Routine measurement of plasma catecholamines in clinical pharmacology by high-performance liquid chromatography with electrochemical detection

A method for the simultaneous determination of epinephrine, norepinephrine and dopamine in human plasma is described, which combines the advantages of liquid-liquid extraction sample preparation, high-performance liquid chromatography on weak cation-exchange stationary phases and dual-electrode coulometric detection. The limits of quantification are less than 5 pg/ml (at a signal-to-noise ratio greater than 5) for each analyte. The influence of various experimental parameters (e.g., composition of the mobile phase, pretreatment of the assay buffer, components of the re-extraction system) on the performance of the assay is reported in detail. A number of applications are presented, which demonstrate the quality of the data obtained in terms of sensitivity, reproducibility and significance.     

Measurement of plasma catecholamines by high-performance liquid chromatography with electrochemical detection in intensive care patients after dobutamine infusion

A procedure for the determination of plasma catecholamine concentrations in critical care patients after dobutamine infusion is presented. A modified chromatographic system is required with an additional washing procedure to achieve maximum sensitivity and stable chromatographic conditions. The influence of storage time on the catecholamine concentrations of plasma samples is reported in detail. A time-dependent decrease in catecholamine concentrations of up to 12 and 39% was found within two and ten months, respectively.     

Routine determination of urinary free catecholamines by high-performance liquid chromatography with electrochemical detection

A simple and reliable high-performance liquid chromatographic method is described for the routine determination of the free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines are isolated from urine samples using small affinity chromatography columns prepacked with immobilised m-aminophenylboronic acid, separated by ion-pair reversed-phase liquid chromatography and quantified by electrochemical detection. Total analysis, including sample preparation time, is achieved in less than 30 min with analytical recoveries of 92-96% for all three catecholamines. Long-term stability and reproducibility of the liquid chromatographic system is attained by selection of optimised conditions for chromatographic separation with a formate mobile phase and produces detection limits of 1.4, 1.8 and 2.2 nmol/l for norepinephrine, epinephrine and dopamine, respectively, in urine samples and day-to-day coefficients of variation of less than 6%. Furthermore, the affinity isolation gels can be reused a minimum of ten times providing a rapid and cost-effective means of sample preparation.     

Neurochemical studies on catecholamines and indoleamines by high-performance liquid chromatography with electrochemical detection

Summary This communication reports the HPLC separation and quantitative ECD assay of dopamine (DA) and its metabolites DOPAC and HVA, 5-HT and its metabolite 5-HIAA and the noradrenaline metabolites MHPG and VMA, in samples of rat brain extracts and human CSF. The separation is carried out by reversed-phase with a methanolic phosphate/citrate buffer as mobile phase. Response is linear within 10pg-20 ng. Rat brain homogenates of cortex plus striatum were centrifuged and 10–20 l aliquots injected in the column. CSF samples were directly injected without any further manipulation. The method has been applied to the study of the possible neuromodulating role of T on the catecholaminergic and serotonergic transmission. For this purpose rats are injected intraperitoneally (ip) with T (150 mg/kg) and killed after 30 min. Relative to control rats, the results show that for n=12, T does not affect the basal level of DA and DOPAC whereas HVA increases a 99.3% and 5HT and 5HIAA show variations of 23% and — 4.1%, respectively. Aside from the fall of 5HIAA, it is interesting to note that the turnover rate of 5HT decreases, which might prove of functional significance.Presented at the 14th International Symposium on Chromatography London, September, 1982     

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

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitative determination of diclofenac potassium in plasma was developed. Naproxen was used as the internal standard. The drug and internal standard were isolated from plasma by extraction with dichloromethane and 2 M hydrochloric acid. Chromatographic separation was performed on a C18 column with methanol-water (68:32, v/v) adjusted to pH 3.2 with phosphoric acid as mobile phase. The oxidation potential for detection was established by constructing a voltammogram for diclofenac. The quantification limit for diclofenac in plasma was 5 ng mL(-1). Linearity of the method was confirmed in the range 5-2000 ng mL(-1), correlation coefficient 0.9998. Within-day relative standard deviations (RSDs) ranged from 0.66 to 14.00% and between-day RSDs from 0.59 to 15.78%. The method was successfully applied for the determination of pharmacokinetic parameters after ingestion of a 50 mg dose of diclofenac. Studies were performed on 18 healthy volunteers of both sexes.     

固相萃取-高效液相色谱电化学法检测大鼠血浆儿茶酚胺

建立了一种Oasis HLB固相萃取-高效液相色谱（HPLC）电化学检测大鼠血浆儿茶酚胺（CAs）的方法。血浆样本在形成二苯基硼酸-儿荼酚胺复合物后经优化的固相提取技术,得到较高样本回收率。以Atlantis C18色谱柱为固定相,确定了各种影响色谱的参数,如流动相组成、pH范围及检测器的设定。儿茶酚胺所有组分肾上腺素（E）、去甲肾上腺素（NE）和多巴胺（DA）的平均提取回收率在90％～95％之间。E、NE和DA的质量浓度在0．25～30ng／mL时与峰面积呈良好的线性关系（r值分别为0．9989,0．9992和0．9984）;检出限为0．4pg。该法灵敏、准确、重现性好、结果可靠。     

Quantitation of melphalan in plasma of patients by reversed-phase high-performance liquid chromatography with electrochemical detection

An analytical procedure for the separation and determination of melphalan in human plasma was carried out. A simple high-performance liquid chromatographic method with electrochemical detection was developed taking advantage of the high sensitivity of the electrode redox reaction. The sample pretreatment consisted of a direct extraction of the interferents rather than of melphalan, owing to the difficulty of extraction of the drug, and was very simple, rapid and reproducible.     

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

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of exifone in human plasma and urine. Exifone was extracted from acidified plasma or neutralized urine with diethyl ether and the evaporated extracts were analysed on a C18 reversed-phase column. The compound was eluted in about 8 min with acetonitrile-0.3 M orthophosphoric acid (15:85, v/v) at a flow-rate of 0.9 ml/min. This method gave accurate and reproducible results; the calibration graphs were linear (r greater than 0.99) over the range of 2.8-360 nmol/l for plasma and 0.18-36 mumol/l for urine, and concentrations as low as 1 nmol/l in plasma could be quantified. These results allowed this assay to be used for determinations in single-dose pharmacokinetic studies.     

Determination of cabergoline in plasma and urine by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of cabergoline in plasma and urine has been developed. After buffering plasma and urine samples, cabergoline was extracted with a methylene chloride-isooctane mixture, back-extracted into 0.1 M phosphoric acid, then analysed by reversed-phase high-performance liquid chromatography. Quantitation was achieved by electrochemical detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the biological matrices (human plasma and urine) was observed. The assay was still inadequate in terms of sensitivity for the quantitation of cabergoline plasma concentrations after a single oral dose of 1 mg of the drug to humans, but was successfully used in the determination of the urinary excretion of the drug.     

Gunshot residue determination by high-performance liquid chromatography with electrochemical detection

A procedure for the detection of gunshot residue via the organic constituent diphenylamine is described. The method incorporates high-performance liquid chromatography with electrochemical detection.