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Spark-Holland Automated on-line DPS Extraction and Analysis with a Q-Exactive Orbitrap

Lecture 9, Page 41

Developing DPS Methods

•Normal regulated bioanalysis protocol:

› Centrifuge collected blood to get plasma

› Fortify control plasma (not blood) with standards and QC’s to create the calibration curve

› Fortify test plasma samples with IS at a fixed level

› Extract plasma standards and QC’s along with test samples and analyze by LC/MS

Lecture 9, Page 42

Towards Higher Throughput DPS

On-line SPE-LC-HRAMS DPS Extraction (Exactive Plus)

DPS Card

Exactive Plus

2 mm

Spark-Holland DPS autosampler

Lecture 9, Page 43

The Q Exactive: Hardware Innovations

Orbitrap Mass Analyzer

HCD Cell

S-lens Ion Source

Enhanced FT

C-Trap Quadrupole Mass Filter

Lecture 9, 4

RP = 15,000 RP = 56,700

High Resolution and Accurate Mass – Example In

ten

sity

(%

)

0

20

40

60

80

100

Mass [amu]

516.65 516.70 516.75 516.80 516.85 516.90

Inte

nsi

ty (

%)

0

20

40

60

80

100

Mass [amu] 516.65 516.70 516.75 516.80 516.85 516.90

516.77581 (observed)

516.76671

(correct) 516.78490

(correct)

516.78490 516.76671

Wrong Answer for Both Peptides

Peptide mixture: [Val5]-Angiotensin II Lys-des-Arg9-Bradykinin

Sequence: DRVYVHPF KRPPGFSPF

Formula: C49H69N13O12 C50H73N13O11

Exact mass: [M+2H]2+ = 516.76671 [M+2H]2+ = 516.78490

Dm (mmu): 18.2 mmu

Right Answer for Both Peptides

Lecture 9, Page 45

Representative XICs (5 ppm) with Orbitrap HRAMS

Lecture 9, Page 46

RBC Filtration Workflow 1

•Whole blood standards, QCs, and blanks were subjected to RBC filtration device to produce DPS

Standard Curve Prepared Using Filtration Device

Offline RBC Filtration card

Lecture 9, Page 47

RBC Filtration Workflow 1

• Acceptable accuracy and precision

Drawbacks

• Time-consuming to prepare all standards and QCs using filtration device

• What is the best way to approach this?

• Stay tuned

Accuracy and Precision

Guanfacine Concentration (ng/mL)

LLOQ QC LQC MQC HQC

0.50 0.75 200 400

Mean 0.49 0.65 211 428

CV (%) 10.6 14.5 2.9 5.1

RE (%) -1.4 -12.8 5.3 6.9

n 5 6 6 6

Lecture 9, Page 48

Summary

• Q-Exactive provides excellent selectivity in a small molecule quantitation assay providing a linear dynamic range over three orders of magnitude.

• A novel membrane-based device capable of filtering RBCs out of whole blood without the need for centrifugation has been presented for offline quantitative bioanalysis.

• Acceptable accuracy and precision for whole blood QCs subjected to our filtration device can be achieved using a fortified plasma standard curve.

• The DPS card is compatible with commercially available online-extraction dried blood spot robotic autosamplers.

RBC filtration device for generating dried plasma spots

Lecture 9, Page 49

Lecture 9, Page 50

Lecture 9, Page 51

Lecture 9, Page 52

Lecture 9, Page 53

The Advion TriVersa NanoMate A Robotic Workstation For Automated nano ESI

Lecture 9, Page 54

TriVersa + MS

MS sample orifice

Automated Direct ‘Infusion’ nano ESI of Protein Samples

Infusion can be for short or long times; the latter allowing for simultaneously screening for

hundreds of compounds! Lecture 9, Page 55

Lecture 9, Page 56

Lecture 9, Page 57

Lecture 9, Page 58

Another Form of Selectivity coupled with Mass Spectrometry

• Ion mobility Spectrometry

Lecture 9, Page 59

Differential Mobility Spectrometry (DMS) ‘Selexion’ from AB Sciex

(DMS is not mass spectrometry)

An orthogonal separation mechanism based upon charge-to-mass and

molecular shape

Lecture 9, Page 60

Mass Spec Vacuum draws ions and gas through

planar DMS electrodes

2.3 torr

2.8 L/min

1 mm

3 cm

760 torr

Millisecond flight times

Lecture 9, Page 61

Chemical Enhanced Separations Driven by Mobility Changes Due to Clustering / Declustering.

