Compounds Using Carbon HPLC Columns

Dwight Stoll, Clayton V. McNeff, and Peter W. Carr,

ZirChrom Separations, Inc.

Polymer

40 Synthetic Rubbers: Polybutadiene

Wyatt Technology


TABLE OF CONTENTS


General

41 Generating Make-Up Gas for GC

with an In-House Nitrogen Generator

Corky Belobraydich, Parker Hannifin Corporation

Articles

42 Translations Between Differing Liquid Chromatography

Formats: Advantages, Principles, and Possible Pitfalls

Patrik Petersson*, Melvin R Euerby†,‡, and Matthew A James‡

*Novo Nordisk A/S; †Strathclyde Institute of Pharmacy and Biomedical

Sciences, University of Strathclyde; and ‡Hichrom Ltd.

Departments

51 Call for Application Notes

Cover Photography: Getty Images


TABLE OF CONTENTS



MEDICAL/BIOLOGICAL

Utilization of CESI Technology for Comprehensive Characterization of BiologicsRajeswari Lakshmanan, PhD, AB Sciex

Monoclonal antibodies (mAbs) form a major class of biologics and

recently biosimilars and biobetters are being added to the grow-

ing inventory of therapeutics. In-depth characterization of mAbs at

various stages of development and manufacturing is essential to

maintain product safety and eff cacy. However, analysis of mAbs is

challenging due to their high molecular weight, the microheteroge-

neity presented by the glycans, and degradative modif cations that

occur during production. Any analytical technique that provides

greater depth of information without a time penalty is an advantage.

A recent advancement to meet this need was the introduction of

CESI–MS. CESI is the integration of capillary electrophoresis (CE)

and electrospray ionization (ESI) in a dynamic process, within the

same device. In this technology, the analytes are separated inside

an open nontapered capillary based on their electrophoretic mobil-

ity, and electrosprayed directly into the MS (2). At operating f ow

rates less than 30 nL/min, very eff cient desolvation and, thus, ion-

ization is achieved.

Though high speed CESI separations reduce analysis time, it also

necessitates the use of high speed MS to preserve the separation

eff ciency. The TripleTOF® 5600+ system (AB Sciex) offers the

necessary high acquisition speed, high resolution, and high mass

accuracy, in both MS and MS-MS modes. CESI performance was

evaluated by analyzing a tryptic digest of trastuzumab using the

SCIEX CESI 8000 - TripleTOF® 5600+ MS platform.

Figure 1: (a) Extracted ion electropherogram of N-terminal peptide with Glu and pyroGlu separated by CESI and (b) MS-MS identif cation of N-terminal peptide with pyroGlu.

4.0e6

3.5e6

3.0e6

2.5e6

2.0e6

1.5e6

1.0e6

5.0e5

0.0e0

6500

6000

5500

5000

4500

4000

3500

3000

2500

2000

1500

1000

500

0

Inte

nsit

yIn

tens

ity

29.5

Spectrum from 6Dec13Her2ugPerul_100mMLE_250TOF501DA30ions5sec500cps_1to5WithChargeSel_2.wiff (sample 1) - Her10, Experiment 10, +TOF MS^2 (100 - 2500) from 35.322 minPrecursor: 932.5 Da

150 200

b2

250 300

*424.2592

*339.1698

*452.2540

b5

*551.3233

b6

b7

*680.3647

b10

b11

b9

*938.4616

350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z

30.0 31.0 32.0 33.0Time (min)

34.0 35.0 36.0 37.036.535.534.533.532.531.530.5

N-terminal Glu

N-terminal pyroGlu

(a)

(b)

b4

*586.3339

y6

*714.3923

*813.4611

y7

y8

*983.5663

y10 *1040.5852

y11

*1097.6123

y12

*1184.6458

y13

*1313.6853

*1314.6921

y9

b3

y14

*1412.7647

y15

Experimental Conditions

Trastuzumab was reduced, alkylated, and digested with trypsin.

After drying, it was resuspended in the leading electrolyte (100

mM ammonium acetate at pH 4) and 50 nL (100 fmol) was

injected into the separation capillary. The background elec-

trolyte used was 10% acetic acid and a separation voltage of

20 kV (normal polarity) was applied for 60 min. Information

dependent acquisition (IDA) was utilized to trigger MS-MS. IDA

parameters were optimized so that the duty cycle of the MS

was matched to the high speed CE separation. Data analysis

was performed using BioPharmaView™ software (AB Sciex,

Massachusetts).

Results and Discussion

Primary Sequence Coverage: 100% primary sequence coverage of

both the light and heavy chains of the antibody were obtained. Pep-

tides ranging from 4 to 63 amino acids in length without any missed

cleavages were detected. Electrophoretic separation is based on the

charge-to-mass ratio of the peptides and is not dependent on rela-

tive hydrophobicity. Thus, small hydrophilic peptides often lost in

the LC void volume, and large hydrophobic peptides, which tend to

be retained on the column, can be identif ed by CESI–MS, resulting

in the high sequence coverage observed.

PTM Characterization: Data from CESI-MS analysis showed the

presence of several PTM hotspots such as N-terminal pyroGlu

formation, methionine oxidation, and asparagine deamidation.

Pyroglutamination leads to loss of a positive charge which re-

sults in the electrophoretic mobility of the modif ed peptide being

lower than the unmodif ed one. This is advantageous since the

modif ed and unmodif ed forms can be separated by CESI–MS

and the MS-MS spectra conf rmed the presence of the pyroGlu-

tamate residue (Figure 1). Oxidative degrada-

tions at Met255 and Met431 and deamidations at

Asn55 and Asn387 in the heavy chain and at Asn30

in the light chain were also identif ed. A typical

identif cation from the CESI–MS data is shown

in Figure 2.

Glycosylation Heterogeneity: Trastuzumab pos-

sesses one N-glycosylation site at Asn300 in

the HC where different glycoforms such as a-

fucosylated or fucosylated glycans can be pres-

ent (3). By using CESI-MS, the G0F, G1F, and

G2F forms of the peptide TKPREEQYNSTYR

were separated well as shown in Figure 3. Fur-

thermore, the identification of G0F, G1F, and

G2F forms of peptide EEQYNSTYR without the

missed cleavage (at the arginine residue in the

peptide TKPREEQYNSTYR) also confirmed the

presence of these glycoforms. In addition, the

a-fucosylated forms of this peptide, such as G0

and G1, were identified, but it has to be further



MEDICAL/BIOLOGICAL

AB Sciex

500 Old Connecticut Path, Framingham, MA 01701

tel. (877) 740-2129, fax (800)343-1346

Website: www.sciex.com/cesi

separation eff ciency and high sensitivity allows the

analysis of all peptides including modif ed and low

abundant species, in addition to conf rming the

amino acid sequence of the antibody.

References

(1) J.M. Busnel, B. Schoenmaker, R. Ramautar, A.

Carrasco-Pancorbo, C. Ratnayake, J. Feitelson, J. Chap-

man, A. Deelder, and O. Mayboroda, Anal. Chem. 82,

9476–9483 (2010).

(2) A. Beck, S. Sanglier-Cianferani, and A. Van Dorsselaer,

Anal. Chem. 84, 4637–4646 (2012).


confirmed that the a-fucosylated forms were not generated due

to source fragmentation of fucosylated counterparts.

Conclusions

We have presented CESI–MS, a robust ultra-low f ow and highly

eff cient separation technology in combination with TripleTOF MS,

a high resolution accurate mass measurement system for qualita-

tive analysis of biopharmaceuticals. CESI–MS is attractive for simul-

taneous analysis of primary sequence coverage and glycopeptide

prof ling, without carry-over concerns. The combination of high

Figure 2: MS-MS identif cation of asparagine deamidation with (a) showing unmodif ed peptide and (b) deamidation at Asn55 in the heavy chain of trastuzumab.

Figure 3: Extracted ion electropherograms of peptide TKPREEQYNSTYR with G0F, G1F, and G2F modif cations.

4.5e4

4.0e4

3.5e4

3.0e4

2.5e4

2.0e4

1.5e4

1.0e4

5.0e3

0.0e0

Inte

nsit

y

3.0e4

2.5e4

2.0e4

1.5e4

1.0e4

5.0e3

0.0e0

Inte

nsit

y

Spectrum from 6Dec13Her2ugPerul_100mMLE_250TOF501DA30ions5sec500cps_NOChargeSel_2.wiff (sample 1) - Her8, Experiment 19, +TOF MS^2 (100 - 2500) from 27.027 min

Precursor: 542.8 Da

Spectrum from 6Dec13Her2ugPerul_100mMLE_250TOF501DA30ions5sec500cps_NOChargeSel_2.wiff (sample 1) - Her8, Experiment 9, +TOF MS^2 (100 - 2500) from 28.180 min

Precursor: 543.3 Da

Unmodifed peptide

Deamidated at Asn55

*136.0803

*171.1181*199.1135

*249.1643

*486.2354*496.2534

*611.2995

*610.2927

*711.3387 (1)

712.3390 (1)

*790.3809 (1)

809.3908 (1)

971.4573

*808.3885 (1)

*277.1595 (1)

*404.7045 (2)

405.2075 (2)

278.1583 (1)

b2

y1

*136.0805

*249.1646

*171.1177

*277.1594

*405.1974

*405.7015

*486.7298

*496.2536

*611.2788 (1)

612.2888 (1)

*712.3253

*722.3113

*810.3828

*809.3766

b2

y1

y4

y5

y6

y7

y4

y5

y6

y8

971.4457y

8

y7

N T P Y

N* T P Y

150 200 250 300 400 500 600 700 800 900 950850750650550450350

m/z

150 200 250 300 400 500 600 700 800 900 950850750650550450350

m/z

(a)

(b)

5.2e5

5.0e5

4.8e5

4.6e5

4.4e5

4.2e5

4.0e5

3.8e5

3.6e5

3.4e5

3.2e5

3.0e5

2.8e5

2.6e5

2.4e5

2.2e5

2.0e5

1.8e5

1.6e5

1.4e5

1.2e5

1.0e5

8.0e4

6.0e4

4.0e4

2.0e4

0.0e0

Inte

nsit

y

24.8 24.9 25.0 25.1

25.10

25.42G0F

25.7

25.95

G1F

G2F

25.2 25.3 25.4 25.5 25.6 25.7Time (min)

25.8 25.9 26.0 26.1 26.2 26.3 26.4 26.5

XIC from 09292013_Tras1mgmL_100mMLE_R7.wiff(sample1)-09292013_Tras1mgmL_100mMLE_R7, Experiment1, +TOF MS (100-2000): 1039.800+/-0.025DaXIC from 09292013_Tras1mgmL_100mMLE_R7.wiff(sample1)-09292013_Tras1mgmL_100mMLE_R7, Experiment1, +TOF MS (100-2000): 1093.780+/-0.025DaXIC from 09292013_Tras1mgmL_100mMLE_R7.wiff(sample1)-09292013_Tras1mgmL_100mMLE_R7, Experiment1, +TOF MS (100-2000): 1147.8200+/-0.025Da


MEDICAL/BIOLOGICAL

Polypeptide hormones play key endocrine functions in humans.

In particular, angiotensins are oligopeptide hormones, critical in

the regulation of body f uids which ultimately affect blood pres-

sure levels in individuals. A UHPLC method based on reversed-

phase chromatography was created for the analysis of angiotensins

using EternityXT C18 UHPLC columns, which can work under a

wide range of pH and withstand exposure to solutions of NaOH

beyond typical clean-in-place conditions.


The sample mixture used in this study consisted of three angiotensins:

Angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu

Angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Angiotensin III Arg-Val-Tyr-Ile-His-Pro-Phe

These samples were analyzed under low and high pH reversed

phase gradient chromatography.

Low pH conditions: mobile phase A 0.1% TFA in water (pH 1.9),

mobile phase B 0.1% TFA in acetonitrile, gradient 0 min 9% B, 10

min 36% B, f ow rate 0.7 mL/min, monitored at 220 nm.

High pH conditions: mobile phase A 0.1% ammonium hydroxide

in water (pH 11.0), mobile phase B acetonitrile, gradient 0 min 5%

B, 10 min 40% B, f ow rate 0.7 mL/min, monitored at 225 nm.

Results and Conclusions

Chromatographic retention changes and selectivity under low and

high pH conditions were evaluated using EternityXT UHPLC C18 for

the separation of angiotensins under linear gradient reversed phase

conditions. Figure 1 shows two chromatographic results, the top

chromatogram was obtained using pH 1.9, while the bottom chro-

matogram resulted from using pH 11. As seen in the f gure, while

the mixture used in this study contained three peptides, the analy-

sis at pH 1.9 resulted in a co-elution of two out of three compounds.

On the other hand, by increasing the pH of the mobile phase to 11,

complete resolution among the three peaks was achieved. Further,

evaluating the top and bottom chromatographic results, it is seen

that selectivity reversal between Antiotensin I and III can occur by

adjusting the pH of the analysis. Finally, the results shown in the

f gure illustrate the utility of using EternityXT UHPLC columns where

a wide range of pH can be explored to achieve the best separation

possible and to benef t analysts working with oligopeptides in the

laboratory.

Analysis of Oligopeptide Hormones at Low and High pH on EternityXT ColumnsFredrik Limé, Robert Fredriksson, and Cecilia Mazza, Akzo Nobel

AkzoNobelStrawinskylaan 2555, 1077 ZZ Amsterdam, The Netherlands

tel. +31 20 502 7555

Website: www.akzonobel.com

Figure 1: Separation of Angiotensin I, II, and III using a 50 × 2.1 mm EternityXT 1.8-C18 column. Conditions (low pH): mobile phase A 0.1% TFA in water (pH 1.9), mobile phase B 0.1% TFA in acetonitrile, gradient 0 min 9% B, 10 min 36% B, f ow rate 0.7 mL/min, detection UV @ 220 nm. Conditions (high pH): mobile phase A 0.1% ammonium hydroxide in water (pH 11.0), mobile phase B acetonitrile, gradient 0 min 5% B, 10 min 40% B, f ow rate 0.7 mL/min, detection UV @ 225 nm.

Low pHAll + Alll

Time (min)0 2 4 6 8

0 2 4 6 8

Time (min)

AI

AI

AII

AIII

High pH



MEDICAL/BIOLOGICAL

Oligonucleotides are in the center of many recent research projects

including genetic testing, polymerase chain reactions (PCR), foren-

sics, and DNA/RNA silencing. Two major challenges for large-scale

applications of oligonucleotides in these f elds include determina-

tion of molecular weight of the individual chains and the detail of

the base sequence as in the case where single base changes could

have major implications in function.

Current common methods for oligonucleotides separations typi-

cally include ion-pair reagents in reversed phase chromatography

or anion-exchange HPLC. Both of these strategies are not ideal for

chromatographers because they often suffer from poor reproduc-

ibility, and short column life times. These strategies also add the

cost of additional reagents as in the case of ion-pairing, and often

require high pHs which cause rapid deterioration of silica-based

columns. Furthermore, these techniques are typically not suff cient-

ly selective to separate oligonucleotide isomers.

In this article, we show the successful application of Scherzo

SW-C18 column for oligonucleotide separation, addressing the two

main analytical goals of chromatographers in these f elds — chain

size discrimination and base pair substituted isomer separation. The

Scherzo SW-C18 is a unique multi-mode column that contains re-

versed phase ODS ligands, as well as both cation and anion exchange

(Figure 2). This places Scherzo SW-C18 at a distinct advantage in the

analysis of oligonucleotides because you get the selective retention

characteristics required for this diff cult separation without the need

of an ion-pairing reagent. Also, since this column has built-in ion

exchange sites, you can have the advantage of IEX with the ease and

reliability of reverse phase mobile phase conditions.

New Solution for Oligonucleotides Separation Using Scherzo SW-C18 ColumnPiotr Macech, Robert Puryear, and Itaru Yazawa, Imtakt USA

Figure 1: Eff cient separation of oligonucleotides using Scherzo SW-C18.

Figure 2: Schematic comparison of the unique stationary phase composi-tion of Scherzo SW-C18 and Scherzo SM-C18.

Imtakt USA1104 NW Overton St., Portland OR, 97209

tel. (888) 456-HPLC, (215) 665-8902, fax (501)646-3497

Website: www.imtaktusa.com

Experimental

A Scherzo SW-C18, 150 × 3 mm column was used. Elution condi-

tions were: (A) 10 mM ammonium acetate, (B) 200 mM ammo-

nium acetate/acetonitrile (85/15), 40–70% B in 0–65 min, injection

volume 1 µL (1 µg), 1 mL/min f ow rate, 20 MPa, and 50 °C column

temperature. Eluent was analyzed using UV detector at 260 nm.

Discussion

Figure 1 shows separation of oligothymidylic acid ammonium salt with

12 to 18 mers. A very good speciation is observed based only on a single

mer difference in oligonucleotides’ size and mass. It should be noted

the separation presented in Figure 1 was acquired without using ion-

pairing reagents or high pH regime. That indicates not only immediate

savings in time and resources in lab, but also increased column lifetime.

The ideal column for oligonucleotide separation would provide not

only excellent separation based on number of mers, but also would

exhibit superior selectivity for isomers. As seen in Figure 1, our Scherzo

SW-C18 column shows excellent size resolution, however in a recent

work described in detail elsewhere, it was shown that a sister column to

the SW-C18, the Scherzo SM-C18, can separate isomers with structural

changes as small as a single base for a 21 mer oligonucleotide. Signif -

cant structural changes such as N-x deletion are also separated using

this column (Biba et al., J. Chromatogr. A 1304, 69–77 [2013]).

In summary, Scherzo columns exhibit excellent capabilities for ex-

tremely challenging separations of oligonucleotides. Individual species

are separated based on their size as well as structural differences; an

accomplishment not available by ion-pair reversed-phase LC or strong-

anion-exchange LC alone.



MEDICAL/BIOLOGICAL

Characterization of Amyloid Fibrils Formed by the Cell Death Regulator Bcl-2 Malvern Instruments Ltd.

Proteins of the Bcl-2 family are molecular transducers sensitive to inter-

nal and external apoptotic signals that play a key role in the regulation of

apoptosis. Their aggregation can lead to the formation of amyloid f bers

due to protein misfolding which are associated with numerous diseases.

The work described in this application note focused on the in vitro

formation of aggregates by a Bcl-2 protein initiated by incubation of the

protein at 37 °C. Multi-detection size exclusion chromatography (SEC)

was used to characterize the early events occurring during the aggrega-

tion process following incubation for 1 day and 1 week. SEC was per-

formed using a Superose 6HR (GE Healthcare) with a buffer of 20 mM

sodium phosphate, pH 8, and 150 mM sodium chloride. The Viscotek

TDA with UV, RI, light scattering, and viscometer detectors was used to

determine the molecular weight (MW) and intrinsic viscosity (IV).


Figure 1 is a typical chromatogram following 1 day of incubation at

37 °C. Three main species were detected, with molecular weights

of 74 kDa, 51 kDa, and 25 kDa. The main population of this sample

was found to be the protein’s monomer. An additional species was

detected by the light scattering detector eluting at 11 mL, which is

a high molecular weight aggregate but at very low concentration.

The samples were also analyzed after incubation for 1 week

at 37 °C. Following this extended incubation period more aggre-

gates were found to have formed. The SEC experiments showed

that the f rst step of the f ber growth is the formation of small ag-

50

45

40

35

30

25

20

15

10

5

0

20

5 10 15 20 25 30

22 24 26

0

20

40

60

80

100

Mw, KDa

Mo

lecu

lar

Weig

ht

(kD

a)

RI

RALS

Figure 1: Chromatogram of Bcl-2 from the Viscotek TDAmax. RALS (green line) and refractive index detector (red line). Inset shows the molecular weight distribution of the different species within the sample.


Enigma Business Park, Groveland Road, Malvern, UK

tel. +44 (0) 1684 892456, Email: [email protected]

Website: www.malvern.com

gregates, mostly dimers and trimers, which further assemble into

larger aggregates. This information was only possible to obtain

with the use of multi-detector SEC using the TDA system.

Work performed in conjunction with the Institut Pasteur, Paris, France

References

(1) A. Chenal, C. Vendrely, H. Vitrac, J.C. Karst, A. Gonneaud, C.E. Blanchet,

S., Pichard, E. Garcia, B. Salin, P. Catty, D. Gillet, N. Hussy, C. Marquette,

C. Almunia, and V. Forge, “Amyloid Fibrils Formed by the Programmed Cell

Death Regulator Bcl-xL.,” J. Mol. Biol. 415, 584–599 (2012).

