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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 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, or . 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 , 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 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 Phytate for testing for pesticides that was developed by Michelangelo Calcium Pantothenate (B-5) Anastassiades (Anastassiades et al., J. AOAC Int., 86:412 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 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. D-Biotin Pure Sodium chloride and magnesium sulfate) and buffering agents DHEA () (e.g. citrate) to induce liquid separation and stabilize acidic Di-Calcium Phosphate Dihydrate Di-Calcium Phosphate Powder and basic labile pesticides, respectively. After shaking and Di-magnesium Malate Granular centrifugation, an aliquot of the organic phase is subjected to Dimethylaminoethanol (DMAE) further clean up using DSPE. The resulting mixture is centri Dimethylglycine HCL (DMG) fuged and the resulting Supernatant can either be analyzed Disodium EDTA Elderberry PE directly or Subjected to a concentration and solvent exchange Ferrous Fumarate step, if necessary, prior to analysis. Folic Acid 10% Trituration Folic Acid USP 0015 The extracted samples are then subjected to GC-MS GABA analysis using methods well known to those of skill in the art Glucosamine Sulfate Potassium Chloride and described below. The total ion chromatogram is decon Glycine USP Voluted as necessary using publicly available software. Such Golden Seal Extract Grape Seed Extract as AMDIS (Automated Mass Spectral Deconvolution and Grapefruit Seed Powdered Extract Identification System; available from the National Institute of debenone Standards and Technology (NIST)), ChemistationTM and/or nositol Granular DRS (Agilent Technologies, Inc. Santa Clara, Calif.). nositol Hexanicotinate ron Amino Acid Chelate (Ferrochel) 0016. The resulting spectra are compared with standard ron Choline Citrate Powder L-Arginine HCL mass spectra for the contaminants of interest that are con L-Asparagine Monohydrate FCC tained within a database that includes internal calibrations, L-Carnosine such as a retention time-locked (RTL) database. Methods for L-Creatine Monohydrate automated retention time-locking are known in the art and L-Glutamine L-Glutamine include, for example those taught in U.S. Pat. No. 5,987,959. L-Glutathione Reduced In certain embodiments, the resulting spectra are compared Lipoic Acid with those contained in a RTL database, such as the L-Isoleucine RTL Pesticide Library available from Agilent Technologies, Lithium Citrate L-Leucine Inc. This database contains locked retention time, compound L-Lysine Mono HCL name, CAS number, molecular weight and mass spectrum for L-Methionine 927 compounds, including pesticides, metabolite and endo L-Phenylalanine crine disrupters, and other known contaminants. Using a RTL L-Proline L-Serine database eliminates the need to re-calibrate the GC-MS sys L-Threonine tem for each potential contaminant and thus significantly L-Tyrosine reduces the time required to test a sample for the presence or L-Valine absence of multiple contaminants. Magnesium Alpha Ketoglutarate Magnesium Ascorbate 0017 Raw materials that can be analyzed using the meth Magnesium Chloride Hexahydrate ods disclosed herein include, but are not limited to, those Magnesium Citrate shown in Table 1, below. US 2014/01161 12 A1 May 1, 2014

TABLE 1-continued Disruptors by GC/MS with Deconvolution Reporting Soft ware and a New Pesticide Library' Application Note, Agilent Magnesium Citrate Anhydrous Magnesium Citrate Tribasic Technologies, Inc., 2006. Magnesium Glycinate Buffered Magnesium Oxide Powder TABLE 2 Magnesium Oxide USP Magnesium Stearate 1,2,4-Trichlorobenzene Magnesium Sulfate USP 1,2-Dibromo-3-chloropropane Magnesium Taurinate 1,3,5-Tribromobenzene Manganese Citrate 1,3-Dichlorobenzene Manganese Sulfate Monohydrate 17a-Ethynylestradiol Marshmallow Root 1-naphthaleno Melatonin 2-(1-naphthyl)actamido Mesodimercaptosuccinic Acic (DMSA) 2-(2-Butoxyethoxy)ethylthiocyanate Methylcobalamin (Vit. B-12) 2-(Octylthio)ethanol Methylcobalamin (Vit. B-12) 1% Trit in DCP 2,3,4,5-Tertrachloronitrobenzene Methylcobalamin (Vit. B12) Pure 2,3,4,5-Tetrachlorophenol Methylparaben 2,3,4,6-Tetrachlorophenol Milk Thistle Powder 2,3,5,6-Tetrachlorophenol N-Acetyl Glucosamine 2,3,5,6-Tetrachloro-p-terphenyl N-Acetyl L-Cysteine 2,3,5-Trichlorophenol Natural Beta Caroteine in Sunflower 2,3,5-Trimethacarb Niacinamide (Vit. B3) Rocoat 2,3,6-Trichloroanisole Niacinamide Granular USP (Vit. B-3) 2,3,7,8-Tetrachlorodibenzofuran Olive Leaf Extract 2,3,7,8-Tetrachlorodibenzo-p-dioxin Oregano Extract 2,4,5,6-Tetrachloro-m- Para Amino Benzoic Acid (PABA) 2,4,5-T methyl ester Paul D'Arco Bark Extract 2,4,5-Trichloroaniline Potassium Ascorbate Powder 2,4,5-Trichlorophenol Potassium Iodide 2,4,5-Trichloro-p-terphenyl Potassium Sorbate 2,4,5-Trimethylaniline Propylparaben 2.4.6-Tribromoanisole Pycnogenol Extract 2.4.6-Tribromophenol Pyridoxal-5-Phosphate (P5P) 24,8-Trichloroanisoln Pyridoxine (Vit. B-6) HCL Powder 24,8-Trichlorophenol Pyridoxine HCL (Vit. B6) Granular 2,4-D methyl ester Pyrodoxine HCL (Vit. B-6) Rocoat 2,4-D sac-butyl ester Dihydrate 2,4-DB methyl ester 2,4'-Dichlorobenzophenone (2,4'-Dicalol Riboflavin (Vit. B-2) Phosphate decomposition product) Riboflavin (Vit. B-2) Rocoat 2,4-Dichlorophenol Riboflavin USP (Vit. B-2) 2,4-Dichlorophenylbenzenesulfonate Slippery Elm Bark 2,4-Dimethylaniline Sodium Ascorbate Crystalline 2,4-Dimethylphenol Sodium Ascorbate Powder 2.8-Dichlorobenzamide Sodium Benzoate Powder NFFCC 2.8-Dichlorobenzonitrile Sodium Citrate Dihydrate 2,6-Dimethylaniline Sodium CMC 2-3-Chlorophenoxypropionamide Sodium Fluoride USP 2-Chlorophenol Stearic Acid (Veg. Grade) 2-Ethyl-1,3-hexanediol Stevia Leaf Extract 2-ethyl-8-methylaniline Taurine (Ajinomoto) 2-Hydroxyestradiol Taurine (Pharmline) 2-Methyl-4,6-dinitrophenol Thiamine (Vit. B-1) Mononitrate 2-Methylphenol Thiamine HCL Powder USP 2-Nitrophenol Thiamine Mononitrate (Vit. B-1) Rocoat 2-Phenoxypropionic acid Thiamine Mononitrate Powder (Vit. B-1) 3,4,5-Trimethacarb Trimethylglycine Powder (TMG) 3,4-Dichloroaniline Turmeric Root Extract 3,4-Dichloroaniline Vitacel 3-Aminophenol Vitamin AAcetate 3-Chloro-4-fluoroaniline Vitamin A Palmitate 3-Chloro-4-methoxyaniline Vitamin B-121% Trit in Mannitol 3-Chloroaniline Vitamin D-3 100 MIUg 3-Hydroxycarbofuran Vitamin D-3 Pure 3-Indolylacetonitrile Vitamin EAcetate 50% 3-Trifluorimethylaniline Vitamin EAcetate 75% 4,4'-Dichlorobenzophenone Vitamin E. Acetate Oil 4,4'-Oxydianiline Vitamin K-1 5% SD, Dry 4,6-Dinitro-o-cresol (DNDC) Amino Acid Chelate 4-Aminodiphenyl Zinc Citrate Dihydrate 4-Bromoaniline Zinc Ketoglutarate 4-Chloro-2-methylaniline Zinc Picolinate Powder 4-Chloro-3-methylphenol Zinc Sulfate 4-Chloroaniline 4-Chlorophenyl-isocyanate 4-Isopropylaniline 0.018 Table 2 shows a list of potential contaminants that 4-Methylphenol can be detected using the methods disclosed herein, as pub 4-Nitrophenol lished in Wylie, “Screening for 926 Pesticides and Endocrine US 2014/01161 12 A1 May 1, 2014

TABLE 2-continued TABLE 2-continued 4- Binapacryl 5,7-Dihydroxy-4'-methoxyisoflavone 9,10-Anthraquinone Bioallethrin S-cyclopentenyl isomer Acenaphthane Bioresmethrin Acenaphthylene Biphenyl Bis(2,3,3,3-tetrachloropropyl) ether Acequinocyl Bis(2-butoxyethyl) Bis(2-ethylhexyl)phthalate Acetochlor A Acifluorfen methyl ester Bitertanol I Aclariffen Bitertanol II (CAS # 551 79-31-2) Accinathrin Boscalid (Niobilfan) Alachlor Bromocil Bromfenvinphos-(E) Alidochlor Bromfenvinphos-(Z) Amelyn Bromobutide Amidithion Bromocyclan Bromophos Bromophos-ethyl Amitraz metabolite Methanimidamide, N Bromopropylate (2,4-dimethylphenyl)-N'-methyl Bromoxynil Ancymidol Bromoxynil octanoic acid ester Anilazine Bromoconazole I Aniline Bromoconazole II (CAS # 116255-48-2) Anilofos Bufencarb Anthracene Bupiliniate Aramite I Aramite II (CAS # 140-57-8) Butachlor Atraton Butafenacil Butamilos Atrazine-desethyl Butoxycarboxim AZaconazole Butralin Butylbenzyl phthalate Azibenzolar-S-methyl Butylate AZinphos-ethyl AZinphos-methyl Cadusafos AZiprotryn metabolite (2-Amino Cafenstrole 4-isopropylamino-6-methylthio 1,3,5-triazine Captafol AZiprotryne Captain Azobenzene Carboryl AZOxybenzene Carbetamide AZOxystrobin Barben Carbofuran-3-keto Belubutamid Carbofuran-7- Benalaxyl Carbophenethion Benazolin-ethyl Carbosulfen Carboxin Benfluralin Carfentrazone-ethyl Benfuracarb Carpropamid Benferesate Carvone Benetanil Cashmeran Benoxacor Cekalix Bentazone Celestolide Bentazone methyl derivative hinomethionet Benthiocarb hloramben methyl ester Benzene, 1,3-bis(bromomethyl)- hloronocryl Benzenesulfonamide hlorbenside Benzidine hlorbenside sulfone Benzo(a)anthracene hlorbicyclen Benzo(a)pyrene hlorbromuron Benzob fluoranthene hlorbulam Benzog,h,iparylene hlordecone Benzokfluoranthene hlordene, trans Benzophenone hlordimeform Benzoximate metabolite hlorethoxyfos Benzoylprop ethyl l orfeinapyr Benzyl benzoate l orfenethol b- l orfenprop-methyl BHC alpha isomer l orfenson BHC beta isomer l orfenvinphos BHC delta isomer l orfenvinphos, cis BHC epsilon isomer l orfenvinphos. trans Bifenazate metabolite l orflurecol-methyl ester (5-Phenyl-o-anisidine) l ormefos Bifenox l omitrofen l orobenzilate US 2014/01161 12 A1 May 1, 2014

TABLE 2-continued TABLE 2-continued Chloraneb Dichlofenthion Chloropropylate Dichlofuanid Chlorothalonil Dichlofluanid metabolite (DMSA) Chlorotoluron Dichlone Chlorpropham Dichlormid Dichlorophen Chlorpyrifos Methyl Dichlorprop Chlorthal-dimethyl Dichlorprop methyl ester Chlorthiamid Dichlorves Chlorthion Diclobutrazol Chlorthiophos Diclocymet I Chlorthiophos sulfone Diclocymet II (CAS # 139920-32-4) Chlorthiophos sulfoxide Diclofop methyl Chlozolinate Dicloran Chrysene Cinerin I Dicyclohexyl phthalate Cinerin II Dicyclopentadieno Cinidon-ethyl cis-Chlodane Diethatyl ethyl Clodinafop-propargyl Diethofencarb ClornaZone Diethyl dithiobis(thionoformate) (EXD) Cloquintocet-methyl Diethyl phthalate Diethylene glycol Crinkline Crotoxyphos Dilfenoconazol I Crufomate Difenoconazol II (CAS # 119446-68-3) Cyanazine Dilfenoxuton Cyanofenphos Diflufenican Diisobutyl phthalate Cyclafuramid Dimelox Cycloate Dimepiperate Cyclopentadecanone Dimethachlor Cycluron Dimethametryn Cyllufenamid Dimethenomid Cylluthrin I Dimethipin Cyllurhrin II (CAS # 68359-37-5) Cylluthrin III (CAS # 68359-37-5) Dimethomorph-(E) Cylluthrin IV (CAS # 68359-37-5) Dimethomorph-(Z) (CAS # 110488-70-5) Cyhalofop-butyl Dimethylphthalate I (lambda) Dimethylvinphos(z) Cyhalothrin (Gamma) Cymiazole Dimoxystrobin Cymoxanil Di-n-butylphthalate I Di-n-hexyl phthalate Cypermethrin II (CAS # 52315-07-8) Diniconazole Cypermethrin III (CAS # 52315-07-8) Dinitramine Cypermethrin IV (CAS # 52315-07-8) Di-n-nonyl phthalate cis Dinobuton Cyphenothrin trans-(CAS #39515-40-7) Dinocap I Cyprazine Dinocap II (CAS #39300-45-3) Dinocap III (CAS # 39300-45-3) Cyprodinil Dinocap IV (CAS # 39300–45-3) Cyprofuram Di-n-octyl phthalate Dinoseb d-(cis-trans)--I Dinoseb acetate d-(cis-trans)-Phenothrin-II Dinoseb methyl ether (CAS # 260002-80-2) Dinoterb Dazomet Dinoterbacetate DDMU 1-Chloro-2,2-bis(4"chlorophenyl) Di-n-propyl phthalate Decachlorobiphenyl Diofenolan I Diofenolan II (CAS # 63837-33-2) Demaphion Dioxabenzofos -S Dioxacarb Demeton-S-methylsulfon Desbromo-bromobutide Diphacinone DeSmediphan Diphenamid Desmetryn Diphenyl pththalate Dialifos Diphenylamine Di-allato I Dipropetryn Di-allate II (CAS # 2303-16-4) Dipropyl isocinchomeronate Diamyl phthalate Disulfoton sulfone Diazinon- Ditalimfos Dibenza,banthracene Dithiopyr Dicamba Diuron Dicamba methyl ester Diuron Metabolite 3,4-Dichlorophenyl Dicapthon isecyanate US 2014/01161 12 A1 May 1, 2014

TABLE 2-continued TABLE 2-continued Dodemorph I sulfoxide Dodemorph II (CAS # 1593-77-7) Fenthion-sulfone Drazoxolon Fenuron Edifenphos I I Fenvalerate II (CAS # 51630-58-1) Empenthrin II (CAS # 54406-48-3) Fepropimorph Empenthrin III (CAS # 54408-48-3) Empenthrin IV (CAS # 54406-48-3) Fipronil, desulfinyl Empenthrin V (CAS # 54406-48-3) Fipronil-sulfide (alpha isomer) Fipronil-Sulfona Endosulfan (beta isomer) amprop-isoprppyl Endosulfan ether amprop-methyl Endosulfan lactone uacrypyrim Eadosulfan sulfate uazilop-p-butyl uaZinam Endrin aldehyde uaZolate E l rin ketone ubenzimine PN uchloralin poxiconazole ucythrinate I PTC ucythrinate II (CAS # 70 124-77-5) Oil Fludioxonil sfenvalarate Fufenacot S procarb Flumetralin aconazole Flumiclorac-pentyl halfluralin Flumioxazin hidimuron Fluometuron hiofencarb Fluoranthane hiolate Fluorane hion Fluorodifen hofenprox Fluoroglycofen-ethyl hofumesate Fluoroimide hofumesate, 2-Keto Fluotrimazole hoprophos Fluoxastrobin cis hoxyfen-ethyl Fluquinconazole hoxyquin Flurenol-butyl ester hylenethiourea Flurenol-methylester oxazole Fluridone ridiazole Furochloridone I ridiazole, deschloro-(5-ethoxy urochloridone II (CAS # 61213-25-0) 3-dichloromethyl-1,2,4-thiadiazole) urochloridone, deschloroO Etrimfos uroxypyr-1-methylheptyl ester Eugenol urprimidol Exaltolida (1.5-Pentadecanolide) urtainOne Famoxadon usilazole Famphur uthiacat-methyl Fenamidone utolanil sulfoxide utrialol Fenamiphos-Sulfone uValinate-tau-I uvalinate-tau-II (CAS # 102851-06-9) Fenazafor Fenazafor metabolite Fenazaquin Fenbuconazole Fosthiazate I Fenchlorazole-ethyl Fosthiazate II (CAS #98888-44-3) Fenchlorphos Futheridazole Fenchlorphos-oxon Furalaxyl Fenclarim Furathiocarb Fenfuram Furilazole Fenhexamid Furmacyclox Halfenprox Fenitrothion-oxon Haloxyfop-methyl Fenoprop Heptachlor epoxide isomer A Fenoprop methyl ester Heptachlor exo-epoxide isomer B Fenothiocarb Heptenophos Fenoxanil Hexabromobenzene Fenoxaprop-ethyl Hexachlorobenzene Hexachlorophene Fenpiclonil Hexaconazole Hexazinone Fenpropidin Fenson Fensulfothion maZaill Fensulfothion-oxon mazamethabenz-methyl I Fensulfothion-oxon-sulfone mazamethabenz-methyl II ensulfothion-sulfone (CAS # 81405-85-8) Fenthion mibenconazole US 2014/01161 12 A1 May 1, 2014

TABLE 2-continued TABLE 2-continued mibenconazole-desbenzyl Methyl (ndeno1,2,3-cdpyrene Methyl (ndoxacarb and Dioxacarb decomposition Methyl-1-naphthalene acetate product Phenol, 2-(1,3-dioxolan-2-yl)- Methyldymron oxynil Metobromuron oxynil octanoate Metolachlor pconazole prohenfos Metominostrobin (E) prodiene Metominostrobin (Z) provalicarb I (CAS # 133408-50-1) provalicarb II (CAS # 140923-25-7) Metrafenone Lrgaro Metribuzin saZophos Sobenzon Sobornylthiocyanoacetate Molinate socarbamide Monalide Monolinuron Muskamberalta sofenphos-oxon Musk Ketone somethiozin Musk Moskene soprocarb Musk Tibetene (Moschustibeten) sopropalin Musk xylene soprothiolane Myclobutanil soproturon N,N-Diethyl-m-toluamide soxaben N-1-Naphthylacetamide soxadifen-ethyl Nalad soxaflutole Naphthalene soxathion Naphthalic anhydride aSmolin I Naproanilide aSmolin II Napropamide odfenphos Nicotine Kinoprene Nitralin Kresoxim-methyl Nitrapyrin Lactolen Nitrofen Lenacil Nitrothal-isopropyl N-Methyl-N-1-naphthyl acetamide Leptophos Oxon Nonachlor, cis Nonachlor, trans Norflurazon Norflurazon, desraethyl Malathion-o-analog Nuarimol MCPA methyl ester MCPA-butoxyethyl ester op'-DDE MCPB methyl ester op'-DDT m-Cresol Octachlorostyrene Mecarbam O-Dianisidine Mecoprop methyl ester o-Dichlorobenzene Mefenacet Ofurace Mefenpyr-diethyl Melluidide o-Phenylphenol Menazon Orbencarb Mepanipyrim ortho-Aminoazotoluene Mephosfalen Oryzalin Mepronil Oxabetrinil Metalaxyl Oxadiazon Metamitron Oxadixyl MetaSystox thiol Metazachlor Oxycarboxin Metconazole I Oxychlordane Metconazole II (CAS # 1251 16-23-6) Oxydemeton-methyl MethabenzthiaZuron decomposition Oxyfluorfen product Methacrillos p.p'-DDE p.p'-DDM bis(4-chlorophenyl)methane Methfuroxam p.p'-DDT p.p'-Dibromobenzophenone p.p'- Methiocarb sulfone Paclobutrazol Methiocarb sulfoxide Paraoxon Parathion I PBB 52 Tetrabrombiphenyl Methoprene II (CAS # 40596-69-8) PBB 101 Methoprotryne PBB 15 PBB 169 Hexabrombiphenyl Methoxychlor olelin PCB 101 Methyl (2-naphthoxy)acetate PCB 105 US 2014/01161 12 A1 May 1, 2014

TABLE 2-continued TABLE 2-continued PCB 110 PCB 118 Promecarb artifact 5-isopropyl PCB 126 3-methylphenol PCB 127 Prometon PCB 131 Prometryn PCB 136 Propachlor PCB 138 PCB 153 Propanil PCB 169 Propaphos PCB 170 Propargite PCB 18O Propargite metabolite (Cyclohexanol, PCB 30 2-(4-tert-butylphenoxy) PCB 3 Propazine PCB 49 Propetamphos PCB 77 Prophem PCB 8 Propiconazole-I p-Dichlorobenzene Propiconazole-II (CAS # 80207-90-1) Pebulate Propisochlor Penconazole Pendimethalin PropyZamide Pentachloroaniline Prosulfocarb Pentachloroanisole Prothioconazofe-dosthio Pentachlorobenzene Prothiofos Pentachloronitrobenzene Pyracarbolid Pentanochlor Pyraclofos I Pyraflufen-ethyl Permethrin II (CAS # 52645-53-1) Pyrazon Perthane Pyrazophos hantolide Pyrazoxyfen henamiphos Pyrene henanthrene I henanthrene-d10 Pyrethrin II henkapton Pyributicarb henol Pyridaben henothiazine Pyridaphenthion henothrin I Pyridate henothrin II Pyridinitril henoxyacetic acid Pyrifenox I henthoate Pyrifenox II (CAS # 88283-41-4) horate Pyriltalid horate sulfone Pyrimethanil horate sulfoxide Pyrimidifen horalta-OXOn Pyriminobac-methyl (E) hosalone Pyriminobac-methyl (Z) hosfolan (CAS # 136191-64-5) OSle hosphamidon I Pyroquilon hosphamidon II (CAS # 13171-21-6) Quinallphos hthalide Quinoclamine hthalimide Quinoxyfen Picloram methyl ester Quintozene metabolite (pentachlorophenyl Picolinefen methylsulfide) Picoxystrobin Quinzalofop-ethyl Pindone Rabenzazole Piperalin Piperonyl butoxide Resmethrine I Piperophos Resmethrine II (CAS # 10453-86-8) Pirimiphos-ethyl S.S.S-Tributylphosphorotrithioate Pirimiphos-methyl Plifenat Sebuthylazine p-Nitrotoluene Sebuthylazine-desethyl Potasan Secbumeton , cis Prallethrin, trans-(CAS # 23031-36-9) Silthiopham Pretilachlor Simazine Probenazole Simeconazole Simetryn Spirodiclofen Prodiamine Spiromesifen Spiroxamine I Profenofos metabolite (4-Bromo Spiroxamine II (CAS # 118134-30-8) 2-chlorophenol) Spioxamine metabolite (4-tert-butylcyclo Profluralin hexanone) Prohydrojasmon I Sudan I Prohydrojasmon II (CAS # 158474-72-7) Sudan II US 2014/01161 12 A1 May 1, 2014

TABLE 2-continued TABLE 2-continued Sudan Red Tricresylphosphate, para Sulfallate Tricyclazole Sulfanilamide Tridemorph, 4-tridecyl Sulfentrazone Tridiphane Trietazine Sulfur (SB) Triethylphosphate Sulprofos Trilenmorph Swep Trilloxystrobin Trillumizole TCMTB Trilluralin Tebuconazole Triphenyl phosphate e bufenpyrad Tris(2-butoxyethyl) phosphate e bupirimifos Tris(2-chloroethyl) phosphate e butan Tris(2-ethylhexyl) posphate Ta buthiuron Triticonazole e Cl8Zelle Tryclopyrbutoxyethyl e fluthrin, cis Tycor (SMY 1500) Temephos Uniconizole-P Terbacil Vamidithion Terbucarb Vernolate Terbufos-oxon-sulfone XMC (3,4-Dimethylphenyl Terbufos-sulfone N-methylcarbama Terbumeton XMC (3,5-Dimethylphenyl Terbuthylazine N-methylcarbama Terbuthylazine-desethyl Zoxamide Terbutryne Zoxamide decomposition product Tetraconazole 0019. The following examples are intended to illustrate, Tetraethylpyrophosphate (TEPP) Tetrahydrophthalimide, cis-1,2,3,6- but not limit, this disclosure. I Tetramethrin II (CAS # 7696-12-0) EXAMPLES Tetrapropylthiodiphosphate Tetrasul Thenylchlor Materials and Methods Theobromine Thiabendazole 1. Preparation of Samples for GC/MS Analysis Thiazopyr Thifluzamide 0020 Approximately 1.0 g of sample was placed in a 50 Thiomeron mL tube and the exact weight was recorded on a log sheet. For Thionazin each sample, two quality control samples were prepared Thymol using 1 g muffled sand; these were labeled “MB (Method Tiocarbazil I Blank) and “LCS (Laboratory Control Sample). 9.0 mL of Tiocarbazil II (CAS # 36756-79-3) Tolclofos-methyl deionized water was added to each of the tubes. Quality Tolfenpyrad control standards were added as follows: olylfluanid 0021 (a) 50 uL of 20 ppm GC surrogate (tetrachloromet olylfluanid metabolite (DMST) Tolyltriazole (1H-Benzotriazole, 4-methyl axylene (TCMX), Decachlorobiphenyl (DCB), Tributyl Tolyltriazole (1H-Benzotriazole, 5-methyl phosphate and Triphenyl phosphate) in acetonitrile was Tonalide added to all samples including the MB and LCS: Parlar 26 Toxaphene Parlar 50 0022 (b) 100 uL of 20 ppm OC pest spiking solution Toxaphene Parlar 62 (Organochlorine Pesticide Mix AB #1 (Restek Corp., Belle trans- fonte, Pa.) containing aldrin, C.-BHC, 3-BHC, 8-BHC, Y-BHC (lindane), cis-chlordane, trans-chlordane, 4,4'-DDD, Trassalide Triadimefon 4,4'-DDE, 4,4'-DDT dieldrin, endosulfan I, endosulfan II, Triadimenol endosulfan Sulfate, endrin, endrin aldehyde, endrin ketone, Tri-allate heptachlor, heptachlor epoxide (isomer B) and methoxy Triamiphos Trispenthenol chlor) in acetonitrile was added to the LCS; and Triazamate 0023 (c) 100 uL of 20 ppm Internal Standard solution in Triazophos acetonitrile was added to all samples. Tributyl phosphate Tributyl phosphorotrithioite 0024. The samples were shaken vigorously and allowed to Trichlamide equilibrate for 2 hours at room temperature. Extraction of the Trichlorfon samples was then performed by adding 10 mL of acetonitrile and shaking for one minute, adding the contents of an extrac Triclopyr methyl ester tion salt packet (Q-SepTM Q110 QuEChERS extraction salt Triclosan-methyl packet containing 4 g. MgSO4, 1 g NaCl, 1 g trisodium citrate Tricresylphosphate, meta dehydrate, 0.5g disodium hydrogen citrate sesquihydrate; Tricresylphosphate, ortho Restek Corp.), shaking again for one minute and then centri fuging for 5 minutes. US 2014/01161 12 A1 May 1, 2014

0025. For samples needing clean-up (as determined for spectrum very similar to the library/database spectrum, example, by previous difficulties with analysis, difficult shows a strong, sharp peak shape in both the ChemistationTM matrix or darkly colored residues), the solvent extract was and AMDIS peak viewer windows, has a very high MF value, placed in a cleanup tube (Q-SepTM dSPE 15 mL sample and will likely be identified by both the ChemistationTM and cleanup centrifuge tubes containing 900 mg MgSO4, 150 mg AMDIS Softwares. PSA and 45 mg GCB), shaken vigorously and centrifuged for 0031. There are times when ChemistationTM integrated a 5 minutes, before being placed in an evaporation tube. The different peak than AMDIS. Because AMDIS has been rig samples were then evaporated to near dry (less than 1 mL orously developed to remove erroneous background Suppres solvent) using a Turbo VapTM evaporator, and 5 mL methylene sion and use several statistical models to effectively “mine' chloride was added using a solvent pump. This process was the data, AMDIS was given higher priority than Chemista repeated until the acetonitrileportion had been exchanged out tionTM when interpreting data. When a peak was accepted as for methylene chloride and the volume had reached less than genuine, it was either left alone or manually integrated. Since 1 mL. Methylene chloride was then added to raise the volume the GC/MS employed for these studies only uses a single in the sample back to 1 mL, and the sample was transferred quadrupole, it often has trouble resolving overlapping peaks. into a labeled vial and cap using a crimper and aluminum cap. In Such instances, the range of the correct peak was manually 0026. Blanks were run with every set of samples to ensure integrated. Once all of the peaks had been reviewed, the data that laboratory media or equipment was not leading to false was saved and a report generated. positives in any contaminants. These were made following the same process as for the extracts for analysis. 0032 Reports were reviewed after analysis to ensure that data met specifications, specifically sequence data informa 2. GC/MS Analysis tion, internal standard recovery, Surrogate recovery percent ages, calibrated analyte concentrations and semiquant com 0027 Samples prepared as described above were analyzed pounds (i.e. those compounds found using AMDIS and DRS for the presence of contaminants using an Agilent Technolo that have not been calibrated for) hits were checked. Due to gies 5975C gas chromatograph/mass spectrometer (GC/MS) limitations with the software, generated reports often listed in combination with enhanced data analysis as described detections for compounds that had qualifier ion mismatches. below. These peaks do not show up in the default view and are very 0028 Prior to analysis of samples, the GC/MS was difficult to track down and delete, but are always erroneous. checked for any instrument problems that could seriously These were simply deleted from the report. affect the quality of analysis using routine procedures well known to those of skill in the art. Each analysis sequence 0033 Internal standard recovery should have a minimum carried out on the GC/MS was bracketed by calibration veri abundance of 1,000,000 counts, and should generally be fication samples, “initial calibration verification samples or within 50% of the calibrated abundance. Matrix effects can “continuing calibration verification' Samples. These samples cause this number to vary somewhat, so data was generally were made using concentrations equal to 0.1 ppm. The con accepted even if the recovery was outside of the 50% margin. centration of the standards in these samples was within 50% 0034 Surrogate recoveries should also be within 50% of of the expected values. The method blank (MB) and labora calibrated values, but variation may also be due to matrix tory control (LCS) samples were placed at the beginning of effects, thus this was not generally used to reject data unless the sequence. there were clear signs that the ability to generate quality data 0029. The instrument was calibrated for target analytes, or was compromised. contaminants, prior to reporting any target analyte concentra 0035) Semiquant compounds that were found regularly in tion. Calibration was performed by running a set of samples blanks were discarded from the screening list. These com containing a blank and five known concentrations, with the pounds, which included among a few others, were highest level corresponding to the highest expected results, ignored when reporting data. Semiquant hits were not cali through the screening method. The calibration set was quan brated; unless they were calibrated, they can only be reported titated using data analysis and deconvolution employing on a presence/absence basis. Deconvolution Reporting Software (DRS: Agilent Technolo gies, Inc). After each of the five calibration samples were 0036. In order for calibrated compounds to be reported, quantitated, the new values were entered into the database. they must fall within the range of the initial calibration curve. The curve shapes were checked for linearity. R2 values were If they were outside of this range, the sample was diluted to be 0.98 or greater. within this range and rerun. Calibrated compound concentra 0030 Analysis was performed on completed data sets tions were multiplied by the dilution factor and divided by the using DRS and Enhanced Data Analysis software (Agilent sample weight before being reported. Technologies, Inc.). After files were deconvoluted, they were reviewed using QEditTM software. Peaks identified by 3. Quality Control AMDIS (Automated Mass Spectral Deconvolution and Iden tification System; available from the National Institute of (a) Evaluation of Retention Time Windows Standards and Technology (NIST)) and ChemistationTM soft ware were reviewed for quality. More specifically, peaks were 0037. The internal standard retention time was calibrated reviewed based on comparison between library spectra, at 13.726 in accordance with the original AMDIS calibration. AMDIS extracted spectra and ChemistationTM spectra; quali During the initial phase of calibration, the retention time was fier peak match; and retention time. Generally peaks were locked, which allowed AMDIS to acceptor reject peaks based considered true “hits” if they had a MF (molecular formula) upon retention time. If the internal standard fell outside of the match value above 75 and the three qualifier ion values were window and was not integrated by AMDIS, corrective action within 25% of expected. In general, a genuine match has a was taken and the sample was reanalyzed. US 2014/01161 12 A1 May 1, 2014

(b) Sample Cleanup 4. Testing of Sodium Ascorbate 0038 Sample cleanup was performed on samples that 0045 Sodium ascorbate intended for use in nutraceuticals were excessively thick or colored, or that, based on previous for human consumption was tested for the presence of mul history, were expected to cause problems during analysis. tiple pesticide residues as described above. No pesticide resi dues in amounts above the USP <561 > Articles of Botanical (c) Handling and Storage Origin reporting limits were found. 0046 While the present invention has been described with 0.039 All standards were stored at -4° C. or below and reference to the specific embodiments thereof, it should be were allowed to reach room temperature before use. understood by those skilled in the art that various changes (d) Limits of Detection may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In 0040. The limits of detection (LOD) determine the lowest addition, many modifications may be made to adapt a par concentration at which an analyte can be detected in an ticular situation, material, composition of matter, method, extracted sample. Since these measurements are not available method step or steps, for use in practicing the present inven for all compounds, the average of the LODs for calibrated tion. All such modifications are intended to be within the compounds determines the estimated detection limit for Scope of the claims appended hereto. uncalibrated compounds. The LOD is determined by the low 0047 All of the publications, patent applications and pat est concentration compound extracted with a signal to noise ents cited in this application are herein incorporated by ref ratio of 2.5-5. These tests were performed periodically to erence in their entirety to the same extent as if each individual determine any changes in instrument sensitivity. publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its (e) Calibration entirety. 0041. Initial calibration established a calibration curve 1. A method for detecting the presence or absence of a used to determine the concentration of calibrated compounds plurality of contaminants in a sample, comprising: and recoveries of Surrogates. The average internal standard (a) extracting the sample with a water-miscible solvent in response was also used to determine the baseline response the presence of a high concentration of salts to provide a used for the internal standard calibration verification. Cali sample extract; brations were run at seven levels: 0.01, 0.025, 0.05, 0.10, 0.5, (b) shaking and centrifuging the sample extract to provide 1.0, and 5.0 ppm. Using the data from this calibration, each a Supernatant; compound should have a linear or quadratic curve with an R (c) exchanging the water-miscible solvent in the Superna sq value of 0.95 or greater. The lowest calibration level tant for an organic non-water-miscible solvent to pro determines the limit of quantification (LOO). If a compound vide a treated Supernatant; failed calibration (i.e. did not have an R sq. value of 0.95 or (d) analyzing the treated Supernatant using gas chromatog greater) it was noted and corrected before any detections of raphy-mass spectrometry to provide a total ion chro this compound were quantitated. matogram: 0042 Internal standard calibration verification (ISCV) (e) deconvoluting the total ion chromatogram to provide was used to verify instrument performance and internal stan non-overlapping spectra; and dard response. This was prepared with 1.0 ppm internal stan (f) comparing the non-overlapping spectra with standard dard in methylene chloride. The internal standard abundance mass spectra for the plurality of contaminants, should be 70%-170% of the response established with the wherein the sample is a raw material for use in the prepa initial calibration and within the AMDIS retention time win ration of a nutraceutical and wherein the standard mass dow. If the response fell outside of this window, the aberration spectra are contained in a retention time-locked data was investigated and corrected before analysis took place. base. The ISCV was also used to determine column condition. The 2. The method of claim 1, wherein the raw material is a abundance of ion 207 (siloxane bleed) at 40 minutes should mineral or plant-based material. be under 25,000. If the background did not improve in sub sequent analyses, the column was replaced and the instrument 3. The method of claim 1, wherein the raw material is recalibrated before sample analysis. selected from those listed in Table 1. 0043. Initial calibration verification (ICV) and continuing 4. The method of claim 1, wherein the water-miscible calibration verification (CCV) samples were used to verify Solvent is selected from the group consisting of acetonitrile, that the analysis performance was within the parameters of ethyl acetate or acetone. the initial calibration. The CCV was run at the end of a set of 5. The method of claim 1, wherein the organic non-water samples to bracket either an initial calibration or ICV sample. miscible solvent is selected from the group consisting of The ICV was run in place of a set of calibration samples methylene chloride; hexane; and toluene. unless there were measures outside of limits requiring a new 6. The method of claim 1, wherein the high concentration calibration set. of salts is sufficient to provide a 60-70% composition by mass 0044 Recovery control limits for surrogates and other salt solution. calibrated compounds were set at 70-170%. These laboratory 7. The method of claim 1, wherein the plurality of contami control spikes calculated percent recovery. If these fall out nants comprises at least50, 100, 150,200,250,300,350, 400, side of limits, it could be due to matrix Suppression, problems 450, 500, 550, 600, 650, 700, 750, 800, 850 or 900 com with analysis or extraction. These issues were addressed as pounds listed in Table 2. necessary. k k k k k