Low field

clustering-

mobility decreases

+

HV=3kV

Schneider BB, Covey TR, Coy SL, Krylov EV, and Nazarov EG, “Chemical Effects in the Separation Process of a Differential Mobility/Mass

Spectrometer System”, Anal. Chem., 2010, 82, 1867-1880.

High field

declustering;

mobility increases

L V=0.3 kV

F ~ 1 MHz, T= 10-6 s

Lecture 9, Page 62

DMS Hardware on AB Sciex 5500 QTRAP

Lecture 9, Page 63

Testosterone Female #3 Plasma Sample: LLE

DMS OFF/

289/109

DMS OFF/

289/97

DMS ON/

289/109 DMS ON/

289/97

Interference

Ion ratio is Peak of interest Interference

Peak of interest

Lecture 9, Page 64

Testosterone Female #3 Plasma Sample: PPT

DMS OFF/

289/109

DMS OFF/

289/97

DMS ON/

289/109

DMS ON/

289/97

Peak of interest

Peak of interest @ RT 3.37 min

Lecture 9, Page 65

•Question: If we consider the entire known organic chemical space, including proteins, peptides, RNA, DNA, as well as polar small molecule chemicals, what % of these are amenable to GC/MS?

•Answer: Let’s take a look….

GC/MS or LC/MS/MS Which Technique Handles the Wider Range of Chemical

Compounds?

Lecture 9, Page 66

GC/MS vs. LC/MS/MS for Range of Pesticides Detected

Lutz Alder,* Kerstin Greulich, Gu¨nther Kempe, and Ba¨rbel Vieth, RESIDUE ANALYSIS OF 500 HIGH PRIORITY PESTICIDES, Mass Spectrometry Reviews, 2006, 25, 838– 865

Lecture 9, Page 67

• This overview evaluates the capabilities of mass spectrometry (MS) in combination with gas chromatography (GC) and liquid chromatography (LC) for the determination of a multitude of pesticides. The selection of pesticides for this assessment is based on the status of production, the existence of regulations on maximum residue levels in food, and the frequency of residue detection. GC–MS with electron impact (EI) ionization and the combination of LC with tandem mass spectrometers (LC–MS/MS) using electrospray ionization (ESI) are identified as techniques most often applied in multi-residue methods for pesticides at present. Therefore, applicability and sensitivity obtained with GC–EI–MS and LC–ESI–MS/MS is individually compared for each of the selected pesticides. Only for one substance class only, the organochlorine pesticides, GC-MS achieves better performance. For all other classes of pesticides, the assessment shows a wider scope and better sensitivity if detection is based on LC–MS.

• Ref: (Mass Spec Rev 25:838–865, 2006)

GC/MS vs. LC/MS/MS

Lutz Alder,* Kerstin Greulich, Gu¨nther Kempe, and Ba¨rbel Vieth, RESIDUE ANALYSIS OF 500 HIGH PRIORITY PESTICIDES, Mass Spectrometry Reviews, 2006, 25, 838– 865

Lecture 9, Page 68

Smaller Mass Spectrometers

Mini 12

Expression CMS

Lecture 9, Page 69

Commercially Available Small Mass Spectrometers

Lecture 9, Page 70

Mini 12 mass spectrometer

Lecture 9, Page 71

Control Tablet

Electronics

Cassette

Loading

system

Bar Code Reader

Vacuum

System

Solvent

Container

Solvent Pump

Mini 12: Internal components

Lecture 9, Page 72

Paper Spray: Immediate Point-of-Care Analysis

kV

1. Prick Finger

2. Load Sample

3. Apply Solution 4. Apply HV

5. Acquire Data

0 s

15 s

30 s 40 s

50 s

60 s

[M+H]+

6. Report Results

• One drop of blood • 60 second analysis of biological samples

Lecture 9, Page 73

Atenolol

MW=266

m/z 267 Atenolol 10 ppm spiked in whole blood

0.4μl blood loaded for each sampling spot

10 μl methanol/H2O added for spraying

m/z 267

MS paper spray of blood spiked with

atenolol (4ng/spot)

MS/MS spectrum to identify atenolol

Paper spray/mini MS EESI (direct detection, Atenolol in blood)

