MSACL 2024 Abstract

Introduction: Porphyria refers to a group of diseases associated with hereditary and acquired deficiencies in the heme biosynthetic pathway. The laboratory diagnosis of porphyrin disorders is performed by identification of pathway intermediates, which present in excessive concentrations. Porphobilinogen (PBG) is formed during the second step of the pathway; in the third step of the pathway, the enzyme hydroxymethylbilane synthase (HMBS) catalyzes tetramerization of PBG to form hydroxymethylbilane. Deficiency of HMBS can result in a condition known as acute intermittent porphyria (AIP). Clinical diagnosis of AIP is confirmed by detection of elevated urinary PBG concentrations. The most common method currently in use for PBG analysis in urine is ion exchange chromatography followed by spectrophotometry (SPh).

Objectives: Our aims were to develop a simple, sensitive, and specific method for measurement of PBG in urine by LC-MS/MS, and to evaluate the method’s performance.

Methods: Sample preparation was performed as follows: 50 µL of calibrators, quality control and urine samples were aliquoted in tubes, and 200 µL of buffer and 20 µL of internal standard were added to the tubes. PBG was extracted from the samples using strong anion exchange solid phase extraction. The extracts were analyzed using an LC-MS/MS system, consisting of a TripleQuadTM 5500 (Sciex) equipped with an series 1260 LC (Agilent) and an autosampler (Leap Technologies). Chromatographic separation was performed using a Waters XBridgeTM PREMIER BEH C18 column. PBG was measured using positive electrospray ionization in MRM mode; mass transitions were 210.2>122.1 (primary) and 210.2>94.0 (secondary); the stable isotope labeled internal standard was 13C215N-PBG. Quantitation was performed using a five-point calibration curve with concentrations calculated based on analyte peak areas normalized to internal standard peak areas; injection-to-injection time for instrumental analysis was 6 min.

Results: The lower limit of quantification (LLOQ) and upper limit of linearity for PBG were 0.1 µmol/L and 100 µmol/L, respectively. Total imprecision of the assay was <14%; accuracy at LLOQ was 88%. Good agreement was observed with a comparative LC-MS/MS assay of another laboratory (slope 0.91, R2=0.995, n=13). In comparison with the new LC-MS/MS method, the SPh method overestimated PBG concentrations (slope 0.55, R2=0.860, n=114). Poor agreement between the SPh method and LC-MS/MS was observed in samples containing PBG below 10 µmol/L. Mean, median, and range of the PBG/creatinine ratio in urine samples from self-reported healthy adults tested by this LC-MS/MS method were 0.059, 0.054, and 0.001-0.25 µmol PBG/mol creatinine. The reference interval (RI) for 24h excretion established using LC-MS/MS was <1.41 µmol (0.318 mg); the RI was confirmed by analysis of a set of clinical samples (n=129). Based on the data from analysis of neat clinical urine samples (n=114), clinical sensitivity and specificity of the SPh assay were 0.85 and 0.84, respectively. PBG concentrations in urine samples of healthy individuals were below the detection limit of the SPh assay; interference of the SPh assay was observed in ~8% of routinely tested samples. PBG concentrations in urine samples from self-reported healthy subjects were above the LOQ of the LC-MS/MS assay in over 97% of samples (n=256); no signs of interferences were observed in neat urine patient samples analyzed by this LC-MS/MS method (n>1000). Measurement of PBG using LC-MS/MS in urine samples of healthy adults demonstrated direct correlation between concentrations of PBG and creatinine, and a trend to inverse correlation between PBG concentrations and body mass index.

Conclusions: Analytical sensitivity and specificity of this LC-MS/MS method for PBG are sufficient for routine measurement of PBG in urine samples of healthy individuals and patients with porphyria. Compared to published methods for PBG, this method uses simple sample preparation and allows high throughput testing of PBG in a clinical laboratory setting.