A simple and sensitive assay for the quantitative analysis of rivastigmine and its metabolite NAP 226-90 in human EDTA plasma using coupled liquid chromatography and tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the determination of rivastigmine and its major metabolite NAP 226-90 is presented. A 100 microL plasma aliquot was spiked with a structural analogue of rivastigmine as internal standard (PKF214-976-AE-1) and proteins were precipitated by adding 200 microL of methanol. After centrifugation a volume of 100 microL of the clear supernatant was mixed with 100 microL of methanol/water (30:70, v/v) and volumes of 25 microL were injected onto the HPLC system. Separation was acquired on a 150 x 2.0 mm i.d. Gemini C18 column using a gradient system with 10 mM ammonium hydroxide and methanol. Detection was performed by using a turboionspray interface and positive ion multiple reaction monitoring by tandem mass spectrometry. The assay quantifies rivastigmine from 0.25 to 50 ng/mL and its metabolite NAP 226-90 from 0.50 to 25 ng/mL, using human plasma samples of 100 microL. Validation results demonstrate that rivastigmine and metabolite concentrations can be accurately and precisely quantified in human EDTA plasma. This assay is now used to support clinical pharmacologic studies with rivastigmine.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of zafirlukast, a selective leukotriene antagonist in human plasma: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of zafirlukast (ZFK) with 500 microL human plasma using valdecoxib as an internal standard (IS). The API-4,000 LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of ZFK and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with 10 mm ammonium acetate (pH 6.4):acetonitrile (20:80, v/v) on a Hypersil BDS C(18) column. The total chromatographic run time was 2.0 min and the elution of ZFK and IS occurred at approximately 1.11 and 1.58 min, respectively. The MS/MS ion transitions monitored were 574.2 --> 462.1 for ZFK and 313.3 --> 118.1 for IS. The method was proved to be accurate and precise at a linearity range of 0.15-600 ng/mL with a correlation coefficient (r) of >or=0.999. The method was rugged with 0.15 ng/mL as lower limit of quantitation. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 20 mg ZFK tablet.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization using multiple ions for quantitation of torcetrapib in hamster and dog plasma

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of torcetrapib (TTB) with 100 microL hamster/dog plasma using DRL-16126 as an internal standard (IS). The API-4000 Q Trap LC-MS/MS was operated under multiple-reaction monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of TTB and IS from plasma with acetonitrile, which yielded consistent recoveries of 65.73 and 94.01% for TTB and 79.68 and 90.70% for IS in hamster and dog plasma, respectively. The total chromatographic run time was 3.0 min and the elution of TTB and IS occurred at approximately 2.25 and 2.20 min, respectively. The resolution of peaks was achieved with 0.01 m ammonium acetate:acetonitrile (15:85, v/v) at a flow rate of 0.40 mL/min on an Inertsil ODS-3 column. The method was proved to be accurate and precise at linearity range of 1.00-200 ng/mL with a correlation coefficient (r) of > or = 0.993. The method was rugged with 1.00 ng/mL as the lower limit of quantitation. TTB was stable in the battery of stability studies. The application of the assay to preclinical pharmacokinetic studies confirmed the utility of the assay to derive hamster/dog pharmacokinetic parameters.     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

A rapid and sensitive high-performance liquid chromatography assay for rofecoxib in human serum

Rofecoxib is a selective cyclooxygenase (COX)-2 inhibitor that is approved for the treatment of acute pain and osteoarthritis in adults. A sensitive and rapid high-performance liquid chromatographic (HPLC) method of determining rofecoxib in human serum is described. Alkalinized plasma samples are extracted into an organic solvent containing an internal standard and evaporated under nitrogen. The dried sample residues are reconstituted with mobile phase and analyzed by HPLC. The method uses 100 microL of the sample and is linear from 20 to 2000 ng/mL of rofecoxib. Precision and accuracy studies are performed. Stability of the drug in serum over four weeks is documented. This new method is simple, sensitive, precise, and accurate. Its use will translate into faster laboratory turnaround time, and the small sample volume required (100 microL) makes this assay suitable for pediatric patients. This assay will expedite pharmacokinetic studies and the therapeutic drug monitoring of rofecoxib and possibly other COX-2 inhibitors.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of rhein in human plasma: application to a pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of rhein with 100 microL human plasma using celecoxib as an internal standard (IS). The API-4,000 Q-Trap LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of rhein and IS from human plasma with acetonitrile, which yielded consistent recoveries of 36.01 and 65.85% for rhein and IS, respectively. The total chromatographic run time was 5.0 min and the elution of rhein and IS occurred at approximately 1.60 and 3.96 min, respectively. The resolution of peaks was achieved with 0.01 m ammonium acetate (pH 6.0):acetonitrile:methanol (30:58:12, v/v) on an Inertsil ODS-3 column. The method was proved to be accurate and precise at a linearity range of 0.005-5.00 microg/mL with a correlation coefficient (r) of >or=0.995. The lower limit of quantitation was 0.005 microg/mL. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. Rhein was found to be stable in the battery of stability studies. The application of the assay to pre-clinical pharmacokinetic studies confirmed the utility of the assay to derive pharmacokinetic parameters.     

