Determination of Plasma Concentration of Cinnamic Acid by High-Performance Liquid Chromatography and Its Pharmacokinetics in Rats after Oral Administration of Zi-Shen Pill

Aim To develop a simple and sensitive high-performance liquid chromatographicmethod for determination of plasma concentration of cinnamic acid and pharmacokinetic study in ratsafter a single oral dose of traditional Chinese medicinal preparation Zi-Shen pill. Method Plasmasamples were acidified with hydrochloric acid and extracted with ethyl acetate . Cinnamic acid wasdetermined by HPLC using a G_(18) column. A mobile phase ofmethanbl-acetonitrile-water-triethyl-amine (7:22:73 = 0.2, V/V), with the pH adjusted to 4.0 withphosphoric acid, and with a UV detector set at 340 nm. Results The standard curve was linear overthe range of 1.92- 192.0 μg·mL^(-1). The LLOQ was 1.92 μg·mL^(-1) . The RSDs of within-day andbetween-day precision were < 8%. The mean recovery was 82.0% . Conclusion After validation, themethpd has been used to investigate the pharmacokinetic profiles of the traditional Chinesemedicinal preparation Zi-Shen pill.

Pharmacokinetics of Recombinant E. coli L-asparaginase in Rats

The distribution of ^(125)I recombinant E. coli L-asparaginase in tissues ororgans and the excretion in urine, feces and bile were studied with in vivo radioactive tracertechnique. The amount of radioactivity excreted in urine, feces and bile within 24 h afterintravenous administration of ^(125)I recombinant E. col L-asparaginase to rats was 68.95% ,4.44%and 5.36% of the dose respectively. ^(125)I recombinant E. coli L-asparaginase in plasma samples wasdetermined. The levels of structural intact molecule in plasma samples were evaluated by SDS-PAGEand bio-imaging analyzer system. Pharmacokinetic parameters were assessed with a model-dependentmethod. The concentration-time curves of recombinant E. coli L-asparaginase after intravenousinjection at 1 250 IU·kg^(-1), 2 500, IU·kg^(-1), 5 000 IU·kg^(-1) to rats were consistent withthe two-compartment model. The first and terminal elimination t_(1/2) were 0.52 ~ 0.63 h and 2.39 ~2.76 h respectively. AUC was linearly related to the doses. The results of distribution in tissuesor organs and excretion in urine suggested that the metabolites of the enzyme were cleared bymechanisms of urinary excretion. Pharmacokinetics parameters of recombinant E. coli L- asparaginasein rats are warranted for the design of future clinical trials.

Pharmacokinetics and Bioavailability of 2-Amino-6-Cyclopropylamino-9-(2,3 -Dideoxy-β-D-glyceropent-2-enofuranosyl) Purine (Cyclo-D4G) in Rats

AimTo characterize the pharmacokinetics of 2 -amino-6-cyclopropylamino-9-(2,3-dideoxy-β-D-glyceropent-2-enofuran osyl) purine (Cyclo-D4G) following intravenous administration and oral administ ration to rats. MethodsThe concentrations of Cyclo-D4G in rat (Sprague-Dawley male rats) plasma and urine were analyzed by high performance liquid chromatography (HPLC). ResultsFollowing intravenous adm inistration to rats, concentrations of Cyclo-D4G in plasma declined with a term inal phase half-life of 0 78±0 14 h (±s). Total clearance was 0 90±0 21 L·h -1 ·kg -1 . Renal excretion of unchanged Cyclo-D4G accounted for approximately 20% of total clearance. Steady state volume of distr ibution was 0 91±0 07 L·kg -1 . After oral administration to rats, conce ntrations of Cyclo-D4G in plasma declined with a terminal phase half-life of 0 83±0 13 h (±s). Total clearance was 3 81±2 03 L·h -1 ·kg -1 . Renal excretion of unchanged Cyclo-D4G accounted for approximat ely 9% of total clearance. Oral bioavailability of Cyclo-D4G in rat was 26 9%. ConclusionThe favorable pharmacokinetic profiles and lower to xicity provide support for further development of Cyclo-D4G clinical trials.

Pharmacokinetics and Biodegradation of Chitosan in Rats

Chitosan, an excellent biomedical material, has received a widespread in vivo application. In contrast, its metabolism and distribution once being implanted were less documented. In this study, the pharmacokinetics and biodegradation of fluorescein isothiocyanate(FITC) labeled and muscle implantation administrated chitosan in rats were investigated with fluorescence spectrophotometry, histological assay and gel chromatography. After implantation, chitosan was degraded gradually during its distribution to diverse organs. Among the tested organs, liver and kidney were found to be the first two highest in chitosan content, which was followed by heart, brain and spleen. Urinary excretion was believed to be the major pathway of chitosan elimination, yet 80% of chitosan administered to rats was not trackable in their urine. This indicated that the majority of chitosan was degraded in tissues. In average, the molecular weight of the degradation products of chitosan in diverse organs and urine was found to be <65 k Da. This further confirmed the in vivo degradation of chitosan. Our findings provided new evidences for the intensive and safe application of chitosan as a biomedical material.

LC-MS/MS法测定干血斑样品中阿戈美拉汀

目的：建立快速、灵敏的液相色谱-串联质谱（LC-MS/MS）法,测定大鼠干血斑（DBS）样品中的阿戈美拉汀,并应用于阿戈美拉汀大鼠药动学研究。方法：给药后的大鼠新鲜全血40μL点于Whatman 903滤纸上,室温下干燥2 h,放入含干燥剂的密封袋保存。取直径为6 mm的干血斑3片,加入甲醇500μL,超声15 min后,进行液-液萃取,以甲醇-甲酸铵-甲酸为流动相,流速0.20 m L·min-1,采用Grom-Sil 120 ODS-5 ST柱分离,通过电喷雾三重四极杆串联质谱,以多反应监测（MRM）方式进行检测,用于定量的离子反应为m/z 244.1→m/z 185.1（阿戈美拉汀）和m/z 249.2→m/z 187.1（d5-阿戈美拉汀）。结果：测定阿戈美拉汀DBS的线性范围为0.025 0-25.0 ng·m L-1;定量下限为0.025 0 ng·m L-1;精密度和准确度均符合要求。结论：DBS采样法全血用量少,样品储存、运输便利,可用于阿戈美拉汀大鼠药动学研究。

Development of a method for the quantitative determination of geniposide in rat plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application in a pharmacokinetic study in rats

Geniposide is a major bioactive constituent isolated from Gardeniajasminoides Ellis. To evaluate the pharmacokinetics of geniposide in pre-clinical studies, a rapid and specific liquid chromatography-tandem mass spectrometry （LC-MS/MS） method was developed and validated. After simple protein precipitation, geniposide was analyzed on a DiamonsilR C18 column with a mobile phase of 10 mM ammonium acetate and methanol （20：80, v/v） at a flow rate of 0.6 mL/min. Detection was performed in ＂Truncated＂ multiple-reaction monitoring （MRM） mode with positive electrospray ionization （ESI） at m/z 411→411 for geniposide, and MRM mode with negative ESI ionization at m/z 415→295 for puerarin （internal standard, IS）. Linearity was established in the concentration range from 10.0 to 5000 ng/mL. The extraction recoveries ranged from 84.8% to 90.5% at concentrations of 10.0, 500 and 4.5x 103 ng/mL. The lower limit of quantification （LLOQ） was 10.0 ng/mL with 50 ~tL plasma. The validated method was successfully applied to the pharmacokinetic study of geniposide in rats at a dose of 200 mg/kg by oral administration.

A high performance liquid chromatography method for the quantitative determination assay of sitagliptin in rat plasma and its application in pharmacokinetics study

A new high-performance liquid chromatography （HPLC） method for the quantitative determination of sitagliptin in rat plasma was developed and validated for pharmacokinetics study. The plasma was spiked with the internal standard （hydrocortisone, IS）, treated with sodium hydroxide, and extracted with ethyl acetate. The extracted analyte was injected into an Agilent Zorbax Extend-C18 column （250 mm×4.6 mm, 4 μm） maintained at 30℃ and monitored at 267 nm. The mobile phase consisting of methanol-water （60：40, v/v, containing 10 mM Tris and 10 mM triethylamine） was titrated to pH 9.0 using 1 mol/L hydrochloric acid. The flow rate was 1.0 mL/min. The method showed high specificity. Calibration curves of the peak area ratio of each analyte/IS versus sitagliptin concentration were linear in the range of 0.75-100.0μg/mL （r〉0.9957）. The lower limit of quantification （LLOQ） was 0.75 μg/mL. The intra-day and inter-day coefficient of variation was lower than 10%. The accuracy （relative recovery） at three levels was 105.3% （0.75 μg/mL）, 99.8% （10.0 μg/mL） and 99.0% （100.0 μg/mL）. The extraction recovery was 81.5%, 82.4% and 84.5% at the concentrations of 0.75, 10.0 and 100.0 μg/mL, respectively. The short-term, long-term, freeze-thaw storage stability of plasma samples and the stability of standard solutions were satisfactory. This HPLC method is suitable for determining the concentration of sitagliptin in rat plasma and it was applied to determine the concentration-time profiles of sitagliptin in rat plasma following oral administration of sitagliptin.

