Analysis of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC–MS/MS method

Fat‐soluble vitamins play a pivotal role in the progression of atherosclerosis and the development of cardiovascular disease. Therefore, plasma monitoring of their concentrations may be useful in the diagnosis of these disorders as well as in the process of treatment. The study aimed to develop and validate an HPLC–MS/MS method for determination of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease. The analytes were separated on an HPLC Kinetex F5 column via gradient elution with water and methanol, both containing 0.1% (v/v) formic acid. Detection of the analytes was performed on a triple‐quadrupole MS with multiple reaction monitoring via electrospray ionization. The analytes were isolated from plasma samples with liquid–liquid extraction using hexane. Linearity of the analyte calibration curves was confirmed in the ranges 0.02–2 μg/mL for retinol, 0.5–20 μg/mL for α‐tocopherol, 5–100 ng/mL for 25‐hydroxyvitamin D2 and 2–100 ng/mL for 25‐hydroxyvitamin D3. Intra‐ and inter‐assay precision and accuracy of the method were satisfactory. Short‐ and long‐term stabilities of the analytes were determined. The HPLC‐MS/MS method was applied for the determination of the above fat‐soluble vitamin concentrations in patient plasma as potential markers of the cardiovascular disease progression.     

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.     

Validated UHPLC–MS/MS assay for quantitative determination of etoposide,gemcitabine, vinorelbine and their metabolites in patients with lung cancer

A fully valid UHPLC–MS/MS method was developed for the determination of etoposide, gemcitabine, vinorelbine and their metabolites (etoposide catechol, 2′,2′‐difluorodeoxyuridine and 4‐O ‐deacetylvinorelbine) in human plasma. The multiple reaction monitoring mode was performed with an electrospray ionization interface operating in both the positive and negative ion modes per compound. The method required only 100 μL plasma with a one‐step simple de‐proteinization procedure, and a short run time of 7.5 min per sample. A Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) provided chromatographic separation of analytes using a binary mobile phase gradient (A, 0.1% formic acid in acetonitrile, v /v; B, 0.1% formic acid in water, v /v). Linear coefficients of correlation were >0.995 for all analytes. The relative deviation of this method was <10% for intra‐ and inter‐day assays and the accuracy ranged between 86.35% and 113.44%. The mean extraction recovery and matrix effect of all the analytes were 62.07–105.46% and 93.67–105.87%, respectively. This method was successfully applied to clinical samples from patients with lung cancer.     

Application of a LC–MS/MS method developed for the determination of p‐phenylenediamine,N‐acetyl‐p‐phenylenediamine and N,N‐diacetyl‐p‐phenylenediamine in human urine specimens

Cases of poisoning by p‐phenylenediamine (PPD) are detected sporadically. Recently an article on the development and validation of an LC–MS/MS method for the detection of PPD and its metabolites, N‐acetyl‐p‐phenylenediamine (MAPPD) and N,N‐diacetyl‐p‐phenylenediamine (DAPPD) in blood was published. In the current study this method for detection of these compounds was validated and applied to urine samples. The analytes were extracted from urine samples with methylene chloride and ammonium hydroxide as alkaline medium. Detection was performed by LC–MS/MS using electrospray positive ionization under multiple reaction‐monitoring mode. Calibration curves were linear in the range 5–2000 ng/mL for all analytes. Intra‐ and inter‐assay imprecisions were within 1.58–9.52 and 5.43–9.45%, respectively, for PPD, MAPPD and DAPPD. Inter‐assay accuracies were within ?7.43 and 7.36 for all compounds. The lower limit of quantification was 5 ng/mL for all analytes. The method, which complies with the validation criteria, was successfully applied to the analysis of PPD, MAPPD and DAPPD in human urine samples collected from clinical and postmortem cases.     

