Determination of spinosad and its metabolites in food and environmental matrices. 3. Immunoassay methods

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidopteran larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing a magnetic particle-based immunoassay (IA) test kit have been developed and validated for determining spinosad in environmental and food matrices. These methods involve an extraction of the residues from the matrices with appropriate solvents. For some matrices, the sample extracts can be diluted and measured directly by IA without any cleanup. For other matrices, sample extracts are purified using liquid-liquid partitioning and/or solid phase extraction prior to measurement by IA. The methods determine the total residue of spinosad, which includes the active ingredients (spinosyns A and D) and several minor metabolites, including spinosyn B, spinosyn K, and N-demethylspinosyn D. The methods have validated limits of quantitation of 0.0001 microgram/mL in water, 0.05 microgram/g in sediment, and 0.010 microgram/g in crops, crop processed commodities, and animal tissues. This paper briefly summarizes the residue methodology and method validation data for spinosad in 34 food, feed, and environmental matrices.     

Liquid chromatography-tandem mass spectrometric ion-switching determination of chlorantraniliprole and flubendiamide in fruits and vegetables

The anthranilic and phthalic diamides, chlorantraniliprole (CAP) and flubendiamide (FLU), respectively, represent a new class of very effective insecticides that activate the ryanodine-sensitive intracellular calcium release channel (ryanodine receptor). This paper reports an analytical method for the simultaneous determination of the two insecticides on fruits and vegetables by liquid chromatography-electrospray tandem mass spectrometry operated in the positive and negative ionization switching mode. The two diamides were extracted with acetonitrile and separated on a Zorbax Column Eclipse XDB C8 (4.6 mm x 150 mm i.d., 3 microm) by isocratic elution with a mobile phase consisting of acetonitrile and water with 0.1% formic acid pumped at a flow rate of 0.4 mL/min. The diamides were selectively detected by multiple reaction monitoring for transitions of proton adduct precursor ions simultaneously: positive m/z 484.3-->285 for CAP, m/z 445.5-->169 for internal standard, and negative m/z 681.4-->253 for FLU. For CAP calibration in the positive mode was linear over a working range of 2 to 1000 microg/L with r > 0.992. The limit of detection (LOD) and limit of quantification (LOQ) for CAP were 0.8 and 1.6 microg/kg, respectively. For FLU in the negative mode the corresponding values were 1-1000 microg/L for linear working range, with r > 0.996 and 0.4 and 0.8 microg/L for LOD and LOQ, respectively. Moreover, the presence of interfering compounds in the fruit and vegetable extracts was found to be minimal. Due to the linear behavior of the MS detector response for the two analytes, it was concluded that the multiple reaction transitions of molecular ions in the ion-switching mode can be used for analytical purposes, that is, for identification and quantification of diamides in fruit and vegetable extracts at trace levels.     

Determination of spinosad and its metabolites in food and environmental matrices. 2. Liquid chomatography-mass spectrometry

A selective and sensitive method utilizing liquid chromatography-mass spectrometry (LC-MS) has been developed for determining residues of the natural insect control agent spinosad in several crop matrices that are difficult to analyze by HPLC with UV detection. The method determines the active ingredients (spinosyns A and D) and three minor metabolites (spinosyns B and K and N-demethylspinosyn D) in alfalfa hay, wheat hay, wheat straw, sorghum fodder, and corn stover. The analytes are extracted from the samples with an acetonitrile/water solution, and the extracts are purified by solid phase extraction with a C(18) disk and a silica cartridge. All five analytes are determined simultaneously in a single injection using positive atmospheric pressure chemical ionization LC-MS with selected ion monitoring. The average recoveries ranged from 69 to 96% with standard deviations ranging from 4 to 15%. The method has a validated limit of quantitation of 0. 01 microgram/g and a limit of detection of 0.003 microgram/g. The LC-MS method can also provide residue confirmation in addition to quantitation.     