(2) J.C. Karst, A.C. Sotomayor-Pérez D. Ladant, and A. Chenal, “Estimation of

Intrinsically Disordered Protein Shape and Time-Averaged Apparent Hydra-

tion in Native Conditions by a Combination of Hydrodynamic Methods,”

Methods Mol. Biol. 896, 163–77 (2012).



MEDICAL/BIOLOGICAL

Methadone is a commonly prescribed pain management type drug.

As such, patients are monitored on a routine basis. This applica-

tion note presents a simple and effective method for the extraction

and analysis of the parent compound as well as its primary analyte

EDDP. The method utilizes mixed mode SPE for urine sample ex-

traction followed by GC–MS analysis.

Sample Preparation

1) To 1 mL of 100 mM pH 6 phosphate buffer in a sample tube add

internal standards (methadone-d9, EDDP-d3). To this add 1 mL of

urine and mix. Add an additional 2 mL of 100 mM ph 6 phosphate

buffer and mix. Centrifuge this mixture for 10 min at 3000 rpm.

2) Condition the SPE cartridge with sequential additions of the fol-

lowing liquids in the order specif ed: 3 mL methanol, 3 mL DI water,

3 mL 100 mM pH 6 phosphate buffer. Draw each solvent through

the cartridge (< 3 in. Hg) until it reaches the top of the sorbent bed;

do not allow the cartridge to go dry between solvent additions.

3) Load the previously prepared (step 1) clear sample onto the

cartridge.

4) Wash the SPE cartridge with 3 mL of DI water, followed by 3

mL of 1 M acetic acid, and then with 3 mL of methanol. Apply full

vacuum (> 10 in. Hg) and allow the column to dry for 5 min.

5) Elute the sample with 3 mL of a freshly prepared methylene

chloride:isopropanol:ammonium hydroxide (78:20:2) mixture. The

eluate is collected at 1–2 mL/min and then vortex mixed.

6) The extract is evaporated to dryness under nitrogen (< 40 °C).

Then reconstituted with 100 µL methanol.

Instrumental

GC-MS: Thermo Scientif c Trace 1300 GC–MS

GC column: Thermo Scientif c TG1-MS; 30 m × 0.25 mm; 0.25 μm

Injector: 1 μL at 250 ºC

Split Ratio: 10:1

Determination of Methadone and Metabolite (EDDP) in Urine Samples Using Solid Phase Extraction and GC–MS Analysis Jeffery Hackett, UCT, LLC

UCT, LLC 2731 Bartram Road, Bristol, PA 19007

tel. (800) 385-3153; email: [email protected]

Website: www.unitedchem.com

Figure 1: Calibration curves of methadone and EDDP

SPE Materials

CSDAU206 Clean Screen® DAU 200 mg, 6 mL tube

SPHPHO6001-5Select pH buffer pouch, 100 mM phosphate buffer,

pH 6

Transfer Line: 280 °C

Carrier gas: Helium at a constant f ow of 1.2 mL/min

Oven: Initial temperature at 50 ºC, hold for 1 min; ramp at 30 ºC/min

to 320 ºC, hold for 3.6 min

Table I: Mass spec table

Compound RT / min. Primary Ion Secondary Ion Tertiary Ion

Methadone 10.74 72 294 223

Methadone-d9 10.70 78 303

EDDP 10.32 276 277 278

EDDP-d3 10.30 279 280

Conclusion

This simple and eff cient method analyzes both methadone and its

primary metabolite (EDDP) in urine samples. Recov-

eries for both compounds were found to be >90%,

the method was linear from 5 ng/mL to 500 ng/mL.

The limits of detection and quantif cation were found

to be 1 ng/mL and 5 ng/mL, respectively. The GC–MS

run time was less than 15 min.

Linearity



MEDICAL/BIOLOGICAL

Analysis of Monoclonal Antibody Aggregates by SEC Using MS-Friendly Mobile PhasesJustin Steve and Atis Chakrabarti, PhD, Tosoh Bioscience LLC

The use of mass spectrometry as a means of detection is becoming

increasingly more common among research laboratories in the f eld

of proteomics. After more than 15 years of research, LC–MS sys-

tems are now more robust, and used more often for routine analy-

ses which are nearly unachievable by any other mode of detection.

Separation of protein aggregates from their native species is of-

ten performed using size exclusion chromatography (SEC), as this

mode allows for the analysis of various components in a sample

on the basis of their hydrodynamic radius in solution. Conventional

SEC separations make use of relatively high ionic strength mobile

phase compositions in an effort to minimize ionic interactions be-

tween the analyte and stationary phase. Due to the substantial

amount of salt present in the mobile phase, on-line interfacing with

mass spectrometry is not feasible due to the inevitable contamina-

tion of the MS ion source by the mobile phase salts. In order to

make SEC-MS an applicable technique, volatile, MS-friendly mobile

phase compositions must be implemented to avoid damage to the

MS system. The challenge of using such mobile phase composi-

tions exists due to the absence of salts which hinder ionic interac-

tions during a separation.

This application note illustrates the effective use of MS-friendly

mobile phase compositions in the analysis of monoclonal antibody

aggregates using a TSKgel UltraSW Aggregate SEC column. The

TSKgel® UltraSW Aggregate column demonstrates high stability and

low reactivity via ionic interactions even in low salt and no salt envi-

ronments most likely due to the diol coating of the silica stationary

phase.

Materials and Methods

Instrumentation: Agilent 1200 HPLC system run by

Chemstation® (ver. B.04.02)

Column: TSKgel UltraSW Aggregate, 3 μm,

7.8 mm ID × 30 cm

Mobile phase: 100 mmol/L PO4/100 mmol/L SO

4, pH 6.7

20% CH3CN/0.1% TFA/0.1% FA

100 mmol/L ammonium bicarbonate, pH 7.0

Gradient: Isocratic

Flow rate: 1 mL/min

Detection: UV @ 280 nm

Temperature: 25 °C

Injection vol.: 10 μL

Sample: TBL mAb 1 (4.0 mg/mL)


Figure 1 illustrates the separation of mAb 1 using three different

mobile phase compositions. All three mobile phase compositions

yielded highly similar results for all peak parameters of the mAb 1

monomer. Additionally, slightly later elution of the mAb 1 monomer

was observed when separated using the salt-based mobile phase.

The observed shift in retention time of the mAb 1 monomer peak

represents a %RSD of 0.1% among the three mobile phase con-

ditions. Similarly average peak area of the mAb 1 monomer was

found to be highly reproducible as well, yielding a %RSD of 0.53%

across the three mobile phase systems evaluated in this work.

This illustrates that the observed differences in retention time and

peak areas obtained from the three mobile phase systems are not

statistically signif cant.

As characterization of protein aggregates is of increasing impor-

tance in research, and with signif cant work being directed towards

analysis by MS, the mAb 1 antibody was subjected to thermal stress

for forced aggregation to evaluate the various mobile phase systems

in this context. As shown in Figure 2, aggregates of mAb 1 are

clearly separated from the monomeric species using all three mo-

bile phase compositions. Similar to Figure 1, results for critical peak

parameters of the mAb 1 monomer are highly reproducible

Figure 1: Separation of mAb 1 using volatile and salt-based mobile phase compositions on the TSKgel UltraSW Aggregate column.

7 7.5 8

0 5 10 15

8.5 9 9.5 100

10

20

30

40

50

60

Retention time (minutes)

De

tec

tor

resp

on

se (

mA

U) Average retention time: 9.03 min (%RSD: 0.10)

Average peak area: 709.71 mAU*s (%RSD: 0.53)

100 mmol/L PO4/100 mmol/L SO

4, pH 6.7


20% CH3CN/0.1% TFA/0.1% FA

Figure 2: Separation of forced aggregated mAb 1 using volatile and salt-based mobile phase compositions on the TSKgel UltraSW Aggregate column.

7 7.5 8 8.5 9 9.5 100

10

20

30

40

50

Retention time (minutes)

De

tec

tor

resp

on

se (

mA

U)

Average retention time: 9.03 min (%RSD: 0.12)

Average peak area: 671.04 mAU*s (%RSD: 3.54)100 mmol/L PO

4/100 mmol/L SO

4, pH 6.7


20% CH3CN/0.1% TFA/0.1% FA

7 7.5 8 8.5



MEDICAL/BIOLOGICAL

Tosoh Bioscience LLC

3604 Horizon Drive, Suite 100, King of Prussia, PA 19406

tel. (484) 805-1219, fax (610) 272-3028

Website: www.tosohbioscience.com

regardless of the mobile phase composition. Additionally, the total

peak area (monomer and aggregate) obtained using the three mo-

bile phase systems was highly similar to one another (avg: 671.04

mAU*s), and yielded a %RSD of 3.54%, illustrating the differences

are not statistically signif cant. It is also of note that the observed

shift in retention time of the mAb 1 monomer peak only corresponds

to a %RSD of 0.12% for all three mobile phase compositions.

Conclusions

The growing interest in both protein aggregate analysis and mass

spectrometry in the f eld of proteomics demand effective SEC-MS

methods utilizing suitable mobile phases. The use of volatile mobile

phase systems, such as 20% acetonitrile, 0.1% trif uoroacetic acid,

and 0.1% formic acid or 100 mmol/L ammonium bicarbonate at

pH 7.0 yield highly reproducible separation of mAb 1 aggregates,

with similar or better performance over traditional salt-based mo-

bile phase compositions. These results also show the effectiveness

of the diol coating on the TSKgel UltraSW Aggregate column to

assist in minimizing ionic interaction which are frequently present

between the sample and stationary phase in low salt environments.

Tosoh Bioscience and TSKgel are registered trademarks of Tosoh Corporation.

Chemstation is a registered trademark of Agilent Technologies Inc.



ENVIRONMENTAL

Keeping Water Safe: Detecting Pharmaceutical and Personal Care Products in Water Using Liquid Chromatography–Mass SpectrometryJoe Anacleto, Zicheng Yang, Helen (Qingyu) Sun, and Kefei Wang, Bruker Daltonics

The Problem with PPCPs

Pharmaceutical and personal care products (PPCPs) are products

used for personal health or cosmetic reasons. This category includes

a broad group of chemical substances such as human and veterinary

medicines and cosmetics. The presence of PPCPs in environmental

and potable water is a widespread concern due to the potentially harm-

ful environmental effects. Evidence suggests PPCPs are linked to some

ecological damage such as the delayed development in f sh (1).

To ensure the safety of water, PPCP concentrations are stringently

monitored by environmental regulatory bodies, including the United

States Environmental Protection Agency (US EPA) (2). Detection

of PPCPs is traditionally a complicated process due to the range of

substances potentially present. Here we explore a simple, more con-

venient method than traditional solid phase extraction (SPE) based

methods for highly sensitive PPCP detection, using triple quadrupole

liquid chromatography–mass spectrometry (LC–MS-MS).

Detecting PPCPs

Conventional methods of PPCP detection in clean water have

followed the defined EPA 1694 “template” for analysis which

requires the pre-concentration of large volume water samples

and tedious solid phase extraction cleanup, followed by liq-

uid chromatography–mass spectrometry analysis in order to

achieve the low ng/L (ppt) level detection necessary to comply

with regulations (3).

Tables Ia & Ib: Instrumentation set up for analysis of PPCPs

in clean water

Mass Spectrometer Parameters (EVOQ Elite)

HV 4000 V

Cone gas f ow 15 units

Cone gas temperature 300 °C

Heated probe gas f ow 40 units

Heated probe temperature 450 °C

Nebulizer gas f ow 50 units

Exhaust gas On

Q2 pressure 1.5 mTorr (Argon)

Chromatography Parameters (Advance UHPLC)

Trap columnYMC-Pack ODS-AQ, 3 µm, 35 mm

× 2.0 mm I.D.

Column temperature 40 °C

Injection volume 400 µL

Flow rate 400 µL/min

Solvent A2 mM ammonium formate, 0.1%

FA in water

Solvent B2 mM ammonium formate, 0.1%

FA in MeOH

Solvent C2 mM ammonium formate, 0.1%

FA in water

Gradient conditions 0.0 min, 10% B

0.2 min, 10% B

0.8 min, 25% B

8.0 min, 95% B

9.0 min, 95% B

9.1 min, 10% B

12.0 min, 10% B

Figure 1: PPCPs in environmental water and nearby soil is a widespread concern.

Trimethoprim; (+) 291.1 > 230.0Hydroxy Atrazine; (+) 198.0 > 156.0

Thiabendazole; (+) 202.0 > 175.0Caffeine; (+) 195.1 > 138.0

Sildenafil; (+) 355.0 > 143.9Sulfamethoxazole; (+) 254.1 > 156.0

Cyanazine; (+) 241.0 > 213.9Hexazinone; (+) 253.0 > 171.0Dapoxetine; (+) 306.0 > 156.9

Bentazone; (-) 239.0 > 132.1Carbamazepine; (+) 237.1 > 194.1

Atrazine; (+) 216.0 > 173.9Alpazolam; (+) 309.0 > 280.8Prometryn; (+) 242.0 > 157.9

MCPA; (-) 199.2 > 141.1Metolachlor; (+) 284.0 > 176.0

1

0

10

20

30

40

2 3 4 5 6 7 8

Minutes

kCps

Figure 2: Selected MRM chromatograms for PPCPs at 2 ppt.



ENVIRONMENTAL

Bruker has explored how LC–MS-MS can be employed specif -

cally for the analysis of PPCPs in clean water. PPCPs were detected

at 1–2 ppt with a linear response up to 200 or 500 ppt. Excellent

system robustness was obtained throughout the extended method

development and sample analysis period.

Case Study: Using LC–MS-MS

to Analyze PPCPs in Clean Water

The study was carried out using ultrahigh-performance liquid chro-

matography (UHPLC) with an integrated on-line extraction (OLE)

option coupled to a triple quadrupole mass spectrometer. The OLE

module enables convenient method-driven on-line sample cleanup

or sample pre-concentration.

Several water samples were analyzed for a range of PPCP spe-

cies, including tap water samples along with bottles and creek water.

Samples were analyzed targeting a wide range of PPCP species

representing compounds displaying varied properties and concen-

trations. Tables Ia and Ib illustrate the Advance UHPLC and EVOQ

instrumentation set up respectively.

All of the PPCPs were detected at 2 ppt or better with the injec-

tion of 0.4 mL samples with a linear response range up to 200 or

500 ppt. The fast polarity switch can analyze positive and nega-

tive PPCPs in the same analytical segment with excellent linear

response for both polarities (Figures 2 and 3). The results for the

analysis of tap, creek, and bottled waters are shown in Table II.

Conclusion

The Bruker Advance UHPLC with OLE coupled to EVOQ LC–MS-

MS detected PPCP samples at 2 ppt or better within 0.4 mL sam-

ples. Excellent linearity, sensitivity, and robustness were achieved

throughout. The technique presents a more convenient and simpler

approach to PPCP analysis than traditional SPE-based methods.

References

(1) K. Hirsch, “Pharmaceuticals and Personal Care Products,” (2013). Avail-

able at: http://serc.carleton.edu/NAGTWorkshops/health/case_studies/

pharmaceutical.html.

(2) US EPA, “PPCPs Basic Information,” (2010). Available at: http://www.epa.

gov/ppcp/basic2.html.

(3) US EPA, “EPA Method 1694: Pharmaceuticals and Personal Care Products

in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS,” (2007).

Figure 3: Selected calibration curves.

100 200Amount (xxx)

400300100

5

0 0

1

2

3

4

5

10

15

20

11 13 11113 1 1 1Replicates Replicates1 1

11 13 11112 1 1 1Replicates Replicates1 1

M M

200Amount (xxx)

Peak S

ize

Peak S

ize

400300

100 200Amount (xxx)

40030050

5.0

0.0

2.5

0

5

10

15

20

7.5

10.0

12.5M M

100Amount (xxx)

Peak S

ize

Peak S

ize

150

BentazoneCurve Fit Linear, 1/0X2Resp. Fact. RSD: 10.46%. CoeII. Det. (r2):0.998783y = +6.7794e+4x + 1.7995e+4

DepoxetineCurve Fit Linear, Ignore, 1/nX2Resp. Fact. RSD: 11.45%. CoeII. Det. (r2):0.998810y = +4.9513e+4x + -1.9343e+4

BentazoneCurve Fit Linear, Ignore, 1/nX2Resp. Fact. RSD: 7.925%. CoeII. Det. (r2):0.996309y = +4.2244e+4x -940.3596

DepoxetineCurve Fit Linear, Ignore, 1/nX2Resp. Fact. RSD: 6.052%. CoeII. Det. (r2):0.999951y = +1.0610e+4x + -2882.1665

Table II: Test results for selected PPCPs in real water samples

Compound NameTap Water

1

Tap Water

2

Creek

Water

Bottle

Water

Trimethoprim <2 <2 5 <2

Hydroxyatrazine 4 <2 7 <2

Thiabendazole ND <2 <2 <2

Ciproxacin ND ND ND ND

Caffeine ND <2 <2 10

Sildenaf l ND ND ND <2

Sulfamethoxazole <2 <2 ND <2

Cyanazine ND ND ND <2

Simazine 3 <2 5 ND

Metribuzin ND ND ND ND

Hexazinone 17 3 3 ND

Dapoxetine ND ND ND ND

Bentazone ND ND ND ND

Ametryn ND ND <2 ND

Carboxine ND ND ND ND

Carbamazepine <2 <2 <2 ND

Atrazine <2 ND ND ND

Alpazolam ND ND ND ND

Diuron 9 <2 6.2 ND

Prometryn ND ND ND <2

2,4-D 9 <2 13 <2

MCPA <2 <2 <2 ND

Mecoprop <2 <2 11 2

Metolachlor 22 <2 <2 <2

Pyriproxifen ND <2 ND <2

Bruker Daltonics Inc.40 Manning Road, Billerica, MA 01821

tel. (978) 663-366-, fax (978) 667-5993

Website: www.bruker.com



ENVIRONMENTAL

In recent years, the increasing cost and uncertain availability of he-

lium have prompted many labs to explore the use of hydrogen as an

alternate carrier gas. While switching from helium to hydrogen can

be problematic for methods using a mass spectrometer, methods

that employ an electron capture detector (ECD) are generally good

candidates for transfer. For commonly used ECD methods that use

helium, such as organochlorine pesticides analysis following EPA

Method 8081, conversion to hydrogen provides a signif cant cost

savings opportunity.

In this application note we evaluate the chromatographic per-

formance of Rtx®-CLPesticides and Rtx®-CLPesticides2 columns

using both helium and hydrogen carrier gas in order to deter-

mine if using hydrogen is a viable alternative to helium for or-

ganochlorine pesticides analysis. These columns were selected

for the comparison because they were developed specif cally for

organochlorine pesticides analysis by GC-ECD, and their unique

column selectivities provide enough separation between critical

peaks to allow analysis times to be reduced using an accelerated

oven program.


A mixed standard solution was prepared using the following individ-

ual Restek® standards: organochlorine pesticide mix AB #2 (cat.#

32292); hexachlorobenzene (cat.# 32231); 2,4,5,6-tetrachloro-m-

xylene (cat.# 32027); decachlorobiphenyl (BZ #209) (cat.# 32029).

Analyte concentrations in the mixed standard solution are given in

Figure 1. The mixed standard was analyzed using helium and then

again using hydrogen carrier gas on an Agilent® 6890 GC equipped

with a micro-ECD set at 340 °C. To convert from helium to hydrogen

carrier gas, we adjusted the f ow rate such that the linear velocity in-

creased and kept the same aggressive oven program. A dual column

set up was used and chromatographic conditions are as follows.

GC columns: Dual column analysis

Rtx®-CLPesticides2 30 m, 0.32 mm ID,

0.25 μm (cat.# 11324)

Rtx®-CLPesticides 30 m, 0.32 mm ID,

0.32 μm (cat.# 11141)

Inj. vol.: 2 μL (splitless, hold 0.3 min)

Liner: Sky® 4 mm single taper w/wool (cat.# 23303)

Inj. temp.: 250 °C

Oven temp.: 120 °C to 200 °C at 45 °C/min to 230 °C

at 15 °C/min to 330 °C at 30 °C/min (hold 2 min)

Carrier gas: Helium (constant f ow), linear velocity: 71 cm/s

Hydrogen (constant f ow),

linear velocity: 81 cm/s


Despite cost and availability concerns, helium continues to be

commonly used as a carrier gas because it provides excellent sep-

arations in a reasonable analysis time. Hydrogen is a popular al-

ternative to helium carrier gas because it is less expensive and its

higher optimal linear velocity means faster f ow rates can be used.

Faster f ow rates result in shorter analysis times and increased

sample throughput. Analytical columns that perform well using

either helium or hydrogen are advantageous as they give labs the

f exibility to use either carrier gas.

