Determination of metoprolol and its alpha-hydroxylated metabolite in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of metoprolol and its alpha-hydroxylated metabolite in plasma, Metoprolol, alpha-hydroxymetoprolol and alprenolol (internal standard) are extracted from plasma at alkaline pH with diethyl ether-dichloromethane (4:1, v/v) and back-extracted with 0.01 N sulfuric acid. A 100-microliter volume of the acidic extract is injected into the chromatographic system. The compounds are eluted in about 12 min with acetonitrile-acetate buffer (75:25, v/v) on a LiChrosorb RP-8 (5 micron) column. The quantitative determinations are made fluorometrically. Concentrations down to 35 nmol/1 (10 ng/ml) of metoprolol base and 30 nmol/1 (8 ng/ml) of alpha-hydroxymetoprolol base in plasma can be determined with good precision and accuracy.     

Determination of etretinate and its main metabolite in human plasma using normal-phase high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of astemizole and its primary metabolite in plasma and animal tissues. Both compounds and the internal standard were extracted from alkalinized plasma with heptane--isoamyl alcohol and analyzed using a reversed-phase column and UV monitoring at 254 nm. The detection limits for both compounds were 1 ng/ml of plasma and 5 ng/g of tissue and extraction recoveries were sufficiently high (71-84%). The method was applied to plasma and tissue samples from dogs after repeated oral administration, and to plasma samples from a volunteer taking a 300-mg oral dose of the drug. The results were compared with those obtained by a formerly developed radioimmunoassay.     

Determination of fluoxetine and its N-desmethyl metabolite in human plasma by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of the antidepressant fluoxetine and its active metabolite norfluoxetine in human plasma using paroxetine as internal standard. After liquid-liquid extraction, the compounds were separated on a C18 column using as mobile phase acetonitrile and 40 mM potassium dihydrogen phosphate buffer (pH 2.3) in the ratio 31:69 (v/v). The quantification of fluoxetine and norfluoxetine was made by fluorescence detection at Ex/Em 230/312 nm. The assay for each analyte was linear over the ranges 1-39 and 0.9-36 ng/ml, respectively. For both compounds intra- and inter-day accuracy and precision ranged between −7.9-12.4 and 0.7-14.7%, respectively. The method was applied to the analysis of plasma samples obtained from healthy subjects treated with one single oral dose of 40 mg fluoxetine.     

Determination of bisaramil and its metabolite in plasma using high-performance liquid chromatography with electrochemical detection.

A column liquid chromatographic method using electrochemical detection has been developed for determination of an antiarrhythmic agent, bisaramil, and its metabolite in plasma. The plasma was fractionated by extraction with chloroform and chloroform-ethanol, and each fraction was dried and dissolved in ethyl acetate. After back-extraction into an acidic buffer, bisaramil was chromatographed on a reversed-phase column, and the metabolite, which has a higher polarity, was analysed by ion-pairing chromatography. Calibration curves were linear over the concentration range 2-200 ng/ml with coefficients of variation, within-day or day-to-day, not exceeding 5% at any level. The limits of detection of bisaramil and its metabolite were 0.5 and 1 ng/ml, respectively, using 0.5 ml of plasma. The dual-electrode detector was operated in the screening mode of oxidation (electrode 1, +0.55 V; electrode 2, +0.8 V), providing a greater specificity and reducing the background noise. This procedure was applied to a large number of samples in a pharmacokinetic study at the therapeutic dose.     

Determination of benzyl alcohol and its metabolite in plasma by reversed-phase high-performance liquid chromatography.

A study undertaken following recent reports of deaths in neonatal children associated with the use of benzyl alcohol resulted in the development of a stability-indicating high-performance liquid chromatographic assay of benzyl alcohol in plasma using benzocaine as internal standard. Thawed plasma samples were diluted and subjected to solid-phase extraction using Extrelut and eluted with ethyl acetate. The evaporated eluate was reconstituted with mobile phase and chromatographed on a C18 column with water-acetonitrile-glacial acetic acid as mobile phase and detection at 254 nm. Baseline separation was achieved within 12 min for benzyl alcohol, benzaldehyde, benzoic acid, hippuric acid and benzocaine. Peak-height ratios were linear over 80-640 ng of benzyl alcohol injected (r = 0.998) and over 10-80 ng of benzoic acid injected (r = 0.999). Benzaldehyde and hippuric acid were not quantitated because these compounds were not detectable in actual dog plasma. Validation studies by spiking dog plasma with benzyl alcohol and benzoic acid gave overall percent recoveries (+/- relative standard deviation, n = 4) of 98.3 +/- 3.0 and 101.4 +/- 7.6%, respectively. The method was applied to the assay of actual plasma samples. Since benzyl alcohol is very susceptible to oxidation to benzaldehyde and benzoic acid, its purity in bulk liquid samples can be determined by this method.     

