Analytical and Bioanalytical Chemistry (v.405, #13)

Certified Reference Material recipe challenge by Catherine A. Rimmer; Melissa M. Phillips (4321-4322).

Functional foods and dietary supplements by Melissa M. Phillips; Catherine A. Rimmer (4323-4324).
are research chemists at the National Institute of Standards and Technology in Gaithersburg, MD, USA. They are involved in the certification efforts for food and dietary supplement Standard Reference Materials and are co-coordinators of the Dietary Supplement Laboratory Quality Assurance Program. Their interests include development of new analytical methods for the determination of marker compounds and vitamins in dietary supplements and foods, as well as improving the measurement capabilities of the dietary supplement and food communities. Here, they are enjoying the views from the steps of St. Stephen's Basilica in Budapest, Hungary.

Standard reference materials for food analysis by Melissa M. Phillips; Katherine E. Sharpless; Stephen A. Wise (4325-4335).
is a research chemist at the National Institute of Standards and Technology (NIST) in Gaithersburg, MD, USA. She is involved in the certification efforts for food and dietary supplement Standard Reference Materials (SRMs) and is the co-coordinator of the Dietary Supplement Laboratory Quality Assurance Program (DSQAP). Her interests include development of new analytical methods for the determination of marker compounds and vitamins in dietary supplements and foods, as well as improving the measurement capabilities of the dietary supplement and food communities. is a scientific advisor in the Chemical Sciences Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), in Gaithersburg, MD, USA. She has been responsible for production of many of NIST’s food-matrix Standard Reference Materials. is the Associate Chief of the Chemical Sciences Division in the Material Measurement Laboratory at the National Institute of Standards and Technology (NIST). He is responsible for coordinating the Food and Nutrition Program within the Material Measurement Laboratory at NIST. He is also an Editor of Analytical and Bioanalytical Chemistry.

Standard reference materials for dietary supplement analysis by Catherine A. Rimmer; Katherine E. Sharpless; Stephen A. Wise; Joseph M. Betz; Paul M. Coates (4337-4344).
received her Ph.D. in analytical chemistry from Florida State University and completed a National Research Council postdoctoral fellowship at the National Institute of Standards and Technology (NIST). In addition to coordinating a dietary supplement quality assurance program she has contributed to the certification of several dietary supplement Standard Reference Materials including Ginkgo biloba, Carrot Extract in Oil, Saw Palmetto Extract, and a Multivitamin/multielement tablet. is a scientific advisor in the Chemical Sciences Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), in Gaithersburg, MD, USA. She has been responsible for production of many of NIST’s food-matrix Standard Reference Materials. the Associate Chief of the Chemical Sciences Division in the Material Measurement Laboratory at the National Institute of Standards and Technology (NIST). He is responsible for coordinating the Food and Nutrition Program within the Material Measurement Laboratory at NIST. He is also an Editor of Analytical and Bioanalytical Chemistry. joined the ODS as Director of the Analytical Methods and Reference Materials Program in 2001. Prior to joining NIH, he spent 12 years as a research chemist at FDA’s Center for Food Safety and Applied Nutrition and 2 years as the Vice President for Scientific and Technical Affairs at the American Herbal Products Association. He earned his Ph.D. in Pharmacognosy at the Philadelphia College of Pharmacy and Science. His research interests lie in the areas of analytical methods for determination of botanical quality. He has served as AOAC International General Referee for Botanical Supplements and on three USP Expert Committees. directs the Office of Dietary Supplements (ODS) at the National Institutes of Health in its mission to strengthen knowledge and understanding of dietary supplements. In 2011, he received the prestigious Conrad A. Elvehjem Award from the American Society for Nutrition for public service in nutrition. Prior to his career at NIH, he was on the faculty of the Children’s Hospital of Philadelphia and the University of Pennsylvania School of Medicine.

Development challenges in the preparation of solution-based phytochemical and vitamin certified reference materials by Derrell Johnson; Mitzi Rettinger; Tamara Tarbox; Beth Marek; Sherri Pogue; Isil Dilek; Uma Sreenivasan (4345-4352).
is New Product Development Manager with Cerilliant Corporation. He joined Cerilliant in December 1999 as a synthesis chemist and, after six years in this position, accepted an opportunity in 2005 to work as an analytical chemist in Cerilliant’s Analytical Laboratory. In 2008 Derrell was promoted to his current position and serves on the New Product Executive Committee and the New Product Design Committee. He is responsible for initiating, researching, and planning new products for Cerilliant’s catalog offering. is Vice President, Sales and Marketing, for Cerilliant. She is responsible for management and oversight of all sales and marketing functions and serves on Cerilliant’s New Product Executive Committee. She began her career at Cerilliant in 1989 as a synthesis and analytical chemist. She is actively involved in the industry as a guest lecturer and expert panelist and has been published in many industry publications. She is a member of the American Association for Clinical Chemistry, American Association of Pharmaceutical Scientists, AOAC International, American Society for Mass Spectrometry, Society of Forensic Toxicologists and serves on the NSF International Joint Committee on Dietary Supplements. , a member of Cerilliant from 2007 to 2012, worked as an analytical chemist in Cerilliant’s Analytical Laboratory. Her responsibilities included research and development for Cerilliant’s catalog, custom projects for a variety of clients, development of analytical methods and standard operating procedures, and the performance of method transfers, verifications and validations. joined Cerilliant as a synthesis chemist in 2007 and has participated in the synthesis of numerous Cerilliant products, both catalog and custom. In early 2012 she moved to Cerilliant’s Analytical Laboratory where she contributes to research and development for Cerilliant catalog products and custom work principally utilizing NMR and LC-MS/MS techniques. She also serves on Cerilliant’s Safety Committee. became Cerilliant’s Chief Executive Officer and President in September 2005. Upon the acquisition by Sigma-Aldrich December 2010, she was named President of Cerilliant Corporation and serves as Site Director in the Sigma-Aldrich system. Today, she is responsible for all aspects of Cerilliant operations and serves on the New Product Executive Committee and is executive sponsor of the Safety Committee. began her career at Cerilliant in 2004 as an analytical chemist focusing on method development working with HPLC, LCMS, FT-IR, and NMR techniques. In 2008, she was promoted to Manager of the Analytical Laboratory. She is responsible for management and technical oversight of all Cerilliant analytical operations and also serves on Cerilliant’s New Product Design Committee. came to Cerilliant in 2000 as a synthesis chemist. She has worked in biomedical research and academic settings and has experience in synthetic and analytical chemistry, project design and management, and process development. In 2003, she was promoted to Manager of Synthesis Operations and in 2006 she was named Chief Science Advisor overseeing technical issues relating to Cerilliant products. She is a member of Cerilliant’s New Product Executive Committee, its New Product Design Committee, and the Management Safety Committee.

