Liquid chromatographic determination of St. John's wort components in functional foods

A method was developed for determination of St. John's wort marker compounds hypericin, pseudohypericin, hyperforin, and adhyperforin in functional foods. Solid-phase extraction provided analyte extraction and significant sample cleanup prior to analysis using liquid chromatography (LC) with UV and fluorescence detection. In addition to quantification using LC-UV, confirmation was made with electrospray ionization LC mass spectrometry (LC/MS). Several commercially available tea and drink products claiming to contain St. John's wort were tested. Recoveries ranged from 51 to 98% for the liquid samples. Comparison of the concentrations in 4 St. John's wort teas showed a variation in analyte concentration (1044-10 ng/mL marker compounds in brewed tea) and composition. No marker compounds were found in the beverages, indicating possible decomposition of the marker compounds caused by low pH and/or exposure to light. A solvent extraction procedure was developed for analysis of the marker compounds from solid samples. Analytes were detected at low parts per million, with an average recovery of 75%. No St. John's wort components were found in the 2 solid functional food samples analyzed.     

Evaluation of major active components in St. John's Wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation

A RP-HPLC method with photodiode array detection and LC-electrospray ionization (ESI) MS confirmation was established for the determination of major active components in St. John's Wort dietary supplement capsules. The samples alternatively were extracted with ethanol-acetone (2:3) using a 55 degrees C water-bath shaker or an ambient temperature ultrasonic bath. Extracts were separated by RP-C18 chromatography using a 95-min water-methanol-acetonitrile-trifluoroacetic acid gradient. The major components were identified by photodiode array detection and then confirmed by LC-ESI-MS. The quantification of components was performed using an internal standard (luteolin). This method may serve as a valuable tool for the quality evaluation of St. John's Wort dietary supplement products.     

Determination of six kavalactones in dietary supplements and selected functional foods containing Piper methysticum by isocratic liquid chromatography with internal standard

Kava (Piper methysticum) dietary products have been sold worldwide for treatment of nervous anxiety, tension, and restlessness. Recent reports showed potential association of kava usage and liver injuries. This study was conducted to develop simple and reliable methodologies for the extraction and determination of 6 major kavalactones: (+)-methysticin, (+)-dihydromethysticin, (+)-kavain, (+)-dihydrokavain, yangonin, and desmethoxyyangonin. Ultrasonic extraction techniques and isocratic reversed-phase liquid chromatography (LC) were optimized for different types of samples, including capsules containing kava root extract or root powder, raw root material, tea bags, and snack bar. A suitable internal standard, 5,7-dihydroxyflavone, was used for LC calibration. Kavalactones were completely separated in 30 min using a Luna C18-2 column at 60 degrees C with an isocratic mobile phase consisting of 2-propanol-acetonitrile-water-acetic acid (16 + 16 + 68 + 0.1, v/v/v/v). Within-laboratory, intraday, and interday method variation (% relative standard deviation) for most samples extracted by methanol or methanol-water mixture were <5%. Lower levels of kavalactone contents and higher variations were observed for tea bags from water extraction or infusion as compared to methanol extraction. Labeling information of tea bags based on methanol extraction could be misleading to consumers. Analytical recoveries of snack bar fortified at 10 and 20 microg/g were >84% with RSD values <8%. Methods developed in this study offer a simple and reproducible means for analysis of kavalactones in various matrixes of dietary products.     

Sample preparation and determination of ginkgo terpene trilactones in selected beverage, snack, and dietary supplement products by liquid chromatography with evaporative light-scattering detection

Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. Sample preparation procedures for determination of ginkgo terpene trilactones (including bilobalide and ginkgolides A, B, C, and J) in various sample matrixes were developed in this study. Ginkgo leaves and capsules were extracted with 5% KH2PO4 aqueous solution under sonication. Tea bags were extracted with boiling water, whereas drink samples were taken directly from the bottles. After filtration and the addition of NaCl to approximately 30% (w/v), the terpene trilactones in aqueous solutions were quantitatively extracted with ethyl acetate-tetrahydrofuran (4 + 1, v/v). Puff samples (a cereal-based fried snack item) were first defatted by using hexane or by using supercritical fluid extraction and then extracting under sonication with methanol-acetic acid (99 + 1, v/v). After evaporation of the organic phase, the terpene trilactones were redissolved in methanol and determined on a C18 reversed-phase column by liquid chromatography (LC) with evaporative light-scattering detection. The method of standard additions and gas chromatography with flame ionization detection were used for method validation. For most samples, the relative standard deviation was <10%. The identities of target compounds in ginkgo leaves and drink samples were confirmed by LC/electrospray ionization-tandem mass spectrometry.     

Development and evaluation of methods for determination of naphthodianthrones and flavonoids in St. John's wort

Several major constituents in St. John's wort were determined for a homogenized plant sample. Three extraction techniques were evaluated: Soxhlet extraction, pressurized-fluid extraction (PFE), and sonication extraction. Levels of nine constituents (chlorogenic acid, rutin, hyperoside, isoquercitrin, quercitrin, quercetin, amentoflavone, pseudohypericin, and hypericin) were measured using liquid chromatography with ultraviolet/visible absorbance, mass spectrometric, and fluorescence detection. Levels of total naphthodianthrones determined by liquid chromatography (LC) with absorbance detection at 590 nm were compared with levels determined by direct spectrophotometry at the same wavelength. Additionally, the methods described in this paper were applied to several brands of St. John's wort finished products.     

Simultaneous determination of the predominant hyperforins and hypericins in St. John's Wort (Hypericum perforatum L.) by liquid chromatography

Hypericin and hyperforin are believed to be among the active constituents in common St. John's wort (Hypericum perforatum L.). Presently, dietary supplements are generally standardized to contain specified levels of hypericin and hyperforin, and the related compounds, pseudohypericin and adhyperforin. A rapid method was developed for simultaneous determination of these 4 active constituents by liquid chromatography (LC). A 1 g portion of dried, finely ground leaf/flower sample is extracted with 20 mL methanol for 2 h. A 0.6 mL aliquot of the crude extract is combined with 5.4 mL acetonitrile-methanol (9 + 1) and passed through a mixed solid-phase cleanup column. The eluate is examined by LC for hyperforin, adhyperforin, hypericin, and pseudohypericin on a Hypersil reversed-phase column by using simultaneous ultraviolet (284 nm) and fluorescence detection (excitation, 470 nm; emission, 590 nm). The compounds are easily separated isocratically within 8 min with a mobile phase of acetonitrile-aqueous 0.1 M triethylammonium acetate (8 + 2). Average recoveries of hyperforin and adhyperforin were 101.9 and 98.4%, respectively, for 3 sample mixtures containing concentrations ranging from approximately 0.2 to 1.5% combined hyperforins per gram dry weight. Average relative standard deviation (RSD) values for hyperforin and adhyperforin for all 3 mixtures were 18.9 and 18.0%, respectively. Average recoveries of hypericin and pseudohypericin were 88.6 and 93.3% respectively, from 3 sample mixtures containing concentrations ranging from approximately 0.2 to 0.4% combined hypericins per gram dry weight. Average RSD values for hypericin and pseudohypericin for all 3 mixtures were 3.8 and 4.2%, respectively.     

Rapid screening for synthetic antidiabetic drug adulteration in herbal dietary supplements using direct analysis in real time mass spectrometry

Adulteration of herbal supplements with synthetic drugs is illegal. A rapid and reliable method which utilizes direct analysis in real time mass spectrometry (DART-MS) was developed for the identification of seven synthetic antidiabetic drugs used as adulterants in herbal dietary supplements. The supplement sample was simply extracted with methanol/water by manually shaking several times and directly analyzed using DART-MS. The presence of synthetic drug adulterants was confirmed through the accurate m/z values and MS/MS data obtained via quadruple time of flight mass spectrometry (QTOF MS). Parameters for the DART source were systematically optimized, and the limits of detection (LODs) in herbal supplement matrices were measured. This method was successfully applied to examine five commercial herbal dietary supplements, and two of them proved to be adulterated with metformin without labeling.     

Determination of total soy isoflavones in dietary supplements, supplement ingredients, and soy foods by high-performance liquid chromatography with ultraviolet detection: collaborative study

An interlaboratory study was conducted to evaluate a method for determining total soy isoflavones in dietary supplements, dietary supplement ingredients, and soy foods. Isoflavones were extracted using aqueous acetonitrile containing a small amount of dimethylsulfoxide (DMSO) and all 12 of the naturally occuring isoflavones in soy were determined by high-performance liquid chromatography (HPLC) with UV detection using apigenin as an internal standard. Fifteen samples (6 pairs of blind duplicates plus 3 additional samples) of soy isoflavone ingredients, soy isoflavone dietary supplements, soy flour, and soy protein products were successfully analyzed by 13 collaborating laboratories in 6 countries. For repeatability, the relative standard deviations (RSDr) ranged from 1.07 for samples containing over 400 mglg total isoflavones to 3.31 for samples containing 0.87 mg/g total isoflavones, and for reproducibility the RSDR values ranged from 2.29 for samples containing over 400 mg/g total isoflavones to 9.36 for samples containing 0.87 mg/g total isoflavones. HorRat values ranged from 1.00 to 1.62 for all samples containing at least 0.8 mg/g total isoflavones. One sample, containing very low total isoflavones (< 0.05 mg/g), gave RSDR values of 175 and a HorRat value of 17.6. This sample was deemed to be below the usable range of the method. The method provides accurate and precise results for analysis of soy isoflavones in dietary supplements and soy foods.     

