Analysis of pesticides in dried hops by liquid chromatography-tandem mass spectrometry

An analytical method was developed for the determination of eleven agrochemicals [abamectin (as B1a), bifenazate, bifenthrin, carfentrazone-ethyl, cymoxanil, hexythiazox, imidacloprid, mefenoxam, pymetrozine, quinoxyfen, and trifloxystrobin] in dried hops. The method utilized polymeric and NH2 solid phase extraction (SPE) column cleanups and liquid chromatography with mass spectrometry (LC-MS/MS). Method validation and concurrent recoveries from untreated dried hops ranged from 71 to 126% for all compounds over three levels of fortification (0.10, 1.0, and 10.0 ppm). Commercially grown hop samples collected from several field sites had detectable residues of bifenazate, bifenthrin, hexythiazox, and quinoxyfen. The control sample used was free of contamination below the 0.050 ppm level for all agrochemicals of interest. The limit of quantitation and limit of detection for all compounds were 0.10 and 0.050 ppm, respectively.     

Gas chromatographic-mass spectrometric method for the analysis of dimethomorph fungicide in dried hops

An analytical method for the determination of dimethomorph [(E,Z)-4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine] residues in dried hops was developed utilizing liquid-liquid partitioning, automated gel permeation chromatography (GPC), Florisil and aminopropyl solid phase extraction (SPE) column cleanups, and gas chromatography (GC) with mass selective detection (MSD). Method validation recoveries from dried hops ranged from 79 to 103% over four levels of fortification (0.1, 1.0, 5.0, and 20 ppm). Control and dimethomorph-treated hop samples collected from three field sites had residue levels of <0.10 and 4.06-17.32 ppm, respectively. The method was validated to the limit of quantitation at 0.10 ppm. The limit of detection for this method was 0.045 ppm.     

Development of a gas chromatographic method for fungicide cymoxanil analysis in dried hops

An analytical method for detecting cymoxanil [2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyimino)acetamide] residues in dried hops was developed utilizing liquid-liquid partitioning, automated gel permeation chromatography (GPC), solid phase extraction (SPE) cleanup, and gas chromatography (GC). Method validation recoveries from dried hops were 96 +/- 12, 108 +/- 11, and 136 +/- 8% over three levels of fortification (0.05, 0.5, and 1.0 ppm, respectively). The hop samples from three field sites, which were treated with cymoxanil, had residue levels ranging from 0.146 to 0.646 ppm. The detection limit and the quantitation limit of the method developed in the present study were 0.022 and 0.050 ppm, respectively.     

Method development and fate determination of pesticide-treated hops and their subsequent usage in the production of beer

The fate of residues of seven agrochemicals (chlorfenapyr, quinoxyfen, tebuconazole, fenarimol, pyridaben, and E- and Z-dimethomorph) from the treatment on hops to the brewing of beer was studied. First, a multi-residue analytical method was developed for the determination of pesticide residues in spent hops, trub, wort, and beer. Each matrix was validated over at least two levels of fortification, for all seven compounds, in the ranges 0.05-5.0, 0.001-1.0, 0.001-0.05, and 0.0005-1.0 ppm for spent hops, trub, wort, and beer, respectively. Recoveries ranged from 73 to 136%. Second, the matrixes prepared from hops, which were treated under commercial practices with each compound, were analyzed using the method developed. The use of treated hops resulted in the carryover of 0.001 ppm of tebuconazole, 0.008 Z-dimethomorph, and 0.005 ppm of E-dimethomorph into the wort. The bulk of the remaining residues of all seven compounds was found on the spent hops. Following fermentation, all compounds were found in levels less than 0.0005 ppm in beer, except Z- (0.006 ppm) and E-dimethomorph (0.004 ppm). Third, when all seven pesticides were spiked prior to the pitching of yeast into clean wort, most of the nonpolar compounds (chlorfenapyr, quinoxyfen, and pyridaben) partitioned into the organic material (trub) which settled to the bottom, while the more polar compounds (fenarimol, tebuconazole, and E- and Z-dimethomorph) were generally distributed evenly between the beer and the trub.     

