Analysis of pesticides residues in fresh produce using buffered acetonitrile extraction and aminopropyl cleanup with gas chromatography/triple quadrupole mass spectrometry, liquid chromatography/triple quadrupole mass spectrometry, gas chromatography/ion trap detector mass spectrometry, and GC with a halogen-specific detector.

A rapid and inexpensive multiresidue method for determining pesticides in fruits and vegetables is presented. Extraction of a 15 g sample with 15 mL acetonitrile was followed by buffering with magnesium sulfate, sodium chloride, sodium citrate dihydrate, and disodium citrate. Acidification with formic acid prior to dispersive cleanup with aminopropyl sorbent and magnesium sulfate was used to stabilize base-sensitive pesticides such as chlorothalonil. Extracts were concentrated to 2 g/mL. Analyses were conducted by GC/ion trap detector MS, GC-halogen-specific detector, and LC/triple quadrupole MS. Accuracy and repeatability for 166 compounds in tomato, potato, and cabbage were 70-120% and <20% CV in 85% of the compounds, respectively. Reproducibility over a 4 month period in multiple commodities and analytical conditions was 62-124%, with CVs better than 30% for 91% of the compounds. Supply cost/sample was reduced 66%, and time to extract a batch of 24 samples was reduced by half compared to the prior method that used a 50 g sample, 100 mL acetonitrile, multiple SPE columns, and additional instrumentation. Additional extraction studies were conducted in tomatoes, oranges, and green beans at 4 g/mL using a GC/MS triple quadrupole system with a programmable temperature vaporization inlet. Recoveries of 70-120% were achieved in 93% of all compounds in green beans, 95% in tomatoes, and 97% in oranges.

Issues in mass spectrometry between bench chemists and regulatory laboratory managers: summary of the roundtable on mass spectrometry held at the 123rd AOAC International Annual Meeting.

At the 123rd AOAC International Annual Meeting in Philadelphia, PA, 45 residue chemists gathered for a roundtable discussion of mass spectrometry (MS) used for regulatory chemical residues analysis. The session was conceived to address current technical and communication issues about MS between "bench chemists and their bosses". The topics covered a range of practical, routine, and recurring issues on capabilities and limitations of MS techniques, and suggestions on how chemists may better communicate their MS results with customers. The customers in this sense include laboratory managers, quality assurance officers, laboratory clients, regulatory officials, policy-makers, lawyers, and others who have interest in the data. The stated goals devised by the roundtable panelists were to provide independent advice, describe limitations, give practical tips, help set realistic expectations, and answer questions from the attendees. The panelists divided the topics into three main themes: practical aspects in routine analysis using MS, choice of MS technique depending on the purpose for analysis, and qualitative identification and confirmation concepts. This report was written to summarize and expand upon the discussion, frame the current issues, and provide advice on handling common situations in MS analysis and reporting of results. Topics included LODs, data quality objectives, quantification and reporting results, matrix effects, calibration, terminology, differences in performance across MS platforms, proficiency testing, qualitative analysis, and laboratory accreditation. Conclusions are presented as a set of questions for structuring a dialog between bench chemists and laboratory managers.

Accurate Quantitation and Analysis of Nitrofuran Metabolites, Chloramphenicol, and Florfenicol in Seafood by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry: Method Validation and Regulatory Samples.

We developed and validated a method for the extraction, identification, and quantitation of four nitrofuran metabolites, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), semicarbazide (SC), and 1-aminohydantoin (AHD), as well as chloramphenicol and florfenicol in a variety of seafood commodities. Samples were extracted by liquid-liquid extraction techniques, analyzed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), and quantitated using commercially sourced, derivatized nitrofuran metabolites, with their isotopically labeled internal standards in-solvent. We obtained recoveries of 90-100% at various fortification levels. The limit of detection (LOD) was set at 0.25 ng/g for AMOZ and AOZ, 1 ng/g for AHD and SC, and 0.1 ng/g for the phenicols. Various extraction methods, standard stability, derivatization efficiency, and improvements to conventional quantitation techniques were also investigated. We successfully applied this method to the identification and quantitation of nitrofuran metabolites and phenicols in 102 imported seafood products. Our results revealed that four of the samples contained residues from banned veterinary drugs.

UHPLC-MS/MS method for the quantitation of penicillin G and metabolites in citrus fruit using internal standards.

Penicillin G has been applied to citrus trees as a potential treatment in the fight against Huanglongbing (HLB). Here, we have developed and validated a method to identify and quantitate penicillin G and two of its metabolites, penillic acid and penilloic acid, in citrus fruit using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). This method improves upon a previous method by incorporating isotopically labeled internal standards, namely, penillic acid-D5, and penilloic acid-D5. These standards greatly enhanced the accuracy and precision of our measurements by compensating for recovery losses, degradation, and matrix effects. When 2g of citrus fruit sample is extracted, the limits of detection (LOD) were determined to be 0.1ng/g for penicillin G and penilloic acid, and 0.25ng/g for penillic acid. At fortification levels of 0.1, 0.25, 1, and 10ng/g, absolute recoveries for penillic and penilloic acids were generally between 50-70%. Recoveries corrected with the isotopically labeled standards were approximately 90-110%. This method will be useful for the identification and quantitation of drug residues and their degradation products using isotopically labeled standards and UHPLC-MS/MS.

LC-MS/MS Validation of a Residue Analysis Method for Penicillin G and Its Metabolites in Commercial Orange Juice.

Florida citrus depends on a breakthrough in the fight against citrus greening disease. Of the antibiotics used to treat this disease, penicillin G has been one of the most effective. Because orange fruit grown in the state of Florida are mainly used to produce orange juice, we have validated an ultra-HPLC tandem MS method to screen for penicillin G and its metabolites (penillic and penilloic acids) at the chemical residue level after treatment. In this method, three spike levels (0.25, 1, and 20 ng/g) were tested in triplicate. Absolute recoveries for penillic and penilloic acids were 60-75% depending on the matrix used, whereas corrected recoveries of penicillin G using an isotopically labeled internal standard were ~100%. Two product ion transitions per analyte were required for identification, which contributes to a high degree of selectivity.

Identification of Penicillin G Metabolites under Various Environmental Conditions Using UHPLC-MS/MS.

In this work, we investigate the stability of penicillin G in various conditions including acidic, alkaline, natural acidic matrices and after treatment of citrus trees that are infected with citrus greening disease. The identification, confirmation, and quantitation of penicillin G and its various metabolites were evaluated using two UHPLC-MS/MS systems with variable capabilities (i.e., Thermo Q Exactive Orbitrap and Sciex 6500 QTrap). Our data show that under acidic and alkaline conditions, penicillin G at 100 ng/mL degrades quickly, with a determined half-life time of approximately 2 h. Penillic acid, penicilloic acid, and penilloic acid are found to be the most abundant metabolites of penicillin G. These major metabolites, along with isopenillic acid, are found when penicillin G is used for treatment of citrus greening infected trees. The findings of this study will provide insight regarding penicillin G residues in agricultural and biological applications.

LC-MS/MS Method for the Determination and Quantitation of Penicillin G and Its Metabolites in Citrus Fruits Affected by Huanglongbing.

In this study, we developed and validated a method for the extraction, identification, and quantitation of penicillin G and its metabolites (penilloic acid and penillic acid) in a variety of citrus fruits by employing sequential liquid/liquid and solid-phase extraction techniques in conjunction with UHPLC-MS/MS. Two product ion transitions per analyte were required for identification, which contributes to a high degree of selectivity. Corrected recoveries of penicillin G using an isotopically labeled internal standard were 90-100% at fortification levels of 0.1, 0.25, 1, and 10 ng/g. Absolute recoveries for penillic acid and penilloic acid were 50-75% depending on the matrix used. The limit of detection (LOD) of penicillin G and its metabolites was found to be 0.1 ng/g when 2 g of citrus was extracted. This method is useful in determining residue levels of penicillin G and its metabolites in citrus trees infected with huanglongbing bacteria after antibiotic treatment.

Multilaboratory validation of a method to confirm chloramphenicol in shrimp and crabmeat by liquid chromatography-tandem mass spectrometry.

An existing method for chloramphenicol (CAP) determination in shrimp using a gas chromatograph with electron capture detector was adapted for confirmation of CAP with a liquid chromatograph interfaced to a triple quadrupole mass spectrometer. CAP residues are extracted from tissue with ethyl acetate, isolated via liquid-liquid extraction, and concentrated by evaporation. Extracts are chromatographed by using a reversed-phased column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions (m/z 152, 176, 194, and 257) of precursor m/z 321 were monitored. Moving from gas chromatography to liquid chromatography-tandem mass spectrometry improved the sensitivity of the method greatly, enabling reliable confirmation of CAP residues at 0.3 microg/kg (ppb). The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 3 laboratories.

Interlaboratory comparison of a general method to screen foods for pesticides using QuEChERs extraction with high performance liquid chromatography and high resolution mass spectrometry.

An interlaboratory comparison of a multipesticide residue analytical method is reported. The goal of the comparison was to evaluate the potential for liquid chromatography/high resolution mass spectrometry along with a specific automated screening procedure to allow the determination of the presence or absence of a set of targeted compounds without additional manual review. The method utilized an off the shelf QuEChERs based extraction followed by analysis with an orbitrap mass spectrometer with the data evaluated by ToxID. The method was tested at three laboratories, with three produce matrices (spinach, carrots, and oranges), and three levels of spiked pesticides with all analyses in triplicate. A series of 247 compounds were tested, and it was found that the three laboratories produced consistent data; however, manual review was still necessary. The data was shown to have no false negatives for 211 compounds in the three produce matrixes at 200 ppb. Of these 211 compounds, 189 had no false negatives at 50 ppb, and 129 had no false negatives at 10 ppb. The HRMS method was shown to be robust with similar data being achieved by all three laboratories and detectable concentrations only slightly above the range shown for triple quadrupole MS/MS.