RP-HPLC Determination and Pharmacokinetic Comparison of Cinnamic Acid in Rat Plasma After Administration of Di-Gu-Pi Decoction and Pure Cinnamic Acid

IntroductionTraditionally,Di-Gu-Pi has been used as a naturaltherapeutic agent for the treatment of diabetes,hemor-rhagic inframmation,hypertension,ulcers,and feverunder the guidance of the theory of Traditional ChineseMedicinal(TCM)science[1].This fact h…     

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies

A simple and sensitive method was developed for the simultaneous quantification of harpagoside and cinnamic acid in rat plasma using high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile. The analytes were separated on an Intersil C8-3 column (2.1 mm i.d.x250 mm, 5 microm) with acetonitrile-5 mm ammonium formate aqueous solution (60:40, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was performed on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source operated under selected ion monitoring (SIM) mode. [M+HCOO]- at m/z 539 for harpagoside, [M-H]- at m/z 147 for cinnamic acid and [M-H]- at m/z 137 for salylic acid (internal standard) were selected as detecting ions, respectively. The method was validated over the concentration range 7-250 ng/mL for harpagoside and 5-500 ng/mL for cinnamic acid. The lower limits of quantitation for harpagoside and cinnamic acid were 7 and 5 ng/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.5% and the assay accuracies (RE%) ranged from -5.3 to 3.0% for both analytes. Their average recoveries were greater than 86%. Both analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the pharmacokinetic study of harpagoside and cinnamic acid following oral administration of Radix Scrophulariae extract to rats.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of mangiferin in plasma of rats having taken the traditional Chinese medicinal preparation Zi-Shen pill

A high-performance liquid chromatographic method for the determination and pharmacokinetic study of mangiferin in the plasma of rats that have been orally administered the traditional Chinese medicinal preparation Zi-Shen pill is established. Plasma samples taken from rats are pretreated by protein precipitation with acetonitrile. Separation of the main effective constituent mangiferin is accomplished on a C18 stationary phase and a mobile phase of methanol&-water (25:75, v/v) with 0.6% glacial acetic acid. The UV detection wavelength is set at 320 nm, and the detection limit for mangiferin in plasma is 0.163 micro g/mL. After validation, the method is used to take a limited view of pharmacokinetic profiles of the traditional Chinese medicinal preparation Zi-Shen pill.     

HPLC determination of ferulic acid in rat plasma after oral administration of Rhizoma Chuanxiong and its compound preparation

An HPLC method is described for determination of ferulic acid in rat plasma. The concentration of ferulic acid in rat plasma was determined after deproteinization with acetonitrile using sulfamethoxazole as internal standard. Chromatographic separations were performed on a C(18) stationary phase with a mobile phase composed of acetonitrile-water (16:84, v/v) with 1% glacial acetic acid. The UV detection wavelength was set at 320 nm. The method was successfully applied to the determination of pharmacokinetic parameters in rat plasma after oral administration of Rhizoma Chuanxiong and and its compound preparation Suanzaoren decoctions. The calibration curve was linear over the range 0.0510-4.08 micro g/mL in rat plasma. Within-day and between-day precisions were less than 4.5% RSD. Mean recovery was determined as 96.9%. The limit of quantitation was 0.0510 micro g/mL. The pharmacokinetic parameters of the two preparations were different significantly (p < 0.05), which may attribute to the effects of other ingredients present in Suanzaoren decoction.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

Determination of cinnamic acid and paeoniflorin in traditional Chinese medicinal preparations by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of cinnamic acid in Cinnamomi ramulus and paeoniflorin in Paeoniae radix was established. The samples were separated by a LiChrospher RP-18 column with water-acetonitrile-methanolacetic acid (61:34:5:0.1 or 80:15:5:0.1, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Cinnamic acid and paeoniflorin were determined by UV detection at 280 and 250 nm, respectively. The method was applied to determine the optimum conditions for the extraction of the traditional Chinese medicinal preparation Huang Chi Chien Chung Tong, which contains Cinnamomi ramulus and Paeoniae radix. The results indicate that the best extraction conditions involved the use of an ultrasonic bath at 60 degrees C for 30 min. In this experiment, butyl paraben and methyl paraben were used as the internal standards for cinnamic acid and paeoniflorin, respectively. A good and reproducible separation of cinnamic acid and paeoniflorin was obtained within 15 min. The method was also applicable to other preparations that contain Cinnamomi ramulus and Paeoniae radix such as Guey Chi Chia Long Ku Muu Li Tong, Kuei Chi Chien Chung Tong and Tang Kuei Chien Chung Tong.     