The Rtx®-CLPesticides column set effectively separated critical

organochlorine compounds using either helium (Figure 1) or hy-

drogen (Figure 2) carrier gas. The selectivity of the column set

provided enough resolution between peaks that an aggressive

multi-ramp oven program could be used to obtain sub-7 min run

times. In order to achieve the separations shown here, it may be

necessary to use an oven pillow or 220 V instrument; otherwise,

Fast Organochlorine Pesticides GC-Micro-ECD Analysis Using Either Helium or Hydrogen Carrier Gas Jason Thomas, Chris English, Jack Cochran, and Gary Stidsen, Restek Corporation

Time (min)

21

22

23

1

2

3

4

5

6 7 89

10

11

12 13 1415

1617 18

1920

21

22

23

1

2

3

4

5

67

89 10

1112

13 1415

16 17 18

19

20

2 3 4 5 6 7

Time (min)2 3 4 5 6 7

Rtx®-CLPesticides2

Rtx®-CLPesticides

6.95 min.

Figure 1: Rtx®-CLPesticides columns provide fast analysis times us-ing helium, allowing sample throughput to be maximized. Peak iden-tif cation: 1. Tetrachloro-m-xylene (20 μg/mL), 2. Hexachlorobenzene (5 μg/mL), 3. α-BHC (5 μg/mL), 4. γ-BHC (5 μg/mL), 5. β-BHC (5 μg/mL), 6. δ-BHC (5 μg/mL), 7. Heptachlor (5 μg/mL), 8. Aldrin (5 μg/mL), 9. Heptachlor epoxide (5 μg/mL), 10. trans-Chlordane (5 μg/mL), 11. cis-Chlordane (5 μg/mL), 12. Endosulfan I (5 μg/mL), 13. 4,4’-DDE (10 μg/mL), 14. Dieldrin (10 μg/mL), 15. Endrin (10 μg/mL), 16. 4,4’-DDD (10 μg/mL), 17. Endosulfan II (10 μg/mL), 18. 4,4’-DDT (10 μg/mL), 19. Endrin aldehyde (10 μg/mL), 20. Endosulfan sulfate (10 μg/mL), 21. Methoxychlor (50 μg/mL), 22. Endrin ketone (10 μg/mL), 23. Decachlorobiphenyl (20 μg/mL).



ENVIRONMENTAL

acceptable resolution may not be obtained because the actual

oven temperatures and ramp rates may be lower and slower than

the instrument settings.

Note that the analysis time was reduced from 6.95 min us-

ing helium to just 6.75 min when using hydrogen. Usually when

switching from helium to hydrogen a greater difference in time

savings is seen; however, the analysis times are comparable in this

case because the optimized selectivity of the Rtx®-CLPesticides

column pair allowed helium to be used at an extremely fast f ow

rate that was well above its optimal linear velocity.

Since good chromatographic separations were achieved in rela-

tively short analytical run times on the Rtx®-CLPesticides column

set, either carrier gas could be used and the choice can be based

on lab priorities. If the expense and availability of helium is a con-

cern, hydrogen is a viable option. However, safety must also be

considered when switching to hydrogen, and hydrogen generators

are recommended as they minimize much of the risk.

One issue with using hydrogen that receives less attention than

safety concerns is the potential for chemical reactions that create

active sites and decreased peak response. This can be caused

by metal shavings in the liner catalyzing a hydrogenation reac-

tion between the hydrogen carrier gas and the pesticides. Since

metal shavings are generated by the needle scraping a metal

needle guide, a Merlin Microseal septum and a 23-gauge needle

should be used with hydrogen carrier gas in order to minimize this

problem. The wear-resistant f uorocarbon elastomer construction

of the Merlin Microseal, in combination with its two-stage sealing

mechanism and rounded needle, greatly reduce the generation of

metal particles in the liner.

Conclusions

The Rtx®-CLPesticides column set used here provided good peak

separations and fast analytical run times for organochlorine pes-

ticides analysis when using either helium or hydrogen carrier gas.

Since all compounds were well resolved and could be accurately

integrated with both gases, this column pair gives labs the f ex-

ibility to improve productivity using either carrier gas.

To learn more, visit www.restek.com/alt-gas

20

21

22

23

1

2

34

5

67 8 9 10 1211

13 1415

16 17 1819

21

22

23

1

2

34

5

6

7 8

9 10 1112

13 14

15 16 17 18

19 20

Time (min)2 3 4 5 6 7

Time (min)2 3 4 5 6 7

Rtx®-CLPesticides

Rtx®-CLPesticides2

6.75 min

Figure 2: Using hydrogen carrier gas saves money, reduces helium dependence, and speeds up analysis times for organochlorine pesti-cides analysis. Peak identif cation: See Figure 1.

Restek Corporation110 Benner Circle, Bellefonte, PA 16823

tel. (800) 356-1688, fax (814) 353-1309

Website: www restek.com/alt-gas



ENVIRONMENTAL

Organophosphate pesticides (OPPs) are widely used globally for ag-

ricultural pest control. Unlike chlorinated pesticides (OCPs), OPPs

are less persistent in the environment but offer greater acute toxicity.

Additionally, OPPs are highly water soluble which makes them of

particular concern in municipal drinking water supplies.

Analytically, OPPs can pose some challenges due to the wide di-

versity of physical structures and behavioral characteristics of indi-

vidual analytes. This often results in individual extraction protocols

being implemented for labs to meet regulatory compliances. With

the FMS, Inc. TurboTrace® ABN Multi Cartridge SPE system, and its

ability to incorporate dual cartridge extractions, it is possible to pair

analytes with dissimilar characteristics into a single, fully automated

extraction process.

• FMS, Inc. TurboTrace® ABN multi-cartridge SPE system

• FMS, Inc. SuperVap® 12 Concentrator

• FMS, Inc. 50 mL direct to vial concentrator tubes

• Thermo Trace Ultra GC with Polaris Q

• Agilent 7890 GC with FPD detector

Consumables

• FMS, Inc. 1 g C18 cartridges

• Restek Resprep 2 g coconut charcoal cartridges (Cat# 26032)

Sample Prep

1. Liter drinking water samples were spiked with target analytes of interest.

2. 1 mL of 6N HCl is added to each sample to bring the pH <2.

3. Sample bottles are f tted onto SPE extraction system

4. 1 g C18 cartridges (position #1) and coconut charcoal cartridges

(position #2) f tted on SPE system.

SPE Procedure

1. SPE cartridges are pre-conditioned with 5 mL MeCl each

2. SPE Cartridges are pre-conditioned with 10 mL MeOH each

3. SPE Cartridges are pre-conditioned with 20 mL H2O each

4. Samples are loaded across cartridge at a 20 mL/min rate via

vacuum

5. SPE cartridges are Nitrogen dried for 10 min to remove excess

water.

6. DVB cartridge eluted with 5 mL EtAC.

7. Sample containers are rinsed with 15 mL MeCl and eluted though

DVB cartridge (10 mL/min).

8. 5 mL additional MeCl eluted through the C18 cartridge.

9. Coconut charcoal cartridges eluted with 15 mL MeCl.

10. Residual water removed by NaSO4 in-line cartridge f ltration and

eluate emptied directly into Super Vap concentrator tubes.

Automated Multi-Cartridge Solid Phase Extractionof Phosphorus Containing Pesticides in Drinking Water FMS

Conclusions

Extraction of the drinking wa-

ter samples on the Turbo Trace

ABN system enabled a fully au-

tomated extraction process due

to its dual cartridge capabilities.

Samples were loaded simultane-

ously across the C18 and coconut

charcoal cartridges in succession.

Cartridges were then able to be

eluted independently with no

manual transferring or handling of

cartridges. Elutions then could be

either independently fractionated

for individual analysis, or com-

bined for a single GC run by uti-

lizing the system’s 3 fraction lines.

Analysis of water extracts

showed consistent instrumental performance by both GC–MS-MS and

GC–FPD detection. Recoveries for C18 bonding pesticides displayed

excellent precision with all falling between 80–120%. Methamidophos

consistently recovered >60% from the coconut charcoal cartridge when

using 1 L samples. Higher recoveries were observed when using re-

duced sample volumes indicating 2 g coconut charcoal cartridges were

better suited for samples ≤500 mL.

Mean Recoveries

Analyte FPD MS-MS Dev.

Analyte FPD MS-MS Dev

Methamidophos 63 67 2.7

Monocrotophos 96 91 3.8

Diazinon 112 97 14.6

Parathion 99 113 7.3

EPN 85 103 16.8

Results run in triplicate by both GC/Ion Trap and GC/FPD.

SuperVap

1. Preheat temp: 20 min at 60 °C

2. Evap. mode w/sensor temp: 60 °C

3. Nitrogen pressure: 10 psi

4. Samples reduced to 1 mL

5. Samples reduced to 1mL f nal volume

FMS Inc. Turbo Trace ABN Multi cartridge system.

FMS, Inc.580 Pleasant Street, Watertown, MA 02472

tel. (617) 393-2396, fax (617) 393-0194

Website: www.fms-inc.com



FOOD & BEVERAGE

Organic arsenic compounds added to feed stocks of chicken and

poultry pose serious environmental and ecological threats. Nation-

al and worldwide health organizations, such as the United States

Environmental Protection Agency and the US Food and Drug

Administration, have recently implemented more stringent concen-

tration limits for arsenic species in drinking water and foodstuffs.

Analysis of arsenic species is made challenging due to diverse

sample matrices. To circumvent these problems, an HPLC ICP-MS

method has been developed. Resolution of arsenic compounds is

achieved by ion exchange chromatography on a Hamilton PRP-X100

column, a 55% cross-linked polystyrene-divinylbenzene copolymer

functionalized with quaternary ammonium anion-exchanger group.

Detection is accomplished by inductively coupled plasma-mass

spectrometry.

Hamilton Company4970 Energy Way, Reno, NV 89502

tel. (775) 858-3000, (800) 648-5950

Website: www.hamiltoncompany.com

Arsenic Speciation on Hamilton PRP-X100 Hamilton Company

149.811.41

DMA∨ + DMA

108.091.20

66.371.00

24.650.79

-17.069.41

Time (min)

Inte

nsi

ty (

cps)

0.0 3.39 6.79 10.18 13.57 16.97

=

MMA + MMA∨

MMTA

AS∨

=

AS ≡

Figure 1: Borrowed and adapted (with permission) from P. Alava et al., Biomed. Chromatogr. 26, 524–533 (2012).

Table I: Chromatographic conditions

Column: Hamilton PRP-X100, 5 µm, 4.6 × 250 mm

Part Number: 79181

Flow Rate: 1.0 mL/min

Mobile Phase:

2 mM (NH4)2CO3 for 0–3 min



Injection Volume: 50 µL, 100 µg/L of each standard

Detection: ICP-MS

Ordering Information

Part

Number

Product

Name

Particle

SizeDimensions Material

79852 PRP-X100 5 µm 2.1 × 150 mm PEEK

79190 PRP-X100 5 µm 2.1 × 250 mm Stainless Steel




79174 PRP-X100 5 µm 4.6 × 150 mm PEEK

79181 PRP-X100 5 µm 4.6 × 250 mm PEEK







79354 PRP-X100 10 µm 4.6 × 150 mm PEEK

79455 PRP-X100 10 µm 4.6 × 250 mm PEEK

79353 PRP-X100 12-20 µm 21.2 x 250 mm Stainless Steel



FOOD & BEVERAGE

Deoxynivalenol is a common mycotoxin found in agricultural grain

crops and f nal consumer processed products. Most impacted are

wheat, barley, and corn. Deoxynivalenol, also known as Vomitoxin,

has the ability to withstand high processing temperatures, creating

the need for rapid and accurate determination methodology. Current-

ly, the United States has an advisory limit for deoxynivanol of 1 ppm

(or 1000 μg/kg) in f nished foodstuffs. Current European Union (EU)

legislation sets the maximum level of deoxynivalenol in foodstuffs at

0.75 ppm (750 μg/kg) for unprocessed cereals marketed for direct

consumer consumption and 0.2 ppm (200 μg/kg) for processed

cereal-based foods and foods intended for babies and small children.

The standard AOAC methodology uses liquid-liquid extraction, fol-

lowed by solid phase extraction with immunoaff nity cartridges. In this

application, a sample of shredded wheat cereal was tested for the

presence of deoxynivalenol and spiked at the EU legislation level of

200 μg/kg for recovery. Simple automation of the SPE process us-

ing the VICAM® DONtest WB immunoaff nity cartridges was imple-

mented using the Horizon Technology SmartPrep® Extractor, followed

by evaporation with the XcelVap® and analysis using a Shimadzu

Nexera XR UHPLC with UV detection at 220 nm.

Conclusions

Data presented concludes that deoxynivalenol was effectively re-

covered and passed performance criteria for % recovery at the EU

limits of 200 µg/kg. Implementing automation with the SmartPrep

Extractor also reduces “scientist bias” by implementing uniform

treatment of all samples. Limiting manual preparation for routine

laboratory food safety testing will signif cantly increase and improve

laboratory workf ow and determine deoxynivalenol levels more con-

sistently in processed foods and animal feed.

References

(1) “Determination of Deoxynivalenol in Shredded Wheat Cereal using Auto-

mated Solid Phase Extraction with Immunoaffinity Cartridges,” Toni

Hofhine, Horizon Technology, Inc., Elizabeth K. Krantz, Dr. Pamela Doolittle,

and Dr. Cheri Barta, University of Wisconsin-Madison, Application Note

AN891407_01, available at www.horizontechinc.com.

Determination of Deoxynivalenol in Shredded Wheat Cereal Using Automated Solid Phase Extraction with Immunoaff nity CartridgesToni Hofhine*, Elizabeth K. Krantz†, Pamela Doolittle, PhD†, and Cheri A. Barta, PhD†, *Horizon Technology*, Inc. and †University of Wisconsin-Madison

Figure 1: Shredded wheat cereal control spiked at 200 µg/kg. Some native concentration was observed in the unspiked cereal.

Horizon Technology, Inc.16 Northwestern Drive, Salem, NH 03079

tel. (603) 893-4494

Website: www.horizontechinc.com



FOOD & BEVERAGE

Two species of spearmint oil (Mentha spicata and Mentha graci-

lis) are cultivated in the United States. In 2008, 1.09 million kg of

spearmint oil were produced in the US (1). Approximatley, 45% of

mint oil produced in the US is used to f avor chewing gum. One

55-gallon drum of mint oil can f avor 5,200,000 sticks of gum or

400,000 tubes of toothpaste (2).

Spearmint oil is stored in glands on the underside of leaves. Mature

mint plants are cut and left to dry before being chopped and transferred

to a distillery. Pressurized steam vaporizes the mint oil, which passes

through a condenser to be collected as a liquid. A separator takes mint

oil from the liquid and transfers it into drums that are placed in a tem-

perature-controlled warehouse. Essential oil companies test samples at

this point to decide whether to purchase the oil. Gas chromatography

is commonly used to assess oil quality. Oils containing impurities may

undergo rectif cation, a re-distillation step used to purify the oil.

Volatile sulfur compounds impart undesirable odors to essential

oils and have extremely low olfactory thresholds. Essential oil com-

panies require a rapid screening technique to detect and quantify

volatile sulfur compounds.


Instrumentation used in this study was an Agilent 7890A GC equipped

with an OI Analytical 5380 Pulsed Flame Photometric Detector. Two

different samples of neat spearmint oil were tested. The identity of

sulfur compounds in the sample was unknown, only suspected.

Results

The PFPD provides two independent channels of data. One chan-

nel provides a carbon chromatogram and the second channel a

sulfur chromatogram. The PFPD carbon channel chromatograms

contained 65 peaks which were compared to the client’s current

FID chromatograms. The percent area report obtained for carbon

from the PFPD closely matched the expected carbon percentages

from the FID detector for hydrocarbon peaks.

The PFPD sulfur chromatograms contained nine peaks. Five

peaks were conf rmed as sulfur peaks in the spearmint oil using the

integration time gate function of the PFPD and WinPulse software.

The percent total sulfur of the smallest peak that was detected and

conf rmed was .00206%.

Conclusions

The study demonstrated that low-level sulfur compounds can be

detected and isolated in spearmint oil using an OI Analytical 5380

PFPD detector, dual integration time gates, and comparative carbon

peak matching with FID chromatograms.

References

(1) V.D. Zheljakov, C.L. Cantrell, T. Astatkie, and M.W. Ebelhar, “Productivity,

Oil Content, and Composition of Two Spearmint Species in Mississippi,”

Agronomy Journal, Vol. 102, Issue 1 (2010).

(2) Mint Industry Research Council, www.usmintindustry.org.

Detection of Low-Level Sulfur Compounds in Spearmint Oil Using the Pulsed Flame Photometric Detector (PFPD) Gary Engelhart and Cynthia Elmore, OI Analytical

OI AnalyticalP.O. Box 9010, College Station, TX 77842

tel. (800) 653-1711 or (979) 690-1711, fax (979) 690-0440

Website: www.oico.com

Figure 1: PFPD carbon channel chromatogram of spearmint oil show-ing 65 peaks labeled with retention times.

Figure 2: PFPD sulfur channel chromatogram of spearmint oil show-ing nine peaks labeled with retention times.



FOOD & BEVERAGE

This study illustrates the successful combination of fast GC

and tandem mass spectrometry to determine 360 pesticides

spiked in a QuEChERS apple extract.

To handle the high number of monitored pesticides, a quick, easy,

and cheap cleanup procedure called QuEChERS was used (1).

Although the QuEChERS technique has signif cant advantages,

samples prepared by this method still contain matrix interferences

that can complicate accurate pesticide quantif cation.

Tandem MS instruments using multiple reaction monitoring

(MRM) mode have become the analytical technique of choice for

analysis of pesticides in food matrices, as they provide increased

sensitivity and selectivity by virtually eliminating the background

matrix interference. The following presents an example of analyzing

360 pesticides spiked in apple extract.

Experimental

The apple sample matrix was extracted and subjected to cleanup

using the QuEChERS procedure. A 6-point calibration curve (0.5

ppb to 100 ppb) was created by spiking the blank sample matrix.

The spiking solution contained 360 different pesticides, and used

TPP as internal standard.

A Shimadzu GCMS-TQ8040 equipped with a GL Science Optic-4

multi-mode inlet and an AOC-5000 Plus was used for sample mea-

surement. Method MRMs and collision energies (CE) were taken

from Shimadzu’s Smart Pesticide Database. All compounds were

measured with one quantif er and one qualif er MRM transition.

Results

Figure 1 shows the full chromatogram of the 360 pesticides; all

compounds elute in less than 10 min. Signif cant co-elution of

pesticides within the analysis time is evident.

Matrix-matched calibration curves (0.5–100 ppb) were created

for all 360 pesticides. The linear correlation factor was 0.998 or

better for all compounds. Most components were detected at the

lowest concentration of 0.5 ppb.

To assure repeatability, at least 10 data points per peak are

required (2). To enable this number of data points, a loop time of

0.18 s was chosen. The shortest dwell time per MRM was 3 ms. At

this short dwell time, instrument precision and speed become very

important to obtain good repeatability for all measured transitions,

as illustrated in Figure 2.

Conclusion

This study illustrates the successful combination of fast GC and

tandem mass spectrometry. It was possible to determine 360

pesticides spiked in a QuEChERS apple extract with excellent cali-

bration curve linearity and good repeatability in less than 10 min.

References

(1) QuEChERS, European Standard, EN 15662,

(2) K. Mastovska and S.J. Lehotay, J. Chromatogr. A 1000, 153–180 (2003).

Fast GC–MS-MS Analysis of Multicomponent Pesticide Residues (360) in Food MatrixHendrik J. Schulte*, Hans-Ulrich Baier*, Stéphane Moreau*, Robert H. Clifford†, and Laura Chambers†,

*Shimadzu Europa GmbH, Duisburg, Germany and †Shimadzu Scientif c Instruments, Columbia, Maryland

Shimadzu Scientif c, Inc.7102 Riverwood Drive, Columbia, MD 21046

tel. (800) 477-1227

Website: www.ssi.shimadzu.com

2.01.50

2.0

1.5

1.0

0.5

1.25

1.00

0.75

0.50

0.25

(x100,000)1(#1)1(#2)1(#3)1(#4)1(#5)

(x100,000)1(#1)1(#2)1(#3)1(#4)1(#5)

(x100,000)1(#1)1(#2)1(#3)1(#4)1(#5)

1.5

1.0

0.5

7.00 5.03 5.25 7.00 7.20

Trifoxystrobin, %RSD = 7.6 Isazofos, %RSD = 6.4 Diazinon, %RSD = 4.6

7.20

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.03.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

(x1,000,000)

Figure 2: Overlaid MRM chromatograms of f ve replicate peaks (un-smoothed) for three compounds measured with 3 ms dwell time.

