Targeted Multiresidue Analysis of Veterinary Drugs in Milk-Based

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Targeted Multiresidue Analysis of Veterinary Drugs in Milk-Based Powders Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) James B. Wittenberg, Kelli A. Simon, and Jon W Wong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05263 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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Journal of Agricultural and Food Chemistry

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Targeted Multi-residue Analysis of Veterinary Drugs in Milk-Based Powders Using Liquid

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Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

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James B. Wittenberg*, Kelli A. Simon, and Jon W. Wong

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U.S. Food and Drug Administration

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Center for Food Safety and Applied Nutrition

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5001 Campus Drive, HFS-717

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College Park, MD 20740-3835, USA

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[email protected]

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*Corresponding Author

ACS Paragon Plus Environment


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Abstract

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An analytical method was developed and validated for the determination of 40 veterinary

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drugs in various milk-based powders. The method involves acetonitrile/water extraction, solid-

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phase filtration for lipid removal in fat-containing matrices, and analysis using liquid

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chromatography tandem mass spectrometry (LC-MS/MS). The limits of quantitation (LOQ)

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ranged from 0.02 to 82 ng/g. Acceptable recoveries (70–120%, RSD < 20%) were reached for

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40 out of 52 target compounds at three fortification levels in non-fat milk powder. Similar

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results were obtained for whole milk powder, milk protein concentrate, whey protein

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concentrate, and whey protein isolate. This new method will allow for better monitoring of a

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wide range of veterinary drugs in milk-based powders.

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Keywords: Veterinary drugs; Milk-based powders; LC-MS/MS


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Introduction

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The U.S. Food and Drug Administration (FDA) is responsible for monitoring the

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presence and quantity of veterinary drugs in various food commodities. A number of analytical

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methods have been implemented by the FDA for the screening and determination of veterinary

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drug residues in milk and dairy products and to verify that the foods do not contain amounts in

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excess of the established tolerances.1 However, due to advances in instrumentation, extension of

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methods to different food matrices, and/or expansion of the list of veterinary drugs, new methods

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are constantly developed to improve and expand monitoring programs..

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Liquid chromatography – tandem mass spectrometry (LC-MS/MS) has been utilized in

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the development of many multi-residue methods for the analysis of veterinary (vet) drug

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residues.2 While there are a large number of methods that analyze vet drugs in liquid milk, 3-9

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there are very few methods in literature, and no compendial methods, that monitor for vet drugs

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in milk powder.10-13 Milk powder, both non-fat and whole, is a globally-traded commodity on the

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scale of 6.4 million metric tons in 2015 alone, and used as an ingredient in a wide variety of

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products, so monitoring for vet drugs in this commodity is of significant importance.14

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The three major components that make up liquid milk and its dry forms are protein,

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carbohydrates, and fat. When raw milk and a number of milk-based powders are broken down

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into these three components, it is clear that they are drastically different in amount of potential

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matrix interferences (Table 1). Therefore, sample preparation is very important in order to attain

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acceptable results at the required tolerance levels.

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methods for milk powder included a treatment similar to the liquid milk methods following

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initial reconstitution (hydration) in H2O,10,11 liquid extraction with a subsequent 12 hour chilling

Previously reported sample preparation



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process to precipitate the lipids and proteins,12 or liquid extraction with the McIlvaine buffer and

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subsequent C18 dSPE cleanup.13

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The primary goal of this study was to develop and validate a multi-residue LC-MS/MS

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method using a triple-quadrupole mass analyzer for the determination of 52 veterinary drugs,

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encompassing 12 classes, in both non-fat and whole milk powders.

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amphenicols (2), anthelmintics (1), avermectins (4), imidazoles (9), lincosamides (2), macrolides

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(6), NSAIDs (7), quinolones (2), ß-lactams (8), sulfonamides (6), tetracyclines (2), and 2

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unclassified compounds, ranging in polarity with log P values of 0.87 to 5.8, were monitored.15

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The method was extended to include milk protein concentrate (MPC), whey protein concentrate

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(WPC), and whey protein isolate (WPI). A simple liquid extraction sample preparation along

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with a fast chromatographic method was developed in the process. This new method, that does

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not require reconstitution or a lengthy clean-up procedure, would then allow for better

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monitoring of a wide range of veterinary drugs in various milk-based powders.

