Quantitative Measurement of Porphobilinogen in Urine by Stable-Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Background: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. Methods: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[13C]pyrolle-3-propanoic acid ([2,4-13C]PBG; 2.75 μg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-13C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-13C]PBG was 1.0 min in a 2.3-min analysis. Results: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2–3.5% and 2.6–3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (Sy|x = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02–0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG ≤0.5 by LC-MS/MS, 8 (9.4%) had values ≥2.0 mg/24 h by the anion-exchange method (mean ± SD, 4.3 ± 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. Conclusions: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.

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

“…ALA and PBG are highly polar compounds and are not sufficiently retained using conventional reversed-phase chromatography. Such methods are susceptible to significant matrix effects because of the insufficient retention of ALA and PBG (Ford et al, 2001). A method using a nonvolatile ion-pairing reagent (Crowne et al, 1981) has been reported, but such buffers are incompatible with mass spectrometric operation.…”

Mass spectrometric characterisation of a condensation product between porphobilinogen and indolyl‐3‐acryloylglycine in urine of patients with acute intermittent porphyria

“…(c)). Taking into account that the unknown compound could be related to PBG and that PBG is mainly ionised in positive mode as [M + H−NH 3 ] + , a more detailed study was necessary for establishing the molecular formula of the species.…”

“…[12,13] In the case of AIP, several LC-MS/MS-based methods have been reported for the target quantification of PBG and ALA in human urine. [14][15][16][17] Additionally, both GC-MS and LC-MS/MS have been used in the evaluation of hormonal and metabolic imbalances in AIP patients. [18,19] Inspection of the urine of AIP patients in our centre by LC-MS/MS leads to the observation of an unidentified chromatographic peak that appeared in all urines from patients with porphyria, whereas its presence was undetectable in all control samples.…”

“…In the last decades, these techniques became the gold‐standard approach in the detection, characterisation and elucidation of biomarkers . In the case of AIP, several LC‐MS/MS‐based methods have been reported for the target quantification of PBG and ALA in human urine . Additionally, both GC‐MS and LC‐MS/MS have been used in the evaluation of hormonal and metabolic imbalances in AIP patients …”

Liquid chromatography and mass spectrometry of haem biosynthetic intermediates: a review

“…A stable-isotope dilution HPLC-ESI-MS/MS method has been described for the quantitation of PBG in urine using 2,4-13 C 2 -PBG as internal standard (IS) with a calibration range from 0.05 to 3.0 mg/L (0.22-13 mmol/L; Ford et al, 2001). PBG was extracted using solid-phase extraction and separated using a Supelcosil LC-18 column with a mobile phase of acetonitrile and formic acid in water.…”