Determination of Anthelmintic Drug Residues in Milk Using Ultra High Performance Liquid Chromatography–Tandem Mass Spectrometry

February 11, 2011

The Application Notebook

The Application Notebook, The Application Notebook-02-11-2011, Volume 0, Issue 0

A modified QuEChERS-type extraction method is used with an additional concentration step to detect 38 anthelmintic (nematicides, flukicides, endectocides) drug residues, consisting of benzimidazoles, avermectins, and flukicides in milk. UHPLC–MS-MS instrumentation detects most analytes at <1 µg/kg.

Don Shelly and Craig A. Perman, UCT, LLC

A modified QuEChERS-type extraction method is used with an additional concentration step to detect 38 anthelmintic (nematicides, flukicides, endectocides) drug residues, consisting of benzimidazoles, avermectins, and flukicides in milk. UHPLC–MS-MS instrumentation detects most analytes at <1 µg/kg.

Materials and Instruments

1) Sample Preparation

a) Weigh 10.0 g milk into a 50 mL centrifuge tube

b) Add IS and allow to sit for 15 min

c) Add 12 mL acetonitrile (MeCN) and the contents of ECMSSC50CT-MP (4000 mg anhydrous MgSO4, 1000 mg NaCl) pouch

d) Shake vigorously then centrifuge for 12 min @ 3500 rpm

2) Dispersive Sample Cleanup

a) Add the supernatant to ECMSC1850CT (1500 mg anhydrous MgSO4 and 500 mg C18) in a 50 mL centrifuge tube

b) Vortex sample for 30 s

c) Centrifuge for 10 min @ 2500 rpm (1449g)

d) Transfer 6 mL of supernatant to an evaporation tube

e) Add 0.25 mL DMSO and vortex for 1 min

f) Evaporate the MeCN @ 50 °C using a Turbovap® or similar apparatus to 0.25 mL

g) Filter extract using 0.2 µm PTFE syringe filter

h) Sample is ready for UHPLC analysis

3) Analysis by UPLC–MS-MS

  • Waters Acquity UPLC system (Milford, Massachusetts) or equivalent

  • Analytical column SS HSS T3 (or equivalent) (100 × 2.1 mm, particle size 1.8 µm) with in-line filter unit and 0.2 µm stainless steel filter

  • Maintain column at 60 °C

  • Pump flow rate of 0.6 mL/min

  • Binary gradient:

1) mobile phase A 0.01% HOAc in water: MeCN (90:10, v/v)

2) mobile phase B 5 mM ammonium formate in MeOH: MeCN (75:25 v/v)

Injection volume 5 µL

  • Waters Quattro Premier XE triple quadrupole mass spectrometer

  • 200 electrospray ionization (ESI) interface

  • System control 201 by MassLynx™ software and data was processed using TargetLynx™ Software (Waters)

  • Parent ion masses are calculated from the elementary compositions

Table 1

Information on standards available in full article summary on the UCT website.

*Used with permission from Whelen, M., Kinsella, B., "Determination Of Anthelmintic Drug Residues In Milk Using Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry With Rapid Polarity Switching", doi:10.1016/j.chroma.2010.05.007, CHROMA 351049, J. of Chromatography A.

Listing of instrument manufacturers and standards suppliers does not constitute endorsement by UCT. Equivalent systems may be used.

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