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.     

Screening and determination of drugs in human saliva utilizing microextraction by packed sorbent and liquid chromatography–tandem mass spectrometry

This study presents a new method for collecting and handling saliva samples using an automated analytical microsyringe and microextraction by packed syringe (MEPS). The screening and determination of lidocaine in human saliva samples utilizing MEPS and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) were carried out. An exact volume of saliva could be collected. The MEPS C₈‐cartridge could be used for 50 extractions before it was discarded. The extraction recovery was about 60%. The pharmacokinetic curve of lidocaine in saliva using MEPS‐LC‐MS/MS is reported. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Development of a sensitive LC‐MS/MS method for simultaneous quantification of eleven constituents in rat serum and its application to a pharmacokinetic study of a Chinese medicine Shengmai injection

A sensitive LC‐MS/MS method was developed and validated for simultaneous quantification of 11 constituents, ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B, in rat serum using digoxin as the internal standard (IS). The serum samples were pretreated and extracted with a two‐step liquid–liquid extraction. Chromatographic separation was achieved on a C₁₈ analytical column with a proper gradient elution using 0.02% acetic acid aqueous solution and 0.02% acetic acid–acetonitrile as mobile phase at a flow rate of 0.5 mL/min. MS detection was performed using multiple reaction monitoring via an electrospray ionization source. Good linearity was observed in the validated concentration range for every analyte (r² ≥0.9929), and the lower limits of quantitation of the analytes were in the range of 0.044–1.190 ng/mL in rat serum. Intra‐ and inter‐day precisions were <14.2%. The accuracy expressed as recovery was within the range of 85.1–112.8%. The extraction recoveries were >75.8%.The validated method was successfully applied to a pharmacokinetic study of all analytes in rats after single intravenous administration of Shengmai injection. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Development and application of a LC–MS/MS assay for the simultaneous quantification of edaravone and taurine in beagle plasma

An LC–MS/MS method was developed and validated for the simultaneous quantification of edaravone and taurine in beagle plasma. The plasma sample was deproteinized using acetonitrile containing formic acid. Chromatographic separations were achieved on an Agilent Zorbax SB‐Aq (100 × 2.1 mm, 3.5 μm) column, with a gradient of water (containing 0.03% formic acid) and methanol as the mobile phase at a flow rate of 0.3 mL/min. The analyte detection was carried out in multiple reaction monitoring mode and the optimized precursor‐to‐product transitions of m/z [M+H]⁺ 175.1 → 133.0 (edaravone), m/z [M+H]⁺ 189.1 → 147.0 (3‐methyl‐1‐p‐tolyl‐5‐pyrazolone, internal standard, IS), m/z [M–H]^? 124.1→80.0 (taurine), and m/z [M–H]^? 172.0 → 80.0 (sulfanilic acid, IS) were employed to quantify edaravone, taurine, and their corresponding ISs, respectively. The LOD and the lower LOQ were 0.01 and 0.05 μg/mL for edaravone and 0.66 and 2 μg/mL for taurine, respectively. The calibration curves of these two analytes demonstrated good linearity (r ＞ 0.99). All the validation data including the specificity, precision, recovery, and stability conformed to the acceptable requirements. This validated method has successfully been applied in the pharmacokinetic study of edaravone and taurine mixture in beagle dogs.     

Liquid chromatography–tandem mass spectrometric assay for the determination of tetrabenazine and its active metabolites in human plasma: a pharmacokinetic study

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and fully validated for the simultaneous quantification of tetrabenazine and its active metabolites α‐dihydrotetrabenazine and β‐dihydrotetrabenazine in human plasma. Tetrabenazine d7 was used as internal standard (IS). The analytes were extracted from 200 μL aliquots of human plasma via solid‐phase extraction using C₁₈ solid‐phase extraction cartridges. The reconstituted samples were chromatographed on a Zorbax SB C₁₈ column using a 60:40 (v/v) mixture of acetonitrile and 5 mm ammonium acetate as the mobile phase at a flow rate of 0.8 mL/min. The API‐4000 LC‐MS/MS in multiple reaction‐monitoring mode was used for detection. The calibration curves obtained were linear (r² ≥ 0.99) over the concentration range of 0.01–5.03 ng/mL for tetrabenazine and 0.50–100 ng/mL for α‐dihydrotetrabenazine and β‐dihydrotetrabenazine. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a validated UPLC–MS/MS method for determination of humantenmine in rat plasma and its application in pharmacokinetics and bioavailability studies

Humantenmine (HMT), the most toxic compound isolated from Gelsemium elegans Benth , is a well‐known active herbal compound. A rapid and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated to estimate the absolute oral bioavailability of HMT in rats. Quantification was performed by multiple reaction monitoring using electrospray ionization operated in positive ion mode with transitions of m/z 327.14 → m/z 296.19 for HMT and m/z 323.20 → m/z 236.23 for gelsemine (internal standard, IS). The linear range of the calibration curve was 1–256 nmol/L, with a lower limit of quantification at 1 nmol/L. The accuracy of HMT ranged from 89.39 to 107.5%, and the precision was within 12.24% (RSD). Excellent recovery and negligible matrix effect were observed. HMT remained stable during storage, preparation and analytical procedures. The pharmacokinetics of HMT in rats showed that HMT reached the concentration peak at 12.50 ± 2.74 min with a peak concentration of 28.49 ± 6.65 nmol/L, and the corresponding area under the concentration–time curve (AUC_0–t ) was 1142.42 ± 202.92 nmol/L min after 200 μg/kg HMT was orally administered to rats. The AUC_0–t of HMT given at 20 μg/kg by tail vein administration was 1518.46 ± 192.24 nmol/L min. The calculated absolute bioavailability of HMT was 7.66%.     

Noninvasive determination of human cortisol and dehydroepiandrosterone sulfate using liquid chromatography-tandem mass spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurements of steroids in human saliva has garnered increased interest in the area of clinical psychoneuroendocrinological research. However, performance characteristics of LC-MS/MS methods for the analysis of steroids in saliva are limited. Human saliva samples were collected via passive drool. Cortisol and dehydroepiandrosterone sulfate (DHEA-S) in the samples were extracted together, resolved on a C18-A column, and analyzed using tandem mass spectrometry. The LC-MS/MS method had limits of quantitation of 0.03 and 0.06 ng/mL for DHEA-S and cortisol, respectively. Method evaluations showed coefficient variation (%CV) of inter-assay ranging 4.6–17.9% for DHEA-S and cortisol, recoveries of 102.4–109.5% for DHEA-S and 94.6–98.3% for cortisol, and assay linearity with R² = 0.9964 for DHEA-S (1.0–25.0 ng/mL) and R² = 0.997 (1.0–25.0 ng/mL) for cortisol. No cross contamination among samples was observed. Human saliva showed 20% and 18% ion enhancement effect for DHEA-S and cortisol assay, respectively. No interference by ten common steroids was detected. Regression analysis of method comparisons with laboratory-developed test (LDT) method revealed R² = 0.9688 (LC-MS/MS = 0.9665 LDT-LC-MS/MS − 0.7355) for cortisol, and R² = 0.9039 (LC-MS/MS = 1.0173 LDT-LC-MS/MS + 3.6797) for DHEA-S. Reference ranges for young adults were determined to be 0.3–5.9 ng/mL for females and 0.1–5.6 ng/mL for males for salivary cortisol, and 0.6–7.4 ng/mL for females and 0.6–10.1 ng/mL for males for salivary DHEA-S. An LC-MS/MS method for quantifying cortisol and DHEA-S in human saliva was developed and validated for clinical and psychoneuroendocrinological research that require noninvasive means of measuring these hormones.

     

Application of nanofiber‐packed SPE for determination of salivary‐free cortisol using fluorescence precolumn derivatization and HPLC detection

Salivary cortisol has emerged as an easy‐to‐collect biologic marker of stress in many researches. In this study, we present a method for the determination of salivary‐free cortisol using HPLC method with fluorescence precolumn derivatization, which is based on a novel extraction from the strongly acidic medium (fluorescent derivatives of cortisol in sulfuric acid medium) by electrospun polystyrene nanofibers packed SPE. For high‐throughput sample extraction, an array pretreatment device based on nanofibers packed SPE micro‐column was designed. The LOD of cortisol was 0.01 μg/L (S/N=3). The RSDs (n=6) for all analytes were below 8.0%, and the recoveries were 110, 102.4, and 99.4% (n=3) for saliva spiked with 0.1, 10, and 20 μg/L of cortisol, respectively. The proposed method was then successfully applied in the determination of free cortisol in human saliva. The salivary cortisol concentrations in the real samples ranged from 0.22 to 7.45 μg/L. The nanofiber‐packed SPE overcame the low extraction recovery and bad clean‐up effect of the conventional methods, and increased the sensitivity and selectivity of the method.     

Rapid and simple determination of psychotropic phenylalkylamine derivatives in urine by direct injection liquid chromatography–electrospray ionization–tandem mass spectrometry

A direct injection liquid chromatography–electrospray ionization–tandem mass spectrometric method (LC‐ESI‐MS/MS) was developed and validated for the rapid and simple determination of 13 phenylalkylamine derivatives. Eight deuterium‐labeled compounds were prepared for use as internal standards (ISs) to quantify the analytes. Urine samples mixed with ISs were centrifuged, filtered through 0.22 µm filters and then injected directly into the LC‐ESI‐MS/MS system. The mobile phase was composed of 0.2% formic acid and 2 mM ammonium formate in distilled water and 0.2% formic acid and 2 mM ammonium formate in acetonitrile. The analytical column was a Capcell Pak MG‐II C₁₈ (150 × 2.0 mm i.d., 5 µm, Shiseido). Separation and detection of the analytes were accomplished within 10 min. The linear ranges were 5–750 ng/mL (ephedrine and fenfluramine), 10–750 ng/mL (3,4‐methylenedioxyamphetamine, phendimetrazine, methamphetamine, 3,4‐methylenedioxyethylamphetamine and benzphetamine), 20–750 ng/mL (norephedrine, amphetamine, phentermine and ketamine) and 30–1000 ng/mL (3,4‐methylenedioxymethamphetamine and norketamine), with determination coefficients, R², ≥ 0.9967. The intra‐day and inter‐day precisions were within 19.1%. The intra‐day and inter‐day accuracies ranged from ?16.0 to 18.7%. The lower limits of quantification for all the analytes were lower than 26.5 ng/mL. The applicability of the method was examined by analyzing urine samples from drug abusers (n = 30). 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.     

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.     

Development and validation of a rapid and high‐sensitivity liquid chromatography–tandem mass spectrometry assay for the determination of neostigmine in small‐volume beagle dog plasma and its application to a pharmacokinetic study

A simple, rapid and high sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the determination of neostigmine in small‐volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96‐well filtration plate, the filtrate was directly injected into the LC‐MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) with a mobile phase composed of methanol–water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 μL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor‐to‐product ion transitions m/z 223.0 → 72.0 and 306.0 → 140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1–100ng/mL for neostigmine (r ≥ 0.998). All the validation data, such as accuracy, intra‐run and inter‐run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Isolation and characterization of free haem from the shell gland of quail and hen

Free haem was isolated from the shell gland of the quail, Coturnix coturnix japonica, and of the fowl, Galinus domesticus, and characterized by HPLC‐ESI‐MS/MS. Quantification by HPLC gave values of 1.17–1.40 nmol/mg quail shell gland protein for haem, 1.66–2.17 nmol/mg protein for protoporphyrin and 0.25–0.40 nmol/mg protein for biliverdin. Possible implications of this previously unreported finding are discussed but they are not considered incompatible with the conclusion that all eggshell pigments are endogenously synthesized in the oviduct system. Copyright © 2011 John Wiley & Sons, Ltd.     

A UFLC–MS/MS method for the simultaneous determination of eight bioactive constituents from red wine and dealcoholized red wine in rat plasma: Application to a comparative pharmacokinetic study

To explore whether alcohol has an effect on the pharmacokinetic behavior of phenolic acids, the main bioactive constituents in red wine, a highly sensitive and simple ultra‐fast liquid chromatography coupled with triple quadrupole mass spectrometry (UFLC–MS/MS) method was developed for simultaneous quantitation of eight phenolic acids in plasma samples. Plasma samples were extracted by liquid–liquid extraction and the chromatographic separation was achieved on a Zorbax SB‐C₁₈ column within 7.0 min. Results of the validated method revealed that all of the calibration curves displayed good linear regression (r > 0.99). The intra‐ and inter‐day precisions of the analytes were <14.0% and accuracies ranged from ?8.5 to 7.3%. The extraction recoveries of the analytes were from 71.2 to 110.2% and the matrix effects ranged from 86.2 to 105.5%. The stability of these compounds under various conditions satisfied the requirements of biological sample measurement. The method was successfully applied to a comparative pharmacokinetic study of phenolic acids in rat plasma. For gallic acid and gentisic acid, the parameters AUC_0–t and AUC_0–∞ increased remarkably (p < 0.05) after oral administration of red wine, which suggested that alcohol might enhance their absorption. This is the first report to compare the pharmacokinetic behavior of phenolic acids in red wine and dealcoholized red wine.     

Residual detection of naproxen,methyltestosterone and 17α‐hydroxyprogesterone caproate in aquatic products by simple liquid–liquid extraction method coupled with liquid chromatography–tandem mass spectrometry

In the present study, we aimed to develop a reliable screening method based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the detection and quantification of naproxen, methyltestosterone and 17α‐hydroxyprogesterone caproate residues. The target analytes were extracted from samples of eel, flatfish and shrimp using acetonitrile with 1% acetic acid, followed by liquid–liquid purification with n‐hexane. Chromatographic separation was achieved on a reversed‐phase analytical column using 0.1% formic acid containing 10 mm ammonium formate in distilled water (A) and methanol (B) as mobile phases. All the matrix‐matched calibration curves were linear (R² ≥ 0.99) over the concentration range of the tested analytes. Recovery at three spiking levels (0.005, 0.01 and 0.02 mg/kg) ranged from 68 to 117% with intra‐ and inter‐day precisions <10%. Five market samples for each matrix (eel, flatfish and shrimp) were collected and tested for method application. In summary, the proposed method is feasible to screen and quantify the analytes with high selectivity in aquatic food products meant for human consumption.     

Investigation of bacterial viability from incubated saliva by application of flow cytometry and hyphenated separation techniques

The aim of the study was determination of bacterial viability in saliva samples and finding a correlation between microbiological and volatile profiles of saliva depending on incubation time. Bacteria colonizing healthy oral cavities were also identified. Twelve healthy adults donated unstimulated saliva samples. Flow cytometry, optical density measurements and colony‐forming unit (CFU) counting method were employed for analyses of native and inoculated saliva after 0, 1, 2, 24, and 48 h of incubation. Volatile profiles were acquired using headspace‐solid phase microextraction‐gas chromatography/mass spectrometry (HS‐SPME‐GC/MS). Oral bacteria were the most viable within 2 h after collection of saliva. Extension of incubation time to 48 h caused considerable decrease in live bacteria counts and sharp increase in dead bacteria counts. The most prevalent strain was Sphingomonas paucimobilis (26.67%). The number of volatiles raised from 5 to 27 with incubation time and most of them were putrefaction products, such as methanethiol, indole and pyrrole. HS‐SPME‐GC/MS method is insufficient for volatile profiling of “fresh” saliva and should be directed rather to investigation of bacterial metabolites.     

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.     

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.