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Direct and simultaneous determination of 5-aminolaevulinic acid and porphobilinogen in urine by hydrophilic interaction liquid chromatography-electrospray ionisation/tandem mass spectrometry.

Urinary concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatisation steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, for the direct and simultaneous quantitation of ALA and PBG in urine following simple dilution with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive electrospray ionisation. Urine samples (N = 46) from active and latent mutation-confirmed acute hepatic porphyria patients and normal subjects (N = 45) were analysed and the results compared with those of a commercially available spectrophotometric method. The validated calibration range was 3-3000 µmol/L for ALA and 2-2000 µmol/L for PBG. For both analytes, imprecision (relative standard deviation) was less than 5% and accuracy (percentage nominal concentrations) was between 88 and 109%. The lower limit of quantitation was 0.1 μmol/L for both analytes. The calculated LC-MS/MS and spectrophotometric results from patient samples compared well [Pearson correlation (r²) of 0.99 and 0.95, for ALA and PBG, respectively]. The method was successfully applied to the measurement of ALA and PBG in urine samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.