Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection.

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid-liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3-0.8 pg injected) and reproducible (coefficients of variation 1-10%), and shows good accuracy (93-98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

Simultaneous determination of several amino acids, including homocysteine, cysteine and glutamic acid, in human plasma by isocratic reversed-phase high-performance liquid chromatography with fluorimetric detection

A method for the simultaneous measurement of two biologically important thiol compounds cysteine and homocysteine and five amino acids including neurotransmitters aspartate and glutamate is reported. This method utilized derivatization of compounds with o-phthalaldehyde in the presence of 2-mercaptoethanol following alkylation of the free sulfydryl group with iodoacetic acid followed by separation using reversed-phase high-performance liquid chromatography. These o-phthalaldehyde-2-mercaptoethanol-labeled compounds were separated within 30 min on a Spherisorb ODS-2 column with isocratic elution using 17% methanol, 0.04 M sodium phosphate buffer (pH 7.0), 0.002 M Na2EDTA and detected fluorimetrically (excitation 340 nm, emission 450 nm). Using this method, the concentrations of homocysteine, cysteine, glutamic acid. aspartic acid, asparagine, serine and glutamine in human plasma were determined.     

Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine-N-oxide in human plasma has been developed and validated. The isocratic HPLC assay uses a mobile phase consisting of an acetonitril-buffered aqueous solution containing 146 microL of triethylamine and 200 microL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydroxide solution (400:600, v/v) at a flow-rate of 0.8 ml/min and a Lichrospher 100 RP-18 reversed-phase column and UV detection at 215 nm. Doxepine was used as the internal standard. Mean recoveries for clozapine, norclozapine, clozapine-N-oxide and doxepine were 95%, 98%, 96% and 94%, respectively, whereas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1.8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concentration range of 100-1600 ng/mL was 0.9998 and 0.9997, respectively; for clozapine-N-oxide (20-200 ng/mL) it was found to be 0.9986. The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, norclozapine and clozapine-N-oxide, respectively.     

Simultaneous determination of itraconazole and hydroxyitraconazole in human plasma by high-performance liquid chromatography

The development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of itraconazole and its metabolite, hydroxyitraconazole, in human plasma is described. The method involved liquid-phase extraction of itraconazole and hydroxyitraconazole using a hexane-dichloromethane (70:30) mixture, after addition of loratidine as an internal standard (IS). Separation was achieved with a reversed-phase C18 column (250 mm x 4.6 mm) employing fluorescence detection (excitation: 264 nm, emission: 380 nm). The mobile phase consisted of [0.01% triethylamine solution adjusted to pH 2.8 with orthophosphoric acid-acetonitrile (46:54)]-isopropanol (90:10, v/v) at a flow rate of 1.0 ml/min. For both the drug and metabolite, the standard curve was linear from 5.0 to 500 ng/ml with goodness of fit (r2) greater than 0.98 observed with four precision and accuracy batches during validation. An observed recovery was more than 70% for drug, metabolite and internal standard. The applicability of this method to pharmacokinetic studies was established after successful application during 35 subjects bioavailibity study. The method was found to be precise, accurate and specific during the study.     

Simultaneous determination of flucytosine and fluorouracil in human plasma by high-performance liquid chromatography

A validated, sensitive and precise reversed-phase high-performance liquid chromatographic method for the simultaneous determination of 5-flucytosine (5-FC) and 5-fluorouracil (5-FU) in human plasma is described. Two compounds, 5-methylcytosine (5-MC) and 5-chlorouracil (5-CU), were used as internal standards for the determination of 5-FC and 5-FU, respectively. Plasma samples were deproteinized with trichloroacetic acid and chromatographed on an octylsilica column, maintained at 30 degrees C during elution, using a 0.04 M phosphate buffer, pH 7.0, as eleunt. Spectrophotometric diode array detection was used at 266 nm. 5-FC, 5-FU, 5-MC and 5-CU were found to have retention times of 4.8, 5.8, 7.7 and 11.0 min respectively. Recoveries of 91-120% with reproducibility and repeatability coefficients of variation of 0.8-6% were obtained. Mean correlation coefficients of 0.99989 and 0.9995 were found for the linear calibration curves (n = 2) of 5-FC (4.816-192.6 mg/l) and 5-FU (0.05368-5.368 mg/l), respectively. The limits of quantitation were 0.3 mg/l for 5-FC and 0.05 mg/l for 5-FU.     