Aminocoumarins (1),

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Materials and Methods

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Chemicals

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LC-MS grade water (H2O), methanol (MeOH), and acetonitrile (ACN) were purchased

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from Fisher Scientific (Pittsburgh, PA). The majority of the analytical standards were purchased

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from Sigma Aldrich (St. Louis, MO). The remaining standards purchased were from U.S.

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Pharmacopeia (Rockville, MD), Toronto Research Chemicals (Toronto, ON, Canada), BOC

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Sciences (Shirley, NY), or U.S. Biological (Salem, MA). The seven milk-based powders were

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purchased either from a grocery store or via the internet.

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Instrumentation

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Liquid chromatography was carried out using a Waters Acquity UPLCTM (Waters,

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Milford, MA), where the autosampler was maintained at 10 °C during operation, and the

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separation was carried out using a Kinetex 2.6 µm Biphenyl 100 Å column (100 × 2.1 mm i.d.,

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Phenomenex, Torrance, CA) coupled to a SecurityGuard ULTRA UHPLC Biphenyl guard

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column (2.1 mm i.d., Phenomenex).

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operation. Elution was completed using a 10 min gradient program at a flow rate of 0.5 mL/min

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with a 5 µL injection volume. Mobile phase A was composed of 5 mM ammonium formate and

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0.1% formic acid in H2O, and mobile phase B was composed of 5 mM ammonium formate and

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0.1% formic acid in MeOH. The gradient parameters were: Initial-0.5 min, 5% B; 0.5-7 min,

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5% B to 100% B; 7-10 min, 100% B. To prevent contamination, the LC eluent was introduced

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to the ion source only between 1 and 8 min during the run using a Valco® valve switch.

The column oven was maintained at 40 °C during

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The LC was interfaced to an AB Sciex QTrap 6500 (AB Sciex, Foster City, CA)

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equipped with an IonDriveTM Turbo V ion source (electrospray). LC-MS/MS operation and data

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acquisition were controlled by the AB Sciex Analyst software version 1.6.2, and quantitation was

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completed using the AB Sciex MultiQuant software version 2.1.1. The ion spray voltage was set

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to 5500 V. The source temperature was maintained at 400 °C. Nitrogen was used as the curtain

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gas and collision gas. The curtain gas, ion source gas 1, and ion source gas 2 were set to

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pressures of 30, 50, and 70 psi, respectively. The collision gas was set to “Medium.” The mass

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spectrometer was operated in scheduled multiple reaction monitoring (scheduled MRMTM)

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mode. The entrance potential and cell exit potential were set to 10 V for all transitions. The

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declustering potentials and collision energies were optimized for each transition (Table 2).

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Preparation of Standard Solutions

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Stock solutions for each individual standard were prepared by weighing a small amount

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(typically 25 mg) of the standard into a 50 mL centrifuge tube and dissolving the standard into a

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stable and soluble solvent, such as ACN, MeOH, or DMF (typically 25 mL via volumetric

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pipette). Three stock standard mixture solutions (one “Negative” ion mixture and two “Positive”

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ion mixtures) were then prepared by combining known amounts of each stock standard solution

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with ACN to a reach the desired concentration. Then, the highest concentration working solution

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was prepared by combining the stock mixtures and diluting with ACN. Finally, the remaining

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six working solutions were prepared by serial dilution of the highest concentration working

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solution.

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Sample Preparation

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Extraction. The bulk milk powders were first transformed into a fine powder in

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approximately 25 g batches using a tube mill operating at 24,900 rpm for 30 s (Ika-Works, Inc.,

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Wilmington, NC). Then, a 1.0 ± 0.1 g sample of the fine powder was weighed into a 50 mL

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conical centrifuge tube (Corning, Inc., Corning, NY). The powder (in triplicate) was fortified

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with 100 µL of working spike solution and 100 µL of working internal standard solution. The

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tube was vortexed on a Vortex Maxi Mixer (Thermo Scientific, Waltham, MA) for 10 s to

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incorporate the liquid into the solid. After 15 min, 9 mL of ACN were added, and the tube was

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vortexed for 10 s. Then, 1 mL of H2O was added along with a stainless steel (SS) ball bearing,

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and the tube was capped. The tube was shaken on a large capacity mixer (Glas-Col, LLC., Terre

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Haute, IN) operating at 80% power and maximum pulse frequency for 30 min. The SS ball

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bearing was removed, and the tube was centrifuged (Thermo Scientific) for 10 min at 4700 × g.

