Determination of paeoniflorin in rat plasma by a liquid chromatography-tandem mass spectrometry method coupled with solid-phase extraction

A rapid, sensitive and selective liquid chromatography-tandem spectrometry method was developed and validated for determination of paeoniflorin in rat plasma using geniposide as the internal standard. The samples were pretreated with solid-phase extraction using Extract-Clean cartridges. Separation of paeoniflorin and IS was achieved on a reversed-phase C18 column (50x4.6 mm i.d.) with a mobile phase made up of acetonitrile and 0.05% formic acid (25:75, v/v) at a flow rate of 0.5 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by multiple-reaction monitoring and an electrospray ionization source was employed as the ionization source. The lower limit of quantification obtained was 4 ng/mL (n=6) using 200 microL plasma with an accuracy of -3.67% (relative error) and a precision of 4.13% (relative standard deviation). A good linearity was found in the range of 4-1000 ng/mL. The intra- and inter-day relative standard deviations in the measurement of quality control samples 10, 150 and 800 ng/mL ranged from 3.73 to 4.94% and from 4.31 to 6.56%, respectively. The accuracy was from -3.93 to -1.11% in terms of relative error. The analyte and IS were stable in the battery of stability studies. This method was successfully applied to a pharmacokinetic study of paeoniflorin after a single oral administration of 53.36 mg/kg paeoniflorin to rats.     

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).     

Simultaneous quantitation of d7-nefazodone, nefazodone, d7-hydroxynefazodone, hydroxynefazodone, m-chlorophenylpiperazine and triazole-dione in human plasma by liquid chromatographic-mass spectrometry

A rapid and sensitive LC-MS assay was developed and validated for the simultaneous determination of d7-nefazodone (d7-NEF), nefazodone (NEF), d7-hydroxynefazodone (d7-OH-NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (IS). A 0.1 mL aliquot of the plasma sample was precipitated with 0.1 mL of acetonitrile and vortexed for 2 min. After centrifugation, 50 microL of supernatant was mixed with 100 microL of 10 mM ammonium formate (pH = 4.0), and a 50 microL aliquot was injected onto a BDS Hypersil C18 column at a flow rate of 0.3 mL/min. The mobile phase consisting of 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, was used in an isocratic system. The mass spectrometer was programmed to admit the protonated molecules at m/z 477.2 (d7-NEF), 493.3 (d7-OH-NEF), 197.0 (mCPP), 372.0 (IS), 470.4 (NEF), 458.0 (Dione) and 486.2 (OH-NEF). Standard curves were linear (r(2) >/= 0.994) over the concentration range of 4-1000 ng/mL for Dione and 2-500 ng/mL for all other analytes. The lowest standard concentrations were the lower limits of quantitation for each analyte. The mean predicted quality control concentrations for all analytes deviated by less than 14.3% from the corresponding nominal values; the intra-assay and inter-assay precisions of the assay for all analytes were within 10.5% relative standard deviation. All analytes including the internal standard were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 79.2 to 109.1%. The validated assay was applied to the analysis of clinical samples obtained from a human subject who simultaneously received d7-NEF and NEF orally.     

Simultaneous determination and pharmacokinetic study of metformin and rosiglitazone in human plasma by HPLC-ESI-MS

A fast, sensitive and specific high-performance liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of metformin and rosiglitazone in human plasma. With phenformin as an internal standard, the analysis was carried out on a C(18) column (50 mm × 2.1 mm, 3.5 μm) using a mobile phase consisting of acetonitrile-10 mM ammonium acetate (20:80, v/v). The detection was performed by tandem mass spectrometry via electrospray ionization. Linear calibration curves were obtained in the concentration of 1.054-263.5 ng/mL for rosiglitazone and 4.040-5050 ng/mL for metformin. The method was applicable to clinical pharmacokinetic study of metformin and rosiglitazone in healthy volunteers following oral administration.     

Determination of lipoic acid in rat plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a pharamcokinetic study

