Determination of the oral platelet aggregation inhibitor Sibrafiban in rat, dog, and human plasma utilising HPLC-column switching combined with turbo ion spray single quadrupole mass spectrometry

A sensitive and selective HPLC-column switching method with single quadrupole mass spectrometric detection was developed for the simultaneous determination of the oral platelet aggregation inhibitor Sibrafiban (double protected prodrug), its prodrug and the active metabolite in rat, dog, and human plasma. The three analytes together with their tri-deuterated internal standards were isolated from plasma by protein precipitation (0.5 M perchloric acid). The de-proteinated samples were injected onto a standard-bore trapping column (4.0 mm i.d., LC-ABZ) of an HPLC-column switching system. Polar plasma components were removed by flushing the trapping column with ammonium formate (pH 3.6; 5 mM). Enriched compounds (including the analytes of interest) were backflushed onto a narrow-bore analytical column (2.1 mm i.d., Inertsil ODS-2) and separated by gradient elution (formic acid/ methanol). The whole effluent (200 microl/min) from the analytical column was passed to the turbo ion spray interface without splitting. Selected ion monitoring (SIM) was used for mass spectrometric detection. The limit of quantification for all three analytes was 1 ng/ml, using a 250-microl specimen of plasma. The mean precision and inaccuracy for the three analytes in all species were < 6 and < 5%, respectively. The practicability of the new analytical method was demonstrated by the analysis of about 500 rat and dog plasma and about 14,000 human plasma samples. The new method represents a successful example for the application of LC single MS with ionspray ionisation to the analysis of small molecule drugs in biological matrices from toxicokinetic studies and large clinical trials.     

葫芦素B对肝癌细胞内阿霉素浓度的影响

目的建立肝癌细胞H22和HepG2中阿霉素含量测定的高效液相色谱方法,以明确葫芦素B对阿霉素在细胞内浓度的影响。方法色谱柱为Waters Sunfine C18柱(4.6 mm×150 mm,5μm),流动相为甲醇-0.01%醋酸(40∶60,V/V),流速为1.0 mL/min,柱温为30℃,荧光检测激发波长为495 nm,发射波长为560 nm,以盐酸表阿霉素为内标。结果在HepG2细胞液中,阿霉素线性范围为0.1～6.4μg/mL(r=0.993 4,n=7),加样回收率为107.4%,RSD为3.77%;在H22细胞液中,阿霉素线性范围为0.625～20μg/mL(r=0.996,n=7),加样回收率为105.93%,RSD为5.64%。结论在肝癌细胞中,葫芦素B能明显促进阿霉素内流,减少外排。葫芦素B使肝癌细胞中阿霉素的浓度升高,两者联合可能起到更好的抗肝癌作用,葫芦素B可能逆转肝癌细胞的阿霉素耐药性。     

高效液相色谱法测定大鼠血浆及组织中多柔比星含量

目的：建立高效液相色谱法测定大鼠血浆及组织样品中多柔比星的含量,并研究其在大鼠体内的分布特征。方法：生物样品经过液-液萃取后,采用HPLC-FLD法,色谱柱为Zorbax SB-C1（8150mm×4.6mm,5μm）,流动相为乙腈和0.01mol·L-1磷酸二氢钾缓冲液（pH2.3）,梯度洗脱,流速为1mL·min-1,柱温为35℃;荧光检测器,激发波长为237nm,发射波长为555nm。结果：该法测定血浆中多柔比星的线性范围为2.5-500ng·mL-1,r=0.9991。其他组织的分析方法也均满足生物样品的测定要求。静脉给予5mg·kg-1多柔比星,结果显示其在大鼠体内分布广泛且持久,肾脏和脾脏及毒性靶器官心脏中浓度较高。结论：该方法准确度、灵敏度高,可以测定72h内多柔比星在主要组织中的分布,可用于研究心脏毒性与多柔比星＂量＂之间相关性。     

维生素K3体外降低Ehrlich腹水癌细胞对阿霉素的抗药性

AIM: To study the effect of menadione (Men) reducing doxorubicin (Dox) resistance in Ehrlich ascites carcinoma (EAC) cells resistant to Dox (EAC/Dox cells). METHODS: Glutathione (GSH) content and membrane fluidity were measured by fluorometric assay and fluorescence depolarization assay, respectively. Glutathione S-transferase (GST) activity was measured with 1-chloro-2,4-dinitrobenzene as the substrate. Cell viability was determined by 3-(4, 5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide assay. RESULTS: GSH content, GST activity, and membrane fluidity in EAC/Dox cells were higher than those in EAC cells (P < 0.01). The IC50 (95% confidence limits) for Dox on EAC/Dox cell was 22.3 (15.8-28.8) mg.L-1. Relative resistance of Dox in EAC/Dox cells was 42-fold. Pretreatment of EAC/Dox cells with Men 5 or 10 mg.L-1 decreased intracellular GSH content (P < 0.01). Men 1 mg.L-1 had no obvious effect on GSH content in EAC/Dox cells (P > 0.05), but decreased the elevated membrane fluidity efficiently (P < 0.05). Men had no obvious effect on GST activity in EAC/Dox cells (P > 0.05). IC50 of Dox was reduced to 9.6 (7.8-11.3), 6.0 (2.8-9.2), or 5.3 (3.9-6.7) mg.L-1 in EAC/Dox cells pretreated with Men 1, 5, or 10 mg.L-1. CONCLUSION: Men reduced Dox resistance effectively due in part to its depletion of GSH content in EAC/Dox cells.     

HPLC-MS/MS法同时测定Beagle犬血浆中槲皮素、山萘酚和异鼠李素(英文)

目的:建立同时测定犬血浆中槲皮素、山萘酚和异鼠李素浓度的HPLC-MS/MS方法。方法:血浆样品经酸水解后用乙醚提取,采用选择性反应检测方法测定其血药浓度。仪器为Finni-ganLC-MS/MS二级四极杆质谱检测器,色谱柱为LunaC18(150mm×2.00mmi.d.,5μm,Luna,USA);流动相为乙腈-0.1%甲酸,梯度洗脱。结果:槲皮素、山萘酚和异鼠李素的线性范围均为0.5～100.0ng/mL,三种黄酮的最低定量限均为0.5ng/mL,各自日内日间精密度分别小于7.3%,6.2%和6.4%,回收率分别大于70%,66%和70%。结论:该测定方法经验证符合血浆样品的测定要求,可以应用于临床前药代动力学研究。     

吗丙嗪对阿霉素作用下大鼠脂质过氧化的影响

采用比色法测定GSH-Px,SOD活性及丙二醛含量,注射阿霉素后,大鼠全血、心肌中GSH-Px及红细胞SOD活性明显下降,心肌丙二醛含量显著升高,给予不同剂量的吗丙嗪后,GSH-Px及SOD活性有明显回升,并呈剂量依赖关系;而心肌丙二醛含量明显下降,提示阿霉素可能通过自由基途径对机体产生影响,而吗丙嗪通过提高体内抗氧化酶的活力,减轻脂质过氧化损伤而对心肌起保护作用。     

葫芦素B和阿霉素联合应用在体内外对肝癌细胞H22的生长抑制作用

目的研究葫芦素B和阿霉素联合应用在体内外对肝癌细胞H22的生长抑制作用。方法将40只雌性昆明小鼠随机分为4组,每组10只,包括阴性对照组(生理盐水组)、阿霉素组、葫芦素B组、联合组。分别以阿霉素、葫芦素B、葫芦素B联合阿霉素处理H22细胞,培养48 h后MTT法检测细胞增殖情况。计算抑瘤率、白细胞数、胸腺指数、脾脏指数和血药浓度。结果 MTT结果显示,与阿霉素组相比,联合组对H22细胞的增殖抑制作用明显增强,联合组与阿霉素组之间、高剂量组与低剂量组之间的差异均有统计学意义(P<0.05或P<0.01)。联合组与阿霉素组相比,抑瘤率明显增强,并且联合组阿霉素在心脏中的浓度明显降低(P<0.05)。结论葫芦素B和阿霉素联合应用具有增强抗H22肝癌作用。     