251

225

208 190

145

173

180

He Wang, Jiangjiang Liu, Guangming Huang

CID

Lecture 9, Page 74

http://upload.wikimedia.org/wikipedia/commons/4/47/Atenolol.svg

The Mini 12 From Purdue University A Miniature Mass Spectrometer for Clinical and Other

Applications Introduction and Characterization

Linfan Li,† Tsung-Chi Chen,† Yue Ren,† Paul I. Hendricks,‡ R. Graham Cooks,*,‡,§ and Zheng Ouyang*,†,§ dx.doi.org/10.1021/ac403766c | Anal. Chem. 2014, 86, 2909−2916 Lecture 9, Page 75

Mini 12 MS and MS/MS nano ESI Mass Spectra for Clenbuterol, Thiabendazole and Amitryptyline

Lecture 9, Page 76

Cart-Based MS System Advion, Inc.

Lecture 9, Page 77

• Little or no sample preparation • No chromatography • Data in < 1 minute The closed end of a glass capillary is dipped into the sample of interest or scraped on a solid surface, and then placed into the CMS for analysis. Ideal for: • Reaction monitoring • Compound ID • Food safety • Natural products

ASAP® – Atmospheric Solids Analysis Probe

Lecture 9, Page 78

SPME Sample Preparation for Drugs in Urine

SPME: Solid-Phase Micro Extraction. • Insert a fused-silica fiber coated

with an HPLC stationary phase into a biological sample

• Allow to equilibrate with the sample

• Remove and rinse with water • Place fiber containing extracted

sample into hot nitrogen gas • Ionize by APCI • Obtain mass sectra

Lecture 9, Page 79

Amlodipine in Urine

Amlodipine C20H25ClN2O5

MW: 408.87

Preparation of fortified urine sample The stock solution of amlodipine was 1.0 mg/ml in methanol by dissolving a 2.5 mg tablet in 2.5 mL Methanol. The 50ng/ul and 100ng/ul amlodipine in urine was made by spiking the stock solution in urine sample.

Lecture 9, Page 80

ASAP-SPME-MS Analysis of 50 ng/ul Amlodipine in urine Extract time: 5 mins, SPME LC Tips SCX/C18

Mass spectrum of Amlodipine in urine

XIC for m/z 406

Total ion current

[M+H]+

C20H25ClN2O5

MW: 408.87

Lecture 9, Pages 81

Copyright Jack Henion 2016

SPME-ASAP-SIM of Incurred urine, two hours after 2.5mg dose Extract time: 60 mins, SPME LC Tips C18

XIC for m/z 409 after background subtraction

Total ion current

Did not detect the m/z 409 in the full-scan mode. However, with the SIM mode, it clearly detected the m/z 409. SCX/C18 tips has similar result to C18 tips

Lecture 9, Pages 82

Real-Time Monitoring of Suzuki Reaction with ASAP®-MS

Changtong Hao, Ph.D Advion Inc.


Lecture 9, Pages 83

Experimental and Materials

Reactants A and B were mixed at equi-molar amounts in a round-bottom reaction flask and stirred at room temperature. 2mL aliquots was transferred to the capillary tip of ASAP® probe for ASAP®-MS analysis.

NaOH, PdCl2; 4 mol% EtOH/H2O 4:1 5ml Room temp NH2

BrB

OH

OH

NH2

+

A B

C D

Suzuki Reaction

Reactants and Catalyst Product

A B C D E

compound 4-bromaniline Phenyl Boronic acid

Sodium hydroxide

Palladium chloride

4-aminobiphenyl

Chemical Formula

C6H6BrN C6H7BO2 NaOH PdCl2 C12H11N

MW 171 122 40 176 169

moles 0.005 0.005 0.0065 0.005 0.005

mg 855 610 260 35 845

E

Chemicals were purchased from Sigma-Aldrich with a purity greater than 99%. MS solvent was LC/MS grade.


Lecture 9, Pages 84

Figure 1. Mass spectra of Suzuki reaction mixture at different time

BrNH2

4-bromaniline C6H6BrN

MW: 172.02

[M+H]+:

NH2

4-aminobiphenyl C12H11N

MW: 169.22

[M+H]+:

0 min 10 min 15 min 30 min 60 min 180 min

The bromine ‘doublet’ in the reactant at m/z 172/174 diminishes while the product ion increases at m/z 170!

m/z 79/81 doublet

NH2

Br

NH2


Lecture 9, Pages 85

Reactant is consumed while product is produced: Know when the reaction is finished!

