A validated LC-MS/MS method for the determination of vinflunine in plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive LC‐MS/MS method for the quantification of vinflunine in plasma was developed and validated. The analysis involved a simple liquid–liquid extraction. After making alkaline with NaOH, plasma was extracted with methyl tert‐butyl ether and the organic extract was then evaporated and the residue was reconstituted in mobile phase. The reconstituted solution was injected into an HPLC system and was subjected to reverse‐phase HPLC on a 5 µm ODS‐3 column at a flow‐rate of 0.2 mL/min. The mobile phase consisted of ammonium acetate (0.02 mol/L, pH = 3.0) and acetonitrile (20:80). Vinflunine was detected in the single ion monitoring mode using target ions at m/z 817.4/160.1/142.3 for vinflunine and m/z 447.2/128.3/112.1 for gefitinib (internal standard). Standard curves were linear over the concentration range of 5–1000 ng/mL. The mean predicted concentrations of the quality control samples deviated by less than 2% from the corresponding nominal values; the intra‐assay and inter‐assay precisions of the assay were within 7% relative standard deviation. The extraction recovery of vinflunine was more than 80%. The validated assay was applied to a pharmacokinetic study of vinflunine in plasma following the administration of a single vinflunine injection (2 mg/kg). Copyright © 2011 John Wiley & Sons, Ltd.     

LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of paeonol in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies following oral administration of Moutan cortex decoction

Quantification of paeonol, the principal bioactive component of Moutan cortex, in rat plasma following oral administration of Moutan cortex decoction was achieved by using a simple and sensitive high-performance liquid chromatographic method. The calibration curves for paeonol were linear in both the low (25-200 ng/mL) and the high concentration range (200-4000 ng/mL) with r(2) values of 0.9928 and 0.9993, respectively. The coefficients of variation of intra- and inter-day assays were 14.36, 6.52, 1.76, 1.25, 5.36, 3.30 and 1.42% and 12.70, 1.19, 2.98, 1.91, 1.75, 1.78 and 0.96% at concentrations of 25, 50, 100, 200, 500, 1000 and 2000 ng/mL, respectively. The recoveries of paeonol from rat plasma were found to be 101.9, 104.5, 105.4 and 101.2% for concentrations of 50, 500, 1000 and 2000 ng/mL, respectively. The paeonol plasma concentrations were fitted to two-compartment model with fi rst order absorption. The mean terminal half-lives (t(1/2)) of paeonol was 80.9 min.     

Rapid LC‐MS/MS determination and pharmacokinetic application of linarin in rat plasma

A sensitive, selective and robust liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the rapid determination of linarin in rat plasma. Separation of the analyte and warfarin as internal standard (IS) from 100 μL rat plasma was carried out by simple protein precipitation treatment. Chromatographic separation of the analyte was performed on a Diamonsil® C₁₈ column (150 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of methanol–0.5% formic acid (80:20, v/v). The flow rate was 0.6 mL/min and the total run time was not more than 4.0 min. The method was validated over a wide dynamic concentration range of 1.00–1000 ng/mL for linarin. The precision and accuracy values for linarin met the acceptance criteria according to US Food and Drug Administration guidelines. Linarin was stable in the stability studies including a long‐term test (?80°C for 43 days), a short‐term test (ambient for 2 h and autosampler for 8 h) and three freeze–thaw cycles (?80–25°C). The developed assay method was applied to the pharmacokinetic study in rats after a single intramuscular administration of 713 µg/kg linarin. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of metacavir and its metabolites in rat plasma using high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS)

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one‐step liquid–liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse‐phase C₁₈ column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product‐ion transitions for metacavir, 2′,3′‐dideoxyguanosine, O‐methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1–1000 ng/mL for metacavir, 5–5000 ng/mL for 2′,3′‐dideoxyguanosine and 1–1000 ng/mL for O‐methylguanine in rat plasma. The precision and accuracy for both within‐ and between‐batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of glaucocalyxin A and glaucocalyxin B in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study after oral administration of Rabdosia japonica extract

