Agilent Quantitative Screening of Multiresidue Application Note

Application Note

Food Testing and

Agriculture

Quantitative Screening of Multiresidue Veterinary Drugs in Milk and Egg Using the Agilent 6495C Triple Quadrupole LC/MS

Authors

Siji Joseph, Aimei Zou,

LimianZhao, Patrick Batoon,

and Chee Sian Gan Agilent Technologies, Inc.

Abstract

This application note demonstrates the use of the Agilent Comprehensive

Veterinary Drug dMRM Solution for the screening of 210 target residues in milk and eggmatrices. The workflow specifies conditions for chromatographic separation,

MS detection, and data processing, using a slightly modified sample preparation

procedure. Workflow performance was assessed based on limit of detection (LOD), limit of quantitation (LOQ), calibration curve linearity, accuracy, precision, recovery, and repeatability. Over 93% of veterinary drugs showed LOD of ≤1 μg/kg in milk samples. Calibration curves for all targets ranged from the LOQ to 100 μg/kg with a coefficient of correlation (R2) ≥0.99. Target peak area response (%RSD) was <15%, and retention time (RT) %RSD was <0.5%. Method accuracy values, based on matrix-matched calibration were within 87 to 117%. The average recovery of 95% of targets was within 60 to 120%, with repeatability %RSD of ≤15%. Both milk and egg matrices showed similar quantitative results. Injection-to-injection robustness results demonstrated excellent target peak area and RT reproducibility across 400injections, confirming the workflow capability for routine multiresidue screening with large-scale sample sets.

Introduction

The Agilent Comprehensive Veterinary Drug dMRM Solution is an end-to-end

workflow solution for targeted screening

or quantitation of 210 veterinary drug residues in animal origin matrices,

which accelerates and simplifies routine laboratory testing. The solution includes

comprehensive sample preparation, chromatographic separations, optimized MS detection method conditions, data analysis methods, and reporting templates for 210 veterinary drugs in various food matrices. The Comprehensive Veterinary Drug dMRM Solution minimizes method development

time and combines multiple food matrix analyses into one easy-to-follow

protocol. Agilent MassHunter data

acquisition software, together with the dMRM database offers easy customization of dMRM submethods based on preferred target list or regulation, as determined by the user. The solution is available and has been verified with two mass spectrometers (Agilent 6470 triple quadrupole LC/MS and the Agilent 6495C triple quadrupole LC/MS) to address diverse sensitivity demands based on the choice of sample

matrix and specific regulations that

varyglobally.

The solution was originally developed

for the quantitative screening of

210multiclass veterinary drugs in chick, beef, and pork.1 It was then demonstrated to be effective for

seafood using salmon and shrimp as example matrices.2 This study further

demonstrates the applicability for milk and eggs using the 6495C triple quadrupole LC/MS. For the 210 target analytes screened in this study, 103

of them had maximum residue limits

(MRL) established in milk regulated by the AOAC3—with an additional 16targets regulated by US FDA-CFR4,

US FSIS5, or EU6 regulations/guidelines.

The MRL values are typically lower in milk compared to meat and seafood,

thus requiring a higher MS detection sensitivity. Additionally, the high fat

and protein content in milk demands

effective sample preparation and a sensitive detector to monitor trace levels

of drug residues. Compared to milk, the number of MRL-established targets for the egg matrix is fewer and the residue

limits are more relaxed.

Experimental

Standards and reagents

Veterinary drug standards were purchased from Sigma-Aldrich (St. Louis, MO, USA), Toronto Research Chemicals (Ontario, Canada), and Alta Scientific (Tianjin, China). Agilent LC/MS-grade acetonitrile (ACN, partnumber 5191-4496), methanol (MeOH, partnumber 5191-4497), and water (part number 5191-4498) were used

for the study. All other solvents used

were HPLC-grade from Sigma-Aldrich. LC/MS additives for mobile phases were

also purchased from Sigma-Aldrich.

Individual stock solutions of veterinary drugs were prepared from powdered or

liquid veterinary drug standards at 1,000

or 2,000 µg/mL using an appropriate solvent (MeOH, dimethyl sulfoxide (DMSO), ACN, or water or solvent mixture). A few stock standard solutions (100 µg/mL) were obtained from the suppliers listed above.

A comprehensive standard mix

(1µg/mL of each target analyte in 50/50 ACN/water) was prepared from individual stock solutions and used for

this experiment.

Sample preparation

Milk and egg samples were purchased

from a local grocery. For the analysis

of milk, a 2.0 ±0.1 mL portion of milk was transferred in a 50 mL conical polypropylene tube. For the analysis

of egg, a 2.0 ±0.1 g portion of the homogenized sample was weighed in a 50 mL conical polypropylene tube. If not analyzed immediately, the samples were

stored at –20 °C.

Sample preparation was performed

as per the procedure defined in the Comprehensive Veterinary Drug

dMRM Solution (G5368AA) using solvent extraction followed by Agilent Captiva EMR—Lipid cleanup (partnumber5190-1003), aided by

the Agilent positive pressure manifold

processor (PPM-48, part number 5191-4101).7 The sample preparation procedure is summarized in Figure1. The aqueous extraction step was modified

to adjust the target dilution due to

increased water content in milk andegg.

The following deviations from the

protocol defined in the Comprehensive Veterinary Drug dMRM Solution are recommended for the aqueous extraction step:

– Milk: Concentration of EDTA solution:

1 M, volume added: 200 µL.

– Egg: Concentration of EDTA solution:

0.1M (same as workflow guide),

volume added: 1 mL

Matrix-spiked (pre-extraction) QC samples were prepared by spiking the

appropriate veterinary standard solution

into the milk and egg matrices at various levels: 1 μg/kg for low-range QC (LQC), 10 μg/kg for mid-range QC (MQC), and 25 μg/kg for high-range QC (HQC), respectively. An additional QC level lower than the LQC of 0.1 μg/kg (LLQC) was included in the milk analysis to verify the analytical characteristics of a few targets, and to meet the very low MRL requirement. After spiking standards, the samples were vortexed for 30seconds, then equilibrated for 15 to 20 minutes to allow the spiked standards to infiltrate the sample matrix before sampleextraction.

Matrix-matched calibration standards

Two 50 mL tubes 2.0 g sample

for QC samples

Blank matrix eluent

Matrix-matched (postextraction) calibration standards were prepared as per the workflow protocol by spiking appropriate standards into the blank matrix extract.7 The targeted

concentrations of matrix-matched

calibration levels were 0.1, 0.25, 0.5, 1.0,

2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 μg/kg (10 levels). An additional matrix-matched calibration level of 0.05μg/kg was added for milk analysis for similar consideration of few targets with very low MRL requirement. Considering the

10x dilution factor introduced during

sample preparation, the actual spiking

concentrations of postextraction

calibration standards were 0.005, 0.01,

0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.5, 5.0,

and 10.0 μg/L (ppb) in the milk blank

matrix extract.

Neat standards at 2.5 μg/L in a 50/50 ratio of ACN/water was used to evaluate matrix effects by comparing

the responses in the corresponding

postextraction-spiked calibration

standards.

Chromatographic separation was performed using an Agilent InfinityLab Poroshell 120 EC-C18 column (partnumber 695575-302) installed on an Agilent 1290 Infinity II LC, including

Agilent 1290 Infinity II flexible pump (G7104C), Agilent 1290 Infinity II multisampler (G7167A), and Agilent1290

Infinity II multicolumn thermostat

(G7116A).

Mobile phase A was water with 4.5 mM

ammonium formate, 0.5 mM ammonium

fluoride, and 0.1% formic acid; mobile phase B was 50/50 ACN/MeOH with

4.5 mM ammonium formate, 0.5 mM

ammonium fluoride, and 0.1% formic acid. The LC system was equipped with a 20 µL injection loop and multiwash capability. Please see the workflow guide included with the Agilent Comprehensive

Veterinary Drug dMRM Solution

(G5368AA) for additional details.

The “6495 Veterinary Drug

Comprehensive” method included in the Comprehensive Veterinary Drug dMRM

Solution for the 6495C triple quadrupole LC/MS (G6495C) was used directly for acquisition. The 6495C LC/MS triple quadrupole with an Agilent Jet Stream (AJS) ion source was operated in dynamic MRM (dMRM) mode. Autotune was performed in unit resolution with report m/z below 100 mode enabled. MassHunter acquisition software version

10.0 was used for data acquisition,

and MassHunter quantitative analysis

software version 10.0 was used to

process the data.

Results and discussion

Workflow performance in milk

Chromatographic separation using the

Agilent InfinityLab Poroshell EC-C18

column resulted in good separation and

RT distribution of 210 veterinary drugs within a 13-minute elution window.

Target-specific MRM transitions included in the dynamic MRM method helped to meet regulatory requirements for compound identification and confirmation. The default dynamic MRM method utilized a cycle time of

750ms, and dwell times for each dMRM transition ranged from 7 to 370ms,

offering more than 10 data points

across any given peaks. Figure2 shows

a representative MRM chromatogram for all 210 veterinary drug targets,

postextraction spiked at 1.0 μg/L in the milk blank matrix extract. Considering

the dilution factor during sample

preparation was 10x, this 1.0 μg/L postextraction spike was equivalent to a 10 μg/kg spike in milk. The symmetrically sharp peaks demonstrate the efficient

chromatographic separation of targets

within the elution window. Table 1 lists the name, chemical class, CAS number,

and RT of all 210 targets covered in

thiswork.

Figure 1. Sample extraction procedure using solvent extraction followed with Agilent Captiva EMR—Lipid cleanup.

Blank matrix

(no spike)

Pre-extraction spike

2-Step solvent

extraction

Centrifuge

Sample cleanup using

Agilent Captiva EMR—Lipid 3 mL

cartridges on Agilent PPM-48

Homogenized

eluents

for matrix blank

and post-extraction

spike calibration levels

LC/MS analysis using

Agilent 6495C TQ

Pre-extraction spike

eluents as QC samples

×10

Acquisition time (min)

Counts

Acquisition time (min)

Counts

2.0

1.5

1.0

0.5

1 2 3 4 5 6 7 8 9 10 11 12

Figure 2. Representative MRM chromatogram of 210 veterinary drug targets postextraction spiked at 1.0 μg/L in the milk blank matrix extract using the Agilent 6495C triple quadrupole LC/MS).

From the AOAC MRL established

list, the early eluted analytes,

including amoxicillin, baquiloprim, deacetylcefapirin, diminazene, imidocarb,

norgestomet, sulfaguanidine, and

tilmicosin showed split peaks due

to solvent effects. The “spectrum

summation” integrator algorithm was used to reliably and automatically

integrate these targets for consistent RT, and thus eliminated the need for manual reintegrations.8 The peak shape for these

targets can be improved by converting

samples into a higher aqueous mixture

prior to LC/TQ injection.

LOD, LOQ and calibration

curvelinearity

LOD and LOQ were established using various low level matrix-matched calibration standards.

1,2

The

signal-to-noise ratio (S/N) was calculated using the peak height for signal and an

auto-RMS algorithm for noise, included in the MassHunter quantitative analysis

software. The method sensitivity using the 6495C LC/TQ system offered a LOD ≤1 μg/kg for over 93% of analytes tested in both milk and egg. The low detection limits achieved allowed the

high sensitivity demand for screening trace level veterinary drug residues in

milk. As an example, AOAC regulated

MRL of 0.05μg/kg for clenbuterol in milk. The 6495C TQ-based workflow provided a clean, symmetrical peak with S/N of

calibration level, thus enabling confident

target identification and quantitation

(Figure 3).

32 at the 0.05 μg/kg matrix-matched

×10

RT: 5.42 (min)

5.1 5.2 5.3 5.4 5.5 5.6 5.7

Figure 3. MRM chromatogram of clenbuterol (MRM 277.1 & 202.9) postextraction-spiked at 0.005 μg/L (black trace) and 0.01 μg/L (blue trace) in the milk matrix extract, overlaid with matrix blank (red trace). The defined LOD of clenbuterol is 0.05 μg/kg (S/N: 32) and LOQ is 0.1 μg/kg (S/N: 76).

A calibration curve for each target

Acquisition time (min)

Counts

was generated using matrix-matched calibration standards at levels ranging from the defined LOQ to the highest-spiked level. The linear regression was used with ignored origin and 1/x or 1/x2 weight. All targets met the calibration curve linearity

requirement of R2 ≥0.99. The LOD, LOQ,

and calibration curve data of all targets in the milk are shown in Table1.

Instrument method precision

andaccuracy

Precision was determined by calculating the %RSD of the target response and

RT using triplicate injections of the

matrix-matched calibration levels.

The average accuracy value for each

matrix-matched calibration level was also calculated from the triplicateinjections.

Good precision and accuracy values

were obtained for all targets in milk. Target response %RSD for all targets in the milk matrix at 10 μg/kg was <15%, and the RT %RSD of all targets were within 0.5%.9 The accuracy values of all targets at 10 µg/kg within a range of 87 to 117%. These results confirm the reproducibility of chromatographic

separation and MS detection.

average recovery was calculated from

duplicate injections of four technical

preparations. The intrabatch recovery repeatability was measured as %RSD

of recovery, calculated using four

technical preparations of matrix-spiked QCsamples.

The results showed that recoveries of about 93% of MRL-established targets reached the acceptable range of 60 to 120% with an excellent intrabatch RSD ≤20%.9 Recoveries of the remaining seven targets, baquiloprim,

chlortetracycline, deacetylcefapirin,

diclofenac, imidocarb, oxytetracycline, and trichlorfon [DEP], were within a range of 30 to <60% or >120 to 124%. However, for these targets, the workflow still provided good recovery repeatability values within a %RSD of

×10

RT: 5.40 (min)

9%, demonstrating consistent extraction behavior. These results confirmed the entire workflow reproducibility using Captiva EMR—Lipid sample

extraction and cleanup protocol in the

6495-TQ-based instrument detection. The recovery and repeatability results of all 210 targets are included in Table 1 (see Appendix).

The workflow performance combined with the 6495C LC/TQ detection helped confident recovery and repeatability assessment at trace levels in milk. Figure 4 shows an example of workflow recovery and repeatability for clenbuterol at 0.1 μg/kg in milk. The average recovery of this target using the LLQC sample is 118% with good recovery repeatability of %RSD <5%.

Target recovery and intrabatchrepeatability

The impact of sample preparation on

target recovery was assessed using matrix-spiked QC samples. Each QC level was prepared with four technical preparations and was injected for

instrument analysis in duplicates. An

appropriate level of matrix-spiked QC sample based on MRL was selected

to evaluate target-specific recovery

and repeatability. Recovery was

calculated using target response in

matrix-spiked QCs, and measured

response using matrix-matched

calibration curve equations. The

5.1 5.2 5.3 5.4 5.5 5.6 5.7

Figure 4. MRM chromatograms of clenbuterol (MRM 277.1 & 202.9) using four technical preparations of LLQC samples in milk (green traces) overlaid with matrix blank (red trace).

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