Quantification and Confirmation of Fifteen Carbamate Pesticide

molecules

Article Quantiﬁcation and Conﬁrmation of Fifteen Carbamate Pesticide Residues by Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes via LC-MS/MS QTRAP System

Ying Zhou, Jian Guan, Weiwei Gao, Shencong Lv and Miaohua Ge *

Jiaxing Center for Disease Control and Prevention, Zhejiang 314050, China; [email protected] (Y.Z.); [email protected] (J.G.); [email protected] (W.G.); [email protected] (S.L.) * Correspondence: [email protected]; Tel.: +86-0573-83683808

Academic Editor: Alessandra Gentili Received: 29 August 2018; Accepted: 27 September 2018; Published: 29 September 2018

Abstract: In this research, fifteen carbamate pesticide residues were systematically analyzed by ultra-high performance liquid chromatography–quadrupole-linear ion trap mass spectrometry on a QTRAP 5500 system in both multiple reaction monitoring (MRM) and enhanced product ion (EPI) scan modes. The carbamate pesticide residues were extracted from a variety of samples by QuEChERS method and separated by a popular reverse phase column (Waters BEH C18). Except for the current conformation criteria including selected ion pairs, retention time and relative intensities from MRM scan mode, the presence of carbamate pesticide residues in diverse samples, especially some doubtful cases, could also be confirmed by the matching of carbamate pesticide spectra via EPI scan mode. Moreover, the fragmentation routes of fifteen carbamates were firstly explained based on the mass spectra obtained by a QTRAP system; the characteristic fragment ion from a neutral loss of CH3NCO (−57 Da) could be observed. The limits of detection and quantification for fifteen carbamates were 0.2–2.0 µg kg−1 and 0.5–5.0 µg kg−1, respectively. For the intra- (n = 3) and inter-day (n = 15) precisions, the recoveries of fifteen carbamates from spiked samples ranged from 88.1% to 118.4%, and the coefficients of variation (CVs) were all below 10%. The method was applied to pesticide residues detection in fruit, vegetable and green tea samples taken from local markets, in which carbamates were extensively detected but all below the standard of maximum residue limit.

Keywords: carbamates; multiple reaction monitoring (MRM); enhanced product ion (EPI); mass fragmentation; conﬁrmatory method; pesticide residues

1. Introduction Carbamate pesticides, namely, N-methyl carbamate esters not only share with organophosphates the capacity to kill insects by inhibiting the enzyme acetylcholinesterase (AChE) but also show lower toxicity to human being [1–3]. Gradually, they have become one kind of most popular pesticides in agriculture to guarantee food production. However, the improper use of carbamate pesticides have adversely affected food safety g [4,5]. Carbamate pesticide residues are known to be frequently present in fruits, vegetables and green teas on the market at levels close to or below the standard of maximum residue limit [6]. The legitimate content has brought chronic and continuous intake of carbamate residues that could be relevant to multiple ailments and further pose a threat to public health [7–9]. Meeting the challenge of many samples in daily work, developing a best concise, highly speciﬁc and sensitive analytical method to detect the carbamate residues in diverse samples from the area of food safety is important.

Molecules 2018, 23, 2496; doi:10.3390/molecules23102496 www.mdpi.com/journal/molecules Molecules 2018, 23, 2496 2 of 16