Simple multiresidue method for monitoring of trimethoprim and sulfonamide residues in buffalo meat by high-performance liquid chromatography

A simple, specific, and rapid analytical method for the determination of trimethoprim (TMP) and three sulfonamide (SA) antimicrobial drug residues in buffalo meat is developed and validated. This method is based on a solid-phase extraction technique followed by high-performance liquid chromatography (HPLC)-photodiode array (PDA) detection. Target compounds were extracted from the meat by acetonitrile and water, cleaned up on a Bond Elute C 18 cartridge column, and separated on a RP-C 18 column during HPLC analysis. Acetonitrile along with water appears to be an excellent extractant as recovery of the analytes at maximum residues levels (MRLs) in spiked sample was in the range of 75-108%, with coefficient of variations (CVs) ranging between 1.34 and 22%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.031 and 0.062 microg/g, respectively, for all of the compounds. Intra- and interday assay precisions of the method at 0.125 microg/g concentrations for any drug ranged between 3 and 4%. The linearities of the TMP, sulfadimidine (SDM), sulfadoxine (SDO), and sulfamethoxazole (SMX) were 0.9989, 0.9999, 0.9998, and 0.9997, respectively. For robustness, the analytical method was applied to 122 buffalo meat samples obtained from export meat processing plants.     

Determination of spinosad and its metabolites in food and environmental matrices. 1. High-performance liquid chromatography with ultraviolet detection

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidoptera larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection have been described for determining spinosad and its metabolites in environmental and food matrices. These residue methods typically involve an extraction with organic solvents, followed by purification using liquid-liquid partitioning and/or solid phase extraction prior to measurement by HPLC-UV. The residue methods determine the active ingredients (spinosyns A and D) and up to three minor metabolites (spinosyn B, spinosyn K, and N-demethylspinosyn D). The methods have validated limits of quantitation ranging from 0.010 to 0.040 microgram/g. This paper briefly reviews the residue methodology for spinosad and metabolites in food and environmental matrices and provides a summary of method validation results for 61 different sample types, including newly published results for 37 additional crop matrices and processed commodities.     

Cryptoxanthin structural isomers in oranges, orange juice, and other fruits

Citrus fruits contain a wide range of bioactive compounds. Their carotenoid fraction is inter alia dominated by structural cryptoxanthin isomers as beta-cryptoxanthin and zeinoxanthin. Both xanthophylls were identified in saponified citrus fruit extracts by comparison to reference compounds extracted from corn and by their typical fragmentation pattern in LC-(APCI)MS analyses. alpha-Cryptoxanthin, another structural cryptoxanthin isomer usually found in carrot leaves, was not identified in the citrus fruits studied. Cryptoxanthin concentrations of direct orange juices (D) and reconstituted juices (C) were compared. Although the respective mean values [beta-cryptoxanthin, 62 (C) versus 110 microg/100 g (D); zeinoxanthin, 22 (C) versus 37 microg/100 g (D)] were statistically distinguishable (P < 0.05%), a doubtless classification is not possible because the concentration ranges overlap. To identify esters of structural cryptoxanthin isomers in native orange juice extracts, four saturated acyl esters were synthesized. LC-(APCI)MS studies revealed for the first time that the dominant acylation partners of both xanthophylls were C12:0, C14:0, and C16:0 in nearly equal amounts of roughly one-third, whereas C10:0 and C18:1 were present at lower extents of 5-14%; other acylation partners were not identified. The presented method is appropriate to gain deeper insight into the pattern of structural cryptoxanthin isomers of citrus fruits. Knowledge of acylated cryptoxanthin isomers may be important in the evaluation of the bioavailability of individual esters in future human digestion studies.     

Determination of oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with tandem mass spectrometry

A new methodology is described for rapidly determining the herbicide oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with negative ion electrospray ionization tandem mass spectrometry (LC/MS/MS). Oryzalin is extracted from water using a polymeric sorbent solid phase extraction (SPE) column and from fruit using methanol. The water samples require no further purification, but an aliquot of the fruit sample extracts is diluted with water and purified using a polymeric 96 well SPE plate. Purified extracts are concentrated prior to determination by LC/MS/MS at m/z 345 (Q1) and m/z 281 (Q3) using an external standard for calibration. The validated limits of quantitation were 0.05 microg/L in water (drinking water, surface water, and groundwater) and 0.01 microg/g in citrus fruits (oranges and lemons) and stone fruits (peaches and cherries). Recoveries averaged 102% for water samples and 85-89% for the various types of fruit samples. For all fortification levels combined, the relative standard deviations ranged from 4 to 6% for water and from 2 to 4% for fruit.     

Determination of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection

Two liquid chromatographic methods that involve precolumn derivatization with o-phthaladehyde (OPA) and phenylisothiocyanate (PITC) with fluorescence and diode array UV detection for the determination of theanine have been developed. The chromatographic separations were achieved by reverse-phase high-performance liquid chromatography using octadecyl columns and gradient elution. The methods were applied to evaluate the theanine content of commercial tea leaves. The coefficient of variation of the peak area repeatability for within day (n = 8) and between day (n = 8 over 10 days) was lower than 3% for both of the methods. The estimated limit of detection (LOD) and limit of quantitation (LOQ) for the OPA method was 0.12 and 0.35 microg theanine, respectively. The PITC method was 500-fold more sensitive with LOD and LOQ values of 0.25 and 0.75 ng, respectively. The theanine content of the commercial tea samples varied from 2-5 mg/g leaf. The overall % recoveries for these methods ranged from 93-99.3. The sensitivity and simplicity of the method render them suitable for use in quality control laboratories.     

Simple and sensitive determination of o-phenylphenol in citrus fruits using gas chromatography with atomic emission or mass spectrometric detection

In this work, a simple and sensitive method for the analysis of the pesticide o-phenylphenol (OPP) on citrus fruits was developed. OPP is extracted with dichloromethane by ultrasonication and derivatized with ferrocenecarboxylic acid chloride. Using ferrocene as a label, residues of OPP are determined by gas chromatography with atomic emission detection in the iron selective mode or with mass spectrometric detection. Sample cleanup is simple and rapid and merely involves a removal of excess reagent on an alumina minicolumn. The method detection limit is 2 ng of OPP/g of fruit, and recoveries from lemon samples fortified at levels of 35 and 140 ng/g are 101 and 106%, respectively. The citrus fruits analyzed (oranges, grapefruits, lemons) contained between 60 ng/g and 0.37 microg/g OPP (RSD = 8-13%), and the results were in good agreement with results obtained when OPP was analyzed using an established HPLC-FLD method. Several alcohols could also be identified in the fruit peel.     

Determination of ethalfluralin in canola seed, meal, and refined oil by capillary gas chromatography with mass selective detection

Ethalfluralin is a herbicide that is effective for weed control on a wide variety of crops, including canola. A method is described for the determination of ethalfluralin residues in canola seed, meal, and refined oil. Residues are extracted from canola sample matrixes with acetonitrile. An aliquot of the extract is diluted with water and purified by C(18) solid-phase extraction prior to analysis by capillary gas chromatography with mass selective detection. For all three sample matrixes, the method has a validated limit of quantitation of 0.02 microg/g and a limit of detection of 0.006 microg/g. Recoveries averaged 96 +/- 7% for canola seed, 87 +/- 6% for canola meal, and 89 +/- 5% for refined oil. In a magnitude-of-residue study, canola seed from field plots that had been treated with ethalfluralin at one to three times the maximum label rate for weed control were found to contain no detectable residue of the herbicide.     

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.)

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.     

Determination of traces of fipronil and its metabolites in pollen by liquid chromatography with electrospray ionization-tandem mass spectrometry

Fipronil is a pesticide suspected of having harmful effects on honey bees at microgram per kilogram levels. Considering the lack of methodology, it thus appeared to be necessary to develop a method for the determination of the lowest amounts of fipronil and its metabolites in pollen. This paper describes a new analytical method with a limit of quantification (LOQ) of 0.1 microg/kg for a representative sample weight of 5 g. In the case of a field study, this tool was used for checking the possible existence of fipronil and/or metabolites in pollen samples, but none of them contained residues higher than the LOQ. This three-step rapid method uses liquid-solid solvent extraction with mechanical grinding, followed by liquid-liquid partitioning and Florisil solid-phase extraction for the two cleanup steps. The quantification is made by liquid chromatography with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Indeed, combined with an adequate sample treatment, this technique offers good sensitivity and selectivity in such a complex matrix. The method has given good recoveries of 74-104% with relative standard deviations of 5.6-18.2%.     

Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

Validation of methods used in the Florida Department of Agricultural and Consumer Services' Chemical Residue Laboratory.

Very few methods for detecting residues of pesticides in food or agricultural samples have undergone rigorous colloborative study and possess official AOAC status. The Chemical Residue Laboratory has formalized a method validation scheme to use when incorporating or developing new, unofficial methods. These methods are validated by assessing certain performance parameters: scope, specificity, linear range, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). For accuracy and precision assessment, 12 replicate fortifications must yield recoveries within the range of 70-120% with a coefficient of variation (CV) that compares favorably to the Horwitz CV. LOD and LOQ are equivalent to 3 and 10 times, respectively, the background signal contributed by a sample matrix blank. This criterion that we use for LOD/LOQ is not universal. In fact, because of differing definitions, we have encountered difficulties in enforcing a tolerance by using a registrant's method. This paper also presents an example of our method validation scheme, using a recent method development project for detecting sulfamethazine in raw milk. The sulfamethazine project also revealed unanticipated personnel problems, underscoring the importance of the human factor in quality assurance.     

Direct aqueous injection LC-ESI/MS/MS analysis of water for 11 chloro- and thiomethyltriazines and metolachlor and its ethanesulfonic and oxanilic acid degradates

A multianalyte method is reported for the determination of atrazine, simazine, propazine, and their respective dealkylated chlorotriazine metabolites; ametryn and prometryn and their respective dealkylated thiomethyltriazine metabolites; and S-metolachlor and its ethanesulfonic and oxanilic acid degradates in deionized, ground, surface, and finished drinking water. Water samples are analyzed using direct aqueous injection (DAI) liquid chromatography-electrospray ionization/mass spectrometry/mass spectrometry (LC-ESI/MS/MS). No preanalysis sample manipulation is required other than transfer of a small portion of sample to an injection vial. The lower limit of the method validation is 0.050 microg/L (ppb) for all analytes except 2,4-diamino-6-chloro- s-triazine (didealkylatrazine, DDA, or G-28273). For this compound the LLMV is 0.50 microg/L (ppb). The overall mean procedural recoveries (and percent relative standard deviations) for all water types for all analytes ranged from 95 to 101% (4.5-11%). The method validation was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160.     

Single-drop and nanogram level determination of sulfite (SO3(2-)) in alcoholic and nonalcoholic beverage samples based on diffuse reflectance fourier transform infrared spectroscopic (DRS-FTIR) analysis on KBr matrix

The diffuse reflectance Fourier transform spectroscopic (DRS-FTIR) method, using potassium bromide matrix, has been developed for the one-drop microdetermination of sulfite in beverage samples. The present method is very simple, rapid, and precise for the determination of sulfite. The nanogram level of sulfite determination is based on the selection of a quantitative analytical peak at 495 cm (-1) among the three observed vibrational peaks obtained by diffuse reflectance Fourier transform infrared spectroscopy (DRS-FTIR). As little as a single drop of sample is sufficient for quantitative analysis of sulfite. The limit of detection (LOD) and the limit of quantification (LOQ) of the method are found to be 8 and 40 ng of SO 3 (2-) 0.1 g (-1) of KBr matrix, respectively. The linear range of the method (LR) as well as the LOD based on the concentration of sulfite in the solution are 5-500 and 0.8 microg/mL, respectively. The precision in terms of standard deviation and relative standard deviation value at a level of 100 ng of SO 3 (2-) 0.1 g (-1) of KBr for n = 10 are found to be 2 ng of SO 3 (2-) and 2.3%, respectively. The relative standard deviation ( n = 10) for the determination of sulfite in beverage samples available in the local market was observed to be in the range of 2.4-7.8%. The method is free from interionic effects of foreign species. No sample pretreatment is required in this method. The proposed method avoids the requirement of large numbers and bulk amounts of reagents. The method is well-suited to the need of green chemistry.     

Development of an enzyme-linked immunosorbent assay for the fungicide imazalil in citrus fruits

Imazalil has been widely used in citrus fruits such as lemons, oranges, and grapefruits. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of residual imazalil in citrus fruits. A monoclonal antibody (MoAb) generated to the synthetic imazalil hapten (EIT-0073)-protein conjugate was used. This assay was applied to lemon, orange, and grapefruit matrices for an imazalil analysis. The acceptable residue level for lemons, oranges, and grapefruits in Japan is 5 ppm. The matrix interference was minimized by direct dilution of the sample homogenate. No further cleanup was needed. The detection limit for imazalil in these citrus fruits was 0.1 ng/mL. The recovery of each fortified citrus fruit sample was >81.0%. The imazalil recovery measured by the proposed ELISA was compared to the recovery determined by a conventional HPLC. A good correlation was observed between the proposed ELISA and the HPLC. This proposed ELISA would be useful for monitoring for residual imazalil.     

Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection.

