Buparvaquone assay method development and validation

Summary

Theileria orientalis is a blood-borne protozoon which infects cattle and has been known to occur in Australia (mainly in Queensland) for more than a century. The infection was known as Benign Theileriosis and seldom caused clinical signs in infected animals. For approximately the past decade, cattle on the eastern seaboard of Australia have become ill, showing signs of anaemia, icterus, depression, weakness, abortion and death. They have been diagnosed as being infected with T orientalis, of which there is now known to be at least three subtypes in Australia, one of which is consistently found in clinical cases. This disease has been given the name Bovine Anaemia caused by T orientalis and has since been found to occur in cattle in Victoria and Western Australia.

There is no remedy registered for treating this disease in Australia. Buparvaquone (BPQ) has been registered elsewhere in the world and was found to be effective against induced infections with two isolates in splenectomised calves. Veterinarians can apply to the Australian Pesticides and Veterinary Medicines Authority (APVMA) for Consent to Import the remedy for treating patients under their care, but they will then bear the responsibility for any residues found in edible tissues from such animals. There is currently no information on which veterinarians can base a recommendation of a suitable with-holding period (WHP) to avoid violative residues in BPQ treated cattle slaughtered for human consumption. Prior to conducting a tissue residue depletion study on which to base WHP recommendations, an analytical method for BPQ residues in edible tissue had to be developed, because the method used previously was not readily available and was deemed to have used obsolete technology.

The principle of the method is that BPQ residues are extracted from bovine tissues with acetonitrile/acetone (80:20). An aliquot of the extract is removed and cleaned up by solid phase extraction (SPE) and BPQ residues determined by reverse-phase high performance liquid chromatography (HPLC), coupled with tandem mass spectrometry (MS) (HPLC/MS/MS) (multiple reaction monitoring – MRM). BPQ residues are extracted from milk by vortexing the sample with acetonitrile/acetone.NaCl, MgSO4 and other selected salts are added to the extract to create a partition between the acetonitrile/acetone and water. An aliquot of the acetonitrile/acetone layer is taken and diluted and BPQ residues determined by HPLC/MS/MS (MRM).

A series of matrix matched external reference standard solutions of BPQ is prepared: a stock standard solution containing 1,000 mg/L in acetone, three intermediate standards containing 0.1, 1 and 10 mg/L in acetone, and quantitation standards in untreated control matrices of muscle, fat, liver, kidney (spiked to concentrations ranging from 0.2 to 0.0025 mg/kg) and milk (0.1 to 0.002 mg/kg) for producing calibration curves.

Sample preparation consists of weighing 5g of the tissue into a 250mL flask, (adding reference standards to control matrices for spiking of quantitation standards,) adding 50mL 80:20 acetonitrile/acetone, homogenising, mixing and centrifuging prior to SPE clean-up.

LC/MS/MS analysis was done on an ACQUITY UPLC System (Waters) with a Xevo TQ-S MassLynx 4.1 XP Workstation in a BEH C18 1.7 μm 50 x 2.1 mm (Waters) column. Quantification and identification were made by comparison with matrix matched external standards using comparative peak areas. A linear regression equation was generated for analyte calibration standards with 1/x weighting using the concentration of the analyte (X-axis) versus the analyte peak area (Y-axis), excluding the origin. Concentrations of analyte in the final extracts were determined by substituting the peak area responses into the linear regression equation.

Quality assurance for this method consists of recoveries (untreated control samples fortified with the BPQ). Recoveries are run concurrently with every batch of test samples. Average recoveries must be between 70% and 110% with relative standard deviations (RSD) < 20% acceptable. The method of analysis was validated using the following parameters: linearity, system precision, accuracy and precision.

It is foreseen that this method can be used for determining BPQ residues in the tissues of treated cattle, e.g. for determining the tissue residue depletion pattern for the setting of with-holding periods (WHP) for treated cattle destined for slaughter for human consumption. This will benefit veterinarians wishing to treat cattle infected with T orientalis and recommending an appropriate WHP, as well as the owners of those cattle, who may otherwise suffer the loss of untreated cattle, or of having to with-hold treated cattle from entry into the human food chain.