Simultaneous determination of rofecoxib and celecoxib in human plasma by high-performance liquid chromatography

An innovative reversed-phase high-performance liquid chromatographic method is validated for the simultaneous determination of rofecoxib and celecoxib in human plasma. The internal standard is 4-n-pentyl-phenyl-acetic acid. Good chromatographic separation is achieved using a Zorbax SB-CN (5 microm) analytical column operated at room temperature and mobile phase consisting of acetonitrile and water containing 0.1M potassium dihydrogen orthophosphate buffer adjusted to pH 2.4 with 85% orthophosphoric acid (42:58, v/v). UV detection is performed at 254 nm, and the flow rate is maintained at 1.0 mL/min. Plasma samples are extracted into an organic solvent (1-chlorobutane) and evaporated under an air flow. The calibration curve for rofecoxib is linear over the range of 10 to 500 microg/L, and the celecoxib calibration curve is linear over the range of 20 to 2000 microg/L. The lower limit of quantitation for rofecoxib and celecoxib is 10 and 20 microg/L, respectively, using 1.0 mL of human plasma. The validation data show that the assay is sensitive, accurate, specific, and reproducible for the determination of rofecoxib and celecoxib. This method is therefore appropriate for pharmacokinetic studies to quantitate these therapeutic agents in patients with arthritis conditions.     

Measurement of vitamin K in human liver by gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection

A sensitive and precise method for measuring endogenous phylloquinone (K1) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K1 and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n = 10) and cirrhosis (n = 22) were significantly lower than in normal livers (n = 6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.     

Determination of alpidem and its metabolites in human plasma by high-performance liquid chromatography and fluorimetric detection

A high-performance liquid chromatographic method has been developed for the simultaneous determination of alpidem and its metabolites in human plasma. The method involved a single extraction of the parent drug and metabolites into diethyl ether from alkalinized plasma, evaporation of the organic solution and chromatography of the extracts on a C18 column coupled to a fluorimetric detector. An internal standard was used for the quantitative determination of the compounds. The method was selective for alpidem and three of its metabolites and has a limit of detection of less than 1 ng ml-1 for all the compounds. Since the chromatographic run took more than 20 min, the chromatographic process was fully automated and performed overnight.     

Assay for dopamine beta-hydroxylase in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection

A highly sensitive assay for dopamine beta-hydroxylase activity in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection is described. Norepinephrine, formed enzymatically from the substrate dopamine, and epinephrine (internal standard), after clean-up with a cation-exchange cartridge (Toyopak IC-SP M), are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-80TM. The detection limit for norepinephrine formed enzymatically is 5 pmol per assay tube.     

Simultaneous determination of lamotrigine, zonisamide, and carbamazepine in human plasma by high-performance liquid chromatography

A high-performance liquid chromatography assay with ultraviolet detection was developed for the simultaneous determination of the anti-epileptic drugs lamotrigine, carbamazepine and zonisamide in human plasma and serum. Lamotrigine, carbamazepine, zonisamide and the internal standard chloramphenicol were extracted from serum or plasma using liquid-liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 1-30 microg/mL for lamotrigine, 2-20 microg/mL for carbamazepine, and 1-40 microg/mL for zonisamide. Within- and between-run precision studies demonstrated coefficient of variation <10% at all tested concentrations. Other anti-epileptic medications tested did not interfere with the assay. The method is appropriate for determining lamotrigine, carbamazepine and zonisamide serum or plasma concentrations for therapeutic monitoring.     

Simultaneous determination of vinburnine and 6-hydroxyvinburnine in human plasma by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method has been developed to allow the simple and rapid determination of both vinburnine (I) and its main metabolite, 6-hydroxyvinburnine (II), in heparinized human plasma (0.5 ml). Compounds I and II and p-chlorodisopyramide (internal standard) were first extracted with alkalinized ethyl acetate and then with sulphuric acid. Separation was achieved on a reversed-phase muBondapak C18 column with a mobile phase of acetonitrile-water-0.1 M heptanesulphonate in acetic acid and with detection at 254 nm. Each run required 20 min. The within-day coefficients of variation for identical samples (20 ng/ml) were 7 and 6% and between-day coefficients of variation 8 and 26% for I and II, respectively. The detection limit was 5 ng/ml (normal therapeutic concentration, 10-300 ng/ml). The application of the method to drug monitoring was compared to that of a thin-layer chromatographic procedure.     

Separation and sensitive determination of i-urobilin and 1-stercobilin by high-performance liquid chromatography with fluorimetric detection.

i-Urobilin and 1-stercobilin were separated by high-performance liquid chromatography on a reversed-phase octadecylsilane-bonded column and detected fluorimetrically through formation of phosphor with zinc ions in the eluent. The separation and the intensity of the fluorescence response were affected by concentrations of zinc acetate and sodium borate buffer, pH and methanol content in the eluent. The optimal eluent used consisted of 0.1% zinc acetate in 75 mM boric acid buffer (pH 6.0)-methanol (25:75). The detection limit was 0.2 microgram/l for both i-urobilin and 1-stercobilin (signal-to-noise ratio 2), which makes the method 250-2500 times more sensitive than conventional methods.     

Determination of dopamine-3- and 4-O-sulphate in human plasma and urine by anion-exchange high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method is reported for the determination of dopamine-3- and -4-O-sulphate isomers in human plasma and urine using an anion exchanger coupled with post-column hydrolysis and fluorimetric detection. Samples of plasma or urine are partially purified on Dowex 1 and Dowex 50 columns and separated using HPLC. These compounds are then hydrolysed and determined automatically by the p-aminobenzoic acid method in a continuous-flow reaction system. As the p-aminobenzoic acid method is very specific for dopamine, it is also possible to determine the isomers by injecting 5-20 microliter of urine or 100-200 microliter of deproteinized plasma directly into the HPLC system without clean-up. The detection limit of the method for both isomers is 0.3 pmol. In normal subjects, the plasma levels of dopamine-3- and -4-O-sulphate are 26.5 (S.D. 11.1) and 2.68 (S.D. 0.34) pmol/ml, and their urinary excretion rates are 1.73 (S.D. 0.56) and 0.27 (S.D. 0.04) nmol/min, respectively. Thus the two isomers are present in both plasma and urine and their urinary excretions reflect directly their plasma levels.     

Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection

A selective, accurate and precise high-performance liquid chromatographic assay coupled to fluorescence detection was developed for the detection of some angiotensin II receptor antagonists (ARA II): Losartan, Irbesartan, Valsartan, Candesartan cilexetil and its metabolite Candesartan MI. The analytes and the internal standard (bumetanide, a high-ceiling diuretic) were extracted from plasma under acidic conditions by means of solid-phase extraction using C8 cartridges. This procedure allowed recoveries close to 80% for all these drugs excluding Candesartan cilexetil (70%) which presented adsorption processes on glass and plastic walls. The analytes and potential interferences were separated on a reversed-phase column, muBondapak C18, at room temperature. A gradient elution mode was used to carry out the separation, the optimal mobile phase being composed of acetonitrile-5 mM acetate buffer, pH 4, at variable flow-rates (from 1.0 to 1.2 ml/min). Fluorescence detector was set at an excitation wavelength of 250 nm and an emission wavelength of 375 nm. Intra- and inter-day relative standard deviations for all the compounds were lower than 8% except for Losartan (12%) and the method assesses a quite good accuracy (percentage of relative error approximately 6% in most of the cases). The limit of quantitation for these compounds was 3 ng/ml for Candesartan cilexetil and M1, 16 ng/ml for Losartan and 50 ng/ml for Irbesartan and Valsartan, which allows their determination at expected plasma concentration levels. This assay method has been successfully applied to plasma samples obtained from hypertensive patients under clinical studies after oral administration of a therapeutic dose of some of these ARA II compounds.