Demonstration of the capabilities of a parallel high performance liquid chromatography tandem mass spectrometry system for use in the analysis of drug discovery plasma samples.

There is a continuing need for increased throughput in the evaluation of new drug entities in terms of their pharmacokinetic (PK) parameters. This report describes an alternative procedure for increasing the throughput of plasma samples assayed in one overnight analysis: the use of parallel high performance liquid chromatography (HPLC) combined with tandem mass spectrometry (parallel LC/MS/MS). For this work, two HPLC systems were linked so that their combined effluent flowed into one tandem MS system. The parallel HPLC/APCI-MS/MS system consisted of two Waters 2690 Alliance systems (each one included an HPLC pump and an autosampler) and one Finnigan TSQ 7000 triple quadrupole mass spectrometer. Therefore, the simultaneous chromatographic separation of the plasma samples was carried out in parallel on two HPLC systems. The MS data system was able to deconvolute the data to calculate the results for the samples. Using this system, 20 compounds were tested in one overnight assay using the rapid rat PK screening model which includes a total of 10 standards plus samples and two solvent blanks per compound tested. This application provides an additional means of increasing throughput in the drug discovery PK assay arena; using this approach a two-fold increase in throughput can be achieved in the assay part of the drug discovery rat PK screening step.     

A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the determination of paclitaxel and its 3'p- and 6-alpha-hydroxy metabolites is presented. A 200 microL plasma aliquot was spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1.0 mL tert-butylmethylether. Dried extracts were reconstituted in 0.1 M ammonium acetate-acetonitrile (1:1, v/v) and 25 microL volumes were injected onto the HPLC system. Separation was performed on a 150 x 2.1 mm C18 column using an alkaline eluent (10 mm ammonium hydroxide-methanol, 30:70, v/v). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range for paclitaxel from 0.25 to 1000 ng/mL and metabolites from 0.25 to 100 ng/mL using 200 microL human plasma samples. Validation results demonstrate that paclitaxel and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with paclitaxel.     

Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard

To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 microL of mobile phase and 50 microL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 x 4.6 mm C(18) with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow-rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r(2) > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5-1000 ng/mL. The intra-run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were <+/-10.69 and <-12.35%, respectively across the QC levels (50-1000 ng/mL). The extraction efficiency was >80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS.     

Development and validation of a liquid chromatography/tandem mass spectrometry method for the determination of the novel anticancer agent 17-DMAG in human plasma

An accurate, sensitive, robust and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin hydrochloride (17-DMAG) in human plasma has been developed and validated. Plasma samples were prepared by liquid/liquid extraction with ethyl acetate. The chromatographic separation was achieved within 9 min on a Synergy Polar column with a linear gradient and a mobile phase consisting of methanol and 0.1% formic acid in water. Detection of 17-DMAG and the internal standard (IS), olomoucine, was achieved by MS/MS with electrospray ionisation in positive ion mode. The calibration curve, ranging from 1.89 to 1890 nM, was linear r > 0.994 using a 1/y2 weighted linear regression. The assay showed no significant interferences from endogenous compounds. The lower limit of quantitation (LLOQ) was 1.89 nM, using 250 microL of plasma, with inter-assay precision (%RSD) and accuracy (%RE) values of 11.6% and -5.8%, respectively. Intra-assay precision ranged from 7.8-13.6%. The method described here is being used to evaluate the pharmacokinetic profiles of 17-DMAG given as a once weekly infusion in patients with advanced solid tumours.     

Liquid chromatographic-tandem mass spectrometric assay for the simultaneous quantification of Camptosar and its metabolite SN-38 in mouse plasma and tissues

A simple, rapid and sensitive LC-MS/MS bioanalytical method has been developed to simultaneously quantify Camptosar (CPT-11) and its active metabolite, SN-38, in mouse plasma and tissues. A single step protein precipitation with acetonitrile in 96-well plates was used for sample preparation. Camptothecin (CPT) was used as the internal standard. Fast separation of SN-38, CPT-11 and CPT was carried out isocratically on a C18, 2 mm x 50 mm, 5 microm HPLC column with a mobile phase containing acetonitrile and 20 mM ammonium acetate (pH 3.5) and a 2.5 min chromatographic run time. The API 4000 MS/MS system was operated in positive ionization multiple reaction monitoring mode, and the transitions for SN-38, CPT-11 and CPT were 393.4 --> 349.3, 587.6 --> 167.2 and 349.3 --> 305.3, respectively. The SN-38 and CPT-11 concentrations in samples were calculated from a standard curve of peak area ratios of the analyte to that of the internal standard using a 1/chi2 weighted linear regression. The quantitation limit of 0.5 ng/mL was achieved by using a low sample volume (100 microL) of plasma or tissue homogenates. The assay was linear over the concentration range of 0.5-500 ng/mL with acceptable precision and accuracy. The method was used for the quantification of CPT-11 and SN-38 in plasma and tissues to support a preclinical pharmacokinetics and tissue distribution study of CPT-11 in mice.     

Trapping 4-fluorobenzyl chloride in human plasma with chemical derivatization followed by quantitative bioanalysis using high-performance liquid chromatography/tandem mass spectrometry

A quantitative bioanalytical method involving chemical derivatization, solid phase extraction (SPE) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was developed for the determination of 4-fluorobenzyl chloride (4FBCl) in human plasma. 4FBCl is a volatile and reactive molecule that is very unstable in human plasma. In order to stabilize 4FBCl in plasma samples prior to storage, 4-dimethylaminopyridine (DMAP) was added, forming a stable quaternary amine salt derivative. A three-step weak cation-exchange SPE procedure was then employed to remove excess DMAP. The plasma extracts were analyzed by HPLC/MS/MS using a TurboIonspray interface and multiple reaction monitoring. Unlike 4FBCl, the quaternary amine derivative shows excellent sensitivity in electrospray mass spectrometry. The method was validated over a concentration range of 0.5-500 ng/mL using 45 microL of plasma. The maximum within-run and between-run precision observed in a three-run validation for quality control (QC) samples was 12.5 and 7.6%, respectively. The maximum percentage bias observed at all QC sample concentrations was 11.9%. The method has proven to be robust and compatible with high-throughput bioanalysis.     

Determination of drug concentrations in plasma by a highly automated, generic and flexible protein precipitation and liquid chromatography/tandem mass spectrometry method applicable to the drug discovery environment

This report presents a highly automated procedure for the determination of drug concentrations in plasma samples. The method is generic, in that it has been applied without adaptation to many different drug candidate molecules, but is also flexible, in that variations in the nature and number of samples to be analyzed can be readily accommodated. The method includes preparation of dilutions of analyte stock solutions, spiking these into control plasma to generate analytical standards, and preparation of samples suitable for analysis by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) by precipitation of plasma proteins with acetonitrile, centrifugation, and dilution of the supernatants with HPLC buffer. All of these steps, apart from centrifugation, are performed without manual intervention on an automated liquid-handling workstation using 96-well plates. Analysis is by HPLC/MS/MS, using a generic HPLC gradient. Commercially available software was used for optimization of parameters for analysis by HPLC/MS/MS, integration of chromatographic peaks, and quantification of drug concentrations. The use of this methodology in our laboratory has greatly facilitated the analysis of small sample sets for a large number of analytes, a situation regularly encountered in an early drug discovery environment.     

Liquid chromatography/tandem mass spectrometry for pharmacokinetic studies of 20(R)-ginsenoside Rg3 in dog

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-ginsenoside Rg3 in dog. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. Dioscin was used as the internal standard. The method had a lower limit of quantitation of 0.5 ng/mL for Rg3 in 200 microL of plasma or 2 ng/mL in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of Rg3 in plasma. The intra- and inter-day precisions were measured to be below 8% and accuracy between -1.5 and 1.4% for all quality control samples. This quantitation method was successfully applied to pharmacokinetic studies of Rg3 after both an oral and an intravenous administration to beagle dogs. No Rh2 and protopanaxadiol were detected in plasma.     

Simultaneous quantitation of enalapril and enalaprilat in human plasma by 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive and rapid method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with rapid solid-phase extraction (SPE) has been developed and validated for the quantitative determination of enalapril and its active metabolite enalaprilat in human plasma. After addition of internal standard to human plasma, samples were extracted by 96-well SPE cartridge. The extracts were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the simultaneous determination of enalapril and enalaprilat was accurate and reproducible, with respective limits of quantitation of 0.2 and 1.0 ng/mL in plasma. The standard calibration curves for both enalapril and enalaprilat were linear (r(2) = 0.9978 and 0.9998) over the concentration ranges 0.2-200 and 1.0-100 ng/mL in human plasma, respectively. The intra- and inter-day precision over the concentration range for enalapril and enalaprilat were lower than 13.3 and 15.4% (relative standard deviation, %RSD), and accuracy was between 89.2-105.0 and 91.9-104.7%, respectively.     