The role of good manufacturing practices for preventing dietary supplement adulteration by Victoria Whitsitt; Cindy Beehner; Cara Welch (4353-4358).
is the Scientific and Regulatory Affairs Manager for the Natural Products Association (NPA). In her position, she works with the association’s self-regulatory quality assurance and regulatory education programs, and manages the NPA GMP Certification Program. In this capacity, she works closely with program advisors, third-party auditors, and companies seeking NPA GMP certification. She has been involved in the development and revisions of the NPA GMP Standard and related program materials and GMP education for the past 13 years. is the President of QSD Consulting. Cindy has more than 25 years’ experience in quality assurance/quality control, regulatory affairs, and process/product development activities in the pharmaceutical, veterinary, dietary ingredient, dietary supplement, and food industries. Since the company’s start in 1997, Cindy has been actively performing corporate and college-level GMP training; third-party auditing, technical writing, and preparation of quality systems for her US and international clients. serves as Senior Vice President of Scientific and Regulatory Affairs at the NPA. She assists industry members to implement policies in response to government initiatives in the regulatory and policy arena, and works with members of Congress and their staff, and officials in the FDA and other agencies whose actions have direct impact on the natural products industry. She directs strategy development and provides guidance for the NPA scientific and regulatory programs, including the NPA’s Natural Seal certification and Dietary Supplement GMP certification.

Fiber is known to be an important part of our nutrition and has many positive health benefits, including weight management and maintaining heart health. In recent years, a number of new ingredients have been manufactured or isolated that are being used to increase the health benefits of a product. Some are used as prebiotics that stimulate the growth of the beneficial bacteria in the gut, or are used as replacements for sugars, starch, or fat in manufactured foods. Fiber supplements have also been produced that can be taken to provide additional fiber to the diet. The term “fiber” does not relate to a single analyte or entity, but instead relates to a multitude of components. This adds to the complexity of analytical testing as there are a number of AOAC International and AACC International official methods which have been validated and can be used. Although methods have been developed for specific fiber ingredients, a number of methods have also been developed to capture just “fiber”. These “fiber” methods will capture differing degrees of the different fiber ingredients, so knowledge of the fiber sources is critical. The net result is that a variety of testing approaches may be used, but caution must be exercised in order to ensure that the total fiber result is accurately determined. A critical review of available fiber methodology and possible testing approaches is presented, along with how to accurately interpret and understand results.
Keywords: Fiber; Oligosaccharide; Food analysis; Supplement; Nutrition

Updates on chemical and biological research on botanical ingredients in dietary supplements by Rahul S. Pawar; Hemlata Tamta; Jun Ma; Alexander J. Krynitsky; Erich Grundel; Wayne G. Wamer; Jeanne I. Rader (4373-4384).
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.
Keywords: Botanicals; Dietary supplements; Geranium; 1,3-Dimethylamylamine; Bitter melon; Momordicosides; Hepatotoxicity

Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health by Christian G. Krueger; Jess D. Reed; Rodrigo P. Feliciano; Amy B. Howell (4385-4395).
The “A-type” proanthocyanidins in cranberry fruit (Vaccinium macrocarpon Ait.) are bioactive components associated with prevention of urinary tract infections (UTI). Cranberry juice, fruit (fresh and dried), functional foods, and cranberry dietary supplements are promoted for prevention of UTI and for maintenance of urinary tract health (UTH), on the basis of their content of cranberry proanthocyanidins (c-PAC) with “A-type” interflavan bonds. With increasing consumer use of cranberries for maintenance of UTH and an expanding number of commercial cranberry products of different types, the availability of unified methods for measuring levels of c-PAC is important. This review discusses quantitative and qualitative analysis of c-PAC with “A-type” interflavan bonds in relation to their biological activity for UTI prevention. The integrity (including authenticity, standardization, efficacy, and safety) of cranberry fruit, juices, and dietary supplements may now be measured by using recent advances in mass spectrometry, liquid chromatography, production of c-PAC standards, and improved simple quantitative techniques.
Keywords: Cranberry; DMAC; Mass spectrometry; Urinary tract health; Proanthocyanidins

Sweeteners from plants—with emphasis on Stevia rebaudiana (Bertoni) and Siraitia grosvenorii (Swingle) by Rahul S. Pawar; Alexander J. Krynitsky; Jeanne I. Rader (4397-4407).
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.
Keywords: Natural sweeteners; Stevia rebaudiana ; Steviol glycosides; Siraitia grosvenorii ; Mogrosides; Dietary supplements; Generally recognized as safe status

Dietary supplements containing dried roots or extracts of the roots and/or rhizomes of blue cohosh (Caulophyllum thalictroides) are widely available. This botanical has a long history of use by Native Americans and its use continues to the present day. The primary constituents of blue cohosh are its alkaloids and saponins. The structures of the alkaloids magnoflorine, baptifoline, anagyrine, and N-methylcytisine have been known for many years. The last 10 years have seen a great increase in isolation and identification of the large number of saponins present in blue cohosh. Important developments in nuclear magnetic resonance techniques have contributed substantially to the increase in elucidation of the structures of the complex saponins. Several authors have described quantitative methods for both the alkaloids and saponins in blue cohosh. Such methods have made it possible to quantify these constituents in dietary supplements containing this botanical ingredient. Concentrations of both alkaloids and saponins vary substantially in dietary supplements of blue cohosh. The nicotinic alkaloid, N-methylcytisine, a potent toxicant, has been found in all dietary supplements of blue cohosh analyzed. The teratogenic alkaloid anagyrine has been found in some but not all dietary supplements.
Keywords: Blue cohosh; Caulophyllum thalictroides ; Alkaloids; Saponins; Dietary supplements

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.
Keywords: Adulteration; Authentication; Botanical identification; Infraspecific variation; Quality control

The content of total and inorganic arsenic was determined in 16 dietary supplements based on herbs, other botanicals and algae purchased on the Danish market. The dietary supplements originated from various regions, including Asia, Europe and USA. The contents of total and inorganic arsenic was determined by inductively coupled plasma mass spectrometry (ICP-MS) and anion exchange HPLC-ICP-MS, respectively, were in the range of 0.58 to 5.0 mgkg−1 and 0.03 to 3.2 mg kg−1, respectively, with a ratio between inorganic arsenic and total arsenic ranging between 5 and 100 %. Consumption of the recommended dose of the individual dietary supplement would lead to an exposure to inorganic arsenic within the range of 0.07 to 13 μg day−1. Such exposure from dietary supplements would in worst case constitute 62.4 % of the range of benchmark dose lower confidence limit values (BMDL01 at 0.3 to 8 μg kg bw−1 kg−1 day−1) put down by European Food Safety Authority (EFSA) in 2009, for cancers of the lung, skin and bladder, as well as skin lesions. Hence, the results demonstrate that consumption of certain dietary supplements could contribute significantly to the dietary exposure to inorganic arsenic at levels close to the toxicological limits established by EFSA.
Keywords: Dietary supplements; Inorganic arsenic; Speciation analysis; HPLC-ICP-MS; Dietary exposure

The development and implementation of quality assurance programs to support nutritional measurements by L. C. Sander; M. Bedner; D. L. Duewer; K. A. Lippa; M. M. Phillips; K. W. Phinney; C. A. Rimmer; M. M. Schantz; K. E. Sharpless; S. S. -C. Tai; J. B. Thomas; S. A. Wise; L. J. Wood; J. M. Betz; P. M. Coates (4437-4441).
The National Institute of Standards and Technology administers quality assurance programs devoted to improving measurements of nutrients and related metabolites in foods, dietary supplements, and serum and plasma samples. These programs have been developed in collaboration with the National Institutes of Health to assist measurement communities in their efforts to achieve accurate results that are comparable among different laboratories and over time. Targeted analytes include micronutrients, botanical markers, nutritional elements, contaminants, fatty acids, and vitamin D metabolites.
Keywords: Quality assurance; Accuracy; Precision; Comparability; Concordance; Nutrients; Dietary supplements; Vitamin D metabolites; Fatty acids

Isolation and structural elucidation of a new sildenafil analogue from a functional coffee by Lin Li; Min-Yong Low; Xiaowei Ge; Bosco C. Bloodworth; Hwee-Ling Koh (4443-4450).
A sildenafil analogue was detected in a functional coffee sample labelled to have male sexual performance enhancement effects. This analogue was isolated and purified by flash chromatography and preparative high-performance liquid chromatography. Its structure was elucidated using high-resolution mass spectrometry; electrospray ionization-tandem mass spectrometry; and nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and infrared spectroscopy. Compared with sildenafil, instead of an N-methylpiperazinyl moiety, ring opening of the piperazine ring with the loss of a carbon atom resulted in a substituted benzenesulfonamide. The chemical name of this analogue is N-[2-(dimethylamino)ethyl]-4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)benzenesulfonamide. It is named descarbonsildenafil because it has one less carbon atom when compared with sildenafil.
Keywords: Coffee; Functional food; Food contaminants; Sildenafil; Adulteration; Erectile dysfunction

Developing qualitative LC-MS methods for characterization of Vaccinium berry Standard Reference Materials by Mark S. Lowenthal; Melissa M. Phillips; Catherine A. Rimmer; Paul A. Rudnick; Yamil Simón-Manso; Stephen E. Stein; Dmitrii Tchekhovskoi; Karen W. Phinney (4451-4465).
Standard Reference Materials (SRMs) offer the scientific community a stable and homogenous source of material that holds countless application possibilities. Traditionally, the National Institute of Standards and Technology (NIST) has provided SRMs with associated quantitative information (certified values) for a select group of targeted analytes as measured in a solution or complex matrix. While the current needs of the SRM community are expanding to include non-quantitative data, NIST is attempting to broaden the scope of how and what information is offered to the SRM community by providing qualitative information about biomaterials, such as chromatographic fingerprints and profiles of untargeted identifications. In this work, metabolomic and proteomic profiling efforts were employed to characterize a suite of six Vaccinium berry SRMs. In the discovery phase, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data was matched to mass spectral libraries; a subsequent validation phase based on multiple-reaction monitoring LC-MS/MS relied on both retention time matching of authentic standards along with fragmentation data for a qualitative overview of the most prominent organic compounds present. Definitive and putative identifications were determined for over 70 metabolites based on reporting guidelines set forth by the Metabolomics Standards Initiative (Metabolomics 3(3):211–221, 2007), and the capability of electrospray ionization mass spectrometry (ESI-MS) to profile untargeted metabolites within a complex matrix using mass spectral matching is demonstrated. Bottom-up proteomic analyses were possible using peptide databases translated from expressed sequence tags (ESTs). Homology searches provided identification of novel Vaccinium proteins based on homology to related genera. Chromatographic fingerprints of these berry materials were acquired for supplemental qualitative information to be provided to users of these SRMs. An unbounded set of qualitative data about a biomaterial is a valuable complement to quantitative information traditionally provided in NIST Certificates of Analysis.
Keywords: Vaccinium ; LC-MS; Qualitative analysis; Reference materials; Mass spectral library; Metabolites