Cardiotoxic effects and the influence on the beta-adrenoceptor function of doxorubicin (Adriamycin) in the rat

The possible relationship between the effect of the anthracycline-cytostatic doxorubicin (Dox) on the cardiac beta-adrenoceptor function in vitro and the development of delayed cardiotoxicity in vivo has been investigated in the rat. Dox (10(-5)-10(-4) M) blocked the chronotropic effect of isoprenaline on isolated atria in a competitive manner. Treatment with a single dose of Dox 5 mg/kg intravenously caused marked ECG changes manifested by progressive prologations of the Q alpha T and S alpha T-intervals, which amounted to 37% and 58% respectivity 5 weeks after the medication. At this time no beta-blocking action was detectable when tested on the isolated atria in the same rats. The results indicate that the delayed cardiotoxicity induced by Dox is not mediated by an interference with the cardiac beta-adrenoceptor function.     

Determination of thiencynonate by liquid chromatographic-mass spectrometry and its application to pharmacokinetics in rats

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry method (LC/ESI/MS) was developed and validated for the identification and quantification of the novel lead compound of anticholinergic drug thiencynonate in rat plasma. The analytes were determined using positive electrospray ionization mass spectrometry in the selected reaction ion monitoring (SRM). The chromatography separation was on BetaBasic-18 column (150 mm x 2.1 mm i.d., 3 microm). The mobile phase was composed of methanol-water (70:30, v/v), containing 0.5 per thousand formic acid, which was pumped at a flow rate of 0.2 ml/min. Phencynonate was selected as the internal standard (IS). Simultaneous MS detection of thiencynonate and IS was performed at m/z 364.4 (thiencynonate), m/z 358 (phencynonate), and the SRM of the two compounds were both at 156. Thiencynonate eluted at approximately 2.8 min, phencynonate eluted at approximately 2.9 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1-100 ng/ml in rat plasma. The lower limit of quantification (LLOQ) was reproducible at 1 ng/ml in rat plasma. The precision measured was obtained from 2.47 to 9.28% in rat plasma. Extraction recoveries were in the range of 67.63-76.76% in plasma. This method was successfully applied to the identification and quantification of thiencynonate in pharmacokinetic studies.     

An improved HPLC method for therapeutic drug monitoring of daunorubicin, idarubicin, doxorubicin, epirubicin, and their 13-dihydro metabolites in human plasma.

A single high-performance liquid chromatography (HPLC) method, suitable for the analysis of daunorubicin, idarubicin, doxorubicin, epirubicin, and their 13-dihydro metabolites is validated in the present study. Preparation of plasma samples was performed by a first extraction of analytes with a chloroform/1-heptanol mixture (9:1) and reextraction with ortophosphoric acid 0.1 M. The chromatographic analysis was carried out by reversed-phase isocratic elution of anthracyclines with a Supelcosil LC-CN 5 mm column (25 cm x 4.6 mm internal diameter; Supelco) and detection was accomplished by spectrofluorimetry at excitation and emission wavelengths of 480 and 560 nm, respectively. All anthracyclines eluted within 15 minutes of injection and the method appeared to be specific, because the chromatographic assay did not show interferences at the retention time of analytes. The linearity, evaluated over a concentration range of 0.4-10,000 ng/mL, gave regression coefficients better than 0.999, with recoveries of doxorubicin-doxorubicinol and epirubicin-epirubicinol of 67%-109% and 61%-109% respectively, and 93%-109% for the other compounds. The limits of detection and quantification were 0.4 ng/mL in a 50-mL sample (40 pg/injection) for all anthracyclines tested. The method proved to be precise and accurate, as the within-day and between-day coefficients of variation were less than 10% and the accuracy of the assay was in the range of 91%-107%. Overall results indicate that it is feasible to analyze all the anthracyclines used in clinical practice and their major metabolites with a single optimized method, thereby simplifying their monitoring in chemotherapeutic regimens of cancer patients.     

Direct simultaneous determination of drug discovery compounds in monkey plasma using mixed-function column liquid chromatography/tandem mass spectrometry.

A direct injection method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for the simultaneous determination of two drug candidates in monkey plasma samples in support of pharmacodynamic studies. Each diluted monkey plasma sample containing internal standard was directly injected into a mixed-function column for sample cleanup, enrichment and chromatographic separation. The proteins and macromolecules first passed through the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column with an aqueous liquid mobile phase were then chemically eluted by switching to a strong organic mobile phase at a constant flow rate of 1.0 ml/min. The column effluent was also diverted from waste to mass spectrometer for analyte detection. Samples from two different analyte studies were assayed in one analytical procedure and the calibration curves were prepared using both analytes. The calibration curves were linear over the range of 5-2500 ng/ml for both analytes. The retention times for analytes and the internal standard were found to be consistent and no column deterioration was observed after 200 injections. The apparent on-column recoveries for the test compounds in monkey plasma samples were greater than 90% with 6% CV (N=5). The total analysis time was less than 5 min per sample.     

Determination of diclofenac sodium, flufenamic acid, indomethacin and ketoprofen by LC-APCI-MS

A sensitive, selective and accurate high-performance liquid chromatography-mass spectrometry (LC-MS) assay for the determination of selected non-steroidal anti-inflammatory drugs (NSAIDs), namely diclofenac sodium (DIC), flufenamic acid (FLU), indomethacin (IND) and ketoprofen (KET), either individually or in mixtures, was developed. The examined drugs were injected onto Shim-pack GLC-CN column and were eluted with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate solution (5:1 v/v)/pH 7.4 at a flow rate l ml min(-1). The mass spectrometer, operated in the single ion monitoring mode, was programmed to admit the negative ions [M-H] at m/z 295.9 (DIC), 280.1 (FLU), 355.8 (IND) and 252.9 (KET), respectively. The calibration curves were linear (r > or = 0.9993) over the concentration range 50-300 ng ml(-1) (FLU, DIC) and 100-500 ng ml(-1) (KET, IND) with detection limits of 0.5-4.0 ng. The mean predicted concentrations for the analytes were in the range -5.9 and 5.2% of the nominal concentrations. Within-day and between-day precision were in the range of 0.8-9.1% of the R.S.D. Mean recovery percentages of the individual compounds from laboratory-made mixtures and pharmaceutical formulations were (99.5-101.5%) and (100.6-102.2%), respectively.     

他莫昔芬体外降低EAC/ADR细胞对阿霉素的耐药性

目的研究他莫昔芬体外降低EAC／ADR细胞对阿霉素耐药性的作用及耐药性与细胞膜流动性的相关性。方法细胞内谷胱甘肽含量及细胞膜流动性分别以荧光分光光度法及荧光偏振法测定，细胞存活力以甲基四唑蓝法测定。结果EAC／ADR细胞膜流动性及谷胱甘肽含量均比敏感EAC细胞明显提高。他莫昔芬2mg·L-1及5mg·L-1不影响EAC／ADR细胞谷胱甘肽含量（P＞0.05），但可明显降低其细胞膜流动性（P＜0.05）。他莫昔芬2mg·L-1或5mg·L-1与阿霉素体外合用于EAC／ADR细胞可降低其对阿霉素的耐药性，同时发现他莫昔芬不影响阿霉素对敏感EAC细胞的毒性。结论EAC／ADR细胞膜流动性增加与其对阿霉素的耐药性有着密切关系，他莫昔芬在降低EAC／ADR细胞膜流动性的同时降低细胞对阿霉素的耐药性。     

Determination of doxorubicin and its metabolites in rat serum and bile by LC: application to preclinical pharmacokinetic studies

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the simultaneous determination of doxorubicin and its three metabolites, including doxorubicinol, doxorubicinolone and doxorubicinone, in rat serum and bile. Following a single protein precipitation step, chromatographic separation was accomplished using a C-18 column with a mobile phase consisting of 50 mM sodium phosphate buffer-acetonitrile-1-propanol (65:25:2, v/v), pH 2.0. The analytes were measured by fluorescence detection with excitation wavelength of 480 nm and emission wavelength of 560 nm. The lower limits of quantitation were 10 ng/ml for doxorubicin, and 5 ng/ml for the three metabolites. The calibration curves were linear over a concentration range of 10-2500 ng/ml for doxorubicin, and 5-1250 ng/ml for its three metabolites. The average recoveries were greater than 89% for all analytes. The within-day and between-day coefficients of variation were generally less than 13%. Doxorubicin and its metabolites were stable in the precipitated serum and bile samples at room temperature in darkness for at least 48 h. This method permitted the analysis of samples without the presence of the anticoagulant sodium citrate and thus was applied to serum and bile samples collected from rats that were administered doxorubicin intravenously in a pharmacokinetic study.     

Quantitative determination of atorvastatin and ortho-hydroxy atorvastatin in human plasma by liquid chromatography tandem mass spectrometry and pharmacokinetic evaluation

A specific, sensitive and simple method was developed to determine the levels of both atorvastatin and ortho-hydroxy atorvastatin in human plasma. The analytes and internal standard pitavastatin were extracted from plasma by liquid-liquid extraction, separated on a Zorbax SB-C18 column, eluted with a mobile phase of water:acetonitrile (45:55 v/v), both containing 5% methanol and 0.01% formic acid. Detection was performed with an electrospray ionization triple quadrupole mass spectrometer in positive ion mode using multiple reaction monitoring. The standard calibration curves of atorvastatin and ortho-hydroxy atorvastatin were linear in the concentration range of 0.2-20 and 0.1-20 ng/mL, respectively. The intra- and inter-day precisions were < 7.7% and the accuracy was within ± 5.9%. The method has been successfully used for the study of the pharmacokinetics of atorvastatin and ortho-hydroxy atorvastatin in Chinese patients with coronary heart disease after a single oral dose of 20 mg atorvastatin. The mean values for the area under the plasma concentration-time curve for atorvastatin and ortho-hydroxy atorvastatin were 63.1 and 46.9 ng.h/mL, respectively.     

Determination of caderofloxacin lactate in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography–electrospray ionization mass spectrometric (LC–ESI-MS) method for the quantification of a newly active quinolone carboxylic acid caderofloxacin lactate in rat plasma was developed and validated after precipitation method with methanol. Chromatographic separation was achieved on a reversed-phase Shimadzu 2.0 μm C18 column (150 mm × 2.00 mm) with the mobile phase of methanol–0.02% formic acid and step gradient elution resulted in a total run time of about 10.0 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (5–2000 ng/mL) (r = 0.9998). The lowest limit of quantification (LLOQ) was 5 ng/mL and the lowest limit of detection (LLOD) was 2 ng/mL. Average recoveries ranged from 88.80 to 93.05% in plasma at the concentrations of 10, 100 and 1000 ng/mL. Intra- and inter-day relative standard deviations were 4.01–7.30 and 4.15–7.51%, respectively. This method was successfully applied in the pharmacokinetic studies in rats.     

