A Complementary, Quantitative, and Confirmatory Method to UHPLC/ESI Q-Orbitrap Screening Based on UPHLC/ESI-MS/MS for Analysis of 416 Pesticides in Fruits and Vegetables.

BACKGROUND: Triple quadrupole (MS/MS) and high-resolution mass spectrometry (HRMS), coupled with ultra-high performance (UHP) liquid chromatography (LC) or gas chromatography (GC), are technologies used to analyze pesticide residues in fruits and vegetables. LC-MS/MS has been the gold standard for analysis of pesticides, offering reliable performance and sensitivity, while LC-HRMS is expanding in application to serve as another benchmark. Method development and testing scope expansion are constantly required with new generation mass spectrometers. OBJECTIVE: This article discusses the development and validation of a quantitative and confirmatory method that can analyze over 400 pesticide residues using a state-of-the-art UHPLC/electrospray ionization (ESI)-MS/MS system. METHODS: Homogenized fruit and vegetable samples were fortified with pesticides and were extracted using a modified quick, easy, cheap, effective, rugged, and safe method. Samples were injected onto a UHPLC/ESI-MS/MS system, and data were acquired in multiple reaction monitoring (MRM) mode. The method was validated using a nested experimental design, and was able to quantify and confirm 416 pesticides in fruits and vegetables. It was also complimentary to the UHPLC/ESI Q-Orbitrap quantitative and screening methods previously developed in the authors' laboratory. RESULTS: The method demonstrated good performance. In all matrixes, 92% of pesticides yielded recoveries between 81-110%, more than 95% of pesticides yielded intermediate precision ≤20%, about 65% of pesticides yielded measurement uncertainties ≤20%, and 96% of pesticides yielded measurement uncertainties ≤50%. CONCLUSION: This method shows great potential to be a standalone method or as part of a laboratory workflow for quantitative and confirmatory analysis of pesticide residues in fruits and vegetables. HIGHLIGHTS: This method was developed using the same mobile phases, analytical columns, and extraction procedure, as UHPLC/ESI Q-Orbitrap methods. Extracts can be run on either system, streamlining monitoring programs and offering high sample throughput.

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.

Applications of nDATA for screening, quantitation, and identification of pesticide residues in fruits and vegetables using UHPLC/ESI Q-Orbitrap all ion fragmentation and data independent acquisition.

High sample throughput and effective multiresidue methods for screening, quantitation, and identification are desired for the analysis of a large number of pesticides in routine monitoring programs for food safety. This study was designed to explore the use of an UHPLC/ESI Q-Orbitrap nontarget data acquisition for target analysis (nDATA) workflow for screening 655 pesticides and quantifying a small group of 46 most likely incurred pesticide residues in fruits and vegetables in a single analysis. High-resolution mass spectrometers such as the Q-Orbitrap offer unique applications for pesticide analysis using full MS scan with data independent acquisition (DIA) or all ion fragmentation (AIF) scan. The experiments were designed to achieve a balance between selectivity and cycle time by considering parameter settings such as mass resolution and the number of mass isolation windows or isolation window widths. Coupled with ultra-high performance liquid chromatography (UHPLC), both full MS/DIA and full MS/AIF nDATA workflows were evaluated for screening, quantification, and identification in a single analysis. In general, UHPLC/ESI full MS/vDIA detected more fragment ions per pesticide than AIF when one to four fragments were compared. UHPLC/ESI full MS/vDIA and AIF generated comparable quantitative results, but the latter provided slightly better repeatability likely due to its shorter cycle time and more scans across a chromatographic peak. UHPLC/ESI full MS/vDIA may be preferable for screening, quantitation and identification when the testing scope covers a few hundreds of pesticides in a single analysis.

UHPLC/ESI Q-Orbitrap Quantitation of 655 Pesticide Residues in Fruits and Vegetables-A Companion to an nDATA Working Flow.

BACKGROUND: Effective and expansive methods for multiresidue pesticide analysis are desired for routine monitoring programs. These methods are complex, especially when several hundred pesticides are involved. OBJECTIVE: Two approaches to sort data and identify isomers and isobaric ions in pesticide mixtures were evaluated to determine whether they could be differentiated by mass resolving power and/or chromatographic resolution. METHOD: This study presents an application of ultra-high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap) along with QuEChERS for the quantitation of 655 pesticide residues in fruits and vegetables. RESULTS: From the developed method, 94.7% of the 655 pesticides in fruits and 93.9% of those in vegetables had recoveries between 81% and 110%; 98.3% in both fruits and vegetables had an intermediate precision of ≤20%; and 97.7% in fruits or 97.4% in vegetables showed measurement uncertainty of ≤50%. When the retention time difference (ΔtR) of two isomers was ≥0.12 min, they were chromatographically resolved. Twenty five out of 35 pairs or groups of isomers were chromatographically separated (ΔtR ≥ 0.12 min), but 14 pairs were not resolved (ΔtR < 0.12 min). There were 493 pairs of pesticides with a mass-to-charge difference of <1 Da. Only one pair of isobaric ions could not be separated by mass and chromatographic resolution. HIGHLIGHTS: UHPLC/ESI Q-Orbitrap along with QuEChERS sample preparation offers a practical quantitative companion method to a non-target data acquisition for target analysis workflow for pesticide residue analysis in routine monitoring programs for food safety.

Non-target data acquisition for target analysis (nDATA) of 845 pesticide residues in fruits and vegetables using UHPLC/ESI Q-Orbitrap.

A non-target data acquisition for target analysis (nDATA) workflow based on accurate mass measurements using UHPLC/ESI Q-Orbitrap full MS-data-independent acquisition and a compound database was developed to screen pesticide residues in fruit and vegetable samples. The compound database of 845 pesticides was built from dd-MS2 (data-dependent acquisition) product ion spectral data and LC retention times of individual pesticide standards. MS2 spectra of samples were acquired using multiplexing data-independent acquisition (mDIA) and variable data-independent acquisition (vDIA). Screening of pesticides in samples was based on either the retention time (± 0.5 min) and the mass accuracy (± 5 ppm) of a precursor (RTP by full MS) or the retention time (± 0.5 min) and the mass accuracy (± 5 ppm) of a precursor and its fragment ion (RTFI by full MS/DIA). In validation studies involving mDIA and vDIA analysis of 10 fruits and vegetables spiked with pesticides prior to QuEChERS sample preparation, RTP correctly found up to 765 and 796 pesticides at 10 and 100 µg/kg, respectively, whereas RTFI correctly identified up to 729 and 764 pesticides at the same respective concentrations. UHPLC/ESI Q-Orbitrap full MS/mDIA or vDIA proved to be a comprehensive detection technique and has potential for pesticide residue screening in fruits and vegetables. Graphical Abstract.

Perspectives on Liquid Chromatography-High-Resolution Mass Spectrometry for Pesticide Screening in Foods.

This perspective discusses the use of liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for multiresidue analysis of pesticides in foods and agricultural commodities. HRMS has the important distinction and advantage of mass-resolving power and, therefore, requires different concepts, experiments, and guidance for screening, identification, and quantitation of pesticides in complex food matrices over triple quadrupole mass spectrometry. HRMS approaches for pesticide screening, including full-scan experiments in conjunction with tandem mass spectrometry (MS/MS) experiments, are described. This approach results in the generation of chromatographic retention times and high-resolution mass spectra with accurate mass measurements that can be used to create compound databases. New data processing tools can create an efficient and optimized screening approach that can speed the analysis and identification of compounds, reduce the need for chemical standards, and harmonize pesticide analytical procedures.

Target screening of 105 veterinary drug residues in milk using UHPLC/ESI Q-Orbitrap multiplexing data independent acquisition.

This paper presents a multi-class target screening method for the detection of 105 veterinary drug residues from 11 classes in milk using ultra-high performance liquid chromatography electrospray ionization quadrupole Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap). The method is based on a non-target approach of full mass scan and multiplexing data-independent acquisition (Full MS/mDIA). The veterinary drugs include endectocides, fluoroquinolones, ionophores, macrolides, nitroimidazole, NSAIDs, ß-lactams, penicillins, phenicols, sulfonamides, and tetracyclines. Veterinary drug residues were extracted from milk using a salting-out and solid-phase extraction (SOSPE) procedure, which entailed the precipitation of milk proteins by an extraction buffer (oxalic acid and EDTA, pH 3) and acetonitrile, a salting-out acetonitrile/water phase separation using ammonium sulfate, and solid-phase extraction for clean-up using polymeric reversed-phase sorbent cartridges. The Q-Orbitrap Full MS/dd-MS2 (data-dependent acquisition) was used to acquire product-ion spectra of individual veterinary drugs to build a compound database and a mass spectral library, whereas its Full MS/mDIA was utilized to acquire sample data from milk for target screening of veterinary drugs fortified at 1.0 or 10.0 µg/kg. The in-spectrum mass correction or solvent background lock-mass correction was used to minimize mass error when building the compound database from experimental dd-MS2 accurate mass data. Retention time alignment and response threshold adjustment were used to eliminate or reduce false negatives and/or false positive rates. The validated method was capable of screening 58% and 96% of 105 veterinary drugs at 1.0 and 10.0 µg/kg, respectively, without manually evaluating every compound during data processing, which will reduce the workload in routine practice.

Development and Validation of a Qualitative Method for Target Screening of 448 Pesticide Residues in Fruits and Vegetables Using UHPLC/ESI Q-Orbitrap Based on Data-Independent Acquisition and Compound Database.

A semiautomated qualitative method for target screening of 448 pesticide residues in fruits and vegetables was developed and validated using ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC/ESI Q-Orbitrap). The Q-Orbitrap Full MS/dd-MS2 (data dependent acquisition) was used to acquire product-ion spectra of individual pesticides to build a compound database or an MS library, while its Full MS/DIA (data independent acquisition) was utilized for sample data acquisition from fruit and vegetable matrices fortified with pesticides at 10 and 100 µg/kg for target screening purpose. Accurate mass, retention time and response threshold were three key parameters in a compound database that were used to detect incurred pesticide residues in samples. The concepts and practical aspects of in-spectrum mass correction or solvent background lock-mass correction, retention time alignment and response threshold adjustment are discussed while building a functional and working compound database for target screening. The validated target screening method is capable of screening at least 94% and 99% of 448 pesticides at 10 and 100 µg/kg, respectively, in fruits and vegetables without having to evaluate every compound manually during data processing, which significantly reduced the workload in routine practice.

Development and Validation of a Multiclass Method for Analysis of Veterinary Drug Residues in Milk Using Ultrahigh Performance Liquid Chromatography Electrospray Ionization Quadrupole Orbitrap Mass Spectrometry.

This paper presents the development and validation of a multiclass method for the analysis of veterinary drug residues in milk using ultrahigh performance liquid chromatography electrospray ionization quadrupole Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap). The 12 classes of veterinary drugs (a total of 125) included in this study were endectocides, fluoroquinolones, ionophores, macrolides, nitroimidazole, NSAIDs, ß-lactams, penicillins, phenicols, sulfonamides, tetracyclines, and aminoglycosides. Veterinary drug residues in milk were extracted using a modified salting-out supported liquid extraction (SOSLE) method, which entailed the precipitation of milk proteins using an extraction buffer (oxalic acid and EDTA, pH 3) and acetonitrile, a salting-out acetonitrile/water phase separation using ammonium sulfate, and solid-phase extraction (SPE) using polymeric reversed-phase sorbent cartridges. The final extracts were concentrated and reconstituted into a buffer solution and analyzed using UHPLC/ESI Q-Orbitrap mass spectrometry. The developed method was validated using a nested experimental design to evaluate the method performance characteristics, such as overall recovery, intermediate precision, and measurement uncertainty. The method was able to quantify or screen up to 105 veterinary drugs from 11 different classes, except aminoglycosides. The limits of quantification were as low as 1.0 µg/kg, with an analytical range from 1.0 to 100.0 µg/kg in milk.

Ultrahigh-performance liquid chromatography electrospray ionization Q-Orbitrap mass spectrometry for the analysis of 451 pesticide residues in fruits and vegetables: method development and validation.

This paper presents an application of ultrahigh-performance liquid chromatography electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC/ESI Q-Orbitrap MS) for the determination of 451 pesticide residues in fruits and vegetables. Pesticides were extracted from samples using the QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure. UHPLC/ESI Q-Orbitrap MS in full MS scan mode acquired full MS data for quantification, and UHPLC/ESI Q-Orbitrap Full MS/dd-MS(2) (i.e., data-dependent scan mode) obtained product ion spectra for identification. UHPLC/ESI Q-Orbitrap MS quantification was achieved using matrix-matched standard calibration curves along with the use of isotopically labeled standards or a chemical analogue as internal standards to achieve optimal method accuracy. The method performance characteristics include overall recovery, intermediate precision, and measurement uncertainty evaluated according to a nested experimental design. For the 10 matrices studied, 94.5% of the pesticides in fruits and 90.7% in vegetables had recoveries between 81 and 110%; 99.3% of the pesticides in fruits and 99.1% of the pesticides in vegetables had an intermediate precision of ≤20%; and 97.8% of the pesticides in fruits and 96.4% of the pesticides in vegetables showed measurement uncertainty of ≤50%. Overall, the UHPLC/ESI Q-Orbitrap MS demonstrated acceptable performance for the quantification of pesticide residues in fruits and vegetables. The UHPLC/ESI Q-Orbitrap Full MS/dd-MS(2) along with library matching showed great potential for identification and is being investigated further for routine practice.

Ultra-high performance liquid chromatography/ electrospray ionization-tandem mass spectrometry determination of 151 pesticides in soybeans and pulses.

This paper presents the application of ultra-high performance LC (UHPLC) and MS for the determination of 151 pesticides in soybeans and pulses. A core-shell particle (2.6 micro m particle size) column and a fully porous sub-2 microm (1.7 microm particle size) column showed comparable performance in chromatographic resolution and separation, increasing selectivity, and reducing analysis time. UHPLC was coupled with either a triple quadrupole mass analyzer (MS/MS) or a quadrupole Orbitrap (namely Orbital trap) mass spectrometer (Q-Orbitrap MS), which possesses fast data acquisition capability. Both configurations yielded analytical run times of < or =14 min. Soybean and pulse samples were analyzed and quantitated for pesticide residues using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure, UHPLC/electrospray ionization (ESI)-MS/MS, and matrix-matched standard calibration curves (in an analytical range of 5-500 microg/kg) with isotopically-labeled standards or a chemical analog as internal standards. The method performance parameters that included overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a nested design experiment. Approximately 89% of the pesticides studied had recoveries between 81 and 110%; 95%, had intermediate precision < or =20%; and 93% showed measurement uncertainty < or =40%. From a pilot study of 100 samples, eight tested positive by UHPLCIESI-MS/MS for carbendazim, methomyl, or imidacloprid. These pesticides were further confirmed using UHPLC/ESI-Q-Orbitrap MS based on accurate mass measurement with mass error < or =5 ppm.

Application of ultrahigh-performance liquid chromatography and electrospray ionization quadrupole orbitrap high-resolution mass spectrometry for determination of 166 pesticides in fruits and vegetables.

This paper presents an application of ultrahigh-performance liquid chromatography and electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC/ESI Q-Orbitrap) for determination of 166 pesticide residues in fruits and vegetables. Pesticides were extracted from the samples using the QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure. UHPLC/ESI Q-Orbitrap MS (i.e., full MS scan) acquired full MS data for quantification, and UHPLC/ESI Q-Orbitrap dd-MS(2) (i.e., data-dependent scan) obtained product-ion spectra for confirmation. UHPLC/ESI Q-Orbitrap MS quantification was achieved using matrix-matched standard calibration curves with isotopically labeled standards or chemical analogues as internal standards. The method performance characteristics that included overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a nested experimental design. For the matrices studied, about 90.3-91.5% of the pesticides had recoveries between 81 and 110%, 92.1-97.6% had intermediate precision ≤20%, and 89.7-95.2% had measurement uncertainty ≤40%. Confirmation was based on mass accuracy ≤5 ppm and LC retention time tolerance within ±2.5%. Overall, the UHPLC/ESI Q-Orbitrap has demonstrated great performance for quantification and confirmation of pesticide residues in fresh fruits and vegetables.

Application of a tandem mass spectrometer and core-shell particle column for the determination of 151 pesticides in grains.

