Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5 > 166.1 for itopride and m/z 342.3 > 111.6 for IS, respectively. Analytes were chromatographed on an YMC C₁₈ reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r² = 0.9999) over the studied range (0.5-1000 ng mL⁻¹) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.     

Derivatization of beta-dicarbonyl compound with 2,4-dinitrophenylhydrazine to enhance mass spectrometric detection: application in quantitative analysis of houttuynin in human plasma

Houttuynin (decanoyl acetaldehyde), a beta-dicarbonyl compound, is the major antibacterial constituent in the volatile oil of Houttuynina cordata Thunb. In the present work, detection of houttuynin in human plasma based on the chemical derivatization with 2,4-dinitrophenylhydrazine (DNPH) coupled with liquid chromatography/tandem mass spectrometry was described. The primary reaction products between the beta-dicarbonyl compound and DNPH in aqueous phase were identified as heterocyclic structures, of which the mass spectrometric ionization and fragmentation behavior were characterized with the aid of high-resolution multistage mass spectral analysis. For quantification, houttuynin and internal standard (IS, benzophenone) in plasma were firstly converted to their DNPH derivatives without sample purification, then extracted from human plasma with n-hexane and detected by liquid chromatography tandem mass spectrometry performed in selected reaction monitoring (SRM) mode. This method allowed for a lower limit of quantification (LLOQ) of 1.0 ng/ml using 100-microl plasma. The validation results showed high accuracy (%bias < 2.1) and precision (%CV < 7.2) at broad linear dynamic range (1.0-5000 ng/ml). The simple and quantitative derivatization coupled with tandem mass spectrometric analysis facilitates a sensitive and robust method for the determination of plasma houttuynin in pharmacokinetic studies.     

Development and validation of a liquid chromatography with tandem mass spectrometry method for the determination of isomangiferin in rat plasma with application to a preclinical pharmacokinetic study

A sensitive and specific liquid chromatography with tandem mass spectrometry method was firstly developed for the measurement of isomangiferin in rat plasma. Chloramphenicol was selected as the internal standard. Sample preparation was carried out through a simple one‐step protein precipitation procedure with methanol. Negative electrospray ionization was performed using multiple reaction monitoring mode with transitions of m/z 421.1/301.1 for isomangiferin, and 321.1/151.9 for chloramphenicol. The calibration curve was linear over the range of 0.1–600 ng/mL, with a lower limit of quantification at 0.1 ng/mL. The intra‐ and interday precisions (relative standard deviation) were no more than 8.2% and accuracies (relative error) were within the range of –8.4 to 2.2%. The recovery, matrix effect and stability under different conditions were all proved acceptable. The validated method has been successfully applied to a preclinical pharmacokinetic study of isomangiferin in rats for the first time.     

Stereospecific radical addition of isopropanol andn-butanal to (5R)-(l-menthyloxy)furan-2(5H)-one

The radical addition of PrⁱOH andn-butanal to (5R)-5-(l-menthyloxy)furan-2(5H)-one occurs regio- and diastereospecifically at position 4 of the furanone ring.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 345–536, March, 1995.This work was carried out with the financial support of the Russian Foundation for Basic Research (Project No. 93-03-04497).     

Determination of thymopentin in beagle dog blood by liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study

The pentapeptide thymopentin (Arg‐Lys‐Asp‐Val‐Tyr, RKDVY) corresponds to amino acids 32–36 of the 49 amino acid immunomodulatory polypeptide, thymopoietin, whose biological activity is partially reproduced. Thymopentin is widely used in the clinic and represents a promising target for drug design but bioanalytical and pharmacokinetic data are limited due to its enzymatic instability. This paper reports a rapid and sensitive method based on liquid chromatography with tandem mass spectrometry for the determination of thymopentin in beagle dog blood. To inactivate peptidases and stabilize thymopentin, acetonitrile was added to blood samples immediately after collection followed by addition of stable isotope‐labeled thymopentin as internal standard and washing with dichloromethane. Chromatography was carried out on an Ascentis Express Peptide ES‐C₁₈ column using gradient elution with methanol and aqueous 0.1% formic acid at a flow rate of 0.6 mL/min. Positive electrospray ionization mass spectrometry with selected reaction monitoring achieved linearity in the range of 1.5–800 ng/mL with good accuracy/precision and minimal matrix effects. The method was successfully applied to a pharmacokinetic study in beagle dogs after intravenous administration of 0.2 mg/kg thymopentin.     

