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Pharmacokinetics of rosmarinic acid in rats by LC-MS/MS: absolute bioavailability and dose proportionality

a Rosmarinic acid (RA), one of the main bioactive compounds of Rosmarinus o ﬃ cinalis L., exhibits diverse pharmacological e ﬀ ects. However, its oral absolute bioavailability and dose proportionality in vivo have not been comprehensively studied. In the present study, a validated LC-MS/MS method was developed for the determination of RA in rat plasma. Pharmacokinetic parameters were obtained following oral and intravenous dosing using DAS 3.0 software. Absolute bioavailability in rats was determined by comparing pharmacokinetic data after administration of single oral (12.5, 25 and 50 mg kg (cid:1) 1 ) and intravenous (0.625 mg kg (cid:1) 1 ) doses of RA. The dose proportionality of AUC and C max were analyzed using a power model. After single-dose oral administration of RA, C max values for groups with 12.5, 25 and 50 mg kg (cid:1) 1 doses ranged from 215.29 to 790.96 ng mL (cid:1) 1 , with AUC 0 – t values from 41 789.84 to 96 070.00 min ng mL (cid:1) 1 , while T max and t 1/2 values ranged from 8.33 to 18.33 and from 332.34 to 295.32 min, respectively. The power model showed RA lacked dose proportionality over the dose range from 12.5 – 50 mg kg (cid:1) 1 . Oral absolute bioavailability was calculated to range from 0.91% to 1.69%. All the results demonstrated that the pharmacokinetic properties of RA in rats after oral administration were characterized as rapid absorption, middle-speed elimination and poor absolute bioavailability. Systemic exposure exhibited lack of dose proportionality over the dose range from 12.5 to 50 mg kg (cid:1) 1 . These presented data could provide useful information for the rational clinical application and optimal dosage form design of RA.


Introduction
Rosemary, Rosmarinus officinalis L. (Labiatae), is used in folk medicine worldwide for initial health care among a variety of plants. It is an evergreen perennial shrub native to Asia Minor and southern Europe. Today it has been cultivated in many parts of the world. 1,2 The powerful antioxidant activity of its constituents supports protection against damage induced by free radicals. 3,4 Furthermore, it has been demonstrated that Rosmarinus officinalis produces an antinociceptive effect in experimental models of pain like acetic acid and formalin, but also in the pain-induced functional impairment model in rats. 5 According to phytochemical reports, rosmarinic acid (RA) (Fig. 1) is the main effective, water-soluble polyphenolic acid from Rosemary, which was found to have various curative activities, such as anti-inammatory, 6,7 antioxidant, 8 anticancer, 9,10 antimicrobial 11 and anti-diabetes 12 properties. Recent studies also showed that it could improve cognitive performance. 13 For safety and efficacy reasons, it is important to understand the real-time effective levels of phytochemicals in the systemic circulation and the time course concentrations following oral administration. Besides, research on bioavailability plays a very important role in the development of a drug and its clinical use.
To date, several pharmacokinetic studies for RA have been reported in human or animals. Most of these studies on pharmacokinetics of RA were accomplished by oral administration of a single herb, 14,15 compound prescription extract 16,17 or mixture containing RA. 18,19 On the other hand, the pharmacokinetics of RA aer pure RA treatment only has been roughly or partially carried out. 20,21 These pharmacokinetics studies have enabled us to understand the absorption characteristics of RA in vivo. But a systemic pharmacokinetic study of RA, especially with gradient dosages, aer single RA treatment still remains to be done.
Phenolic acids were usually reported with low oral bioavailability. 22,23 In Caco-2 cells' model, RA transport was mainly via paracellular diffusion with low intestinal absorption efficiency. 24,25 Intestinal ora and intestinal epithelial cells usually transform polyphenols into low molecular weight metabolites, which decrease the intestinal absorption of polyphenols, such as catechins found in tea. 26 In order to achieve rational clinical application and optimal dosage form design of drug, the study of absolute bioavailability is indispensable. Furthermore, dose proportionality of a drug is important for its safety and efficiency, because it could indicate whether dose-response relation is linear or not, which may affect effectiveness and toxicity. To the best of our knowledge, there is no report concerning bioavailability or dose proportionality of RA in vivo.
In the present paper, a validated LC-MS/MS method was developed for the determination of RA in rat plasma. Pharmacokinetic parameters were obtained following oral and intravenous dosing using DAS 3.0 soware. Absolute bioavailability in rats was determined by comparing pharmacokinetic data aer administration of single oral (12.5, 25 and 50 mg kg À1 ) and intravenous (0.625 mg kg À1 ) doses of RA. The dose proportionality of AUC and C max were analyzed by power model.

Materials
Rosmarinic acid (raw material) was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China) and had a purity of mass fraction of more than 98.0%. Rosmarinic acid and silibinin (purity > 99%), used as a reference standard and an internal standard (I.S.), were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile and formic acid (HPLC grade) were purchased from Merck and Tedia Company Inc. (Merk, Darmstadt, Germany). Puried water was prepared using a Milli-Q purication system (Millipore, Billerica, MA, USA). Other chemical reagents of analytical grade or better were obtained from Sinopharm Chemical Reagent Co. (Nanjing, China).

Animal treatment
Twenty-four Sprague Dawley male rats weighing 220-250 g were supplied by the Animal Center of Nanjing Medical University. Animals were housed in environmentally controlled conditions at 25 AE 2 C and 50 AE 10% relative humidity under a 12 h darklight cycle. The rats were kept with free access to food and water until 12 h prior to the experiments. All procedures and experiments of this study were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine, and its approved protocol is "Scientic Protocol (2007) Number 16 of Nanjing University of Chinese Medicine".

LC-MS/MS conditions
The LC-MS/MS system consisted of a Shimadzu LC-20A series liquid chromatograph (Shimadzu) and a QTRAP 5500 mass spectrometer (Applied Bio-systems, AB Sciex) equipped with an electro-spray ionization (ESI) source. The data acquisition and processing were performed using Analyst 1.5.2 soware. The separation was performed on a Waters Acquity UHPLC BEH C 18 column (100 Â 2.1 mm, i.d., 1.7 mm, Waters) protected by a Van Guard BEH C 18 column (5 Â 2.1 mm, 1.7 mm, Waters) maintained at 40 C. The mobile phase consisting of a 0.5% formic acid aqueous solution and 0.1% formic acid acetonitrile solution (60 : 40, v/v) was run at a ow rate of 0.3 mL min À1 . The injection volume was 3 mL. The total run time for a LC-MS/MS analysis was 6 min.
An ESI interface in the negative mode was used. The ion spray voltage was set at À4.5 kV and the turbo spray temperature was maintained at 500 C. Nebulizer gas (gas 1) and heater gas (gas 2) were set at 160 and 260 psi, respectively. The curtain gas was kept at 40 psi and the interface heater was on. Nitrogen gas was used in all cases. Quantication was performed using MRM mode.

Preparation of calibration and quality control (QC) samples
Stock solutions of RA and silibinin (I.S.) were prepared in methanol at a concentration of 0.1122 and 0.1126 mg mL À1 , respectively. Stock solutions were then diluted with methanol to produce a series of standard or QC working solutions at desired concentrations. The calibration standards were prepared by adding 10 mL of the series of standard working solutions into 100 mL of blank plasma to provide nal concentrations at 8.976, 17.95, 44.88, 89.76, 224.4, 448.8, 1122, 2244, 5610, and 11 220 ng mL À1 . Low, medium and high levels of QC samples were prepared at 17.95, 448.8 and 5610 ng mL À1 , respectively. The I.S. working solution of 2.252 mg mL À1 was also prepared by dilution of the stock solution with methanol. All the solutions were kept at 4 C and brought to room temperature before use.

Sample preparation
A simple liquid-liquid extraction method was applied to extract RA and I.S. from the rat plasma. In an aliquot of 100 mL of rat plasma, 10 mL of the I.S. and 10 mL of methanol (volume of the corresponding working solution for calibration curve and QC samples) and 16 mL of formic acid/water (1 : 3, v/v) were added. Then the mixture was vortexed for 1 min and 0.2 mL of acetone/ water (70 : 30, v/v) solution was added, followed by adding 1 mL of ethyl acetate to each tube. Extraction was performed by vortex mixing the tubes for 2 min, followed by centrifugation at 16 000 rpm for 3 min. The organic layer was transferred to Eppendorf tubes and dried using a centrifugal concentrator. The residue was reconstituted in 100 mL of water containing 25% acetone. Aer centrifugation at 16 000 rpm for 3 min, aliquots of 3 mL were injected into the UPLC-MS/MS system for analysis.

Method validation
The method was validated for selectivity, linearity, precision, accuracy, extraction recovery and stability according to the FDA guidelines for the validation of bio-analytical methods. 27 Specicity. To investigate whether or not endogenous constituents interfered with the assay, the specicity of the method was tested by comparing the chromatograms of a blank rat plasma sample, a blank rat plasma sample spiked with RA (448.8 ng mL À1 ) and I.S. (2.252 mg mL À1 ), a rat plasma sample 10 min aer intravenous administration of RA, and a rat plasma sample 10 min aer receiving oral doses of 50 mg kg À1 .
Linearity and lower limit of quantication (LLOQ). Calibration curves were prepared by assaying standard plasma samples at ten concentration levels. Calibration curves were prepared by plotting the peak area ratios between the analytes and the I.S. against the concentration of analytes.
The lower limit of quantication is dened as the lowest concentration on the calibration curve with an acceptable precision (RSD) not exceed 20% and RE (relative error) of accuracy should not deviate by more than 20%.
Precision and accuracy. The intra-day precision and accuracy were evaluated by repeated analyses of QC samples at three different concentration levels (17.95, 448.8 and 5610 ng mL À1 ) from six replicates on the same day, while the inter-day precision and accuracy were evaluated on three independent days. The concentration of each sample was determined using the calibration curve prepared and analyzed on the same batch. Precision was evaluated by intra-day and inter-day RSD, which should not exceed 15% except for the LLOQ. Accuracy was evaluated by comparing the observed concentration with the actual concentration and RE of accuracy should not deviate by more than 15% except for the LLOQ.
Matrix effect and recovery. The matrix effects were measured by comparing the peak areas of the analytes dissolved in the pretreated blank plasma with that of analytes working solutions containing equivalent amounts of the analytes. Recovery was determined by comparing the peak areas of extracted RA from the QC samples to those for RA prepared in methanol. This procedure was repeated six times at three QC concentration levels, 17.95, 448.8 and 5610 ng mL À1 . The extraction recovery and matrix effect of the I.S. were also evaluated using the same procedure.
Stability. The stability of the analytes in rat plasma was assessed by analyzing QC samples at three concentration levels under three different sets of conditions. The short-term stability was determined with untreated QC samples stored for 24 hours at room temperature. The long-term stability was assessed aer the untreated QC samples had been stored at À20 C for 15 days. The freeze-thaw stability was determined aer three freeze-thaw cycles (À20 C to room temperature as one cycle).

Bioavailability and pharmacokinetics of RA in rats
The validated method was applied to analyze the plasma concentrations of RA in rats aer oral and intravenous administration of RA. Twenty-four male rats were randomly divided into four groups with six in each. Three groups were given RA at a single oral dose of 12.5, 25 and 50 mg kg À1 , and one group received a single dose of RA of 0.625 mg kg À1 by intravenous injection via caudal vein aer an overnight fasting period. The rats were fasted for the rst 2 h with free access to water aer dosing. Serial blood samples (approximately 0.2 mL) were collected from the eyeground vein into 1.5 mL heparinized tubes at 0, 2, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, 480 and 720 min aer oral administration and at 0, 2, 5, 10, 20, 30, 45, 60, 75, 90, 120, 180, 240 and 360 min aer intravenous injection. Plasma was isolated from the blood samples by cooling them for 2 h at 4 C, followed by centrifugation at 3000 rpm for 10 min. The collected plasma was transferred into 1.5 mL Eppendorf tubes and then stored at À20 C for analysis.
Pharmacokinetic parameters were estimated using Drug and Statistics 3.0 (DAS 3.0, Mathematical Pharmacology Professional Committee of China) employing the noncompartmental model. The oral bioavailability (F) of RA is calculated by comparing the respective AUC aer oral and intravenous administration according to the following equation:

Statistical analyses
Descriptive statistics were used to summarize the pharmacokinetic parameters for RA. Statistical comparisons of AUC 0-t , AUC 0-N , C max between the three doses were performed using an analysis of variance (ANOVA). Multivariable regression analysis was used to test associations between AUC 0-t , AUC 0-N , C max and the dose. Dose proportionality for AUC 0-t , AUC 0-N and C max was assessed by linear regression of ln-transformed parameters on the natural ln-transformed dose [ln(PK) Values of the proportionality constant, b 1 , and its corresponding 90% CI were estimated with the SPSS 19.0 soware. The primary methodology used to assess dose proportionality from 12.5 to 50 mg kg À1 was a comparison of the 90% CI of the slopes with the modied acceptance range [1 + (ln(q L )/ln(r)), 1 + (ln(q H )/ ln(r))] based on a power model, 28,29 where q L and q H are the lower and upper limits of the condence interval and r is the maximal dose ratio for the study. Dose proportionality was assumed if the plot of pharmacokinetic parameter vs. dose indicated linearity and the 90% CI for the slope fell within the modied acceptance range.

LC-MS/MS development
In the full scan mass spectra, the deprotonated molecular ions [M À H] À of RA and silibinin (I.S.) (m/z at 359.0 and 480.6) were stable and exhibited higher abundance. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions of m/z 359.0 / 160.9 for RA and 480.6 / 301.0 for I.S., respectively. Declustering potential, collision energy and cell exit potential of RA and I.S. were optimized as À70, À22, À15 V and À210, À26, À21 V, respectively. The optimized mass spectrometric parameters had a higher signal for both precursor ions and product ions mentioned above. Selection of the LC conditions was key in order to obtain chromatograms with better peak responses. We found that acetonitrile-water can achieve higher peak responses and shorter analysis times for the target compounds in chromatograms. It was found that the peak shapes and responses of analytes were improved with an eluent consisting of water containing 0.5% formic acid and acetonitrile containing 0.1% formic acid (60 : 40, v/v). Satisfactory separation was achieved in 6 min by isocratic elution using the LC conditions described above.

Method validation
Specicity. Under the above LC-MS/MS conditions, the retention times of RA and I.S. were 1.08 and 1.61 min, respectively. The typical chromatograms of a blank rat plasma sample, a blank rat plasma sample spiked with RA (448.8 ng mL À1 ) and I.S., a rat plasma sample 10 min aer intravenous administration of RA, a rat plasma sample 10 min aer receiving oral doses of 50 mg kg À1 , and the chemical structures of RA and I.S. are shown in Fig. 1. No obvious interferences were observed in these typical chromatograms of a blank plasma sample at the retention times of the analyte and the I.S.
Linearity and lower limit of quantication (LLOQ). Linearity for RA was obtained over the concentration range of 8.976-11 220 ng mL À1 . A typical standard curve was y ¼ 0.0089x À 0.3320 (r 2 ¼ 0.9980), where y represents the peak area ratios of RA to the I.S. and x represents the plasma concentrations of RA.
The linearity and lower limit of quantication (LLOQ) of RA in rat plasma was 8.976 ng mL À1 with an intra-and inter-day relative standard deviation (RSD) of less than 3.85%, and an intra-and inter-day relative error (RE) of À8.74% and À7.88%, which was sufficient for the pharmacokinetic studies of RA in rats.
Precision and accuracy. The intra-and inter-day precisions of this method for RA ranged from 5.70 to 6.32% and from 2.39 to 6.54% for each quality control (QC) level, respectively. The intraand inter-day accuracies for RA were À9.18% to 5.57% and À12.57% to 7.04%, respectively. The results indicated that the present method was reliable and reproducible for the quantitative determination of RA.
Extraction recovery. The extraction recoveries of RA were 78.19 AE 8.42, 80.18 AE 11.84 and 72.03 AE 6.18% at concentrations of 17.95, 448.8 and 5610 ng mL À1 , respectively. The recovery of I.S. was 84.58 AE 6.79%. The results of the matrix effect of RA were in the range of 87.30-95.47%. These are shown in Table 1.
Stability. Short-term stability showed good stability as responses varied no more than AE8% at three QC concentrations. The long-term stability showed no obvious substance loss (RE < 7%, RSD < 8%). The concentration variation aer three freeze-thaw cycles was within AE14% of nominal concentrations, which indicated that the analytes in rat plasma were all stable for 24 hours at room temperature, 15 days at À20 C and three cycles of freeze-thaw.

Pharmacokinetic parameters and bioavailability study
The validated analytical method was successfully applied to a pharmacokinetic study of RA in rats. The mean plasma concentration-time curves of RA aer oral administration of 12.5, 25, 50 mg kg À1 and intravenous administration of 0.625 mg kg À1 to rats are presented in Fig. 2. The corresponding pharmacokinetic parameters calculated using non-compartmental analysis are listed as mean AE SD in Table 2.
As seen from Table 2 and Fig. 2, it was found that aer single oral administration (12.5, 25 and 50 mg kg À1 ), RA could be absorbed rapidly into blood, and reached its peak concentration in plasma within 19 min. Aer that RA eliminated slowly with t 1/ Table 1 The matrix effects and extraction recoveries of RA and I.S. in rat plasma (n ¼ 6)

Analytes
Concentration (ng mL À1 ) Oral bioavailability is a complicated parameter that involves a number of chemical and physical processes. Poor bioavailability of RA may result from several different causes such as its intestinal absorption and metabolism. Its high hydrophilicity makes RA exhibit poor permeation across intestinal epithelial cells. 30 Besides, in rats orally administered with RA, the polyphenol was absorbed, degraded, and/or conjugated as m-hydroxyphenylpropionic acid, m-coumaric acid, and sulphated forms of caffeic acid and ferulic acid, 20 which were then absorbed by a specic intestinal monocarboxylic acid transporter-mediated active process, 21 before being excreted gradually in the urine.
Considering the low absolute bioavailability of RA and its derivatives' efficacy, the effects of RA may be explained not solely by the established absorption of RA, but also by the absorption of its microbial phenolic acid metabolites. The assigned effect of RA and its metabolites is a need for further research. [31][32][33][34] Dose proportionality According to One-Way ANOVA analysis, the results of homogeneity of variance test statistics indicated that the variances of C max , AUC 0-t and AUC 0-N were not homogenous. The C max , AUC 0-t and AUC 0-N need to be ln-transformed. The results are summarized in Table 3. Aer curve estimation analysis with different models, relationships between ln C max and dose, ln AUC 0-t and dose, ln AUC 0-N and dose could be well tted with power model, as showed in Table 4. Plots of the tted function for the power model with associated 90% condence intervals are presented for AUC 0-t , AUC 0-N and C max in Fig. 3. Results of the assessment based on the power model are presented in Table 5. The slopes (90% condence intervals (CIs)) were 0.586 (0.466, 0.707) for AUC 0-t , 0.553 (0.433, 0.673) for AUC 0-N and 0.887 (0.679, 1.095) for C max . The corresponding

Conclusions
In summary, it has been rstly carried out to comprehensively investigate absolute bioavailability, pharmacokinetics, and dose proportionality on the pharmacokinetics of single oral dose of RA. The absolute bioavailability of RA in rats was estimated as 1.69%, 1.28% and 0.91% aer oral administration of RA at the doses of 12.5, 25 and 50 mg kg À1 . The relationships between pharmacokinetic parameters (C max , AUC 0-t and AUC 0-N ) of RA and dose could be well tted with power model. Pharmacokinetic properties of RA in rats aer oral administration were characterized as rapid absorption, middle-speed elimination and poor absolute bioavailability. Analysis by power model demonstrated that systemic exposure exhibited lack of dose proportionality over the dose ranged from 12.5 to 50 mg kg À1 . The data presented in this study could provide useful information for its rational clinical application and optimal dosage form design.

Declaration of interest
The authors declare no conict of interest.