Characterizing Vaccinium berry Standard Reference Materials by GC‐MS using NIST spectral libraries by Mark S. Lowenthal; Nirina R. Andriamaharavo; Stephen E. Stein; Karen W. Phinney (4467-4476).
A gas chromatography–mass spectrometry (GC-MS)-based method was developed for qualitative characterization of metabolites found in Vaccinium fruit (berry) dietary supplement Standard Reference Materials (SRMs). Definitive identifications are provided for 98 unique metabolites determined among six Vaccinium-related SRMs. Metabolites were enriched using an organic liquid/liquid extraction, and derivatized prior to GC-MS analysis. Electron ionization (EI) fragmentation spectra were searched against EI spectra of authentic standards compiled in the National Institute of Standards and Technology’s mass spectral libraries, as well as spectra selected from the literature. Metabolite identifications were further validated using a retention index match along with prior probabilities and were compared with results obtained in a previous effort using collision-induced dissociation (CID) MS/MS datasets from liquid chromatography coupled to mass spectrometry experiments. This manuscript describes a nontargeted metabolite profile of Vaccinium materials, compares results among related materials and from orthogonal experimental platforms, and discusses the feasibility and development of using mass spectral library matching for nontargeted metabolite identification.
Keywords: Vaccinium ; GC-MS; Qualitative analysis; Reference materials; Mass spectral library; Metabolites

Angelica sinensis (Oliv.) Diels (“Danggui” in Chinese) is one of the most commonly used traditional Chinese medicines. It has been used to invigorate blood circulation for the treatment of anemia, hypertension, chronic bronchitis, asthma, rheumatism, and cardiovascular diseases. There are a number of A. sinensis-derived dietary supplements in the US markets. However, no study have been conducted to investigate the quality of these dietary supplements. In this paper, high-performance liquid chromatographic and flow-injection mass spectrometric fingerprints were both evaluated to assess the consistency of A. sinensis-derived dietary supplements. Similarity analysis was carried out on the high-performance liquid chromatographic (HPLC) fingerprints. Meanwhile, principal component analysis (PCA) was performed on the data obtained from flow-injection mass spectrometric (FIMS) fingerprints, which can analyze each sample in 2 min, compared with 30 min required for the chromatographic fingerprint. Both methods show significant chemical differences between samples that may be due to differences in growing locations, growing conditions, harvesting times, and/or botanical processing. The loading plots obtained from PCA singled out the discriminatory ions that were responsible for chemical differences of A. sinensis-derived dietary supplements. Fig In the present study, HPLC and flow-injection mass spectrometric fingerprints as well as chemometrics were applied to assess the consistency of A. sinensis-derived dietary supplements from U.S. markets in order to understand the variability of the products and to provide useful information with customers.
Keywords: Angelica sinensis (Oliv.) Diels; Dietary supplements; Chromatographic Fingerprint; Flow-injection mass spectrometric fingerprint; Principal component analysis

Liquid chromatography coupled to multistage mass spectrometry (LC-MSn) is being used increasingly in pharmaceutical research and for quality control in herbal medicines because of its superior sensitivity and selectivity. In this study, a rapid, high-resolution liquid chromatography-mass spectrometry (LC-MSn) method was developed to separate and identify alkaloids in the root extract of goldenseal, which is one of the 20 most popular herbal supplements used worldwide. In total, 28 alkaloids were separated and characterized including one novel compound and 21 identified, or tentatively identified, for the first time in goldenseal. The current high-resolution LC-MSn method provides a rapid and definitive means of profiling the composition of goldenseal root and will provide a useful tool in understanding the bioactivity of this medicinal plant. Figure Extraction and Orbitrap LC-MSn analysis of Goldenseal root for alkaloid identification
Keywords: Hydrastis canadensis ; Alkaloid; Structural characterization; LC-ESI-MSn

Distinguishing Ontario ginseng landraces and ginseng species using NMR-based metabolomics by Jimmy Yuk; Kristina L. McIntyre; Christian Fischer; Joshua Hicks; Kimberly L. Colson; Ed Lui; Dan Brown; John T. Arnason (4499-4509).
The use of 1H-NMR-based metabolomics to distinguish and identify unique markers of five Ontario ginseng (Panax quinquefolius L.) landraces and two ginseng species (P. quinquefolius and P. ginseng) was evaluated. Three landraces (2, 3, and 5) were distinguished from one another in the principal component analysis (PCA) scores plot. Further analysis was conducted and specific discriminating metabolites from the PCA loadings were determined. Landraces 3 and 5 were distinguishable on the basis of a decreased NMR intensity in the methyl ginsenoside region, indicating decreased overall ginsenoside levels. In addition, landrace 5 was separated by an increased amount of sucrose relative to the rest of the landraces. Landrace 2 was separated from the rest of the landraces by the increased level of ginsenoside Rb1. The Ontario P. quinquefolius was also compared with Asian P. ginseng by PCA, and clear separation between the two groups was detected in the PCA scores plot. The PCA loadings plot and a t-test NMR difference plot were able to identify an increased level of maltose and a decreased level of sucrose in the Asian ginseng compared with the Ontario ginseng. An overall decrease of ginsenoside content, especially ginsenoside Rb1, was also detected in the Asian ginseng’s metabolic profile. This study demonstrates the potential of NMR-based metabolomics as a powerful high-throughput technique in distinguishing various closely related ginseng landraces and its ability to identify metabolic differences from Ontario and Asian ginseng. The results from this study will allow better understanding for quality assessment, species authentication, and the potential for developing a fully automated method for quality control. Figure Principal component analysis scores and loadings plot for differentiating between closely-related ginseng landraces in Ontario, Canada
Keywords: Metabonomics; Metabolite profiling; Quality control; Botanicals; Nuclear Magnetic Resonance and authenticity

New approaches for the recovery of ginsenosides are presented that greatly simplify the liquid chromatographic (LC) determination of the total content of eight ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg2) in powdered Panax ginseng rhizomes. The extraction protocols not only recover the neutral ginsenosides, but also simultaneously incorporate base-catalyzed hydrolysis of the malonyl-ginsenosides using dilute potassium hydroxide added to the methanol–water extractant. This eliminates the need for an independent extraction step followed by acid- or base-catalyzed hydrolysis. Both ultrasonically-assisted and microwave-assisted extraction methods are developed. The optimization of these simplified methods to remove pendant malonate esters, while retaining the glycosidic linkages, was determined by LC through variation of the extraction/hydrolysis time, order of hydrolysis reagent addition, and evaluation of multiple extractions. A comparison of the ginsenoside profiles obtained with and without addition of base to the extractant solution was made using LCMS with positive-mode electrospray ionization (ESI+) detection. A number of malonyl-ginsenosides were tentatively identified by their mass spectral fragmentation spectra and indicating that they were converted to the free ginsenosides by the new extraction/hydrolysis procedure. Figure LCUV chromatograms for different extraction solvents
Keywords: Ginsenoside; Liquid Chromatography with Ultraviolet/Visible Detection (LCUV); LC with Electrospray Mass Spectrometry (LCMS); Panax ginseng ; Malonyl-ginsenoside; Ultrasonically-assisted solvent extraction; Microwave-assisted solvent extraction

Ginseng (Panax ginseng C. A. Meyer) has been one of the most popular herbs used for nutritional and medicinal purposes by the people of eastern Asia for thousands of years. Ginsenosides, the mostly widely studied chemical components of ginseng, are quite different depending on the processing method used. A number of studies demonstrate the countercurrent chromatography (CCC) separation of ginsenosides from several sources; however, there is no single report demonstrating a one-step separation of all of these ginsenosides from different sources. In the present study, we have successfully developed an efficient CCC separation methodology in which the flow-rate gradient technique was coupled with a new solvent gradient dilution strategy for the isolation of ginsenosides from Korean white (peeled off dried P. ginseng) and red ginseng (steam-treated P. ginseng). The crude samples were initially prepared by extraction with butanol and were further purified with CCC using solvent gradients composed of methylene chloride–methanol–isopropanol–water (different ratios, v/v). Gas chromatography coupled with flame ionization detector was used to analyze the components of the two-phase solvent mixture. Each phase solvent mixture was prepared without presaturation, which saves time and reduces the solvent consumption. Finally, 13 ginsenosides have been purified from red ginseng with the new technique, including Rg1, Re, Rf, Rg2, Rb1, Rb2, Rc, Rd, Rg3, Rk1, Rg5, Rg6, and F4. Meanwhile, eight ginsenosides have been purified from white ginseng, including Rg1, Re, Rf, Rh1, Rb1, Rb2, Rc, and Rd by using a single-solvent system. Thus, the present technique could be used for the purification of ginsenosides from all types’ ginseng sources. To our knowledge, this is the first report involving the separation of ginsenoside Rg2 and Rg6 and the one-step separation of thirteen ginsenosides from red ginseng by CCC.
Keywords: Countercurrent chromatography; Fourteen ginsenosides; Stepwise gradient; Evaporative light-scattering detection

Development of botanical and fish oil standard reference materials for fatty acids by Michele M. Schantz; Lane C. Sander; Katherine E. Sharpless; Stephen A. Wise; James H. Yen; Agnes NguyenPho; Joseph M. Betz (4531-4538).
As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed Standard Reference Material (SRM) 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids and SRM 3275 Omega-3 and Omega-6 Fatty Acids in Fish Oil. SRM 3274 consists of one ampoule of each of four seed oils (3274-1 Borage (Borago officinalis), 3274-2 Evening Primrose (Oenothera biennis), 3274-3 Flax (Linium usitatissimum), and 3274-4 Perilla (Perilla frutescens)), and SRM 3275 consists of two ampoules of each of three fish oils (3275-1 a concentrate high in docosahexaenoic acid, 3275-2 an anchovy oil high in docosahexaenoic acid and eicosapentaenoic acid, and 3275-3 a concentrate containing 60 % long-chain omega-3 fatty acids). Each oil has certified and reference mass fraction values for up to 20 fatty acids. The fatty acid mass fraction values are based on results from analyses using gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS). These SRMs will complement other reference materials currently available with mass fractions for similar analytes and are part of a series of SRMs being developed for dietary supplements. Figure Components of SRM 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids
Keywords: Fatty acid; Omega-3; Omega-6; Plant oil; Fish oil; Reference material

Characterization of NIST food-matrix standard reference materials for their vitamin C content by Jeanice B. Thomas; James H. Yen; Katherine E. Sharpless (4539-4548).
The vitamin C concentrations in three food-matrix Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) have been determined by liquid chromatography (LC) with absorbance detection. These materials (SRM 1549a Whole Milk Powder, SRM 1849a Infant/Adult Nutritional Formula, and SRM 3233 Fortified Breakfast Cereal) have been characterized to support analytical measurements made by food processors that are required to provide information about their products’ vitamin C content on the labels of products distributed in the United States. The SRMs are primarily intended for use in validating analytical methods for the determination of selected vitamins, elements, fatty acids, and other nutrients in these materials and in similar matrixes. They can also be used for quality assurance in the characterization of test samples or in-house control materials, and for establishing measurement traceability. Within-day precision of the LC method used to measure vitamin C in the food-matrix SRMs characterized in this study ranged from 2.7 % to 6.5 %.
Keywords: Ascorbic acid; Food; Reference material; Standard reference material; Liquid chromatography; Absorbance detection

A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product by Zia Fazili; Ralph D. Whitehead Jr; Neelima Paladugula; Christine M. Pfeiffer (4549-4560).
Small specimen volume and high sample throughput are key features needed for routine methods used for population biomonitoring. We modified our routine eight-probe solid phase extraction (SPE) LC-MS/MS method for the measurement of five folate vitamers [5-methyltetrahydrofolate (5-methylTHF), folic acid (FA), plus three minor forms: THF, 5-formylTHF, 5,10-methenylTHF] and one oxidation product of 5-methylTHF (MeFox) to require less serum volume (150 μL instead of 275 μL) by using 96-well SPE plates with 50 mg instead of 100 mg phenyl sorbent and to provide faster throughput by using a 96-probe SPE system. Total imprecision (10 days, two replicates/day) for three serum quality control pools was 2.8–3.6 % for 5-methylTHF (19.5–51.1 nmol/L), 6.6–8.7 % for FA (0.72–11.4 nmol/L), and ≤11.4 % for the minor folate forms (<1–5 nmol/L). The mean (±SE) recoveries of folates spiked into serum (3 days, four levels, two replicates/level) were: 5-methylTHF, 99.4 ± 3.6 %; FA, 100 ± 1.8 %; minor folates, 91.7–108 %. SPE extraction efficiencies were ≥85 %, except for THF (78 %). Limits of detection were ≤0.3 nmol/L. The new method correlated well with our routine method [n = 150, r = 0.99 for 5-methylTHF, FA, and total folate (tFOL, sum of folate forms)] and produced slightly higher tFOL (5.6 %) and 5-methylTHF (7.3 %) concentrations, likely due to the faster 96-probe SPE process (1 vs. 5 h), resulting in improved SPE efficiency and recovery compared to the eight-probe SPE method. With this improved LC-MS/MS method, 96 samples can be processed in ∼2 h, and all relevant folate forms can be accurately measured using a small serum volume. Figure High-throughput LC-MS/MS method for population monitoring of serum folate forms
Keywords: Automated solid phase extraction; MeFox; hmTHF; Method comparison; Microbiologic assay; Anticoagulant types

The National Institute of Standards and Technology (NIST) is developing a wide variety of Standard Reference Materials (SRMs) to support measurements of vitamins and other nutrients in foods. Previously, NIST has provided SRMs with values assigned for the folate vitamer, folic acid (pteroylglutamic acid), which is fortified in several foods due to its role in prevention of neural tube defects. In order to expand the number of food-based SRMs with values assigned for folic acid, as well as additional endogenous folates, NIST has developed methods that include trienzyme digestion and isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Sample preparation was optimized for each individual food type, but all samples were analyzed under the same LC-MS/MS conditions. The application of these methods resulted in folic acid values for SRM 1849a Infant/Adult Nutritional Formula and SRM 3233 Fortified Breakfast Cereal of (2.33 ± 0.06) μg/g and (16.0 ± 0.7) μg/g, respectively. In addition, the endogenous folate vitamer 5-methlytetrahydrofolate (5-MTHF) was detected and quantified in SRM 1849a Infant/Adult Nutritional Formula, candidate SRM 1549a Whole Milk Powder, and candidate SRM 1845a Whole Egg Powder, resulting in values of (0.0839 ± 0.0071) μg/g, (0.211 ± 0.014) μg/g, and (0.838 ± 0.044) μg/g, respectively. SRM 1849a Infant/Adult Nutritional Formula is the first food-based NIST SRM to possess a reference value for 5-MTHF and the first certified reference material to have an assigned 5-MTHF value based on LC-MS/MS. The values obtained for folic acid and 5-MTHF by LC-MS/MS will be incorporated into the final value assignments for all these food-based SRMs.
Keywords: Folate; Fortification; Reference materials; LC-MS/MS

Breakfast cereal sampling study for nutritional elements by Laura J. Wood; Katrice A. Lippa; Melissa M. Phillips; Catherine A. Rimmer; N. Alan Heckert; Stefan D. Leigh; Amanda J. Moors; Rebecca S. Pugh; Lauren B. Rust (4569-4578).
The National Institute of Standards and Technology (NIST) has established a Dietary Supplement Laboratory Quality Assurance Program (DSQAP) in collaboration with the National Institutes of Health Office of Dietary Supplements (NIH-ODS). The DSQAP invites laboratories twice annually to participate in interlaboratory studies where participants elect to measure concentrations of nutritional and/or toxic elements as well as active and/or marker compounds. One of these studies was designed to determine the effects of material granularity and sample processing techniques on measurement variability (precision) as well as to provide participating laboratories information on their performance relative to the NIST assigned values (bias) and to the other participants (concordance). Participants were asked to determine the mass fractions of Ca, Fe, and Zn, in mg/kg, in six breakfast cereal samples. Cereal samples consisted of three ground materials (homogenized wheat, wheat, and rice), two flake materials (wheat and rice) and a partially crushed material (a wheat/rice mixture). In general, approximately 25 % of the laboratories processed and analyzed the suite of six cereal materials with adequate to exemplary measurement precision. Over half of the laboratories (60 %) experienced measurement issues related to only a particular type of cereal matrix or for only a single element. A small number (15 %) of laboratories experienced significant sample processing or measurement problems. Future studies planned by the DSQAP may be designed to use commercial products to aid laboratories with their sampling and analytical techniques. Figure Cereal processing method using mortar & pestle
Keywords: Sampling; Nutritional analysis; Food; Reference materials; Nutritional elements

The potential effect of spectral interference on the accurate measurement of the cadmium (Cd) mass fraction in fortified breakfast cereal and a variety of dietary supplement materials using inductively coupled plasma quadrupole mass spectrometry was studied. The materials were two new standard reference materials (SRMs)—SRM 3233 Fortified Breakfast Cereal and SRM 3532 Calcium Dietary Supplement—as well as several existing materials—SRM 3258 Bitter Orange Fruit, SRM 3259 Bitter Orange Extract, SRM 3260 Bitter Orange-containing Solid Oral Dosage Form, and SRM 3280 Multivitamin/Multielement Tablets. Samples were prepared for analysis using the method of isotope dilution and measured using various operating and sample introduction configurations including standard mode, collision cell with kinetic energy discrimination mode, and standard mode with sample introduction via a desolvating nebulizer system. Three isotope pairs, 112Cd/111Cd, 113Cd/111Cd, and 114Cd/111Cd, were measured. Cadmium mass fraction results for the unseparated samples of each material, measured using the three instrument configurations and isotope pairs, were compared to the results obtained after the matrix was removed via chemical separation using anion exchange chromatography. In four of the six materials studied, measurements using the standard mode with sample introduction via the desolvating nebulizer gave results for the unseparated samples quantified with the 112Cd/111Cd isotope pair that showed a positive bias relative to the matrix-separated samples, which indicated a persistent inference at m/z 112 with this configuration. Use of the standard mode, without the desolvating nebulizer, also gave results that showed a positive bias for the unseparated samples quantified with the 112Cd/111Cd isotope pair in three of the materials studied. Collision cell/kinetic energy discrimination mode, however, was very effective for reducing spectral interference for Cd in all of the materials and isotope pairs studied, except in the multivitamin/multielement matrix (SRM 3280) where the large corrections for known isobaric interferences or unidentified interferences compromised the accuracy. For SRM 3280, matrix separation provided the best method to achieve accurate measurement of Cd.
Keywords: Foods; Mass spectrometry; ICP-MS; Metals; Heavy metals; Reference materials; Trace elements; Separations; Instrumentation

Nutraceuticals and separations by Luigi Mondello (4589-4590).
is Full Professor of Analytical Chemistry at the University of Messina, Italy, and Campus Bio-Medico University in Rome, Italy. He is the author of 200 scientific articles, 29 book chapters, and 11 reviews, he is the editor and co-editor of two books. He was chairman of the 36th International Symposium on Capillary Chromatography, the 9th GC×GC Symposium (Riva del Garda, Italy, 2012), and the 14th International Symposium on Advances in Extraction Technologies (Messina, Italy, 2012). He is a member of the Steering Committee of the Italian Separation Science Group of the Italian Chemical Society, a member of the Chromedia (Chromatography Knowledge Base) expert team, and co-president and co-founder of the Mediterranean Separation Science Foundation Training and Research Center. His research interests include the development of multidimensional and comprehensive techniques (LC–GC–MS, GC–GC, GC×GC, LC×LC, LC×GC), and their application for the analysis of natural complex matrices.

Innovative analytical tools to characterize prebiotic carbohydrates of functional food interest by Claudio Corradini; Claudia Lantano; Antonella Cavazza (4591-4605).
Functional foods are one of the most interesting areas of research and innovation in the food industry. A functional food or functional ingredient is considered to be any food or food component that provides health benefits beyond basic nutrition. Recently, consumers have shown interest in natural bioactive compounds as functional ingredients in the diet owing to their various beneficial effects for health. Water-soluble fibers and nondigestible oligosaccharides and polysaccharides can be defined as functional food ingredients. Fructooligosaccharides (FOS) and inulin are resistant to direct metabolism by the host and reach the caecocolon, where they are used by selected groups of beneficial bacteria. Furthermore, they are able to improve physical and structural properties of food, such as hydration, oil-holding capacity, viscosity, texture, sensory characteristics, and shelf-life. This article reviews major innovative analytical developments to screen and identify FOS, inulins, and the most employed nonstarch carbohydrates added or naturally present in functional food formulations. High-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED) is one of the most employed analytical techniques for the characterization of those molecules. Mass spectrometry is also of great help, in particularly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is able to provide extensive information regarding the molecular weight and length profiles of oligosaccharides and polysaccharides. Moreover, MALDI-TOF-MS in combination with HPAEC-PED has been shown to be of great value for the complementary information it can provide. Some other techniques, such as NMR spectroscopy, are also discussed, with relevant examples of recent applications. A number of articles have appeared in the literature in recent years regarding the analysis of inulin, FOS, and other carbohydrates of interest in the field and they are critically reviewed.
Keywords: Fructooligosaccharides; High-performance anion-exchange chromatography with pulsed electrochemical detection; Inulin; Matrix-assisted laser desorption/ionization time of flight-mass spectrometry; Prebiotics

Optimization of clean extraction methods to isolate carotenoids from the microalga Neochloris oleoabundans and subsequent chemical characterization using liquid chromatography tandem mass spectrometry by María Castro-Puyana; Miguel Herrero; Iratxe Urreta; Jose A. Mendiola; Alejandro Cifuentes; Elena Ibáñez; Sonia Suárez-Alvarez (4607-4616).
A novel experimental design was used to optimize the extraction of carotenoids from Neochloris oleoabundans using pressurized liquid extraction with food-grade solvents such as ethanol and limonene. Experimental factors, including the extraction temperature and the solvent composition, were optimized using a three-level factorial design. The response variables extraction yield and total amount of carotenoids were assessed. The statistical analysis of the results provided mathematical models to predict the behavior of the responses as a function of the factors involved in the process. The optimum conditions predicted by the model developed in this study were 112 °C as the extraction temperature and 100 % ethanol as the extraction solvent. Chemical characterization of the extracts obtained was performed by means of high-performance liquid chromatography–tandem mass spectrometry. The results obtained demonstrated that, under certain growth conditions (photoautotrophically cultured in a medium supplemented with 0.3 g L−1 KNO3), N. oleoabundans accumulated significant total amounts of the carotenoids (from 57.4 to 120.2 mg carotenoids per gram of extract depending on the extraction conditions), mainly lutein, cantaxanthin, zeaxanthin, and astaxanthin monoesters and diesters.
Keywords: Pressurized liquid extraction; Carotenoids; Microalga; Neochloris oleoabundans ; Experimental design; Limonene

Profiling and quantifying polar lipids in milk by hydrophilic interaction liquid chromatography coupled with evaporative light-scattering and mass spectrometry detection by Marina Russo; Filomena Cichello; Carla Ragonese; Paola Donato; Francesco Cacciola; Paola Dugo; Luigi Mondello (4617-4626).
In this work, a high-performance liquid chromatography with evaporative light scattering detection method has been developed and applied for quantification of the polar content of the lipid fraction in milk samples of different origin. From a chromatographic stand-point, a 4.6-mm I.D. hydrophilic interaction liquid chromatography column was employed to attain a baseline separation of major phospholipid classes contained in the various milk samples tested. Quantitative analysis was performed by the external calibration method using reference material solutions in the 5–100 mg/L concentration range. Analytical recoveries ranging from 57 to 100 %, and repeatability data lower than 8.04 % were obtained on a skimmed cow’s milk sample. The crude cow milk was the most abundant (0.04 %) in phospholipids and donkey milk was the poorest (0.004 %). Quantitative differences were determined in the phospholipid content of the milk samples tested. Finally, characterization of phospholipid profile and fatty acid composition of the different samples was carried out by an ion trap-time of flight mass spectrometer and gas chromatography coupled to flame ionization and mass spectrometry detection. A thorough screening of the polar lipid composition of milk samples of different origin is here outlined, for the first time.
Keywords: Phospholipids; Milk; HILIC; ELSD; MS-IT-TOF

In this work, the development and optimization of a new methodology to analyze grape seed procyanidins based on the application of two-dimensional comprehensive LC is presented. This two-dimensional method involves the use of a microbore column containing a diol stationary phase in the first dimension coupled to either a C18 partially porous short column or a C18 monolithic column in the second dimension. The orthogonal hydrophilic interaction × reversed phase liquid chromatography (HILIC×RP-LC) system is interfaced through a ten-port two-position switching valve. The optimized HILIC×RP-LC separation followed by diode array and tandem mass spectrometry detection (HILIC×RP-LC-DAD-MS/MS) made possible the direct analysis of a complex grape seed extract and allowed the tentative identification of 43 flavan-3-ols, including monomers and procyanidin oligomers till a polymerization degree of 7 units with different galloylation degrees. To the best of our knowledge, this is the first time that this powerful analytical technique is employed to characterize complex procyanidin samples. This work successfully demonstrates the great capabilities of the HILIC×RP-LC-DAD-MS/MS coupling for the direct analysis of very complex natural samples like grape seeds. Figure Two-dimensional HILIC x RP plot (280 nm) of grape seed procyanidins.
Keywords: Flavan-3-ols; Grape seed procyanidins; HILIC; LC × LC; Mass spectrometry; Procyanidins; Two-dimensional comprehensive LC

Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation—application to the quantitative analysis of furanocoumarins in apiaceous vegetables by Elliot D. Larson; Stephen R. Groskreutz; David C. Harmes; Ian C. Gibbs-Hall; Sabrina P. Trudo; Robert C. Allen; Sarah C. Rutan; Dwight R. Stoll (4639-4653).
Various implementations of two-dimensional high-performance liquid chromatography are increasingly being developed and applied to the analysis of complex materials, including those encountered in the analysis of foods, beverages, and nutraceuticals. Previously, we introduced the concept of selective comprehensive two-dimensional liquid chromatography (sLC × LC) as a hybrid between the more conventional, but extreme opposite sampling modes of heartcutting (LC–LC) and fully comprehensive (LC × LC) 2D separation. The sLC × LC approach breaks the link between first dimension (1D) sampling time and second dimension (2D) analysis time that is faced in LC × LC and allows very rapid (as low as 1 s) sampling of highly efficient 1D separations, while at the same time allowing efficient 2D separations on the timescale of tens of seconds. In this paper, we improve upon our previous sLC × LC work by demonstrating the ability to perform the processes of 1D sampling and 2D separation in parallel. This significantly improves the flexibility of the technique and allows targeted analysis of analytes that elute close together in time in the 1D separation. To demonstrate the value of this added capability, we have developed a sLC × LC method using multi-wavelength ultraviolet absorbance detection for the quantitative analysis of six target furanocoumarin compounds in extracts of celery, parsley, and parsnips. We show that 2D separations of 1D effluent containing the target compounds of interest reveal the presence of unanticipated interferent peaks that would otherwise compromise the quantitative accuracy of the method. We also demonstrate the application of the chemometric method iterative key set factor analysis with alternating least-squares to sLC × LC to mathematically resolve target compounds that are only slightly separated chromatographically but not sufficiently resolved for accurate quantitation.
Keywords: Liquid chromatography; Two-dimensional; Furanocoumarin; Chemometrics; Selective comprehensive; Targeted analysis

Qualitative and quantitative analysis of the unsaponifiable fraction of vegetable oils by using comprehensive 2D GC with dual MS/FID detection by Peter Q. Tranchida; Simona Salivo; Flavio A. Franchina; Ivana Bonaccorsi; Paola Dugo; Luigi Mondello (4655-4663).
The present investigation is focused on the development of a comprehensive two-dimensional GC (GC × GC) method, with dual MS/FID detection, for the qualitative and quantitative analysis of the entire unsaponifiable fraction of vegetable oils. The unsaponifiable fraction forms a minor, highly specific part of a vegetable oil, and can be used as an indicator of genuineness. The column set used consisted of a low-polarity first dimension, and a medium-polarity secondary one, both characterized by a high thermal stability. The use of dual detection enabled the attainment of both mass spectral information and relative % FID data. The complexity of the fingerprint, generated by the unsaponifiable fraction, fully justified the employment of the two-dimensional GC technology. Furthermore, two other GC × GC benefits contributed greatly to the attainment of promising results, namely sensitivity enhancement and the formation of group-type patterns. The method herein proposed could potentially open a new opportunity for the more in-depth knowledge of the unsaponifiable fraction of vegetable oils.
Keywords: Comprehensive two-dimensional gas chromatography; Vegetable oil; Unsaponifiable fraction; Food analysis