Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection

A HPLC method with UV detection at 262nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0mL/min and a reverse-phase XTerra MS C(18) column (4.6mmx150mm, 3.5microm). The standard curve was linear over a concentration range of 1.5-100.0microg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55-4.30% and 2.69-21.5%, respectively. The intra- and inter-day biases were -0.75 to 19.8% and 2.58-22.0%, respectively. At plasma concentrations of 1.5-100microg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24h at 4 degrees C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.     

Quantification of doripenem in human plasma and peritoneal fluid by high-performance liquid chromatography with ultraviolet detection

A simple and rapid HPLC method that includes ultrafiltration to remove plasma and peritoneal fluid protein was developed to determine doripenem concentrations in human plasma and peritoneal fluid. Doripenem was stabilized by immediate mixing of the plasma or peritoneal fluid with 1M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Doripenem and an internal standard were detected by measuring their ultraviolet absorbance at 300 nm. The calibration curves for doripenem in human plasma and peritoneal fluid were linear from 0.05 to 100 microg/mL. For plasma, both the intra- and the interday precision were less than 3.41% (CV), and the accuracy was between 97.4 and 101.7% above 0.05 microg/mL. For peritoneal fluid, the intra- and the interday precision were less than 2.98% (CV), and the accuracy was between 94.4 and 103.9% above 0.05 microg/mL. The limit of detection was 0.02 microg/mL in both plasma and peritoneal fluid. The assay has been applied to the therapeutic drug monitoring of doripenem in both plasma and peritoneal fluid.     

Determination of phenethyl isothiocyanate in human plasma and urine by ammonia derivatization and liquid chromatography-tandem mass spectrometry

Phenethyl isothiocyanate (PEITC) is a dietary compound present in cruciferous vegetables that has cancer-preventive properties. Our objective was to develop and validate a novel liquid chromatography-tandem mass spectrometry procedure to analyze PEITC concentrations in human plasma and urine. Following hexane extraction, ammonia was added to samples to derivatize PEITC to phenethylthiourea. Chromatographic separation was achieved on a C(18) column with acetonitrile/5 mM formic acid (60:40, v/v) as the mobile phase followed by tandem mass spectrometry detection in multiple reaction monitoring mode. Deuterium-labeled PEITC was used as the internal standard. The detection limit was 2 nM and calibration curves were linear from 7.8 to 2000 nM. The intra- and inter-day coefficients of variation were less than 5 and 10%, respectively. The intra- and inter-day accuracies ranged from 101.0 to 104.2% and from 102.8 to 118.6%, respectively. The recovery from spiked human plasma and urine ranged from 100.3 to 113.5% and from 98.3 to 103.9%, respectively. The assay was used to measure PEITC in plasma and urine samples obtained from subjects after consumption of 100g of watercress. This novel assay represents the first analytical method with the sensitivity and specificity to determine plasma and urine concentrations of PEITC.     

Method development and validation of a high-performance liquid chromatographic method for tramadol in human plasma using liquid-liquid extraction

An HPLC system using a simple liquid-liquid extraction and HPLC with UV detection has been validated to determine tramadol concentration in human plasma. The method developed was selective and linear for concentrations ranging from 10 to 2000 ng/ml with average recovery of 98.63%. The limit of quantitation (LOQ) was 10 ng/ml and the percentage recovery of the internal standard phenacetin was 76.51%. The intra-day accuracy ranged from 87.55 to 105.99% and the inter-day accuracy, 93.44 to 98.43% for tramadol. Good precision (5.32 and 6.67% for intra- and inter-day, respectively) was obtained at LOQ. The method has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Simultaneous determination of methylephedrine and noscapine in human plasma by liquid chromatography-tandem mass spectrometry

A selective and sensitive method has been developed and validated for simultaneous quantification of methylephedrine and noscapine in human plasma. Analytes were extracted from human plasma samples by liquid-liquid extraction, separated on a Diamonsil C18 column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization (APCI) interface. Diphenhydramine was used as the internal standard (I.S.). The method was found to be precise and accurate within the linear range 0.1-100 ng/ml for each analyte. The intra- and inter-day relative standard deviations (R.S.D.s) were below 5.2% for methylephedrine and 6.7% for noscapine. The inter-day relative error (RE) as determined from quality control samples (QCs) was less than 3.0% for each analyte. The assay was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation containing 20 mg DL-methylephedrine hydrochloride, 16 mg noscapine, 300 mg paracetamol and 1mg of chlorpheniramine maleate.     

High-performance liquid chromatography spectrometric analysis of trans-resveratrol in rat plasma

A HPLC method for determination of trans-resveratrol concentrations in rat plasma was developed. Plasma samples were treated with acetonitrile to deposit proteins. The analysis used a Hypersil ODS(2) C(18) column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow-rate=1 mL/min). The UV detection wavelength was 303 nm, and chlorzoxazone was used as the internal standard. The calibration curve was linear over the range of 0.02-40 microg/mL with a correlation coefficient of 0.9997. This concentration range corresponds well with the plasma concentrations of resveratrol in pharmacokinetic studies. There was 98.7%, 91.3% and 84.4% recovery from 0.02, 0.4 and 40 microg/mL plasma samples respectively. The R.S.D. of intra- and inter-day assay variations were all less than 12%. This HPLC assay is a quick, precise and reliable method for the analysis of resveratrol in pharmacokinetic studies.     

Determination of scopoletin in rat plasma by high performance liquid chromatographic method with UV detection and its application to a pharmacokinetic study

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for determination of scopoletin in rat plasma using psoralen as internal standard. Chromatographic separation was achieved on a C(18) column using methanol and distilled water (49:51, v/v) containing 0.05% (v/v) phosphoric acid as mobile phase. The UV detector was set at 345 nm. The calibration curve was linear over the range of 0.165-9.90 microg/ml with a correlation coefficient of 0.9994. The recovery for plasma samples of 0.165, 1.32 and 6.60 microg/ml was 93.2%, 95.9% and 95.5%, respectively. The RSD of intra- and inter-day assay variations was less than 6.7%. This HPLC assay is a precise and reliable method for the analysis of scopoletin in pharmacokinetic studies.     

Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: application to studies of oxidative stress

A sensitive, reproducible, and robust high-performance liquid chromatography (HPLC) method has been validated for simultaneously determining total concentrations of the aminothiols homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma. Plasma aminothiols are reduced via incubation with tris-(2-carboxyethyl)-phosphine hydrochloride, followed by protein precipitation with trichloroacetic acid and derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid. Separation of aminothiols and the internal standard mercaptopropionylglycine is achieved using reversed-phase HPLC conditions and fluorescence detection. Excellent linearity is observed for all analytes over their respective concentration ranges with correlation coefficients (r) > 0.99. The intra- and inter-day precision and accuracy were within +/-10%. This method utilizes an internal standard, employs phosphate buffered saline-based standards and quality controls, and demonstrates excellent plasma recovery and improved sensitivity. This assay is well suited for high-throughput quantitative determination of aminothiols in clinical studies, and is currently being used to support investigations of oxidative stress in patients with chronic kidney disease.     

Rapid determination of the active leflunomide metabolite A77 1726 in human plasma by high-performance liquid chromatography

A simple method for the measurement of the active leflunomide metabolite A77 1726 in human plasma by HPLC is presented. The sample workup was simple, using acetonitrile for protein precipitation. Chromatographic separation of A77 1726 and the internal standard, alpha-phenylcinnamic acid, was achieved using a C(18) column with UV detection at 305 nm. The assay displayed reproducible linearity for A77 1726 with determination coefficients (r2) > 0.997 over the concentration range 0.5-60.0 microg/ml. The reproducibility (%CV) for intra- and inter-day assays of spiked controls was <5%. The limit of quantification was 0.8 microg/ml. The average absolute recovery was approximately 100%. This assay is suitable for the determination of A77 1726 in plasma of patients taking leflunomide, and is simpler to use than other HPLC methods reported previously.     

High performance liquid chromatographic measurement of iothalamate in human serum and urine for evaluation of glomerular filtration rate

A simple and sensitive HPLC-UV assay was developed for the measurement of iothalamate (IOT) in human serum and urine. Chromatographic separation was achieved using an embedded-carbamate-group bonded RP18 column and mobile phase consisting of 50 mM monobasic sodium phosphate and methanol (90:10, v/v) without the addition of ion-pair reagents. The assay demonstrated a high analytical reliability within the IOT concentration range of 1-150 microg/ml in serum and 25-1500 microg/ml in urine. The relative standard deviations (RSDs) for intra- and inter-day analysis were less than 5.1% in all cases. This method has been used for the evaluation of glomerular filtration rate (GFR) in subjects participating in a phase I clinical trial of a novel antimalarial medicine. The average baseline GFR was 100.41+/-19.99 ml/min/1.73 m(2) in 119 healthy volunteers. The assay may also allow the simultaneous measurements of p-aminohippuric acid (PAH), N-acetyl PAH (aPAH), and IOT with some modification. PAH, IOT, aPAH, and beta-hydroxyethyl-theophylline internal standard peaks appeared approximately at 2.5, 3.7, 5.9, and 11.8 min, respectively, in an isocratic run.     

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

A high performance liquid chromatographic method for determination of moxifloxacin in human saliva 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 0.25 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 saliva was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Reversed-phase liquid chromatography method to determine COL-3, a matrix metalloproteinase inhibitor, in biological samples

A reversed-phase high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline (COL-3), a matrix metalloproteinase (MMPs) inhibitor, in rat serum. This simple, sensitive, rapid and reproducible assay involved a preliminary serum deproteinization by adding a mixture of acetonitrile-methanol-0.5 M oxalic acid (70:20:10 (v/v)), as the combined precipitant and metal blocking agent, into serum samples (2:1 (v/v)). An aliquot (20 microl) of the supernatant was injected into the HPLC system linked to a Waters XTerra RP(18) column (150 mm x 4.6 mm i.d., particle size 5 microm). The compound was eluted by a mixture of acetonitrile-methanol-0.01 M oxalic acid (40:10:50 (v/v), pH 2.00), as the mobile phase, and detected at the wavelength of 350 nm. The total running time was 10 min. The low and high concentration calibration curves were linear in the range of 50-1200 ng/ml and 1200-12,000 ng/ml, respectively. The intra- and inter-day coefficients of variation at three quality control concentrations of 100, 1200, and 12,000 ng/ml were all less than 6%, while the percent error ranged from -2.5 to 6.6%. The limit of quantitation (LOQ) for COL-3 in serum was 50 ng/ml. This assay was successfully employed to study the serum concentration-time profiles of COL-3 after its intravenous and oral administration in rats. The method with some minor modifications in sample pretreatment was also applicable to the determination of the concentrations of COL-3 in rat bile, urine and feces.     

Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).     

Analysis of perfluorinated chemicals in umbilical cord blood by ultra-high performance liquid chromatography/tandem mass spectrometry

Perfluorinated compounds (PFCs) can cross the placental barrier and enter fetal circulation. This study aimed at developing a fast and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry method for the determination of twelve perfluorinated compounds in cord blood. Samples were processed with protein precipitation using formic acid and methanol, mixed with stable isotope labeled standard, followed by sonication and centrifugation, and were analyzed using a Waters ACQUITY UPLC coupled with a Waters Quattro Premier XE triple-quadrupole mass spectrometer. The instrument was operated in selected reaction monitoring (SRM) with negative electrospray ionization. Using BEH C(18) column (2.1 mm×50 mm, 1.7 μm) with 10-mM N-methylmorpholine/methanol gradient elution provided a fast chromatographic separation (5.5 min) and sharp peaks. Intra- and inter-day calibration bias was less than 7% and intra- and inter-day calibration of relative standard deviations were within 0.02-8.22% for all the analytes and concentrations. The recoveries of PFCs spiked into bovine serum ranged from 85 to 104% with relative standard deviations from 0.02 to 6.37%. The limits of quantitation (LOQs), defined as a signal-to-noise ratio of ten, ranged from 0.15 to 3.1 ng/mL for the twelve PFCs. Perfluorooctanoic acid (PFOA), perfluorooctyl sulfonate (PFOS), perfluoroundecanoic acid (PFUA) and perfluorononanoic acid (PFNA) were detected in up to 68% of umbilical cord plasma (n=444) in Taiwan Birth Panel Study and the health effect of these chemicals on children developmental deserves further investigation.     