Analysis of methoxyfenozide residues in fruits, vegetables, and mint by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Methoxyfenozide [3-methoxy-2-methylbenzoic acid 2-(3,5-dimethylbenzoyl)-2-(1,1-dimethylethyl) hydrazide; RH-2485], in the formulation of INTREPID, was applied to various crops. Analysis of methoxyfenozide was accomplished by utilizing liquid-liquid extraction and partitioning, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method validations for fruits, vegetables, and mint are reported. Methoxyfenozide mean recoveries ranged from 72 to 129% over three levels of fortification. The overall average of mean recoveries is 97 +/- 10%. The limit of quantitation for fruits, artichoke, cucumber, squash, and refined sugar was 0.010 ppm, with a detection limit of 0.005 ppm. For all other crops, the limit of quantitation was 0.050 ppm, with a detection limit of 0.025 ppm. No residues were found greater than the limit of quantitation in control samples. Residues above the limit of quantitation were found in all matrices except refined sugar. Foliage (bean, beet, pea, and radish) had greater residue levels of methoxyfenozide residue than their corresponding roots or pods. Other crop matrices contained <1.0 ppm of methoxyfenozide except artichoke, which had a mean of 1.10 ppm.     

Determination of volatile compounds in different hop varieties by headspace-trap GC/MS--in comparison with conventional hop essential oil analysis

A headspace (HS)-trap method in combination with gas chromatography (GC)-mass spectrometry (MS) was developed for the determination of volatile constituents in hops. The highly sensitive HS-trap system reduces the detection limit by using up to four trap enrichment cycles. Seventy hop samples of different varieties and cultivation regions, from the 2008 harvest, were examined using the HS-trap-GC-MS method and the established "European Brewery Convention" (EBC) method for hop essential oil analysis. Twenty-one different volatiles were quantified for each hop sample. For all compounds, except caryophyllene oxide, a strong correlation was found between the results of the HS-trap method and the EBC method. Experiments have revealed that the EBC method using steam distillation is not appropriate for thermolabile compounds, such as caryophyllene oxide, due to decomposition during boiling. The HS-trap method is fast and sensitive, requires small sample amounts and minimal sample preparation, and is easy to apply.     

Comparison of the most odor-active compounds in fresh and dried hop cones (Humulus lupulus L. variety spalter select) based on GC-olfactometry and odor dilution techniques

Application of aroma extract dilution analysis on the volatiles obtained from dried cones of Spalter Select hops grown in the German hop-growing area of Hallertau revealed 23 odorants in the flavor dilution (FD) factor range of 16-4096, 20 of which could be identified. On the basis of high FD factors, trans-4, 5-epoxy-(E)-2-decenal, linalool, and myrcene were identified as the most potent odorants, followed by ethyl 2-methylpropanoate, methyl 2-methylbutanoate, (Z)-1,5-octadien-3-one, nonanal, (E,Z)-1,3, 5-undecatriene, 1,3(E),5(Z),9-undecatetraene, propyl 2-methylbutanoate, 4-ethenyl-2-methoxyphenol, and 1-octen-3-one. Ten of the high-impact hop aroma compounds had previously not been identified as hop constituents and, in particular, 1,3(E),5(Z), 9-undecatetraene has not yet been reported as a food odorant. In an extract obtained from fresh hops, in addition to the odorants found in dry hops, (Z)-3-hexenal was characterized as a further key odorant rendering an additional green aroma note to the fresh material.     

Analysis of fenhexamid in caneberry, blueberry, and pomegranate by liquid chromatography-tandem mass spectrometry

An analytical method for the determination of fenhexamid [N-(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide] in caneberry, blueberry, and pomegranate was developed utilizing acetone extraction, column cleanup, liquid-liquid partitioning, and liquid chromatography-tandem mass spectroscopy (LC-MS/MS) for detection. Method validation recoveries ranged from 91 to 96% for caneberry, from 80 to 91% for blueberry, and from 74 to 95% for pomegranate. Control samples collected from IR-4 trials for all matrixes had residue levels of <0.020 ppm. Fenhexamid-treated field samples had residue levels that ranged from 0.46 to 16.11 ppm (caneberry), from 0.87 to 2.91 ppm (blueberry), and from 1.59 to 1.85 ppm (pomegranate). The method was validated to a limit of quantitation of 0.020 ppm, and the limit of detection was 0.009 ppm.     

U.S. market basket study to determine residues of the insecticide chlorpyrifos.

