Development and validation of a UPLC–MS method for the determination of galantamine in guinea pig plasma and its application to a pre‐clinical bioavailability study of novel galantamine formulations

To evaluate the bioavailability and pharmacokinetic profiles of two novel galantamine formulations as medical countermeasure products, an ultra‐performance liquid chromatography–single quadrupole mass spectrometry (UPLC–MS) method was developed and validated for quantifying galantamine in guinea pig plasma using solid‐phase extraction with a mixed mode strong cation exchange reversed‐phase cartridge. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C₁₈ column maintained at 40°C. The mobile phases were solution A, acetonitrile–water, 5:95 (v/v) and solution B, acetonitrile–water 90:10 (v/v), both containing 2 mM ammonium formate and 0.2% formic acid. The mobile phase was delivered utilizing a 3 min gradient program start with 95%A–5%B at a flow rate of 0.6 mL/min. The analyte and internal standard, galantamine‐d3, were detected by selected ion monitoring mode on a Waters 3100 single quadrupole mass spectrometer with positive electrospray ionization. The method was validated according to the US Food and Drug Administration bioanalytical guidance. The method was selective and was linear over the analytical range of 2–2000 ng/mL. Accuracy and precision were acceptable with intra‐ and inter‐day accuracies between 96.8 and 101% and precisions (RSD) <4.88%. The method was successfully implemented to measure galantamine plasma levels in a series of pre‐clinical bioavailability studies for the evaluation of novel galantamine formulations.     

Development and validation of bioanalytical method for quantification of cycloserine in human plasma by liquid chromatography–tandem mass spectrometry: Application to pharmacokinetic study

A selective, sensitive and high‐throughput liquid chromatography–tandem mass spectrometry bioanalytical method has been developed for the estimation of cycloserine in human plasma, employing cytosine as the internal standard. The extraction of the analyte was facilitated by solid‐phase extraction using 100 μL of human plasma. The separation was carried out on a BDS Hypersil C₁₈ (150 × 4.6 mm, 5 μm) column using a mixture of 0.2% formic acid in HPLC‐grade water, methanol and acetonitrile (70:15:15, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was linear over the range of 0.20–20 μg/mL with r² > 0.99. Complete validation of the method was performed as per US Food and Drug Administration guidelines and the results met acceptance criteria. Applying the present method, the clinical pharmacokinetics of cycloserine following oral administration of 250 mg cycloserine was studied under fasting conditions. Assay reproducibility was also verified by incurred sample reanalysis.     

Determination of Fulvestrant in Rat Plasma by LC�CMS�CMS: Application to a Pharmacokinetic Study

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC?CMS?CMS) method was developed and validated for the determination of fulvestrant in rat plasma. Sample preparation involved a liquid-liquid extraction using 1.0 mL of n-hexane?Cisopropanol (90:10, v/v) to extract the analyte from 0.1 mL of rat plasma. The analytes were separated on a phenyl-based column using the mobile phase consisting of methanol/water containing 5 mM ammonium acetate at the flow rate of 0.3 mL min^?1. The analytes were monitored by tandem mass spectrometry under electrospray negative ionization mode. Linear calibration curves were generated over the fulvestrant concentration ranges of 0.05?C10.0 ng mL^?1 in rat plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical methods (<15%). This developed and validated assay method was successfully employed to characterize the plasma concentration-time profile of fulvestrant after its intramuscular administration in rats at a dose of 10 mg kg^?1.     

A highly sensitive and efficient UPLC‐MS/MS assay for rapid analysis of tedizolid (a novel oxazolidinone antibiotic) in plasma sample

Tedizolid (TDZ) is a novel oxazolidinone class antibiotic, indicated for the treatment of acute bacterial skin and skin structure infections in adults. In this study a highly sensitive UPLC‐MS/MS assay was developed and validated for the determination of TDZ in rat plasma using rivaroxaban as an internal standard (IS). Both TDZ and IS were separated on an Acquity UPLC BEH? C₁₈ column using an isocratic mobile phase comprising of acetonitrile–20 mm ammonium acetate (85:15, v/v), eluted at 0.3 mL/min flow rate. The plasma sample was processed by liquid liquid extraction technique using ethyl acetate as an extracting agent. The analyte and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.09 > 343.10 for TDZ and m/z 435.97 > 144.94 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 0.74–1500 ng/mL and the lower limit of quantification was 0.74 ng/mL only. The developed assay was validated following standard guidelines for bioanalytical method validation (US Food and Drug Administration) and all the validation results were within the acceptable limits. The developed assay was successfully applied into a pharmacokinetic study in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative determination of rosuvastatin in human plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for determining rosuvastatin in human plasma, a new synthetic hydroxymethylglutaryl-coenzyme A reductase inhibitor. The analyte and internal standard (IS; cilostazol) were extracted by simple one-step liquid/liquid extraction with ether. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The chromatographic separation was performed on an Atlantis C18 column (2.1 mm x 150 mm, 5.0 microm) with a mobile phase consisting of 0.2% formic acid/methanol (30:70, v/v) at a flow rate of 0.20 mL/min. The analyses were carried out by multiple reaction monitoring (MRM) using the precursor-to-product combinations of m/z 482 --> 258 and m/z 370 --> 288. The areas of peaks from the analyte and the IS were used for quantification of rosuvastatin. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification (LLOQ) was 0.2 ng/mL and the assay exhibited a linear range of 0.2-50.0 ng/mL and gave a correlation coefficient (r) of 0.9991 or better. Quality control samples (0.4, 8, 25 and 40 ng/mL) in six replicates from three different runs of analysis demonstrated an intra-assay precision (RSD) 7.97-15.94%, an inter-assay precision 3.19-15.27%, and an overall accuracy (relative error) of < 3.7%. The method can be applied to pharmacokinetic or bioequivalence studies of rosuvastatin.     

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.     

Development and validation of a high performance liquid chromatography quantification method of levo‐tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study

Levo ‐tetrahydropalmatine (l‐ THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l‐ THP and its desmethyl metabolites l‐ corydalmine (l‐ CD) and l‐ corypalmine (l‐ CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid–liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed‐phase Symmetry® C₁₈ column (4.6 × 150 mm, 5 μm) at 25°C. The mobile phase consisted of acetonitrile–methanol–10 mm ammonium phosphate (pH 3) (10:30:60, v /v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1–10,000 ng/mL. The intra‐ and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l‐ THP in rats. Taken together, the developed method can be applied for bioanalysis of l‐ THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.     

Quantification of metronidazole in human plasma using a highly sensitive and rugged LC–MS/MS method for a bioequivalence study

A highly sensitive, selective and rugged method has been described for the quantification of metronidazole (MTZ) in human plasma by liquid chromatography–tandem mass spectrometry using metronidazole‐d4 as the internal standard (IS). The analyte and the IS were extracted from 100 μL plasma by liquid–liquid extraction. The clear samples obtained were chromatographed on an ACE C₁₈ (100 × 4.6 mm, 5 μm) column using acetonitrile and 10.0 mm ammonium formate in water, pH 4.00 (80:20, v/v) as the mobile phase. A triple quadrupole mass spectrometer system equipped with turbo ion spray source and operated in multiple reaction monitoring mode was used for the detection and quantification of MTZ. The calibration range was established from 0.01 to 10.0 μg/mL. The results of validation testing for precision and accuracy, selectivity, matrix effects, recovery and stability complied with current bioanalytical guidelines. A run time of 3.0 min permitted analysis of more than 300 samples in a day. The method was applied to a bioequivalence study with 250 mg MTZ tablet formulation in 24 healthy Indian males.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of febuxostat in human plasma

A rapid and sensitive analytical method based on liquid chromatography coupled to tandem mass spectrometry detection with positive ion electrospray ionization was developed for the determination of febuxostat in human plasma using d₇‐febuxostat as the internal standard (IS). A simple protein precipitation was performed using acetonitrile. The analyte and IS were subjected to chromatographic analysis on a Capcell PAK C₁₈ column (4.6 × 100 mm, 5 µm) using acetonitrile–5 mm ammonium acetate–formic acid (85:15:0.015, v/v/v) as the mobile phase at a flow rate of 0.6 mL/min. An Agilent 6460 electrospray tandem mass spectrometer was operated in the multiple reaction monitoring mode. The precursor‐to‐product ion transitions m/z 317 → m/z 261 (febuxsotat) and m/z 324 → m/z (261 + 262) (d₇‐febuxostat, IS) were used for quantitation. The results were linear over the studied range (10.0–5000 ng/mL), and the total analysis time for each chromatograph was 3 min. The intra‐ and inter‐day precisions were less than 7.9 and 7.2%, respectively, and the accuracy was within ±4.2%. No evidence of analyte instability in human plasma was observed storage at ?20°C for 31 days. This method was successfully applied in the determination of febuxostat concentrations in plasma samples from healthy Chinese volunteers. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of ginsenosides and bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill for pharmacokinetic study by liquid chromatography tandem mass spectrometry following solid‐phase extraction

A sensitive and reliable bioanalytical method was established for quantitati\ve and pharmacokinetic investigation of nine ginsenosides and seven bufadienolides in rat plasma after the oral administration of Shexiang Baoxin Pill by liquid chromatography–electrospray ionization tandem mass spectrometry, using tinidazole and digoxin as internal standards (ISTDs). All of the analytes and ISTDs obtained satisfactory recoveries by solid‐phase extraction using an Oasis HLB μElution Plate, which was eluted with methanol and ethyl acetate successively, and chromatographic separation was achieved on a Shim‐pack XR‐ODSIIcolumn (75 × 2.0 mm, 2.2 μm) with gradient elution using a mixture of acetonitrile–0.1% formic acid solution (v /v) as the mobile phase at a flow rate of 0.3 mL/min. Detection was carried out by a triple‐quadrupole tandem mass spectrometry with positive/negative ion switching multiple reaction monitoring mode. All analytes showed good linearity over a wide concentration range (r ² > 0.99). The lower limit of quantification was in the range 0.625–12.5 ng/mL for bufadienolides and 2–5.5 ng/mL for ginsenosides, and the mean recoveries of all analytes were in the range 78.29–99.35%. The intra‐ and inter‐day precisions (RSD) were in the range 0.08–12.38% with the accuracies between 86.09 and 99.40%. The validated method was then successfully applied to pharmacokinetic study of the above 16 compounds in rat plasma. Pharmacokinetic results indicated that the developed extraction and analytical method could be employed as a rapid, effective technique for pharmacokinetic study of multiple components, especially various polarity that are difficult to extract simultaneously.     

Development and validation of a high‐performance liquid chromatographic method for the determination of stemoninine in rat plasma after administration of Stemona tuberosa extracts

For the first time, an HPLC method was developed and validated for the determination of stemoninine in plasma after oral and intravenous administration of the extract of the roots of Stemona tuberosa to rats. Plasma samples were analyzed on a Waters reversed‐phase C₁₈ column using a gradient mobile‐phase of eluent A (water containing 0.1% formic acid and 0.2% triethylamine, pH 3.68) and eluent B (acetonitrile–water, 50:50, v/v). The flow rate was 1.0 mL/min and the detector wavelength was 210 nm. The Waters Oasis solid‐phase extraction cartridge was applied for the preparation of plasma samples with high recovery. A good linear relationship was obtained in the concentration range of 1.55–124 µg/mL (r = 0.9995). The limits of quantification and detection were 1.55 and 0.42 µg/mL, respectively. The average recoveries ranged from 91.11 to 96.43% in plasma at stemoninine concentrations of 3.10, 62.0 and 99.2 µg/mL. Intra‐ and inter‐batch coefficient of variations were 3.27–5.37% and 2.49–3.92%, respectively. This method was successfully applied to pharmacokinetic studies after oral and intravenous administration of Stemona tuberosa extract in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

A simple,selective and rapid validated method for estimation of anastrazole in human plasma by liquid chromatography–tandem mass spectrometry and its application to bioequivalence study

A rapid, simple and specific method for estimation of anastrazole in human plasma was validated using letrozole as internal standard. The analyte and internal standard were extracted from plasma using simple solid‐phase extraction. The compound were separated on a reverse‐phase column with an isocratic mobile phase consisting of 0.1% formic acid in water and acetonitrile (12 : 88, v/v) and detected by tandem mass spectrometry in positive ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 294.1 → 225.1 for anastrazole and m/z 286.1 → 217.1 for internal standard. Linearity in plasma was observed over the concentration range 0.3–30 ng/mL for anastrazole. The mean recovery for anastrazole was 83.7% with a lower limit of quantification of 0.3 ng/mL. The coefficient of variation of the assay was less than 6.8% and the accuracy was 96.1–102.2%. The validated method was applied to a bioequivalence study of 1 mg anastrazole tablet in healthy human volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid–liquid extraction with n-hexane–ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C₁₈, 150 mm × 2.0 mm, 5 μm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10–10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 μL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients.     

A simple and sensitive HPLC-UV method for the determination of ponicidin in rat plasma and its application in a pharmacokinetic study

A simple, rapid, selective and sensitive HPLC‐UV method has been developed and validated for the determination of ponicidin in rat plasma. The analyte was extracted from rat plasma by liquid–liquid extraction with ethyl acetate as the extraction solvent. The LC separation was performed on a Zorbax Eclipse XDB C₁₈ analytical column (150 × 4.6 mm i.d., 5 µm) with an isocratic mobile phase consisting of methanol–water–phosphoric acid (45:55:0.01, v/v/v) at a flow rate of 1.0 mL/min. There was a good linearity over the range of 0.1–25 µg/mL (r = 0.9995) with a weighted (1/C²) least square method. The lower limit of quantification was proved to be 0.1 µg/mL. The accuracy was within ±10.0% in terms of relative error and the intra‐ and inter‐day precisions were less than 9.1% in terms of relative standard deviation. After validation, the method was successfully applied to characterize the pharmacokinetics of ponicidin in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of Pregabalin in Human Plasma Using LC-MS-MS

A bioanalytical method has been developed and validated for determination of pregabalin in human plasma. The analytical method consists in the precipitation of plasma sample with trichloro acetic acid (20% v/v solution in water), followed by the determination of pregabalin by an LC-MS-MS method using gabapentin as internal standard. Separation was achieved on a Gemini C₁₈ 50 mm × 2.0 mm (3 μm) column with an isocratic mobile phase consisting of methanol–water (98:2, v/v) with 0.5% v/v formic acid. Protonated ions formed by a turbo ionspray in positive mode were used to detect analyte and internal standard. The MS-MS detection was by monitoring the fragmentation of 160.2→55.1 (m/z) for pregabalin and 172.2→67.1 (m/z) for gabapentin on a triple quadrupole mass spectrometer. The assay was calibrated over the range 0.1–15.0 μg mL⁻¹ with correlation coefficient of 0.9998. Validation data showed intra-batch (n = 6) CV% ≤ 6.89 and RE (%) between −4.17 and +3.08 and inter-batch (n = 18) CV% < 9.09 and RE (%) between −3.0 and +10.00. Mean extraction recovery were 80.45–89.12% for three QC samples and 87.56% for IS. Plasma samples were stable for three freeze–thaw cycles, or 24 h ambient storage, or 1 and 3 months storage at −20 °C. Processed sample (ready for injection) were stable up to 72 h at autosampler (4 °C). This method has been used for analyzing plasma samples from a bioequivalence study with 18 volunteers.     

A rapid HPLC‐ESI‐MS/MS method for determination of β‐asarone,a potential anti‐epileptic agent,in plasma after oral administration of Acorus calamus extract to rats

β‐Asarone (BAS), a phenylpropanoid from Acorus calamus Linn., has shown biological effects in the management of cognitive impairment conditions such as Alzheimer's disease. The present paper describes a selective and sensitive liquid chromatography–tandem mass spectrometric method (HPLC‐MS/MS) using electrospray ionization source (ESI) for quantification of BAS in rat plasma. Briefly, the plasma samples were pre‐treated using a simple solid‐phase extraction method. The separation of BAS and the internal standard, caffeine, was achieved on an Agilent Zorbax XDB C₁₈ column (50 × 2.1 mm i.d., 5 µm) using 0.2 mL/min isocratic mobile phase flow. The detection was performed using an Applied Biosystems Hybrid Q‐Trap API 2000 mass spectrometer equipped with an ESI source operated in positive mode. Also, the developed bioanalytical method was validated as per the US FDA bioanalytical guidelines over the concentration range of 9.79–4892.50 ng/mL (r² ≥ 0.9951) for BAS from rat plasma. The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 86.92 ± 3.89, 85.30 ± 1.09 and 87.24 ± 4.03%, respectively. The applicability of the validated HPLC‐MS/MS method was demonstrated by successful measurement of BAS from plasma following oral administration of Acorus calamus rhizome extracts to three female albino Wistar rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

HPLC‐UV method for quantifying etoposide in plasma and tumor interstitial fluid by microdialysis: application to pharmacokinetic studies

A simple and sensitive bioanalytical method was developed and validated for determination of etoposide in plasma and microdialysis samples of Walker‐256 tumor‐bearing rats. A microdialysis probe was implanted in the center of a subcutaneous tumor and Ringer's solution was used as perfusion medium. Chromatographic separation was conducted on a Shimadzu CLC‐C₈ column using a mobile phase consisting of water–acetonitrile (70:30; v/v) adjusted to pH 4.0 ± 0.1 with formic acid at a gradient flow rate of 1.0–0.6 mL/min, an injection volume of 30 μL and UV detection at 210 nm. Microdialysate samples were analyzed without processing and plasma samples (100 μL) were spiked with phenytoin as internal standard (IS) (1 µg/mL) followed by extraction with tert‐butyl methyl ether. The organic layer was evaporated and reconstituted with 100 μL of mobile phase before injection. The methods for plasma and microdialysate were linear in the ranges of 25–10,000 ng/mL and of 10–1500 ng/mL, respectively. All the validation parameters such as intra‐ and inter‐day precision and accuracy and stability were within the limits established by international guidelines. The present method was successfully applied in the investigation of etoposide pharmacokinetics in rat plasma and microdialysate tumor samples following a single 15 mg/kg intravenous dose. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous quantification of two steroidal glycosides after oral gavage of Marsdenia tenacissima extract in rats using a LC‐MS/MS method

A specific, sensitive and accurate analytical LC‐MS/MS assay was developed for the simultaneous determination of two steroidal glycosides, tenacissoside H and tenacissoside I, in rat plasma. An Agilent ZORBAX SB‐C₁₈ column was used with an isocratic mobile phase system composed of methanol–water–formic acid (70:30:0.1, v/v/v) at a flow rate of 0.3 mL/min. The analysis was performed on a positive ionization electrospray mass spectrometer via selected reaction monitoring mode scan. One‐step protein precipitation with acetonitrile was chosen to extract the analytes from plasma. The lower limits of quantification were 0.9 ng/mL for tenacissoside H and tenacissoside I. The intra‐ and inter‐day precisions were 2.03–11.56 and 3.76–11.62%, respectively, and the accuracies were <110.28% at all quality control levels. The validated method was applied to a pharmacokinetic study in rats after oral gavage of Marsdenia tenacissima extract. Copyright © 2014 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.