3-BROMOPROPIONIC ACID in URINE
8324
metabolite of 1-bromopropane
FORMULA: C3H5BrO2 METHOD: 8324, Issue 1
MW: 152.97 CAS: 590-92-1 EVALUATION: FULL
Published limits and guidelines using this compound as a marker: OSHA: None NIOSH: None Other OELs: [1,2,3,4]*
BIOLOGICAL INDICATOR OF:
RTECS: UE7875000 Issue 1: 29 August 2014
Exposure to 1-bromopropane
PROPERTIES: Solid; d 1.48 g/mL at 25 oC; MP 58-62 oC
SYNONYMS: β-Bromopropionic acid; 2-carboxyethylbromide. SAMPLING SPECIMEN: Urine VOLUME:
MEASUREMENT TECHNIQUE:
GAS CHROMATOGRAPHY, MASS SPECTROMETRY with SELECTED ION MONITORING (GC/MS-SIM)
ANALYTE:
tert-butyldimethylsilane derivative of 3-bromopropionic acid
EXTRACTION:
Liquid-liquid extraction (LLE)
INJECTION VOLUME:
0.5 μL, splitless
At least 15 mL
PRESERVATIVE:
None added. Refrigerate or freeze upon collection.
SHIPMENT: Ship cold or frozen with ice or dry ice. Freeze upon receipt at the laboratory. SAMPLE STABILITY: Stable in frozen urine for at least 30 days. CONTROLS: Urine specimens obtained from non-exposed individuals. ACCURACY
TEMPERATURES INJECTION: 200 oC COLUMN:
60 oC initial, 4 oC/min to 180 oC,15 oC/min to 255 oC, post-run at 270 oC for 5 min
DETECTOR:
Source 230 oC, Quadrupole 150 oC; Solvent delay, 15 min
RANGE STUDIED:
RUN TIME:
Approximately 50 minute cycle time
Table 1
CARRIER GAS:
Helium, 0.8 mL/min constant flow
BIAS:
None established
COLUMN:
Capillary, fused silica, 100% dimethylpolysiloxane, 50 m X 0.20 mm (ID), 0.33 μm film thickness
CALIBRATION:
Analyte in control urine; with internal standard
PRECISION: Table 1 RECOVERY ACCURACY: Full recovery (95% overall) was established by a spiked urine recovery, overall RSD was +3.1% (Table 1). The definitions of precision and accuracy in this method are those utilized by the US Food and Drug Administration [5].
QUALITY CONTROL:
RANGE:
At least one level of spiked urine sample prepared from a separately weighed stock solution 2.0 to 100 μg/mL
ESTIMATED LOD: Approximately 0.01 μg/mL (IUPAC) [6]. 0.1 μg/mL is the lowest calibration standard. APPLICABILITY: 3-Bromopropionic acid (3-BPA) has been reported to be a rat metabolite [7] and is a potential human biomarker for exposure to 1-bromopropane. This method measures the quantity of free 3-BPA in urine. 1-Bromopropane is used as an industrial solvent. In one limited study, 3-BPA was not detected in individuals exposed to low levels of 1-bromopropane [8]. INTERFERENCES: None found or identified. OTHER METHODS: This method is from the one described by B’Hymer and Cheever [9] and further investigated by Mathias, et al. [8].
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 2 of 9
REAGENTS:
EQUIPMENT:
1. 3-Bromopropionic acid (3-BPA) reference standard as a 1.0 mg/mL stock solution in deionized water. Store in a refrigerator. 2. 3-Chloropropionic acid (3-CPA) reference standard, 20 μg/mL in deionized water, internal standard solution. Store in a refrigerator. 3. Ethyl acetate, HPLC grade or better* 4. N-Methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) with 1% tertbutyldimethylchlorosilane (TBDMCS) 5. Magnesium sulfate, anhydrous, powdered, ACS reagent grade 6. Hydrochloric acid, concentrated, ACS reagent grade 7. Water, deionized (ASTM type II) 8. Nitrogen, prepurified grade or better 9. Urine, non-exposed*
- See SPECIAL PRECAUTIONS
1. Gas chromatograph with mass spectrometric detector, column, autosampler, and data collector (page 8324-1) 2. Bottles, polypropylene, 125-mL with caps 3. Analytical balance, to ±0.0001 g 4. Analytical evaporator with nitrogen gas sweep 5. Test-tube vortex mixer 6. Heating block or oven 7. Automatic pipettor with disposable tips 8. Repeating pipet dispenser, 1 L 9. Tubes, disposable screw-top culture (16 X 100 mm and 16 X 150 mm), with polytetrafluoroethylene (PFTE)-lined caps 10. Metal spatula 11. Flasks, volumetric; 10, 50 and 100-mL 12. Autosampler vials, 2 mL, silanized, with caps and septa 13. Glass funnels 14. Glass wool, silanized 15. Disposable glass pipets 16. Dry ice or bagged refrigerant
SPECIAL PRECAUTIONS: Standard precautions should always be used when handling bodily fluids and/or extracts of bodily fluids [10]. Handle urine samples and urine extracts using powder-free latex or nitrile gloves. Ethyl acetate and MTBSTFA are flammable; handle with care and use in a chemical fume hood. Handle all chemicals using the required safety precautions. Manufacturer expiration dates for reagents should be observed. SAMPLING: 1. Collect at least 15 mL urine in an appropriate polypropylene tube or bottle and cap. Refrigerate or freeze immediately after collection. Collect at least two urine specimens for each worker: one before the work shift and one after. 