Urinary porphyrins are separated, according to number of carboxyl groups, in a system consisting of a mu-Bondapak C18 stationary phase and a mobile phase of methanol and aqueous sodium phosphate (pH 3.5) in a linear gradient. The specimen is prepared simply by adjusting the pH of a 5-mL sample to 2.0 and removing solids by centrifugation. The eluted porphyrins are measured fluorometrically. Naturally occurring non-porphyrin fluorescent substances are eluted ahead of the porphyrins. Chromatography requires about 20 min, and a re-establishment of initial conditions requires an additional 15 min.

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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.

A method has been developed for the extraction, separation, and measurement of 3,4-dihydroxyphenylalanine (dopa) and several of its metabolites in brain tissue. These compounds are extracted from tissue homogenates by adsorption on activated aluminum oxide. Lyophylization of the alumina prior to elution with acidic methanol reduced loss and provided anhydrous conditions for subsequent derivative formation. Pentafluoropropionyl derivatives were measured by gas-liquid chromatography employing an electron capture detector. Relative recovery of dopa, dopamine, norepinephrine, and 3,4-dihydroxyphenylacetic acid added to tissue samples averaged in excess of 90%; relative standard deviation = 15.43%. Dopamine concentrations as low as 0.05 pg/g tissue could be accurately determined using 500-mg tissue samples. Analysis time for six samples averaged 4-5 hours. SINCE ITS INCEPTION in 1952, gas-liquid chromatography (GLC) has been employed for the analysis of a wide variety of compounds of biological importance. Moreover, the applications of GLC have been greatly expanded in the past decade by the use of derivatives which now allow the GLC of an increased number of different compounds (1-5). While

Again, by analogy with the binary case, a semiquantitative, a priori, prediction can be made of the accuracy of these results. In Equation 12, the ratio of the cross product term to the sum of the first two is defined as : = 100 '(1) "Beryllium, Its Industrial Hygiene Aspects," .