oasis® prime hlb - introducing a new sorbent for the sample cleanup of food matrices - waters...

©2016 Waters Corporation 1

Oasis® PRiME HLB - Introducing A New Sorbent

for the Sample Cleanup of Food Matrices


Agenda

Why do sample preparation

Considerations for sample preparation

Oasis PRiME HLB Introduction

Application of Oasis PRiME HLB for the Cleanup Food Samples

Prior to Multi-Residue Veterinary Drug LC-MS/MS Analysis

Summary


Sample Preparation

How can I turn this?

Into this?


Why Do Sample Preparation?

Need to remove matrix interferences

– Increase signal to noise by simplifying the chromatographic separation

o Enables better, more consistent quantitation

– Reduce variability in analytical results due to matrix inconsistencies

o Higher, more consistent recovery

o Minimize matrix effects

o Less rework

– Increase column lifetime

o Fewer columns need to be replaced

– Reduces system downtime

o Less time spent with wrenches or waiting for service

Need to concentrate analyte of interest

– Present in low levels = difficult to quantitate

Need to transfer (extract) analytes of interest into a solution that can be tested


Sample Preparation Techniques: Which One?

How do you choose a technique to clean up complex sample

matrices?

– Filtration / Dilution

– Protein precipitation (PPT)

– Liquid-liquid extraction (LLE)

– QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe)

– Solid-phase extraction (SPE)

Objectives: Simplest technique

Fastest preparation procedure Cleanest extracts

“Good Enough” Sample Preparation


Method Requirements: Multi-residue vs. Compound-specific analysis

Multi-Residue/ Multi-Class

Compound or Class Specific

Entire procedure (sample prep & analytical method)

Generic to a diverse set of analytes

Specific for one compound or class of compounds

Sample Preparation Protocol

Simple (one or two steps) Multi-step

Goal of Sample Cleanup

Speed

Recovery & cleanup may be compromised for a large

number of analytes

Maximizing recovery & matrix cleanup

Minimizing interference/ion

suppression

Level of Sample Cleanup Minimum/moderate Maximum

Detection Techniques Tandem MS, Time-of-Flight

(LC-MS and GC*-MS) Single quad MS, UV, FLR,

ELS, GC* (FID or MS)

*GC typically requires a higher level of sample cleanup even for multi-residue analyses


Waters Mass Spectrometry Instruments SQ, TQ, QTof, & QTof with Ion Mobility

QDa SQD2 Xevo TQD Xevo TQ-S micro Xevo TQ-S

Xevo G2-XS Tof Xevo G2-XS QTof

Vion

Synapt G2-Si


Inlets: Direct Analysis and Chromatographic


Introducing Oasis PRiME HLB

PROCESS ROBUSTNESS improvements in… MATRIX EFFECTS EASE of USE


Oasis PRiME HLB – What is it?

A reversed-phase solid phase extraction device

– PATENT PENDING

Simpler

– Streamlined protocols, no condition and equilibration steps are

required

Faster

– Faster and more even flows through devices with less plugging and

faster overall processing

Cleaner

– Reduce matrix effect by removing interferences, fats and

phospholipids

Why should we care about the phospholipid cleanup from the

sample?


Phospholipid Build Up Can Lead to Matrix Effects and Unpredictable Results

Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, 720004453en; Danaceau et al. 2014 Bioanalysis 6(6) 761-771

50th Injection Phospholipid buildup


Phosphate group

“hydrophilic head”

Fatty acids

“hydrophobic tails”

A class of lipids served as a

major component of most

biologic membranes (plant or

animal cells)

Amphipathic, surfactant-like

property

Form lipid bi-layer in cell

membranes of organisms

Example: phosphatidylcholine or

lecithin

– Often found in animal and plant

tissues, such as egg yolks, milk,

soy beans, sunflower and canola

seeds

What Are Phospholipids? - A Brief Introduction











Speed


number of analytes



suppression





√


3 Step Catch and Release Protocol - Suitable for Compound/Class Specific Analysis

No SPE expertise required

Reversed-phase

retention for

aqueous samples

Wash and elution steps can be

adjusted if desired

Load:

Pre-Treated Sample

Wash:

5% MeOH

Elute:

