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 by high-performance liquid chromatography of hydroxyurea in human plasma

An assay using reversed-phase high-performance liquid chromatography with electrochemical detection was developed to measure hydroxyurea in plasma at concentrations suitable for pharmacokinetic studies. The sample preparation is simple, the analysis rapid and assays can be batched. The between-run precision is excellent (coefficient of VARIATION = 2.8–4.5%) and the limit of detection is 0.02 mmol/l. Preliminary studies have shown that the method is suitable for pharmacokinetic studies.     

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.     

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 5-methyltetrahydrofolic acid in plasma and spinal fluid by high-performance liquid chromatography,using on-column concentration and electrochemical detection

Chromatographic conditions for the determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid are described, involving simple pretreatment of the sample. Electrochemical detection was used. The linear range of the method is more than 10³. Recovery from plasma and spinal fluid is 100%, and the detection limit of the method is 2·10^?9M, sufficient for the detection of endogenous plasma and spinal fluid levels. The detection conditions are discussed. Endogenous concentrations of the compound in plasma and spinal fluid were determined and correlated with a folate bioassay. Plasma concentrations have been shown after the administration of leucovorin which is used in anticancer therapy.     

Determination of amikacin in cerebrospinal fluid by high-performance liquid chromatography with pulsed electrochemical detection

A highly sensitive and fast reversed-phase liquid chromatographic (LC) method combined with pulsed electrochemical detection (PED) was developed for the direct quantification of the aminoglycoside antibiotic amikacin in cerebrospinal fluid (CSF). The limit of quantification obtained was 0.06 microg/ml and linearity was established over the concentration range 0.06-4.00 microg/ml. The recovery was found to be close to 100%. This method was developed in order to study CSF pharmacokinetics of amikacin in neonates. The narrow therapeutic range calls for monitoring to ensure optimal therapy and to minimize the risk of toxic side effects such as nephro- and ototoxicity, especially in populations like preterm neonates at birth, where the predictability of amikacin clearance is limited. Typical problems to be solved were the low amikacin concentrations and the limited sample volume of CSF.     

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.     

Quantification of doripenem in human plasma and peritoneal fluid by high-performance liquid chromatography with ultraviolet detection

A simple and rapid HPLC method that includes ultrafiltration to remove plasma and peritoneal fluid protein was developed to determine doripenem concentrations in human plasma and peritoneal fluid. Doripenem was stabilized by immediate mixing of the plasma or peritoneal fluid with 1M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Doripenem and an internal standard were detected by measuring their ultraviolet absorbance at 300 nm. The calibration curves for doripenem in human plasma and peritoneal fluid were linear from 0.05 to 100 microg/mL. For plasma, both the intra- and the interday precision were less than 3.41% (CV), and the accuracy was between 97.4 and 101.7% above 0.05 microg/mL. For peritoneal fluid, the intra- and the interday precision were less than 2.98% (CV), and the accuracy was between 94.4 and 103.9% above 0.05 microg/mL. The limit of detection was 0.02 microg/mL in both plasma and peritoneal fluid. The assay has been applied to the therapeutic drug monitoring of doripenem in both plasma and peritoneal fluid.     

Determination of non-protein-bound iron in human synovial fluid by high-performance liquid chromatography with electrochemical detection

Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron—diethylenetriaminepentaacetic acid (DPTA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 μM Fe(III) to be determined with an injection volume of 10 μl. A mixture of synovial fluid, 20 μM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O⁻₂) generated by a xanthine—xanthine oxidase system and was ultrafiltered through a 30 000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O⁻₂ and decreased pH, iron may be released into the synovial fluid.     

Determination of thiols and disulfides using high-performance liquid chromatography with electrochemical detection

Low-molecular-mass thiols, such as glutathione (GSH), and their associated disulfides are ubiquitous in nature, and based upon the many known functions of these compounds, their identification and accurate measurement is essential. Our objectives were to develop a simple method for the simultaneous measurement of thiols and disulfides in biological samples using HPLC with dual electrochemical detection (HPLC-DED). Particular emphasis was placed on the applicability to a wide variety of important GSH-related thiols and disulfides, including γ-Glu-Cys, Cys-Gly, their disulfides, and the mixed disulfide of glutathione and cysteine (CSSG), validation on different types of biological samples, maintenance of chromatographic resolution and reproducibility with routine and extended use, and enhancement of assay sensitivity. To this end, optimal HPLC conditions including mobile phase, column, and electrode polishing procedures were established and the method was applied to, and validated on a variety of biological samples. This improved methodology should prove to be a useful tool in studies on the metabolism of GSH and other thiols and disulfides and their role in cellular homeostasis and disease processes.     

Determination of (+)-catechin in plasma by high-performance liquid chromatography using fluorescence detection

A high-performance liquid chromatographic method, using fluorescence detection, was developed for the determination of (+)-catechin in rabbit plasma. The procedure involved the precipitation of plasma protein using acetonitrile, followed by solid-phase adsorption onto alumina. After washing with water and methanol, the residue was vortex-mixed with perchloric acid solution to release the adsorbed (+)-catechin. Separation was performed on a reversed-phase column using an eluent consisting of phosphoric acid solution with 12% acetonitrile. The excitation and emission wavelengths were set at 280 and 310 nm, respectively. The retention times for (+)-catechin and the internal standard (deoxyhigenamine) were 6.87 and 8.47 min respectively, without any interference. Validations of accuracy and precision were satisfactory in both within- and between-run assays. All coefficients of variance were less than 6% and mean relative errors were within ± 3.75%. The average recovery was 73.77%. The limit of detection and quantitation were 1 ng and 0.02 μg/ml, respectively. Application of this method was successfully assessed by intravenous administration of a 15 mg/kg dose of (+)-catechin in rabbits. This new method provides a simple, specific and sensitive determination for (+)-catechin in rabbit plasma and is suitable for pharmacokinetic studies.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution–acetonitrile (80:20). The fluorescence of the column effluent was monitored at λ_ex=338 and λ_em=425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r²=0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

Determination of olanzapine in plasma by high-performance liquid chromatography using ultraviolet absorbance detection

A rapid method for the determination of olanzapine in plasma using high-performance liquid chromatography with ultra violet detection is described. Olanzapine was extracted from plasma with a mixture of hexane/dichloromethane (85:15), and then back extracted into phosphate buffer pH 2.8. Separation was achieved on a RP Select B C(18) column and commonly administered drugs did not interfere with the assay. The limit of quantitation was 1.5 microg/l and the inter-day and intra-day relative standard deviations were less than 10%. Olanzapine was shown to be stable in plasma for up to 7 days when stored at 4 degrees C. Moreover, the addition of ascorbic acid was not necessary for the achievement of chemical stability during storage, or during the assay procedure. The method has been used to measure olanzapine concentrations in patients treated with various doses of the drug varying from 5 to 40 mg/day.     

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 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.