Stability-indicating TLC-densitometric method for estimation of dexrabeprazole and domperidone in pharmaceutical dosage form

A stability-indicating thin layer chromatographic (TLC) method for determination of dexrabeprazole (DEX) and domperidone (DOM) in combined dosage form has been developed and validated. The mobile phase selected was acetone:toluene:methanol (4.5:4.5:0.5, v/v/v) with ultraviolet (UV) detection at 285 nm. The retention factors for DEX and DOM were found to be 0.49 ± 0.02 and 0.24 ± 0.03, respectively. The method was validated with respect to linearity, accuracy, precision, and robustness. The linearity range for DEX was 50-350 ng/band (r2 = .9960) and for DOM was 100-700-n /band (r2 = .9982), respectively. The method was successfully applied for the analysis of drugs in pharmaceutical formulation.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of huperzine A in dog plasma

A rapid and sensitive LC-MS-MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane-dichloromethane-2-propanol (300:150:15, v/v/v), chromatographed on a C(18) column (5 microm, 50 mm x 4.6 mm i.d.) with a mobile phase consisting of acetonitrile-methanol-10mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05-20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration-time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 microg/kg per day for 15 days) of a sustained-release formulation of huperzine A.     

Simultaneous quantification of alpha-/beta-diastereomers of arteether, sulphadoxine and pyrimethamine: a promising anti-relapse antimalarial therapeutic combination, by liquid chromatography tandem mass spectrometry

A rapid, sensitive, selective and specific HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of alpha-/beta-diastereomers of arteether (AE), sulphadoxine (SDX) and pyrimethamine (PYR) in rat blood plasma using propyl ether analogue of beta-arteether as internal standard. The method involved a single-step, liquid-liquid extraction with ethyl acetate and the analytes were chromatographed on a C18 chromatographic column by isocratic elution with methanol:ammonium acetate buffer (10 mM, pH 4) (90:10%, v/v) and analyzed by tandem mass spectrometry. The run time was 4.5 min and the weighted (1/x2) calibration curves were linear over a range of 0.78-400 ng ml-1. The method was validated fully and the lower limit of quantification (LLOQ) in plasma was 0.78 ng ml-1 for all the analytes. The intra- and inter-day precision and accuracy were found to be well within the acceptable limits (<15%) and the analytes were stable after three freeze-thaw (f-t) cycles. The absolute recoveries were consistent and reproducible. The assay method was applied to pre-clinical pharmacokinetic interaction studies of alpha-/beta-AE, SDX and PYR in rats.     

Determination of nimodipine in human plasma by a sensitive and selective liquid chromatography-tandem mass spectrometry method

A sensitive and highly selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine nimodipine in human plasma. The analyte and internal standard nitrendipine were extracted from plasma samples by n-hexane-dichloromethane-isopropanol (300:150:4, v/v/v), and chromatographed on a C(18) column. The mobile phase consisted of methanol-water-formic acid (80:20:1, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source. The method has a limit of quantification of 0.24 ng/ml. The linear calibration curves were obtained in the concentration range of 0.24-80 ng/ml. The intra- and inter-day precisions were lower than 4.4% in terms of relative standard deviation (R.S.D.), and the accuracy ranged from 0.0 to 5.8% in terms of relative error (RE). This validated method was successfully applied for the evaluation of pharmacokinetic profiles of nimodipine tablets administered to 18 healthy volunteers.     

Method development and validation of the simultaneous determination of a novel topoisomerase 1 inhibitor, the prodrug, and the active metabolite in human plasma using column-switching LC-MS/MS, and its application in a clinical trial

A robust and sensitive method using liquid chromatography-tandem mass spectrometry was developed and validated for the simultaneous determination of a novel topoisomerase 1 inhibitor CH0793076 (3076), the prodrug CH4556300 (TP300), and the active metabolite CH0793011 (3011) in human plasma. All plasma analyzed with this method was acidified with 1M HCl and 46% citric acid solution in a ratio of 100:10:1 (v:v:v) to avoid the pH-based degradation of TP300 and to shift the equilibria of 3076 and 3011 between the lactone and carboxylate forms towards the lactone forms. After the plasma proteins were precipitated with methanol:acetonitrile:HCl 1M (50:50:1, v:v:v) containing stable isotopic internal standards, the analytes were trapped on an Xterra MS C18 column (10×2.1 mm i.d., 5 μm) and separated on a Gemini C18 column (50×2.0 mm i.d., 5 μm) using column-switching liquid chromatography. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to quantify the analytes with transitions m/z 587.2>441.2 for TP300, 459.1>415.2 for 3076, and 475.1>361.1 for 3011. The inter- and intra-day precisions were below 12%, and the accuracy was between -16% and 16% at the lower limit of quantitation (LLOQ) and between -11% and 14% at the other quality controls. The LLOQs of TP300, 3076, and 3011 were 0.8, 0.04, and 0.04 ng/mL, respectively. The validated method was successfully applied to clinical sample analysis and incurred sample reanalysis was also conducted.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

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.     

Liquid chromatography-tandem mass spectrometric assay with a novel method of quantitation for the simultaneous determination of bulaquine and its metabolite,primaquine, in monkey plasma

A sensitive and selective liquid chromatography-tandem mass spectrometry method (LC-MS-MS) for the simultaneous estimation of bulaquine and primaquine has been developed and validated in monkey plasma. The mobile phase consisted of acetonitrile/ammonium acetate buffer (20 mM, pH 6) (50:50 v/v) at a flow-rate of 1 ml/min. The chromatographic separations were achieved on two spheri cyano columns (5 microm, 30 x 4.6 mm I.D.) connected in series. The quantitation was carried out using a Micromass LC-MS-MS with an electrospray source in the multiple reaction monitoring (MRM) mode. The analytes were quantified from the summed total ion value of their two most intense molecular transitions. This is another novel method leading to increased sensitivity and precision. A simple liquid-liquid extraction with 2 x 1.0 ml n-hexane/ethyl acetate/dimethyloctyl amine (90:10:0.05, v/v) was utilized. The method was validated in terms of recovery, linearity, accuracy and precision (within- and between-assay variation). The recoveries from spiked control samples were >or=90 and 50% for bulaquine and primaquine, respectively. Linearity in plasma was observed over a dynamic range of 1.56-400 and 3.91-1000 ng/ml for bulaquine and primaquine, respectively.     

Determination of beraprost in human plasma by a high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of beraprost, a stable, orally active prostacyclin analogue with vasodilatory, antiplatelet and cytoprotective effects. The analyte and internal standard, indomethacin, were extracted by solid-phase extraction using OASIS HLB cartridge. The chromatographic separation was performed on a C18 column with a mobile of 0.1% formic acid-methanol (30:70, v/v). The highest daughter ion of deprotonated analyte was quantitated in negative ionization by multiple reactions monitoring with a mass spectrometer. The mass transitions m/z 397>269 and m/z 356>312 were used to measure beraprost and internal standard, respectively. The assay exhibited a linear range from 0.02 to 2 ng/mL for beraprost in human plasma. The lower limit of quantitation was 20 pg/mL with a relative standard deviation of less than 20%. The method was validated with respect to linearity, sensitivity, specificity, recovery, accuracy and precision. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic study.     

Simultaneous determination of hydrochlorothiazide, quinapril and quinaprilat in human plasma by liquid chromatography-tandem mass spectrometry

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous estimation of hydrochlorothiazide, quinapril and its metabolite quinaprilat in human plasma. After solid phase extraction (SPE), the analytes and IS were chromatographed on a hypurity C8 (100mmx2.1mm i.d., 5mum particle size) column using 2muL injection volume with a run time of 2.8min. An isocratic mobile phase consisting of 0.5% (v/v) formic acid:acetonitrile (25:75, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The proposed method was validated over the range of 5-500ng/mL for hydrochlorothiazide method and 5-1500ng/mL for quinapril and quinaprilat. Inter-batch and intra-batch precision (coefficient of variation - % CV) across five validation runs lower limit of quantitation (LLOQ), lower quality control (LQC), middle quality control (MQC), higher quality control (HQC) and upper limit of quantitation (ULOQ) was less than 15. The accuracy determined at these levels was within +/-13% in terms of relative percentage error.     

Quantitation of niflumic acid in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection and its application to a bioequivalence study of talniflumate tablets

A rapid and simple HPLC method with UV detection (288 nm) was developed and validated for quantitation of niflumic acid in human plasma, the active metabolite of talniflumate. After precipitation with 100% methanol containing the internal standard, indomethacin, the analysis of the niflumic acid level in the plasma samples was carried out using a reverse phase C18 CAPCELL PAK (5 microm, 4.6 mm x 250 mm) column. The chromatographic separation was accomplished with an isocratic mobile phase consisting of a mixture of 0.1M sodium acetate in water and acetonitrile (37:63, v/v), adjusted to pH 6.4. This HPLC method was validated by examining its precision and accuracy for inter- and intra-day runs in a linear concentration range of 0.02-5.00 microg/mL. Stability of niflumic acid in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method was successfully applied to the bioequivalence study of talniflunate in healthy volunteers.     

Development and validation of a liquid chromatography-mass spectrometry method for the quantitation of naltrexone and 6beta-naltrexol in guinea pig plasma

A quantitative liquid chromatographic-electrospray ionization mass spectrometry method for the determination of naltrexone and 6beta-naltrexol in guinea pig plasma has been developed and validated using naloxone as an internal standard. A single step precipitation-extraction technique was carried out to extract the plasma samples using acetonitrile:ethyl acetate (1:1, v/v). The chromatographic separation was performed on a C(18) column using a mobile phase consisting of 35:65 (v/v) acetonitrile:2 mM ammonium acetate with 0.01 mM ammonium citrate at a flow rate of 0.25 mL/min. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The mean recoveries for naltrexone, naltrexol, and naloxone were 91.7, 89.3, and 99.0%, respectively. The lower limit of quantification (LLOQ) for naltrexone and 6beta-naltrexol was 1.25 ng/mL, and the limit of detection (LOD) was 0.75 ng/mL. The method was applied to a pharmacokinetic study in order to assess the drug disposition of naltrexone in guinea pigs.     

High-performance liquid chromatographic method for the determination of pioglitazone in human plasma using ultraviolet detection and its application to a pharmacokinetic study

An analytical method based on high-performance liquid chromatography (HPLC) with ultraviolet detection (269 nm) was developed for the determination of pioglitazone in human plasma. Rosiglitazone was used as an internal standard. Chromatographic separation was achieved with a reversed-phase Apollo C18 column and a mobile phase of methanol-acetonitrile-mixed phosphate buffer (pH 2.6; 10mM) (40:12:48, v/v/v) with a flow rate of 1.2 ml/min. The calibration curve was linear over the range of 50-2000 ng/ml (r(2)>0.9987) and the lower limit of quantification was 50 ng/ml. The method was validated with excellent sensitivity, accuracy, precision, recovery and stability. The assay has been applied successfully to a pharmacokinetic study with human volunteers.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic study

Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug-drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C(18) column (50 mm x 4.6mm i.d., 5 microm) using acetonitrile: 2mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC-MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20-2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug-drug interaction study in Wistar rats.     

Liquid chromatography-positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography-mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid-liquid extraction with a mixture of hexane-heptane-isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C(8) micro-bore column (250 mm x 2.1 mm i.d.; 3.5 microm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate-formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min(-1). The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50-250 ng ml(-1) (r(2) > 0.996). The limit of quantitation was 0.50 ng ml(-1). Accuracy and precision were below the acceptance limits of 15%.     

Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography

A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred microl of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90:10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/ml, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively.     

High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease (K_i=2 nM), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 μl of human plasma with a mixture of ethyl acetate–acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C₁₈ analytical column and a mobile phase consisting of 25 mM monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)–acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 μg/ml to 10 μg/ml, with a minimum quantifiable level of 0.0500 μg/ml for nelfinavir.     

Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.     

Liquid chromatographic determination of urinary 2-thiothiazolidine-4-carboxylic acid, a biomarker of carbon disulphide exposure

An effective gradient high-performance liquid chromatographic method for baseline separation of urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), with photodiode array detection at 271 nm was described. o-Methylhippuric acid was used as an internal standard (I.S.). A 1-ml urine sample was saturated with 300 mg of sodium sulphate, acidified with 100 μl of 6 M hydrochloric acid, extracted twice with 2 ml of diethyl ether, and after evaporation, the residue was taken up in 1 ml of 0.1% (v/v) phosphoric acid. The two mobile phases used for gradient elution were: (A) 10 mM ammonium dihydrogenphosphate (pH 3.5) and (B) same concentration of buffer but containing 20% (v/v) of methanol (pH 4.8). The flow-rate was set at 1.0 ml/min. TTCA and I.S. were detected at 2.2 and 9.1 min, respectively. The method was validated with urine samples collected from normal subjects and workers occupationally exposed to carbon disulphide. The present method enables the detection of urinary TTCA at a concentration of 0.025 mg/l. Analytical recovery and reproducibility generally exceeded 90%. The proposed method is considered more sensitive, specific and reliable than other existing methods.     

Determination of 3'-amino-3'-deoxythymidine, a cytotoxic metabolite of 3'-azido-3'-deoxythymidine, in human plasma by ion-pair high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of 3'-amino-3'-deoxy-thymidine (AMT), a cytotoxic metabolite of 3'-azido-3'-deoxy-thymidine (AZT, zidovudine), in human plasma. The sample pretreatment involved solid-phase extraction using cation-exchange extraction columns. Chromatography was carried out on a C₈ column, using a mobile phase of methanol—0.01 M ammonium acetate (pH 5)—0.25 M sodium dioctylsulfosuccinate (60:40:4, v/v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The lower limit of quantitation is 5 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used for the determination of AMT in patients with AIDS who used AZT.