Roddey E Ford, Mark J Magera, Karen M Kloke, Paul A Chezick, Abdul Fauq, Joseph P McConnell, Quantitative Measurement of Porphobilinogen in Urine by Stable-Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry, Clinical Chemistry, Volume 47, Issue 9, 1 September 2001, Pages 1627–1632, https://doi.org/10.1093/clinchem/47.9.1627
Navbar Search Filter Mobile Enter search term Search Navbar Search Filter Enter search term SearchBackground: 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-[ 13 C]pyrolle-3-propanoic acid ([2,4- 13 C]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- 13 C]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- 13 C]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.
The porphyrias are a heterogeneous group of inherited or acquired disorders resulting from a deficiency of one of the eight enzymes that control the heme biosynthesis pathway. Clinical manifestations of specific enzyme deficiencies are attributable to the overproduction of specific heme precursors and their accumulation in tissues and body fluids. The porphyrias can be subdivided into acute and nonacute forms based on the presence of acute recurrent neurologic attacks. Acute attacks often follow exposure to precipitating factors such as porphyrinogenic drugs and chemicals, hormones, ethanol ingestion, and reduced caloric intake, which influence the rate of heme biosynthesis, leading to expression of the underlying genetic trait ( 1)( 2)( 3). Clinically, the acute porphyrias, which include acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, present intermittently with neurovisceral signs and symptoms that may be severe, possibly leading to paralysis, and in some cases can be life threatening ( 4)( 5). Symptoms may include mental, neurologic, and abdominal manifestations ( 6). The signs and symptoms, however, are often variable and nonspecific, and may be confused with other medical conditions ( 5). Laboratory analysis is thus required to confirm or rule out acute porphyria.
Porphobilinogen (PBG) 1 excretion in urine increases in acute porphyric attacks, and the measurement of PBG is particularly important in diagnosing the acute porphyrias (acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria) during the acute stage ( 7)( 8). Markedly increased urine PBG is diagnostic for acute porphyria ( 5). Neurologic symptoms without a concomitant increase in PBG excretion cannot be attributed to acute porphyria, and other causes for the symptoms should be investigated ( 9).
Methods currently used to measure PBG in urine require derivatization of PBG, typically with Ehrlich reagent, to form a chromogen that is quantified colorimetrically. The Watson–Schwartz method is a screening test that is still widely used today ( 10). However, the method is not quantitative and has poor sensitivity and specificity. Other qualitative and quantitative methods have been reported ( 11)( 12)( 13)( 14), many of which include prepurification of PBG with anion-exchange resins, but all rely on the derivatization of PBG with Ehrlich-type reagent, which is not specific for PBG, and thus have the potential for false positives. We have developed an alternative method for PBG analysis that takes advantage of the versatility, sensitivity, and specificity of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) using stable-isotope dilution, which does not require derivatization of PBG.
PBG monohydrate was purchased from Frontier Scientific. The labeled PBG internal standard H-2,4- 13 C-pyrrole-3-propanoic acid ([2,4- 13 C]PBG)> was synthesized by Frontier Scientific (Logan, UT) from 5-amino-4- 13 C-oxopentanoic acid (4- 13 C-aminolevulinic acid), which was custom made by the Organic Synthesis Core Facility, Mayo Clinic Jacksonville (Jacksonville, FL). All other reagents were of the highest purity available from commercial sources and used without purification. Oasis HLB ® solid-phase extraction columns were obtained from Waters Corporation.
Urine (5 mL) was acidified with 6 mol/L HCl to pH 2.0. One milliliter of the acidified urine was placed in a 10 × 75-mm glass culture tube and mixed with 100 μL of internal standard solution (2.75 mg/L). Extraction was performed on a Gilson ASPEC ® automated SPE sample processor. Oasis HLB columns (1 mL, containing 30 mg of packing) were preconditioned with 1 mL of 1 mol/L glacial acetic acid and 1 mL of reverse osmosis water. One milliliter of sample was added to the column, followed by a 500-μL water wash. PBG and [2,4- 13 C]PBG were then eluted with 1 mL of 1 mol/L glacial acetic acid. The eluate was transferred to a sealed, amber glass autosampler vial for injection onto the LC-MS/MS instrument. Calibrators were prepared in urine of known PBG concentration (blank) by the addition of a 100 mg/L working solution to produce PBG concentrations of 0.1, 0.2, 0.5, 1.0, and 2.0 mg/L. Calibrators were treated and extracted like samples, and the endogenous PBG concentration of the blank sample was subtracted from the calibrators after visual verification of results.
An API 2000 LC-MS/MS triple quadrupole mass spectrometer (Perkin-Elmer Sciex) with a TurboIonSpray ionization probe source (operated at 5800 V) was used. Peripherals included a Perkin-Elmer Series 200 pump and an autosampler. PBG and [2,4- 13 C]PBG were separated from the bulk of the specimen matrix on a short HPLC column [Supelcosil LC-18; 3.3 cm × 4.6 mm (i.d.); 3-μm bead size; Supelco] to enhance the stability of the signal. Autosampler injections of 10 μL were made using a mobile phase composed of 300 mL/L acetonitrile in 1.4 g/L formic acid in water. The flow rate was 1.0 mL/min. The column was directly connected to the TurboIonSpray ionization probe operating with the turbo gas on (6 L/min; sensor temperature, 250 °C) with the LC column effluent flow-splitting set at 1:5. PBG and [2,4- 13 C]PBG eluted apart from the bulk of the specimen matrix at a retention time of ∼1.0 min. Total instrument acquisition cycle time was 2.3 min/sample.
