Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control

The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Analytes were extracted from equine plasma by liquid-liquid extraction using a mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v) and separated on a sub-2 micron C(18) column. Detection of analytes was achieved on a triple-quadrupole mass spectrometer by positive electrospray ionization mode with selected reaction monitoring (SRM). Mobile phase comprised 2 mM ammonium formate and methanol. Deuterium-labeled testosterone enanthate and testosterone undecanoate were used as dual-internal standards for quantification. Limits of detection (LOD) and quantification (LOQ) were 25-100 pg/mL and 100-200 pg/mL, respectively. The linear dynamic range of quantification was 100-10,000 pg/mL. For confirmation of the presence of these analytes in equine plasma, matching of the retention time with mass spectrometric ion ratios from MS/MS product ions was used. The limit of confirmation (LOC) was 100-500 pg/mL. The method is sensitive, robust, selective and reliably reproducible.     

Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry

Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-nortestosterone-17beta-sulphate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) had potential for the detection of 19-nortestosterone misuse in the male horse. The high concentrations of sulphate conjugates of the female sex hormones naturally present in male equine urine were overcome by selective hydrolysis of the aryl sulphates using glucuronidase from Helix pomatia; this was shown to have little or no activity for alkyl sulphates such as 19-nortestosterone-17beta-sulphate. The 'free' phenolic steroids were removed by solid-phase extraction (SPE) prior to LC/MS/MS analysis. The method also allowed for the quantification of the sulphate conjugate of boldenone, a further anabolic steroid endogenous in the male equine with potential for abuse in sports. The method was applied to the quantification of these analytes in a population of samples. This paper reports the results of that study along with the development and validation of the LC/MS/MS method. The results indicate that while 19-nortestosterone-17beta-sulphate is present at low levels as an endogenous substance in urine from 'normal' male horses, its use as an effective threshold substance may be viable. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Hair analysis of anabolic steroids in connection with doping control-results from horse samples

Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses. In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to detect anabolic steroids and also to obtain further information about previous abuse. Moreover, the process of incorporation of steroids into hairs is described and the consequences on interpretation are discussed, e.g. on the retrospective estimation of the application date. The chosen examples deal with the detection of the anabolic agent testosterone propionate. Hair samples of an application study, as well as a control sample taken from a racing horse, were referred to. Hair material was investigated by a screening procedure including testosterone, nandrolone and several esters (testosterone propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate, dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg). Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).     

Resolution, quantification and confirmation of betamethasone and dexamethasone in equine plasma by liquid chromatography/tandem mass spectrometry

This method describes the simultaneous separation, identification, quantification and confirmation of betamethasone (BTM) and dexamethasone (DXM) in equine plasma by liquid chromatography (LC) integrated with multidimensional tandem mass spectrometry. Analytes were directly extracted from equine plasma by methyl tert-butyl ether (MTBE). The residues were reconstituted with sample solvent. LC separation of the analytes was performed on a Hypercarb column using acetonitrile/water/formic acid (95:5:0.5, v/v/v) as the mobile phase. Sample screening, quantification and confirmation were performed in multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1-75 ng/mL for both analytes. Limit of detection (LOD) was 50 pg/mL and that of quantification (LOQ) was 100 pg/mL for both analytes. The limit of confirmation (LOC) for the presence of BTM or DXM by MRM was 0.5 ng/mL. The intra-and inter-day precisions expressed as coefficient of variation (CV) for quantification of DXM and BTM from 0.1 to 50 ng/mL were less than 7% and the accuracy was in the range of 97-105%. This method is capable of distinguishing BTM from DXM when both analytes are simultaneously present in equine plasma. Measurement uncertainty for both analytes was estimated at less than 16%. The method is rapid, specific, selective, sensitive, simple and reliable. The importance of this method is its usefulness in directly identifying and differentiating BTM from DXM without derivatization.     

Simultaneous analysis of twenty-one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.     

Mass spectrometry of steroid glucuronide conjugates. II-Electron impact fragmentation of 3-keto-4-en- and 3-keto-5alpha-steroid-17-O-beta glucuronides and 5alpha-steroid-3alpha,17beta-diol 3- and 17-glucuronides

The steroid glucuronide conjugates of 16,16,17-d(3)-testosterone, epitestosterone, nandrolone (19-nortestosterone), 16,16,17-d(3)-nortestosterone, methyltestosterone, metenolone, mesterolone, 5alpha-androstane-3alpha,17beta-diol, 2,2,3,4,4-d(5)-5alpha-androstane-3alpha,17beta-diol, 19-nor-5alpha-androstane-3alpha,17beta-diol, 2,2,4,4-d(4)-19-nor-5alpha-androstane-3alpha,17beta-diol and 1alpha-methyl-5alpha-androstane-3alpha/beta,17beta-diol were synthesized by means of the Koenigs-Knorr reaction. Selective 3- or 17-O-conjugation of bis-hydroxylated steroids was performed either by glucuronidation of the corresponding steroid ketole and subsequent reduction of the keto group or via a four-step synthesis starting from a mono-hydroxylated steroid including (a) protection of the hydroxy group, (b) reduction of the keto group, (c) conjugation reaction and (d) removal of protecting groups. The mass spectra and fragmentation patterns of all glucuronide conjugates were compared with those of the commercially available testosterone glucuronide and their characterization was performed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. For mass spectrometry the substances were derivatized to methyl esters followed by trimethylsilylation of hydroxy groups and to pertrimethylsilylated products using labelled and unlabelled trimethylsilylating agents. The resulting electron ionization mass spectra obtained by GC/MS quadrupole and ion trap instruments, full scan and selected reaction monitoring experiments are discussed, common and individual fragment ions are described and their origins are proposed.     

Development of a sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of apomorphine in canine plasma

A sensitive, rapid and specific quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of apomorphine (APO) in canine plasma. The analytes were prepared using one-step liquid-liquid extraction, and analyzed on a Waters Symmetry C(18) column interfaced with triple quadrupole tandem mass spectrometer. A mixture of methanol/0.1% formic acid in water (70: 30, v/v) was employed as the isocratic mobile phase. Positive electrospray ionization was utilized as the ionization source. The analyte and clenbuterol (internal standard) were both detected using multiple reaction monitoring (MRM) mode. The limit of detection (LOD) obtained was 0.03 ng/mL. The assay was linear over the concentration range of 0.1-100 ng/mL, and provided good precision (RSD) and good accuracy (RE). The analyte was stable by using antioxidants throughout the whole study. The experimental results show that LC/MS/MS is a rapid and sensitive method to analyze APO in plasma. Finally, the proposed method was successfully applied to a pharmacokinetic study of APO after intranasal administration of 0.5 mg apomorphine to 10 healthy beagle dogs.     

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of YM155, a novel small-molecule survivin suppressant, in dog plasma

This paper describes a sensitive and selective liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for determination of the novel survivin suppressant YM155, 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium, which is developed for the treatment of solid tumors. This method uses a liquid-liquid extraction from 0.25 mL of dog plasma. LC separation was carried out on a Genesis Silica column (50 mm x 3.0 mm i.d.) at a flow-rate of 0.5 mL/min. Compounds were eluted using a mobile phase of 5 mm ammonium acetate and 0.1% formic acid in water-0.1% formic acid in acetonitrile, 17:83 (v/v). MS/MS detection was carried out with an MDS-Sciex API3000 triple quadrupole mass spectrometer in positive electrospray ionization mode. The standard curve was linear from 0.05 to 50 ng/mL (r > or = 0.9968). The lower limit of quantitation was 0.05 ng/mL. Good intra- and inter-day assay precision (within 7.4% RSD) and accuracy (within +/-12.3%) were obtained. The extraction recovery was 66.2%. The method was successfully applied to preclinical pharmacokinetic studies in dogs.     

Determination of faropenem in human plasma and urine by liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed-phase column with 0.1% formic acid-methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r=0.9991 for plasma sample and r=0.9993 for urine sample) were obtained in the range 5-4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC-MS/MS.     

Determination of a selective Na+/H+ exchanger inhibitor, 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method was developed for the determination of a selective Na(+)/H(+) exchanger inhibitor 4-cyano(benzo[b]thiophene-2-carbonyl)guanidine (KR-33028) in rat plasma. KR-33028 and the internal standard, linezolid, were extracted from rat plasma with ethyl acetate at neutral pH. The analytes were separated on an XBridge C(18) column with a mixture of methanol-0.1% formic acid (35:65, v/v) as mobile phase and detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.9998) over the concentration range of 2.0-1000 ng/mL. The coefficients of variation of intra- and inter-assay were 1.3-6.8% and the relative error was 0.8-5.0%. The recoveries of KR-33028 and linezolid were 70.5 and 84.6%, respectively. The lower limit of quantification for KR-33028 was 2.0 ng/mL using 50 microL plasma sample. This method was successfully applied to the pharmacokinetic study of KR-33028 in rats.     

超高效液相色谱-串联质谱法测定血浆与尿液中14种麻痹性贝类毒素北大核心CSCD

人体生物基质中麻痹性贝类毒素的检测对其引起的食物中毒诊断和救治具有重要意义。研究建立了超高效液相色谱-串联质谱法测定血浆、尿液中14种麻痹性贝类毒素的分析方法。实验比较了不同固相萃取柱的影响,优化了前处理条件和色谱条件,血浆样品采用0.2 mL水、0.4 mL甲醇、0.6 mL乙腈提取后直接上机测定,尿液样品采用0.2 mL水、0.4 mL甲醇、0.6 mL乙腈提取,聚酰胺(PA)固相萃取柱净化后上机测定。采用Poroshell 120 HILIC-Z色谱柱(100 mm×2.1 mm,2.7μm)对14种贝类毒素进行分离,流动相为含0.1%(v/v)甲酸的5 mmoL/L甲酸铵缓冲溶液和0.1%(v/v)甲酸乙腈溶液,流速为0.50 mL/min。在电喷雾模式(ESI)下进行正负离子扫描,采用多反应监测(MRM)模式检测,外标法定量。结果表明,对于血浆和尿液样品,14种贝类毒素分别在0.24~84.06 ng/mL范围内线性关系良好,相关系数均大于0.995。尿液检测的定量限为4.80~34.40 ng/mL,血浆检测的定量限为1.68~12.04 ng/mL。尿液和血浆样品在1、2和10倍定量限加标水平下平均回收率为70.4%~123.4%,日内精密度为2.3%~19.1%,日间精密度为4.0%~16.2%。应用建立的方法对腹腔注射14种贝类毒素小鼠血浆和尿液进行测定,20份血浆样本中检出含量分别为19.40~55.60μg/L和8.75~13.86μg/L。该方法操作简便,样品取样量少,方法灵敏度高,适用于血浆和尿液中麻痹性贝类毒素的快速检测。     

Rapid and sensitive determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the internal standard. The collision-induced transition m/z 380 --> 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 --> 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.1-20.0 ng/mL. The lower limit of quantification of the LC/MS/MS method was 0.1 ng/mL. The intra- and inter-day precisions were below 10.2% and the accuracy was between -2.3% and +2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to fit the donepezil plasma concentration-time curve. Maximum plasma concentration was 12.3 +/- 2.73 ng/mL which occurred at 3.50 +/- 1.61 h post-dosing. The apparent elimination half-life and the area under the curve were, respectively, 60.86 +/- 12.05 h and 609.3 +/- 122.2 ng . h/mL. LC/MS/MS is a rapid, sensitive and specific method for determining donepezil in human plasma samples.     

A rapid LC-MS/MS method for quantitation of eszopiclone in human plasma: application to a human pharmacokinetic study

A highly reproducible, specific and cost-effective LC-MS/MS method was developed for simultaneous estimation of eszopiclone (ESZ) with 50 μL of human plasma using paroxetine as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract ESZ and IS from human plasma. The total run time was 1.5 min and the elution of ESZ and IS occurred at 0.90 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (15:85, v/v) at a flow rate of 0.50 mL/min on a Discover C(18) (50 × 4.6 mm, 5 μm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for ESZ. A linear response function was established for the range of concentrations 0.10-120 ng/mL (r > 0.998) for ESZ. The intra- and inter-day precision values for ESZ were acceptable as per FDA guidelines. Eszopiclone was stable in the battery of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Validation and application of a high-performance liquid chromatography/tandem mass spectrometry assay for sumatriptan in human plasma

A sensitive and convenient high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) assay is described for the (5-HT(lB/lD)) receptor agonist sumatriptan in human plasma. Sumatriptan was recovered from plasma (81.8 +/- 6.8%) by liquid-liquid extraction. The mobile phase flow rate was 0.3 mL/min and consisted of methanol:water:formic acid (90:10:0.1, v/v/v). The analytical column (4.6 x 100 mm) was packed with Partisil C(8) (5 micro m). The standard curve was linear from 0.7 to 70.4 ng/mL (r(2) > 0.99). The lower limit of quantitation was 0.7 ng/mL. The assay was specific, accurate (percentage deviation from nominal concentrations were <15%), precise and reproducible (within- and between-day coefficients of variation <10.3%). Sumatriptan in plasma was stable over three freeze/thaw cycles and at room temperature for one day. The utility of the assay was demonstrated by following sumatriptan plasma concentrations in two healthy subjects for 8-12 h following a single 20 mg intranasal dose.     

Liquid chromatographic-electrospray tandem mass spectrometric determination of bisoprolol in human plasma

An analytical method for the determination of bisoprolol in human plasma has been developed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (IS) diphenhydramine were cleaned up by protein precipitation with acetonitrile, reconstituted in mobile phase and separated by reversed-phase high-performance liquid chromatography (HPLC) using methanol:10 mm ammonium acetate:formic acid (70:30:0.1 v/v/v) as mobile phase. Detection was carried out by multiple reaction monitoring (MRM) on an LC-MS/MS system and was completed within 2.5 min. The assay was linear over the range 0.5-100 ng/mL with a limit of quantitation (LOQ) of 0.5 ng/mL. The intra- and inter-day precision levels were within 5.54 and 9.95%, respectively, while the accuracy was in the range 89.4-113%. This method has been utilized in a pharmacokinetic study, where healthy volunteers were treated with an oral dose of 5 mg bisoprolol.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

LC‐ESI‐MS/MS analysis of quercetin in rat plasma after oral administration of biodegradable nanoparticles

A simple, rapid and sensitive LC‐MS/MS method was developed and validated for the determination of free quercetin in rat plasma, using fisetin as internal standard. The detection was performed by negative ion electrospray ionization under selected reaction monitoring. Chromatographic separation (isocratic elution) was carried out using acetonitrile–10 m m ammonium formate (80:20, v/v) with 0.1% v/v formic acid. The lower limit of quantification (4.928 ng/mL) provided high sensitivity for the detection of quercetin in rat plasma. The linearity range was from 5 to 2000 ng/mL. Intra‐ and inter‐day variability (RSD) of quercetin extraction from rat plasma was <4.19 and 1.37% with accuracies of 98.77 and 99.67%. The method developed was successfully applied for estimating free quercetin in rat plasma, after oral administration of quercetin‐loaded biodegradable nanoparticles (QLN) and quercetin suspension. QLN (C_max, 1277.34 ± 216.67 ng/mL; AUC, 17,458.25 ± 3152.95 ng hr/mL) showed a 5.38‐fold increase in relative bioavailability as compared with quercetin suspension (C_max, 369.2 ± 108.07 ng/mL; AUC, 3276.92 ± 396.67 ng hr/mL). Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of atorvastatin,amlodipine, ramipril and benazepril in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

A rapid, simple, sensitive and specific LC‐MS/MS method has been developed and validated for the simultaneous estimation of atorvastatin (ATO), amlodipine (AML), ramipril (RAM) and benazepril (BEN) using nevirapine as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple‐reaction monitoring mode using electrospray ionization. Analytes and IS were extracted from plasma by simple liquid–liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on C₁₈ column by pumping 0.1% formic acid–acetonitrile (15:85, v/v) at a flow rate of 1 mL/min. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.26–210 ng/mL for ATO; 0.05–20.5 ng/mL for AML; 0.25–208 ng/mL for RAM and 0.74–607 ng/mL for BEN with mean correlation coefficient of ≥0.99 for each analyte. The intra‐day and inter‐day precision and accuracy results were well with in the acceptable limits. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The developed assay method was successfully applied to a pharmacokinetic study in human male volunteers. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination and pharmacokinetics of manidipine in human plasma by HPLC/ESIMS

A sensitive HPLC/ESIMS method was established for the determination of manidipine in human plasma and pharmacokinetics study. After basified plasma with ammonia, manidipine and the internal standard (IS) (felodipine) were extracted with n-hexane and separated on a Hypersil ODS2 column with a mobile phase of methanol-5 mm ammonium acetate solution containing 0.1% acetic acid (85:15, v/v). MS determination was performed by electrospray ionization in the selected ion monitoring mode. Manidipine was monitored at m/z 611.4 and IS at m/z 384. The assay had a calibration range from 0.2 to 20 ng/mL and a lower limit of quantification of 0.1 ng/mL. The method has been successfully applied to the pharmacokinetic study in healthy volunteers.     

Highly sensitive method for the determination of JI-101, a multi-kinase inhibitor in human plasma and urine by LC-MS/MS-ESI: method validation and application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of JI-101 in human plasma and urine using LC-MS/MS-ESI in the positive-ion mode. The assay procedure involves extraction of JI-101 and alfuzosin (internal standard, IS) from human plasma/urine with a solid-phase extraction process. Chromatographic resolution was achieved on two Zorbax SB-C(18) columns connected in series with a PEEK coupler using an isocratic mobile phase comprising acetonitrile-0.1% formic acid in water (70:30, v/v). The total run time was 2.0 min. The MS/MS ion transitions monitored were 466.20 → 265.10 for JI-101 and 390.40 → 156.10 for IS. The method was subjected to rigorous validation procedures to cover the following: selectivity, sensitivity, matrix effect, recovery, precision, accuracy, stability and dilution effect. In both matrices the lower limit of quantitation was 10.0 ng/mL and the linearity range extended from ~10.0 to 1508 ng/mL in plasma or urine. The intra- and inter-day precisions were in the ranges 1.57-14.5 and 6.02-12.4% in plasma and 0.97-15.7 and 8.66-10.2% in urine. This method has been successfully applied for the characterization of JI-101 pharmacokinetics in cancer patients.