90/10

Acetonitrile/MeOH

Generic 3-Step Protocol


3-Step Protocol Example: Test Analyte Properties

Name pKa Log P Comments

1B Azidothymidine (AZT) 9.68 0.05 Antiretroviral drug for HIV/AIDS

2B 7-Hydroxycoumarin 7.8 1.6 Gradient in sunscreen, absorb UV

3A Phenacetin 2.2 1.6 Pain, fever reducer

4N Betamethasone -- 1.1 Anti-inflammatory and

immunosuppressive

5B Protriptyline 8.2 4.4 Antidepressant

6A Alprazolam 2.4 4.9 Panic and anxiety disorders

7B Amitriptyline 9.7 4.8 Antidepressant

8A Naproxen 4.2 3.2 Pain, fever reducer

9B Propranolol 9.5 2.5 Hypertension

Drug Panel Mixture: Highly variable hydrophobicities, wide pKa

range and Log Ps


3-Step Protocol Example: Recovery and Matrix Effects

-20

0

20

40

60

80

100

120

Luckycat 1cc plasma 500-500 Luckycat 1cc MERecovery Matrix Effects

3-Step Protocol CONCLUSION HIGH analyte recoveries (>80%) and

LOW (<15%) matrix effects











Speed


number of analytes



suppression





*GC typically requires a higher level of sample cleanup even for multi-residue analyses

√


Pass-Through Protocol Suitable for Multi-Class Compounds, Multi-Residue Analysis

Pass-Thru Protocol – Ideal for

high organic (ACN) samples,

like meat or milk extracts

Analytes not retained by the

sorbent

Rinse step is optional

Load – Collect

Interferences Retained

Analytes Pass Through

Rinse – Collect

Analytes Pass Through

Pass-Thru Protocol


Recovery of Multi-residue Veterinary from Milk (60 compounds in 9 drug classes)

One single method replaces 9 separate methods!!! Excellent recoveries ranging from 70% to 120% with precision (RSD) < 20% (n=5) for all compounds (Average recovery 91%, %RSD @ 6 (n=5)) Recovery values are a subject to the initial milk extraction efficiency

0

20

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Cim

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Cle

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Racto

pam

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Salb

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Terb

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Tulo

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Zilpate

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Clindam

ycin

Ery

thro

mycin

kitasam

ycin

Lin

com

ycin

Spiram

ycin

Tilm

icosin

Tylo

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Sulfachlo

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Sulfaclo

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Sulfadim

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Sulfaguanid

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Sulfam

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Sulfam

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sulfam

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Sulfam

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sulfanilaceta

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sulfaphenazole

Sulfapyridin

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sulfis

om

idin

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Trim

eth

oprim

Cin

oxacin

Cip

rofloxacin

Danofloxacin

Diflo

xacin

Enoxacin

Enro

floxacin

Flu

mequin

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Lom

efloxacin

Marb

ofloxacin

Nalidix

ic a

cid

Norf

loxacin

Ofloxacin

Orb

iflo

xacin

Oxolinic

acid

Pefloxacin

Sara

floxacin

Chlo

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phenic

ol

florf

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ol

Thia

mphenic

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penic

illin V

Beta

meth

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Cort

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Dexam

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Hydro

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Mepre

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Meth

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Triam

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Applications

Using Oasis PRiME HLB for the Cleanup of

Food Samples Prior to Multiresidue

Veterinary Drug Analysis


Multi-Residue Analysis - Composition of Milk and Meat

Typical Cow’s Milk

– Approximately 14 % solids

o 4 % fat

o 0.3-1 % phospholipid

o 4 % protein

o 5 % sugar (lactose)

o 85 % water

Typical Pork Muscle

—Approximately 30 % solids

o 5-20 % fat

o 1-3 % phospholipid

o 15-25 % protein

o ~70% water

√

√

√

√


Veterinary Drug Classes

tetracycline

Tetracycline Fluoroquinolone

enrofloxacin

Sulfonamide

sulfamethazine

Macrolide

erythromycin

Beta-Lactam

oxacillin

Beta-adrenergic

salbutamol

Steroid

dexamethasone

Amphemicol

Chloramphemicol

LogP 2.05, pKa 3.75

LogP -1.3, pKa -2.2

LogP 0.44, pKa (basic) 9.4

LogP 2.37 , pKa(basic)8.3


Challenges Creating a Single Extraction Method

Compound diversity

– Large number of diverse group and classes

– Different physical and chemical properties

o Polarity

o pKa

– Parent drugs and their metabolites

Matrix complexity

– Matrix effects – proteins, fats, phospholipids, salts

– Protein binding

– Low limits of detection vs. co-extracted material – ion suppression

– Chelating - Tetracycline and similar drugs can form bonds with calcium (milk matrix)


US DA Residue Methods for Veterinary Drug Analysis

Method Analytes Sample Matrix

R04b β-Adrenergic agonists Muscle

R06 β-Lactam Kidney and muscle

R08b Chloramphenicol Muscle

R18 Macrolide Liver, kidney, and muscle

R27d Sulfonamides Liver and muscle

R32 Fluoroquinolone Liver and muscle

R46 Penicillin G Kidney, liver, and muscle

Using Oasis PRiME HLB -Enable multi-class compounds to be analyzed using one sample prep method.

Combined


Multi-Residues Veterinary Drug Analysis - Sample Extraction

MILK

Protein Precipitation

Add 4 mL of 0.2 % formic acid ACN to

1 mL of sample

Pass-Through SPE

Cleanup

TISSUE

Extraction/Precipitation

Add 10 mL of 0.2 % formic acid in 80:20 ACN/water to

2.5 g of sample

Provides good recovery of

most analytes

Removes proteins sugars

and salts

Waters Application Notes Optimized Extraction and Cleanup Protocols for LC/MS/MS Multi-Residue Determination of Veterinary Drugs in Milk (720004089en) Optimized Extraction and Cleanup Protocols for LC-MS/MS Multi-Residue Determination of Veterinary Drugs in Edible Muscle Tissues (720004144en)

Centrifuge

Take aliquot


SPE Strategy for Vet Drugs Pass-Through Cleanup*

Pass-Through SPE *

Cleanup

Removes fats and non-polar pigments

Sep-Pak

C18

Oasis

PRiME HLB

Removes fats, non-polar

pigments and phospholipids

*Also known as “Chemical Filtration”


Time1.00 2.00 3.00 4.00 5.00 6.00

%

0

1.00 2.00 3.00 4.00 5.00 6.00

%

0

LC050615_3 1: MRM of 12 Channels ES+ TIC (Phospholipid)

1.87e8

4.58

4.33

LC050615_5 1: MRM of 12 Channels ES+ TIC (Phospholipid)

1.87e8

4.33

Cleanup by Silica C18

Cleanup by Oasis PRiME HLB

CLEANER - Pork ACN Extract Phospholipids Remaining after Pass-Through

Only remove fats, Not phospholipids

Remove fats and phospholipids

Oasis PRiME HLB removes more than 90% of hexane-extractable total lipids (determined gravimetrically).

> 90% removal of phospholipids


Multi-Residue Veterinary Drugs in Milk and Tissue

Sample pre-treatment (Milk):

To 1mL of milk, add 4 mL of 0.2% formic acid (FA) in acetonitrile (ACN), mix well. Centrifuge for 5 min at 10000 rpm. An aliquot of the supernatant is taken for SPE cleanup.

Sample pre-treatment (Tissue):

Extract 2.5 g tissue with 10 mL of 0.2% formic acid (FA) in 80:20 ACN/Water and mix well. Centrifuge for 5 min at 10000 rpm. An aliquot of the supernatant is taken for SPE cleanup.

Pass-Thru Cleanup:

The vacuum was set to 1-2 psi. Approximately 0.5 mL of the supernatant was passed-through the Oasis PRiME cartridge and collected. A 0.3 mL aliquot of the pass-thru cleanup sample was taken and diluted three-fold with aqueous 10 mM ammonium formate buffer (pH 4.5) prior to UPLC-MS/MS analysis.