2. Ship the specimens stored in either wet or dry ice in an insulated container. Store frozen upon arrival at the laboratory. A reminder: commercial shippers have special labeling requirements for packages containing dry ice. SAMPLE PREPARATION: 3. Thaw the urine specimen to room temperature. 4. Mix thoroughly to ensure urine homogeneity. 5. Transfer 2.0 mL of urine into a 16 X 100 mm (or larger) screw-capped culture tube. 6. Acidify by adding 40 μL of concentrated hydrochloric acid. 7. Add 0.5 mL of deionized water. 8. Add 0.5 mL of the 20 μg/mL 3-CPA internal standard solution. 9. Ethyl Acetate Extraction: Dispense 4 mL of ethyl acetate into the culture tube, cap, and vortex (or vigorously shake) for 1 minute. 10. Allow layers to separate, collect the ethyl acetate (top) layer.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 3 of 9
11. Repeat the extraction (steps 9 and 10) three more times. Collect and combine all ethyl acetate extracts using a glass pipet into a 16 X 150 mm culture tube. 12. Dry the ethyl acetate extract by adding approximately 100 to 200 mg of anhydrous magnesium sulfate and swirl for about 15 seconds. 13. Transfer the extract solution into a 16 X 150 mm culture tube by means of a glass funnel with silanized glass wool patch to remove the wet magnesium sulfate. The glass wool patch must be packed tightly enough to prevent particles of magnesium sulfate from passing through. 14. Rinse the tube and the funnel with ethyl acetate to ensure complete transfer. 15. Concentrate the combined ethyl acetate extract for each sample to 1 mL using a nitrogen sweep at room temperature and transfer the solution to a 2-mL GC autosampler vial. 16. tert-Butyldimethylsilane derivatization: Add 50 μL of MTBSTFA with 1% TBDMCS silanizing reagent to each autosampler vial and cap immediately. 17. Heat the solution for 1.5 hours at 70 oC in a heating block or oven. CALIBRATION AND QUALITY CONTROL: 18. 3-Bromopropionic acid (3-BPA) standards are prepared in blank, non-exposed urine. The 1 mg/mL stock 3-BPA solution is diluted in deionized water to make 0.4, 1, 2, 4, 8, 20, 80, 200, 400, 600, and 800 μg/mL 3-BPA solutions for spiking. 19. Transfer 2.0 mL of non-exposed urine into a 16 X 100 mm (or larger) screw-capped culture tube. 20. Acidify by adding 40 μL of concentrated hydrochloric acid. 21. Add 0.5 mL of the 20 μg/mL 3-CPA internal standard solution. 22. Add 0.5 mL of the appropriate 3-BPA spiking solution described in step 18 to make urine samples equivalent to 0.1, 0.25, 0.5, 1, 2, 5, 20, 50,100, 150 and 200 μg/mL of 3-BPA in the original 2.0 mL volume of urine. 23. Prepare at least one blank urine without a 3-BPA spike to verify the source of blank urine contains no detectable quantity of 3-BPA. 24. Prepare at least two levels of quality control (QC) standard of 3-BPA fortified urine using a separately weighed and prepared 3-BPA stock solution. One level should be within the lower 25% of the calibration curve and one level within the upper 25% of the calibration curve. More than two QC levels can be used. QC samples should be analyzed with every batch such that they constitute at least 5% of the sample batch. 25. QC values should be within ±20% of the spiked values. If not, the batch is considered out of control, the batch data discarded, and corrective actions should be taken before more samples are analyzed. 26. Ethyl acetate extraction: Prepare the spiked standard urine samples, the blank urine sample(s), and the QC standards the same as described in the preceding Sample Preparation section using Steps 9 through 17. MEASUREMENT: 27. Set the gas chromatograph according to the manufacturer’s recommendations and to the conditions listed on page 8324-1. 28. Set the mass selective detector to selected ion monitoring mode for ions m/z 211 (derivative of 3-BPA) and 165 (derivative of 3-CPA). NOTE: The use/non-use of qualifier ions for this method is discussed in the literature [9]. 29. Inject 0.5 μL of each sample, standard, blank, and QC standard extract from Steps 17 and 26. 30. Measure the peak areas of the tert-butyldimethylsilane derivatives of 3-BPA and 3-CPA in the chromatograms of the standards. 31. Divide the peak area of the derivative of 3-BPA by the peak area from the derivative of 3-CPA in the same chromatogram.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition 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 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 5 of 9
