• Application Note

Optimized Extraction and Cleanup Protocols for LC-MS/MS Multi-Residue Determination of Veterinary Drugs in Edible Muscle Tissues

Optimized Extraction and Cleanup Protocols for LC-MS/MS Multi-Residue Determination of Veterinary Drugs in Edible Muscle Tissues

  • Michael S. Young
  • Kim Van Tran
  • Waters Corporation

Abstract

In order to ensure public health and safety, a reliable screening analysis is necessary to determine veterinary drug residue levels in meat and other edible tissue samples. The compounds of interest range from highly polar water-soluble compounds to very non-polar, fat-soluble compounds. There exist very effective extraction and cleanup procedures for individual compounds or compound classes, but these methods are not well suited for a multi-class, multi-residue screening analysis.

  • Solvent extraction (with excess acetonitrile or methanol) can be effective for many veterinary drug residues in milk, but highly water soluble drugs such as salbutamol are not well recovered using this approach.
  • Aqueous buffer extraction can also be effective for many compounds, but fat-soluble compounds, such as dexamethasone, are not well recovered using this approach.
  • Traditional solid-phase extraction (SPE) enrichment and cleanup (retention/ wash/elution) has limited utility for multi-residue analysis. Because the range of acidity/polarity/solubility among the compounds is so broad, dispersive, or pass-through SPE is preferred for multi-residue methods.

Benefits

  • Efficient, timesaving multi-class/multi-residue methodology
  • Straightforward sample preparation for diverse range of analytes
  • Fast, sensitive UPLC-MS analysis

Introduction

Optimized sample preparation and analysis protocols were developed for tandem LC/MS determination of a wide variety of veterinary drug residues in tissue samples. Three types of muscle tissue samples (pork, chicken, and salmon) were chosen to demonstrate the suitability of the methodology. Samples are treated with an acidified acetonitrile/water solvent to precipitate proteins and to extract the veterinary drugs of interest. Then, a simple SPE cleanup is performed using a Sep-Pak C18 cartridge or 96-well plate. After evaporation and reconstitution, the sample is analyzed using tandem LC-MS. Representative compounds were chosen from major classes of veterinary drugs including tetracyclines, fluoroquinolones, sulfonamides, macrolides, beta-lactams, NSAIDS, steroids, and beta-andrenergids.

Experimental

LC Conditions

System:

ACQUITY UPLC system

Column:

ACQUITY UPLC CSH C18,

1.7μm, 100 mm x 2.1 mm

(i.d.)

Mobile Phase:

A: 0.1% formic in water

B: 0.1% formic acid

in acetonitrile

Injection Volume:

7 μL

Injection Mode:

Partial loop injection

Column Temp.:

30 °C

Weak Wash:

10:90 acetonitrile:water

(600 μL)

Strong Wash:

50:30:40

water:acetonitrile:IPA

(200 μL)

Seal Wash:

10:90 acetonitrile:water

Gradient

MS Conditions

Detector:

Waters Xevo TQ

Ionization:

Positive Electrospray (except

negative for chloramphenicol)

Source Temp.:

150°C

Desolvation Temp.:

500°C

Desolvation Gas Flow:

1,000 L/hr

Cone Gas Flow:

30 L/hr

Collision Gas Flow:

0.15 mL/min

Data Management:

MassLynx v4.1

Sample Preparation

1. Initial Extraction/Precipitation

Place a 5 g sample of homogenized tissue into a 50 mL centrifuge tube. Add 10 mL 0.2% formic acid in 80:20 acetonitrile/water. Vortex for 30 seconds and place on mechanical shaker for 30 minutes. Centrifuge at 12000 rpm for 5 minutes.

The extraction/precipitation step gives good recovery of most compounds of interest but also extracts significant amounts of fat.

2. SPE Cleanup

Take 1 mL of the supernatant (from step 1) for SPE cleanup using a Sep-Pak C18 cartridge or plate (see SPE details in Figure 1).

This step removes fats and non-polar interferences.

Figure 1. SPE Cleanup Protocol

Table 1 summarizes the MRM transitions and instrument parameters used for this study. Also presented in Table 1 are typical matrix matched calibration data for each compound (calculated using the primary transition in pork matrix).

Table 1. MRM Transitions and Calibration Data Obtained in Chicken Matrix (Other Matrixes Similar).

Results and Discussion

Figure 2 shows a typical LC-MS chromatogram obtained from analysis of a matrix matched standard of erythromycin at 10 ng/g. Performance of the other compounds was similar. Table 2 shows the recovery data obtained from replicate analysis of spiked tissue samples. Table 3 shows the observed matrix effects for the multi-residue tissue analysis.

Figure 2. Typical LC-MS/MS Chromatogram Obtained from Pork Spiked with Erythromycin at 10 ng/g (Primary MRM Transition on Top).

The procedure utilized in this study was developed from methods presented by Lehotay et.al.1 and Martos et. al.2. The method used in reference 1 uses no acid in the tissue extraction solvent; under these conditions we observed recovery of tetracyclines below 5% and the RSD for recovery of fluoroquinolones was greater than 50%. The method used in reference 2 prescribes the acidification of the extract to 1% formic acid prior to centrifugation; under these conditions penicillin recovery was under 10% compared with 48% using our approach. Our extraction procedure is a compromise of the methods presented in reference 1 and 2. A similar acetonitrile/water based extraction is used but is acidified only to 0.2% with formic acid; more balanced recovery and minimized degradation of labile compounds was achieved.

Another approach was considered based on the method of Kauffman et. al.3 by which two separate extractions were performed. The first extraction, for the water soluble compounds, was accomplished using aqueous succinic buffer. The second, performed on the re-suspended pellet, was with acetonitrile. This approach requires that each fraction be worked up independently before ultimately combining fractions for a single injection. Performance was only marginally better than the chosen procedure but at a much greater cost of time and materials. The extraction, cleanup, and analysis protocols chosen for this study provide a good balance of preparative time, cost and method performance.

Table 2. Recovery data obtained from three types of spiked tissue samples
Table 3. Matrix effects (% ion-suppression) observed for three types of spiked tissue samples (negative value indicates ion enhancement).

Conclusion

  • A simple extraction/protein precipitation procedure was developed and demonstrated for analysis of meat, chicken and salmon muscle tissue.
  • The procedure was suitable for screening for a wide range of veterinary drug residues.
  • Recoveries averaged 60% and were similar for all tissues.
  • A pass-through SPE cleanup protocol using Sep-Pak C18 was utilized for effective removal of residual fats.
  • The sample preparation methodology produced an extract that was free of particulates and required no subsequent filtration prior to LC/MS analysis.

References

  1. S. Lehotay, “High-Throughput Screening Analysis by UHPLC-MS/MS of >60 Veterinary Drugs in Animal Tissues”, 125th AOAC Annual Meeting, Presentation 2303, 21 September, 2011.
  2. P. A. Martos, F. Jayasundara, J. Dolbeer, W. Jin, L. Spilsbury, M. Mitchell, C. Varilla, B. Shurmer, “Multiclass, Multiresidue Drug Analysis, Including Aminoglycosides, in Animal Tissue Using Liquid Chromatography Coupled to Tandem Mass Spectrometry”, J. Agric. Food Chem. 58, (2010), 5932–5944.
  3. A. Kaufmann, P. Butcher, K. Maden, M. Widmer, “Quantitative Multiresidue Method for About 100 Veterinary Drugs in Different Meat Matrices by Sub 2-Micron Particulate High-Performance Liquid Chromatography Coupled to Time of Flight Mass Spectrometry”, J. Chromatogr. A. 1194 (2008), 66–79.

720004144, November 2011

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