* Typically, an exact portion of the pass-thru fraction is evaporated or simply diluted with mobile phase, depending on the instrumental conditions


UPLC Conditions

LC system: ACQUITY UPLC I-Class

ACQUITY UPLC CSH™ C18, 1.7µm, 2.1 x 100 mm

Column temperature 30 °C

Mobile phase:

– A: 0.1% formic in water

– B: 0.1% formic acid in acetonitrile

Gradient

Injection volume: 5 µL

615850.47.0

69550.43.9

69550.44.9

615850.45.0

40

15

% B

6600.42.5

Initial850.4Initial

Curve% AFlow

(mL/min)

Time

(min)

615850.47.0

69550.43.9

69550.44.9

615850.45.0

40

15

% B

6600.42.5

Initial850.4Initial

Curve% AFlow

(mL/min)

Time

(min)


MS Conditions

Mass Spectrometer: Waters Xevo TQ-S

Positive Ion Electrospray

Source Temperature: 150°C

Desolvation Temperature: 500°C

Desolvation Gas Flow: 1000 L/Hr

Cone Gas Flow: 30 L/Hr

Collision Gas Flow: 0.15 mL/Min

Data Management: MassLynx v4.1


MRM Parameters

Compounds MRM Cone CID Compounds MRM Cone CID

Carbadox 263.0>231.0 25 15 Oxytetracyline 461.2>426.2 30 21

263.0>145.0 25 20 461.2>408.11 30 13

Ceftiofur 524.3>241.1 30 16 Pennicillin 335.16>160.1 20 30

524.3>285.0 30 16 335.15>176.1 20 30

Chlortetracycline 479.3>444.2 30 21 Phenylbutazone 309.4>160.0 37 20

479.3>462.2 30 18 309.4>103.9 37 20

Ciprofloxacin 332.1>288.1 30 18 Ractopamine 302.2.164.1 30 15

332.1>231.1 30 40 302.2>107.0 30 27

Cortisol 363.2>121.0 42 52 Salbutamol 240.2>148.1 30 20

363.2>91.03 30 22 240.2>222.1 30 12

Dexamethasone 393.2>373.2 30 10 Sulfamerazine 265>92.0 30 28

393.2>355.3 30 15 265>156.0 30 15

Enrofloxacin 360.4>245.0 50 25 Sulfamethazine 279.1>186.0 30 16

360.4>316.1 50 25 279.1>92.0 30 28

Erythromycin 734.4>158.1 30 32 Sulfanilamide 156>92.0 30 15

734.4>576.5 30 20 156>65.0 30 25

Lincomycin 407.2>126.1 36 34 Tetracycline 445.3>154.0 30 26

407.2>359.3 36 20 445.3.410.2 30 21

Lomefoxacin 352.1>265.1 31 22 Tylosin 916.5>174.1 57 40

352.1>308.1 31 16 916.5>101.1 57 45

Oxacillin 402.2>160.0 30 12

402.2>243.1 30 15


Phospholipids Removal From Milk

Milk sample processed by protein precipitation only

Precipitated milk sample cleaned up with

Oasis PRiME HLB

Oasis PRiME HLB removed many more phospholipids in comparison with protein precipitation


Phospholipid Removal From Shrimp Extract

No Cleanup

Waters Application Notes Rapid, Simple and Effective Cleanup of Seafood Extrats Prior to UPLC-MS/MS Analysis of Multi-Residue Veterinary Drugs Method (720005488en)


Recovery of Veterinary Drugs from Shrimp Tissue (9 Classes of Drugs)

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120

140

Reco

very

Total Method Recovery from Shrimp



Recovery of Veterinary Drugs from Salmon Tissue (9 Classes of Drugs)

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120

Recovery

Total Method Recovery from Salmon



Applications

Using OASIS PRiME HLB for Cleanup of Infant

Formula Extract prior to UPLC-MS/MS Multi-

Residue Veterinary Drugs Analysis


Nutrients of Infant Formula Powder - Complicated Sample Matrix

Nonfat Milk, Lactose, Whey Protein Concentrate, High Oleic Safflower Oil, Soy Oil, Coconut

Oil, Galactooligosaccharides. Less than 2% of: C. Cohnii Oil, M. Alpina Oil, Beta-Carotene,

Lutein, Lycopene, Potassium Citrate, Calcium Carbonate, Ascorbic Acid, Soy Lecithin,

Potassium Chloride, Magnesium Chloride, Ferrous Sulfate, Choline Bitartrate, Choline

Chloride, Ascorbyl Palmitate, Salt, Taurine, m-Inositol, Zinc Sulfate, Mixed Tocopherols, d-

Alpha-Tocopheryl Acetate, Niacinamide, Calcium Pantothenate, L-Carnitine, Vitamin A

Palmitate, Cupric Sulfate, Thiamine Chloride Hydrochloride, Riboflavin, Pyridoxine

Hydrochloride, Folic Acid, Manganese Sulfate, Phylloquinone, Biotin, Sodium Selenate,

Vitamin D3, Cyanocobalamin, Calcium Phosphate, Potassium Phosphate, Potassium

Hydroxide, and Nucleotides (Adenosine 5’-Monophosphate, Cytidine 5’-Monophosphate,

Disodium Guanosine 5’-Monophosphate, Disodium Uridine 5’-Monophosphate).

Contains milk and soy ingredients.

Major Nutrients: (% in powder)

Carbohydrates: 50% ~ 60%

Proteins: ~10%

Fats/Oils: ~25%

Phospholipids: ~3%

Vitamins: ~8.5%

Minerals: ~2%

Cleaned up by Oasis PRiME HLB


Infant Formula - Calibration Curve & Spiking Levels

Low Level High Level

Calibration Corr.

Compounds (ppb) (ppb) RT Range (ppb) R2

Carbadox 25 100 1.37 12.5-200 0.9990

Ceftiofur 250 1000 2.79 125-2000 0.9980

Chlortetracycline 25 100 1.53 12.5-200 0.9977

Ciprofloxacin 25 100 0.84 12.5-200 0.9929

Cortisol 50 200 2.93 25-400 0.9961

Dexamethasone 25 100 3.39 12.5-200 0.9917

Enrofloxacin 50 200 0.98 25-400 0.9991

Erythromycin 2.5 10 2.16 1.25-20 0.9978

Lincomycin 12.5 50 0.57 6.25-100 0.9962

Lomefoxacin 50 200 0.90 25-400 0.9991

Oxacillin 25 100 3.75 12.5-200 0.9990

Oxytetracyline 25 100 0.96 12.5-200 0.9971

Pennicillin 12.5 50 3.41 6.25-100 0.9956

Phenylbutazone 25 100 4.22 12.5-200 0.9962

Ractopamine 75 300 0.90 37.5-600 0.9990

Salbutamol 25 100 0.55 12.5-200 0.9941

Sulfamerazine 25 100 1.50 12.5-200 0.9998

Sulfamethazine 25 100 1.67 12.5-200 0.9996

Sulfanilamide 25 100 0.86 12.5-200 0.9964

Tetracycline 25 100 1.05 12.5-200 0.9964

Tylosin 5 20 2.38 2.5-40 0.9920

Place 0.5 g infant milk powder into 50-mL tube, then add 3 mL of 0.2% formic

acid in 70/30 ACN/water. Vortex for 30 send and then shake for 30 mins

After centrifugation, take 0.5 mL supernatant and pass-thru 3cc/60mg HLB

PRiME cartridge


Phospholipid Removal by PRiME HLB from Infant Formula

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

4.34

4.28

4.51

4.59

Extract Cleanup by Pass-Thru PRiME HLB

Extract with no SPE Cleanup

99% Removal of Phospholipids

TIC for Total Phospholipids


Spike and Recovery

Observe low

recoveries for

tetracyclnes and

lincomycin.

Is the loss due to

SPE retention by

Oasis PRiME HLB,

or extraction loss?

Low Level

High Level

Compounds REC (%) %RSD REC (%) %RSD

Carbadox 110 5

115 3

Ceftiofur 84 4

71 10

Chlortetracycline 47 9

46 9

Ciprofloxacin 107 8

102 6

Cortisol 111 8

117 5

Dexamethasone 113 14

121 5

Enrofloxacin 113 4

110 5

Erythromycin 126 6

125 6

Lincomycin 50 7

52 5

Lomefloxacin 120 2

111 2

Oxacillin 117 6

114 4

Oxytetracycline 29 13

27 13

Penicillin 120 10

116 7

Phenylbutazone 105 13

94 6

Ractopamine 115 2

117 5

Salbutamol 82 7

83 4

Sulfamerazine 130 4

126 4

Sulfamethazine 128 2

129 3

Sulfanilamide 105 12

120 5

Tetracyline 41 13

40 17

Tylosin 110 13

116 7


Total Method Recovery versus

SPE Cleanup Recovery

0

20

40

60

80

100

120

140

160

Total Method Recovery SPE Clean up Recovery

SPE Clean up Recoveries (>90%) >> Total Method Recoveries

Low recoveries of tetracyclines are due to extraction loss


Options for Compounds Like Tetracycline for Improved Recovery

Option A – Based on the method validation result, build in concentration factor when

considering the reporting levels. Use the result for screening purpose, and perform confirmation test if needed.

