Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

A flow-injection mass spectrometry fingerprinting method for authentication and quality assessment of Scutellaria lateriflora-based dietary supplements

Scutellaria lateriflora, commonly known as skullcap, is used as an ingredient in numerous herbal products. However, it has been occasionally adulterated/contaminated with Teucrium canadense and/or Teucrium chamaedrys, commonly known as germander, due to the morphological similarities between the two genera. The latter contains hepatotoxic diterpenes. Despite the potential hepatotoxicity introduced by germander contamination, analytical methodologies for the authentication and quality assessment of S. lateriflora-based dietary supplements have not been reported. In this study, a flow-injection/mass spectrometry fingerprinting method in combination with principal component analysis was used to survey S. lateriflora-based dietary supplements sold in the USA.     

Mass spectrometric analysis of pharmaceutical adulterants in products labeled as botanical dietary supplements or herbal remedies: a review

The increased availability and use of botanical dietary supplements and herbal remedies among consumers has been accompanied by an increased frequency of adulteration of these products with synthetic pharmaceuticals. Unscrupulous producers may add drugs and analogues of various classes, such as phosphodiesterase type 5 (PDE-5) inhibitors, weight loss, hypoglycemic, antihypertensive and anti-inflammatory agents, or anabolic steroids, to develop or intensify biological effects of dietary supplements or herbal remedies. The presence of such adulterated products in the marketplace is a worldwide problem and their consumption poses health risks to consumers. Analytical methods that allow rapid and reliable testing of dietary supplements for the presence of synthetic drugs are needed to address such fraudulent practices. Mass spectrometry (MS) and hyphenated techniques such as liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) have become primary tools in this endeavor. The present review critically assesses the role and summarizes the applications of MS in the analysis of pharmaceutical adulterants in botanical dietary supplements and herbal remedies. The uses of MS techniques in detection, confirmation, and quantification of known pharmaceutical adulterants as well as in screening for and structure elucidation of unexpected adulterants and novel designer drugs are discussed.     

A flow-injection mass spectrometry fingerprinting method for authentication and quality assessment of <Emphasis Type="Italic">Scutellaria lateriflora</Emphasis>-based dietary supplements

Scutellaria lateriflora, commonly known as skullcap, is used as an ingredient in numerous herbal products. However, it has been occasionally adulterated/contaminated with Teucrium canadense and/or Teucrium chamaedrys, commonly known as germander, due to the morphological similarities between the two genera. The latter contains hepatotoxic diterpenes. Despite the potential hepatotoxicity introduced by germander contamination, analytical methodologies for the authentication and quality assessment of S. lateriflora-based dietary supplements have not been reported. In this study, a flow-injection/mass spectrometry fingerprinting method in combination with principal component analysis was used to survey S. lateriflora-based dietary supplements sold in the USA.     

Determination of isoflavones in dietary supplements containing soy, Red Clover and kudzu: extraction followed by basic or acid hydrolysis

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to beta-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Dietary supplement preparations contain extracts from soy, Red Clover and kudzu. Soy products contain primarily genistein, daidzein, and glycitein, while Red Clover products contain primarily formononetin and biochanin A. Kudzu extracts contain puerarin and daidzein among other components. Previous methods of analysis focused on the determination of isoflavones from a single botanical source, while dietary supplements are often a blend of extracts from different plants. We developed a method for the analysis of isoflavones in dietary supplements regardless of their botanical composition, using HPLC-PDA because of its applicability to routine analysis. Isoflavones are found as free compounds, glucoside derivatives, 6'-O-malonyl-beta-d-glucoside and 6'-O-acetyl-beta-d-glucoside derivatives. In this study, the samples were extracted at room temperature with 50:50 (v/v) MeCN/water, and then analyzed before and after hydrolyzing the isoflavones by acid or basic digestion. 2'-Methoxy-flavone and 6-methoxy-flavone were used as internal standards and were added together to every sample. Daidzein, glycitein, genistein, puerarin, calycosin, pratensein, pseudobaptigenin, formononetin, biochanin A and prunetin were among the isoflavones determined.     

