Determination of neostigmine in human plasma and cerebrospinal fluid by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic assay coupled with ultraviolet detection has been developed for the determination of neostigmine in human plasma and cerebrospinal fluid. A novel solid-phase extraction procedure was first used for this analyte and allowed good recovery (89+/-4.5%) together with ease and speed of execution. The method was sensitive, reproducible (C.V.<4.5%) and accurate (100+/-6.6%) over the range 2.6-167.0 ng/ml neostigmine concentrations in plasma or cerebrospinal fluid, and was applied successfully to study the pharmacokinetics of neostigmine in patients suffering from chronic postoperative abdominal pain.     

Determination of the cephalosporin antibiotic cephradine in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic method, for the determination of cephradine in human plasma samples has been developed and validated. Cephradine and cephaloridine (internal standard) were extracted from human plasma by perchloric acid protein precipitation followed by centrifugation. Aliquots of the extracts were analysed by reversed-phase high-performance liquid chromatography (HPLC) utilising a polymeric reversed-phase PLRP-S column, followed by ultraviolet detection at 260 nm. The method has a working dynamic range from 0.2 to 30.0 microg/ml from 200 microl human plasma. The precision of the method at 0.2 microg/ml was 4.9% (intra-assay) and negligible (inter-assay) as calculated by one-way analysis of variance and the accuracy of the method at 0.2 microg/ml was -4.1% in terms of percentage bias. This method has been successfully applied to clinical studies including an oral bioequivalence study comparing the pharmacokinetics of 500 mg tablets of Kefdrin with 500 mg tablets of Velosef in healthy human volunteers.     

Simultaneous quantitative determination of amprenavir and indinavir in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the determination of the HIV protease inhibitors amprenavir (Agenerase) and indinavir (Crixivan) in human plasma is described, using a mobile phase consisting of 0.50 M phosphate buffer (adjusted to pH 5,5) - Milli-Q water - acetonitrile (120: 1,080: 800, v/v/v). A solid-phase extraction using C18 extraction columns (Discovery columns 100 mg, 1 ml Supelco) and a liquid-liquid extraction with 0.5 ml hydrogenocarbonate/carbonate buffer (adjusted to pH 10.6) and 6 ml methyl ter-butyl ether have been compared. The liquid-liquid extraction has been chosen to be easier and cheaper. The method has been validated over the range of 60 to 3,000 ng/ml for amprenavir and 20 to 3,000 ng/ml for indinavir using a 0.5 ml sample volume. The specificity, linearity, accuracy and precision have been studied. The limit of detection was respectively for amprenavir and indinavir 15 and 4 ng, and the limit of quantification was 60 and 20 ng/ml. Stability tests under various conditions were performed. This assay can readily be used in a hospital laboratory for the routine monitoring of plasma concentrations of amprenavir in HIV-infected patients. The trough plasma concentrations average has been determined in patients treated by amprenavir and indinavir for seven months.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid-liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1-5000 ng (r2>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83 +/- 6 and 92 +/- 6% in plasma, respectively, and 79 +/- 7 and 89 +/- 7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid-liquid extraction of citalopram with hexane-isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45 x 4.6 mm, 5 microm particles), the mobile phase consisted of an acetonitrile-phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Fully automated analytical method for codeine quantification in human plasma using on-line solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

A simple, sensitive and fully automated analytical method for the analysis of codeine in human plasma is presented. Samples are added with oxycodone, used as internal standard (I.S.), and directly loaded in the autosampler tray. An on-line sample clean-up system based on solid-phase extraction (SPE) cartridges (Bond-Elut C2, 20 mg) and value switching (Prospekt) is used. Isocratic elution improved reproducibility and allowed the recirculation of the mobile phase. A Hypersil BDS C18, 3 microns, 10 x 0.46 cm column was used and detection was done by UV monitoring at 212 nm. Retention times of norcodeine (codeine metabolite), codeine and oxycodone (I.S.) were 5.5, 6.4 and 9.1 min, respectively. Morphine was left to elute in the chromatographic front. Detection limit for codeine was 0.5 microgram l-1 and inter-assay precision (expressed as relative standard deviation) and accuracy (expressed as relative error) measured at 2 micrograms l-1 were 5.03% and 1.82%. Calibration range was 2-140 micrograms l-1.     

Determination of rutin in human plasma by high-performance liquid chromatography utilizing solid-phase extraction and ultraviolet detection

The degradation kinetics of aplidine were investigated using reversed-phase high-performance liquid chromatography combined with UV detection. Aplidine consists of at least two isomers that undergo interconversion at a low rate. Influences of pH, temperature, buffer ions and ionic strength on the degradation kinetics were studied. The log kobs) -pH profile can be divided into three parts, a proton, a solvent and a hydroxyl-catalysed section. The stability-indicating properties of the used analysis technique as well as the identities of the main degradation products were checked using gradient liquid chromatography and mass spectrometric detection. The overall degradation rate constant as a function of the temperature under acidic and alkaline conditions obeys the Arrhenius equation. No catalytic influences were observed with phosphate and carbonate buffers and, in addition, the ionic strength showed no substantial effect on the stability, as expected. Results from gradient LC-MS indicated that hydrolysis of the ester groups present in the ring structure was the main degradation route. There is no difference in degradation rate constants for the individual isomers.     

Determination of celecoxib in human plasma by normal-phase high-performance liquid chromatography with column switching and ultraviolet absorbance detection

A method is described for the determination of celecoxib in human plasma. Samples were extracted using 3M Empore membrane extraction cartridges and separated under normal-phase HPLC conditions using a Nucleosil-NO2 (150x4.6 mm, 5 microm) column. Detection was accomplished using UV absorbance at 260 nm. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. The assay was linear in the concentration range of 25-2000 ng/ml when 1-ml aliquots of plasma were extracted. Recoveries of celecoxib were greater than 91% over the calibration curve range. Intraday precision and accuracy for this assay were 5.7% C.V. or better and within 2.3% of nominal, respectively. The assay was used to analyze samples collected during human clinical studies.     

Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC-UV determination of loratadine in human plasma is presented. After simple liquid-liquid extraction with 2-methylbutane-hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH3CN-water-0.5 M KH2PO4-H3PO4 (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5-50 ng/ml) with relative standard deviations of 2.3-6.3 and 5.2-14.1% for intra- and inter-assays, respectively.     

Determination of ketorolac in human plasma by reversed-phase high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

An improved high-performance liquid chromatographic method has been developed to measure human plasma concentrations of the analgesic nonsteroidal anti-inflammatory drug ketorolac for use in pharmacokinetic studies. Samples were prepared for analysis by solid-phase extraction using Bond-Elut PH columns, with nearly complete recovery of both ketorolac and the internal standard tolmetin. The two compounds were separated on a Radial-Pak C18 column using a mobile phase consisting of water-acetonitrile-1.0 mol/l dibutylamine phosphate (pH 2.5) (30:20:1) and detected at a UV wavelength of 313 nm. Using only 250 microl of plasma, the standard curve was linear from 0.05 to 10.0 microg/ml.     

Determination of roxithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple and reproducible method for the determination of roxithromycin in human plasma is presented. This method is based on liquid-liquid extraction with hexane-isoamylalcohol (98:2, v:v) and reversed-phase chromatography with spectrophotometric detection at 220 nm. The mobile phase consists of methanol-15 mM dihydrogen potassium phosphate (70:30, v:v), pH of the aqueous part of the mobile phase is 6.0. The column is operated at 60 degrees C. Clarithromycin is used as the internal standard. The limit of quantitation is 0.5 microg/ml and the calibration curve is linear up to 30 microg/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 9%. The assay was used for pharmacokinetic studies.     

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

Dihydroergotamine, a 5-hydroxytryptamine antagonist, is used for the treatment of vascular headaches. A high-performance liquid chromatography assay with fluorescence detection is described for the determination of dihydroergotamine in plasma. The assay was validated over the concentration range 0.1-10 ng/ml plasma and applied to the analysis of plasma samples from subjects treated intramuscularly and intranasally with 2 mg of dihydroergotamine.     