Simultaneous determination of major type‐B trichothecenes and the de‐epoxy metabolite of deoxynivalenol in chicken tissues by HPLC–MS/MS

In this study, a specific and sensitive LC–MS/MS method for the simultaneous analysis of type‐B trichothecenes (deoxynivalenol, 3‐acetyldeoxynivalenol, and 15‐acetyldeoxynivalenol) and the de‐epoxy metabolite of deoxynivalenol (de‐epoxy‐deoxynivalenol) in chicken muscle, liver, kidney, and fat tissues was developed and validated. The method involved an extraction step using ethyl acetate, followed by the evaporation of the supernatant, which was further purified by an Oasis HLB SPE cartridge (Waters, Milford, MA, USA). Chromatographic separation was performed on a C₁₈ column by detection with MS in multiple‐reaction monitoring mode and using a gradient elution program with 0.1% formic acid in water and methanol. The correlation coefficients (r) for each calibration curve were >0.99 within the experimental concentration range. The extraction recoveries ranged from 73.7 to 106.4%, with intraday and interday RSD < 11.6% at three levels of concentrations of 2, 10, and 100 μg/kg. The decision limits and the detection capabilities of the analytes in the chicken tissues ranged from 0.16 to 0.92 and 0.68 to 2.07 μg/kg, respectively. The results demonstrated the applicability of this sensitive procedure to the determination of trichothecenes in chicken tissue samples.     

Residue analysis of tebufenozide and indoxacarb in chicken muscle,milk, egg and aquatic animal products using liquid chromatography–tandem mass spectrometry

We developed an analytical method using liquid–liquid extraction (LLE) and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) to detect and quantify tebufenozide (TEB) and indoxacarb (IND) residues in animal and aquatic products (chicken muscle, milk, egg, eel, flatfish, and shrimp). The target compounds were extracted using 1% acetic acid (0.1% acetic acid for egg only) in acetonitrile and purified using n‐hexane. The analytes were separated on a Gemini‐NX C₁₈ column using (a) distilled water with 0.1% formic acid and 5 mm ammonium acetate and (b) methanol with 0.1% formic acid as the mobile phase. All six‐point matrix‐matched calibration curves showed good linearity with coefficients of determination (R²) ≥0.9864 over a concentration range of 5–50 μg/kg. Intra‐ and inter‐day accuracy was expressed as the recovery rate at three spiking levels and ranged between 73.22 and 114.93% in all matrices, with a relative standard deviation (RSD, corresponding to precision) ≤13.87%. The limits of quantification (LOQ) of all target analytes ranged from 2 to 20 μg/kg, which were substantially lower than the maximum residue limits (MRLs) specified by the regulatory agencies of different countries. All samples were collected from different markets in Seoul, Republic of Korea, and tested negative for tebufenozide and indoxacarb residues. These results show that the method developed is robust and may be a promising tool to detect trace levels of the target analytes in animal products.     

Determination of spinosad at trace levels in bee pollen and beeswax with solid–liquid extraction and LC–ESI‐MS

This paper reports the use of a new LC method with a fused‐core analytical column coupled to ESI‐MS to determine residues of the biopesticide spinosad in bee pollen and beeswax. The method analyzes the active ingredients, spinosyns A and D, with a simple and efficient sample treatment (recovery between 90 and 105%) consisting of a solid–liquid extraction with acetone (bee pollen) or acetonitrile (beeswax). The method was validated in terms of selectivity, LOD, LOQ, linearity, and precision. The LOD and LOQ values ranged between 0.1–0.2 and 0.4–0.7 μg/kg, respectively. Moreover, the precision obtained within the linear concentration range (LOQ 500 μg/kg) was satisfactory (RSD lower than 5%). Finally, the proposed method was applied to analyze bee pollen and beeswax samples collected from apiaries located close to fruit orchards in two Spanish regions.     

A rapid MCM‐41 dispersive micro‐solid phase extraction coupled with LC/MS/MS for quantification of ketoconazole and voriconazole in biological fluids

A rapid dispersive micro‐solid phase extraction (D‐μ‐SPE) combined with LC/MS/MS method was developed and validated for the determination of ketoconazole and voriconazole in human urine and plasma samples. Synthesized mesoporous silica MCM‐41 was used as sorbent in d ‐μ‐SPE of the azole compounds from biological fluids. Important D‐μ‐SPE parameters, namely type desorption solvent, extraction time, sample pH, salt addition, desorption time, amount of sorbent and sample volume were optimized. Liquid chromatographic separations were carried out on a Zorbax SB‐C₁₈ column (2.1 × 100 mm, 3.5 μm), using a mobile phase of acetonitrile–0.05% formic acid in 5 mm ammonium acetate buffer (70:30, v /v). A triple quadrupole mass spectrometer with positive ionization mode was used for the determination of target analytes. Under the optimized conditions, the calibration curves showed good linearity in the range of 0.1–10,000 μg/L with satisfactory limit of detection (≤0.06 μg/L) and limit of quantitation (≤0.3 μg/L). The proposed method also showed acceptable intra‐ and inter‐day precisions for ketoconazole and voriconazole from urine and human plasma with RSD ≤16.5% and good relative recoveries in the range 84.3–114.8%. The MCM‐41‐D‐μ‐SPE method proved to be rapid and simple and requires a small volume of organic solvent (200 μL); thus it is advantageous for routine drug analysis.     

Simultaneous quantitative analysis of phosphocreatine,creatine and creatinine in plasma of children by HPLC–MS/MS method: Application to a pharmacokinetic study in children with viral myocarditis

A simple and rapid HPLC–MS/MS method was developed and validated for simultaneous measurement of phosphocreatine and its metabolites creatine and creatinine in children's plasma. A 50 μL aliquot of plasma was prepared by protein precipitation with acetonitrile–water (1000 μL, 1:1, v/v) followed by separation on a Hypersil Gold C₁₈ column (35°C) with gradient mobile phase consisting of 2 mm ammonium acetate aqueous solution (pH 10) and methanol at a flow rate of 0.3 mL/min and analyzed by mass spectrometry in both positive (phosphocreatine) and negative (creatine and creatinine) ion multiple reaction monitoring mode. Good linearity (r > 0.99) was obtained for the three analytes. The intra‐day and inter‐day values of CV were <5.46% (?13.09% ≤ RE ≤ 2.57%). The average recoveries of the three analytes were 70.9–97.5%. No obvious impact was found for the quantitation of three analytes in normal, hemolyzed and hyperlipemic plasma. In the end, this method was successfully applied to a pharmacokinetic study of phosphocreatine in children (six cases) with viral myocarditis of children after intravenous infusion of 2 g of the test drug. The pharmacokinetc parameters of phosphocreatine/creatine were as follows: t_1/2 0.24/0.83 h, T_max 0.49/0.55 h, C_max 47.34/59.29 μg/mL, AUC_last 17.07/59.63 h μg/mL, AUC_inf 17.16/79.01 h μg/mL and MRT 0.29/0.67 h.     

Development and validation of a sensitive UHPLC‐MS/MS method for the simultaneous analysis of tramadol,dextromethorphan chlorpheniramine and their major metabolites in human plasma in forensic context: application to pharmacokinetics

The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to European Union guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. In the present study, a fast, sensitive and robust method to quantify tramadol, chlorpheniramine, dextromethorphan and their major metabolites, O‐desmethyltramadol, dsmethyl‐chlorpheniramine and dextrophan, respectively, in human plasma using ibuprofen as internal standard (IS) is described. The analytes and the IS were extracted from plasma by a liquid–liquid extraction method using ethyl acetate–diethyl‐ether (1:1). Extracted samples were analyzed by ultra‐high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC‐ESI‐MS/MS). Chromatographic separation was performed by pumping the mobile phase containing acetonitrile, water and formic acid (89.2:11.7:0.1) for 2.0 min at a flow rate of 0.25 μL/min into a Hypersil‐Gold C₁₈ column, 20 × 2.0 mm (1.9 µm) from Thermoscientific, New York, USA. The calibration curve was linear for the six analytes. The intraday precision (RSD) and accuracy (RE) of the method were 3–9.8 and ?1.7–4.5%, respectively. The analytical procedure herein described was used to assess the pharmacokinetics of the analytes in 24 healthy volunteers after a single oral dose containing 50 mg of tramadol hydrochloride, 3 mg chlorpheniramine maleate and 15 mg of dextromethorphan hydrobromide. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of saflufenacil and its two metabolites in soil samples by ultra‐performance liquid chromatography with tandem mass spectrometry

Saflufenacil is a new protoporphyrinogen‐IX‐oxidase inhibitor herbicide. When used, it can enter the soil and has a high risk to reach and contaminate groundwater and aquatic systems. A rapid and sensitive method of ultra‐performance LC with MS/MS was developed for the simultaneous determination of saflufenacil and its two metabolites in soil samples. A modified quick, easy, cheap, effective, rugged, and safe method was applied as the pretreatment procedure. The method was validated by five types of soil samples collected from several regions of China, which all showed good linearity (R² ≥ 0.9914) and precision (RSD ≤ 26.2%). The average recoveries of the three analytes ranged between 74.1 and 118.9% at spiking levels of 3–300 μg/kg. The method limits of detection (S/N 3:1) and method limits of quantification (S/N 10:1) achieved are in the ranges of 0.25–2.75 and 0.83–9.16 μg/kg, respectively. This indicated that the developed ultra‐performance LC with MS/MS method is a promising analytical tool for monitoring the environmental risks posed by saflufenacil.     

Developing a robust LC–MS/MS method to quantify Zn‐DTPA,a zinc chelate in human plasma and urine

Quantitation of Zn‐DTPA (zinc diethylenetriamene pentaacetate, a metal chelate) in complex biological matrix is extremely challenging on account of its special physiochemical properties. This study aimed to develop a robust and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of Zn‐DTPA in human plasma and urine. The purified samples were separated on Proteonavi (250 × 4.6 mm, 5 μm; Shiseido, Ginza, Tokyo, Japan) and a C₁₈ guard column. The mobile phase consisted of methanol–2 mm ammonium formate (pH 6.3)–ammonia solution (50:50:0.015, v/v/v), flow rate 0.45 mL/min. The linear concentration ranges of the calibration curves for Zn‐DTPA were 1–100 μg/mL in plasma and 10–2000 μg/mL in urine. The intra‐ and inter‐day precisions for quality control (QC) samples were from 1.8 to 14.6% for Zn‐DTPA and the accuracies for QC samples were from −4.8 to 8.2%. This method was fully validated and successfully applied to the quantitation of Zn‐DTPA in plasma and urine samples of a healthy male volunteer after intravenous infusion administration of Zn‐DTPA. The result showed that the concentration of Zn‐DTPA in urine was about 20 times that in plasma, and Zn‐DTPA was completely (94.7%) excreted through urine in human.     

MEKC–MS/MS method using a volatile surfactant for the simultaneous determination of 12 synthetic cannabinoids

This study describes a method for the simultaneous determination of 12 synthetic cannabinoids by MEKC–MS/MS using a volatile surfactant (ammonium perfluorooctanoate) as a constituent of the micellar pseudostationary phase. Although most synthetic cannabinoids comigrated by a CZE method, sufficient separation could be achieved by the proposed method. The best separation was made possible by 50 mM ammonium perfluorooctanoate in 20% v/v acetonitrile/water (apparent pH* 9.0) as the BGE, followed by MS detection using a sheath liquid composed of 5 mM ammonium formate in 50% v/v methanol/water mixed hydro‐organic solvent. The standard calibration curve for all analytes showed good linearity (r > 0.99). Satisfactory recoveries, ranging from 89.5 to 101.7%, were obtained. The LODs were 6.5–76.5 μg/g for the target analytes. This method appears to be a useful tool for the identification of synthetic cannabinoids in illegal herbal incense blends.     

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.     

Determination of silodosin and its active glucuronide metabolite KMD‐3213G in human plasma by LC–MS/MS for a bioequivalence study

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method is described for the simultaneous determination of silodosin (SLD) and its active metabolite silodosin β‐d ‐glucuronide (KMD‐3213G) in human plasma. Liquid–liquid extraction of plasma samples was carried out with ethyl acetate and methyl tert‐butyl ether solvent mixture using deuterated analogs as internal standards. The extraction recoveries of SLD and KMD‐3213G were in the ranges 90.8–93.4 and 87.6–89.9%, respectively. The extracts were analyzed on a Symmetry C₁₈ (50 × 4.6 mm, 5 μm) column under gradient conditions using 10 mm ammonium formate in water and methanol–acetonitrile (40:60, v/v), within 6.0 min. For MS/MS measurements, ionization of the analytes was carried out in the positive ionization mode and the transitions monitored were m/z 496.1 → 261.2 for SLD and m/z 670.2 → 494.1 for KMD‐3213G. The method showed good linearity, accuracy, precision and stability in the range 0.10–80.0 ng/mL for SLD and KMD‐3213G. The IS‐normalized matrix factors obtained were highly consistent, ranging from 0.962 to 1.023 for both analytes. The method was used to support a bioequivalence study of SLD and its metabolite in healthy volunteers after oral administration of 8 mg silodosin capsules.     

Determination of phthalate esters in bottled water using dispersive liquid–liquid microextraction coupled with GC–MS

Dispersive liquid–liquid microextraction method was developed for the determination of the amount of phthalate esters in bottled drinking water samples and dispersive liquid–liquid microextraction samples were analyzed by GC–MS. Various experimental conditions influencing the extraction were optimized. Under the optimized conditions, very good linearity was observed for all analytes in a range between 0.05 and 150 μg/L with coefficient of determination (R²) between 0.995 and 0.999. The LODs based on S/N = 3 were 0.005–0.22 μg/L. The reproducibility of dispersive liquid–liquid microextraction was evaluated. The RSDs were 1.3–5.2% (n = 3). The concentrations of phthalates were determined in bottled samples available in half shell. To understand the leaching profile of these phthalates from bottled water, bottles were exposed to direct sunlight during summer (temperature from 34–57°C) and sampled at different intervals. Result showed that the proposed dispersive liquid–liquid microextraction is suitable for rapid determination of phthalates in bottled water and di‐n‐butyl, butyl benzyl, and bis‐2‐ethylhexyl phthalate compounds leaching from bottles up to 36 h. Thereafter, degradation of phthalates was observed.     

Development and validation of a solid‐phase extraction method coupled with LC–MS/MS for the simultaneous determination of 16 antibiotic residues in duck meat

The present study was carried out to determine 16 antibiotics belonging to seven different groups (tetracyclines, sulfonamides, penicillins, fluoroquinolones, macrolides, lincosamides and trimethoprims) in duck meat. A solid‐phase extraction method based on Oasis HLB cartridges coupled with liquid chromatography–electrospray ionization tandem mass spectrometry was developed. Solutions of 0.1 m ethylenediaminetetraacetic acid disodium salt and 2% trifluoroacetic acid were used for the preliminary extraction of the target antibiotics from duck meat and n‐hexane was used for purification prior to solid‐phase extraction. Mobile phases composed of 0.1% trifluoroacetic acid in distilled water (solvent A) and 0.1% trifluoroacetic acid in methanol (solvent B), combined with a reversed‐phase C₁₈ analytical column, provided the optimal separation and signal intensity. The linearity of the method was assessed using six concentrations (5, 10, 20, 30, 40, and 50 μg/kg), and the recoveries, which were calculated at three spiking concentrations (5, 10 and 20 μg/kg), were in the range 69.8–103.3% with relative standard deviations (RSDs) ≤ 6.9% for the 16 tested antibiotics. Matrix effects ranging from ?47.2 to ?13.5% were observed for all the analytes, and the limits of quantitation (LOQ), which ranged from 4.93 to 26.21 μg/kg, were much lower than the maximum residue limits (MRLs) set by various regulatory authorities. Ten samples from a market were tested, and none of the target analytes were detected. Thus, a simple and versatile protocol has been developed to detect and quantify 16 antibiotics in duck meat samples.     

Liquid chromatography–tandem MS/MS method for simultaneous quantification of paracetamol,chlorzoxazone and aceclofenac in human plasma: An application to a clinical pharmacokinetic study

A facile, fast and specific method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous quantitation of paracetamol, chlorzoxazone and aceclofenac in human plasma was developed and validated. Sample preparation was achieved by liquid–liquid extraction. The analysis was performed on a reversed‐phase C₁₈ HPLC column (5 μm, 4.6 × 50 mm) using acetonitrile–10 mM ammonium formate pH 3.0 (65:35, v/v) as the mobile phase where atrovastatin was used as an internal standard. A very small injection volume (3 μL) was applied and the run time was 2.0 min. The detection was carried out by electrospray positive and negative ionization mass spectrometry in the multiple‐reaction monitoring mode. The developed method was capable of determining the analytes over the concentration ranges of 0.03–30.0, 0.015–15.00 and 0.15–15.00 μg/mL for paracetamol, chlorzoxazone and aceclofenac, respectively. Intraday and interday precisions (as coefficient of variation) were found to be ≤12.3% with an accuracy (as relative error) of ±5.0%. The method was successfully applied to a pharmacokinetic study of the three analytes after being orally administered to six healthy volunteers.     

Simultaneous quantitation of rosuvastatin and ezetimibe in human plasma by LC–MS/MS: Pharmacokinetic study of fixed‐dose formulation and separate tablets

A simple, high‐throughput and highly sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous estimation of rosuvastatin and free ezetimibe. Liquid–liquid extraction was carried out using methyl‐tert butyl ether after prior acidification from 300 μL human plasma. The recovery for both the analytes and their deuterated internal standards (ISs) ranged from 95.7 to 99.8%. Rosuvastatin and ezetimibe were separated on Symmetry C₁₈ column using acetonitrile and ammonium formate buffer, pH 3.5 (30:70, v/v) as the mobile phase. The analytes were well resolved with a resolution factor of 3.8. Detection and quantitation were performed under multiple reaction monitoring using ESI(+) for rosuvastatin (m/z 482.0 → 258.1) and ESI(−) for ezetimibe (m/z 407.9 → 271.1). A linear response function was established in the concentration ranges of 0.05–50.0 ng/mL and 0.01–10.0 ng/mL for rosuvastatin and ezetimibe, respectively, with correlation coefficient, r² ≥ 0.9991. The IS‐normalized matrix factors for the analytes ranged from 0.963 to 1.023. The developed method was successfully used to compare the pharmacokinetics of a fixed‐dose combination tablet of rosuvastatin‐ezetimibe and co‐administered rosuvastatin and ezetimibe as separate tablets to 24 healthy subjects. The reliability of the assay was also assessed by reanalysis of 115 subject samples.     

Application of a sensitive and specific LC‐ESI‐MS/MS method for the simultaneous quantification of twelve bioactive components in dog plasma for an intravenous pharmacokinetic study of Yiqifumai Injection in beagle dogs

Yiqifumai Injection is a lyophilized powder preparation widely used to treat coronary heart disease. However, its in vivo bioactive components and pharmacokinetic behavior remain unknown. Therefore a sensitive and specific LC–MS/MS was developed and validated for the simultaneous quantification of eight saponins and four lignans in beagle dog plasma. The plasma samples were pretreated by protein precipitation with methanol–acetonitrile (1:1, v/v). Chromatographic separation of all the 12 analytes and estazolam (internal standard, IS) was successfully accomplished on an Ultimate® XB‐C₈ column (100 × 2.1 mm, 3 μm) with a gradient elution system. The total running time was 8 min with a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed via positive electrospray ionization in multiple reaction monitoring mode. The assay was fully validated in terms of selectivity, linear range, lower limit of quantitation, precision, accuracy, matrix effect, recovery and stability. This validated method was successfully applied to the pharmacokinetics of 12 bioactive components after intravenous administration of Yiqifumai Injection to beagle dogs at a dose of 0.541 g/kg.