BUT OX YAC ETIC IN UR INE: ME TH OD 8316, Issue 1, dated 1 5 Ma rch 2003 - Page 3 of 5 SAMPLING: 1. Collect a spot urine sam ple in one or several 250-mL polypropylene bottles. Measure and record the volume of the whole voiding and transfer ap proxim ate ly 20 mL to a 30-mL polypropylene bottle. Label the bottle with the code unique to that specimen. Freeze imm ediately with dry ice. 2. Ship in a polystyrene-foam shipping container kept frozen with dry ice. 3. Store the samples at -76 °C until time of analysis.
SAMPLE PREPARATION: 4. Th aw th e urine sa m ple to ro om tem pera ture. R em ove a portion for creatinine ana lysis [8]. 5. Transfer 0.200 mL of the sample to a culture tube. 6. Add 1.80 mL of tetrabutylamm onium hydrogen sulfate in phosphate buffer, 2.00 mL of methylene chloride, and then 10 :L of pentafluorobenzyl bromide. Cap the tube. 7. W rap the tube with aluminum foil to prevent exposure to light, and tumble with the rotator for 20 hr at 30 rev/m in. Tim ing is critical, bec aus e an alyte rec overy reac hes a m axim um at 20 hr then de crease s. 8. At 20 hours, centrifuge culture tubes for 5 min at 3000 rpm. 9. Transfer 1.5 mL of the methylene chloride (lower layer)to a clean culture tube. Evapo rate to dryness under nitrogen at 30 / C. 10. Add 1.00 mL of 1:1 isopropanol-toluene to the residue, vortex for 1 min, then sonicate for 1 min. 11. Transfer the solution to an auto inje cto r vial, cap, and seal.
CALIBRATION AND QUALITY CONTRO L: 12.
13.
14.
15.
Calibrate the GC for each batch of samples. a. Prepare 9 working standards by diluting the PFB-B AA stoc k solution with 1:1 toluen e/pro pan ol to 9 equally-spaced concentrations between 0.8 and 68 :mol/L (equivalent to urina ry levels of 5.3 to 453 :m ol/L). b. Analyze these standards with the unknowns in step 17 in random order but after every third unknown in the batch. c. Prepare a calibration gra ph of p eak a rea versus analyte concentratio n, using, if necess ary, quadratic regression to fit the data. Prepare and analyze a minimum of 4 BAA-in-urine quality-control samples per batch. a. Prepare control samples at 0, 20, 90, and 400 :mol/L by diluting aliquots of BAA-in-urine stock solution with urine from unexposed individuals. b. Analyze quality control samples and five samples of the unspiked urine with the unknowns. c. Correct the nom inal values for the control samples for the background level of BAA in the blank urine. d. Calculate the recoveries and plot them on a control chart for the method. Re-analyze two field samples from the previous batch with each batch. a. If possible, select field sam ples with levels at both ends o f the analytical range, but above the dete ction lim it. b. Calculate the percent difference for each duplicate and plot it on a control chart for the method. Analyze at least one sample of pure water with each batch as a check for background interferences.
MEASUREMENT: 16. 17.
18.
Set the gas chrom atograph system according to the manufacturer’s recomm endations and the conditions given on page 8316-1. Inject 5 :L of sample from step 11 or standard from step 12. Note: W ith the chrom atog raph ic system use d for m etho d de velop m ent, the PFB-B AA peak had an efficiency of 124,000 plates, an asymm etry factor of 1.4, and a retention time of 31.7 min. The inten sity and precision of the detector’s response to PFB-BAA were sensitive to temperature, with an optimal precision obtained at 177 / C. Measure peak area.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
