Stereoselective determination of R-(+)- and S-(−)-remoxipride, a dopamine D2-receptor antagonist, in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic (HPLC) method is described for the selective and sensitive quantitation in human plasma of R-(+)- and S-(−)-enantiomers of remoxipride. Remoxipride was extracted from basified plasma into hexane-methyl-tert.-butyl ether (20:80, v/v), washed with sodium hydroxide (1.0 M), then back-extracted into phosphoric acid (0.1 M). A structural analog of remoxipride was used as an internal standard. The sample extracts were chromatographed using a silica-based derivatized cellulose chiral column, Chiralcel OD-R, and a reversed-phase eluent containing 30–32% acetonitrile in 0.1 M potassium hexafluorophosphate. Ultraviolet (UV) absorbance detection was performed at 214 nm. Using 0.5-ml plasma aliquots, the method was validated in the concentration range 0.02-2.0 μg/ml and was applied in the investigation of systemic inversion of remoxipride enantiomers in man.     

Determination of succinylcholine in human plasma by high-performance liquid chromatography with electrochemical detection

An alternative HPLC method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine in human plasma is described. Drug spiked plasma and patient plasma samples were extracted using a C₁ solid-phase cartridge. Succinylcholine was separated on a Cyano column and quantitated using electrochemical detection at a potential of 450 mV and 750 mV. Mobile phase consisted of a mixture of phosphoric acid–acetonitrile–methanol (45:35:25) adjusted to an apparent pH of 5. Standard curves for the quantitation were linear in the range of 250–8000 ng/ml. Between-day and within-day relative standard deviations were 5.1% and 1.7%, respectively. Mean drug recovery and accuracy was 68% and 104%, respectively.     

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92–98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.     

Resolution of catecholic tetrahydroisoquinoline enantiomers and the determination of R- and S-salsolinol in biological samples by gas chromatography-mass spectrometry

Tetrahydroisoquinolines (TIQs) might be formed endogenously and can act centrally to promote a mechanism governing alcohol drinking behaviour. The possibility that biosynthesis occurs through a stereospecific enzymatic reaction is considered. Several TIQs were transformed into diastereomers by a two-step derivatization with N-methyl-N-trimethylsilyltrifluoracetamide and R-(−)-2-phenylbutyrylic acid and were analyzed by gas chromatography-mass spectrometry (GC-MS). High resolution of the TIQ enantiomers was achieved. This method was applied to the quantification of the enantiomers of salsolinol (SAL) in urine and plasma of healthy humans. Deuterated SAL was used as the internal standard. SAL was extracted from biological material using phenulboronic Deuterated SAL was used as the internal standard. SAL was extracted from biological material using phenylboronic phase cartridges and transformed into diastereomers. The sensitivity and specificity of the assay permit the determination of the enantiomeric composition of SAL in plasma and urine. The limit of quantification was found to be 100 pg/ml for each enantiomer. The described method has the advantage that optimal resolution of the SAL enantiomers without peak overlapping between analyte and other compounds can be achieved. Contrary to other findings, our GC-MS studies have demonstrated that endogenously formed SAL is racemic in plasma as well as in urine of healthy subjects.     

Determination of lipoic acid in human plasma by high-performance liquid chromatography with electrochemical detection

A selective and sensitive method for the determination of lipoic acid in human plasma samples has been developed. After enzymatic hydrolysis of the sample, the liberated lipoic acid was extracted by a solid-phase cartridge and measured by HPLC using electrochemical detection. The detection limit was 1 ng/ml lipoic acid in plasma. The calibration curve was non-linear in the range 0.01–50 μg/ml but could be described by a power function. The average extraction recoveries were 82.5 and 85.1% at the 25 and 2500 ng/ml levels, respectively. Coefficients of variation for both within-day and day-to-day analysis were between 2.1 and 9.4%. The assay method is sensitive, reproducible and suitable for disposition studies of lipoic acid in humans.     

Determination of R- and S-3-methyl-2-oxopentanoate enantiomers in human plasma: suitable method for label enrichment analysis

A sensitive method for the determination of S- and R-3-methyl-2-oxopentanoate enantiomers (KMV, (α-keto-β-methylval-erate) in physiological fluids suitable for isotope enrichment analysis is described: after extraction with acid, 2-oxo acids are separated from interfering amino acids by cation-exchange chromatography. Reductive amination of the branched-chain 2-oxo acids by use of - leucine dehydrogenase yields the corresponding -amino acids. -Isoleucine and -alloisoleucine which are formed from S- and R-3-methyl-2-oxopentanoate, respectively, are then quantified by amino acid analysis. The method was used for determination of the R-/S-3-methyl-2-oxopentanoate ratio in plasma of healthy subjects and patients with diabetes mellitus and maple syrup urine disease. Applicability for gas chromatographic-mass spectrometric analysis of ¹³C-label enrichment in plasma S-3-methyl-2-oxopentanoate is demonstrated.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C₁₈ column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 μl of the mobile phase. A 10-μl aliquot of the resulting solution is injected onto a 150 mm × 4.6 mm I.D. column packed with 5-μm octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E₁ = +0.25 V and E₂ = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90–95% which allows the determination of 0.5 μg/l analyte.     

Determination of etoposide in human plasma and leukemic cells by high-performance liquid chromatography with electrochemical detection

This paper describes a high-performance liquid chromatographic method with electrochemical detection for the determination of etoposide levels in plasma, total and non-protein bound concentration, and in leukemic cells. The precision for between-runs (n=6) was 7.0, 4.9, and 9.5%, the accuracy was 3.7, 7.1 and 6.3%, and within-runs precision (n=6) was 3.9, 2.9 and 5.1% for total plasma, non-protein bound plasma fraction and leukemic cells, respectively. The correlation coefficients (R²) were 1.00 for all calibration curves. These assays have been applied to analyze samples from one patient with acute myelogenous leukemia during 24 h after i.v. infusion of etoposide (100 mg/m²).     

Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH>8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5–50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.     

Determination of captopril in human blood by high-performance liquid chromatography with electrochemical detection

A new method for quantitation of captopril in human blood is described. Captopril was derivatized with N-(4-dimethylaminophenyl)maleimide into the electrochemically active adduct. The derivative was separated and determined by high-performance liquid chromatography with an electrochemical detector on a reversed-phase column. The proposed method was satisfactory for determination of captopril in whole blood with respect to accuracy and precision. The detection limit of captopril thereby obtained was 10 ng/ml. The blood levels of captopril in patients orally given an officinal dose were measured by the present method.     

Measurement of total plasma cysteamine using high-performance liquid chromatography with electrochemical detection

Cysteamine is currently used to treat children with the inherited disorder nephropathic cystinosis. A method for the quantitative determination of this aminothiol in human plasma is presented. Whole plasma was reduced with sodium borohydride to convert disulfides to thiols. Cysteamine was then separated by high-performance liquid chromatography and detected electrochemically. The recovery of standard cysteamine added to plasma was 96.6 +/- 1.9%. In a patient with cystinosis, an oral dose of cysteamine was absorbed rapidly, with plasma cysteamine reaching a maximum of 56 microM 1 h after the dose. By 1.8 h the plasma cysteamine concentration had decreased to one-half the maximum value.     

Determination of melatonin in rat pineal,plasma and retina by high-performance liquid chromatography with electrochemical detection

A sensitive method for the routine measurement of endogenous melatonin (MEL) in pineal, retina and plasma rat tissues has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Quantification limit for MEL was 0.2 ng/mg protein in pineal, 15 pg/ml in plasma and 2.0 pg/mg protein in retina. To improve both MEL quantification and the reproducibility of the assay, an internal standard was used when an extraction in organic solvent was required, in contrast with other available chromatographic methods. MEL values and the circadian profile obtained in this study from both rat pineal and plasma agree with those reported previously. This method allows MEL detection in mammal retina, particularly in rat, where MEL levels are very low.     

Determination of midazolam in human plasma by liquid chromatography with mass-spectrometric detection

A liquid chromatographic assay with mass-spectrometric detection was developed for the quantitative determination of the cytochrome p450 3A phenotyping probe midazolam in human plasma. Sample pretreatment involved a one-step extraction of 600 microl aliquots with ethyl acetate. Midazolam and the internal standard, lorazepam, were separated on a column (150 mm x 4.6mm, i.d.) packed with 5 microm Zorbax Eclipse XDB-C8 material, using a mobile phase composed of methanol and 10mM aqueous ammonium acetate (60:40, v/v). Column effluents were analyzed using mass-spectrometry with an atmospheric pressure chemical ionization source. Calibration curves were linear in the concentration range of 1.00-200 ng/ml. The accuracy and precision ranged from 92.8 to 112% and 0.056 to 13.4%, respectively, for four different concentrations of quality control samples analyzed in triplicate on eight separate occasions. The developed method was subsequently applied to study the pharmacokinetics of midazolam in a group of 35 human subjects at a single dose of 25 microg/kg.     

Determination of morphine in cerebrospinal fluid and plasma by high-performance liquid chromatography with electrochemical detection

Two methods for the extraction of morphine from cerebrospinal fluid or plasma with quantitation by high-performance liquid chromatography with electrochemical detection were compared for accuracy, precision and ease of preparation. One procedure was a standard extraction procedure and the other utilized a commercially available liquid—liquid extraction column. Both methods produced linear calibration curves over the concentration range of 1–200 ng/ml with coefficients of correlation of 0.999. Since the electrochemical detector is capable of detecting 20 pg of morphine, biological samples as small as 0.1 to 0.4 ml can be quantified with an average relative precision of 4.1 ± 3.9% over the concentration range 1–200 ng/ml. The potential clinical importance of the assay is demonstrated using a time course distribution study of morphine in the cerebrospinal fluid and plasma of a Rhesus monkey.     

Determination of benperidol and its reduced metabolite in human plasma by high-performance liquid chromatography and electrochemical detection

An isocratic high-performance liquid chromatographic method with electrochemical detection for the quantification of benperidol and its suggested reduced metabolite TVX Q 5402 in human plasma is described. The method included a two-step solid-phase extraction on reversed-phase and cation-exchange material, followed by separation on a cyanopropyl silica gel column (5 μm; 250 mm × 4.6 mm I.D.). The eluent was 0.15 M acetate buffer (pH 4.7) containing 25% acetonitrile (w/w). Spiperone served as internal standard. The inclusion of the cation-exchange step provided sample purity higher than those achieved with other methods. After extraction of 1 ml of plasma, concentrations as low as 0.5 ng/ml were detectable for both benperidol and the metabolite. In plasma samples collected from a schizophrenic patient treated with a single oral dose of 6 mg of benperidol, plasma levels of benperidol and of the metabolite could be measured from 20 min to at least 12 h after administration.     

Determination of nalbuphine in human plasma by high-performance liquid chromatography with electrochemical detection Application to a pharmacokinetic study

A high-performance liquid chromatographic procedure has been developed for the measurement of nalbuphine in plasma. Separation is performed on a reversed-phase analytical column (Ultrasphere ODS, 250 × 4.6 mm I.D., particle size, 5 μm). Mobile phase consisted of methanol-phosphate buffer (20:80, v/v) (pH 3.4). Electrochemical detection was performed using an ESA Coulochem II Model 5200 electrochemical detector equipped with a Model 5020 guard cell working at 550 mV and a Model 5021 analytical cell operating in the oxidation screening mode, with the potential of the first electrode set at 60 mV and the second electrode set at 450 mV. The method involves sample clean-up by liquid-liquid extraction using a chloroform-isopropanol mixture. After centrifugation, the organic phase was back-extracted with 17 mM phosphoric acid and then the aqueous phase was injected onto the column. The limits of quantitation and detection were 0.3 and 0.1 ng/ml, respectively. The extraction recovery was 91.1±3.7%. The intra- and inter-assay coefficients of variation were below 10%. Stability tests under various conditions have been performed. This method has been used to determine the pharmacokinetic parameters of nalbuphine in children.     

Assay of plasma catecholamines by liquid chromatography with electrochemical detection

A method was developed for the simultaneous assay of noradrenaline and adrenaline in 2 ml of human plasma. The method involves adsorption of the catechols onto alumina, desorption, lyophilizing, reconstitution, and injection into a reverse-phase ion-pairing liquid chromatography system. Sensitivity and selectivity are introduced using direct electrochemical detection of the column eluant.