Microbial metabolism of loganin by intestinal bacteria and identification of new metabolites in rat

Loganin is an important constituent of the traditional Chinese medicine Fructus Corni, with several bioactivities. Microbial metabolism of loganin by intestinal bacteria was investigated. Two metabolites (log-1 and log-2) were isolated from anaerobic culture and their structures were identified by means of their ESI-MS, (1)H-NMR, (13)C-NMR and 2D-NMR spectral data. Log-1 was an aglycone of loganin and log-2 was proved to be a new compound. In vivo metabolites of loganin were detected in rat urine, bile and feces after oral administration of loganin and the structures were proved to be identical with that of the microbial metabolites log-1 and log-2 by HPLC-PDA analysis and comparison with the reference standards. Therefore we can prepare metabolites by anaerobic culture with intestinal bacteria.     

Pharmacokinetics and tissue distribution of pefloxacin mesylate in chickens

A specific, sensitive and stable high‐performance liquid chromatography (HPLC)‐based analytical method was established to determine the level of pefloxacin mesylate (PM) in the plasma and various tissues of chickens. Chickens were randomly assigned to 12 equal experiment groups, including 11 treatment groups and one control group. The chickens in the treatment groups received oral administration of PM and were sacrificed at different pre‐determined time points, with their blood and various organs harvested, extracted and analyzed by HPLC to quantify the level of the residual antibiotic. Method validation studies indicated that the HPLC measurement showed excellent precision, reproducibility, stability and robustness. The obtained pharmacokinetic parameters suggested that PM reached peak levels in various tissues within 1–2 h after its oral administration, and was mainly concentrated in liver and kidney. The antibiotic was also found to be cleared from chicken crureus, brain, testes, ovaries and pancreas at higher rates compared with other organs. Overall, the rapid accumulation of PM could at least be partially attributed to its relatively slow organ clearance. These results could serve as a useful guidance for the rational use of PM and other quinolone‐derived antimicrobials in the treatment of infectious diseases in chickens and other animals.     

Tissue distribution study of columbianadin and its active metabolite columbianetin in rats

Columbianadin, one of the main bioactive constituents of the roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan, has been found to possess obvious pharmacological effects in previous studies. In this study, a valid and sensitive reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated for the determination of columbianadin (CBN) and its active metabolite columbianetin (CBT) in rat tissue samples. Sample separation was performed on an RP‐HPLC column using a mobile phase of MeOH–H₂O (75:25, v/v) at a flow rate of 1.0 mL/min. The UV absorbance of the samples was measured at the wavelength 325 nm. The calibration curves for CBN were linear over the ranges of 0.5–20 µg/g for brain, testes and muscle, 1.0–10.0 µg/g for stomach and intestine, and 0.2–20.0 µg/g for heart, liver, spleen, lung and kidney. The calibration curves for CBT were linear over the ranges of 0.5–25 µg/g for stomach and intestine, and 0.1–10.0 µg/g for heart, liver, spleen, lung and kidney. The analysis method was successfully applied to a tissue distribution study of CBN and CBT after intravenous administration of CBN to rats. The results of this study indicated that CBN could be detected in all of the selected tissues after i.v. administration. CBN was distributed to rat tissues rapidly and could be metabolized to CBT in most detected tissues. Of the detected tissues, heart had the highest uptake of CBN, which suggested that heart might be one of the main target tissues of CBN. Concentrations of CBT were obviously higher in the digestive system than in other assayed tissues. The information provided by this research is very useful for gaining knowledge of the capacities of CBN and CBT to access different tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

枸杞子中铜的测定及其浓度分布特征

用超声波辅助水提取-醇沉-溶剂萃取方法分离枸杞子得Ⅰ-Ⅷ8个组分,用石墨炉原子吸收分光光度法测定其中铜的浓度,并探讨了铜在枸杞子不同组分中的浓度分布特征。结果表明,枸杞子及其分离组分Ⅰ-Ⅷ中铜的质量分数分别是13.20、4.03、9.01、6.41、3.41、5.66、3.96、16.70、8.20μg/g,枸杞子中的铜呈现不均匀的浓度分布特征,在组分Ⅱ中浓度最高,组分Ⅳ中浓度最低。     

HPLC Determination of Lovastatin in Rat Tissue

A simple HPLC method has been developed and validated for the determination of lovastatin in rat tissues. Samples were prepared by a simple protein precipitation. Separation was carried out on a C18 column with a mobile phase of acetonitrile:0.05 M ammonium acetate, a flow rate of 1.0 mL min⁻¹ and with detection at 238 nm. There was no interference from endogenous tissue compounds. The calibration curve was linear from 0.0175 to 7.0 μg mL⁻¹ with a limit of detection of 0.006 μg mL⁻¹. The method was used to measure the concentration of lovastatin in rat tissue after a single oral dose. The highest level was observed in the liver, then in kidney, heart and spleen; the lowest level was found in the brain. These results suggest that lovastatin distributes rapidly into all tissues and particularly the liver.     

Determination of fluoroquinolones in edible animal tissue samples by high performance liquid chromatography after solid phase extraction

In the present work, a rapid, accurate, and sensitive method has been developed for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in edible animal tissues (muscle tissue, liver, kidney, and eggs). The separation was accomplished on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature within 15 min. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v). UV detection at 275 nm yielded the following limits of detection: 100 pg per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 20 pg for ofloxacin, and 200 pg for enrofloxacin. Peaks in real samples were identified by means of a photodiode array detector. The method was validated in terms of intra-day (n = 8) and inter-day (n = 8) precision and accuracy. Tissue samples were purified from endogenous interference by solid-phase extraction using Oasis HLB cartridges. The solid-phase extraction protocol was optimized in terms of retention and elution. Recovery rates at fortification levels of 40, 60, and 80 ng/g ranged from 82.5% to 111.1%. The applicability of the method was examined using real samples from a chicken treated orally with the five studied fluoroquinolones.     

Pharmacokinetics and tissue distribution study of clevidipine and its primary metabolite H152/81 in rats

This present study was designed to investigate the pharmacokinetic profiles and tissue distribution characteristics of clevidipine and its primary metabolite H152/81 in rats following a single intravenous administration of clevidipine butyrate injectable emulsion. For this study, a sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established and validated for the simultaneous quantitation of clevidipine and H152/81 in rat whole blood and various tissues. A Hedera ODS‐2 column with two gradient elution programs was employed for the troubleshooting of matrix effect on the detection of analytes among different biological samples. The experimental data showed that clevidipine represented quick elimination from blood with a half‐life of about 4.3 min and rapid distribution in all of the investigated tissues after administration; the highest concentration of clevidipine was found in the heart whereas the lowest concentration was detected in the liver. In addition, clevidipine was almost undetectable in most tissues except for heart and brain at 90 min post‐dosing, suggesting that there was no apparent long‐term accumulation in rat tissues. For H152/81, the peak concentration of 3714 ± 319 ng/mL occurred at 0.129 ± 0.048 h, the half‐life was 10.08 ± 1.45 h and area under the concentration–time curve was 42091 ± 3812 ng h/mL after drug administration. In addition, H152/81 was found at significant concentration levels in all tissues, in descending order of lung, kidney, heart, liver, spleen and brain at each time point. The results of current study offer useful clues for better understanding the distribution and metabolism of clevidipine butyrate injectable emulsion in vivo.     

The determination of raloxifene in rat tissue using HPLC

We report a rapid and reliable HPLC-UV method for determination of raloxifene, a kind of selective estrogen receptor modulator (SERM), in rat tissue. Proteins were precipitated by adding 200 microL of acetonitrile and 50 microL of methanol to 100 microL of the tissue homogenates, following vortex mixing and centrifugation. Separation was carried out on a reversed-phase C(18) column (150 x 4.6 mm, 5 microm) with a mobile phase of acetonitrile:0.05 m ammonium acetate (pH 4.0 +/- 0.1; 33:67, v/v) at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 289 nm and the temperature of column was kept at 23 degrees C, without interference from endogenous tissue compounds. The calibration curve was linear from 0.0125 to 10.0 microg/mL with correlation coefficient of over 0.994, while the limit of quantification was 0.008 microg/mL. The intra- and inter-day coefficients of variation were less than 10% (RSD). The recovery of assay was between 95.8 and 104.5%. Furthermore, the method was used to measure the concentration of raloxifene in rat tissue after a simple oral dose. The highest level was observed in liver, lung, spleen, then heart and kidney. The lowest level was found in brain. These results suggest that raloxifene distributes rapidly and moderately into tissues such as liver, lung and spleen.     

HPLC analysis and pharmacokinetics of icariin in rats

As a prerequisite to the determination of pharmacokinetic parameters of icariin in rats, an HPLC method using UV detection was developed and validated. Icariin and the internal standard, quercetin, were extracted from plasma samples using ethyl acetate after acidification with 0.05 mol/L NaH2PO4 solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB Cls column (250 x 4.6 mm id, 5 microm) equipped with a Shim-pack GVP-ODS C18 guard column (10 x 4.6 mm id, 5 microm) using a mobile phase of ACN/water/acetic acid (31:69:0.4 v/v/v) at a flow rate of 1.0 mL/ min. Detection was at 277 nm. The calibration curve was linear from 0.05 to 100.0 microg/mL with 0.05 microg/mL as the lower LOQ (LLOQ) in plasma. The intra- and interday precisions in terms of RSD were lower than 5.7 and 7.8% in rat plasma, respectively. The accuracy in terms of relative error (RE) ranged from -1.6 to 3.2%. The extraction recoveries of icariin and quercetin were 87.6 and 80.1%, respectively. The main pharmacokinetic parameters for rats were determined after a single intravenous administration of 10 mg/kg icariin: t1/2, 0.562 +/- 0.200 h; AUC0-infinity, 8.73 +/- 2.23 microg x h/mL; CLToT, 20.10 +/- 5.80 L/kg x h; Vz, 1.037 +/- 0.631 L/kg; MRT0-infinity, 0.134 +/- 0.040 h; and Vss, 0.170 +/- 0.097 L/kg.     

芳香异羟肟酸二丁基锡类抗癌化合物DBDCT和DBDMT在大鼠体内组织分布的比较

系统比较研究芳香异羟肟酸二丁基锡抗癌先导化合物二-(苯甲酰异羟肟酸)二正丁基合锡(DBDBT)配体苯环上引入不同电负性取代基团得到的化合物二-(4-氯苯甲酰异羟肟酸)二正丁基合锡(DBDCT)和二-(4-甲氧基-苯甲酰异羟肟酸)二正丁基合锡(DBDMT)在大鼠体内的组织分布规律及其差异,探讨结构-组织分布-抗癌活性间的关系.静注3min后,DBDCT可迅速分布到各组织,主要分布在肾上腺,其次是十二指肠和肾,给药10min后肝脏、肠、胃壁、肺、肌肉和脂肪等组织浓度较高;而DBDMT只能分布于脑、心脏、肾、脾等少数几种组织.前期研究中DBDCT对人肝癌Bel-7402、人胃腺癌SGC-7901、卵巢癌Hela等细胞株具有比DBDMT更强的抗癌活性可能与DBDCT在肝脏、胃壁和卵巢中有较高分布、但这三个组织中几乎检测不到DBDMT有关.可见先导化合物DBDBT的配体苯环上引入的取代基团的电负性可明显影响其相应衍生物在大鼠体内的组织分布,从而可能导致对不同组织肿瘤的抗癌活性的较大差别,该研究为先导化合物DBDBT的进一步结构优化提供参考依据.     

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze‐dried powder

A simple and rapid liquid chromatography–mass spectrometry (LC‐MS) method was developed and validated for analysis of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze‐dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C₁₈ column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion‐pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra‐ and inter‐day RSDs were all less than 10% with the relative error (RE) within ±9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze‐dried powder. Copyright © 2011 John Wiley & Sons, Ltd.     

Biodistribution study of free and microencapsulated 6‐methylcoumarin in Wistar rats by HPLC

A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of 6‐methylcoumarin (6MC) in plasma and other tissues in Wistar rats. A C₁₈ column was used with UV detection at 321 nm and a gradient system consisting of methanol‐deionized water was used as mobile phase. The retention time for 6MC was 14.921 min and no interfering peaks were observed for any of the matrices. Linear relationships (r² > 0.997) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range 0.4–12.8 µg/mL. Precision and accuracy were evaluated; the coefficient of variation and the relative error for all of the organs were <2 and 7%, respectively. The limit of quantitation was 0.20 µg/mL for the heart and 0.30 µg/mL for the other tissues evaluated. This HPLC method was successfully used in the determination of 6MC in the biodistribution study after administration of 200 mg/kg of both 6MC‐free and 6MC‐loaded polymeric microparticles. In this study, extensive 6MC was found, in both free and microencapsulated forms, in all the organs tested. The 6MC‐free showed a range of between 1.7 and 11.5 µg/g, while the microencapsulated 6MC showed concentrations of between 6.35 and 17.7 µg/g, suggesting that 6MC improved absorption rate. Copyright © 2014 John Wiley & Sons, Ltd.     

HPLC method for comparative study on tissue distribution in rat after oral administration of salvianolic acid B and phenolic acids from Salvia miltiorrhiza

A sensitive and selective high-performance liquid chromatography method was developed and validated to determine the prototype of salvianolic acid B and the metabolites of phenolic acids (protocatechuic acid, vanillic acid and ferulic acid) in rat tissues after oral administration of total phenolic acids and salvianolic acid B extracted from the roots of Salvia miltiorrhiza, respectively. The tissue samples were treated with a simple liquid-liquid extraction prior to HPLC. Analysis of the extract was performed on a reverse-phase C(18) column with a mobile phase consisting of acetonitrile and 0.05% trifluoracetic acid. The calibration curves for the four phenolic acids were linear in the given concentration ranges. The intra-day and inter-day relative standard deviations in the measurement of quality control samples were less than 10% and the accuracies were in the range of 88-115%. The average recoveries of all the tissues ranged from 78.0 to 111.8%. This method was successfully applied to evaluate the distribution of the four phenolic acids in rat tissues after oral administration of total phenolic acids of Salvia miltiorrhiza or salvianolic acid B and the possible metabolic pathway was illustrated.     

Establishment of a HPLC method for preclinical pharmacokinetic study of the novel anti-Parkinson's disease candidate drug FLZ in rats

An HPLC method was established and validated for the determination of compound FLZ, a synthetic novel anti-Parkinson's disease candidate drug, in rat plasma. FLZ and the internal standard bicyclol were extracted from plasma by solid-phase extraction method and analyzed on a Restek C18 column (4.6 x 250 mm, 5 microm) with a mobile phase consisting of methanol and water (60:40, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was set at 320 nm. The calibration curve was linear within the concentration range from 25 to 500 ng/mL (r2 > 0.999), the limit of quantitation was 25 ng/mL and the average recovery was 92.0% with the RSD less than 5.9%. The relative standard deviation for intra- and inter-day precision was less than 3.8 and 6.9%, respectively. The established HPLC method was validated to be a simple, rapid and reliable procedure and applied to study the preclinical pharmacokinetics of FLZ in rat plasma, and it was the first time that the pharmacokinetics of FLZ had been investigated.     

高效液相色谱法测定大鼠血浆中防己诺林碱和粉防己碱

建立了高效液相色谱(HPLC)快速测定血浆中2种生物活性碱-防己诺林碱和粉防己碱的方法.血浆经乙腈沉淀蛋白,稀释后直接进样,采用ODS C18柱,乙腈-磷酸缓冲液-三乙胺(80∶30∶0.3,V/V/V)为流动相,紫外检测波长282 nm,外标法定量.为研究防己诺林碱和粉防己碱在生物体内的药物代谢机理研究提供了简便、灵敏、准确的分离测定方法.     

A new validated HPLC method for the determination of quercetin: Application to study pharmacokinetics in rats

A simple, accurate, and reproducible high‐performance liquid chromatography (HPLC) method has been developed and validated for the quantification of quercetin (QR) in rat plasma. The method involves a simple protein precipitation procedure to extract both QR and thymoquinone (TQ), the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20 A HPLC system equipped with a Supelcosil LC‐18 T C₁₈ column and an isocratic mobile phase consisting of 0.3% trichloroacetic acid in water and acetonitrile HPLC‐grade (50:50, v /v) run at a flow rate of 0.9 mL/min for 13 min. The UV detection wavelength was set at 254 nm. The method exhibited good linearity (R ² > 0.994) over the assayed concentration range (0.10–25 μg/mL) and demonstrated good intra‐day and inter‐day precision and accuracy (relative standard deviations and the deviation from predicted values were <20%). This method was also successfully applied for studying the pharmacokinetics of QR in rats following a single oral dose of QR to evaluate its pharmacokinetic parameters in rats.     

Determination of dehydrodiisoeugenol in rat tissues using HPLC method

Dehydrodiisoeugenol (DDIE) is a bioactive neolignan from the seeds of Myristica fragrans Houtt., which exhibits good anti-inflammatory activity. A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of DDIE in rat tissues after intravenous administration. The tissue samples were processed by liquid-liquid extraction. The analyses were successfully carried out on a Diamonsiltrade mark ODS C18 column (250 x 4.6 mm i.d., 5 microm) equipped with a C18 guard column (8 x 4.6 mm i.d., 5 microm). The mobile phase was the system of methanol-water (4:1, v/v). The UV detection was set at 270 nm. The calibration curves were linear from 0.4 to 200.0 microg/g with the correlation coefficients (r(2)) greater than 0.998. The intra- and inter-day precisions in quality control samples were less than 10% and the accuracies were in the range 85.4-110.3%. The average recoveries from all the tissues were between 84.4 and 106.0%. This assay method has been successfully used to study the tissues distribution of DDIE in rats after intravenous administration. The result suggests that DDIE is distributed to rat tissues rapidly with possibly greater initial concentrations in liver and brain than in other tissues.     

A new validated HPLC method for the determination of sulforaphane: application to study pharmacokinetics of sulforaphane in rats

A simple, accurate and reproducible high‐performance liquid chromatography (HPLC) method has been developed and validated for the quantification of sulforaphane (SF) in rat plasma. The method involves a simple liquid–liquid extraction procedure to extract both SF and 7‐hyrdoxycoumarin, the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20A HPLC system equipped with a Zorbax Eclipse XDB C₁₈ column and an isocratic mobile phase consisting of 10 mm KH₂PO₄ (pH 4.5) and acetonitrile HPLC grade (40:60, v/v) run at a flow rate of 1 mL/min for 10 min. The UV detection wavelength was set at 202 nm. The method exhibited good linearity (R² > 0.999) over the assayed concentration range (0.05–2 μg/mL) and demonstrated good intra‐ and inter‐day precision and accuracy (relative standard deviations and the deviation from predicted values were <15%). This method was also successfully applied for studying the pharmacokinetics of SF in spontaneously hypertensive rats following single oral dietary doses of SF. The pharmacokinetics of SF show linear behavior at the dose range investigated in this study. Copyright © 2015 John Wiley & Sons, Ltd.