Systematic approach to the development of plasma amino acid analysis by high-performance liquid chromatography with ultraviolet detection with precolumn derivatization using phenyl isothiocyanate.

A reversed-phase liquid chromatographic method for the separation of 26 phenylthiocarbamyl derivatives of amino acids in human plasma in ca. 35 min. is described. The method used a C18 column (150 x 4.6 mm I.D., 3 micron) thermostatted at 41 degrees C, and a simple multistep linear gradient of two solvents. Solvent A was 0.05 M sodium acetate (pH 5.1)-acetonitrile (98:2, v/v), and solvent B was water-acetonitrile (40:60, v/v). A simple and successful approach to the optimization of the conditions for the separation of the 26 amino acid derivatives was realized. In the initial phase of development, the composition of the gradient, its timings, the column temperature, the flow-rate and the mobile phase compositions were optimized. At the end the influence of pH was studied, and this approach led to a clear resolution of the 26 amino acids. The method was validated by accuracy, precision, and recovery studies, by analyzing patient samples, and by comparing the quality control sample results with the classical ion-exchange method.

Assay of human plasma cortisone by liquid chromatography: normal plasma concentrations (between 8 and 10 a.m.) of cortisone and corticosterone.

A high-performance liquid chromatographic method using ultraviolet detection to quantitate human plasma concentrations of cortisone simultaneously with cortisol and corticosterone is described. The method is based on the use of an octadecyl silica column (100 mm x 2 mm I.D., 3 microns), an ultraviolet absorbance detector (242 nm) with a 10 mm path length flow-cell, and a mobile phase composed of water-tetrahydrofuran-acetonitrile (82:10:8, v/v) containing 5 ml/l triethylamine and citric acid to adjust the pH of the buffer to 6.5. Flumethasone is used as the internal standard. The detection limit of the method for the three steroids is 300 ng/l using a 1-ml sample. The average inter-assay coefficient of variation for cortisone is 3.3% and the average recovery is 100.8%. Possible interferences from common drugs and endogenous and exogenous steroids in the method have been studied. Plasma concentrations (drawn from 8 to 10 a.m.) of cortisone and corticosterone for 43 normal volunteers have been determined.

Simultaneous assay of corticosterone and cortisol in plasma by reversed-phase liquid chromatography.

We have developed a simple, specific, and sensitive reversed-phase liquid-chromatographic method for accurate and simultaneous analysis of corticosterone and cortisol in human plasma. We achieved a detection limit of 300 ng/L for both steroids by modifying the old solid-phase extraction method to make use of "Tef Elutor" C18 columns, using a minibore (100 x 2 mm) analytical column, and using an ultraviolet detector with a 10-mm-pathlength flow cell. With the new extraction method absolute extraction efficiencies were greater than 90% for all the analytes, including the internal standard, flumethasone. The mobile phase was water (containing 5 mL of triethylamine per liter and citric acid to adjust the pH to 6.5), tetrahydrofuran, and acetonitrile (82/10/8 by vol). The average interassay CV for corticosterone at 0-25 micrograms/L was 6.5%; that for cortisol at 0-300 micrograms/L was 3.8%. The analytical recovery relative to the internal standard was 100.2% for cortisol and 102.6% for corticosterone. Possible interferences from drugs and other steroids were studied.

Liquid-chromatographic determination of nicotine and cotinine in urine from passive smokers: comparison with gas chromatography with a nitrogen-specific detector.

We describe a simple, sensitive, and specific high-performance liquid-chromatographic method with ultraviolet detection (256 nm) for the simultaneous analysis of nicotine and cotinine in urine of passive smokers. The analytes are extracted and purified from the complex and impure matrix in two stages; first, by liquid-liquid extraction and followed by solid-phase extraction (C2 column). We used a "DB" C8 5-microns-particle column (25 x 0.46 cm) and a mobile phase of phosphate-citrate buffer and acetonitrile (91:9 by vol) containing 5 mL of triethylamine and 600 mg of heptanesulfonate per liter, adjusted to pH 4.4, to separate the compounds. Two internal standards (2-phenylimidazole and N-ethylnorcotinine) were used. The detection limit of the HPLC assay was less than 1 microgram/L for both analytes. The average interassay CV for nicotine was 7.6%, for cotinine 6.5%, in the concentration range 0-60 micrograms/L. The mean analytical recovery of nicotine with respect to the internal standard N-ethylnorcotinine was 102% and that for cotinine was 99%; the mean absolute recoveries of the compounds were as follows: nicotine 85%, cotinine 87%, and N-ethylnorcotinine 87%. To compare the HPLC assay results of 20 samples with the more-sensitive in-laboratory gas-chromatographic method with a nitrogen-phosphorus detector, we used both N-ethylnornicotine and N-ethylnorcotinine as internal standards. The mean correlation coefficient for nicotine values between the two methods was 0.934; for cotinine, 0.987.

A simple, sensitive liquid chromatographic assay of cis-thiothixene in plasma with coulometric detection.

A novel, simple, and very sensitive liquid-chromatographic assay with a coulometric detector has been developed for quantitating cis-thiothixene (CTX) in human plasma. A reverse phase, 5-microns cyano column (25 x 0.46 cm), a mobile phase of phosphate buffer (pH 2.5) and acetonitrile (40/60 by vol), and a coulometric detector are used for the separation of CTX, and the internal standard, trifluoperazine. CTX and trifluoperazine are extracted from alkalinized plasma into n-pentane-isopropanol (95/5 by vol) and purified by back extraction into perchloric acid. The optimum oxidation potential for the analytes is +0.8 V versus an Ag/AgCl electrode. The detection limit for CTX is 200 pg using 1 mL of plasma and CTX concentrations are linear from 0 to 40 micrograms/L. The average interassay CV is 9%, and the mean recovery is 99% relative to the internal standard. Possible interferences from various psychiatric and common drugs in the assay have been studied. The assay method was validated by determining the concentration of CTX in the plasma of 100 schizophrenic patients.

An improved sensitive assay for simultaneous determination of plasma haloperidol and reduced haloperidol levels by liquid chromatography using a coulometric detector.

A simple, highly sensitive and specific high-performance liquid chromatographic method that uses a coulometric detector for the simultaneous assay of haloperidol and reduced haloperidol in human plasma has been developed, using bromoperidol as the internal standard. A reversed phase C8 5-microns column (25 x 0.46 cm) and a mobile phase of phosphate buffer (pH 7.0), acetonitrile, and methanol (40:20:40 vol/vol) are used for separating the analytes. The analytes are extracted from alkalinized plasma using a mixture of pentane and isopropanol (95:5 vol/vol) and purified by back extraction into a perchloric acid solution. Teflon tubes with screw caps are used throughout the extraction work. The compounds are oxidized at a potential of +0.90 V against a Ag/AgCl reference electrode. The detection limit of the assay is 50 ng/L using 1 mL of plasma. The average interassay coefficient of variation for samples of concentration 1-40 micrograms/L is approximately 8%. Possible drug interferences in the assay have been studied. The absolute recovery of the method is approximately 80%. The assay has been validated by quantitating 150 schizophrenic patients' samples.

Free 3-methoxy-4-hydroxyphenylglycol determined in plasma by liquid chromatography with coulometric detection.

We describe a simple "high-performance" liquid-chromatographic method for determining free 3-methoxy-4-hydroxyphenylglycol (MHPG) in plasma, with coulometric detection and with 4-methoxy-3-hydroxyphenylglycol (iso-MHPG) as the internal standard. MHPG and iso-MHPG are extracted from plasma into ethyl acetate and the extract is washed with a sodium bicarbonate solution, evaporated, reconstituted, and injected into a 25 x 0.4 cm column packed with 3-micron particles of C18 material. We used a mobile phase of acetate buffer (100 mmol/L, pH 5.0) and methanol (92:8 by vol) and an oxidation voltage of 0.5 V. The detection limit of the assay is 0.1 micrograms/L. The interassay CV for a sample with a mean MHPG concentration of 3.84 micrograms/L was 5.2%. The average absolute recovery for the method was 37%.

A high-performance liquid-chromatographic method for routine simultaneous determination of nicotine and cotinine in plasma.

We describe a rapid, sensitive method for the routine simultaneous determination of nicotine and cotinine in 1 mL of plasma. Extraction in 10-mL screw-capped Teflon tubes with methylene chloride after deproteinization with trichloroacetic acid eliminated emulsion formation. The extract, after evaporation and reconstitution in 30 microL of mobile phase, is injected into a reversed-phase C-18 ion-pair column of an isocratic high-performance liquid-chromatographic unit. Absorbance is monitored at 256 nm. The mobile phase is a citrate-phosphate (30 mmol each per liter) buffer mixture containing 50 mL of acetonitrile and 1 mmol of sodium heptanesulfonate per liter. 2-Phenylimidazole is the internal standard. The detection limit is 1 microgram/L for nicotine and 3 micrograms/L for cotinine. The standard curve is linear from 0 to 700 micrograms/L for both compounds. The average CV for nicotine in the concentration range 0-100 micrograms/L is 6.5%, and that for cotinine in the concentration range 50-700 micrograms/L is 4%.