longer time frame of two months or more. Derivatized sample extracts appeared to be stable during a one week time frame. Range: This method should be considered accurate for the estimation of 3-BPA in human urine within the 2.0 to 100 μg/mL method validation range. REFERENCES: [1] ACGIH [2013]. TLVs and BEIs based on the documentation of the Threshold Limit Values for chemical substances and physical agents and Biological Exposure Indices. Cincinnati, Ohio: American Conference of Governmental Industrial Hygienists. www.acgih.org [Date accessed: April 2014.] [2] Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area [2013]. List of MAK and BAT values 2013; Maximum Concentrations and Biological Tolerance Values at the Workplace. DFG: Deutsche Forschungsgemeinschaft, Wiley-VCH Verlag GmbH & Co. http:// www.dfg.de/en/dfg_profile/publications/senate_commissions/index.html#micro7535782 [Date accessed April 2014.] [3] SUVA (Swiss National Accident Insurance Fund) [2013]. Protection de la santé au poste de travail. Valeurs limtes d’exposition aux postes de travail. http://www.suva.ch/english/startseite-en-suva/ praevention-en-suva/publications-en-suva.htm [Date accessed: April 2014.] [4] Institut für Arbeitsschutz der Deutschen Gesetzlichen Unfallversicherung (IFA) http://www.dguv. de/ifa/Gefahrstoffdatenbanken/GESTIS-Internationale-Grenzwerte-für-chemische-Substanzenlimit-values-for-chemical-agents/index-2.jsp [Date accessed: April 2014.] [5] US FDA [2001]. Guidance for industry: Bioanalytical method validation. U.S. Food and Drug Administration, Washington, DC. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107. pdf [Date accessed April 2014]. [6] B’Hymer C [2004]. 3-Bromopropionic acid in urine, Backup data report, Method 8324. Internal report. [7] Jones AR, Walsh DA [1979]. The oxidative metabolism of 1-bromopropane in the rat. Xenobiotica 9(12): 763-772. [8] Mathias PI, Cheever KL, Hanley KW, Marlow KL, Johnson BC, B’Hymer C [2012]. Comparison and evaluation of urinary biomarkers for occupational exposure to spray adhesives containing 1-bromopropane. Toxicol Mech Methods 22(7): 526-532. [9] B’Hymer C, Cheever KL [2004]. Development of a gas chromatographic test for the quantification of the biomarker 3-bromopropionic acid in human urine. J Chromatogr B 802: 361-366. [10] CDC [2007]. 2007 Guidelines for isolation precautions: preventing transmission of infectious agents in healthcare settings. http://www.cdc.gov/hicpac/2007IP/2007isolationprecautions.html [Date accessed: April 2013.] METHOD WRITTEN BY: Clayton B’Hymer, PhD, NIOSH/DART/BHAB. Final editing performed by Dale A. Shoemaker, PhD, NIOSH/ DART/CEMB. Disclaimer: Mention of any company or product does not constitute endorsement by the National Institute for Occupational Safety and Health. In addition, citations to Web sites external to NIOSH do not constitute NIOSH endorsement of the sponsoring organizations or their programs or products. Furthermore, NIOSH is not responsible for the content of these Web sites. All Web addresses referenced in this document were accessible at the time of publication.
- Note: Because exposure limits and guidelines may change over time, NIOSH recommends referring to the following sources
for updated limits and guidelines on the use of this compound as a marker for 1-bromopropane [1,2,3,4].
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 6 of 9
FIGURE 1
The major ions monitored for the tert-butyldimethylsilane derivatives were the molecular ions less the tert-butyl group (m/z = 57). The ions used for quantitation are m/z (mass to charge) 165 for the internal standard derivative and m/z 211 for the 3-bromopropionic acid derivative.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 7 of 9
FIGURE 2
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 8 of 9
FIGURE 3
A total ion chromatogram of a (A) blank non-exposed volunteer urine specimen and (B) 20 µg/mL spiked 3-BPA urine solution with 5 µg/mL equivalent 3-CPA as the internal standard. No interfering peaks were evident in any of the group of 20 non-exposed volunteer specimens.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition 3-BROMOPROPIONIC ACID in URINE: METHOD 8324, Issue 1, dated 29 August 2014 - Page 9 of 9
TABLE 1. Recovery studies of 3-bromopropionic acid:
(A) multilevel recovery study of 3-BPA from spiked urine samples1, (B) recovery of 20 μg/mL 3-BPA spikes from individual urines of 20 non-exposed volunteers2 Note: Overall recovery of all samples was 95% and overall RSD was 3.1% (n = 47.)
(A) Spike level (μg/mL)
Mean 3-BPA recovered, (n = 9, μg/mL)
Average % Recovery
Standard Deviation (μg/mL)
% Relative Standard Deviation
2
1.91
96
0.11
5.7
10
9.32
93
0.13
1.4
50
48.9
98
0.36
0.7
(B) Volunteer urine Spike level (μg/mL) 20
Mean 3-BPA recovered (n = 20, μg/mL)
Average % Recovery
19.0
95
Standard Deviation (μg/ mL)
% Relative Standard Deviation
0.48
2.5
1 Three different spiked urine samples were prepared at each level and chromatographed on three separate experimental trial runs (a total of nine samples at each spike level were analyzed.) 2 All non-spiked specimens showed no 3-BPA derivative peak in the chromatograms.
NIOSH Manual of Analytical Methods (NMAM), Fifth Edition