Development and Optimization of a Multi-Class, Multi

Development and Optimization of a Multi-Class, Multi-Residue Method
for Veterinary Drug Analysis in Infant Formula Ingredients and Products
Hui Zhao, John Zulkoski, Azeem Hasan and Katerina Mastovska
Nutritional Chemistry and Food Safety, Covance Laboratories Inc., Madison, WI, USA
hui.zhao@covance.com, katerina.mastovska@covance.com
Method Development Plan
A modern multi-class, multi-residue method for detection, identification
and quantitation of veterinary drugs using liquid chromatographytandem mass spectrometry (LC-MS/MS) is a highly effective approach
for residue monitoring with the ultimate goal of protecting consumers
from potentially unsafe exposure to drug residues in foods of animal
origin (milk, meat, poultry, seafood, fish, egg and honey). In this study,
we developed and optimized a method for over 150 compounds
belonging to a variety of veterinary drug classes, including
amphenicols, anthelmintics, antibiotics (beta-lactams – penicillins and
cephalosporins, lincosamides, macrolides, quinolones, sulfonamides,
tetracyclines, and others), antimicrobial growth promoters,
antiprotozoals, beta-agonists, coccidiostats, dyes, NSAIDs, and
tranquilizers. Infant formula was chosen to establish a robust, efficient
and reliable method for screening, identification and quantitation of the
included veterinary drug residues.
MRM (dMRM) Acquisition Method
Optimization
Optimization of MS/MS conditions for
individual compounds
▶ Mass spectrometer: Agilent® Triple Quadrupole 6495A
MS/MS
Optimization of LC conditions
Main Phases
Introduction
Development of the final LC-MS/MS method
the best two MRMs for the method
Sample preparation procedure development
and optimization
▶ Verify the selectivity for the final sample preparation procedure
for multiple matrices
Validation, data acceptance criteria and
implementation in routine analysis
Optimization of MS/MS Conditions for Individual
Compounds
Modern Multi-Class, Multi-Residue Method
Using LC-MS
▶ Product ion scans
▶ Selection of MRMs and collision energy optimization
▶ Up to 10 MRMs per analyte
Benefits
Example: Sulfadoxin, m/z 311.1
▶ Cost-effective
▶ Time-effective
▶ Selective detection of individual analytes
▶ Improved sensitivity for low LODs/LOQs
▶ Identification/confirmation
▶ Cell accelerator voltage (CAV)
▶ ESI source conditions
▶ Evaluate the MRMs for sensitivity and selectivity to choose
+ESI Product Ion:1 (rt: 3.765-3.877 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d
x10 6
CE = 0
4
311.1000
2
0
x10 6
156.0000
108.2000
+ESI Product Ion:2 (rt: 3.770-3.882 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 CID@15.0 (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d
CE = 15
3
2
Challenges
– Water and acetonitrile solvent mixture
– Ratio between aqueous and organic solvent
– Addition of acid
– Addition of EDTA buffer
▶ Clean-up (optional)
– Hexane defatting
– Modified QuEChERS (dSPE)
– Supported liquid extraction (SLE)
– Enhanced Matrix Removal-Lipid (EMR)
– SPE
Ratio of Extraction Solvent
311.2000
140.2000
65.1000
+ESI Product Ion:3 (rt: 3.774-3.886 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 CID@30.0 (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d
245.0000
218.0000
CE = 30
1.5
1
▶ A large spectrum of drug classes
▶ Parent drugs and metabolites
▶ Different physical/chemical properties
107.9000
92.0000
0
▶ Extraction
156.1000
1
x10 6
Sample Preparation
92.0000
108.1000
156.1000
140.1000
65.2000
80.3000
+ESI Product Ion:4 (rt: 3.779-3.891 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 CID@45.0 (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d
0.5
0
x10 5
5
64.9000
2.5
– Hydrophilic to hydrophobic
– Acidic, neutral and basic
– Stability
– Interaction with matrix components
▶ Compromise between analyte scope and performance
characteristics
▶ Matrix effects and potential interference from co-extractives
Matrices of Interest for Our Method Development
▶ Infant formula and its relevant ingredients
Veterinary Drugs of Interest for Our Method
Development
Total Numbers of Interest
0
x10 5
92.0000
230.0000
CE = 45
154.1000
201.2000
CE = 60
80.2000
53.1000
50
60
70
80
90
100
110
120
132.1000
130
140
150
154.2000
160
170
180
190
200
210
220
230
240
250
260
270
280
290
▶ 102 analytes, 9 sub-classes
▶ Aminoglycoside (7, analyzed in separate method), Amphenicol (5),
Beta Lactom-cephalosporin (12), Beta lactam-penicillin (8),
Macrolides and lincosamides (12), Other (9), Quinolones (16),
Sulfonamides (24), Tetracyclines (9)
Structures of Representative Antibiotics and
Other Drugs
Streptomycin (Aminoglycoside)
