This paper features a first-time reported sample preparation chemistry for analysis of MIC-tyrosine adducts.

Base-catalyzed hydrolysis of hemoglobin-MIC-tyrosine adducts forms phenylmethyl carbamate.

LC-MS/MS was used to detect the analyte from MIC-exposed rats.

The method has excellent sensitivity, linearity, and dynamic range.

The method also produced remarkable accuracy and precision compared to existing methods.

Methyl isocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, is a toxic industrial chemical that causes irritation and damage to the eyes, respiratory tract, and skin. Due to the high reactivity of MIC, it binds to proteins to form protein adducts. While these adducts can be used as biomarkers to verify exposure to MIC, methods to detect MIC adducts are cumbersome, typically involving enzymatic (pronase) or strong acid (Edman degradation) hydrolysis of hemoglobin. Hence, in this study, a simple method was developed which utilizes base hydrolysis of MIC-tyrosine adducts from isolated hemoglobin to form phenyl methyl carbamate (PMC), followed by rapid liquid-liquid extraction, and liquid chromatography tandem mass spectrometry analysis. The hydrolysis chemistry is the first report of base hydrolysis of a tyrosine-β-C-hydroxo phenol bond in aqueous solution. The method produced excellent sensitivity (detection limit of 0.02 mg/kg), linearity (R2 = 0.998, percent residual accuracies > 96), and dynamic range (0.06‒15 mg/kg). The accuracy and precision (100 ± 9% and < 10% relative standard deviation, respectively) of the method were outstanding compared to existing techniques. The validated method was able to detect significantly elevated levels of PMC from hemoglobin isolated from MIC-exposed rats.

Amino acids

Phenyl methyl carbamate

Tyrosine adduct

LC-MS/MS

Method development

Data will be made available on request.

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