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

Abstract

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. Copyright © 2012 John Wiley & Sons, Ltd.

Direct and simultaneous quantitation of 5‐aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography–atmospheric pressure chemical ionization/tandem mass spectrometry

“…Although ALA and PBG can be monitored in both positive and negative ionization modes (Benton and Lim, ; Benton et al ., ), positive ionization mode was used for this study as it offered increased sensitivity. The protonated [M + H] + PBG ion ( m/z 227) showed significant loss of NH 3 in the ion source ([M + H − NH 3 ] + ) to give a stable, positively charged methylenepyrrolenine ion ( m/z 210; Lord et al ., ; Benton et al ., 2012a, 2012b), as shown in Fig. .…”

“…We have recently described a hydrophilic interaction liquid chromatography (HILIC)–MS/MS method for the direct and simultaneous analysis of ALA and PBG in human urine (Benton et al ., 2012a). We report here a sensitive LC‐MS/MS method for the simultaneous quantitation of ALA and PBG in human plasma or serum, without the need for extensive sample preparation and/or analyte derivatization.…”

Application of HPLC-ELSD for the Quantification of 5-Aminolevulinic Acid after Penetration into Human SkinEx Vivo

“…The LLOQ for these methods was approximately 0.001-0.4 mg=ml Namjoshi et al 2007;De Rosa et al 2003). The LOQ for HPLC or UPLC coupled with mass spectrometry was 1 Â 10 À3 -5 Â 10 À3 mg=ml Benton et al 2012); for CE, it was about 100 attomoles (Lord et al 2012). These low values were not necessary for skin samples, as the content of 5-ALA is at least 100-fold higher in them.…”

Simultaneous Determination of Phenol and p-Cresol in Human Urine by an HPLC Method

“…The supernatant was directly injected to HPLC system. In various methods, we see such a simple pretreatment step for analysis of some chemical substances in urine samples [6][7][8].…”