Page:NIOSH Manual of Analytical Methods - 8326.pdf/1

 S-Benzylmercapturic acid and S-phenylmercapturic acid in urine

8326

Metabolites of toluene and benzene

1. S-Benzylmercapturic acid: 2. S-Phenylmercapturic acid: METHOD:

FORMULA: C12H15NO3S FORMULA: C11H13NO3S

8326, Issue 1

MW: 253.3 MW: 239.3

EVALUATION: Full

Published limits and guidelines using these compounds as markers:

1. Toluene: OSHA and NIOSH: None 2. Benzene: OSHA and NIOSH: None Other OELs: Because data on exposure limits and guidelines may change over time, NIOSH recommends referring to the following sources for updated limits and guidelines concerning the use of these compounds as markers for toluene and benzene [1,2,3,4].

BIOLOGICAL INDICATOR OF:

CAS:  19542-77-9 CAS:  20640-68-0 Issue 1:	 20 May 2014

Exposure to 1. toluene and 2. benzene

PROPERTIES: 1. Solid; 1.246 g/cm3; mp 162-163 °C 2. Solid; 1.28 g/cm3; mp 155 °C

SYNONYMS (not all inclusive): 1.	 S-Benzylmercapturic Acid (BMA): S-benzyl-N-acetyl-L-cysteine; (2R)-2-Acetamido-3-(phenylmethylsulfanyl)propanoic acid; Alanine, N-acetyl-3(benzylthio)-; S-phenylmethyl-N-acetyl-L-cysteine 2.	 S-Phenylmercapturic acid (PMA): S-phenyl-N-acetyl-L-cysteine; (2R)-2-Acetamido-3-(phenylsulfanyl)propanoic acid; (2R)-2-Acetylamino-3(phenylthio)propionic acid

SAMPLING

MEASUREMENT

SPECIMEN:	 Urine VOLUME:

At least 8 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 periods of a month or more and for several freeze/thaw cycles [5,6]. CONTROLS:	 Urine specimens obtained from non-exposed or low level exposed individuals.

TECHNIQUE:

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (HPLC/MS/MS)

ANALYTES:

S-Benzylmercapturic acid and S-phenylmercapturic acid

EXTRACTION:

Solid-Phase Extraction (SPE) C18

COLUMN:

C18 [dimethyloctadecylsilane solid phase type, 3.5 µm particle size] (150 mm by 3 mm)

MOBILE PHASES: FLOW RATE:

0.3 mL/min (0.4 mL/min post run)

GRADIENT:

Time (min) vs. Mobile Phase Composition 0 to 10 0 to 40% B 10 to 18 40 to 100% B 18 to 20 100% B 20 to 21 100% B Flow increased to 0.4 mL/min 21 to 28 100% B (0.4 mL/min flow) 28 to 30 100 to 0% B (re-equilibration, 0.3 mL/min) 30 to 37 0% B (re-equilibration)

ACCURACY* RANGE STUDIED:

See Table 2

BIAS:

Negligible

OVERALL PRECISION (ŜrT)*:

See Table 2

ACCURACY*:

A = 5/95/0.1% (v/v/v) acetonitrile/water/acetic acid B = 75/25/0.1% (v/v/v) acetonitrile/water/acetic acid

INJECTION VOLUME: 8 μL IONIZATION SOURCE:

Overall recoveries (103% overall) obtained from spiked urine samples (n=48) were 103% and 106% for S-benzylmercapturic acid and S-phenylmercapturic acid, respectively. The precision as relative standard deviation was no greater than 5.0% at any concentration level (n=9, Table 2).

those utilized by the US Food and Drug Administration [7].
 * The definitions of precision and accuracy in this method are

Electrospray at 3500 Volts and negative scan mode, nebulizer gas at 35 psi and 10 L/min flow

DETECTOR (MS/MS): Dwell time = 200 msec; Fragmentor at 80 Volts; Collision energy at 8 Volts; Collision gas: nitrogen at 0.06 L/min MULTIPLE REACTION MODE: Quantification mass transitions; BMA = m/z 252 → 123, PMA = 238 → 109, d5-BMA = 257 → 128, d5-PMA = 243 → 114 TOTAL RUN TIME:

Approximately 37 minute cycle time

CALIBRATION:

BMA and PMA solutions with internal standards

QUALITY CONTROL: At least one level of spiked urine specimen prepared from a separately weighed stock solution RANGE:

0.5 to 50 ng/mL for BMA and PMA

ESTIMATED LOD:

Approximately 0.2 ng/mL for BMA and PMA; by lowest standard levels, BMA and PMA = 0.5 ng/mL

PRECISION (Ŝr):

See Table 2

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition