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.     

Measurement of carvedilol in plasma by high-performance liquid chromatography with electrochemical detection

Carvedilol is a beta/alpha1-adrenoceptor blocker. A sensitive method for measuring plasma levels of carvedilol in human administrated low doses is needed since its plasma concentration is low. We measured carvedilol and carvedilol M21-aglycon using high-performance liquid chromatography (HPLC) with electrochemical detection. The amperometric detector was operated at 930 mV versus Ag/AgCl. Mean coefficients of variation (n = 5) for carvedilol and M21-aglycon were 4.0 and 7.7% (intra) and 6.1 and 6.7% (inter), respectively. The lower limit of quantification for each analyte was 0.10 ng/ml (signal-to-noise ratio = 3). This lower limit of quantification for carvedilol was sufficient for clinical use.     

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.     

Pineal and plasma melatonin as determined by high-performance liquid chromatography with electrochemical detection.

A rapid and sensitive method for the routine quantitative determination of melatonin in pineal and plasma is described. The assay used reversed-phase high-performance liquid chromatography (RP-HPLC) separation combined with either amperometric (system A) or coulometric (system B) detection. The method gave satisfactory reproducibility and accuracy, and detection limits for melatonin were as low as 8.5 pg (system A) and 1 pg (system B). This high sensitivity, together with the short analysis time (less than 10 min), and the simplicity of sample procedure make the present RP-HPLC method suitable for a wide range of studies concerning melatonin measurements. Melatonin values obtained in this study from both rat pineal and human plasma agree with those reported previously, and clearly determined a circadian pattern.     

High-performance liquid chromatography with electrochemical detection for the determination of 7-monohydroxyethylrutoside in plasma

MonoHER (7-monohydroxyethyl rutoside) is a semisynthetic flavonoid, which can be used as a modulator for doxorubicin-induced cardiotoxicity. To study the pharmacokinetics of monoHER in mice and human an HPLC procedure was developed to measure the level of monoHER in plasma. After extraction of monoHER with methanol, the supernatant was equally diluted (v/v) with 25 mM phosphate buffer (pH 3.33). This solution was analysed by HPLC, using a reversed-phase ODS column, with a mobile phase consisting of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogen phosphate (pH 3.4), 10 mM acetic acid and 36μM EDTA. The retention time of monoHER was about 5.2 min. The lower limit of quantification of monoHER was set at 0.3 μM and the calibration line was linear up to 75 μM. The within-day accuracy and precision of the quality control samples (0.45, 1.0, 10 and 40 μM) were better than 15 and 13%, respectively. The between-day accuracy and precision were less than 3, 20%, respectively. The recovery of monoHER (using quality control concentrations) was concentration independent and ranged from 90.5 to 95.3% except for the lowest quality control, 0.45 μM, of which the recovery was 85%. The concentration of monoHER in plasma decreased with 10% when stored at −80°C for one month and with 20% when stored at −20°C for 3 weeks. The repeated injection of monoHER in aliquots of 10 μM, stored in the autosampler tray (4°C), showed a consistent decrease during a run: 15% over 24 h. To compensate for this decrease, sample duplicates were analysed in a mirror image sequence.     

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 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 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.     

Whole blood serotonin and plasma tryptophan using high-pressure liquid chromatography with electrochemical detection

Methods are detailed for whole blood serotonin (5-HT), equivalent to platelet 5-HT, and plasma tryptophan. These assays may be carried out on the same blood sample which need be no more than 1 ml, are rapid, and avoid the difficulties, such as protein precipitation and fluorophor formation, encountered in other methods. The linearity of the methods is extensive and allows for accurate measurement of low concentrations. Data are given for normal humans and for patients receiving psychiatric treatment for depression.     

Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection

A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (%C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma.     

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.     

Sensitive determination method of estradiol in plasma using high-performance liquid chromatography with electrochemical detection

The improvement in the sensitive determination method of estradiol using HPLC with electrochemical detection is described. The improvement was due to the optimization of the potential applied to the electrode of the analytical cell and employment of a guard cell. The detection conditions were optimized from the electrochemical properties of estradiol in acidic and alkaline eluents. The employment of the guard cell drastically decreased the background noise without any reduction in the response of estradiol, and contributed to improvement in the sensitivity. The optimized method combined with pretreatment by liquid-liquid extraction was applied to the determination of estradiol in rat plasma. The detection limit of 8 pg for the standard solution and 24 pg for the plasma sample, which was about 6-8-fold more sensitive compared to the previous reports, was attained.     

Stability of apomorphine in plasma and its determination by high-performance liquid chromatography with electrochemical detection

Dilute solutions (50 ng/ml) of apomorphine in plasma are unstable at 37°C and pH 7.4. The chemical half-life is only 39 min. Mercaptoethanol (0.01%) is effective in stabilizing these samples while sodium metabisulphite (1%), which is generally used, is not effective. Biological samples are extracted with diethyl ether (recovery 96.5%) and analysed using HPLC with coulometric detection (oxidation potential 0.25 V). The stationary phase employed was C₁₈ material (4 μm) and the mobile phase was phosphate buffer (pH 3)—acetonitrile (70:30, v/v). The flow-rate was 1.8 ml/min. This bioanalytical method presents a reliable tool for pharmacokinetic studies in man.     

A sensitive method for the determination of plasma catecholamines using liquid chromatography with electrochemical detection

Simple and sensitive methods for the determination of plasma catecholamines are of great interest since the level of catecholamines in plasma reflects the activity of the sympatho-adrenal system. In the present work a previously described procedure based on high pressure liquid chromatography with electrochemical detection has been adapted for assay of plasma catecholamines. This method permits simultaneous detection of noradrenaline, adrenaline and dopamine in concentrations down to 0.1 nmol/1 in less than one ml plasma.     

Measurement of free and bound 5-hydroxytryptophan in plasma by liquid chromatography with electrochemical detection

A new method for the measurement of concentrations of 5-hydroxytryptophan (5HTP) in plasma is described. This method, which uses high-performance liquid chromatography with electrochemical detection, was used to measure 5HTP in plasma of patients with the carcinoid syndrome, and also in the plasma of rats after injection of 5HTP (30 mg/kg). A significant proportion of 5HTP was bound to macromolecules in plasma both in the rats and in the patients.     

Neurochemical applications of liquid chromatography with electrochemical detection

Liquid chromatography with electrochemical detection (LCEC) has been shown to have unique advantages for the determination of many substances of neurochemical interest. The technique is rapid, sensitive, and relatively inexpensive. In addition, it avoids the need for radiolabelled substances, the formation of volatile derivatives, or reactions which generate fluorescent products. LCEC is widely used for the measurement of the catecholamines and their metabolites and has recently gained acceptance for determination of the neurochemically important tryptophan metabolites. The method is also capable of assessing the activity of a number of neurologically important enzymes. The review which follows is intended to provide a brief overview of the LCEC technique and a guide to recent literature exemplifying its neurochemical applications.     

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.