The challenge to modify this analytical method could be ascribed to two main factors: the existence of a range of carbamate pesticides and samples with complex composition that depend on the instrumental analysis and the sample pretreatment. With the efforts of researchers for half a century, the sample pretreatment of pesticide residues has been developed from Soxhlet extraction, liquid–liquid partition chromatography and column chromatography to solid-phase extraction, solid-phase micro-extraction, molecularly imprinted solid-phase extraction, super critical fluid extraction, matrix solid phase dispersion and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methods [10–12]. Developed in 2003 by Anastassiades, QuEChERS sample preparation has been readily accepted by many pesticide residue analysts over the years [13,14]. In the section of instrumental analysis, a literature survey (Table1) shows several methods for the detection of carbamate pesticide residues have been developed at low concentration levels in various samples, such as electrochemical detection [15], liquid chromatography [16–24], high-performance thin-layer chromatography [25], liquid chromatography with post-column fluorescence derivatization [26,27] and liquid chromatography with different mass spectrometry [28–32]. Of all the instrumental methods, the LC-MS system that combines the separation ability of liquid chromatography along with the sensitivity and specificity of detection from mass spectrometry and abandoned the additional derivation procedure of whole analysis process has gone mainstream for pesticide residue analysis. Among the different mass spectrometers, the ion trap triple quadrupole mass spectrometer allows for multiple reaction monitoring (MRM) scan modes is the ascendant instrument to quantify the residues of pesticides at trace amount. In MRM mode, the selected precursor ion of relevant compound is pre-screened in the first quadrupole (Q1), dissociated in the second quadrupole (Q2) and identified and quantified in the third quadrupole (Q3). During the analysis procedure, both Q1 and Q3 are set at several specific masses, allowing only those distinct fragment ions from the certain precursor ions to be detected. The setting of certain precursor ions and specific masses results in increased sensitivity and the structural specificity of the analyte [33,34].Compared with traditional triple quadrupole mass spectrometry, the Triple Quadrupole Linear Ion Traps system (QTRAP, AB SCIEX) equips an extra linear ion trap (LIT) located in the third quadrupole set and represents the new generation of LC-MS/MS system with unique scan mode of multiple reaction monitoring–information dependent acquisition–enhanced product ion (MRM-IDA-EPI) scan mode, which could maximize the information of the analytes in a single run, including selected ion pairs, retention time, relative intensities and the mass spectra. In this mode, the ions of analytes enter Q3 and are stopped from leaving this region by the changing of voltages. Then, the trap is allowed to fill with target ions with a set amount of time. The voltages are changed to stop any more ions from entering. Finally, the packet of ions is scanned out from the trap in a controlled manner. The target analytes are quantified as ever, and further identified on the automatically acquired MS/MS spectra while the precursor ions intensity exceeds the fixed area threshold setting. In particular, the linear ion trap could keep on capturing the interested precursor ions and product ions for a few milliseconds. This accumulation signifies a 500-times increase in the scanning sensitivity and brings a high intensity of the mass spectra recorded in EPI mode. It double checks the doubtful samples by comparing the acquired mass spectra with the mass spectral library built from standards [35]. This study developed a new analytical strategy to combine QuEChERS sample preparation and QTRAP LC-MS/MS system to improve the efficiency of quantification and confirmation of carbamate residues while ensuring high degree of reproducibility. In addition, this method should be stable enough to avoid false negative or false positive results. Molecules 2018, 23, 2496 3 of 16

Table 1. Comparative study between the published analysis methods for carbamates.

Target Object Sample Pretreatment Instrumental Analysis Analysis Limits Disadvantage Ref. ionic liquid-dispersive Ionic liquids are not commercially HPLC-FD Carbaryl soil liquid–liquid 0.63–4.0 ng g−1 available [11] (fluorescence detector) microextraction No confirmation spectra Large amount of Solvents gram, wheat, lentil, soybean, Seven carbamates column chromatography HPLC-UV 0.08–1.16 mg L−1 Lack of sensitivity [17] fenugreek leaves apple No confirmation spectra HPLC post-column No application Seven carbamates / / derivatization and fluorescence 0.2–0.7 ng [26] No confirmation spectra detection The tempestuously hydrolyzation of HPLC post-column standards in dilute hydrochloric acid Eleven carbamates / / derivatization and fluorescence 0.5 ng mL−1 [27] solution detection No confirmation spectra Lack of sensitivity; expensive Fifteen carbamates corn, cabbage, tomato QuEChERS method LC-MS (QDa) 1 µg mL−1 [28] No confirmation spectra orange, grape, onion, Matrix solid-phase Thirteen carbamates LC-MS (ESI/APCI) 0.001–0.01 mg kg−1 No confirmation spectra [29] tomatoes dispersion (concentration) Thirteen carbamates Traditional Chinese Medicine QuEChERS method UPLC-MS (MRM) 5.0–10.0 µg kg−1 No confirmation spectra [30] MoleculesMolecules2018 2018, 23, ,23 2496, x 4 of 416 of 16

2. Results2. Results and and Discussion Discussion

2.1.2.1. Optimization Optimization of of Sample Sample Preparation Preparation AsAs shown shown in in Figure Figure1, 1, the the scheme scheme of of analysisanalysis method, extraction extraction process process and and purification purification process process areare the the two two main main components components of the of QuEChERS the QuEChERS pretreatment. pretreatment. In this In study, this study, acetonitrile acetonitrile with different with concentrationsdifferent concentrations of formic acid of formic supplement acid supplement was compared was compared for extraction for extraction efficiency. efficiency. However, However, acetonitrile andacetonitrile acidified acetonitrile and acidified did acetonitrile not show any did obvious not show distinction any obvious in the distinction recovery in experiment. the recovery Thus, it wasexperiment. more convenient Thus, it was to adopt more convenient acetonitrile to in adopt the extraction acetonitrile process. in the extraction In the purification process. In process, the purification process, 1.0 g of sodium chloride and 2.0 g of anhydrous sodium sulfate were applied to 1.0 g of sodium chloride and 2.0 g of anhydrous sodium sulfate were applied to separate the aqueous separate the aqueous solution and acetonitrile part. In this study, the traditional anhydrous solution and acetonitrile part. In this study, the traditional anhydrous magnesium sulfate was replaced magnesium sulfate was replaced by anhydrous sodium sulfate because of the heat release in the by anhydrous sodium sulfate because of the heat release in the moisture absorbing process. In the moisture absorbing process. In the purification process, the commonly used cleaning agents are purificationethylenediamine-N-propylsilane process, the commonly (PSA), used graphitized cleaning agents carbon are black ethylenediamine-N-propylsilane (GCB) and C18. PSA is pertinent (PSA), to graphitizedorganic acids, carbon some black pigments, (GCB) and and some C18. sugars PSA is and pertinent fats; GCB to organichas a strong acids, adsorption some pigments, effect on and somepigments; sugars and and C18 fats; aims GCB to remove has a strong non-polar adsorption impurities effect such on as pigments;fats. Different and proportions C18 aims toof PSA, remove non-polarGCB, and impurities C18 were such combined as fats. Different to find the proportions best recipe of with PSA, the GCB, best and average C18 were recoveries. combined It was to find theproventhat best recipe the with combination the best average of 50 mg recoveries. C18 and 150 It was mg proventhatPSA as cleaning the combination agent was most of 50suitable mg C18 for and 150determining mg PSA as cleaning quantitatively agent carbamatewas most suitable residues for in determining multi-matrices quantitatively by recovery carbamate tests and samplesresidues in multi-matricesdetermination by (Figure recovery S1). tests and samples determination (Figure S1).

FigureFigure 1. Scheme 1. Scheme of analysis of analysis for the for simultaneous the simultaneous determination determination and identiﬁcation and identification of ﬁfteen of carbamate fifteen pesticidecarbamate residues pesticide in multi-matrices. residues in multi-matrices.

2.2.2.2. Optimization Optimization of of Mass Mass Spectrometry Spectrometry andand ChromatographicChromatographic Conditions Conditions ToTo confirm confirm the the ion ion pairs pairs for for quantification, quantification, the mixed standard standard solution solution of offifteen fifteen carbamate carbamate pesticidespesticides with with a concentrationa concentration of of 100 100µ μgg L L−−11 waswas infused infused into into the the QTRAP QTRAP mass mass spectrometer spectrometer at a at flow a flow raterate of 7ofµ 7L μL min min−1−1to to obtain obtain automaticautomatic analytes optimization optimization by by the the ESI ESI positive positive mode. mode. In the In the optimal optimal mass spectrometry conditions, all carbamates except tsumacide acquired two pairs of optimum ions for quantification and identification. MoleculesMolecules2018 2018, 23, ,23 2496, x 5 of 516 of 16