Option B – Exclude tetracycline from the list of this screening method. Analyze

tetracyclines using the current analytical method optimized for recovery.

o Tetracyclines are strong chlelating agents – EDTA or oxalic acid is added to the McIlvaine buffer to sequester calcium. Follow by Oasis MAX cleanup

Option C – Extract sample again using the optimized method for tetracycline, and

combine the extracts together. After adjusting the solvent by adding ACN, then pass through Oasis PRiME HLB.

– Procedures may be complicated and time-consuming


Applications

Quantitative Determination of Veterinary

Drug Residues in Eggs by UPLC-MS/MS

Using a Simple, Rapid and Effective

Sample Cleanup Approach


Structures of Some Veterinary Drugs

Antiprotozoal agents Polypeptide antibiotics Tetracyclines

amprolium bacitracin A chlortetracycline HCl

Benzimidazoles Fluorinated Aminoglycoside Macrolides

Flubendazole florfenicol hygromycin B erythromycin

Antifungal Antimicrobia Macrolides Quinolone

nystatin A1 tiamulin Tylosin oxolinic acid


Sample Preparation Protocol - Chicken Eggs

Extraction Add 8 mL 0.2% formic acid in 80:20

ACN/water to 2.0 g of scrambled egg and shake for 30 min

Centrifuge Take 0.5 mL supernatant for cleanup

Pass-Thru SPE Cleanup (No Conditioning step !) Oasis PRiME HLB 3cc 60 mg

UPLC-MS/MS Take 0.2 mL filtrate and dilute three-fold with 10 mM ammonium formate buffer at pH 4.5

Provides good recoveries for most analytes Removes proteins, sugars and salts

Removes fats, phospholipids and non-polar pigments


Total Method Recovery versus


SPE Clean up Recoveries ≥ 90% except Nystatin A1 at 80%

SPE Cleanup Recoveries >> Total Method Recoveries for all analytes

20% to 40% analytes were lost during the extraction step

0

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40

60

80

100

120

140

Total Method Recovery



Phospholipid Removal by PRiME HLB from Chicken Eggs

Oasis PRiME HLB Cartridge

Pass-Thru Cleanup

95% removal of phospholipid

No Cleanup


Published Validated Method Using Oasis PRiME HLB

Anal. Methods, 2016,8, 1457-1462


New Applications Currently in Development


Pesticide Compounds Registered in US, EU or China for avocado

Abamectin LC Metalaxyl LC GC

Azoxystrobin LC GC Methomyl LC

Bifenazate LC Methoxyfenozide LC

Boscalid LC Novaluron LC

Buprofezin LC Oryzalin LC

Carfentrazone-ethyl LC GC Oxyfluorfen GC

Chlorantraniliprole LC Permethrin GC

Chlorothalonil GC Propiconazole LC

Cyprodinil LC GC Pyraclostrobin LC

Cypermethrin GC Pyriproxyfen LC GC

Dichlorvos GC Simazine LC GC

Etofenprox LC Spinetoram LC

Etoxazole LC Spinosad A LC

Fenpropathrin GC Spinosad D LC

Fenpyroximate LC Spirodiclofen LC

Fludioxonil LC GC Spirotetramat LC

Folpet GC Thiabendazole LC

Imidacloprid LC Thiamethoxam LC

Lambda Cyhalothrin GC Thiram LC

Malathion LC GC

23 compounds by LC-MS only 8 compounds by GC-MS only 8 compounds by LC-MS and/or GC-MS


Instruments for Avocado Analysis

UPLC-MS/MS

– Xevo TQ-S with I-Class

– Acquity BEH C18 2.1 x 100

– Pesticide method mobile phases (ammonium acetate in MeOH, pH 5)

APGC-MS/MS

– Xevo TQ-S with A7890 in charge transfer mode

– Restek RTX-5MS (30M x 0.25mm, 0.25 µ film)

– Carrier gas He @ 1 mL min


Instrument Setup - Two Analysis done on one MS

UPLC with TQ-S APGC with TQ-S


Pesticide Spike in Avocado APGC-MS

avocado mix 3ppm

Time6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100

LC_16March2016_05 Scan API+ TIC

3.82e8

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chlo

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Multi-Residue Mycotoxin Analysis in Grains


Using Modified QuEChERS for Analysis of Mycotoxin in Grains – Sample Prep

QuEChERS sample extraction

– 5 g homogenized sample in 50 mL centrifuge tube

– Add 10 mL water and 10 mL 10:90 formic acid/acetonitrile

– Shake for 1 hr at max speed with horizontal shaker

– Add CEN QuEChERS salt (pn 186006813), and then shake vigorously

for 1 min

– Centrifuge at 2200 rcf for 5 min

d-SPE cleanup

– Transfer 5 mL upper acetonitrile layer into 15-mL tube containing the

following: 750 mg MgSO4, 250 mg PSA, 150 mg C18 , 150 mg

Alumina-N, pn 186008080

– Shake for 1 min, centrifuge at 2200 rcf for 5 min

– Remove 100 µL extract and diluted to 1.0 mL with mobile phase A


Multiresidue Mycotoxin Analysis- Maize

Oasis PRiME HLB

dSPE

Oasis PRiME HLB

dSPE

The left photo shows modified QuEChERS extracts of a maize sample after after cleanup using Oasis PRiME HLB (left tube) compared with dSPE (right tube); the right photo shows the

sample (0.5 mL) after evaporation and reconstitution (0.2 mL)


Phospholipids Removal from Maize Extract

LC-MS/MS chromatograms showing effective removal of =95% of

phospholipids (lecithins) from QuEChERS extract of a maize sample

Time4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

myco09082034a 1: MRM of 12 Channels ES+ TIC (phospholipid)

1.21e7

10.34

10.15

10.95

11.35

myco09082033a 1: MRM of 12 Channels ES+ TIC (phospholipid)

1.21e7

dSPE Cleanup

Oasis PRiME HLB Cleanup


Summary

Oasis PRiME HLB effectively removes majority of fatty matrix

and phospholipids from milk and tissues, and gives the cleanest

possible sample extracts for multi-residue veterinary drug

analysis.

PRiME HLB operated as “chemical filter” in Pass-Thru protocol.

Interferences such as fatty matrices and phospholipids are

retained by the sorbent, and analytes are allowed to pass

through.

Using Pass-Thru protocol, the generic cleanup method could be

used for a wide variety of residues, such as veterinary drugs,

pesticides, mycotoxins, or other analytes, with little or no

modification.


Questions?


Additional Slides


Why not QuEChERS for Sample Prep of Veterinary Drugs?

Some classes of veterinary drugs have good recovery using QuEChERS

(i.e., avermectins)

– However, too many have low recovery (< 50%)

First step of veterinary drug extraction is similar

– However, no salts are added for veterinary drug analysis and pass

through SPE is preferred

Pesticides are generally more non-polar and less water-soluble than

veterinary drugs

– At least 80% partition into ACN layer when DisQuE salts are added

In comparison, a larger portion of veterinary drugs partition into the

salt-saturated water layer


Recovery of Veterinary Drugs Discussion

Total Method recovery averages ~ 80% with range from

50-103%

– Most of any recovery loss occurs in the initial extraction/precipitation

step

o Not all classes of compounds are extracted equally

• Penicillins > 70%

• Sulfonamides > 85%

• Fluroquinolones> 75%

• Tetracyclines > 40%

Were there recovery losses caused by the pass-thru cleanup?


Oasis PRiME HLB Pass-Thru Cleanup Recovery

Salmon matrix blank extracts were spiked with the veterinary drugs and then cleaned up by passing through the Oasis PRiME HLB cartridge. This experiment eliminates the recovery loss contribution from the initial protein precipitation/extraction.

0

20

40

60

80

100

120

140

Recovery

Recovery from Salmon

Method

Recovery

Pass-Thru

Recovery