Page:NIOSH Manual of Analytical Methods - 8326.pdf/3

 S-Benzylmercapturic acid and S-phenylmercapturic acid in urine: METHOD 8326, Issue 1, dated 20 May 2014 - Page 3 of 10

SAMPLING: 1.	Collect at least 8 mL of urine in an appropriate polypropylene tube or bottle and cap. Refrigerate at 4 °C or freeze after collection. Collect at least two urine specimens for each worker, one before the work shift and one after. 2.	Ship the specimen stored in either wet or dry ice in an insulated container. Freeze specimens upon arrival at the laboratory and store frozen. A reminder: commercial shippers have special labeling requirements for packages containing dry ice. SAMPLE PREPARATION: NOTE: BMA and PMA are somewhat light sensitive (see Evaluation of Method section). Perform the sample preparation steps in a low light environment. Extreme measures are not required. 3.	Thaw the urine specimen to room temperature. 4.	Mix urine specimen thoroughly to ensure homogeneity. 5.	Transfer 4.0 mL of urine into a 16 X 150 mm (or larger) screw-capped culture tube. 6.	Add 0.5 mL of deionized water to aid in the dissolution of solids. 7.	Add 0.5 mL of the 30 ng/mL deuterated BMA/PMA internal standard solution. 8.	Solid-Phase Extraction NOTE: All SPE steps are performed using the vacuum manifold apparatus. The flow rate should not exceed 1 mL/min. The SPE cartridges should not be allowed to go to dryness until the end of Step 8d. Refer to manufacturers’ recommendations for use of specific SPE cartridges. a.	 Pre-wash the C18 SPE cartridge with 2 mL of acetone. b.	 Equilibrate the SPE cartridge with 2 mL of HPLC grade water. c.	 Load the 5-mL urine mixture and draw the sample through the cartridge. d.	 Wash the cartridge with 1 mL of HPLC grade water. Discard any liquid collected up to this point. e.	 Apply (or increase) the vacuum to pull most of the water from the cartridge. f.	 Elute the analytes with 3 mL of acetone three times, collecting all of the acetone washes into a 15-mL plastic screw-capped tube. 9.	Evaporate the 9 mL of acetone from the extracts to dryness by means of a vacuum rotary concentrator or by using a nitrogen sweep. 10.	Cap the plastic tubes containing the dry extract and store in a refrigerator/freezer until ready for chromatographic analysis. 11.	Prior to chromatographic analysis, dissolve the extract in 1 mL of HPLC mobile phase A and transfer the sample into an amber HPLC autosampler vial. CALIBRATION AND QUALITY CONTROL: 12.	Prepare a BMA/PMA standard mixture by combining 2.0 mL of each stock solution and diluting to 100 mL with deionized water to make an approximately 4 µg/mL solution concentration for each. 13.	BMA/PMA standard solutions: The 4 µg/mL BMA/PMA solution is diluted in water to make 4, 8, 16, 40, 80, 320 and 400 ng/mL BMA/PMA solutions. 14.	Accurately transfer 0.500 mL of each BMA/PMA solution from step 13 into a separate HPLC autosampler vial. [This delivers 2, 4, 8, 20, 40, 160 and 200 ng of each analyte to each autosampler vial.] 15.	Add 0.5 mL of the deuterated internal standard spiking solution [30 ng/mL] to each vial. [This delivers 15 ng of the internal standards to each autosampler vial.]

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition