Simultaneous determination of tetrahydropalmatine,protopine, and palmatine in rat plasma by LC-ESI-MS and its application to a pharmacokinetic study

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the simultaneous quantification of tetrahydropalmatine, protopine and palmatine in rat plasma using phenacetin as the internal standard (IS). Two hundred microliters plasma samples were extracted by dichloromethane under a strong basic condition. The analytes were separated by a C18 column and detected with a single quadrupole mass spectrometer. The used mobile phase was acetonitrile–water (40:60, v/v) containing 5 mM ammonium acetate and 0.2% glacial acetic acid. Detection was carried out by positive electrospray ionization in selected ion reaction (SIR) mode at m/z 356.6 for tetrahydropalmatine, 354.6 for protopine, 352.6 for palmatine and 180.4 for the IS, respectively. The method was validated over the concentration range of 1.00–500 ng mL⁻¹ and the lower limit of quantification (LLOQ) was 1.00 ng mL⁻¹ for all three analytes. The intra- and inter-day precision values were less than 9% relative standard deviation (R.S.D.), and the relative error ranged from −7.4 to 4.8%. The extraction recoveries were on average 91.42% for tetrahydropalmatine, 84.75% for protopine, 57.26% for palmatine, and 83.18% for IS. The validated method was successfully applied to a pharmacokinetic study of tetrahydropalmatine, protopine and palmatine in rats after oral administration of Rhizoma Corydalis Decumbentis extract.     

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.     

LC-MS method for determination and pharmacokinetic study of chimaphilin in rat plasma after oral administration of the traditional Chinese medicinal preparation Lu xian cao decoction

Lu xian cao (Hebra pyrolae) is a traditional Chinese medicine used for the treatment of several major diseases. Chimaphilin has been demonstrated one of the major active components in Lu xian cao by modern pharmacological studies. In this study, a liquid chromatography-mass spectrometry method for the determination of chimaphilin in rat plasma was developed and validated. The separation was carried out on a C18 column using methanol and water as mobile phase after the plasma sample was extracted with diethyl ether. Atmospheric pressure chemical ionization in negative ion mode and selected ion monitoring method were developed to determine [M]- at 186 and 210 for chimaphilin and benzil (internal standard), respectively. The lower limit of quantitation was 10 ng/ml and the calibration curve was linear (r>0.9964) over the concentration range 10-1000 ng/ml. The method was demonstrated reproducible and reliable with intra-day precision<11.5%, inter-day precision<7.6%, accuracy in the range of 88.4-113.0%, and mean extraction recovery excess of 83.0%, which were all calculated from the quality control samples at concentrations of 20, 100, and 500 ng/ml. The method was successfully applied to pharmacokinetic study of chimaphilin in rat plasma following oral administration of a 30-mg/kg dose of chimaphilin in Lu xian cao decoction to male Wistar rats.     

SPE-HPLC method for the determination and pharmacokinetic studies on paeoniflorin in rat serum after oral administration of traditional Chinese medicinal preparation Guan-Xin-Er-Hao decoction

A new HPLC method for the determination of paeoniflorin in rat serum with solid-phase extraction (SPE) for preconcentration is introduced. Paeoniflorin and an internal standard (pentoxifylline) were extracted from serum by means of SPE using cartridges with octadecyl chemically bound phase. The HPLC separation was then performed on a reversed-phase C(18) column using acetonitrile-water (18:82, v/v) as eluting solvent system, and UV detection at 230 nm to measure the analyte with a limit of quantitation about 10 ng ml(-1). The calibration curve for paeoniflorin was linear (r=0.9938) in the concentration range of 10-1200 ng ml(-1), both intra- and inter-day precision of the paeoniflorin were determined and their coefficience of variation did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of paeoniflorin from rat serum after oral administration of Guan-Xin-Er-Hao decoction.     

传统中药汤剂与免煎中药饮片的对比

免煎中药饮片是中药汤剂改革的一种现代化的尝试,由于对免煎中药饮片的相关研究还比较薄弱,目前也没有免煎中药饮片的专用质量控制标准,在免煎中药饮片的临床应用中也出现了很多问题,这些都需要我们立足于传统中医药理论,拓宽思路,结合现代实验研究的手段,使免煎中药饮片向合理化、普遍化的方向发展.     

传统中药汤剂在糖尿病并发症治疗中的应用分析

目的：评价中药汤剂治疗糖尿病并发症的有效性。方法计算机检索中国生物医学文献数据库（CBM）、中国知网（CNKI）、维普中文科技期刊数据库（VIP）和万方数据资源系统（WF）。收集各种中药汤剂治疗糖尿病并发症的临床随机对照试验,筛选资料并采用RevMan5.2软件进行系统分析。结果共纳入12篇临床随机对照试验, Meta分析结果显示：六味地黄汤辅助治疗糖尿病肾病,血府逐瘀汤治疗糖尿病周围神经病变,中药汤剂治疗糖尿病冠心病均较常规西医治疗具有明显优势。结论传统中药汤剂治疗糖尿病并发症具有良好效果,优势明显。     

Simultaneous quantitative determination of 9 active components in traditional Chinese medicinal preparation ShuangDan oral liquid by RP-HPLC coupled with photodiode array detection

A simple, accurate and reliable method for the simultaneous separation and determination of 9 active components (danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, rosmarinic acid, salvianolic acid B, paeonol, paeoniflorin and gallic acid) in traditional Chinese medicinal preparation ShuangDan (SD) oral liquid was developed using reverse phase high-performance liquid chromatography (RP-HPLC) coupled with photodiode array (PDA) detection. The chromatographic separation was performed on a SinoChrom ODS-BP C₁₈ column with gradient elution using methanol (A) and 3% glacial acetic acid aqueous solution (B) at a flow rate of 1.0 mL min⁻¹, and with a PDA detection. Good linear behaviors over the investigated concentration ranges were observed with the values of r² higher than 0.9992 for all the analytes. The recoveries and relative standard deviation (RSD), measured at three concentration levels, varied from 98.21% to 101.82% and 0.07% to 1.37%, respectively. The proposed method enables the simultaneous identification and determination of 9 active components in a single run for the quality control of ShuangDan oral liquid.     

Simultaneous determination and pharmacokinetics of five bufadienolides in rat plasma after oral administration of Chansu extract by SPE-HPLC method

A sensitive and rapid solid-phase extraction-high performance liquid chromatography (SPE-HPLC) method has been developed for the determination of five bufadienolides, arenobufagin, teliocinobufagin, cinobufotalin, cinobufagin and resibufogenin in rat plasma and applied to a pharmacokinetic study in rats after oral administration of Chansu extract (Venenum Bufonis). Plasma samples were pretreated with solid-phase extraction using Extract-Clean cartridges, and the extracts were analyzed by a reversed-phase C(18) column on a HPLC system with photodiode array detection (DAD). The calibration curves were linear over the range of 0.10-1.66 microg/ml for arenobufagin, 0.03-1.20 microg/ml for telocinobufagin, 0.01-0.62 microg/ml for cinobufotalin, 0.03-0.70 microg/ml for cinobufagin and 0.02-2.57 microg/ml for resibufogenin, respectively. The limit of quantification was 1.1 ng/ml for arenobufagin, 0.3 ng/ml for telocinobufagin, 9.7 ng/ml for cinobufotalin, 8.8 ng/ml for cinobufagin, 7.7 ng/ml for resibufogenin, respectively. The established method could be easily applied to the determination and pharmacokinetic studies of five bufadienolides in rat plasma after oral administration of Chansu extract.     

LC-MS determination and pharmacokinetic studies of ursolic acid in rat plasma after administration of the traditional chinese medicinal preparation Lu-Ying extract

Sambucus chinensis L. is a native perennial herb distributed throughout China. In traditional Chinese medicine (TCM), this herb is known as Lu-Ying. Ursolic acid is the major effective constituent of Lu-Ying. A rapid, sensitive, and accurate liquid chromatography-mass spectrometry (LC-MS) method for the determination of ursolic acid in rat plasma was developed and validated. Plasma samples taken from rats that had received Lu-Ying extract orally were acidified with acetic acid and then extracted with a mixture of hexane-dichloromethane-2-propanol (20:10:1, v/v/v). Separation of ursolic acid was accomplished on a C(18) column interfaced with a single quadrupole mass spectrometer. The mobile phase consisting of methanol and water (95:5, v/v) was delivered at a flow rate of 1.0 ml/min. Atmospheric pressure chemical ionization was operated in negative-ion mode. Using selected ion-monitoring mode, the deprotonated molecules [M-H](-) at m/z 455 and 469 were used to quantify ursolic acid and glycyrrhetic acid (internal standard), respectively. The assay was shown to be linear over the range of 10-1000 ng/ml (r> or =0.9960) with a lower limit of quantification of 10 ng/ml. The method was shown to be reproducible and reliable with intraday precision below 7.8%, interday precision below 8.1%, accuracy within +/-4.3%, and mean extraction recovery excess of 83.6%, which were all calculated from the blank plasma sample spiked with ursolic acid at three concentrations of 20, 200, and 800 ng/ml. The LC-MS method has been successfully applied to pharmacokinetic studies of ursolic acid after oral administration of Lu-Ying ethanolic extract (at a dose containing 80.32 mg/kg ursolic acid) to rats. The main pharmacokinetic parameters were: t(1/2), 4.3 h; K(e), 0.16 1/h; t(max), 1.0 h; C(max), 294.8 ng/ml; AUC(0-t) and AUC(0-infinity), 1007.1 ng.h/ml and 1175.3 ng.h/ml, respectively.     