A comparison of ultrahigh performance liquid chromatography (UHPLC) with a 2.6 µm core-shell particle column (Kinetex C(18)) and conventional liquid chromatography (LC) with a 3 µm porous particle column (Atlantis dC(18)), coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS), for the determination of 151 pesticides in grains is presented in this study. Pesticides were extracted from grain samples using a procedure known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). Quantification, with an analytical range from 5 to 500 µg/kg, was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analogue as internal standards. The method performance parameters that included overall recovery, intermediate precision, and measurement uncertainty were evaluated using a designed experiment, that is, the nested design. The UHPLC (Kinetex C(18)) was superior to conventional LC (Atlantis dC(18)) as it yielded a shorter analytical run time, increased method sensitivity, and improved method performance. For UHPLC/ESI-MS/MS (Kinetex C(18)), 90% of the pesticides studied had recoveries between 81 and 110%, 88% of the pesticides had intermediate precision ≤20%, and 84% of the pesticides showed measurement uncertainty ≤40%. As compared to UHPLC/ESI-MS/MS (Kinetex dC(18)), the LC/ESI-MS/MS (Atlantis dC(18)) showed a relatively lower sensitivity, less repeatability, and larger measurement uncertainty. UHPLC/ESI-MS/MS with 2.6 µm core-shell particle column and scheduled MRM proved to be a good choice for quantification or determination of pesticides in grains.

Applications of LC/ESI-MS/MS and UHPLC/Qq-TOF-MS for the determination of 141 pesticides in tea.

This paper presents the applications of LC-electrospray ionization (ESI)/MS/MS and ultra-HPLC (UHPLC)/ESI quadrupole (Qq)-time-of-flight (TOF) MS for the determination of 141 pesticides in tea. Pesticides were extracted and cleaned up from tea with a modified quick, easy, cheap, effective, rugged, and safe method using graphitized carbon black and primary-secondary amine sorbents. Quantification was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analog as internal standards in an analytical range from 5 to 500 microg/kg. The LC/ESI-MS/MS served as a reliable tool to quantify the pesticides due to its superior sensitivity and good repeatability. Its method performance characteristics that include overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a statistically designed experiment, i.e., a nested design. About 87% of the pesticides had recoveries between 81 and 110%; 94% had an intermediate precision < or = 20%; and 90% showed measurement uncertainty < or = 40%. About 92% of the pesticides were able to be detected at 5 microg/kg with an S/N > or = 3. The UHPLC/Qq-TOF-MS showed much less sensitivity and poorer repeatability compared to the LC/ESI-MS/MS, and, therefore, it was primarily used for confirmatory purposes based on the accurate mass measurement and isotopic patterns.

Applications of LC/ESI-MS/MS and UHPLC QqTOF MS for the determination of 148 pesticides in fruits and vegetables.

This paper presented the applications of liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and ultra-high-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC QqTOF MS) for the determination of 148 pesticides in fruits and vegetables. Pesticides were extracted from fruits and vegetables using a buffered QuEChERS method. Quantification was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analog as internal standards in an analytical range from 5 to 500 microg/kg. The method performance parameters including overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a statistically designed experiment, i.e., a nested design. For LC/ESI-MS/MS, 95% of the pesticides had recoveries between 81% and 110%; 97% had an intermediate precision < or = 20%; and 95% (in fruits) or 93% (in vegetables) showed measurement uncertainty < or = 40%. Compared to LC/ESI-MS/MS, UHPLC QqTOF MS showed a relatively poor repeatability and large measurement uncertainty. About 93% (in fruits) or 94% (in vegetables) of the pesticides had recoveries between 81% and 110%; 86% (in fruits) or 90% (in vegetables) had an intermediate precision < or = 20%; and 79% (in fruits) or 88% (in vegetables) showed measurement uncertainty < or = 40%. LC/ESI-MS/MS proved to be the first choice for quantification or pre-target analysis due to its superior sensitivity and good repeatability. UHPLC QqTOF MS provided accurate mass measurement and isotopic patterns, and was an ideal tool for post-target screening and confirmation.

Applications of LC/ESI-MS/MS and UHPLC QqTOF MS for the determination of 148 pesticides in berries.

Applications of liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and ultrahigh-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC QqTOF MS) for the determination of 148 pesticides in berry fruits are presented in this study. Pesticides were extracted from berries using a procedure known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). Quantification, with an analytical range from 5 to 500 microg/kg, was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analogue as internal standards. The method performance parameters, which included overall recovery, intermediate precision, and measurement uncertainty, were evaluated according to a designed experiment, that is, the nested design. For LC/ESI-MS/MS, 95% of the pesticides studied had recoveries between 81 and 110%, 98% of the pesticides had intermediate precision of