Bioanalytical technique for determination of tasimelteon in human plasma by LC–MS/MS and its application to pharmacokinetic study in humans

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry technique for the quantification of tasimelteon in human plasma has been developed and validated using tasimelteon‐d₅ as internal standard. Liquid–liquid extraction technique with ethyl acetate was used for extraction of tasimelteon from the plasma. The chromatographic separation was achieved on an Agilent Zorbax, Eclipse, C₁₈ (4.6 × 50 mm, 5 μm) column using a mobile phase of acetonitrile and 0.02% formic acid buffer (85:15, v/v) with a flow rate of 0.5 mL/min. A detailed method validation was performed as per the United States Food and Drug Administration guidelines. The linear calibration curve was obtained over the concentration range 0.30–299 ng/mL. The API‐4000 liquid chromatography–tandem mass spectrometry was operated under multiple reaction monitoring mode during analysis. The validated method was successfully applied to estimate plasma concentration of tasimelteon after oral administration of a single dose of a 20 mg capsule in healthy volunteers under fasting conditions. The maximum concentration of the drug achieved in the plasma was 314 ± 147 ng/mL and the time at which this concentration was attained was 0.54 ± 0.22 h.     

Structure of (Z)-(5R)-methyl-2-(4-phenylbenzylidene)cyclohexanone as chiral component of liquid-crystalline systems

The spatial structure of (Z)-(5R)-methyl-2-(4-phenylbenzylidene)cyclohexanone prepared by photochemical isomerization of the E-isomer was studied by analyzing the magnitudes and temperature dependence of the proton spin-spin coupling constants obtained by ¹H NMR spectroscopy and the results of molecular modeling using semiempirical quantum chemical AM1 and PM3 methods and the density functional theory (DFT). Comparison of the results obtained for the Z-and E-isomers shows that in both cases the conformational equilibrium for both isomers is characterized by significant preference of the chair conformer having an equatorial methyl group, namely, − ΔH (chair a ⇌ chair e) = 1.98–2.12 and 1.36–1.54 kcal mole⁻¹ for the Z-and E-isomers, respectively. Distinctions in the non-planarity of the enone fragment and cyclohexanone ring in the Z-and E-isomers under study following from the results of mathematical modeling were confirmed by the experimental values of the geminal spin-spin coupling constants of protons of the methylene groups in α,α ′-positions with respect to the enone group. Quantum chemical calculations of the Z-isomer revealed the existence of intramolecular hydrogen bond between the carbonyl oxygen and the nearest aromatic proton in ortho-position of the benzene ring. Possible reasons for different helical twisting power of (Z)-(5R)-methyl-2-(4-phenylbenzylidene)cyclohexanone and the E-and Z-arylidene derivatives of 1R, 4R-isomenthone in the mesophase are discussed based on the results of molecular structure studies for these compounds. In the text below the unsaturated ketones under study will be called “arylidene cyclohexanone derivatives” for convenience of comparing the characteristics of methylcyclohexanone and isomenthone derivatives. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 962–972, June, 2006.     

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.     

