Anal. Chem. 2003, 75, 6084-6088
Determination of Hyperforin in Mouse Brain by High-Performance Liquid Chromatography/Tandem Mass Spectrometry Jan-Henning Keller,† Michael Karas,† Walter E. Mu 1 ller,‡ Dietrich A. Volmer,*,§ Gunter P. Eckert,‡ † | Mona Abdel Tawab, Henning H. Blume, Theodor Dingermann,⊥ and Manfred Schubert-Zsilavecz*,†
Institute of Pharmaceutical Chemistry, Biocenter, Institute of Pharmacology, Biocenter, and Institute of Pharmaceutical Biology, Biocenter, J.W. Goethe-University, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany, Institute for Marine Biosciences, National Research Council, 1411 Oxford St., Nova Scotia B3H 3Z1, Canada, and Socra Tec R&D GmbH, Feldbergstrasse 59, 61440 Oberursel, Germany
Hyperforin is one of the essential active ingredients of St. John’s wort extract, which is used as an antidepressant for mild to moderately severe depressions. In vitro and in vivo data as well as several clinical studies and meta analyses have confirmed the pharmacological effect of treatment with hyperforin-containing preparations. However, little is known about the brain availability of hyperforin until now. Accordingly, a highly sensitive and selective LC/MS method for this purpose was developed and validated. This method proved suitable for the determination of hyperforin in mouse brain, after oral administration of hyperforin sodium salt and St. John’s wort extract. This method involves liquid-liquid extraction of hyperforin with ethyl acetate followed by separation with rapid reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using electrospray ionization. Excellent linearity was obtained for the entire calibration range from 0.25 to 10 ng/mL (corresponding to 2.5-100 ng/g brain tissue concentration, calculated with the factor derived from sample processing) with an average coefficient of correlation of 0.9992. The recovery of hyperforin from mouse brain homogenates was between 71.4 and 75.3% with a relative standard deviation of less than 3%. Validation assays for the lower limit of quantitation yielded an accuracy of 5.8%. Intraday accuracy and precision for the developed method were between 4.6 and 10.6% and 4.3-8.4%, respectively, while the interday parameters varied between 6.7 and 12.2% for accuracy and 2.0-5.0% for precision. After the method validation, hyperforin brain levels in mice, treated with 15 mg/kg hyperforin (either as the sodium salt or as 5% St. John’s wort extract), were investigated. The average concentration of hyperforin found for the sodium salt group was 28.8(10.1 ng/g of brain (n ) 8), which was somewhat higher than the hyperforin concentration of 15.8(10.9 ng/g of brain (n ) 8), determined in the extract-treated group. This method is robust, selective, and highly sensitive and represents an appropriate tool 6084 Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
to further prove the occurrence and distribution of hyperforin in mouse brain. St. John’s wort (Hypericum perforatum L.) extracts are well established as alternatives to synthetic antidepressants for treating mildly and moderately severe depressions.1-6 Biochemical as well as behavioral pharmacological models confirm the preclinical antidepressant profile of St. John’s wort preparations.1,3,5,6 Furthermore, most, but not all, clinical studies have approved the antidepressant efficacy.2,4,5,7-10 The mechanism of its antidepressant activity, however, has not yet been completely elucidated. While some antidepressant activity could be demonstrated for naphthodianthron derivates such as hypericin, as well as for biflavonoids and procyanidins,11-13 the lipophilic phloroglucinol derivate hyperforin was recently identified as probably the major * Corresponding authors. E-mail:
