Simple method for the analysis of tenoxicam in human plasma using high-performance liquid chromatography

A simple, rapid and cost-effective method for the determination of tenoxicam in human plasma is described, using ketorolac as the internal standard. The extraction procedure utilised 5% zinc sulphate and methanol. A nucleosil C₁₈ column and 35:65 acetonitrile-water phosphate buffered mobile phase (pH 2.8) were used, with ultraviolet detection at 355 nm. The assay was linear in the range 40 ng/ml-10 μg/ml, with recovery of extraction ranging from 87 to 102%. The intra- and inter-assay reproducibility had coefficients of variation of 3.9–7.7 and 1.6% respectively. The limit of detection for this method was 40 ng/ml.     

High-performance liquid chromatography method for the quantification of rabeprazole in human plasma using solid-phase extraction

A simple, sensitive and selective HPLC method with UV detection (284 nm) was developed and validated for quantitation of rabeprazole in human plasma, the newest addition to the group of proton-pump inhibitors. Following solid-phase extraction using Waters Oasistrade mark SPE cartridges, the analyte and internal standard (Pantoprazole) were separated using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH adjusted to 7.4 with sodium hydroxide solution)/acetonitrile/methanol (45/20/35, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 8%. A linear range of 20-1000 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 2.4-7.2% and 2.2-7.3%, respectively. The between- and within-batch bias was -1.7 to 2.6% and -2.6 to 2.1%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of rabeprazole in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 3 months storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of pantoprazole in human plasma

A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid-liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20-5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3-3.2% and 0.7-3.3%, respectively. The between-batch and within-batch bias was -0.5 to 8.2 % and -2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of entacapone in human plasma

A simple, sensitive and selective HPLC method with UV detection (315 nm) was developed and validated for quantitation of entacapone in human plasma, the newest addition to the group of antiparkinsonian agents. Following a single-step liquid-liquid extraction (LLE) with ethyl acetate/n-hexane (30/70, v/v), the analyte and internal standard (rofecoxib) were separated using an isocratic mobile phase of 30 mM phosphate buffer (pH 2.75)/acetonitrile (62/38, v/v) on a reverse phase C18 column. The lower limit of quantitation was 25 ng/mL, with a relative standard deviation of less than 8%. A linear range of 25-2500 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 2.2-4.2% and 1.7-7.8%, respectively. The between-batch and within-batch accuracy was 98.7-107.5% and 97.5-106.0%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of entacapone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Simple and sensitive method for quantification of fludarabine triphosphate intracellular concentration in leukemic cells using isocratic liquid chromatography

A simple, isocratic HPLC method was newly developed for quantitating intracellular fludarabine triphosphate (F-ara-ATP). Samples (500 microl) were injected onto an anion-exchange column and eluted isocratically with phosphate-acetonitrile buffer (flow rate: 0.7 ml/min) at an ambient temperature. F-ara-ATP was quantitated according to its peak area at the absorbance of 261 nm. The standard curve was linear with minimal within-day and inter-day variability. The low and high quantification limits were 50 pmol and 20 nmol, respectively. The method was capable of measuring F-ara-ATP generated in cultured leukemic cells in vitro. Thus, our method will be useful because of its sensitivity and simplicity as well as applicability to biological materials.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in plasma

A high performance liquid chromatographic method for determination of moxifloxacin in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.125 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Simple and rapid liquid chromatography method for determination of efavirenz in plasma

A simple and rapid high performance liquid chromatographic method for determination of efavirenz in human plasma was developed. The method involved extraction of sample with ethyl acetate and analysis using a reversed-phase C(18) column (150 mm) with UV detection. The assay was linear from 0.0625 to 10.0 microg/ml. The method was specific for efavirenz estimation and the drug was stable in plasma up to one month at -20 degrees C. The average recovery of efavirenz from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring of efavirenz.     

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: application to pharmacokinetic studies

A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC-MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water-acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C(18) (100 mm x 2.0 mm i.d., 3 microm) column connected to a guard column MS-pursuit (0.20 mm x 0.20 mm i.d., 5 microm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r(2) values of 0.995 (CV=0.24%, n=10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C(max) (1667.25 ng/mL) and T(max) (3.89 h).     

Simultaneous quantification of emtricitabine and tenofovir in human plasma using high-performance liquid chromatography after solid phase extraction

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of emtricitabine and tenofovir in human blood plasma is described. Using 200 microL of plasma and BOND ELUT-C18 Varian columns, the solid phase extraction (SPE) method results in a clean baseline and high extraction efficiencies (100% for emtricitabine and 98.6% for tenofovir). An Atlantistrade mark dC-18 analytical column is used along with an 18 min linear gradient elution of phosphate buffer (pH 5.7) and methanol to provide sharp peaks for emtricitabine at 280 nm, tenofovir at 259 nm, and the internal standard 2',3'didoxyuridine (DDU) at 262 nm. The method was validated over the range of 10-10,000 ng/mL for both analytes, and is accurate (average accuracies of three different concentrations ranged from 98 to 105% for emtricitabine and 97 to 103% for tenofovir) and precise (within- and between-day precision ranged from 1.7 to 3.7% and 3.7 to 5.2%, respectively). This method is suitable for use in clinical pharmacokinetic studies and is nimble enough for therapeutic drug monitoring.     

Simple and sensitive method for the quantitative analysis of lometrexol in plasma using high-performance liquid chromatography with electrochemical detection

Previously described methods for the determination of lometrexol in plasma used either fluorescence or ultraviolet detection. An alternative method for the determination of lometrexol utilizing electrochemical detection is described, having comparable sensitivity to fluorometric methods but no requiring pre-analytical oxidation. Following sample clean-up, separation is achieved on a phenyl column with a mobile-phase of 8% acetonitrile in 50 mM sodium acetate buffer, pH 4.0. The calibration curve in plasma is linear from 10 to 200 ng/ml, with inter- and intra-day precision of 5.4 and 5.5%, respectively. The recovery of lometrexol from plasma is 81 ± 1.5%, and the lower limit of detection is 5 ng/ml, using a signal-to-noise ratio of 3.     

Simple and sensitive fluorimetric liquid chromatography method for the determination of valproic acid in plasma

A simple and sensitive liquid chromatographic method is described for the analysis of valproic acid in human plasma. The method is based on the derivatization of valproic acid extracted from acidified plasma with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate. The resulting derivative is highly responsive to a fluorimetric detector (excitation at 230 nm and emission at 350 nm), giving a low detection limit of 0.6 microM (S/N = 3, 10 microl injected). The relative standard deviations of the method for intra- and inter-day analyses (n = 5) are below 3.3 and 4.1%, respectively. Toluene was used for the extraction of valproic acid from plasma and the toluene extract obtained was subjected to subsequent derivatization without solvent replacement. The simple method was applied to the analysis of valproic acid in plasma of dosed patients using only small amount of sample (10-50 microl plasma).     

High-performance liquid chromatographic method for the determination of atracurium and laudanosine in human plasma: Application to pharmacokinetics

A high-performance liquid chromatographic method coupled with fluorimetric detection has been developed for the determination of atracurium and its major metabolite, laudanosine, in human plasma. The detection is performed at 240 nm for excitation and 320 nm for emission. Verapamil was used as the internal standard. The proposed technique, involving the direct precipitation of plasma proteins is reproducible, selective and sensitive. Linear detector responses were observed for the calibration curve standards in the range of 40 to 2000 ng/ml. Precision, expressed as C.V., was in the range 1 to 14%. The limit of quantification for both atracurium and laudanosine was 40 ng/ml. The method has been validated and stability tests under various conditions have been performed. This method has been used to determine the pharmacokinetic profile of atracurium and laudanosine in patients with acute respiratory distress syndrome.     

A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-β glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.     

High-performance liquid chromatographic method for quantification of busulfan in plasma after derivatization by tetrafluorothiophenol

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of busulfan in plasma. Busulfan was extracted in toluene, derivatized by 2,3,5,6-tetrafluorothiophenol to obtain di-TFTP-butane, the derivatization product was then re-extracted in toluene and injected into the HPLC system with ultraviolet detection (wavelength: 275 nm). Recovery from extraction was 80%, the limit of quantification was 50 ng/ml and linearity ranged from 50 to 2000 ng/ml. In addition, forty-two samples obtained from pediatric patients treated with busulfan were analyzed by the HPLC and GC–MS assays based on the same derivatization procedure. The correlation between the di-TFTP-butane concentrations was highly significant (p<0.0001), demonstrating that the two methods were in good agreement.     

A rapid and sensitive method for the quantitation of montelukast in sheep plasma using liquid chromatography/tandem mass spectrometry

A rapid LC-MS/MS method was developed and partially validated for the quantitation of montelukast in spiked sheep plasma. A total run time of 1.5 min was achieved using a short monolithic column and employing a rapid gradient. Sample preparation involved protein precipitation with twofold acetonitrile by volume during which a deuterated internal standard (montelukast D-6) was incorporated. The MRM transitions for montelukast and the deuterated internal standard were 586/422 and 592/427, respectively. A linear dynamic range of 0.25-500 ng/mL with a correlation coefficient of 0.9999 was achieved. Precision was below 5% at all levels except at the LOQ (0.36 ng/mL) which demonstrated an overall of R.S.D. of 8%. Post-column infusion experiments were performed with precipitated plasma matrix and showed minimal interference with the peaks of interest.     

A validated method for the quantification of enterodiol and enterolactone in plasma using isotope dilution liquid chromatography with tandem mass spectrometry

Enterolactone and enterodiol are phytoestrogens with structural similarity to endogenous estrogens. Because of their biological activities, they may affect the development of several diseases. To quantify enterodiol and enterolactone in plasma, we developed and validated a liquid chromatography-tandem mass spectrometry method with electrospray ionization using 13C3 labeled isotopes. The method consists of a simple enzymatic hydrolysis and ether extraction followed by a rapid LC separation (run-time of 11 min). Detection limits as low as 0.15 nM for enterodiol and 0.55 nM for enterolactone were achieved. The within-run R.S.D. ranges from 3 to 6% and the between-run R.S.D. ranges from 10 to 14% for both enterolignans. This method allows simple, rapid, and sensitive quantification, and is suitable for measuring large numbers of samples.     

Enantioselective determination of metoprolol acidic metabolite in plasma and urine using liquid chromatography chiral columns: applications to pharmacokinetics

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.     

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.     

Simple and sensitive determination of 5-fluorouracil in plasma by high-performance liquid chromatography: Application to clinical pharmacokinetic studies

5-Fluorouracil (5-FU) is an antineoplastic agent widely employed in the treatment of many types of cancer. Recent studies have proved the need for individual adjustment of 5-FU dosage based on pharmacokinetics. A simple and sensitive high-performance liquid chromatographic method for the determination of 5-FU in plasma and their preliminary clinical pharmacokinetics is described. After sample acidification with 20 μl of orthophosphoric acid (5%), the drug is extracted from plasma using n-propanol–diethyl ether (16:84). The organic layer is evaporated to dryness, the residue dissolved in 100 μl of mobile phase and 20 μl of this mixture is injected into a LiChrospher 100RP-18 (5 μm, 250×4.0 mm) analytical column. Mobile phase consisted of potassium dihydrogenphosphate (0.05 M, adjusted to pH 3). The limit of quantitation was 2 ng/ml. The method showed good precision: the within-day relative standard deviation (RSD) for 5-FU (10–20 000 ng/ml) was 3.75% (2.57–5.93); the between-day RSD for 5-FU, in the previously described range, was 5.74% (4.35–7.20). The method presented here is accurate, precise and sensitive and it has been successfully applied for 5-FU pharmacokinetic investigation and therapeutic drug monitoring.     

Determination of aesculin in rat plasma by high performance liquid chromatography method and its application to pharmacokinetics studies

A sensitive and reproducible high performance liquid chromatography method with UV detection was described for the determination of aesculin in rat plasma. After deproteinization by methanol using metronidazole as internal standard (I.S.), solutes were evaporated to dryness at 40 degrees C under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of mobile phase and a volume of 20 microl was injected into the HPLC for analysis. Solutes were separated on a Diamonsil C18 column (250 mm x 4.6 mm i.d., 5 microm particle size, Dikma) protected by a ODS guard column (10 mm x 4.0 mm i.d., 5 microm particle size), using acetonitrile-0.1% triethylamine solution (adjusted to pH 3.0 using phosphoric acid) (10:90, v/v) as mobile phase (flow-rate 1.0 ml/min), and wavelength of the UV detector was set at 338 nm. No interference from any endogenous substances was observed during the elution of aesculin and internal standard (I.S., metronidazole). The retention times for I.S and aesculin were 10.4 and 12.4 min, respectively. The limit of quantification was evaluated to be 57.4 ng/ml and the limit of detection was 24.0 ng/ml. The method was used in the study of pharmacokinetics of aesculin after intraperitoneal injection (i.p.) administration in rats.