Determination of tubuloside B by LC‐MS/MS and its application to a pharmacokinetic study in rats

Tubuloside B, a novel neuroprotective phenylethanoid, is a major active constituent of Cistanche tubulosa and Cistanche deserticola. A specific and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for the quantification of tubuloside B in rat plasma. Sample preparation was conducted through a protein‐precipitation extraction with methanol using tubuloside A as internal standard (IS). Chromatographic separation was achieved using a Capcell Pak C₁₈ column (2.0 × 50 mm, 5 μm) with a mobile phase of methanol–10 mm ammonium acetate buffer (70:30, v/v) in an isocratic elution. Mass spectrometry analysis was performed in negative ionization mode with selected reaction monitoring transitions at m/z 665.1 → 160.9 for tubuloside B, and m/z 827.1 → 160.9 for IS. Calibration curves were linear over the range of 1.64–1640 ng/mL for plasma samples samples (R² > 0.990). The lower limit of quantification (LLOQ) was 1.64 ng/mL. The intra‐ and inter‐day accuracy was between 92.3 and 113.0% with the RSD <9.23% at all LLOQ and quality control levels. Finally, this method was successfully applied in the pharmacokinetics study of tubuloside B after intravenous administration.     

Determination of lansoprazole enantiomers in dog plasma by column‐switching liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study

Lansoprazole, a selective proton pump inhibitor, has a chiral benzimidazole sulfoxide structure and is used for the treatment of gastric acid hypersecretory related diseases. To investigate its stereoselective pharmacokinetics, a column‐switching liquid chromatography with tandem mass spectrometry method was developed for the determination of lansoprazole enantiomers in dog plasma using (+)‐pantoprazole as an internal standard. After a simple protein precipitation procedure with acetonitrile, matrix components left behind after sample preparation were further eliminated from the sample by reversed‐phase chromatography on a C₁₈ column. The fluent was fed to a chiral column for the separation of lansoprazole enantiomers. Baseline separation of lansoprazole enantiomers was achieved on a Chiralcel OZ‐RH column using acetonitrile/0.1% formic acid in water (35:65, v/v) as the mobile phase at 40°C. The linearity of the calibration curves ranged from 3 to 800 ng/mL for each enantiomer. Intra‐ and inter‐day precisions ranged from 2.1 to 7.3% with an accuracy of ±1.7% for (+)‐lansoprazole, and from 1.6 to 6.9% with an accuracy of ±3.5% for (–)‐lansoprazole, respectively. The validated method was successfully applied for the stereoselective pharmacokinetic study of lansoprazole in beagle dog after intravenous infusion.     

(R)-4-Menthenone in the synthesis of optically pure sex pheromone of the peach leafminer moth (Lyonetia clerkella)

The synthesis of (14S)-methyloctadec-1-ene, sex pheromone of the peach leafminer moth (Lyonetia clerkella), is described to demonstrate a new potential of the synthetic use of (R)-4-menthenone.     

Determination of cilnidipine, a new calcium antagonist, in human plasma using high performance liquid chromatography with tandem mass spectrometric detection

A rapid, sensitive and reliable high performance liquid chromatographic method coupled with tandem mass spectrometry (HPLC–MS/MS) has been developed and validated for the determination of cilnidipine, a relatively new calcium antagonist, in human plasma. The reversed-phase chromatographic system was interfaced with a TurboIonSpray (TIS) source. Nimodipine was employed as the internal standard (IS). Sample extracts following protein precipitation were injected into the HPLC–MS/MS system. The analyte and IS were eluted isocratically on a C18 column, with a mobile phase consisting of CH₃OH and NH₄Ac (96:4, v/v). The ions were detected by a triple quadrupole mass spectrometric detector in the negative mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 for cilnidipine and for the IS, respectively. The analysis time for each run was 3.0 min. The calibration curve fitted well over the concentration range of 0.1–10 ng mL⁻¹, with the regression equation Y = (0.103 ± 0.002)X + (0.014 ± 0.003) (n = 5), r = 0.9994. The intra-day and inter-day R.S.D.% were less than 12.51% at all concentration levels within the calibration range. The recoveries were between 92.71% and 97.64%. The long-term stability and freeze-thaw stability were satisfying at each level. The present method provides a modern, rapid and robust tool for pharmacokinetic studies of cilnidipine.     

Quantification of chlordesmethyldiazepam by liquid chromatography–tandem mass spectrometry: application to a cloxazolam bioequivalence study

A rapid, sensitive and specific LC‐MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid‐liquid extraction (diethyl‐ether/hexane, 80/20, v/v) procedure. The LC‐MS/MS method on a RP‐C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5–50 ng/mL (R > 0.999). The between‐run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification—LLOQ (0.500 ng/mL). The between‐run accuracies were 0.1, –1.5, –2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam—test, Eurofarma Lab. Ltda and Olcadil®— reference, Novartis Biociências S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUC0‐inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of apomorphine in canine plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to a pharmacokinetic study

An LC-ESI-MS method was developed and validated for the assay of apomorphine in canine plasma using one-step liquid-liquid extraction. The analytes were separated on a Phenomenex Gemini C18 (150 mm x 2.0 mm id 3 microm) column and determined by MS in the positive ion mode. The linear range was 0.4-40 ng/mL with an LOD of 0.2 ng/mL for apomorphine in plasma. The intraday and interday precision and accuracy of quality control samples were < 5.9% RSD and < 7.5% bias for apomorphine. Extraction recoveries were > 80%. The validated method was successfully applied to analyze canine plasma samples in a pharmacokinetic study of apomorphine in dogs and detailed pharmacokinetic parameters were calculated.     

多羟基甾醇25(R)-异螺甾环-5-烯-2β,3α,19-三醇的合成

以薯蓣皂素为原料, 经过磺酰酯化、N-溴代丁二酰亚胺(NBS)氧化加成、Pb(OAc)₄远程氧化关环、消去反应、间氯过氧苯甲酸(mCPBA)氧化、高氯酸开环及锌粉还原7步反应, 合成多羟基甾体25(R)-异螺甾环-5-烯-2β,3α,19-三醇. 并用IR, ¹H NMR, ¹³C NMR, MS及元素分析对各中间体及目标化合物进行了表征.     

Determination of didanosine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A method based on solid-phase extraction coupled to liquid chromatography with positive ion electrospray ionization and tandem mass spectrometric detection was developed for the determination of didanosine in human serum, using lamivudine as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions m/z 237 --> 136.7 for didanosine and m/z 230 --> 111.7 for lamivudine. The method was linear over the range studied (10-1500 ng ml(-1)), with r(2) > 0.98, and the run time was 5 min. The intra- and inter-assay precisions were < or =10% and the intra- and inter-assay accuracies were >95%. The absolute recoveries were 99.8% (10 ng ml(-1)), 98.4% (30 ng ml(-1)), 91.5% (700 ng ml(-1)) and 94.7% (1200 ng ml(-1)). The limits of detection and quantitation were 5 and 10 ng ml(-1), respectively. The method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (200 mg) of reference and test didanosine formulations (buffered powder for oral solutions), in an open, two-way, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (area under the serum concentration versus time curve from time zero to infinity) were within the range 80-125%, which supports the conclusion that the two formulations are bioequivalent regarding the rate and extent of didanosine absorption.     

LC-MS/MS methods for determination of unstable pivmecillinam and mecillinam in acidified human plasma: Application to a pharmacokinetic study

Pivmecillinam, the ester of biologically active antibiotic mecillinam, is an effective oral preparation to treat urinary tract infections. To study pharmacokinetics in humans, LC-MS/MS methods were developed to quantify pivmecillinam and mecillinam in human plasma, respectively. Considering cephalexin as internal standard, analytes were separated on UltimateXB-C18 columns after protein precipitation by acetonitrile. The mobile phase was composed of water containing 0.1% formic acid and methanol. The multiple reactions monitoring transitions of m/z 440.2→167.1, 326.1→167.1, and 348.1→158.1 were selected to inspect pivmecillinam, mecillinam, and the internal standard in positive ion mode. No apparent matrix effect was perceived. Linearities were obtained over calibration ranges of 0.0500–12.0 and 10.0–15,000 ng/mL, respectively. The intraday precisions were below 5.5%, the interday precisions were below 6.1%, and accuracies were within –8.1 to 13.0%. Stability tests were conducted and an acidification step was explored to enhance the stability of pivmecillinam and mecillinam. Further stability was validated under various storage and processing conditions. Both methods were applied to a pharmacokinetic study of pivmecillinam and mecillinam after oral administration of 400 mg pivmecillinam hydrochloride tablets in healthy Chinese subjects.     