mass spectrometry conditions, all carbamates except tsumacide acquired two pairs of optimum ions forFor quantification the high sensitivity, and identification. symmetric shape of the ionic peaks and minimal retention time, this study examinedFor the high the sensitivity, elution type, symmetric flow rate, shape gradient of the andionic thepeaks type and of minimal the chromatographic retention time, column. this Oncestudy the examined most general the elution chromatographic type, flow rate, column gradient C18 and was the decided, type of the the separationchromatographic and ionization column. of theOnce analytes the most were general mainly chromatographic affected by the column compositions C18 was of decided, the elution the typeseparation and the and elution ionization gradient. of Therefore,the analytes several were classical mainly affected compositions by the ofcompositions the mobile of phase the elution were performedtype and the including elution gradient. methanol, acetonitrile,Therefore, waterseveral and classical water compositions with ammonium of the acetates mobile orphase formic were acid. performed Finally, including water and methanol, acetonitrile, acetonitrile, water and water with ammonium acetates or formic acid. Finally, water and acetonitrile, both supplemented with 0.1% formic acid, were chosen as the optimal mobile phases. The final both supplemented with 0.1% formic acid, were chosen as the optimal mobile phases. The final gradient elution with the total flow rate of 0.3 mL/min was as follows: 0–2.5 min, 15% A; 2.5–5 min, gradient elution with the total flow rate of 0.3 mL/min was as follows: 0–2.5 min, 15% A; 2.5–5 min, 15–50% A; 5–7 min, 50% A; 7–8 min, 50–15% A and 8–10 min, 15% A. The column oven was maintained 15–50% A; 5–7 min, 50% A; 7–8 min, 50–15% A and 8–10 min, 15% A. The column oven was at 40 ◦C and the injection volume was 5 µL. The representative LC-QTRAP-MS/MS chromatograms maintained at 40 °C and the injection volume was 5 μL. The representative LC-QTRAP-MS/MS werechromatograms merged (Figure were2). merged The retention (Figure 2). time The retention (RT) and time MS (RT) information and MS information for each analyte for each includinganalyte precursorincluding and precursor product and ions, product DP and ions, CE DP are and shown CE are in Tableshown2. in Table 2.

1.2x108 3

8 1.0x10 12

8.0x107

6.0x107 14 11 4.0x107 13 Intensity (cps)

4/5/6 10 15 2.0x107 89 2 1 7 0.0 0 2 4 6 8 10 Time (min) FigureFigure 2. LC-MS/MS2. LC-MS/MS chromatograms chromatograms of fifteen ﬁfteen carbamate carbamate pesticides pesticides (50 (50 ng ng mL mL−1).− 1).

TheThe EPI EPI survey survey scans scans in in IDA IDA experiment experiment should be be triggered triggered when when the the ionic ionic intensity intensity exceeded exceeded thethe threshold threshold of of 1000 1000 cps. cps. The The total total scanscan timetime (including (including pauses) pauses) was was 1.08 1.08 s for s for all allMRM MRM transitions. transitions. TheThe range range of of the the mass mass spectraspectra was set set between between 50 50 and and 300 300 amu amu with with a scan a scanrate of rate 10,000 of 10,000 Da s−1. DaThe s −1. Themass mass spectra spectra of of fifteen ﬁfteen carbamates carbamates and and their their characteristic characteristic fragmentations fragmentations are are shown shown in thein the SupplementarySupplementary Materials. Materials.

Table 2. Retention time and MS parameters of the fifteen carbamates pesticides.

Molecules 2018, 23, 2496 6 of 16

Table 2. Retention time and MS parameters of the ﬁfteen carbamates pesticides.

Retention Precursor Product Declustering Collision No. Compound 1 CAS No. Time (min) Ion (m/z) Ion (m/z) Potential (V) Energy (eV) 132.0 130 8 1 Aldicarb-sulfoxide 3.81 1646-87-3 206.9 89.0 * 130 17 166.1 * 140 11 2 Aldicarb-sulfone 4.17 1646-88-3 222.8 148.0 140 18 182.1 * 80 20 3 Pirimicarb 5.45 23103-98-2 239.1 72.0 80 30 181.0* 150 15 4 Carbofuran-3-hydroxy 5.57 16655-82-6 237.8 163.0 150 19 135.0 180 26 5 Methomyl 5.58 16752-77-5 162.8 106.0 * 180 28 72.0 40 18 6 Oxamyl 5.59 23135-22-0 220.0 163.1 * 40 10 89.0 * 150 18 7 Aldicarb 6.40 116-06-3 212.7 156.0 150 19 8 Tsumacide 6.60 1129-41-5 166.0 109.0 * 50 13 168.0 * 60 18 9 Propoxur 6.87 114-26-1 210.0 153.0 60 10 165.0 * 120 15 10 Carbofuran 6.98 1563-66-2 221.7 123.0 120 27 145.0 * 60 12 11 Carbaryl 7.04 63-25-2 202.0 117.0 80 35 95.0 100 19 12 Isoprocarb 7.18 2631-40-5 194.0 137.0 * 100 11 169.0 * 55 12 13 Methiocarb 7.33 2032-65-7 226.0 121.0 55 24 95.0 * 70 19 14 Fenobucarb 7.84 3766-81-2 208.0 152.0 70 11 157.1 * 60 12 15 Banol 8.29 671-04-5 214.1 121.2 60 16 1 CAS: chemical abstracts service; *: quantitative ion.

2.3. Fragmentation Manner of Carbamate Pesticides Based on the structure, the fifteen carbamates can be divided into three kinds: N-methyl amino formic acid aromatic ester, N-methyl amino formic acid oxime ester and heterocyclic N-methyl amino formic acid ester. The former two include carbaryl, tsumacide, methiocarb and methomyl; and aldicarb sulfone, aldicarb sulfoxide, etc., respectively, while the latter includes pirimicarb, isolan (highly regulated and unavailable), etc. Generally speaking, in the positive mode of electrospray ionization mass spectrometry, the proton first attaches to the protonation site, and then triggers the cleavages by migrating to the reactive center [36]. With the common structure of amino nitrogen and carbonyl oxygen, the protons could gravitate to these preferred protonation sites, and then trigger the dissociation reaction by migrating to the reactive center. Therefore, the oxygen of ester near the carbonyl carbon is a suitable active site to induce this fragmentation of carbamates. For example, 3-hydroxyl carbofuran belonging to N-methyl amino formic acid aromatic ester, could generate a stable fragment ion m/z 181.1 by a neutral loss of CH3NCO (−57 Da). The resulting spectrum is shown in Figure3. According to this fragment mechanism, all fifteen carbamate pesticides were investigated and the vast majority of carbamate pesticides, except pirimicarb, could dissolve at the same active site and obtain the same characteristic loss of 57 Da (Figures4 and5). Molecules 2018, 23, x 6 of 16

168.0 * 60 18 9 Propoxur 6.87 114-26-1 210.0 153.0 60 10 165.0 * 120 15 10 Carbofuran 6.98 1563-66-2 221.7 123.0 120 27 145.0 * 60 12 11 Carbaryl 7.04 63-25-2 202.0 117.0 80 35 95.0 100 19 12 Isoprocarb 7.18 2631-40-5 194.0 137.0 * 100 11 169.0 * 55 12 13 Methiocarb 7.33 2032-65-7 226.0 121.0 55 24 95.0 * 70 19 14 Fenobucarb 7.84 3766-81-2 208.0 152.0 70 11 157.1 * 60 12 15 Banol 8.29 671-04-5 214.1 121.2 60 16 1 CAS: chemical abstracts service; *: quantitative ion.

2.3. Fragmentation Manner of Carbamate Pesticides Based on the structure, the fifteen carbamates can be divided into three kinds: N-methyl amino formic acid aromatic ester, N-methyl amino formic acid oxime ester and heterocyclic N-methyl amino formic acid ester. The former two include carbaryl, tsumacide, methiocarb and methomyl; and aldicarb sulfone, aldicarb sulfoxide, etc., respectively, while the latter includes pirimicarb, isolan (highly regulated and unavailable), etc. Generally speaking, in the positive mode of electrospray ionization mass spectrometry, the proton first attaches to the protonation site, and then triggers the cleavages by migrating to the reactive center [36]. With the common structure of amino nitrogen and carbonyl oxygen, the protons could gravitate to these preferred protonation sites, and then trigger the dissociation reaction by migrating to the reactive center. Therefore, the oxygen of ester near the carbonyl carbon is a suitable active site to induce this fragmentation of carbamates. For example, 3- hydroxyl carbofuran belonging to N-methyl amino formic acid aromatic ester, could generate a stable fragment ion m/z 181.1 by a neutral loss of CH3NCO (−57 Da). The resulting spectrum is shown in Figure 3. According to this fragment mechanism, all fifteen carbamate pesticides were investigated Moleculesand the2018 vast, 23, 2496majority of carbamate pesticides, except pirimicarb, could dissolve at the same active7 of 16 site and obtain the same characteristic loss of 57 Da (Figures 4 and 5).

4x106 181.1 163.1 3x106

2x106 135.1 220.1 107.0 Intensity (cps) 1x106 117.1 237.8 72.6 196.0 0 50 100 150 200 250 m/z (Da)

Figure 3. Product ion spectrum and probable fragmentation routes of Carbofuran-3-hydroxy. Molecules 2018, 23, x 7 of 16 Molecules 2018, 23, 2496 8 of 16 Figure 3. Product ion spectrum and probable fragmentation routes of Carbofuran-3-hydroxy.

4x106 (1) Carbofuran-3-hydroxy 181.1 163.1 3x106

6 135.1 2x10 220.1 107.0 6 Intensity (cps) Intensity 117.1 1x10 237.8 72.6 196.0 0 50 100 150 200 250 m/z (Da)

1.0x107 (2)Tsumacide 109.1

8.0x106

6.0x106

4.0x106

Intensity (cps) Intensity 91.1 2.0x106 94.0 166.0 0.0 60 80 100 120 140 160 180 m/z (Da)

1.2x107 (3) Propoxur 111.0

1.0x107

8.0x106

6.0x10 92.9 168.0 4.0x106

Intensity (cps) Intensity 64.7 6 2.0x10 153.0 210.0 0.0 40 60 80 100 120 140 160 180 200 220 m/z (Da)

1.2x107 (4) Carbofuran 165.1 1.0x107

6 123.0 8.0x10

6.0x10

4.0x106

Intensity (cps) Intensity 221.7 2.0x106

0.0 50 100 150 200 250 m/z (Da)

2.0x107 (5) Carbaryl 145.1 1.6x107

1.2x107

8.0x106 202.1 Intensity (cps) Intensity 6 174.2 4.0x10 117.1 127.0 0.0 50 100 150 200 m/z (Da)

1.5x107 (6) Isoprocarb94.9 1.2x107

9.0x106

6.0x106 152.2 194.0 Intensity (cps) Intensity 6 3.0x10 76.7 137.2

0.0 60 80 100 120 140 160 180 200 m/z (Da)

Figure 4. Cont. Molecules 2018, 23, x 8 of 16

Molecules 2018, 23, 2496 9 of 16 Molecules 2018, 23, x 8 of 16 7 (7) Methiocarb 1.4x10 169.1 1.2x107

7 1.0x10 7 1.4x10 (7) Methiocarb 121.1 6 169.1 8.0x101.2x107

6 6.0x101.0x107 121.1 6 6 Intensity (cps) Intensity 4.0x108.0x10

6 6 90.8 2.0x106.0x10 184.9 226.0 0.0 6 Intensity (cps) Intensity 4.0x1060 80 100 120 140 160 180 200 220 240 6 90.8 2.0x10 m/z (Da) 184.9 226.0 0.0 60 80 100 120 140 160 180 200 220 240 m/z (Da) (8) Fenobucarb 8.0x106 94.9 O 6.0x106 (8) Fenobucarb 8.0x106 94.9 N

6 O 4.0x10 6 6.0x10 -57 Da N

6 Intensity (cps) Intensity 2.0x104.0x106 151.0 -57 Da 208.0

6 0.0 (cps) Intensity 2.0x10 40 60 80 100 120 140151.0 160 180 200208.0 220 m/z (Da) 0.0 40 60 80 100 120 140 160 180 200 220 m/z (Da) 8 3.0x10 (9) Banol 8 8 O 2.5x103.0x10 157.1 (9) Banol 8 2.0x102.5x108 157.1O O

1.5x102.0x108 8 N O N H 1.5x108 8 O N 1.0x10 -57 Da N H Intensity (cps) Intensity 57.1 8 OH+ 5.0x101.0x107 -57 Da

