S-Benzylmercapturic acid and S-phenylmercapturic acid in urine: METHOD 8326, Issue 1, dated 20 May 2014 - Page 4 of 10
16. Add 0.1 mL of the chromatographic matrix adjustment solution to each standard vial. NOTE: These standard samples are equivalent to 0.5 to 50 ng/mL BMA/PMA urine samples [based on the original 4.0 mL urine volume extracted and placed in each autosampler vial.] 17. Prepare one blank urine sample without analyte spikes; alternatively, prepare one blank using a urine substitute by following steps 14-16 using 0.5 mL of the unspiked urine or substitute in step 14. 18. Prepare at least one quality control (QC) standard of urine or urine substitute fortified with PMA/ BMA using separately prepared stock solutions. A 10 ng/mL equivalent spike level is suggested and more than one level can be used if desired. MEASUREMENT: 19. Set the high-performance liquid chromatograph according to the manufacturer’s recommendations and to the conditions listed on page 8326-1. A needle rinse with 50/50% (v/v) acetonitrile/water is required to eliminate sample carry-over by the autosampler. 20. Set the mass spectrometric detector to multiple reaction mode (MRM) according to the manufacturer’s recommendations and the conditions listed on page 8326-1. Example conditions are summarized below: Table 1. MS/MS CONDITIONS USING NEGATIVE ELECTROSPRAY IONIZATION Dwell Collision Analyte Precursor MS1 Product MS2 Fragmentor Time Energy Ion Resolution Ion Resolution Voltage (msec) (volt) 257 unit 128 unit 200 80 8 d5-BMA BMA 252 unit 123 unit 200 80 8 d5-PMA 243 unit 114 unit 200 80 8 PMA 238 unit 109 unit 200 80 8 21. Inject 8 μL of each sample extract, standard, QC standard, and blank. Sample chromatograms for each compound are illustrated in Figure 1. 22. Measure the peak areas of the two analytes (BMA and PMA) and those for the deuterated internal standards (d5-BMA and d5-PMA) in the chromatograms. Divide the peak area of the analytes by the peak area from the matching deuterated internal standard. 23. Prepare calibration curves of the peak Area Std./Area Internal Std. (ratio calculated in step 22) versus the urine equivalent concentration of the standards for the two analytes. CALCULATIONS: 24. Determine the concentration of the two analytes in the extracts from the original urine (4.0 mL specimen) from the curves obtained in step 23. The results can be expressed as ng/mL of each analyte in urine. EVALUATION OF METHOD: This method was evaluated and described in detail by B’Hymer [9,10]. A general summary of this published information is given below: Accuracy and Precision. Three recovery studies using multiple columns over several days demonstrated the accuracy and precision of this test method. The first recovery study was performed using fortified urine samples over three separate experimental batch runs, and these data are presented in Table 2. Average recoveries were between 102 and 106% for the two analytes over the four spiked
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition
