Large Differences in Urinary Benzene Metabolite S-Phenylmercapturic Acid Quantitation: A Comparison of Five LC–MS-MS Methods

Abstract

Benzene is a known genotoxic carcinogen linked to many hematological abnormalities. S-phenylmercapturic acid (PHMA, N-Acetyl-S-(phenyl)-L-cysteine, CAS# 4775-80-8) is a urinary metabolite of benzene and is used as a biomarker to assess benzene exposure. Pre-S-phenylmercapturic acid (pre-PHMA) is a PHMA precursor that dehydrates to PHMA at acidic pH. Published analytical methods that measure urinary PHMA adjust urine samples to a wide range of pH values using several types of acid, potentially leading to highly variable results depending on the concentration of pre-PHMA in a sample. Information is lacking on the variation in sample preparation among laboratories regularly measuring PHMA and the effect of those differences on PHMA quantitation in human urine samples. To investigate the differences in PHMA quantitation, we conducted an inter-laboratory comparison that included the analysis of 50 anonymous human urine samples (25 self-identified smokers, 25 self-identified non-smokers), quality control samples, and commercially available reference samples in five laboratories using different analytical methods to determine which sample preparation methods are currently in use and compare PHMA results. Observed urinary PHMA concentrations were proportionally higher at lower pH and results for anonymous urine samples varied widely among the methods. The method with the neutral preparation pH yielded results about 60% lower than the method using the most acidic conditions. Samples spiked with PHMA showed little variation, suggesting that the variability in results in human urine samples across methods is driven by the acid-mediated conversion of pre-PHMA to PHMA.

Increased Levels of the Acrolein Metabolite 3-Hydroxypropyl Mercapturic Acid in the Urine of e-Cigarette Users

“…TNE, 3-hydroxypropyl mercapturic acid (3-HPMA), hydroxymethylpropyl mercapturic acid (HMPMA), S -phenyl mercapturic acid (SPMA), CEMA, and total NNAL were analyzed as described previously; however, in the total NNAL assay 0.5% aqueous HCOOH was used instead of HCl in all the SPE steps, and the final elution was with 5% (v/v) NH 4 OH in MeOH. The HMPMA analysis includes mercapturic acids of crotonaldehyde, methyl vinyl ketone, and methacrolein .…”

Urinary Volatile Organic Compound Metabolites are Associated with High Blood Pressure Among Non-smoking Participants in the National Health and Nutrition Examination Survey (2011-2018)

“…Limited, consistent evidence exists of urinary benzene metabolites and ambient benzene and cardiovascular outcomes due to ongoing analytical issues. 51,52 In the Sister Study, ambient benzene was significantly associated with a 1.06 (95% CI 1.01, 1.10) greater risk of hypertension among never smokers. 41 The crotonaldehyde metabolite, HPMMA, was associated with higher prevalence of hypertension.…”