Simultaneous determination of viloxazine,venlafaxine, imipramine,desipramine, sertraline,and amoxapine in whole blood: comparison of two extraction/cleanup procedures for capillary gas chromatography with nitrogen-phosphorus detection

A comparative study for the simultaneous gas chromatographic (GC) resolution and detection of the six antidepressants viloxazine, venlafaxine, imipramine, desipramine, sertraline, and amoxapine in whole blood at concentration levels of 100-2000 ng/mL was developed. Two extraction/cleanup analytical procedures were compared regarding their recovery, precision, sensitivity and matrix purification efficiency. The first procedure consists of the employment of Chem Elut columns (diatomaceous earth) and is based on the principle of liquid-solid absorption extraction that is closely related to conventional liquid-liquid extraction. The second focuses on the use of Bond Elut Certify columns and a mixed SPE, reversed-phase and cation-exchange sorbent, more recently developed for the market. Each procedure required 2.0 mL of whole blood extraction and injection into a capillary GC equipped with a nitrogen-phosphorus detector. Mepivacaine was used as the extraction standard (surrogate), and prazepam was used as the chromatographic standard. No interferences were found, and the time for the chromatographic analysis was 16 min for one sample. Recoveries of the compounds using Chem Elut columns at 500 ng/mL were in the range of 28-74% with intra-assay and interassay precisions of less than 7% and 19%, respectively. Limits of detection (LOD) and quantitation (LOQ) ranged from 39 to 153 ng/mL and from 128 to 504 ng/mL, respectively. Recoveries of the compounds using Bond Elut Certify columns at 500 ng/mL were in the range of 64-86% with intra-assay and interassay precisions of less than 4% and 10%, respectively. LODs and LOQs ranged from 21 to 100 ng/mL and from 70 to 330 ng/mL, respectively. An excellent linearity was observed with both procedures from the LOQs up to 2000 ng/mL. The use of the reversed-phase and cation-exchange sorbent Bond Elut Certify showed advantages compared with Chem Elut columns for the screening of these antidepressants such as higher recoveries, cleaner extracts, better sensitivity, better precision, and less solvent consumption and disposal.     

A comparative solid-phase extraction study for the simultaneous determination of fluoxetine, amitriptyline, nortriptyline, trimipramine, maprotiline, clomipramine, and trazodone in whole blood by capillary gas-liquid chromatography with nitrogen-phosphorus detection

This paper reports the simultaneous detection of the seven antidepressants fluoxetine, amitriptyline, nortriptyline, trimipramine, maprotiline, clomipramine, and trazodone in whole blood at concentration levels of 100-2000 ng/mL by gas chromatography with a nitrogen-phosphorus detector (GC-NPD). A comparative and validation study using two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, were developed regarding their recovery, precision, sensitivity, and matrix purification efficiency. The Chem Elut columns, a diatomaceous earth, are closely related to conventional liquid-liquid extraction. The Bond Elut Certify columns, more recently developed in the market, are a mixed SPE: reversed-phase and cation exchange sorbent. Recoveries of the compounds using Chem Elut columns at 500 ng/mL were in the range 30-50%, with intra- and interassay precisions of less than 9% and 17%, respectively. Limits of detection (LODs) and quantitation (LOQs) ranged from 13 to 146 ng/mL and from 44 to 485 ng/mL, respectively. Recoveries of the compounds using Bond Elut Certify columns at 500 ng/mL were in the range 59-84% with intra- and interassay precisions of less than 8% and 11%, respectively. LODs and LOQs ranged from 8 to 67 ng/mL and from 25 to 223 ng/mL, respectively. An excellent linearity was observed with both extraction procedures from the LOQs up to 2000 ng/mL. Higher recoveries, cleaner extracts, better sensitivity, better precision, and less solvent consumption and disposal were achieved for the screening of these antidepressants with the use of the mixed SPE Bond Elut Certify compared with Chem Elut columns.     

A comparative solid-phase extraction study for the simultaneous determination of fluvoxamine, mianserin, doxepin, citalopram, paroxetine, and etoperidone in whole blood by capillary gas-liquid chromatography with nitrogen-phosphorus detection

This paper reports a simple and reliable gas chromatographic method with nitrogen-phosphorus detection without derivatization for the simultaneous detection of fluvoxamine, mianserin, doxepin, citalopram, paroxetine, and etoperidone in whole blood as part of a systematic toxicological analysis (STA). All drugs were studied at concentration levels of 100-2000 ng/mL, except paroxetine for which it was necessary to study at concentration levels of 400-8000 ng/mL. A comparative and validation study using two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, was developed regarding their recovery, precision, sensitivity, and matrix purification efficiency. The Chem Elut columns, diatomaceous earth, are closely related to conventional liquid-liquid extraction. The Bond Elut Certify columns, more recently developed in the market, are mixed SPE (reversed-phase and cation exchange sorbent). Recoveries for the antidepressants using Chem Elut columns at 500 ng/mL (2000 ng/mL for paroxetine) were in the range 43-72% with intra- and interassay precisions of less than 10% and 16%, respectively. Limits of detection (LODs) and quantitation (LOQs) for fluvoxamine, mianserin, doxepin, citalopram, and etoperidone ranged from 18 to 236 ng/mL and 60 to 786 ng/mL, respectively. LOD and LOQ for paroxetine were 303 and 1009 ng/mL, respectively. Recoveries of these compounds using Bond Elut Certify columns at 500 ng/mL (2000 ng/mL for paroxetine) were in the range 52-83% with intra- and interassay precisions of less than 6% and 8%, respectively. LODs and LOQs for fluvoxamine, mianserin, doxepin, citalopram, and etoperidone ranged from 7 to 28 ng/mL and 23 to 93 ng/mL, respectively. LOD and LOQ for paroxetine were 113 and 376 ng/mL, respectively. An excellent linearity was observed with both procedures from the LOQs up to the upper studied concentration level. In general, higher recoveries, cleaner extracts, better sensitivity, better precision, and reduced solvent consumption and disposal were achieved for the screening of these antidepressants with the use of the mixed SPE Bond Elut Certify compared with Chem Elut columns. The application of these methods on a forensic case study is also presented.     

LC/MS/MS plasma assay for the peptidomimetic VLA4 antagonist I and its major active metabolite II: for treatment of asthma by inhalation.

In vitro and in animals, I is a potent and specific peptidomimetic for the potential treatment of airway inflammation in the pathogenesis of asthma. Preclinical studies indicated extensive conversion of I to an active metabolite II, and thus, a very sensitive assay for I and II was needed to support an inhalation ascending-dose study in man. The LC/MS/MS plasma/urine assay method (1.0 ml of sample) involves the following: liquid-liquid extraction of acidified plasma into pentane-ethyl acetate (90:10 v/v); evaporation of the organic extract, reconstitution into methanol; addition of water to the methanolic extract and freezing. After thawing, the extract is centrifuged and the clear supernatant injected for chromatography. Extract is chromatographed on a YMC ODS-AM column (50 x 2.0 mm). For detection, a Sciex 365 LC/MS/MS with an electrospray inlet and used in the positive ion, multiple reaction monitoring mode was used to monitor precursor-->fragment ions of m/z 709-->594 for I and m/z 513-->380 for II. The plasma assay was linear over the concentration range of 0.1-100 ng/ml in plasma for I and II. Accuracy and precision for I ranged from 97.9 to 102.1% of nominal with a 0.84-10.65% CV; similarly for II, 98.0-101.7% and 1.39-9.28% CV, respectively. Extraction recovery averaged 63.7% for I and 64.9% for II. This general assay methodology may be applied to assay small acidic peptides and peptidomimetics from biological fluids by LC/MS/MS.     

Recent advances in high-throughput quantitative bioanalysis by LC-MS/MS

Liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) has played an important role in pharmacokinetics and metabolism studies at various drug development stages since its introduction to the pharmaceutical industry. This article reviews the most recent advances in sample preparation, separation, and the mass spectrometric aspects of high-throughput quantitative bioanalysis of drug and metabolites in biological matrices. Newly introduced techniques such as ultra-performance liquid chromatography with small particles (sub-2 microm) and monolithic chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed-phase LC-MS/MS. Sample preparation formatted to 96-well plates has allowed for semi-automation of off-line sample preparation techniques, significantly impacting throughput. On-line solid-phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications to reduce both time and labor required to produce bioanalytical results. Extraction sorbents for on-line SPE extend to an array of media including large particles for turbulent flow chromatography, restricted access materials (RAM), monolithic materials, and disposable cartridges utilizing traditional packings such as those used in Spark Holland systems. In the end, this paper also discusses recent studies of matrix effect in LC-MS/MS analysis and how to reduce/eliminate matrix effect in method development and validation.     

Determination of SR 49059 in human plasma and urine by LC-APCI/MS/MS

SR 49 059 ((2S 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide) is an orally active non-peptide vasopressin V_1a antagonist. A sensitive, selective, and robust LC-MS/MS method was developed to determine the plasma and urine concentrations of SR 49 059 in support of clinical studies. Plasma samples were prepared based on a rapid extraction procedure using Chem Elut™ cartridges. The extracted samples were analyzed on a C₁₈ HPLC column interfaced with a Finnigan TSQ 700 mass spectrometer. Positive atmospheric chemical ionization (APCI) was employed as the ionization source. The analyte and its internal standard (²H₆-SR 49 059) were detected by use of multiple reaction monitoring (MRM) mode. The plasma matrix had a calibration range 0.2−20 ng ml⁻¹, with within and between run accuracy and precision both less than 10%. The chromatographic run time was approximately 3 min. Urine samples were prepared based on a simple dilution with water, followed by analysis under the same conditions as plasma. The calibration range for urine matrix was 20–5000 ng ml⁻¹, with within and between run accuracy and precision less than 11%. The method has been successfully applied to the clinical sample analysis. The plasma assay was also evaluated on a Finnigan TSQ 7000 mass spectrometer. The performance based on precision and accuracy was virtually identical to that on the TSQ 700, with the exception of linearity in calibration curve (the TSQ 700 was linear, the TSQ 7000 was quadratic).     

High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC-MS/MS

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d₇-nefazodone, d₇-hydroxynefazodone, d₄-BMS-180492 and d₄-mCPP as internal standards (IS). The plasma samples were injected into the LC–MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis^® HLB column (on-line extraction column, 1 mm × 50 mm, 30 μm) and a conventional Luna C8 column (analytical column, 4.6 mm × 50 mm, 5 μm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC–MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.     

Quantitation of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide and 4-amino-3,5-dichloropyridine in rat and mouse plasma by LC/MS/MS

The metabolism of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (RP73401), a phosphodiesterase IV (PDE IV) inhibitor is extensive (unpublished); however, until recently, studies for this compound did not report 4-amino-3,5-dichloropyridine (ADCP) as a metabolite either in vitro or in vivo. This prompted a reinvestigation into the metabolism of RP73401 in rats and mice using mass spectrometry. The results of the reinvestigation confirmed that 4-amino-3,5-dichloropyridine was formed via the metabolism of RP73401 both in vitro and in vivo. In order to further investigate RP73401 hydrolysis in vivo, a liquid chromatography/mass spectrometry assay was developed and validated for the simultaneous determination of RP73401 and ADCP in rat and mouse plasma. The method used Waters Oasis HLB brand solid phase extraction cartridges to isolate the analytes (RP73401 and ADCP) and internal standard from the plasma. HPLC chromatographic separation was achieved using a Zorbax SB C18 HPLC column and detection was accomplished using positive ion atmospheric pressure chemical ionization tandem mass spectroscopy in multiple reaction monitoring (MRM) mode. The assay was developed and validated over the range of 0.5-100 ng ml(-1) for RP73401 and 5-500 ng ml(-1) for ADCP using 0.050 ml of plasma. The assay proved to be sensitive, accurate, precise and specific for RP73401 and ADCP. Intraday and interday quality control results routinely showed accuracy and precision to be within +/- 20%. This LC/MS/MS method was subsequently employed to investigate the hydrolysis of RP73401 in the rat and mouse, and determine the effects of tri-o-tolyl phosphate (TOTP, a carboxylesterase inhibitor) preadministration on the hydrolysis reaction in the rat.     

High-throughput pharmacokinetics screen of VLA-4 antagonists by LC/MS/MS coupled with automated solid-phase extraction sample preparation

Automation of plasma sample preparation for pharmacokinetic studies on VLA-4 antagonists has been achieved by using 96-well format solid-phase extraction operated by Beckman Coulter Biomek 2000 liquid handling system. A Biomek 2000 robot is used to perform fully automated plasma sample preparation tasks that include serial dilution of standard solutions, pipetting plasma samples, addition of standard and internal standard solutions, performing solid-phase extraction (SPE) on Waters OASIS 96-well plates. This automated sample preparation process takes less than 2 h for a typical pharmacokinetic study, including 51 samples, 24 standards, 9 quality controls, and 3-6 dose checks with minimal manual intervention. Extensive validation has been made to ensure the accuracy and reliability of this method. A two-stage vacuum pressure controller has been incorporated in the program to improve SPE efficiency. This automated SPE sample preparation approach combined with liquid chromatography coupled with the high sensitivity and selectivity of tandem mass spectrometry (LC/MS)/MS has been successfully applied on both individual and cassette dosing for pharmacokinetic screening of a large number of VLA-4 antagonists with a limit of quantitation in the range of 1-5 ng/ml. Consequently, a significant throughput increase has been achieved along with an elimination of tedious labor and its consequential tendency to produce errors.     

Application of a novel ultra-low elution volume 96-well solid-phase extraction method to the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma

A novel extraction method has been utilized in the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma. In this method, 300 microl of plasma sample was loaded onto a Waters Oasis 96-well HLB microElution plate, the stationary phase was washed using 2 x 400 microl of 5% methanol in water, and the analytes were eluted using 35 microl of 95/5 acetonitrile/H(2)O twice. The sample extracts were diluted with 40 microl of methyl ammonium acetate (1mM, pH 4.5). Chromatography was performed on a Phenomenex Synergi Max-RP column (2.0 mm x 50 mm, 4 microm). A PE Sciex API 3000 tandem mass spectrometer interfaced with a turbo ionspray source was used for mass detection. Compared to solid-phase extraction, liquid-liquid extraction and solid-supported liquid-liquid extraction methods that were developed and previously used in our laboratory, this method reduced the labor cost and was less time consuming in sample preparation, due to the fact that post-extraction solvent evaporation and reconstitution steps were avoided using this microElution solid-phase extraction plate. The method has been proved to be fast, reliable and reproducible.     

Sensitive determination of erdosteine in human plasma by use of automated 96-well solid-phase extraction and LC-MS/MS

A sensitive and selective method for quantitation of erdosteine in human plasma was established by use of 96-well solid-phase extraction (SPE) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). Plasma samples were transferred into 96-well OASIS HLB extraction plate using an automated sample handling system and the drugs were eluted with methanol. The eluents were then evaporated and reconstituted with mobile phase. All sample transfer and SPE was automated through the application of both the Perkin-Elmer MultiPROBE II HT and TOMTEC Quadra 96 workstation. Compounds were separated on a C18 column with 1 mM ammonium acetate-acetonitrile (80:20, pH 3.2), as mobile phase at a flow rate of 0.3 ml/min. The limit of quantitation (LOQ) was 0.2 ng/ml, using a sample volume of 0.2 ml for the analysis. The reproducibility of the method was evaluated by analyzing three at 14 quality control (QC) levels over the nominal concentration range from 0.2 to 5000 ng/ml. The intraday accuracy was found to range from 99.6 to 105.0% with precision (% RSD) of less than 4.76% at five QC levels. The interday accuracy was found to range from 95.0 to 100.5% with precision of less than 5.26% at five QC levels. Erdosteine produced a protonated precursor ion ([M+H]+) at m/z 250, and a corresponding product ion at m/z 204. Internal standard (letosteine) produced a protonated precursor ion ([M+H]+) at m/z 280 and a corresponding product ion at m/z 160. The high sample throughput of the method has been successfully applied to a pharmacokinetic study of erdosteine in human plasma.     

