Simple and rapid analysis of lamotrigine, a novel antiepileptic, in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A simple and rapid method for the quantitation of concentrations of lamotrigine, a novel antiepileptic, in human serum was developed with high-performance liquid chromatography, using a solid-phase extraction technique. The mobile phase was composed of acetonitrile-10 mM phosphate buffer (pH 3.5) containing 5 mM sodium octanesulphonate (27:73, v/v), and components were detected at 265 nm. Retention times of acetanilide as an internal standard and lamotrigine were 3.4 and 10.3 min, respectively. The coefficients of variation were 3.1–4.5% and 4.4–9.8% for the within-day and between-day precision estimates, respectively. The extraction recovery of lamotrigine added to blank serum was 86–107%. The quantitation limit of lamotrigine was ca.0.2 μg/ml in 100 μl of serum. These results suggest that the method employed in this study is useful for the routine monitoring of sereum concentrations of lamotrigine in epileptic patients.     

Separation of cobalt binding proteins by immobilized metal affinity chromatography

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.     

Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry

The validation of a method for the determination of mevalonic acid (MVA; after conversion to the lactone, MVAL) in human plasma, using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS-MS), is reported. MVAL and deuterated internal standard were extracted from human plasma samples using automated solid-phase extraction. Analysis was conducted by column-switching, reversed-phase LC-MS-MS, using two hyper-cross-linked styrene-divinylbenzene copolymer sorbent reversed-phase columns. An assay range of 0.2-35 ng/ml and a lower limit of quantitation (LLOQ) of 0.2 ng/ml were achieved with acceptable accuracy and precision. MVA was stable in plasma under a variety of storage conditions.     

Micellar electrokinetic capillary chromatography determination of +S and -R arotinolol in serum using UV detection and solid phase extraction.

A method for the simultaneous determination of +S and -R arotinolol in serum by micellar electrokinetic capillary chromatography is described. Stereoselective resolution of the arotinolol enantiomers was achieved using 5 mM sodium taurocholate in 10 mM sodium dihydrogen phosphate buffer of pH 2.5. A 72-cm uncoated fused-silica capillary at a constant voltage of 15 kV was used for the analysis. The analytes of interest were extracted from serum using solid phase extraction. An octadecyl cartridge gave good recoveries in excess of 87% for both +S and -R arotinolol without any interference. The calibration curves were linear over the range of 50-500 ng ml(-1) with +S propranolol as the internal standard and the coefficient of determination was greater than 0.999 (n = 3). The limit of quantitation was 50 ng ml(-1) for each enantiomer and the detection limit using 1 ml serum and a UV detection set et 220 nm was 25 ng ml(-1) (S/N = 2). Precision and accuracy of the method were in the range 0.8-2.7% and 1.2-6.4%, respectively, for +S arotinolol and 1.1-3.9% and 2.2-6.5%, respectively, for -R arotinolol.     

Determination of isosorbide-5-mononitrate in human plasma by high-resolution gas chromatography

A method for the determination of isosorbide-5-mononitrate (5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed and applied to clinical samples. 9-Fluorenone was used as an internal standard, ethyl acetate was employed for liquid-liquid extraction. The advantage of the extraction procedure is the possibility of a direct injection of the plasma extract, without solvent removal/reconstitution of the sample. The precision and accuracy of the method were satisfactory in the concentration range 10-1600 ng/ml. The lower limit of quantification was 10 ng/ml.     

High-performance thin-layer chromatographic determination of 5-methoxypsoralen in serum from patients

A simple and rapid high-performance thin-layer chromatographic (HPTLC) determination of 5-methoxypsoralen in serum is necessary for the therapeutic survey of patients treated with Puvatherapy (psoralen+UV A). The assay for this biological fluid involves an extraction with heptane-dichloromethane (4:1, v/v). The analytical method is linear from 50 to 250 ng/ml. This assay range is adequate for analysing human serum, as it corresponds to psoralen concentrations measured in serum from patients treated with psoralen and UV A against psoriasis and vitiligo. The limit of detection is 15 ng/ml. The coefficient of variation was less than 7%.     

High-throughput bioanalytical method using automated sample preparation and liquid chromatography-atmospheric pressure ionspray mass spectrometry for quantitative determination of glybenclamide in human serum

A liquid chromatographic-mass spectrometric (LC-MS) method with rapid automated sample preparation was developed and validated for determination of glybenclamide in human serum. Glybenclamide and its deuterated labelled internal standard were extracted from human serum samples by automated solid-phase extraction. The extract was injected into the LC-MS system for analysis. Glybenclamide and its internal standard were measured in multiple ion monitoring mode. The method was validated over a range of 10-1000 ng/ml with good accuracy and precision and was applicable for pharmacokinetic studies.     

