TRICHLOROACETIC ACID IN URINE: METHOD 8322, Issue 1, dated 17 April 2015 - Page 3 of 5
CALIBRATION AND QUALITY CONTROL: 11. Prepare a stock solution by accurately weighing a known quantity of sodium trichloroacetate into a volumetric flask. Add a known volume of deionized water and mix. Convert the weight of the sodium trichloroacetate to TCAA by multiplying by 0.8814 (MW TCAA divided by MW sodium trichloroacetate = 0.8814). As an example, 34 mg of sodium trichloroacetate into a 10-mL flask makes a 3 mg/mL stock solution to be used in preparing the calibration standards. NOTE: Sodium trichloroacetate was used instead of trichloroacetic acid for all phases of this method development as well as in the preparation of standards. Trichloroacetic acid is very hygroscopic; the salt is much less so. 12. Prepare working (calibration) standards by serial dilution to cover the analytical range. A suggested working standard concentration range is 0.08 to 300 µg/mL. Withdraw 200 µL of each calibration standard and follow steps 4 through 10. 13. Determine the retention time for the analyte of interest. 14. Prepare at least one blank urine specimen without an analyte spike to verify whether the source (of blank urine) contained no detectable quantity of TCAA. 15. Prepare at least two levels of quality control spikes of TCAA, sodium salt to be analyzed with each analysis batch. These levels should be at ~10 X the limit of quantitation (LOQ) and 200 X LOQ, but can be adjusted to better suit the anticipated levels of the set of specimens. QC samples must be analyzed with every batch such that they constitute 10% of the sample batch. 16. QC values must be within ±20% of the spiked values. If not, the batch is considered out of control, the batch data discarded, and corrective actions taken before more samples are analyzed. 17. Calibrate daily with at least six liquid working standards covering the expected concentration range of the samples. MEASUREMENT: PRECAUTION: SYRINGE-RINSE SOLUTIONS: Toluene will extract material from some urine specimens that may eventually clog the syringe and cause injection errors unless the syringe is rinsed with the following solutions following each injection.
First rinse solution: 1:3 glacial acetic acid:deionized water
Second rinse solution: 1:1 acetone:methanol 18. Set the gas chromatograph according to manufacturer’s recommendations and to conditions given on page 8322-1. With the chromatographic conditions listed, the retention time of the methyl ester of TCAA was 8.87 min [6]. 19. Inject each of the samples, standards, blanks, and quality control samples. 20. Measure peak area or peak height; peak area is recommended. NOTE: If the sample peak area or height is greater than that of the highest calibration standard, dilute with toluene and reanalyze. Apply the appropriate dilution factor in the calculations. 21. Prepare a calibration curve by plotting instrument responses (usually peak area) for the standards vs. concentration. The simplest model that adequately describes the data should be used, but either a linear (mostly likely 1/x weighted because of the range of the calibration curve) or a quadratic model may be utilized in processing the analytical results. The standard curve must have a coefficient of determination (r2) of equal to or greater than 0.98 to be acceptable for use. Furthermore, when each standard is plugged back into the calibration equation, the measured value must be within ±20% of the expected value. CALCULATION OF ANALYTE PER SAMPLE: 22. Determine the concentration of TCAA in µg/mL (mg/L) using the response of each sample and the calibration curve prepared in step 21. Apply any dilution factor if applicable.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition
