Oasis® Prime HLB - introducing a new sorbent for the sample cleanup of Food matrices - Waters Corporation Food Safety

©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

Multi-residue vs. Compound-specific analysis
Multi-Residue/
Multi-Class
Compound or
Class Specific
analytes
class of compounds
Sample Preparation
Protocol
Speed
number of analytes
cleanup
suppression
(LC-MS and GC*-MS)
√

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
Luckycat1cc plasma 500-500 Luckycat1cc MERecovery Matrix Effects
3-Step Protocol CONCLUSION
HIGH analyte recoveries (>80%) and
LOW (<15%) matrix effects

Multi-residue vs. Compound-specific analysis
Multi-Residue/
Multi-Class
Compound or
Class Specific
analytes
class of compounds
Sample Preparation
Protocol
Speed
number of analytes
cleanup
suppression
(LC-MS and GC*-MS)
*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
40
60
80
100
120
140
Cimaterol
Clenbuterol
Ractopamine
Salbutamol
Terbutaline
Tulobuterol
Zilpaterol
Clindamycin
Erythromycin
kitasamycin
Lincomycin
Spiramycin
Tilmicosin
Tylosin
Sulfachlorpyridazine
Sulfaclozine
Sulfadimethoxine
Sulfaguanidine
Sulfamerazine
sulfameter
Sulfamethazine
sulfamethizole
Sulfamethoxypyridazi…
sulfanilacetamide
sulfaphenazole
Sulfapyridine
sulfisomidine
Trimethoprim
Cinoxacin
Ciprofloxacin
Danofloxacin
Difloxacin
Enoxacin
Enrofloxacin
Flumequine
Lomefloxacin
Marbofloxacin
Nalidixicacid
Norfloxacin
Ofloxacin
Orbifloxacin
Oxolinicacid
Pefloxacin
Sarafloxacin
Chloramphenicol
florfenicol
Thiamphenicol
penicillinV
Betamethasone
Cortisone
Dexamethasone
Hydrocortisone
Meprednisone
Methylprednisolone
Prednisolone
Triamcinolone
Triamcinolone…
Cefotaxime
Ceftiofur
cephapirin

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”

Time
1.00 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
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)
0
20
40
60
80
100
120
140
Recovery
Total Method Recovery from Shrimp

Recovery of Veterinary Drugs from
Salmon Tissue (9 Classes of Drugs)
0
20
40
60
80
100
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
Time
0.50 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

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
20
40
60
80
100
120
140

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
Time
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
%
0
100
LC_16March2016_05 Scan API+
TIC
3.82e8
dichlorvos
chlorothalonil
folpet
cypermethrin
azoxystrobin
simazine
metalaxyl
cyprodinil
oxyfluorfen
fenpropathrin
cyhalothrin

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
Time
4.00 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

Oasis® Prime HLB - introducing a new sorbent for the sample cleanup of Food matrices - Waters Corporation Food Safety

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