Improved high-performance liquid chromatographic detection of paclitaxel in patient's plasma using solid-phase extraction, and semi-micro-bore C18 separation and UV detection

Although a number of analytical methods for taxanes have been published, none of them are sufficiently suitable for use in a medical setting. In this study, we established an improved analytical HPLC/UV detection method using a Sep-Pak C18 cartridge for extraction and a semi-micro-borecolumn for separation. This method employed here reduced chromatographic background signals, and allowed a more sensitive analysis of taxanes in human blood sample. The recovery of taxanes after the solid-phase extraction procedure was over 90%. Chromatographic separation of paclitaxel and docetaxel was achieved within 30 min with no interference peak by a semi-micro-bore column, packed either with C18 (Wakosil 5C18 RS) or pentafluorophenyl (Curosil/Taxol) materials. The method was reproducible with coefficients of variation less than 6%. This analytical procedure was simple and sensitive with lower quantification limit of 3 ng/ml. The improved sensitivity achieved by the popular HPLC/UV apparatus, which is available in hospitals, would vouch safer and more efficient therapy with taxane.     

Determination of hyperforin in human plasma using solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

The role of the Mg2+ cation on antihypertensive molecule binding on human serum albumin (HSA) was studied by affinity chromatography. The thermodynamic data corresponding to this binding were determined for a wide range of Mg2+ concentrations (c). For the nifedipine molecule, an increase in the Mg2+ concentration produced a decrease in binding due to a decrease in the electrostatic interactions. For verapamil and diltiazem, which have the highest solvent accessible surface area, the solute binding on HSA was divided into two Mg2+ concentration regions. For a low c value below c(c) (approximately 1.6 mmol/l), the binding dependence with c was similar to that of nifedipine. For c above c(c) the hydrophobic effect created in the bulk solvent associated with a decrease in the van der Waals interactions between the solute molecule and the HSA implied a decrease in its binding. These results showed that for patients with hypertension, an Mg2+ supplementation during treatment with these antihypertensive molecules can increase the active pharmacological molecule concentration.     

Assay for etoposide in human serum using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

An HPLC assay for etoposide in human serum was developed. Serum, spiked with podophyllotoxin (internal standard), was treated with sodium dodecyl sulphate prior to solid phase extraction. Analysis was performed on a 300×3.9 mm Bondclone 10 C18 column coupled with a fluorometric detector (λ_ex 230 nm, λ_em 330 nm). The retention times for etoposide and podophyllotoxin were 14 and 28 min respectively. The range of assay was 0.5 to 20 μg/ml with a detection limit of 0.2 μg/ml. This assay is suitable for use in clinical studies with etoposide.     

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 μl of plasma, the standard curve was linear from 0.05 to 10.0 μg/ml.     

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

A new, simple and rapid high-performance liquid chromatography (HPLC) method with UV detection has been developed for the determination of apovincaminic acid in human plasma. Apovincaminic acid and internal standard were isolated from plasma samples by solid-phase extraction with OASIS HLB cartridges. The chromatographic separation was accomplished on a reversed-phase C(18) column and UV detection was set at 311 nm. The calibration curves were linear in the concentration range of 2.4-240.0 ng/ml, and the limits of quantification was 2.4 ng/ml. The precision and accuracy ranged from 0.84 to 8.54% and 91.5 to 108.3%, respectively. The developed method was subsequently applied to study the pharmacokinetics of apovincaminic acid in a group of 20 human subjects at a single oral dose of 10mg of vinpocetine tablet.     

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 valve switching (Prospekt) is used. Isocratic elution improved reproducibility and allowed the recirculation of the mobile phase. A Hypersil BDS C₁₈, 3 μm, 10×0.46 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 μg l⁻¹ and inter-assay precision (expressed as relative standard deviation) and accuracy (expressed as relative error) measured at 2 μg l⁻¹ were 5.03% and 1.82%. Calibration range was 2–140 μg l⁻¹.     

Determination of amosulalol in human plasma using solid-phase extraction combined with liquid chromatography and ultraviolet detection

Amosulalol is an antihypertensive drug with selective postsynaptic alpha 1 and non-selective beta blocking effects. A simple solid-phase extraction and high-performance liquid chromatographic (HPLC) method has been developed and validated for the quantitative determination of amosulalol in human plasma. A reversed phase C18 column was used for the separation of amosulalol and ethyl paraben (internal standard) with a mobile phase composed of 0.025 M phosphate buffer (pH 6.0).acetonitrile (73:27, v/v) at a flow rate of 1.5 mL/min. The ultraviolet detector was operated at the 272 nm wavelength. Intra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification of 30 ng/mL. Recovery of amosulalol from human plasma was >95.6%. Amosulalol was stable in human plasma under various storage conditions. This method was used successfully for a pharmacokinetic study in plasma after oral administration of a single 20 mg dose of amosulalol hydrochloride to 16 healthy volunteers.     

Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 μm particle size and 250×4 mm I.D., together with a mobile phase consisting of acetonitrile-12 μM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.     

Determination of penicillin-V in human plasma by high-performance liquid chromatography and solid-phase extraction

A high-performance liquid chromatographic method has been developed for the determination of penicillin-V concentrations between 0.1 and 19 μg/ml in human plasma. Penicillin-V was isolated from plasma by solid-phase extraction on a C₁₈/OH cartridge. The extracts were injected onto a reversed-phase HPLC system. A 125×4 mm C₁₈ column was used to separate penicillin-V from its main metabolites, 5R- and 5S-penicilloic acid and endogenous compounds. The eluent consisted of 66% 0.02 M phosphoric acid buffer, to which tetrabutylammonium dihydrogenphosphate and 34% acetonitrile were added. The column effluent was monitored by ultraviolet spectrophotometry at 269 nm. Using this method, penicillin-V concentrations in plasma could be determined with an accuracy between −5.4 and 5.2% and a precision between 0.8 and 1.6%. The method has proved to be reliable and was used in biovailability studies for the development of a new oral penicillin-V formulation.     

Simultaneous solid-phase extraction and chromatographic analysis of morphine and hydromorphone in plasma by high-performance liquid chromatography with electrochemical detection

High-performance liquid chromatography has become an important analytical tool for the quantitation of opioid drugs. Using solid-phase extraction and coulometric electrochemical detection, we have developed a chromatographic method for the simultaneous measurement of morphine and hydromorphone which is both sensitive and specific. Using 1 ml of plasma, intra-assay and inter-assay data show that the detection limit for accurate quantitation of these compounds is about 1.2 ng/ml (coefficient of variation 11.6%) for morphine and 2.5 ng/ml (coefficient of variation 10.5%) for hydromorphone. The method is simple and readily adaptable to most pharmacokinetic studies and toxic screens involving these drugs.     

Determination of sumatriptan succinate in human plasma by high-performance liquid chromatography with coulometric detection and utilization of solid-phase extraction

Sumatriptan succinate (the analyte) and naloxone (the internal standard) were extracted from plasma with a solid-phase extraction technique. Chromatography and detection were performed by isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The standard curve was linear over the range 0–100 ng/ml of sumatriptan succinate in plasma. The reproducibility (as defined by the coefficient of variation, C.V.) over the range of the standard curve was 4.9–7.3%. The recovery averaged 83%. The sensitivity was 0.25 ng of sumatriptan on column (allowing a concentration of 0.5 ng/ml to be determined from a 1-ml plasma sample volume). Plasma profiles of the analyte following subcutaneous (s.c.) administration in eight normal male volunteers, are presented.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

Quantification of tipranavir in human plasma by high-performance liquid chromatography with UV detection

A simple method for the quantification of tipranavir, a new non-peptidic protease-inhibitor, was developed. An internal standard, prazepam, was added to 100 microl of plasma before a liquid-liquid extraction by 3 ml of tert-butyl methyl ether. The extracts were evaporated to dryness and reconstituted with 100 microl of mobile phase before being injected in the chromatographic system. The separation was made on a C8 column using sodium acetate buffer (pH 5):methanol:acetonitrile (35:30:35, v/v/v) as mobile phase. The detection was performed at a wavelength of 260 nm. The method was linear and has been validated over a concentration range of 2-80 mg/l. The mean precision and accuracy of the method were respectively, 10.5 and -9.1%. The mean recovery was 70.8%.     

Quantitation of efletirizine in human plasma and urine using automated solid-phase extraction and column-switching high-performance liquid chromatography

A heart-cut column-switching, ion-pair, reversed-phase HPLC system was used for the quantitation of efletirizine (EFZ) in biological fluids. The analyte and an internal standard (I.S.) were extracted from human EDTA plasma by C₁₈ solid-phase extraction (SPE) using a RapidTrace^® workstation. The eluent from the SPE was evaporated, reconstituted and injected onto the HPLC column. Urine samples were diluted and injected directly without the need of extraction. The compounds of interest were separated from most of the extraneous matrix materials by the first C₁₈ column, and switched onto a second C₁₈ column for further separation using a mobile phase of stronger eluting capability. Linearity range was 10–2000 ng ml⁻¹ for plasma and 0.05–10 μg ml⁻¹ for urine. The lower limit of quantitation (LOQ) was 10 ng from 1 ml of plasma, with a signal-to-noise ratio of 15:1. Inter-day precision and bias of quality control samples (QCs) were <5% for plasma and <7% for urine. Selectivity was established against six other antihistamines, three analogs of efletirizine, and on 12 control plasma lots and nine control urine lots. Recovery was 90.0% for EFZ and 89.5% for I.S. from plasma. One hundred samples can be processed in every 2.75 h on a 10-module RapidTrace^® workstation with minimal human attention. Method ruggedness were tested on three brands of SPE and six different lots of one SPE brand. Performance ruggedness was demonstrated by different analysts on multiple HPLC systems. Analyte stability through sample storage, extraction process (benchtop, freeze–thaw, refrigeration after extraction) and chromatography (on-system, reinjection) was established.     

Determination of mitiglinide in rat plasma by high-performance liquid chromatography with UV detection

A selective and sensitive high-performance liquid chromatography method has been developed and validated for determination of mitiglinide (MGN) in rat plasma using 2-(4-biphenylyl) propionic acid (BPA) as internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and 0.02 mol/l KH(2)PO(4) buffer (pH 4.0) (45:55, v/v) as mobile phase delivered at 1.0 ml/min. The UV detector was set at 210 nm. The assay was linear over the range 0.1-20 microg/ml for MGN. The average extraction recoveries of MGN and BPA from rat plasma were 98.6 and 97.4%, respectively. The developed method has been applied to the pharmacokinetic study of MGN in rats.     

Determination of A 3,4-diaminopyridine in plasma by liquid chromatography with electrochemical detection using solid-phase extraction

In order to quantify a small amount of a drug, 3,4-diaminopyridine (3,4-DAP), in animal plasma samples, an analytical method was developed. It involved an extraction of 3,4-DAP and phenylephrine, used as internal standard (IS), from plasma with solid-phase extraction (SPE) on C18 cartridges. This analytical method is a hyphenated technique based on high-performance liquid chromatography with electrochemical detection (HPLC-EC) whose purpose is to obtain first a sensitive method and second a satisfying separation between 3,4-DAP and phenylephrine. The analytical method is accurate, specific, and linear between 10 and 500 g of 3,4-DAP per litre. The recovery of 3,4-DAP is estimated at 70.8% with a 95% confidence interval of (66.0 -75.6%). Intermediate precision was evaluated on three quality control samples; the intra-day precision was estimated at 13.5, 9.1, 7.8% and the inter-day precision at 17.9, 8.4, 9.3%. The limit of quantification of the method was evaluated at 10 g l-1. First toxicokinetic parameters determined on dogs plasma samples after one 3,4-DAP oral administration of 1 mg kg-1 were: Cmax=395.7 microg l-1; Tmax =15 min; t1/2=113.6 min; Clearance/F=16.8 ml kg-1 min-1 and Vd/F=2.7 l kg -1.     

Validation of a high-performance liquid chromatography method for tramadol and o-desmethyltramadol in human plasma using solid-phase extraction

An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36±12.53% and 93.52±7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Quantitation of psilocin in human plasma by high-performance liquid chromatography and electrochemical detection: comparison of liquid–liquid extraction with automated on-line solid-phase extraction

Two modifications of the HPLC–ED method with respect to extraction procedure used have been developed for psilocin, the active metabolite of psilocybin, in human plasma using either liquid–liquid extraction (LLE) or automated on-line solid-phase extraction (on-line SPE). Each type of the sample preparation required a different HPLC system followed by electrochemical detection at 650 to 675 mV. The limit of quantitation of both modifications was 10 ng/ml psilocin. There was no significant difference observable between the LLE and the on-line SPE in terms of method standard deviation (LLE 1.82%, on-line SPE 1.13%) and the analytical results. However, the advantages of on-line SPE in addition to different selectivity were less manual effort, smaller plasma volumes of 400 μl (LLE 2 ml) and a recovery of psilocin in human plasma of nearly 100% (LLE 88%). In contrast to a previous procedure both methods were rapid, simple and reliable and yielded high plasma recoveries. They were used successfully in the quantitation of psilocin in plasma samples obtained from healthy volunteers after p.o. administration of 0.2 mg psilocybin per kg body mass. Plasma concentration curves and pharmacokinetic parameters were calculated.     

Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection

A simple, sensitive, selective, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of lorazepam (LZP) in human plasma, using oxazepam (OZP) as internal standard. LZP and OZP were extracted from alkalinized (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with a mixture of n-hexane-dichloromethane (70:30%; v/v). Chromatographic separation was performed on a reversed-phase Synergi Max RP analytical column (150 mmx4.6 mm i.d.; 4 microm particle size), using an aqueous mobile phase (10 mM KH2PO4 buffer (pH 2.4)-acetonitrile; 65:35%, v/v) delivered at a flow-rate of 2.5 ml/min. Retention times for OZP and LZP were 10.2 and 11.9 min, respectively. Calibration curves were linear from 10 to 300 ng with correlation coefficients (r2) better than 0.99. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 10 ng/ml, respectively, using 0.5 ml samples. The mean relative recoveries at 20 and 300 ng/ml were 84.1+/-5.5% (n=6) and 72.4+/-5.9% (n=7), respectively; for OZP at 200 ng the value was 68.2+/-6.8% (n=14). The intra-assay relative standard deviations (R.S.D.) at 20, 150 and 270 ng/ml of LZP were 7.8%, 9.8% (n=7 in all cases) and 6.6% (n=8), respectively. The inter-assay R.S.D. at the above concentrations were 15.9%, 7.7% and 8.4% (n=7 in all cases), respectively. Intra- and inter-assay accuracy data were within the acceptance interval of +/-20% of the nominal values. There was no interference from other commonly co-administered anticonvulsant, antimicrobial, antipyretic, and antimalarial drugs. The method has been successfully applied to a pharmacokinetic study of LZP in children with severe malaria and convulsions following administration of a single intravenous dose (0.1 mg/kg body weight) of LZP.     

Determination of ketamine and norketamine enantiomers in plasma by solid-phase extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method is described for determination of sub-anaesthetic concentrations of the enantiomers of ketamine and its metabolite norketamine in plasma. The samples are purified by reversed-phase solid-phase extraction. The enantiomers are separated on a Chiral AGP column with a mobile phase containing 16% methanol and a 10 mM phosphate buffer at pH 7.0, and measured by UV-detection at a wavelength of 220 nm. Linear calibration curves with correlation coefficients better than 0.995 have been obtained in the range 10–320 ng/ml. Minimum detectable concentrations were about 2 ng/ml.