Validation of an LC-MS/MS method for analysis of anti-diabetic drugs in botanical dietary supplements labeled for blood sugar management.

We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantitate 14 anti-diabetic, 2 anti-obesity, and 3 cholesterol-lowering drugs in botanical dietary supplements marketed for blood sugar management. Many botanical dietary supplements which carry label statements related to blood sugar management are available over the Internet. Potential adulteration of such dietary supplements with anti-diabetic and other prescription drugs, some of which have been removed from the market due to adverse events, is of concern. No significant matrix effects were observed and mean recoveries of all 19 analytes from a single product matrix were 88 to 113% at spiking concentrations from 500 to 2000 µg/g. Mean recoveries of metformin, phenformin, and sibutramine from matrices prepared from multiple product composites ranged from 93 to 115% at a spiking concentration of 100 µg/g. The relative standard deviations (RSD) (%) of intra-day analyses ranged from 0.2 to 13 for all recovery studies. Eighty dietary supplements obtained in the USA and carrying label statements related to blood sugar management were analyzed using this method and none were found to be adulterated with the above 19 drugs. Two products obtained outside of the USA and known to be adulterated were also analyzed by this method and found to contain phenformin, glibenclamide, and sibutramine. This method provided satisfactory selectivity, linearity, accuracy, precision, and sensitivity for rapid determination of 19 drugs and has broad applicability for the analysis of dietary supplements for possible adulteration with these compounds.

Assessment of the Authenticity of Herbal Dietary Supplements: Comparison of Chemical and DNA Barcoding Methods.

About 7 % of the U. S. population reports using botanical dietary supplements. Increased use of such supplements has led to discussions related to their authenticity and quality. Reports of adulteration with substandard materials or pharmaceuticals are of concern because such substitutions, whether inadvertent or deliberate, may reduce the efficacy of specific botanicals or lead to adverse events. Methods for verifying the identity of botanicals include macroscopic and microscopic examinations, chemical analysis, and DNA-based methods including DNA barcoding. Macroscopic and microscopic examinations may fail when a supplement consists of botanicals that have been processed beyond the ability to provide morphological characterizations. Chemical analysis of specific marker compounds encounters problems when these compounds are not distinct to a given species or when purified reference standards are not available. Recent investigations describing DNA barcoding analysis of botanical dietary supplements have raised concerns about the authenticity of the supplements themselves as well as the appropriateness of using DNA barcoding techniques with finished botanical products. We collected 112 market samples of frequently consumed botanical dietary supplements of ginkgo, soy, valerian, yohimbe, and St. John's wort and analyzed each for specific chemical markers (i.e., flavonol glycosides, total isoflavones, total valerenic acids, yohimbine, and hypericins, respectively). We used traditional DNA barcoding techniques targeting the nuclear ITS2 gene and the chloroplast gene psbA-trnH on the same samples to determine the presence of DNA of the labelled ingredient. We compared the results obtained by both methods to assess the contribution of each in determining the identity of the samples.

Overview of regulation of dietary supplements in the USA and issues of adulteration with phenethylamines (PEAs).

The multi-billion dollar dietary supplement industry is global in reach. The industry has been criticized for problems related to poor quality control, safety, misbranding, and adulteration. In this review, we describe how the US Food and Drug Administration (FDA) regulates dietary supplements within the framework of the Federal Food, Drug, and Cosmetic Act (FD&C Act). The Dietary Supplement Health and Education Act of 1994 (DSHEA), which amended the FD&C Act, gave the FDA the authority to promulgate Good Manufacturing Practices for dietary supplements and required that manufacturers provide the FDA information supporting a conclusion that the ingredients are reasonably expected to be safe if the dietary ingredients were not marketed in the USA before 15 October 1994. Recent amendments to the FD&C Act require that serious dietary-supplement-related adverse events be reported to the FDA and provide the agency with mandatory recall authority. We discuss the presence of naturally occurring (e.g. Ephedra, Citrus aurantium, Acacia) and synthetic (e.g. ß-methylphenethylamines, methylsynephrine, α-ethyl-phenethylamine) biologically active phenethylamines (PEAs) in dietary supplements and of PEA drugs (e.g. clenbuterol, fenfluramine, sibutramine, lorcaserin) in weight-loss products. Regulatory actions against manufacturers of products labelled as dietary supplements that contain the aliphatic amines 1,3-dimethylamine and 1,3-dimethylbutylamine, and PEAs such as ß-methylphenethylamine, aegeline, and Dendrobium illustrate the FDA's use of its authority under the FD&C Act to promote dietary supplement safety. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Sesquiterpenoid tropolone glycosides from Liriosma ovata.

