are lipids weighing you down? - agilent · 2017-06-15 · are lipids weighing you down? -...

Are lipids weighing you down? -optimizing sample prep for MS analysis

Xiaomi Xu Ph.D.

Global sample preparation

product manager

June 13, 2017

For Research Use Only; Not for Diagnostic Procedures

1

Outline

June 13, 2017


2

Why does sample preparation matter?

Lipids – the most challenging interferences in various sample matrices

EMR-Lipid: an innovative and novel technology to effectively remove lipids

• What is it?

• QuEChERS workflow with application example.

• Protein precipitation workflow with application example.

Summary

Why does sample preparation matter?

Remove interferences

Accurate and consistent data

Keep the instruments running

Process as many samples for as little cost as possible

1 1

2

3

4

June 13, 2017


3

Insights

VOC – customer pain points

Customers want:

• Sample preparation with fewer steps…ideally a single step

• Reduced method development

Good results include:

• Effective removal of matrix interferences

• High recoveries of a broad range of analytes in complex matrices

• Rugged and reproducible

Complex and unwanted matrices include –

Lipids, proteins, carbohydrates, pigments, etc.

June 13, 2017


4

Matrix removal

instead of

analyte isolation

Matrix versus analytes

Matrix = high levels

Analytes = trace levels

The analytes of interest are many and vary significantly.

1,000s of pesticides used to improve crop yield.

A variety of veterinary drugs are used to keep animals healthy and alive to become profitable food supplies.

Grains may also be treated with pesticides but can also develop mycotoxins during transport or storage

Analyzing various drug compounds in biological samples

Matrix

Analyte

June 13, 2017


5

Matrices and Approximate Total Lipid Content

<2% 4-12% >12%

Beef Liver (4%) Nut Butters (16%) Spinach (0%)

Strawberry (0%)

Onion (0%)

Canned Pet Food (~10%)

Avocado (21%)

Chocolate (8%)

Edible Oil (100%)

Paprika (1%)

Hops (2%)

Cumin (2%)

Salmon (27%) Pork (8%)

Serum, Plasma or whole blood

(10 – 20 %)

Cow’s Milk (5%)

Low Med High

6

June 13, 2017


Current Procedures for Lipid Removal

Method How Lipids are removed Weakness

Dilute and Shoot No lipid removal, only dilution No lipid removal

Protein

Precipitation

PPT followed by centrifugation Insufficient lipid removal

PPT followed by filtration with

or without sorbent

Insufficient lipid removal; low

analyte recovery

QuEChERS PSA/C18 sorbent (dSPE) Not selective; insufficient lipid

removal; analyte loss

Zr-containing sorbent Low total lipid capacity; analyte

recovery

Freeze sample Time needed; loss of analyte

SPE/SLE Load and elute Time needed; solvent usage;

extensive method development

SEC/GPC Chromatographic separation Uses copious amounts of solvent

and time; capital expense

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June 13, 2017


Our answer

We heard you!

- A unique, differentiated EMR-Lipid technology to address these needs

Reduce time spent on poor data quality

• LC/MS and GC/MS

• Data review/processing time reduced

• Recalibration and re-runs reduced

Reduced instrument maintenance (by up to 50%)

• GC Liner change

• Column change / GC column trim

• MS maintenance / source cleaning

• 2500 high fat sample injections without cleaning the source

Outcome

June 13, 2017


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9

June 13, 2017


EMR-Lipid (enhanced matrix removal)

Captiva EMR-Lipid

Bond Elut dSPE EMR-Lipid

EMR-Lipid

2016 R&D100

Award Finalist!

Coming Soon!

BOND ELUT DSPE EMR-LIPID

June 13, 2017


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Bond Elut QuEChERS dSPE EMR-Lipid

June 13, 2017

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Extraction Tube

(EN or AOAC

extraction salts) EMR-Lipid

Dispersive SPE

(p/n 5982-1010)

Polish Pouch

MgSO4

(p/n 5982-0102)


EMR-Lipid Sorbent - What is it?

When “activated” by water…

- Suspension of particles with high surface area rapidly interacts with and retain lipids.

- Centrifugation to separate precipitate from solution.

12

1.0 g EMR in 15 mL tube

June 13, 2017


EMR sorbent technology

effectively traps lipids through

two mechanisms:

Size exclusion – Unbranched

hydrocarbon chains (lipids) enter

the sorbent; bulky analytes do not

Sorbent chemistry – Lipid chains

that enter the sorbent are trapped

by hydrophobic interactions

… and what does it do?

EMR sorbent removes Lipids

What are Lipids?

A class of naturally occurring hydrocarbon containing compounds commonly known as fats and oils

Free Fatty Acids

Triglycerides

Cholesterol Phospholipids

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June 13, 2017


What Does EMR NOT Interact With?

EMR does NOT remove analytes of interest

Fluoroquinolones Tetracyclines

14

β-Lactams

Tranquilizers Sulfonamides Macrolides

Exceptions? Compounds containing long aliphatic functional groups (e.g. prostaglandins)

June 13, 2017


http://en.wikipedia.org/wiki/File:Amoxicillin.svg

http://en.wikipedia.org/wiki/File:Ketoprofen.svg

http://en.wikipedia.org/wiki/File:Sulfamethoxypyridazine.png

http://en.wikipedia.org/wiki/File:Tylosin_structure.png

EMR – Lipid – dSPE Cleanup

A B C D

E

A. QuEChERS or Liquid Extract

B. Add H2O to EMR tube (“activation”)

C. Transfer extract

D. Vortex and centrifuge

E. Supernatant (1:1; extract: H2O)

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June 13, 2017


EMR – Polish – ACN/H2O Phase Separation

A B C D

E

A. Mix supernatant with EMR Polish (MgSO4)

B. Vortex immediately

C. Phase separation after centrifuge

D. Transfer upper layer for analysis

E. Final samples split for GC and LC analysis

Note: An additional drying step using anhydrous

MgSO4 is recommended after polish step to removes

residual water for GC applications - 300mg

anhydrous MgSO4 per 1mL ACN sample extract

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June 13, 2017


Applications of EMR – Lipid:

Pesticide Analysis

Challenging matrix: Avocado

June 13, 2017


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67 Pesticides Analysis in Avocado by LC and GC-QQQ


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June 13, 2017

Comparison of GC/MS Full-scan Chromatogram for Matrix Background

The use of EMR material cleanup provides significantly cleanup chromatographic

sample background.


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June 13, 2017

Chromatographic Benefits of Matrix Removal Provided by EMR-Lipid Cleanup


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ExampleEPN in Avocado on

LC-QQQ

+ MRM (151.0 -> 79.1) Post-S 50ppb Fatty dSPE C1-3.D

Acquisition Time (min)

12.8 12.9 13 13.1 13.2 13.3 13.4 13.5 13.6

Coun

ts 5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

*13.174 min.

Captan in Avocado on

GC-QQQPermethrin in Avocado on

GC-QQQ

EMR-Lipid

cleanup

Zirconia

sorbent

cleanup

C18/PSA

cleanup

BenefitReduced matrix

suppressionImproved S/N ratio Less interferences for

accurate integration

+ MRM (324.1 -> 296.1) AV EMR PO-Q 4.d


14.2 14.4 14.6 14.8 15 15.2 15.4 15.6

Co

un

ts 3x10

0

0.5

1

14.863 min.

+ MRM (324.1 -> 296.1) AV C18 PR-Q 3.d


14.2 14.4 14.6 14.8 15 15.2 15.4 15.6

Co

un

ts 3x10

0

0.5

1

14.855 min.

+ MRM (324.1 -> 296.1) AV Z-Sep+ PO-Q 4.d


14.2 14.4 14.6 14.8 15 15.2 15.4 15.6

Co

un

ts 3x10

0

0.5

1

14.863 min.

June 13, 2017

Method Accuracy and Precision


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June 13, 2017

Veterinary Drugs in Bovine Liver


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June 13, 2017

Vet Drugs List for Evaluation

Representative

Vet DrugDrug Class

Representative Vet

DrugDrug Class

Representative

Vet DrugDrug Class

Amoxicillin Pednisone Corticosteroid Tylosin Macrolide

Difloxacin Oxyphenylbutazone NSAID Oxytetracline

Florfenicol 2-Thiouracil Thyreostat Doxycycline

Chloramphenicol Metronidazole-OH Nitroimidazole Chlortetracycline

Sulfamethizole Fenbendazole Acepromazine

Sulfamethoxypri

dazineLavamisole

Chlorpromazine

Lincomycin Lincosamide Morantel Ketoprofen

Ciprofloxacin Bithionol Cefazolin Cephalosporin

Norfloxacin Clorsulon Melengesterol other

Danofloxacin Niclosamide Ractopamine β-Agonist

Flukicide

β-Lactam

Phenicol

Sulfonamide

Fluoroquinolone

Tetracycline

TranquilizerAnthelmintic


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June 13, 2017

GC-MS Fullscan Beef Liver

Beef Liver – Untreated


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June 13, 2017

GC-MS Fullscan Beef Liver

Beef Liver – Untreated

Beef Liver – EMR Treated


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June 13, 2017

Selected Problematic Vet Drugs for Recovery Comparison


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0

20

40

60

80

100

120

2-Thiouracil Lincomycin Norfloxacin Ciprofloxacin Cefazolin Chlortetracycline Doxycycline

Ave

rage

Rec

ove

ry%

(n

=6)

in 5

0p

pb

Vet

Dru

gs s

pik

ed B

ovi

ne

Live

r Sa

mp

les

Selected Vet Drugs Recovery Comparison from Multi-residue Vet Drugs in Bovine Liver by LC-QQQ

EMR-Lipid QuEChERS w/ C18 dSPE QuEChERS w/ Zirconia sorbent

June 13, 2017

Statistical Recovery Comparison for Vet Drugs in Bovine Liver


27

0

5

10

15

20

25

30

EMR-Lipid QuEChERS w/ C18 dSPE QuEChERS w/ zirconia sorbent

Nu

mb

er o

f V

et D

rugs

tes

ted

in B

ovi

ne

Live

r

Statistical Recovery Results for Multi-residue Vet Drugs in Bovine Liver by LC-QQQ

REC <70%

REC in 70-120%

REC > 120

June 13, 2017

Peer reviewed publication – Comparison of veterinary drug residue results in animal tissues by UHPLC coupled to QQQ or QTOF after different sample preparation methods, including use of a commercial lipid removal product.

June 13, 2017


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Amount of co-

extractives (in

percent) in each

bovine matrix for

both sample

preparation

techniques

Anumol T, Lehotay SJ, Stevens J, Zweigenbaum J. Anal Bioanal Chem. 2017 Apr; 409 (10): 2639-2653.

More customer outcomes

UC Davis customer webinar

– July 2016

2 Peer-reviewed J. Chrom A articles on EMR-Lipid

• Evaluation of different cleanup sorbent for multi-residue pesticides analysis in fatty vegetable

matrices by liquid chromatography tandem mass spectrometry. Rafael López-Blancoa et al.

• Evaluation of a recent product to remove lipids and other matrix co-extractives in the analysis of

pesticide residues and environmental contaminants in foods. Steven J. Lehotay et al.

Optimizing the Extraction of Pesticide Residues in Edible Oils

Eugene Chang

National Food Expert

United States Food and Drug Administration

Live event - presented through Separation Science

Date: Wednesday, 19 October 2016

Start Time: 8 am PDT / 11 am EDT / 4 pm BST / 5 pm CEST

USFDA customer webinar

– October 2016

June 13, 2017


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Benefits to Instrumental Flow-path


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June 13, 2017


Comparison of Analytes Response Consistency over Multiple Avocado Sample Injections

Dichlovos

OP pesticide

RT: 4.7 min

June 13, 2017

Comparison of Analytes Response Consistency over Multiple Avocado Sample Injections

TPP

Commonly

used IS

RT: 18.3 min


32

June 13, 2017

Analytes Responses Reproducibility on GC/MS/MS over 100 Injections of Avocado Samples


33

June 13, 2017

MS Source Critical Tuning Parameters

0

200

400

600

800

1000

1200

1400

1600

0

10

20

30

40

50

60

70

80

90

100

110

120

130

0 500 1000 1500 2000 2500

EM V

ola

tage

Re

lati

ve A

bu

nd

ance

fo

r T

un

ing

Ion

(%

)

Injections of Avocado Samples with EMR cleanup on GC/MS

MS (GC/MS) Performance Monitoring over Multiple Avocado Sample Injections

Rel Abundance for Ion 219, Minimum of 70 needed

Rel Abundance for Ion 502, minimum of 5 needed

EM voltage, higher than 1400 indicated needs to replace


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June 13, 2017

CAPTIVA EMR-LIPID

Coming soon…

June 13, 2017


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• In response to VOC

• EMR technology with

modification:

o SPE cartridge/well

plate format

o “pass through” clean

up workflow

o polish step eliminated

June 13, 2017


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Captiva EMR – Lipid Releasing July 1st 2017

Ideal for protein precipitation workflow

Option 1 – Active in-well mixing

with pipette

Option 2 – Passive mixing - let

stand for 5 min

Add crash solvent

then sample Mix to Precipitate

Protein

Filter

Lipids

Proteins

Analytes

June 13, 2017


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In-well protein precipitation workflow

Solvent and sample

/ vortex

Transfer

Filter

Lipids

Proteins

Analytes

June 13, 2017


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Off-line protein precipitation workflow

Phospholipid Removal

Plasma heparin 99.53% Removal

June 13, 2017


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Ion suppression / Reproducibility

Standard protein precipitation

25-OH D3

Protein Precipitation

25-OH D2

RSD > 25%

Captiva EMR - Lipid

25-OH D3

Captiva EMR

25-OH D2

RSD < 3%

June 13, 2017


40


Post Column Infusion – supporting suppression results

Captiva EMR-Lipid Protein precipitation only

Captiva EMRProtein Precipitation

June 13, 2017


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Better Linearity with EMR-Lipid

Agilent EMR-Lipid Training

42

Relative Concentration

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1

R e

l a t i v e

R e

s p o n s e s

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

R^2 = 0.9989

Relative Concentration

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1

R e

l a t i v e

R e

s p o

n s e s

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

R^2 = 0.9854

PPT cleanup only PPT + Captiva EMR-Lipid cleanup

Sample 25-OH D2

(ng/mL)

25-OH D3

(ng/mL)

LLOQ (lower limit of quantitation) 10 20

LQ (low QC) 20 30

MQ (mid QC) 250 250

HQ (high QC) 500 500

ULOQ (upper limit of quantitation) 750 750


Data quality - Absolute Recovery and Precision

25-OH D2 25-OH D3

Absolute

Recovery (%) %RSD

Absolute

Recovery (%) %RSD

10 ng/mL 106.4 4.8 106.3 10.2

20 ng/mL 88.8 9.5 94.2 9.2

30 ng/mL 90.8 5.0 94.7 6.3

250 ng/mL 96.9 14.6 96.6 4.2

500 ng/mL 97.1 11.5 100.1 9.5

750 ng/mL 96.9 6.4 100.3 5.0

Absolute recovery and %RSD values (n = 6) for 25-OH D2 and D3.

June 13, 2017


43


Method robustness: Intra-day Accuracy and Precision

Day 1 25-OH D2 25-OH D3

Accuracy (%) %RSD

Accuracy

(%) %RSD

LLOQ 97.2 4.7 106.8 9.9

LQ 97.8 5.8 104.1 5.9

MQ 96.0 5.3 93.4 2.8

HQ 107.3 4.9 105.1 2.7

ULOQ 101.9 2.7 101.5 3.1

Day 2

LLOQ 108.4 5.8 108.3 6.6

LQ 101.2 4.8 99.3 7.4

MQ 108.7 5.6 108.1 4.7

HQ 105.4 3.6 108.7 2.7

ULOQ 104.3 2.9 109.1 1.5

Day 3

LLOQ 99.1 5.6 109.4 8.1

LQ 92.9 4.8 99.2 5.4

MQ 105.0 4.6 104.0 4.0

HQ 106.0 3.1 105.8 3.0

ULOQ 105.6 2.7 109.1 3.7

Intra-day accuracy

and precision from

method verification

of 25-OH D2 and

D3 using d3 in

human serum (n =

6 at each QC level)

June 13, 2017


44


Method robustness: Inter-day Accuracy and Precision

25-OH D2 25-OH D3

Accuracy (%) %RSD

Accuracy

(%) %RSD

LLOQ 101.6 5.4 108.2 8.2

LQ 97.3 5.1 100.9 6.2

MQ 103.2 5.2 101.9 3.8

HQ 106.2 3.9 106.5 2.8

ULOQ 103.9 2.8 106.6 2.8

Accuracy and Precision results for inter-day QC samples (n = 18).