Figure 1: MRM chromatogram of 360 pesticides in apple matrix.



FOOD & BEVERAGE

A New HILIC Column for Saccharide AnalysisMelissa Turcotte* and Naoya Nakajima†,

*Showa Denko America, Inc. and †Showa Denko K.K.

Due to the highly polar nature of saccharides, the analysis of sugars

is typically achieved using hydrophilic interaction chromatography

(HILIC). Using a high polarity packing material, separation is based

on the hydrophilic interactions of the sugars on the stationary phase

surface typically using an eluent with a majority of polar organic solvent.

HILIC columns with amino functional groups are often used to

prevent the separation of anomers; however, one drawback of the

amino functional group on the packing material is the low recovery

rate of reducing sugars, such as glucose and mannose. Reducing

sugars are able to adhere to the packing material through the forma-

tion of a Schiff base, and must be hydrolyzed with acid for removal.

Shodex introduces the NGP-50 4D column packed with a durable

polymer based packing material modif ed with chemically stable tertiary

amino functional groups. Shodex NGP-50 4D is suitable for saccharide

analysis, has demonstrated the separation of reducing sugars, and has

been compared to two competitor silica-base amino columns.


The analysis of four saccharides is accomplished with Shodex NGP-

50 4D (4.6 mm ID × 150 mm, 5 μm), a polymer-based amino

HILIC column. The separation is compared to the same analysis

Shodex™/Showa Denko America, Inc.420 Lexington Avenue Suite 2335A, New York, NY, 10170

tel. (212) 370-0033 x109, fax: (212) 370-4566

Website: www.shodex.net

TM

Figure 1: The analysis of sugars using NGP-50 4D and two silica-based amino columns. Column: Shodex NGP-50 4D (top) and silica-based amino columns (middle and bottom), column temperature: 40 °C, injection volume: 5 µL, eluent: CH3CN/H2O = 80/20, f ow rate: 0.4 mL/min for NGP-5- 4D and 1.0 mL/min for silica columns. Detector: Shodex RI. Sample: 1. fructose, 2. mannose, 3. glucose, 4. sucrose.

Figure 2: Comparison of Shodex NGP-50 4D and silica-based amino columns demonstrating recovery ratio of mannose. Recovery ratio = (peak area mannose)/(peak area sucrose).

1

1

1

2

2

2

3

3

3

4

4

4

5 10 15 20 25

NGP-50

Silica-based amino column 1

Silica-based amino column 2

100

Re

cove

ry r

ate

(%

) 90

8070

605040

3020100

NPG–50 amino column 1 amino column 2

using two silica-based amino columns under the same conditions.

Column temperature was 40 °C and f ow rate was 0.4 mL/min for

the NGP-50 4D analysis and 1.0 mL/min for the silica-based amino

analyses. Eluent conditions are 80% acetonitrile in water. Injection

volume of 5 μL of 5 mg/mL of each sugar was used for each experi-

ment. The HPLC system was coupled with RI detector.

Results

The saccharides, fructose, mannose, glucose, and sucrose, were

analyzed successfully by HPLC and RI detection with NGP-50 4D

and two silica-based amino columns (Figure 1). A comparison

of the recovery ratio of mannose, a reducing sugar, in relation to

sucrose, a non-reducing sugar, is demonstrated in Figure 2. Shodex

NGP-50 4D demonstrates baseline separation of the four sugars

with an elution volume of 20 min and allows up to 90% recovery

rate of mannose. Silica-based amino column 1 shows baseline sep-

aration of the four compounds; however, the recovery ratio of reduc-

ing sugars is 40%. Silica-based amino column 2 cannot separate

the four sugars, and the recovery ratio is less than 20%.

Conclusions

Shodex NGP-50 4D, a polymer-based hydrophilic interaction

(HILIC) chromatography column suitable for saccharide analysis,

has demonstrated the recovery ratio of reducing sugars compared

with silica-based amino columns.



FOOD & BEVERAGE

Determination of Carbendazim in Orange Juice Using an Automated QuEChERS SolutionTyler Trent, Teledyne Tekmar

Carbendazim is a widely used, broad-spectrum fungicide to control

mold on citrus crops. This fungicide is approved for use in Brazil and

several other countries, but is not approved for use in the United States

or on imported products. This has lead to increased testing of orange

juice for pesticide residues.

With recent advancement in multiresidue pesticide screening, meth-

ods have been simplif ed by the introduction of QuEChERS. Both the

AOAC Off cial Method 2007.01 and EN15662:2008 require several

manual steps to extract the pesticides of interest (1,2). The AutoMate-

Q40 streamlines the QuEChERS method by automating the addition of

acetonitrile (ACN) and buffering salts; shaking, mixing, and centrifuging

the sample; transferring the sample to a dispersive solid phase extrac-

tion (d-SPE) tube using an air displacement pipetter (ADP); and f nally

measuring and delivering the extract. The intent of this poster is to eval-

uate the performance of the AutoMate-Q40 by monitoring carbendazim

in orange juice. The target pesticide will be analyzed using LC–MS-MS.

Extraction Procure

Figure 1 shows the f ow chart for the AOAC QuEChERS extraction

procedure using the AutoMate-Q40 without the dSPE cleanup step.

Teledyne Tekmar4736 Socialville Foster Rd., Mason, OH 45040

tel. (513) 229-7000

Website: www.teledynetekmar.com Figure 1: AutoMate-Q40 extraction procedure for orange juice.

Figure 2: AutoMate-Q40 automated QuEChERS platform.

Table I: Carbendazim recovery data

Spike Level (ng/mL) % Recovery %RSD

10.0 94.7 3.9

60.0 92.3 3.3

100.0 91.4 6.2

Conclusion

A precision and accuracy study was performed using the AutoMate-

Q40. A 6 µg/mL stock pesticide standard was used to fortify the

samples through the AutoMate-Q40 spiking system. The orange

juice samples were spiked with 25, 150, and 250 µL of the pesti-

cide standard yielding 10 ng/mL, 60 ng/mL and 100 ng/mL check

samples. These QC samples were quantitated against their corre-

sponding matrix-matched calibration curve.

Table I demonstrates the excellent recoveries achieved when using

the AutoMate-Q40, ranging from 91.4% to 94.7%. The AutoMate-Q40

also demonstrates greater precision ranging from 3.3% to 6.2% RSD.

References

(1) AOAC Official Method 2007.07 Pesticide Residues in Food by Acetonitrile

Extraction and Partitioning with Magnesium Sulfate. Gas Chromatography/

Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry,

First Action 2007.

(2) European Committee for Standardization/Technical Committee CEN/TC275

(2008), Foods of plant origin: Determination of pesticide residues using

GC–MS and/or LC–MS-MS following Acetonitrile extraction/ partitioning and

cleanup by dispersive SPE QuEChERS-method.



INDUSTRIAL

Starch is used for a variety of industrial and nutritional purposes. Its

functional properties are inf uenced by the ratio and molar masses

of its macromolecular constituents, which vary with source, crop

year, and climate. Starch contains large homopolymers of amylose

(AMY) and amylopectin (AMP).

Linear AMY consists of long chains of (1⇒4)-α-D-glucose link-

ages, while the higher molar mass AMP is a branched structure con-

taining a mixture of (1⇒4)-α- and (1⇒6)-α-D-glucose linked resi-

dues. The goal of this work was to apply AF4-MALS-RI to separate

AMY and AMP in order to calculate a mass ratio, to determine the

molar mass distributions, the average molecular weights (Mw), and

the mean-square radius (Rz) of the AMP component. We applied the

technique to starches with AMY:AMP ratios covering a wide range.

An Eclipse AF4 system (Wyatt Technology) was equipped with a

short (18 cm) channel, a 350 μm spacer, and a regenerated cel-

lulose (10 kDa cutoff) membrane. Detection was accomplished with

DAWN Multi Angle Light Scattering (MALS) and Optilab RI detectors

(both instruments Wyatt Technology). The channel f ow was main-

tained at 1.0 mL/min and the cross-f ow was varied linearly from 1.0

to 0.1 mL/min for 10 min, then abruptly switched to 0.0 mL/min.

Integration of RI peak areas enabled calculation of the AMY:AMP

ratios, in excellent agreement with the nominal values. The values

for Mw and R

z fall within the generally accepted limits found in the

literature. Conformational plots for the AMP component verify its

branched nature.

This note graciously submitted by Rick White and Eija Chiaramonte, Global Analytical Sciences—

Personal Health, The Procter & Gamble Company, Mason, OH.

DAWN®, miniDAWN®, ASTRA®, Optilab® and the Wyatt Technology logo are registered trade-

marks of Wyatt Technology Corporation. ©2013 Wyatt Technology Corporation 4/4/13

AFFF-MALS-RI for Determining the Mass and Size Distributions of Amylose and Amylopectins in Starch Wyatt Technology

Wyatt Technology6300 Hollister Avenue, Santa Barbara, CA 93117

tel. +1 (805) 681-9009, fax +1 (805) 0123

Website: www.wyatt.com

Figure 2: Conformation plot (log Rz versus log Mw) for the amylopectin component of f ve starches (Slopes 0.39–0.41 indicative of branching).

Figure 1: AF4-MALS-RI results for f ve native starches of varying AMY:AMP ratio: AF4-RI fractograms with molar mass distributions overlayed. (Cross-f ow (Vx) = 1.0 to 0.1 mL/min in 10 min, then Vx = 0.0 mL/min).



PHARMA/DRUG DISCOVERY

Optimizing separation of ionizable compounds in order to find

robust conditions has become an important part of method

development in liquid chromatography. In this work, adjustment

of pH is used to control selectivity using a Poroshell HPH-C18

column that is designed to be stable in high pH mobile phases.

Use of pH to Affect Selectivity

Figure 1 depicts how the elution order of a mixture consisting of

acidic, basic, and neutral compound changes as pH of the mobile

phase is changed. In this work a generic gradient is used with the

organic modif er (acetonitrile) concentration changing from 10% to

90% over 4 min. Chromatograms at pH 3 (ammonium formate), pH

4.8 (ammonium acetate), and pH 10 (ammonium bicarbonate) are

shown using mass spec compatible buffers. The f ow rate used in

this work is 2 mL/min.

As can be seen, the three chromatograms use the same gradient and

column, the neutral (hexanophenone) and non-ionized compounds

(caffeine) remain at the same elution time. They are not affected by

the change in pH. As the mobile phase pH is increased from pH 4.8

to pH 10, the acidic compounds become charged and their retention

time decreases. This is depicted by the red arrows in Figure 2. As the

pH is increased the retention time of the bases increases as shown with

the blue arrows. The peak elution order changes dramatically as does

the spacing. In all three chromatograms the peak shape is excellent. In

this case, the spacing of the compounds is more even using the pH 10

buffer than either of the other buffers. In addition to longer retention

of bases, better peak shape is also found when using high pH mobile

phases as compared to low pH mobile phase.

Another way to look at selectivity is by plotting retention time

using two different conditions for a group of acids, bases, and neutral

compounds. In this case, 117 compounds are run using the Poroshell

HPH-C18 column using identical gradients using two organic modif ers

(methanol and acetonitrile) and in two pHs (pH 3 and pH 10). The

generic gradient used in this work was 0.42 mL/min, starting at 5%

organic and increasing to 95% organic over four min, and holds at this

concentration for 2 min.

As can be seen in Figure 2a, a subgroup of analytes line up per-

fectly with a slope of 1, these compounds are neutral or non-ion-

izable compounds with methanol as the organic modif er. These

include substituted benzenes, steroids, phenols, and phenones,

Use of Poroshell HPH-C18 Columns at Elevated pH as a Tool for Method DevelopmentWilliam Long, Agilent Technologies, Inc.

min0 0.5 1.51 2 2.5 3 3.5

min0 0.5 1.51 2 2.5 3 3.5

min0 0.5 1.51 2 2.5 3 3.5

mA

Um

AU

mA

U

0

50

100

150

200

250

300

0

50

100

150

200

250

300

0

50

100

150

200

250

300

8

8

8

7

7

7

6

6

6

5

5

5

4

4

4

3

3

3

2

2

2

1

1

1

Poroshell HPH C-18 4.6 × 50 mm, 2.7 µm

Bases

Acids

1. Procainamide 2. Caffeine3. Acetyl Salicylic Acid4. Hexanophenone Deg.5. Dipyrimadole 6. Diltiazem 7. Difunisal 8. Hexanophenone

pH 3 10 mM HCO2NH4

pH 10 10 mM NH4HCO3

pH 4.8 10 mM NH4C2H3O2

Time % Buffer % MeCN

0 10 90

5 90 10

7 10 90

2 ml/min 254 mn

Figure 1: Selectivity control by altering pH with an Agilent Poroshell HPH-C18, 4.6 × 50 mm, 2.7 µm LC column at pH 3, 4.8, and 10.




the retention time of these materials is not affected by the pH

of the mobile phase as expected. This methodology was applied

and discussed in previous work where two highly similar columns

(Poroshell 120 EC-C18 and ZORBAX Eclipse Plus C18) were

compared under similar chromatographic conditions (2). Analytes

that appear above the line are bases. At pH 3 these compounds

are charged, as they become uncharged with the pH increased

to 10, the retention time increases. The correlation coeff cient of

retention times is a measure of the difference of the separation

under two different pH conditions. A highly correlated plot

would have a value close to 1. This would indicate that the

chromatographic separations are very similar. On the other hand,

a very low correlation value (close to 0.5 or lower) indicates a

more orthogonal or dissimilar separation. A second comparison is

also shown in Figure 3b, where a comparison of low and high pH

gradients is made using acetonitrile as the organic modif er. In

this case the correlation coeff cient is smaller than when using

methanol (2,3,4).

Stability of Poroshell HPH-C18 at High pH

HPLC column stability is one of the critical factors impacting method

performance and has been widely studied. Column stability can be

affected by temperature, type of aqueous buffer and their

concentration, choice of organic solvents, additives, and mobile phase

pH. Prescreening of compounds and columns should enable scientists

to arrive at successful separations more quickly. HPLC column stability

is one of the critical factors affecting method performance. A robust

HPLC method using a durable column leads to successful support of

new clinical and manufacturing projects. A column that is not stable

during method development leads to inaccurate results and frustration.

Column degradation is caused by silica dissolution, bonded-phase

removal, or through the exposure of silanols through the removal of

end capping (hydrolysis). Both dissolution and hydrolysis of silica

columns are known to be related to pH and temperature (increased

degradation rate at higher pH/temperatures). Other causes of

column degradation include poor sample preparation (dirty samples)

and column bed instability.

A good criterion for column stability under a given pH is 500

injections. This allows development, adjustment, and use for a

column under established method. In this work, a Poroshell HPH-C18

column was evaluated in a gradient using ammonium bicarbonate and

acetonitrile at pH 10. In this experiment, acidic compounds, neutral

compounds, and basic compounds were used. In order to evaluate

columns from a variety of manufacturers, a common stress gradient

is used while changing the analytes to accommodate differences in

selectivity. In all cases at least two acid, base and neutral compounds

are used.

The impact of sample solution was very minimal, as typically only

a few µg of sample were loaded. The test mixture was chosen to

assess column performance, not to assess the impact of the test probes

themselves on column stability. A low f ow rate was used to minimize

column bed stability problems during development. As can be seen,

the retention time of all compounds remain stable throughout the 2000

injection run with the exception of nortryptyline. This compound, with

a pKa very close to the pH of the mobile phase, moves slowly to longer

retention times.

Retention Time Correlation at Low and High pH(Methanol)

Retention Time Correlation at Low and High pH(Acetonitrile)

Retention Time pH 3 Methanol(min)

R2 = 0.4939 R2 = 0.3996

mHpH_pH10 aHpH_pH10

Rete

nti

on

Tim

e p

H 1

0 M

eth

an

ol

(min

)

7

6

5

4

3

2

1

0 Rete

nti

on

Tim

e p

H 1

0 A

ceto

nit

rile

(min

)

7

6

5

4

3

2

1

00 1 2 3 4 5 6 7

Retention Time pH 3 Acetonitrile(min)

0

(a) (b)

1 2 3 4 5 6 7

Instrument : Agilent 1260 Infnity LC System Gradient:Column : Poroshell HPH C-18 2.1 x 50 mm, 2.7 µm Time 0 min. 5% BMobile Phase : A: 10 mM Ammonium formate adjusted to pH 3 Time 4 min. 95% B in water or 10 mM ammonium bicarbonate adjusted to Time 5 min 95% B pH 10.0 in water, Time 6 min 5 %B B: Methanol (fgure 2a) or acetonitrile (fgure 2b). Time 8 min 5 %BFlow rate : 0.4 mL/min.

Figure 2: Retention time correlation with an Agilent Poroshell HPH-C18, pH 3 versus pH 10, (a) methanol and (b) acetonitrile.




A second column from a major competitor is subjected to the same

experimental conditions. Most of the analytes remain at the same

retention time throughout the 2000 injections. Nortryptyline moves

rapidly to later elution times. Within 500 injections, nortryptyline

begins to coelute with the next compound, neutral hexanophenone.

The peak continues to migrate through this peak, totally coeluting

by injection 2000. This experiment indicates greater degradation

of the competitor column than Poroshell 120 HPH-C18 column.

Differences in peak height occur as the sample is changed.

Conclusion

Using Poroshell HPH C-18, pH can be used to adjust selectivity

without sacrif cing column lifetime at elevated pH conditions. By

keeping a gradient constant and altering pH the elution order of a

group eight acid, base, and neutral compounds can be dramatically

changed, and in this case chromatographic resolution. In a second

experiment, the correlation coeff cient of the retention times was

determined using a generic gradient plotted for pH 3 and pH 10.

Using the R2 as a measure of orthogonally, we f nd that the two

conditions offer different selectivity. Using pH as a method

development tool is very effective, especially when the sample

contains acidic or basic compounds. We determined that

Poroshell HPH-C18 can be used for extended periods (over 2000

injections) at pH 10 in ammonium bicarbonate at 25 °C. By

utilizing pH as a method development tool with Poroshell HPH-C18,

chromatographers can maximize the f exibility in their method

development and analyses, while still benef ting from the rugged,

long lifetime with the Poroshell 120 family.

For more details, and to see additional analysis involving LC–MS,

download Agilent publication 5991-4893EN.

References

(1) Transfer of Methods between Poroshell 120 EC-C18 and ZORBAX Eclipse

Plus C18 Columns, Agilent Technical Report 5990-6580 February 2011.K.

(2) A. Steffens Croes, D. Marchand, and L. Snyder, “Relevance of π–π and

Dipole–Dipole Interactions for Retention on Cyano and Phenyl Columns

in Reversed-Phase Liquid Chromatography,” J. Chromatogr. A, 1098(1–2),

123–130 (2005).

(3) W. Long and A. Mack, “Comparison of Selectivity Differences Among Dif-

ferent Agilent ZORBAX Phenyl Columns Using Acetonitrile or Methanol,”

Agilent Technologies Publication 5990-4711EN, 2009.

Agilent Technologies2850 Centerville Road, Wilmington, DE 19808

Website: www.agilent.com/discoverporoshell

(a) (b)

Instrument : Agilent 1260 Infnity LC System Gradient:Column : Poroshell HPH C-18 2.1 x 50 mm, 2.7 µm (3a); Non-Agilent High Time 0 min. 5% B pH column, 2.1 x 50 mm, 3 µm (3b) Time 4 min. 95% B Mobile Phase : A: 10 mM Ammonium bicarbonate adjusted to pH 10.0 Time 5.1 min 5% B in water or 10 mM ammonium bicarbonate adjusted to pH 10.0 in water, B: Acetonitrile. Flow rate : 0.4 mL/min.

Injection 11

12

2

1.5 2.5 3.5 4.52 3 4 1.5 2.5 3.5 4.52 3 4

1.5 2.5 3.5 4.52 3 4

1.5 2.5 3.5 4.52 3 4

1.5 2.5 3.5 4.52 3 4

1.5 2.5 3.5 4.52 3 4

1.5 2.5 3.5 4.52 3 4

1.5 2.5 3.5 4.52 3 4

334

45 5

6

6

7

7

Injection 500

Injection 2000

pH 10

min

min

min

min

min

min

min

min

Injection 1000

Injection 1

Injection 500

Injection 2000

pH 10

Injection 1000

1. Methyl Salicylate2. 4 Chlorocinnamic acid3. Acetophenone4. Quinine

5. Nortryptyline6. Heptanophenone7. Amitriptylin

Non-Agilent High pH columnAgilent Poroshell 120 HPH-C18

Figure 3: (a) Excellent retention on the Agilent Poroshell HPH-C18, 2.1 × 50 mm, 2.7 µm column even under high pH bicarbonate conditions (total method run time = 7 min, f ow rate 0.4 mL/min). (b) A competitor 3 µm column suffered greater degradation under high pH bicarbonate.




