Development and validation of an LC-MS/MS method for measurement of porphobilinogen in urine

Measurement of urinary porphobilinogen (PBG) is used to diagnose acute porphyria, which results from disorders of heme biosynthesis. In response to clinical demand and to provide optimal patient care, we developed a simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for measurement of PBG in urine.

Aliquots (50 µL) of urine were spiked with a stable isotope-labeled internal standard (¹³C₂,¹⁵N-PBG); PBG was extracted using anion exchange solid phase extraction, and the extracts were analyzed using reverse phase chromatographic separation with mass spectrometry detection. Injection-to-injection time was 5.5 min. Measured concentrations were normalized to creatinine (Cr) concentration in random specimens; PBG excretion per day was calculated for 24-h urine collections.

Mean recovery was 99.7 %; total imprecision of analysis was < 16 %. Limit of quantification of the method was 0.1 µmol/L and the upper limit of linearity was 100 µmol/L. Reference intervals (RI) for the ratio of PBG to Cr concentration in urine samples from apparently healthy adults were < 0.19 and < 0.17 mmol PBG/mol Cr for women and men, respectively. The RI for 24-h excretion established using LC-MS/MS was 0.39-1.40 µmol/24-h. Percent positivity determined following implementation of the LC-MS/MS method (7.6 %) compared well with the positivity rate of the spectrophotometric method (7.7 %).

This LC-MS/MS method has sufficient specificity and sensitivity for analysis of urinary PBG in pathologic samples and samples from healthy subjects. RIs for PBG in random and 24-h urine collections were established and the clinical performance of the assay was evaluated.