• Application Note

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

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

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

Abstract

In order to insure public health and safety, a reliable screening analysis is necessary to determine veterinary drug residue levels in milk samples. The compounds of interest range from highly polar water-soluble compounds to very non-polar fat-soluble compounds. There exists 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 fatsoluble 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/MS determination of a wide variety of veterinary drug residues in milk samples. Samples are initially precipitated and extracted with an equal volume of acetonitrile. After the resulting extract is treated with acidified acetonitrile to precipitate remaining proteins, a simple SPE cleanup is performed using a Sep-Pak C18 cartridge. 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

LC system:

ACQUITY UPLC system

Column:

ACQUITY UPLC CSH C18, 1.7 μm, 100 mm x 2.1 mm (i.d.)

Mobile phase A:

0.1% formic in water

Mobile phase B:

0.1% formic acid acetonitrile

Injection volume:

7 μL

Injection mode:

Partial loop injection

Column temperature:

30 °C

Weak needle wash:

10:90 acetonitrile:water (600 μL)

Strong needle wash:

50:30:20 water:acetonitrile:IPA (200 μL)

Seal wash:

10:90 acetonitrile:water

Gradient:

Time(min)

Flow(mL/min)

%A

%B

Curve

Initial

0.4

85

15

6

2.5

0.4

60

40

6

3.9

0.4

5

95

6

4.9

0.4

5

95

6

5.0

0.4

85

15

6

7.0

0.4

85

15

6

MS conditions

Mass spectrometer:

Waters ACQUITY TQD

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

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

Table 1. MRM transitions and calibration data.

Sample Preparation Protocol

Figure 1. SPE cleanup protocol.

Results and Discussion

Figure 2 shows a typical LC-MS chromatogram obtained from analysis of a matrix matched standard of erythromycin at 6.7 ng/g. Performance of the other compounds was similar. Table 2 shows the recovery and matrix effects observed for multiresidue milk analysis.

Figure 2. Typical LC-MS/MS Chromatogram obtained from milk spiked with erythromycin at 6.7 ng/g (primary MRM transition on top).

 Table 2. Recovery and matrix effects.

* Negative number signifies matrix enhancement

The procedure chosen for the milk analysis was to initially extract and precipitate a milk sample with an equivalent amount of acetonitrile and then to precipitate the remaining protein from the supernatant with acidified acetonitrile. A single step procedure was also considered by which the milk sample was directly precipitated with 0.2% formic acid in 80% acetontrile. This procedure was more straightforward and produced a final extract of similar cleanliness compared with the chosen protocol. However, there was significantly lower recovery for the most polar compounds, such as sulfanilamide, and virtually no recovery of chlorotetracycline. Another approach was considered, 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 marginally better than the chosen procedure but at a much greater cost of time and materials. Although the chosen procedure requires two precipitation steps, it provided the best balance of preparative time with good method performance.

Conclusion

  • A two step extraction/protein precipitation procedure was developed and demonstrated for milk analysis

The procedure was suitable for screening for a wide  range of veterinary drug residues

Recoveries averaged 67% (22-110) with the lowest  values for tetracyclines.

  • A pass-thru SPE cleanup protocol using Sep-Pak C18 was utilized for effective removal of residual fats. 
  • The sample preparation methodology for milk produced an extract for LC/MS that was free of particulates and required no subsequent filtration prior to LC-MS analysis.

720004089, August 2011

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