Chemical fingerprint of Hoodia species,dietary supplements,and related genera by using HPTLC

A HPTLC method was developed for simple and rapid chemical fingerprint analysis of four Hoodia species, dietary supplements that claim to contain Hoodia gordonii, and plants from genera related to Hoodia. HPTLC was performed on precoated silica 60F₂₅₄ plates with dichloromethane/methanol/water 75:17:2.2 by volume, as mobile phase. Evaluation of the HPTLC plates was done by using the CAMAG DigiStore2 digital system with winCATS software. The authentication of H. gordonii was achieved by comparing the band colors and R_f values for TLC fingerprints with those of 11 standard compounds including P57. The developed method was successfully applied for the identification of the 11 pregnane glycosides for four different species of Hoodia, 24 related genera and 13 dietary supplements that claim to contain H. gordonii. Different sample matrices were successfully analyzed, providing a wide range of applicability for this method, including gels, capsules, tablets, sprays, teas, snack bars, powders, and juices. The developed method was validated for specificity, stability, repeatability, and robustness. The results of HPTLC method were verified by LC‐UV‐MS method.     

Extraction and determination of biologically active components of St. John’s wort and its pharmaceutical preparations

A procedure is developed for the determination of biologically active substances (BAS) of common St. John’s wort (Hypericum perforatum L.) by HPLC using two columns, Luna C18, 100 Å (for the determination of phenolcarboxylic acids and flavonoids), and Onyx Monolithic C18 (for the determination of phloroglucinols and naphthodianthrones), in the gradient elution mode with diode array detection. The detection limits for analytes are 0.05–0.20 μg/mL. To optimize the conditions, we studied the extraction of biologically active substances from St. John’s wort by a water–ethanol solution at different temperatures and pressures and under the effect of microwave radiation and ultrasound. The maximum amounts of substances were extracted in a dynamic mode under heat and pressure. The procedure was applied to the St. John’s wort samples of different brands and some pharmaceutical products based on it. The components of extracts were identified by retention times, UV spectra, and mass spectra. It was shown that the content of biologically active substances in pharmaceutical samples of St. John’s wort depends on the herb habitat. It was shown that hyperforin decomposed in pharmaceutical formulations based on St. John’s wort during storage.     

Essential oil and volatile components of the genus Hypericum (Hypericaceae)

The flowering plant genus Hypericum (Hypericaceae) contains the well-known medicinally valuable species Hypericum perforatum (common St. John's wort). Species of Hypericum contain many bioactive constituents, including proanthocyanins, flavonoids, biflavonoids, xanthones, phenylpropanes and naphthodianthrones that are characterized by their relative hydrophilicity, as well as acylphloroglucinols and essential oil components that are more hydrophobic in nature. A concise review of the scientific literature pertaining to constituents of Hypericum essential oils and volatile fractions is presented.     

Simultaneous determination of water-soluble vitamins in SRM 1849 Infant/Adult Nutritional Formula powder by liquid chromatography–isotope dilution mass spectrometry

Assessing dietary intake of vitamins from all sources, including foods, dietary supplements, and fortified foods, would be aided considerably by having analytical methodologies that are capable of simultaneous determination of several vitamins. Vitamins naturally present in foods may occur in different chemical forms, with levels ranging over several orders of magnitude. Vitamins in dietary supplements and fortified foods, however, are typically added in a single chemical form, and matrix issues are usually not as complex. These sources should thus be relatively amenable to approaches that aim for simultaneous determination of multiple vitamins. Our recent work has focused on development of liquid chromatography (LC)–UV/fluorescence and LC–tandem mass spectrometry methods for the simultaneous determination of water-soluble vitamins (thiamine, niacin, pyridoxine, pantothenic acid, folic acid, biotin, and riboflavin) in dietary supplement tablets and fortified foods, such as formula powders and breakfast cereals. As part of the validation of our methods and collaboration in characterization of a new NIST SRM 1849 Infant/Adult Nutritional Formula powder, we report data on SRM 1849 using isotope dilution mass spectrometric methods. Use of available NIST Standard Reference Materials® as test matrices in our method development and validation gives a benchmark for future application of these methods. We compare three chromatographic approaches and provide data on stability of vitamin standard solutions for LC-based multiple vitamin determinations.

