Rapid and sensitive quantitative analysis of the new antimalarial N4-[2,6-dimethoxy-4-methyl-5-[(3-trifluoromethyl)phenoxy]-8- quinolinyl]-1,4-pentanediamine in plasma by liquid chromatography and electrochemical detection

A rapid, sensitive and simple method was developed for the quantitation of the plasma concentration of N4-[2,6-dimethoxy-4-methyl-5-[(3-trifluoromethyl)phenoxy]-8- quinolinyl]-1,4-pentanediamine, a new antimalarial active against Plasmodium vivax. N4-(5-Hexoxy-6-methoxy-4-methyl-8-quinolinyl)-1,4- pentanediamine diphosphate, a similar 8-aminoquinoline, was used as an internal standard. The method involves sample clean-up by a prepacked cyano solid-phase column followed by reversed-phase liquid chromatography and oxidative electrochemical detection at +0.95 V. The assay has been validated to 5 ng/ml of plasma and is sensitive to 1 ng/ml of plasma. The results of a pilot study assessing the relative oral bioavailability of two different salt forms of the new antimalarial in dogs show the usefulness of the method for animal and human pharmacokinetic studies.     

Enantioselective drug monitoring of (R)- and (S)- propranolol in human plasma via derivatization with optically active (R,R)-O,O-diacetyl tartaric acid anhydride

A sensitive high-performance liquid chromatographic method was developed for the stereoselective assay of (R)- and (S)-propranolol in human plasma. The method involves diethyl ether extraction of the drugs and a racemic internal standard, N-tert.-butylpropranolol, followed by derivatization of the compounds with the chiral reagent (R,R)-O,O-diacetyl tartaric acid anhydride. The resulting diastereomeric derivatives were separated isocratically on a reversed-phase column. Quantitation was achieved by the peak-height ratio method with reference to the internal standard. The assay was accurate and reproducible in the concentration range 1-100 ng of (R)- and (S)-propranolol per ml plasma, using fluorescence detection at lambda ex 290 nm and lambda em 335 nm. The applicability of this method was demonstrated for the determination of concentration-time profiles of propranolol enantiomers in the course of comparative pharmacokinetic studies.     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

Determination of the (+)- and (-)-enantiomers of pirprofen in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed to determine the (+)- and (-)-enantiomers of pirprofen, an anti-inflammatory drug. After addition of an internal standard, the plasma sample was brought onto a glass column pre-packed with silica and eluted with dichloromethane. The extracts were derivatized with 1,1'-carbonyldiimidazole and R (+)-1-methylbenzylamine to form the two diastereomeric amides. The diastereoisomers were separated on a chiral column by HPLC with ultraviolet detection at 272 nm using n-hexane-dichloromethane (64:36, v/v) as the mobile phase. The limit of quantitation was 0.992 mumol/l (0.25 microgram/ml) for each enantiomer.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17 X 4.6 mm I.D.), filled with C18 Corasil 37-53 micron. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-100 ng/ml was between 1.0 and 4.7% for all compounds.     

High Performance Liquid Chromatographic Determination of (-)-N-Formylnorephedrine in Plasma

Abstract

An HPLC procedure for the detection and quantitative estimation of (-)-N-formylnorephedrine in rabbit plasma had been developed. The procedure involved the extraction of (-)-N-formylnorephedrine from plasma spiked with the internal standard (phenacetin), using ethyl acetate. The ethyl acetate extract is evaporated under nitrogen and the residue is reconstituted in water and injected onto the column. A u-Bondapak-C18 column 30 cm × 3.9 mm ID was used. The mobile phase is 20% acetonitrile in water; at a flow rate of 1.5 ml/min and uv detection at 256 nm. A linear relationship between concentration and peak height ratio (I/internal standard) was obtained (r = 1.00). The reported procedure allows the measurement of (-)-formylnorephedrine in concentrations as low as 150 ng/ml of plasma with total procedure time of about 10 min. The applicability of the procedure to pharmacokinetic studies is illustrated and metabolites are shown not to interfere with the assay procedure.     

