LC-MS/MS 法测定人尿液中氯法拉滨的浓度

目的建立高效液相色谱-串联质谱（LC-MS/MS）方法测定人尿样中氯法拉滨的浓度。方法采用AB SCIEX QTRAP 5500串联质谱仪及Agilent1200高效液相色谱仪进行检测。尿样经甲基叔丁基醚提取处理,以克拉屈滨为内标。色谱柱为ThermoC18柱（150mm×4.6mm,5μm）,流动相为乙腈—4mM乙酸铵（含0.3%的甲酸）（250∶3,v/v）;流速为0.5mL·min-1。氯法拉滨和克拉屈滨的MRM扫描离子通道m/z分别为304.2→170.0,286.1→170.0。进样量：10μL。结果氯法拉滨和克拉屈滨分离良好,保留时间分别为3.77min,3.88min。氯法拉滨在2.5~500ng·mL-1范围内线性关系良好（r=0.9995）,日内、日间RSD均低于6.39%,准确度（RE）均低于10.17%。结论本法样品预处理简便快捷,检测结果专属性强,灵敏度好,准确度高,适用于氯法拉滨药代动力学的研究。     

Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A highly sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the quantitative determination of buprenorphine and its active metabolite norbuprenorphine in human plasma. Automated solid phase extraction (SPE) on disposable extraction cartridges (DEC) is used to isolate the compounds from the biological matrix and to prepare a cleaner sample before injection and analysis in the LC-MS/MS system. After conditioning, the plasma sample (1.0 ml) is loaded on the DEC filled with octyl silica (C8) and washed with water. The analytes are, therefore, eluted by dispensing methanol containing 0.1% of acetic acid. The eluate is collected and evaporated to dryness. The residue is dissolved in mobile phase and an aliquot is injected in the LC-MS/MS system. On-line LC-MS/MS system using atmospheric pressure chemical ionization (APCI) has been developed for the determination of buprenorphine and norbuprenorphine. The separation is obtained on a RP-18 stationary phase using a mobile phase consisting in a mixture of methanol and 50 mM ammonium acetate solution (50:50, v/v). Clonazepam is used as internal standard (IS). The MS/MS ion transitions monitored are m/z 468-->468, 414-->414 and 316-->270 for buprenorphine, norbuprenorphine and clonazepam, respectively. The method was validated regarding recovery, linearity, precision and accuracy. The limits of quantification (LOQs) were around 10 pg/ml for buprenorphine and 50 pg/ml for norbuprenorphine.     

Development and validation of an automated SPE-LC-MS/MS assay for valdecoxib and its hydroxylated metabolite in human plasma

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantitate valdecoxib (I) and its hydroxylated metabolite (II) in human plasma. The analytes (I and II) and a structurally analogue internal standard (IS) were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile:water (50:50, v/v) containing 10 mM ammonium acetate. The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring (MRM) with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118 and m/z 329-->196 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/ml of I and II in human plasma with absolute recoveries from plasma at 91 and 86%, respectively. The lower limit of quantitation was 0.5 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.5-200 ng/ml). Sample analysis time for each injection was 5 min, a throughput of 70 human plasma standards and samples per run was achieved. The assay has been successfully used to analyze human plasma samples to support clinical phase I and II studies.     

Determination of ambroxol in human plasma using LC-MS/MS

A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) was developed for the quantification of ambroxol in human plasma. Domperidone was used as internal standard, with plasma samples extracted using diethyl ether under basic condition. A centrifuged upper layer was then evaporated and reconstituted with 200 microl methanol. The reconstituted samples were injected into a C(18) XTerra MS column (2.1 x 30 mm) with 3.5 microm particle size. The analytical column lasted for at least 600 injections. The mobile phase was composed of 20 mM ammonium acetate in 90% acetonitrile (pH 8.8), with flow rate at 250 microl/min. The mass spectrometer was operated in positive ion mode using turbo electrospray ionization. Nitrogen was used as the nebulizer, curtain, collision, and auxiliary gases. Using MS/MS with multiple reaction monitoring (MRM) mode, ambroxol was detected without severe interferences from plasma matrix. Ambroxol produced a protonated precursor ion ([M+H](+)) at m/z 379 and a corresponding product ion at m/z 264. And internal standard (domperidone) produced a protonated precursor ion ([M+H](+)) at m/z 426 and a corresponding product ion at m/z 174. Detection of ambroxol in human plasma was accurate and precise, with quantification limit at 0.2 ng/ml. This method has been successfully applied to a study of ambroxol in human specimens.     

Determination of cetirizine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: application to a bioequivalence study

A rapid, sensitive and selective HPLC-MS/ MS method was developed and validated for the quantification of cetirizine dihydrochloride (CAS 83881-51-0) in human plasma using mosapride citrate as internal standard (IS, CAS 112885-42-4). Following liquid-liquid extraction, the analytes were separated using a mobile phase consisting of methanol and aqueous ammonium acetate solution (10 mM) (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 398 --> 201 for cetirizine and m/z 422 --> 198 for mosapride. The analysis time for each run was 8.0 min. The assay exhibited a linear dynamic range of 0.5-500 ng/ml for cetirizine dihydrochloride in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/ml with a relative standard deviation of less than 15% (all the concentration data in this study related to the salt (cetirizine dihydrochloride)). Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC-MS/MS method has been successfully applied to a bioequivalence study in 20 healthy male Chinese volunteers.     

High performance liquid chromatography-tandem mass spectrometric determination of rupatadine in human plasma and its pharmacokinetics

A simple, rapid, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of rupatadine in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-ammonium acetate (pH 2.2; 5mM) (50:50, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the positive ion and multiple reaction monitoring (MRM) mode, m/z 416-->309 for rupatadine and m/z 295-->267 for the IS. The assay exhibited a linear dynamic range of 0.1-100 ng/ml for rupatadine in human plasma. The lower limit of quantification (LLOQ) was 0.1 ng/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of rupatadine in healthy volunteers.     

Simultaneous quantitation of 17alpha-hydroxyprogesterone caproate, 17alpha-hydroxyprogesterone and progesterone in human plasma using high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS)

A sensitive and specific assay method for the simultaneous quantitation of 17alpha-hydroxyprogesterone caproate (17-OHPC), 17alpha-hydroxyprogesterone (17-OHP), and progesterone (P) in human plasma using high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis((R)) HLB extraction cartridge prior to chromatography. Medroxyprogestrone acetate (MPA) was used as the internal standard. The compounds were separated using Waters C18 Symmetry analytical column (3.5 microm, 2.1 mm x 50 mm) using a gradient elusion with a mobile phase consisting of 5% methanol in water [A] and methanol [B], with ammonium acetate (2mM) and formic acid (0.1%) being added to both [A] and [B], at a flow rate 0.3 ml/min. The retention times for 17-OHPC, 17-OHP, P and MPA were 4.5, 1.5, 2.5 and 2.2 min, respectively, with a total run time of 7 min. The analytes were detected by a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 429.10-->313.10 for 17-OHPC, m/z 331.17-->97.00 for 17-OHP, m/z 315.15-->109.00 for P and m/z 387.15-->327.25 for MPA (IS). The assay was linear over the range 1-200 ng/ml for 17-OHPC and 17-OHP, and 2-400 ng/ml for P, when 0.4 ml of plasma was used in the extraction. The overall intra- and inter-day assay variation was <15%. No significant variation in the concentration of 17-OHPC, 17-OHP or P was observed with different sample processing and/or storage conditions. This method is simple, allows easy, accurate and reproducible measurement of 17-OHPC, 17-OHP and P simultaneously in human plasma, and is used to evaluate the pharmacokinetics of 17-OHPC in pregnant subjects.     

Liquid chromatography-negative ion electrospray tandem mass spectrometry method for the quantification of ezetimibe in human plasma

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of free and total ezetimibe in human plasma. The analyte and internal standard (13C6-ezetimibe) were extracted by liquid-liquid extraction with methyl tert-butyl ether. The reversed-phase chromatographic separation was performed on a Capcell C18 column, and the plasma extract was eluted with a gradient consisting of acetonitrile and 5 mM ammonium acetate. The analyte was detected using negative ionization by multiple reaction monitoring mode. The mass transition pairs of m/z 408.5-->270.8 and m/z 414.5-->276.8 were used to detect ezetimibe and internal standard, respectively. The assay exhibited linear ranges from 0.02 to 20 ng/ml for free ezetimibe and 0.25 to 250 ng/ml for total ezetimibe in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully used to analyze human plasma samples for application in a pharmacokinetic study.     

液相色谱-串联质谱法同时测定人血清与尿液中伏立诺他及其代谢物4-苯胺基-4-氧代丁酸的浓度

目的分别建立人血清和尿液中液相色谱-串联质谱法同时测定伏立诺他及其代谢物4-苯胺基-4-氧代丁酸(M2)的方法。方法血清样品经甲醇沉淀蛋白后取上清液进样分析,以Amethyst C_(18)-P(150mm×2.1mm,5μm)为分析柱,流动相为2%的甲酸(含5mmol·L~(-1)醋酸铵水溶液)-甲醇(55:45,V/V);尿液样品以流动相稀释后进样分析,以Hedera ODS-2(150mm×2.1mm,5μm)为分析柱,流动相为2%的甲酸(含30mmol·L~(-1)醋酸铵水溶液)-甲醇(55:45,V/V)。质谱采用气动辅助电喷雾离子化和正离子多反应监测,定量分析的反应离子对分别为m/z 265.2→232.2(伏立诺他)、m/z 194.1→176.1(M2)和m/z 180.1→110.1(内标非那西丁)。结果伏立诺他血药浓度在2.004～1503μg·L~(-1)范围内线性关系良好,平均回收率大于98.3%,M2血药浓度在5.015～5015μg·L~(-1)范围内线性关系良好,平均回收率大于96.8%;伏立诺他尿药浓度在0.03006～20.04mg·L~(-1)范围内线性关系良好,平均回收率大于96.8%,M2尿药浓度在1.003～300.9mg·L~(-1)范围内线性关系良好,平均回收率大于96.5%。尿样测定中,由伏立诺他的另一代谢物O-葡萄糖醛酸苷发生源内裂解而对伏立诺他的测定造成的干扰通过色谱分离得到解决。结论建立的两个方法快速、简便,可应用于人体样本测定。     

Identification of phase I and phase II metabolites of Guanfu base A hydrochloride in human urine.

Guanfu base A is a novel arrhythmic drug candidate isolated from the tuber of a traditional Chinese herb. Phase I and Phase II metabolites of Guanfu base A (GFA) Hydrochloride were studied in human urine by means of liquid chromatography mass spectrometry (LC/MSD) and tandem mass spectrometry (MS/MS). For phase I metabolites, Guanfu base I (GFI) was separated by HPLC and identified by comparison with authentic reference for their retention times, molecular ion peaks, fragment ions, and UV spectra. GFA oxide was also indicated to exist in human urine. For phase II metabolites, after human urine was treated either with glucuronidase or sulfatase, GFA occured in the chromatograms. It was suggested that there were GFA glucuronide and GFA sulfate in human urine. Further more, positive molecular ions, m/z 606 and m/z 510, of the two conjugates were detected in human urine by LC/MSD. In addition, characteristic ion of m/z 606 was identified as the precursor ion of m/z 177 [Glucuronic acid+H]+ by using MS/MS. Characteristic ion of m/z 430 [GFA+H]+ was also identified as a product ion of m/z 606 [GFA glucuronide+H]+. It was concluded that there were GFI. GFA oxide, GFA glucuronide and GFA sulfate in human urine.     

Simultaneous extraction of acetylsalicylic acid and salicylic acid from human plasma and simultaneous estimation by liquid chromatography and atmospheric pressure chemical ionization/tandem mass spectrometry detection. Application to a pharmacokinetic study

A simple analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in atmospheric chemical ionization mode (APCI) for the simultaneous estimation of acetylsalicylic acid (ASA, CAS 50-78-2) and its active metabolite salicylic acid (SA, CAS 69-72-7) in human plasma has been developed and validated. ASA and SA were analyzed simultaneously despite differences in plasma concentration ranges of ASA and SA after oral administration of ASA. In spite of having different chemical, ionization and chromatographic properties, ASA and SA were extracted simultaneously from the plasma sample using acetonitrile protein precipitation followed by liquid-liquid extraction. The analytes were separated on a reversed phase column with rapid gradient program using mobile phase consisting of ammonium acetate buffer and methanol. The structural analogue diclofenac was used as an internal standard. The multiple reaction monitoring (MRM) transitions m/z 179 --> 137 for ASA, m/z 137 --> 65 for SA and m/z 294 --> 250 for IS were used. The assay exhibited a linear dynamic range of 0.02-10 microg/mL for ASA and 0.1-50 microg/mL for SA. The between-batch precision (%CV) ranged from 2.1 to 7.9% for ASA and from 0.2 to 5.2% for SA. The between-batch accuracy ranged from 95.4 to 96.7% for ASA and from 94.6 to 111.3% for SA. The validated method was successfully applied for the evaluation of pharmacokinetics of ASA after single oral administration of 650 mg test formulation versus two 325 mg reference formulations of ASA in human subjects.     

