W. Vogt

Diagnosis of porphyrias by ion-pair high-performance liquid chromatography

Ion-pair reversed-phase high-performance liquid chromatography together with fluorescence detection is useful in the analysis of urinary porphyrin carboxylic acids. The sensitive and quantitative detection facilitates the clinical diagnosis of porphyrias. The method described permits the detection of porphyrins down to 0.1 ng directly from urine without laborious sample pre-treatment. A linear response curve was obtained from 0.2 up to 200 ng for coproporphyrin I. The within-assay correlation coefficients ranged from 2.5 to 10.1%. Recovery experiments gave an accuracy of 89-109%. The rapidity and simplicity of the method allows its application to the routine analysis of urinary porphyrins in the clinical laboratory.

Improved separation and detection of free porphyrins by high-performance liquid chromatography

Porphyrins were separated using ion-pair reversed-phase high-performance liquid chromatography. The eluents were aqueous potassium phosphate buffer and tetrabutylammonium phosphate in methanol. The influences of pH value and ionic strength of the phosphate buffer and molarity of the ion-pair reagent in methanol were investigated to improve separation and detection. A linear response curve was obtained from 0.38 to 7.64 pmol for coproporphyrin I. The detection limits were determined to be 0.12 pmol for coproporphyrin I and 0.22 pmol for uroporphyrin I.

Ion-pair high-performance liquid chromatographic separation of porphyrin isomers

Abstract A novel ion-pair reversed-phase high-performance liquid chromatographic system for the simultaneous separation of type I, II, III and IV isomers of coproporphyrin has been developed. Complete separation of all isomers was obtained on a 7 μm LiChrosorb RP-18 column. The mobile phase consisted of an aqueous phosphate buffer and a mixture of methanol and acetonitrile containing the ion-pair reagent tetrabutylammonium phosphate. The method was applied to the analysis of the isomeric purity of numerous synthetic coproporphyrins. Separation of type I and III isomers of uroporphyrin was achieved by a modified ion-pair system using both isocratic and gradient elution. The latter elution technique served for the simultaneous separation of the naturally occurring type I and type III isomers of octacarboxylic through tetracarboxylic porphyrins. Urine samples originating from porphyric patients were analysed with this highly selective profiling technique.

Analysis of free stool porphyrins by high-performance liquid chromatography.

The determination of stool porphyrins is necessary for the diagnosis of some porphyrias in clinical laboratories. Quantitative methods for the analysis of faeces for porphyrins are unpleasant and difficult to perform. An extraction and ion-pair reversed-phase high-performance liquid chromatographic procedure is described for the separation and determination of individual free stool porphyrins. The within-assay coefficients of variation range from 2 to 6%. A linear response curve is observed between 38 and 380 nmol/g for coproporphyrin I in dry stool The method can be applied to the routine analysis of free stool porphyrins in the clinical laboratory.

Highly sensitive method for the quantitation of homovanillic acid in cerebrospinal fluid

Homovanillic acid in minute samples (50-100 microliters) of cerebrospinal fluid can be quantitated as its O-dimethylthiophosphinate methyl ester. The derivative is determined after glass capillary gas chromatography (GC) with high sensitivity by using a phosphorus-specific thermionic detector or by mass fragmentography, combined with a large sample volume split-splitless injector. The dimethylthiophosphinic esters show excellent stability against moisture and air. The precision of the overall procedure is 5.4% (GC) and 3.8% (GC-mass spectrometry). The method shows good linearity (r = 0.9999) over three orders of magnitude, from 500 pg to 500 ng. The lowest detectable concentration of homovanillic acid is 2-5 ng/ml. Concentrations of homovanillic acid determined in cerebrospinal fluid were 9.9-63.1 ng/ml (n = 10, mean = 30.9 ng/ml).

Quantitation of adrenaline and noradrenaline from human plasma by combined gas chromatography—high-resolution mass fragmentography

A gas chromatographic--high-resolution mass fragmentographic method for the simultaneous determination of adrenaline and noradrenaline from human plasma is presented. The catecholamines are separted by adsorption on alumina and converted by a selective, two-step procedure to the corresponding N-trifluoroacetyl-N-trimethylsilyl derivatives. The benzylic fragment C16H31O3Si3 (m/e 355.1568) of these derivatives is detected at a mass spectrometric resolving power of 5000. This high resolution detection was necessary to differentiate this fragment from others with the same nominal mass of 355 originating from the biological matrix and/or the bleeding from column and septum.

Ion-pair-reversed-phase high-performance liquid chromatographic determination of porphyrins from red blood cells

SummaryAn extraction and ion-pair reversed-phase HPLC procedure is described for the separation and determination of protoporphyrin and its zinc chelate from red blood cells. The recoveries were greater than 90% with coefficients of variation between 8 and 10%. A linear response curve was obtained from 800 to 4000 μg/l erythrocytes for the porphyrins.

Dimethylthiophosphinic esters for the gas chromatographic determination of monohydroxy-steroids with the alkali flame detector : VI. Steroid phosphorus compounds

Abstract Dimethylthiophosphinic chloride has been found to react readily at 70–90° with monohydroxy-steroids to yield dimethylthiophosphinic esters. These derivatives have good gas chromatographic properties and can be detected at the lower picogram level (10–15 pg) with the alkali flame detector. The reaction kinetics were examined, and depend on steric influences and the chemical nature of the hydroxyl group. The esters are stable to heat and hydrolysis, and since their formation is highly reproducibile and they offer a very low detection limit, they are suitable for gas-liquid chromatographic determination of various compounds of biological interest.

Performance of various mathematical methods for computer-aided processing of radioimmunoassay results.

Interpolation and regression methods are available for computer aided determination of radioimmunological end results. We compared the performance of 6 algorithms (weighted and unweighted linear logit log regression; quadratic logit log regression, smoothing spline interpolation with a large and small smoothing factor, respectively, and polygonal interpolation and the manual curve fitting on the basis of three radioimmunoassays with different reference curve characteristics (digoxin, estriol, human chorionic somatomammotrophin (HCS)). Great store was set by the accuracy of the approximation at the intermediate points on the curve, i.e. those points that lie midway between two standard concentrations. These concentrations were obtained by weighing and inserted as unknown samples. In the case of digoxin and estriol the polygonal interpolation provided the best results, while the weighted logit log regression proved superior in the case of HCS.

Polygonal interpolation, a simple, rapid, and versatile approximation method requiring minimal computing facilities

Abstract A method is described for interpolation of data points by a polygon of arbitrarily high subdivision, the breakpoints of which are obtained by a simple and rapid process, which can be performed on programmable desk calculators. When compared with classical interpolation methods in quality of approximation and computational expense, polygonal interpolation proves as a useful and versatile tool for approximation of general functions.