Improved rapid procedure for simultaneous determinations of hemoglobins A1a, A1b, A1c, F, C, and S, with indication for acetylation or carbamylation by cation-exchange liquid chromatography.

Although the clinical utility of glycohemoglobin (HbA1c) as an indicator of blood glucose control is generally accepted, the use of cation-exchange liquid chromatography for its quantification is controversial, given numerous studies that show no correlation between HbA1c and other indicators of blood glucose control in uremic subjects. We report a specific cation-exchange liquid-chromatographic method that measures simultaneously HbA1a, HbA1b, HbA1c, HbF, HbA0, HbC, and HbS quickly and reproducibly. Carbamylation or acetylation is indicated by extra peaks. Carbamylated hemoglobin increased the result for HbA1c by as much as 1% above the nonuremic value. The method is a fast (approximately 15 min per sample), easy to perform, reproducible, and sensitive screening method for abnormal hemoglobins. Within-assay CVs were < 0.7% in all cases and between-assay CVs were < 1.4%.

Optimized determination of thiochrome derivatives of thiamine and thiamine phosphates in whole blood by reversed-phase liquid chromatography with precolumn derivatization.

A liquid chromatographic method involving precolumn derivatization for determining thiamine and its phosphate esters in human blood has been optimized. Blood sample stored at - 20 degrees C were haemolysed and deproteinized by perchloric acid. The supernatants of the samples were oxidized by addition of potassium ferricyanide-sodium hydroxide, and phosphoric acid was added to obtain a neutral pH in order to extend the column life. The samples were stable after derivatization for at least 24 h, if protected from light and kept at room temperature. Gradient separation with 140 mmol phosphate buffer (pH 7.0), and methanol, tert-butylammonium hydroxide and dimethylformamide as modifiers, on a 3-microns Chromsphere octadecylsilica column gave an analysis time of 15 min. The method was found to be very suitable for the determination of thiamine components in whole blood. The minimal detectable amount is 0.5 nmol/l and the method is linear to at least 1000 nmol/l. The recovery (98 +/- 3%) and precision are very good.

Determination of serum triglycerides by capillary on-column gas chromatography.

We report an accurate method, based on capillary on-column gas chromatography, for determining triglycerides in human serum. After serum extraction and chemical hydrolysis, glycerol is directly measured by gas chromatography (GC). With our extraction method no free glycerol is extracted from serum. The accuracy of this method was compared with that of a method based on the original procedure of Carlson (J. Atheroscler. Res. 3, 334-336 (1963)) and which is standardized by the Centers for Disease Control (CDC, Atlanta, U.S.A.). Orthogonal regression analysis of the GC method (y) and the CDC reference method (X) resulted in y = 0.996 X + 0.000 with a correlation coefficient of 0.999. The variances of analytical data, collected over a two month period showed that, for the GC method, the within-day coefficient of variation (CV) was less than 1.43%; the between-day CV less than 1.35%. The data for the CDC method were less than 3.36% (within-day) and less than 6.38% (between-days). The CDC method is linear up to 3.7 mmol/l and the GC method to 22 mmol/l.

Direct determination of total serum cholesterol by on-column gas-liquid chromatographic analysis without previous derivatisation compared with WHO-CDC reference method.

A method for the direct determination of total serum cholesterol by on-column gas-liquid chromatography is described. The French reference method developed by Gambert et al. served as a model for our method, which is fast and less laborious than the well-known CDC reference method of Cooper et al. based on the Abell-Kendall technique. The accuracy and precision of our on-column gas-liquid chromatographic method and the CDC reference method are comparable. The obvious advantage of this proposed gas-liquid chromatographic reference method is its increased analytical speed. The problem of the specificity of our method in relation to cholestanol is discussed.