中药汤剂配合针灸推拿在肩周炎患者治疗中的应用效果

目的 评价中药汤剂配合针灸推拿用于肩周炎的临床疗效.方法 将2013年11月至2015年10月我院收治的96例肩周炎患者随机分为对照组和观察组各48例,所有患者人院后均采取针灸推拿治疗,观察组加服中药汤剂,3个疗程后比较两组的治疗效果.结果 与治疗前相比,两组Neer评分和VAS评分均明显改善(P＜0.05),而观察组改善幅度更大(P＜0.05).观察组治疗愈显率显著高于对照组(87.50％ vs.68.75％,P＜0.05).结论 中药汤剂配合针灸推拿可有效改善肩周炎患者的肩关节功能,缓解疼痛,提高治疗效果,是一种安全高效的肩周炎治疗方法.     

观察中药汤剂对慢性腹泻患者的治疗效果

目的探究中药汤剂对慢性腹泻患者的临床治疗效果。方法66例慢性腹泻患者,按照不同治疗方法分为观察组与对照组,各33例。对照组采取常规西药治疗,观察组采取中药汤剂治疗。比较两组治疗前后症状积分及临床效果。结果治疗后,两组患者症状积分均低于治疗前,且观察组症状积分(10.06±3.11)分低于对照组的(14.53±3.77)分,差异有统计学意义(P<0.05)。观察组治疗总有效率为93.94%,高于对照组的72.73%,差异有统计学意义(P<0.05)。结论中药汤剂对慢性腹泻患者具有十分显著的疗效,可改善临床症状,值得临床推荐。     

Simultaneous determination and pharmacokinetic analysis of seven alkaloids and two flavonoids from rat plasma by HPLC-DAD after oral administration of Wuzhuyu decoction

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic analysis of seven alkaloids dehydroevodiamine (DHED), 10-hydroxyrutaecarpine (HDR), evodiamine (EDM), rutaecarpine (RCP), 1-methyl-2-n-nonyl-4(1H)quinolone (MNQ), evocarpine (ECP), and dihydroevocarpine (DHE), and two flavonoids isorhamnetin-7-O-rutinoside (RIM) and diosmetin-7-O-β-d-glucopyranoside (GRD) in rat plasma after oral administration of Wuzhuyu decoction. The flow rate was kept at 1.0?ml/min and the detection wavelength was set at 300?nm. The calibration curves were linear in the range of 0.5013-30.076?μg/ml for DHED, 0.2161-21.608?μg/ml for RIM, 0.161-12.876?μg/ml for HDR, 0.2146-21.457?μg/ml for GRD, 2.0464-40.928?μg/ml for EDM, 1.0398-31.194?μg/ml for RCP, 0.5970-35.818?μg/ml for MNQ, 0.8371-20.928?μg/ml for ECP, and 0.5167-31.003?μg/ml for DHE. The precision (relative standard deviation (RSD), %) for all was less than 10% and the accuracy (relative error (RE), %) was within ±?10%. The results demonstrated that the assay had remarkable reproducibility with acceptable accuracy and precision. The lower limit of quantifications for the compounds in plasma ranged from 0.12 to 0.23?μg/ml and the lower limit of detections ranged from 0.024 to 0.076?μg/ml. This validated method has been successfully applied in the pharmacokinetics study of seven alkaloids and two flavonoids after orally administrating the Wuzhuyu decoction to rats.     

HPLC程序波长法同时测定黄连解毒汤中栀子苷和4种黄酮类成分的含量

目的:建立一种同时检测黄连解毒汤中栀子苷、黄芩苷、汉黄芩苷、黄芩素和汉黄芩素含量的 HPLC 方法。方法:采用Kromasil C_(18)色谱柱(150 mm×4.6 mm,5 μm),以乙腈(A)-5 mmol·L~(-1)磷酸二氢钠溶液(含0.05%磷酸,pH 3.0)(B)为流动相,线性梯度洗脱(0～8.5 min,86.5%B;8.5～17.0 min,86.5%B→80%B;17～30 min,80%B;30～40 min,80%B→60%B;40～51 min,60%B→52%B;51～55 min,52%B→42%B;55～60 min,42%B→86.5%B;60～65 min,86.5%B),流速1.0 mL·min~(-1),程序可变波长紫外检测(0～15 min,238 nm;15～50 min,276 nm)。结果:在58 min 内黄连解毒汤中栀子苷、黄芩苷、汉黄芩苷、黄芩素和汉黄芩素5种成分被完全分离;回归方程显示5种成分的峰面积与其浓度呈良好的线性;5种成分的加样叫收率(n=15)分别为102.2%,97.56%,98.61%,97.85%,98.41%;RSD 小于5.0%。结论:利用程序可变波长检测,建立了一种可靠的同时测定黄连解毒汤中5种标志性成分含量的 HPLC 方法。     

LC/ESI-MS method for the determination of trimetazidine in human plasma: application to a bioequivalence study on Chinese volunteers

A rapid liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) method with good sensitivity and specificity has been developed and validated for the identification and quantification of trimetazidine in human plasma. Trimetazidine and lidocaine (internal standard) were isolated from plasma samples by protein precipitation with methanol. The chromatographic separation was accomplished on a Xterra MS C₁₈ Column (150 mm × 4.6 mm, 5 μm particle size) with the mobile phase consisting of methanol and water (40:60, v/v) (pH 2.0, adjusted with trifluoroacetic acid), and the flow rate was set at 0.6 mL/min. Detection was performed on a single quadruple mass spectrometer by selected ion monitoring (SIM) mode (m/z 267.0 for trimetazidine and m/z 235.0 for lidocaine) with the retention time at about 3.47 and 5.05 min, respectively. The calibration curve for trimetazidine was satisfactory with regression coefficient 0.9995 over the range of 2.5–100 ng/mL in the plasma. The LOQ (S/N = 10) was accordingly 2.5 ng/mL. The intra-day and inter-day precision expressed as relative standard deviation was 2.83–6.10% and 4.83–5.82%. The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference product) in 19 healthy male Chinese volunteers. After a single 20 mg dose for the test and reference product, the resulting mean of major pharmacokinetic parameters such as AUC_0–24, AUC_0−∞, C_max, T_max and t_1/2 of trimetazidine were (673.1 ± 117.6 ng h mL⁻¹ versus 652.3 ± 121.9 ng h mL⁻¹), (717.1 ± 120.9 ng h mL⁻¹ versus 692 ± 128.6 ng h mL⁻¹), (74.85 ± 12.13 ng mL⁻¹ versus 71.93 ± 14.32 ng mL⁻¹), (2.312 ± 0.663 h versus 2.211 ± 0.608 h) and (4.785 ± 0.919 h versus 4.740 ± 0.823 h), respectively, indicating that these two kinds of tablets were bioequivalent in the Chinese population.     

中药熏蒸在膝关节镜术后的早期应用与疗效观察

目的探讨桃红四物汤加减中药熏蒸在膝关节镜手术后的早期应用效果。方法将120例膝关节镜手术后患者（拆线后均I级愈合）随机分成治疗组与对照组,每组各60例,治疗组于膝关节镜术后第3天给予养血、祛风、活血、通络方药为基础的桃红四物汤加减,熏蒸膝关节30min,每日1剂,2次／d,15d为1个疗程,每次熏蒸膝关节后再循序渐进行膝关节的弯曲活动等功能锻炼;对照组于膝关节镜术后第1天行患肢肌肉的舒缩活动,循序渐进行膝关节的弯曲活动。结果术后2、4周及3、6、12个月治疗组晨僵、关节肿胀、下蹲困难、行走疼痛情况的发生率低于对照组;术后3、12个月,治疗组的HSS评分高于对照组（P〈0．05）。结论膝关节镜手术后早期应用桃红四物汤加减熏蒸膝关节,能促进患肢膝关节的血液循环,减轻疼痛,减少肌肉纤维组织的粘连,增加膝关节的活动度,能使患者早日康复,疗效好。     

Ultra-performance liquid chromatography-tandem mass spectrometry for the determination of lacidipine in human plasma and its application in a pharmacokinetic study