A sensitive human bone assay for quantitation of tigecycline using LC/MS/MS

Tigecycline (Tygacil,Wyeth) is a first-in-class, broad spectrum antibiotic with activity against multiple-resistant organisms. In order to address the unexpectedly low tigecycline concentrations in human bone samples analyzed using a LC/MS/MS method developed elsewhere, we have developed and validated a new and sensitive human bone assay for the quantitation of tigecycline using LC/MS/MS. The new method utilizes the addition of a stabilizing agent to the human bone sample, homogenization of human bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation by liquid chromatography, and detection of tigecycline by mass spectrometry. Linearity was demonstrated over the concentration range from 50 to 20,000 ng/g using a 0.1g human bone sample. The intra- and inter-day accuracy of the assay was within 100+/-15%, and the corresponding precision (CV) was <15%. The stability of tigecycline was evaluated and shown to be acceptable under the assay conditions. The extraction recovery of tigecycline with the current method was 79% when using radio-labeled rat bone samples as a substitute for human bone samples. Twenty-four human bone samples collected previously from volunteers without infections who had elective orthopedic surgery after receiving a single dose of tigecycline were re-analyzed using the current validated method. Tigecycline concentrations in these samples ranged from 238 to 794 ng/g with a mean value 9 times higher than the mean concentration previously reported. The data demonstrated that the current method has significantly higher extraction efficiency than the previously reported method.     

Simultaneous determination of desloratadine and its active metabolite 3-hydroxydesloratadine in human plasma by LC/MS/MS and its application to pharmacokinetics and bioequivalence

A rapid and simple liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method was developed and validated for the simultaneous determination of desloratadine and its active metabolite 3-hydroxydesloratadine concentrations in human plasma. After liquid-liquid extraction with ethyl ether for sample preparation, the chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mm x 2.0mm, 5 microm, Shiseido). [(2)H(4)]desloratadine and [(2)H(4)]3-OH desloratadine were used as internal standards. A mobile phase consisted of 5mM ammonium formate in water, methanol and acetonitrile (50:30:20). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. A quadratic regression (weighted 1/concentration) gave the best fit for calibration curves over the concentration range 0.05-10 ng/mL for both desloratadine and 3-OH desloratadine. The method was shown to be accurate, rapid and sufficiently sensitive to be successfully applied to a pharmacokinetic and bioequivalent study.     

A sensitive LC/MS/MS bioanalysis assay of orally administered lipoic acid in rat blood and brain tissue

A sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) bioanalytical method was developed and validated to analyze lipoic acid (LA) in rat blood and brain samples. Ten mobile phase combinations were investigated during method development. Mobile phase combination of 0.1% acetic acid (pH 4 adjusted with ammonia solution)/acetonitrile was most optimum in terms of sensitivity and peak shape of LA and the internal standard, valproic acid. Sample extraction method was explored using liquid–liquid extraction and protein precipitation methods. Protein precipitation yielded the highest recovery of the analytes from blood and brain ranging from 92 to 115%. The lower limit of quantitation (LLOQ) of LA was 0.1 ng/mL (0.485 nM) in both blood and brain while on-column lower limit of detection (LLOD) was 0.03 pg. The precision (% R.S.D.) ranged from 1.49 to 26.39% and 1.49 to 10.89% for intra- and inter-day assays, respectively. The accuracy ranged from 91.2 to 116.17% for intra-day assay and 102.68 to 114.33% for inter-day assay.     

A simple 96-well liquid-liquid extraction with a mixture of acetonitrile and methyl t-butyl ether for the determination of a drug in human plasma by high-performance liquid chromatography with tandem mass spectrometry

A simple 96-well plate liquid-liquid extraction (LLE), liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a basic drug candidate in human plasma. Against the wisdom of conventional approaches, an aqueous/organic miscible solvent, acetonitrile, was used for liquid-liquid extraction along with methyl t-butyl ether. The use of acetonitrile effectively eliminated the formation of the irregular emulsion between aqueous/organic interfaces and modulated the polarity of the extraction solvents to achieve the desired recovery. This approach, which solved the emulsion problem, permitted the method to be automated using standard 96-well plate technology. A practical application was demonstrated through the use of this technique in the measurement of a novel drug in human plasma samples by LC/MS/MS. Chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm x 50 mm, 5 microm). The mobile phase contained 60% of 0.1% formic acid and 40% acetonitrile. Detection was by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 1.22 to 979ng/ml, was fitted to a 1/x2 weighted quadratic regression model. The validation results show that this method was very rugged and had excellent precision and accuracy. The actual sample analysis results further demonstrated that this extraction procedure is well suited for real life applications. It is expected that with some modifications, this approach can be applied for the extraction of similar compounds from various biological fluids.     

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.     

Rapid determination of granisetron in human plasma by liquid chromatography coupled to tandem mass spectrometry and its application to bioequivalence study

A simple, sensitive and rapid method for analysis of granisetron in human plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and internal standard (IS) were isolated from 100microl plasma samples by liquid-liquid extraction (LLE). A Varian 1200l tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 313.4/138 for granisetron and m/z 270/201 for the IS used for quantitation. The assay exhibited a linear dynamic range of 0.02-20ng/ml for granisetron in human plasma. The lower limit of quantification (LLOQ) was 0.02ng/ml with a relative standard deviation of less than 15%. The mean extraction recovery from spiked plasma samples was 97.9%. The intra-day accuracy of the assay was within 10% of nominal and intra-day precision was better than 15% C.V. A run time of 2.0min for each sample made it possible for high-throughput bioanalysis. The method was employed in a bioequivalence study of two formulations of granisetron hydrochloride 1mg rapidly disintegrating tablets/1mg capsules.     

液相色谱-串联质谱法测定人血浆中米格列奈浓度及其药动学研究

目的建立一种快速分析测定人血浆中米格列奈的液相色谱-串联质谱色谱法,用以研究米格列奈在健康人体内的药动学。方法以那格列胺为内标,血浆酸化后经液液萃取后,采用液相色谱-串联质谱法以多反应检测方式进行测定,选择监测的离子为m/z 316.2→298.2(米格列奈)和m/z 318.2→120.2 (那格列胺)。流动相以甲醇-10 mmol·L~(-1)醋酸铵水溶液(75:25,V/V),流速0.3 mL·min~(-1),色谱柱为Agilent Zorbax Eclipse Plus C_(18)(1.8μm,3 mm×150 mm);柱温:30℃。结果米格列奈在0.502 0～4 016μg·L~(-1)浓度范围内呈良好的线性关系(r=0.995),最低检测浓度为0.502 0μg·L~(-1),精密度和准确度试验均符合生物分析要求,应用此法测得5、10、20 mg剂量组不同给药方式、多个时间点的米格列奈血药浓度,结果呈线性动力学特征。结论该方法灵敏度高、专属性强、准确、简便,适用于米格列奈的人体药动学研究。     

Development of a rapid and sensitive LC-MS/MS assay for the determination of sorafenib in human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of sorafenib in human plasma. Sample pretreatment involved simple protein precipitation by the addition of 0.5 mL acetonitrile, containing internal standard ([2H3, 15N] sorafenib), to 50 microL of plasma sample volume. Separation was achieved on a Waters SymmetryShield RP8 (2.1 mm x 50 mm, 3.5 microm) column at room temperature using an isocratic elution method with acetonitrile/0.1% formic acid in water: 65/35 (v/v) at a flow rate of 0.25 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 464.9 --> 252.0 (sorafenib) and m/z 469.0 --> 259.0 (internal standard). Calibration curves were linear in the concentration range of 5-2000 ng/mL. The accuracy and precision values, calculated from three different sets of quality control samples analyzed in quintuplicate on six different days, ranged from 92.86% to 99.88% and from 1.19% to 4.53%, respectively.