[email protected]. Tel: +(902) 426-4356. Fax: +(902) 426-9413. E- mail: Schubert-Zsilavecz@ pharmchem.uni-frankfurt.de. Tel: +49-69-798-29339. Fax: +49-69-798-29352. † Institute of Pharmaceutical Chemistry, Biocenter, J.W. Goethe-University. ‡ Institute of Pharmacology, Biocenter, J.W. Goethe-University. § National Research Council. | Socra Tec R&D GmbH. ⊥ Institute of Pharmaceutical Biology, Biocenter, J.W. Goethe-University. (1) Nathan, P. Mol. Psychiatry 1999, 4, 333-338. (2) Kasper, S. Pharmacopsychiatry 2001, 34 (Suppl. 1), S51-55. (3) Greeson, J. M.; Sanford, B.; Monti, D. A. Psychopharmacology (Berlin) 2001, 153, 402-414. (4) Gaster, B.; Holroyd, J. Arch. Intern. Med. 2000, 160, 152-156. (5) Di Carlo, G.; Borrelli, F.; Ernst, E.; Izzo, A. A. Trends Pharmacol. Sci. 2001, 22, 292-297. (6) Muller, W. E. Pharmacol. Res. 2003, 47, 101-109. (7) Lecrubier, Y.; Clerc, G.; Didi, R.; Kieser, M. Am. J. Psychiatry 2002, 159, 1361-1366. (8) Shelton, R. C.; Keller, M. B.; Gelenberg, A.; Dunner, D. L.; Hirschfeld, R.; Thase, M. E.; Russell, J.; Lydiard, R. B.; Crits-Cristoph, P.; Gallop, R.; Todd, L.; Hellerstein, D.; Goodnick, P.; Keitner, G.; Stahl, S. M.; Halbreich, U. JAMA, J. Am. Med. Assoc. 2001, 285, 1978-1986. (9) Philipp, M.; Kohnen, R.; Hiller, K. O. BMJ [Br. Med. J.] 1999, 319, 15341538. (10) Whiskey, E.; Werneke, U.; Taylor, D. Int. Clin. Psychopharmacol. 2001, 16, 239-252. (11) Butterweck, V.; Petereit, F.; Winterhoff, H.; Nahrstedt, A. Planta Med. 1998, 64, 291-294. (12) Nahrstedt, A.; Butterweck, V. Pharmacopsychiatry 1997, 30 (Suppl, 2), 129134. (13) Wonnemann, M.; Singer, A.; Siebert, B.; Muller, W. E. Pharmacopsychiatry 2001, 34 (Suppl, 1), S148-151. 10.1021/ac034520z CCC: $25.00
© 2003 American Chemical Society Published on Web 10/09/2003
antidepressant constituent,13,14 as it shows many effects on different brain neurotransmitter systems that are comparable to the extract.6 In contrast to classical antidepressants, alcoholic St. John’s wort extracts and hyperforin not only inhibit the synaptosomal uptake of serotonin, norepinephrine and dopamine but also show the same effect on the uptake of γ-aminobutyric acid and L-glutamate.15 These effects are probably based on an increase of the intracellular Na+ concentration, due to an altering of sodium conductive pathways.14,16-20 To rationalize these pharmacological data, large efforts have been made to investigate the pharmacokinetic behavior of hyperforin using appropriate analytical methods. The use of an HPLCtandem mass spectrometry method after liquid-liquid extraction with hexane-ethyl acetate (70/30, v/v) allowed the detection of hyperforin down to a lower limit of quantification of 1 ng/mL. With this method, maximum plasma concentrations of 370 ng/ mL in rats could be determined 3 h after oral administration of 300 mg/kg hypericum extract (WS 5572, containing 5% hyperforin).21 Furthermore, several validated HPLC methods were developed for routine analysis of hyperforin with emphasis on robustness and reproducibility.22-25 Modifications of the early method were mainly conducted to improve the sample preparation. Solid-phase extraction was used in order to reduce stability problems of hyperforin primarily observed in organic solutions.26 In addition, liquid-liquid extraction procedures were improved to recover naphthodianthrons as well as phloroglucinols in a single step, thus reducing the time of analysis.27 Recently, the first investigation on the direct relationship between antidepressant activity and bioavailability of hyperforin in rats after peritoneal administration of St. John’s wort extract (4,5% hyperforin) provided a positive correlation between pharmacological activity (immobilization time) and plasma analysis.28 However, even after administration of the highest dose (3 × 6.25 mg/kg), no hyperforin could be detected in the brain. By increasing the dosage to 12.5 mg/kg hyperforin given as the (14) Jensen, A. G.; Hansen, S. H.; Nielsen, E. O. Life Sci. 2001, 68, 1593-1605. (15) Chatterjee, S. S.; Bhattacharya, S. K.; Wonnemann, M.; Singer, A.; Muller, W. E. Life Sci. 1998, 63, 499-510. (16) Muller, W. E.; Singer, A.; Wonnemann, M. Pharmacopsychiatry 2001, 34 Suppl 1, S98-102. (17) Singer, A.; Wonnemann, M.; Muller, W. E. J. Pharmacol. Exp. Ther. 1999, 290, 1363-1368. (18) Wonnemann, M.; Singer, A.; Muller, W. E. Neuropsychopharmacology 2000, 23, 188-197. (19) Gobbi, M.; Moia, M.; Pirona, L.; Morizzoni, P.; Mennini, T. Pharmacopsychiatry 2001, 34 (Suppl. 1), S45-48. (20) Gobbi, M.; Valle, F. D.; Ciapparelli, C.; Diomede, L.; Morazzoni, P.; Verotta, L.; Caccia, S.; Cervo, L.; Mennini, T. Naunyn Schmiedebergs Arch. Pharmacol. 1999, 360, 262-269. (21) Biber, A.; Fischer, H.; Romer, A.; Chatterjee, S. S. Pharmacopsychiatry 1998, 31 (Suppl. 1), 36-43. (22) Mauri, P.; Pietta, P. Rapid Commun. Mass Spectrom. 2000, 14, 95-99. (23) Fuzzati, N.; Gabetta, B.; Strepponi, I.; Villa, F. J. Chromatogr., A 2001, 926, 187-198. (24) Tolonen, A.; Uusitalo, J.; Hohtola, A.; Jalonen, J. Rapid Commun. Mass Spectrom. 2002, 16, 396-402. (25) de los Reyes, G. C.; Koda, R. T. J. Pharm. Biomed. Anal. 2001, 26, 959965. (26) Bauer, S.; Stormer, E.; Graubaum, H. J.; Roots, I. J. Chromatogr., B: Biomed. Sci. Appl. 2001, 765, 29-35. (27) Pirker, R.; Huck, C. W.; Bonn, G. K. J. Chromatogr., B: Analyt Technol. Biomed. Life Sci. 2002, 777, 147-153. (28) Cervo, L.; Rozio, M.; Ekalle-Soppo, C. B.; Guiso, G.; Morazzoni, P.; Caccia, S. Psychopharmacology (Berlin) 2002, 164, 423-428.
dicyclohexylammonium salt, hyperforin could be detected for the first time in the brain. Unfortunately, the mean whole rat brain concentration of ∼30 ng/g hyperforin was at or below the quantitation limit of the solid-phase extraction and LC/UV method and so did not allow a reliable quantification in that study.28 Simultaneously, the present study was initiated to extend the preliminary pharmacological data obtained in our laboratories and to prove the occurrence of hyperforin in mouse brain after a single oral administration of hypericum extract and hyperforin sodium salt. For this purpose, a more sensitive and selective method was developed and validated, allowing the determination of hyperforin down to a concentration level of 250 pg/mL (2.5 ng/g of brain respectively). EXPERIMENTAL SECTION Extracts and Materials. St. John’s wort extracts, containing 5% hyperforin (WS 5572), and isolated hyperforin sodium salt, including traces of adhyperforin, were kindly provided by Dr. Wilmar Schwabe Arzneimittel, Karlsruhe. HPLC-grade methanol and acetonitrile were purchased from Caledon (Georgetown, ON, Canada). Ethyl acetate, Tris buffer, ascorbic acid, formic acid, and ammonium formate were obtained from Sigma-Aldrich (Mississauga, ON, Canada). Water was purified by a Milli-Q system (Millipore, Bedford, MA). Animal Study. Female NMRI mice with body weights ranging between 15 and 20 g were supplied by Harlan-Winkelmann GmbH (Borchen, Germany). Animals were housed under standard conditions with standard chow diet and water freely available. Hyperforin and extract suspensions were prepared in 0.2% (w/v) aqueous agarose gel. The suspension was homogenized and tempered in a water bath of 37 °C prior to treatment. Control treatment was prepared by simply tempering 0.2% (w/v) agarose gel (vehicle). Groups of eight animals were administered the extract (300 mg/kg), hyperforin (15 mg/kg), respectively, which corresponds to the average dose used in behavioral experiments.11,12,15,28 Treatment was given once by oral gavage via a pharyngeal tube (diameter, 1 mm) with maximal application volume of 0.5 mL. Oral application was chosen, as it is the standard administration of hyperforin as well as of the extract. 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 experience. Three hours after oral administration, the mice were dissected and the brains were isolated. Both cerebellum and brain stem were removed and carefully washed in ice-cold Tris buffer. After weighing, the whole brain was homogenized in 5 mM Tris-HCl buffer, pH 7.4 (1 mL of buffer/ 100 mg of brain). Finally, homogenates were stored at -20 °C until analysis. Sample Preparation. Concentrated stock solutions of hyperforin sodium salt and St. John’s wort extract were prepared for standards and quality controls at a concentration of 100 ng/mL in 70% methanol. Different spike solutions were prepared by diluting the stock solution with 70% methanol. Calibration curves were obtained by spiking brain homogenates with methanolic solutions of hyperforin to achieve the following standard concentrations: 0.25, 0.5, 0.75, 2.5, 5, 7.5, and 10 ng/mL. Brain homogenates were thawed and vortexed at 2500 rpm for 30 s. For the preparation of calibration solutions, 50 µL Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
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Figure 1. Chemical structures of the phloroglucinol derivates (a) hyperforin and (b) adhyperforin.
of the particular spike solution were added to 450 µL of blank brain homogenate. In the animal study, 500 µL of brain homogenate was used. Following the addition of 50 µL of ascorbic acid (200 mg/mL) and vortexing (1 min), hyperforin was extracted with ethyl acetate (750 µL) and vortexed again for 2 min. The tubes were centrifuged for 5 min at 13 400 rpm, and then an aliquot of the organic layer was removed and evaporated to dryness under a stream of nitrogen at 30 °C. The residue was redissolved in 200 µL of a mixture of 70% methanol and 30% water. After vortexing for 1 min and treatment in an ultrasonic bath for another minute, the insoluble residue was separated by centrifugation for 1 min. Finally, 20 µL was injected into the LC/MS system. Liquid Chromatography. Liquid chromatography was carried out on an Agilent 1100 instrument (Palo Alto, CA) using a monolithic reversed-phase column (Chromolith PerformanceRod, 100 mm, i.d. 4.6 mm. Merck, Darmstadt, Germany) at a flow rate of 3 mL/min. Separation of the analytes was achieved using a mixture of acetonitrile/water (88:12) containing 3.5 mM formic acid and 2 mM ammonium formate. Solvents were degassed by the on-line degasser of the Agilent 1100 system. Electrospray Mass Spectrometry. Electrospray data were acquired using an MDS-Sciex (Concord, ON, Canada) API 4000 triple-quadrupole mass spectrometer in the negative ion mode with a spray voltage of -4.5 kV and a declustering potential of -60 V. The orthogonal Turbo-V source’s injectors were heated to 700 °C to allow connection to the HPLC without mobile-phase splitting. The curtain gas (30 psig), nebulizer gas (70 psig), and turbo gas (50 psig) were slightly increased above normal values as well. The dwell time for the multiple reaction monitoring (MRM) was 200 ms. MS/MS was performed using nitrogen as collision gas (CAD gas setting 6) at a collision offset voltage of -41 V. Both, Q1 and Q3 were operated at unit resolution.