A simple and reliable semipreparative high-performance liquid chromatography technique for the isolation of marker-grade hyperforin from Hypericum perforatum L extract

The present work describes isolation of bioactive lipophilic constituent [namely, hyperforin from St. John's wort (Hypericum perforatum L.)], of approximately 98% purity by semipreparative high-performance liquid chromatography (LC). The extraction, isolation, and analysis of the collected compound is performed without the use of antioxidants and inert gas atmospheres at all the stages. Hyperforin, separated isocratically on a 12microm semiprep column, is obtained in high purity, lyophilized after the removal of the organic phase, and preserved at a low temperature. The purity of the collected marker compound is estimated by the use of LC-mass spectrometry and spectroscopic techniques.     

Precolumn derivatization liquid chromatography method for analysis of dietary supplements for glucosamine: single laboratory validation study

A precolumn derivatization liquid chromatography (LC) method was developed for the analysis of various dietary supplement formulations and raw materials for glucosamine. A 1 mL sample or standard water solution (containing about 0.05 mg glucosamine) was mixed with 1 mL pH 8.3 buffer, 1 mL 5% phenylisothiocyanate methanolic solution, and derivatized at 80 degrees C in a water bath for 30 min. After derivatization, the solution was cooled in a cold water bath and centrifuged at 3000-5000 rpm. The clear upper layer was ready for injection. The LC system was equipped with a C18 reversed-phase column and an ultraviolet detector set at 240 nm. The column was developed with a linear gradient composed of 0.1% phosphoric acid in deionized water and 0.1% phosphoric acid in methanol. The method was subjected to Single Laboratory Validation. The method precision was 0.50% relative standard deviation, accuracy was less than +/-1.5%, method linearity in the range 0-2 mg glucosamine/mL was 1.00, the detection limit was 0.0705 microg/mL, and the quantitation limit was 0.235 microg/mL. Chondroitin sulfate, amino acids, and excipients did not interfere with glucosamine testing. After derivatization, both standard and sample preparations were stable for at least 48 h. Due to its high sensitivity, this method can be used to assay glucosamine in functional foods and pet foods. The validation data will be published separately.     

Simultaneous determination of vitamin A and beta-carotene in dietary supplements by liquid chromatography

Several liquid chromatography (LC) methods for analysis of vitamin A in foods and feeds have been previously reported but only a few have been applied in non-food matrixes. A validated LC method is needed for determination of vitamin A and beta-carotene in the various matrixes presented by dietary supplements. The performance of a reversed-phase method with methanol-isopropanol gradient elution was evaluated with standard retinyl derivatives and beta-carotene. The reversed-phase method is capable of separating retinol from other derivatives such as retinyl acetate, retinyl palmitate, and beta-carotene. Two types of extraction were used to extract the analytes from the dietary supplements: a hexane-methylene chloride extraction for soft-gel capsules containing beta-carotene, and a direct solvent extraction for dietary supplements in tablet form. The direct solvent extraction consisted of treatment with ethanol and methylene chloride following addition of hot water (55 degrees C). Results with the reversed-phase method for vitamin A and beta-carotene in the products examined (n = 8) indicated excellent method performance. The main form of vitamin A or beta-carotene in dietary supplements was the all-trans isomer. The reversed-phase method avoids saponification and is rapid, accurate, precise, and suitable for simultaneous determination of retinyl derivatives and beta-carotene in dietary supplements.     

Single-laboratory validated method for determination of nordihydroguaiaretic acid in chaparral-containing dietary supplements

Nordihydroguaiaretic acid (NDGA) occurs naturally in chaparral (Larrea tridentate Coville), a plant which commonly grows in the Southwest United States and has been used for medicinal purposes by Native Americans indigenous to that region. In addition to its traditional use as a tea, manufacturers of dietary supplements have marketed chaparral-containing products in a variety of formulations. Because of the hepatotoxicity of NDGA, and its occurrence in regulated products, we have developed a method for the determination of NDGA in dietary supplements and have tested this method in several dietary supplement formulations. Products were extracted with 80% methanol, filtered, and analyzed by high-performance liquid chromatography. NDGA was detected and determined with both a diode array detector and negative-ion electrospray. Fragmentation in the triple-quadrupole mass spectrometer was obtained by collisional activation of the [M-H](-) ion. Collisional activation produced sufficient fragmentation to provide unambiguous identification. Lack of a stable isotope labeled internal standard has led us to compare quantitations based on UV detection with quantitations based on tandem mass spectrometry (MS/MS). Presence of NDGA was confirmed in several dietary supplement products. Quantitative results from the 2 detection methods were comparable for most products. The limit of quantitation using MS/MS was lower and fewer interferences were observed, although UV detection provided better linearity.     

Determination of total iodine in foods and dietary supplements using inductively coupled plasma-mass spectrometry

A method was developed and validated for the determination of total iodine in a wide variety of food products and dietary supplements. The method involves a unique sample digestion with a KOH solution in an oven or by using an open-vessel microwave system. After digestion, a stabilizer is added and the solution is taken to volume, then filtered and analyzed either directly or after dilution. The amount of iodine is determined with inductively coupled plasma-mass spectrometry (ICP-MS). The method was validated by experiments to determine its precision, accuracy, linearity, specificity, ruggedness, and robustness. The LOQ of this method is 25-50 microg/kg. The method demonstrated an average RSD of 2.27% during analysis of milk powder and 4.30% during analysis of a dietary supplement tablet reference material. The accuracy of the method as determined with these same reference materials was 100 and 94.2%, respectively. The method has been used successfully on commodity foods, processed foods, dairy products, pet food, infant formula, animal feed, mineral premixes, and a variety of dietary supplements.     

Simultaneous Determination of Isoquinoline Alkaloids in Medicinal Asiatic Plants by Ultrasound-Assisted Extraction and High-Performance Liquid Chromatography – Mass Spectrometry with Principal Component Analysis

Isoquinoline alkaloids (papaverine, noscapine, berberine, emetine, and quinine) were determined in medicinal plants and herbs used in Traditional Chinese Medicine and Ayurveda by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). The analyzed alkaloids were separated at gradient conditions using methanol and 2% acetic acid within 12?min. The validated method was successfully applied for 17 herbal samples: Ashwagandha, Astragalus membranaceus, Emblica officinalis, Mucuna pruriens, Pueraria lobata, Ocimum sanctum, Rehmannia glutinosa, Schisandra sinensis, Terminalia arjuna, Terminalia chebula, and dietary supplements. The highest concentration of studied alkaloids was observed for berberine in Puearia lobata (6.68?±?0.62?mg 100?g^?1 d.m.), while the lowest value was obtained for noscapine in a dietary supplement containing Terminalia arjuna (0.09?±?0.01?mg 100?g^?1 d.m.). Principal component analysis, cluster analysis, and one-way ANOVA tests were also performed. The results indicate the need to control plant materials and dietary supplements in terms of the content of alkaloids and toxic components.     

Chromatographic performance of a new polar poly(ethylene glycol) bonded phase for the phytochemical analysis of Hypericum perforatum L

The aim of this study was to evaluate the chromatographic performance of a poly(ethylene glycol) (PEG) stationary phase for the HPLC analysis of the secondary metabolites (chlorogenic acid, flavonoids, phloroglucinols and naphthodianthrones) in methanolic extracts of Hypericum perforatum L. (St. John's Wort) flowering tops, herbal medicinal products and dietary supplements. A fast and reliable method was developed. The analyses were carried out on a Supelco Discovery HS PEG column (150 mm x 4.6 mm i.d., 5 microm). A gradient mobile phase, composed of 0.1 M aqueous acetic acid solution (pH 2.8) and methanol-acetonitrile (5:4, v/v), was used. The flow rate was 1 mL/min. The photodiode array detector monitored the eluent at 270 (for chlorogenic acid, flavonoids and phloroglucinols) and 590 nm (for naphthodianthrones). The column was maintained at room temperature. The total running time was 40 min. The method was validated and showed good linearity, precision, accuracy, sensitivity and specificity. Through the above described phytochemical markers, this technique allowed the unequivocal identification and standardization of H. perforatum plant material and phytoproducts. The quantification data highlighted the fact that the products on sale, in particular those labeled as dietary supplements, varied widely in the quantitative composition of the active constituents. The developed method could be considered suitable for the quality control of H. perforatum herb and derivatives.     

Method for the determination of beta-carotene in supplements and raw materials by reversed-phase liquid chromatography: single laboratory validation

A single laboratory validation (SLV) study was conducted for a liquid chromatography (LC) method for the determination of total and all-trans-beta-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of beta-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane-ethanol. Oily suspensions were directly dissolved in dichloromethane-ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of alpha-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1-50 microg beta-carotene/mL. The main geometrical isomers of beta-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as a-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total beta-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2-4.4%. Recoveries determined for supplements and beadlet raw material spiked with beta-carotene levels of 10 microg to 100 mg/test portion and 0.2-40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-beta-carotene in dietary supplements.