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

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25-18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Determination of metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the hypoglycemic agent metformin is described. Acidified samples of plasma were deproteinated with acetonitrile, washed with dichloromethane and the resulting supernatant injected. Chromatography was performed at 40 degrees C by pumping a mobile phase of acetonitrile (250 ml) in pH 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) at a flow-rate of 1 ml/min through a silica column. Metformin and the internal standard (atenolol) were detected at 240 nm and were eluted 7.8 and 6.8 min, respectively, after injection. No endogenous substances were found to interfere. Calibration curves were linear (r>0.999) from 10 to 2000 ng/ml. The absolute recovery of both metformin and atenolol was greater than 76%. The detection limit and limit of quantitation were 2.5 and 10 ng/ml, respectively. The intra- and inter-day precision (C.V.) was 12%, or less, and the accuracy was within 6.2% of the nominal concentration. This method is suitable for clinical investigation and monitoring metformin concentration.     

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection.

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92-98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.     

Determination of indinavir in human cerebrospinal fluid and plasma by solid-phase extraction and high-performance liquid chromatography with column switching

A rapid, sensitive and robust sample preparation procedure for the quantitative determination of indinavir in human cerebrospinal fluid (CSF) and plasma is described. Indinavir and the internal standard were isolated from CSF or plasma samples by cation-exchange solid-phase extraction with SCX cartridges, while the chromatographic separation was adopted from a previous method, using a cyano column connected by a switching valve to a C18 column. UV detection was set at 210 nm. The standard curve was linear over the concentration range of 2 to 2000 ng/ml in CSF and 5 to 2000 ng/ml in plasma. The intra-day coefficients of variation at all concentration levels were < or = 5.9%. The inter-day consistency was assessed by running QC samples during each daily run. The coefficients of variation for quality control samples in both matrixes were < or = 6.1%. The method has been utilized to support clinical pharmacokinetic studies.     

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20x3.9 mm I.D.) and analytical column (Symmetry C18, 75x4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile-water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40 degrees C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r2>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate-triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a microbondapak C18 column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20-1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Determination of new 2,3-benzodiazepines in rat plasma using high-performance liquid chromatography with ultraviolet detection.

A method for the analysis of [1-(4-aminophenyl)-3,5-dihydro-7, 8-dimethoxy-4H-2,3-benzodiazepin-4-one] (CFM-2) and its analogues CFM-3, CFM-4 and CFM-5 in rat plasma was developed. The 2,3-benzodiazepines (2,3-BZs) were extracted by liquid-liquid extraction and analyzed using high-performance liquid chromatography (HPLC) with ultraviolet detection (UV) at 240 nm. The method exhibited a large linear range from 0.05 to 2 micrograms/ml with an intra-assay accuracy for all studied compounds ranging from 92 to 105.5%; whereas the intra-assay precision ranged from 0.59 to 8.16% in rat plasma. The inter-assay accuracy of CFM-2, CFM-4 and their 3-methyl derivatives, CFM-3 and CFM-5 ranged from 92.2 to 107% and the inter-assay precision ranged from 2.17 to 11.9% in rat plasma. The lower limit of detection was 5.5 ng/ml for CFM-2, 6.5 ng/ml for CFM-3, 7 ng/ml for CFM-4 and 8.5 ng/ml for CFM-5 in rat plasma. The pharmacokinetic study demonstrated that 2,3-BZs achieved a peak plasma concentration between 45 and 75 min after drug administration. Moreover, we observed that plasma chromatograms of rats treated with CFM-3, CFM-4 and CFM-5, respectively, showed a peak consistent with CFM-2. Our study suggests that CFM-4, CFM-5 and CFM-3 are prodrugs of CFM-2, in which they are biotransformed in vivo via different metabolic pathways. In particular, CFM-2 has been proven to possess anticonvulsant activity in various models of seizures, acting as alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist.