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

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed to determine cefixime ((6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxyimino)acetamido]-8-oxo-3-vinyl-5-thia-1-azabicyclo-[4,2,0]-oct-2-ene-2-carboxylic acid) in human plasma. After a simple protein precipitation using acetonitrile, the post-treatment samples were analyzed on a C(8) column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water-formic acid (40:60:0.5, v/v/v). The analyte and internal standard cefetamet were both detected by use of selected reaction monitoring mode. The method was linear in the concentration range of 0.05-8.0 microg/ml. The lower limit of quantification was 0.05 microg/ml. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.7%. The accuracy determined at three concentrations (0.05, 0.80 and 7.2 microg/ml for cefixime) was within +/-2.0% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of cefixime capsule in 24 healthy volunteers.     

Sensitive liquid chromatography-tandem mass spectrometry method for the determination of clarithromycin in human plasma

A sensitive method for the determination of clarithromycin in plasma is described, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Samples were prepared using liquid-liquid extraction and separated on a Supelco Discovery C18 column with a mobile phase consisting of acetonitrile, methanol and acetic acid. Detection was performed by a PE SCIEX API 2000 mass spectrometer in the multiple reaction monitoring (MRM) mode (LC-MS-MS) using TurbolonSpray ionization and monitoring the transition of the protonated molecular ion for clarithromycin at m/z 748.5 (M+1) to the predominant product ion of m/z 158.2. The mean recovery of clarithromycin was 87.3%, with a lower limit of quantification of 2.95 ng/ml when using 0.3-ml plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.     

Sensitive method for the determination of ibutilide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for determination of ibutilide in human plasma. The analyte and internal standard sotalol were extracted from plasma samples by liquid-liquid extraction, and separated on a C(18) column, using acetonitrile-water-10% butylamine-10% acetic acid (80:20:0.07:0.06, v/v/v/v) as the mobile phase. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via TurboIonSpray ionization (ESI). Linear calibration curves were obtained in the concentration range of 20-10,000 pg/ml, with a lower limit of quantitation of 10 pg/ml. The intra- and inter-day precision values were below 8% and accuracy was within +/-3% at all three QC levels. The method was utilized to support clinical pharmacokinetic studies of ibutilide in healthy volunteers following intravenous administration.     

Sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of meloxicam in human plasma

Meloxicam was quantified in human plasma after a single 15 mg oral dose of the drug was given to 26 healthy volunteers. An Applied Biosystems Sciex API 2000 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) in the positive ion mode, was used. Protein precipitation with acetonitrile was followed by C(18) reverse phase liquid chromatography and tandem mass spectrometry. The mean recovery for meloxicam was 92% with a lower limit of quantification of 8.96 ng/ml. Piroxicam was used as the internal standard. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometry (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of meloxicam in human plasma than has previously been described.     

Sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of stavudine in human plasma

A sensitive method for the determination of stavudine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak Vac, 100 mg, tC(18) solid-phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery C(18), 5 microm, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M)-acetonitrile-methanol (800:100:100, v/v/v) at a flow-rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for stavudine was 94% with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been described.     

Sensitive method for the quantitative determination of bromocriptine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive LC-MS-MS assay for the quantitative determination of bromocriptine has been developed and validated and is described in this work. The assay involved the extraction of the analyte from 1 ml of human plasma using a solid phase extraction on Oasis MCX cartridges. Chromatography was performed on a Symmetry C18 (2.1 mm x 100 mm, 3.5 microm) column using a mobile phase consisting of 25:75:01 acetonitrile-water-formic acid with a flow rate of 250 microl/min. The linearity was within the concentration range of 2-500 pg/ml. The lower limit of quantification was 2 pg/ml. This method has been demonstrated to be an improvement over existing methods due to its greater sensitivity and specificity.     

