Multilaboratory Collaborative Study of a Nontarget Data Acquisition for Target Analysis (nDATA) Workflow Using Liquid Chromatography-High-Resolution Accurate Mass Spectrometry for Pesticide Screening in Fruits and Vegetables.

Nontarget data acquisition for target analysis (nDATA) workflows using liquid chromatography-high-resolution accurate mass (LC-HRAM) spectrometry, spectral screening software, and a compound database have generated interest because of their potential for screening of pesticides in foods. However, these procedures and particularly the instrument processing software need to be thoroughly evaluated before implementation in routine analysis. In this work, 25 laboratories participated in a collaborative study to evaluate an nDATA workflow on high moisture produce (apple, banana, broccoli, carrot, grape, lettuce, orange, potato, strawberry, and tomato). Samples were extracted in each laboratory by quick, easy, cheap, effective, rugged, and safe (QuEChERS), and data were acquired by ultrahigh-performance liquid chromatography (UHPLC) coupled to a high-resolution quadrupole Orbitrap (QOrbitrap) or quadrupole time-of-flight (QTOF) mass spectrometer operating in full-scan mass spectrometry (MS) data-independent tandem mass spectrometry (LC-FS MS/DIA MS/MS) acquisition mode. The nDATA workflow was evaluated using a restricted compound database with 51 pesticides and vendor processing software. Pesticide identifications were determined by retention time (tR, ±0.5 min relative to the reference retention times used in the compound database) and mass errors (δM) of the precursor (RTP, δM ≤ ±5 ppm) and product ions (RTPI, δM ≤ ±10 ppm). The elution profiles of all 51 pesticides were within ±0.5 min among 24 of the participating laboratories. Successful screening was determined by false positive and false negative rates of <5% in unfortified (pesticide-free) and fortified (10 and 100 µg/kg) produce matrices. Pesticide responses were dependent on the pesticide, matrix, and instrument. The false negative rates were 0.7 and 0.1% at 10 and 100 µg/kg, respectively, and the false positive rate was 1.1% from results of the participating LC-HRAM platforms. Further evaluation was achieved by providing produce samples spiked with pesticides at concentrations blinded to the laboratories. Twenty-two of the 25 laboratories were successful in identifying all fortified pesticides (0-7 pesticides ranging from 5 to 50 µg/kg) for each produce sample (99.7% detection rate). These studies provide convincing evidence that the nDATA comprehensive approach broadens the screening capabilities of pesticide analyses and provide a platform with the potential to be easily extended to a larger number of other chemical residues and contaminants in foods.

Influence of dough composition on the formation of processing contaminants in yeast-leavened wheat toasted bread.

The influence of dough composition on acrylamide, 3-monochloropropane-1,2-diol (3-MCPD) esters, and glycidyl esters (GE) formation during bread toasting was investigated. The doughs differed in added amounts of soy lecithin, salt, and reducing agents (l-cysteine and glutathione). The toasting of bread for 2.5 min considerably enhanced the formation of acrylamide and 3-MCPD esters. The addition of lecithin (1%, w/w) resulted in four times higher content of 3-MCPD esters in toasted bread slices. No distinct relationship between dough composition and GE formation in untoasted and toasted bread was found. The addition of reducing agents (0.05%, w/w) mitigated during toasting not only the formation of 3-MCPD esters (more than six times) but also the extent of Maillard reaction that resulted in three times lower amounts of acrylamide and predominant formation of alcohol-like compounds. Toasted bread without reducing agents contained typical Maillard reaction compounds such as aldehydes, alkyl pyrazines, and derivatives of furan.

Quantitation of Cannabinoids in Cannabis Dried Plant Materials, Concentrates, and Oils Using Liquid Chromatography-Diode Array Detection Technique with Optional Mass Spectrometric Detection: Single-Laboratory Validation Study, First Action 2018.11.

This paper describes a single-laboratory validation of a liquid chromatography-diode array detection (LC-DAD) method for quantification of 12 major cannabinoids in Cannabis dried plant materials, concentrates, and oils. The method met Standard Method Performance Requirements for quantitative analysis of cannabinoids in Cannabis concentrates and Cannabis dried plant materials. The LOQs were in the range 0.003-0.10% (w/w), depending on the analyte and matrix. Spike recoveries were between 96.7 and 101.3% with relative SDs (RSDs) ≤2.3%. Precision expressed as repeatability and intermediate precision was within 0.3-4.8 and 1.1-5.1%, respectively. The chromatographic separation conditions used in this versatile method are compatible with both DAD-UV and MS detection. During method validation, high-resolution quadrupole time-of-flight MS was employed as a secondary detector (connected in series to the LC-DAD instrument) to provide high confidence identification of target analytes and as a tool for monitoring other cannabinoids for which reference standards were not available. The obtained results demonstrate applicability of the method to quantitative analysis of important cannabinoids in dried plants, concentrates, and oils. Limited data were generated for a food matrix (Cannabis-containing cookies) using this method with LC coupled to a compact single quadrupole mass spectrometer.

Determination of Bisphenol A (BPA) in Commercially Packaged Ready-to-Consume Carbonated and Noncarbonated Water and Nonalcoholic Beverages: A Single-Laboratory Validation Study, First Action 2017.15.

Background: Bisphenol A (BPA) is an endocrine-disrupting compound that could migrate to beverages from packaging materials. AOAC INTERNATIONAL issued a call for methods for determination of free BPA in beverages based on the AOAC Standard Method Performance Requirement (SMPR®) 2017.018. Objective: The objective of the single-laboratory validation (SLV) study was to validate a method for the analysis of BPA in relevant beverages and evaluate its performance characteristics against the AOAC SMPR 2017.18. Methods: The analytical method involves extraction of a beverage sample (10 mL) using 10 mL 1% acetic acid in acetonitrile after the addition of isotopically labeled internal standard. Sodium chloride is added to salt out BPA into the acetonitrile phase. After centrifugation, a freeze-out step is used to remove coextracted lipids. An aliquot of the supernatant upper layer is then analyzed using high-pressure LC coupled to tandem mass spectrometry in electrospray negative ionization mode. Results: Accuracy, repeatability, and intermediate precision were determined by spiking 10 representative nonalcoholic beverage matrixes at three concentration levels. Individual percent recoveries ranged between 76.5 and 113.2%, 86.2 and 114.8%, and 90.9 and 109.3% for the spike levels of 0.3, 1.5, and 20 µg/L, respectively. The repeatability and intermediate precision results were in the range of 0.2-11.3% and 1.8-11.1%, respectively. The LOD was estimated at ≤0.1 µg/L and LOQ was validated at 0.3 µg/L. Conclusions: The validated method performance characteristics met the requirements stated in the AOAC SMPR 2017.018. This method was approved AOAC First Action Official MethodsSM 2017.15. Highlights: SLV study evaluating free BPA at three concentration levels (0.3, 1.5, and 20 µg/L) in 10 types of commercially packaged nonalcoholic beverages (as recommended for the method validation in the AOAC SMPR 2017.018). Validated method performance characteristics (LOD, LOQ, analytical range, accuracy, and precision) met the AOAC SMPR 2017.018. Method approved as the AOAC First Action Official Method 2017.15.

Method development and validation for low-level propineb and propylenethiourea analysis in baby food, infant formula and related matrices using liquid chromatography-tandem mass spectrometry.

Two simple, selective and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for determination of propineb and propylenethiourea (PTU) in infant formula, fruit-based and cereal-based baby food and raw materials used in production of infant formula, including carbohydrates, protein isolates, vegetable oils and emulsifiers. The sample preparation procedure for propineb analysis was based on streamlined derivatisation to form and stabilise the target analyte (propylenebisdithiocarbamate-dimethyl), followed by extraction using a modified QuEChERS procedure with a dispersive solid phase extraction (d-SPE). The PTU determination employed an aqueous extraction with optimised protein precipitation and single-step SPE clean-up. To achieve maximum sensitivity, electrospray ionisation and atmospheric-pressure chemical ionisation were employed for LC-MS/MS analysis of propineb and PTU, respectively. Validation of the developed methods was performed in accordance with Document SANTE/11813/2017. Mean recoveries were in the range of 86-120% for propineb and PTU, respectively, with interday and intraday repeatabilities below 13%. A limit of quantification (LOQ) of 0.003 mg kg-1 was validated for most of the evaluated analyte/sample matrix combinations with the exception of PTU in soy protein isolate and soybean oil, for which an LOQ of 0.01 mg kg-1 was obtained. This is the first report that provides validated methods for monitoring propineb and PTU in infant formula and baby foods at concentrations compliant with the maximum residue levels established in the EU legislation.

Method development and validation for total haloxyfop analysis in infant formulas and related ingredient matrices using liquid chromatography-tandem mass spectrometry.

According to the European Commission directive 2006/141/EC, haloxyfop residue levels should not exceed 0.003 mg/kg in ready-to-feed infant formula, and the residue definition includes sum of haloxyfop, its esters, salts, and conjugates expressed as haloxyfop. A simple method for total haloxyfop analysis in infant formula and related ingredient matrices was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of an alkaline hydrolysis with methanolic sodium hydroxide to release haloxyfop (parent acid) from its bound forms prior to the extraction with acetonitrile. A mixture of magnesium sulfate (MgSO4) and sodium chloride (NaCl) (4:1, w/w) was added to the extract to induce phase separation and force the analyte into the upper acetonitrile-methanol layer and then a 1-mL aliquot was subsequently cleaned up by dispersive solid phase extraction with 150 mg of MgSO4 and 50 mg of octadecyl (C18) sorbent. The analytical procedure was developed and carefully optimized to enable low-level, total haloxyfop analysis in a variety of challenging matrices, including infant formulas and their important high-carbohydrate, high-protein, high-fat, and emulsifier ingredients. The final method was validated in two different laboratories by fortifying samples with haloxyfop and haloxyfop-methyl, which was used as a model compound simulating bound forms of the analyte. Mean recoveries of haloxyfop across all fortification levels and evaluated matrices ranged between 92.2 and 114% with repeatability, within-lab reproducibility, and reproducibility RSDs ≤ 14%. Based on the validation results, this method was capable to convert the haloxyfop ester into the parent acid in a wide range of sample types and to reliably identify and quantify total haloxyfop at the target 0.003 mg/kg level in infant formulas (both powdered and ready-to-feed liquid forms). Graphical abstract LC-MS/MS-based workflow for the determination of the total haloxyfop in infant formula and related ingredients.

Development and Validation of a Multiclass, Multiresidue Method for Veterinary Drug Analysis in Infant Formula and Related Ingredients Using UHPLC-MS/MS.

A multiclass, multiresidue method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed and validated for the analysis of around 150 veterinary drugs in infant formula and related dairy ingredients. The included analytes belong to the following veterinary drug classes: anthelmintics, antibiotics (aminoglycoside, amphenicols, ß-lactams-penicillins and cephalosporins, lincosamides, macrolides, quinolones, sulfonamides, tetracyclines, and others), antimicrobial growth promoters, antiprotozoals, ß-agonists, coccidiostats, dyes, pesticides, and tranquilizers. The sample preparation procedure involves dispersing the sample in 0.05 M EDTA solution in water, followed by extraction with 0.1% formic acid in acetonitrile, drying down an aliquot of the extract, and reconstituting it in a water-acetonitrile mixture. The analyte detection, identification, and quantitation are performed by UHPLC-MS/MS using positive electrospray ionization mode. The method was validated in infant formula powder, whole milk powder, and whey protein isolate, typically achieving limits of quantitation (meeting acceptable recovery and precision validation criteria) at 1-10 ng/g.

Single-Laboratory Validation Study of a Method for Screening and Identification of Phosphodiesterase Type 5 Inhibitors in Dietary Ingredients and Supplements Using Liquid Chromatography/Quadrupole-Orbital Ion Trap Mass Spectrometry: First Action 2015.12.

A single-laboratory validation study of a method for screening and identification of phosphodiesterase type 5 (PDE5) inhibitors in dietary ingredients and supplements is described. PDE5 inhibitors were extracted from the samples using a 50:50 (v/v) mixture of acetonitrile and water and centrifuged. Supernatant was diluted, filtered, and analyzed by LC-high-resolution MS. Data were collected in MS acquisition mode that combined full-scan MS experiment with all-ion fragmentation and data-dependent MS/MS product from the ion scan experiment. This approach enabled collection of MS and tandem MS (MS/MS) data for both targeted and nontargeted PDE5 inhibitors in a single chromatographic run. Software-facilitated identification of targeted analytes was performed based on the retention time, accurate mass, and isotopic pattern of pseudomolecular ions, and accurate masses of fragment ions using an in-house compound database. Detection and identification of other PDE5 inhibitors and novel analogs were performed by retrospective evaluation of MS and MS/MS experimental data. The method validation results obtained for evaluated matrixes fulfilled the probability of identification requirements and probability of detection requirements (for the pooled data) set at 90% (95% confidence interval) in the respective AOAC Standard Method Performance Requirements for identification and screening methods for PDE5 inhibitors. Limited data demonstrating the quantification capability of the method were also generated. Mean recovery and repeatability obtained for the evaluated PDE5 inhibitors were in the range 69-90% and 0.4-1.8%, respectively.