A simple, sensitive and specific LC-MS/MS method for the determination of lipoic acid was developed and validated over the linearity range 5-1000 ng/mL (r2 > 0.99) with 200 microL rat plasma using rosigliatzone as an internal standard (IS). The assay procedure involved a simple one-step liquid-liquid extraction of lipoic acid and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Hichrom RPB column (4.6 x 250 mm, 5 microm). Separation of lipoic acid and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min. The API-3000 LC-MS/MS was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique. Positive and negative ion acquisition within the same chromatographic run was used in the present method. For lipoic acid a pseudo-molecular ion transition pair was acquired in negative polarity, whereas for IS the transition pair was acquired in positive polarity. Quantitation was determined for both analyte and IS in MRM scan mode. Absolute recovery of lipoic acid and IS was >70 and 97%, respectively. The lower limit of quantification (LLOQ) of lipoic acid was 5.0 ng/mL. The inter- and intra-day precision in the measurement of quality control (QC) samples 5, 15, 400 and 800 ng/mL were in the range 2.18-5.99% relative standard deviation (RSD) and 0.93-13.77% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.40-114.40% of the nominal values. Analyte and IS were stable in the battery of stability studies, viz. bench-top, auto-sampler and freeze-thaw cycles. Stability of lipoic acid was established for 1 month at -80 degrees C. The application of the assay to a pharmacokinetic study in rats confirmed the utility of the assay.     

A rapid and highly sensitive method for the determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry: application to a pre-clinical pharmacokinetic study

A sensitive and specific liquid chromatography-positive electrospray ionization-tandem mass spectrometry method has been developed and validated for the determination of glimepiride (GPD) in human plasma. GPD and the internal standard (IS, glibenclamide) were extracted from a small aliquot of human plasma (200 microL) by a simple liquid-liquid extraction technique using ethyl acetate as extraction solvent. The compounds were separated on a YMC Propack, C18, 4.6x50 mm column using a mixture of ammonium acetate buffer, acetonitrile and methanol (30:60:10, v/v) as mobile phase at 0.5 mL/min on an API 4000 Sciex mass spectrometer connected to an Agilent HPLC system. Method validation and pre-clinical sample analysis was performed as per FDA guidelines and the results met the acceptance criteria. GPD and IS were detected without any interference from human plasma matrix. The method was proved to be accurate and precise at linearity range of 0.02-100.00 ng/mL with a correlation coefficient of 0.999. The method was robust with a lower limit of quantitation of 0.02 ng/mL. Intra- and inter-day accuracies for GPD were 88.60-113.50 and 96.82-103.93%, respectively. The inter-day precision was better than 12.21%. This method enabled faster and reliable determination of GPD in a pre-clinical study.     

Simultaneous determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using ultra-performance liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive, and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method for the determination of udenafil and its active metabolite, DA-8164, in human plasma and urine using sildenafil as an internal standard (IS) was developed and validated. Udenafil, DA-8164 and IS from a 100 microL aliquot of biological samples were extracted by protein precipitation using acetonitrile. Chromatographic separation was carried on an Acquity UPLC BEH C(18) column (50 x 2.1 mm, i.d., 1.7 microm) with an isocratic mobile phase consisting of acetonitrile and containing 0.1% formic acid (75:25, v/v) at flow rate of 0.4 mL/min, and total run time was within 1 min. Detection and quantification was performed by the mass spectrometer using multiple reaction-monitoring mode at m/z 517 --> 283 for udenafil, m/z 406 --> 364 for DA-8164 and m/z 475 --> 100 for IS. The assay was linear over a concentration range of 1-600 ng/mL with a lower limit of quantification of 1 ng/mL in both human plasma and urine. The coefficient of variation of this assay precision was less than 13.7%, and the accuracy exceeded 92.0%. This method was successfully applied for pharmacokinetic study after oral administration of udenafil 100 mg to healthy Korean male volunteers.     

Determination of a selective Na+/H+ exchanger inhibitor, 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed for the determination of a selective Na(+)/H(+) exchanger inhibitor 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma. KR-33028 and the internal standard, linezolid, were extracted from rat plasma with ethyl acetate at neutral pH. The analytes were separated on an XBridge C(18) column with a mixture of methanol-0.1% formic acid (35:65, v/v) as mobile phase and detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.9998) over the concentration range of 2.0-1000 ng/mL. The coefficients of variation of intra- and inter-assay were 1.3-6.8% and the relative error was 0.8-5.0%. The recoveries of KR-33028 and linezolid were 70.5 and 84.6%, respectively. The lower limit of quantification for KR-33028 was 2.0 ng/mL using 50 microL plasma sample. This method was successfully applied to the pharmacokinetic study of KR-33028 in rats.     

Selective and sensitive determination of bis(7)-tacrine, a high erythrocyte binding acetylcholinesterase inhibitor, in rat plasma by high-performance liquid chromatography-tandem mass spectrometry

The current study aims to develop a specific and sensitive LC-MS/MS method for determination of bis(7)-tacrine (B7T) in rat plasma. A 100 microL plasma sample was extracted with ethyl acetate. B7T and the internal standard (IS), pimozide, in the samples were then analyzed with LC-MS/MS in positive electrospray ionization condition. Chromatographic separation of B7T and IS was achieved in a C(18) reversed-phase HPLC column (150 x 2.1 mm i.d.) by isocratic elution with a mobile phase consisting of 0.05% formic acid in water and acetonitrile (1:1, v/v) at a flow rate of 0.35 mL/min. Multiple-reaction monitoring (MRM) mode was employed to measure the ion transitions: m/z 247 to 197 for B7T and m/z 462 to m/z 328 for IS, respectively. The method was linear over the studied ranges of 100-5000 and 10-100 ng/mL. The intra-day and inter-day variations of the analysis were less than 6.8% with standard errors less than 9.0%. The detection limit of B7T in rat plasma was 1 ng/mL. The developed method was successfully applied to the pharmacokinetic study of B7T after intravenous administration of 1 mg/kg B7T and further proved to be readily utilized for determination of B7T in rat plasma samples.     

The use of liquid chromatography/mass spectrometry for quantitative analysis of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue

Sensitive and reproducible methods for the determination of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue by liquid chromatography/mass spectrometry are described. Deuterated analogs of the substances were used as internal standards. Samples in Ringer solution were analyzed by direct injection of 10 microL Ringer solution diluted by an equal volume of water. The limit of quantification was 0.5 ng/mL and the method was linear in the range of 0.5-150 ng/mL for all substances. To analyze oxycodone and oxymorphone in rat plasma, 50 microL of plasma were precipitated with acetonitrile, and the supernatant was directly injected onto the column. To analyze oxycodone, oxymorphone and noroxycodone in rat plasma, 100 microL of rat plasma were subjected to a C18 solid-phase extraction (SPE) procedure, before reconstituting in mobile phase and injection onto the column. For both methods the limit of quantification in rat plasma was 0.5 ng/mL and the methods were linear in the range of 0.5-250 ng/mL for all substances. To analyze the content of oxycodone, oxymorphone and noroxycodone in rat brain tissue, 100 microL of the brain homogenate supernatant were subjected to a C18 SPE procedure. The limit of quantification of oxycodone was 20 ng/g brain, and for oxymorphone and noroxycodone 4 ng/g brain, and the method was linear in the range of 20-1000 ng/g brain for oxycodone and 4-1000 ng/g brain for oxymorphone and noroxycodone. All methods utilized a mobile phase of 5 mM ammonium acetate in 45% acetonitrile, and a SB-CN column was used for separation. The total run time of all methods was 9 min. The intra-day precision and accuracy were <11.3% and <+/-14.9%, respectively, and the inter-day precision and accuracy were <14.9% and <+/-6.5%, respectively, for all the concentrations and matrices described.     

Solid phase extraction of oxcarbazepine and its metabolites from plasma for analysis by high performance liquid chromatography.

A rapid, sensitive and simple-to-operate high performance liquid chromatographic method for the simulataneous determination of oxcarbazepine, 10-hydroxycarbazepine and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octadecylsilane bonded-silica columns (Bond Elut C18, 1 mL capacity). Chromatographic separation of oxcarbazepine and its metabolites was achieved using a mobile phase consisting of acetonitrile/methanol/water (13:25:62 by volume) at a flow rate of 1.2 mL/min in conjunction with a Waters Associates Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a LiChrospher 5 microns C18 guard column. By measuring the UV absorbance at 214 nm, plasma levels in the region of 50-100 ng/mL for the drug and its metabolites can be detected with only 100 microL of plasma. The method has been applied to pharmacokinetic studies of oxcarbazepine and its metabolites in children with epilepsy; preliminary pharmacokinetic findings in two patients at steady-state are presented.     

High-performance liquid chromatography analysis of curcumin in rat plasma: application to pharmacokinetics of polymeric micellar formulation of curcumin

A simple, rapid and reliable high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of curcumin in rat plasma. Plasma was precipitated with acetonitrile after addition of the internal standard (IS), 4-hydroxybenzophenone. Separation was achieved on a Waters muBondapak C(18) column (3.9 x 300 mm, 5 microm) using acetonitrile (55%) and citric buffer, pH 3.0 (45%) as the mobile phase (flow rate = 1.0 mL/min). The UV detection wavelength was 300 and 428 nm for IS and curcumin, respectively. The extraction efficiencies were 97.08, 95.69 and 94.90% for 50, 200 and 1000 ng/mL of curcumin in rat plasma, respectively. The calibration curve was linear over the range 0.02-1 microg/mL with a correlation coefficient of r(2) > 0.999. The intra- and inter-day coefficients of variation were less than 13%, and mean intra- and inter-day errors were less than +/-6% at 50, 200 and 1000 ng/mL of curcumin. This assay was successfully applied to the pharmacokinetic studies of both solubilized curcumin and its polymeric micellar formulation in rats. It was found that polymeric micelles increased the half-life of curcumin 162-fold that of solubilized curcumin and increased the volume of distribution (Vd(ss)) by 70-fold.     

