Bioanalytical method development,validation and quantification of dorsomorphin in rat plasma by LC‐MS/MS

The present investigation describes the development and validation of a sensitive liquid chromatography–mass spectrometry/mass spectrometry (LC‐MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC‐MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C₁₈ column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r² ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter‐day and intra‐day precision and accuracy were found to be within 85–115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the quantification of tigecycline in rat brain tissues

Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra‐abdominal infections. A selective, accurate and reversed‐phase high‐performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel‐Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C₁₈ column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150–1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time‐points. Copyright © 2015 John Wiley & Sons, Ltd.     

A simple and sensitive LC‐MS/MS method for the determination of sotalol in rat plasma

A sensitive, rapid and robust HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the quantification of sotalol in rat plasma. Plasma samples were precipitated with acetonitrile before analysis. The chromatographic separation was performed on an Atlantis hydrophilic interaction liquid chromatography Silica column (50 × 2.1 mm, 3 µm) with a gradient mobile phase of 10 mm NH₄COOH (containing 0.2% of formic acid) as buffer A and acetonitrile as mobile phase B. Sotalol (m/z 273.2 → 255.1) and atenolol (the internal standard, IS, m/z 267.2 → 190.1) were monitored under positive ionization mode with 5500 QTRAP. Retention time of sotalol and the IS were 2.69 and 3.43 min, respectively. The linear range was 5–500 nm based on the analysis of 0.1 mL of plasma. The intrabatch precision ranged from 1.2 to 6.1%, and the inter‐batch precision was from 3.3 to 6.5%. The coefficient of variation of IS‐normalized matrix factor was 7.6%. Experiments for stability were performed and the analyte was sufficiently stable. A run time of 6 min for each injection made it possible to analyze a high throughput of plasma samples. The assay was successfully applied to the determination of sotalol in rat plasma after a micro‐dose oral administration. Copyright © 2014 John Wiley & Sons, Ltd.     

A rapid LC‐MS/MS method for quantification of CSUOH0901, a novel antitumor agent,in rat plasma

CSUOH0901, a novel anticancer derivative of nimesulide, exhibits very promising anticancer activities in various cancer cell lines. In order to support further pharmacological and toxicological studies of this promising anticancer drug candidate, an LC‐MS/MS method was developed and validated in accordance with the US Food and Drug Administration guidelines. The drug molecules were extracted from plasma samples by protein precipitation and then analyzed with LC‐ESI‐MS/MS. An excellent analyte separation was achieved using a phenomenex C₁₈ column with a mobile phase of 90% methanol and 5 m m of ammonium formate. The validated linear dynamic range was between 0.5 and 100 ng/mL and the achieved correlation coefficient (r²) was >0.9996. The results of inter‐ and intra‐day precision and accuracy were satisfactory, that is, <12% for accuracy and within ±5% for precision at a low and high quality control concentrations, respectively. In addition, the analyte and internal standard (JCC76) were found to be stable under the storage conditions at ?20°C for about 2 months. Hence, the acquired results proved that the LC‐ESI‐MS/MS method developed is precise, accurate and selective for the quantification of CSUOH0901 in plasma, and can be used for pharmacokinetic studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

A simple LC‐MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development,validation and application in the CUMS rat model

Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC‐MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4‐aminohippurate, 5‐HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25–12,800 ng mL^?1 with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of bullatine A in rat plasma: application to a pharmacokinetic study

Bullatine A is a diterpenoid alkaloid of Xue‐Shang‐Yi‐Zhi‐Hao (Aconitum brachypodum), which is widely used in traditional Chinese medicine for the treatment of rheumatism and pain. The plasma levels of bullatine A were measured by a rapid and sensitive LC‐MS/MS method. Samples were prepared using acetonitrile precipitation and the separation of bullatine A was achieved on a Capcell Pak MG‐C₁₈ column by isocratic elution using acetonitrile (phase A) and 0.1% formic acid (phase B, pH 4.0; A:B, 30:70, v/v) as the mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple‐quadrupole tandem mass spectrometer by multiple‐reaction monitoring of the transitions at m/z 344.2 → 105.2 for bullatine A and m/z 256.2 → 167.1 for the internal standard. The linearity was found to be within the concentration range of 1.32–440 ng/mL with a lower limit of quantification of 1.32 ng/mL. Only 1.3 min was needed for an each analytical run. This method was successfully applied in the determination of the active component bullatine A in rat plasma after intramuscular administration of A. brachypodum injection. Copyright © 2015 John Wiley & Sons, Ltd.     

