Application of dried blood spots combined with high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of vincristine and actinomycin-D

A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile–methanol–water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm ID Xbridge C₁₈ column using elution with 1 mM ammonium acetate–acetonitrile (70:30, v/v) adjusted to pH 10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycine-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.     

Development and validation of a gas chromatography-mass spectrometric method for the determination of sildenafil and desmethyl-sildenafil in whole blood

Sildenafil (SDL) is a phosphodiesterase type 5 inhibitor and it is approved for the treatment of erectile dysfunction and pulmonary hypertension. SDL is extensively metabolized to its pharmacologically active metabolite, desmethyl‐sildenafil (DSDL). A sensitive and specific GC/MS method for the determination of SDL and DSDL in whole blood was developed and validated to support therapeutic drug monitoring of SDL patients. The combination of solid‐phase extraction with derivatization using BSTFA with 1% TMCS in acetonitrile efficiently reduced matrix effect and improved sensitivity of the method. In this assay, protriptyline was used as internal standard for both analytes. The LODs were 1.50 and 5.00 ng/mL for SDL and DSDL, respectively, whereas the respective LOQs were 5.00 and 15.0 ng/mL. The calibration curves were linear up to 500.0 ng/mL (SDL: R² 0.992, DSDL: R² 0.990). Absolute recovery values for both analytes ranged from 83.1 to 93.2%. Within‐ and between‐batch accuracy was less than 11.8 and 10.2%, respectively, whereas within‐ and between‐batch precision was less than 8.1 and 10.8%, correspondingly. The developed method is suitable for the determination of SDL and DSDL concentrations in blood samples obtained from patients under Viagra^® treatment, for pharmacokinetic studies or for the investigation of related forensic cases.     

Development of a rapid and sensitive SPE-LC-MS/MS method for the simultaneous estimation of fluoxetine and olanzapine in human plasma

A high‐performance liquid chromatographic assay with tandem mass spectrometric detection was developed to simultaneously quantify fluoxetine and olanzapine in human plasma. The analytes and the internal standard (IS) duloxetine were extracted from 500 μL aliquots of human plasma through solid‐phase extraction. Chromatographic separation was achieved in a run time of 4.0 min on a Hypersil Gold C₁₈ column (50 × 4.6 mm, 5 µm) using isocratic mobile phase consisting of acetonitrile–water containing 2% formic acid (70:30, v/v), at a flow‐rate of 0.5 mL/min. Detection of analytes and internal standard was performed by electrospray ionization tandem mass spectrometry, operating in positive‐ion and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions monitored for fluoxetine, olanzapine and IS were m/z 310.01 → 147.69, 313.15 → 256.14 and 298.1 → 153.97, respectively. The method was validated over the concentration range of 1.00–150.20 ng/mL for fluoxetine and 0.12–25.03 ng/mL for olanzapine in human plasma. The intra‐batch and inter‐batch precision (%CV) across four quality control levels was ≤6.28% for both the analytes. In conclusion, a simple and sensitive analytical method was developed and validated in human plasma. This method is suitable for measuring accurate plasma concentration in bioequivalence study and therapeutic drug monitoring as well, following combined administration. Copyright © 2011 John Wiley & Sons, Ltd.     

A chiral liquid chromatography method for the simultaneous determination of oxcarbazepine, eslicarbazepine, R-licarbazepine and other new chemical derivatives BIA 2-024, BIA 2-059 and BIA 2-265, in mouse plasma and brain

Recently, in silico models have been developed to predict drug pharmacokinetics. However, before application, they must be validated and, for that, information about structurally similar reference compounds is required. A chiral liquid chromatography method with ultraviolet detection (LC‐UV) was developed and validated for the simultaneous quantification of BIA 2–024, BIA 2–059, BIA 2–265, oxcarbazepine, eslicarbazepine (S‐licarbazepine) and R‐licarbazepine in mouse plasma and brain. Compounds were extracted by a selective solid‐phase extraction procedure and their chromatographic separation was achieved on a LiChroCART 250–4 ChiraDex column using a mobile phase of water–methanol (92:8, v/v) pumped at 0.7 mL/min. The UV detector was set at 235 nm. Calibration curves were linear (r² ≥ 0.996) over the concentration ranges of 0.2–30 µg/mL for oxcarbazepine, eslicarbazepine and R‐licarbazepine; 0.2–60 µg/mL for the remaining compounds in plasma; and 0.06–15 µg/mL for all the analytes in brain homogenate. Taking into account all analytes at these concentration ranges in both matrices, the overall precision did not exceed 9.09%, and the accuracy was within ±14.3%. This LC‐UV method is suitable for carrying out pharmacokinetic studies with these compounds in mouse in order to obtain a better picture of their metabolic pathways and biodistribution. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a RP-HPLC method for simultaneous quantification of bedaquiline (TMC207), moxifloxacin and pyrazinamide in a pharmaceutical powder formulation for inhalation

A reversed phase high performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of bedaquiline (TMC207), moxifloxacin and pyrazinamide in a pharmaceutical powder formulation for inhalation has been developed and validated. The powder was simply dissolved in methanol and the analytes separated in a run time of 20?min on a Luna C18 (2) (150?×?4.6?mm, 5?µm) column using gradient elution with methanol and triethylamine phosphate buffer (pH 2.5) delivered at 1.2?mL/min. The detection (with retention time) was carried out at 269?nm (2.9?min) for pyrazinamide, 296?nm (7.0?min) for moxifloxacin and 225?nm (16.3?min) for bedaquiline, respectively. The method was linear for all analytes in the concentration range 1-100?µg/mL with correlation coefficients >0.998. Lower limits of quantitation (µg/mL) of bedaquiline, moxifloxacin and pyrazinamide were 0.56, 0.43 and 0.24, respectively. The method was accurate (relative error in the range ?0.2 to 2.2) and precise (%RSD ≤6.2) with recovery in the range 100.0–104.7%. The method was successfully applied to determine the drug content and content uniformity of the three analytes in a spray-dried combination powder formulation for inhalation containing L-leucine.     

Simultaneous quantification of olanzapine and its metabolite N‐desmethylolanzapine in human plasma by liquid chromatography tandem mass spectrometry for therapeutic drug monitoring

A simple, sensitive, and selective liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of olanzapine (OLZ) and its metabolite N‐desmethylolanzapine (DMO) in human plasma for therapeutic drug monitoring. Sample preparation was performed by one‐step protein precipitation with methanol. The analytes were chromatographed on a reversed‐phase YMC‐ODS‐AQ C₁₈ Column (2.0 × 100 mm,3 µm) by a gradient program at a flow rate of 0.30 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in positive ion mode. The method was validated for selectivity, linearity, accuracy, precision, matrix effect, recovery and stability. The calibration curve was linear over the concentration range 0.2–120 ng/mL for OLZ and 0.5–50 ng/mL for DMO. Intra‐ and interday precisions for OLZ and DMO were <11.29%, and the accuracy ranged from 95.23 to 113.16%. The developed method was subsequently applied to therapeutic drug monitoring for psychiatric patients receiving therapy of OLZ tablets. The method seems to be suitable for therapeutic drug monitoring of patients undergoing therapy with OLZ and might contribute to prediction of the risk of adverse reactions. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

Development of hollow fiber‐supported liquid‐phase microextraction and HPLC‐DAD method for the determination of pyrethroid metabolites in human and rat urine

A simple hollow fiber liquid‐phase microextraction method for the determination of synthetic pyrethroid metabolites, 3‐phenoxybenzoic acid and 4‐hydroxy‐3‐phenoxybenzoic acid, in human and rat urine was developed and validated. A polypropylene hollow fiber tightly fitted onto a Nylon rod and impregnated with organic solvent served as a disposable extraction device. Desorption of analytes was carried out in NaOH solution, analyzed further by gradient HPLC and diode array detection method. Important factors were identified using Taguchi OA16 (4⁵) orthogonal array design and further optimized using univariate approach. The optimum method performance was observed when 1 mL of urine hydrolyzed with 0.2 mL of concentrated HCl was further supplemented with 100 mg of NaCl and extracted for 120 min into dihexyl ether immobilized in the pores of the hollow fiber. Metabolites were desorbed into 0.1 mL of 0.1 M NaOH for another 120 min. Limits of detection and quantitation of 15 and 50 ng/mL were obtained for both analytes. Relative standard deviations of 1.6–12.6% over the linear range (50–10,000 ng/mL, r > 0.9906) were observed. Intra‐ and inter‐day accuracies of the method ranged from 98.3 to 109.5% and from 93.3 to 110.9%, respectively. The optimized method was applied to the analysis of real urine samples collected from rats exposed orally to cypermethrin. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of five bioactive constituents in the traditional Chinese medicinal preparation refined xue-fu-zhu-yu-tang by high-performance liquid chromatography with diode-array UV detection

A high-performance liquid chromatographic method for the simultaneous determination of oxypaeoniflorin, paeoniflorin, ferric acid, naringin, and neohesperidin in a traditional Chinese medicinal preparation, refined xue-fu-zhu-yu-tang, was developed. Separations were carried out with a ZORBAX SB-C₁₈ column along with an Agilent C₁₈ precolumn by linear gradient elution using 1% acetic acid-methanol (v/v: 0 min, 80/20; 15 min, 70/30; 35 min, 60/40; 55 min, 60/40) as the mobile phase at a flow rate of 0.5 mL/min. The analytes were detected with a diode-array detector at their maximum UV wavelengths. The correlation coefficients of the calibration curve for the analytes exceeded 0.9999. The recoveries were in the range 95.95–104.42 % with RSD less than 1.85%, and the precision of this method was better than 1.80%. The effects of several factors on the chromatographic behavior and sample extraction of the analytes were compared. In addition, the contents of these bioactive constituents in six different samples of this preparation were analyzed by using the optimized high-performance liquid chromatographic method. The text was submitted by the authors in English.     

Determination of β-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic-tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1 mL of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, v/v) as organic phase with an extraction time of 30 min. After extraction, the analytes were resolved within 5 min using a mobile phase consisting of methanol-ammonium acetate (10 mmol L⁻¹, pH 5.0, 80:20, v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000 ng mL⁻¹ for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation.     

An automated method for the simultaneous measurement of azole antifungal drugs in human plasma or serum using turbulent flow liquid chromatography-tandem mass spectrometry

Azole antifungal drugs are important in the prophylaxis and treatment of invasive aspergillosis. Therapeutic drug monitoring may be indicated to (1) monitor adherence, (2) guide dosage and (3) minimise the risk of drug–drug interactions and dose-related toxicity. TurboFlow^TM technology offers online, automated sample preparation. An Aria Transcend^TM TLX-II coupled with a TSQ Vantage^TM MS was used. Centrifuged samples (25 μL) were mixed with internal standard solution (975 μL) and 30 μL injected directly onto a C18-P-XL TurboFlow column. Analytes were focussed onto a Phenomenex Gemini Phenyl analytical column and eluted using a methanol/water gradient (flow-rate, 0.8 mL/min). Analytes were monitored in selected reaction monitoring mode (two transitions per analyte, positive mode APCI). Calibration ranges were as follows: itraconazole, hydroxyitraconazole, and posaconazole 0.05–5.0 mg/L; voriconazole and fluconazole 0.1–10 mg/L. Total analysis time was 12 min. TurboFlow column recovery was >77% for all analytes. Calibration was linear (R ²?>?0.99) for all analytes. Inter- and intra-assay imprecision (% RSD) was <8% and accuracy (nominal internal quality control values) 90–105% for all analytes. The limit of detection was 0.01 mg/L for all analytes. No matrix effects were observed. This method is simple, robust and suitable for measuring these compounds at concentrations attained during therapy.     

Simultaneous determination of methotrexate and metoclopramide in physiological fluids using RP-HPLC with ultra-violet detection; application in evaluation of polymeric nanoparticles

Methotrexate (MTX) is an anticancer drug while metoclopramide (MCP) is an antiemetic agent. Both the drugs are commonly coprescribed to avoid the emesis caused by anticancer drug. In this study, a novel, rapid, sensitive, and cost-effective reverse-phase high-performance liquid chromatography method was developed and validated for simultaneous determination of the methotrexate and metoclopramide in biological and pharmaceutical samples using sparfloxacin as internal standard. The analytes were separated on a Kromasil 100-5C18 RP (250?×?4.6?mm, 5?µm) column, methanol, and 0.05% trifloroacetic acid (36:64?v/v) as mobile phase with a flow rate of 1?mL/min, detection wavelength of 290?nm, and column oven temperature at 40°C. Both the analytes were extracted from physiological fluids (bovine aqueous humor, vitreous humor, and human plasma) using mixture of methanol and 10% perchloric acid (50:50 v/v). The method was linear over the concentration range of 0.025–1.0?µg/mL for methotrexate and 0.030–1.0?µg/mL for metoclopramide. The % recovery from human plasma was 98.57 and 96.74% for MTX and MCP, respectively, while from aqueous humor and vitreous humor was 95.84 and 98.51% for MTX.

The developed method was applied for in vitro release of MTX from polymeric nanoparticles and can be applied for analysis of pharmaceutical and biological samples containing both the drugs.     

Simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by LC‐MS/MS using supported liquid extraction

A rapid, sensitive and selective bioanalytical method was developed for the simultaneous determination of fluoxetine and its primary metabolite norfluoxetine in human plasma. Sample preparation was based on supported liquid extraction (SLE) using methyl tert‐butyl ether to extract the analytes from human plasma. Chromatography was performed on a Synergi 4 μ polar‐RP column using a fast gradient. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM functions, m/z 310 → 44 for fluoxetine, m/z 296 → 134 for norfluoxetine and m/z 315 → 44 for fluoxetine‐d₅ (internal standard). The method is linear over the range of 0.05–20 ng/mL (using a human plasma sample volume of 0.1 mL) with a coefficient determination of greater than 0.999. The method is accurate and precise with intra‐batch and inter‐batch accuracy (%bias) of <±15% and precision (%CV) of <15% for both analytes. A run time of 4 min means a high throughput of samples can be achieved. To our knowledge, this method appears to be the most sensitive one reported so far for the quantitation of fluoxetine and norfluoxetine and can be used for routine therapeutic drug monitoring or pharmacokinetic studies. Copyright © 2011 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.     

Solid phase extraction for GC-FID determination of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and methamphetamine (MA) in human urine

A simple solid phase extraction method was developed for estimating the amounts of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and methamphetamine (MA) in urine by using the GC-FID technique. The urine sample was alkalinized prior to undergoing solid phase extraction using Oasis HLB®. A 5% methanol-water mixture containing 2% ammonium hydroxide was used for washing, whereas a 70% methanol-water mixture containing 2% acetic acid was used for elution. The compounds were analyzed using the standard GC-FID conditions previously established for ecstasy samples, i.e., column: CP-SIL 24 CB WCOT (30 m × 0.32 mm i.d., 0.25 μm film thickness); carrier gas: N₂ (2.6 mL/min); injector temperature: 290°C; detector temperature: 300°C; oven temperature: initial 80°C, final 270°C (1 min), ramp rate 20°C/min. Validation demonstrated the linearity of the calibration curves between 1 and 20 μg/mL (r > 0.99) for all analytes. The precisions (% RSD) were approximately 3–17%, 6–16% and 7–17% for MDMA, MDA and MA, respectively. The accuracies (% DEV) were (?)17-(+)5%, (?)18-(+)15% and (?)18-(+)0.6% for MDMA, MDA and MA, respectively. The recovery ranged from 80 to 107% and the lower limit of quantification (LLOQ) was 1 μg/mL. The method was successfully applied to determine the levels of these compounds in the urine of drug abuse suspects.     

Development of a sensitive ultra high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of nine active compounds in rat plasma and its application to a pharmacokinetic study after administration of Viscum coloratum extracts

A simple, specific, and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous quantification of nine compounds including a new compound, rhamnazin‐3‐Ο‐β‐d ‐(6″‐β‐hydroxy‐β‐methyglutaryl)‐β‐d ‐glucoside‐4′‐Ο‐β‐d ‐glucoside, in rat plasma using baicalin as an internal standard. The plasma samples were pretreated and extracted by protein precipitation with 0.2% formic acid in acetonitrile. The analytes were separated on a Thermo Syncronis C₁₈ column by gradient elution with a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at a flow rate of 0.25 mL/min. The detection of the analytes was performed on an electrospray ionization interface operating in positive‐ion and multiple reaction monitoring acquisition modes. The calibration curves of these analytes showed good linearity (r > 0.99) within the test ranges. The lower limit of quantification ranged from 0.4 to 20.1 ng/mL for the analytes. The intra‐ and interday precision and accuracy were all within ±15%, and the recoveries were higher than 80.0%. The validated method was successfully applied to a pharmacokinetic study of the nine flavonoids after administration of the Viscum coloratum extracts by intravenous injection.     

High‐throughput LC–MS/MS method with 96‐well plate precipitation for the determination of arotinolol and amlodipine in a small volume of rat plasma: Application to a pharmacokinetic interaction study

A rapid, sensitive, and selective liquid chromatography with tandem mass spectrometry method was developed and fully validated for the simultaneous quantification of arotinolol and amlodipine in rat plasma. Two internal standards were introduced with metoprolol as the internal standard of arotinolol and (S)‐amlodipine‐d4 as the internal standard of amlodipine. The analytes were isolated from 50.0 μL plasma samples by a simple protein precipitation using acetonitrile. The chromatographic separation was achieved in 5 min on a C18 column. The mobile phase consisted of phase A 5% methanol and phase B 95% methanol (both containing 0.5% formic acid and 5 mM ammonium acetate) and was delivered in gradient elution at 0.300 mL/min. Quantification was performed in multiple reaction monitoring mode with the transition m/z 372.1 → 316.1 for arotinolol, m/z 268.2 → 116.2 for metoprolol, m/z 409.1 → 238.1 for amlodipine and m/z 413.1 → 238.1 for (S)‐amlodipine‐d4. Linearity was obtained over the range of 0.200–40.0 ng/mL for arotinolol (r² = 0.9988) and 0.500–100 ng/mL for amlodipine (r² = 0.9985) in rat plasma. The validated data have met the acceptance criteria in FDA guideline. This method was successfully applied to a pharmacokinetic interaction study in rats, and the results indicated that there was no significant drug–drug interaction between arotinolol and amlodipine.     

Liquid chromatography tandem mass spectrometry method for the simultaneous stereoselective determination of venlafaxine and its major metabolite,O‐desmethylvenlafaxine,in human plasma

A sensitive, accurate and highly stereoselective assay for the simultaneous determination of venlafaxine (VEN) and its equipotent metabolite, O‐desmethyl venlafaxine (ODV), in human plasma was developed and validated. Analytes were simultaneously extracted from plasma using solid‐phase extraction and detected by tandem mass spectrometry in positive ion mode with a turbo ion spray interface. Deuterium‐labeled VEN and ODV were used as internal standards. Chromatographic separation was performed on a Chiral AGP column, using a time programmed gradient flow with a total run time of 16 min. The method has a lower limit of quantitation of 0.60 ng/mL. The assay was linear over a range 0.60–300.00 ng/mL for both the enantiomers of VEN and ODV, respectively, with coefficient of correlation > 0.99. The extraction recoveries were >77.0% on an average for all the four analytes. The analytes were found stable in plasma through three freeze (?15 °C) and thaw cycles and under storage at room temperature for 8 h, and also in mobile phase at 10 °C for 54 h. The method has shown good reproducibility, with intra‐ and inter‐day variation coefficients < 9%, for all the analytes, and has proved to be very reliable for analysis of VEN and its metabolite in clinical study samples. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Simultaneous determination of sunitinib and its two metabolites in plasma of Chinese patients with metastatic renal cell carcinoma by liquid chromatography–tandem mass spectrometry

A simple and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous determination of sunitinib and its two metabolites in plasma of Chinese patients with metastatic renal cell carcinoma (mRCC). After simple one‐step protein precipitation with methanol–acetonitrile (1:1, v/v), all three analytes were separated on an Agilent Zorbax SB‐C₁₈ column using a gradient mobile phase consisting of water (0.1% formic acid)–acetonitrile (0.1% formic acid) at a flow rate of 0.50 mL/min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 399.0 → 326.2, m/z 371.0 → 283.1, m/z 343.0 → 283.1 and m/z 386.3 → 122.2 for sunitinib, M1, M2 and buspirone, respectively. The method was linear over the range of 0.10–100 ng/mL for all three analytes using only 50 μL of plasma and the lower limit of quantifications for the three analytes were all 0.10 ng/mL. The intra‐day and inter‐day precisions were all less than 15% and the accuracies were within the range of ±15%; recoveries were between 85.0 and 115%. The validated method was successfully applied to an explorative pharmacokinetic study of sunitinib in Chinese patients with mRCC following multi‐dose oral administration. Copyright © 2012 John Wiley & Sons, Ltd.