The Agilent 1200 Infinity Series HDR-DAD Impurity Ana-

lyzer System covers a wide linear range typically up to

6000 mAU and enables the analysis of impurities typically

down to 0.2 mAU. Very high and very low concentrations

are determined in one run, saving time and labor.

The determination of very high and very low concentrations in one run

using the same injection volume is often not possible due to the lim-

ited linear range of conventional UV detectors. The Agilent 1200 Inf nity

Series HDR-DAD Impurity Analyzer covers a wide linear range, typically

up to 6000 mAU. In addition, the system typically exhibits low noise and

provides for the quantif cation of peaks with heights below 0.5 mAU. As

examples, the determination of paracetamol and pindolol over a wide

linear range and the analysis of doxycycline and its very low concen-

trated impurities and degradation products were chosen.

Results

The determination of log P, the distribution of a compound be-

tween water and octanol, is frequently used in the pharmaceutical

industry and needs a high linear range to be able to determine

the low respectively high concentrated compound in the water or

octanol phase or vice versa in one run (Figure 1). To test the linear-

ity, paracetamol and pindolol were analyzed with good correlation

from 0.77 ng/µL to 7700 ng/µL and from 0.367 ng/µL to 3670 ng/

µL respectively (1).

When exposed to heat and light, tetracyclines undergo epimer-

ization and degradation. Often, the degradation products have very

HPLC UV Determination of Very High and Very Low Concentrations of Compounds in One RunA.G. Huesgen-Gratzfeld, Agilent Technologies

Figure 1: Three example runs with different concentrations, linearity from 8000 mAU down to < 1 mAU.

Figure 2: Analysis of doxycycline impurities.

2 3

mAU

0

1000

2000

3000

4000

5000

6000

7000

8000

Pa

race

tam

ol

Pin

do

lol

7700ng/µl

3670ng/µl

1.6 1.8 2

6

8

10

12

7.7ng/µl

min 2.2 2.4 2.6 2.8 3

mAU

14

Pa

race

tam

ol

Pin

do

lol

0.77ng/µl

3.67ng/µl

0.367ng/µl

min 1 2 3 4 5

mAU

0

100

200

300

400

500

600

700

800

DAD1 C, Sig=280,10 Ref=off (SOP 210 KI...XY_HDR_START_21JAN2 2014-01-21 15-12-24.SC.SSIZIP\DOXY_START3.D)

Do

xycy

clin

e

min 0.94 0.96 0.98 1 1.02 1.04 1.06 1.08 1.1

mAU

-0.058

-0.056

-0.054

-0.052

-0.05

-0.048

-0.046

-0.044

-0.042PtoP noise ~ 0.013mAU

min 1.6 1.8 2 2.2 2.4

mAU

-0.5

0

0.5

1

1.5

2

2.5

1.5

41

1.7

51

1.9

59

2.1

91

2.3

34

2.3

91

Peak height =0.125mAU

Agilent Technologies Inc.5301 Stevens Creek Blvd., Santa Clara, CA 95051

Website: www.agilent.com

low antibiotic activity, and some are toxic. Doxycycline was exposed

to light and elevated temperature for days to determine decomposi-

tion products. After 5.5 days of exposure, the percentage of doxy-

cycline was reduced by 5.3%, while additional impurities occurred

and the amount of impurities already present in the original solution

increased proportionally. It was possible to detect impurities down

to < 0.2 mAU of peak height (Figure 2) (2).

Conclusions

Quantif cation of signif cantly different concentration levels present

in the water and the octanol phase for determination of log P were

performed without the need of injection volume adaption. Peak

heights up to 8000 mAU were calibrated and quantif ed by means

of the wide linear range of the Agilent 1200 Inf nity Series HDR-DAD

Analyzer System. In addition, the 1200 Inf nity Series HDR-DAD

typically exhibits low noise and provides for the quantif cation of

peaks with peak heights below 0.125 mAU as are present for impu-

rities after decomposition of doxycycline.

References

(1) A.G. Huesgen, “Determination of Log P for Compounds of Different Polarity

Using the Agilent 1200 Infinity Series HDR-DAD Impurity Analyzer System,”

Agilent Application Note, publication number 5991-4121EN, 2014.

(2) A.G. Huesgen, “Analysis of Degradation Products of Doxycycline in Solution

Exposed to Light and Elevated Temperatures Using the Agilent 1200 Infinity

Series High Dynamic Range Diode Array Detector Impurity Analyzer Sys-

tem,” Agilent Application Note, publication number 5991-4044EN, 2014.




Diamond Analytics1260 S. 1600 W., Orem, UT 84058

tel. (801) 235-9001, fax (801) 235-9141

Website: www.diamond-analytics.com

Separation of Apo-Transferrin and Bovine Serum Albumin (BSA) ProteinsJanusz Zukowski, Diamond Analytics

Figure 1: FLARE versus commercial silica column.

2.0 2.5 3.0 3.5 4.0

0

50

100

150

200

250

300

UV

at

230 n

m

min

FLARE

Commercial Silica Column

FLARE vs. Commercial Silica Column

1 1

2

2

Resolved Impurity Peak

1- BSA2- Apo-Transferrin

The unique surface chemistry of the FLARE diamond core-

shell column combines ionic and hydrophobic separation

mechanisms to effectively retain a variety of chemical

species in a single run.

HPLC Conditions

Columns: FLARE 4.6 × 33 mm, Commercial Silica Column

4.6 × 50 mm

Temperature: 55.0 °C

Mobile Phase: Solvent A: 50 mL ACN + 950 mL water + 2 mL TFA

Solvent B: 1000 mL ACN + 2 mL TFA

Flow Rate: 1 mL/min

Injection: 2 μL, ca. 0.2 mg/mL in solvent A

Detection: UV @ 230 nm

Gradient: 0 min 100% A, 4 min 10% A, 4.1 min 100% A,

10 min END

Notes

Proteins are complex structures that are essential in modern drug

development and discovery. Proteins are vital building blocks in our

bodies and play a major role in creating overall health and wellness.

In laboratory settings, separating complex proteins from other chemi-

cal structures, impurities, and excipients is a major challenge for the

pharmaceutical industry. The problem is that proteins may be com-

plex, bulky, and fragile. They may also denature under certain condi-

tions. In addition, these compounds are notorious for “sticking” onto

columns, making their purif cation, analysis, and quantitation diff cult.

Conclusions

Recent drug development is creating a growing interest in the sepa-

ration and purif cation of a variety of proteins, such as monoclo-

nal antibodies (mAb) and antibody drug conjugates (ADC). These

challenging separations require more robust platforms for improved

analytical results. Diamond-based columns provide an exciting new

tool that can handle complex protein mixtures even under elevated

pH and temperature conditions, and can also deliver a more cost-

effective solution that will save time and money to laboratory scien-

tists. Diamond-based columns can also be regenerated under high

pH conditions for a longer, useful life.




Advanced Analytical SEC Measurements of Anthrolysin Molecular Weight and Structure Malvern Instruments Ltd.

Anthrolysin (ALO) is a pore forming cholesterol-dependent cytolysin

(CDC) secreted by Bacillus antracis. Research suggests that ALO

plays a role in the pathogenesis of Anthrax. An SEC experiment

was performed using a Superdex 200 (GE Healthcare) with a buffer

containing 20 mM Tris, 150 mM NaCl, pH 7.3. The Viscotek TDA

with UV, RI, light scattering, and viscometer detectors was used to

determine the molecular weight (MW) and intrinsic viscosity (IV) of

ALO in solution with the results shown in Figure 1.


The absolute MW was calculated to be 53.6 kDa indicating that ALO ex-

ists as a monomer when in solution. A retention volume of 22.2 mL was

recorded for the peak and if a traditional column calibration method had

been used this would have corresponded to a MW of only 15–20 kDa.

This means that without the advanced detectors used in this work, the

underestimation of protein MW frequently goes unnoticed.

In addition to measuring the absolute MW of ALO the addition of

the viscometer detector allowed the IV to be measured. The IV is

inversely proportional to the molecular density of a protein so any

changes in structure, shape or hydration (that is, f exibility) will lead

to changes in the volume of that protein and consequently the density

and IV. For ALO (53.6 kDa) the IV was measured as 0.51 dL/g. As a

comparison the monomer of a BSA (66.5 kDa) has an IV of approxi-

mately 0.4 dL/g, indicating that BSA has a more compact structure

than ALO. This is paradoxical when considered with its late elution

from the column but may indicate column interaction at some level.

This application note shows that the use of multi-detection TDA-SEC

is essential to obtain accurate and insightful data from SEC experiments.

Work performed in conjunction with the Institut Pasteur, Paris, France

References

(1) R.W. Bourdeau, E. Malito, A. Chenal, B.L. Bishop, M.W. Musch, M.L. Ville-

real, E.B. Chang, E.M. Mosser, R.F. Rest, and W.J. Tang, “Cellular Functions

and X-ray Structure of Anthrolysin O, a Cholesterol-Dependent Cytolysin

Secreted by Bacillus anthracis,” J. Biol. Chem. 284, 14645–14656, (2009).

(2) A. Chenal, C. Vendrely, H. Vitrac, J.C. Karst, A. Gonneaud, C.E. Blanchet,

S. Pichard, E. Garcia, B. Salin, P. Catty, D. Gillet, N. Hussy, C. Marquette,

C. Almunia, and V. Forge, “Amyloid Fibrils Formed by the Programmed Cell

Death Regulator Bcl-xL.,” J. Mol. Biol. 415, 584–599 (2012).

(3) J.C. Karst, A.C. Sotomayor-Pérez, D. Ladant, and A. Chenal, “Estimation of

Intrinsically Disordered Protein Shape and Time-Averaged Apparent Hydra-

tion in Native Conditions by a Combination of Hydrodynamic Methods,”

Methods Mol. Biol. 896, 163–77 (2012).


Enigma Business Park, Groveland Road, Malvern, UK

tel. +44 (0) 1684 892456, Email: [email protected]

Website: www.malvern.com

300

250

200

UVRALSLALS

DP

150

100

50

0

300lV, mL/g

Mw, KDa

UV 250

200

150

100

50

0

70

60

50

40

30

20

10

0

19 21 2523

Ch

an

ne

l re

spo

nse

s, m

V

Retention Volume, mL

Mo

lecu

lar

We

igh

t, In

trin

sic

Vis

cosi

tya

nd

UV

re

spo

nse

19 21 2523

Retention Volume, mL

A

B

Figure 1: A: Chromatograms of ALO from the Viscotek TDAmax. Data lines show UV (purple), RALS (green), LALS (black), and DP (blue). B: Molecular weight (Mw) and intrinsic viscosity (IV) patterns of ALO. Data lines show molecular weight (black), UV (purple), and IV (grey).




Amino Acid Analysis According to European Pharmacopoeia 8.0 Pickering Laboratories

The European Pharmacopoeia (Ph. Eur.) def nes requirements for

the qualitative and quantitative composition of medicines, as well

as the tests to be carried out on medicines and on substances and

materials used in their production.

It covers active substances, excipients, and preparations of

chemical, animal, human or herbal origin, homoeopathic prepa-

rations and homoeopathic stocks, antibiotics, as well as dosage

forms and containers. It also includes tests on biologicals, blood

and plasma derivatives, vaccines, and radiopharmaceutical prepa-

rations. The European Pharmacopoeia and its requirements are le-

gally binding in the member states of the European Pharmacopoeia

Convention and the European Union.

All manufacturers of medicines or substances for pharmaceutical

use therefore must apply the Ph. Eur. quality standards in order to

be able to market and use these products in Europe.

Amino acids analysis can be used for:

• Identiå cation tests on biopharmaceutical active ingredients (such as

peptides, proteins) by means of amino acids composition analysis;

• Impurities and related substances determination on active

pharmaceutical ingredients (APIs) (such as free amino acids) and

intermediates;

• Single or total amino acids quantiå cation in drug products,

including markers determination in complex matrixes

(such as phytopharmaceuticals).

The following Ph. Eur. monographs have already off cially intro-

duced the amino acid analysis method with post-column ninhydrin

derivatization as the analytical procedure required for the determina-

tion of the ninhydrin-positive substances, and additional papers are

expected to be published in upcoming months:

Cysteine HCl Monohydrate 01/2014:0895, Isoleucine

07/2013:0770, Leucine 07/2013:0771, Lysine HCl 07/2013:0930,

Serine 01/2014:0788, Proline 01/2014:0785, Threonine

01/2014:1049, Valine 01/2014:0796, Arginine 07/2014:0806

Pickering Laboratories, Inc. offers a complete solution for

amino acids analysis according to European Pharmacopoeia

8.0. This includes the Pinnacle PCX post-column derivatization

instrument, analytical columns and GARDs, buffers and Trione®

Ninhydrin reagent. The Pinnacle PCX is capable of performing

column temperature gradients that allow easily modif ed condi-

tions and improved run times and amino acids separations. The

methods presented in this application note were optimized to

comply with system suitability requirements of Pharmacopoeia

8.0 methods.

Each Pharmacopoeia monograph describes the preparation of

the test and reference solutions specif c for each amino acid. The

solutions are used for calculations of percentage contents, impu-

rity levels as well as parameters of system suitability. Resolution of

1.5 is required between Leucine and Isoleucine peaks.

For all amino acids, except Cysteine, Sodium-based and Lithium-

based methods are available. For Cysteine analysis, only Lithium-

based methods are suitable. Sodium-based methods have shorter

run times and are preferable for all amino acids except Cysteine.

Equipment:

Quaternary HPLC pump, autosampler, UV-vis detector

Pinnacle PCX post-column derivatization system

Analytical columns and Eluants:

For Sodium-based methods: High-eff ciency Sodium cation-ex-

change column, 4.6 × 110 mm, Catalog Number 1154110T. Elu-

ants: Na315, Na425, Na640, RG011

Figure 1: Sodium chromatogram of amino acids analyzed using Pharmacopoeia 8.0 methods (3 µg/mL each, 50 µL injection).

Figure 2: Sodium chromatogram of alternative amino acids analyzed using Pharmacopoeia 8.0 methods (3 µg/mL each, 50 µL injection).

Threonine

Serine

Proline

Ammonia

Arginine

Lysine

Isoleucine

Leucine

Valine

Cystine

Alanine

0 10 20 30 40 min

Threonine

Proline

Ammonia

Lysine

Isoleucine

Leucine

Valine

0 10 20 30 40 min

Phenylalanine

Methionine




Pickering Laboratories, Inc.1280 Space Park Way, Mountain View, CA 94043

tel. (800) 654-3330, (650) 694-6700

Website: www.pickeringlabs.com

For Lithium-based methods: High-eff ciency Lithium cation-

exchange column, 4.6 × 75 mm, Catalog Number 0354675T.

Eluants: 1700-1125, Li365, Li375, RG003

Post-column reagent

Trione® Ninhydrin Reagent

To make it easier to start using Pickering Laboratories methods,

we offer chemistry kits that include: analytical column, GARD, buf-

fers, and reagents for amino acids analysis. All parts of the kit could

be ordered individually if needed. Please contact Pickering Labora-

tories if you have any questions regarding this application.

Pickering Laboratories will keep updating its Pharmacopoeia

8.0 methods as new monographs are released. Please contact

[email protected] for the latest methods and chromatograms.

Figure 3: Lithium chromatogram of amino acids used as reference solutions for Cysteine analysis (3 µg/mL each, 50 µL injection).

Figure 4: Lithium chromatogram of amino acids analyzed using Pharmacopoeia 8.0 methods (3 µg/mL each, 50 µL injection)

Cysteine

Ammonia

Isoleucine

Leucine

Proline

Cystine

0 10 20 30 40min

50

Threonine

Proline

Ammonia

Lysine

Isoleucine

Leucine

Valine

Methionine

Phenylalanine

0 10 20 30 40min

50




Screening of Drugs of Abuse Using the Velox 360™ Paper Spray SystemJoseph H. Kennedy and Justin M. Wiseman, Prosolia Inc.

Paper spray mass spectrometry (MS) allows for the direct anal-

ysis of pharmaceuticals, drugs of abuse, and other small mol-

ecules from blood, urine, and other biof uids (1,2). Described

herein is the application of a Velox 360 paper spray autosam-

pler and ion source for the direct analysis of drugs of abuse in

urine samples.

Paper spray is performed by depositing the sample directly onto a po-

rous cellulosic substrate contained within a disposable cartridge (Ve-

lox Sample Cartridge). The sample is allowed to dry, and the cartridge

is inserted into the automated ion source for analysis. The Velox 360

autosampler and ion source performs all of the steps necessary to per-

form the analysis, including loading the cartridge, depositing extraction

solvent onto the cartridge, positioning the cartridge in front of the mass

spectrometer inlet, and applying the ionization voltage. Ions are gener-

ated from the sharp tip of the porous substrate contained within the

cartridge. The Velox 360 paper spray system provides an automated,

cost effective, and eff cient method for screening DOAs using mass

spectrometry with minimal sample preparation.

This application note describes the use of the Velox 360 System

and high resolution mass spectrometry for rapid screening of illicit

drugs and pharmaceuticals from urine samples

Experimental

Anonymous urine samples from a methadone clinic were obtained

from Clinitox Dx (Ontario, Canada). First, 5 µLs of urine were depos-

ited onto the Velox sample cartridge using a pipette. The sample was

allowed to dry for 30 min at room temperature or was dried in a 40°

incubator for 10 min. The cartridges were then analyzed automati-

cally using the Velox 360 coupled to a Thermo Scientif c ExactiveTM.

Results

A typical mass spectrum from a urine sample collected at a methadone

clinic is shown in Figure 1. The intense signals from various drugs of

abuse, along with creatine, are shown in the mass spectrum and are

within 3 ppm of the theoretical mass-to-charge ratio. The presence of

these drugs in the urine sample was further conf rmed by HPLC–MS-

MS (data not shown).

Conclusion

This application note demonstrates the suitability of paper spray mass

spectrometry for the rapid screening of illicit drugs and drug metabolites

in urine, particular when used in combination with high resolution MS

instrumentation. Compared to more traditional approaches, the Velox

360 offers several compelling advantages for this application, including:

1) No sample preparation. Analysis is done directly from urine.

2) Rapid analysis. Less than 2 min per sample for the entire analyti-

cal procedure (excepting time required to dry samples).

3) No carry-over. Because the analysis is done from a single use

cartridge, there is no worry that high concentration samples will

cause carry-over.

4) No chromatography. That means no columns to replace, no

carry-over on the column, and no chromatography methods to

develop.

References

(1) H. Wang, J. Liu., R.G. Cooks, and Z. Ouyang, Angew. Chem., Int. Ed. 49,

877–880 (2010).

(2) N.E. Manicke, Q.A. Yang, H. Wang, S. Oradu, Z. Ouyang, and R.G. Cooks,

Int. J. Mass Spectrom. 300, 123–129 (2011).

Prosolia Inc.6500 Technology Center Drive, Suite 200, Indianapolis, IN 46278

tel. +1(866) 241-0239, fax +1(317) 873-3175

Website: www.prosolia.com

Figure 1: Full mass spectrum recorded using paper spray directly from a dried urine sample showing the presence of various substances includ-ing the (M+H+) ions of: creatine (m/z 114.066), methamphetamine (m/z

150.158), tramadol/desmethyl-venlafaxine (m/z 264.195), EDDP (m/z

278.189), and methadone (m/z 310.215).




Statistic copolymers of methyl methacrylate (MMA) and methacrylic acid (MAA) are widely used in pharmaceutical applications. Aside from the molar mass distribution, the chemical composition and the amount of comonomers in the copolymer is of importance. Separations by con-ventional gradient high performance liquid chromatography (HPLC) failed, since the polar eluents required to dissolve polymers with a high acid content prevent adsorption onto the stationary phase, resulting in pronounced breakthrough peaks. The problem can be avoided by ap-plying size-exclusion chromatography (SEC)-gradients, resulting in the desired separation according to the amount of methacrylic acid. The system can be calibrated using reference materials of known compo-sition. This allows the average copolymer composition as well as the compositional heterogeneity to be determined.

Experimental

GPC–SEC analysis was performed on a PSS SECcurity GPC Systemcomprising a 1260 binary pump, a 1260 autosampler, and anELSD1000.

