Analytical determination of virginiamycin drug residues in edible porcine tissues by LC-MS with confirmation by LC-MS/MS.

A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the determination and confirmation of virginiamycin (VMY) M1 residues in porcine liver, kidney, and muscle tissues at concentrations > or =2 ng/g. Porcine liver, kidney, or muscle tissue is homogenized with methanol-acetonitrile. After centrifugation, the supernatant is diluted with phosphate buffer and cleaned up on a C18 solid-phase extraction cartridge. VMY in the eluate is partitioned into chloroform and the aqueous upper layer is removed by aspiration. After evaporating the chloroform in the residual mixture to dryness, the dried extract is reconstituted in mobile phase and VMY is quantified by LC-MS. Any samples eliciting quantifiable levels of VMY M1 (i.e., at concentrations > or =2 ng/g) are subjected to confirmatory analysis by LC-MSIMS. VMY S1, a minor component of the VMY complex, is monitored but not quantified or confirmed.

Alternative methodology for the analysis of progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle.

Research has shown that traditional solvent extraction procedures used for the analysis of endogenous steroids often give inconsistent recoveries and results. However, a single-laboratory validation of a liquid chromatography/tandem mass specrometry method using 2 product ions per transition for progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle showed accuracy and precision to within 23% at concentrations ranging from 0.5 to 2.0 microg/kg. Homogenized samples were pretreated with methanol to denature endogenous enzymes. Following removal of methanol, samples were treated overnight with Helix pomatia beta-glucuronidase to deconjugate glucuronide conjugates. Alkali digestion of the samples in KOH solutions was done under shaking at 37 degrees C for 30 min. The digestate was extracted with methyl tert-butyl ether, and the extracts were cleaned by partitioning between acetonitrile-hexane, followed by solid-phase extraction cleanup on silica cartridges. In bovine liver, average recoveries exceeded 54% for all analytes, and the within-run assay coefficients of variations were < 6 and 13% for high (2.0 microg/kg) and low (0.3 microg/kg) analyte concentrations, respectively. In veal muscle, average recoveries exceeded 60%, and the analysis of blind spikes gave accuracy estimates of over 85%, with coefficients of variation (CVs) < 15% for all analytes. The CVs for the multiple reaction monitoring ion ratios for all compounds were < 22% for all validation data. The method meets the requirements for confirmatory methods as outlined in 2002/657/EC. An analyst is capable of processing up to 20 samples within 5 days.

A determinative and confirmatory method for residues of the metabolites of carbadox and olaquindox in porcine tissues.

Carbadox (CBX) and olaquindox (OLQ) are used in swine feed for growth promotion, to improve feed efficiency, increase the rate of weight gain, control swine dysentery and bacterial enteritis in young swine. In 1991, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) recommended maximum residue limits (MRLs) of 30 and 5mugkg(-1) in liver and muscle tissues of pigs, respectively, based on the concentration of, and expressed as, quinoxaline-2-carboxylic acid (QCA) as marker residue. In 1998, the European Commission (EC) banned the use of CBX and OLQ in food animal production together with four other feed additives, following reports that CBX and desoxycarbadox (DCBX) are suspect carcinogens and mutagens. In 2001, the sale of CBX was halted in Canada. In 2003, JECFA recommended the withdrawal of the previously recommended acceptable daily intake (ADI) and MRLs and concluded that QCA was not a suitable marker residue for CBX, based on new sponsor studies reporting that DCBX, the suspect carcinogen, persisted in animal tissues much longer than had previously been thought. This paper presents a very sensitive LC-MS/MS method that was developed by CFIA scientists for the simultaneous determination and confirmation of DCBX residues at concentrations >/=0.050 ngkg(-1) and QCA and mQCA residues at concentrations >/=0.50 ngkg(-1)in bovine muscle, pork liver and muscle tissues.