All results were generated in positive-ion mode with the orifice voltage set at 6 V, automatically optimized using the protonated PBG ion. Mass calibration and resolution adjustments (at 0.7 atomic mass units, full width at half-height) on both the resolving quadrupoles were automatically optimized using a 1 × 10 −4 mol/L poly(propylene)glycol solution introduced via the built-in infusion pump on the API/2000. Collisionally activated decomposition MS/MS was performed through the closed-design Q2 collision cell, operating with nitrogen at 0.06 kPa as collision gas. The 15-eV (lab frame) collision energy was adjusted automatically by the AutoTune algorithm.
MS/MS spectra were collected in continuous flow mode by connecting the built-in infusion pump directly to the TurboIonSpray probe. For MS/MS optimization, solutions of 20 μmol/L PBG and [2,4- 13 C]PBG were prepared in 10 mL of 1 g/L formic acid and infused separately at a rate of 10 μL/min. In SRM mode, the instrument was optimized by the built-in algorithm to monitor the m/z 227 to 210 and m/z 229 to 212 transitions for PBG and [2,4- 13 C]PBG, respectively. Data were acquired and processed using the Mass-Chrom software (Ver 1.1.1; Perkin-Elmer Sciex) including Multiview, Ver. 1.4, for chromatographic and spectral interpretation and Turboquan for Windows NT (Ver 1.0; Perkin-Elmer Sciex) for the quantitative processing. To calculate sample PBG concentration, the ratio of the area of the PBG analyte to the area of the internal standard was compared to area ratios of calibrators with known PBG concentrations. PBG values were reported in units of mg/24 h.
LC-MS/MS results were compared with a modified Watson–Schwartz screening method and with quantitative anion-exchange PBG analysis. Briefly, the screening method involved addition of 1 mL of urine or PBG calibrator (3.0 mg/L) to 1 mL of Ehrlich reagent (5.6 g; p-dimethyl-amino-benzaldehyde in 1000 mL of concentrated HCl and 800 mL of water). After mixing, 2 mL of chloroform was added, and the resulting solution was centrifuged for 5 min at 2000g. The upper aqueous layer was added to 2 mL of n-butanol, mixed, and centrifuged for 5 min at 2000g. The upper butanol layer was discarded, and the absorbance (555 nm) of the aqueous layer was measured using a Beckman DU 650 spectrophotometer. If the absorbance of the sample was greater than that of the calibrator, the sample was considered positive for the presence of PBG. The sample was considered negative for PBG if its absorbance was less than that of the calibrator. PBG quantification by anion-exchange column was accomplished using the ALA/PBG Column Test from Bio-Rad Laboratories, which is based on the method of Mauzerall and Granick ( 12). The manufacturer provided instructions for analysis as well as reagents and supplies.
The MS/MS spectrum obtained by infusion of 20 μmol/L PBG is shown in Fig. 1A . This spectrum was acquired by transmitting the protonated molecular ion via Q1 and scanning the second resolving quadrupole, Q3, for products resulting from fragmentation in the collision cell. The autotune algorithm provided in the system software was used to optimize the instrument for transmission of the protonated molecular ion and for maximum intensity of the selected fragment. These results were used to design the SRM experiment to sequentially transmit the m/z 227 protonated molecular ion and the m/z 210 fragment via Q1 and Q3, respectively. Fig. 1B shows the MS/MS spectrum obtained by infusion of 20 μmol/L [2,4- 13 C]PBG, which was optimized for the transmission of the m/z 229 protonated molecular ion and the m/z 212 fragment.
MS/MS product ion scans of standard protonated ions.
(A), PBG optimized to favor the m/z 227 to 210 transition. (B), [2,4- 13 C]PBG optimized to favor the m/z 229 to 212 transition.
The extracted SRM chromatogram obtained from a specimen in which the calculated PBG concentration was 0.10 mg/24 h is shown in Fig. 2A . The extracted SRM chromatogram obtained from a specimen from a patient with acute intermittent porphyria in which the calculated PBG concentration was 110 mg/24 h is shown in Fig. 2B (this sample was diluted 1:100 before analysis).
LC-MS/MS SRM-extracted chromatograms of PBG and [2,4- 13 C]PBG from a urine sample containing PBG at 0.10 mg/24 h (A) and a urine sample containing PBG at 110 mg/24 h, diluted 1:100 before analysis (B).
Three urine samples with high PBG concentrations obtained from patients with acute porphyria were serially diluted to test assay linearity. In the range of 0.05–3.0 mg/L, the coefficients of linear regression (r 2 ) were 0.9987, 0.9996, and 0.9999. Calibrators with PBG concentrations of 0.1, 0.2, 0.5, 1.0, and 2.0 mg/L prepared in urine matrix were assayed on 6 different days. The mean slope and r 2 were 1.07 and 0.9978, respectively. The CVs for the slope and r 2 of the PBG calibration curves were 4.2% and 0.2%, respectively. For routine sample analysis, samples with PBG concentrations above the highest calibrator (2 mg/L) were diluted to produce values that fell within the range of the calibration curve. The SRM-extracted chromatogram signal-to-noise ratios for two specimens with calculated PBG concentrations of 0.10 and 0.06 mg/L were 34.8 and 21.5, respectively. There was no appreciable carryover of PBG on the HPLC column until the PBG concentrations exceeded 5.0 mg/L in the final solutions. Specimens analyzed immediately after samples with PBG concentration >5.0 mg/L were reinjected to ensure accurate quantification.
The recovery and precision data for the method are summarized in Table 1 . These experiments were conducted using a urine matrix (blank). We added PBG to aliquots of the blank urine to produce final PBG concentrations of 0.26, 1.20, and 2.15 mg/L. Six aliquots of each sample were analyzed on each of 6 consecutive days. The method demonstrated quantitative recovery and CVs
Precision and recovery of the LC-MS/MS PBG method.