Florfenicol amine (Amphenicol)
Ceftiofur (Cephalosporin)
MF C21H39N7O12 MW=581.57
MF C10H14FNO3S MW=247.291
MF C19H17N5O7S3 MW=523.56
320
Note: 0.1% formic acid
added to ACN to assist
with protein precipitation
▶ Anthelmintic (Mix A-22)
▶ Beta lactam of cephalosporin and penicillin (antibiotic) (Mix B-20)
▶ Macrolides and lincosamides (antibiotic) (Mix C-13)
▶ Quinolones and others (antibiotic) (Mix D-23)
▶ Sulfonamides (antibiotic) (Mix E-24)
▶ Tetracyclines (antibiotic) (Mix F-9)
▶ Beta agonists, coccidiostat, and antimicrobial growth promoter
Spike recovery of 60-130% for the
majority of analytes/classes
Lower spike recoveries (<60%) for
tetracycline and some quinolones
Addition of EDTA Buffer
Aqueous EDTA
buffer
0.05M EDTA
0.025M EDTA
0.1M EDTA
0.1M EDTA0.05M succinate
▶ To improve the elution profile
▶ To reduce potential interferences between compounds
▶ To separate compounds, which share the same precursor and even
Majority of analytes/classes maintained
spike recovery of 60-130%
LC Conditions
Tetracyclines improved spike recoveries
from 0-15% to 60-85%
product ions
▶ Column:
C18
▶ Column temperature: 40oC
▶ Flow rate: 0.5 mL/min
▶ Injection volume: 5 µL
Agilent®
Zorbax®
Eclipse®
Mobile Phase A
Mobile Phase B
0.1M EDTAMcIlvaine
Plus, 2.1x100 mm, 1.8 μm
Problematic quinolones improved spike
recoveries from 50-60 % to 70-120%
Initial Mobile Phases
and Gradients
Gradient
Antibiotics
310
Analytes in Positive Mode (~150) Divided into
9 Groups
Optimization of LC Conditions
Antibiotic
300
Counts vs. Mass-to-Charge (m/z)
Multi-Class
Antiprotozoal (1), Beta agonist (8), Coccidiostat (10), Dye (5), NSAID
(1), Other-no class (1), Pesticide (2) and Tranquilizer (5)
1:1 water - ACN
1:3 water - ACN
107.9000
▶ 161 veterinary drug analytes and 10 internal standards
▶ All the analytes included to cover for the needs of global regulation
▶ 11 classes
▶ Anthelmintic (23), Antibiotic (102), Antimicrobial Growth Promoter (3),
Two steps:
1:1 water - ACN
followed by 1:1 extract
dilution with ACN
65.2000
(Mix G-22)
▶ Tranquilizers, Dyes and Pesticides (Mix H-12)
▶ Amphenicol, aminoglycoside, and other (antibiotic) (Mix I-4)
and different government monitoring programs
Aqueous to organic
solvent ratio
245.2000
108.2000
80.1000
126.1000
58.0000
184.2000
+ESI Product Ion:5 (rt: 3.783-3.895 min, 6 scans) Frag=380.0V CF=0.000 DF=0.000 CID@60.0 (311.1000 -> **) Sulfadoxin_Optimizer_5uL_P2.d
5
0
212.1000
Optimum Mobile Phases
and Gradients
0.1% formic acid in water
0.1% formic acid in acetonitrile
Sample Preparation General Procedure
0.1% formic acid in water
0.1% formic acid in methanol
Min
%A
%B
Min
%A
%B
0
0.5
9
11
11.1
17
98
98
10
10
98
98
2
2
90
90
2
2
0
0.75
7
11
13
13.1
17
98
98
60
0
0
98
98
2
2
40
100
100
2
2
Weigh 2 g sample
Add internal standard mix
Add 10 mL 0.05M EDTA buffer in water, shake to homogeneous
Add 10 mL 0.1% formic acid in acetonitrile, shake 5 minutes
Centrifuge 5 minutes
Clean-up (optional)
Amoxicillin (Penicillin)
MF C16H19N3O5S MW=365.4
Tylosin A (Macrolide)
Lincomycin (Lincosamide)
MF C46H77NO17 MW=915.52
MF C18H34N2O6S MW=406.21
Elution Profile Comparison for All Analytes
Reconstitute in 3:1 (v/v) water-acetonitrile
LC-MS/MS analysis
Initial
Ciprofloxacin (Quinolone)
MF C17H18FN3O3 MW=331.34
Sulfamethizole (Sulfonamide)
MF C9H10N4O2S2 MW=270.02
Next Steps
Trimethoprim (other)
▶ Evaluate the extraction procedure in other relevant matrices
▶ Evaluate short-term and long-term effects of clean-up
▶ Determine analyte stability in standard mixtures and sample extracts
MF C14H18N4O3 MW=262.22
Other Drugs
Cambendazole (Anthelmintic)
Ractopamine (β-agonist)
Ipronidazole (Coccidiostat-nitroimidazole)
MF C14H14N4O2S MW=302.35
MF C18H23NO3 MW=301.17
MF C7H11N3O2 MW=169.09
Decoquinate (Coccidiostat)
MF C24H35NO5 MW=417.54
Malachite Green (Dye)
Chlorpromazine (Tranquilizer)
MF C23H25ClN2 MW=364.91
MF C17H19ClN2S MW=318.10
Virginiamycin (M1) (Antimicrobial Growth Promoter)
Strychnine (Pesticide)
Isometamidium (Antiprotozoal)
MF C28H35N3O7 MW=525.59
MF C21H22N2O2 MW=334.41
MF C28H25N7 MW=460.55
Presented at RAFA 2015
Optimized
References
Robert C., Gillard N., Brasseur P. Y., Pierret G., Ralet N., Dubois M., and Delahaut Ph., “Rapid
multi-residue and multi-class qualitative screening for veterinary drugs in foods of animal origin
by UHPLC-MS/MS”, Food Additives and Contaminants, Part A 30(3):443-457 (2013).
Kaufmann A., Butcher P., Maden K., Walker S., and Widmer M., “Multi-residue quantification of
veterinary drugs in milk with a novel extraction and cleanup technique: Salting out supported
liquid extraction (SOSLE)”, Analytical Chimica Acta 820:56-68 (2014).