A rapid HPLC method with fluorometric detection for determination of plasma itraconazole and hydroxy-itraconazole concentrations in cystic fibrosis children with allergic bronchopulmonary aspergillosis

The development and validation of a simple, rapid and selective high-performance liquid chromatography (HPLC) method is described for the quantitation of itraconazole and hydroxy-itraconazole in 100 microL of plasma from a paediatric population. The mobile phase of methanol (75% v/v) and water (25% v/v) was pumped at 1 mL/min through a C18 Symmetry (3.9 mm i.d. x 150 mm) cartridge. Using a protein-precipitation method, 100 microL internal standard (IS) solution (R051012, 555 microg/L in acetonitrile) were added to 100 microL of plasma followed by 10 microL zinc sulphate solution (20% w/v). Itraconazole, hydroxy-itraconazole and IS eluted at 4.7, 8.3 and 12.5 min, respectively and were detected fluorometrically at 250 nm (excitation) and 380 nm (emission). Recoveries were 87.1-96.7%. Calibrations in drug-free plasma were linear (r2 > 0.99) from 50 to 2000 microg/L, using 1/c2 (c = concentration) weighting. Intraday and interday imprecision (CV%) was 4.8-17.3 and 6.3-16.6% for itraconazole, and 4.6-17.9 and 7.02-18.4% for hydroxy-itraconazole. Inaccuracy was -7.1 to -14.7% for itraconazole and -0.1 to -9.7% for hydroxy-itraconazole. The clinical application of this method was demonstrated by measurement of itraconazole and hydroxy-itraconazole in plasma samples drawn from paediatric cystic fibrosis patients, who were prescribed itraconazole for treatment of allergic bronchopulmonary aspergillosis.     

Quantitative analysis of D-24851, a novel anticancer agent, in human plasma and urine by liquid chromatography coupled with tandem mass spectrometry

The development of a liquid chromatography/tandem mass spectrometric assay for the quantitative analysis of the novel tubulin inhibitor D-24851 in human plasma and urine is described. D-24851 and the deuterated internal standard were extracted from 250 microL of plasma or urine using hexane/ether (1:1, v/v). Subsequently, 10-microL aliquots of reconstituted extracts were injected onto an Inertsil ODS analytical column (50 x 2.0 mm i.d., 5 microm particle size). An eluent consisting of methanol/5 mM ammonium acetate, 0.004% formic acid in water (80:20, v/v) was pumped at a flow rate of 0.2 mL/min. An API 365 triple quadrupole mass spectrometer was used in the multiple reaction monitoring mode for sensitive detection. For human plasma a dynamic range of 1-1000 ng/mL was validated, and for human urine a range of 0.25-50 ng/mL. Validation was performed according to the most recent FDA guidelines and all results were within requirements. The assay has been successfully applied to support a phase I clinical trial with orally administered D-24851.     

Direct determination of five fluoroquinolones in chicken whole blood and in veterinary drugs by HPLC

A direct, accurate, and sensitive chromatographic analytical method for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in chicken whole blood is proposed in the present study. For quantitative determination lamotrigine was used as internal standard at a concentration of 20 ng/microL. The developed method was successfully applied to the determination of enrofloxacin, as the main component of commercially available veterinary drugs. Fluoroquinolone antibiotics were separated on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v) leading to retention times less than 14 min, at a flow rate 1.4 mL min(-1). UV detection at 275 nm provided limits of detection of 2 ng/mL per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 0.4 ng/mL for ofloxacin, and 4 ng/mL for enrofloxacin. Preparation of chicken blood samples is based on the deproteinization with acetonitrile while the pharmaceutical drug was simply diluted with water. Peaks of examined analytes in real samples were identified by means of a photodiode array detector. The method was validated in terms of within-day (n=6) precision and accuracy after chicken whole blood sample deproteinization by CH3CN. Using 50 microL of chicken blood sample, recovery rates at fortification levels of 40, 60, and 80 ng ranged from 86.7% to 103.7%. The applicability of the method was evaluated using real samples from chicken under fluoroquinolone treatment.     

Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.     

