Page:NIOSH Manual of Analytical Methods - 8324.pdf/4

 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 4 of 9

32.	Prepare a linear calibration curve of the peak Area Std./Area Int. Std. versus the concentration of the standard for 3-BPA. 33.	Measure the peak areas of the derivatives of 3-BPA and 3-CPA in the remainder of the chromatograms. 34.	Divide the peak area of the derivative of 3-BPA by the peak area from the derivative of 3-CPA in the same chromatogram. CALCULATIONS: 35.	Determine the concentration of 3-BPA in the extracts from the original urine (2.0 mL specimen) from the curve obtained in step 32. The results are expressed as μg/mL of 3-BPA in urine. EVALUATION OF METHOD: This method was evaluated and described by B’Hymer [6] and in detail by B’Hymer and Cheever [9]. A general summary of this published information is given below: Accuracy and Precision: Two recovery studies using multiple GC columns over several days demonstrated the accuracy and precision of this test method. The first recovery study was performed over three separate experimental batch runs, and these data are presented in Table 1A. Average recovery was between 93 and 98% for the three 3-BPA spiked level urine samples investigated. For each batch run, the experimental trial consisted of three samples at three different concentration levels. The recovery for each level (n=9 samples) is displayed in Table 1A. The second recovery study (data shown in Table 1B) used spiked urine samples from 20 non-exposed volunteers and demonstrated that the procedure was accurate (95% average recovery) and precise (2.5% relative standard deviation.) No interferences were detected in the unspiked urine from the 20 volunteer specimens. Both recovery studies generated a total of 47 spiked urine samples at 2, 10, 20 and 50 μg/mL 3-BPA levels. Precision expressed as percent relative deviation (% RSD) was as high as 5.7% on the 2 μg/mL recovery samples (n = 9). Overall recovery was 95% and overall RSD was 3.1% (n = 47). Linearity: All calibration curves used during the development of this method were linear and had correlation coefficients of 0.98 and greater. The concentration range was 0.1 to 200 μg/mL 3-BPA in urine with 2.0 mL urine sample size. Calibration curves were run at the beginning and end of all sample batch runs; calibration curve slope drift was found to be acceptable. Specificity: The optimized chromatographic conditions developed for this procedure proved to be specific and have no major interferences. The mass spectrometric detector was useful in adding additional specificity to the method. The ion m/z 211 was chosen for monitoring the calibration curve used in the calculations because of its greater abundance, and it was a characteristic fragment for the tert-butyldimethylsilane (TBDMS) derivative of 3-BPA. This is the molecular ion less the tert-butyl group, m/z 57 (Figure 1). Ion m/z 165 was used to monitor the TBDMS derivative of 3-CPA, the internal standard, for the same reasons. Full-scan mass spectra of the TBDMS derivatives are presented in Figure 2. An example chromatogram for blank urine and urine spiked with 3-BPA and 3-CPA is shown in Figure 3. Robustness: Multiple HP-1 (100% dimethylpolysiloxane) columns of different manufacturing lots were used during the recovery studies. Accuracy and precision did not appear to be affected; therefore, the method appears to be reproducible with any normal functioning HP-1 capillary column. Recovery results from individual urine samples spiked with 3-BPA indicate that the method is accurate and not significantly affected by individual urine sample matrix differences during analyte extraction. Stability: While sample stability was not exhaustively evaluated, an aqueous stock standard solution of 3-BPA stored for two weeks at 4 oC gave full recovery assay values when compared to a freshly prepared 3-BPA standard. The 3-BPA in the urine specimens appears to be stable in frozen urine for a much

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition