Determination of L-3,4-dihydroxyphenylalanine in blood by high-performance liquid chromatography after solvent extraction

The use of high-performance liquid chromatography combined with solvent extraction for sample purification is described for the determination of L-3,4-dihydroxyphenylalanine in blood plasma. It is extracted into n-hexanol via complexation of its catechol moiety with diphenyl borate and ion-pair formation of its carboxylic group with tetrapentylammonium ion in an alkaline buffer. Under optimal extraction conditions, L-3,4-dihydroxyphenylalanine and 3,4-dihydroxybenzylamine used as an internal standard are extracted from blood plasma by a simple procedure and in a short time and then separated by reversed-phase ion-pair chromatography. The analytical recovery (100.8%) and reproducibility (coefficient of variation = 2.3% for n = 6) from plasma samples are good enough for routine analysis. L-3,4-Dihydroxyphenylalanine levels in blood can be monitored by this method after oral intake of the substance.     

Determination of dextromethorphan and metabolites in human plasma and urine by high-performance liquid chromatography with fluorescence detection

Sensitive methods were developed for the analysis of dextromethorphan (I) and two metabolites, (+)-17-methyl-morphinan-3-ol (II) and (+)-morphinan-3-ol (III), in plasma as well as dextromethorphan and three metabolites II, III and (+)-3-methoxymorphinan (IV) in urine using high-performance liquid chromatography followed by detection with a fluorometer. Dextromethorphan and its metabolites were extracted from plasma and urine and separated in the reversed-phase mode. The practical lower limits of determination for I, II, and III in plasma were 0.5, 5, and 5 ng/ml, respectively; for I, II, III, and IV in urine, the limits were 20 ng/ml, 0.6 microgram/ml, 0.5 microgram/ml, and 15 ng/ml, respectively. The linearity of the calibration graphs was excellent (r varied from 0.9994 to 0.9999) over concentration ranges of two orders of magnitude.     

Determination of oestrogens in pregnancy urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of three oestrogens (oestriol, oestrone and oestradiol) in pregnancy urine. Free oestrogens are extracted with chloroform from the urine sample. The phenolic group of each oestrogen in chloroform is formylated in the presence of an alkaline aqueous solution, and the resulting aldehyde is converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. In order to determine free and conjugated oestrogens, conjugated oestrogens are hydrolysed by heating in hydrochloric acid before the extraction and then treated in the same way as free oestrogens. The fluorescent derivatives are separated on a reversed-phase column, TSKgel ODS-120T, with stepwise gradient elution using an aqueous methanol-containing phosphate buffer (pH 2.2). The lower limit of detection for each oestrogen is ca. 200 fmol per 100-microliters injection volume.     

Determination of cisapride in human plasma by high-performance liquid chromatography with fluorescence detection

Summary A new sensitive HPLC-FLD method has been developed and validated for the determination of cisapride in human plasma for a bioequivalence study. A gradient method was used to remove late-eluting plasma components of no interest. The separation was performed on a Li-ChroCART 250-4 Purospher RP-18 (5 μm particle) analytical column fitted with a LiChroCART 4-4 Purospher RP-18 endcapped (5 μm particle) guard column. The excitation and emission wavelengths were 295 and 350 nm during fluorescence detection. The calibration plot was linear in the range of 5–200 ng mL⁻¹. A demethoxy analogue of cisapride was used as internal standard.     

Determination of 21-hydroxycorticosteroids in human urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method with fluorescence detection for the determination of nineteen 21-hydroxycorticosteroids is described. The corticosteroids are oxidized by cupric acetate to form the corresponding glyoxal derivatives. The derivatives are converted into fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-dicarbonyl compounds. The quinoxalines are separated within 70 min on a reversed-phase column (TSK gel ODS-120T) by stepwise elution with mixtures of methanol, acetonitrile, and 1.0 M ammonium acetate. The detection limits are 0.14-29.4 pmol at a signal-to-noise ratio of 3 in a 50-microliter injection volume. This sensitivity permits precise determination of hydrocortisone, cortisone, corticosterone, and their tetrahydro derivatives in 500 microliters of normal human urine.     

Determination of amidepin in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of amidepin has been developed. The method is based on the extraction of alkaline plasma with diethyl ether-dichloromethane, and the injection into the Supelcosil LC-18 column of the evaporated and reconstituted organic phase. After separation, detection is carried out by a fluorescence detector (excitation at 195 nm with no filter). The limit of detection is 10 ng/ml of plasma. The mean coefficient of variation is 12%. The plasma levels after oral administration and after intravenous administration are shown.     

Determination of orbifloxacin in rabbit plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive high-performance liquid chromatography method is developed for the determination of orbifloxacin (ORB) in rabbit plasma. Sample preparations are carried out by adding phosphate buffer (pH 7.4, 0.1 M) and extracting with trichloromethane. ORB and the internal standard, norfloxacin (NOR), are separated on a reversed-phase column using an aqueous phosphate buffer-acetonitrile (80:20, v/v) mobile phase. The concentrations of ORB and NOR eluting from the column with retention times of 2.16 and 3.09 min, respectively, are monitored by fluorescence detection at 338 (excitation) and 425 nm (emission). The method is shown to be linear from 4 to 1500 ng/mL (regression coefficient r2 = 0.999). The quantitation and detection limits are 4 and 9 ng/mL, respectively. Mean recovery is determined as 92% by the analysis of plasma standards containing 150, 750, and 1500 ng/mL. Inter- and intra-assay precisions were 4 and 3%, respectively.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with UV-Vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV-Vis detection has been developed and validated for the determination of vigabatrin (VG) in human plasma and urine. The samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A good chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) gradient elution. Tranexamic acid was used as an internal standard (I.S.). The method was linear over the concentration range of 0.8-30.0 microg/ml for both samples. The method is precise (relative standard deviation (R.S.D.) <9.13%) and accurate (relative mean error (RME) <-8.75%); analytical recoveries were 81.07% for plasma and 83.05% for urine. The assay was applied to pharmacokinetic study in a healthy volunteer after a single oral administration of 1 g of vigabatrin.     

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.     

Determination of midodrine in human plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive fluorometric high-performance liquid chromatographic method was developed for the determination of midodrine in human plasma. After liquid-liquid extraction from plasma, the drug and 2-phenylglycinol (internal standard) were convened into the corresponding fluorescent derivatives by reaction with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives were separated within 30 min on a reversed-phase column using isocratic elution with acetonitrile-methanol-water (10:30:60, v/v) and were detected spectrofluorometrically at 485 nm with excitation at 400 nm. The detection limit for midodrine was 0.3 pmol (76 pg) per mL plasma at a signal-to-noise ratio of 3.     

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 pindolol in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

This paper presents a rapid, simple and economical method for assaying pindolol concentrations in plasma and urine by high-performance liquid chromatography using ultraviolet detection. It is sensitive enough for use in single-dose pharmacokinetic studies and may also be used to determine pindolol concentrations in the plasma from patients taking the drug, provided that the patient is not taking any of the drugs which interfere with the method. Drugs which were found to interfere with the pindolol peak are quinidine, n-acetylprocainamide and lidocaine. Disopyramide, oxprenolol and levobunolol interfered with the internal standard peak.     

Determination of morphine and 6-acetylmorphine in plasma by high-performance liquid chromatography with fluorescence detection

A method is described for the simultaneous determination of morphine and 6-acetylmorphine in small volumes of human plasma by normal-phase high-performance liquid chromatography using solid-phase extraction, dansyl derivatisation and fluorescence detection. The lower limits of quantitation in a 0.1-ml plasma sample are 10 ng/ml for morphine and 25 ng/ml for 6-acetylmorphine. The method has been applied to determine concentrations of morphine and 6-acetylmorphine in plasma samples from premature babies administered an intravenous infusion of diamorphine.