Analytical method for the determination of atrazine and its dealkylated chlorotriazine metabolites in water using gas chromatography/mass selective detection

A multiresidue method is reported for the determination of atrazine and its dealkylated chlorotriazine metabolites in water. Water samples are buffered to pH 10 and partitioned in ethyl acetate. Final analysis is accomplished using gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring (SIM) mode. The limit of detection (LOD) is 0.050 ng and the limit of quantification (LOQ) is 0.10 ppb for 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 2-amino-4-chloro-6-(isopropylamino)-s-triazine (G-30033), 2-amino-4-chloro-6-(ethylamino)-s-triazine (G-28279), and 2, 4-diamino-6-chloro-s-triazine (G-28273). The mean procedural recoveries were 90, 92, 98, and 85% and the standard deviations were 12, 13, 16, and 20% for atrazine, G-30033, G-28279, and G-28273, respectively (n = 30). The study was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160 for method validation. The reported procedure accounts for residues of G-28273 in water that are not included in EPA Method 507.     

Determination of the 1'S and 1'R diastereomers of metolachlor and S-metolachlor in water by chiral liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS)

An enantioselective method for the separation and quantification of the diastereomer pairs of metolachlor and S-metolachlor in surface and ground waters is presented. Samples are purified and concentrated using a C18 (octadecyl silica) solid-phase extraction (SPE) procedure and analyzed by chiral column liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS) interfaced with either atmospheric pressure chemical ionization (APcI) or atmospheric pressure photoionization (APPI) sources. The overall mean percent procedural recoveries (percent relative standard deviations) are 89% (10.6%) for surface water and 80% (9.1%) for ground water. The method limit of quantitation (LOQ) is 0.10 ppb. The method validation was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160.     

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.     

高效液相色谱法同时测定土壤中环丙氨嗪和三聚氰胺

本试验研究建立了同时测定土壤中环丙氨嗪和三聚氰胺残留量的高效液相色谱法.红壤、潮土等5种土壤样品经氨水/甲醇 (5/95,v/v)超声提取3次,浓缩处理后上机检测.环丙氨嗪和三聚氰胺的标准曲线在0.1 ～ 15.0 μg/ml浓度范围内线性关系良好,绝对系数(R2)分别为1.0000和0.9998;在0.5 ～ 5 mg/kg添加范围内,环丙氨嗪和三聚氰胺在土壤中的平均回收率分别为87.2% ～ 101.1% 和 75.3% ～ 101.6%,变异系数分别为3.3% ～ 8.1%、1.6% ～ 9.9%,最低检测限分别为0.05 mg/kg、0.07 mg/kg.与国际上气相/液相色谱-质谱连用法相比,操作简单,经济方便易于普及.     

Analytical method for the determination of atrazine and its dealkylated chlorotriazine metabolites in water using SPE sample preparation and GC-MSD analysis

A method is reported for the determination of atrazine and its dealkylated chlorotriazine metabolites in ground, surface, and deionized water. Water samples are adjusted to pH 3-4 prior to loading onto two SPE cartridges in series: C-18 and C-18/cation exchange mixed-mode polymeric phases. The analytes are eluted from each of the two cartridges separately, and the pooled and concentrated fraction is analyzed using gas chromatography-mass selective detection in the selected ion monitoring mode. The lower limit of method validation is 0.10 micrograms/L (ppb) for 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 2-amino-4-chloro-6-(isopropylamino)-s-triazine (G-30033, deethylatrazine), 2-amino-4-chloro-6-(ethylamino)-s-triazine (G-28279, deisopropylatrazine), and 2,4-diamino-6-chloro-s-triazine (G-28273, didealkyatrazine). The overall mean procedural recoveries (and standard deviations) are 96 (6.9), 96 (5.5), 95 (6.8), and 100% (10%) for atrazine, G-30033, G-28279, and G-28273, respectively (n = 49). The method validation study was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160. The reported procedure accounts for residues of G-28273 in water.     

Direct aqueous injection liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry analysis of water for atrazine, simazine, and their chlorotriazine metabolites

A method is reported for the determination of atrazine, simazine, and their respective dealkylated chlorotriazine metabolites in ground, surface, and finished drinking water. Water samples are diluted 1:4 in an injection vial prior to analysis using liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS). The lower limit of method validation is 0.10 microg/L (ppb) for 2-chloro-4-(ethylamino)-6-isopropylamino)-s-triazine (atrazine, G-30027), 2-chloro-4, 6-(diethylamino)-s-triazine (simazine, G-27692), 2-amino-4-chloro-6-(isopropylamino)-s-triazine (deethylatrazine, DEA, or G-30033), 2-amino-4-chloro-6-(ethylamino)-s-triazine (deisopropylatrazine, DIA, or G-28279), and 2,4-diamino-6-chloro-s-triazine (didealkylatrazine, DDA, or G-28273). The overall mean procedural recoveries (and % relative standard deviations) for atrazine, simazine, DEA, DIA, and DDA are 98 (4.4), 102 (3.6), 99 (4.8), 103 (4.0), and 109% (4.8%), respectively, in finished drinking water; 108 (2.7), 104 (5.4), 113 (4.5), 111 (5.2), and 105% (5.3%), respectively, in groundwater; and 96 (6.9), 103 (4.2), 102 (4.4), 102 (5.2), and 102% (8.2%), respectively, in surface water. The method validation was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160.     

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.     

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.     

Single-run electrochemical determination of melamine in dairy products and pet foods

A simple electrochemical approach, which does not require any expensive and complex instruments, is established for the selective and quantitative recognition of melamine in diary products and pet foods. During a preconcentration step (at 1.8 V versus Ag/AgCl), the formation of a polymer film from melamine on a preanodized screen-printed carbon electrode was identified by SEM and XPS. The as-formed polymer was found to be electroactive with a reversible redox peak, and hence square-wave voltammetry was applied to further increase the detection sensitivity to meet the detection limit for application in real sample analysis. Simply with a medium exchange procedure, melamine was selectively detected with a detection limit (S/N=3) of 0.8 μM (i.e., 98.3 ppb) by square-wave voltammetry. Lower than 1 ppm of melamine in real samples can be easily detected with good recoveries of 98.7-100.9% by the proposed approach. The recovery tests established for external calibration and standard addition techniques verified that the analysis can be done in a single-run measurement.     

Determination of sulfonylurea herbicides in water and food samples using sol-gel glass-based immunoaffinity extraction and liquid chromatography-ultraviolet/diode array detection or liquid chromatography-tandem mass spectrometry

Immunoaffinity supports (IAS) were prepared using broad specific polyclonal anti-sulfonylurea (SU) antibodies immobilized in sol-gel glass. Two different kinds of supports were applied, crushed sol-gel monoliths and sol-gel-coated highly porous silica particles. Both were used for the quantitative enrichment of SUs in natural water and food samples followed by high-performance liquid chromatography-ultraviolet/diode array detection (HPLC-UV/DAD) and tandem mass spectrometry (LC-MS/MS), respectively. Loading, washing, and elution conditions of IAS were optimized. The capacity of supports was determined for 30 SUs and compared with the cross-reactivity pattern of the direct competitive enzyme-linked immunosorbent assay. The capacities correlated well with the affinity to individual SU compounds. Even analytes to which the polyclonal antibodies showed only a lower cross-reactivity could be enriched to a certain degree, if a sufficient capacity of IAS was provided. The IAS could be reused at least 10 times without a loss of effectiveness. Recovery of 16 selected SUs extracted from spiked water and food samples was dependent on the affinity of both immobilized antibodies to single compounds and matrix interferences. In water, 13 SUs showed recoveries higher than 80% when immunoaffinity extraction was used in combination with LC-UV/DAD. On the basis of the enrichment of 200 mL of aqueous sample, corresponding limit of detection (LOD) values ranged between 20 and 100 ng/L. The recoveries of 10 SUs, which were extracted from 10 g of potato spiked at a 10 microg/kg level, were higher than 75%. For grain samples, recoveries were at the same order for at least five SU herbicides. The LOD of LC-MS/MS measurements was about 1 order of magnitude higher, i.e., gave LODs between 1.1 and 6.9 microg/kg of food sample, depending on the compound and extraction procedure. These LODs provide evidence that the main advantage of the prepared IAS is their high selectivity for group specific recognition of SUs as compared to other nonspecific solid phase extraction materials.     

Multiresidue pesticide analysis in wines by solid-phase extraction and capillary gas chromatography-mass spectrometric detection with selective ion monitoring

A method was developed to determine pesticides in wines. The pesticides were extracted from the wine using solid-phase extraction on a polymeric cartridge, and the coextractives were removed with an aminopropyl-MgSO(4) cartridge. Analysis was performed using capillary gas chromatography with electron impact mass spectrometric detection in selective ion monitoring mode (GC-MSD/SIM). Three injections are required to analyze all 153 organohalogen, organonitrogen, organophosphate, and organosulfur pesticides and residues. Pesticides were confirmed by retention times of the target ions and three qualifier-to-target ion ratios. Detection limits for most of the pesticides were less than 0.005 mg/L, and quantitation was determined from approximately 0.01 to 5 mg/L. Spike recoveries were performed by fortifying red and white wines at 0.01 and 0.10 mg/L. At the 0.01 ppm level, the spike recoveries were greater than 70% for 116 and 124 pesticides (out of 153) in red and white wines, respectively, whereas at the higher spike concentration of 0.10 mg/L, the recoveries were greater than 70% for 123 and 128 pesticides in red and white wines, respectively. The recoveries of less than 70% were most likely from pesticide polarity or lability, resulting in the inefficient adsorption of the pesticide to the polymeric sorbent, ineffective elution of the pesticide from the sorbent, or thermal degradation of the pesticide under GC-MSD conditions.     

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method extension to quantify simultaneously melamine and cyanuric acid in egg powder and soy protein in addition to milk products

As a consequence of the adulteration of infant formulas and milk powders with melamine (MEL) in China in 2008, much attention has been devoted to the analysis of MEL [and cyanuric acid (CA)] in dairy products. Several methods based on high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR), or Raman spectroscopy have been described in the literature. However, no method is available for the simultaneous determination of MEL and CA in other raw materials, which are considered as high-risk materials for economically motivated adulteration. The present paper reports the results of an interlaboratory-based performance evaluation conducted with seven laboratories worldwide. The purpose was to demonstrate the ability of a cleanup-free LC-MS/MS method, originally developed for cow's milk and milk-powdered infant formula, to quantify MEL and CA in egg powder and soy protein. Limit of detection (LOD) and limit of quantification (LOQ) were 0.02 and 0.05 mg/kg for MEL in egg powder and soy protein, respectively. For CA, LOD and LOQ were 0.05 and 0.10 mg/kg in egg powder and 1.0 and 1.50 mg/kg in soy protein, respectively. Recoveries ranged within a 97-113% range for both MEL and CA in egg powder and soy protein. Reproducibility values (RSD(R)) from seven laboratories were within a 5.4-11.7% range for both analytes in the considered matrices. Horwitz ratio (HorRat) values between 0.4 and 0.7 indicate acceptable among-laboratory precision for the method described.     

Efficient fluorescence energy transfer system between CdTe-doped silica nanoparticles and gold nanoparticles for turn-on fluorescence detection of melamine

We here report an efficient and enhanced fluorescence energy transfer system between confined quantum dots (QDs) by entrapping CdTe into the mesoporous silica shell (CdTe@SiO?) as donors and gold nanoparticles (AuNPs) as acceptors. At pH 6.50, the CdTe@SiO?-AuNPs assemblies coalesce to form larger clusters due to charge neutralization, leading to the fluorescence quenching of CdTe@SiO? as a result of energy transfer. As compared with the energy transfer system between unconfined CdTe and AuNPs, the maximum fluorescence quenching efficiency of the proposed system is improved by about 27.0%, and the quenching constant, K(sv), is increased by about 2.4-fold. The enhanced quenching effect largely turns off the fluorescence of CdTe@SiO? and provides an optimal "off-state" for sensitive "turn-on" assay. In the present study, upon addition of melamine, the weak fluorescence system of CdTe@SiO?-AuNPs is enhanced due to the strong interactions between the amino group of melamine and the gold nanoparticles via covalent bond, leading to the release of AuNPs from the surfaces of CdTe@SiO?; thus, its fluorescence is restored. A "turn-on" fluorimetric method for the detection of melamine is proposed based on the restored fluorescence of the system. Under the optimal conditions, the fluorescence enhanced efficiency shows a linear function against the melamine concentrations ranging from 7.5 × 10?? to 3.5 × 10?? M (i.e., 1.0-44 ppb). The analytical sensitivity is improved by about 50%, and the detection limit is decreased by 5.0-fold, as compared with the analytical results using the CdTe-AuNPs system. Moreover, the proposed method was successfully applied to the determination of melamine in real samples with excellent recoveries in the range from 97.4 to 104.1%. Such a fluorescence energy transfer system between confined QDs and AuNPs may pave a new way for designing chemo/biosensing.     

Poly- and monoclonal antibody-based ELISAs for fipronil

An enzyme-linked immunosorbent assay (ELISA) for fipronil was developed by using polyclonal antibodies (pABs) or monoclonal antibodies (mABs), and its suitability of the determination of this analyte in spiked water samples was studied. The pABs-based assay showed I50 = 17.95 ppb, I90 = 203.40 ppb, and I10 = 0.066 ppb, whereas the mABs-based assay showed I50 = 5.99 ppb, I90 = 485.40 ppb, and I10 = 0.074 ppb. The recoveries of fipronil from tap water samples by pABs-based ELISA were 93.00-124.00% in the range of 0-500 ng/mL, and those obtained from the samples by mABs-based ELISA were 94.70-108.00%. Different types of water from pool, river, and sea were spiked at different levels (ranging form 0.1 to 10 microg/L) and were assayed by the indirect ELISA with mABs. The recoveries of fipronil by this ELISA were in the range of 80-120%. The results demonstrate that this assay is suitable for the quantitative detection of fipronil at trace levels in water samples.     

Application and fate of Cyromazine in a closed-cycle hydroponic cultivation of bean (Phaseolus vulgaris L.)

The fate of cyromazine applied via the nutrient solution (20, 40, and 60 mg of active ingredient per plant) in a closed-cycle soilless cultivation of bean with zero discharge of effluents was traced in both the recycled drainage solution and the plant tissues for 99 days. The insecticide was applied once, 15 days after planting (16 days prior to the first harvest). In addition to cyromazine, the residues of melamine, its metabolite, in the drainage solution and plant tissues were also regularly determined during the 99 days. The two higher application doses induced toxicity symptoms on the leaves of the bean plant. The maximum cyromazine levels were measured 8 days after application in the drainage solution (17-46 mg l(-1)), 16 days in the roots (1.1-2.4 mg kg(-1) fresh weight [f. wt.]) and the vegetative shoot (4.5-9.5 mg kg(-1) f. wt.), and 24 days after application in the pods (2.6-4.1 mg kg(-1) f. wt.). However, the cyromazine residues in pods were clearly below the maximum acceptable levels for bean. The half-life of cyromazine in the drainage solution ranged from 16 to 19 days for the three doses. The melamine residues in the drainage solution and in the roots reached a concentration peak 16 days after cyromazine application, whereas in the vegetative shoot and the pods they were constantly increasing over the 99 days after application. Nevertheless, the melamine residues were constantly much lower than those of cyromazine, although on the last sampling day (99) they tended toward convergence. Cyromazine proved to be highly persistent, as indicated by the remarkably high residues measured in both the drainage solution and the plant tissues, even 99 days after application. Nevertheless, the application of cyromazine via the nutrient solution to beans grown in closed-cycle hydroponic systems at doses not exceeding 20 mg per plant seems to be safe with respect to both phytotoxicity and residue levels in the edible pods.     

U.S. EPA regulation of plant-incorporated protectants: assessment of impacts of gene flow from pest-resistant plants

The U.S. Environmental Protection Agency licenses pesticide-expressing plants under the authority of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). Transgenes and their pesticidal products represent pesticides under FIFRA and are referred to as plant-incorporated protectants (PIPs). When sexually compatible wild relatives (SCWR) are sympatric with PIP crops, there is a need to assess the potential for adverse effects to man and the environment resulting from transgene introgression in accord with FIFRA requirements. Genetic compatibility, introgression, weediness of SCWR × PIP hybrids, seed dispersal, and dormancy, among other parameters, as well as effects on other species (herbivores and beneficial insects), all need to be considered as part of the risk assessment for experimental use under Section 5 or registration under Section 3 of FIFRA. EPA is currently developing data requirements and guidance toward addressing potential gene flow impacts from PIPs.     