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。     

Quantitative determination of a novel insulin sensitizer and its para-hydroxylated metabolite in human plasma by LC-MS/MS

I, 5-[3-[3-(4-phenoxy-2-propylphenoxy)-propoxy]-phenyl]-2,4-thiazolidinedione sodium salt, is a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist for potential use in diabetic patients. The compound has a para-hydroxylated metabolite, II, which has also been shown to exhibit PPAR activity. An LC-MS/MS method for the simultaneous determination of I and its active metabolite (II) in human plasma has been successfully developed. The method consists of treating 0.5 ml plasma with ammonium acetate (pH 9.6; 50mM) and extracting I, II and internal standard (III, Fig. 2) with 5 ml ethyl acetate. The ethyl acetate is evaporated and the samples are reconstituted in 0.1 ml acetonitrile:0.1% formic acid (65:35, v/v). The entire extraction procedure, as well as sample collection, was performed in glass tubes and vials to overcome the analytes adherence to polypropylene. A linear HPLC gradient was used to separate the analyte, metabolite, internal standard, and other interfering, non-quantitated metabolites. Detection was by negative ionization MS/MS on a turbo ionspray probe. Precursor-->product ion combinations were monitored in multiple reaction monitoring (MRM) mode. The linear range is 0.05-20 ng/ml for I and 0.1-20 ng/ml for II. Recoveries were 59.4, 90.1 and 56.8% for I, II and III, respectively. Intraday variation using this method was <==7.0% for I and <==9.2% for II. The method exhibits good linearity and reproducibility for each analyte and good sensitivity, selectivity and robustness when used for the analysis of clinical samples.     

LC-MS/MS 法测定人血浆中倍他米松

建立测定人血浆中倍他米松的LC-MS/MS方法。采用Venusil XBP C₈ (200 mm×3.9 mm ID, 5 μm)色谱柱，流动相为甲醇-水(含甲酸铵5 mmol·L^-1)(80∶20)，流速0.4 mL·min^-1；质谱仪离子源为电喷雾离子源(ESI)，正离子模式检测，监测离子为393.3→355.2(倍他米松)和361.3→343.2(泼尼松龙,内标)。血浆样本用乙酸乙酯处理。倍他米松在0.5~80.0 ng·mL^-1线性关系良好(r=0.999 2)， 血浆低、 中、 高3种浓度(1.0， 10.0， 60.0 ng·mL^-1)平均提取回收率为88.24%，定量限为0.5 ng·mL^-1。本方法操作简便、准确、灵敏，适用于复方倍他米松注射液人体药代动力学研究。     

High-performance liquid chromatography-mass spectrometric analysis of furosemide in plasma and its use in pharmacokinetic studies

This study presents a rapid, specific and sensitive high-performance liquid chromatography-mass spectrometric (LC-MS) assay for the determination of furosemide in human plasma using diclofenac as an internal standard (IS). Both compounds were extracted from human plasma with ethyl acetate at pH 1 and were chromatographed using Shim-Pack GLC-CN column and a mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer solution pH 7, 4:1 (v/v) at a flow rate 1 ml min(-1). Furosemide and IS were detected by mass spectrometer operated in the negative single ion monitoring mode using APCI as an ionization process at m/z 329.2 and 294.1, respectively. The assay linearity of furosemide was confirmed over the range 50-2,000 ng ml(-1). Detection limit for furosemide in plasma was 10 ng ml(-1). The selected concentration range corresponds well with the plasma concentrations of furosemide for pharmacokinetic study. Intraday and interday relative standard deviations were 1.3-4.7 and 2.7-11.5%, respectively. The extraction recovery percentages of furosemide and IS from plasma were in the range 89.3-97.1%. The developed LC-MS procedure was applied for the determination of the pharmacokinetic parameters of furosemide after an oral administration of tablet formulation (40 mg) to two healthy male volunteers. The calculated parameters were in good agreement with the reported values.     

Determination of vinflunine in rat plasma by liquid chromatography-electrospray ionization mass spectrometry for a pharmacokinetic study

A rapid, simple and sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the quantification of vinflunine in rat plasma was developed and validated. After making alkaline with NaOH, plasma was extracted with ethyl acetate and determined by LC-MS. The analysis was carried out on a Shimadzu VP-ODS column (150 mmx4.6 mm ID, packed with 5 microm C18 Silica RP particle). The mobile phase consisted of methanol-10 mM ammonium acetate buffer (80:20, v/v) with the flow rate of 1.0 ml/min. LC-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z: 817.3 for vinflunine and m/z: 373.2 for finasteride (IS). Chromatographic separation was achieved in less than 6 min and the calibration curve was linear over a concentration range of 0.025-6.25 microg/ml. The intra-assay and inter-assay variability values were less than 8.6%. The accuracy ranged from 91.5 to 105.6%. The established method has been successfully applied to a pharmacokinetic study of vinflunine in rats.     

LC-MS/MS法定量测定人血浆中普罗布考的浓度

目的：建立一种特异、灵敏的液质联用方法定量测定人体血浆中普罗布考的浓度。方法：在25.0μL含待测药物的血浆中加入内标Pr-1312及萃取液混匀沉淀蛋白。采用X-Terra RPC（82.1mm×50mm,3.5μm）色谱柱;柱温50℃;流动相为10mmol·L-1碳酸氢铵水溶液-乙腈（20：80）;进样量5μL;流速0.2mL·min-1。质谱检测方式为ESI-,MRM扫描,监测普罗布考m/z515.3→236.1,内标Pr-1312m/z382.2→161.0,分析时间3.3min。结果：普罗布考在10.0～5000.0ng·mL-1的浓度范围内线性关系良好（r〉0.99）,日内与日间精密度（RSD）〈10.2%,准确度（RE）〈±15.0%。所有稳定性考察项目结果均符合要求。结论：本文采用蛋白沉淀样品前处理以及液质联用定量测定人体血浆中普罗布考的浓度方法,能够满足临床试验中生物样品分析的需要。     

Liquid chromatographic-electrospray tandem mass spectrometric method for the quantification of nimodipine in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of nimodipine, a calcium channel blocker, in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 419/343 for nimodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.2-50 ng/mL for nimodipine in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

替加氟有关物质的色谱-质谱结构鉴定

目的采用色谱-质谱联用技术鉴定替加氟有关物质,为其工艺和质量控制提供参考依据。方法采用十八烷基硅烷键合硅胶的填充剂,流动相为乙腈-甲醇-10 mmol·L-1醋酸铵缓冲溶液,对替加氟有关物质进行分离;采用电喷雾正离子化-飞行时间质谱法测定各有关物质的准确质量,三重四极杆质谱测定子离子特征,并经解析鉴定各杂质结构。结果与结论替加氟与其有关物质分离良好,检测出4个主要有关物质均为替加氟合成的起始原料,是母核基本未发生变化,仅5位被不同基团取代的衍生物。     

Selective determination of tenofovir in human plasma by LC-MS-MS method

In the present study, we developed and validated a selective, specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) method for the determination of tenofovir in human plasma. Entecavir was used as an internal standard, and plasma samples were prepared by solid-phase extraction performed on Phenomenex Strata cartridges (30 mg). The mobile phase consisted of 10 mM ammonium acetate in water and methanol (60:40, v/v). The chromatographic separation was performed isocratically on a Phenomenex C₁₈ (4.6 mm×150 mm, 5 μm), and analytes were analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H]⁺ ions, m/z 288.2→m/z 176.1 for tenofovir and m/z 278.1→m/z 152 for entecavir. The calibration curve (r² = 0.9962) of tenofovir was established within the range of 4.096–1000 μg/L. The intra- and inter-day precisions were less than 10%. This validated method was successfully applied to a pharmacokinetic study in 12 healthy Chinese volunteers after the oral administration of tenofovir disoproxil fumarate.