Quantitation of paclitaxel and its two major metabolites using a liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of paclitaxel (Taxol) and its two major metabolites in human plasma has been developed. Samples were prepared after liquid-liquid extraction and analyzed on a C(18) column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.05% formic acid) (65:35) at the flow rate of 0.25 mL/min. The analytes and internal standard docetaxel were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 0.5-500.0 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The lower limit of quantification (LLOQ) was 0.5 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3'-hydroxypaclitaxel, respectively. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 8.18%. The accuracy determined at three concentrations was within ±10.8% in terms of relative error. The total run time was 7.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of paclitaxel and its metabolites in various biological fluids.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.     

Validation of a sensitive assay for thiocoraline in mouse plasma using liquid chromatography-tandem mass spectrometry

A sensitive high-performance liquid chromatography-tandem mass spectrometry assay for thiocoraline, an anti-tumor depsipeptide, in mouse plasma is described. Echinomycin, a quinoxaline peptide, was used as an internal standard. Thiocoraline was recovered from the mouse plasma using protein precipitation with acetonitrile and followed by solid-phase extraction of the supernatant. The mobile phase consisted of methanol (0.1% formic acid)-water (0.1% formic acid) (90:10, v/v). The analytical column was a YMC C(18). The standard curve was linear from 0.1 to 50 ng/ml (R(2)>0.99). The lower limit of quantitation was 0.1 ng/ml. The assay was specific based on the multiple reaction monitoring transitions at m/z 1157-->215 and m/z 1101-->243 for thiocoraline and the internal standard, echinomycin, respectively. The mean intra- and inter-day assay accuracies remained below 5 and 12%, respectively, for all calibration standards and quality control (QC) samples. The intra- and inter-day assay precisions were less than 11.4 and 9.5% for all QC levels, respectively. The utility of the assay was demonstrated by a pharmacokinetic study of i.v. (bolus) thiocoraline on CD-1 mice. Thiocoraline was stable in mouse plasma in an ice-water bath for 6 h and for three freeze-thaw cycles. The reconstituted thiocoraline after extraction and drying sample process was stable in the autosampler for over 24 h. The assay was able to quantify thiocoraline in plasma up to 48 h following dose. Pharmacokinetic analysis showed that thiocoraline has distinct pharmacokinetic profiling when dosed in different formulation solutions. The assay is currently used to measure thiocoraline plasma concentrations in support of a project to develop a suitable formulation with a desirable pharmacokinetic profile.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantitation of colchicine in human plasma

A rapid and sensitive method to determine colchicine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Colchicine and the internal standard (I.S.), tegafur, were extracted from the matrix with n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) and separated by reversed-phase high-performance liquid chromatography (HPLC) using formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) as the mobile phase in a run time of 2.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 0.050-10 ng/ml with intra- and inter-day precision (as relative standard deviation (R.S.D.)) of <2 and <7%, respectively. The method was applied to a pharmacokinetic study of colchicine in healthy volunteers given an oral dose of 2.0 mg.     

Simple and rapid high-performance liquid chromatography method for the determination of ofloxacin concentrations in plasma and urine

A high-performance liquid chromatographic method for the determination of ofloxacin in human plasma and urine was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was linear from 0.5 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 5%. The average recovery of ofloxacin from plasma was 93%. The method was evaluated in samples from healthy subjects whose drug levels were already measured by microbiological assay.     

Sensitive liquid chromatography-tandem mass spectrometry method for the determination of scutellarin in human plasma: Application to a pharmacokinetic study

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of scutellarin in human plasma has been developed. Samples were prepared using solid phase extraction and analyzed on a C(18) column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol-water (0.1% formic acid), using gradient procedure. The analyte and internal standard baicalin were both detected by use of selected reaction monitoring mode. The method was linear in the concentration range of 0.2-20.0 ng/mL. The lower limit of quantification (LLOQ) was 0.2 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.4%. The accuracy determined at three concentrations (1.0, 5.0 and 10.0 ng/mL for scutellarin) was within +/-5.0% in terms of relative error. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of scutellarin guttate pills in 20 healthy volunteers.