Monitoring propofol serum levels by rapid and sensitive reversed-phase high-performance liquid chromatography during prolonged sedation in ICU patients.

A quick and sensitive reversed-phase high-performance liquid chromatography (HPLC) method has been developed in order to determine the concentration of Propofol (2,6 diisopropylphenol) in human serum. Propofol can be isolated from serum by adding 0.5 mL precipitating solution. This consists of an acetonitrile and perchloric acid (67:33, v/v) mixture, which also contains dibutylphthalate (2 mg/100 mL) as internal standard. The sample is then mixed for 1 min on a vortex-mixer. The endogenous serum substances precipitated by acetonitrile and perchloric acid are further separated by centrifugation. The supernatant is directly injected into the HPLC system. A 250- x 4.6-mm column, packed with 10-microns Spherisorb reversed-phase octadecylsilane particles (C18), is used for chromatographic separation. The mobile phase consists of an acetonitrile-water mixture (67:33 ratio) with 0.4 mL acetic acid (pH 4). Propofol is monitored by a UV-visible detector at 270 nm and 0.1-0.002 absorbance units full scale (AUFS). The detection limit of Propofol (in human serum) is 0.1 mg/L for a 20-microL injection volume. The time of the assay is less than 20 min, including sample preparation.     

High-performance liquid chromatographic method for simultaneous determination of sophoridine and matrine in rat plasma

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of sophoridine and matrine in rat plasma. Sophoridine and matrine in the resulting supernatant of the plasma deproteinized with acetonitrile containing an internal standard (acetanilide) were directly determined by reversed-phase HPLC and ultraviolet detection. The result of limits of quantitation for matrine and sophoridine were 200 and 350 ng/mL in plasma, respectively, and recovery of both analytes was greater than 98%. The assay was linear from 250 to 4000 ng/mL for matrine and from 500 to 8000 ng/mL for sophoridine. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of matrine and sophoridine in the plasma following oral administration of Kexieling tablets, which is one of the preparations of Kudouzi at a dose equivalent to 30 and 60 mg/kg of matrine and sophoridine, respectively.     

High-performance liquid chromatography-electrospray ionization mass spectrometric determination of nisoldipine in human plasma

A selective and sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (MS) for the determination of nisoldipine in human plasma is first presented. With nimodipine as the internal standard, nisoldipine is extracted from plasma with ethyl acetate. The organic layer is evaporated and the residue is resuspended in the mobile phase of methanol-water (80:20, v/v). An aliquot of 40 microL is chromatographically analyzed on an Agilent ODS C18 reversed-phase column (5 microm, 250- x 4.6-mm i.d.) by means of selected-ion monitoring mode MS. The calibration curve of nisoldipine in plasma exhibits a linear range from 0.5 to 20.0 ng/mL with a correlation coefficient of 0.9995. The limit of detection is 0.2 ng/mL. The within- and between-day variations (relative standard deviation) are less than 9.28% and 11.13% (n = 5), respectively. The developed method is validated through successful use for the analysis of nisoldipine contained in biological fluids resulting from a phase-I human pharmacokinetic study.     

An automated method for the determination of diclofenac sodium in human plasma

An automated method utilizing laboratory robotics has been developed for quantifying diclofenac sodium concentrations in human plasma. The robotic system aliquots the biological sample, adds the internal standard (CGP 4287), extracts the compounds from the acidified biological matrix (pH less than 2) into an organic phase (hexane-isopropyl alcohol), and concentrates the extracts for reversed-phase, high-performance liquid chromatographic (HPLC) analysis. The laboratory robot is directly interfaced to the HPLC system, and the data are automatically collected and results calculated. Separation is achieved on a 3-microns ODS (6.2-mm x 8.0-cm) column with ultraviolet (UV) detection of the drug and internal standard at 280 nm. Recovery and reproducibility assessments indicate good accuracy (overall mean relative recovery of 99.8%) and precision (coefficient of variation from 0.5 to 11.1%) over the diclofenac sodium concentration range of 5.0-1000 ng/mL, with a quantification limit of 5.0 ng/mL. The method has been successfully applied to a pharmacokinetic study in which normal volunteers received 150 mg of a prototype controlled-release formulation of diclofenac sodium.     

Simple and rapid high-performance liquid chromatographic method for endogenous alpha-tocopherol determination in human plasma

A simple and rapid reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of endogenous alpha-tocopherol in human plasma. Following addition of alpha-tocopheryl acetate as the internal standard, the plasma was deproteinized using acetonitrile and isopropanol mixture prior to HPLC analysis. Methanol was used as the mobile phase and the effluent was quantitated at 292 nm. By this developed method, the concentrations of alpha-tocopherol were linearly related to their responses in the range of 0.8-30 microg/mL. The relative standard deviations intra-day and inter-day for alpha-tocopherol in plasma were less than 10%. The percentage of bias was within +/-4%, which confirmed the accuracy of the method. The method has been successfully applied for determining endogenous alpha-tocopherol in healthy Thai male volunteers.     

Quantification of zolpidem in human plasma by high-performance liquid chromatography with fluorescence detection

A simple, reliable HPLC method with fluorescence detection (excitation 320 and emission 388 nm) was developed and validated for quantitation of zolpidem in human plasma. Following a single-step liquid-liquid extraction, the analyte and internal standard (quinine) were separated using an isocratic mobile phase on a reversed-phase C(18) column. The lower limit of quantitation was 1.8 ng/mL, with a relative standard deviation of less than 5%. A linear dynamic range of 1.8-288 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.7-4.8 and 1.2-2.3%, respectively. The between-batch and within-batch accuracy was 95.3-100.4 and 95.5-102.7%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zolpidem in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is simple and repeatable enough to be used in pharmacokinetic studies.     

Selective and sensitive determination of bis(7)-tacrine, a high erythrocyte binding acetylcholinesterase inhibitor, in rat plasma by high-performance liquid chromatography-tandem mass spectrometry

The current study aims to develop a specific and sensitive LC-MS/MS method for determination of bis(7)-tacrine (B7T) in rat plasma. A 100 microL plasma sample was extracted with ethyl acetate. B7T and the internal standard (IS), pimozide, in the samples were then analyzed with LC-MS/MS in positive electrospray ionization condition. Chromatographic separation of B7T and IS was achieved in a C(18) reversed-phase HPLC column (150 x 2.1 mm i.d.) by isocratic elution with a mobile phase consisting of 0.05% formic acid in water and acetonitrile (1:1, v/v) at a flow rate of 0.35 mL/min. Multiple-reaction monitoring (MRM) mode was employed to measure the ion transitions: m/z 247 to 197 for B7T and m/z 462 to m/z 328 for IS, respectively. The method was linear over the studied ranges of 100-5000 and 10-100 ng/mL. The intra-day and inter-day variations of the analysis were less than 6.8% with standard errors less than 9.0%. The detection limit of B7T in rat plasma was 1 ng/mL. The developed method was successfully applied to the pharmacokinetic study of B7T after intravenous administration of 1 mg/kg B7T and further proved to be readily utilized for determination of B7T in rat plasma samples.     

Development of a method for the determination of ibafloxacin in plasma by HPLC with flourescence detection and its application to a pharmacokinetic study

A simple, rapid, and sensitive high-performance liquid chromatographic method is developed for the determination of ibafloxacin in rabbit plasma. Plasma proteins are precipitated with acetonitrile, and after extraction with methylene chloride followed by desecation, ibafloxacin is determined by reversed-phase chromatography with fluorescence detection exciting at 330 nm and emission at 368 nm. Peaks corresponding to ibafloxacin and the internal standard (salycilic acid) are obtained at 9.8 and 5.2 min, respectively. The method is validated for a limit of quantitation of 10 ng/mL. The intraday relative standard deviation ranges from 4.78-7.15%, and the interday precision ranges from 1.32-4.03%. The method shows linearity for the two calibration curves used (10-100 ng/mL and 100-2000 ng/mL). The procedure described is applied successfully to a pharmacokinetics study of ibafloxacin in rabbits.     

Quantitation of zopiclone and desmethylzopiclone in human plasma by high-performance liquid chromatography using fluorescence detection

A simple, reliable HPLC method using fluorescence detection (excitation 307 and emission 483 nm) was developed and validated for simultaneous quantitation of zopiclone and its metabolite desmethylzopiclone in human plasma. Following a single-step liquid-liquid extraction, the analytes and internal standard (zaleplon) were separated using an isocratic mobile phase on a reversed-phase C18 column. The lower limit of quantitation was 3 ng/mL for zopiclone and 6 ng/mL for desmethylzopiclone with a relative standard deviation of less than 5%. A linear dynamic range of 3-300 ng/mL for zopiclone and of 6-500 ng/mL for desmethylzopiclone was established. This HPLC method was validated with between-batch precision of 1.7-4.2% and 3.2-7.5% for zopiclone and desmethylzopiclone respectively. The between-batch accuracy was 99.4-111.5% and 101.6-104.8% for zopiclone and desmethylzopiclone, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zopiclone and desmethylzopiclone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days' storage in a freezer. This validated method is simple and repeatable enough to be used in pharmacokinetic studies.     

Simultaneous determination of oxycodone and its major metabolite, noroxycodone, in human plasma by high-performance liquid chromatography

Oxycodone (14-hydroxy-7,8-dihydrocodeinone) is a potent opioid receptor agonist. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of oxycodone and its major metabolite, noroxycodone, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile and phosphate buffer (8:92, v/v) at a flow rate of 1 mL/min, and UV detection at 205 nm. The retention times for oxycodone, noroxycodone and codein (internal standard) were 14.7, 13.8 and 10.2 min, respectively. The validated quantitation range of the method was 2-100 ng/mL for oxycodone and 10-100 ng/mL for noroxycodone. The developed procedure was applied to assess the pharmacokinetics of oxycodone and its metabolite following administration of a single 20 mg oral dose of oxycodone hydrochloride to one healthy male volunteer.     

High-throughput quantification of perindopril in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of perindopril in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 369/172 for perindopril and m/z 417/234 for the internal standard. The method exhibited a linear dynamic range of 0.1-100 ng/mL for perindopril in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6.1%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 450 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.     

Simultaneous determination of niflumic acid and its prodrug, talniflumate in human plasma by high performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of niflumic acid and its prodrug, talniflumate, in human plasma. Niflumic acid and talniflumate were eluted isocratically with methanol-water (73:27, v/v, adjusted to pH 3.5 by acetic acid) at a fl ow rate of 1 mL/min. Indomethacin was used as an internal standard. Signals were monitored by an UV detector at 288 nm. Retention times of indomethacin, niflumic acid and talniflumate were 5.9, 7.2 and 13.5 min, respectively. Calibration plots were linear over the range 50-5000 ng/mL for niflumic acid and 100-5000 ng/mL for talniflumate. The limits of quantitation were 50 ng/mL for niflumic acid and 100 ng/mL for talniflumate. The intra- and inter-day relative standard deviations (RSD) of niflumic acid and talniflumate were less than 10% and the accuracies were higher than 90%. This method is rapid, sensitive and reproducible for the determination of niflumic acid and talniflumate in human plasma.     

Simultaneous determination of pantoprazole and its two metabolites in dog plasma by HPLC

A simple and sensitive high-performance liquid chromatography (HPLC) method is developed and validated for simultaneous determination of pantoprazole and its two metabolites (pantoprazole sulfone and pantoprazole thioether) in dog plasma and applied to a pharmacokinetic study in Beagle dogs. Following a protein precipitation procedure, the samples are separated using reversed-phase HPLC (C18) by a gradient of acetonitrile and ammonium acetate (pH 6.0) at a flow rate of 1.0 mL/min and quantitated using UV detection at 290 nm. Omeprazole is selected as the internal standard. The method has a lower limit of quantitation of 0.025 microg/mL for pantoprazole and its two metabolites, using 0.1-mL aliquots of plasma. The linear calibration curves are obtained in the concentration range of 0.025-10.0 microg/mL for three analytes. The intra- and interrun precision (relative standard deviation), calculated from quality control (QC) samples, is less than 13% for three analytes. The accuracy determined from QC samples is between -6.4% and 12%.     

Improved HPLC method for doxorubicin quantification in rat plasma to study the pharmacokinetics of micelle-encapsulated and liposome-encapsulated doxorubicin formulations

An improved simple, rapid and accurate HPLC method for quantification of doxorubicin derived from micelle-encapsulated or liposome-encapsulated doxorubicin formulation in rat plasma was described. The mobile phase consisting of a mixture of methanol-water [containing 0.1% formic acid anhydrous and 0.1% ammonia solution (25%), pH 3.0], 60:40, was delivered at a flow rate of 1.0 mL/min. Sample preparation for micelle- or liposome-encapsulated doxorubicin in rat plasma were achieved directly by protein precipitation with acetonitrile. Doxorubicin and daunorubicin (internal standard, IS) were separated on a C(18) reversed-phase HPLC column and quantified by a fluoresence detection with an excitation wavelength of 475 nm and an emission wavelength of 580 nm. The linearity was obtained over the range of 5.0-1000.0 ng/mL and 1.0-200.0 microg/mL for doxorubicin and the lower limit of quantitation was 5.0 ng/mL. For each level of quality control samples, inter- and intra-assay precision was less than 9.6 and 5.1% (relative standard deviation), respectively, and percentage error was within +/-2.6%. The extraction recoveries of doxorubicin in the range of 10 ng/mL to 100 microg/mL in rat plasma were between 94.1 and 105.6%. This method was successfully applied to the pharmacokinetic study of doxorubicin formulations after i.v. administration to rats.     

Quantification of granisetron in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of granisetron in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 313/138 for granisetron and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for granisetron in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 5%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Determination of imatinib mesylate and its main metabolite (CGP74588) in human plasma and murine specimens by ion-pairing reversed-phase high-performance liquid chromatography

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of imatinib, a tyrosine kinase inhibitor, and its main metabolite N-desmethyl-imatinib (CGP74588) in human plasma and relevant murine biological matrices. A simple HPLC assay for the individual quantification of imatinib and CGP74588 in murine specimens has not been reported to date. Sample pre-treatment involved liquid-liquid extraction with tert-butyl-methyl ether. Imatinib, CGP74588 (metabolite) and the internal standard 4-hydroxybenzophenone were separated using a narrow bore (2.1 x 150 mm) stainless steel Symmetry C(18) column and detected by UV at 265 nm. The mobile phase consisted of 28% (v/v) acetonitrile in 50 mM ammonium acetate buffer pH 6.8 containing 0.005 M 1-octane sulfonic acid and was delivered at 0.2 mL/min. The calibration curve was prepared in blank human plasma and was linear over the dynamic range 10 ng/mL to 10 microg/mL). The accuracy was close to 100% and the within-day and between-day precisions were within the generally accepted 15% range. The validation results showed that the assay was selective and reproducible. This method was applied to study the pharmacokinetics of imatinib and its main metabolite in human and mice.     

Liquid chromatography/negative ion electrospray tandem mass spectrometry method for the quantification of fluvastatin in human plasma: validation and its application to pharmacokinetic studies

A simple, sensitive and rapid high-performance liquid chromatography/negative ion electrospray tandem mass spectrometry method was developed and validated for the assay of fluvastatin in human plasma. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M-H]- ions, m/z 410/348 for fluvastatin and m/z 480/418 for the internal standard. The assay exhibited a linear dynamic range of 2-500 ng/mL for fluvastatin in human plasma. The lower limit of quantification was 2 ng/mL with a relative standard deviation of less than 5%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

High-performance liquid chromatography-electrospray ionization-mass spectrometric determination of emedastine difumarate in human plasma and its pharmacokinetics

A selective and sensitive method employing high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry is developed and validated for the determination of emedastine difumarate in human plasma. With naphazoline hydrochloride as the internal standard, emedastine difumarate is extracted from plasma with ethyl acetate. The organic layer is evaporated, and the residue is redissolved in the mobile phase. An aliquot of 10 microL is chromatographically analyzed on a prepacked Phenomenex Luna 5u CN 100A (150 x 2.0-mm i.d.) column, using a mobile phase comprised of methanol-water (20 mM CH(3)COONH(4), pH 4.0) (80:20, v/v). Standard curves are linear (r(2) = 0.9990) over the concentration range of 0.05-30 ng/mL and had good accuracy and precision. The within- and between-batch precisions did not exceed 15% for the relative standard deviation. The lower limit of detection is 0.01 ng/mL. The validated HPLC-ESI-MS method is successfully used to study emedastine difumarate pharmacokinetics in 12 healthy volunteers.     

Simultaneous determination of leflunomide and its active metabolite,A77 1726, in human plasma by high-performance liquid chromatography

The isoxazol derivative leflunomide [N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide] is an inhibitor of de novo pyrimidine synthesis used for the treatment of rheumatoid artrithis. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of leflunomide and its active metabolite, A77 1726, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile, water and formic acid (40/59.8/0.2, v/v), at a flow rate of 0.5 mL/min, and UV detection at 261 nm. The retention times for A77 1726, leflunomide and warfarin (internal standard) were 8.2, 16.2 and 12.2 min, respectively. The validated quantification range of the method was 0.05-100 micro g/mL for leflunomide and 0.1-100 micro g/mL for A77 1726. The developed procedure was applied to assess steady-state plasma concentrations of A77 1726 in patients with rheumatoid arthritis treated with 10 or 20 mg leflunomide per day.     

Usefulness of the chaotropic effect in sample preparation for chromatographic analysis of acidic xenobiotics in human plasma

In this study a new RP‐HPLC with photo‐diode array detection method for the determination of ibuprofen ((RS)‐2‐(4‐isobutylphenyl)propionic acid) in human plasma samples was developed. Samples were prepared by SPE and analyzed by an isocratic elution mode over a C₁₈ column using 80% methanol. A novel sample pretreatment method, based on the addition of ionic liquids possessing chaotropic ions to small human plasma sample (100 μL), was elaborated. 1‐Butyl‐3‐methylimidazolium chloride and 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMIM BF₄) were tested from the point of view of extraction yield. Quantification was based on calibration curve applying diclofenac as the internal standard. Owing to dilution of plasma sample by 2 mM aqueous solution of BMIM BF₄ before SPE, appropriate sample purification and extraction yields higher than 95% with precision lower than 2% can be achieved. Linear coefficients of correlation (r²) were >0.99 in the range of 0.3–5 μg/mL ibuprofen concentration in plasma. The limit of quantification was 65 ng/mL and the detection limit for ibuprofen was 19.5 ng/mL.