Determination of zolmitriptan in human plasma by liquid chromatography-tandem mass spectrometry method: application to a pharmacokinetic study

A sensitive and selective liquid chromatography-tandem spectrometry method for the determination of zolmitriptan was developed and validated over the linearity range 0.05-30 ng/ml with 0.5 ml of plasma using diphenhydramine as the internal standard. Liquid-liquid extraction using a mixture of diethyl ether and dichloromethane was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring (SRM) mode using the atmospheric pressure chemical ionization (APCI) technique. The instrument parameters were optimized to obtain 3.0 min run time. The mobile phase consisted of acetonitrile-water-formic acid (70:30:0.5), at a flow rate of 0.5 ml/min. In positive mode, zolmitriptan produced a protonated precursor ion at m/z 288 and a corresponding product ion at m/z 58. And internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The inter- and intra-day precision (%R.S.D.) were less than 8.5% and accuracy (%error) was less than -2.5%. The method had a lower limit of quantification of 0.05 ng/ml for zolmitriptan, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokinetic study of zolmitriptan after an oral administration of 5 mg zolmitriptan to 20 healthy volunteers.     

Sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of paracetamol and guaifenesin in human plasma

A rapid and sensitive method for the simultaneous determination of paracetamol and guaifenesin in human plasma was developed and validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. After extracted from plasma samples by diethyl ether-dichloromethane (3:2, v/v), the analytes and internal standard osalmide were chromatographed on a C18 column. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method was linear in the concentration range of 0.05-20.0 microg/ml for paracetamol and 5.0-2000.0 ng/ml for guaifenesin. The intra- and inter-day precision was within 14% for both paracetamol and guaifenesin. The assay accuracy was within +/-2.4% for the analytes. This is the first assay method described for the simultaneous determination of paracetamol and guaifenesin in plasma using one chromatographic run. The method was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation, containing 650 mg paracetamol, 200 mg guaifenesin, 60 mg pseudoephedrine and 20 mg dextrorphan.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Enantioselective determination of lercanidipine in human plasma for pharmacokinetic studies by normal-phase liquid chromatography-tandem mass spectrometry

We describe here the first method for the enantioselective analysis of the calcium antagonist lercanidipine in human plasma by high performance liquid chromatography (HPLC) employing tandem mass spectrometric (MS) detection. Routine determination of lercanidipine enantiomers in human plasma in the working range of 0.025-50.0 ng ml(-1) plasma for each enantiomer with an accuracy and precision less than 15% was possible. Application of the method to a stereospecific study of the pharmacokinetics showed that plasma levels after an oral dose of rac-lercanidipine administered to a healthy volunteer were found to be higher for the (S)-enantiomer.     

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the quantification of aniracetam in human plasma

A rapid, sensitive and selective LC-MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 220-->135 for aniracetam and m/z 295-->205 for the IS. The assay exhibited a linear dynamic range of 0.2-100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.     

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.     

Sensitive quantification of rifaximin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-mass spectrometry method (LC-MS/MS) for the quantitative determination of rifaximin in human plasma was developed and validated. In the developed procedure, metoprolol was added to human plasma as an internal standard (IS) and acetonitrile was used to precipitate the plasma proteins before LC-MS/MS analysis. Chromatographic separation was obtained on a RESTEK Pinnacle C18 column (50 mm x 2.1mm, 5 microm) with a mobile phase consisted of ammonium acetate solution (15 mM, pH 4.32) as buffer A and methanol as mobile phase B. Quantification was performed in positive mode using multiple reaction monitoring (MRM) of the transitions m/z 786.1-->754.1 for rifaximin and m/z 268.3-->116.1 for the IS. The assay has been validated over the concentration range of 0.5-10 ng/ml (r=0.9992) based on the analysis of 0.2 ml of plasma. The assay accuracy was between 98.2% and 109%. The within-day and between-day precision was better than 3.9% and 8.9% at three concentration levels. The freeze-thaw stability was also investigated and it was found that both rifaximin and the IS were quite stable. This method provides a rapid, sensitive, specific and robust tool for the quantitative determination of rifaximin in human plasma, which is especially useful for the pharmacokinetic study of rifaximin.     

Simultaneous determination of dronedarone and its active metabolite debutyldronedarone in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

Dronedarone is a derivative of amiodarone--a popular antiarrhythmic drug. It was developed to overcome the limiting iodine-associated toxicities of amiodarone. Debutyldronedarone is a major circulating active metabolite of dronedarone in humans. To investigate the pharmacokinetics of dronedarone, a rapid, simple, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine dronedarone and debutyldronedarone in human plasma using amiodarone as internal standard (IS). Acetonitrile with IS was used to precipitate proteins from a 50-μL aliquot of plasma. Effective chromatographic separation was performed on a CAPCELL PAK C(18) MG (100 mm × 4.6 mm, 5 μm) column with gradient elution (5 mmol/L ammonium acetate-acetonitrile, with each phase containing 0.2% acetic acid) at a flow rate of 0.7 mL/min. Complete separation was achieved within 5.5 min. Detection was carried out on an tandem mass spectrometer in multiple reaction monitoring mode using a positive atmospheric pressure chemical ionization interface. A lower limit of quantification of 0.200 ng/mL was achieved for both dronedarone and debutyldronedarone, with acceptable precision and accuracy. The linear range of the method was from 0.200 to 200 ng/mL for each analyte. Intra- and inter-day precisions were lower than 7.2% in relation to relative standard deviation, while accuracy was within ±5.1% in terms of relative error for analytes. Our findings demonstrate the successful application of the validated LC-MS/MS method to a pharmacokinetic study after a single oral administration of 400mg dronedarone to six healthy volunteers.     

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.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of levosulpiride in human plasma

A rapid, sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric (HILIC-MS/MS) method for the determination of levosulpiride in human plasma was developed. Levosulpiride and internal standard, tiapride were extracted from human plasma with ethyl acetate at pH 11 and analyzed on an Atlantis HILIC silica column with the mobile phase of acetonitrile-ammonium formate (190 mM, pH 3.0) (94:6, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.999) over the concentration range of 1.00-200 ng/ml. The lower limit of quantification for levosulpiride was 1.00 ng/ml using 100 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at three quality control (QC) levels were 3.8-9.1 and -2.9 to -0.1%, respectively. The recoveries of levosulpiride ranged from 80.5 to 87.4%, with that of tiapride (internal standard) being 84.6%. This method was successfully applied to the pharmacokinetic study of levosulpiride in humans.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of hydroxysafflor yellow A in human plasma: application to a pharmacokinetic study

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of hydroxysafflor yellow A (HSYA) in human plasma. HSYA was extracted from human plasma by using solid-phase extraction technique. Puerarin was used as the internal standard. A Shim-pack VP-ODS C(18) (150mm x 4.6mm, 5 microm) column and isocratic elution system composing of methanol and 5mM ammonium acetate (80:20, v/v) provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 611.19-->491.19 for HSYA and m/z 415.19-->295.10 for puerarin. The proposed method has been validated with a linear range of 1-1000 ng/ml for HSYA with a correlation coefficient >/=0.999. The lower limit of quantitation was 1 ng/ml. The intra-batch and inter-batch precision and accuracy were within 10%. The average extraction recovery was 81.7%. The total run time was 5.5 min. The validated method was successfully applied to the study on pharmacokinetics of HSYA in 12 healthy volunteers after a single oral administration of safflower oral solution containing 140 mg of HSYA.     

Enantioselective determination of azelnidipine in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [(2)H(6)]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing alpha(1)-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583-->167 for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589-->167 for [(2)H(6)]-azelnidipine. The standard curve was linear over the studied range (0.05-20 ng/mL), with r(2)>0.997 using weighted (1/x(2)) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2-8.2% and 2.4-5.8% for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2-117.0% for (R)-(-)-azelnidipine and 100.0-107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine were 71.4-79.7% and 71.7-84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.