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.

Quantification of aristolochic acids I and II in herbal dietary supplements by ultra-high-performance liquid chromatography-multistage fragmentation mass spectrometry.

A rapid, selective and sensitive ultra-high-performance liquid chromatography-multistage fragmentation mass spectrometry (UHPLC-MS³) method was developed and evaluated for the determination of aristolochic acids I and II (AA I and II) in herbal dietary supplements. A hybrid triple quadrupole/linear ion-trap mass spectrometry was used to monitor MS³ ion transitions m/z 359.2 > 298.1 > 268.0 and m/z 329.2 > 268.2 > 238.0 to detect AA I and II, respectively. The extraction and clean-up of target analytes from dry powdered samples was performed using the quick, easy, cheap, effective, rugged and safe (QuEChERS) procedure. Herbal liquid extracts were analysed directly. Average recoveries ranged from 89% to 112%, with relative standard deviations (RSDs) ranging from 3% to 16%. Limits of quantification (LOQs) estimated for three selected matrices were as follows (AA I/II): 5/10 ng g⁻¹ (tablets); 25/50 ng g⁻¹ (capsules); and 2.5/5.0 ng ml⁻¹ (liquid herbal extract). The method was applied in a limited survey of 30 herbal products marketed in the United States via the Internet. AA I and II were detected in 20% and 7%, respectively, of tested samples.

Targeted analysis of multiple pharmaceuticals, plant toxins and other secondary metabolites in herbal dietary supplements by ultra-high performance liquid chromatography-quadrupole-orbital ion trap mass spectrometry.

In this study, an ultra-high performance liquid chromatography-quadrupole-orbital ion trap mass spectrometry (UHPLC-Q-orbitrap MS) method was developed and validated for simultaneous determination of 96 pharmaceuticals, plant toxins, and other plant secondary metabolites in herbal dietary supplements. Target analytes were extracted from samples using the QuEChERS (quick easy cheap effective rugged safe) procedure. The instrument was operated in full MS-data dependent tandem mass spectrometry (full MS-dd-MS/MS) acquisition mode which enabled collection of quantitative high resolution (HR) full mass spectral data and confirmatory HR MS/MS data in a single run. The method provided excellent selectivity in both full MS and dd-MS/MS mode. Under optimized collision energy settings, product ion spectra containing both precursor and two or more product ions were obtained for most of the analytes. Limits of detection (LODs) and limits of quantification (LOQs) for the method differed significantly for the examined matrices. LODs≤10µg kg(-1) and LOQs≤50µg kg(-1) were obtained for 48 to 81% of target compounds across five different matrices. With the exception of highly polar analytes, the optimized QuEChERS extraction procedure provided acceptable recoveries in the range 70%-120%. The precision of the method, characterized as the relative standard deviation (RSD, n=5), was ≤25% and ≤18% at spiking concentrations of 50µg kg(-1) and 500µg kg(-1), respectively. Because of variations in matrix effects in extracts of herbal dietary supplements that differed in composition, the method of standard additions and an approach based on dilution of matrix components followed by quantification using solvent standards were applied for quantification. The procedure was used to examine commercial dietary supplements for the 96 analytes of interest. To the best of our knowledge, this is the first report of an integrated analysis and quantification of this wide range of compounds.

Determination of multiple mycotoxins in dietary supplements containing green coffee bean extracts using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 34 mycotoxins in dietary supplements containing green coffee bean (GCB) extracts was developed, evaluated, and used in the analysis of 50 commercial products. A QuEChERS-like procedure was used for isolation of target analytes from the examined matrices. Average recoveries of the analytes were in the range of 75-110%. The precision of the method expressed as relative standard deviation was below 12%. Limits of detection (LODs) and limits of quantitation (LOQs) ranged from 1.0 to 50.0 µg/kg and from 2.5 to 100 µg/kg, respectively. Due to matrix effects, the method of standard additions was used to ensure accurate quantitation. Ochratoxin A, ochratoxin B, fumonisin B1 and mycophenolic acid were found in 36%, 32%, 10%, and 16% of tested products, respectively. Mycotoxins occurred in the following concentration ranges: ochratoxin A, <1.0-136.9 µg/kg; ochratoxin B, <1.0-20.2 µg/kg; fumonisin B1, <50.0-415.0 µg/kg; mycophenolic acid, <5.0-395.0 µg/kg. High-resolution mass spectrometry operated in full MS and MS/MS mode was used to confirm the identities of the reported compounds.

Sweeteners from plants--with emphasis on Stevia rebaudiana (Bertoni) and Siraitia grosvenorii (Swingle).

In addition to their widely recognized use as dietary supplement ingredients, plant-derived compounds are increasingly used as natural sweeteners. The search for nonnutritive sweeteners has been stimulated over the last 20-30 years by concern over demonstrated or suspected relationships between consumption of sucrose and high-fructose corn syrups and a variety of health-related conditions. In the USA, there is increased use of plant extracts known to contain highly sweet terpenoids. Purified extracts of Stevia rebaudiana (Bertoni) containing the diterpene glycosides stevioside and rebaudioside A are popular as sweeteners and are also used as dietary supplements, and soft drinks and nutritional and energy shakes incorporating extracts of Siraitia grosvenorii (Swingle) fruits containing sweet triterpene glycosides such as mogroside V are also on the market. Here, we review recent studies on these two important sources of noncaloric natural sweeteners, including analytical methods used to identify and quantify specific constituents and structural features relating to their sweetness. We also review the generally recognized as safe status of specific components and their status with respect to review by the Joint FAO/WHO Expert Committee on Food Additives.

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.

Simultaneous analysis of steviol and steviol glycosides by liquid chromatography with ultraviolet detection on a mixed-mode column: application to Stevia plant material and Stevia-containing dietary supplements.

Simultaneous separation of steviol and steviol glycosides is challenging because of differences in their polarity and chemical structure. In this study, simultaneous analysis of steviol and steviol glycosides was achieved by LC with UV detection using a mixed-mode RP weak anion exchange chromatography column. Steviol and seven steviol glycosides were analyzed on an Acclaim Mixed-Mode Wax-1 (Dionex) column with a linear gradient of deionized water adjusted to pH 3.00 with phosphoric acid and acetonitrile. The extraction was performed by sonicating dry plant material at 40 degreesC in acetonitrile-water (30 + 70, v/v). LOQ values (mg/g dry weight of plant material) were rebaudioside B, 0.50; steviol, 0.70, dulcoside A, 1.0; steviolbioside, 1.2; stevioside and rebaudioside C, 2.0; rebaudioside D, 3.3; and rebaudioside A, 5.0. The method demonstrated suitable performance for all analytes tested with respect to accuracy (mean recoveries 95-99%), intraday and interday precision for retention times (0.070-0.28% and 0.33-1.0% RSD, respectively), and linearity. The method was used to authenticate steviol glycosides in several samples of Stevia plant material as well as to quantitate steviol glycosides in dietary supplements containing Stevia.

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.

Cucurbitane-type triterpenoids from Momordica charantia.

One new cucurbitane-type triterpenoid glycoside, momordicoside U (1), together with five known cucurbitane-type triterpenoids and related glycosides, 3ß,7 ß,25-trihydroxycucurbita-5,23 (E)-dien-19-al (2), momordicine I (3), momordicine II (4), 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-ß-glucopyranoside (5), and kuguaglycoside G (6), were isolated from the whole plant of Momordica charantia. Their structures were determined by chemical and spectroscopic methods. Momordicoside U (1) was evaluated for insulin secretion activity in an in vitro insulin secretion assay and displayed moderate activity.

Multiresidue pesticide analysis of ginseng powders using acetonitrile- or acetone-based extraction, solid-phase extraction cleanup, and gas chromatography-mass spectrometry/selective ion monitoring (GC-MS/SIM) or -tandem mass spectrometry (GC-MS/MS).

A multiresidue method for the analysis of 168 pesticides in dried powdered ginseng has been developed using acetonitrile or acetone mixture (acetone/cyclohexane/ethyl acetate, 2:1:1 v/v/v) extraction, solid-phase extraction (SPE) cleanup with octyl-bonded silica (C(8)), graphitized carbon black/primary-secondary amine (GCB/PSA) sorbents and toluene, and capillary gas chromatography-mass spectrometry/selective ion monitoring (GC-MS/SIM) or -tandem mass spectrometry (GC-MS/MS). The geometric mean limits of quantitation (LOQs) were 53 and 6 microg/kg for the acetonitrile extraction and 48 and 7 microg/kg for the acetone-based extraction for GC-MS/SIM and GC-MS/MS, respectively. Mean percent recoveries and standard deviations from the ginseng fortified at 25, 100, and 500 microg/kg using GC-MS/SIM were 87 +/- 10, 88 +/- 8, and 86 +/- 10% from acetonitrile extracts and 88 +/- 13, 88 +/- 12, and 88 +/- 14% from acetone mixture extracts, respectively. The mean percent recoveries from the ginseng at the 25, 100, and 500 microg/kg levels using GC-MS/MS were 83 +/- 19, 90 +/- 13, and 89 +/- 11% from acetonitrile extracts and 98 +/- 20, 91 +/- 13, and 88 +/- 14% from acetone extracts, respectively. Twelve dried ginseng products were found to contain one or more of the following pesticides and their metabolites: BHCs (benzene hexachlorides, alpha-, beta-, gamma-, and delta-), chlorothalonil, chlorpyrifos, DDT (dichlorodiphenyl trichloroethane), dacthal, diazinon, iprodione, quintozene, and procymidone ranging from <1 to >4000 microg/kg. No significant differences were found between the two extraction solvents, and GC-MS/MS was found to be more specific and sensitive than GC-MS/SIM. The procedures described were shown to be effective in screening, identifying, confirming, and quantitating pesticides in commercial ginseng products.

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.

Analysis of organophosphorus pesticides in dried ground ginseng root by capillary gas chromatography-mass spectrometry and -flame photometric detection.

A method was developed to determine organophosphorus pesticides (OPs) in dried ground ginseng root. Pesticides were extracted from the sample using acetonitrile/water saturated with salts, followed by solid-phase dispersive cleanup, and analyzed by capillary gas chromatography with electron ionization mass spectrometry in selective ion monitoring mode (GC-MS/SIM) and flame photometric detection (GC-FPD) in phosphorus mode. The detection limits for most of the pesticides were 0.025-0.05 microg/g using GC-FPD but were analyte-dependent for GC-MS/SIM, ranging from 0.005 to 0.50 microg/g. Quantitation was determined from 0.050 to 5.0 microg/g with r 2 > 0.99 for a majority of the pesticides using both detectors. Recovery studies were performed by fortifying the dried ground ginseng root samples to concentrations of 0.025, 0.1, and 1.0 microg/g, resulting in recoveries of >90% for most pesticides by GC-FPD. Lower (<70%) and higher (>120%) recoveries were most likely from complications of pesticide lability or volatility, matrix interference, or inefficient desorption from the solid-phase sorbents. There was difficulty in analyzing the ginseng samples for the OPs using GC-MS at the lower fortification levels for some of the OPs due to lack of confirmation. GC-FPD and GC-MS/SIM complement each other in detecting the OPs in dried ground ginseng root samples. This procedure was shown to be effective and was applied to the analysis of OPs in ginseng root samples. One particular sample, a ground and dried American ginseng (Panax quinquefolius) root sample, was found to contain diazinon quantified at approximately 25 microg/kg by external calibration using matrix-matched standards or standard addition using both detectors. The advantage of using both detectors is that confirmation can be achieved using GC-MS, whereas the use of a megabore column in GC-FPD can be used to quantitate some of the nonpolar OPs without the use of matrix-matched standards or standard addition.