The analysis conditions were:Columns: PSS PROTEEMA, 3-μm, 100 Å (8 × 300 mm) + precolumnSolvent: Gradient: Chloroform/DMAc, f ow-rate 1 mL/min 0–3 min: 50% DMAc 3–8 min: 100% DMAc 8–23 min: 100% CHCl3 23–26 min: 5% DMAc 26–32 min: linear increase from 5% to 50% DMAcTemperature: 60 °CCalibration: PSS MMA-MAA copolymers of different acid content (MAA: 9%, 25%, 31%, 42%, 48% wt)Concentration: 1 g/LInject volume: 100 μL, injection interval 32 minSoftware: PSS WinGPC UniChrom 8.0

Procedure, Results, and Discussion

The experimental procedure for SEC-gradients differs from con-ventional gradient HPLC. In conventional HPLC the sample is dis-solved in a weak eluent and injected at adsorbing conditions; in SEC-gradient the sample is dissolved in a strong mobile phase and injected at the end of the SEC-gradient. In the present application the copolymers were dissolved in DMAc.

Figure 1 shows the separation of f ve statistical MMA-MAA copolymers with MAA contents between 9% and 48% wt using a Chloroform/DMAc gradient from 5% to 50% DMAc. The sample components elute before the injected solvent and the different samples are clearly separated.

A nearly linear dependence between MAA content and elution volume is observed. The use of this calibration curve allows the determination of the chemical composition distribution. Figure 2 shows the chemical composition distribution for the f ve samples with the PMAA content between 48% and 9%. It is observed that an increase in MAA content results in a signif cantly broader chemical composition distribution (CCD).

Separation of StatisticMMA-MAA CopolymersUsing Gradient SEC PSS Polymer Standards Service

Figure 1: Separation of 5 MMA-MAA copolymers with different PMMA content using a Chloroform - DMAc gradient on PSS PROTEEMA columns.

Figure 2: Chemical composition distribution for 5 MMA-MAA copoly-mers. For MAA content see Figure 1.

1.2

0.9

0.6

0.3

0.012 11 10

Gradient 5% - 50% DMAc

48% PMAA 42% 31% 25% 9%

9 8 7 6

Elution volume (mL)

No

rmali

zed

ELSD

sig

nal

Content methacrylic acid (wt%)

w(%

)

0.3

0.2

0.1

0.0100 20 30 40 50 60 70

PSS-USA, Inc.Amherst Fields Research Park,

160 Old Farm Road, Ste. A, Amherst, MA 01002

tel. (413) 835-0265, fax: (413) 835-0354

Website: www.pss-polymer.com




Improved USP Chlorhexidine Gluconate Assay on YMC-Triart C18 and YMC Meteoric Core HPLC Packing MaterialsJeffrey A. Kakaley, YMC America, Inc.

USP assay methods are widely used for quality control and stability testing.

Older methods can often be improved to take advantage of newer instrument

design and column technologies. Improvements can include increases in

sample throughput, method reproducibility, and solvent savings. This appli-

cation note illustrates how run time and solvent consumption can be reduced

when compared against the USP assay method for chlorhexidine gluconate.

The improved method is demonstrated on two different columns: YMC-

Triart C18 (hybrid particle) and YMC-Meteoric Core C18 (core-shell particle).

Original USP Method ParametersDetection: 239 nm

Column: 250 × 4.6 mm, base-deactivated 5 µm packing L1

Column Temp: 40 °C

Flow rate: 1.5 mL/min

Inj. Volume: 50 µL

Total Runtime: 21 min

Mobile Phase A: 0.1M NaH2PO

4, 0.5% Triethylamine, pH=3.0

Mobile Phase B: Acetonitrile

Gradient: 0–9 min 0% B, 9–10 min 0–55% B, 10–15 min hold 55% B, 15–16 min

55–0% B, 16–21min hold 0% B

Resolution: NLT 3.0 between chlorhexidine and p-chloroaniline

Improved Method ParametersDetection: 239 nm

Columns: Test done on two different columns: YMC-Triart C18, 3 µm, 2.0 × 100 mm

YMC-Meteoric Core C18, 2.7 µm, 2.1 × 100 mm

Column Temp: 40 °C


Inj. Volume: 4 µL

Total Runtime: 8.8 min

Mobile Phase A: 0.1M NaH2PO

4, 0.5% Triethylamine, pH=3.0

Mobile Phase B: Acetonitrile

Gradient: 0–3.6 min 0% B, 3.6–4 min 0–55% B, 4–6 min hold 55% B, 6–6.4 min

55–0% B, 6.4–8.5 min hold 0% B

Resolution: NLT 3.0 between chlorhexidine and p-chloroaniline (same as original method)

Results

YMC America, Inc.941 Marcon Blvd., Suite 201, Allentown, PA 18109

tel. (610) 266-8650, fax (610) 266-8652

Website: www ymcamerica.com

Figure 2: YMC Meteoric Core, Part number = CAS08SQ7-10Q1PT.

Figure 1: YMC-Triart C18, Part number = TA12S03-1002WT.

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Method Transfer Considerations: Scale-down for YMC L1 Packing Materials• Injection volume was decreased to 4 µL due to changes in column I.D. and length

• Flow rate was decreased to 0.3 mL/min due to changes in column I.D.

• The gradient was adjusted to take into account the changes in ý ow rate, column I.D., and column length.

• Total run time for this method was reduced from 21 min to 8.8 min.

Separation with YMC-Triart C18

Highlights:

• Solvent usage for TA12S03-1002WT Method = (0.3 mL/min) × (8.8 min) = 2.64 mL per injection

• USP Resolution between CHX and pCA = 6.2

• USP Tailing CHX = 1.0

• USP Tailing pCA = 1.0

• Backpressure = 2570 psi

Separation with YMC-Meteoric Core C18

Highlights:

• Solvent usage for CAS08SQ7-10Q1PT Method = (0.3 mL/min) × (8.8 min) = 2.64 mL per injection

• USP Resolution between CHX and pCA = 8.5

• USP Tailing CHX = 1.1

• USP Tailing pCA = 1.0

• Backpressure = 2832 psi

Reproducibility

The method was repeated on both YMC-Triart and YMC-Meteoric

Core for 25 consecutive injections each. Both types exhibit excellent

reproducibility for peak shape and retention time. Space here does

not allow illustration, but the details are available from YMC America.

Conclusions

YMC-Triart C18 and YMC Meteoric Core C18 columns exhibit the

selectivity and reproducibility necessary to deliver meaningful im-

provements to the USP method. The smaller particle sizes, shorter

column length, and smaller column inner diameter allow for a much

faster method (1/3 the runtime) that

uses 12× less solvent. The method also

meets the original method’s resolution

requirements of NLT 3.0 without issue.




Carbon-clad zirconia phases offer dramatically different

chromatographic selectivity for structurally similar com-

pounds when compared to traditional silica-based bonded

phases. This note shows baseline separation of six structur-

ally similar sulfate-steroid conjugates using a zirconia-based

ZirChrom®-CARB HPLC column.

Method development in reversed-phase liquid chromatography

(RPLC) has traditionally been diff cult for molecules which are geo-

metric isomers or structurally very similar. In bonded-phase silicas,

the partition mechanism responsible for retention in RPLC often does

not offer adequate chemical selectivity for such compounds. On the

other hand, carbon-based phases can provide retention in RPLC

through an adsorption mechanism which increases the chromato-

graphic selectivity, especially for differences involving aromatic rings,

and dramatically increase the chances of resolving critical pairs.

The ZirChrom®-CARB phase is a zirconia particle that has been

carbon-clad, using patented chemical vapor deposition technology,

with an elemental layer of graphitized carbon. The resulting phase

contains only C-C surface bonds, which are extremely resistant to

chemical and thermal attack, allowing ZirChrom®-CARB to be stable

throughout the pH range (1–14) and up to 200 °C.

Experimental

A mixture of sulfate-steroid conjugates was separated at elevated

temperature using a ZirChrom®-CARB column. The separation

conditions were as follows:

Column: ZirChrom®-CARB, 100 mm × 4.6 mm

Mobile Phase: Gradient elution from 55/5/40 to 90/5/5 A/B/C

from 0 to 4.5 min.

A: Acetonitrile

B: Tetrahydrofuran

C: 25 mM Ammonium f uoride, 10 mM

Ammonium acetate, pH 5.6

Temperature: 80 °C

Injection Vol.: 10 μl


Pressure Drop: 195 bar

Detection: UV at 270 n

This method is an excellent example of how the enlarged zirconia

method development “tool box” can be used to overcome the

toughest separation challenges. Using temperature, the unique

surface chemistry of ZirChrom®-CARB, and a gradient, baseline

resolution of these six structurally similar sulfate-steroid conjugates

was achieved in under 5 min (see Figure 1). The critical pair, dihy-

droequilin sulfate and equilin sulfate, which differ in structure by

only a hydrogen, are well resolved.

This method can be tailored to your specif c application needs.

ZirChrom method developers can help you optimize and transfer

this method to your site. Please contact ZirChrom technical support

at 1-866-STABLE1 or [email protected] for details.

ZirChrom phases offer unique selectivity for ionic compounds,

high eff ciency, and excellent chemical and thermal stability.

Fast Methods for Structurally Similar Compounds Using Carbon HPLC ColumnsDwight Stoll, Clayton V. McNeff, and Peter W. Carr, ZirChrom Separations, Inc.

Figure 1: Steroid conjugate selectivity. 1 = Dihydroestradiol sulfate, 2 = Dihydroequilin sulfate, 3 = Equilin sulfate, 4 = Estrone sulfate, 5 = Equilenin sulfate, 6 = Dihydroequilenin sulfate.

ZirChrom Separations, Inc.617 Pierce Street, Anoka, MN 55303

tel. 1-866-STABLE-1, email: [email protected]

Website: www.zirchrom.com



POLYMER

Synthetic elastomers have replaced natural rubber to an astonishing

degree, and account for more than 70% of the rubber used today. In

the United States alone, 5 million tons of synthetic rubber are pro-

duced annually. The principal synthetic rubber elastomer is a copoly-

mer of butadiene and styrene. The latex form of rubber and synthetic

elastomers has applications in carpet and gloves, and coagulated

latex is used for the production of tires and mechanical goods. It is

of critical importance to know the absolute molar mass and its dis-

tribution, as well as to gain insight into the conformation of synthetic

rubber — which are indicative of the product’s end-use performance.

Typically, polystyrene standards are used to estimate the mo-

lar masses of these polymers in SEC experiments, but by using a

DAWN or miniDAWN multi-angle light scattering (MALS) detector,

standards and column calibration are no longer needed. Here, the

absolute molar mass and polydispersity, as well as the rms radius

of two synthetic rubber samples were measured directly using SEC

combined with a DAWN.

The synthetic rubber samples were analyzed in toluene, and Wyatt

Technology’s Optilab was used as the refractive index detector for the

SEC line. The refractive index increment dn/dc of polybutadiene in

toluene is relatively low, and the dn/dc value of the butadiene/styrene

copolymer in toluene increases with the ratio of styrene present. In

Figure 1, the molar mass and its distribution are determined abso-

lutely — without using any standards or calibration routines.

In addition to the weight-average molar mass and the polydis-

persity, the DAWN can also determine the shape of the polymer

Synthetic Rubbers: Polybutadiene Wyatt Technology

Wyatt Technology6300 Hollister Avenue, Santa Barbara, CA 93117

tel. +1 (805) 681-9009, fax +1 (805) 0123

Website: www.wyatt.com

Figure 2: RMS radius versus molar mass (“conformation plot”) for the two samples. The slope is indicative of the conformation (rod, coil, or sphere) of the molecule.

Figure 1: Molar mass versus elution time for the synthetic rubber sample superimposed upon the signal from one of the light scattering detector channels.

by measuring the rms radius directly at each elution volume. It is

well-known that butadiene forms highly branched polymers and that

styrene forms linear polymers — and this is revealed by the DAWN.

Figure 2 shows a logarithmic plot of the molar mass versus the

radius of the two synthetic rubber samples. The slope of such a plot

is indicative of the shape of a polymer. A slope between 0.5 and 0.6

is usually found for linear polymers with a random coil conforma-

tion, while spheres have a slope of approximately 0.3. The values

obtained for polybutadiene (0.25) and for the butadiene/styrene co-

polymer (0.38) indicate that this polybutadiene is more branched

than the styrene/butadiene copolymer.

DAWN®, ASTRA®, AURORA®, and the Wyatt Technology logo are registered

trademarks of Wyatt Technology Corporation. ©2014 Wyatt Technology Corpora-

tion 4/14/2014



GENERAL

Since the gas f ow required for the separation step in gas chroma-

tography is frequently lower than that required to optimize the de-

tection, nitrogen is used as a make-up gas to increase the gas f ow

for detection. In many facilities, zero grade nitrogen make-up gas

is provided from a cylinder or tank. While this approach works, an

in-house “make-up” gas generator can provide the desired nitrogen

with a higher level of purity than bottled nitrogen. In addition, the

use of an in-house make-up gas generator can provide a consider-

ably safer, more convenient, and less expensive approach to supply

the required gas.

Design of an In-House Zero-Air Generator

Zero grade nitrogen for make-up gas can be readily obtained from

laboratory compressed air using an in-house generator (Parker

Hannif n FID MakeUpGas Generator) that includes a heated cata-

lytic converter in which a proprietary catalyst blend is combined

with platinum to remove all hydrocarbons by converting them to

CO2 and water vapor. The converter is followed by a hollow f ber

membrane separator which preferentially allows oxygen and water

vapor to quickly permeate the membrane wall while nitrogen travels

through the hollow f ber out the end (Figure 1). The hollow f ber

has a small internal diameter and thousands of f bers are bundled

together to provide a large surface area to provide the desired f ow

of nitrogen. The makeup gas generator can provide nitrogen with

purity of better than 99.9999% with respect to hydrocarbons (< 1

ppm) and greater than 99% with respect to oxygen.

Performance

A chromatographic comparison of the nitrogen that was produced

by the MakeUpGas generator and gas that was obtained from bot-

Generating Make-Up Gas for GC with an In-House Nitrogen GeneratorCorky Belobraydich, Parker Hannif n Corporation

Parker Hannif n CorporationFiltration and Separation Division

260 Neck Road, Haverhill MA 01835

tel. (800) 343-4048, (978) 858-0505

Website: www.parker.com/gasgeneration.

0.5

0.25

0

-0.25

-0.5

0 8.33 16.67 25 33.33

Time in Hours

TH

C (

PPM

)

0.5

0.25

0

-0.25

-0.5

0 8.33 16.67 25 33.33

Time in Hours

TH

C (

PPM

)

BaselineMGG-2500NA Makeup Gas Generator

BaselineBottled Fuel Air

Figure 1: Oxygen and water vapor permeate the membrane, providing high purity nitrogen.

Figure 2: The MakeUpGas generator (left f gure) provides gas of sig-nif cantly higher purity than bottled fuel gas (right f gure).

tled fuel air from a commercial supplier is shown in Figure 2. The

gas generated by the MakeUpGas generator is much purer than

that from bottled fuel air; and provides an extremely f at baseline

with essentially no signal due to hydrocarbons, while the zero grade

bottled air provided an irregular baseline with a signif cant level of

hydrocarbons, which could impact the analysis.

Conclusions

In addition to the extremely high level of purity provided by the gen-

erator, the use of an in-house generator provides benef ts in safety,

cost, and convenience. When a MakeUpGas generator is employed,

only a small amount of nitrogen is generated at a given instant and

a leak would lead to a negligible change in the composition of the

laboratory air. In contrast, a leak from a full tank could cause prob-

lems. When an in-house generator is employed, gas is available

on a 24/7 basis and the possibility of injury or damage during the

transportation and installation of a heavy gas tank is eliminated. In

addition to the signif cant safety and convenience benef ts, there is

an economic benef t from using a MakeUpGas generator. The run-

ning cost of operation maintenance of the MakeupGas generator is

extremely low; as the raw materials to prepare the required gas are

air and electricity.



Translations Between Differing Liquid Chromatography Formats: Advantages, Principles, and Possible PitfallsThe numerous advantages of translating gradient chromatographic methods between the differing formats of liquid chromatography (LC) have been explored and discussed. Although translations in principle obey well-defined chromatographic theories, the authors investigate a number of potential pitfalls that may result in poor translations as exhibited by selectivity differences, changes in efficiency, and hence failure to meet resolution system suitability criteria. The consequences of these pitfalls are examined and the regulatory implications of method translation are explored.

Patrik Petersson*, Melvin R Euerby†,‡, and Matthew A James‡ *Novo Nordisk A/S; †Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde; and ‡Hichrom Ltd.

As a result of the introduction of commercially available sub-2-µm porous particles (1), sub-2-µm, 3-µm, and 5-µm superficially porous (2,3) particles, and ultrahigh-

pressure liquid chromatography (UHPLC) instrumentation (4,5) from 2004 onward, there has been an increasing interest in the ability to perform accurate translations between different liquid chromatography (LC) formats. An example would be translating between 150 mm × 4.6 mm, 5-µm dp formats on standard high performance liquid chromatography (HPLC) systems and 50 mm × 2.1 mm, sub-2-µm formats on UHPLC systems while maintain-ing the same resolution. The findings of a recent survey of major chromatographic users predicted that the use of standard HPLC systems is expected to steadily decline from 2011 to 2015 with a concomitantly higher usage and purchase of UHPLC systems predicted over the same time frame (6).

There are a plethora of reasons for this shift in LC format usage and purchase, all of which are based on sound chromatographic theory (5,7,8). From the extensive experience of the authors within the pharmaceutical industry, the major drivers for this shift appear to be increased productivity (that is, reduced analy-sis time) coupled with minimal loss of information quality or an increased quality of data with no loss of productivity.

Advantages and DriversIncreased Resolution

Reduction of the packing material particle size by a factor of two (that is, substitution of 3–3.5 µm particles by 1.7-µm particles), while keeping other operation factors constant, should result in an increase of resolution of approximately 30–40%.

Speed of Analysis

A reduction in column length (L) and particle size (dp) while keep-ing the L/dp ratio constant (for example, substitution of a 150-mm

column with 3–3.5 µm particles for a 75-mm column with 1.7-µm particles) should maintain the same chromatographic efficiency and hence resolution, while reducing the gradient analysis time by 50% (same velocity typically used for large molecules) to 70% (higher velocity typically used for small molecules) and substan-tially increasing productivity. This approach is vitally important for the analysis of increasingly larger numbers of samples (that is, to better describe a process or formulation performance), increased utilization of instruments, the analysis of labile samples, and rapid at-line analysis (that is, process analytical technology).

A compromise between the approaches of increased resolution and speed of analysis has been the use of 100-mm columns with sub-2-µm particles, which results in a 60% reduction in gradient time and an approximate 10% increase in resolution for small molecules.

Reduced Solvent Consumption

Converting a standard HPLC method that uses a 150 mm × 4.6 mm, 3–3.5 µm column to a 100 mm × 2.1 mm, 1.7-µm or 100 mm × 1.0 mm, 1.7-µm column in theory offers a possible reduction in solvent consumption of approximately 86% to 97%. In practice, it is less often because of the necessity to prime the LC lines. During a global implementation of UHPLC within AstraZeneca (during the period of 2007–2010) involving 41 UHPLC systems, a reduc-tion in solvent consumption of 63% was realized compared to the theoretical reduction of 77% (9).

Ease of Method Transfer

Within many industries it is often standard practice to transfer the chromatographic testing from the research and development (R&D) laboratory to a contract research organization (CRO), operation, or quality control (QC) sites. This method transfer exercise can be made even more problematic because it is now

ES495546_LCGCAN0914_042.pgs 08.29.2014 16:46 ADV blackyellowmagenta


quite common for many R&D depart-ments to develop only UHPLC methods. However, not all receiving laboratories have sufficient UHPLC capacity or ex-perience and, thus, method translations become necessary. The reverse of this is becoming true in that QC laboratories, which have moved predominantly to UHPLC, may have to use UHPLC meth-ods for the analysis of legacy products or methods that use HPLC columns.

Increased Instrument Utilization

The drive for increased productivity and efficiency has necessitated an increased flexibility and utilization of available instru-mentation. Many companies have a rolling program to replace their worn out HPLC systems with a reduced number of UHPLC systems capable of running LC methods based on both 5-µm and sub-2-µm par-ticles. In addition, valve arrangements are used that allow queuing of both HPLC and UHPLC methods on the same LC system, hence a reduced number of LC systems allow continuous operation.