Two new sesquiterpenoid tropolone glycosides, liriosmasides A (1) and B (2), along with two known compounds, secoxyloganin and oplopanpheside C, were isolated from a methanol extract of the roots of Liriosma ovata. The structures of 1 and 2 were elucidated by spectroscopic methods including 1D and 2D NMR and by high-resolution mass spectrometry involving an ultra-high-performance liquid chromatography-quadrupole-orbital ion trap mass spectrometric (UHPLC-Q-Orbitrap MS) method. Compound 1 showed weak inhibitory activity against HIV RNase H.

Determination of selected biogenic amines in Acacia rigidula plant materials and dietary supplements using LC-MS/MS methods.

Extracts of Acacia rigidula leaves are used in weight-loss products sold in vitamin shops and over the internet with little or no published data about their potential biological effects. In our chemical investigations on authenticated A. rigidula plant material, we established a rapid and sensitive LC-MS/MS method for the quantitative determination of several phenethylamine, tyramine and tryptamine derivatives. Stable isotopically labeled compounds were used as internal standards for quantitative analysis. We found total calculated contents of 6 biogenic amines in A. rigidula leaf of 18.6 and 32.9µg/g. The content of selected amines in 21 dietary supplements labeled as containing A. rigidula was determined by a second LC-MS/MS method. Our study revealed significant differences in the amine profiles of authenticated plant materials and dietary supplements. ß-Methylphenethylamine, a non-natural compound, was found in 9 of the 21 dietary supplement products. ß-Methylphenethylamine was found at levels of 960-60,500µg/g while phenethylamine was found at levels of 710-171,620µg/g. ß-Methylphenethylamine is a positional isomer of amphetamine and our results showed that it can be misidentified as amphetamine during LC-MS analysis. An independent GC-MS analysis was used to confirm the presence of ß-methylphenethylamine and the absence of amphetamine in dietary supplements labeled as containing A. rigidula. This study demonstrates that confirmations by independent analytical methods are essential to verify findings of unusual or unexpected compounds in dietary supplements.

Updates on chemical and biological research on botanical ingredients in dietary supplements.

Increased use of dietary supplements is a phenomenon observed worldwide. In the USA, more than 40% of the population recently reported using complementary and alternative medicines, including botanical dietary supplements. Perceptions that such dietary supplements are natural and safe, may prevent disease, may replace prescription medicines, or may make up for a poor diet, play important roles in their increased use. Toxicity of botanical dietary supplements may result from the presence of naturally occurring toxic constituents or from contamination or adulteration with pharmaceutical agents, heavy metals, mycotoxins, pesticides, or bacteria, misidentification of a plant species in a product, formation of electrophilic metabolites, organ-specific reactions, or botanical-drug interactions. The topics discussed in this review illustrate several issues in recent research on botanical ingredients in dietary supplements. These include (1) whether 1,3-dimethylamylamine is a natural constituent of rose geranium (Pelargonium graveolens), (2) how analysis of the components of dietary supplements containing bitter melon (Momordica charantia) is essential to understanding their potential biological effects, and (3) how evolving methods for in vitro studies on botanical ingredients can contribute to safety evaluations. The virtual explosion in the use of botanical ingredients in hundreds of products presents a considerable challenge to the analytical community, and the need for appropriate methods cannot be overstated. We review recent developments and use of newer and increasingly sensitive methods that can contribute to increasing the safety and quality of botanical ingredients in dietary supplements.

Comparison of metabolism-mediated effects of pyrrolizidine alkaloids in a HepG2/C3A cell-S9 co-incubation system and quantification of their glutathione conjugates.

1. Toxicity of pyrrolizidine alkaloids (PAs) largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrrolic derivatives. These then spontaneously release the esterifying acids to generate carbonium ions that form covalent adducts with cellular nucleophiles to exhibit toxicity. 2. In our investigation, metabolism-mediated toxicity of monocrotaline, retrorsine, lycopsamine, echimidine (retronecine-type PAs), heliotrine (a heliotridine-type PA) and senkirkine (an otonecine-type PA) was studied using an in vitro co-incubation assay. 3. Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver S9 fraction and the toxicity was assessed as lowered mitochondrial activity. 4. Bioactivation potential was measured by incubating PAs with rat liver S9 fraction, NADPH and GSH in a cell free system. Pyrrolic metabolites generated were entrapped as glutathione conjugates (7-GSH-DHP and 7,9-di-GSHDHP) which were quantified using LC-MS-MS analysis. 5. Our results indicated that PAs were metabolized by rat liver S9 fraction into reactive pyrrolic derivatives which were toxic to HepG2/C3A cells. This approach can be used to determine and compare bioactivation potential and metabolism-mediated toxicity of various PAs.

Quantitative determination of cucurbitane-type triterpenes and triterpene glycosides in dietary supplements containing bitter melon (Momordica charantia) by HPLC-MS/MS.

Momordica charantia L. (Cucurbitaceae), commonly known as bitter melon, is widely cultivated in many tropical and subtropical areas of the world. It is a common food staple; its fruits, leaves, seeds, stems, and roots also have a long history of use in traditional medicine. In the United States, dietary supplements labeled as containing bitter melon can be purchased over-the-counter and from Internet suppliers. Currently, no quantitative analytical method is available for monitoring the content of cucurbitane-type triterpenes and triterpene glycosides, the major constituents of bitter melon, in such supplements. We investigated the use of HPLC-electrospray ionization (ESI)-MS/MS for the quantitative determination of such compounds in dietary supplements containing bitter melon. Values for each compound obtained from external calibration were compared with those obtained from the method of standard additions to address matrix effects associated with ESI. In addition, the cucurbitane-type triterpene and triterpene glycoside contents of two dietary supplements determined by the HPLC-ESI-MS/MS method with standard additions were compared with those measured by an HPLC method with evaporative light scattering detection, which was recently developed for quantification of such compounds in dried fruits of M. charantia. The contents of five cucurbitane-type triterpenes and triterpene glycosides in 10 dietary supplements were measured using the HPLC-ESI-MS/MS method with standard additions. The total contents of the five compounds ranged from 17 to 3464 microg/serving.

Chiral stationary phases for separation of intermedine and lycopsamine enantiomers from Symphytum uplandicum.

Enantioseparation of the pyrrolizidine alkaloid isomers intermedine and lycopsamine, isolated from Symphytum uplandicum, is discussed. The separatory power of two immobilized carbohydrate-based chiral HPLC columns, Chiralpak IA and IC, in different chromatographic conditions is compared. The study demonstrated the importance of solvent and column selection while developing such chiral HPLC separation methods. The baseline HPLC separation of the two alkaloid isomers in preparatory scale is reported for the first time. The optimized separations were achieved on a Chiralpak IA column with mobile phases of ACN/methanol (80:20) and methanol/methyl-t-butyl ether (90:10), both containing 0.1% diethylamine.

Alkaloids and saponins in dietary supplements of blue cohosh (Caulophyllum thalictroides).

Preparations of blue cohosh (Caulophyllum thalictroides) have been used traditionally by Native Americans for medicinal purposes. Dietary supplements containing dried roots or extracts of blue cohosh rhizomes are available as dietary supplements. The safety and efficacy of these preparations have not been systematically evaluated. Recent studies indicate that ingestion of specific alkaloids in blue cohosh preparations can produce birth defects and neonatal heart failure. Blue cohosh also contains saponins, which may be responsible for uterine-stimulating effects. We determined the amounts of major alkaloids and saponins in preparations of blue cohosh by high-performance liquid chromatography (HPLC). Alkaloids and saponins were monitored with a photodiode array detector and an evaporative light-scattering detector, respectively. Profiles were compared with those of authenticated blue cohosh root extracts. Identities of the alkaloids and saponins were confirmed by HPLC/mass spectrometry and nuclear magnetic resonance spectrometry. Calculations based on the results of analyses of dietary supplements showed that maximum daily intake of alkaloids and saponins will vary with the form (e.g., root, liquid extract) and doses recommended in product labeling. Intakes may vary from < 1 to 75 mg/day for alkaloids and from about 9 to 420 mg/day for saponins.

Isolation and characterisation of selected germander diterpenoids from authenticated Teucrium chamaedrys and T. canadense by HPLC, HPLC-mS and NMR.

Teucrium species, such as germander, are rich in neo-clerodane diterpenoids and have been used in traditional folk medicine for their stimulant, diuretic, antipyretic and antiseptic properties. However, the furano neo-clerodane diterpenoids present in germander have been implicated in the in vivo hepatotoxicity of this botanical. In this study, authenticated germander (Teucrium chamaedrys L. and Teucrium canadense L.) was used as the source material. Methanol extracts of powdered plant mate rial were prepared and analysed by HPLC using Synergi Max-RP columns with monitoring at 220 nm. Limited amounts of teucrin A and other diterpenoid standards were analysed on a Synergi Max-RP column in order to determine their retention times and to generate calibration curves. The same standards were subjected to concurrent mass spectral analysis. Teucrin A and diterpenoids such as dihydroteugin, teuflin, teuflidin and teucvidin were tentatively identified in the plant extracts by HPLC-MS and 1H-NMR experiments. For the isolation of teucrium diterpenoids on a semipreparative scale, a solid-phase extraction method was developed for the first time using styrene divinylbenzene and strata-X sorbents for teucrin A and teuflin, respectively. Semi-preparative HPLC of the methanol extract of the powdered aerial parts of Teucrium plants was carried out on a semipreparative Synergi Max-RP column with photodiode array detection in order to confirm the identities of some diterpenoids by HPLC-MS and NMR.

Effects of dietary taurocholate, fat and protein on the storage and metabolism of dietary beta-carotene and alpha-tocopherol in ferrets.

Dietary factors affecting tissue storage of beta-carotene (BC), alpha-tocopherol (alpha-T), and retinol (ROL) in mammals include taurocholate, protein, and fat. Few studies have examined the effects of these factors on the storage of BC, retinyl esters, and alpha-T in a mammalian system that is similar to humans. The main objective of the study was to investigate the effects of taurocholate (TC), fat, and protein on the absorption and metabolism of BC and alpha-T in ferret tissues. Three 4-week experiments were conducted using groups of 5-6 ferrets per treatment. All diets contained 0.2% BC. In Experiment 1, taurocholate was fed at concentrations of 0, 0.5, or 1%. Effects of two concentrations of dietary fat (6 and 23%) and three concentrations of protein (10, 20, and 40%) were also studied in Experiments 2 and 3, respectively. Tissues were analyzed for BC, retinoids, and alpha-T by high-pressure liquid chromatography (HPLC). Taurocholate enhanced hepatic and plasma concentrations of BC (2.3- to 3-fold), retinyl palmitate [(RP) 3.2- to 9.5-fold], retinyl stearate [(RS) 2.9- to 6- fold], and hepatic alpha-T (6- to 13- fold) at p < 0.05. High-fat diets elevated hepatic BC, RP, RS, and retinyl linoleate (RL) concentrations (2- to 3.6-fold, p < 0.05). In contrast, high-protein diets lowered hepatic RL 1.8-fold and alpha-T 8-fold (p < 0.05). Our results indicate the importance of taurocholate, fat, and protein in achieving adequate levels of vitamins A and E in mammals.

Effects of androstenedione on in utero development in rats.

This study was conducted to characterize the effect of androstenedione on estrous cyclicity, mating behavior and fetal development. Thirty-day old rats received corn oil alone or androstenedione (in corn oil) at one of four concentrations (0, 1.0, 5.0, 10.0 or 30.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and throughout gestation. Dose related increases in serum androstenedione, estradiol and estrone were observed in all androstenedione treated animals at gestation day 20. A statistically significant increase in serum testosterone concentration was observed in the 30 mg/kg dose group. Feed and fluid consumption were not affected by androstenedione treatment during the pre-mating or gestational periods, however a statistically significant decrease in the number of females with regular estrous cycles was observed in the 10.0 and 30.0 mg/kg dose groups. Exposure to androstenedione did not affect mean body weight gain during pre-mating or gestation. Slight not statistically significant reductions in the number of implants, number of viable fetuses and number of viable male fetuses were observed in the 30.0 mg/kg androstenedione group. Reductions were not observed in the number of corpora lutea. Fetal growth in terms of fetal weight, crown-rump length, anogenital distance and the number of external abnormalities was not affected by androstenedione exposure. At the doses given, androstenedione had no specific effect on the development of individual bones, including sternebrae. Dose related effects of androstenedione were not observed on the development of soft tissues. A statistically significant increase in moderately enlarged ureter at the kidney was observed in both the 1.0 and 5.0 mg/kg dose groups. Organ weights (expressed per gram of body weight or per gram of brain weight) were not affected by androstenedione treatment.

Lutein interacts with ascorbic acid more frequently than with alpha-tocopherol to alter biomarkers of oxidative stress in female zucker obese rats.

The influence of dietary lutein, with and without moderate amounts of vitamin C (VC) or vitamin E (VE), on biomarkers of oxidative stress was examined in rats. Nine groups of immature Zucker obese (fa/fa) and lean female rats (8/group) consumed ad libitum for 8 wk the AIN-93G diet (Control) to which was added either dl-alpha-tocopherol acetate (VE) at 0.60 mg/kg or ascorbic acid (VC) at 0.75 mg/kg diet. Each of these diets contained lutein oil (FloraGlo) at 0.5 (Lut0.5) or 1.0 (Lut1.0) mg/kg diet. Weight gain, food efficiency and relative liver weight were higher in obese than in lean rats. Although liver malondialdehyde (MDA) concentrations were significantly higher in obese than in lean rats, levels were significantly lower in obese rats fed VE, VE-Lut and VC-Lut0.5 compared with other obese groups. The accumulation of alpha-tocopherol in liver was 6- and 3-times greater in the VE and VE-Lut1.0 groups, respectively, compared with the obese and lean control groups. Lutein reduced the activity of superoxide dismutase (SOD) in obese rats, independent of VC or VE, and raised the activity of glutathione peroxidase to higher levels in lean rats when combined with VC. Plasma insulin levels were dramatically higher in obese compared with lean rats, but significantly lower in obese rats fed VC-Lut0.5, VE-Lut1.0 and Lut1.0 compared with the Control group. These results suggest that lutein independently reduces the activity of SOD and alters more biomarkers of oxidative stress when combined with vitamin C than with vitamin E, and that vitamin E reduces liver lipid peroxidation in obese rats when the accumulation of liver alpha-tocopherol is very high.

Measurement of cis and trans isomers of vitamin K1 in rat tissues by liquid chromatography with a C30 column.

The purpose of this study was to validate a method for measuring vitamin K isomers in rat tissues by liquid chromatography (LC) with fluorescence detection after simple solvent extraction. This method uses separation on a C30 column, followed by zinc reduction and fluorescence measurement (243 nm, excitation; 430 nm, emission) to detect and quantitate vitamin K isomers. We were able to separate cis- and trans-vitamin K1 in methylene chloride extracts of homogenized rat livers and in hexane extracts of rat plasma. Tissue extracts were evaporated and rediluted with tetrahydrofuran-methanol (1 + 1) or methanol before being injected under isocratic conditions onto the LC column. Liver tissue of Fischer 344 rats fed a vitamin K1-containing diet ad libitum contained approximately 20 and 60 ng/g cis- and trans-vitamin K1, respectively. Mean recoveries of vitamin K1 isomers from spiked liver were 92 +/- 11% for cis-vitamin K1 and 106 +/- 5% for trans-vitamin K1. We recovered 96 +/- 8% of trans-vitamin K1 added at 1, 3, and 6 ng/mL to plasma (containing an endogenous level of 4 ng/g) from the same rats; we recovered 112 +/- 5% when trans-vitamin K1 was added to human serum (National Institute of Standards and Technology Standard Reference Material 968C). This direct method shows significant potential for the selective measurement of vitamin K1 isomers in tissues.