Development and validation of an RP-HPLC-UV method for the determination of BOL-303225-A, a new coumarin-based anti-inflammatory drug, in rat plasma

A simple and rapid high-performance liquid chromatographic method was developed and validated for the analysis in rat plasma of BOL-303225-A, a new coumarin-based anti-inflammatory drug. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and water containing 1% triethylamine pH 3.5, adjusted with orthophosphoric acid (35.5:64.5 v/v) as mobile phase. The UV detector was set at 324 nm. The method proved to be linear (r(2) > 0.99) and precise (RSD < 7%) over the concentration range 29-940 ng/mL, and was suitable for the support of pharmacokinetic studies in rats.     

Simultaneous determination and pharmacokinetic studies of aloe emodin and chrysophanol in rats after oral administration of Da-Cheng-Qi decoction by high-performance liquid chromatography

A validated high-performance liquid chromatography (HPLC) method was developed for simultaneous determination and pharmacokinetic study of aloe emodin and chrysophanol in rats. It was performed on a reverse-phase C(18) column and a mobile phase made up of methanol and 0.2% acetic acid (83:17, v/v). The ultraviolet detection was 254 nm. 1,8-dihydroxyanthraquinone was used as the internal standard. The assay was linear over the range 28-2800 ng/mL (r(2) = 0.9993) for aloe emodin and 25.6-2560 ng/mL (r(2) = 0.9991) for chrysophanol. The average percentage recoveries of three spiked plasmas were 98.8-104.8% and 97.7-103.2% for aloe emodin and chrysophanol, respectively. Their RSD of intra-day and inter-day precision at concentrations of 56, 280 and 1400 ng/mL for aloe emodin and 51.6, 258 and 1290 ng/mL for chrysophanol were less than 3.5%. This method was applied for the first time to simultaneously determinate aloe emodin and chrysophanol in rats following oral administration of traditional Chinese medicine of Da-Cheng-Qi decoction. The pharmacokinetic parameters showed that chrysophanol was better absorbed with higher concentrations in plasma than aloe emodin did. They both eliminated slowly in male rats. The assay is suitable for identifying the plasma and tissue levels of aloe emodin and chrysophanol in preclinical investigations.     

A sensitive UPLC–MS/MS method for simultaneous determination of polyphenols in rat plasma: Application to a pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs

This study established a rapid and reliable approach using liquid chromatography–tandem mass spectrometry for the simultaneous determination of cinnamic acid, vanillic acid and protocatechuic acid in rat plasma. This is the first report on a comparative pharmacokinetic study of dispensing granules and standard decoction of Cinnamomum cassia twigs in rats. After liquid–liquid extraction by ethyl acetate, the plasma samples were subjected to LC–MS/MS for multiple reaction monitoring. The standard curves showed good linear regression (r² > 0.9991) in the range of 10.0–16000 ng/mL. The intra‐ and inter‐day accuracy and precision were found to be within 15% of the nominal concentration. The recoveries of the three phenolics ranged from 88.7 to 105.7%. Finally, this approach was successfully applied to pharmacokinetic analysis of the three phenolics after oral administration of standard decoction and dispensing granules of C. cassia twigs in rats. Although the values of AUC_0–t of vanillic acid and protocatechuic acid in standard decoction group were larger than those of the dispensing granule group, no significant difference was observed for the two groups. Of note, the elimination rates of vanillic acid were slower in the standard decoction group than the dispensing granule group.     

高效液相色谱法测定家兔血浆中桂皮酸的浓度及其药代动力学初探

 建立了采用高效液相色谱(HPLC)测定家兔血浆中桂皮酸含量的方法,并应用此法进行了桂皮酸的药代动力学研究。 采用的色谱柱为Kromasil C18柱(250 mm×4.6 mm i.d.,5 μm)；流动相为甲醇-乙腈-水-冰醋酸(体积比为10∶22∶55 ∶0.5),流速0.8 mL/min；检测波长为270 nm;柱温为室温；内标物为苯丙酸。实验结果表明,低、中、高浓度的提取回 收率分别为84.9%,84.4%,87.7%,方法回收率分别为98.4%,99.2%,100.1%,相对标准偏差(RSD)分别为5.5%,3.6%,3.7%, 日内及日间测定值的RSD均小于6%。所建立的HPLC方法灵敏、专一、准确、精密,可作为桂皮酸在家兔体内药代动力学研 究的检测手段。口服冠心苏合丸和冠心苏合胶囊后,桂皮酸在家兔体内的代谢呈一级吸收双室模型。     

Determination of mangiferin, jateorrhizine, palmatine, berberine, cinnamic acid, and cinnamaldehyde in the traditional Chinese medicinal preparation Zi-Shen pill by high-performance liquid chromatography

High-performance liquid chromatography is employed to determine the contents of six marker components such as mangiferin, jateorrhizine, palmatine, berberine, cinnamic acid, and cinnamaldehyde in the traditional Chinese medicinal preparation Zi-Shen pill. The separation is performed on a C(18) column by stepwise gradient elution with water (0.2%, v/v, triethylamine adjusted to pH 4 with phosphoric acid)-methanol-acetonitrile (0.01 min, 98:0:2; 20 min, 80:5:15; 30 min, 65:13:22; and 55 min, 65:13:22) as the mobile phase at a flow rate of 0.9 mL/min, with UV detection at 280 nm. Six regression equations show good linear relationships between the peak area of each marker and concentration. The recoveries of the markers listed are 95.5%, 98.3%, 96.8%, 99.5%, 101.7%, and 102.1%, respectively. The repeatability and reproducibility (relative standard deviation) of the method are less than 2.5% and 3.3%, respectively.     

HPLC determination of safflor yellow A and three active isoflavones from TCM Naodesheng in rat plasma and tissues and its application to pharmacokinetic studies

A high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic studies of safflor yellow A, puerarin, 3'-methoxyl-puerarin, and puerarinapioside in the plasma and tissues of rats that had been administered with the traditional Chinese medicine (TCM) preparation Naodesheng via the caudal vein. Samples taken from rats were subjected to protein precipitation with acetone. Separation of these four compounds was accomplished on a Kromisil C18 stationary phase using a mobile phase of acetonitrile-0.1% phosphoric acid-tetrahydrofuran (8:92:2, v/v/v) at a flow-rate of 1.0 mL/min. The detection wavelength was set at 250 nm. The calibration curves of the four components were linear in the given concentration ranges. The intra- and inter-day precisions in plasma and tissues were less than 15% and the extraction recoveries were higher than 60%. The lower limits of quantitation of four components were low enough to determine the four components. These four components all exhibited kinetics that fitted a two-compartment model in rats. The elimination half-life was 1.19 h for safflor yellow A, 2.69 h for puerarin, 2.94 h for 3'-methoxyl-puerarin, and 0.87 h for puerarinapioside, respectively. Following administration of a single injection of Naodesheng, the concentration (C) of the four components in the tissues showed C(kidney) > C(lung), C(liver) > C(spleen), C(stomach), C(heart), approximately. The method is a reliable tool for performing studies of safflor yellow A and three puerarin isoflavones in different biological material.     

Comparative pharmacokinetic studies of Ephedra herba in common cold and nephrotic syndrome rat models

Ephedra herba is a conventional Chinese medicine to treat cold, fever, asthma, edema, and lung diseases in the clinic. At present, most pharmacokinetic studies focus on the pharmacokinetic process of alkaloids in normal animals. However, the non-alkaloid components are also active. In addition, the pharmacokinetic studies under pathological state make more sense for clarifying the material basis of efficacy. In this study, a sensitive and rapid ultra-high-performance–tandem mass spectrometry method was developed and applied to determine nine bioactive components (ephedrine, pseudoephedrine, methylephedrine, (+)-catechin, epicatechin, vitexin, vicenin-2, cinnamic acid, and ferulic acid) in normal, common cold and nephrotic syndrome rats after the oral administration of Ephedra herba. Compared to the normal group, except for ferulic acid, the exposure levels of the other eight components were significantly increased and the plasma clearance clearly declined in common cold rats. Similarly, the exposure levels of seven components other than cinnamic acid and ferulic acid were also significantly augmented and the plasma clearance decreased significantly in nephrotic syndrome rats. In brief, the pathological conditions of the common cold and nephrotic syndrome could lead to alterations in the pharmacokinetics profiles of the nine components, which provide a reference for further exploration of the pharmacodynamics basis of Ephedra herba.     

Sensitive liquid chromatography/mass spectrometry assay for the quantification of azithromycin in human plasma

A simple, rapid, sensitive and specific liquid chromatography/electrospray ionization mass spectrometry method was developed and validated to quantify azithromycin in human plasma, using erythromycin as the internal standard (IS). A simple sample preparation method of protein precipitation with methanol was employed. Methanol, acetonitrile and water (12:30:58, v/v/v) were used as the isocratic mobile phase, with 0.1% formic acid and 0.1% ammonium acetate in water. Selected ion monitoring was specific for azithromycin and erythromycin. The assay was linear over the concentration range 4.69-600 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9994 to 0.9998. The intra- and inter-day precisions, calculated from quality control samples, were less than 8.24%. The method was employed in a pharmacokinetic study after oral administration of 500 mg azithromycin dispersible tablet to 20 healthy volunteers.     

Development and validation of an LC-MS-MS method for the simultaneous determination of sulforaphane and its metabolites in rat plasma and its application in pharmacokinetic studies

A highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay is developed and validated for the quantification of sulforaphane and its metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione (SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from rat plasma by methanol-formic acid (100:0.1, v/v) and analyzed using a reversed-phase gradient elution on a Develosil 3 μm RP-Aqueous C(30) 140? column. A 15-min linear gradient with acetonitrile-water (5:95, v/v), containing 10 mM ammonium acetate and 0.2% formic acid, as mobile phase A, and acetonitrile-water (95:5, v/v), containing 10 mM ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The calibration curves are linear over the concentration range of 25-20,000 ng/mL of plasma for each analyte. This novel LC-MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in rats.     

Development and validation of high-performance liquid chromatography/electrospray ionization mass spectrometry for assay of madecassoside in rat plasma and its application to pharmacokinetic study

A rapid, sensitive and specific high-performance liquid chromatography/electrospray ionization mass spectrometric (LC-ESI-MS) method was developed and validated for the quantification of madecassoside, a major active constituent of Centella asiatica (L.) Urb. herbs, in rat plasma. With paeoniflorin as an internal standard (IS), a simple liquid-liquid extraction process was employed for the plasma sample preparation. Chromatographic separation was achieved within 6 min on a Shim-pack CLC-ODS column using acetonitrile and water (60:40, v/v) containing 0.1% (v/v) formic acid as the mobile phase. The detection was performed by MS with electrospray ionization interface in negative selected ion monitoring (SIM) mode. The linear range was 11-5500 ng/mL with the square regression coefficient (r(2) ) of 0.9995. The lower limit of quantification was 11 ng/mL. The intra- and inter- day precision ranged from 4.99 to 9.03%, and the accuracy was between 95.82 and 111.80%. The average recoveries of madecassoside and IS from spiked plasma samples were >92%. The developed method was successfully applied to the pharmacokinetic study of madecassoside in rats after an oral administration.     

Liquid chromatography/tandem mass spectrometry assay for the quantification of troxerutin in human plasma

A simple, rapid, sensitive and specific liquid chromatography/tandem mass spectrometry method was developed and validated to quantify troxerutin in human plasma. The analyte and rutin, used as the internal standard, were analyzed on a Phenomenex Synergi Fusion RP column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. Acetonitrile/water (20:80 v/v) was used as the isocratic mobile phase, with 0.1% formic acid in water. A simple sample preparation method of protein precipitation with perchloric acid was employed. The assay was linear over the concentration range 31.25-4000 pg/mL. Correlation coefficients generated by linear regression with a 1/x(2) weighting factor ranged from 0.9991 to 0.9996. The intra- and inter-day precision over the entire concentration range were less than 12.28%. The method was successfully applied to a pharmacokinetic study after oral administration of a 300 mg troxerutin drop pill to 18 healthy volunteers.     

LC determination and pharmacokinetic study of vitexin-4″-O-glucoside in rat plasma after oral administration

A simple and specific high-performance liquid chromatography (HPLC) method was developed for the pharmacokinetic study of vitexin-4″-O-glucoside (VOG) in rats after oral administration. The plasma samples were deproteinised with methanol after the addition of an internal standard, hesperidin. HPLC analysis was performed on a Diamonsil C(18) analytical column, using methanol -0.5% aqueous phosphoric acid (45:55, v/v) as the mobile phase with ultraviolet detection at 330?nm. The calibration curve was linear over the range of 5-450?μg?mL(-1) in rat plasma. The average extraction recovery of VOG was 98.74%?±?0.44%, and the relative standard deviations of the intra- and inter-day precisions were not greater than 4.1% and 2.0%, respectively. The validated method was successfully applied during a pharmacokinetic study in rats after oral administration of VOG at different doses, and all the results indicated that the pharmacokinetics of VOG in rats obeyed nonlinear processes.     

Simultaneous determination of five triterpene acids in rat plasma by liquid chromatography–mass spectrometry and its application in pharmacokinetic study after oral administration of Folium Eriobotryae effective fraction

Folium Eriobotryae effective fraction (FEA), the extract of Folium Eriobotryae, had been used as anti‐hyperglycemia and anti‐hyperlipemia medicine in China. A previous study indicated that euscaphic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid, the five structurally similar triterpene acids (containing two groups of structural isomers), are the major components of FEA. In the present study, we developed a specific and reliable LC‐MS method for simultaneous determination of the five triterpene acids in rat plasma, and further investigated their pharmacokinetic properties after oral administration of FEA. Following a simple sample preparation, chromatographic separation was achieved on a C₁₈ column with a mobile phase composed of methanol–0.1% ammonium acetate (80:20, v/v). Quantification was achieved by monitoring the selected ions at m/z 487.6 for euscaphic acid, m/z 471.5 for maslinic acid and corosolic acid, m/z 455.5 for oleanolic acid and ursolic acid and m/z 469.5 for internal standard. The method was validated to be specific, accurate and precise over the concentration ranges of 10–3000 ng/mL with limits of detections of 5 ng/mL for the five triterpene acids. Finally, the method was successfully applied to the pharmacokinetic study of the five structurally similar triterpene acids in rats after oral administration of FEA. Copyright © 2015 John Wiley & Sons, Ltd.