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.     

高效液相色谱-四极杆/静电场轨道阱高分辨率质谱法快速筛查中成药和保健食品中非法添加的42种化学药物

建立了高效液相色谱-四极杆/静电场轨道阱高分辨率质谱法快速筛查中成药和保健品中非法添加化学药物的方法。中成药和保健品样品,经提取溶剂提取,以12000 r/min离心后进行质谱分析。采用Phe-nomenex C18色谱柱(100 mm×4.6 mm,2.6μm)进行分离,以乙腈和0.1%甲酸溶液作为流动相,进行梯度洗脱。质谱采用正离子和负离子同时扫描,Fullms-dd-MS2模式进行分析。在所建立的色谱条件下,42种化学药物能够得到较好分离,在线性范围内线性关系良好,相关系数均大于0.99。通过加标验证,在20,50和100 ng/g加标水平下,所有分析物的平均回收率为69.3%~105.2%,相对标准偏差(RSD)小于8.9%。运用本方法对31种保健品和中成药进行快速筛查,发现其中3种添加了盐酸二甲双胍,1种添加了西地那非,1种添加了羟基莫豪西地那非。本方法样品处理过程简单,分析时间短,准确可靠,灵敏度高。适用于中成药和保健品中非法添加化学药品的定性与定量检测,可用于非法添加药物的筛查。     

Thermal characterization of dried extract of medicinal plant by DSC and analytical techniques

The increased search for herbal products has generated an increasing interest in improving the quality control of dried extracts by pharmaceutical industry since these are raw materials of great importance by their quality and versatility. This work aimed at the application of various analytical techniques (thermal analysis, X-ray diffraction, scanning electron microscopy, and infrared Fourier transform spectroscopy) in the characterization of dried extracts of two plants from the Brazilian semiarid region with medicinal properties. The DSC curves for the dried extracts of Ximenia americana L. and Schinopsis brasiliensis Engl. showed that thermal processes occur between 33.50 and 118.58 °C and between 39.17 and 126.14 °C. The X-ray powder diffraction revealed high degree of amorphization, but the dried extract of X. americana L. showed some diffraction peaks of high intensity. The IR spectra showed high variety of metabolites in the extracts dried. Through this study it was possible to verify the feasibility of applying these techniques in the characterization of raw materials from medicinal plants for use in the herbal medicines production.     

Selection and authentication of botanical materials for the development of analytical methods

Herbal products, for example botanical dietary supplements, are widely used. Analytical methods are needed to ensure that botanical ingredients used in commercial products are correctly identified and that research materials are of adequate quality and are sufficiently characterized to enable research to be interpreted and replicated. Adulteration of botanical material in commerce is common for some species. The development of analytical methods for specific botanicals, and accurate reporting of research results, depend critically on correct identification of test materials. Conscious efforts must therefore be made to ensure that the botanical identity of test materials is rigorously confirmed and documented through preservation of vouchers, and that their geographic origin and handling are appropriate. Use of material with an associated herbarium voucher that can be botanically identified is always ideal. Indirect methods of authenticating bulk material in commerce, for example use of organoleptic, anatomical, chemical, or molecular characteristics, are not always acceptable for the chemist’s purposes. Familiarity with botanical and pharmacognostic literature is necessary to determine what potential adulterants exist and how they may be distinguished.     

Improved method for the determination of anatoxin-a and two of its metabolites in blue-green algae using liquid chromatography with fluorescence detection

Anatoxin-a, a neurotoxin produced by blue-green algae (BGA) species, can cause death to exposed organisms. In North America, BGA are harvested and sold as food supplements, some of which contain elevated levels of other algal toxins, such as microcystins. Concern that elevated levels of anatoxin-a also may be present in BGA food supplements has led to the development of a simple method to determine the presence of anatoxin-a in BGA. Some researchers have successfully analyzed this compound using liquid chromatography with fluorescence detection by forming a fluorescent derivative with 4-fluoro-7-nitrobenzofurazan (NBD-F) in water and phytoplankton extracts. With this method, the background noise is high in BGA extracts due to the presence of co-extractives. Addition of o-phthaldialdehyde (OPA) and mercaptoethanol to the extract before addition of the NBD-F resulted in the successful removal of primary amines from the background noise when the NBD-F derivatives were detected with fluorescence. Improved chromatograms were obtained when extracts were cleaned up in this manner, leading to a lower detection limit (approximately 50 microg/kg) for anatoxin-a. The detection limits obtained for the 2 degradation products dihydroanatoxin-a and epoxyanatoxin-a in BGA extracts were similarly low (55 and 65 microg/kg, respectively).     

A strategy for the identification of plants in illegal pharmaceutical preparations and food supplements using chromatographic fingerprints

The detection of regulated and forbidden herbs in pharmaceutical preparations and nutritional supplements is a growing problem for laboratories charged with the analysis of illegal pharmaceutical preparations and counterfeit medicines. This article presents a feasibility study of the use of chromatographic fingerprints for the detection of plants in pharmaceutical preparations. Fingerprints were developed for three non-regulated common herbal products—Rhamnus purshiana, Passiflora incarnata L. and Crataegus monogyna—and this was done by combining three different types of detection: diode-array detection, evaporative light scattering detection and mass spectrometry. It is shown that these plants could be detected in respective triturations of the dry extracts with lactose and three different herbal matrices as well as in commercial preparations purchased on the open market.

Development of the analytical methods and reference materials program for dietary supplements at the National Institutes of Health

The need for validated analytical methods and reference materials to identify and measure constituents in dietary supplements is essential. Such methods allow for the documentation that products meet manufacturer's specifications and contain what their labels declare. In March 2003, the U.S. Food and Drug Administration issued a proposed rule that would establish specific current good manufacturing practices for dietary supplements. This proposed rule requires that companies create a quality control unit to set specifications and ensure compliance with these specifications using scientifically valid procedures. This report provides insights and lessons learned from 3 meetings the Office of Dietary Supplements (ODS) at the National Institutes of Health helped organize. These meetings were structured to (1) educate individuals about the importance and need for validated analytical methods and reference materials to identify and quantify constituents of dietary supplements; (2) identify resources required to fulfill this need; and (3) serve as a platform to obtain input from interested parties to help frame the research agenda for the Dietary Supplements Methods and Reference Materials Program within ODS. Stakeholder's opinions and views expressed at these 3 meetings are outlined in this report.     

Determination of the appetite suppressant P57 in Hoodia gordonii plant extracts and dietary supplements by liquid chromatography/electrospray ionization mass spectrometry (LC-MSD-TOF) and LC-UV methods

Hoodia gordonii is traditionally used in South Africa for its appetite suppressant properties. P57AS3 (P57), an oxypregnane steroidal glycoside, is the only reported active constituent from this plant as an appetite suppressant. Effective quality control of these extracts or products requires rapid methods to determine P57 content. New methods of liquid chromatography/mass spectrometry (LC/MS) and LC-UV for analysis of P57 from H. gordonii have been developed. The quantitative determination of P57 was achieved with a Phenomenex Gemini (Torrance, CA) reversed-phase column using gradient mobile phase of water and acetonitrile, both containing 0.1% acetic acid. The method was validated for linearity, repeatability, and limits of detection and quantification. Good results were obtained in terms of repeatability (relative standard deviation <5.0%) and recovery (98.5-103.5%). The developed methods were applied to the determination of P57 for H. gordonii plant samples, one related genus (Opuntia ficus-indica), and dietary supplements that claim to contain H. gordonii.     

Chromatographic analysis of Fritillaria isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb.

Bulbus Fritillariae derived from plants of various Fritillaria species is the most commonly used antitussive traditional Chinese medicinal herb and is called Beimu. Herbs derived from similar and/or different species of Fritillaria are also used in Japan and Turkey as traditional or folk medicines. Isosteroidal alkaloids are the main bioactive ingredients in Fritillaria species. As the contents and structure types of these bioactive alkaloids vary in different Fritillaria species, quality control of these active principles in herbal Beimu is very important to ensure its safe and effective clinical use. This review describes the development of chromatographic analyses for the simultaneous qualitative and quantitative determination of the main bioactive Fritillaria isosteroidal alkaloids in herbal and biological samples. The recently developed direct HPLC-evaporative light scattering detection method is the most simple, selective and sensitive assay, and is readily used as a suitable quality control method for the analysis of the active principles of herbal Beimu.     

中药材提取物的混批勾兑研究

采用非线性最小二乘拟合计算中药材提取物的勾兑系数,不同批的中药材提取物经过勾兑后与参照样品的差异减小,各成分含量稳定。采用数据预处理的方法,并对数据预处理方法进行改进,使峰面积较小的色谱峰可以实现较小的相对差异。引入误差控制系数,可实现对特定色谱峰的控制要求。实验结果表明,非线性最小二乘拟合可以用于计算中药材提取物的勾兑系数。     

Recent discovery of plant-derived anti-diabetic natural products

Covering: 2005 to 2010. This review covers recent discoveries of anti-diabetic compounds. Diabetes mellitus (DM) is a complex disease affecting patients' daily life and elevating patients' risk of developing other diseases. There are several forms of diabetes, including type-1 diabetes (insulin-dependent), type-2 diabetes (noninsulin-dependent), and gestational diabetes. Type-2 diabetes is the most common form and the patient population with type-2 DM rises every year. Current treatments meet some but not all patients' needs. Therefore, new anti-diabetic drugs are in great demand. Traditional herbal medicine provides a rich source for new drug discovery. In this review, recent discoveries of anti-diabetic compounds have been summarized according to their chemical structures and mechanisms of action. Anti-diabetic plant extracts, many of which have been used and marketed as dietary supplements, were also included and discussed, and are classified according to the positive control used in the anti-diabetic animal studies. New anti-diabetic natural products found in the recent patent literature are also summarized.     

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.     

Biosensoric flow-through method for the determination of cysteine sulfoxides

Cysteine sulfoxides are known for their canceroprotective and antioxidant potential and have been detected in several plants, mainly those belonging to the leek family (Alliaceae). A number of these plants are used as herbal remedies, spices, or vegetables, including garlic (Allium sativum), onion (Allium cepa) and leek (Allium porrum). Determination of the quality and pharmacological activity of garlic-containing herbal remedies produced from garlic powder is by measurement of their content of the cysteine sulfoxide alliin. A convenient method allowing reliable routine estimation of the alliin levels present in the fresh plant material through to the final remedy is urgently required. The newly developed flow-through method described in this paper was designed in order to fulfill this demand. In contrast to conventional HPLC methods, neither a pre-column derivatization nor a chromatographic separation are required allowing a high throughput of samples. This technique is based on immobilized alliinase (EC 4.4.1.4), which was combined with an ammonia gas electrode mounted in a miniaturized flow-through cell. The lower detection limit obtained for alliin was 3.7×10⁻⁷ M alliin. High specificity for alliin was demonstrated for the electrode and a variety of garlic samples including garlic powders, dry extracts and garlic tablets were analyzed successfully. Results obtained with the new method showed a good correlation with those obtained with conventional HPLC methods. In conclusion, the flow-through method provides a valuable method for quality control of garlic preparations and screening plant materials for their cysteine sulfoxide-related health potential.     

Identification of thiosildenafil in a health supplement

The presence of a sildenafil derivative, the thiosildenafil, in an herbal product has been evidenced first by HPTLC and later determined by isolation and analysis of spectroscopic data. The analyzed product is nowadays marketed as dietary supplement containing herbal extracts and claimed for male and female sexual improvement. This report is noteworthy since it is clear that adulterated materials can cause serious health problems if they are consumed as herbal "natural" products, generally considered deprived of toxicity by the consumers. The use of a simple and reliable method, based on HPTLC, to determine synthetic adulterations is reported in this paper.