Reaction Time(mins)

0 50 100 150 200

Rela

tive R

ati

o t

o T

oa

l P

eak H

eig

ht

of

Reac

tan

t an

d P

rod

uct

(%)

0

20

40

60

80

100

Product

Reactant

Suzuki Reaction Monitoring by ASAP/MS

NH2

Br

NH2


Lecture 9, Pages 86

Thin Layer Chromatography/Mass Spectrometry

TLC/MS

The Advion PlateExpressTM


Lecture 9, Pages 87

Thin Layer Chromatography

Wouldn’t it be nice to know what each of those spots really is?


Lecture 9, Pages 88

A Compact Mass Spectrometer (CMS) connected to the Plate Express™ sample extraction device

TLC-CMS set-up


Lecture 9, Pages 89

TLC-CMS Workflow Schematic

200 µL/min Methanol with 0.1% Formic Acid

SSI Isocratic Pump

6 port valve

Plate Express sample extraction device

TLC Plate

Sealing Face

Elution Head

• Controlled by MS operation software (Mass Express)

• Programmable sealing force for different media (eg TLC)

• Interchangeable heads for different bed heights


Lecture 9, Pages 90

TLC/MS for the detection of chemical compounds related to cannabis

Daniel Eikel and Simon Prosser

Advion Inc., Ithaca, NY, USA


Lecture 9, Pages 91

Cannabinoids used in this study

OH

OH CH3

CH3

CH3CH3

Cannabigerol Mw=316.24 (C21H32O2)

CH3 CH3O

OH

CH3

CH3

Cannabichromene Mw=314.22 (C21H30O2)

Cannabinol (CBN) Mw=310.19 (C21H26O2)

CH3

CH3OH

OH

CH3

HHCH2

OH

OCH3

CH3OH

OH

CH3

HHCH2 Cannabidiolic acid (CBD-A)

Mw=358.21 (C22H30O4) Cannabidiol (CBD) Mw=314.22 (C21H30O2)

CH3

CH3

CH3

CH3O

OH

CH3

CH3

CH3

CH3O

OHH

HCH3

CH3

CH3

CH3O

OHH

H

OH

O

-Δ9-THC acid (THC-A) Mw=358.21 (C22H30O4) -Δ9-THC (THC) Mw=314.22 (C21H30O2)


Lecture 9, Pages 92

93

6

TLC-FIA-MS (TIC)

TLC-CMS example:

THC (M-H)- at m/z 313.3

In-source CID-MS

CH3

CH3

CH3

CH3O

OHH

HCH3

CH3

CH3

CH3O

OHH

H

OH

O

(M+H)+


References: Mini 12


• Linfan Li,† Tsung-Chi Chen,† Yue Ren,† Paul I. Hendricks,‡ R. Graham Cooks,*,‡,§ and Zheng Ouyang*,†, Mini 12, Miniature Mass Spectrometer for Clinical and Other Applications Introduction and Characterization, Anal. Chem. 2014, 86, 2909−2916

Lecture 9, Pages 94

1. Ramagiri, S. and F. Garofolo, Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges. Bioanalysis, 2012. 4(5): p. 529-540.

2. Zhang, J., et al., Proteoform analysis of lipocalin-type prostaglandin D-synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with Fourier transform mass spectrometry. Proteomics, 2014. 00: p. 1–9.

3. Ruan, Q., et al., Strategy and Its Implications of Protein Bioanalysis Utilizing High-Resolution Mass Spectrometric Detection of Intact Protein. Analytical Chemistry, 2011. 83(23): p. 8937-8944.

4. Li, H., et al., Native Top-Down Electrospray Ionization-Mass Spectrometry of 158 kDa Protein Complex by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Analytical Chemistry, 2013. 86(1): p. 317-320.

5. Sun, L., et al., Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry. Analytical Chemistry, 2013. 85(12): p. 5989-5995.

6. Zhang, J., et al., Top-Down Mass Spectrometry on Tissue Extracts and Biofluids with Isoelectric Focusing and Superficially Porous Silica Liquid Chromatography. Analytical Chemistry, 2013. 85(21): p. 10377-10384.

7. Ref: Hongcheng Liu, Georgeen Gaza-Bulseco, and Chris ChumsaeJ Am Soc Mass Spectrom 2009, 20, 2258–2264.

Relevant Publications

Lecture 9, Pages 95

The End

Thank you

Jack Henion

Lecture 9, Pages 96

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