Simultaneous determination of hypericin and hyperforin in human plasma with liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the simultaneous determination of hypericin and hyperforin--the two main active ingredients of St. John's Wort (SJW) extract--in human plasma depending on liquid/liquid-extraction and LC/MS/MS detection has been developed, validated after specifying the stability of the photosensitive hypericin in plasma samples during light exposure and applied to samples of a patient. After extraction with ethyl acetate/n-hexane in the darkness, sample extracts were chromatographed isocratically within 6 min on a Kromasil RP-18 column. The analytes were detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source. The limit of quantification was 0.05 ng/mL for hypericin and 0.035 ng/mL for hyperforin. The accuracy of the method varied between 101.9 and 114.2% and the precision ranged from 4.7 to 15.4% (S.D., batch-to-batch) for both analytes. The method was linear at least between 0.05 and 10 ng/mL for hypericin and between 0.035 and 100 ng/mL for hyperforin. Using this method hypericin and hyperforin were determined successfully in a patient over seven days following discontinuation of exposure with therapeutic doses of St. John's Wort extract.     

Fast high-performance liquid chromatographic analysis of naphthodianthrones and phloroglucinols from Hypericum perforatum extracts

Hypericum perforatum L. (St. John's Wort) has been used in modern medicine for treatments of depression and neuralgic disorders. An HPLC method with photodiode array detection for the rapid determination of the major active compounds, naphthodianthrones and phloroglucinols, has been developed. The method permits the determination of hypericin, protohypericin, pseudohypericin, protopseudohypericin, hyperforin and adhyperforin in an extract in less than 5 min. Good linearity over the range 0.5-200 microg/mL for hyperforin and 0.02-100 microg/mL for hypericin was observed. Intra-assay accuracy and precision varied from 0.1 to 17% within these ranges. Lower levels of quantitative determination were 2 microg/mL for hyperforin and 0.5 microg/mL for hypericin, while detection limits were 0.1 and 0.02 microg/mL, respectively.     

Simultaneous determination of total hypericin and hyperforin in St. John's wort extracts by HPLC with electrochemical detection

An analytical procedure was developed for the simultaneous determination of total hypericin (protopseudohypericin, pseudohypericin, protohypericin and hypericin) and hyperforin in Hypericum perforatum (St. John's wort) extracts and its preparations. The determination of total hypericin and hyperforin in one step was achieved by exposing the samples to artificial daylight in amber glass vials. This procedure allows both the photoconversion of the protoforms into the appropriate hypericins and the protection of the photosensitive hyperforin. For quantification, an HPLC method with electrochemical detection was applied. As an example of the application of the principle, two preparations containing St. John's wort were assayed.     

In vitro photochemical and phototoxicological characterization of major constituents in St. John's Wort (Hypericum perforatum) extracts

Extracts from St. John's Wort (SJW: Hypericum perforatum) have been used for the treatment of mild-to-moderate depression. In spite of the high therapeutic potential, orally administered SJW sometimes causes phototoxic skin responses. As such, the present study aimed to clarify the phototoxic mechanisms and to identify the major phototoxins of SJW extract. Photobiochemical properties of SJW extract and 19 known constituents were characterized with focus on generation of reactive oxygen species (ROS), lipid peroxidation, and DNA photocleavage, which are indicative of photosensitive, photoirritant, and photogenotoxic potentials, respectively. ROS assay revealed the photoreactivity of SJW extract and some SJW ingredients as evidenced by type I and/or II photochemical reactions under light exposure. Not all the ROS-generating constituents caused photosensitized peroxidation of linoleic acid and photodynamic cleavage of plasmid DNA, and only hypericin, pseudohypericin, and hyperforin exhibited in vitro photoirritant potential. Concomitant UV exposure of quercitrin, an SJW component with potent UV/Vis absorption, with hyperforin resulted in significant attenuation of photodynamic generation of singlet oxygen from hyperforin, but not with hypericin. In conclusion, our results suggested that hypericin, pseudohypericin, and hyperforin might be responsible for the in vitro phototoxic effects of SJW extract.     

Oral bioavailability of active principles from herbal products in humans. A study on Hypericum perforatum extracts using the soft gelatin capsule technology.

The oral absorption of two known active principles of Hypericum perforatum, namely hyperforin and hypericin, was studied in an open, single dose, two-way, randomized, cross-over study involving 12 healthy subjects (six males and six females). Alcoholic Hypericum extract (300 mg, containing 5% hyperforin and 0.3 % hypericin) was administered in the morning after 12 hours fasting. The formulation was administered as softgel capsules containing, inter alia, soya oil together with the herbal extract. A second standard formulation in two piece hard gelatin capsules was also used for comparison purposes. Blood was sampled from the subjects at different times after drug administration and the plasma was analysed according to published analytical methods for the determination of hyperforin and hypericin. Peaks of plasma concentration, Cmax of hyperforin were 168.35 ng/ml +/- 57.79 for the soft gelatin formulation (CV=34.32, n=12) and 84.25 ng/ml +/- 33.51 for the hard gelatin capsule (CV=39.77, n=12). The Tmax values for hyperforin were 2.50 h +/- 0.83 for the soft gelatin formulation compared to 3.08 h +/- 0.79 for the reference formulation, whereas the total AUC were respectively 1482.7 h x ng/ml +/- 897.13 and 583.65 h x ng/ml +/- 240.29. As for hypericin, plasma levels were detectable in approximately half of the subjects treated. However also in this case the soft gelatin capsules exhibited a higher individual absorption when compared with the corresponding data for the hard gelatin capsules.     

Step by step removal of hyperforin and hypericin: activity profile of different Hypericum preparations in behavioral models

Herbal extracts of Hypericum perforatum L. (St. John's wort, SJW) are now successfully competing for status as a standard antidepressant therapy. Because of this, great effort has been devoted to identifying the antidepressive active compounds. In the present study we used the following strategy to evaluate the relative pharmacological importance of various extract components: 1. preparation of an hydroalcoholic SJW extract containing both hyperforin (3.2%) and hypericin (0.15%) (extract A); 2. step by step removal of hyperforin and hypericin led to the following extracts: Extract B, devoid of hyperforin but still containing hypericin (0.14%) and Extract C, free of hypericin and hyperforin but enriched in flavonoids ( approximately 12%). We characterized the in vivo activity profile of all three preparations using the tail suspension test (TST) in mice and the forced swimming test (FST) in rats as screening models. We further investigated the activity of pure hyperforin. Extract B and C (500 mg/kg each) as well as pure hyperforin (8 mg/kg) significantly shortened immobility time in the TST after acute pre-treatment whereas extract A was inactive. In the FST all three extracts decreased immobility time in a dosage of 500 mg/kg after acute as well as after repeated treatment. The present results clearly show that an SJW extract free of hyperforin and hypericin exerts antidepressant activity in behavioral models, supporting our working hypothesis that flavonoids are part of the constituents responsible for the therapeutic efficacy of SJW extracts. We also could show that hyperforin contributes to the beneficial properties of SJW extract, confirming the hypothesis that the crude SJW extract contains several constituents with antidepressant activity.     

Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.     

Effect of eluent pH on the HPLC-UV analysis of hyperforin from St. John's Wort (Hypericum perforatum L.)

The effect of the pH of the mobile phase in HPLC analysis of hyperforin was investigated. Working with an extract of St. John's Wort (Hypericum perforatum L.) that is rich in hyperforin, significant differences were observed in conventional chromatograms depending on whether the mobile phase was acidic or alkaline. Chromatogram changes were paralleled by changes in the UV spectrum of the hyperforin peak. The structural changes in hyperforin occur in the chromatographic column itself, as has been confirmed by UV spectroscopy performed on a sample of purified hyperforin, which showed that the UV spectrum is indeed dependent on the pH of its environment.     

Production of St. John's wort plants under controlled environment for maximizing biomass and secondary metabolites

Summary St. John's wort (Hypericum perforatum L.) is a medicinal plant used in the treatment of neurological disorders and has been recently shown to have anticancer potential. The principle medicinal components of St. John's wort are hypericin. pseudohypericin, and hyperforin. One of the problems associated with medicinal plant preparations including St. John's wort is the extreme variability in the phytochemical content, mostly due to environmental variability, and biotic and abiotic contamination during cropping. The current study was undertaken to transplant St. John's wort plants from in vitro bioreactors in a closed controlled environment system (CCES) with CO₂ enrichment for the optimized production of biomas and phytochemicals. The growth and levels of hypericin, pseudohypericin, and hyperforin in plants grown in CCES were compared with those of the greenhouse and in vitro-grown plants. The environmental parameters in the greenhouse were found to be variable whereas in the CCES these parameters were controlled. Generally, all the growth parameters and hypericin and psendohypericin levels were significantly higher in the plants grown in the CCES. These results provide the first indication that growing St. John's wort plants, under CO₂ enrichment in a closed environment system can enhance the biomass and medicinal contents. The adaptation of this growing system may be useful for the production of optimized products of St. John's wort and other medicinal species.     

Quantitation of psilocin in human plasma by high-performance liquid chromatography and electrochemical detection: comparison of liquid–liquid extraction with automated on-line solid-phase extraction

Two modifications of the HPLC–ED method with respect to extraction procedure used have been developed for psilocin, the active metabolite of psilocybin, in human plasma using either liquid–liquid extraction (LLE) or automated on-line solid-phase extraction (on-line SPE). Each type of the sample preparation required a different HPLC system followed by electrochemical detection at 650 to 675 mV. The limit of quantitation of both modifications was 10 ng/ml psilocin. There was no significant difference observable between the LLE and the on-line SPE in terms of method standard deviation (LLE 1.82%, on-line SPE 1.13%) and the analytical results. However, the advantages of on-line SPE in addition to different selectivity were less manual effort, smaller plasma volumes of 400 μl (LLE 2 ml) and a recovery of psilocin in human plasma of nearly 100% (LLE 88%). In contrast to a previous procedure both methods were rapid, simple and reliable and yielded high plasma recoveries. They were used successfully in the quantitation of psilocin in plasma samples obtained from healthy volunteers after p.o. administration of 0.2 mg psilocybin per kg body mass. Plasma concentration curves and pharmacokinetic parameters were calculated.     

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: a beneficial "side effect" of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic-phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.     

Rapid and simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical herb-drug interaction study

Nifedipine (NIF), a calcium channel antagonist, is metabolized primarily by cytochrome P450 (CYP3A4) to dehydronifedipine (DNIF). As such, NIF is often used as a probe drug for determining CYP3A4 activity in human studies. A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine NIF and DNIF in human plasma using nitrendipine as the internal standard (IS). After extraction of the plasma samples by ether-n-hexane (3:1, v/v), NIF, DNIF and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C(18) column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of approximately 2.5 min and linear calibration curves over the concentrations of 0.5-100 ng/mL for NIF and DNIF. The recoveries of the one-step liquid extraction method were 81.3-89.1% for NIF and 71.6-80.4% for DNIF. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for both analytes. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL. The validated LC/MS/MS method has been successfully used to study pharmacokinetic interactions of NIF with the herbal antidepressant St. John's wort in healthy volunteers. These results indicated that the developed LC/MS/MS method was efficient with a significantly shorter running time (2.5 min) for NIF and DNIF compared to those methods previously reported in the literature. The presented LC/MS/MS method had acceptable accuracy, precision and sensitivity and was used in a clinical pharmacokinetic interaction study of NIF with St. John's wort, a known herbal inducer of CYP3A4. St. John's wort was shown to induce NIF metabolism with increased plasma concentrations of DNIF.     

A high-performance liquid chromatography with electrochemical detection for the determination of total hypericin in extracts of St. John's Wort

An HPLC method for the quantitation of hypericin using a new and sensitive amperometric detection is presented. Hypericin was eluted isocratically using a mobile phase consisting of ammonium acetate, methanol and acetonitrile. The oxidation was carried out with a glassy carbon electrode at a potential of + 1.1 V vs. an Ag-AgCl-KCl reference electrode. Under the conditions described, hypericin was separated at a retention time (Rt) of 12 min. Linearity was obtained over the range 0.035-1.30 microg/mL (r = 0.9994). The limit of detection was determined to be 0.010 ng on-column for hypericin. The method was applied to the determination of total hypericin (hypericin, pseudohypericin, protohypericin and protopseudohypericin) in extracts of St. John's wort using hypericin as an external standard. The protoforms were converted into hypericin and pseudohypericin by subjecting the sample to artificial light prior to chromatographic analysis. For the evaluation of total hypericin, the peak areas of pseudohypericin (Rt 3.7 min) and hypericin (Rt 12.0 min) were combined. The relative standard deviation in analysing samples containing Hypericum ranged from 2.5 to 5.4%.     

Quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry

The method describes quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS/MS). Samples were screened by enzyme-linked immunosorbent assay (ELISA) and only those samples presumptively declared positive were subjected to quantification and confirmation for the presence of flunixin by this method. The method is also readily adaptable to instrumental screening for the analyte. Flunixin was recovered from plasma by liquid-liquid extraction (LLE). The sample was diluted with 2 ml saturated phosphate buffer (pH 3.10) prior to LLE. The dried extract was reconstituted in acetonitrile:water:formic acid (50:50:0.1, v/v/v) and subsequently analyzed on a Q-TOF tandem mass spectrometer (Micromass) operated under electrospray ionization positive ion mode. The concentration of flunixin was determined by the internal standard (IS) calibration method using the peak area ratio with clonixin as the IS. The limits of detection (LOD) and quantification (LOQ) for flunixin in equine plasma were 0.1 and 1 ng/ml, respectively, whereas the limit of confirmation (LOC) was 2.5 ng/ml. The qualifying ions for the identification of flunixin were m/z 297 [M+H](+), 279 (BP), 264, 259, 239 and those for clonixin (IS) were m/z 263 [M+H](+), 245 (BP) and 210. The measurement uncertainty about the result was 8.7%. The method is simple, sensitive, robust and reliably fast in the quantification and confirmation of flunixin in equine plasma. Application of this method will assist racing authorities in the enforcement of tolerance plasma concentration of flunixin in the racehorse on race day.     

Measurement of hyperforin a constituent of St. John’s wort in plasma by high-performance liquid chromatography

Hyperforin is a constituent of Hypericum perforatum extracts (St. John’s wort, H. perforatum), which have antidepressant action. Hyperforin was extracted from plasma utilising a solid-phase extraction procedure. Chromatography was performed by isocratic reversed-phase high-performance liquid chromatography with UV end-point detection. The calibration curve was linear over the range 0.15–3 μg per ml of plasma. The sensitivity for hyperforin was 4.5 ng on-column. Mean inter- and intra-assay relative standard deviations over the range of the standard curve were less than 5%. The absolute recovery for hyperforin averaged 97.8%.     

Seasonal variation in hypericin content of Hypericum perforatum L. (St. John's Wort)

Hypericin and pseudohypericin, bioactive constituents in St. John's Wort (Hypericum perforatum), have been determined in the soft tops of the plant that are most likely to be browsed by foraging livestock. In two consecutive seasons, the hypericin/pseudohypericin concentration in a broad leaf biotype varied from a winter minimum of less than 100 ppm to a summer maximum approaching 3000 ppm. In contrast the narrow leaf biotype increased from similar winter values to summer maxima approaching 5000 ppm. The latter biotype was slower in returning to low levels of hypericin/pseudohypericin.     

Short term treatment with St. John's wort, hypericin or hyperforin fails to induce CYP450 isoforms in the Swiss Webster mouse

This investigation was designed to determine whether St. John's wort (SJW)(435 mg/kg/d), a readily available antidepressant, or its purported active constituents hypericin (1 mg/kg/d) and hyperforin (10 mg/kg/d) were able to induce various hepatic cytochrome P450 (CYP450) isoforms. SJW, hypericin and hyperforin were administered to male Swiss Webster mice for four consecutive days and hepatic microsomes were prepared on day 5. None of the three treatments resulted in a statistical change in total hepatic CYP450 (SJW treated 0.95 +/- 0.09 nmol/mg vs control 1.09 +/- 0.14 nmol/mg). Furthermore, the catalytic activities of CYP1A2. CYP2E1 and CYP3A were unchanged from control following all three treatments as determined by ethoxyresorufin O-deethylation, p-nitrophenol hydroxylation and erythromycin N-demethylation respectively. Additionally, western immunoblotting demonstrated that there was no significant change in the polypeptide levels of any of the three isoforms. These results indicate that four days of treatment with moderate to high doses of SJW, hyperforin or hypericin fails to induce these CYP450 isoforms in the male Swiss Webster mouse.     

Structural investigations of isomeric oxidised forms of hyperforin by HPLC-NMR and HPLC-MSn

The prenylated phloroglucinol hyperforin, thought to be an essential component for the anti-depressant activity of St. John's Wort (Hypericum perforatum), is unstable. The facile oxidative degradation of hyperforin poses serious problems for standardisation, and may also dramatically affect the pharmacological activity of the extracts. Hyperforin was dissolved in hexane and stored at room temperature for 3 days and yielded various closely related degradation products which, although difficult to isolate on the preparative scale, have been analysed by on-flow and stop-flow HPLC-NMR and HPLC-MS/MS. From on-line spectroscopic data, and with the aid of complementary in-mixture standard NMR two-dimensional correlation experiments, the different oxidised forms of hyperforin were found to be phloroglucinol derivatives in which a hydroxy-dihydrofuran ring is formed involving the enol OH at C-7 or C-9 (tautomeric form) and the prenyl chain at C-8 of the core nucleus of hyperforin. The strategy followed for the on-line identification of these constituents is discussed.     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a “chelating” agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid–liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 μl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid–liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from −8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC–MS–MS are discussed in this paper.