Rapid determination of rosmarinic acid and its two bioactive metabolites in the plasma of rats by LC-MS/MS and application to a pharmacokinetics study.

Rosmarinic acid (RA), an ester compound of caffeic acid (CA) and 3,4-dihydroxyphenyllacic acid, is widely distributed in the herbs of the Lamiaceae family and has shown a wide spectrum of pharmacological properties. CA and FA (ferulic acid) are two bioactive metabolites in vivo after oral administration of RA; however, a rapid and robust analytical approach that can enable the quantitative assay of RA and two bioactive metabolites is still lacking. A liquid chromatography/tandem mass spectrometry method was established that was capable of the quantitative determination of RA, CA and FA by negative-mode multiple reaction monitoring within 7 min using a Zorbax SB-C18 column and an isocratic elution. This assay method was validated as linear over the investigated ranges with correlation coefficients (r) > 0.9950. The intra- and inter-day precision was <10.65%, and the accuracies (relative error, %) <-6.41%. The validated approach was applied to a pharmacokinetics study of RA and its two metabolites in rats after oral and intravenous administration. RA was rapidly metabolized in both administration modes, whilst the metabolites CA and FA were only detectable by oral administration. The absolute availability of RA was calculated to be 4.13%.

Pharmacokinetics of salvianolic acid B, rosmarinic acid and Danshensu in rat after pulmonary administration of Salvia miltiorrhiza polyphenolic acid solution.

A sensitive and accurate LC-MS/MS method was established for quantifying salvianolic acid B (Sal B), rosmarinic acid (Ros A) and Danshensu (DA) in rat plasma. Salvia miltiorrhiza polyphenolic acid (SMPA), active water-soluble ingredients isolated and purified from Salvia miltiorrhiza Bge included Sal B, Ros A and DA. The pharmacokinetic analysis of Sal B, Ros A and DA after pulmonary administration of SMPA solution to rat was performed by LC-MS/MS. Results from the pharmacokinetic studies showed that the peak concentration of DA was 21.85 ± 6.43 and 65.39 ± 3.83 ng/mL after pulmonary and intravenous administration, respectively. DA was not detected at 2 h after administration. The absolute bioavailabilities of Sal B and Ros A were respectively 50.37 ± 27.04 and 89.63 ± 12.16% after pulmonary administration of 10 mg/kg SMPA solution in rats. The absolute bioavailability of Sal B increased at least 10-fold after pulmonary administration, compared with oral administration. It was concluded that the newly established LC-MS/MS method was suitable for describing the pharmacokinetic characteristics of Sal B, Ros A and DA in rat after pulmonary administration of SMPA solution. The data from this study will provide a preclinical insight into the feasibility of pulmonary administration of SMPA.

Pharmacokinetic and Pharmacodynamic Properties of Rosmarinic Acid in Rat Cholestatic Liver Injury.

The objective of this study was to evaluate the hepatoprotective and metabolic effects of rosmarinic acid (RA) in rats. RA [100 mg/kg body weight (BW)] was intragastrically (i.g.) administered to Sprague-Dawley (SD) rats once a day for seven consecutive days. The rats were then i.g. administered α-naphthylisothiocyanate (ANIT) (80 mg/kg once on the 5th day) to induce acute intrahepatic cholestasis after the last administration of RA. Blood samples were collected at different time points (0.083 h, 0.17 h, 0.33 h, 0.5 h, 0.75 h, 1 h, 1.5 h, 3 h, 4 h, 6 h, 8 h, 12 h, 20 h) after administration, and the levels of RA were estimated by HPLC. Plasma and bile biochemical analysis, bile flow rate, and liver histopathology were measured to evaluate the hepatoprotective effect of RA. The PK-PD curves showed obviously clockwise (AST and ALT) or anticlockwise (TBA, TBIL). Pretreatment with RA at different doses significantly restrained ANIT-induced pathological changes in bile rate, TBA, TBIL, ALT, AST (p < 0.05 or p < 0.01). The relationship between RA concentration and its hepatoprotective effects on acute cholestasis responses was assessed by PK-PD modeling.

A sensitive LC-MS/MS-based bioanalytical method for quantification of salviaflaside and rosmarinic acid in rat plasma and its application in a pharmacokinetic study.

A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of salviaflaside and rosmarinic acid in rat plasma. Sample preparation was carried out through liquid-liquid extraction with ethyl acetate using curculigoside as internal standard (IS). The analytes were determined by selected reaction monitoring operated in the positive ESI mode. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column (100 × 4.6 mm, 1.8 µm) with a mobile phase consisting of methanol-water-formic acid (50:50:0.1, v/v/v) at a flow rate of 0.3 mL/min. The run time was 1.9 min per sample and the injection volume was 5 µL. The method had an LLOQ of 1.6 ng/mL for salviaflaside and 0.94 ng/mL for rosmarinic acid in plasma. The linear calibration curves were fitted over the range of 1.6-320 ng/mL for salviaflaside and 0.94-188 ng/mL for rosmarinic acid in plasma with correlation coefficients (r2 ) >0.99. Intra- and inter-day precisions (relative standard deviation) were < 13.5%, and accuracies (relative error) were between -8.6% and 14.5% for all quality control samples. The method was validated and applied to the pharmacokinetics of salviaflaside and rosmarinic acid in plasma after oral administration of Prunella vulgaris extract to rats.

Effect of piperine on the bioavailability and pharmacokinetics of rosmarinic acid in rat plasma using UPLC-MS/MS.

1. The purpose of the present study was to investigate the effect of piperine (PP) on the pharmacokinetics of rosmarinic acid (RA) in rat plasma and to determine whether PP could enhance the oral bioavailability of RA via inhibition of its glucuronidation. 2. The pharmacokinetic profiles of RA between oral administration of RA (50 mg/kg) alone and in combination with different oral dose PP (20, 40, 60, and 80 mg/kg) to rats were investigated via a validated UPLC/MS/MS method. 3. The AUC and Cmax of RA were significantly increased in combination with different dose PP dose dependently, especially in the presence of 60 and 80 mg/kg PP (p < 0.01). The relative bioavailability of RA in the presence of 20, 40, 60, and 80 mg/kg PP was 1.24-, 1.32-, 2.02-, and 2.26-folds higher, respectively, compared with the control group given RA alone. Compared with RA, the pharmacokinetic modulations of RA glucuronide were even more apparent, and the glucuronidation of RA was remarkedly inhibited. 4. This study demonstrated that PP significantly improved the in vivo bioavailability of RA partly attributing to the inhibition of gut and hepatic metabolism enzymes of RA.

Pharmacokinetic Assessments of Liquiritin, Protocatechuic Aldehyde and Rosmarinic Acid in Rat Plasma by UPLC-MS-MS After Administration of ZibuPiyin Recipe.

The three analytes of the Traditional Chinese Medicine ZibuPiyin Recipe (ZBPYR), namely, liquiritin, protocatechuic aldehyde and rosmarinic acid, may synergistically play an important role in regulating memory and learning. However, the pharmacokinetic behaviors of these compounds after their co-administration remain unclear. To this end, a selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated in rat plasma for the study of these three major bioactive ingredients in ZBPYR. The analytes in the plasma samples were separated on a Shiseido Capcell core C18 column using bendrofluazide as an internal standard, with a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid. Electrospray ionization in the negative-ion mode and multiple reaction monitoring were used to identify and quantify the three analytes. All of the calibration curves showed good linearity (r > 0.992) over the concentration range, with a lower limit of quantification of 5 ng/mL. The precision of the analytical method was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation (SD) was within 15%. Satisfactory extraction efficiency (between 83.4 and 99.4%) and matrix effects (76.4-107.4) were obtained by liquid-liquid extraction. The pharmacokinetic results showed that the three bioactive ingredients were rapidly absorbed and had a short terminal half-life in rats after oral administration of ZibuPiyin recipe. This UPLC-MS-MS study method used in this study may be useful for assessing the pharmacokinetic characteristics of various compounds, which would be helpful in determining their clinical potential.

Determination of quantitative retention-activity relationships between pharmacokinetic parameters and biological effectiveness fingerprints of Salvia miltiorrhiza constituents using biopartitioning and microemulsion high-performance liquid chromatography.

In this study, we analyzed danshen (Salvia miltiorrhiza) constituents using biopartitioning and microemulsion high-performance liquid chromatography (MELC). The quantitative retention-activity relationships (QRARs) of the constituents were established to model their pharmacokinetic (PK) parameters and chromatographic retention data, and generate their biological effectiveness fingerprints. A high-performance liquid chromatography (HPLC) method was established to determine the abundance of the extracted danshen constituents, such as sodium danshensu, rosmarinic acid, salvianolic acid B, protocatechuic aldehyde, cryptotanshinone, and tanshinone IIA. And another HPLC protocol was established to determine the abundance of those constituents in rat plasma samples. An experimental model was built in Sprague Dawley (SD) rats, and calculated the corresponding PK parameterst with 3P97 software package. Thirty-five model drugs were selected to test the PK parameter prediction capacities of the various MELC systems and to optimize the chromatographic protocols. QRARs and generated PK fingerprints were established. The test included water/oil-soluble danshen constituents and the prediction capacity of the regression model was validated. The results showed that the model had good predictability.

Simultaneous determination of eight bioactive components of Qishen Yiqi dripping pills in rat plasma using UFLC-MS/MS and its application to a pharmacokinetic study.

In this study, a rapid and reliable ultra-fast liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of eight active ingredients, including astragaloside IV, ononin, tanshinol, protocatechualdehyde, protocatechuic acid, salvianolic acid D, rosmarinic acid and ginsenoside Rg1 , in rat plasma. The plasma samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was performed on a Waters Acquity UPLC® BEH C18 column (1.7 µm particles, 2.1 × 100 mm). The mobile phase consisted of 0.1% aqueous formic acid (A)-acetonitrile with 0.1% formic acid (B) at a flow rate of 0.4 mL/min. Quantification was performed on a triple quadruple tandem mass spectrometry with electrospray ionization by multiple reaction monitoring both in the negative and in the positive ion mode. The lower limit of quantification of tanshinol was 2.0 ng/mL and the others were 5.0 ng/mL. The extraction recoveries, matrix effects, intra- and inter-day precision and accuracy of eight tested components were all within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of the eight active constituents after intragastric administration of three doses (1.0, 3.0, 6.0 g/kg body weight) of Qishen Yiqi Dripping Pills to rats.

Simultaneous determination of three active components in rat plasma by UPLC-MS/MS: Application to pharmacokinetic study after oral administration of Herba Sarcandrae extract.

A rapid, specific and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determination of isofraxidin, rosmarinic acid and kaempferol-3-O-glucuronide in rat plasma using warfarin as an internal standard (IS). Separation was conducted on a Thermo Hypersil GOLD C18 column with linear gradient elution using methanol and water. Mass spectrometric detection was conducted using selected reaction monitoring (SRM) via an electrospray ionization (ESI) source. All analytes exhibited good linearity within their concentration ranges (r > 0.9990). The lower limits of quantitations of isofraxidin, rosmarinic acid, and kaempferol-3-O-glucuronide were 1.31, 0.67 and 0.92 ng/mL, respectively. Intra- and inter-day precisions of these investigated components exhibited an RSD within 11.7%, and the accuracy ranged from -12.5 to 15.0% at all QC levels. The developed method was successfully applied to a pharmacokinetic study of isofraxidin, rosmarinic acid, and kaempferol-3-O-glucuronide in rats after oral administration of Herba Sarcandrae Extract.

Simultaneous determination of tanshinones and polyphenolics in rat plasma by UPLC-MS/MS and its application to the pharmacokinetic interaction between them.

The aim of this study was to investigate the pharmacokinetic interaction between tanshinones and polyphenolics which act as the main bioactive compounds in Saliva miltiorrhiza Bunge (SMB). Thus, a rapid and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine the concentrations of Tanshinone IIA (TSIIA), Tanshinone I (TI), Cryptotanshinone (CT), Salvianolic acid B (Sal B), Protocatechuic aldehyde (PAL), Rosmarinic acid (RA), and Danshensu (DSS) in rat plasma. The Sprague-Dawley rats were allocated to three groups which orally administered tanshinones (DST), polyphenolics (DFS), and a mixture of tanshinones and polyphenolics (DTF). These samples were processed by a simple liquid-liquid extraction (LLE) method with ethyl acetate. Chromatographic separation was achieved on an Acquity BEH C18 column (100 mm × 2. 1 mm, 1.7 µm) with the mobile phase consisting of 0.1% (v/v) formic acid and acetonitrile by gradient elution at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole-tandem mass spectrometer TQ-MS/MS equipped with negative and positive electrospray ionization (ESI) interface in multiple reaction monitoring (MRM) mode. The statistical analysis was performed by the Student's t-test with P ≤ 0.05 as the level of significance. The method showed good precision, accuracy, recovery, sensitivity, linearity, and stability. The pharmacokinetic profiles and parameters of these polyphenolics changed when co-administrated with tanshinones. The tanshinones improved the bioavailability of DSS, accelerated the eliminating rate of RA and Sal B and promoted their distribution in vivo. They also contributed to promoting the biotransformation of Sal B to DSS. The polyphenolics could affect the pharmacokinetic of tanshinones, especially CT and TSIIA. Furthermore, the biotransformation of CT to TSIIA and the bioavailability of TSIIA were both improved. This study may provide useful information to avoid unexpected increase of the plasma drug concentration in the clinical practice. Copyright © 2015 John Wiley & Sons, Ltd.

A UPLC-MS/MS method for simultaneous determination of danshensu, protocatechuic aldehyde, rosmarinic acid, and ligustrazine in rat plasma, and its application to pharmacokinetic studies of Shenxiong glucose injection in rats.

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of the four major active ingredients, danshensu, protocatechuic aldehyde, rosmarinic acid, and ligustrazine, in the traditional Chinese medicine Shenxiong glucose injection in rat plasma. Acidified and alkalized plasma samples were extracted using ethyl acetate, and separated on a Waters C18 column (2.1mm×50mm, 1.7µm) by using a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid and luteoloside as an internal standard. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to identify and quantitate the active components. All calibration curves showed good linearity (r>0.994) over the concentration range, with a lower limit of quantification (LLOQ) between 0.02 and 0.21µg/mL. The precision of the in vivo study was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation was within 15%. Moreover, satisfactory extraction efficiency was obtained (between 83.94 and 117.81%) by liquid-liquid extraction. The validated method was successfully applied in a pharmacokinetic study in rats after intravenous administration of Shenxiong glucose injection. The results showed that the four bioactive ingredients in Shenxiong glucose injection have linear pharmacokinetic properties in rats after intravenous injection within the administered dose range and partially different ones compared to single ingredient.

Pharmacokinetics, Safety and Tolerability of Melissa officinalis Extract which Contained Rosmarinic Acid in Healthy Individuals: A Randomized Controlled Trial.

The aim of this study was to evaluate the safety, tolerability and pharmacokinetics of single dose of Melissa officinalis extract which contained rosmarinic acid, including food-effects in healthy individuals. A total of eleven healthy individuals were randomly assigned to treatment arms in the two studies [Study 1 (fasted state) and Study 2 (fed state)]. Rosmarinic acid in serum was measured by a coulometric detection method using High-Performance Liquid Chromatography electrochemical detector. The serum concentration of total rosmarinic acid peaked at 1 hour after administration of Melissa officinalis extract containing 500mg rosmarinic acid in fasted state, with a maximum serum concentration 162.20 nmol/ L. The area under the curve for intact rosmarinic acid was calculated from the serum concentration-time profile to be 832.13 nmol • hour/ L. Food intake increases area under the curve and delayed time at which the maximum serum concentration. Rosmarinic acid supplementation did not affect liver, kidney, or blood cell function parameters. No adverse event was reported by any of the participants due to the study treatment. Single dose of Melissa officinalis extract containing 500 mg rosmarinic acid appears to be safe and tolerable in healthy individuals. Food intake increased the exposure of rosmarinic acid and delayed absorption of rosmarinic acid in healthy individuals.

Pharmacokinetic investigation on interaction between hydrophilic lithospermic acid B and lipophilic tanshinone IIA in rats: an experi- mental 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 and 20.2 to 49.1 h. The concomitant use of tanshinone II A magnified the volume of distribution at steady state (Vss) and time for the drug in the plasma to reduce the highest concentration by half (t½) of lithospermic acid B, while at the same time the Vss and t½ of tanshinone II A changed significantly in the presence of lithospermic acid B. CONCLUSION: Lithospermic acid B and tanshinone II A interact with each other following simultaneous intravenous injection in rats and this observation may expand the clinical use of Danshen (Radix Salviae Miltiorrhizae).

[Pharmacokinetic study on peoniflorin, astilbin, rosmarinic acid, isofraxidin and liquiritin in rat blood after oral administration of shaolin xiaoyin tablets].

To establish a method for the determination of astilbin, peoniflorin, rasmarinci acid, isofraxidin and liquiritin contained in Shaolin Xiaoyin tablets, in order to lay a foundation for designing late-stage dosage forms and clinical medication schemes. In this paper, efforts were made to establish a method for the determination of the blood concentration of the five components and study the in vivo pharmacokinetics in rats. The blood concentration was determined by HPLC. Phenomenex C18 column (4.6 mm x 250 mm, 5 microm) was adopted and eluted with methanol-acetonitrile-0.05% formic acid, the flow rate was 0.8 mL x min(-1), and the wavelength was 275 nm. The samples were processed by the solid phase extraction method. After oral administration of Shaoling Xiaoyin tablets, the rat bloods were collected at different time points to determine the blood concentrations. The experimental results showed that the baseline separation could be adopted for the five components, and astilbin, peoniflorin, rasmarinci acid, isofraxidin and liquiritin showed good linear relations within ranges of 2.48-248, 0.213 6-21.36, 0.531-53.1, 0.704-70.4, 0.253-25.3 mg x L(-1). All the five components could be absorbed in blood and excreted quickly. The method established in this paper is rapid and accurate, and could be used for in vivo analysis on preparations containing similar components. The main components in Shaoling Xiaoyin tablets could be absorbed and excreted quickly, and thus suitable to be made into sustained release tablets. Common preparations are required to be taken for 4-6 times a day.

Validation of determination of plasma metabolites derived from thyme bioactive compounds by improved liquid chromatography coupled to tandem mass spectrometry.

In the present study, a selective and sensitive method, based on microelution solid-phase extraction (µSPE) plate and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was validated and applied to determine the plasma metabolites of the bioactive compounds of thyme. For validation process, standards of the more representative components of the phenolic and monoterpene fractions of thyme were spiked in plasma samples and then the quality parameters of the method were studied. Extraction recoveries (%R) of the studied compounds were higher than 75%, and the matrix effect (%ME) was lower than 18%. The LODs ranged from 1 to 65 µg/L, except for the thymol sulfate metabolite, which was 240 µg/L. This method was then applied for the analysis of rat plasma obtained at different times, from 0 to 6h, after an acute intake of thyme extract (5 g/kg body weight). Different thyme metabolites were identified and were mainly derived from rosmarinic acid (coumaric acid sulfate, caffeic acid sulfate, ferulic acid sulfate, hydroxyphenylpropionic acid sulfate, dihydroxyphenylpropionic acid sulfate and hydroxybenzoic acid) and thymol (thymol sulfate and thymol glucuronide). The most abundant thyme metabolites generated were hydroxyphenylpropionic acid sulfate and thymol sulfate, their respective concentrations in plasma being 446 and 8464 µM 1h after the intake of the thyme extract.

Simultaneous determination of six phenolic constituents of Danshen injection in rat plasma by LC-ESI-MS and its application to a pharmacokinetic study.

Salvianolic acid A, salvianolic acid B, danshensu, protocatechuic aldehyde, rosmarinic acid and lithospermic acid are the six major active constituents in Danshen injection. In this study, a rapid, sensitive and specific liquid chromatographic-electrospray ionization-mass spectrometry method for the simultaneous quantitative determination of these compounds in rat plasma was developed. After a single step of liquid-liquid extraction with ethyl acetate, they were eluted by a Hypersil C18 column (5 µm, i.d. 4.6 × 200 mm) within 4 min with a mobile phase consisting of acetonitrile and 0.1% formic acid water solution (35:65, v/v). The assay was linear in the concentration range of 0.05-10 µg mL(-1). Absolute recoveries were above 60%. The precisions and accuracies determined within three consecutive days were within acceptable limits. The method was successfully applied to a pharmacokinetic study in rats after an intravenous administration of Danshen injection.

Function of Plectranthus barbatus herbal tea as neuronal acetylcholinesterase inhibitor.

This study aims to determine the function of Plectranthus barbatus (Lamiaceae) herbal tea as inhibitor of the brain acetylcholinesterase (AChE) activity. To accomplish this objective the herbal tea as well as its main component, rosmarinic acid were administered to laboratory animals (rats) and the effect on the brain AChE activity was evaluated. The study of the herbal tea metabolites in the plasma and also in the brain was undertaken. The herbal water extract was administered intragastrically and also intraperitoneally. When the plant extract was intragastrically administered, vestigial amounts of metabolites from P. barbatus extract compounds were present in rat plasma, but none were found in brain, although inhibition of brain acetylcholinesterase activity was detected. However, when P. barbatus extract was administered intraperitoneally, all its compounds were found in plasma, and rosmarinic acid was found in brain. The highest concentrations of compounds/metabolites were found 30 min after administration. An inhibition of 29.0 ± 2.3% and 24.9 ± 3.7% in brain acetylcholinesterase activity was observed 30 and 60 min after intraperitoneal administration, respectively. These values were higher than those expected, taking into account the quantity of rosmarinic acid detected in the brain, which suggests that other active extract compounds or metabolites may be present in non-detectable amounts. These results prove that the administration of P. barbatus aqueous extract can reach the brain and act as AChE inhibitor.

High performance liquid chromatography-mass spectrometry analysis for rat metabolism and pharmacokinetic studies of lithospermic acid B from danshen.

Metabolism and pharmacokinetic studies on rat were conducted for lithospermic acid B, one of the components from Radix Salviae Miltiorrhizae (danshen) that shows many bioactivities. Liquid chromatography-electrospray ionization mass spectrometry method was applied for the determination of lithospermic acid B and its metabolites in samples from in vitro and in vivo metabolism studies. Rat plasma samples collected after intravenous administration were analyzed for obtaining pharmacokinetic data of lithospermic acid B. Four O-methylated metabolites, namely one monomethyl-, two dimethyl- and one trimethyl-lithospermic acid B, were detected when lithospermic acid B was incubated in rat hepatic cytosol. These four metabolites were also detected in rat bile, plasma and feces samples after intravenous administration of lithospermic acid B. The in vitro and in vivo results indicate that the methylation is the main metabolic pathway of lithospermic acid B. The danshen component and its methylated metabolites were excreted to rat bile and feces.

HPLC analysis of rosmarinic acid in feed enriched with aerial parts of Prunella vulgaris and its metabolites in pig plasma using dual-channel coulometric detection.

This paper describes a sensitive isocratic HPLC/ECD method developed for the determination of rosmarinic acid (RA) in plant material, animal feed, and pig plasma. The plasma sample preparation only includes protein precipitation and adjustment of the pH. The applicability of the method was tested on plasma samples of pigs that were exposed to a 91-day oral intake of RA via feed enriched by aerial parts of Prunella vulgaris. The plasma was directly analyzed using the method described as well as after enzymatic hydrolysis. When no hydrolysis step was included, RA and caffeic acid (CA) were quantified in the plasma. In hydrolyzed plasma samples, several other metabolites were determined, including dihydrocaffeic, ferulic, and dihydroferulic acid. The dual-channel coulometric detection employed, as an alternative to mass spectrometry, offers good selectivity and sensitivity owing to the electrochemical properties of the phenolic constituents.

In vitro and in vivo stability of caffeic acid phenethyl ester, a bioactive compound of propolis.

The in vitro biochemical stability of caffeic acid phenethyl ester in rat and human plasma was investigated and compared with the stability of other caffeic acid esters (chlorogenic acid and rosmarinic acid). The incubation of the compounds in rat plasma for up to 6 h showed that caffeic acid phenethyl ester, but not the other compounds, was hydrolyzed, whereas human plasma did not affect the stability of all the assayed compounds. The products in rat plasma were caffeic acid and an unknown compound, which was identified by mass spectrometry as caffeic acid ethyl ester, produced by transesterification in the presence of ethanol used as vehicle for standard compounds. Specific inhibitors of different plasma esterases allowed the identification of a carboxylesterase as the enzyme involved in the metabolism of caffeic acid phenethyl ester. The oral administration in rats of caffeic acid phenethyl ester in the presence of both ethanol and 2-(2-ethoxyethoxy)ethanol gave rise to a dramatic increase of caffeic acid, as well as low levels of caffeic acid phenethyl ester, caffeic acid ethyl ester, and caffeic acid 2-(2-ethoxyethoxy)ethyl ester, in urine collected within 24 h after treatment. These results suggest that caffeic acid phenethyl ester is hydrolyzed also in vivo to caffeic acid as the major metabolite and that its biological activities should be more properly assayed and compared with those of caffeic acid, its bioactive hydrolysis product. Moreover, alcohols should be carefully used in vivo as solvents for caffeic acid phenethyl ester, since they can give rise to new bioactive caffeic acid esters.