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Quantification of 4‐OH‐2,6‐Xylidine and its Conjugates in Human Urine Samples Utilising Microextraction in Packed Syringe On‐line with Liquid Chromatography and Electrospray Tandem Mass Spectrometry (MEPS‐LC‐MS/MS)

Abstract A method has been developed to quantify the major metabolite, 4‐OH‐2,6‐xylidine (4‐OH‐XYL), of lidocaine in human urine samples after acid hydrolysis of its conjugates. Detemination of 4‐OH‐XYL was performed using micro extraction in packed syringe (MEPS) as a sample preparation method on‐line with liquid chromatography and tandem mass spectrometry (MEPS‐LC‐MS‐MS). MEPS is a new technique for miniaturised solid‐phase extraction that can be connected, online, to gas chromatography (GC) or liquid chromatography (LC) without any modifications. The validation of the method was performed in the range 17 to 8700 nmol/L. The inter‐day accuracy and precision values were determined from six replicates of quality control (QC) samples at three different concentrations in human urine. The nominal urine concentrations of 4‐OH‐XYL in the QC samples were 80.5, 805, and 8050 nmol/L. The mean accuracy for each concentration level, reported as the percentage difference from the nominal value, was within ±8%. The precisions, given as coefficient of variation, were in the range 6 to 8%. The validated method was used for the quantification of 4‐OH‐XYL in urine samples from subjects receiving lidocaine. It was found that 4‐OH‐XYL is stable under highly acidic conditions (pH≤1), and is highly unstable in neutral and alkaline solutions. Our investigation showed that 4‐OH‐XYL is stable for at least 24 hours in the refrigerator in 0.1 M HCl (pH≈1). At pH 9.1, more than 40% decomposition of 4‐OH‐XYL occurs within 6 h at room temperature compared to 20% at pH 2.0. Therefore, our approach was to use acid hydrolysis of 4‐OH‐XYL conjugates and on‐line sample preparation for the quantification of 4‐OH‐XYL in humane urine.