A market basket study was conducted to measure residues of the insecticide chlorpyrifos in samples of apples, applesauce, apple juice, fresh orange juice, tomatoes, peanut butter, whole milk, ground beef, and pork sausage collected during a 12-month period from 200 grocery stores across the United States. Approximately 90% of the samples contained no detectable levels of chlorpyrifos, and all residues detected were below tolerances, the legal limits for the United States. No values greater than the limit of quantitation (LOQ) were found in applesauce (LOQ = 0.008 ppm), apple juice (LOQ = 0.003 ppm), whole milk (LOQ = 0.006 ppm), ground beef (LOQ = 0.005 ppm), or pork sausage (LOQ = 0.007 ppm) samples. Only one fresh orange juice sample contained residues greater than the LOQ at 0.015 ppm. Only about 20% of the apples (maximum = 0.052 ppm), 20% of the tomato samples (maximum = 0.058 ppm), and 50% of the peanut butter samples (maximum = 0.021 ppm) contained quantifiable residues.     

Organochlorine pesticide residues in strawberries from integrated pest management and organic farming

A rapid, specific, and sensitive method based on the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method and a cleanup using dispersive solid-phase extraction with MgSO(4), PSA, and C18 sorbents has been developed for the routine analysis of 14 pesticides in strawberries. The analyses were performed by three different analytical methodologies: gas chromatography (GC) with electron capture detection (ECD), mass spectrometry (MS), and tandem mass spectrometry (MS/MS). The recoveries for all the pesticides studied were from 46 to 128%, with relative standard deviation of <15% in the concentration range of 0.005-0.250 mg/kg. The limit of detection (LOD) for all compounds met maximum residue limits (MRL) accepted in Portugal for organochlorine pesticides (OCP). A survey study of strawberries produced in Portugal in the years 2009-2010 obtained from organic farming (OF) and integrated pest management (IPM) was developed. Lindane and β-endosulfan were detected above the MRL in OF and IPM. Other OCP (aldrin, o,p'-DDT and their metabolites, and methoxychlor) were found below the MRL. The OCP residues detected decreased from 2009 to 2010. The QuEChERS method was successfully applied to the analysis of strawberry samples.     

Gas-liquid chromatographic and mass spectrometric indentification of chlorinated trifluorotoluene residues in Niagara River fish.

Several unknown halogenated compounds were detected in Niagara River fish using a method similar to the AOAC multiresidue method for chlorinated pesticides in high-moisture foods. From gas-liquid chromatographic-mass spectrometric (GLC/MS) data and GLC retention times on 3 columns, 7 of the compounds were identified as 4-chloro-alpha,alpha,alpha-trifluorotoluene (0.17--2.0 ppm), 2-chloro-alpha,alpha,alpha-trifluorotoluene (0.002--0.1 ppm), 3,4-dichloro-alpha,alpha,alpha-trifluorotoluene (0.02--0.28 ppm), 2,4-dichloro-alpha,alpha-alpha-trifluorotoluene (0.02--0.17) ppm), 2,3-dichloro-alpha,alpha,alpha-trifluorotoluene (trace-0.005 ppm), 2,6-dichlorotoluene (not quantitated), and 2,4,5-trichlorotoluene (0.31 ppm was found in the only sample quantitated). Other isomers of tri- and tetrachloro-alpha,alpha,alpha-trifluorotoluene and di-,tri-, and tetrachlorotoluene were also present in these samples. Recoveries of the specific chlorinated trifluorotoluenes identified in these samples ranged from 86 to 108%.     

Microsatellite DNA Analysis of Wild Hops, Humulus lupulus L.

To study the relationships and genetic diversity among wild hops, Humulus lupulus, we analyzed 133 samples of wild hops collected from Europe, Asia and North America using polymorphism on 11 microsatellite loci. Although only three primers showed bands in Japanese hops, all other samples showed polymorphic bands at most loci. There were no duplicate genotypes among samples of European, Chinese and North American hops, and each individual hop could be distinguished completely. The phylogenetic tree constructed from DA distance with the UPGMA method showed a large cluster comprised of European hops, although Russian hops from the Caucasus and Altai regions were separate from the European cluster. Chinese and North American samples gave distinct clusters suggesting genetic differentiation. This study has indicated that hop microsatellite DNA is differentiated, and is dependent upon the origin in regions of Europe, Asia and North America.     

Use of GC-olfactometry to identify the hop aromatic compounds in beer

This paper describes a sensorial aroma extract dilution analysis (AEDA) approach to the analysis of beer aromas derived from hops. To obtain an extract with an odor representative of the original product, the XAD extraction procedure was applied and the experimental conditions were optimized. The aromagrams of three beers were compared: one brewed without hops, one brewed with Saaz hop pellets, and one brewed with Challenger hop pellets. One spicy/hoppy compound, unmodified from hop to beer, proved responsible for the most intense odor in both hopped beer extracts. Another flavoring compound in hops, linalool, also survives through the process to the final beer. Other compounds such as gamma-nonalactone and humuladienone, although not found in our extracts of hop, significantly modify beer aromagrams after hopping. Sulfur compounds characteristic of Challenger hops proved to be at least partially responsible for the unpleasant flavor found in the corresponding beer.     

Occurrence of resveratrol and piceid in American and European hop cones

The recent discovery in hops of trans-resveratrol and its glucoside, trans-piceid, both famous in wine for their potential role in the "French paradox", opens new doors to understanding hop health benefits. In the present work, trans-resveratrol and trans-piceid were quantified by HPLC-APCI-MS/MS in 40 American and European hop cone samples from harvests of 2004, 2005, and 2006. Their content varies greatly, in the range 0.5-12 mg/kg. All German varieties revealed low amounts of stilbenes. The highest concentrations were found in Cascade (2004) and Willamette (2004 and 2006), two American low-alpha-acid varieties. Yet, probably because trans-resveratrol is a phytoalexin, a strong influence of harvest year was also observed.     

Formation and accumulation of alpha-acids,beta-acids,desmethylxanthohumol, and xanthohumol during flowering of hops (Humulus lupulus L.)

Important secondary metabolites, present in hops (Humulus lupulus L.), include alpha-acids and beta-acids, which are essential for the brewing of beer, as well as the prenylated chalcones, desmethylxanthohumol, and xanthohumol, which exhibit interesting bioactive properties. Their formation and accumulation in five selected hop varieties, Wye Challenger, Wye Target, Golding, Admiral, and Whitbread Golding Variety, were quantitatively monitored by high-performance liquid chromatography using UV detection. All target compounds were present from the onset of flowering, not only in female hop cones but also in male inflorescences, albeit in low concentrations. During development from female inflorescences to cones, levels of alpha-acids, beta-acids, desmethylxanthohumol, and xanthohumol gradually increased, while each hop variety exhibited individual accumulation rates. Furthermore, these compounds were present in leaves of fully grown hops as well. The study demonstrated that key compounds for flavor and potential beneficial health effects associated with beer not only reside in the glandular lupulin structures but also are distributed over various parts of the hop plant.     

Direct thermal desorption-gas chromatography and gas chromatography-mass spectrometry profiling of hop (Humulus lupulus L.) essential oils in support of varietal characterization

The use of direct thermal desorption-gas chromatography-mass spectrometry (DTD-GC-MS) and DTD-GC-flame ionization detection (DTD-GC-FID) for characterization of hop essential oils is described. Four hop varieties (Nugget, Galena, Willamette, and Cluster) from the Yakima valley (Yakima, WA) 1998 harvest were analyzed by DTD-GC-MS and DTD-GC-FID methodology. Approximately 1 g of hops was needed for the analysis. Hop samples were prepared for GC-MS and/or GC-FID profiling in approximately 20 min. More than 100 volatile compounds have been identified and quantified for each hop variety. The results were found to be in good agreement with conventional steam distillation-extraction (SDE) data. A calibration curve for determination of essential oil content in hops by DTD-GC-FID has been generated. Quantitation of hop oil content by DTD-GC-FID was shown to be in good agreement with conventional SDE data. The recovery of key oil components valuable for varietal identification was demonstrated to be highly reproducible and characteristic of each variety analyzed when DTD-GC-FID was used for analysis.     

Determination of the hop-derived phytoestrogen, 8-prenylnaringenin, in beer by gas chromatography/mass spectrometry

A method was developed to determine 8-prenylnaringenin, a novel hop-derived phytoestrogen, in beer. Matrix purification involved solid-phase extraction on octadecyl silica followed by liquid/liquid extraction on a ChemElut 1010 column connected to a Florisil adsorption/desorption cartridge. 8-Prenylnaringenin was eluted from the tandem columns using a 1:1 mixture of diethyl ether and ethyl acetate and subsequently determined as tris(trimethylsilyl) ether by GC/MS-SIM. The recovery of 8-prenylnaringenin in beer samples was between 61.1 +/- 6.6 and 82.2 +/- 8.8% for levels of 37 and 92.5 microg L(-1), respectively, and the detection limit was approximately 5 microg L(-1). Although most beers do not contain 8-prenylnaringenin in detectable quantities, the highest concentration found was 19.8 microg L(-1). The concentration of 8-prenylnaringenin in beers and, possibly, its absence depend on the selection of particular hop varieties, the hopping rate, or the type of hop product used in brewing. The efficiency of transfer of 8-prenylnaringenin from hops to beer is between 10 and 20%.     