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For non-fat milk-based powder extracts, a 5 mL aliquot of the supernatant was transferred to a 15


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mL conical centrifuge tube (Corning, Inc.) and the sample was concentrated and filtered without

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additional clean-up steps (see below). For extracts of fat-containing milk-based powder, the

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extract was processed by solid phase extractin prior to concentration filtration(see below).

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Solid phase extraction. A Waters Oasis PRiME HLB 6 cc (200 mg) cartridge (Waters

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Corp., Milford, MA) was setup for pass-through filtration using a Visiprep 24 DL SPE vacuum

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manifold (Supelco, St. Louis, MO). The cartridge was preconditioned with 2.5 mL 90:10

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ACN:H2O. A 5 mL aliquot of the supernatant from the extraction procedure was passed through

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the cartridge under gravity and collected in a 15 mL centrifuge tube. The cartridge was rinsed

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with 1 mL 90% ACN which was also collected in the tube.

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Concentration and Filtration. The extract was concentrated to near-dryness (approx. 50

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min) on an ExcelVap (Horizon Technology, Inc., Salem, NH) at 45 °C under a 24 psi stream of

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N2. The extract was reconstituted with 1 mL of 20% ACN in 0.1 M ammonium acetate and

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vortexed for 30 s. Approximately 400 µL was then transferred to an outer vial (Thomson

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Instrument Company, Oceanside, CA), pressed through a 0.2 µm PVDF filter membrane

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plunger, and immediately placed in the sample manager for injection and analysis by LC-

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MS/MS.

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Results and Discussion

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LC-MS/MS Optimization

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Each solution of standards used for MS optimization contained five or six veterinary

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drugs dissolved in MeOH.

For the majority of the compounds, the MS parameters were

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optimized by infusing a ~0.1-1 µg/mL solution at a flow rate of 10 µL/min. Some of the

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problematic compounds (i.e. avermectins, macrolides, and ß-lactams) required an alternative



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approach for MS parameter optimization. For these, an LC flow of a 50:50 mixture of MP A and

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MP B at 0.5 mL/min was combined with the infusion solution using a tee in order to simulate the

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conditions during sample analysis and to better optimize the MS parameters for these compounds

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that exhibited poor ionization. Two product ions were optimized for each precursor: a primary

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transition for quantitation and a secondary transition for confirmation. The MS parameters for

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all 52 compounds are shown in Table 2. The primary/secondary transition ion ratio was also

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used for further confirmation, which was determined by the calculated ion ratio being within

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20% of the expected ratio.

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switching were utilized for the analysis of all 52 compounds in a single chromatographic run.

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The initial source conditions were optimized by monitoring the total ion current (TIC) of the LC

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flow/infusion solution mixture.

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chromatographic separation and then re-optimized once the chromatography was established.

Electrospray ionization (ESI) and positive/negative polarity

These conditions were used while optimizing the

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Once the MS parameters for all 52 compounds were established, and the initial source

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conditions were set, the chromatographic separation was optimized. A Kinetex 2.6 µm C18 100

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Å column (100 × 2.1 mm i.d., Phenomenex) was first chosen due to the widespread use of C18

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columns for veterinary drug separations.

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shapes, poor retention, and slight fronting no matter the LC gradient program employed. A

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Kinetex 2.6 µm Biphenyl 100 Å (100 × 2.1 mm i.d.) column was then tried in an effort to better

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retain the relatively polar compounds. Better peak shape and retention was gained with the

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biphenyl column, most likely due to the π-effects of the biphenyl groups interacting with the

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compounds. Initially, a mobile phase combination of 0.1% formic acid in H2O and 0.1% formic

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acid in MeOH was used. When ACN was used, an overall decrease in signal response and

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retention was observed. Eventually, 5 mM ammonium formate was added to both mobile phase

However, some compounds exhibited poor peak


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A and B to help stabilize the pH and reduce the formation of unwanted adducts, particularly with

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the macrolides and avermectins. Once the chromatographic separation was established, the

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source conditions were re-optimized by injecting a mixture of all compounds, multiple times,

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and with different conditions (source temperature, curtain gas, source gases, and ion voltage) and

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then by monitoring the change in overall response as well as the individual response of the “non-

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sensitive” compounds (particularly the macrolides and avermectins).