To routinely assay the concentrations of ochratoxin A (OTA) in wines and beers, two new methods were developed and evaluated. The first utilized solid-phase extraction on a C(18) cartridge to achieve a 100-fold sample concentration followed by high-performance liquid chromatography on a C(18) column with gradient elution and quantitation at 333 nm by means of a photodiode array detector. Positive confirmation can be carried out by purity and match-factor analysis as well as peak shift following esterification with BF(3). Total run time is 28 min. The limits of detection (LOD) and quantitation (LOQ) are 0.05 and 0.10 microg/L, respectively. Recovery and imprecision ranged from 83 to 94% and from 4.0 to 8.9%, respectively. With a throughput of 35 assays per working day, this method is ideal for routine OTA analysis. It was used to survey the concentrations of OTA in 942 wines (2 of which gave values between 0.1 and 0.2 microg/L) and 107 beers (2 of which gave values between 0.05 and 0.1 microg/L). OTA was detected more frequently in red than white wines, with the highest incidence in red wines from Spain and Argentina. There was no association between OTA and country of origin or beverage type among the beers analyzed. The second method utilized gas chromatography with mass selective detection monitoring eight specific ions, preceded by extraction in dichloromethane and derivatization with bis[trimethylsilyl]trifluoroacetamide. LOD and LOQ were 0.1 and 2 microg/L, respectively; recovery and imprecision were 69-75 and 9.0-11.1%, respectively. The method is not suitable for routine quantitation but is potentially useful as a confirmatory tool for samples with OTA > or =0.1 microg/L.     

Improved high-performance liquid chromatography (HPLC) method for qualitative and quantitative analysis of allantoin in Zea mays

A high-performance liquid chromatography (HPLC) method for the qualitative and quantitative analysis of allantoin in silk and seed of Zea mays has been developed. Allantoin separation in crude extract was achieved using a C 18 column and phosphate buffer solution (pH 3.0) as a mobile phase at ambient temperature at a flow rate of 1.0 mL/min and detected at 210 nm. The results showed that the amount of allantoin in samples was between 14 and 271 mg/100 g of dry plant material. A comprehensive validation of the method including sensitivity, linearity, repeatability, and recovery was conducted. The calibration curve was linear over the range of 0.2-200 microg/mL with a correlation coefficient of r2>0.999. Limit of detection (LOD, S/N=3) and limit of quantification (LOQ) values of the allantoin were 0.05 and 0.2 microg/mL (1.0 and 4.0 ng) respectively. The relative standard deviation (RSD) value of the repeatability was reported within 1.2%. The average recovery of allantoin added to samples was 100.6% with RSD of 1.5%.     

Determination of 4-ethylphenol and 4-ethylguaiacol in wines by LC-MS-MS and HPLC-DAD-fluorescence

Volatile phenols produced by Brettanomyces dekkera have been associate with off-flavors of wines. A versatile liquid chromatography-tandem mass spectrometry together with an HPLC-DAD-fluorescence methods were developed for the quantitation of two phenols, 4-ethylphenol (4EP) and 4-ethylguaiacol (4EG), in red and white wines. For LC-MS-MS analysis, fortified wines were directly injected after a dilution with methanol, and levels of phenols were measured by monitoring the multiple reaction (MRM) transitions of precursor ions mass charge (m/z) 121 --> 106 for 4EP and (m/z) 151 --> 136 for 4EG. Qualitative and quantitative confirmation data were acquired simultaneously by monitoring alternative MRM transitions following an external standard method. Calibration was linear over a working range of 10 and 5000 microg/L. Limit of determination (LOD) and limit of quantification (LOQ) were 10 and 50 microg/L for both 4EG and 4EP. HPLC analysis phenols were separated with a gradient system of acetonitrile-water and detected using a diode array detector (DAD) at 280 nm, and for the fluorescence analysis, excitation and emission wavelengths of 260 and 305 nm were used. Quantitative analysis of 4EP and 4EG was performed by the standard addition method to avoid matrix interferences. Calibration was linear over a concentration range from 10 to 5000 microg/L for HPLC-DAD, from 1 to 10,000 microg/L for 4EP, and from 10 to 10,000 for 4EG for fluorescence analysis. LOD and LOQ for the DAD analysis were 10 and 50 microg/L for both 4EG and 4EP. For fluorescence analysis, LOD and LOQ were 1 and 5 microg/L for 4EP and 10 and 50, respectively, for 4EG. The proposed methods can be easily used for the qualitative and quantitative determination of 4EP and 4EG in wines affected by microbial contamination with yeasts of the Brettanomyces genus.