June 13, 2017


45


Captiva

EMR-Lipid

Competitor

product 3

Competitor

Product 2

Competitor

product 4

Competitor

product 5

Human

plasma

K3EDT

A

Human

plasma

NaEDTA

Human

plasma

LiHep

Human

plasma

NaCit

Human

Serum

Horse

plasma

NaCit

Porcine

plasma

K3EDT

A

Canine

plasma

K3EDTA

Rat

plasma

NaHep

June 13, 2017


46


Sample clarity comparison

Agilent Confidential

Neat standard

(no matrix)

Plasma

Captiva EMR - Lipid

Plasma

Competitor product A

Plasma

Competitor product B

Warfarin Atorvastatin + MRM (559.3 -> 440.2) CD STD-3.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2

Cou

nts 3x10

0

0.5

1

1.5

2

6.279 min.

+ MRM (309.1 -> 163.0) Human plasma Ostra-CL 1ppb PO-Q 3.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7

Cou

nts 3x10

1

2

3

4

6.146 min.

+ MRM (559.3 -> 440.2) Human plasma Ostra-CL 1ppb PO-Q 3.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2

Cou

nts 3x10

0.25

0.5

0.75

1

1.25

1.5

1.75

2

6.279 min.

+ MRM (309.1 -> 163.0) Human plasma PLD+-CL 1ppb PO-Q 3.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7

Cou

nts 3x10

0

1

2

3

4

6.167 min.

+ MRM (559.3 -> 440.2) Human plasma PLD+-CL 1ppb PO-Q 3.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2

Cou

nts 3x10

0.5

1

1.5

2

6.279 min.

+ MRM (309.1 -> 163.0) 96-well EMR-CL 1ppb PO-Q 2.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7

Cou

nts 3x10

0

1

2

3

46.156 min.

+ MRM (309.1 -> 163.0) CD STD-1.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7

Cou

nts 3x10

0

1

2

3

4

6.157 min.

+ MRM (559.3 -> 440.2) 96-well EMR-CL 1ppb PO-Q 8.d


5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2

Cou

nts 3x10

0

0.5

1

1.5

2

6.279 min.

Complete lipid removal leads to superior analytical performance (LC-MS ion suppression comparison)


Summary

June 13, 2017


48

Sample preparation (just enough) is important to achieve desired

analytical result and protect your GC/MS and LC/MS instrumentation.

Removing matrix (instead of analyte concentration) is a simpler

approach and couples will highly sensitive and selective QQQ method.

EMR-Lipid is an innovative cleanup sorbent that removes the most

challenging matrix – Lipid.

EMR-Lipid technology is compatible with various workflow, including

QuEChERS, protein precipitation and modified liquid extraction.

We are here to help!

ASMS posters on EMR-Lipid technology

June 13, 2017


49

MP 395 Analysis of Per/Polyfluoroalkyl Substances (PFASs) in Biological Fluid

using a Novel Lipid Removing Sorbent and LC/MSMS

TP 451 Enhanced Lipids Removal from Biological Matrices to Prepare Samples for

LC/MS/MS Analysis

TP 459 Improved LC/MS/MS Analysis of Vitamin D Metabolites in Biological

Samples Using Enhanced Matrix Removal Sample Preparation

Acknowledgement

• Joan Stevens – Sample prep Application chemist

• Limian Zhao – Sample prep R&D chemist

• Derick Lucas – Sample prep R&D chemist

• Dave Long – Sample prep R&D chemist

• Tarun Anumol – LC Application chemist

• Diana Wong – GC Application chemist

• Our external collaborators!

June 13, 2017


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June 13, 2017


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are lipids weighing you down? - agilent · 2017-06-15 · are lipids weighing you down? -...

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