Structural identification and distribution of proanthocyanidins in 13 different hops

Ten newly isolated hop proanthocyanidin oligomers and flavan-3-ol monomers from 13 different hops have been identified as gallocatechin, gallocatechin-(4alpha-->8)-catechin, gallocatechin-(4alpha-->6)-catechin, catechin-(4alpha-->8)-gallocatechin, catechin-(4alpha-->6)-gallocatechin, afzelechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-catechin-(4alpha-->8)-catechin, epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin, catechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, and gallocatechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, together with seven previously isolated oligomers, namely, catechin, epicatechin, epicatechin-(4beta-->8)-catechin, epicatechin-(4beta-->8)-epicatechin, catechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-epicatechin, and epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin. These compounds were subjected to acid-catalyzed degradation in the presence of phloroglucinol or by partial or complete acid-catalyzed degradation and reaction with benzyl mercaptan followed by desulfurization. The resultant adducts when compared to authentic samples by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry served to identify the precursors. The composition of proanthocyanidins from 13 different hops was similar, but the concentration of individual compounds showed some differences, which indicated that hop proanthocyanidin profiles are affected by geographic origin and are variable depending on the cultivars.     

Comparison of odor-active compounds in the spicy fraction of hop (Humulus lupulus L.) essential oil from four different varieties

The "spicy" character of hops is considered to be a desirable attribute in beer, associated with "noble hop aroma". However, the compounds responsible have yet to be adequately identified. Odorants in four samples of the spicy fraction of hop essential oil were characterized using gas chromatography-olfactometry (GC-O) and CharmAnalysis. Four hop varieties were compared, namely, Target, Saaz, Hallertauer Hersbrucker, and Cascade. Odor-active compounds were tentatively identified using comprehensive two-dimensional gas chromatography (GCxGC) combined with time-of-flight mass spectrometry (TOFMS). An intense "woody, cedarwood" odor was determined to be the most potent odorant in three of the four spicy fraction samples. This odor coincided with a complex region where between 8 and 13 compounds were coeluting in each of the four spicy fractions. The peak responsible was determined by (i) correlating peak areas with Charm values in eight hop samples and (ii) heart-cut multidimensional gas chromatography-olfactometry (MDGC-O). The compound responsible was tentatively identified as 14-hydroxy-beta-caryophyllene. Other important odorants identified were geraniol, linalool, beta-ionone, and eugenol.     

Modified gas chromatographic/mass spectrometric method for determination of daminozide in high protein food products

A modified version of the Conditt and Baumgardner gas chromatographic/mass spectroscopic (GC/MS) method for determination of daminozide in peanut butter and raw peanuts is described. Daminozide in the food product is hydrolyzed to unsymmetrical dimethylhydrazine (UDMH) by sodium hydroxide digestion. The generated UDMH is distilled from the food matrix and captured by reaction with salicylaldehyde in a condensation trap. Resulting high pH distillates generated by peanuts and peanut products are adjusted back to a pH of 5-6 through addition of glacial acetic acid. After thermal incubation and extraction into methylene chloride, salicylaldehyde dimethylhydrazone is separated from interferences by capillary GC and quantitated by MS using the selective ion monitoring (SIM) mode. Quantitation of daminozide is based on the ratio of the salicylaldehyde dimethylhydrazone molecular ion (m/z 164) to the molecular ion (m/z 153) of the internal standard, 4-nitroanisole. Confirmation of daminozide identity is determined by relative intensity of the m/z 164 ion to the m/z 120 (C7H4ON) ion. Improved m/z 164 ion intensity and reduction of neighboring interferences due to acetic acid treatment permitted a daminozide detection limit of 0.005 ppm in a 50 g sample and an associated 0.02 ppm limit of quantitation. This modification is specific for high protein samples that generate high pH distillates such as peanuts and peanut products and is not specifically intended for analysis of low protein samples.