Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous determination of venlafaxine and its active metabolite O-desmethyl venlafaxine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous quantification of venlafaxine (VEN) and O-desmethyl venlafaxine (ODV) in human plasma. The analytes were extracted from human plasma by using solid-phase extraction (SPE) technique. Escitalopram (ESC) was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair has been followed as m/z 278.27-->121.11 for VEN, m/z 264.28-->107.10 for ODV and m/z 325.00-->262.00 for ESC. The method involves a solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated with linear range of 3-300 ng/ml for VEN and 6-600 ng/ml for ODV. The intrarun and interrun precision and accuracy values are within 10%. The overall recoveries for VEN and ODV were 95.9 and 81.7%, respectively. Total elution time as low as 3 min only.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of rivastigmine 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 rivastigmine in human plasma. Rivastigmine was extracted from human plasma by using solid-phase extraction technique. Zolpidem was used as the internal standard. A Betabasic-8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 251.20-->206.10, 86.20 for rivastigmine and m/z 308.10-->235.10 for zolpidem. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatographic conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.2-20.0 ng/ml with a correlation coefficient > or =0.9988. The intra-run and inter-run precision and accuracy were within 10.0%. The overall recoveries for rivastigmine and zolpidem were 86.28% and 87.57%, respectively. The total run time was 2.0 min. The developed method was applied for the determination of the pharmacokinetic parameters of rivastigmine following a single oral administration of a 3 mg rivastigmine capsule in 20 healthy male volunteers.     

Development and validation of a liquid chromatography-tandem mass spectrometric assay for pitavastatin and its lactone in human plasma and urine

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with electrospray ionization (ESI) was developed and validated for the simultaneous determination of pitavastatin and its lactone in human plasma and urine. Following a liquid-liquid extraction, both the analytes and internal standard racemic i-prolact were separated on a BDS Hypersil C(8) column, using methanol-0.2% acetic acid in water (70: 30, v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 422.4-->m/z 290.3 for pitavastatin, m/z 404.3-->m/z 290.3 for pitavastatin lactone and m/z 406.3-->m/z 318.3 for the internal standard, respectively. Linear calibration curves of pitavastatin and its lactone were obtained in the concentration range of 1-200 ng/ml, with a lower limit of quantitation of 1 ng/ml. The intra- and inter-day precision values were less than 4.2%, and accuracies were between -8.1 and 3.5% for both analytes. The proposed method was utilized to support clinical pharmacokinetic studies of pitavastatin in healthy subjects following oral administration.     

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

Simultaneous determination of metoprolol succinate and amlodipine besylate in human plasma by liquid chromatography-tandem mass spectrometry method and its application in bioequivalence study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol succinate (MPS) and amlodipine besylate (AM) using hydrochlorothiazide (HCTZ) as IS in human plasma. Both the drugs were extracted by simple liquid-liquid extraction with chloroform. The chromatographic separation was performed on a reversed-phase peerless basic C18 column with a mobile phase of methanol-water containing 0.5% formic acid (8:2, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 1-100ng/ml for MPS and 1-15ng/ml AM in human plasma. The MRM transition of m/z 268.10-103.10, m/z 409.10-334.20 and m/z 296.00-205.10 were used to measure MPS, AM and HCTZ (IS), respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of MPS and AM formulation product after an oral administration to Indian healthy human volunteers.     

Sensitive quantification of ranolazine in human plasma by liquid chromatography--tandem mass spectrometry with positive electrospray ionization

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.     

Determination of etoricoxib in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionisation

The validation of a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of the selective cyclooxygenase-2 inhibitor etoricoxib in human plasma with phenazone as internal standard is described. The plasma samples were extracted by solid-phase extraction using polymer-based cartridges. Chromatography was carried out on a short, narrow bore RP C(18) column (30x2 mm). Detection was achieved by a Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source working in positive ion mode. The respective mass transitions used for quantification of etoricoxib and phenazone were m/z 359.2-->280.2 and m/z 189.0-->104.1. The analytical method was validated over the concentration range 0.2-200 ng/ml. The limit of quantification was 0.2 ng/ml. The method is applicable to pharmacokinetic studies in humans.     

A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of bisoprolol in human plasma using multiplexing technique

A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of bisoprolol in human plasma using multiplexing technique (two HPLC units connected to one MS). Bisoprolol was extracted from human plasma using solid-phase extraction technique using metoprolol as internal standard. A Betabasic 8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 326.2-->116.1 for bisoprolol and m/z 268.2-->191.0 for metoprolol. The method involves a simple multiplexing, rapid solid-phase extraction, simple isocratic chromatography conditions and mass spectrometric detection which enable detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.5-70.0 ng/mL with correlation coefficient > or =0.9991. The precision and accuracy were within 10% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for bisoprolol and metoprolol were 93.89% and 77.65%, respectively. Total MS run time was 0.90 min only. The developed method was applied for the determination of pharmacokinetic parameters of bisoprolol following a single oral administration of a 10mg bisoprolol tablet in 18 healthy male volunteers.     

Simultaneous determination of fixed dose combination of nebivolol and valsartan in human plasma by liquid chromatographic-tandem mass spectrometry and its application to pharmacokinetic study

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometry method is described for the simultaneous determination of nebivolol and valsartan in human plasma. Nebivolol and valsartan were extracted from plasma using acetonitrile and separated on a C18 column. The mobile phase consisting of a mixture of acetonitrile and 0.05 mM formic acid (50:50 v/v, pH 3.5) was delivered at a flow rate of 0.25 ml/min. Atmospheric pressure ionization (API) source was operated in both positive and negative ion mode for nebivolol and valsartan, respectively. Selected reaction monitoring mode (SRM) using the transitions of m/z 406.1-->m/z 150.9; m/z 434.2-->m/z 179.0 and m/z 409.4-->m/z 228.1 were used to quantify nebivolol, valsartan and internal standard (IS), respectively. The linearity was obtained over the concentration range of 0.01-50.0 ng/ml and 1.0-2000.0 ng/ml and the lower limits of quantitation were 0.01 ng/ml and 1.0 ng/ml for nebivolol and valsartan, respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of nebivolol and valsartan formulation product after an oral administration to healthy human subjects.     

Simultaneous determination of metformin and rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: application to a pharmacokinetic study

A selective and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin and rosiglitazone in human plasma using phenformin as internal standard (IS) has been first developed and validated. Plasma samples were precipitated by acetonitrile and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A (150 mm x 2.0 mm I.D.) column using a mobile phase comprised of methanol:30 mM ammonium acetate pH 5.0 (80:20, v/v) delivered at 0.2 ml/min. Detection was performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in positive ion selected reaction monitoring (SRM) mode using electrospray ionization. The ion transitions monitored were m/z 130.27-->71.11 for metformin, m/z 358.14-->135.07 for rosiglitazone and m/z 206.20-->105.19 for the IS. The standard curves were linear (r(2)>0.99) over the concentration range of 5-3000 ng/ml for metformin and 1.5-500 ng/ml for rosiglitazone with acceptable accuracy and precision, respectively. The within- and between-batch precisions were less than 15% of the relative standard deviation. The limit of detection (LOD) of both metformin and rosiglitazone was 1 ng/ml. The method described is precise and sensitive and has been successfully applied to the study of pharmacokinetics of compound metformin and rosiglitazone capsules in 12 healthy Chinese volunteers.     

Determination of duloxetine in human plasma by liquid chromatography with atmospheric pressure ionization-tandem mass spectrometry and its application to pharmacokinetic study

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometry (LC-MS-MS) method is described for the determination of duloxetine in human plasma. Duloxetine was extracted from plasma using methanol and separated on a C18 column. The mobile phase consisting of a mixture of acetonitrile and 5mM ammonium acetate (45:55, v/v, pH 3.5) was delivered at a flow rate of 0.3 ml/min. Atmospheric pressure ionization (API) source was operated in positive ion mode. Multiple reaction monitoring (MRM) mode using the transitions of m/z 298.1-->m/z 44.0 and m/z 376.2-->m/z 123.2 were used to quantify duloxetine and internal standard (I.S.), respectively. The linearity was obtained over the concentration range of 0.1-50.0 ng/ml and the lower limit of quantitation (LLOQ) was 0.1 ng/ml. This method was successfully applied to pharmacokinetic study of a duloxetine formulation product after oral administration to healthy human subjects.     

Liquid chromatography tandem mass spectrometry method for simultaneous determination of metoprolol tartrate and ramipril in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol tartrate (MT) and ramipril, in human plasma. Both the drugs were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v). The chromatographic separation was performed on a reversed-phase C8 column with a mobile phase of 10 mM ammonium formate-methanol (3:97, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 5-500 ng/ml for metoprolol and ramipril in human plasma. The precursor to product ion transitions of m/z 268.0-103.10 and m/z 417.20-117.20 were used to measure metoprolol and ramipril, respectively.     

Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study

A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.     

Liquid chromatography-tandem mass spectrometry method for determination of N-acetylglucosamine concentration in human plasma

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of N-acetylglucosamine in human plasma. Plasma samples were pretreated with acetonitrile for protein precipitation. The chromatographic separation was performed on Hypersil Silica column (150mmx2mm, 5microm). The deprotonated analyte ion was detected in negative ionization mode by multiple reaction monitoring mode. The mass transition pairs of m/z 220.3-->118.9 and m/z 226.4-->123.2 were used to detect N-acetylglucosamine and internal standard 13C6-N-acetylglucosamine, respectively. The assay exhibited a linear range from 20 to 1280ng/ml for N-acetylglucosamine in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully applied to analyze human plasma samples in a pharmacokinetic study.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid determination of omeprazole in human plasma by protein precipitation and liquid chromatography-tandem mass spectrometry

A rapid, sensitive and reliable method was developed to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The assay is based on protein precipitation with acetonitrile and reversed-phase liquid chromatography performed on an octadecylsilica column (55 mm x 2mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (60:40, v/v). Omeprazole and flunitrazepam, the internal standard, elute at 0.80+/-0.10 min with a total run time 1.35 min. Quantification was through positive ion mode and selected reaction monitoring mode at m/z 346.1-->197.9 for omeprazole and m/z 314.0-->268.0 for flunitrazepam, respectively. The lower limit of quantitation was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 12%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Development of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of flurogestone acetate in ovine plasma

Flurogestone (FGA) is a synthetic progesterone, with a progestational action higher than that of progesterone itself. It is intended for vaginal use in large animals to induce oestrus synchronization. A quantitative method for the analysis of flurogestone acetate (FGA) in ovine plasma by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed. After the incorporation of megestrol acetate (MGA) as internal standard (IS) and followed by a liquid-liquid extraction from plasma, FGA and MGA were chromatographed using a reverse-phase HPLC column and detected by tandem mass spectrometry with a TurboIonSpray source. Multiple reaction-monitoring (MRM) mode was used for the quantitative determination of FGA in ovine plasma. The precursor ions [M+H](+) at m/z 407.2 and 385.1 for FGA and MGA, respectively, produced product ions at m/z 267.1/285.1 for FGA and m/z 267.1/224.0 for MGA. The validated concentration range was 0.2-5.0 ng/ml based on 500 microl plasma aliquots. The lower limit of quantitation was 0.2 ng/ml. Fully validated selectivity, accuracy, precision and reproducibility criteria for routine use in pharmacokinetic studies were demonstrated.     

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.     

Determination of carbamazepine and carbamazepine 10,11-epoxide in human plasma by tandem liquid chromatography-mass spectrometry with electrospray ionisation

A sensitive method for the determination of carbamazepine and carbamazepine 10,11-epoxide in plasma is described, using high-performance liquid chromatographic separation with tandem mass spectrometry. Samples were purified using liquid-liquid extraction and separated on a Phenomenex Luna C18 5 microm. 150 x 2 mm column with a mobile phase consisting of acetonitrile, methanol and formic acid (0.1%) (10:70:20, v/v). Detection was performed by a Micromass Quattro Ultima mass spectrometer in the MRM mode (LC-MS-MS) using electro spray ionisation (ESI+), monitoring the transition of the protonated molecular ion for carbamazepine at m/z 237.05 and carbamazepine 10,11-epoxide at m/z 253.09 to the predominant ions of m/z 194.09 and 180.04, respectively. The mean recovery was 95% for carbamazepine and 101% for carbamazepine 10,11-epoxide, with a lower limit of quantification of 0.722 ng/ml for carbamazepine and 5.15 ng/ml for carbamazepine 10,11-epoxide, when using 0.5 ml plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.