Quantitation of iptakalim in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using sildenafil as internal standard. Sample preparation involved liquid-liquid extraction with dichloromethane-diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile-water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5-100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range +/-5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.     

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

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

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

This paper describes a simple, robust and cost-effective assay for the determination of ecabet in human plasma. After a simple step of protein precipitation using methanol, plasma samples were analyzed by reverse phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) with valsartan as the internal standard (I.S.). Ecabet and the I.S. valsartan were separated on a Venusil MP C18 analytical column using methanol-10mM ammonium acetate (75:25, v/v, pH 3.0) as mobile phase at a flow rate of 1.0 mL/min. Ecabet and I.S. were eluted at 0.91 and 0.92 min, respectively, ionized in negative mode, and then detected by multiple reaction monitoring (MRM) essay. The MRM transitions of m/z 379.1-->m/z 277.1 and m/z 434.3-->m/z 350.1 were used to quantify ecabet and I.S., respectively. The assay was linear over the concentration range of 10-6000 ng/mL and was successfully applied to a pharmacokinetic study in healthy volunteers.     

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

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

Quantification of zolmitriptan in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of zolmitriptan in human plasma. After the addition of the internal standard (IS) and 1.0 M sodium hydroxide solution, plasma samples were extracted with methylene chloride:ethyl acetate mixture (20:80, v/v). The organic layer was evaporated under a stream of nitrogen at 40 degrees C. The residue was reconstituted with 100 microl mobile phase. The compounds were separated on a prepacked Lichrospher CN (5 microm, 150 mm x 2.0 mm) column using a mixture of methanol:water (10 mM NH(4)AC, pH 4.0) = 78:22 as mobile phase. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 0.30-16.0 ng/ml with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (R.S.D.%) were lower than 15% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.30 ng/ml. The proposed method enables the unambiguous identification and quantification of zolmitriptan for pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid determination of tafenoquine in small volume human plasma samples by high-performance liquid chromatography-tandem mass spectrometry

A method was developed for the determination of tafenoquine (I) in human plasma using high-performance liquid chromatography-tandem mass spectrometry. Prior to analysis, the protein in plasma samples was precipitated with methanol containing [2H3(15N)]tafenoquine (II) to act as an internal standard. The supernatant was injected onto a Genesis-C18 column without any further clean-up. The mass spectrometer was operated in the positive ion mode, employing a heat assisted nebulisation, electrospray interface. Ions were detected in multiple reaction monitoring mode. The assay required 50 microl of plasma and was precise and accurate within the range 2 to 500 ng/ml. The average within-run and between-run relative standard deviations were < 7% at 2 ng/ml and greater concentrations. The average accuracy of validation standards was generally within +/- 4% of the nominal concentration. There was no evidence of instability of I in human plasma following three complete freeze-thaw cycles and samples can safely be stored for at least 8 months at approximately -70 degrees C. The method was very robust and has been successfully applied to the analysis of clinical samples from patients and healthy volunteers dosed with I.     

Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap LC-MS/MS system with an ESI interface. The assay was linear over the range 0.5-600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9-111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.     

Determination of solifenacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of tiropramide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of tiropramide in human plasma was developed. Tiropramide and internal standard, cisapride were extracted from human plasma by liquid-liquid extraction and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10mM, pH 4.5) (50:50, v/v). The analytes was detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.998) over the concentration range of 2.0-200 ng/ml. The intra- and inter-assay coefficients of variation ranged from 2.8 to 7.8 and 6.7 to 8.9%, respectively. The recoveries of tiropramide ranged from 50.2 to 53.1%, with that of cisapride (internal standard) being 60.9+/-5.3%. The lower limit of quantification for tiropramide was 2.0 ng/ml using 100 microl plasma sample. This method was applied to the pharmacokinetic study of tiropramide in human.     

Quantification of isosorbide 5-mononitrate in human plasma by liquid chromatography-tandem mass spectrometry using atmospheric pressure photoionization

Isosorbide 5-mononitrate (5-ISMN) is an organic nitrate widely used for its vasodilating properties in the treatment of angina pectoris. In the present study, an efficient, sensitive, robust method was developed for the determination and quantification of isosorbide 5-mononitrate, in human plasma, by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), using photospray ionization. Isosorbide 5-mononitrate was extracted from 0.5 mL human plasma by liquid-liquid extraction (LLE). The method had a chromatographic run of 2.0 min using a C(8) analytical column (100 mm x 2.1 mm i.d.) and the linear calibration curve over the range was linear from 20 to 2000 ng mL(-1) (r(2)>0.995). The between-run precision, based on the relative standard deviation replicate quality controls, was 7.9% (60 ng mL(-1)), 5.2% (300 ng mL(-1)) and 7.0% (1800 ng mL(-1)). The between-run accuracy was 94.9%, 94.1% and 88.8% for the above-mentioned concentrations, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of isosorbide 5-mononitrate 40 mg.     

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry

Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography-tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1-250 pg per 50 microL. The recoveries of fat-soluble vitamins were 91-105%. Inter-assay CV values of each vitamin were 1.9-11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, alpha-tocopherol, phylloquinone and menaquinone-4 were 0.455 microg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 microg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n=82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.     

