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.     

Rapid determination of metformin in human plasma by liquid chromatography-tandem mass spectrometry method

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method is described for quantitation of metformin in human plasma. After a simple, one-step protein precipitation using acetonitrile, metformin and the internal standard diphenhydramine were chromatographed on a C(8) column and detected by tandem mass spectrometry. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The method has a chromatographic total run time of 3.4 min and was linear within the range 2-2000 ng/ml. Intra- and inter-day precision, expressed as the relative standard deviation (R.S.D.), ranged from 4.4 to 5.7% and from 1.3 to 2.8%, respectively. Assay accuracy was less than 1% in terms of %RE (relative error). The assay was used to evaluate the pharmacokinetics of metformin after an oral administration of multicomponent formulation containing 500 mg metformin and 2.5 mg glyburide to 20 healthy volunteers.     

Extractionless and sensitive method for high-throughput quantitation of cetirizine in human plasma samples by liquid chromatography-tandem mass spectrometry

Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.     

Selective and sensitive liquid chromatography-tandem mass spectrometry method for the determination of levonorgestrel in human plasma

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of levonorgestrel in plasma was developed. An Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, using atmospheric pressure photospray ionisation (APPI) in the positive mode. Using 17-alpha-methyltestosterone as internal standard (IS), liquid-liquid extraction was followed by reversed phase liquid chromatography using a phenyl-hexyl column and tandem mass spectrometric detection. The mean recovery for levonorgestrel and 17-alpha-methyltestosterone was 99.5 and 62.9%, respectively. The method was validated from 0.265 to 130 ng levonorgestrel/ml plasma with the lower limit of quantification (LLOQ) set at 0.265 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection, allowing for a rapid (extraction and chromatography) and selective method for the determination of levonorgestrel in human plasma. The assay method was used in a pharmacokinetic study to quantify levonorgestrel in human plasma samples generated after administrating a single oral dose of 1.5 mg levonorgestrel to healthy female volunteers for up to five half lives. The total chromatographic runtime of this method was 5.0 min per sample, allowing for analysis of a large number of samples per batch.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of clonidine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of clonidine in human plasma. Clonidine was extracted from human plasma by using solid-phase extraction technique. Nizatidine was used as the internal standard. A Hypurity C18 (50 mm x 4.6 mm i.d., 5 microm particle size) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 50-2500 pg/mL. The absolute recoveries for clonidine (71.86%) and IS (69.44%) achieved from spiked plasma samples were consistent and reproducible.     

A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of mometasone furoate in human plasma

A robust, rapid, selective and sensitive liquid chromatography-negative atmospheric pressure chemical ionization (LC-(APCI(-))-MS-MS) method has been developed for the quantification of mometasone furoate (MF) in human plasma utilizing a solid-phase extraction clean-up step and 13C-fluticasone propionate as internal standard. The intra- and inter-day coefficients of variation were < or = 15% and the lower limit of quantification (LLOQ) was 15 pg/ml. This method is ideally suited for pharmacokinetic investigations of low MF levels following inhalation of MF.     

Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of alpha-methyltyrosine in human plasma

A sensitive and selective LC-MS-MS method for the isolation and quantification of alpha-methyltyrosine (AMT) from human plasma is described. The method employs a simple protein precipitation using zinc sulfate and sodium hydroxide. This precipitation procedure produced samples with high aqueous content that could be directly injected into a LC-MS-MS system without compromising reverse-phase chromatographic performance. Chromatographic separation was performed on a MetaChem MonoChrom C(18) column (2.0 mm x 50 mm; 5 microm) at a flow rate of 1 mL/min. Compounds were eluted using a gradient mixture of water-acetic acid (100:0.1, v/v) and acetonitrile-acetic acid (100:0.1, v/v). The structural analog alpha-hydroxymethyltyrosine was used as the internal standard. Mass spectrometric detection was carried out with a triple quadrupole mass spectrometer. The method was validated and used to determine human plasma AMT concentrations, and has been implemented to derive pharmacokinetic parameters.     

Highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method for the analysis of dextromethorphan in human plasma

A stable-isotope-dilution HPLC-tandem mass spectrometry-based method was developed for the determination of dextromethorphan in human plasma. Plasma samples were prepared for analysis by solid-phase extraction on octadecylsilane extraction cartridges. Dextromethorphan and the deuterium-labeled dextromethorphan internal standard were chromatographed on a short reversed-phase column and detected by a selected-reaction-monitoring scheme. Linear standard curves were obtained over three orders of magnitude and the limit of quantitation for dextromethorphan was 50 pg/ml, using a 1-ml plasma sample. The combination of HPLC and electrospray tandem mass spectrometry resulted in a rapid, selective and sensitive method for the analysis of dextromethorphan in plasma. The method was applied for the evaluation of the pharmacokinetic profile of dextromethorphan in human volunteers following peroral administration.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of sildenafil in human plasma

A method to determine sildenafil in human plasma involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Sildenafil and the internal standard (I.S.), diazepam, are extracted from human plasma with ether-dichloromethane (3:2, v/v) at basic pH and analyzed by reversed-phase high-performance liquid chromatography (HPLC) using methanol-10mM ammonium acetate pH 7.0 (85:15, v/v) as the mobile phase. Detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was linear over the concentration range 0.125-40.0 ng/ml. Intra- and inter-day precision of the assay at four concentrations within this range were 2.5-8.0%. The method was used to evaluate plasma concentration-time profiles in healthy volunteers given an oral dose of 20mg sildenafil as a combination tablet also containing apomorphine.     

A mixed-mode liquid chromatography-tandem mass spectrometric method for the determination of cytarabine in mouse plasma

A novel mixed-mode high performance liquid chromatographic system (HPLC) interfaced with an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) was developed for the determination of cytarabine (ara-C) in mouse plasma to support pharmacodynamic studies. The mixed-mode reversed-phase ion-exchange chromatography column was adapted for sufficient retention and separation of a small and polar analyte. The impact of the mobile phase composition on both chromatographic separation and the ionization efficiency of the test compound in the positive mode was investigated. The potential of ionization suppression from endogenous biological matrices on the mixed-mode LC-APCI/MS/MS method was evaluated using the post-column infusion technique. Furthermore, the feasibility of using the mixed-mode HPLC-MS/MS method for the determination of the plasma concentrations of cytarabine in mice was demonstrated by comparing those obtained by the ion-pairing HPLC-MS/MS method.     

Ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of strychnine and brucine in mice plasma

A selective, simple and efficient method-ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C(18) column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48-496.4 ng/ml for strychnine and 2.64-528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucine in vivo.     

Liquid chromatography-tandem mass spectrometric assay for the light sensitive tyrosine kinase inhibitor axitinib in human plasma

A bioanalytical assay for the new tyrosine kinase inhibitor axitinib was developed and validated. In addition, the light mediated trans to cis isomerization of this drug was investigated. For the quantitative assay, human plasma samples were pre-treated under light protection using protein precipitation with acetonitrile containing erlotinib as the internal standard. The extract was diluted with water and injected into the chromatographic system. The system consisted of a trifunctional bonded octadecyl silica column with isocratic elution using formic acid in a water-methanol mixture. The eluate was transferred into an electrospray interface with positive ionization and the analyte was detected and quantified using the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.2–200 ng/ml concentration range, the lowest level of this range being the lower limit of quantification. Within day precisions were 2.5–6%, between day precisions 4–9% and accuracies were between 91 and 106% for the whole calibration range. Light protected axitinib showed no isomerization and was shown to be chemically stable under all relevant conditions. Finally, the assay was successfully applied for a mouse tissue distribution study using mouse samples diluted with human plasma.     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

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.     

Determination of cefaclor in human plasma by a sensitive and specific liquid chromatographic-tandem mass spectrometric method

A sensitive and specific liquid chromatographic-tandem mass spectrometric method is described for the determination of cefaclor in human plasma. The plasma samples were treated by two sample preparation procedures, i.e. protein precipitation (PPT) and solid-phase extraction (SPE). The pretreated samples were analyzed on a C(18) HPLC column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization (ESI) was employed as the ionization source. The analyte and internal standard ampicillin (for PPT) or cefetamet (for SPE) were detected by use of selected reaction monitoring (SRM) mode. The lower limit of quantitation obtained as a result of the PPT procedure was 100 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 12% for cefaclor. The accuracy as determined from QC samples was within +/-3% for the analyte. The SPE procedure could provide the lower limit of quantitation of 2 ng/ml. The precision and accuracy were measured to be below 7.1% and between -3.6% and 1.1%, respectively, for all QC samples. The method was applied for the evaluation of the pharmacokinetic profiles of cefaclor sustained-release formulation.     

Rapid and sensitive determination of vinorelbine in human plasma by liquid chromatography-tandem mass spectrometry and its pharmacokinetic application

Vinorelbine is a semi-synthetic vinca alkaloid with demonstrated high activities against various types of advanced cancer. To support a clinical pharmacokinetic study, a simple, rapid and sensitive method to determine vinorelbine in human plasma was developed using reversed phase liquid chromatography (LC) coupled with electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). Vinorelbine and vinblastine (the internal standard) were extracted from human plasma by one-step liquid-liquid extraction (LLE) with methyl-t-butyl ether. The chromatographic separation was achieved on a Spursil polar-modified C(18) column (50 mm×2.1 mm, 3 μm, Dikma Technologies) with an isocratic mobile phase of a 75:25 (v/v) acetonitrile-4 mmol/L ammonium formate (pH 3.0) mixture at a flow-rate of 0.4 mL/min. The MS/MS detection was performed in the positive ion multiple reaction monitoring (MRM) mode by monitoring the precursor→product ion transitions at m/z 779.4→122.0 and m/z 811.3→224.2 for vinorelbine and the internal standard, respectively. The assay was validated in the range 0.1-200 ng/mL (r>0.997), the lowest level of this range being the lower limit of quantification (LLOQ) based on 50 μL of plasma. The intra- and inter-day precisions were within 6.0%, while the accuracy was within ±4.7% of nominal values. Detection and quantification of both analytes within 2 min make this method suitable for high-throughput analyses. The method was successfully applied to evaluate the systemic pharmacokinetics of vinorelbine after a 20-min intravenous infusion of 25 mg/m(2) of vinorelbine to patients with metastatic breast cancer.     

Rapid and selective liquid chromatographic/tandem mass spectrometric method for the determination of fosfomycin in human plasma

A rapid and selective liquid chromatographic/tandem mass spectrometric method for determination of fosfomycin was developed and validated. Following protein-precipitation, the analyte and internal standard (fudosteine) were separated from human plasma using an isocratic mobile phase on an Ultimate XB-CN column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 137-->79 and m/z 178-->91 was performed to quantify fosfomycin and fudosteine, respectively. The method was linear in the concentration range of 0.10-12.0 microg/mL using 50 microL of plasma. The lower limit of quantification was 0.10 microg/mL. The intra- and inter-day relative standard deviation over the entire concentration range was less than 10.6%. Accuracy determined at three concentrations (0.25, 1.00 and 8.00 microg/mL for fosfomycin) ranged from -1.0% to -4.2% in terms of relative error. Each plasma sample was chromatographed within 5.0 min. The method was successfully used in a bioequivalence study of fosfomycin in human plasma after an oral administration of capsules containing 1.0 g fosfomycin (approximately 1.3g calcium fosfomycin).     

Stable isotope dilution assay for liquid chromatography-tandem mass spectrometric determination of L-homoarginine in human plasma

Nitric oxide (NO), the endogenous modulator of vascular tone and structure, originates from oxidation of L-arginine catalysed by NO synthase (NOS). The L-arginine derivative L-homoarginine serves as an alternative NOS substrate releasing NO, competing with L-arginine for NOS, arginase, and arginine transport. In the present article we report a liquid chromatography-tandem mass spectrometric (LC-tandem MS) method for the determination of L-homoarginine in human plasma by stable-isotope dilution. L-[(13)C(6)]-Homoarginine was used as internal standard. This method provides high sample throughput of 25-μl aliquots of plasma with an analysis time of 4 min using LC-tandem MS electrospray ionisation in the positive mode (ESI+). Specific transitions for L-homoarginine and L-[(13)C(6)]-homoarginine were m/z 245 → m/z 211 and m/z 251 → m/z 217, respectively. The mean intra- and interassay CVs were 7.4 ± 4.5% (±SD) for 0.1-50 μmol/L and 7.5 ± 2.0% for 2 and 5 μmol/L, respectively. Applying this method, a mean plasma concentration of L-homoarginine of 2.5 ± 1.0 μmol/L was determined in 136 healthy humans.     

A liquid chromatography-tandem mass spectrometric method for quantification of curcuminoids in cell medium and mouse plasma

Curcumin and tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors, but they suffer from low oral bioavailability and rapid metabolism in vivo. To circumvent these problems, two curcumin analogs: 1,7-bis(3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics has yet to be performed. In this paper, a simple liquid chromatography-tandem mass spectrometric method was developed for the determination of these four curcuminoids in cell medium and mouse plasma. The method showed linearity from 1 to 1000 ng/mL with the lower limit of quantification of 1 ng/mL in cell medium, and 5 ng/mL in mouse plasma for all test curcuminoids. The within-day coefficients of variation were found to be below 15% and the accuracy was in the range of 85-115%. This method was subsequently used to evaluate their stability in these matrices and a pilot pharmacokinetics of curcumin, DMCHC and TMC in mice after an intraperitoneal (i.p.) cassette dosing of 10mg/kg each. Curcuminoids degraded in two phases with terminal half lives of 186, 813, 724, and 2000 min for curcumin, THC, TMC, and DMCHC, respectively, in cell culture medium. In plasma, their respective half lives were 111, 232, 1202 and 3000 min. These data demonstrated that their stability is in the order curcumin相似文献   

Development and validation of high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of loratadine and desloratadine in human plasma

As a continuation of effort to improve our high flow on-line bioanalytical approach for high-throughput quantification of drugs and metabolites in plasma by high-throughput liquid chromatography tandem mass spectrometry (HTLC-MS/MS), we have developed a simple, sensitive and reliable method for simultaneous quantification of loratadine and desloratadine in human plasma. We have performed on-line coupling of extraction with Cyclone P 50 mm x 0.5 mm 50 microm HTLC column and chromatographic separation is performed with Zorbax XDB C18 50 mm x 2.1 mm 5 microm, followed by quantification with mass detector. The method is validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. A marked improvement in sample throughput efficiency is realized with this method and the proposed method will be useful for pharmacokinetic and/or bioequivalence studies.