A specific and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the analysis of glaucocalyxin A and glaucocalyxin B in rat plasma using praeruptorin A as an internal standard. Separation was performed on a Hypurity C₁₈ column (2.1 × 50 mm, 5 μm) with isocratic elution using 0.2% formic acid in water–acetonitrile (20:80, v/v). Mass spectrometric detection was conducted using selected reaction monitoring via an electrospray ionization source. Both analytes exhibited good linearity within their concentration ranges (r² > 0.9932). The lower limit of quantitation of glaucocalyxin A and glaucocalyxin B was 1.10 ng/mL. Intra‐ and inter‐day precision exhibited an RSD within 14.5%, and the accuracy (RE) ranged from –12.1 to 15.0% at the lower limit of quantitation and three quality control levels. The developed assay was successfully applied to a pharmacokinetic study of glaucocalyxin A and glaucocalyxin B in rats after oral administration of Rabdosia japonica extract.     

Validated liquid chromatography mass spectrometry method for quantitative determination of strictosamide in dog plasma and its application to pharmacokinetic study

A simple, sensitive and specific high‐performance liquid chromatography mass spectrometry (LC‐MS) method was developed and validated for the quantification of strictosamide in dog plasma. Strictosamide and internal standard (IS, ranolazine) extracted by liquid–liquid extraction with ethyl acetate were separated on a C₁₈ column using a gradient elution program. The detection was performed by selected ion monitoring mode via a positive electrospray ionization interface. The LLOQ was 1.0 ng/mL and the method exhibited acceptable precision, extraction efficiency and matrix effect. Finally, this proposed method was successfully applied to dog pharmacokinetic study and yielded the most comprehensive data on systemic exposure of strictosamide to date. Copyright © 2011 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of 1‐O‐acetylbritannilactone in rat plasma and its application to a preclinical pharmacokinetic study

A novel, rapid and sensitive LC‐MS/MS method for the determination of 1‐O‐Acetylbritannilactone (ABL), a sesquiterpene lactone abundant in Inula britannica, was developed and validated using heteroclitin D as internal standard. Separation was achieved on a reversed phase Hypersil Gold C₁₈ column (50 × 4.6 mm, i.d., 3.0 µm) using isocratic elution with methanol–5 mM ammonium acetate buffer aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min. Calibration curve was linear (r > 0.99) in a concentration range of 1.60–800 ng/mL with the lower limit of quantification of 1.60 ng/mL. Intra‐ and inter‐day accuracy and precision were validated by relative error (RE) and relative standard deviation (RSD) values, respectively, which were both less than ±15%. The validated method has been successfully applied to a pharmacokinetic study of ABL in rats. The elimination half‐lives were 0.412 ± 0.068, 0.415 ± 0.092 and 0.453 ± 0.071 h after a single intravenous administration of 0.14, 0.42, and 1.26 mg/kg ABL, respectively. The area under the plasma concentration–time curve from time zero to the last quantifiable time point and from time zero to infinity and the plasma concentrations at 2 min were linearly related to the doses tested. Copyright © 2015 John Wiley & Sons, Ltd.     

A validated LC‐MS/MS method for rapid determination of brazilin in rat plasma and its application to a pharmacokinetic study

Brazilin is a major homoisoflavonoid component isolated from the dried heartwood of traditional Chinese medicine Caesalpinia sappan L., which is a natural red pigment used for histological staining. Herein a sensitive, specific and rapid analytical LC‐MS/MS method was established and validated for brazilin in rat plasma. After a simple step of protein precipitation using acetonitrile, plasma samples were analyzed using an LC‐MS/MS system. Brazilin and the IS (protosappanin B) were separated on a Diamonsil C₁₈ analytical column (150 × 4.6 mm, 5 µm) using a mixture of water and 10 mm ammonium acetate in methanol (20:80, v/v) as mobile phase at a flow rate of 0.6 mL/min. The method was sensitive with a lower limit of quantitation of 10.0 ng/mL, with good linearity (r² ≥ 0.99) over the linear range 10.0–5000 ng/mL. All the validation data, such as accuracy and precision, matrix effect, extraction recovery and stability tests were within the required limits. The assay method was successfully applied to evaluate the pharmacokinetics parameters of brazilin after an oral dose of 100 mg/kg brazilin in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

LC‐MS/MS determination of pogostone in rat plasma and its application in pharmacokinetic studies

Pogostone is an important constituent of Pogostemon cablin (Blanco) Benth., and possesses various known bioactivities. A rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the analysis of pogostone in rat plasma using chrysophanol as internal standard (IS). The analytes were extracted with methanol and separated using a reversed‐phase YMC‐UltraHT Pro C₁₈ column. Elution was achieved with a mobile phase consisting of methanol–water (75:25, v/v) for 5 min at a flow rate of 400 μL/min. The precursor/product transitions (m/z) under MS/MS detection with negative electrospray ionization (ESI) were 223.0 → 139.0 and 253.1 → 224.9 for pogostone and IS, respectively. The calibration curve was linear over the concentration range 0.05–160 µg/mL (r = 0.9996). The intra‐ and inter‐day accuracy and precision were within ±10%. The validated method was successfully applied to the preclinical pharmacokinetic investigation of pogostone in rats after intravenous (5, 10 and 20 mg/kg) and oral administration (5, 10 and 20 mg/kg). Finally, the oral absolute bioavailability of pogostone in rats was calculated to be 70.39, 78.18 and 83.99% for 5, 10 and 20 mg/kg, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination and pharmacokinetic study of oxymatrine and matrine in beagle dog plasma after oral administration of Kushen formula granule, oxymatrine and matrine by LC-MS/MS

A rapid, specific and sensitive LC-MS/MS method was developed for the determination of oxymatrine (OMT) and matrine (MT) in beagle dog plasma. The method was applied to study the pharmacokinetics of OMT and MT after oral administration of OMT, MT and Kushen formula granule (KFG) containing equivalent amounts of OMT and MT in a three-period crossover design. The analysis was carried out on an Acquity UPLC BEH C(18) column by linear gradient elution with 0.01% acetic acid-water-methanol as mobile phase. Detection was by positive ion electrospray ionization (ESI) mass spectrometry with multiple-reaction monitoring (MRM). Linear calibration curves were both obtained over the concentration range 15-2000 ng/mL, with a limit of quantification of 15 ng/mL. The matrix effect was minimized. The intra- and inter-day precisions (RSDs) were less than 12.4 and 14.7%, respectively, and the accuracy (RE) was from -2.1 to 2.7%. The validated method was used to determine the concentration-time profiles of OMT and MT. The results indicated that the absorption of OMT and MT after oral administration of KFG was significantly greater than that after oral administration of pure components.     

Validated LC-MS/MS assay for the quantitative determination of clematichinenoside AR in rat plasma and its application to a pharmacokinetic study

This study aimed to develop and validate a liquid chromatography tandem mass spectrometry method for measuring clematichinenoside AR in rat plasma. Clematichinenoside AR was extracted by solid‐phase extraction and chromatographed on an XTerra MS C₈ column. Pulchinenoside B4 was used as the internal standard. Elution was achieved using an isocratic mobile phase of acetonitrile with 0.1% acetic acid (21:79, v/v) at a flow‐rate of 0.2 mL/min. The detection was performed by multiple reaction monitoring mode via a negative electrospray ionization interface. Standard curves were linear, ranging from 2.5 to 500 ng/mL. The intra‐ and inter‐day precision values were <14.0% and the accuracy was within ±13%. Extraction recovery ranged from 93.2 to 93.9%. This proposed method was successfully applied to a pharmacokinetic study on clematichinenoside AR in rats after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

LC‐MS/MS methods for the determination of edaravone and/or taurine in rat plasma and its application to a pharmacokinetic study

Three liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methods were respectively developed and validated for the simultaneous or independent determination of taurine and edaravone in rat plasma using 3‐methyl‐1‐p‐tolyl‐5‐pyrazolone and sulfanilic acid as the internal standards (IS). Chromatographic separations were achieved on an Agilent Zorbax SB‐Aq (100 × 2.1 mm, 3.5 µm) column. Gradient 0.03% formic acid–methanol, isocratic 0.1% formic acid–methanol (90:10) and 0.02% formic acid–methanol (40:60) were respectively selected as the mobile phase for the simultaneous determination of two analytes, taurine or edaravone alone. The MS acquisition was performed in multiple reaction monitoring mode with a positive and negative electrospray ionization source. The mass transitions monitored were m/z [M + H]⁺ 175.1 → 133.0 and [M + H]⁺ 189.2 → 147.0 for edaravone and its IS, m/z [M ? H]^? 124.1 → 80.0 and [M ? H]^? 172.0 → 80.0 for taurine and its IS, respectively. The validated methods were successfully applied to study the pharmacokinetic interaction of taurine and edaravone in rats after independent intravenous administration and co‐administration with a single dose. Our collective results showed that there were no significant alterations on the main pharmacokinetic parameters (area under concentration–time curve, mean residence time, half‐life and clearance) of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of six C21 steroids of Xiao‐Ai‐Ping injection in rat plasma by LC‐MS/MS

Xiao‐Ai‐Ping injection (XAPI) is a traditional Chinese medicine that has been widely used to treat cancer. Modern pharmacological studies have demonstrated that C₂₁ steroids are the main active compounds in XAPI. In this study, a sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated the first time for simultanenous determination of three isomeric pregnane genins (17β‐tenacigenin B, tenacigenin B and tenacigenin A) and their corresponding glycosides (tenacigenoside A, tenacissoside F and marsdenoside I) from XAPI in rat plasma. A simple liquid–liquid extraction technique was used after the addition of dexamethasone acetate as internal standard. The chromatography separation of analytes was achieved on an Agilent Zorbax Eclipse XDB‐C₁₈ column (3.5 µm, 150 × 3 mm i.d.) using methanol–water as mobile phase in a gradient elution program. Detection was performed in multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method showed satisfactory linearity over a concentration range 5.00–2000.00 ng/mL for tenacigenin B, tenacigenin A, marsdenoside I and tenacissoside F (r² > 0.99), 10.00–4000.00 ng/mL for 17β‐tenacigenin B and tenacigenoside A (r² > 0.99). Intra‐ and inter‐day precisions (valued as relative standard deviation) were <9.00% and accuracies (as relative error) in the range ?6.31 to 7.23%. Finally, this validated method was successfully applied to the pharmacokinetic study of XAPI after intravenous administration to rats. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive determination of hypoxic radiosensitizer agent nimorazole in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

A highly sensitive, accurate and robust LC‐MS/MS method was developed and validated for determination of nimorazole (NMZ) in rat plasma using metronidazole (MNZ) as internal standard (IS). The analyte and IS were extracted from plasma by precipitating protein with acetonitrile and were chromatographed using an Agilent Poroshell 120, EC‐C₁₈ column. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid (85:15 v/v). The total run time was 1.5 min and injection volume was 5 μL. Multiple reaction monitoring mode using the transitions of m/z 227.1 → m/z 114.0 for MNZ and m/z 172.10 → m/z 128.1 for IS were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The calibration curve was linear in the range of 0.25–200 ng/mL (r² > 0.9996) and the lower limit of quantification was 0.25 ng/mL in the rat plasma samples. Recoveries of NMZ ranged between 88.05 and 95.25%. The precision (intra‐day and inter‐day) and accuracy of the quality control samples were 1.25–8.20% and ?2.50–3.10, respectively. The analyte and IS were found to be stable during all sample storage and analysis procedures. The LC‐MS/MS method described here was validated and successfully applied to pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

An improved LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study

A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed. After a liquid-liquid extraction procedure, samples were chromatographed on an Agilent TC-C(18) (150 × 4.6 mm, 5 μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. After single-dose administration of 0.5, 1 and 2 mg metolazone, the t(1/2) values were 6.6 ± 2.8, 7.9 ± 1.2 and 7.6 ± 1.9 h, respectively. The pharmacokinetic parameters of multiple doses (1 mg metolazone) were as follows: t(1/2) was 8.9 ± 1.0 h; C(max) was 22.4 ± 5.0 ng/mL; and AUC(0-48) was 156.8 ± 31.6 ng h/mL.     

Determination of plasma topiramate concentration using LC-MS/MS for pharmacokinetic and bioequivalence studies in healthy Korean volunteers

A rapid, simple and validated liquid chromatography coupled to tandem mass spectrometric method (LC-MS/MS) for topiramate analysis in human plasma has been applied to pharmacokinetic and bioequivalence studies in 24 healthy male Korean volunteers. The procedure involves a simple liquid extraction of topiramate and prednisone (internal standard) with acetonitrile and separation by HPLC equipped with a Capcell Pak C18 column using acetonitrile-0.1% triethylamine (80:20, v/v) as a mobile phase. Detection was carried out on an API 2000 MS system by multiple reactions monitoring mode. The ionization was optimized using ESI(-) and selectivity was achieved by MS/MS analysis, m/z 338.0 --> 77.5 and m/z 357.1 --> 327.2 for topiramate and prednisone, respectively. The method had a total run time of 2.5 min and showed good linearity over a working range of 20-5000 ng/mL in human plasma with a lower limit of quantification of 20 ng/mL. No metabolic compounds were found to interfere with the analysis. The inter-day and intra-day accuracy were in the ranges of 99.24-116.63 and 93.45-108.68%, respectively, and inter-day and intra-day precisions were below 6.24 and 5.25%, respectively. This method was successfully applied for pharmacokinetic and bioequivalence studies by analysis of blood samples taken up to 96 h after an oral administration of 100 mg of topiramate in 24 healthy Korean volunteers.     

Development and validation of liquid chromatography-mass spectrometric method for simultaneous determination of moxifloxacin and ketorolac in rat plasma: application to pharmacokinetic study

A highly sensitive, selective and rapid liquid chromatography–electrospray ionization mass spectrometry (LC‐MS) method has been developed and validated for simultaneous determination of moxifloxacin (MFX) and ketorolac (KTC) in rat plasma. Gemifloxacin (GFX) was used as an internal standard (IS). A simple protein precipitation method was used for the extraction of analytes from rat plasma. Effective chromatographic separation of MFX, KTC and GFX was achieved on a Kromasil C₁₈ column (100 × 4.6 mm, 5 µm) using a mobile phase consisting of acetonitrile–10 mm ammonium acetate (pH 2.5)–0.1% formic acid (50:25:25) in an isocratic elution, followed by detection with positive ion electrospray ionization mass spectrometry using target ions of [M + H]⁺ at m/z 402 for MFX, m/z 256 for KTC and m/z 390 for GFX in selective ion recording mode. The method was validated over the calibration range of 5–100 ng/mL for MFX and 10–6000 ng/mL for KTC. The method demonstrated good performances in terms of intra‐ and inter‐day precision (0.97–5.33%) and accuracy (93.91–101.58%) for both MFX and KTC, including lower and upper limits of quantification. The recoveries from spiked control samples were >75% for MFX and >79% for KTC. The matrix effect was found to be negligible and the stability data were within acceptable limits. Further, the method was also successfully applied to a single‐dose pharmacokinetic study in rats. This method can be extended to measure plasma concentrations of both drugs in human to understand drug interaction and adverse effects. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of HPLC‐MS/MS method for the determination of lixivaptan in mouse plasma and its application in a pharmacokinetic study

The aim of the present study was to develop a simple, sensitive and accurate liquid chromatography–electrospray ionization tandem mass spectrometry (ESI‐MS/MS) method for the determination of lixivaptan (LIX) in mouse plasma using vildagliptin as the internal standard (IS). A precipitation procedure was used for the extraction of LIX and vildagliptin from mouse plasma. Chromatographic separation of LIX was achieved using a C₁₈ analytical column (50 × 2.1 mm, 1.8 μm) at 25°C. The mobile phase comprised acetonitrile and ammonium formate (10 mm , pH 3.1; 40:60, v /v) pumped at a flow rate of 0.3 mL min⁻¹. A tandem mass spectrometer with an electrospray ionization source was used to perform the assay. Quantification of LIX at m/z 290 → 137 and IS at 154 → 97 was attained through multiple reaction monitoring. The investigated method was authenticated following the bio‐analytical method of validation guidelines of the US Food and Drug Administration. The developed method showed a good linearity over the concentration range from 5 to 500 ng mL⁻¹, and the calibration curve was linear (r = 0.9998). The mean recovery of LIX from mouse plasma was 99.2 ± 0.68%. All validation parameters for LIX were within the levels required for acceptance. The proposed method was effectively used for a pharmacokinetic study of LIX in mouse plasma.