氟哌啶醇对人红白血病耐多柔比星细胞株多药耐药性的逆转及其机制

目的　从临床常用药物中探寻逆转肿瘤耐药性的活性物质。方法　应用MTT法测定不同浓度Hal处理的瘤细胞对 0～ 2 0 μmol·L- 1多柔比星 (Dox)的敏感性的影响。RT PCR法分析 12 .5 μmol·L- 1氟哌啶醇 (Hal)处理后多药耐药基因 (MDR1) ,多药耐药相关蛋白 (MRP)和谷胱甘肽S转移酶Pi(GSTπ)mRNA表达的变化。通过流式细胞术观察 0 ,6 .2 5 ,12 .5 ,2 5 μmol·L- 1Hal对细胞内药物蓄积和细胞周期进程的影响。结果　Hal对K5 6 2 /Dox的耐药性具有明显的逆转作用。在 12 .5 ,6 .2 5及 3.12 5 μmol·L- 1时的逆转倍数分别为 8.35 ,4 .2 1及 2 .16。用 12 .5μmol·L- 1Hal处理后 ,MDR1及MRP的mRNA表达水平均呈现时间依赖性明显降低 ,分别较原水平下降76 .3%及 6 4.6 %。药后d 2GSTπmRNA表达下降6 6 .1% ,于d 3回升。Hal处理细胞lh后 ,Dox在细胞内蓄积量明显增加 ,并呈浓度依赖性 ;此外 ,Hal可明显增强Dox对K5 6 2 /Dox细胞的G2 /M阻滞作用 ,12 .5 μmol·L- 1浓度可以使 5 μmol·L- 1Dox的G2 /M阻断由单独应用时的 9.9%± 4 .3%增加到2 3.4 %± 3.0 %。结论　Hal具有较强的逆转K5 6 2 /Dox细胞MDR的作用 ,其逆转机制为多种途径 ,包括相关基因mRNA的表达下调 ,增加细胞内药物蓄积 ,增强Dox对K5 6 2 /Dox在G2     

Liquid chromatographic/mass spectrometry assay of triptolide in dog plasma and its application to pharmacokinetic study

A rapid and sensitive method for the determination of triptolide in dog plasma was developed and validated, using high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI/MS). Sample preparation consisted of liquid-liquid extraction with ethyl acetate from dog plasma. The analytes and internal standard prednisolone were well separated on a Zorbax Extend-C18 analytical column. Detection was performed on a triple quadrupole mass spectrometer using selected-ion monitoring (SIM) mode on the deprotonated ions [M-H]- at m/z 359. Calibration curves were linear over the concentration range of 0.5-200 ng/mL of triptolide with the intra- and inter-day precision (the relative standard deviation values) were being less than 7%. Triptolide was stable under different conditions. The intra-day and inter-day accuracy were 99.3-105.2% and 101.3-107.0%, respectively. The lower limit of quantification was 0.5 ng/mL. The method was successfully applied to a pharmacokinetic study after an intragastric administration (i.g.) of triptolide to dogs with a dose of 0.05 mg/kg. The results confirm that the assay is suitable for the pharmacokinetic study of triptolide.     

A simple and sensitive gradient elution liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the quantification of doxorubicin in rabbit plasma

In the present study, we developed and validated a simple and sensitive gradient elution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of doxorubicin in rabbit plasma. Daunorubicin was used as an internal standard (IS). The doxorubicin and IS were extracted with ethyl acetate from plasma samples. The chromatographic separations were achieved on a C₁₈ column (2.1 mm×50 mm, 2.5 μm) configured with a C₁₈ guard column (2.1 mm×10 mm, 2.5 μm). The mobile phase of 0.1% formic acid-water solution and acetonitrile was delivered using a gradient elution program at a flow rate of 0.4 mL/min. The temperature for column was maintained at 40 ºC. The electrospray ionization (ESI) source was operated in the positive ion mode, and the quantification was conducted using multiple reaction monitoring (MRM) of the transitions m/z 544.07→396.96 and m/z 528.06→321.05 for doxorubicin and IS, respectively. The calibration curve of doxorubicin was linear (r > 0.999) within the range of 2-600 ng/mL. The lower limit of quantification was 2 ng/mL. The relative errors of intra­day and inter-day accuracies ranged from -2.48% to 0.18% and from -3.78% to 1.94%, respectively. The relative standard deviations of intra­day and inter-day precisions were less than 8.65% and 6.41%, respectively. The method exhibited satisfactory results in terms of specificity, sensitivity, matrix effect, recovery and stability. The newly developed LC-MS/MS method was reliable to monitor doxorubicin concentrations in rabbit plasma.