Liquid Chromatography-Electrospray Quadrupole Linear Ion Trap Mass Spectrometric Method for Quantitation of Domperidone in Chinese Healthy Volunteers

A rapid,sensitive,and accurate method based on LC/MS/MS was developed and validated for the determination of domperidone in human plasma.Domperidone and internal standard,tramadol,were extracted from plasma with diethyl ether-dichloromethane(60∶40,volume ratio)and separated by reversed-phase HPLC with methanol-water-ammonia solution(80∶20∶0.2,volume ratio)as the mobile phase.Detection was carried out via multiple-reaction monitoring(MRM)on a Q-trapTM LC/MS/MS system(Q-trapTM).The assay result was linear over a concentration range of 0.1-30 ng/mL with a limit of quantitation(LOQ)of 0.1 ng/mL.The inter-and intra-day precision levels were within 7.52% and 12.9%,respectively,whereas the accuracy was within a range of 87.3%-114%.This method has been successfully applied to evaluate the pharmacokinetics of domperidone in Chinese healthy volunteers given an oral dose of 10 mg.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of doxofylline in human serum: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of doxofylline (DFL) with 300 microL human serum using imipramine as the internal standard (IS). The API-3,000 LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved direct precipitation of DFL and IS from human serum with acetonitrile. The resolution of peaks was achieved with formic acid (pH 2.5): acetonitrile (10:90, v/v) on an Amazon C(18) column. The total chromatographic run time was 3.0 min and the elution of DFL and IS occurred at approximately 1.46 and 2.15 min, respectively. The MS/MS ion transitions monitored were 267.5 --> 181.1 for DFL and 281.1 --> 86.2 for IS. The method was proved to be accurate and precise at linearity range of 1.00-5,000 ng/mL with a correlation coefficient (r) of >or=0.999. The method was rugged with 1.00 ng/mL as lower limit of quantitation. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of DFL tablet.     

High-performance liquid chromatography/tandem mass spectrometry method for the determination of arbidol in human plasma

An HPLC/MS/MS method for the determination of arbidol in human plasma was developed. Arbidol and internal standard (loratadine) were extracted from alkaline plasma with tert-butyl methyl ether and analyzed on a Zorbax SB C18 column (30 x 2.1 mm id, 3.5 microm particle size). The detection was by monitoring arbidol at m/z 479.1 --> 434.1 and the internal standard at m/z 383.2 --> 337.2. The method was validated according to U.S. Food and Drug Administration guidelines. The calibration curve was linear over the range of 0.5-500 ng/mL using a 100 microL sample volume. The intraday and interday precisions were less than 6.5%, and acceptable values were obtained for accuracy, recovery, and sensitivity. The developed method was selective, simple, sensitive, and easily applicable.     

高效液相色谱法测定吲哚美辛控释胶囊的血药浓度和生物利用度

采用ＯＤＳ柱,甲醇－稀磷酸溶液（７６２４）为流动相,２６０ｎｍ为检测波长,建立了测定血浆中吲哚美辛浓度的高效液相色谱法,并测定了吲哚美辛控释胶囊炎痛康的血药浓度。结果表明,血浆中吲哚美辛浓度在０．１２５～５．０ｍｇ／Ｌ范围内线性关系良好（ｒ＝０．９９９６）,检测限６２．５μｇ／Ｌ（Ｓ／Ｎ＝３１）,平均回收率为１００．４％,日内和日间ＲＳＤ均小于５％。１１位受试者单剂量口服炎痛康后的相对生物利用度为１０２．３８％。     

Analysis of kanamycin A in human plasma and in oral dosage form by derivatization with 1-naphthyl isothiocyanate and high-performance liquid chromatography

A simple and sensitive HPLC method has been developed for trace determination of kanamycin A by derivatization. Plasma proteins are precipitated by acetonitrile and chemical derivatization is performed on the supernatant containing kanamycin A with 1-naphthyl isothiocyanate in pyridine at 70 degrees C. After the derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess of derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Lichrocart Purospher STAR RP-18e column and water/methanol (33:67, v/v) as a mobile phase (detection at 230 nm). Optimization conditions for the derivatization of kanamycin A were investigated by HPLC. The linear range for the quantitation of kanamycin A in spiked plasma was over 1.2-40 microg/mL; the detection limit (signal to noise ratio = 3; injection volume, 10 microL) was about 0.3 microg/mL. The relative standard deviation was less than 2.9% for intra-day assay (n = 6) and inter-day assay (n = 6) and relative recoveries were found to be greater than 98%. Preliminary application of the method for monitoring kanamycin A in humans upon intramuscular injection of the injection product demonstrated the usefulness of the assay for clinical studies. The proposed method can also be used to analyze the compound in pharmaceutical formulations.