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An alternative calibration procedure for use when performing carbon isotope ratio measurements by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) has been developed. This calibration procedure does not rely on the corrections in-built in the instrument software, as the carbon isotope ratios of a sample are calculated from the measured raw peak areas. The method was developed for the certification of a urine reference material for sports drug testing, as the estimation of measurement uncertainty is greatly simplified. To ensure that the method is free from bias arising from the choice of calibration material and instrument, the carbon isotope ratios of steroids in urine extracts were measured using two different instruments in different laboratories, and three different reference materials (CU/USADA steroid standards from Brenna Laboratory, Cornell University; NIST RM8539 mineral oil; methane calibrated against NIST RM8560 natural gas). The measurements were performed at LGC and the Australian National Measurement Institute (NMI). It was found that there was no significant difference in measurement results when different instruments and reference materials were used to measure the carbon isotope ratio of the major testosterone metabolites androsterone and etiocholanolone, or the endogenous reference compounds pregnanediol, 11- ketoetiocholanolone and 11β-hydroxyandrosterone. Expanded measurement uncertainties at the 95% coverage probability ranged from 0.21‰ to 1.4‰, depending on analyte, instrument and reference material. The measurement results of this comparison were used to estimate a measurement uncertainty of δ(13)C for the certification of the urine reference material being performed on a single instrument using a single reference material at NMI.

An electrospray ionization–liquid chromatography–tandem mass spectrometry (ESI/LC/MS/MS) method was developed for the simultaneous determination of the β-agonists clenbuterol, salbutamol, and cimaterol in bovine retina. The tissue was homogenized in alkaline buffer and spiked to give 10, 15, and 20 ng/g each of the 3 analytes together with the internal standards d6-salbutamol and d6-clenbuterol. The mixture was incubated with protease enzyme to release any protein-bound analytes and then made alkaline before extraction with isobutanol. The extract was dissolved in water and transferred to a clenbuterol immunoaffinity column. After washing, the analytes were eluted and analyzed by ESI/LC/MS/MS using a C18 column with acetic acid–methanol as mobile phase. No interferences were observed from the spiked retina extract at the various single-reaction monitoring modes. Average recoveries for clenbuterol, salbutamol, and cimaterol were 94, 85, and 87% with coefficients of variation (CVs) of 9.4, 9.9, and 8.6%, respectively. A correlation coefficient of r2 = 0.9999 was obtained for all analytes. The limits of detection for clenbuterol, salbutamol, and cimaterol, determined from 3 times the standard deviation of 7 replicates of the lowest spike, were 2.5, 3.5, and 2.0 ng/g with CVs of 8.9, 11.6, and 7.2%, respectively.