A selective, rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–ESI-MS/MS) method was developed and validated for the quantification of lacidipine in human plasma. With nifedipine as an internal standard, sample pretreatment involved a simple liquid–liquid extraction with tert-butyl methyl ether of 1 ml plasma. The analysis was carried out on an Acquity™ UPLC BEH C₁₈ column (50 mm × 2.1 mm, 1.7 μm) with flow rate of 0.28 ml/min. The mobile phase was 30 mM ammonium acetate buffer–acetonitrile (18:82, v/v, pH 5.5). The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 0.025–10.000 ng/ml, with a lower limit of quantification of 0.025 ng/ml. The intra- and inter-day precision (R.S.D.) values were below 15% and accuracy (RE) was −12.7% to 11.9% at all QC levels. The method was successfully applied to a clinical pharmacokinetic study of lacidipine in healthy volunteers following oral administration.     

Determination of berberine, palmatine and jatrorrhizine in rabbit plasma by liquid chromatography-electrospray ionization-mass spectrometry

Incurred rabbit plasmas samples were utilized for method quality assessment in this study, where an optimized protein precipitation method for the preparation of rabbit plasma samples and a rapid and sensitive liquid chromatography-electrospray ionization-mass spectrometry for the simultaneous determination of berberine, palmatine and jatrorrhizine was described. Plasma samples (100 μl) were pretreated by protein precipitation with the mixture of 3% formic acid and 50 ng/ml clozapine (internal standard) in acetonitrile followed by LC analysis using a C₁₈ column and a mobile phase composed of 0.4% formic acid solution and 0.2% formic acid solution of methanol (60:40, v/v) operated at a flow rate of 0.4 ml/min. The analysis was performed in the multiple reaction monitoring mode via electrospray ionization source operating in the positive ionization mode. The method was linear over the concentration range of 0.1-400 ng/ml for all target components. The lower limits of quantification were 0.1 ng/ml for all analytes, all intra- and inter-day precision values were less than 7.10%, and accuracy (bias, %) was within ±7.11%. The mean absolute recovery was more than 72% for all analytes. The validated method has been successfully applied to the pharmacokinetic study of berberine, palmatine and jatrorrhizine in rabbit plasma after oral administration of San-Huang decoction to rabbits.     

Simultaneous determination of six flavonoids in rat plasma by high-performance capillary electrophoresis and its application to a pharmacokinetic study

A high-performance capillary electrophoresis (HPCE) method was developed for the separation and quantification of six flavonoids in rat plasma, which provides the basis for further study and exploitation of the effective parts of Artemisia frigida. To the plasma sample, formic acid was added to precipitate protein first with kaempferol as the internal standard (IS). The organic layer was separated and evaporated to dryness and the supernatant was extracted with ethyl acetate. The residue was dissolved in 500 μL acetonitrile and filtrated through 0.45-μm filters, and then the samples were introduced into the capillary by electrokinetic injection. Separations were performed using 20 mM sodium borate with 10% acetonitrile as a buffer solution with UV detection at 283 nm. Applied voltage was 18 kV and temperature was 25°C. The calibration curves for the six flavonoids were linear (r > 0.9990) over the studied concentration range separately. The intra- and inter-day precision values were <5.2%, and the accuracy values were between ?4.4% and ?1.3% relative error. The extraction recoveries ranged from 90% to 97% across the calibration curve range. The validated method was successfully applied to a pharmacokinetic study of the six flavonoids in rats after oral administration of A. frigida extract.     

Reverse-phase HPLC determination and pharmacokinetic study of vanillic acid in the plasma of rats treated with the traditional Chinese medicinal preparation Di-Gu-Pi decoction

A sensitive, simple, and accurate method for the determination and pharmacokinetic study of vanillic acid in rat plasma was developed using reverse-phase HPLC with UV detection after oral administration of the traditional Chinese medicine preparation of the Di-Gu-Pi decoction. Plasma samples taken from rats were extracted with methanol. The constituent vanillic acid was separated on a C(18) stationary phase and a mobile phase of acetonitrile-water (15:85, v/v) (adjusted to pH 3.0 using phosphoric acid), with a UV detector setting at 260 nm. The validated HPLC method developed was used to determine the pharmacokinetic profile of vanillic acid in rat plasma after administration of the Di-Gu-Pi decoction.     

中药药物代谢动力学在中药新药研究中的作用

虽然中药药物代谢动力学研究在促进中药新药注册中的作用还有限,但在中药剂型改革、阐明中药效应物质基础、指导临床用药方案制定和中药国际化研究中发挥了重要作用,本文以研究实例就中药药物代谢动力学在中药新药研发中的促进作用和进展作一综述。