Determination of a new orally active cephalosporin in human plasma and urine by high-performance liquid chromatography using automated column switching

Analytical procedures have been investigated for the separation, detection, identification and quantitation of some metabolites of N-benzyl-4-substituted anilines. Techniques based on gas-liquid chromatography were investigated and found to be unsatisfactory. By the use of reversed-phase high-performance liquid chromatography with gradient and ion-pairing techniques, methods were devised for the simultaneous analyses of a variety of metabolites. The method involves minimum sample work-up (acetonitrile precipitation) and allows easy and prompt analysis in biological media avoiding undue decomposition of unstable metabolites.     

An automated analyzer for vancomycin in plasma samples by column-switching high-performance liquid chromatography with UV detection

An automated analyzer for vancomycin in rat plasma by column-switching high-performance liquid chromatography (HPLC) with UV detection was developed. The method includes in-line extraction of vancomycin by ion-exchange cartridge column and a separation on a reversed-phase column with UV detection at 215 nm. Plasma samples were diluted by mobile phase solution and directly injected to HPLC. Vancomycin was quantitatively recovered from rat plasma samples. The separation was completed within 15 min. The calibration curve was linear over the range from 0.5 to 100 microg/mL with the detection and quantification limits of 0.5 microg/mL (2.5 ng on column; signal-to-noise ratio = 3). The values of precision in intra- and inter-day assays (n = 3) were less than 1.92 and 3.69%, respectively. This method does not require time-consuming pre-treatment and is suitable for the routine assay of plasma samples.     

Determination of a pyrimido-benzazepine anxiolytic agent and its 5-hydroxy metabolite in whole blood, plasma and urine by gas--liquid chromatography with electron-capture detection and by high-performance liquid chromatography

Electron-capture gas--liquid chromatographic and reversed-phase high-performance liquid chromatographic assays are described for the quantitation of the compound, 9-chloro-7-(2-chlorophenyl)-5H-pyrimido [5,4-d] [2]-benzazepine, [I], a member of the benzazepine class of compounds undergoing clinical evaluation as anxiolytic agents. Studies on the biotransformation of [I] in the rat and dog showed that the compound was metabolized mainly by hydroxylation to yield the 5-hydroxy compound, [II], 9-chloro-7-(2-chlorophenyl)-5H-pyrimido [5,4-d] [2]-benzazepin-5-ol (major metabolite), along with the formation of lesser amounts of the N-oxide, [III], 9-chloro-7-(2-chlorophenyl)-5H-pyrimido [5,4-d] [2]-benzazepine 3-oxide, and the phenolic analogue, [IV], 3-chloro-4-(9-chloro-5H pyrimido-[5,4-d] [2] benzazepin-7-yl)phenol. This report describes the quantitation of [I] and [II] (major metabolite) in plasma using the above analytical techniques, both in preclinical studies in the dog and in clinical pharmacokinetic studies in man.     

Determination of onapristone and its N-desmethyl metabolite in human plasma or serum by high-performance liquid chromatography

An automated reversed-phase high-performance liquid chromatographic method for the determination of the antiprogestin onapristone and its N-desmethyl metabolite in human plasma or serum is described. Ultraviolet detection was performed at 315 nm, with a limit of detection of 1 ng/ml at a signal-to-noise ratio of 3. The intra- and inter-assay precision were less than or equal to 6% and less than or equal to 7%, respectively. Onapristone and its N-desmethyl metabolite were stable in human plasma. The method was successfully applied to serum samples obtained from human volunteers after oral administration of onapristone.     

Determination of amineptine and its main metabolite in plasma by high-performance liquid chromatography after solid-phase extraction

Amineptine and its main metabolite were determined simultaneously in plasma by high-performance liquid chromatography using quinupramine as internal standard. The method comprised adsorption on Extrelut column from alkaline plasma, elution with diethyl ether-methylene chloride, evaporation in the presence of 0.01 M hydrochloric acid and injection of the acid solution onto a mu Bondapak C18 column, using acetonitrile-0.025 M potassium dihydrogenphosphate as mobile phase and ultraviolet detection at 210 nm. Average steady-state concentrations of the two compounds were determined in four patients under treatment regimen (two 100-mg doses of amineptine per day, at 8.00 and 12.00 h). The concentrations determined 20 h after the last dose were undetectable in all cases, whereas the concentrations determined 1 h after the second dose were found to be 780 +/- 96 ng ml-1 for amineptine and 690 +/- 137 ng ml-1 for its metabolite. The technique can also be applied to whole blood with, if necessary, identification on the basis of the ultraviolet spectrum obtained by photodiode-array detection.     

Simultaneous quantitation of loganin and sweroside in plasma using column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of loganin and sweroside, which are the active ingredients of purified herbal extract from Lonicera japonica (SKL JI), in rat plasma using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply filtrated prior to injection to the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of loganin and sweroside was accurate and repeatable, with a limit of quantitation of 0.05 μg ml⁻¹ in plasma. The calibration curves for both loganin and sweroside were linear over the concentration ranges of 0.05-40.0 and 0.02-40.0 μg ml⁻¹ in rat plasma, respectively. The intra- and inter-day precision over the concentration range for loganin and sweroside were lower than 8.1 and 10.9% (relative standard deviation, R.S.D.), and accuracy was between 94.7 and 113.5 and 95.0 and 113.1%, respectively. This method has been successfully applied to determine the levels of loganin and sweroside in rat plasma samples from pharmacokinetics studies.     

Determination of renin activity in human plasma by column-switching high-performance liquid chromatography with fluorescence detection

A column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the determination of the renin activity in human plasma. The method is based on the quantification of the enzymatically produced angiotensin I. Angiotensin I liberated from a synthetic substrate (tridecapeptide of human angiotensinogen) and [Val5]-angiotensin I as an internal standard are converted into fluorescent derivatives by reaction with benzoin. The derivatives are separated from various interfering substances by column-switching HPLC using three reversed-phase columns. The limit of detection (signal-to-noise ratio = 3) of the renin activity is 2.7 pmol of angiotensin I formed per h per ml of plasma, which corresponds to approximately 820 fmol of angiotensin I injected. The column-switching method in combination with pre-column derivatization for the fluorimetric detection permits the sensitive and selective determination of the enzymatically formed angiotensin I. Hence low activities of renin in normal human plasma are readily measured.     

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.     

Determination of the antifungal agent voriconazole in human plasma using a simple column-switching high-performance liquid chromatography and its application to a pharmacokinetic study

A simple column-switching high performance liquid chromatographic (HPLC) method that does not require any complicated pretreatment has been developed to determine voriconazole in human plasma samples. An internal standard (IS) and borate buffer (pH 9.0) were added to plasma samples, which were then injected directly into the column-switching HPLC system using MAYI-ODS as a pre-column. The calibration curve for voriconazole showed good linearity in the range of 0.2-10 mug/ml in human plasma. The mean RSD (%) value of intra-day (n=6) and inter-day (n=5) precision were less than 5.4% and 8.2%, respectively. This system could make more than three hundred successive, accurate measurements when a washing step with ammonium acetate solution was added. This method was successfully applied to measure the therapeutic voriconazole level in patients' plasma, and was used in a study of voriconazole pharmacokinetics after oral administration.     

Determination of benperidol and its reduced metabolite in human plasma by high-performance liquid chromatography and electrochemical detection

An isocratic high-performance liquid chromatographic method with electrochemical detection for the quantification of benperidol and its suggested reduced metabolite TVX Q 5402 in human plasma is described. The method included a two-step solid-phase extraction on reversed-phase and cation-exchange material, followed by separation on a cyanopropyl silica gel column (5 microns; 250 mm x 4.6 mm I.D.). The eluent was 0.15 M acetate buffer (pH 4.7) containing 25% acetonitrile (w/w). Spiperone served as internal standard. The inclusion of the cation-exchange step provided sample purity higher than those achieved with other methods. After extraction of 1 ml of plasma, concentrations as low as 0.5 ng/ml were detectable for both benperidol and the metabolite. In plasma samples collected from a schizophrenic patient treated with a single oral dose of 6 mg of benperidol, plasma levels of benperidol and of the metabolite could be measured from 20 min to at least 12 h after administration.     

Simple determination of azasetron in rat plasma by column-switching high-performance liquid chromatography

For the quantification of azasetron in rat plasma samples, a column-switching HPLC method was developed and validated. Following dilution of plasma samples with mobile phase A (17?mM potassium phosphate buffer (pH 3.0)) and simple protein precipitation by addition of perchloric acid (60%), the mixture was directly injected onto the pre-column. After endogenous plasma substances were eluted to waste, the analyte was transferred to the trap column by switching the system. Then, the analyte was back-flushed to the analytical column for separation with mobile phase B (a 22:78 v/v mixture of acetonitrile and 17?mM potassium phosphate buffer (pH 3.0)) and detected at 250?nm using a photodiode array detector. A linear standard curve was obtained in the concentration range of 10-800?ng/mL with the correlation coefficient (r) of 0.9998. The intra- and inter-day precision and accuracy values for azasetron were in the ranges of 0.3-12.9% and 89.7-101.4%, respectively. The method was valid in terms of specificity, precision, and accuracy. In addition, this efficient analytical method was successfully applied to determine plasma concentrations of azasetron following oral administration of azasetron at a dose of 4.0?mg/kg to rats.     

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 the heroin metabolite 6-acetylmorphine by high-performance liquid chromatography using automated pre-column derivatization and fluorescence detection

An improved method for the determination of 6-acetylmorphine in the urine of drug addicts receiving morphine was developed. A newly introduced reversed-phase high-performance liquid chromatographic system proved to be more sensitive than a normal-phase system used previously. By replacing the earlier manual derivatization procedure with an automated on-line pre-column method, both the reproducibility and efficiency were considerably improved. Coefficients of variation for repeated analyses typically ranged from 6 to 10% in the 1-100 micrograms/l concentration range. The detection limit was 1 microgram/l and the correction for recovery by calibration with blank urine samples spiked with 6-acetylmorphine was satisfactory. The analytical improvements achieved, however, did not increase the chance of detecting heroin use by drug addicts.     

Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatography

We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 microm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile-0.1% phosphoric acid, 36:64, v/v) containing 2 mM sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5-5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from -2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats.