Anal. Chem. 2005, 77, 6640-6645
Determination of Boswellic Acids in Brain and Plasma by High-Performance Liquid Chromatography/Tandem Mass Spectrometry Karen Reising,† Juergen Meins,† Baerbel Bastian,† Gunter Eckert,‡ Walter E. Mueller,‡ Manfred Schubert-Zsilavecz,§ and Mona Abdel-Tawab*,†,§
Central Laboratory of German Pharmacists, Carl-Mannich Strasse 20, D-65760 Eschborn, Germany, and Institutes of Pharmacology and Pharmaceutical Chemistry, ZAFES, Biocenter, J.W. Goethe-University, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany
Peritumoral edema, one of the major causes for neurological disorders in brain tumor patients, is mainly treated with steroids, which unfortunately have significant side effects and interfere with the efficacy of chemotherapy. Boswellic acids, the main active ingredients of Boswellia serrata, are antiinflammatory agents, inhibiting 5-lipoxygenase, the key enzyme of leukotriene biosynthesis and one of the pathophysiological mechanisms of peritumoral edema. Based on positive results in clinical trials and animal studies, B. serrata resin dry extract was designated an orphan drug by the European Commission for the treatment of peritumoral edema resulting from brain tumors. Thus boswellic acids may be alternative drugs to corticosteroids. However, the question of the availability of boswellic acids in brain has not been addressed until now. Accordingly, a highly sensitive LC/MS method has been developed for the simultaneous determination of KBA and AKBA, the most potent boswellic acids, in plasma and brain. This method involves matrix-assisted liquid-liquid extraction on Extrelut NT followed by separation on reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using atmospheric pressure chemical ionization. Excellent linearity was obtained for the entire calibration range from 5 to 1500 ng/mL KBA and AKBA in plasma and 5 to 1000 ng/mL KBA and AKBA in brain. Validation assays of the lower limit of quantification as well as for the intraand interday precision and accuracy met the international acceptance criteria for bioanalytical method validation. Moreover, the interchangeability of calibration curves generated in pork and rat brain homogenates could be demonstrated. Using the developed analytical method, KBA and AKBA could be detected for the first time in brain up to a concentration of 99 and 95 ng/g of brain, respectively, 3 h after the single oral administration of 240 mg/kg of dry B. serrata resin extract to Wistar rats. The developed method represents an appropriate tool to * Corresponding author. Tel.: +(49) 6196-937-955. Fax: +(49) 6196-48 23 67. E- mail:
[email protected]. † Central Laboratory of German Pharmacists. ‡ Institute of Pharmacology, J.W. Goethe-University. § Institute of Pharmaceutical Chemistry, J.W. Goethe-University.
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further study the time-dependent distribution of KBA and AKBA in plasma and brain as well as the absolute brain concentration after multiple doses and contributes thus to the optimization of the dosage regimen and to a better understanding of the therapeutic effects of B. serrata. Gliomas constitute the largest group of intracranial neoplasms accounting approximately for two-thirds of all primary tumors in both children and adults. In the United States alone, 20 000 patients are given a diagnosis of glial neoplasm each year.1,2 Glioblastoma is the most malignant form and represents 15-20% of all intracranial tumors and ∼50% of all gliomas.3 In particular, peritumoral edema being a major cause for neurological disability and morbidity represents a common management problem in brain tumor patients. Until now, steroid medications have been essential for the control of cerebral edema. However, prolonged steroid therapy has significant side effects including immunosuppression, Cushing syndrome, and osteoporosis. Moreover, steroids interfere with the efficacy of chemotherapy by reducing tumor perfusion and inhibiting apoptosis in human malignant glioma cells.4,5 Alternative agents for the treatment of cerebral edema in glioma patients are therefore urgently needed. Boswellic acids are the main active ingredients of the gum resin of Boswellia serrata, which has been used in the treatment of inflammatory diseases in the traditional Ayurvedic medicine. They have been attributed antiinflammatory properties due to the inhibition of 5-lipoxygenase, the key enzyme of leukotriene synthesis.6-10 (1) The gliomas; Berger, M. S., Wilson, C. B., Eds.; W. B. Saunders: Philadelphia, 1998. (2) Ohgaki, H.; Kleihues, P. Acta Neuropathol. 2005, 109 (1), 93-108. (3) Winking, M.; Sarikaya, S.; Rahmanian, A.; Joedicke, A.; Boeker, D.-K. J. Neurooncol. 2000, 46, 97-103. (4) Weller, M.; Schmidt, C.; Roth, W.; Dichgans, J. Neurology 1997, 48, 17041705. (5) Naumann, U.; Durka, S.; Weller, M. Oncogene 1998, 17, 1567-1575. (6) Ammon, H. P. T. Eur. J. Med. Res. 1996, 1, 369-370. (7) Gupta, I.; Parihar, A.; Malhotra, P.; Singh, G. B.; Luedtke, R.; Safayhi, H.; Ammon, H. P. T. Eur. J. Med. Res. 1997, 2, 37-43. (8) Safayhi, H.; Mack, T.; Sabieraj, J.; Anazado, M.; Subramannian, L. R.; Ammon, H. P. T. J. Pharmacol. Exp. Ther. 1992, 261, 1143-1146. (9) Safayhi, H.; Sailer, E. R.; Ammon, H. P. T. Mol. Pharmacol. 1995, 47, 12121216. (10) Sailer, E. R.; Subramannian, L. R.; Rall, B.: Hoernlein, R. F.; Ammon, H. P. T.; Safayhi, H. Br. J. Pharmacol. 1996, 117, 615-618. 10.1021/ac0506478 CCC: $30.25
© 2005 American Chemical Society Published on Web 09/21/2005
Moreover the 5-lipoxygenase pathway was identified as one pathophysiological mechanism for edema formation.11,12 In a clinical trial on glioblastoma patients, the B. serrata extract was able to significantly reduce the peritumoral edema in patients receiving the highest dose (3 × 1200 mg) and to improve the clinical conditions.13 Furthermore, it has been reported that the orally administered gum resin extract (240 mg kg-1) inhibits the growth of intracranially inoculated C6 gliomas in rat.3 Finally, boswellic acids exert cytostatic and apoptosis-inducing activity toward malignant cell lines in vitro.14,15 On the basis of these results, B. serrata resin dry extract was designated an orphan drug by the European Commission for the treatment of peritumoral edema resulting from brain tumors. All these data suggest that boswellic acids may be alternative drugs to corticosteroids in the treatment of cerebral edema. Different analytical methods have been developed to determine the plasma levels of boswellic acids. Two methods concentrated on the determination of a single boswellic acid, the 11-keto-βboswellic acid (KBA), in human plasma. The first method based on a solid-phase extraction step followed by high-performance liquid chromatography and UV detection yielded a limit of quantification of 100 ng/mL for KBA.16 A more sensitive GC/MS method allowed the determination of KBA up to a concentration of 10 ng/mL in plasma; however, it was complicated by the use of time-consuming derivatization procedures.17 Finally, a novel method combining serial extraction on diatomaceous earth and graphitized carbon black followed by RP-HPLC and photodiode array detection allowed the determination of 12 different pentacyclic triterpenic acids including KBA and acetyl-11-keto-β-boswellic acid (AKBA) in plasma, but the limit of quantification did not excced 47 ng/mL with regard to KBA and AKBA.18 Despite various experimental investigations and different clinical studies that have been published, the question of the availability of boswellic acids in brain has not been addressed until now. This is, however, the most important aspect in terms of therapeutic approaches of brain tumors. For a successful treatment of peritumoral edema, the boswellic acids must be able to cross the blood-brain barrier, which serves primarily a protective function constraining the diffusion of drugs. Thus, to be able to evaluate the putative pharmacological and therapeutic potential of boswellic acids in the treatment of peritumoral edema, a highly sensitive LC/MS method has been developed and validated for the determination of trace amounts of the most potent boswellic acids, KBA and AKBA (Figure 1). This method can be applied without any modification to both plasma and brain allowing thus an easy correlation between plasma and brain concentrations. (11) Maskowitz, M. A.; Kiwak, K. J.; Hekimian, K.; Levine, L. Science 1984, 224, 886-889. (12) Simmet, T.; Luck, W.; Winking, M.; Delank, W. K.; Peskar, B. A. J. Neurochem. 1990, 54, 2091-2099. (13) Winking, M.; Boeker, D. K.; Simmet, T. Neurooncology 1996, 30, 39. (14) Glaser, T.; Winter, S.; Groscurth, P.; Safayhi, H.; Sailer, E. R.; Ammon, H. P.; Schabet, M.; Weller, M. Br. J. Cancer 1999, 80 (5-6), 756-765. (15) Hostanska, K.; Daum, G.; Saller, R. Anticancer Res. 2002, 22 (5), 28532862. (16) Abdel Tawab, M.; Kaunzinger, A.; Bahr, U.; Karas, M.; Wurglics, M.; Schubert-Zsialvecz, M. J. Chromatogr., B 2001, 761, 221-227. (17) Kaunzinger, A.; Baumeister, A.; Cuda, K.; Haering, N.; Schug, B.; Blume, H. H.; Raddatz, K.; Fischer, G.; Schubert-Zsilavecz, M. J. Pharm. Biomed. Anal. 2002, 28, 729. (18) Buechele, B.; Simmet, T. J. Chromatogr., B 2003, 795, 355-362.
Figure 1. Chemical structures of (a) KBA (R ) H) and AKBA (R ) acetyl) and (b) asiatic acid.
Furthermore a pilot animal study was initiated to test the suitability of the developed analytical method in practice and to prove the availability of KBA and AKBA in brain tissue. EXPERIMENTAL SECTION Chemicals and Reagents. KBA (Lot No. 02575-033) and AKBA (Lot No. 02570-006) was obtained from LGC Promochem (Wesel, Germany). The internal standard asiatic acid was purchased from Extrasynthese (Lyon, France). All solvents used were of analytical grade or better quality. Methanol, 96% acetic acid, n-hexane, 2-propanol, and Extrelut NT were obtained from VWR (VWR, Darmstadt, Germany), tetrahydrofuran was purchased from Acros Organics, ethyl acetate was from Scharlau (Barcelona, Spain), and Tris buffer was from AppliChem (Darmstadt, Germany). Animal Study. Female albino Wistar rats with a body weight ranging between 247 and 274 g were supplied by Charles River laboratories (Wilmington, United Kingdom). Animals were housed under standard conditions with standard chow diet and water freely available. H15 tablets (Lot. 508, Gufic, India) containing 400 mg of dry B. serrata extract were pulverized in a mortar, and extract suspensions were prepared in 0,2% (w/v) aqueous agarose gel. The control group consisting of three rats was given only 0.2% agarose gel. Groups of nine animals were administered the tablet suspension in a similar dose (240 mg/kg) as mentioned in the animal study reported by Winking et al.3 Treatment was given once by oral gavage via a pharyngeal tube with maximal application volume of 1.2 mL. Oral application was chosen, as it is the standard administration route of frankincense. All experiments were carried out according to the guidelines of the German Protection of Animals Act (Deutsches Tierschutzgesetz, BGBI 1998, Part I, No. 30, S.1105 ff.) by individuals with appropriate training and exercise. One, 2, and 3 h after oral administration, three rats respectively were dissected and the brains were isolated. The brain weight ranged between 725 and 1274 mg. Both cerebellum and brain stem were removed and washed with water. After weighing, the whole brain was homogenized in 5 mM TrisHCL buffer, pH 7,4 (1 mL of buffer/100 mg of brain). Finally, homogenates were stored at -20 °C until analysis. Blood samples were withdrawn from the retrobulbar venous plexus of the anesthetized animals. Blood was collected in tubes containing 0.05 mL of heparin to avoid coagulation and centrifuged at 10000g and 4 °C for 10 min to gain the plasma fraction. Sample Preparation. Concentrated stock solutions of KBA and AKBA for standards and quality controls as well as of the internal standard asiatic acid were prepared at a concentration of 1 mg/mL in methanol and stored at -20 °C. A 200 µg/mL working solution of the internal standard as well as different working Analytical Chemistry, Vol. 77, No. 20, October 15, 2005
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solutions containing KBA and AKBA were prepared by diluting the stock solutions with methanol. Calibration standards were prepared daily by spiking 1 mL of blank plasma and brain homogenate, respectively, with 25 µL of asiatic acid working solution and 40 µL of the appropriate KBAAKBA working solutions, resulting in concentrations of 5, 10, 50, 100, 500, 750, 1000, 1200, and 1500 ng of KBA and AKBA/mL of plasma and 5, 10, 50, 100, 500, 750, and 1000 ng of KBA and AKBA/mL of brain homogenate. Three different concentrations of quality control (QC) samples (15, 800, and 1300 ng/mL) were prepared by spiking 1-mL aliquots of blank plasma with 40-µL spiking solutions of KBA and AKBA freshly diluted from the stock solution. For the analysis in brain tissue, three different pools of QC samples (15, 400, and 800 ng/ mL) were also prepared by spiking 1-mL aliquots of blank brain homogenate with 40-µL spiking solutions of KBA and AKBA freshly diluted from the stock solution. The QC samples were stored at -20 °C until the day of extraction. Based on the method described by Buechele and Simmet,18 0.8 g of Extrelut NT was filled into an 8-mL glass column for matrix-assisted liquid-liquid extraction. Following the addition of 5 µg of the internal standard dissolved in 25 µL of methanol to 1 mL of plasma or brain homogenate, respectively, the samples were thoroughly mixed and 1 mL of undiluted plasma or brain homogenate was transferred onto each column. After adsorption for 15 min on the columns, KBA and AKBA were eluted with 8 mL of a solvent mixture consisting of tetrahydrofuran-n-hexaneethyl acetate-2-propanol (160:160:160:15, v/v/v/v). The solvent was than evaporated to dryness in a gentle stream of nitrogen at 40 °C, and the residue was reconstituted in 150 µL of methanol. Instrumental and Chromatographic Conditions. Liquid chromatography was carried out on a Perkin-Elmer instrument (Ju¨gesheim, Germany) using a Hypersil BDS RP C18 column (150 × 4 mm, 5 µm) (MZ-Analysentechnik, Mainz, Germany) for isocratic chromatographic separation of the analytes at a flow rate of 1 mL/min. The optimal composition of the mobile phase was determined to be methanol-water-acetic acid (80:10:04 v/v/v). During the whole analysis, the autosampler was kept at a constant temperature of 10 °C. MS analysis was performed in the positive atmospheric pressure chemical ionization mode on a PE Sciex API 300 (Toronto, Canada) equipped with a triple quadrupole operating in the multiple-reaction monitoring (MRM) detection mode. The ion source was heated to 425 °C. The precursor ion at m/z 471.2 and the fragment ion of highest intensity at m/z 95.1 were selected for the MRM of KBA; similarly, the ions at m/z 513.4 and 91.1 were used for AKBA and the ions at m/z 489.2 and 205.3 were chosen for asiatic acid (Figure 2a). The dwell time for the MRM was 500 ms. MS/MS was performed using nitrogen as collision gas. Data acquisition and integration of the peak areas were achieved using the standard instruments selected ion recording software. Analyte concentration of KBA and AKBA were evaluated using the internal standard method. The standard curves y ) a + bx (a, intercept; b, slope) were calculated from the peak area ratios of the analyte/internal standard and the nominal analyte concentrations using linear regression with 1/x2 weighting. 6642 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005
Figure 2. (a) MS/MS spectra of KBA and AKBA. (b) HPLC-MRM chromatogram of spiked brain homogenate with 5 ng/mL KBA (t ) 2.9), 5 ng/mL AKBA (t ) 4.2), and 5 µg/mL asiatc acid (t ) 1.6).
Method Validation. To prove the suitability of the developed analytical method, validation was performed according to the current guidelines for method validation.19 The specificity of the method was verified by comparing the chromatograms of six blank plasma and brain homogenate samples of different origins before and after spiking with KBA, AKBA, and asiatic acid. Additionally, six plasma and brain homogenate samples from six different sources were measured after spiking them with the internal standard only. Linearity in plasma and brain was checked using five calibration curves including the quality control samples. The correlation coefficients of the calculated regression curves in plasma and brain and the bias of the resulting concentrations from their nominal values (accuracy) were chosen as parameters to verify the linearity of the mass spectrometer. For validation of the lower limit of quantification (LLOQ), five different plasma and brain homogenate samples were spiked with KBA and AKBA at the lowest level of the calibration curve (5 ng/ mL) and recalculated with a freshly prepared standard curve. The intraday accuracy and precision were determined by measuring five replicates of each QC sample (in the lower, middle, (19) Shah. V.; Midha, K. K.; Findley, J. W. A.; Hill, H. M.; Hulse, J. D.; McGilveray, I. J.; McKay, G.; Miller, K. J.; Patnaik, R. N.; Powell, M. L.; Tonelli, A.; Viswanathan, C. T.; Yacobi, A. J. Pharm. Res. 2000, 17, 1551-1557.
and upper ranges) together with one standard curve in one analytical run within 1 day. Interday accuracy and precision were obtained by comparing the calibration curves including the QC samples on three different days. The mean, the standard deviation, the relative standard deviation and bias were calculated. The relative recovery of KBA and AKBA from plasma and brain homogenates was determined at three concentration levels (n ) 5) by comparing the response of the extracted samples spiked with KBA and AKBA before extraction with the response of extracted blank plasma and brain homogenate samples to which the analytes have been added at the same nominal concentration just before injection. The ratio of these two values in percent was used to calculate the relative recovery. Stability tests of KBA and AKBA in plasma and brain homogenates were performed for three concentration levels (low, medium, high) after storage at room temperature for 24 h, after three freeze and thaw cycles, and after storage at -20 °C for 5 months. Furthermore, the stability of both analytes was assessed in processed samples in the autosampler for 24 h. To minimize the total number of dissected rats, development and validation of the analytical method for the determination of KBA and AKBA in brain tissue was carried out in pork brain homogenates instead of rat brain homogenates. The equivalence and reliability of KBA and AKBA concentrations measured in brain of both species was verified by determining the “interspecies accuracy and precision”. For this purpose, three calibration curves generated in rat and pork brain homogenates, respectively, were compared with each other with regard to their slope. Furthermore, four sets of quality control samples prepared in rat brain homogenates were recalculated with a freshly prepared calibration curve in pork brain homogenates. The biases of the resulting concentrations from their nominal values were chosen as parameters to verify the interspecies accuracy and should not exceed 15%. Similarly, development and validation of the analytical method for the determination of KBA and AKBA in plasma was carried out in human instead of rat plasma. Again, the equivalence and reliability of KBA and AKBA concentrations determined in rat and human plasma were verified by recalculating quality control samples prepared in rat plasma with a freshly prepared calibration curve in human plasma. RESULTS AND DISCUSSION Method Validation. Specificity could be confirmed since no significant interfering signals were detected in blank plasma and brain homogenates at the retention times for KBA (t ) 2.9 min) and AKBA (t ) 4.2 min) as well as in the samples spiked only with the internal standard. Good linearity of the assay was found over the investigated calibration range of 5-1500 ng/mL in plasma and 5-1000 ng/ mL in brain homogenate for KBA and AKBA, respectively. The coefficients of correlation (r) of this method were always above 0.9868, resulting in mean values of 0.9959 and 0.9961 for KBA as well as 0.9965 and 0.9956 for AKBA in plasma and brain homogenates, respectively. The relative deviations of the calculated standard concentrations from their nominal values were below 15% and the LLOQ below 20% in all cases, satisfying the general requirements for bioanalytical method validation.19
Table 1. Results for Intra- and Interday Precision and Accuracy in Plasma and Brain Homogenatesa Plasma intraday (n ) 5) nominal concn (ng/mL) 15 800 1300
KBA RSD bias (%) (%) 12.3 4.3 13.5
-5.2 -11.3 6.3
AKBA RSD bias (%) (%) 4.4 5.9 7.3
-11.7 -6.4 5.5
interday (n ) 8) KBA RSD bias (%) (%) 8.8 5.1 2.7
Brain intraday (n ) 5) nominal concn (ng/mL) 15 400 800
KBA RSD bias (%) (%) 5.3 4.5 6.7
-1.1 4.9 -2.0
AKBA RSD bias (%) (%) 7.0 7.2 9.2
-4.7 2.2 -7.7
-4.4 3.5 10.7
AKBA RSD bias (%) (%) 5.1 9.1 6.0
-10.7 -2.7 7.1
interday (n ) 10) KBA RSD bias (%) (%) 11.4 7.7 6.0
-0.7 6.6 3.4
AKBA RSD bias (%) (%) 9.1 11.3 7.6
3.8 -7.9 5.9
a The average recovery using this method yielded a value of 57.0% for KBA and 54.4% for AKBA in plasma and 62.4% for KBA and 57.9% for AKBA in brain homogenate.
Validation of the LLOQ at 5 ng/mL for KBA and AKBA was carried out for five spiked plasma and brain homogenate samples, yielding an average of 5.1 ( 11.5 (bias 1.4%) and 5.6 ( 7.2% (bias 12.4%) for KBA as well as 5.6( 2 (bias 12.6%) and 4.8( 14.3% (bias -3,3%) for AKBA in plasma and brain homogenates, respectively. Consequently, the criteria for accuracy and precision for the lowest quantification level are fulfilled. An illustrative example at this concentration level is shown in Figure 2b. The values for the precision and accuracy of the intra- and interday assays summarized in Table 1 fulfill the international acceptance criteria for bioanalytical method validation. The stability assays showed no significant degradation of KBA and AKBA in plasma and brain homogenate after storage at room temperature for 24 h, at -20 °C for 5 months, after three freeze and thaw cycles, and after storage in processed samples in the autosampler for 24 h. To verify the reliability of KBA and AKBA concentrations measured within the frame of the animal study in rat brain homogenates, three calibration curves generated in rat and pork brain homogenates have been compared with each other. As can be seen in Figure 3a, the calibration curves generated in the brain homogenates of both species are comparable and no significant difference could be detected with regard to the slope of the standard curves. To confirm these results, additional quality control samples (n ) 10) prepared in rat brain homogenates were recalculated with a freshly prepared calibration curve in pork brain homogenate on three different days (Table 2). The bias of the resulting concentrations from their nominal values and the interspecies precision were determined to be
