Re. Ford et al., Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry, CLIN CHEM, 47(9), 2001, pp. 1627-1632
Background: Measurement of porphobilinogen (PBG) is useful in the diagnosis
of the acute neurologic porphyrias. Currently used colorimetric assays lac
k analytical and clinical sensitivity and specificity.
Methods: We developed a liquid chromatography-electrospray tandem mass spec
trometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, usi
ng 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[C-13] pyrolle-3-propanoic acid
([2,4-C-13]PBG; 2.75 mug) as internal standard. After solid-phase extractio
n, 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 an
d product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). T
he retention time of PBG and [2,4- 13C]PBG was 1.0 min in a 2.3-min analysi
s.
Results: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear an
d reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respe
ctively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regressio
n equation for the comparison between an anion-exchange column method (y) a
nd the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S-y\x = 5.8 mg/24 h; r =
0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lowe
r than reported reference intervals (up to 2.0 mg/24 h) based on colorimetr
ic methods. In 85 samples with PBG less than or equal to 0.5 by LC-MS/MS, 8
(9.4%) had values greater than or equal to 2.0 mg/24 h by the anion-exchan
ge 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 w
ithin the reported reference intervals by a quantitative anion-exchange met
hod.
Conclusions: The quantitative LC-MS/MS method for PBG measurement exhibits
greater analytical specificity and improved clinical sensitivity and specif
icity than currently available methods. (C) 2001 American Association for C
linical Chemistry.