Page:NIOSH Manual of Analytical Methods - 8326.pdf/2

 S-Benzylmercapturic acid and S-phenylmercapturic acid in urine: METHOD 8326, Issue 1, dated 20 May 2014 - Page 2 of 10

APPLICABILITY: BMA and PMA are metabolites of toluene and benzene, respectively. PMA is a very specific biomarker for benzene. BMA can form from exposure to other sources, such as benzyl acetate or benzyl alcohol which can be found in personal care products [8]. Both toluene and benzene are common solvents with multiple occupational uses; furthermore exposure to toluene and benzene (less commonly) can occur from environmental and other sources. This method measures the quantity of the two target metabolite analytes in urine. INTERFERENCES: None found or identified. OTHER METHODS: This method is based on the research of B’Hymer [9,10]. There are numerous literature methods for these two compounds, but standardized methods from governmental agencies or consensus standards organizations are not currently available, though there is also a recent method developed at CDC/National Center for Environmental Health for these analytes and others [11].

REAGENTS:

EQUIPMENT:

1.	S-Benzylmercapturic acid (BMA, N-acetyl-S-benzyl-DLcysteine, CAS no. 19542-77-9) reference standard 0.20 mg/mL stock solution in methanol. Store at 4 °C in amber vials (or in the dark). 2.	S-Phenylmercapturic acid (PMA, phenyl-N-acetyl-DLcysteine, CAS no. 20640-68-0) reference standard 0.20 mg/mL stock solution in methanol. Store at 4 °C in amber vials (or in the dark). 3.	Deuterated S-benzyl-d5-mercapturic acid reference standard (d5-BMA), 0.15 mg/mL stock solution in methanol. Store at 4 °C in amber vials (or in the dark). 4.	Deuterated S-phenyl-d5-mercapturic acid reference standard (d5-PMA), 0.15 mg/mL stock solution in methanol. Store at 4 °C in amber vials (or in the dark). 5.	Internal standard spiking solution, deuterated stock solutions diluted to approximately 30 ng/mL in water 6.	Acetone, HPLC grade or better* 7.	Acetonitrile, HPLC grade or better* 8.	Acetic Acid, glacial, ACS reagent grade or better* 9.	Methanol, HPLC grade or better* 10.	Water, doubly deionized, minimum resistivity of 18 MΩ-cm 11.	Water, HPLC grade 12.	Acetonitrile/water (50/50%, v/v) injector rinse solution 13.	Chromatographic matrix adjustment solution, 49/50/1% (v/v/v) acetonitrile/water/acetic acid 14.	Mobile phase A (5/95/0.1%, v/v/v) acetonitrile/water/ acetic acid, filtered through 0.7 µm glass microfiber filters 15.	Mobile phase B (75/25/0.1%, v/v/v) acetonitrile/water/ acetic acid, filtered through 0.7 µm glass microfiber filters 16.	Synthetic urine substitute, if desired 17.	Nitrogen, UHP

1.	High-Performance Liquid Chromatograph (HPLC) equipped with a tandem mass spectrometric detector with data collection system 2.	HPLC column (C18 [dimethyloctadecylsilane solid phase type], 150 mm X 3 mm, 3.5 µm particle size) 3.	Autosampler 4.	Analytical balance, 0.1 mg readability 5.	Rotary vacuum concentrator with cold trap and vacuum pump or analytical evaporator with nitrogen 6.	Automatic pipet with disposable tips in the 1000 µL volume delivery range 7.	Solid-phase extraction (SPE) cartridges, C18, 500 mg, 3 mL 8.	SPE vacuum manifold apparatus 9.	Culture tubes, disposable screw-top (16 X 150 mm) with PTFE-lined caps 10.	Tubes, polypropylene, disposable screw-top, 15 mL. Polypropylene bottles and caps may also be used for sample collection. 11.	Metal spatula 12.	Volumetric flasks: 10, 50, 100 and 200 mL 13. Autosampler vials, amber, with caps and septa 14.	Disposable glass pipets 15.	Sintered glass filtration apparatus and 1-Liter sidearmed Erlenmeyer flask 16.	Microfiber filters, glass, 0.7 µm; diameter to fit the filtration apparatus 17.	Wet or dry ice

SPECIAL PRECAUTIONS: Standard precautions should always be used when handling bodily fluids and/or extracts of bodily fluids [12]. Handle urine specimens and urine extracts using powder-free latex or nitrile gloves. Acetic acid, acetone, acetonitrile and methanol are flammable; handle with care and use in a chemical fume hood. Handle all chemicals using the required safety precautions. Reagents with manufacturer expiration dates should be observed.
 * See SPECIAL PRECAUTIONS*

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition