Rapid and sensitive LC‐MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study

A rapid, simple and fully validated LC‐MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one‐step liquid–liquid extraction with methyl‐tert‐butyl‐ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C₁₈ column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)–methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal‐to‐noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1–2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra‐ and inter‐day precision and accuracy, recovery, matrix effect and stability. The validated LC‐MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace?) to five healthy Korean male volunteers under fed conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

LC‐ESI‐MS/MS analysis and pharmacokinetics of heterophyllin B,a cyclic octapeptide from Pseudostellaria heterophylla in rat plasma

Heterophyllin B (HB) is a cyclic octapeptide isolated from Pseudostellaria heterophylla. HB is used as the quality control index for evaluating P. heterophylla in the Chinese Pharmacopoeia. A rapid and sensitive LC‐ESI‐MS/MS method was developed and validated for the analysis of HB in rat plasma. Sample preparation consisted of a solid‐phase extraction step for the removal of interference and preconcentration of the target analyte HB and the internal standard N‐acetylcysteine before chromatographic analysis by MS/MS detection. The separation of HB and N‐acetylcysteine was performed using a Hypersil GOLD^TM C₁₈ column and a mixture of methanol–water (60:40, v/v) containing 10 mmol/L ammonium formate and 0.1% formic acid as the mobile phase. The determination step was optimized in the selected reaction monitoring mode for the highly selective and sensitive quantitation of HB in rat plasma. Intra‐ and inter‐assay precision (as relative standard deviation) was ≤9.1%, and accuracy was between 92.6 and 102.7%. The validated method was successfully applied to quantify HB concentrations up to 7 h after tail intravenous injections of 2.08, 4.16 and 8.32 mg/kg HB in rats. The LC‐MS/MS method identified the relevant pharmacokinetic parameters of HB and its studied analog. Copyright © 2015 John Wiley & Sons, Ltd.     

Validated UPLC‐MS/MS method for determination of moclobemide in human brain cell supernatant and its application to bidirectional transport study

A simple and sensitive analytical method based on ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) has been developed for determination of moclobemide in human brain cell monolayer as an in vitro model of blood–brain barrier. Brucine was employed as the internal standard. Moclobemide and internal standard were extracted from cell supernatant by ethyl acetate after alkalinizing with sodium hydroxide. The UPLC separation was performed on an Acquity UPLC^TM BEH C₁₈ column (50 × 2.1 mm, 1.7 µm, Waters, USA) with a mobile phase consisting of methanol–water (29.5:70.5, v/v); the water in the mobile phase contained 0.05% ammonium acetate and 0.1% formic acid. Detection of the analytes was achieved using positive ion electrospray via multiple reaction monitoring mode. The mass transitions were m/z 269.16 → 182.01 for moclobemide and m/z 395.24 → 324.15 for brucine. The extraction recovery was 83.0–83.4% and the lower limit of quantitation (LLOQ) was 1.0 ng/mL for moclobemide. The method was validated from LLOQ to 1980 ng/mL with a coefficient of determination greater than 0.999. Intra‐ and inter‐day accuracies of the method at three concentrations ranged from 89.1 to 100.9% for moclobemide with precision of 1.1–9.6%. This validated method was successfully applied to bidirectional transport study of moclobemide blood–brain barrier permeability. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive LC‐MS/MS method for the determination of osthole in rat plasma: application to pharmacokinetic study

Osthole, a major component isolated from the fruit of Cnidium monnieri (L.) Cusson, has been widely used in traditional Chinese medicine. We developed and validated a rapid and sensitive LC‐MS/MS method for the quantification of osthole in rat plasma. Sample preparation involved simple liquid–liquid extraction by ethyl acetate after addition of imperatorin as internal standard (IS). The analyte was separated using a C₁₈ column with the mobile phase of methanol–0.1% formic acid (80:20, v/v) at a flow rate of 0.4 mL/min. The elutes were detected under positive electrospray ionization in multiple reaction monitoring mode. The method was sensitive with 0.5 ng/mL as the lower limit of detection. Good linearity was obtained over the range of 1.0–500.0 ng/mL. The intra and inter‐batch accuracy for osthole in rat plasma samples ranged from 99.5 to 108.1% and the variation was <8.9%. The stability, extraction efficiency and matrix effect were also acceptable. This method was successfully applied to the pharmacokinetic study of osthole in rat after intravenous and oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

A validated HPLC‐MS/MS method for the quantification of caderofloxacin in human plasma and its application to a clinical pharmacokinetic study

A simple, selective and rapid HPLC‐MS/MS method was developed and validated for the determination of caderofloxacin in human plasma. Sparfloxacin was used as the internal standard (IS). After precipitation with methanol and dilution with the mobile phase, the samples were injected into the HPLC‐MS/MS system. The chromatographic separation was performed on a Zorbax XDB Eclipse C₁₈ column (150 × 4.6 mm, 5 µm) with a mobile phase of ammonium acetate buffer (20 mm, pH 3.0)–methanol, 45:55 (v/v). The MS/MS analysis was done in positive mode. The multiple reaction monitoring transitions monitored were m/z 412.3 → 297.1 for caderofloxacin and m/z 393.2 → 292.2 for the IS. The calibration curve was linear over the range of 50.0–8000 ng/mL with an aliquot of 100 μL plasma. The precision of the assay was 2.0–9.4 and 6.6–11.5% for the intra‐ and inter‐run variability, respectively. The intra‐ and inter‐run accuracy (relative error) was 4.4–10.0 and ?1.2–4.0%. The total run time was 3.5 min. The assay was fully validated in accordance with the US Food and Drug Administration guidance. It was successfully applied to a pharmacokinetic study of caderofloxacin in healthy Chinese volunteers. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatography‐tandem mass spectrometric method for determination of E121 in mouse plasma and its application to pharmacokinetics

A rapid LC‐MS/MS method for quantification of an enaminone analog, E121 in mouse plasma using E118 as an internal standard (IS) has been developed and validated. The analyte was analyzed on C₁₈ column using a mobile phase of acetonitrile/methanol/ammonium acetate/formic acid (60:20:20:0.025, v/v/v/v) at a flow rate of 0.25 mL/min. Quantitation was achieved using ESI+ interface, employing MRM mode at m/z 308>262 and 222>194 for E121 and IS, respectively. The calibration standards were linear over a range of 0.10–20 μg/mL (r²>0.99) with an LLOQ of 0.1 μg/mL (RSD%; 11.4% and bias%; 9.5%). Intra‐ and inter‐run precision of E121 assay ranged from 3.7 to 10.9% with accuracy (bias) that varied between ?10.0 and 12.0%, demonstrating good precision and accuracy. Recoveries of E121 and the IS from plasma were above 80%. Stability of E121 in plasma showed that the analyte was stable under various conditions. The matrix effect study showed a lack of effect. The applicability of the developed method was demonstrated by measuring E121 in mouse plasma samples following intraperitoneal administration of various doses ranging from 10 to 100 mg/kg and this study demonstrates that E121 exhibits linear kinetics in the dose range studied.     

A simple LC–MS/MS method facilitated by salting‐out assisted liquid–liquid extraction to simultaneously determine trans‐resveratrol and its glucuronide and sulfate conjugates in rat plasma and its application to pharmacokinetic assay

A simple LC–MS/MS method facilitated by salting‐out assisted liquid–liquid extraction (SALLE) was applied to simultaneously investigate the pharmacokinetics of trans‐ resveratrol (Res) and its major glucuronide and sulfate conjugates in rat plasma. Acetonitrile–methanol (80:20, v /v) and ammonium acetate (10 mol L⁻¹) were used as extractant and salting‐out reagent to locate the target analytes in the supernatant after the aqueous and organic phase stratification, then the analytes were determined via gradient elution by LC–MS/MS in negative mode in a single run. The analytical method was validated with good selectivity, acceptable accuracy (>85%) and low variation of precision (<15%). SALLE showed better extraction efficiency of target glucuronide and sulfate conjugates (>80%). The method was successfully applied to determine Res and its four conjugated metabolites in rat after Res administration (intragastric, 50 mg kg⁻¹; intravenous, 10 mg kg⁻¹). The systemic exposures to Res conjugates were much higher than those to Res (AUC_0–t , i.v., 7.43 μm h; p.o., 8.31 μm h); Res‐3‐O‐β ‐d ‐glucuronide was the major metabolite (AUC_0–t , i.v., 66.1 μm h; p.o., 333.4 μm h). The bioavailability of Res was estimated to be ~22.4%. The reproducible SALLE method simplified the sample preparation, drastically improved the accuracy of the concomitant assay and gave full consideration of extraction recovery to each target analyte in bio‐samples.     

Development and validation of a rapid LC‐MS/MS method to quantify letrozole in human plasma and its application to therapeutic drug monitoring

A selective, rapid, and sensitive liquid chromatography–tandem mass spectrometry(LC‐MS/MS) method was developed and validated for the determination of letrozole (LTZ) in human plasma, using anastrozole as internal standard (IS). Sample preparation was performed by one‐step protein precipitation with methanol. The analyte and IS were chromatographed on a reversed‐phase YMC‐ODS‐C₁₈ column (2.0 × 100 mm i.d., 3 µm) with a flow rate of 0.3 mL/min. The mobile phase consisted of water containing 0.1% formic acid (v/v) and methanol containing 0.1% formic acid (v/v). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 286.2 → 217.1 for LTZ and m/z 294.1 → 225.1 for IS, respectively. The method was validated for selectivity, linearity, lower limit of quantitation, precision, accuracy, matrix effects and stability in accordance with the US Food and Drug Administration guidelines. Linear calibration curves were 1.0–60.0 ng/mL. Intra‐ and inter‐batch precision (CV) for LTZ were <9.34%, and the accuracy ranged from 97.43 to 105.17%. This method was successfully used for the analysis of samples from patients treated with LTZ in the dose of 2.5 mg/day. It might be suitable for therapeutic drug monitoring of these patients and contribute to predict the risk of adverse reactions. Copyright © 2015 John Wiley & Sons, Ltd.     

LC–MS/MS method for the simultaneous determination of ethyl gallate and its major metabolite in rat plasma

A simple, rapid and sensitive liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method was developed and validated for the determination of ethyl gallate, a pharmacologically active constituent isolated from Lagerstroemia speciosa (Linn.) Pers. This method was used to examine the pharmacokinetics of ethyl gallate and its major metabolite gallic acid in rat plasma using propyl gallate as an internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a Zorbax SB‐C₁₈ column (3.5 μm, 2.1 × 50 mm) with an isocratic mobile phase consisted of methanol–acetonitrile–10 mM ammonium acetate (10 : 25 : 65, v/v/v) containing 0.1% formic acid at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple‐reaction monitoring mode using the electrospray ionization technique in negative mode. The lower limits of quantification of gallic acid and ethyl gallate of the method were 0.5 and 1.0 ng/mL. The intra‐day and inter‐day accuracy and precision of the assay were less than 8.0%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of ethyl gallate to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of isochamaejasmin,neochamaejasmin A and aphnoretinin rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study of Stellera chamaejasme L. extract

Isochamaejasmin, neochamaejasmin A and daphnoretin derived from Stellera chamaejasme L. are important because of their reported anticancer properties. In this study, a sensitive UPLC‐MS/MS method for the determination of isochamaejasmin, neochamaejasmin A and daphnoretin in rat plasma was developed. The analyte and IS were separated on an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm) using gradient elution with the mobile phase of aqueous solution (methanol–water, 1:99, v/v, containing 1 mm formic acid) and organic solution (methanol–water, 99:1, v/v, containing 1 mm formic acid) at a flow rate of 0.3 mL/min. Multiple reaction monitoring mode with negative electrospray ionization interface was carried out to detect the components. The method was validated in terms of specificity, linearity, accuracy, precision, stability, etc. Excellent linear behavior was observed over the certain concentration ranges with the correlation coefficient values >0.99. Intra‐ and inter‐day precisions (RSD) were <6.7% and accuracy (RE) ranged from −7.0 to 12.0%. The validated method was successfully applied to investigate the pharmacokinetics of three chemical ingredients after oral administration of S. chamaejasme L. extract to rats.     

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.     

Quantitative determination of euphol in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C₁₈ short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was <10.83%. This LC‐MS/MS method was successfully applied to investigate the pharmacokinetic study of euphol in rats after intravenous (6 mg/kg) and oral (48 mg/kg) administration. Results showed that the absolute bioavailability of euphol was approximately 46.01%. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Preclinical pharmacokinetic analysis of armillarisin succinate ester in mouse plasma and tissues by LC–MS/MS

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated to determine the concentration of armillarisin succinate ester in mouse plasma and tissues, used for preclinical evaluation. Bavachin was employed as the internal standard. Separation was performed on a 3.5 µm Zorbax SB‐C₁₈ column (30 × 2.1 mm), with a mobile phase consisting of methanol and aqueous 20 mm ammonium acetate. Both analyte and internal standard were determined using electrospray ionization and the MS data acquisition was via selected ion monitoring in negative scanning mode. Quantification was performed using the transitions m/z 333 → 233 and 323 → 221 for armillarisin succinate ester and internal standard, respectively. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic and tissue distribution study after intravenous injection of ASE in mouse in a dose of 10 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

LC‐MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study

A sensitive and selective LC‐MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid–liquid extraction, the analytes were separated on a Zorbax SB‐C₁₈ column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow‐rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry‐over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet. Copyright © 2013 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for simultaneous determination of R‐bambuterol and its active metabolite R‐terbutaline in human plasma and urine with application to a clinical pharmacokinetic study

A sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for simultaneous determination of R‐bambuterol and its active metabolite R‐terbutaline in human plasma and urine was established. The inhibition for the biotransformation of R‐bambuterol in plasma was fully investigated. Plasma samples were prepared on ice and neostigmine metilsulfate added as a cholinesterase inhibitor immediately after sample collection. All samples were extracted with ethyl acetate and separated on a C₁₈ column under gradient elution with a mobile phase consisting of methanol and water containing 5 mm ammonium acetate at a flow rate of 0.6 mL/min. The analytes were detected by an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte in plasma. In urine samples, the LLOQs were 20.00 and 500.0 pg/mL for R‐bambuterol and R‐terbutaline, respectively. The intra‐ and inter‐day precisions were <12.7 and <8.6% for plasma and urine, respectively. The analytical runtime within 6.0 min per sample made this method suitable for high‐throughput determination. The validated method has been successfully applied to the human pharmacokinetic study of R‐bambuterol involving 10 healthy volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

LC–MS/MS method for the simultaneous determination of tenofovir,emtricitabine, elvitegravir and rilpivirine in dried blood spots

A simple, short, and rugged LC–MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine was developed and validated. Dried blood spots were prepared with 25 μL of spiked whole blood. A 3 mm punch was extracted with methanol containing labeled internal standards. Ten microliters was injected into the LC–MS/MS using isocratic mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (45: 55 v/v) at a flow rate of 0.25 mL/min. The method was validated in the range of 10–2000 ng/mL for all four analytes. The intra‐assay accuracy (RE) of the method was −4.73–4.78, 1.35–2.89, −8.89 to −0.49 and − 1.40–1.81 for tenofovir, emtricitabine, elvitegravir and rilpivirine, respectively. The inter‐assay accuracy was within ±15% of nominal and precision (CV) was <15%. The hematocrit effect on quantification was nonsignificant at the tested hematocrit levels (35–70%). The dried blood spot method showed good agreement with the plasma method, and hence can be used as an alternative to plasma method.     

Validation and application of a novel LC/MS/MS method for the determination of isoginkgetin in rat plasma

Isoginkgetin is a biflavonoid compound isolated from the leaf extracts of Ginkgo biloba. In this study, an liquid chromatography–tandem mass spectrometry (LC/MS/MS) with liquid–liquid extraction was developed and validated for the analysis of isoginkgetin in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for isoginkgetin and IS were m/z 566.8 → 134.7 and m/z 430.8 → 269.3, respectively. The validation parameters including selectivity, linearity, LLOQ, accuracy, precision, matrix effect, stability and recovery were satisfactory. The intra‐ and inter‐batch precision (RSD) were <12.1% in plasma, while the accuracy (RE) was within ±14.3%. This method was employed in a pharmacokinetic study on rats after the intravenous administration of isoginkgetin.     

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.