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procedure, the source conditions were set as mentioned above, where the sensitivity of the

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macrolides and avermectins was optimized, and the LC-MS/MS method was finalized (Fig. 1).

By employing this

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Sample Preparation

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The initial sample pretreatment and clean-up employed preparing the milk powder

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similar to liquid milk. The procedure entailed hydrating the milk powder with H2O after spiking

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with the vet drug mixture in ACN. Unfortunately, complete hydration of the powder after

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fortification could not be accomplished. The fortification solutions contained at least some

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ACN, and even a very small amount of ACN (100 µL) added to the powder (1 g) caused

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clumping of the powder which resulted in poor repeatability. Another reason was due to the

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large amount of matrix components (~9× more) that was in 1 g of powder when compared to 1 g

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(~1 mL) liquid milk (Table 1). Since complete hydration of the fortified powder was not

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possible and a large dilution could not be afforded, an alternative extraction procedure was

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developed.

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When the powder was mixed with a solution containing a significant amount of organic

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solvent (70% ACN or greater in H2O) the majority of the powder (protein) would remain out of

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solution thus reducing matrix interferences. Therefore, extraction of the compounds in a high



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percent ACN solution that left the majority of the matrix behind as a precipitate was tested. Four

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different extraction solutions were tested: 70% ACN in H2O, 80% ACN in H2O, 90% ACN in

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H2O, and 100% ACN. Extraction with 80% for the LLOQ and >85% for all other levels. For many of the compounds,

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the upper limit of quantification (ULOQ) was restricted to the calibration levels of interest and

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not by the actual ULOQ.

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Recovery Studies. There are no tolerance levels for veterinary drugs established for milk

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powder, only for liquid milk and animal tissues.21 Since milk powder is commonly used as a

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liquid milk substitute as an ingredient, the tolerance levels for liquid milk were used as a

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reference point for the mass equivalent of milk powder in this study. The dairy-based powders

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were fortified with all 52 veterinary drugs at three different levels: ½X, X, and 2X, where X

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equaled the maximum residue limit (MRL) established for that particular compound in liquid

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milk. The results at the lowest fortification level for one of the non-fat milk powders are shown

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in Fig. 3 (and Table S1 in the Supporting Information). There was very little variation in the

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results when compared to the two higher fortification levels. A large number of the target

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compounds, however, did not have an MRL in milk. In those cases, the lowest fortification

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levels (½X) were determined to be approximately 2-5× LOQ of the compounds. It is important

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to reiterate that the MRLs stated for 1 mL of liquid milk was also utilized for evaluating the

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method for 1 g of dairy-based powder. Typically, liquid milk is made up of about 10-20%


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solids. Therefore, there was about 5-10× more matrix material in the dairy-based powders

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compared to the mass equivalent of liquid milk. Initially, the plan was to use an isotopically-

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labeled internal standard in order to account for variations from sample-to-sample, such as

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volume of extract after concentration, as well as matrix effects. Although the results obtained for

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the six native compounds of the internal standards used were an improvement when compared to

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the results obtained without internal standard correction, the results for the remaining 46

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compounds did not always show improvement with internal standard correction. The matrix

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effects observed for the six isotopically-labeled standards varied greatly from the matrix effects

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observed for a number of the non-native compounds. Therefore, the use of internal standard

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correction was abandoned, and the recoveries were calculated uncorrected against matrix-

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matched standards. The overall uncorrected results were similar, if not better, to the corrected

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results for the majority of the compounds.

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isotopically-labeled standards was no longer necessary.

Furthermore, the purchasing of expensive

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As stated before, a variety of non-fat and fat-containing powders were tested (Table 3).

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For the majority of the powders tested, approximately 80% of the compounds reached an

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acceptable recovery level with the average recovery ranging from 74 to 87% and RSDrs below

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10% (Table S1 in the Supporting Information).22 The current sample preparation method relies

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on the dissolution of the target compounds and leaving behind the majority of the matrix

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interferences (proteins, carbohydrates, etc.).

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interactions with such a large amount of matrix interferences with a simple extraction procedure.

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The production process of the dried milk powder may also be contributing to recovery loss.