Determination of a novel derivative of the PAC-1 anticancer agent in rat plasma by LC-MS/MS and its application to a pharmacokinetics study

A new and sensitive liquid chromatography-tandem mass spectrometry method was developed for the determination of SM-1 in rat plasma. After a simple protein precipitation, SM-1 and internal standard （gefitinib） were separated with gradient elution on a Waters XBridge C18 （50 mm×4.6 mm, 3.5μm） using acetonitrile-methanol-10 mM ammonium acetate （37.5：37.5：25, v/v/v） as mobile phase. The triple quadruple mass spectrometer was set in positive electrospray ionization mode, multiple reaction monitoring was used for quantification. The precursors to produce ion transitions monitored for SM-1 and IS were m/z 407.3→203.4 and 447.3→128.3, respectively. The method validation was conducted over the curve range of 30-6000 ng/mL. The intra- and inter-day precisions were less than 4.7%, the average extraction recoveries ranged from 98.7% to 104.1% for each analyte. SM-1 was proved to be stable during sample storage preparation and analytical procedures. All the results met the acceptance criteria in accordance with the FDA guidance for bioanalytical method. Consequently, this method was successfully applied to determine SM-1 concentrations in rats after oral administrations at the doses of 200, 100 and even 50 mg/kg.

Quantitative determination of ganoderic acid T in rat plasma by a sensitive RP-HPLC method and its application in pharmacokinetic studies

A selective and sensitive HPLC method has been developed and validated for the quantification of ganoderic acid T in rat plasma. A reverse-phase column was used with UV detection at 245 nm. The mobile phase consisted of methanol-water-acetic acid （95.5：4.5：0.5, v/v/v） run at a flow rate of 1 mL/min. Testosterone propionate was used as the internal standard. The standard curve was linear over the range of 0.05-50 ~tg/mL in rat plasma with a linear correlation coefficient greater than 0.999. The lower limit of quantification of this assay was 20 ng/mL. The recoveries of samples at 0.05, 2 and 40 μg/mL were 97.6%, 98.4% and 103.2%, respectively. The relative standard deviations of intra- and inter-day assay variations were less than 8.2%. The usefulness of the assay was confirmed by the successful analysis of plasma samples from a pharmacokinetics study in rats. Following a single dose of ganoderic acid T （5 mg/kg for i.v. or 14 mg/kg for p.o.）, the main pharmacokinetic parameters by intravenous injection were： t1/2α （5.46±1.25） min; t1/2β：（227.18±11.40） min; CL： （1.09±0.16） mL/（kg·min）; A UC0-12 h： （3939.13±311.14） μg.min/mL; AUC0-12h （4681.96±710.70）μg.min/mL and the absolute bioavailability was 41.98%±2.40%. This method is simple, sensitive, and accurate. It is suitable for pharmacokinetic studies of ganoderic acid in rats.

Determination of metformin in diabetic rat plasma by an improved ion-pair high-performance liquid chromatography: application to a pharmacokinetic study

An efficient and sensitive ion-pair HPLC-UV method using atenolol as internal standard （IS） was developed and validated for the determination of metformin in the plasma of diabetic rats. Plasma samples were deproteinated with 10% （v/v） perchloric acid. Separation was achieved on a UltimateTM AQ-C18 column （250 mm×4.6 mm, 5 μm） with a mobile phase （pH 5.05） composed of acetonitrile-water （31：69, v/v, containing 0.002 M sodium dodecyl sulfate, 0.0125 M potassium dihydrogen phosphate, 0.015 M triethylamine） at a flow rate of 1.0 mL/min. The calibration curve was linear （r〉0.994） between 7.5 and 4000 ng/mL. The lower limit of quantification （LLOQ） was 7.5 ng/mL. The precision was validated and the relative standard deviation was in the range of 1.87% to 15.70%; the accuracy was between 93.98%-106.89%. The mean recoveries were 95.40% and 95.31% for metformin and IS, respectively. The relative error （RE） of stability at different storage conditions was within ±9.00%. This method was used to determine the concentration-time profile of metformin in diabetic rat plasma following an oral administration of metformin at the dose of 10 mg/kg. Our results indicated that ion-pair HPLC-UV method using UltimateTM AQ-C18 column was effective for the pharmacokinetic studies of high polarity compounds like metformin.

Simultaneous determination of two bioactive components of Huangqi Guizhi Wuwu Decoction in rat plasma using UPLC-MS/MS and its application to a pharmacokinetic study

A rapid, sensitive and selective UPLC-MS/MS method was developed to determine paeoniflorin and astragaloside IV, This method was validated via a pharmacokinetic study using rat plasma. The internal standard was clarithromycin. A simple one-step deproteinization procedure was used to prepare plasma samples. Separation was achieved on a CAPCELL CORE ADME CI8 column with a gradient mobile phase consisting of solution A （water containing 0.1% formic acid） and solution B （acetonitrile） at a flow rate of 0.3 mL/min. Multiple reaction monitoring （MRM） was used with an electrospray ionization source （ESI） in positive mode. A good linear response was observed within the ranges of 0.01 to 5.00 ~g/mL for paeoniflorin and 0.000l to 0.05 ~tg/mL for astragaloside IV. The accuracy （RE） was within the range of-3.5% to 6.3%, and the intra- and inter-day precisions （RSD） were within 14.2%. The extraction recoveries were all above 78.9%. The pharmacokinetic study of the two analytes in rats after oral administration of Huangqi Guizhi Wuwu Decoction （HGWD） was successfully completed through this method. The method develooed in this studv will fill a gap in oharmacokinetic studies of HGWD.

Pharmacokinetic investigation on nteraction between hydrophilic lithospermic acid B and lipophilic tanshinone IIA in rats:an experimental study

OBJECTIVE:To elucidate the interaction between hydrophilic lithospermic acid B and lipophilic tanshinone Ⅱ A in rats.METHODS:A reliable high-performance liquid chromatography method was adopted for simultaneous determination of lithospermic acid B and tanshinone Ⅱ A in rat plasma,through which the pharmacokinetic interaction between lithospermic acid B and tanshinone Ⅱ A by intravenous injection was investigated.RESULTS:The simultaneous intravenous injection of tanshinone Ⅱ A and lithospermic acid B significantly altered the pharmacokinetic parameters of both compounds when compared with the individual intravenous administration of each compound.The area under the concentration-time curve of tanshinone Ⅱ A and lithospermic acid B increased by 18.35 and 59.31%,respectively.The mean retention time of tanshinone Ⅱ A and lithospermic acid B increased,respectively,from 9.3 to 32.8 h and20.2 to 49.1 h.The concomitant use of tanshinoneⅡ A magnified the volume of distribution at steady state(V_(ss)) and time for the drug in the plasma to reduce the highest concentration by half(t_(1/2)) of lithospermic acid B,while at the same time the V_(ss) and t_(1/2)of tanshinone Ⅱ A changed significantly in the presence of lithospermic acid B.CONCLUSION:Lithospermic acid B and tanshinone D A interact with each other following simultaneous intravenous injection in rats and this observation may expand the clinical use of Danshen(Radix Salviae Miltiorrhizae).

In vitro and in vivo evaluation of cucurbitacin E on rat hepatic CYP2C11 expression and activity using LC-MS/MS

This study explored the effects of cucurbitacin E （CUE）, a bioactive compound from Cucurbitaceae, on the metabolism/ pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chro- matography-（tandem） mass spectrometry （LC-MS/MS） assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE （1.25-100μmol· L-1） competitively inhibited tolbutamide 4-hydroxylation （CYP2C11） activity only in concentration-dependent manner with a Ki value of 55.5 μmol L-1 in vitro. In whole animal studies, no signifi- cant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, mul- tiple pretreatments of CuE （200 μg kg-1 d-1, 3 d, i.p.） significantly decreased tolbutamide clearance （CL） by 25% and pro- longed plasma half-time （T1/2） by 37%. Moreover, CuE treatment （50-200 pg kg-l d-1, i.p.） for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or nat- ural products containing CuE to avoid unnecessary drug-drug interactions.