The precursor ion at m/z 535 and the fragment ion at m/z 383 of hyperforin were selected for the multiple reaction monitoring; similarly, the ions at m/z 549 and 397 were used for adhyperforin. Quantification of hyperforin was based on external standardization using the peak areas. Standard calibration curves were constructed by plotting the corresponding peak area against seven standard concentrations of hyperforin. Method Validation. To prove the suitability of the developed analytical method, validation was performed according to the current guidelines for method validation.29 The specificity of the method was verified by comparing the chromatograms of six blank brain samples of different origin before and after spiking with hyperforin sodium salt. Linearity was checked using five calibration curves. The correlation coefficients of the calculated regression curves and the bias of the resulting concentrations from their nominal values (accuracy) were chosen as parameters to verify the linearity of the mass spectrometer. The lowest concentration of analyte to be measured with acceptable accuracy and precision (e20%) was defined as the lower limit of quantitation (LLOQ). Intraday accuracy and precision were determined by measuring three replicates of each QC sample (in the lower, middle, and upper range) within 1 day. Interday accuracy and precision were obtained by comparing the calibration curves including the QC samples on 3 different days. The mean, the standard deviation, the relative standard deviation, and bias were calculated. The relative recovery of hyperforin from mouse brain homogenates was determined at three concentration levels (n ) 5) by comparing the response of the extracted samples spiked with hyperforin before extraction with the response of extracted blank brain homogenate samples to which analyte has 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 hyperforin in brain homogenate were performed for three concentration levels (low, medium, high) after storage at room temperature and after three freeze-thaw cycles. Furthermore, the stability of hyperforin at room temperature in the final solution after processing was assessed after 24 and 48 h. The stability was estimated by comparison of the obtained value with the nominal value.
Figure 2. Negative ion electrospray LC/MS/MS product ion spectra for (a) hyperforin and (b) adhyperforin. 6086
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Figure 3. Proposed MRM dissociation reactions used for quantification of (a) hyperforin and detection of (b) adhyperforin. Table 1. Results for Intra- and Interday Precision and Accuracy in Mouse Brain Homogenate intraday (n ) 6)
interday (n )6)
nominal conn (ng/mL)
RSD (%)
bias (%)
RSD (%
bias (%)
0.4 4 8
4.6 10.6 7.8
8.4 4.3 4.7
8.8 12.2 6.7
4.7 2.0 5.0
Table 2. Recovery of Hyperforin (n ) 5 at Each Concentration Level) Figure 4. HPLC-MRM chromatogram of spiked brain homogenate with 250 pg/mL hyperforin (t ) 1.26 min) and ∼25 pg/mL adhyperforin (t ) 1.46 min).
RESULTS AND DISCUSSION LC/MS Method. Tandem mass spectrometry analysis of hyperforin and adhyperforin (Figure 1) was carried out to select specific pairs of precursor and product ions for the use of multiple reaction monitoring. The product ion spectra from the collisioninduced dissociation of the [M - H]- ions of hyperforin (m/z 535) and adhyperforin (m/z 549) are shown in Figure 2. The main product ion at m/z 383 was used for quantification of hyperforin. Figure 3 illustrates the proposed dissociation reaction outlining the loss of -152 amu for both precursor ions. The chromatographic conditions were optimized for analysis speed, with special (29) Shah, V. P.; 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, 15511557.
original concn (ng/mL)
recovery (%)
0.4 4 8
75.3 71.4 73.8
mean SD RSD (%)
73.5 2 2.7
emphasis on the separation of the two phloroglucinol derivates as adhyperforin was present in the hyperforin standard and occurs naturally in the extract as well. Therefore, adhyperforin is primarily used in this study as proof for the good chromatographic separation and accordingly for exclusion of interactions in the detection of hyperforin Method Validation. In blank brain homogenates, no interfering signals were detected at the retention times for hyperforin t ) 1.26 min and adhyperforin t ) 1.46 min, supporting the Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
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Table 3. Comparison of Initial Values and Results of the Different Groups in the Animal Study animal group
average body wt (g)
average brain wt (mg)
hyperforin source for oral admin (15 mg/kg)
determined brain concn of hyperforin (ng/g brain)
hyperforin extract
18.60 18.31
300 318
hyperforin sodium St. John’s wort extract (WS 5572, 5% hyperforin)
28.8 ( 10.1 (n ) 8) 15.8 ( 10.9 (n ) 8)
control
18.51
294
specificity of this method and the high selectivity of the developed MRM procedure. Good linearity of the assay was found over the investigated calibration range of 0.25-10 ng/mL, based on five calibration curves. The coefficients of correlation (r2) of this method were always above 0.998, resulting in a mean value of 0.9992. The relative deviations of the LLOQ and the other calculated standard concentrations from their nominal values were below 15.5 and 13.9%, thus satisfying the general requirements for bioanalytical method validation.29 Validation of the LLOQ at 250 pg/mL was carried out for five spiked brain homogenate samples, yielding an average of 235 pg/ mL ( 7.8% with a bias of 5.8%. Accordingly, the criteria for accuracy and precision for the lowest quantification point are fulfilled. An illustrative example at this concentration level is shown in Figure 4. The values for the precision and accuracy of the intra- and interday assays are summarized in Table 1. The intraday precision values of the method ranged from 4.6 to 10.6%; the corresponding accuracy varied within the range of 4.3 to 8.4%. Also, the values for the interday precision and accuracy were between 6.7 and 12.2% and 2.0-5.0%, respectively. Thus, the international acceptance criteria (coefficient of variation and bias better than 15% for precision and accuracy) are clearly met. The average recovery using this method yielded a value of 73.5% (Table 2). The precision of the recovery at each concentration level was 2.7%. Due to the constant recovery over the whole concentration range, it was not necessary to improve the extraction procedure described above. The stability assays showed no significant degradation of hyperforin in mouse brain homogenate (after 4 h at room temperature) or in the final processed solution (48 h). Also, three freeze-thaw cycles did not significantly affect the stability of hyperforin. Animal Study. As the method provided high selectivity and sensitivity as well as good accuracy, precision, and robustness during the validation procedure, the major challenge to determine the occurrence of hyperforin in the brain after oral administration could be approached. The mice fed in this study had an average total weight of 18.5 g and their brains weighed between 272 and 335 mg (Table 3). In the hyperforin group, an average concentration of 2.6 ng/ mL hyperforin, which corresponds to a mean content of 28.8(10.1 ng/g of brain (n ) 8), was determined. In the extract group, the concentration of hyperforin was 1.6 ng/mL, which is equivalent
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to 15.8(10.9 ng/g of brain (n ) 8), significantly lower than in the hyperforin group. No hyperforin could be determined in the control group. These data confirm our preliminary finding and clearly demonstrate substantial levels of hyperforin in mouse brain even after one single oral dosage. Based on these results, further investigations with regard to the time-dependent distribution of hyperforin in plasma and brain as well as the absolute brain concentration after multiple doses will help to complete the knowledge about the functional role of hyperforin in the St. John’s wort extract, especially as the brain availability of hypericin appears to be low.30 CONCLUSIONS Hyperforin represents at least one of the most relevant active constituents of St. John’s wort. So far, the only pharmacokinetic parameters of hyperforin published are related to the determination in plasma. The present work shows, for the first time, a validated, highly sensitive, and selective method for the determination of very low concentrations of hyperforin in mouse brain ranging from 250 pg/mL to 10 ng/mL. Precision and accuracy as major control parameters of the whole validation procedure were within acceptable limits. Furthermore, the suitability of the method for the determination of hyperforin in mouse brain after single oral administration of the isolated sodium salt and the whole extract could be clearly demonstrated. The experimental data make a relevant contribution to the understanding of the pharmacological effects of St. John’s wort extract and its active constituents. ACKNOWLEDGMENT The authors thank Dr. Willmar Schwabe Arzneimittel for the provision of hypericum extracts and standards. J.-H.K. thanks Pearl Blay at the National Research Council’s Institute for Marine Biosciences for her assistance during the sample preparation. The excellent technical assistance by Andrea Wilke, Stefanie SchulteLo¨bbert, Christopher Kirsch, und Kristina Treiber is gratefully acknowledged. This work was supported by a grant from the German Academic Exchange Service (DAAD). Received for review May 16, 2003. Accepted September 5, 2003. AC034520Z (30) Fox, E.; Murphy, R. F.; McCully, C. L.; Adamson, P. C. Cancer Chemother. Pharmacol. 2001, 47, 41-44.
