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US 2014/0116112 A1 HUMPHREY Et Al US 201401 16112A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0116112 A1 HUMPHREY et al. (43) Pub. Date: May 1, 2014 (54) METHODS FOR DETERMINING THE Publication Classification PRESENCE OR ABSENCE OF CONTAMINANTS IN A SAMPLE (51) Int. Cl. GOIN30/72 (2006.01) (71) Applicant: K & D LABORATORIES, INC., Lake (52) U.S. Cl. Oswego, OR (US) CPC .................................. G0IN30/7206 (2013.01) USPC ......................................................... T3/23.37 (72) Inventors: David Kent HUMPHREY, Reno, NV (US); Nicholas Joseph GEISE, Portland, OR (US) (57) ABSTRACT (73) Assignee: K & D LABORATORIES, INC., Lake Oswego, OR (US) Methods are provided for rapidly determining the presence or absence of large numbers of contaminants in a test sample, (21) Appl. No.: 13/830,388 Such as a raw material intended for use in the preparation of a nutraceutical. The disclosed methods employ gas chromatog (22) Filed: Mar 14, 2013 raphy-mass spectrometry techniques together with the spe cific use of software in combination with a database to ana Related U.S. Application Data lyze data collected after ionization of the sample and (60) Provisional application No. 61/718,607, filed on Oct. determine the presence or absence of the contaminants in the 25, 2012. sample. US 2014/01161 12 A1 May 1, 2014 METHODS FOR DETERMINING THE 0007. In one embodiment, methods for detecting the pres PRESENCE OR ABSENCE OF ence or absence of a plurality of contaminants in a sample are CONTAMINANTS IN A SAMPLE provided, such methods comprising: (a) extracting the sample with a water-miscible solvent in the presence of a high con REFERENCE TO RELATED APPLICATIONS centration of salts to provide a sample extract; (b) shaking and centrifuging the sample extract to provide a Supernatant; (c) 0001. This application claims priority to U.S. provisional exchanging the water-miscible solvent in the Supernatant for patent application No. 61/718,607, filed Oct. 25, 2012. an organic, preferably non-water miscible, Solvent methylene chloride to provide a treated Supernatant; (d) analyzing the TECHNICAL FIELD treated Supernatant using gas chromatography-mass spec 0002 The present disclosure relates to methods for rapidly trometry (GC-MS) to provide a total ion chromatogram; (e) determining the presence or absence of multiple contami deconvoluting the total ion chromatogram to provide non nants in a test sample, such as a raw material intended for use overlapping spectra; and (f) comparing the non-overlapping in the preparation of a nutraceutical, using gas chromatogra spectra with standard mass spectra for the plurality of con phy-mass spectrometry techniques. taminants, wherein the standard mass spectra are contained in a retention time-locked database. In certain embodiments, the BACKGROUND water-miscible solvent is selected from the group consisting of acetonitrile, ethyl acetate or acetone. In a preferred 0003. As the use of nutraceuticals, such as multivitamins embodiment, the water-miscible solvent is acetonitrile. In and other dietary Supplements, has become more common certain embodiments, the organic non-water-miscible solvent place, concerns over the levels of purity, quality, consistency is selected from the group consisting of methylene chloride, and potency of Such supplements have increased. Ensuring hexane and toluene. In a preferred embodiment, the non that nutritional and dietary Supplements are free of contami water-miscible solvent is methylene chloride. nants is particularly important when the Supplements are 0008 Such methods can be used to quickly detect the intended for use by children and/or individuals with health problems, environmental sensitivities, etc. The US Food and presence or absence of at least 50, 100, 150, 200, 250, 300, Drug Administration (FDA) regulates dietary Supplements as 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 900 a category of foods and not as drugs, meaning that dietary contaminants. In certain embodiments, the disclosed meth Supplements do not need to be specifically pre-approved by ods are employed to detect the presence or absence of a the FDA. In 2007, the FDA implemented a current Good plurality of compounds selected from those listed in Table 2. Manufacturing Practices (cGMP) policy in an attempt to below. ensure that dietary Supplements do not contain contaminants 0009. In one embodiment, an initial analysis is performed or impurities and are accurately labeled. However, the level of to determine whether or not one or more specific contami non-compliance with the coMP is very high. Based on audits nants are present in a sample (i.e. to give a simple “yes or no completed by the FDAs compliance division in 2011 and result). If this initial analysis indicates that the contaminant is 2012, it has been estimated that nearly 70% of dietary supple indeed present, a second analysis is performed to determine ment manufacturers are non-compliant with the coMP the amount of the contaminant in the sample. In certain policy. Significant concerns thus remain over the quality of embodiments, the second analysis is performed using GC nutritional and dietary Supplements on the market today. MS. 0004. Many supplement companies obtain the raw mate rials for their supplements from a variety of Suppliers, and DEFINITIONS then use those materials to formulate Supplements for sale. 0010. As used herein, the term “deconvolution” refers to a Over 80% of the raw materials used in nutraceuticals sold in mathematical technique that separates overlapping mass the US come from China and other non-US countries, leading spectra (i.e. overlapping peaks in a total ion chromatogram to additional concerns over potential levels of contamination. (TIC)) into clean spectra of individual components. 0005 Methods that are typically employed to check for 0011. As used herein, the term “high concentration of contaminants in raw materials, such as those used in nutra salts' refers to an amount of salts sufficient to provide a ceuticals, are time consuming and expensive. There thus Solution having a percentage composition by mass of salts remains a need for methods that can rapidly and cost-effec between 40% and 90%, such as between 50% and 80% or tively identify the presence or absence of, and/or determine between 60% and 70%. In certain embodiments, the term the levels of a large number of contaminants in raw materials “high concentration of salts' refers to an amount of salts intended for use in one or more dietary Supplements. Sufficient to provide a salt Solution having approximately 65% composition by mass of salts. In one specific embodi SUMMARY ment, the methods disclosed herein employ 4 g. MgSO4, 1 g 0006. The present invention provides methods for rapidly NaCl, 1 g trisodium citrate dehydrate and 0.5g disodium and accurately determining the presence or absence of, and/or hydrogen citrate sesquihydrate in 10 ml of Solution. quantifying the amount of a large number of contaminants, 0012. As used herein, the term “nutraceutical refers to Such as pesticides, in a sample. In certain embodiments, the food, or parts of food, that provide medical or health benefits, methods disclosed herein are employed to test for the pres including the prevention and treatment of disease, and that are ence or absence of contaminants, such as pesticides, in raw intended for consumption by a human or other mammal. The materials intended for use in nutraceuticals. Such as vitamins term nutraceutical encompasses, but is not limited to, dietary and dietary Supplements. Such raw materials include, but are Supplements including botanicals, vitamins, minerals, co-en not limited to, minerals and plant-based materials such as Zyme Q, carnitine, ginseng, gingko biloba, Saint John's Wort, those listed in Table 1, below. saw palmetto, prebiotics and probiotics. US 2014/01161 12 A1 May 1, 2014 0013 As used herein, the term “retention time-locking TABLE 1 refers to the matching of a first set of retention times obtained Acetyl L-Carnitine HCL using a known chromatographic method having a defined set Adipic Acid FCC of column parameters and operating parameters to a second Alpha Ketoglutaric Acid set of retention times obtained using a new, different, chro Ascorbic Acid (Vit. C-90) matographic method having a new, different, set of column Ascorbic Acid (Vit. C-90) Ascorbic Acid Crystals (Vit. C) parameters, wherein the second set of retention times are Astragalus Root Extract Powder matched, or locked, to the first set of retention times. Beta Caroteine Beta Caroteine 10% Beta Caroteine 20% DETAILED DESCRIPTION Calcium Alpha Ketoglutarate Calcium Caprylate 0014. As outlined above, the present disclosure provides Calcium Carbonate Granular rapid and cost-effective methods for detecting the presence or Calcium Carbonate Powder Calcium Carbonate Powder(CE Low Lead) absence of multiple contaminants in a sample, such as raw Calcium Citrate Powder materials for use in the preparation of a nutraceutical. Prior to Calcium Citrate Tetrahydrate Granular analysis using GC-MS, the sample is extracted using a modi Calcium D-Glucarate fied QuEChERS (Quick Easy Cheap Effective Rugged Safe) Calcium Folinate (Folinic Acid) Calcium Glycinate Chelate technique described in detail below. QuEChERS is a method Calcium Magnesium Phytate for testing for pesticides that was developed by Michelangelo Calcium Pantothenate (B-5) Anastassiades (Anastassiades et al., J. AOAC Int., 86:412 Choline Dihydrogen Citrate Citric Acid Anhydrous Granular 431 (2003)). This method entails solvent extraction of Citric Acid Anhydrous Powder samples with acetonitrile, ethyl acetate or acetone, and parti Cobamamide (Vit. B-12 Coenzyme) tioning with magnesium Sulfate, either alone or in combina Cod Liver Oil (Vitamin A Assay) tion with other salts followed by clean-up using dispersive Coenzyme Q10 solid phase extraction (DSPE). More specifically, the sample Cupric Oxide Cyanocobalamin Crystals (Vit. B-12) is first extracted with a water-miscible solvent, such as aceto D-Biotin 196 nitrile, in the presence of a high concentration of salts (e.g.
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