Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

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

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

Modified high-performance liquid chromatography assay for the measurement of 2′-deoxyuridine in human plasma and its application to pharmacodynamic studies of antimetabolite drugs

A new method is presented for the HPLC determination of plasma 2′-deoxyuridine (dUrd). Briefly, 1 ml of human plasma is deproteinised with perchloric acid followed by purification by solid-phase extraction using a non-polar high-capacity polymeric sorbent. The dUrd is separated on a C₁₈ reversed-phase column using a mobile-phase of 0.05% v/v trifluoroacetic acid in water, with a retention time of 8.5 min at a flow-rate of 1.25 ml min⁻¹. Quantitation is by UV detection at 261 nm using a photodiode array detector. The limit of quantitation is 6 nM with a linear response over the measured range 6–400 nM. Both intra- and inter-day RSD and bias are typically less than 13%. Chromatograms and pharmacodynamic data from a Phase 1 Clinical Trial of a new antifolate drug, ZD9331 are included to illustrate the utility of the method. They show the increase in circulating dUrd as a result of drug inhibition of the target enzyme thymidylate synthase. The method has the significant advantages of ease and simplicity over earlier methods and may be applied to the analysis of other nucleoside species.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.     

Determination of the halothane metabolites trifluoroacetic acid and bromide in plasma and urine by ion chromatography

Halothane (CF₃CHClBr), a widely used volatile anesthetic, undergoes extensive biotransformation in humans. Oxidative halothane metabolism yields the stable metabolites trifluoroacetic acid and bromide which can be detected in plasma and urine. To date, analytical methodologies have either required extensive sample preparation, or two separate analytical procedures to determine plasma and urine concentrations of these analytes. A rapid and sensitive method utilizing high-performance liquid chromatography-ion chromatography (HPLC-IC) with suppressed conductivity detection was developed for the simultaneous detection of both trifluoroacetic acid and bromide in plasma and urine. Sample preparation required only ultrafiltration. Standard curves were linear (r²≥0.99) from 10 to 250 μM trifluoroacetic acid and 2 to 5000 μM bromide in plasma and 10 to 250 μM trifluoroacetic acid and 2 to 50 μM bromide in urine. The assay was applied to quantification of trifluoroacetic acid and bromide in plasma and urine of a patient undergoing halothane anesthesia.     

Determination of saquinavir in human plasma by high-performance liquid chromatography

We developed and characterized a high-performance liquid chromatography (HPLC) assay for the determination of saquinavir, an HIV protease inhibitor, in human plasma samples. Extraction of plasma samples with diethyl ether resulted in quantitative recovery of both saquinavir and its stereoisomer Ro 31-8533 which was used as an internal standard. The assay was performed isocratically using 5 mM H₂SO₄ (pH 3.5) and acetonitrile (75.5:24.5, v/v) containing 10 mM tetrabutylammonium hydrogen sulfate (TBA) as a mobile phase, a Nucleosil 3C8 column kept at 45°C and UV detection at 240 nm. Using this method, saquinavir and Ro 31-8533 can be separated from endogenous substances, and in the concentration range of 5–110 ng/ml the relative standard deviations for the determination of saquinavir were below 5%. The detection limit of saquinavir in human plasma was 1 ng/ml. The usefulness of the method was demonstrated by quantification of saquinavir in plasma of human subjects treated with 600 mg of saquinavir per os or 12 mg intravenously.     

Determination of a new oral iron chelator, ICL670, and its iron complex in plasma by high-performance liquid chromatography and ultraviolet detection

ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe–[ICL670]₂ with one ferric iron. A simple and rapid HPLC–UV method for the separate determination of ICL670 and Fe–[ICL670]₂ in the plasma of iron-overloaded patients is described. Plasma samples were prepared as rapidly as possible, the tubes being kept at 4°C. Plasma proteins were precipitated with methanol. The supernatant was diluted with water and placed on the refrigerated sample rack of an autosampler before injection. The chromatographic separations were achieved on an Alltima C₁₈ column using 0.05 M Na₂HPO₄ and 0.01 M tetrabutylammonium hydrogen sulfate–acetonitrile–methanol (41:9:50, v/v/v) as mobile phase. The analytes were detected at 295 nm. Calibration and quality control samples were prepared in normal human plasma. The mean accuracy (n=6) over the entire investigated concentration range 0.25–20 μg/ml ranged from 91 to 109% with a coefficient of variation (C.V.) from 4 to 8% for ICL670, and from 95 to 105% with a C.V. from 2 to 20% for the iron complex. The dissociation of the complex during analysis was shown to be marginal. The iron removal from plasma of iron-overloaded patients by free ICL670 during analysis was low. The in vitro iron transfer from the iron pools of iron-overloaded plasma onto ICL670 was shown to be a slow process.     

High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease (K_i=2 nM), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 μl of human plasma with a mixture of ethyl acetate–acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C₁₈ analytical column and a mobile phase consisting of 25 mM monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)–acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 μg/ml to 10 μg/ml, with a minimum quantifiable level of 0.0500 μg/ml for nelfinavir.     

Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in 100 μl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C₁₈ J’Sphere ODS-M80 (150×4.0 mm) column with mobile phase acetonitrile–phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 μM and 13.5% at 1.00 μM. The inter-assay precision was 3.3% at 12.00 μM and 15.6% at 1.00 μM. The lower limit of quantification was 1.00 μM. The limit of detection was 0.50 μM.     

Rapid, highly sensitive gradient narrow-bore high-performance liquid chromatographic determination of suramin and its analogues

A high-performance liquid chromatography (HPLC) method for the determination of suramin, its precursors and analogues in a aqueous solutions and in plasma samples with advantages compared to earlier methods is described. Due to the method's high sensitivity (detection limit of suramin in plasma samples: 7 ng/ml; in aqueous solutions: 5 ng/ml) and selectivity suramin t_R: 7.05 min, precursor amine 2 t_R: 4.68 min), it is possible to analyze degradation products, impurities and possible metabolites of suramin besides suramin. Tetrabutylammonium hydrogensulfate (TBAHS) (5 mM) is used as ion-pairing reagent in a mixture of 36% methanol and 0.02 M phosphate buffer pH 6.5 is used as the mobile phase. After sample injection, a linear gradient from 36 to 62.9% methanol is run. A C₈ stationary phase (100 × 2.1 mm I.D.) is used and ultraviolet (UV) detection at 238 nm is applied. Plasma extraction is performed with tetrabutylammonium bromide (pH 8.0) and acetonitrile. This procedure allows the determination of suraminn and its precursor amine 2 in the range of 0.05–400 μg/ml with high precision [relative standard deviation of peak areas at 0.05 μg/ml: 2.10% (n = 5)] and nearly complete recovery (>96.5%). Because of the high flexibility of the chromatographic system and subsequently the universality of the method, the analysis of a broad range of suramin analogues is possible. The result of the purity check of two suramin analogues is given.     

Solid-phase extraction and determination of ranitidine in human plasma by a high-performance liquid chromatographic method utilizing midbore chromatography

An improved high-performance liquid chromatographic (HPLC) method utilizing solid-phase extraction (SPE) and midbore chromatography was developed for the determination of ranitidine in human plasma. A mobile phase of 20 mM K₂HPO₄-acetonitrile-triethylamine (87.9:12.0:0.1, v/v) pH 6.0 was used with a phenyl analytical column and ultraviolet detection (UV). The method demonstrated linearity from 25 to 1000 ng/ml in 500 μl of plasma with a detection limit of 10 ng/ml. The method was utilized in a pharmacokinetic study evaluating the effects of pancreatico-biliary secretions on ranitidine absorption.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

High-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma

A simple, rapid and sensitive reversed-phase high-performance liquid chromatographic method with UV detection was developed for the quantification of di(2-ethylhexyl) phthalate (DEHP) in parenteral nutrition admixtures containing fat emulsion and in plasma samples of children daily treated by total parenteral nutrition. The analyte and the internal standard, di-n-heptyl phthalate, were extracted twice using hexane and the organic layer separated and dried under nitrogen. The residues were reconstituted with acetonitrile and 20 μl was injected into a Waters Spherisorb C₁₈ column, the UV detector was set at 202 nm. The mobile phase was acetonitrile–aqueous buffer (triethylamine 0.08% adjusted to pH 2.8 with 1 M phosphoric acid) mixture (88:12, v/v) and it was pumped at 1 ml/min. Average recoveries were 97% or greater. This method was successfully used to investigate the amounts of DEHP which can leach from bags and tubing into fat emulsion and which could contaminate children under long-term parenteral nutrition. On the other hand, the circulating DEHP concentrations were estimated in four children under regular long-term parenteral nutrition.     

High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C₈ cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C₁₈ stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% (n = 10) for plasma (0.2 μg/ml) and lower than 12.4% (n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.     

High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 μg/ml with a 50-μl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 μl of the samples with Bond Elut C₁₈ cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7–9.9 (n=4) and 1.7–7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8–8.1% (n=3) and 2.9–4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.     

Improved assay method for the determination of pyronaridine in plasma and whole blood by high-performance liquid chromatography for application to clinical pharmacokinetic studies

An improved high-performance liquid chromatography method using a diisopropyl-C₁₄ reversed-phase column (Zorbax Bonus-RP column) and a liquid–liquid extraction technique with UV detection is presented for the analysis of pyronaridine in human whole blood and plasma. Tribasic phosphate buffer (50 mM, pH 10.3) and diethyl ether were used for liquid–liquid extraction. The mobile phase consists of acetonitrile–0.08 M potassium dihydrogen phosphate buffer (13:87, v/v) with the pH 2.8 adjusted by orthophosphoric acid. Amodiaquine was found to be a suitable internal standard for the method. The quantification limit with UV detection at 275 nm was 3 ng on-column for both plasma and blood samples. The method was applied to plasma and blood specimens from a rabbit after a single intramuscular dose of pyronaridine tetraphosphate (20 mg/kg as base). From this in vivo study, evidence was found that pyronaridine is concentrated in blood cells, with a blood:plasma ratio ranging from 4.9 to 17.8. We conclude that blood is the preferred matrix for clinical pharmacokinetic studies.     

Determination of citalopram enantiomers in human plasma by liquid chromatographic separation on a Chiral-AGP column

A liquid chromatographic method for the quantitative analysis of S-(+)- and R-(−)-citalopram in human plasma has been developed and validated. The enantiomers of citalopram and the internal standard, R-(+)-propranolol, were extracted from alkaline plasma with 2% n-butanol in n-hexane. After a clean-up step, the organic phase was evaporated and the residues dissolved in 50–100 μl of 0.001 M HCl. The separation was performed on a Chiral-AGP column with 3.0 mM N-dodecyl-N,N-dimethylammonio-3-propanesulfonate and 10 mM hexanoic acid in phosphate buffer pH 6.5 as the mobile phase. The limit of detection was estimated to be 1 ng/ml (S/N≈3) for each enantiomer monitoring UV absorption at 240 nm. In the range studied, 2.31–191 ng/ml, the recoveries were quantitative and the coefficients of variations were between 2.47% and 11.5%.     

Trace analysis of SN-38 in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with fluorescence detection was developed and validated for the quantitation of SN-38, the active metabolite of irinotecan (CPT-11), a new anticancer drug. This method uses solid-phase extraction with a C₁₈ column for sample clean-up and concentration following acidification of human plasma with two volumes of 0.1 M HCl. Using blank plasma spiked with SN-38, we found the assay to be linear over the concentration range of 10–500 pM (3.9–195 pg/ml) with acceptable total and within-day imprecision. The recovery of SN-38 ranged from 48.3% (10 pM) to 91.5% (500 pM) whereas that of the internal standard, 20-(S)-camptothecin, was 96.9% (500 pM). This method represents a sizeable increase in sensitivity over other published methods and is shown to be suitable for the measurement of ‘trough' concentrations of SN-38 during the treatment of patients with a weekly regimen of irinotecan.     

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Summary Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA₃, 15 μM)+N⁶-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA₃ (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA₃ (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.