Determination of the antimitotic agents N-desacetylcolchicine, demecolcine and colchicine in serum and urine

In an effort to characterize the pharmacokinetic behavior of the antimitotic agent N-desacetylcolchicine a selective, sensitive high-performance liquid chromatographic method was developed for the determination of N-desacetylcolchicine, demecolcine and colchicine in serum or urine. To 0.5 ml of serum or 0.1 ml of urine diluted to 0.5 ml were added 50 microliters demecolcine (2 micrograms/ml) which serves as the internal standard. The sample was extracted using a C2 reversed-phase solid extraction column. N-Desacetyl-colchicine, colchicine and the internal standard were eluted from the column with methanol. The combined eluates were evaporated to dryness and the residue was reconstituted with water. The reconstituted sample was injected into a C18 reversed-phase column and eluted using a mobile phase consisting of 0.1 M potassium dihydrogenphosphate, 5 mM 1-pentanesulfonic acid in methanol and acetonitrile with a final pH of 6.0, at a flow rate of 1.5 ml/min. N-Desacetylcolchicine, colchicine and the internal standard were detected using a variable-wavelength ultraviolet detector at 254 nm. The limit of detection was 0.4 ng/ml for desacetylcolchicine and 4.0 ng/ml for colchicine. The method is linear over a concentration range of 1.0-200 ng/ml. The method has been shown to be a rapid, reliable method to monitor N-desacetylcolchicine levels in clinical trials in cancer patients.     

High-performance liquid chromatographic separation and electrochemical or spectrophotometric determination of R(-)N-n-propylnorapomorphine and R(-)10,11-methylenedioxy-N-n-propylnoraporphine in primate plasma

The dopamine receptor agonist R(-)N-n-propylnorapomorphine (NPA) and its proposed pro-drug R(-)10,11-methylenedioxy-N-n-propylnoraporphine (MDO-NPA) were isolated simultaneously from monkey plasma using a solid-phase extraction procedure. R(-)Apomorphine (APO) and R(-)10,11-methylenedioxyaporphine (MDO-APO) were added as internal standards, and separation and quantification were by high-performance liquid chromatography with electrochemical or ultraviolet detection of the free catechol and MDO compounds, respectively. The detection limits for NPA and MDO-NPA in plasma were 0.5 and 10 ng/ml and the coefficient of variation (S.D./mean) within assays and between days of assays for both drugs was 5.6% or less. Quantification of plasma levels of NPA and MDO-NPA was possible at ranges of 2-1000 and 40-5000 ng/ml, respectively, including concentrations found after intravenous administration of these agents.     

Determination of diclofenac in plasma using a fully automated analytical system combining liquid-solid extraction with liquid chromatography

A fully automated analytical system based on liquid-solid extraction combined with column liquid chromatography is described for the determination of diclofenac in plasma. After addition of pH 5 buffer and the internal standard solution to the plasma sample, both sample preparation via a C18 disposable extraction column and injection were performed by a Gilson ASPEC system. Diclofenac and the internal standard were separated on a reversed-phase column, using methanol-pH 7.2 phosphate buffer (56:44, v/v) as mobile phase at a flow-rate of 0.4 ml/min. The reproducibility and accuracy of the method were acceptable over the concentration range 31-3140 nmol/l in plasma.     

Determination of 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol in plasma using liquid chromatography with electrochemical detection

A reversed-phase column liquid chromatographic (LC) method with electrochemical detection (ED) is described for the quantification of 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol (compound 1), a new locally active dual inhibitor of leukotriene and prostaglandin synthesis, in plasma. After a single liquid-liquid extraction of the biological specimen, the extract was analyzed using a liquid chromatograph with an amperometric detector set at an oxidation potential of +0.55 V. The resulting chromatograms are free from endogenous interference and the limit of detection is 0.2 ng/ml. Several other analogous dihydrobenzofuranols were shown to be electrochemically active, permitting their determination using LC with ED. The described analytical method has been fully validated in the concentration range 0.5-20 ng/ml of plasma and utilized in the analysis of plasma samples from human clinical studies. The analytical methodology has also been adapted for analysis of compound 1 in human skin blister fluid after topical administration of 1.     

Simultaneous determination of the anticonvulsants, cinromide (3-bromo-n-ethylcinnamamide), 3-bromocinnamamide, and carbamazepine in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for monitoring plasma concentrations of cinromide (3-bromo-N-ethylcinnamamide) and its de-ethylated metabolite. Carbamazepine levels can be easily measured by the same technique. The N-isopropyl analogue of cinromide is used as internal standard, and all compounds are easily separated on a reversed-phase column operated at 55 degrees with a small-diameter pre-column maintained at the same temperature. The extraction is rapid and generally applicable to plasma and urine samples that are to be analyzed by reversed-phase chromatography. Short- and long-term reproducibility studies show less than 4% relative standard deviation for replicate determinations for all drugs. Limits of quantitation are 10-20 ng/ml with an internal standard concentration of 3 micrograms/ml. Another metabolite of cinromide, 3-bromocinnamic acid, which may have some anticonvulsant effect, can be analyzed simultaneously by buffering the mobile phase and adding an ion-pairing reagent.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.     

Estimation of catecholamines in human plasma by ion-exchange chromatography coupled with fluorimetry

Estimation of catecholamines in human plasma was made by ion-exchange chromatography coupled with fluorimetry. Catecholamines in deproteinized plasma were adsorbed onto Amberlite CG-50 (pH 6.5, buffered with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid. The catecholamine fraction was separated further on a column of Amberlite IRC-50 which was coupled with a device for the automated performance of the trihydroxyindole method (epinephrine and norepinephrine) or the 4-aminobenzoic acid-oxidation method (dopamine). One sample could be analysed within 25 min with either method. The lower detection limits were 0.02 ng for epinephrine and dopamine, and 0.04 ng for norepinephrine. Plasma catecholamine contents of healthy adults at rest were epinephrine 0.07 +/- 0.01 ng/ml (n = 19), norepinephrine 0.27 +/- 0.03 ng/ml (n = 19) and dopamine 0.22 +/- 0.03 ng/ml (n = 26). The procedure of adsorption and elution of the plasma catecholamines by ion-exchange resin was simple, the simplicity contributing to constant recovery. The catecholamine fraction could be analysed without evaporation of the eluate. The analytical column could be used for the analysis of more than 1000 samples before excessive back-pressure developed. Our method of continuous measurement of plasma catecholamine fulfils clinical requirements.     

Determination of an aldosterone antagonist in urine by high-performance liquid chromatography using automated column switching

An automated high-performance liquid chromatographic assay for the determination of an aldosterone antagonist (I) is described using column switching for direct injection of urine samples. After dilution with buffered internal standard solution, the sample was injected onto a clean-up column (17 X 4.6 mm I.D.), dry-packed with C18 reversed-phase material (particle size 30 micron). Polar urine components were removed by flushing the clean-up column with water. Retained substances, including I and the internal standard, were desorbed by backflush elution onto a 5-micron ODS-silica analytical column (125 X 4 mm I.D.), separated with water-methanol-tetrahydrofuran, and detected at 295 nm. After backflushing the analytical column and re-equilibrating the clean-up column, the system was ready for the next injection. The limit of quantification was ca. 100 ng/ml, using a 100-microliter specimen of diluted urine. The mean inter-assay precision of the method up to 25.6 micrograms/ml was 2%. Practicability and accuracy of the new method were demonstrated by the application to excretion studies performed with human volunteers.     

High-performance liquid chromatographic determination of glibenclamide in human plasma and urine

A selective and sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma or urine has been developed. With glibornuride as internal standard, acid-buffered plasma or urine was extracted with benzene. The organic layer was evaporated and the residue was dissolved in equilibrated mobile phase (acetonitrile-phosphate buffer 0.01 M pH 3.5, 50:50). An aliquot of 20 microliters was chromatographed on a Spherisorb ODS reversed-phase column, and quantitation was achieved by monitoring the ultraviolet absorbance at 225 nm. The response was linear (0-1000 ng/ml) and the detection limit was 5-10 ng/ml in plasma or urine. The within-assay variation was less than or equal to 10%. No interferences from metabolites or endogenous constituents could be noted. The utility of the method was demonstrated by analysing glibenclamide in samples from diabetic subjects on therapeutic doses of the drug.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.     

Determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma by high-performance liquid chromatography and fluorescence detection

A high-performance liquid chromatographic method for the simultaneous determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma is described. After extraction with an organic solvent, the compounds and their internal standard (7-amino-methylclonazepam or Ro 5-3384) are derivatized with fluorescamine and chromatographed on a reversed-phase muBondapak C18 column using pH 8 buffer solution-acetonitrile (3:1) as mobile phase. The detection is performed by a fluorometer at excitation and emission wavelengths of 390 and 470 nm, respectively. The sensitivity limit is about 1 ng/ml of plasma for both 7-amino-flunitrazepam and 7-amino-desmethylflunitrazepam. The method has been applied to the determination of plasma levels of these substances during pharmacokinetic studies of flunitrazepam, desmethylflunitrazepam and 7-amino-flunitrazepam.     

Liquid chromatography/negative ion electrospray tandem mass spectrometry method for the quantification of fluvastatin in human plasma: validation and its application to pharmacokinetic studies

A simple, sensitive and rapid high-performance liquid chromatography/negative ion electrospray tandem mass spectrometry method was developed and validated for the assay of fluvastatin in human plasma. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M-H]- ions, m/z 410/348 for fluvastatin and m/z 480/418 for the internal standard. The assay exhibited a linear dynamic range of 2-500 ng/mL for fluvastatin in human plasma. The lower limit of quantification was 2 ng/mL with a relative standard deviation of less than 5%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantitation of a novel antiemetic (ADR-851) in plasma and urine by reversed-phase high-performance liquid chromatography with fluorescence detection

A sensitive and specific bioanalytical method for quantitation of a novel antiemetic (ADR-851) in plasma and urine has been developed and validated. The drug and internal standard (metoclopramide) are extracted from the plasma matrix by solid-phase extraction on cyanopropyl bonded-phase columns. After extraction, samples are separated by isocratic reversed-phase high-performance liquid chromatography. The parent drug, internal standard and a yet unidentified metabolite are detected by fluorescence. The method requires 1.0 ml of plasma or 0.1 ml of urine and has a lower limit of quantitation of 2 ng/ml with 10.9% relative standard deviation (R.S.D.). Method linearity has been established over a 2-800 ng/ml range when 1.0 ml of plasma is used. The intra- and inter-day imprecisions for the method are typically better than 6% and 11% R.S.D., respectively, in both plasma and urine over the entire dynamic range. The pooled estimate of bias is less than 5% and attests to the excellent accuracy.     

Determination of a potential anxiolytic drug (CGS 20625) in human plasma by high-performance liquid chromatography.

An analytical method employing reversed-phase high-performance liquid chromatography is described for the determination of a potential anxiolytic agent in human plasma. This experimental drug candidate has potent and selective affinity for the central benzodiazepine receptor complex. The compound and internal standard are extracted from buffered plasma (pH 9.0) into ethyl acetate. The solvent is evaporated and the residue is reconstituted in chromatographic mobile phase. Separation is achieved on a 5-microns phenyl column with ultraviolet absorbance detection of the drug and internal standard at 270 nm. Recovery and reproducibility assessments indicate good accuracy (overall relative recovery of 101%) and precision (coefficients of variation from 2.0 to 11%) over the concentration range 10-1000 ng/ml. The limit of quantification for the method is 10 ng/ml. The method is suitable for pharmacokinetic analysis following the administration of 80 mg of drug to normal volunteers.