Mycotoxins in Plant-Based Dietary Supplements: Hidden Health Risk for Consumers.

Mycotoxin contamination of dietary supplements represents a possible risk for human health, especially in the case of products intended for people suffering from certain health conditions. The aim of this study was to assess the extent of this problem based on analyses of a wide set of herbal-based dietary supplements intended for various purposes: (i) treatment of liver diseases (milk thistle); (ii) reduction of menopause effects (red clover, flax seed, and soy); and (iii) preparations for general health support (green barley, nettle, goji berries, yucca, etc.) The analytical method including 57 mycotoxins was based on a QuEChERS-like (quick, easy, cheap, effective, rugged, safe) approach and ultrahigh performance liquid chromatography coupled with tandem mass spectrometry. The main mycotoxins determined were Fusarium trichothecenes, zearalenone and enniatins, and Alternaria mycotoxins. Co-occurrence of enniatins, HT-2/T-2 toxins, and Alternaria toxins was observed in many cases. The highest mycotoxin concentrations were found in milk thistle-based supplements (up to 37 mg/kg in the sum).

Determination of polycyclic aromatic hydrocarbons (PAHs) in seafood using gas chromatography-mass spectrometry: collaborative study.

A collaborative study was conducted to determine selected polycyclic aromatic hydrocarbons (PAHs) and their relevant alkyl homologs in seafood matrixes using a fast sample preparation method followed by analysis with GC/MS. The sample preparation method involves addition of (13)C-PAH surrogate mixture to homogenized samples and extraction by shaking with a water-ethyl acetate mixture. After phase separation induced by addition of anhydrous magnesium sulfate-sodium chloride (2 + 1, w/w) and centrifugation, an aliquot of the ethyl acetate layer is evaporated, reconstituted in hexane, and cleaned up using silica gel SPE. The analytes are eluted with hexane-dichloromethane (3 + 1, v/v), the clean extract is carefully evaporated, reconstituted in isooctane, and analyzed by GC/MS. To allow for the use of various GC/MS instruments, GC columns, silica SPE cartridges, and evaporation techniques and equipment, performance-based criteria were developed and implemented in the qualification phase of the collaborative study. These criteria helped laboratories optimize their GC/MS, SPE cleanup, and evaporation conditions; check and eliminate potential PAH contamination in their reagent blanks; and become familiar with the method procedure. Ten laboratories from five countries qualified and completed the collaborative study, which was conducted on three seafood matrixes (mussel, oyster, and shrimp) fortified with 19 selected PAH analytes at five different levels of benzo[a]pyrene (BaP) ranging from 2 to 50 µg/kg. Each matrix had a varying mixture of three different BaP levels. The other studied PAHs were at varying levels from 2 to 250 µg/kg to mimic typical PAH patterns. The fortified analytes in three matrixes were analyzed as blind duplicates at each level of BaP and corresponding other PAH levels. In addition, a blank with no added PAHs for each matrix was analyzed singly. Eight to 10 valid results were obtained for the majority of determinations. Mean recoveries of all tested analytes at the five different concentration levels were all in the range of 70-120%: 83.8-115% in shrimp, 77.3-107% in mussel, and 71.6-94.6% in oyster, except for a slightly lower mean recovery of 68.6% for benzo[ a ]anthracene fortified at 25 µg/kg in oyster (RSDr: 5.84%, RSDR: 21.1%) and lower mean recoveries for anthracene (Ant) and BaP in oyster at all three fortification levels (50.3-56.5% and 48.2-49.7%, respectively). The lower mean recoveries of Ant and BaP were linked to degradation of these analytes in oyster samples stored at -20°C, which also resulted in lower reproducibility (RSDR values in the range of 44.5-64.7% for Ant and 40.6-43.5% for BaP). However, the repeatability was good (RSDr of 8.78-9.96% for Ant and 6.43-11.9% for BaP), and the HorRat values were acceptable (1.56-1.94 for Ant and 1.10-1.45 for BaP). In all other cases, repeatability, reproducibility, and HorRat values were as follows: shrimp: RSDr 1.40-26.9%, RSDR 5.41-29.4%, HorRat: 0.22-1.34; mussel: RSDr 2.52-17.1%, RSDR 4.19-32.5%, HorRat: 0.17-1.13; and oyster: RSDr 3.12-22.7%, RSDR 8.41-31.8%, HorRat: 0.34-1.39. The results demonstrate that the method is fit-for-purpose to determine PAHs and their alkyl homologs in seafood samples. The method was approved by the Expert Review Panel on PAHs as the AOAC Official First Action Method 2014.08.

50th anniversary of the Florida pesticide residue workshop and the birth of the north american chemical residue workshop.

The papers in this special issue of Journal of Agricultural and Food Chemistry were originally presented at the 50th North American Chemical Residue Workshop (NACRW), formerly known as the Florida Pesticide Residue Workshop (FPRW). The 2013 meeting celebrated the rich history of 50 years of the FPRW and the birth of the NACRW, which in its name reflects the increased scope of the workshop to topics related to the analysis of all chemical residues and contaminants in food, feed, and environmental samples.

Rapid analysis of aminoglycoside antibiotics in bovine tissues using disposable pipette extraction and ultrahigh performance liquid chromatography-tandem mass spectrometry.

A high-throughput qualitative screening and identification method for 9 aminoglycosides of regulatory interest has been developed, validated, and implemented for bovine kidney, liver, and muscle tissues. The method involves extraction at previously validated conditions, cleanup using disposable pipette extraction, and analysis by a 3 min ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. The drug analytes include neomycin, streptomycin, dihydrosptreptomycin, and spectinomycin, which have residue tolerances in bovine in the US, and kanamicin, gentamicin, apramycin, amikacin, and hygromycin, which do not have US tolerances established in bovine tissues. Tobramycin was used as an internal standard. An additional drug, paromomycin also was validated in the method, but it was dropped during implementation due to conversion of neomycin into paromomycin. Proposed fragmentation patterns for the monitored ions of each analyte were elucidated with the aid of high resolution MS using a quadrupole-time-of-flight instrument. Recoveries from spiking experiments at regulatory levels of concern showed that all analytes averaged 70-120% recoveries in all tissues, except hygromycin averaged 61% recovery. Lowest calibrated levels were as low as 0.005 µg/g in matrix extracts, which approximately corresponded to the limit of detection for screening purposes. Drug identifications at levels <0.05 µg/g were made in spiked and/or real samples for all analytes and tissues tested. Analyses of 60 samples from 20 slaughtered cattle previously screened positive for aminoglycosides showed that this method worked well in practice. The UHPLC-MS/MS method has several advantages compared to the previous microbial inhibition screening assay, especially for distinguishing individual drugs from a mixture and improving identification of gentamicin in tissue samples.

Evaluation of a new column backflushing set-up in the gas chromatographic-tandem mass spectrometric analysis of pesticide residues in dietary supplements.

This study evaluated the use of a new concurrent backflushing set-up in the multiresidue analysis of pesticides in dietary supplement matrices using gas chromatography-tandem mass spectrometry (GC-MS/MS). The backflushing configuration employed a purged union installed between a short, 5-m long capillary column and a 15-m analytical column of the same column diameter (0.25 mm i.d.), stationary phase type (HP-5MS UI) and film thickness (0.25 µm). This set-up is more time- and cost-effective than the use of post-run or mid-column backflushing configurations because the backflushing starts as soon as the last analyte elutes from the short column, thus preventing the less volatile matrix components from reaching the longer analytical column and MS source. As opposed to the analysis without backflushing, the column does not need to be kept at a higher temperature for an extended period of time, resulting in about 50% increased sample throughput on the instrument (a run time of 20 min). Optimization of the GC-MS/MS method is discussed in detail, especially when it comes to the selection of MS/MS transitions, optimization of injection conditions using a programmable temperature vaporizer (PTV) inlet in solvent vent mode, and optimization of the backflushing parameters. The optimized method showed very good long-term performance, which was evaluated in a 2.5-day uninterrupted sequence (without any system maintenance) of repeated injections of various dietary supplement extracts containing over one hundred pesticides, mainly those with limits set for herbal drugs and preparations by the U.S. and European Pharmacopoeias.