Determination of steroid hormones in a human-serum reference material by isotope dilution--mass spectrometry: a candidate definitive method for cortisol.

We report a method, based on isotope dilution--mass spectrometry, for determining cortisol in a pooled specimen of human serum. Isotopically labeled cortisol is added to 5.0 mL of serum so that the molar concentrations of labeled cortisol and unlabeled cortisol are approximately equal. The specimen and two calibration standards are extracted with dichloromethane, and the extracted cortisol is converted to the methoxime-trimethylsilyl ether derivative. Samples and standards are analyzed by gas chromatography--mass spectrometry by monitoring the peak areas for m/z 605 and 608. The cortisol concentration is calculated by linear interpolation between the two bracketing standards. Variances of data collected during six weeks showed that the overall coefficient of variation (CV) was 0.69% (n = 32); the within-vial CV, 0.63%; the among-vial CV, 0.22%; and the among-day CV, 0.15% (means = 3.973 nmol/vial). Method specificity was demonstrated by liquid chromatographic as well as C8 mini-column cleanup of samples before derivation, by alternative ion monitoring at m/z 636 and 639, and by negative-ion chemical ionization at m/z 459 and 462. Derivatives of all observed degradation products of cortisol under basic, neutral, and acidic conditions did not interfere.

Multivariate and univariate optimization studies of liquid-chromatographic separation of steroid mixtures.

We developed a high-performance liquid-chromatographic separation of five steroids (estriol, estradiol, cortisol, progesterone, and testosterone), eluting with a water-acetonitrile gradient from a reversed-phase (C18) column. By applying a simplex search algorithm to maximize a chromatographic-response function, we sought to optimize the original conditions of the chromatographic analysis, which did not separate two pairs of overlapping peaks. Our chromatographic-response function incorporated both peak separation and total time of analysis. Three factors were varied simultaneously to maximize this function: flow rate, column temperature, and gradient shape. From the simplex optimization, we selected a flow rate of 1.50 mL/min, a temperature of 52 degrees C, and a linear gradient for our analysis. Subsequent univariate studies of the initial mobile phase composition showed that acetonitrile/water (20/80 by vol) gave an adequate separation.

Separation of protoporphyrins and related compounds by reversed-phase liquid chromatography.

We describe the separation of protoporphyrin and related porphyrins by reversed-phase "high-performance" liquid chromatography, with fluorometric detection. We used the method to demonstrate that acid hydrolysis of the dimethyl ester of protoporphyrin IX is complete in 2 to 3 h and is followed by the acid-catalyzed conversion of protoporphyrin IX to a chemical species that chromatographic evidence indicates to be hematoporphyrin IX. In addition, the method was used to evaluate the purity of a commercial preparation of protoporphyrin IX and was also demonstrated to have the sensitivity and specificity needed for measuring the protoporphyrin IX content of whole-blood.

Determination of free, total, and esterified cholesterol by high-performance liquid chromatography.

A method is described for measuring free, total, and esterified cholesterol in blood serum in which reversed-phase liquid chromatography is used and the eluate is monitored at 200 nm. The sample for total cholesterol is prepared according to the Abell-Kendall procedure, and for free cholesterol an extract of serum--isopropanol (1:5, v/v) is used. The column is a muBondapak C18, 10 micrometers, and the mobile phase for total cholesterol is isopropanol--acetonitrile (50:50, v/v); for free cholesterol, it is isopropanol--acetonitrile--water (60:30:10). An approximation of the free cholesterol, triglycerides, and individual cholesteryl esters is obtained from single chromatograms of isopropanol extracts of serum if the first mobile phase is used. In a comparison study with the Abell-Kendall method for total cholesterol, the correlation is excellent and the precision is acceptable.

4-Nitrophenol in 4-nitrophenyl phosphate, a substrate for alkaline phosphatase, as measured by paired-ion high-performance liquid chromatography.

We used paired-ion high-performance liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase analysis. This was done on a reversed-phase column with a mobile phase of methanol/water, 45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per 200 ml of solvent. At a flow rate of 1 ml/min, 4-nitrophenol was eluted at 9 min and monitored at 404 nm; 4-nitrophenyl phosphate was eluted at 5 min and could be monitored at 311 nm. Samples of 4-nitrophenyl phosphate obtained from several sources contained 0.3 to 7.8 mole of 4-nitrophenol per mole of 4-nitrophenyl phosphate.