A validated LC‐MS/MS assay for simultaneous quantification of methotrexate and tofacitinib in rat plasma: application to a pharmacokinetic study

A highly sensitive, specific and rapid LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid‐phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP_18e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow‐gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49–91.0 and 0.40–74.4 ng/mL for MTX and TFB, respectively. The intra‐ and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post‐dosing of MTX and TFB orally and intravenously to rats. Copyright © 2014 John Wiley & Sons, Ltd.     

Bioanalytical LC‐MS/MS method validation for plasma determination of topiramate in healthy Indian volunteers

A LC‐MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge‐coupled solid‐phase extraction and reverse‐phase chromatographic separation was performed on an Ascentis C₁₈ column. Turbo‐spray negative‐ion mode multiple‐reaction monitoring was selected for mass pair detection at m/z 338.3 → 78.0 and m/z 407.3 → 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half‐life matching for test and reference drug was achieved with 73.43 ± 9.68 and 73.06 ± 14.03 h, respectively, and intra‐subject coefficient of variation achieved within 11% for AUCs and C_max evaluated by non‐compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article. Copyright © 2009 John Wiley & Sons, Ltd.     

Combined quantification of paclitaxel,docetaxel and ritonavir in human feces and urine using LC‐MS/MS

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human feces and urine is described. The drugs were extracted from 200 μL urine or 50 mg feces followed by high‐performance liquid chromatography analysis coupled with positive ionization electrospray tandem mass spectrometry. The validation program included calibration model, accuracy and precision, carry‐over, dilution test, specificity and selectivity, matrix effect, recovery and stability. Acceptance criteria were according to US Food and Drug Administration guidelines on bioanalytical method validation. The validated range was 0.5–500 ng/mL for paclitaxel and docetaxel, 2–2000 ng/mL for ritonavir in urine, 2–2000 ng/mg for paclitaxel and docetaxel, and 8–8000 ng/mg for ritonavir in feces. Inter‐assay accuracy and precision were tested for all analytes at four concentration levels and were within 8.5% and <10.2%, respectively, in both matrices. Recovery at three concentration levels was between 77 and 94% in feces samples and between 69 and 85% in urine samples. Method development, including feces homogenization and spiking blank urine samples, are discussed. We demonstrated that each of the applied drugs could be quantified successfully in urine and feces using the described assay. The method was successfully applied for quantification of the analytes in feces and urine samples of patients. Copyright © 2013 John Wiley & Sons, Ltd.     

A highly sensitive LC‐MS/MS method for the determination of S‐citalopram in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive, rapid assay method has been developed and validated for the estimation of S‐citalopram (S‐CPM) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of S‐CPM and phenacetin (internal standard, IS) from rat plasma with t‐butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP₁₈ column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S‐CPM and 180.10 → 110.10 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.14–5.56 and 0.25–12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain‐to‐plasma ratio of S‐CPM in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of piperaquine and its N‐oxidated metabolite in rat plasma using LC‐MS/MS

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N ‐oxidated metabolite (PQ‐M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C₁₈ column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple‐quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0–400.0 ng/mL for PQ and 1.0–50.0 ng/mL for PQ‐M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ‐M, respectively, using 40 μL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co‐administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ‐M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).     

Validated LC‐ESI‐MS/MS method for simultaneous quantitation of felodipine and metoprolol in rat plasma: application to a pharmacokinetic study in rats

A highly sensitive and specific LC‐ESI‐MS/MS method has been developed and validated for simultaneous quantification of felodipine (FDP) and metoprolol (MPL) in rat plasma (50 μL) using phenacetin as an internal standard (IS) as per the FDA guidelines. Liquid–liquid extraction method was used to extract the analytes and IS from rat plasma. The chromatographic resolution of FDP, MPL and IS was achieved with a mobile phase consisting of 0.2% formic acid in water–acetonitrile (25:75, v/v) with a time program flow gradient on a C₁₈ column. The total chromatographic run time was 4.0 min and the elution of FDP, MPL and IS occurred at 1.05, 2.59 and 1.65 min, respectively. A linear response function was established for the range of concentrations 0.59–1148 and 0.53–991 ng/mL for FDP and MPL, respectively, in rat plasma. The intra‐ and inter‐day accuracy and precision values for FDP and MPL met the acceptance as per FDA guidelines. FDP and MPL were stable in battery of stability studies viz., bench‐top, auto‐sampler and freeze–thaw cycles. The validated assay was applied to a pharmacokinetic study in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a sensitive and high‐throughput LC‐MS/MS method for the simultaneous determination of esomeprazole and naproxen in human plasma

A sensitive and high‐throughput LC‐MS/MS method has been developed and validated for the combined determination of esomeprazole and naproxen in human plasma with ibuprofen as internal standard. Solid‐phase extraction was used to extract both analytes and internal standard from human plasma. Chromatographic separation was achieved in 4.0 min on XBridge C₁₈ column using acetonitrile–25 mM ammonium formate (70:30, v/v) as mobile phase. Mass detection was achieved by ESI/MS/MS in negative ion mode, monitoring at m/z 344.19 → 194.12, 229.12 → 169.05 and 205.13 → 161.07 for esomeprazole, naproxen and IS, respectively. The calibration curves were linear from 3.00 to 700.02 ng/mL for esomeprazole and 0.50 to 150.08 ng/mL for naproxen. The intra‐ and inter‐batch precision and accuracy across four quality control levels met established criteria of US Food and Drug Administration guidelines. The assay is suitable for measuring accurate esomeprazole and naproxen plasma concentrations in human bioequivalence study following combined administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a bioanalytical method by LC‐MS/MS for the quantification of the LAFIS 10 – an antimalarial candidate – and its pharmacokinetics first evaluation

A rapid and highly sensitive method by LC‐MS/MS was developed and validated for the quantification of an antimalarial candidate (LAFIS10) in rat plasma using dexamethasone as internal standard (IS). The chromatographic separation was performed with a Poroshell 120 EC‐C₁₈ column. The mobile phase consisted of water (A) and acetonitrile (B), both containing 10 m m of ammonium formate and 0.1% formic acid, delivered in the form of elution gradient. The LAFIS10 was monitored using an electrospray ionization interface operating in the positive mode in multiple reaction monitoring mode, monitoring the transitions 681.47 → 538.2 for LAFIS10 and 393.20 → 355.30 for the IS. The flow rate was 500 μL/min. The column temperature was kept at 40 °C and the injection volume was 2 μL. The lower limit of quantification was of 10 ng/mL and linearity between 10 and 1000 ng/mL was observed, with an R² > 0.99. The accuracy of the method was >90%. The relative standard deviations intra‐ and interday were <8.80 and <6.37%, respectively. The method showed sensitivity, linearity, precision, accuracy and selectivity required to quantify LAFIS 10 in preclinical pharmacokinetic studies according to criteria established by the US Food and Drug Administration and European Medicines Agency. Copyright © 2014 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for quantitation of bivalirudin in human plasma: application to a human pharmacokinetic study

A sensitive, specific and simple LC‐MS/MS method was developed for the identification and quantification of bivalirudin in human plasma using diazepam as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under multiple‐reaction monitoring mode using electrospray ionization. The sample preparation consisted of an easy protein precipitation sample pretreatment with methanol. Chromatographic separation was achieved on a Zorbax Eclipse plus C₁₈ 100 × 2.1 mm column with a mobile phase of water–methanol–0.1% formic acid. The analytes were detected with a triple quadrupole Quantum Access with positive ionization. Ions monitored in the multiple‐reaction monitoring mode were m/z 1091 → 650 for bivalirudin (at 2.70 min) and m/z 285 → 193 for diazepam (at 3.85 min). The developed method was validated in human plasma with a lower limit of quantitation of 20 µg/L for bivalirudin. A linear response function was established for the range of concentrations 20–10,000 µg/L (r > 0.998) for bivalirudin. The intra‐ and inter‐day precision values for bivalirudin met the acceptance criteria as per US Food and Drug Administration guidelines. Bivalirudin was stable in the battery of stability studies, viz. bench‐top, freeze–thaw cycles and long‐term stability. The developed assay method was applied to an intravenous administration study in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and application of an analytical method for curdione quantification in pregnant sprague–dawley rats by LC‐MS/MS

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague–Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C₁₈ MGIII column. A gradient elution profile with 0.5% formic acid (A)–0.5% formic acid–acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5–500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5–101.1%. The intra‐day and inter‐day precisions were between 2.35 and 9.08%. This LC‐MS/MS method provides a simple and sensitive means for determining the plasma concentration. Copyright © 2015 John Wiley & Sons, Ltd.     

Doping control analysis of desmopressin in human urine by LC‐ESI‐MS/MS after urine delipidation

The World Anti‐Doping Agency (WADA) has recently added desmopressin, a synthetic analogue of the endogenous peptide hormone arginine vasopressin, to the Prohibited List, owing to the potential masking effects of this drug on hematic parameters useful to detect blood doping. A qualitative method for detection of desmopressin in human urine by high‐performance liquid chromatography–electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. Desmopressin purification from urine was achieved by means of delipidation with a 60:40 di‐isopropyl ether/n‐butanol and solid‐phase extraction with WCX cartridges. The lower limit of detection was 25 pg/mL. Extraction recovery was determined as 59.3% (SD 29.4), and signal reduction owing to ion suppression was estimated to be 42.7% (SD 12.9). The applicability of the method was proven by the analysis of real urine samples obtained after intravenous, oral and intranasal administration of desmopressin, achieving unambiguous detection of the peptide in all the cases. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.