Principles of Method TranslationBecause many translation guides suc-cessfully describe translations of isocratic LC methodologies (10), this article will focus entirely on the translation of gra-dient LC methodologies, which can be more difficult to perform. In theory, the translation required to maintain the chro-matographic selectivity and performance of either an isocratic or gradient separa-tion is very simple (7,11). The additional consideration that has to be accounted for in gradient separations is that a constant ratio must be maintained between the volume of each segment in the gradient over the column dead volume (8,12–15). After the introduction of sub-2-µm par-ticles and UHPLC in the mid-2000s, sev-eral articles were published that focused exclusively on translations between 4.6- and 2.1-mm i.d. columns (7,8,10,16–18). To assist the practicing chromatographer in all types of translations, several com-mercial and academic computer applica-tions have been developed (19); however, although these computer applications un-doubtedly aid chromatographers, they all possess certain drawbacks to successful method translation.

In contrast to previous publications,

this article only briefly describes the underlying theory and equations relat-ing to chromatographic method transla-tion principles (readers are encouraged to see the sidebar “Theory for Transla-tions” for more information). Instead, this article will focus on how to perform successful translations of gradient chro-matographic methods between HPLC and UHPLC systems, the accuracy that can be expected, and the potential pitfalls that chromatographers must be aware of and how to successfully avoid them. If the necessary precautions are taken, it is the experience of the authors that gradi-ent translations work extremely well. For example, 11 LC methods were translated to or from UHPLC within AstraZeneca; the maximum deviations in relative reten-tion times were only between 0.02 and 0.05, which was deemed acceptable (9,18).

ExperimentalExperimental work was performed on Agilent 1100, 1260, and 1290 LC systems of which the dwell volume and system volumes had been previously well charac-terized. Any experimental data including the injector programs used can be sup-plied by the authors on request. For the delayed injection work on the Agilent LC systems, the injector must be in the bypass mode (that is, no flow through the injec-tor). After the calculated delay time, the flow was returned to the mainpass posi-tion (that is, flow through the injector), then after a predefined time (time = [5 × [injection volume + 5]]/flow rate) to flush the sample onto the column, the flow was returned to the bypass position. At the top of the gradient the flow was returned to the mainpass position to wash the injector with the mobile phase to avoid carryover

Figure 1: An illustration of how a larger VD/VM for the translated method can be com-pensated using a delayed sample injection (∆ negative). The data also illustrates how VD/VMdifferences can affect the selectivity. The chromatograms have been scaled by align-ment of the f rst and last eluted peaks. (a) binary model 1290 Agilent system, VD/VM = 1.8, injection in mainpass; (b) quaternary model 1290 Agilent system, no correction, VD/VM = 9.0, injection in mainpass; (c) quaternary model 1290 Agilent system, 0.93-min injection delay, VD/VM = 8.2, injection in bypass. Other conditions for (a)–(c): column: 50 mm ×2.1 mm, 1.8-µm dp; f ow rate: 0.75 mL/min; mobile-phase A = 0.1% formic acid in water; mo-bile-phase B = 0.1% formic acid in acetonitrile; gradient: 5–27% B in 2 min; temperature: 40 °C. Peaks: 1 = paracetamol, 2 = 4-hydroxybenzoic acid, 3 = caffeine, 4 = 3-hydroxyben-zoic acid, 5 = salicylamide, 6 = acetanilide, 7 = aspirin, 8 = salicylic acid, 9 = phenacetin.

(a)

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3 4 7

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9 tR 2.77 min

1 tR 1.37 min



and subsequently flush the injector with the starting mobile phase composition prior to the next injection. Zorbax Eclipse XDB C18 columns (Agilent Technologies) of 1.8-, 3.5-, and 5-µm particle sizes were selected for the small molecule work be-cause they exhibit a good scalability with respect to efficiency and particle size (R2 = 0.9 for N versus 1/dp) (20).

Translations were made using a Micro-soft Excel spreadsheet and the equations described in the sidebar “Theory for Translations.” A corresponding software application for these types of translations is now freely accessible at the ACD/Labs website (21). In addition, a more complete set of translation tools will be included in version 2014 of the ACD/Chrom Work-book software (22).

Potential Pitfalls in

Method Translations

Observed Selectivity Anomalies as a

Result of Differences in Dwell Volume

The system dwell volume (VD) is defined as the volume from the point where the two solvents A and B first meet to the inlet of the column. For accurate translation of gradient methods it is of critical impor-tance that the ratio between the dwell and dead volume (VM) is kept constant; that is, the difference in ratio between the sys-tem dwell volume and the column dead volume needs to be negligible (Δ = [VD/VM]original – [VD/VM]new must approach 0). If this is not the case, then differences in chromatographic selectivity and relative retention time shifts may be observed. One such example is shown in Figure 1, where a series of analgesic related drugs have been chromatographed on the same 50 mm × 2.1 mm, 1.8-µm dp column using a binary high-pressure mixing system with low dwell volume (that is, 202 µL) and a qua-ternary low-pressure mixing system with a higher dwell volume (that is, 990 µL). Note that in this example a translation from a system with VD/VM = 1.8 (Figure 1a) to a

system with VD/VM = 9 (Figure 1b) resulted in coelution (peaks 7 and 8) as well as a re-versal of elution order (peaks 3 and 4).

To compensate for a larger VD/VM

on the translated method (that is, Δ = [VD/VM]original – [VD/VM]new = nega-tive), the gradient must be started before injecting the sample — that is, so-called injection delay or pre-injection volume — which can be easily implemented on cer-tain LC systems (such as the Agilent 1290 or the Waters Acquity H-class systems). Equations 9 and 10 in the sidebar describe this process in detail. It should be noted that it is only necessary to determine the VD (23) once for a certain LC configura-tion. Figure 1 illustrates how the injection delay principle can be used to compensate for VD differences and thereby maintain the original selectivity and relative reten-tion (Figure 1a versus 1c).

To compensate for a smaller VD/VM

on the translated method (that is, Δ = [VD/VM]original – [VD/VM]new = positive) the translated gradient must possess an isocratic hold before commencing the gradient.

A small VD/VM results in a gradient with sharp changes (a Z-shaped gradi-ent), whereas larger VD/VM will result in smoother, more S-shaped changes. This difference in gradient shape may also result in selectivity changes, mainly for fast separations on short columns (for example, 2-min gradients on 50 mm × 2.1 mm columns). To address this prob-lem with possible selectivity changes, Agi-lent Technologies developed an approach (24) in which the company’s model 1290 UHPLC systems can mimic the gradi-ent shape of systems with a larger system dwell volume.

Observed Selectivity

Anomalies as a Result of

Incorrect Dead Volume Estimation

The column dead volume is a funda-mental parameter that has a significant impact on translations and their subse-quent accuracy (equations 1, 9–13 in the sidebar). To the best of our knowledge, all available translation applications er-roneously assume an equal porosity for all stationary phases irrespective of their nature (equation 2 in the sidebar). In cases where this assumption is not valid, significant errors in translated gradient

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

4+5

1+2

3 6 7

8 9

10

11

5

1+2

3 6 7

8 9 10

11

4

(a)

(b)

Time (min)

Figure 2: An illustration of how a translation from one column to another may af-fect both retention and selectivity if the translation is incorrectly performed (that is, assuming equal porosity between materials). Conditions: (a) Column: 150 mm × 2.1 mm,3.6-µm dp, 3.2 µm d

core, VM = 0.27 mL, 200-Å superf cially porous particle Aeris

widepore XB-C18; f ow rate: 0.3 mL/min; gradient: 27–65% B in 30 min; mobile-phase A: 0.1% trif uoroacetic acid in water; mobile-phase B: 0.08% trif uoroacetic acid in acetonitrile; temperature: 40 °C. Conditions (b): Gradient scaled assuming the same porosity as for 300 Å porous particles, that is, VM = 0.38 mL and a gradient time of 41.8 min. Other conditions same as (a). Sample: proteins with pI ranging from 3.6 to 9.3 (Sigma-Aldrich I3018 IEF mix 3.6–9.3).



times are possible. For example, large differences in porosity and hence dead volume can be expected and observed between porous and superficially porous particles (that is, 10–29% difference was observed for seven different types of superficially porous columns [25] and 13–37% predicted difference according to equations 2–5 in the sidebar). Conse-quently, translated gradient times may have an error of the same magnitude; for nonporous particles the error will be even greater (that is, 52%).

Equations 2–5 in the sidebar describe how VM can be estimated for columns based on superficially porous particles using their reported particle and core diameters. However, these and other VMestimates are all associated with an error that can be quite substantial. The use of quoted particle sizes may be misleading as there are many differing ways that par-ticle size can be determined and reported (20). The pore size of the particles also affects the porosity (100-Å columns have a porosity of ~0.60, whereas 300-Å col-umns have a porosity of ~0.75). VM esti-mations may also be inaccurate because of the fact that columns packed with different particle sizes are often packed with different pressures, which can result in lower porosities for columns designed for UHPLC. Consequently, for columns packed with porous particles, the differ-ence between theoretical and measured dead volumes can be quite substantial. A comparison of 14 brands of modern col-umns from various vendors packed with porous particles exhibited a dead volume prediction error of ±28% (25). This value corresponds to an error in gradient time of up to 28%. Figure 2 exemplifies how a 29% error in VM can affect retention time as well as chromatographic selectivity.

Figure 2a demonstrates the experi-mental chromatogram derived by using a 200-Å superficially porous material with a dp and dcore of 3.6 µm and 3.2 µm, respec-tively, with a measured VM for the column of 0.27 mL. In comparison, when the methodology is translated using the same column (to eliminate any stationary-phase selectivity differences), but assuming that it is 300 Å and fully porous in nature, the VM is calculated as 0.38 mL and the new gradient yields a significant change in selectivity (see Figure 2b).

1

2 3

3

(a)

(b)

(c)

(d)

3+4

3+4

4

4

5

6

78 9

10

11 tR 37.0 min

11 tR 12.9 min

11 tR 12.4 min

11 tR 13.7 min

Figure 3: A translation while maintaining constant linear velocity from an HPLC meth-od based on (a) a 150 mm × 4.6 mm, 5-µm column to the corresponding UHPLC meth-od based on a 50 mm × 2.1 mm × 1.8 µm column (b) without and (c) with dwell volume compensation. The chromatograms have been scaled by alignment of the f rst and last eluted peaks. (a) binary Agilent 1100 system, VD/VM = 0.8 (injection in mainpass), 103 bar; (b) binary Agilent 1290 system, no correction, VD/VM = 1.9 (injection in mainpass), 231 bar; (c) binary Agilent 1290 system, 0.12 min injection delay, VD/VM = 1.0 (injection in bypass), 228 bar; (d) as in (b) but with a postcolumn restrictor, 714 bar. Conditions (a): Flow rate: 1 mL/min; gradient: 5–100% B in 40 min; mobile-phase A: 20 mM monobasic potassium phosphate (pH 2.7) in water; mobile-phase B: 20 mM monobasic potassium phosphate (pH 2.7) in 65:35 (v/v) acetonitrile–water; temperature: 40 °C. Conditions for (b), (c), and (d) are the same as (a) except for a gradient time of 13.3 min, a f ow of 0.208 mL/min. Peaks: 1 = terbutaline, 2 = N-acetylprocainamide, 3 = phenol, 4 = eserine, 5 = quinoxaline, 6 = quinine, 7 = ARD12495, 8 = diphenhydramine, 9 = carvediol, 10 = amitriptyline, 11 = reserpine.



Given the above discussion, the authors strongly recommend that VM values are experimentally determined to obtain accurate translations. VM can be easily determined by injecting a reversed-phase-LC dead time marker such as uracil or thiourea.

Observed Selectivity and Peak

Width Anomalies as a Result of

Differences in Column Thermostat

Design Between LC Instrumentation

Differences in column thermostat design used by differing LC instrument vendors will result in different temperatures being achieved within the column despite identi-cal “set-point and feedback” temperatures being recorded in the column compartment. The extent of this temperature deviation is dependent on instrument design, flow rate, and temperature set point. According to our experience, deviations of 5 °C or more are

commonplace (25). This degree of tempera-ture difference is sufficient to cause signifi-cant selectivity differences when transferring a method from one type of LC instrument to another. Consequently, when translating from HPLC to UHPLC or transferring a method from one type of HPLC system to another, it is very likely that observed selec-tivity differences are related to differences in column temperature.

Column thermostat design affects not only selectivity, but also efficiency. It has been found that a precolumn heat exchanger in combination with a still air column compartment can reduce radial temperature gradients in the col-umn and thereby results in narrower peaks compared to a water bath or a thermostat based on a forced air flow principle (26,27).

To address this problem, we recom-mend that the system suitability test (SST)

section of the method describes how the resolution is affected by temperature and how corrections can be employed; this description can be achieved, for example, by illustrating chromatograms corre-sponding to 30 °C, 35 °C, and 40 °C for a method developed on an instrument at 35 °C or by the construction of van ’t Hoff plots (that is, log k versus 1/temperature). By following this procedure, users can see in what direction and approximately how much the temperature needs to be changed to meet the SST acceptance cri-teria. This approach is supported by the United States, European, and Japanese pharmacopeias in that they allow a ±5 °C adjustment of temperature to obtain the right selectivity (23,40,44).


Result of Differences in Heat of Friction

When the mobile phase is depressurized

Theory for TranslationsIn theory a volumetric translation of a chromatographic method is simply a matter of keeping the ratio between the volume of each segment in the gradient and the column dead volume constanttG1iF1

=

VM1

tG2iF2

VM2

[1]

where subscript 1 corresponds to the method being translated, subscript 2 the translated method, tGi the time for segment i in the gradient, F is the flow rate and VM the column dead volume (12–15). The dead volume can be mea-sured by injecting a dead time marker such as uracil or thiourea for reversed-phase LC. This step is strongly recom-mended to get an accurate translation. The dead volume can also be estimated by the following approximation:

VM≈π Lε

pp

d2

2 [2]

where d is the internal diameter of the column, L the length of the column and εpp is the porosity of the column (typi-cally ~0.6 for 100 Å porous particles).

In previous publications and soft-ware related to translations, it is assumed that the porosity of the col-umns is the same and VM in equation 1 can be replaced with d2L. However,

when translating between a porous and a superficially porous particle it is unacceptable since the dead volume is reduced by the solid core of the lat-ter. Differences in porosity will result in inaccurately scaled gradient times. For example, when translating from a porous particle to a superficially porous wide pore material the error in gradient time will be 36% (for the Aeris phase, dp = 3.6 µm and dcore = 3.2 µm).

Based on the knowledge of the diam-eter of the particle (dp) and the solid core (dcore) it is possible to estimate the poros-ity of the column packed with superfi-cially porous particles.

εspp=(x

inter+x

intra)ε

pp [3]

xinter

=

π

61–

[4]

xintra

=– d 3

core)π(d 3

p

6d 3p

[5]

where xintra and xintra are the fraction of the dead volume corresponding to inter- and intraparticle volume of mobile phase, respectively.

Since the porosity of columns packed with porous particles varies significantly, up to ±28% as described in the text, it is necessary to use experimentally deter-

mined dead volumes to obtain accurate translations.

The flow rate is often scaled against column internal diameter to obtain the same linear velocity for columns with the same porosity.

F2=

F1d 2

2

d 21

[6]

UHPLC is usually operated at higher linear velocities than HPLC because of the more favorable van Deemter curves. Thus, going from HPLC to UHPLC requires a flow higher than the scaled flow and vice versa. To translate to a flow rate scaled for another particle size, the reduced inter-stitial linear velocity (43) should be kept constant. This can be achieved by a scal-ing based on the following equation:

F2=

F1d

p1d 2

2

dp2

d 21

[7]

Translating flow rates, however, is diffi-cult for superficially porous particles since both the A term and the C term in the van Deemter equation are smaller than for porous particles with the same diameter. In addition, it should not be assumed that the original method is running at an opti-mal flow rate. To make a translation to a truly optimal flow rate it is therefore rec-ommended to translate to a series of flow



in the column, frictional heat is generated, resulting in an axial temperature gradient. The heat generated is proportional to both the pressure drop over the column, but also the flow has an impact on heat generation and dissipation (28,29). On a 2.1-mm i.d. column operated at a pressure close to 1000 bar, the difference between the column outlet and inlet temperature can be on the order of 5–10 °C for a 150-mm column and 10–18 °C for a 50-mm column (28,29).

Selectivity differences caused by heat of friction can be compensated for by increasing the temperature when trans-lating from UHPLC to HPLC and thereby compensate for the reduced heat of friction. Alternatively, the tempera-ture is decreased to compensate for an increased heat of friction when translating from HPLC to UHPLC. One approach that is recommended is to change the temperature ±2 °C, 4 °C, 6 °C, and

8 °C and thereby investigate if the observed change in selectivity is related to heat of friction and can be compensated for (18).


Result of Differences in Pressure

A change in pressure affects the molar volume of solvated analytes as well as their degree of ionization (30–34). This effect typically results in an increased retention as pressure increases; however, there are exceptions to this rule (34). Differing degrees of pressure-induced retention changes can result in either en-hanced or reduced chromatographic res-olution. It has been reported that proteins are more sensitive to pressure-induced retention effects than lower-molecular-weight analytes because their secondary and tertiary structures are additionally affected by pressure and flow (35).

The majority of previous studies on

pressure-induced retention effects have been conducted with a postcolumn restrictor to increase the pressure while maintaining the same flow rate and do not reflect typical chromatographic con-ditions. Furthermore, it has been sug-gested that heat of friction should coun-teract pressure-related retention and selectivity changes to some extent (28,35). Nevertheless, pressure-related selectivity differences are also seen during typi-cal chromatographic conditions and for quite modest differences in pressure (for example, 142 bar) (36). Figure 3 illus-trates a typical example of a translation from an HPLC method based on a 150 mm × 4.6 mm, 5-µm column (Figure 3a) to a UHPLC method based on a 50 mm × 2.1 mm, 1.8-µm column (Figure 3c) where a pressure increase of only 125 bar resulted in a significant change in selec-tivity (peaks 3 and 4). Unfortunately, it is

rates using equation 1 and thereby deter-mine the optimal flow experimentally.

An approximate estimation of the max-imal pressure for the translated method can be determined based on a pressure reading of the maximal pressure for the original method. Maximal pressure is obtained at approximately 20% (v/v) ace-tonitrile or 50% (v/v) methanol in water. It should be noted, however, that this esti-mate may be unreliable since both pack-ing procedures and particle size distribu-tions differ between columns and vendors (20). Furthermore, the system’s contribu-tion to the pressure also differs.

Δp2=

Δp1d 2

1d 2

p1 F

2L

2

d 22 d 2

p2F

1L

1

[8]

For an accurate scaling it is of critical importance that the ratio between dwell and dead volume is kept constant; that is, the difference in ratio between the sys-tem’s dwell volume and the column dead volume needs to negligible.

VD1

– =ΔV

M1

VD2

VM2

[9]

where VD is the system dwell volume and Δ the difference in dwell and dead volume ratio between the systems. The dwell vol-ume for an HPLC system is typically 1–2

mL and for a UHPLC system it is 0.1–0.5 mL depending on configuration and model. For an accurate translation it is recommended to measure (23) the dwell volume (only necessary once for a certain instrument model and configuration).

If Δ is positive, it is necessary to intro-duce an initial isocratic step of z min to the translated method corresponding to

z =∣Δ∣

VM2

F2

[10]

If Δ is negative, it is necessary to start the gradient z min before the sample is injected, a function available in certain chromatographic data systems. A delayed injection after the start of the gradient can be easily implemented on certain LC systems (such as the Waters Acquity H-class system or the Agilent 1290 sys-tem). Another alternative that partially solves the problem is to use a larger col-umn internal diameter (equation 9).

If the translated method is not cor-rected for, it may display different relative retention times and potentially also dif-ferent selectivity. The injection volume is scaled against the dead volume to obtain the same relative amount on column.

Vinj2=

Vinj1V

M2

VM1

[11]

Because of the higher efficiency of sub-2-µm and superficially porous columns, asymmetry will be higher for overloaded peaks due to the higher concentration at peak apex. This can be compensated for by a scaling against the number of plates for a nonoverloaded peak chromato-graphed under isocratic conditions.

V ′inj2=

Vinj1

N1V

M2

N2V

M1

[12]

For porous particles the efficiency is proportional to the ratio of column length or particle size. Thus, for porous particles:

V ′inj2≈

Vinj1

L1dp2

VM2

L2dp1

VM1

[13]

To minimize poor efficiency for early peaks it is important to match the column dead volume with the system’s contribution to peak volume:

N =V

M (1+k)

σ2

col+σ

2

ext√

[14]

where k is the retention factor and σ is the contribution to the peak volume from band broadening in column (σcol) and system (σext) expressed as standard deviation.



difficult to compensate for these types of pressure effects. While it does not provide a solution, adding a postcolumn restrictor can enable confirmation that pressure is the cause of the problem (Figure 3d). To

compensate for pressure-related selec-tivity changes, it is probably necessary to reoptimize the method by adjustments of parameters such as gradient shape and temperature.