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Milk is typically spray-dried, and during the spray-drying process some of the proteins can

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become denatured, or undergo lactosylation.23 These denatured proteins could therefore interact

However, it may not be possible to avoid all



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more strongly with certain compounds of interest. Other analytical methods also report having

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recovery issues in milk with the ẞ-lactams and tetracyclines due to compound stability, matrix

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interaction, and extraction inefficiency.3,24 Another potential factor for low recovery of certain

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compounds, particularly the more polar compounds, is lack of solubility in a high % organic

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solution in combination with the matrix. Although the number of acceptable recoveries was low

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for the whey protein isolate a high average recovery was observed. This was due to an ion

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enhancement observed for the imidazoles, where six out of nine imidazoles reached >120%

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recovery. The additional pass-through filtration step used for removing the fat from the fat-

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containing samples did not seem to significantly alter the overall recovery or number of

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acceptable recoveries. In fact, when a non-fat milk powder sample was prepared with the

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additional filtration step, similar results were obtained (37 out of 52 were acceptable). Overall,

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this sample preparation method produces reasonable results no matter the % protein, % fat, or %

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carbohydrate content in the dairy-based powder.

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In conclusion, a multi-residue, LC-MS/MS method was developed and validated for the

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determination of 40 out of 52 veterinary drugs in non-fat milk powder using a simple liquid

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extraction sample preparation.

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fortification recoveries in other non-fat dairy powders (WPC, WPI, and MPC). The method was

292

further applied to accommodate fat-containing diary powders with a single-step addition to the

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sample preparation of using an SPE cartridge in a pass-through filtration format, and this resulted

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in comparable fortification recoveries to the non-fat powders. This new method does not require

295

a lengthy sample preparation time or the hassle of a multi-step procedure as showcased in some

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of the previously reported methods. A relatively short chromatographic separation has also been

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achieved unlike some of the other procedures that take over 30 minutes or require 2 separate

This simple extraction method also resulted in acceptable


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injections to analyze all target compounds. Overall, this method is capable of determining a

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wide range of veterinary drugs and accommodating a variety of dairy-based powders in which

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their percent protein, carbohydrate, and fat contents greatly differ.

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Acknowledgements

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J.B.W. and K.A.S. were supported by an appointment to the research participation program of

304

the FDA administrated by the Oak Ridge Institute for Science and Education (ORISE).

305 306

Supporting Information

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A supplemental table (Table S1) is included in the supporting information which details the

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quantitative recovery results for all 52 veterinary drugs in all 5 milk-based powder matrices at

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the lowest fortification level.



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(23) Guyomarc’h, F.; Warin, F.; Muir, D. D.; Leaver, J. Lactosylation of milk proteins during

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the manufacture and storage of skim milk powders. Int. Dairy J. 2000, 10, 863-872.

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(24) Wang, J.; Leung, D. The challenges of developing a generic extraction procedure to

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analyze multi-class veterinary drug residues in milk and honey using ultra-high pressure

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liquid chromatography quadrupole time-of-flight mass spectrometry. Drug Test. Anal.

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2012, 4 (Suppl 1), 103−111.



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Figure Captions: Table 1. Protein, fat, and carbohydrate content in raw milk and in various milk-based powders studied. Table 2. Scheduled SRM parameters, LODs, LOQs, and linear dynamic ranges for veterinary drugs studied. Table 3. Recovery results for the various milk-based powders studied. Fig. 1. Chromatogram of the primary transition overlay of the 52 veterinary drugs of interest. Fig. 2. Percent recoveries for 52 veterinary drugs at the highest fortification level and extracted from non-fat milk powder using three different % ACN solutions. Error bars represent the standard deviation in results from three samples. Fig. 3. Percent recoveries for 52 veterinary drugs at the lowest fortification level in non-fat milk powder. Error bars represent the standard deviation in results from three samples.


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Table 1. Protein, fat, and carbohydrate content in raw milk and in various milk-based powders studied. Commodity Raw Milk Non-Fat Milk Powder Whole Milk Powder Milk Protein Concentrate Whey Protein Concentrate Whey Protein Isolate

Protein (%) 3.8 35 26 80 80 90

Fat (%) 3.8 0 22 0 6.7 0

Carbohydrate (%) 5.0 52 52 6.7 3.3 3.3

Note: Percentages are estimated values based on the sample nutrition labels.



