High-performance liquid chromatographic method for the determination of gabapentin in human plasma

A sensitive high-performance liquid chromatography (HPLC) method using UV detection for the determination of gabapentin in human plasma has been developed. In this method, gabapentin was extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge followed by derivatization with phenylisothiocyanate. Analysis was achieved by using a HPLC system that was equipped with a UV detector. The quantitation limit of gabapentin in human plasma was 0.03 microg/ml. The method is sensitive with excellent selectivity and reproducibility and it has been applied to a bioequivalence clinical study with great success.     

Determination of chlorpromazine in human breast milk and serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of chlorpromazine in serum and human breast milk. Chlorpromazine in serum and human breast milk was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction, and a reversed-phase HPLC separation technique was developed. Chlorpromazine and levomepromazine as the internal standard were detected by ultraviolet absorbance at 254 nm. Determination was possible for chlorpromazine in the concentration range 10–300 ng/ml. The recoveries of chlorpromazine added to serum and human breast milk were 80.1–87.6 and 80.3–84.4%, respectively, with coefficients of variation of less than 10.2 and 7.8%. The method is applicable to drug level monitoring in the serum and human breast milk of patients treated with chlorpromazine.     

反相HPLC法测定静注人免疫球蛋白中残余胆酸钠含量研究

用反相高效液相色谱法(R—HPLC)测定静注人免疫球蛋白(IVIG)中残余胆酸钠含量。首先用固相提取柱吸附、浓缩制品中残余胆酸钠,然后加入一定量胆酸钠,使样品中胆酸钠含量达到可检测水平后,依外标法进行测定。结果表明,用R—HPLC法测定胆酸钠准确性及重复性较好,是一种相对准确快速的方法。     

Determination of S-1 (combined drug of tegafur, 5-chloro-2,4- dihydroxypyridine and potassium oxonate and 5-fluorouracil in human plasma and urine using high-performance liquid chromatography and gas chromatography-negative ion chemical ionization mass spectrometry

A high-performance liquid chromatography (HPLC) and gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS) method was developed for the analysis of the combined antitumor drug S-1 (tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate[ and active metabolite 5-fluorouracil in human plasma and urine. Tegafur was fractionated from biological fluids by extraction with dichloromethane and analyzed by HPLC. 5-Fluorouracil and 5-chloro-2,4-dihydroxypyridine were extracted with ethyl acetate from the residual layer after extraction of tegafur, and converted to pentafluorobenzyl (PFB) derivatives. Potassium oxonate was cleaned up with an anion-exchange column (Bond Elut NH₂). The extracted potassium oxonate was degraded to 5-azauracil and converted to PFB derivatives. The PFB derivatives were analyzed by GC-NICI-MS. A stable isotope was employed as the internal standard in the GC-NICI-MS analysis. The limits of quantitation of tegafur, 5-fluorouracil, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in plasma were 10, 1, 2 and 1 ng/ml, respectively. The reproducibility of the analytical method according to the statistical coefficients is ∼10%. The accuracy of the method is good; that is, the relative error is <10%. The methods were applied to pharmacokinetic studies of S-1 in patients.     

Development and validation of a high-sensitivity assay for an antipsychotic agent,CP-88,059, with solid-phase extraction and narrow-bore high-performance liquid chromatography

An analytical method has been developed and validated for the quantitation of CP-88,059 in human serum. The compound and internal standard were extracted from serum by solid-phase extraction with a weak cation-exchange phase. The analytes were resolved from endogenous interferences using narrow-bore (2.1 mm I.D.) C₁₈ reversed-phase HPLC. Column effluent was monitored by UV absorbance detection at 215 nm. The standard curve range was 1 to 250 ng/ml. The accuracy and precision values for the method were within ±10% and ±15%, respectively. A four-fold detectability enhancement was achieved using a 2.1 mm I.D. HPLC column relative to the more common 4.6 mm I.D. column. A performance comparison was made between the 2.1 mm I.D. column used for validation and a 4.6 mm I.D. column with the same stationary phase.     

Quantitation of free and conjugated bile acids in human feces using a high-pressure liquid chromatography counterion method

A method has been developed for extraction and purification of the major bile acids in human feces, and for their quantitative estimation using high-pressure liquid chromatography. Freeze-dried fecal material was extracted with alkaline ethanol and, after removal of neutral steroids, was subjected to thin-layer chromatography, followed by reversed-phase C18 silica cartridge (Sep-Pak) purification. The mixture was further separated into free, and glyco and tauro conjugates by ion-exchange chromatography. Subsequent resolution of individual bile acids was accomplished by HPLC using a counterion pairing method.     

Simple high-performance liquid chromatographic determination of the protease inhibitor indinavir in human plasma

Indinavir is a member of a class of protease inhibitors that actively prevent the acquired immunodeficiency syndrome virion from maturing. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of indinavir in human plasma. Indinavir and the internal standard were isolated from the plasma by ether extraction. The residue after evaporation of ether was reconstituted with buffer and injected onto a C₄ reversed-phase column eluted isocratically with a mobile phase consisting of 35:65 (v/v) of acetonitrile and buffer. A wavelength of 210 nm was found to be optimum for detection. The calibration range of this assay was from 10 to 5000 ng/ml and coefficients of variation for the assay ranged from 4.6% to 11.0% for three different drug concentrations and the limit of quantitation was 10 ng/ml. During the validation, short-term stability of the drug in plasma, stability during heat deactivation and on repeated freezing and thawing of plasma was evaluated. The overall recovery of indinavir by the ether extraction method was 91.4%. This HPLC assay was found to be a simple and reproducible method for monitoring indinavir levels in human plasma obtained during clinical trials of the drug.     

Development of a sensitive high-performance liquid chromatography method for the detection of 667 COUMATE in vivo

Steroid sulphatase inhibitors which decrease or prevent the biosynthesis of oestrogens, potentially have an important role in the treatment of breast cancer in postmenopausal women. The non-steroidal sulphatase inhibitor 667 COUMATE has been shown to be active both in vitro and in vivo. The pharmacokinetics of this drug have not been investigated. In preparation for the clinical evaluation of this agent, a sensitive and robust reversed phase high-performance liquid chromatography (HPLC) method was developed for the detection of 667 COUMATE in biological fluids. The sulphatase inhibitor was extracted from plasma with diethyl ether and separated from putative metabolites and endogenous plasma components with a C3-phenyl column. Using this method an extraction efficiency of 76+/-5% and a limit of detection of less than 0.1 ng/ml was achieved. The stability of this agent was investigated under different pH conditions and during storage in plasma at room temperature or -20 degrees C. 667 COUMATE was found to be stable when stored in acidified plasma (pH 4.5) at -20 degrees C. In conclusion, the HPLC method developed is a reproducible and sensitive assay that will enable quantitation of the potent non-steroidal sulphatase inhibitor 667 COUMATE in biological fluids in the forthcoming Phase I clinical trial.     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2′-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid-liquid extraction following the derivatization. The conjugate was analyzed by ion-pair HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.     

High-performance liquid chromatographic determination of oxodipine enantiomers, a new 1,4-dihydropyridine, applied to stereoselectivity studies in man and dog

A specific and reproducible HPLC method using a Chiral-AGP column and UV detection was developed for the evaluation of the pharmacokinetic profile of oxodipine enantiomers in dog and man. Each enantiomer was determined in plasma in the concentration range 1–400 ng/ml using the internal standard calibration method with linear regression analysis. After extraction of oxodipine and the internal standard at alkaline pH with diethyl ether—n-hexane (50:50, v/v), this method permitted the determination of each enantiomer at levels down to 10 ng/ml in dog plasma and 25 ng/ml in human plasma with sufficient accuracy (relative error <11%, n = 6) and precision (coefficient of variation <16%, n = 6). The extracted plasma volume was 500 μl and after evaporation of the organic phase, the dry residue was dissolved in 100 μl of water—2-propanol; an aliquot of 80 μl was injected into the HPLC system.     

Determination of tezosentan,a parenteral endothelin receptor antagonist,in human plasma by liquid chromatography-tandem mass spectrometry

An analytical method was developed for the quantification of tezosentan in human plasma obtained in clinical studies. The method was linear in the range 1 to 512 ng/ml. After liquid-liquid extraction, the samples were analyzed by reversed-phase HPLC with tandem mass spectrometry. The limit of quantification was 1 ng/ml and the extraction recovery was at least 88.2%. Intra- and inter-assay coefficients of variation were below 10%. Stability tests revealed that tezosentan is stable under the different conditions tested.     

Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection

A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (%C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma.     

Simultaneous measurement of fluoroquinolones in eggs by a combination of supercritical fluid extraction and high pressure liquid chromatography

Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO(2) alone, and then the fluoroquinolones were extracted by SFE with supercritical CO(2) containing 20% (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96% in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r(2) value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.     

Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatography assay for the novel antitumour agent CT2584 in human plasma

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the novel anticancer agent CT2584, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, which has recently completed a phase I trial at the Christie Hospital, Manchester under the auspices of the CRC phase I/II committee. Following addition of CT2576, 1-(11-octylamino-10-hydroxylundecyl)-3,7-dimethylxanthine, as internal standard, a solid-phase extraction cartridge (100 mg cyanopropyl) was used to isolate the drug CT2584 from human plasma. Analysis was performed by reversed-phase chromatography. CT2576 was used as internal standard at a concentration of 4 μg ml⁻¹ for the quantification of CT2584 from plasma for the duration of this work. The lower limit of quantification for the drug CT2584 in buffer using this assay was found to be 0.0122 μM (0.008 μg ml⁻¹) and 0.048 μM (0.027 μg ml⁻¹) when extracted from human plasma.     

Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC–RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE–RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC–RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE–RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.     

Determination of metformin in plasma using a new ion pair solid phase extraction technique and ion pair liquid chromatography

This article describes the development of the first ion pair solid phase extraction technique (IPSPE), which has been applied to the extraction of metformin from plasma samples. In addition an ion pair chromatographic method was developed for the specific HPLC determination of metformin. Several extraction and HPLC methods have been described previously for metformin, however, most of them did not solve the problems associated with the high polarity of this drug. Drug recovery in the developed method was found to be more than 98%. The limit of detection and limit of quantification was 3 and 5 ng/ml, respectively. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error, R.E.%) was always less than 6.9%. Stability analysis showed that metformin is stable for at least 3 months when stored at -70 degrees C. The method has been applied to 150 patient samples as part of a medication adherence study.     

Plasma determination of the enantiomers of SL 84.0418, a new antihyperglycaemic drug, by HPLC on a chiral alpha 1-AGP column.

SL 84.0418 is a new antihyperglycaemic drug. It is an orally active and selective alpha 2-adrenoceptor antagonist. This molecule, a pyrroloindole derivative, contains an asymmetric center yielding two enantiomers. In order to evaluate the pharmacokinetic profile of both enantiomers following oral administration of the racemate we have developed an HPLC method for their separation and quantification. The liquid-liquid extraction involved three steps with two salting-out procedures at pH 11.5 before and after a back-extraction with 0.005 M H2SO4. The enantiomers were separated by HPLC on a stainless-steel column (100 x 4.0 mm) packed with a chiral alpha 1-acid. The UV response was linear from 1 to 250 ng/ml for both enantiomers. The relative standard deviation (RSD) for reproducibility was below 10.7%. Using quality control samples, precision was found below 7.8% and accuracy was 108%. Extraction recoveries were ca. 60% for both enantiomers and 94% for the internal standard. Column life was brought up to 2 months, which corresponds to about 1,000 injections of biological extract. No guard column was used, but a daily back-flush was carried out. This method is suitable for routine analysis and 30 to 40 plasma samples a day can be processed. This method allows the definition of the pharmacokinetic profile of both enantiomers of SL 84.0418 in human plasma after single oral administration of doses as low as 20 mg of the racemic drug.     

Analysis of sofalcone in human plasma by high performance liquid chromatography

A simple, rapid, sensitive and reliable high performance liquid chromatography (HPLC) method for the determination of the anti-ulcer drug sofalcone in human plasma was developed. Plasma was extracted with ethyl acetate under acidic conditions and sofalcone was determined by HPLC using a C18 column and (methanol-0.1% formic acid aqueous 80:20) mobile phase. The linear calibration curves of sofalcone in human plasma were obtained over the concentration range of 0.01-5.0 microg/ml. The lower limit of quantitation (LLOQ) was 10 ng/ml in human plasma. The precision measured for plasma was within 15%. Extraction recovery was over 85% in blood. The method was successfully applied to the identification and quantification of sofalcone in pharmacokinetic studies.     

9-fluorenylmethyl chloroformate as a fluorescence-labeling reagent for derivatization of carboxylic acid moiety of sodium valproate using liquid chromatography/tandem mass spectrometry for binding characterization: a human pharmacokinetic study

In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography-tandem MS/MS (LC-MS/MS) method. Following liquid-liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01 μg/mL. Also the method is linear over the concentrations range of 0.01-32 μg/mL of VPA in human serum using 100 μL serum sample and 5 μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.