基于气相色谱-质谱法测定塞来昔布中磺酸酯类基因毒性杂质的残留量

为了建立一种测定塞来昔布原料药及其制剂中塞来昔布磺酸甲酯和塞来昔布磺酸乙酯残留量的气相色谱-质谱(gas chromatography-mass spectrometry,GC-MS)分析方法,采用碘化钠衍生-顶空进样,将两杂质衍生成碘甲烷和碘乙烷,DB624毛细管色谱柱(60 m×0.25 mm,1.4μm)分离,氦气为载气,质谱检测器检测。塞来昔布磺酸甲酯和塞来昔布磺酸乙酯均在10～500 ng·m L^-1浓度范围内线性关系良好;回收率在80.87%～106.52%,RSD小于10%;定量限均为10 ng·m L^-1。所有塞来昔布样品中均未检测出塞来昔布磺酸甲酯和塞来昔布磺酸乙酯杂质。该方法简便准确,可用于塞来昔布中塞来昔布磺酸甲酯和塞来昔布磺酸乙酯2个磺酸酯类基因毒性杂质的检测。     

塞来昔布诱导前列腺癌细胞凋亡及其机制研究

目的:研究塞来昔布对前列腺癌DU-145细胞凋亡及侵袭力的影响,并探讨其可能作用机制.方法:应用Hoechst 33342/PI染色检测细胞凋亡形态;Annexin V-FITC/PI双染色流式细胞术检测不同浓度塞来昔布诱导细胞凋亡能力;RT-PCR法检测塞来昔布作用后Bcl-2、E-cadherin、ICE及COX-2 mRNA表达水平的变化.结果:Hoechst 33342/PI双染色可观察到药物作用后.细胞呈现明显凋亡现象.流式细胞术证实塞来昔布能有效诱导细胞凋亡,0、25、50、100、200μmol/L塞来昔布诱导细胞凋亡率分别为(1.10±0.15)%,(3.87±0.79)%,(10.59±1.58)%,(22.50±3.30)%,(33.85±2.71)%,细胞凋亡率呈现浓度依赖性递增.RT-PCR显示Bcl-2mRNA表达水平下调,E-cadherin mRNA表达水平上调,ICE mRNA表达水平无明显变化,COX-2 mRNA未检测到.结论:塞采昔布能有效诱导前列腺癌DU-145细胞凋亡并使其侵袭力降低.     

塞来昔布诱导HCT-116结肠癌细胞G2/M阻滞

目的:研究选择性COX-2抑制剂塞来昔布诱导结肠癌细胞株HCT-116细胞周期阻滞的作用及其可能的机制。方法:应用流式细胞仪检测塞来昔布对HCT-116细胞周期的影响,定量PCR检测细胞周期素cyclinB1及COX-2 mRNA表达水平,Western-Blot检测细胞周期素cyclinB1的蛋白水平。结果:塞来昔布诱导HCT-116细胞G2/M阻滞的作用呈剂量依赖性,塞来昔布在mRNA及蛋白水平下调HCT-116细胞的cyclinB1。结论:塞来昔布能在体外抑制HCT-116细胞的增殖,诱导G2/M的阻滞,其作用与下调细胞周期素cyclinB1有关。     

塞来昔布对直肠癌HCA-7 细胞放射敏感性的实验研究

目的:观察环氧合酶-2(COX-2)抑制剂塞来昔布对直肠癌HCA-7细胞株的放射敏感性及探讨其机制。方法:采用MTT法检测塞来昔布作用不同时间对直肠癌HCA-7细胞株增殖的影响,计算出塞来昔布的半数抑制浓度IC50;HCA-7细胞克隆形成实验用于检测塞来昔布对HCA-7细胞的放射敏感性,并绘制存活曲线;流式细胞仪(FCM)测定塞来昔布对HCA-7的细胞周期的影响。结果:塞来昔布对HCA-7细胞株的抑制率随时间的延长而升高,48h的IC50是40.19μmol/L;照射组+药物组的SF2、D0、Dq、SER较单纯照射组均有所下降。塞来昔布使HCA-7细胞发生G2和M期阻滞,并抑制S期的比例。结论:塞来昔布能增加直肠癌HCA-7细胞的放射敏感性。     