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Table 2. Scheduled SRM parameters, LODs, LOQs, and linear dynamic ranges for veterinary drugs studied. RT Precursor Product 1 Product 2 Compound Class (min) (m/z) (m/z) (m/z) ● Compounds that met the validation criteria for non-fat milk powder Metronidazole Nitroimidazole 2.36 172.1 128.1 82.1 Lincomycin Lincosamide 2.74 407.2 126.1 359.2 Ronidazole Nitroimidazole 2.80 201.1 140.1 55.1 Ternidazole Nitroimidazole 2.89 186.1 128.0 82.0 Tildipirosin Macrolide 3.14 368.0 98.1 174.1 Levamisole Anthelmintic 3.39 205.0 178.1 91.0 Ornidazole Nitroimidazole 3.57 220.0 128.0 82.0 Florfenicol Amphenicol 3.59 356.1 185.0 119.0 Thiabendazole Benzimidazole 3.62 202.1 175.1 131.1 Tulathromycin Macrolide 3.62 403.8 577.4 72.0 Clorsulon 3.65 379.9 343.8 341.8 Acetylsalicylic acid NSAID 3.76 137.0 93.0 137.0 Chloramphenicol Amphenicol 3.77 321.1 152.0 194.0 Danofloxacin Quinolone 3.85 358.2 340.2 314.0 Sulfachlorpyridazine Sulfonamide 3.96 285.0 156.1 92.1 Sulfamethazine Sulfonamide 3.96 279.2 156.0 92.1 Enrofloxacin Quinolone 4.06 360.3 342.2 316.1 Sulfadimethoxine Sulfonamide 4.48 311.1 156.2 92.1 Sulfaethoxypyridazine Sulfonamide 4.53 295.0 155.9 140.0 Sulfaquinoxaline Sulfonamide 4.63 301.1 156.1 92.1 Gamithromycin Macrolide 4.66 389.0 619.4 619.4 Penicillin G β-lactam 4.67 335.0 160.0 176.1 Tripelennamine 4.84 256.1 210.8 119.0 Oxfendazole Benzimidazole 4.95 316.0 159.0 191.1 5-Hydroxy flunixin NSAID 5.01 313.0 295.2 280.0 Tilmicosin Macrolide 5.02 435.5 695.7 174.1 Penicillin V β-lactam 5.05 349.0 208.0 305.0 Erythromycin Macrolide 5.07 734.6 158.2 83.2 Sulfabromomethazine Sulfonamide 5.30 357.0 264.0 156.0 Flunixin NSAID 5.34 295.1 208.9 191.0 Albendazole Benzimidazole 5.38 266.1 234.1 191.1 Cloxacillin β-lactam 5.40 436.0 277.0 160.0 Naproxen NSAID 5.61 229.0 185.1 169.9 Ketoprofen NSAID 5.63 255.1 209.2 105.1 Meloxicam NSAID 5.73 352.0 115.0 141.0 Novobiocin Aminocoumarin 6.29 613.2 189.2 396.4 Eprinomectin Avermectin 7.09 914.5 186.1 112.1 Ivermectin Avermectin 7.09 897.5 185.9 153.9 Doramectin Avermectin 7.28 899.5 593.3 219.2 Moxidectin Avermectin 7.32 640.4 528.3 498.3 ● Compounds that did not meet the validation criteria for non-fat milk powder Amoxicillin β-lactam 1.59 366.1 349.1 114.1 Desacetyl cephapirin β-lactam 2.22 382.0 152.0 112.0 Ampicillin β-lactam 2.95 350.1 106.0 160.0 Cephapirin β-lactam 3.13 424.1 292.0 363.9 Dimetridazole Nitroimidazole 3.25 142.0 95.9 81.0 Oxytetracycline Tetracycline 3.55 461.2 426.1 201.1 Tetracycline Tetracycline 3.80 445.2 410.1 154.1 Pirlimycin Lincosamide 4.29 411.1 112.0 363.0 Ipronidazole Nitroimidazole 4.49 170.0 124.1 109.0 Ceftiofur β-lactam 4.94 524.0 241.2 210.2 Tylosin Macrolide 5.50 916.6 174.0 772.4 Phenylbutazone NSAID 6.10 309.2 160.2 104.1

DP CE 1 CE 2 LODa LOQa Linearityb (V) (V) (V) (ng/g) (ng/g) (ng/mL) 68 92 34 46 90 74 39 -73 97 84 -90 -20 -81 100 66 97 105 94 56 97 46 28 40 105 90 80 -20 80 75 -104 90 27 -90 86 56 65 72 65 156 40

21 32 18 19 23 29 21 -25 37 19 -16 -24 -23 30 23 26 31 26 23 25 15 13 17 41 30 22 -13 35 24 -42 30 17 -10 22 21 51 20 21 18 12

37 25 38 35 27 45 39 -43 47 61 -16 -9 -17 24 41 41 27 50 25 46 45 15 43 29 46 33 -10 104 27 -44 48 16 -20 35 25 25 100 23 23 15

0.14 0.11 0.50 0.044 0.50 0.040 0.16 2.4 0.066 0.58 4.8 7.4 5.0 0.30 0.036 0.010 0.22 0.018 0.008 0.040 0.086 0.018 0.10 0.078 13 0.46 2.0 0.46 0.080 3.2 0.040 0.11 24 0.24 0.030 0.32 0.082 28 26 8.4

0.42 0.32 1.48 0.14 1.52 0.12 0.50 7.4 0.20 1.7 14 22 15 0.88 0.12 0.020 0.66 0.060 0.020 0.12 0.26 0.060 0.30 0.24 38 1.36 6.0 1.36 0.24 9.4 0.1 0.32 70 0.72 0.1 0.96 0.24 82 78 26

.04-20 .04-20 .02-20 .02-20 5-100 .02-20 .02-10 .25-50 .5-100 1-100 .5-100 1-500 1-500 .5-100 .02-20 .02-20 .5-50 .02-20 .02-20 .02-20 1-100 .02-20 .1-20 .02-20 .6-300 1-100 1.0-50 5-100 .1-50 .5-500 0.04-20 .1-100 5-500 .1-50 .02-20 1-500 .1-20 .5-500 .5-250 .4-40

36 70 59 66 42 75 82 111 105 80 100 100

12 31 21 19 23 39 27 31 23 27 47 31

24 30 17 16 33 54 40 21 31 32 41 50

40* 4.0 19 2.2 1.02 28 9.2 0.46 0.68 0.78 2.4 2.2

120* 12 58 6.6 3.0 84 28 1.34 2.0 2.4 7.4 6.4

25-500 1-100 5-500 0.5-50 .5-500 12-600 5-500 8-800 .1-100 2-200 .1-500 5-500

Note: RT = retention time, DP = declustering potential, CE 1 = collision energy for product 1, CE 2 = collision energy for product 2. aCalculated using method detection limits. bEstimated using solvent-based standards. *Estimated using S/N ratio. Product 1 was used for quantitation, and Product 2 was used for confirmation.


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Table 3. Recovery results for various milk-based powders studied. Commodity 3 non-fat 1 whole fat 1 WPC 1 WPI 1 MPC

Acceptable Recovery Avg. Recovery RSDr (Out of 52) (%) (%) 40 83 6.1 43 80 8.8 39 74 7.3 36 87 6.0 39 78 6.6



Fig. 1. Chromatogram of the primary transition overlay of the 52 veterinary drugs of interest.


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70% ACN

140

90% ACN

100% ACN

120 % Recovery

100 80 60 40 20 0 3.0 4.5 6.0 7.5 1.5 3.0 4.5 6.0 3.0 4.5 6.0 7.5 1.5 Retention Time (min) Retention Time (min) Retention Time (min) Fig. 2. Percent recoveries for 52 veterinary drugs at the highest fortification level and extracted from non-fat milk powder using three different % ACN solutions. Error bars represent the standard deviation in results from three samples. 1.5


7.5


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140

% Recovery

120 100 80 60 40 20 1.5

3.0 4.5 6.0 Retention Time (min)

7.5

Fig. 3. Percent recoveries for 52 veterinary drugs at the lowest fortification level in non-fat milk powder. Error bars represent the standard deviation in results from three samples.


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TOC Graphic:

1. 1 g sample 2. 10 mL of 90% ACN 3. Shake 30 min 4. Concentrate/reconstitute Milk-based powder LC-MS/MS analysis of 52 veterinary drugs


Targeted Multiresidue Analysis of Veterinary Drugs in Milk-Based

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