Quantification of lipoic acid in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of lipoic acid (LA) in human plasma. LA and the internal standard, naproxen, were extracted from a 500 microl plasma sample by one-step deproteination using acetonitrile. Chromatographic separation was performed on a Zorbax SB-C(18) Column (100 mmx3.0mm i.d. with 3.5 microm particle size) with the mobile phase consisting of acetonitrile and 0.1% acetic acid (pH 4, adjusted with ammonia solution) (65:35, v/v), and the flow rate was set at 0.3 ml/min. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method was linear over the concentration range of 5-10,000 ng/ml for LA. The intra- and inter-day precisions were less than 7% and accuracy ranged from -7.87 to 9.74% at the LA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around time. The method has been successfully applied to a clinical pharmacokinetic study of LA in 10 healthy subjects.     

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.     

Quantitative determination of buagafuran in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of buagafuran in human plasma. The analyte was extracted from plasma samples with hexane after addition of isotopic internal standard and chromatographed on a RP-C(8) column. The mobile phase consisted of methanol-water (90:10, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.5-200 ng/mL. Inter- and intra-day precision (RSD%) were all within 15% and the accuracy (RE%) was equal or lower than 9.5%. The lower limit of quantitation (LLOQ) was 0.5 ng/mL. The extraction recovery was on average 38.1% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of buagafuran in healthy Chinese volunteers.     

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

A rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for quantification of cyclizine and its main metabolite norcyclizine in human plasma. Samples were prepared by protein precipitation with acetonitrile and cinnarizine was used as internal standard (recovery >87%). The analytes were eluted from a C8 50 mm×2.0 mm analytical column using a linear gradient of methanol and 0.05% formic acid with a total analysis time of 4 min. Analytes were detected by MS/MS using electrospray ionisation in the positive mode with multiple reactions monitoring (MRM) of the precursor ion/product ion transitions 267.2/167.2 for cyclizine and 253.2/167.2 for norcyclizine. Matrix effects were negligible. Standard curves for cyclizine and norcyclizine were linear (r(2)≥0.996) over the range 2-200 ng/mL, with 2 ng/mL representing the lower limit of quantification. Relative standard deviations were <14% for intra- and inter-day precision and the accuracy was within ±8%. The assay was successfully applied to a clinical study.     

Quantification of dimethyl-ifosfamide and its N-deschloropropylated metabolites in mouse plasma by liquid chromatography-tandem mass spectrometry

Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N(2)- and N(3)-deschloropropyl-dimethylifosfamide (N(2)-DCP-diMeIFO and N(3)-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289→168 for diMeIFO, m/z 213→168 for N(2)-DCP-diMeIFO, m/z 213→92 for N(3)-DCP-diMeIFO and m/z 261→154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20-10,000ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N(2)-DCP-diMeIFO, N(3)-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 μL plasma. The method was accurate with calculated bias from -5.8 to 4.0% for diMeIFO, from -1.1 to 10.6% for N(2)-DCP-diMeIFO and from -6.9 to 9.8% for N(3)-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice.     

Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry

Determination of quantitative changes in the pattern of serum bile acids is important for the monitoring of diseases affecting bile acid metabolism. A sensitive and specific high-performance liquid chromatography (HPLC)-MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)-MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%. The developed method was applied to a group of 21 healthy volunteers and preliminary reference intervals in serum were established. In patients with drug-induced cholestasis, an elevation of primary bile acids has been shown.     

Determination of mildronate in human plasma and urine by liquid chromatography-tandem mass spectrometry

A sensitive and selective analytical method based on liquid chromatography-triple-quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r=0.9998 for plasma sample and r=0.9999 for urine sample) were obtained in the range of 1.0-20,000 ng ml(-1) for mildronate. The detection limit was 1 ng ml(-1). Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC-MS-MS analysis of mildronate in plasma and urine.     

Quantitative determination of ondansetron in human plasma by enantioselective liquid chromatography-tandem mass spectrometry

A sensitive and enantioselective method was developed and validated for the determination of ondansetron enantiomers in human plasma using enantioselective liquid chromatography-tandem mass spectrometry. The enantiomers of ondansetron were extracted from plasma using ethyl acetate under alkaline conditions. HPLC separation was performed on an ovomucoid column using an isocratic mobile phase of methanol-5 mM ammonium acetate-acetic acid (20:80:0.02, v/v/v) at a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 294-->170 for ondansetron enantiomers, and m/z 285-->124 for tropisetron (internal standard). The method was linear in the concentration range of 0.10-40 ng/mL for each enantiomer using 200 microL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.10 ng/mL. The intra- and inter-assay precision was 3.7-11.6% and 5.6-12.3% for R-(-)-ondansetron and S-(+)-ondansetron, respectively. The accuracy was 100.4-107.1% for R-(-)-ondansetron and 103.3-104.9% for S-(+)-ondansetron. No chiral inversion was observed during the plasma storage, preparation and analysis. The method was successfully applied to characterize the pharmacokinetic profiles of ondansetron enantiomers in healthy volunteers after an intravenous infusion of 8 mg racemic ondansetron.     

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.