Poor Efficiency Because of

Differences in Extracolumn

Band Broadening Between

LC Instrumentation

Efficiency is dependent on retention as well as the ratio between column dead volume and the volume of the peak (equation 14 in the sidebar). Since the dead volume of an HPLC column is typically much larger than that of an UHPLC column, it is nec-essary to reduce the UHPLC instrument’s contribution to peak volume significantly (7). This extracolumn band broadening (ECBB) — usually measured as 4σ — is typically in the order of 30–50 µL and 10 µL for HPLC and UHPLC, respectively. Even the low volume associated with modern UHPLC instrumentation has been found to be insufficiently low to compen-sate completely for the extracolumn band broadening contribution associated with peaks of low retention (7,37). This effect is illustrated in Figure 4, where an isocratic translation from HPLC to UHPLC exhib-ited the expected efficiency for late eluted peaks; however, the efficiency observed for early eluted peaks was significantly lower. Figure 5 illustrates the efficiency versus the retention factor for the same separation for other combinations of column and LC system ECBB volumes. In this example as well as in reference 7, the expected plate numbers are not reached until a retention factor of approximately 4 is reached when translating to a 50 mm × 2.1 mm column operated on an UHPLC system.

Modifying the ECBB of an LC system is usually quite difficult (38). A more realistic alternative is to increase the col-umn internal diameter. Today, 3-mm i.d. UHPLC columns are commercially avail-able, and they can significantly reduce this problem. The drawback to this approach is that a flow rate twice as high as for a 2.1-mm i.d. column must be used, resulting in a reduced saving in solvent consumption and a potential for increased heat of fric-tion, which could cause band broadening and selectivity differences (29).

In general, the minimized ECBB asso-ciated with UHPLC systems is advanta-geous; however, there is a possibility that it may cause significant band broadening when performing HPLC methods with the sample dissolved in a high proportion of organic solvent compared to the initial mobile phase. This may appear contradic-

(a)

1

k = 0.1

N = 8727

3

k = 1.3

N = 13,294

4

k = 3.4

N = 14,971

5

k = 5.6

N = 17,707

tR = 17.3 min

k = 0.1

N = 3602

k = 1.4

N = 8571

k = 3.9

N = 17,108 k = 6.4

N = 18,699

tR = 7.7 min

(b)

2

Figure 4: An isocratic translation from (a) a 250 mm × 4.6 mm, 5-µm column and a binary Agilent 1100 HPLC system to (b) a 100 mm × 2.1 mm, 1.8-µm column and a quaternary Agilent 1290 UHPLC system, showing how ECBB becomes critical for peaks with low re-tention when using columns with small dead volumes. The chromatograms have been scaled by alignment of the f rst and last eluted peaks. Flow rate (a): 1.0 mL/min; f ow rate (b): 0.21 mL/min; mobile phase: 2:400:600 (v/v/v) phosphoric acid 85% w/v–acetonitrile–water; temperature: 22 °C. Peaks: 1 = 4-hydroxybenzoic acid, 2 = acetylsalicyclic acid, 3 = salicylic acid, 4 = acetylsalicylsalicylic acid, 5 = salsalate.

Nu

mb

er

of

theo

reti

cal p

late

s (N

)

Retention factor (k)

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

00 2 4 6

100 × 2.1 mm, 1.8μm, ECBB 9 μL

100 × 2.1 mm, 1.8 μm, ECBB 11 μL

100 × 2.1 mm, 1.8 μm, ECBB 23 μL

150 × 4.6 mm, 3.5 μm, ECBB 9 μL

150 × 4.6 mm, 3.5 μm, ECBB 11 μL

150 × 4.6 mm, 3.5 μm, ECBB 23 μL

250 × 4.6 mm, 5 μm, ECBB 23 μL

Figure 5: Number of theoretical plates versus retention factor for the separations shown in Figure 4 and for f ve additional combinations of columns and LC systems with different ECBB contributions.



tory, but it can be explained by the nar-rower capillaries on the UHPLC system reducing the mixing between the sample and the mobile phase and thereby reduc-ing peak focusing of the sample on top of the column. The solution to this problem is to reduce the sample volume or the amount of organic solvent in the sam-ple solution. Alternatively, the capillary between the injection valve and the col-umn can be replaced with a large internal diameter capillary to increase the mixing.

Differences in Linearity, Response, or

Repeatability Related to Injector Design

Different LC systems possess differing injection flow principles and materials in their autosampler construction. For exam-ple, in a loop injector the sample is typically exposed to larger surface areas and other materials than in a flow through needle in-jector design. This larger surface area may result in a more pronounced adsorption and, consequently, also a more pronounced nonlinear response at low concentrations for loop injectors.

Other potential problems can be related to differences in the internal diameter of the injector needle and capillaries. UHPLC systems have significantly narrower injec-tor needle and capillary diameters, which can result in poor injection repeatability because of bubble formation if the draw speed is set too high in relation to the viscosity of the sample. Another related problem is that differences in viscosity between samples and standards because of matrix differences may result in differ-ing amounts being injected, thereby the sample concentration can be under or over-estimated.

Consequently, it is important to validate linearity, response, and repeatability of the LC method when transferring a method from one type of LC system to another.

Differences in Peak Asymmetry

Related to Differences in Efficiency

The transfer of HPLC methodology to UHPLC when overloaded peaks are in-volved results in more-pronounced peak asymmetries being observed even if injec-tion volumes have been correctly scaled against column volume. The explanation for this phenomenon is that the higher efficiency associated with the UHPLC method results in higher concentrations of

the analyte at the apex of the peak and thus a higher degree of overloading is observed (39). Despite this overloading, the resolu-tion of peaks adjacent to the overloaded peak is typically higher in the UHPLC method than in the HPLC method. It is possible to compensate for this overload ef-fect by simply scaling the injection volume against column dead volumes as well as the isocratic efficiencies (equations 12 and 13 in the sidebar).

Other Issues

In the early days of UHPLC, selectivity dif-ferences were commonly observed between columns of nominally the same material but differing particle size. These differences were impossible to explain by differences in the heat of friction or pressure differences (20). It was assumed that the base silica of the smaller particle size material was subtly different compared to its larger particle size counterparts. Today this difference is less of a problem. However, if a selectivity differ-ence is observed that cannot be compen-sated for by either increasing or decreasing the temperature by a few degrees to mimic heat of friction or by adding a postcolumn restrictor to mimic a pressure-induced re-tention change, it is more than likely that it is related to subtle differences in the hetero-geneity of the base silica used.

Regulatory AspectsThe degree of revalidation that is required for a translated method depends on the in-tended purpose for that method and where in the R&D process it is to be used.

For pharmacopeia methods, LC trans-lations can, in principle, be made with-out any formal validation exercise being performed provided that the operat-ing ranges described in the appropriate monographs (23,40,44) are not exceeded. Unfortunately, the current operating ranges limit the ability to translate from HPLC to UHPLC. For example, the parti-cle size can only be reduced by 50% which prevents translation from a 5-µm mate-rial to a sub-2-µm material. It is, however, expected that the pharmacopoeias will be updated in the near future to allow for such translations to take place as long as the specified selectivity and efficiency is maintained (that is, by maintaining or increasing the ratio between column length and particle diameter [41]). Hope-

fully, this will also result in increased reg-ulatory flexibility in general.

For original pharmaceutical products it is necessary to validate all versions of the method that are used for analysis of samples to be used in toxicological or clinical studies. Do both the HPLC and the UHPLC meth-ods require a full International Conference on Harmonisation (ICH) validation (42)? From a scientific point of view, it should be sufficient to submit a full ICH validation for either the HPLC or the UHPLC version of the method. For the other version, it should be sufficient to submit a minimalistic vali-dation report claiming that the translation has been made according to first principles (that is, fundamental chromatographic theory). In principle, it should be enough to validate selectivity and linearity for the translated version of the method. It is advis-able, at least during this transition period, to perform a full ICH validation and be prepared to submit the “missing” parts of the validation if requested by the authori-ties. It may also be prudent to include both the HPLC and UHPLC versions of the method in the experimental design used for validation of intermediate precision.

Postsubmission changes to methods are possible according to both the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) regu-lations. However, it is uncertain what is required in the rest of the world. In addi-tion, the cost associated with a postapproval change is often considered prohibitive.

ConclusionsThis article demonstrates how the prac-tical advantages of translating existing HPLC methodologies to those using newer column formats and UHPLC instrumentation can be realized. Ad-vantages include increased resolution, enhanced speed of analysis, reduced solvent consumption, more efficient utilization of LC equipment, and ease of method transfer between HPLC and UHPLC. The authors describe a number of potential pitfalls that must be taken into consideration and avoided if ac-curate and reliable translations are to be achieved. These pitfalls include dif-ferences in LC instrumentation dwell volumes, which can lead to coelution of peaks and even reversal of elution order. The difference between the original and



new VD/VM ratios must be accounted for: In the case of the new methodology having a larger VD/VM, a delayed injec-tion must be used, or if it has a smaller VD/VM, an isocratic hold must be in-serted before commencing the gradient. Errors in translated gradient times of up ~30% can be encountered unless ex-perimentally determined VM values are used. This may affect both retention and selectivity. Hence, the authors strongly recommend that chromatographers practically determine their VM values. The effect of instrument differences be-tween HPLC and UHPLC models have been highlighted as possible sources of translation error; these include differ-ences in column thermostat design, the effect of extracolumn band broadening, and the influence of differing injector design. In addition, the effect of only moderately elevated pressures on selec-tivity has been demonstrated, coupled with the effect of increased heat of fric-tion associated with smaller particles and the influence of increased efficiency on peak asymmetry has been described.

A free translation tool is available to assist the successful translation between HPLC and UHPLC methods (21). The tool is based on the principles described in this article and permits scaling of gradient times, flow rates, and injection volume as well as accounting for differ-ences in VD/VM ratios between LC sys-tems. A more comprehensive transla-tion tool also will be available (22).

References

(1) J.W. Thompson, J.S. Mellors, J.W. Eschelbach,

and J.W. Jorgenson, LCGC North Am. 24(s4),

16Ð21 (2006).

(2) J.J. Kirkland, F.A. Truszkowski, C.H. Dilks Jr., and

G.S. Engel, J. Chromatogr A 890, 3Ð13 (2000).

(3) J.J. DeStefano, T.J. Langlois, and J.J. Kirkland, J.

Chromatogr. Sci. 46, 254Ð260 (2008).

(4) M. Swartz, LabPlus International 18, 6Ð9 (2004).

(5) J.R. Mazzeo, U.D. Neue, M. Kele, and R.S.

Plumb, Anal. Chem. 77, 460AÐ467A (2005).

(6) Strategic Directions International (SDi) Tacti-

cal Sales and Marketing Report (HPLC: End

Users Influencing Development for Advanced

HPLC Systems, Columns and Chemistry).

(2013).

(7) D. Guillarme, D.T.T. Nguyen, S. Rudaz, and

J.L. Veuthey, Eur. J. Pharm. Biopharm. 66,

475Ð482 (2007).

(8) D. Guillarme, D.T.T. Nguyen, S. Rudaz, and J.L.

Veuthey, Eur. J. Pharm. Biopharm 68, 430Ð440

(2008).

(9) P. Petersson, ÒImplementation of U(H)PLC

within a Global Pharmaceutical Company - A

New Way of Working, Advances in High Reso-

lution and High Speed Separations,Ó Chromato-

graphic Soc. Alderley Park, 2010.

(10) R.E. Majors, LCGC North Am. 29(6), 476Ð484

(2011).

(11) J.W. Dolan, LCGC North Am. 32(2), 98Ð102

(2014).

(12) H. Engelhardt and H. Elgass, J. Chromarogr. A

158, 249Ð259 (1978).

(13) P. Janderra, J. Chromatogr. A 485, 113Ð141

(1989).

(14) J.W. Dolan and L. Snyder, J. Chromatogr. A 799,

21Ð34 (1998).

(15) J.W. Dolan, LCGC North Am. 32(3), 188Ð193

(2014).

(16) A.P. Schellinger and P.W. Carr, J. Chromatogr. A

1077, 110Ð119 (2005).

(17) P. Janderra, J. Chromatogr. A 1126, 195Ð218

(2006).

(18) A.Clarke, J. Nightingale, P. Mukherjee, and P.

Petersson, Chromatography Today, May/June, 4Ð9

(2010).

(19) www.unige.ch/sciences/pharm/fanal/lcap/

telechargement.htm.

(20) P. Petersson and M.R. Euerby, J. Sep. Sci. 30,

2012Ð2024 (2007).

(21) www.acdlabs.com/translatemymethod.

(22) www.acdlabs.com/products/spectrus/workbooks/

chrom/.

(23) European Pharmacopoeia 7th Edition, 2.2.46,

ÒChromatographic separation techniques,Ó Euro-

pean Pharmacopoeia (European Directorate for

the Quality of Medicines, Strasbourg, France,

2011).

(24) A.G. Huesgen, Agilent Technologies, www.agi-

lent.com, Publication Number 5990-5062EN.

(25) P. Petersson and M.R. Euerby, personal commu-

nication; unpublished data; March 16, 2014.

(26) A. de Villiers, H. Lauer, R. Szucs, S. Goodall, and

P. Sandra, J. Chromatogr. A 1113, 84Ð91 (2006).

(27) K. Kaczmarski, F. Gritti, and G. Guiochon, J.

Chromatogr. A 1177, 92Ð104 (2008).

(28) Y. Zhang, X. Wang, P. Mukherjee, and P. Peters-

son, J. Chromatogr. A 1216, 4597Ð4605 (2009).

(29) F. Gritti and G. Guiochon, Anal. Chem. 80,

5009Ð5020 (2008).

(30) M.M. Fallas, U.D. Neue, M.R. Hadley, and

D.V. McCalley, J. Chromatogr. A 1209, 195Ð205

(2008).

(31) M.M. Fallas, U.D. Neue, M.R. Hadley, and

D.V. McCalley, J. Chromatogr. A 1217, 276Ð284

(2010).

(32) F. Gritti and G. Guiochon, Anal. Chem. 81,

2723Ð2736 (2009).

(33) P. Szabelski, A. Cavazzini, K. Kaczmarski, X. Liu,

J. Van Horn, and G. Guiochon, J. Chromatogr. A

950, 41Ð53 (2002).

(34) M.R. Euerby, M. James, and P. Petersson, J. Chro-

matogr. A, 1228, 165Ð174 (2012).

(35) S. Fekete, J.L. Veuthey, D.V. McCalley, and D.

Guillarme, J. Chromatogr. A 1270, 127Ð138

(2012).

(36) M.R. Euerby, M.A. James, and P. Petersson, Anal.

Bioanal. Chem. 405, 5557 (2013).

(37) N. Wu, A.C. Bradley, C.J. Welch, and L. Zhang,

J. Sep. Sci. 35, 2018Ð2025 (2012).

(38) A.J. Alexander, T.J. Waeghe, K.W. Himes, F.P.

Tomasella, and T.F. Hooker, J. Chromatogr. A

1218, 5456Ð5469 (2011).

(39) P.Petersson, P. Forssen, L Edstšm, F. Samie, S.

Tatterton, A. Clarke, and T. Fornstedt, J. Chro-

matogr A 1218, 6914Ð6921 (2011).

(40) United States Pharmacopeia General Chapter

<621> ÒChromatographyÓ (United States Phar-

macopeial Convention, Rockville, MD).

(41) U.D. Neue, D. McCabe, V. Ramesh, H. Pappa,

and J. DeMuth, Pharmacopeial Forum 35, 1622Ð

1626 (2009).

(42) International Conference on Harmonisation,

ICH Q2(R1), Validation of Analytical Procedures:

Text and Methodology (ICH, Geneva, Switzer-

land, 2005).

(43) U.D. Neue, HPLC Columns: Theory, Technology and

Practice (Wiley-ICH, New York, 1997), pp. 14.

(44) Japanese Pharmacopoeia 16th Edition, section

2.01 ÒLiquid ChromatographyÓ (JP XVI 2.01,

Pharmaceuticals and Medical Devices Agency,

Shin-Kasumigaseki Building, 3-3-2 Kasumi-

gaseki, Chiyoda-ku, Tokyo 100-0013 Japan,

2011).

Patrik Petersson is a principal sci-

entist with Novo Nordisk A/S in Måløv,

Denmark. Melvin R. Euerby is a

professor at the Strathclyde Institute of

Pharmacy and Biomedical Sciences in

the University of Strathclyde in Glasgow,

UK and the head of research and devel-

opment and training at Hichrom Ltd. in

Berkshire, UK. Matthew A. James

is a research analyst with Hichrom Ltd.

Direct correspondence to:

[email protected] ◾

For more information on this topic, please visit our homepage at:

www.chromatographyonline.com


THE APPLICATION NOTEBOOK

Call for Application Notes

LCGC is planning to publish the next issue of

T e Application Notebook special supplement in

December. T e publication will include vendor

application notes that describe techniques and

applications of all forms of chromatography and

capillary electrophoresis that are of immediate in-

terest to users in industry, academia, and govern-

ment. If your company is interested in participat-

ing in these special supplements, contact:

Michael J. Tessalone, Group Publisher,

(732) 346-3016

Edward Fantuzzi, Associate Publisher,

(732) 346-3015

Stephanie Shaf er, East Coast Sales Manager,

(774) 249-1890

Lizzy T omas, Account Executive,

(574) 276-2941

Application Note Preparation

It is important that each company’s mate-

rial f t within the allotted space. T e editors

cannot be responsible for substantial editing

or handling of application notes that deviate

from the following guidelines:

Each application note page should be no more

than 500 words in length and should follow

the following format.

Format

• Title: short, specif c, and clear

• Abstract: brief, one- or two-

sentence abstract

• Introduction

• Experimental Conditions

• Results

• Conclusions

• References

• Two graphic elements: one is the company

logo; the other may be a sample chromato-

gram, f gure, or table

• T e company’s full mailing address,

telephone number, fax number,

and Internet address

All text will be published in accordance with

LCGC ’s style to maintain uniformity through-

out the issue. It also will be checked for gram-

matical accuracy, although the content will not

be edited. Text should be sent in electronic for-

mat, preferably using Microsoft Word.

Figures

Refer to photographs, line drawings, and

graphs in the text using arabic numerals in

consecutive order (Figure 1, etc.). Company

logos, line drawings, graphs, and charts must

be professionally rendered and submitted as

.TIF or .EPS f les with a minimum resolution

of 300 dpi. Lines of chromatograms must be

heavy enough to remain legible after reduc-

tion. Provide peak labels and identif cation.

Provide f gure captions as part of the text,

each identif ed by its proper number and title.

If you wish to submit a f gure or chromato-

gram, please follow the format of the sample

provided below.

Tables

Each table should be typed as part of the main

text document. Refer to tables in the text by

Roman numerals in consecutive order (Table I,

etc.). Every table and each column within the

table must have an appropriate heading. Table

number and title must be placed in a continu-

ous heading above the data presented. If you

wish to submit a table, please follow the format

of the sample provided below.

References

Literature citations must be indicated by arabic

numerals in parentheses. List cited references

at the end in the order of their appearance. Use

the following format for references:

(1) T.L. Einmann and C. Champaign, Science

387, 922–930 (1981).

T e deadline for submitting application notes for the December issue of T e Application Notebook is:

October 24, 2014

T is opportunity is limited to advertisers in LCGC North America. For more information, contact:

Mike Tessalone at (732) 346-3016, Ed Fantuzzi at (732) 346-3015, Stephanie Shaf er at (774) 249-1890, or Lizzy T omas at (574) 276-2941.

Table I: Factor levels used in the designs

Factor Nominal value Lower level (−1) Upper level (+1)

Gradient profile 1 0 2

Column temperature (°C) 40 38 42

Buffer concentration 40 36 44

Mobile-phase buffer pH 5 4.8 5.2

Detection wavelength (nm) 446 441 451

Triethylamine (%) 0.23 0.21 0.25

Dimethylformamide 10 9.5 10.5

Figure 1: Chromatograms obtained using the conditions under which the ion sup-pression problem was originally discov-ered. The ion suppression trace is shown on the bottom. Column: 75 mm × 4.6 mm ODS-3; mobile-phase A: 0.05% heptaf uo-robutyric acid in water; mobile-phase B: 0.05% heptaf uorobutyric acid in aceto-nitrile; gradient: 5–30% B in 4 min. Peaks: 1 = metabolite, 2 = internal standard, 3 = parent drug.



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