Qualitative and quantitative event-specific PCR detection methods for oxy-235 canola based on the 3' integration flanking sequence

As more genetically modified plant events are approved for commercialization worldwide, the event-specific PCR method has become the key method for genetically modified organism (GMO) identification and quantification. This study reveals the 3' flanking sequence of the exogenous integration of Oxy-235 canola employing thermal asymmetric interlaced PCR (TAIL-PCR). On the basis of the revealed 3' flanking sequence, PCR primers and TaqMan probe were designed and qualitative and quantitative PCR assays were established for Oxy-235 canola. The specificity and limits of detection (LOD) and quantification (LOQ) of these two PCR assays were validated to as low as 0.1% for the relative LOD of qualitative PCR assay; the absolute LOD and LOQ were low to 10 and 20 copies of canola genomic DNA in quantitative PCR assay, respectively. Furthermore, ideal quantified results were obtained in the practical canola sample detection. All of the results indicate that the developed qualitative and quantitative PCR methods based on the revealed 3' integration flanking sequence are suitable for GM canola Oxy-235 identification and quantification.     

Simultaneous determination of ochratoxin A and cyclopiazonic,mycophenolic, and tenuazonic acids in cornflakes by solid-phase microextraction coupled to high-performance liquid chromatography

A solid-phase microextraction (SPME) method, coupled to liquid chromatography with diode array UV detection (LC-UV/DAD), for the simultaneous determination of cyclopiazonic acid, mycophenolic acid, tenuazonic acid, and ochratoxin A is described. Chromatographic separation was achieved on a propylamino-bonded silica gel stationary phase using acetonitrile/methanol/ammonium acetate buffer mixture (78:2:20, v/v/v) as mobile phase. SPME adsorption and desorption conditions were optimized using a silica fiber coated with a 60 microm thick polydimethylsiloxane/divinylbenzene film. Estimated limits of detection and limits of quantitation ranged from 3 to 12 ng/mL and from 7 to 29 ng/mL, respectively. The method has been applied to cornflake samples. Samples were subjected to a preliminary short sonication in MeOH/2% KHCO(3) (70:30, v/v); the mixture was evaporated to near dryness and reconstituted in 1.5 mL of 5 mM phosphate buffer (pH 3) for SPME followed by LC-UV/DAD. The overall procedure had recoveries (evaluated on samples spiked at 200 ng/g level) ranging from 74 +/- 4 to 103 +/- 9%. Samples naturally contaminated with cyclopiazonic and tenuazonic acids were found; estimated concentrations were 72 +/- 9 and 25 +/- 6 ng/g, respectively.     

Rapid assay for analyzing biogenic amines in cheese: matrix solid-phase dispersion followed by liquid chromatography-electrospray-tandem mass spectrometry

A new rapid and sensitive method based on matrix solid-phase dispersion (MSPD) followed by liquid chromatography-electrospray-tandem mass spectrometry was devised for the determination of biogenic amines at trace levels in cheese samples. The method required 0.25 g of sample, CN-bonded silica as a dispersant sorbent, and a formic acid aqueous solution/methanol mixture as an eluting solvent. Extraction recoveries from soft cheese products were calculated in the 98 +/- 4-110 +/- 6% range. A procedure based on solid-phase extraction was also evaluated for the extraction of these compounds in cheese. Chromatographic separation was performed using a C18 column with an aqueous ammonium acetate/methanol mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision, and trueness. Results in the 0.05-0.25 mg kg(-1) range were obtained for the LOD of histamine, tyramine, and beta-phenylethylamine in soft cheese samples. Linearity was established over 2 orders of magnitude. Excellent precision in terms of intra-day repeatability was calculated (RSD% < 5). The applicability of the method to the determination of biogenic amines in cheese products was demonstrated.     

Determination of spinosad and its metabolites in citrus crops and orange processed commodities by HPLC with UV detection

Spinosad is an insect control agent that is derived from a naturally occurring organism and is effective on a wide variety of crops, including citrus crops. A method is described for the determination of spinosad and its metabolites in citrus crops and orange processed commodities. The method determines residues of the active ingredients (spinosyns A and D) and three minor metabolites (spinosyn B, spinosyn K, and N-demethylspinosyn D). For dried orange pulp and orange oil, the method has a limit of quantitation (LOQ) of 0.02 microg/g and a limit of detection (LOD) of 0.006 microg/g. For all other sample matrices (whole fruit, edible fruit, juice, and peel), the method has an LOQ of 0.01 microg/g and an LOD of 0.003 microg/g. The analytes are extracted from the various sample types using appropriate solvents, and the extracts are purified by liquid-liquid partitioning and/or solid-phase extraction. All five analytes are determined simultaneously in the purified extracts by reversed-phase high-performance liquid chromatography with ultraviolet detection at 250 nm.