Rapid and sensitive high-performance liquid chromatographic determination of four cephalosporin antibiotics in pharmaceuticals and body fluids

A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins, cephalexin and cefadroxil (first-generation), cefaclor (second-generation) and cefataxim (third-generation), in pharmaceuticals as well as in human blood serum and urine. A Spherisorb ODS-2 250 x 4-mm, 5-microm analytical column was used with an eluting system consisting of a mixture of acetate buffer (pH 4.0)-CH(3)OH 78-22% (v/v) at a flow-rate 1.2 ml/min. Detection was performed with a variable wavelength UV-Vis detector at 265 nm resulting in limit of detection of 0.2 ng for cefadroxil and cephalexin, but only 0.1 ng for cefotaxime and cefaclor per 20-microl injection. Hydrochlorothiazide (HCT) (6-chloro-3,4-dihydro-7 sulfanyl-2H-1,2,4-benzothiadiazine-1-1-dioxide) was used as internal standard at a concentration of 2 ng/microl. A rectilinear relationship was observed up to 8, 5, 12 and 35 ng/microl for cefadroxil, cefotaxime, cefaclor, cephalexin, respectively. Analysis time was less than 7 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=9) and was found to be satisfactory with high accuracy and precision. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid-phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked samples was in the range from 76.3 to 112.0%, over the range of 1-8 ng/microl.     

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.     

Determination of Tirofiban in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed and validated for the determination of Tirofiban in biological fluids. Tirofiban in serum samples was extracted and cleaned up by using an automated solid phase extraction method. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode (MRM). A good linear response over the range of 2-200ng/ml was demonstrated. The accuracy for Tirofiban ranged from 94.8 to 110.8% within-day and from 103.0 to 104.7% between-day. The lower limit of quantification was 2ng/ml. This method is suitable for pharmacokinetic studies.     

Determination of Ochratoxin A in small volumes of human blood serum

A new simple and rapid method for analysing Ochratoxin A (OTA) in small volumes of human blood serum using capillary zone electrophoresis coupled to laser-induced fluorescence is described. The clean-up procedure solely consists of a double extraction step. To improve the reproducibility of migration times and quantification, two internal standards were used. The limit of detection was 0.55 ng/ml, with a linear range of 1-100 ng/ml of OTA in spiked human blood serum. The method is used to rapidly screen suspected patients.     

High-performance liquid chromatography-mass spectrometry method for the determination of paroxetine in human plasma

A rapid and specific liquid chromatographic mass spectrometric (LC-MS-MS) method has been developed for the determination of paroxetine in human plasma. The procedure involves a liquid-liquid extraction of paroxetine and fluoxetine (internal standard) with cyclohexane-ethyl acetate. The standard curve was linear over a working range of 0.2-50 ng/ml. The lower limit of quantitation was 0.2 ng/ml. No endogenous compounds were found to interfere with the analysis. The absolute recovery was 70.8% for paroxetine and 84.1% for the internal standard. The accuracy of inter-assay and intra-assay accuracy was in the ranges -4.8 to -0.5% and -3.4 to 4.8%, respectively. This method proved to be suitable for bioequivalence studies by being simple, selective and reproducible.     

Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction

An improved method for the determination of ethyl glucuronide (EtG) in human serum and urine was developed using solid-phase extraction (SPE) and gas chromatography (GC) with mass spectrometric detection (MS). EtG was isolated from serum and urine using aminopropyl SPE columns after deproteination with perchloric acid and hydrochloric acid, respectively. The chromatographic separation was performed on a DB 1701 fused-silica column. At a signal-to-noise ratio of 3:1, a quantification limit of 173 and 560 ng/ml and a detection limit of 37 and 168 ng/ml could be determined for serum and urine, respectively. This indicates high specificity and sensitivity of the described method. The mean absolute recovery was 85%, while intra- and inter-day precision of the assay were all less than 7.5%. The linearity of the calibration curves was satisfying as indicated by correlation coefficients of >0.993. The presented method provides the basis for determination and identification of EtG in human serum and urine samples in a low-concentration range for monitoring alcohol consumption during treatment for alcohol dependence and comorbid alcohol abuse of psychotherapy patients.     

HPLC analysis of the novel antidepressant duloxetine in human plasma after an original solid-phase extraction procedure

Duloxetine is the most recent serotonin and norepinephrine reuptake inhibitor (SNRI) drug introduced for the therapy of depression. Thus, it is evident that there is a need for having on hand new reliable analytical methods for the determination of duloxetine plasma levels in depressed patients. The present paper deals with the development of a rapid and sensitive high-performance liquid chromatographic method for duloxetine analysis in human plasma. The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 60% aqueous phosphate buffer containing triethylamine at pH 3.0 and 40% acetonitrile. The UV detector was set at 230 nm and loxapine was used as the internal standard. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with mixed-mode reversed phase-strong cation exchange cartridges (30 mg, 1 mL). The extraction yields values were higher than 90%. Linearity was found in the 2-200 ng mL(-1) duloxetine concentration range; the limit of quantitation was 2.0 ng mL(-1) and the limit of detection was 0.7 ng mL(-1). The method was applied to plasma samples from depressed patients undergoing therapy with duloxetine. Precision data and accuracy results were satisfactory and no interference from other drugs was found. Thus, the method seems to be suitable for the therapeutic drug monitoring of duloxetine in depressed patients' plasma.     

Determination of metronidazole in vaginal tissue by high-performance liquid chromatography using solid-phase extraction

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of metronidazole in vaginal tissue is reported. The method uses a Zorbax SB phenyl column with a 0.01 M aqueous monobasic potassium phosphate buffer (pH 4.0)-absolute methanol (85:15, v/v) as mobile phase at a flow-rate of 1.0 ml/min and detection at 313 nm. Tinidazole was used as the internal standard. The method employed homogenization of tissue followed by solid-phase extraction. The quantitation was achieved within 30 min with sensitivity in the ng/g range. Metronidazole was linear in the 100–2000 ng/g range. The accuracy and precision were in the 1–4% range for the drug and the limit of detection was approximately 100 ng/g based on a signal-to-noise ratio of 3 and a 100-μl injection.     

High performance liquid chromatographic determination of azithromycin in serum using fluorescence detection and its application in human pharmacokinetic studies

A fast and sensitive high-performance liquid chromatographic method for determination of azithromycin in human serum using fluorescence detection was developed. The drug and an internal standard (clarithromycin) were extracted from serum using n-hexan and subjected to pre-column derivatization with 9-fluorenylmethyl chloroformate as labeling agent. Analysis was performed on a phenyl packing material column with sodium phosphate buffer containing 2 ml/l triethylamine (pH 5.9) and methanol (29:71, v/v) as the mobile phase. The standard curve was linear over the range of 10-500 ng/ml of azithromycin in human serum. The means between-days precision were from 13.3% (for 10 ng/ml) to 2% (500 ng/ml) and the within-day precision from 11.9 to 1.7% determined on spiked samples. The accuracy of the method was 100.7-107.2% (between days) and 100.3-107.8% (within day). The limit of quantification was 10 ng/ml. This method was applied in a bioequivalence study of four different azithromycin preparations in 12 healthy volunteers.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Quantitative liquid chromatographic and tandem mass spectrometric determination of vitamin D3 in human serum with derivatization: a comparison of in-tube LLE, 96-well plate LLE and in-tip SPME

Sensitive and selective methods based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection were developed for the determination of vitamin D(3) in human serum. Derivatization of vitamin D(3) and its stable isotope labeled internal standard provided highly sensitive quantification and selective detection from endogenous compounds. Samples were prepared using the in-tube liquid-liquid extraction (LLE), 96-well plate LLE, and in-tip solid phase micro-extraction (SPME) in 96-well format. In all methods, the MS/MS detection was performed using Applied Biosystems-Sciex API 3000 tandem mass spectrometers interfaced with a heated nebulizer probe and operated in the positive ionization mode. Both tube and plate LLE methods achieved a lower limit of quantitation (LLOQ) of 0.5 ng/mL when 1.0 and 0.4 mL of human serum was processed, respectively, and were validated in the concentration range of 0.5-25 ng/mL; while for the in-tip SPME method, LLOQ was 5 ng/mL with only 0.1 mL of human serum required. Comparisons were made among three different methods, including precision and accuracy, sample throughput, recovery and matrix effects.     

Determination of ractopamine in monkey plasma and swine serum by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of ractopamine (LY031537) in monkey plasma and swine serum. Plasma or serum (0.5 ml) was diluted with phosphate buffer pH 7.0. Ractopamine was isolated from the plasma matrix using ion exchange on a polymeric carboxylic acid solid-phase extraction cartridge followed by partitioning with ethyl acetate. An isocratic HPLC method using electrochemical detection at +700 mV was used to separate and measure ractopamine in the purified extract in 6.5 min of run time. Standard area response was linear with respect to concentration of ractopamine over the range of 0.5 to 40 ng/ml. Validation data were collected using rhesus monkey plasma and swine serum. The method precision and accuracy were evaluated in the range 1.0 to 20 ng/ml using fortified samples of monkey plasma. The method limit of quantitation was estimated at 2 ng/ml as determined in monkey plasma.