Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 9–23

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Development of a SPE-LC/MS/MS method for simultaneous quantification of baicalein, wogonin, oroxylin A and their glucuronides baicalin, wogonoside and oroxyloside in rats and its application to brain uptake and plasma pharmacokinetic studies Sophia Yui Kau Fong, Yin Cheong Wong, Zhong Zuo ∗ School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

a r t i c l e

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Article history: Received 21 January 2014 Received in revised form 18 March 2014 Accepted 22 March 2014 Available online 18 April 2014 Keywords: Scutellariae Radix Brain Plasma Glucuronides LC/MS/MS

a b s t r a c t This study aims to identify and quantify the six major bioactive flavones of the traditional Chinese medicine Scutellariae Radix (RS), including baicalein, baicalin, wogonin, wogonoside, oroxylin A and oroxyloside in rat after oral administration of a standardized RS extract. A novel, sensitive and selective method for simultaneous determination of these six analytes in rat brain and plasma using solid phase extraction-liquid chromatography–tandem mass spectrometry (SPE-LC/MS/MS) was developed and fully validated. The lower limits of quantification (LLOQs) for the six RS flavones in brain tissue were 0.02 nmol/g. The LLOQs in plasma were 0.005 nmol/ml for B, W and OA, 0.025 nmol/ml for WG and OAG, and 0.1875 nmol/ml for BG. The current study provides novel evidence of the presence of all the tested RS flavones and an isoform of BG (BG , probably baicalein-6-O-glucuronide) in the rat brain after oral administration of RS extract, suggesting their ability to permeate through the blood–brain barrier. The method was also successfully applied to the pharmacokinetic study of all these analytes in plasma after oral administration of RS extract (300 mg/kg) to Sprague-Dawley rats. The developed assay method provides a useful tool for both preclinical and clinical investigations on the disposition of RS flavones in brain and plasma. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Scutellariae Radix (RS), the dried root of Scutellariae baicalensis Georgi, is a traditional Chinese medicine that has been used for thousands of years for antifebrile and detoxification purposes. Six flavones are identified as the major bioactive components of RS, namely baicalein (B), baicalin (baicalein-7-O-glucuronide, BG), wogonin (W), wogonoside (wogonin-7-O-glucuronide, WG), oroxylin A (OA) and oroxyloside (oroxylin A-7-O-glucuronide) [1,2]. Modern researches demonstrate that RS and these bioactive flavones have multiple pharmacological actions, including antiinflammatory [3], anti-oxidant [4], anti-cancer [5], and anti-viral [6] activities. They are also active in the central nervous system (CNS) and their effectiveness in CNS disorders such as anxiety [7], epilepsy [8], memory and learning impairment [9], attention-deficit hyperactivity disorder [10] and neurotoxicity [11] have been reported.

∗ Corresponding author. Tel.: +852 3943 6832; fax: +852 2603 5295. E-mail address: [email protected] (Z. Zuo). http://dx.doi.org/10.1016/j.jpba.2014.03.033 0731-7085/© 2014 Elsevier B.V. All rights reserved.

Since RS has such enormous therapeutic potentials, a better understanding of its pharmacokinetics and bioavailability is necessary for extrapolating the data from pharmacological assays to clinical effects, developing clinical regimens and elucidating potential drug interactions [12]. Simultaneous quantification of the six major bioactive flavones in RS is therefore important in both the plasma and its target sites of action such as the brain. We previously published a simple high-performance liquid chromatography/ultraviolet detection (HPLC/UV) method for simultaneous determination of these six flavones in rat plasma. However, the assay was not sensitive enough to quantify B, W, OA and OAG in plasma after oral administration of a commercial RS product to rats [13]. Kim et al. [14] and Chung et al. [15] developed liquid chromatography–tandem mass spectrometric (LC/MS/MS) methods to quantify B, W and OA in rat plasma after intravenous and oral administration of RS extract to rats. Nevertheless, both assays only measured some but not all of the six flavones simultaneously. To our knowledge, four studies have reported the determination of individual RS flavones in brain with full validation data. Zhang et al. developed a HPLC/UV assay to quantify BG in rat brain and thalamus after intravenous administration of RS extract [16,17]. Employing a

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solid phase extraction (SPE)-LC/MS/MS method, Liu et al. detected W and WG in the brain of tumor-bearing nude mice after intravenous administration of 20 mg/kg of W [18]. Hou et al. attempted but failed to quantify B, BG, W and WG simultaneously in rat brain after repeated oral doses of RS decoction at 2 g/kg using a validated HPLC/UV assay [19]. They reported that no traces of these RS flavones were found in the brain despite the high oral dosage of RS extract. Whether the absence of flavones in brain was due to the low sensitivity of the assay method or due to the route of administration remained uncertain. Furthermore, no study has ever reported the presence of OA and its glucuronide OAG in brain. Therefore, it is also novel to develop and validate a sensitive method for the simultaneous determination of the six RS flavones in order to confirm their presence (or absence) in brain after oral administration of RS extract to rats. In the current study, a novel, sensitive and selective method for simultaneous determination of the six flavones B, BG, W, WG, OA, OAG in rat brain tissue and plasma using SPE-LC/MS/MS method is developed and fully validated. The method was applied to investigate the disposition of these flavones in the brain and plasma after oral administration of RS extract to rats. 2. Materials and methods 2.1. Materials and chemicals B and BG with purity over 98% were purchased from Aldrich Chem. Co. (Milwaukee, WI, USA). W and WG with purity over 98% were purchased from AvaChem Scientific LLC (San Antonio, TX, USA). OA (purity over 98%), OAG (purity over 95%) and a standardized RS extract were supplied by Shanghai U-sea Biotech Co., Ltd. (Shanghai, China). 4 ,5,7-Trihydroxylflavanone used as internal standard (IS) was obtained from Aldrich Chem. Co. (Milwaukee, WI, USA). Oasis hydrophilic–lipophilic-balanced copolymer extraction cartridges (HLB, 1 ml and 3 ml) used for SPE were supplied by Waters (Milford, USA). Acetonitrile (ACN) and methanol (MeOH) were obtained from RCI Labscan (Bangkok, Thailand) and Merck (Darmstadt, Germany) respectively. Both of them were of HPLC grade and were used without further purification. Formic acid (analytical grade) was purchased from BDH Laboratory Supplies Ltd. (Kampala, Ukraine). Dimethyl sulfoxide (DMSO, analytical grade) was from Lab-scan Analytical Sciences (Bangkok, Thailand). Distilled and deionized water was used for the preparation of all solutions. All other reagents were of at least analytical grade. 2.2. Preparation of stock solutions, calibration standards and quality control (QC) samples Master stock solutions of B, BG, W, WG, OA, OAG were prepared separately by dissolving each authentic compound in DMSO at 10 mM. Each of the stock solutions was further diluted to 100 ␮M by MeOH in water (1:1, v/v) and was stored at −80 ◦ C. For brain validation assay, working standard mixture solutions were freshly prepared by mixing and diluting the stock solutions of each compound with MeOH in water (1:1, v/v, with 0.1% formic acid and 1% ascorbic acid) to yield concentrations of 0.2, 0.7, 6 and 18 ␮M respectively. For plasma validation assay, a stock mixture containing 1.28 ␮M of B, W and OA, 6.4 ␮M of WG and OAG and 48 ␮M of BG was freshly prepared by mixing the stock solutions of each compound with MeOH in water (1:1, v/v, with 0.1% formic acid and 1% ascorbic acid). The stock mixture was further diluted serially to produce final concentrations of 0.64/3.2/24, 0.32/1.6/12, 0.16/0.8/6, 0.08/0.4/3, 0.04/0.2/1.5, 0.02/0.1/0.75,

0.01/0.05/0.375 and 0.002/0.01/0.075 ␮M (B, W and OA/WG and OAG/BG). Standard solution of IS was prepared by dissolving 4 ,5,7-trihydroxylflavanone in MeOH at a final concentration of 5 ␮g/ml. Calibration standards and QC samples were prepared by spiking working standards to drug-free biological matrices. To 300 mg (wet weight) brain tissues obtained from minced rat whole brain, 20 ␮l of IS and working standard mixture were spiked to yield analyte concentrations of 0.02, 0.04, 0.08, 0.12, 0.4, 0.8, 1.2, 2, 6, 10 nmol/g with 1.2 nmol/g of IS. To 100 ␮l plasma, 10 ␮l IS working standard and 50 ␮l working standard mixture were spiked to yield the concentrations of 0.001, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, and 0.32 nmol/ml for B, W and OA; 0.005, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 nmol/ml for WG and OAG; and 0.0375, 0.1875, 0.375, 0.75, 1.5, 3, 6 and 12 nmol/ml for BG, together with 1.8 nmol/ml IS. QC samples were prepared at three concentrations (low, medium and high) for each analytes. For brain tissue, the QC concentrations were 0.05, 0.2 and 1 nmol/g. For plasma, the QC concentrations were 0.015, 0.12 and 0.28 nmol/ml for B, W, and OA; 0.5625, 4.5 and 10.5 nmol/ml for BG; and 0.075, 0.6 and 1.4 nmol/ml for WG and OAG. All calibration and QC samples were subjected to the sample extraction as described in Section 2.3. 2.3. Sample extraction procedure 2.3.1. Brain tissue The whole brain of each rat was minced (cut into small pieces by scissors) and weighted to obtain 300 mg of tissue. To prepare the homogenate, 0.06 vol. (0.06 ␮l/mg tissue) of IS working standard, 0.6 vol. (0.6 ␮l/mg tissue) of 50% ACN in water (1:1, v/v, with 1% ascorbic acid and 0.01% formic acid), and 6 vol. (6.7 ␮l/mg tissue) of water (with 1% ascorbic acid and 0.01% formic acid) were added as homogenization medium. The brain sample was subjected to homogenization by ultrasonic probe (Microson XL-2000, Misonix, USA) for three times as described below. For the first homogenization step, the sample was homogenized by the probe for 30 s and was centrifuged at 2500 × g for 15 min. The supernatant (water extract) was further centrifuged at 21,000 × g for 10 min and was stored at −20 ◦ C. After transferring the tissue residue back to the sample tube, 6 vol. (6.7 ␮l/mg tissue) of ACN (with 0.04% ascorbic acid) was added, followed by the second homogenization step by the ultrasonic probe for 25 s. The homogenate was then rotated for 15 min by rotary mixer (Stuart Scientific Co. Ltd., UK) to allow time for extraction of the analytes, followed by centrifugation at 1000 × g for 10 min. The ACN extract was collected while the tissue residue underwent the third homogenization step by the ultrasonic probe for 20 s in 5 vol. (5 ␮l/mg tissue) of ACN (with 0.04% ascorbic acid) and was centrifuged at 1000 × g for 10 min. The ACN supernatant was collected and combined with the ACN extract from the second homogenization step and was evaporated to dryness in the Centrivap concentrator (Labconco, MO, USA). The dry residue was re-dissolved by 300 ␮l of ACN in MeOH (1:1, v/v) and by the water extract from the first homogenization step. After centrifuging at 21,000 × g for 10 min, the supernatant was transferred to a new tube and made up to 6 ml with water (with 1% ascorbic acid and 0.01% formic acid). This solution was loaded onto the Oasis® HLB cartridge (3 ml) in two portions, which had been preconditioned with 3 ml of MeOH and 3 ml of 0.1% formic acid in water. The cartridge was then rinsed with 3 ml of 0.1% formic acid, followed by 3 ml of 35% MeOH in water (with 1% ascorbic acid and 0.1% formic acid); and subsequently rinsed with 3 ml of 21% ACN in water (with 1% ascorbic acid and 0.1% formic acid). After the cartridge was dried under vacuum for 20 min, the analytes were eluted by 3 ml of MeOH from the cartridge. The MeOH eluent was then evaporated to dryness in the Centrivap concentrator, and the residue was

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reconstituted by 200 ␮l of ACN in water (1:1, v/v, with 1% ascorbic acid and 0.01% formic acid). After centrifugation at 21,000 × g for 10 min, the supernatant was injected into the LC/MS/MS system for analysis.

2.3.2. Plasma To 100 ␮l of plasma sample obtained from pharmacokinetic study, 10 ␮l of IS working standard (prepared in Section 2.2) and 50 ␮l of MeOH in water (1:1, v/v, with 1% ascorbic acid and 0.1% formic acid) were added. After diluting the mixture with 1 ml of 35% MeOH in water (with 1% ascorbic acid and 0.1% formic acid), the sample was centrifuged at 21,000 × g for 10 min. The supernatant was collected and loaded onto the Oasis® HLB cartridge (1 ml) which had been preconditioned with 1 ml MeOH followed by 1 ml 0.1% formic acid in water. The cartridge was then rinsed with 1 ml of 0.1% formic acid, followed by 1 ml of 35% MeOH in water (with 1% ascorbic acid and 0.1% formic acid). After the cartridge was dried under vacuum for 20 min, the analytes were eluted by 1 ml of MeOH from the cartridge. The MeOH eluent was then evaporated to dryness in the Centrivap concentrator, and the residue was reconstituted by 100 ␮l of MeOH in water (1:1, v/v, with 1% ascorbic acid and 0.01% formic acid). After centrifugation at 21,000 × g for 10 min, the supernatant was injected into the LC/MS/MS system for analysis.

2.4. LC/MS/MS conditions The LC/MS/MS system consisted of Agilent 1200 series LC pumps and auto-sampler (Agilent, CA, USA), coupled with an ABI 2000 Q-Trap triple quadrupole mass spectrometer with an electrospray ionization source (AB Sciex Instruments, CA, USA). The HPLC method including gradient condition and percentages of split tee was optimized for MS detection. Chromatographic separation was achieved by an Alltima C18 column (150 mm × 4.6 mm i.d., 5 ␮m particle size, Alltech) equipped with a guard filter (Unifilter 0.5 ␮m, Thermo). The two mobile phases were (A) ACN and (B) 0.1% formic acid in water. For the plasma assay, the HPLC gradient started at 70% B and decreased linearly to 25% B over 10 min and was maintained for 2 min. The gradient was then returned to the original condition of 70% B in 0.5 min and was held at 70% B for the next 2.5 min to equilibrate the column before next injection. For the brain assay, the HPLC gradient also started at 70% B and decreased linearly to 25% B over 10 min. It further decreased to 10% B over 2 min before returning to the original condition of 70% B in 0.5 min and was held at 70% B for the next 2.5 min for column equilibration. The flow rate was set at 1 ml/min and the total running time was 15 min. The temperatures of the auto-sampler and the column were set at 4 ◦ C and ambient, respectively. Prior to the ionization source, 60% of the HPLC column effluent stream was split off by a split tee and thus only 40% of the effluent was introduced. In addition, the first 3 min of the column effluent was diverted to waste to reduce the amount of unwanted impurities (e.g. inorganic salts) loaded to the MS/MS system. The injection volume was 40 ␮l. The MS/MS system was operated under positive mode and multiple reaction monitoring mode. The MS conditions were: ion spray voltage at +5.5 kV; nitrogen as nebulizer gas, auxiliary gas and curtain gas at 30, 60 and 10 psi, respectively; collision gas set at medium and auxiliary gas temperature at 400 ◦ C. For each analyte their MS parameters such as MRM transitions were optimized by direct infusion of the individual authentic standard into the mass spectrometer (Supplementary Material S1). The data acquisition was performed with Analyst software 1.4.1 (AB Sciex Instruments, CA, USA).

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2.5. Method validation The developed method was validated according to the guidelines for Bioanalytical Method Validation published by the U.S. Food and Drug Administration (FDA) in 2001 [20]. 2.5.1. Specificity The specificity of the method was evaluated by comparing the chromatograms of blank rat brain/plasma samples with that of blank rat brain/plasma spiked with standard solutions and rat brain/plasma samples after oral administration of RS extract. 2.5.2. Linearity and range Calibration samples were prepared by spiking IS and different amounts of analytes to the matrices (Section 2.2) followed by the extraction process stated in Section 2.3. Calibration curves were generated by plotting the peak area ratio of analyte to IS against the analyte concentration. Linearity was considered satisfactory if the coefficient of determination (R2 ) of the plot was higher than 0.99. The lower limit of quantification (LLOQ) was defined as the lowest concentration of the calibration curve at which the accuracy (relative error) was within ±20% of the nominal concentration and the precision (relative standard deviation, RSD) was less than 20%, and with a signal-to-noise peak height ratio greater than 5:1. The lower limit of detection (LLOD), which was the lowest concentration with a signal-to-noise peak height ratio greater than 3:1, was also presented. 2.5.3. Accuracy and precision The intra-day accuracy and precision were determined by analyzing five replicates of the QC samples at three concentrations (low, medium and high) within one day. The inter-day accuracy and precision were determined on three separate days. Accuracy within ±15% of the nominal concentration and the precision with RSD less than 15% were considered to be acceptable. 2.5.4. Extraction recovery The extraction recovery was calculated by comparing the peak area of the analyte spiked to biological matrix followed by sample extraction to that of the analyte spiked to the already extracted biological matrix. 2.5.5. Stability Freeze (−80 ◦ C)–thaw (room temperature) stability was determined by exposing the QC sample to three freeze–thaw cycles before sample extraction. Stability of the analytes in plasma was determined by spiking 50 ␮l of the QC working standard in MeOH in water (1:1) to 100 ␮l of blank plasma before undergoing freeze–thaw cycles. The stability of sample in auto-sampler was evaluated by analyzing the extracted QC sample after being placed in the auto-sampler at 4 ◦ C for 24 h. 2.6. Application to pharmacokinetic studies in rats Male Sprague-Dawley rats with body weight between 240 and 250 g were supplied by the Laboratory Animal Services Centre at The Chinese University of Hong Kong. The experiment was conducted under the approval of the Animal Ethics Committee of The Chinese University of Hong Kong. RS extract (150 mg/g) was prepared as a suspension in water containing 5% (w/w) propylene glycol 400. The content of the six bioactive components in RS extract was measured by the LC/MS/MS method described above and is presented in Fig. 1.

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Fig. 1. Chemical structures of the six major bioactive flavones in Scutellariae Radix and 4 ,5,7-trihydroxylflavanone (internal standard). The content of each flavone per 100 mg of Scutellariae Radix extract was presented as mean (mg) ± SD, n = 3, in parenthesis.

2.6.1. Identification and quantification of RS flavones in rat brain and plasma The disposition of the analytes in brain and plasma at a single time point was first investigated. 480 ␮l of RS extract suspension was administered to rats (n = 6) by oral gavage, equivalent to a RS dose of 300 mg/kg body weight; this is also equivalent to a human dose of 3 g RS extract which is clinically relevant. 6 h after RS extract administration, the rats were anesthetized in a carbon dioxide chamber for 1–2 min. 3 ml of blood was quickly withdrawn by cardiac puncture and was collected in a centrifuge tube containing heparin. Plasma was obtained by centrifugation of the blood at 16,000 × g for 5 min. After the rats were euthanized with excessive carbon dioxide, the whole brain was removed from skull and rinsed with cold normal saline (4 ◦ C). The brain was wiped by tissue paper to remove excess water and meninges and blood vessels were removed. All samples were frozen at −80 ◦ C until analysis as described in Section 2.3.1.

2.6.2. Plasma pharmacokinetic study in rats For the second experiment the pharmacokinetic time profiles of the analytes in plasma were investigated. Rats (n = 4) were anesthetized with an intramuscular dose of ketamine (60 mg/kg) and xylazine (6 mg/kg) followed by cannulation with a polythene tube (0.5 mm i.d., 1 mm, Portex Ltd., Hythe, Kent, England) in the right jugular vein. The rats were allowed to recover overnight and had free access to water and food. In the morning following the cannulation, RS extract at a dose of 300 mg/kg was administered to rats by oral gavage. Blood samples were taken from the jugular vein catheter at 0, 15, 60, 120, 180, 240, 360, 480, 600, 720, 840 and 1440 min and were collected in a centrifuge tube containing heparin. Plasma was obtained by centrifugation of the blood at 16,000 × g for 5 min and stored at −80 ◦ C until analysis. The plasma concentration vs. time profile of each analyte was analyzed by WinNonlin (Pharsight Corporation, Mountain View, CA, USA, Version 2.1). Pharmacokinetic parameters including the area under the curve from zero to infinity (AUC0–∞ ) and elimination half-life (t1/2z ) were calculated by non-compartmental model. The peak plasma concentration (Cmax ) and the time reaching Cmax (Tmax ) were obtained directly from the experimental data. Samples with analyte concentrations higher than the liner range of calibration curve were diluted with reconstitute solvent and measured again.

3. Results and discussion 3.1. Optimization of the extraction conditions for brain tissues 3.1.1. Solvents for extraction Combinations of different solvents and different solvent strengths for optimal extraction of the analytes from rat brain were first tested. A total of seven combinations of solvents for the two separate extraction steps were tested: combination A (ACN; ACN), combination B (ACN; 20% ACN (v/v) and 20% MeOH in water), combination C: (ACN; ethyl acetate), combination D (50% ACN (v/v) in MeOH; 50% ACN (v/v) in MeOH), combination E (20% ACN (v/v) in MeOH; 80% ACN (v/v) in MeOH), combination F (MeOH; ACN), and combination G (ethyl acetate; ACN). Among the seven combinations, double extraction using ACN (combination A) achieved the highest recoveries of most analytes, and the recoveries ranged from 50% to 80% (data not shown). Addition of MeOH appeared to marginally increase the recoveries of the glucuronides (BG and OAG) but severely compromised the recoveries of the aglycones (B, W, OA) which experienced a 30–50% reduction in recovery when switching from combination A to combination D or E. Addition of ethyl acetate before or after ACN extraction (combinations C and G) did not show higher recoveries than that of double ACN extraction but severely reduced the recoveries of B, W and OA. Therefore, double extraction using ACN was selected. Before the two ACN extraction steps, the brain tissues were first homogenized in water in a 1:6 ratio to disperse to brain tissue. This step was necessary since we observed that the homogenized brain tissue would be tightly packed if pure ACN was used at the first homogenization step, which reduced the extraction efficiency of ACN and lowered the recoveries. Zhang et al. employed similar extraction solvent combination to extract BG from different rat brain tissues [16,17]. Furthermore, the effects of ascorbic acid and formic acid on the extract recoveries of the analytes were preliminarily determined since flavanoids were found to be more stable in the presence of ascorbic acid and acidic environment in biological samples [21]. It was found that addition of 1% ascorbic acid could greatly increase the extraction recoveries of all analytes (data not shown). Moreover, the recoveries of analytes were further increased by adding formic acid to the water homogenization step but not to the ACN extraction steps. Among the tested concentrations of formic acid (0.01–1%), 0.01% of formic acid resulted in the best recovery (data

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not shown). Therefore, addition of 1% ascorbic acid and 0.01% formic acid was employed aiming to improve the stabilities of the tested analytes possibly through minimizing their oxidation and enzyme degradation during sample preparation [21]. 3.1.2. Incubation and rotation step At the first ACN extraction step, the brain homogenates were rotated for 15 min in rotary mixer to allow time for extraction of the analytes. Trial tests indicated that a rotation step which lasted for less than 30 min could significantly improve the recoveries of B, W, and OA at their high QC concentrations (data not shown). The recoveries of the above aglycones at their low QC concentrations and the glucuronides (BG, WG and OAG) at both the high and low QC concentrations were not affected by the rotation step. However, if the rotation time lasted for 60 min or longer, the recoveries of all the analytes at their low QC concentrations were reduced compared to their corresponding recoveries without the rotation step. Therefore, a rotation step of 15 min was added to the first ACN extraction step. However, adding a rotation step to the second ACN extraction step would not significantly improve the recoveries (data not shown). 3.1.3. SPE-LC/MS/MS conditions The SPE-LC/MS/MS conditions for brain tissue samples were similar to that for plasma samples with certain modifications. 3 ml SPE cartridge instead of 1 ml cartridge was used to accommodate the larger volume of the brain extract. An additional washing step with 21% ACN was added aiming to further eliminate the hydrophobic impurities originated from the brain tissue. ACN at 21% was used since loss of flavone analytes would occur when higher percentage of ACN was applied (data not shown). Similarly, the HPLC gradient was increased to 90% ACN from 10 to 12 min for brain samples to further eliminate the hydrophobic impurities retained in the column. 3.2. Method validation 3.2.1. Specificity The representative selective reaction monitoring chromatograms of blank rat brain, blank rat brain spiked with analytes at their low QC concentrations and a rat brain sample obtained after oral administration of RS extract are shown in Fig. 2. The representative selective reaction monitoring chromatograms of blank rat plasma, blank rat plasma spiked with analytes at their medium QC concentrations and a rat plasma sample obtained after oral administration of RS extract are shown in Fig. 3. No interfering peak was found at the retention times of the six flavones in RS extract, suggesting good selectivity of the assay. From the selective reaction monitoring chromatograms, crosstalk between BG and B was identified (Figs. 2(II) and 3(II)). In the channel of m/z 271.1 → 122.9, there was a peak eluted at the same time as BG (shown in channel of m/z 447.3 → 271.1), which represented the resulted peak from the fragmentation of BG (m/z 447.3 → 271.1 → 122.9). Similarly, the two peaks appeared in the channel of m/z 285.2 → 270.0, which were eluted at the same time as OAG and WG, were the resulted peaks from the fragmentation of OAG and WG, respectively. Since the retention time of these crosstalk peaks were different from that of the main peaks (B, W and OA), their presence did not affect the detection or quantification of our analytes. 3.2.2. Linearity and range The linear ranges of the calibration curves, the LLOQs and the LLODs of the seven analytes in brain and plasma are shown in Table 1. Within these ranges, the calibration curve of each analyte produced good linearity (R2 > 0.99). The current assay is the

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first fully validated method for simultaneous determination of the six bioactive flavones in RS extract in rat brain. Only limited assay methods for the determination of flavones from RS extract in brain were reported. Zhang et al. validated a HPLC/UV method for the determination of BG in brain tissue with LLOQ of 0.11 nmol/g [16] while Liu et al. determined the LLOQs of W and WG in brain tissue using SPE-LC/MS/MS, which were 0.56 nmol/g and 0.35 nmol/g respectively [18]. Comparing to these reported methods, the current LC/MS/MS method not only allows simultaneous determination of the six bioactive flavones in RS extract in brain tissues but also offers much higher sensitivities (LLOQs of the six flavones were 0.02 nmol/g). Comparing to brain assay, more information is available from the literature for simultaneous determination of RS extract flavones in rat plasma. We previously reported a simple HPLC/UV method that could detect the six flavones simultaneously with LLOQs of 100 ng/ml, which were equivalent to 0.35 nmol/ml of B, W and OA and 0.22 nmol/ml of BG, WG and OAG [13]. Kim et al. employed LC/MS/MS for simultaneous detection of B, W, OA and BG in rat plasma with LLOQs of 0.02 nmol/ml for B, W, OA and 0.01 nmol/ml for BG [14]. Chung et al. also developed a LC/MS/MS method for detection of B, W, BG and WG in rat plasma with LLOQs of 0.09 nmol/ml for B, 0.04 nmol/ml for BG, 0.004 nmol/ml for W and 0.01 nmol/ml for WG [15]. Having LLOQs of 0.005 nmol/ml for the aglycones (B, W and OA), the sensitivities of the current LC/MS/MS method were comparable or higher than those achieved by the previous assays. Since the observed amount of glucuronides present in plasma after taking oral RS extract was much higher than their corresponding aglycones, we developed higher calibration ranges for BG (0.1875–12 nmol/ml), WG and OAG (0.025–4.6 nmol/ml). It should be noted that the LLODs of these glucuronides could be as low as 0.005 nmol/ml.

3.2.3. Accuracy and precision The results for intra-day and inter-day accuracy and precision are shown in Table 2. For the brain and plasma assays, the accuracy (within ±15% bias) and precision (RSD less than 15%) at low, medium and high concentrations of all the analytes met the criteria set by the guidance on Bioanalytical Method Validation from FDA (2001).

3.2.4. Extraction recovery As presented in Table 2, the recoveries of the analytes in brain and plasma were consistent across the concentration range studied. It is noticed that the recoveries of the aglycones in brain samples were generally higher than their corresponding glucuronides: B (73–77%) vs. BG (52–66%); W (83–86%) vs. WG (59–60%); and OA (78–82%) vs. OAG (54–65%). Similar observation was found by Liu et al. who employed SPE-LC/MS/MS method to extract W and WG from rat brain tissue, with recovery of W (92–101%) higher than its glucuronide WG (67–74%) [18]. Under the current extraction method, the recovery was 71% for IS. For plasma samples, recoveries of the analytes ranged from 85–112%, which were comparable to that reported by Chung et al. (88–105%) [15] and higher than that reported by Kim et al. (71–88%) and Li et al. (70–98%) [13,14].

3.2.5. Stability The stabilities of the analytes at three levels of QC concentrations under different conditions are listed in Table 3. After three freeze–thaw cycles, the percentages of analytes remaining were 82–104% and 80–107% in brain and plasma respectively. All the analytes were stable in plasma and brain for 24 h in the auto-sampler (4 ◦ C).

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Fig. 2. Representative selective reaction monitoring chromatograms of (I) a blank rat brain; (II) a blank rat brain spiked with analytes at their low QC concentrations (0.05 nmol/g of B, BG, W, WG, OA, OAG) and 1.2 nmol/g IS; and (III) a rat brain sample obtained after oral administration of RS extract (with 1.2 nmol/g IS).

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Fig. 2. (Continued)

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Fig. 2. (Continued).

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17

Fig. 3. Representative selective reaction monitoring chromatograms of (I) a blank rat plasma; (II) a blank rat plasma spiked with analytes at their medium QC concentrations (0.12 nmol/ml B, W, OA, and 0.6 nmol/ml WG and OAG) and 1.8 nmol/ml IS; and (III) a rat plasma sample obtained after oral administration of RS extract (with 1.8 nmol/ml IS).

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Fig. 3. (Continued)

S.Y.K. Fong et al. / Journal of Pharmaceutical and Biomedical Analysis 97 (2014) 9–23

Fig. 3. (Continued).

19

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Table 1 Linearity, LLOD and LLOQ of the analytes in brain and plasma. Analytes

Matrices Brain

Plasma

Linear range (nmol/g) B BG W WG OA OAG

0.02–2 0.02–2 0.02–2 0.02–2 0.02–2 0.02–2

LLOQ (nmol/g)

LLOD (nmol/g)

0.02 0.02 0.02 0.02 0.02 0.02

0.005 0.01 0.003 0.005 0.003 0.005

Linear range (nmol/ml) 0.005–0.32 0.1875–12 0.005–0.32 0.025–4.6 0.005–0.32 0.025–1.6

3.3. Application to pharmacokinetic study in rats 3.3.1. Identification and quantification of RS flavones in rat brain and plasma The concentrations of the six RS bioactive flavones in brain and plasma at 6 h after oral administration of RS extract to rats are presented in Table 4. Calculated from the content of individual flavones present in RS extract (Fig. 1), from a single dose of RS extract (300 mg/kg) the rat received 6 mg/kg of B, 1.2 mg/kg of W, 0.4 mg/kg of OA, 144 mg/kg of BG, 5.2 mg/kg of WG and 12.4 mg/kg of OAG. All the analytes could be identified in rat brain by the current method, though the concentrations of W and WG were below their LLOQs. To the best of our knowledge, this is the first study reporting the presence of RS flavones in rat brain after oral administration of a single dose of RS extract. In contrast, Hou et al. did not identify B, BG, W and WG in rat brain after seven repeated oral doses of RS decoction (each dose provided 77 mg/kg of BG, 19 mg/kg of B, 6.4 mg/kg of W and 8.2 mg/kg of WG) [19]. The discrepancy might be due to the lower sensitivities of their assay method for the determination of B and W in brain tissue, with LLOQs of B and W at 1.1 nmol/ml and 0.7 nmol/ml respectively. As discussed in Section 3.3.2, our assay method provides sensitive measurement of RS flavones in brain tissue as low as 0.02 nmol/g; this allows detection of traces of RS flavones in brain after oral administration of RS extract.

LLOQ (nmol/ml)

LLOD (nmol/ml)

0.005 0.1875 0.005 0.025 0.005 0.025

0.002 0.005 0.008 0.005 0.008 0.005

Although the CNS activities of OA have been reported [10,22], to our knowledge there is no study confirming the presence of OA in brain. The current study provides novel evidence that OA and OAG could pass through the blood–brain barrier (BBB) as 0.09 nmol/g of OA and 0.02 nmol/g of OAG were quantified in rat brain. Having a chemical structure similar to B and W, the possibility of OA being capable to permeate through the BBB is high. Besides revealing the presence of OA and OAG in brain, the current study also identifies the presence of a BG isoform (BG ) in brain. From the selective reaction monitoring chromatograms of a brain sample after oral administration of RS extract (Fig. 2(III)), two peaks were identified under the m/z transition from 447.3 to 271.1. The one at retention time of 3.68 min was confirmed to be BG (baicalein7-O-glucuronide) as it matched with the retention time of the authentic BG standard. The other peak appeared at the retention time of 5.04 min was probably an isoform of BG since it was also detected at this specific MRM transition which indicated the loss of a glucuronic acid moiety. We previously demonstrated the formation of BG in in vitro rat intestinal and hepatic microsomes, and we proposed that it was more likely to be baicalein-6-Oglucuronide than baicalein-5-O-glucuronide since the conjugation to uridine 5 -diphosphoglucuronic acid at the C5 hydroxyl group was impaired by the steric hindrance and intra-molecular hydrogen bond imposed by the adjacent C4 carbonyl group [23–25].

Table 2 Extraction recoveries of the analytes in brain and plasma with intra- and inter-day accuracy and precision of the assays (n = 3–5). Analytes

Matrices Brain

Plasma

Spiked amount (nmol/g)

Extraction recovery (%)a

Accuracy (%)

RSD (%)

B

0.05 0.2 1

73 ± 10 77 ± 15 76 ± 15

101 102 104

6 9 4

BG

0.05 0.2 1

66 ± 12 52 ± 14 62 ± 14

97 97 100

15 6 1

W

0.05 0.2 1

86 ± 10 83 ± 11 84 ± 12

95 101 105

WG

0.05 0.2 1

59 ± 15 60 ± 13 60 ± 13

OA

0.05 0.2 1

OAG

IS a b

Extraction recovery (%)a

Accuracy (%)

RSD (%)

0.015 0.12 0.28

102 ± 9 112 ± 10 108 ± 7

86 94 90

4 3 6

0.5625 4.5 10.5

101 ± 7 100 ± 13 107 ± 13

98 93 95

15 2 10

7 7 3

0.015 0.12 0.28

85 ± 6 102 ± 13 100 ± 13

91 95 90

7 13 7

90 96 100

6 7 8

0.075 0.6 1.4

89 ± 8 97 ± 11 89 ± 9

104 106 102

10 10 8

78 ± 15 82 ± 15 79 ± 15

90 96 100

6 7 8

0.015 0.12 0.28

93 ± 6 91 ± 11 93 ± 10

92 100 92

12 14 6

0.05 0.2 1

54 ± 15 60 ± 9 65 ± 10

99 99 100

13 5 8

0.075 0.6 1.4

88 ± 5 98 ± 12 100 ± 14

95 99 104

1 13 8

1.2

71 ± 10

NAb

NA

1.8

83 ± 6

NA

NA

Data presented as mean ± SD. Not applicable.

Spiked amount (nmol/ml)

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21

Table 3 Stabilities of the analytes under different conditions in plasma and brain (n = 3).a Analytes

Matrices Brain

Plasma

Spiked amount (nmol/g)

Three freeze–thaw cycle (%)

B

0.05 0.2 1

104 ± 8 101 ± 8 95 ± 11

BG

0.05 0.2 1

84 ± 10 90 ± 8 89 ± 7

W

0.05 0.2 1

109 ± 1 94 ± 12 98 ± 6

WG

0.05 0.2 1

OA

Three freeze–thaw cycle (%)

Auto-sampler (4 ◦ C) for 24 h (%)

108 ± 9 115 ± 3 113 ± 2

102 ± 9 102 ± 3 107 ± 8

0.5625 4.5 10.5

86 ± 9 86 ± 7 80 ± 3

92 ± 6 92 ± 3 99 ± 5

97 ± 4 93 ± 9 86 ± 11

0.015 0.12 0.28

98 ± 7 107 ± 7 102 ± 5

85 ± 8 88 ± 7 102 ± 9

102 ± 8 105 ± 3 88 ± 12

97 ± 9 79 ± 4 67 ± 4

0.075 0.6 1.4

98 ± 7 97 ± 5 89 ± 6

95 ± 6 92 ± 5 97 ± 5

0.05 0.2 1

103 ± 15 94 ± 8 102 ± 13

81 ± 4 60 ± 3 56 ± 7

0.015 0.12 0.28

105 ± 9 104 ± 8 98 ± 8

78 ± 8 79 ± 7 91 ± 5

OAG

0.05 0.2 1

104 ± 5 105 ± 7 100 ± 10

102 ± 4 87 ± 4 77 ± 7

0.075 0.6 1.4

96 ± 7 94 ± 4 88 ± 6

95 ± 4 92 ± 2 94 ± 6

IS

1.2

106 ± 10

1.8

107 ± 5

105 ± 4

a

97 ± 15

Auto-sampler (4 ◦ C) for 24 h (%) 98 ± 11 77 ± 3 72 ± 8 101 ± 5 100 ± 2 86 ± 3

Spiked amount (nmol/ml) 0.015 0.12 0.28

Data presented as mean ± SD.

On the other hand, it is not surprising that W and WG were hardly present in the brain since the plasma concentrations of these flavones were minimal: only 0.02 nmol/ml for W and 0.3 nmol/ml for WG. As a single oral RS extract dose contained very little amount of W and WG, it followed that the amount of these flavones present in plasma and brain was also limited. It should however be noted that although the amount of W and WG present in brain could not be accurately quantified, peaks of W and WG were identified from the selective reaction monitoring chromatograms (Fig. 2(III)) which suggested their presence in the brain. Should a higher initial dose of W and WG administered to the rats, the amount of W and WG could be confidently quantified by the current assay method. Recently, Schaffer and Halliwell raised questions on whether polyphenols can actually enter the brain as the residual blood in brain tissue could be a potential cofounder in the measurement of drug concentrations in brain [26]. Indeed, the amount of analytes present in the residual blood of the brain capillaries could not be neglected and corrections are necessary for accurate

quantification of analytes in brain tissue. The correction could be made by estimating the amount of intravascular drug as the product of the brain residual blood volume and the drug concentration in a systematic blood sample and then subtracting this from the total brain sample concentration [27]. Using radiolabelled 3 H-inulin and spectrophotometric method, Friden et al. reported that the total volume of residual blood in rat brain was 12.4 ␮l/g [27]. Employing this value, we then corrected our analytes concentration in brain by the formula of: [brain concentration (nmol/g) − (0.0124 ml/g × plasma concentration (nmol/ml))] and the results are presented in Table 4. After corrections were made, the brain concentrations of the three glucuronides were greatly reduced while the aglycones (including B, W, and OA) were minimally affected. Even with this conservative correction, 0.02 nmol/g of BG and 0.004 nmol/g of OAG were still detected in the brain. This provided supporting evidence that the glucuronides could penetrate through the BBB. In alignment with our result, Zhang et al. demonstrated the presence of BG and its distribution

Table 4 The brain and plasma concentrations of the analytes at 6 h after oral administration of RS extract (300 mg/kg) to rats (n = 6).a A dose of 300 mg/kg RS extract contains 6 mg/kg of B, 1.2 mg/kg W, 0.4 mg/kg OA, 144 mg/kg BG, 5.2 mg/kg WG and 12.4 mg/kg OAG.

Brain concentration (nmol/g)b Corrected brain concentration (nmol/g)c Plasma concentration (nmol/ml)b Brain to plasma ratiod Log P valuee a b c d e f g

B (6 mg/kg)f

BG (144 mg/kg)

W (1.2 mg/kg)

WG (5.2 mg/kg)

OA (0.4 mg/kg)

OAG (12.4 mg/kg)

0.017 ± 0.018 0.016 ± 0.018 0.08 ± 0.01 0.12 ± 0.03 3.6

0.066 ± 0.027 0.024 ± 0.013 4.6 ± 0.7 0.0050 ± 0.002 1.4

0.0059 ± 0.0029g 0.0056 ± 0.0029 0.02 ± 0.01 0.28 ± 0.10 2.2

0.003 ± 0.001f 0±0 0.3 ± 0.1 0 ± 0.002 −0.1

0.086 ± 0.086 0.085 ± 0.085 0.1 ± 0.1 0.53 ± 0.28 3.0

0.016 ± 0.006 0.0041 ± 0.0019 1.0 ± 0.4 0.0037 ± 0.0030 2.3

Data presented as mean ± SD. Brain and plasma concentration of analytes were determined at 6 h after RS extract administration. Calculated by brain concentration − (0.0124 ml/g × plasma concentration). Calculated by corrected brain concentration/plasma concentration. Log P values obtained from ACD/Laboratories Software (ACD/Laboratories, version 11.02; Advanced Chemistry Development Inc., Toronto, Canada). Calculated dose of individual analytes of 300 mg/kg of RS extract. The brain concentrations of W and WG were below LLOQs.

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Fig. 4. Plasma concentration vs. time profile of the analytes of rats after oral administration of RS (300 mg/kg). Profiles of B, W, WG and OA are enlarged. Data represent mean ± SD (n = 4).

Table 5 Plasma pharmacokinetic parameters of RS flavones after oral administration of RS extract (300 mg/kg) to rats (n = 4).a Parametersb

B(6 mg/kg)c

Tmax (h) Cmax (nmol/ml) AUC0→∞ (nmol h/ml) t1/2 (h)

6.0 0.1 1.0 4.2

± ± ± ±

2.0 0.0 0.3 1.9

BG(144 mg/kg) 6.0 6.8 95 6.2

± ± ± ±

2.0 2.7 30 1.7

W(1.2 mg/kg) 4.0 0.03 0.2 2.1

± ± ± ±

1.7 0.02 0.0 0.8

WG(5.2 mg/kg) 6.7 0.5 5.1 5.0

± ± ± ±

1.2 0.2 2.2 2.4

OA(0.4 mg/kg) 6.0 0.06 0.6 3.7

± ± ± ±

2.0 0.03 0.4 2.9

OAG(12.4 mg/kg) 6.7 1.4 9.0 4.8

± ± ± ±

1.2 0.8 3.4 1.9

Data presented as mean ± SD. Tmax : time to reach the maximum plasma concentration; Cmax : maximum plasma concentration; AUC0→∞ : area under the plasma concentration–time curve from 0 h to infinity; t1/2 : elimination half-life. c Calculated dose of individual analytes of 300 mg/kg of RS extract. a

b

in different brain regions after intravenous administration of RS extract equivalent to 90 mg/kg of BG to Wistar rats [16,17]. Moreover, the brain-to-plasma ratios of B, W, and OA (0.12–0.53) were much higher than that of the glucuronides (0–0.005). The result is expected since the lipophilic aglycones have higher log P values (2.2–4.8) than the hydrophilic glucuronides (−0.1–2.3) (Table 4), which would facilitate passive diffusion across the BBB. 3.3.2. Plasma pharmacokinetic study in rats The pharmacokinetic profiles of the flavones in RS extract in rat plasma after oral administration of RS extract are shown in Fig. 4. By applying our sensitive assay method, all the six analytes were successfully quantified across the 24-h time period. The pharmacokinetic parameters of each analyte are summarized in Table 5. The Cmax and AUC0→∞ values of the analytes were proportional to their administered doses and the Cmax of the glucuronides were around 17–70 fold higher than their corresponding aglycones. Furthermore, the Cmax and AUC0→∞ of BG and WG obtained in the current study were comparable to that reported by Chung et al. after dose adjustment [15]. However, the Tmax values were longer in our study, which might have been resulted from the different administration formulation and the effect of food, in which we allowed free access to food while the rats in Chung’s study were fasted before and on the day of blood sampling. Due to extensive first-pass metabolism [28], the amount of aglycones in plasma was relatively low, with AUC0→∞ of B, W and OA at 1, 0.2 and 0.6 nmol h/ml respectively. Nevertheless, our method was sensitive enough to accurately detect and quantify these aglycones, with LLOQs of B, W and OA at 0.005 nmol/ml. In contrast, Li et al. [13] and Hou et al. [19] were unable to detect B, W and OA

in rat plasma after oral administration of 3200 mg/kg of ShuangHuang-Lian Capsule (a commercially available capsule product of RS) and 2000 mg/kg RS decoction respectively, possibly due to the lower sensitivities of their assay methods [13]. With sensitivities of 0.09 nmol/ml for B and 0.004 nmol/ml for W, Chung et al. identified B and W in rat plasma with Cmax of 0.45 nmol/ml and 0.02 nmol/ml respectively after oral administration of a standardized RS extract, which are comparable to our results after dose adjustment [15]. As discussed in Section 3.3.1, a peak of BG at retention time of 4.98 min under the m/z transition from 447.3 to 271.1 was also found in the plasma sample after oral administration of RS extract (Fig. 3(III)). This indicates the presence of an isoform of BG, probably baicalein-6-O-glucuronide, in the plasma. Similarly, from the selective reaction monitoring chromatograms (Fig. 3(III)), an isoform of WG (WG ) was identified at the retention time of 4.25 min under the m/z transition from 461.2 to 285.2. This observation is in agreement with our previous report and we proposed WG to be wogonin-5-O-glucuronide [13]. 4. Conclusions A LC/MS/MS method has been developed and fully validated for the simultaneous determination of the six major bioactive flavones present in RS extract, including B, W, OA, BG, WG and OAG in rat plasma and brain following a SPE procedure. The current method was successfully applied to determine the concentrations of the flavones in rat brain and plasma after oral administration of RS extract, which provided novel evidence of the ability of all the tested flavones as well as an isoform of BG to cross the BBB after

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oral administration of RS extract. Given its sensitivity and reliability, the present method could be applied for preclinical and clinical investigations on the disposition of RS flavones in brain and plasma. Acknowledgements This work was supported by the Research Grants Council, Hong Kong SAR [CUHK480010 and CUHK479813]. We would like to acknowledge two student interns Miss Sovanrotha Tan from the Université Paris Descartes and Miss Qianwen Wang from the Sun Yat-Sen University for their kind involvements in the project.

[12]

[13]

[14]

[15]

Appendix A. Supplementary data [16]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.03.033.

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