High performance liquid chromatographic-mass spectrometric assay for the quantitation of BMS-204352 in dog K(3)EDTA plasma

A high performance liquid chromatographic-mass spectrometric (LC/MS) assay was developed and validated for the determination of BMS-204352 in dog K(3)EDTA plasma. A 0.5 mL aliquot of control plasma was spiked with BMS-204352 and internal standard (IS) and buffered with 1 mL of 5 mM ammonium acetate. The mixture was then extracted with 3 mL of toluene. After separation and evaporation of the organic phase to dryness using nitrogen at 40 degrees C, the residue was reconstituted in the mobile phase and 25 microL of the sample were injected onto a Hypersil C(18) column (2 x 50 mm; 3 microm) at a flow rate of 0.5 mL/min. The mobile phase was consisted of two solvent mixtures (A and B). Solvent A was composed of 5 mM ammonium acetate and 0.1% triethylamine in 75:25 v/v water:methanol, pH adjusted to 5.5 with glacial acetic acid, and solvent B was 5 mM ammonium acetate in methanol. A linear gradient system was used to elute the analytes. The mass spectrometer was programmed to admit the de-protonated molecules at m/z 352.7 (IS) and m/z 357.9 (BMS-204352). Standard curves of BMS-204352 were linear (r(2) > or = 0.998) over the concentration range of 0.5-1000 ng/mL. The mean predicted quality control (QC) concentrations deviated less than 5.1% from the corresponding nominal values (ie 4, 80, 400 and 2000 ng/mL); the within- and between-assay precision of the assay were within 5.5% relative standard deviation. Stability of BMS-204352 was confirmed after at least three freeze/thaw cycles and BMS-204532 was stable in dog plasma when stored frozen at or below -20 degrees C for at least 16 weeks in spiked QC samples and for at least 4 1/2 weeks for in vivo study samples. BMS-204352 and IS were stable in the injection solvent at room temperature for at least 24 h. The assay was applied to delineate the pharmacokinetic disposition of BMS-204352 in dogs following a single intravenous dose administration. In conclusion, the assay is accurate, precise, specific, sensitive and reproducible for the pharmacokinetic analysis of BMS-204532 in dog plasma.     

A rapid LC-MS/MS method for quantitation of eszopiclone in human plasma: application to a human pharmacokinetic study

A highly reproducible, specific and cost-effective LC-MS/MS method was developed for simultaneous estimation of eszopiclone (ESZ) with 50 μL of human plasma using paroxetine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract ESZ and IS from human plasma. The total run time was 1.5 min and the elution of ESZ and IS occurred at 0.90 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (15:85, v/v) at a flow rate of 0.50 mL/min on a Discover C(18) (50 × 4.6 mm, 5 μm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for ESZ. A linear response function was established for the range of concentrations 0.10-120 ng/mL (r > 0.998) for ESZ. The intra- and inter-day precision values for ESZ were acceptable as per FDA guidelines. Eszopiclone was stable in the battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Simultaneous quantification of fexofenadine and pseudoephedrine in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: method development, validation and application to a clinical study

To support the pharmacokinetic and bioavailability study of a once-daily fexofenadine/pseudoephedrine combination, a high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous quantification of fexofenadine and pseudoephedrine was developed and validated with 500 microL human plasma using mosapride as an internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 502/466 for fexofenadine, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited linear dynamic ranges of 1-500 ng/mL and 2-1000 ng/mL for fexofenadine and pseudoephedrine, respectively, in human plasma. The lower limits of quantification were 1 and 2 ng/mL with a relative standard deviation of less than 10% for fexofenadine and pseudoephedrine, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time was 2 min and more than 400 human plasma samples could be analyzed in one day by running the system overnight. The method is precise and sensitive enough for its intended purpose.     

Sensitive and rapid method to determine trimetazidine in human plasma by liquid chromatography/tandem mass spectrometry

A simple, sensitive, and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method, using electrospray ionization, was developed and validated to quantify trimetazidine in human plasma using propranolol hydrochloride as an internal standard (IS). Samples were prepared by solid-phase extraction and analyzed without drying and reconstitution. The analyte and IS were chromatographed on a C18 reversed-phase column under isocratic conditions using 2 mM ammonium acetate (pH 3.5)-acetonitrile (40 + 60, v/v) as the mobile phase with a run time of 2.0 min. Quantitation was done on a triple-quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring mode to detect parent --> product ion (m/z 267.2 --> 181.4) transition. The method was validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, and stability. Linearity in plasma was observed over the concentration range of 1.5-300 ng/mL. Lower limit of quantification achieved was 1.5 ng/mL with precision < 10% using 10 microL injection volume. The mean relative recovery of analyte (97.36%) and IS (99.93%) was consistent and reproducible. Interbatch and intrabatch precision was < 8.0% and the accuracy determined was within +/- 8% in terms of relative error.