Determination of the novel antiarrhythmic drug sulcardine sulfate in human plasma by liquid chromatography tandem mass spectrometry and its application in a clinical pharmacokinetic study

Sulcardine sulfate (Sul), a novel antiarrhythmic agent, is currently in phase I and phase II clinical trials. To elucidate its clinical pharmacokinetic characteristics, a rapid and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of Sul in human plasma. Plasma samples were precipitated by acetonitrile and isotope‐labeled sulcardine was added as internal standard. The analysis was carried out on a Capcell Pak C₁₈ MG III column (100 × 2.0 mm, 5 μm) with 0.1% formic acid in acetonitrile solution and water (17:83, v/v) as mobile phase. The linear range was 5.0–1000 ng/mL for Sul, with a lower limit of quantification of 5.0 ng/mL. The intra‐ and inter‐batch CVs were within ±11.0% and the accuracies were 4.9–107.3%. Our method, for the first time, allows the rapid (only 3.0 min) and accurate quantification of Sul in human plasma. The method has been successfully applied in the pharmacokinetic study of Sul in a clinical trial following oral administration of Sul to healthy volunteers. Copyright © 2016 John Wiley & Sons, Ltd.     

Novel synthesis of (4<Emphasis Type="Italic">R</Emphasis>)-4-methylpentanolide from (L)-(−)-menthol

A novel synthesis of the promising optically pure chiral (4R)-4-methylpentanolide that is based on several regiospecific oxidative transformations of (4R)-2,4-dimethyl-1-(1-methylethyl)-1-cyclohexene, the product of addition of (–)-menthone and methylmagnesium iodide followed by acid dehydration, was proposed.Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 451–453, November–December, 2004.This revised version was published online in April 2005 with a corrected cover date.     

Determination of chlorpheniramine in human plasma by HPLC‐ESI‐MS/MS: application to a dexchlorpheniramine comparative bioavailability study

In the present study a fast, sensitive and robust validated method to quantify chlorpheniramine in human plasma using brompheniramine as internal standard (IS) is described. The analyte and the IS were extracted from plasma by LLE (diethyl ether–dichloromethane, 80:20, v/v) and analyzed by HPLC‐ESI‐MS/MS. Chromatographic separation was performed using a gradient of methanol from 35 to 90% with 2.5 mm NH₄OH on a Gemini Phenomenex C₈ 5 μm column (50 × 4.6 mm i.d.) in 5.0 min/run. The method fitted to a linear calibration curve (0.05–10 ng/mL, R > 0.9991). The precision (%CV) and accuracy ranged, respectively: intra‐batch from 1.5 to 6.8% and 99.1 to 106.6%, and inter‐batch from 2.4 to 9.0%, and 99.9 to 103.1%. The validated bioanalytical procedure was used to assess the comparative bioavailability in healthy volunteers of two dexchlorpheniramine 2.0 mg tablet formulations (test dexchlorpheniramine, Eurofarma, and reference Celestamine^®, Schering‐Plough). The study was conducted using an open, randomized, two‐period crossover design with a 2 week washout interval. Since the 90% confidence interval for C_max and AUC ratios were all within the 80–125% interval proposed by ANVISA and FDA, it was concluded that test and reference formulations are bioequivalent concerning the rate and the extent of absorption. Copyright © 2009 John Wiley & Sons, Ltd.