分析塞来昔布与不同剂量艾瑞昔布治疗axSpA的效果及对骨代谢的影响

目的:探究塞来昔布和2种剂量艾瑞昔布治疗中轴脊柱关节炎(axSpA)的效果及对患者骨代谢的影响。方法:选取我院96例axSpA患者为研究对象,采用随机数字表法分为A组、B组、C组各32例。A组给予0.2 g/d艾瑞昔布治疗,B组给予0.4 g/d艾瑞昔布治疗C组给予0.4 g/d塞来昔布治疗。比较3组治疗前及治疗12周后疾病活动性[C反应蛋白(CRP)、红细胞沉降率(ESR)、Bath强直性脊柱炎疾病活动指数(BASDAI)]、躯体活动度(踝间距、腰椎侧弯度)、功能状态[Bath强直性脊柱炎功能指数(BASFI)、加拿大脊柱骨关节研究协会评分系统(SPARCC)]、骨代谢[血清骨形成发生蛋白-2(BMP-2)、血管内皮生长因子(VEGF)、Dickkopf相关蛋白1(DKK-1)]差异,并记录3组治疗期间不良反应发生情况。结果:治疗12周后,3组疾病活动性(CRP、ESR、BASDAI)、功能状态(BASFI、SPARCC)、骨代谢(BMP-2、VEGF、DKK-1)均较治疗前降低(P<0.05),躯体活动度(踝间距及左右侧腰椎侧弯度)则较治疗前升高(P<0.05);但B组及C组组间比较,差异无统计学意义(P>0.05),而A组上述指标变化幅度低于B组及C组(P<0.05)。3组治疗期间不良反应发生情况比较,差异均无统计学意义(P>0.05)。结论:较高剂量(0.4 g/d)艾瑞昔布疗效明显优于较低剂量(0.2 g/d),不良反应也未增加,且0.4 g/d艾瑞昔布及同剂量塞来昔布治疗axSpA具有相似的疗效及安全性,适用于临床治疗。     

双氯芬酸钠与塞来昔布治疗类风湿关节炎的疗效及对心血管的影响研究

目的:探讨双氯芬酸钠和塞来昔布治疗类风湿关节炎的临床疗效及用药安全性。方法:将我院2011年1月-2012年1月门诊收治的98例类风湿性关节炎患者随机分为对照组和观察组,每组49例。对照组给予双氯芬酸钠治疗,观察组给予塞来昔布治疗,观察两组临床治疗效果及心血管不良事件的发生情况。结果:观察组总有效率为91.84%显著高于对照组的75.51%,两组比较差异具有统计学意义(P0.05);观察组ESR及CRP分别为(110.65±7.28)mm/h和(10.42±0.98)mg/L显著低于治疗前和对照组,比较差异具有统计学意义(P0.05);观察组心血管不良事件发生率为8.16%显著低于对照组的20.41%,两组比较差异具有统计学意义(P0.05)。结论:塞来昔布治疗类风湿关节炎具有较好的临床疗效,可有效改善患者疼痛、僵硬或功能受限等症状,且心血管不良事件发生率低,值得临床进一步推广和应用。     

盐酸氨基葡萄糖联合塞来昔布对膝关节骨性关节炎的临床疗效观察

目的:观察盐酸氨基葡萄糖联合塞来昔布治疗膝关节骨性关节炎的效果及对不同程度关节炎Lequesne评分的影响,为关节炎的临床治疗提供参考。方法:选取2012年6月至2014年3月我院收治的膝关节骨性关节炎患者60例,根据治疗方法不同,将所选患者分为观察组和对照组,每组30例。对照组患者给予盐酸氨基葡萄糖单药治疗,观察组患者给予盐酸氨基葡萄糖和塞来昔布联合治疗。观察两种治疗方案的不良反应发生率,比较两组患者治疗前后的Lequesne评分。结果:对照组患者不良反应的发生率为26.67%,观察组为23.33%,差异无统计学意义(P0.05)。两组患者治疗后的Lequesne评分均低于治疗前,且观察组患者Lequesne评分显著低于对照组,差异具有统计学意义(P0.05)。观察组不同程度膝关节骨性关节炎患者的Lequesne评分均显著低于对照组,差异具有统计学意义(P0.05)。结论:盐酸氨基葡萄糖联合塞来昔布治疗膝关节骨性关节炎具有良好的临床效果,应进一步推广应用。     

双氯芬酸钠与塞来昔布治疗类风湿关节炎的疗效及对心血管的影响研究

目的：探讨双氯芬酸钠和塞来昔布治疗类风湿关节炎的临床疗效及用药安全性。方法：将我院2011 年1 月-2012 年1 月门诊收治的98 例类风湿性关节炎患者随机分为对照组和观察组,每组49 例。对照组给予双氯芬酸钠治疗,观察组给予塞来昔布治疗,观察两组临床治疗效果及心血管不良事件的发生情况。结果：观察组总有效率为91.84%显著高于对照组的75.51%,两组比较差异具有统计学意义（P〈0.05）;观察组ESR及CRP 分别为（110.65± 7.28）mm/h 和（10.42± 0.98）mg/L显著低于治疗前和对照组,比较差异具有统计学意义（P〈0.05）;观察组心血管不良事件发生率为8.16%显著低于对照组的20.41%,两组比较差异具有统计学意义（P〈0.05）。结论：塞来昔布治疗类风湿关节炎具有较好的临床疗效,可有效改善患者疼痛、僵硬或功能受限等症状,且心血管不良事件发生率低,值得临床进一步推广和应用。     

塞来昔布联合氟尿嘧啶对胰腺癌细胞株SW1990生长抑制作用及其机制研究

目的:研究选择性环氧化酶-2(COX-2)抑制剂塞来昔布联合氟尿嘧啶对胰腺癌细胞株SW1990生长的抑制作用,并对其机制进行初步探讨.方法:实验分为对照组、氟尿嘧啶组、塞来昔布组及两药联合组,将不同浓度的药物分剐作用于胰腺癌细胞株SW1990,MTT法检测各组细胞的生长抑制率,并探索产生最佳细胞生长抑制作用的药物浓度.流式细胞仪检测不同药物作用对肿瘤细胞周期的影响.RT-PCR检测各组细胞Survivin的表达情况.结果:MTT法显示氟尿嘧啶、塞来昔布均能抑制SW1990的生长,且细胞的存活率都随药物浓度的增加而降低.两药联合组对细胞生长的抑制作用更明显.流式细胞仪检测结果显示塞来昔布组、氟尿嘧啶组及两药联合组细胞较对照组G0/G1期细胞比例明显增加,S期和G2/M期细胞比例明显减少.RT-PCR结果显示氟尿嘧啶组、塞来昔布组、联合组都可下调Survivin的表达,以联合组最为明显.结论:塞来昔布联合氟尿嘧啶对胰腺癌SW1990细胞的生长具有抑制作用,其机制可能与下调Survivin的表达从而诱导细胞的凋亡和细胞周期的停滞有关.     

盐酸氨基葡萄糖联合塞来昔布治疗不同程度膝关节骨性关节炎的临床研究

目的:研究盐酸氨基葡萄糖联合塞来昔布治疗不同程度膝关节骨性关节炎的临床价值。方法:选取2011年1月至2014年2月已被收治的符合标准的病例共150例,按照病情轻重分为轻度(64例)、中度(52例)及重度(34例)3组,各组又随机等分为治疗组和对照组两组,治疗组给盐酸氨基葡萄糖联合塞来昔布治疗,对照组仅给予盐酸氨基葡萄糖治疗,分别于给药第2、4、6周以及停药8、12周观察两组患者经两种不同的治疗方案治疗后患者的Lequesne评分有无统计学差异。结果:轻度:两组停药12周后Lequesne评分仍低于治疗前,差异有统计学意义(P0.05),停药12周时治疗组低于对照组,差异有统计学意义(P0.05)。中度:用药2周、4周、6周及停药8周时两组相比差异均有统计学意义(P0.05)。重度:对照组与治疗组在给药4周及给药6周时其Lequesne评分差异有统计学意义(P0.05),停药后均无统计学意义(P0.05)。结论:单用盐酸氨基葡萄糖可改善轻度膝关节骨性关节炎的临床症状,对于中度患者联合非类固醇类抗炎药类有较好疗效,对于重度患者两种方式都无明显效果。     

Simultaneous determination of nicotinic acid and its four metabolites in rat plasma using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS)

A sensitive and specific liquid chromatography electrospray ionization–tandem mass spectrometry method for the simultaneous quantitation of nicotinic acid (NicA) and its metabolites nicotinamide (NA), 1-methylnicotinamide (MNA), 1-methyl-2-pyridone-5-carboxamide (M2PY) and 1-methyl-4-pyridone-5-carboxamide (M4PY) in rat plasma has been developed and validated. As an internal standard, 6-chloronicotinamide was used. The samples (100 μL) were subjected to deproteinization with acetonitrile (200 μL) and then, after centrifugation, 150 μL of the supernatant was transferred into conical vial and evaporated. Dry residue was reconstituted in 100 μL of the ACN/water (10:90, v/v) mixture. Chromatography was performed on a Waters Spherisorb^® 5 μm CNRP 4.6 × 150 mm analytical column with gradient elution using a mobile phase containing acetonitrile and water with 0.1% of formic acid. The full separation of all compounds was achieved within 15 min of analysis. Detection was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer set at unit resolution. The mass spectrometer was operated in the selected reactions monitoring mode (SRM), monitoring the transition of the protonated molecular ions m/z 153–110 for M2PY, 153–136 for M4PY, 124–80 for NicA, 123–80 for NA and 137–94 for MNA. The mass spectrometric conditions were optimized for each compound by continuously infusing the standard solution at the rate of 5 μL/min using a Harvard infusion pump. Electrospray ionization (ESI) was used for ion production. The instrument was coupled to an Agilent 1100 LC system. The precision and accuracy for both intra- and inter-day determination of all analytes ranged from 1.3% to 13.3% and from 94.43% to 110.88%. No significant matrix effect (ME) was observed. Stability of compounds was established in a battery of stability studies, i.e. bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for various applications. In particular using this method we detected increased concentration of MNA and its metabolites in rat plasma after treatment with exogenous MNA (100 mg/kg), as well as increased concentration of endogenous NA and MNA in rat plasma in the early phase of hypertriglyceridemia development in rats fed high-fructose diet.     

Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.     

Simultaneous determination of ipratropium and salbutamol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study

A novel, sensitive and specific LC-MS/MS method with silica-based solid-phase extraction was developed for simultaneous determination of ipratropium (IPR) and salbutamol (SAL) in rat plasma. Chromatographic separation was achieved on a Shiseido Capcell Pak CR column (SCX:C(18)=1:4, 150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of methanol/water (85:15, v/v) containing 20 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. This method was validated in terms of specificity, linearity, accuracy (within ±115.4%), intra- and inter-day precision (<11.4%) over the concentration range of 8-1612 pg/mL for IPR and 50-10,000 pg/mL for SAL. In addition, stability and matrix effects of IPR and SAL in plasma were evaluated. This method has been successfully applied to the pharmacokinetic study of compound ipratropium bromide aerosol mainly containing ipratropium bromide (IB) and salbutamol sulphate (SS) after inhalation in rats.     

Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS

An LC/MS/MS assay for the determination of tenofovir (TNF) was developed and validated for use with the EDTA anticoagulated human plasma matrix. Heparin-treated plasma and serum matrices were also validated. After addition of adefovir as an internal standard, trifluoroacetic acid was used to produce a protein-free extract. Chromatographic separation was achieved with a Polar-RP Synergi, 2.0 mm x 150 mm, reversed-phase analytical column. The mobile phase was 3% acetonitrile/1% acetic acid, aq. Detection of TNF and the internal standard was achieved by ESI MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The method was linear from 10 to 750 ng/ml with a minimum quantifiable limit of 10 ng/ml when 250 microl aliquots were analyzed. The usefulness of this LC/MS/MS method to routinely monitor plasma concentrations of TNF was demonstrated along with its ability to assist in the performance of pharmacokinetic studies.     

Stereoselective analysis of bupropion and hydroxybupropion in human plasma and urine by LC/MS/MS

A sensitive, stereoselective assay using solid phase extraction and LC-MS-MS was developed and validated for the analysis of (R)- and (S)-bupropion and its major metabolite (R,R)- and (S,S)-hydroxybupropion in human plasma and urine. Plasma or glucuronidase-hydrolyzed urine was acidified, then extracted using a Waters Oasis MCX solid phase 96-well plate. HPLC separation used an alpha(1)-acid glycoprotein column, a gradient mobile phase of methanol and aqueous ammonium formate, and analytes were detected by electrospray ionization and multiple reaction monitoring with an API 4000 Qtrap. The assay was linear in plasma from 0.5 to 200 ng/ml and 2.5 to 1000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. The assay was linear in urine from 5 to 2000 ng/ml and 25 to 10,000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. Intra- and inter-day accuracy was >98% and intra- and inter-day coefficients of variations were less than 10% for all analytes and concentrations. The assay was applied to a subject dosed with racemic bupropion. The predominant enantiomers in both urine and plasma were (R)-bupropion and (R,R)-hydroxybupropion. This is the first LC-MS/MS assay to analyze the enantiomers of both bupropion and hydroxybupropion in plasma and urine.     

A sensitive assay for simultaneous determination of plasma concentrations of valganciclovir and its active metabolite ganciclovir by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of valganciclovir and its active metabolite ganciclovir in human plasma. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on an Aquasil C18 column (50 mm x 2.1mm, 5 microm). A linear gradient mobile phase between 0.02% formic acid and methanol was used. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 4 to 512 ng/mL for valganciclovir and from 0.1 to 12.8 microg/mL for ganciclovir, were fitted to a 1/x weighted quadratic regression model. The method was proved to be accurate, specific and sensitive enough and was successfully applied to a pharmacokinetic study.     

Simultaneous determination of tectorigenin, irigenin and irisflorentin in rat plasma and urine by UHPLC-MS/MS: application to pharmacokinetics

A sensitive and reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) has been developed and validated for the simultaneous determination of three active components, i.e., tectorigenin, irigenin and irisflorentin, in rat plasma and urine after oral administration of Rhizoma Belamcandae extract. Chromatographic separation was achieved on a Zorbax SB-C(18) column (50 mm × 2.1 mm, 1.8 μm; Agilent, USA) with gradient elution using a mobile phase that consisted of acetonitrile - 0.1% formic acid in water at a flow rate of 0.4 mL/min. Detection was performed by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between the negative (for tectorigenin and irigenin) and positive (for irisflorentin) ionization modes. The calibration curve was linear over a range of 50-50,000 ng/mL for tectorigenin, 10-5000 ng/mL for irigenin and 0.1-200 ng/mL for irisflorentin, respectively. The intra- and inter-day precisions (RSD %) were within 11.3% for all analytes, whereas the deviation of assay accuracies ranged from -8.7 to +11.1%. All analytes were proven to be stable during all sample storage and analysis procedures. This method was successfully applied to a pharmacokinetic study of the three isoflavones after oral administration of Rhizoma Belamcandae extract to rats.     

Quantitative determination of cyclic phosphatidic acid in human serum by LC/ESI/MS/MS

An LC/ESI/MS/MS method for cyclic phosphatidic acid (cPA) quantification in serum is established in the present report. The limit of quantitation of the assay reaches low nanomolar level in human serum and the CV% are within 10%. Using this method, we successfully quantify the levels of two cPA species, 16:0 and 18:1, in human serum. We find that the concentrations of 16:0 cPA in the serum of normal subjects and post-surgery ovarian cancer patients are significantly higher than its corresponding concentration in pre-surgery ovarian cancer patients, supporting the observation that cPA has anti-cancer activity. Another discovery is that the addition of strong acids (such as hydrochloric acid) in human serum may lead to the production of artificial cPA. Therefore, strong acids should be avoided in the extraction of cPA present in a complex matrix. Based on this observation, a new lipid extraction method was developed and used to extract cPA. The extraction recovery is close to 80%, guaranteeing an accurate quantification of cPA by LC/ESI/MS/MS can be performed.     

Simultaneous determination of five flavonoids from Scutellaria Barbata extract in rat plasma by LC-MS/MS and its application to pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of five flavonoids including scutellarin, naringenin, apigenin, luteoline and wogonin in rat plasma using sulfamethalazole as internal standard (IS). Plasma samples were pretreated with liquid-liquid extraction procedure and acid hydrolysis method was used for converting conjugated flavonoids to their respective free forms. The chromatographic separation was performed on a C(18) column with a linear gradient elution using a mobile phase consisted of 0.01% acetic acid and methanol. The detection was accomplished by multiple-reaction monitoring (MRM) scanning with electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion-pairs (m/z) monitored for scutellarin, naringenin, apigenin, luteoline, wogonin and IS were 461.1/285.1, 271.0/119.0, 269.0/117.0, 285.0/132.9, 283.0/268.0 and 252.0/155.9, respectively. The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.9915. The lower limit of quantitation (LLOQ) of scutellarin was 9.15 ng/mL and other compounds were all less than 2.0 ng/mL. The proposed method showed appropriate accuracy and repeatability and was suitable for pharmacokinetic studies of the five flavonoids after oral administration of Scutellaria Barbata extract.