Intensity (cps) Intensity 121.2 72.257.1 196.1214.1 7 H+ 0.05.0x10 72.2 121.2 214.1 50 100 150 200196.1 250 Cl m/z 214.1 0.0 m/z (Da) 50 100 150 200 250 Cl m/z 214.1 m/z (Da) Figure 4. Product ion spectra and proposed fragmentation pathway of nine N-methyl amino formic FigureacidFigure 4. aromaticProduct 4. Product ionester: spectra ion (1) spectra Carbofuran-3-hydroxy; andproposed and proposed fragmentation fragmentation (2) Tsumacide; pathway pathway ( of3) nineof Propoxur; nine N-methyl N-methyl (4) aminoCarbofuran; amino formic formic (5acid ) acid aromatic ester: (1) Carbofuran-3-hydroxy; (2) Tsumacide; (3) Propoxur; (4) Carbofuran; (5) aromaticCarbaryl; ester: (6) (Isoprocarb;1) Carbofuran-3-hydroxy; (7) Methiocarb; (8 ()2 )Fenobucarb; Tsumacide; and (3) ( Propoxur;9) Banol. (4) Carbofuran; (5) Carbaryl; (6) Isoprocarb;Carbaryl; ( 7(6)) Methiocarb;Isoprocarb; (7 ()8 Methiocarb;) Fenobucarb; (8) Fenobucarb; and (9) Banol. and (9) Banol.

1.2x107 (1) Methomyl 163.1 1.2x107 (1) Methomyl 163.1 1.0x107 1.0x107 8.0x106 8.0x106 6 6.0x10 6

106.9 6.0x10 106.9 135.1 6 107.1 135.1 4.0x10 6 4.0x10 106.4 107.1 Intensity (cps) Intensity 106.4 Intensity (cps) Intensity 6 144.9 6 144.9 2.0x102.0x10 127.0127.0 0.0 0.0 6060 80 80 100 100 120 120 140 140 160 160 180 180 m/z (Da) m/z (Da)

7 7 163.1 1.0x101.0x10(2) Oxamyl(2) Oxamyl 163.1

8.0x108.0x106 6

6 6.0x106.0x106

6 4.0x106 135.1135.1 4.0x10 106.9

Intensity (cps) Intensity 106.9

Intensity (cps) Intensity 6 220.0 2.0x102.0x106 220.0 0.0 0.0 60 80 100 120 140 160 180 200 220 60 80 100 120 140 160 180 200 220 m/z (Da) m/z (Da)

7 1.0x10 1.0x107 (3) Aldicarb 213.0 (3) Aldicarb 213.0 8.0x106 8.0x106 6.0x106 6.0x106

4.0x106 89.0 4.0x106 Intensity (cps) Intensity 89.0 2.0x106 Intensity (cps) Intensity 116.0 2.0x106 70.0 98.0 156.0 0.0 116.0 70.060 8098.0 100 120 140156.0 160 180 200 220 0.0 m/z (Da) 60 80 100 120 140 160 180 200 220 m/z (Da)

Figure 5. Cont. MoleculesMolecules2018 2018, 23,, 23 2496, x 9 of10 16 of 16

5x106 (4) Aldicarb Sulfoxide 4x106 89.0

3x106

207.0 2x10Molecules6 2018, 23, x 9 of 16

Intensity (cps) Intensity 1x106 75.8 132.1 6 104.8 145.0 69.75x10 189.0 0 (4) Aldicarb Sulfoxide 50 100 150 200 4x106 89.0 m/z (Da) 3x106

207.0 6 5 2x10 222.8 7x10 (5) Aldicarb Sulfone

Intensity (cps) Intensity 6 152.0 5 1x10 6x10 75.8 104.8165.8132.1 69.7 145.0 189.0 5 0 149.0 5x10 50 100 150207.0 200 m/z (Da) 4x105

3x105 7x105 133.8 192.6 222.8 5 85.9(5) Aldicarb Sulfone 152.0

Intensity (cps) Intensity 2x10 6x105 96.8 165.8 5 1x10 5x105 149.0 207.0 0 4x105

50 100 150 200 250 5 3x10 m/z (Da)133.8 192.6 5 85.9

Intensity (cps) Intensity 2x10 96.8 1x105 Figure 5. Product0 ion spectra and proposed fragmentation pathway of three N-methyl amino formic Figure 5. Product ion50 spectra 100 and 150 proposed 200 fragmentation 250 pathway of three N-methyl amino formic acid oximeacid ester: oxime (1 )ester: Methomyl; (1) Methomyl; (2) Oxamyl;m/z (Da) (2) Oxamyl; (3) Aldicarb; (3) Aldicarb; (4) Aldicarb-sulfoxide; (4) Aldicarb-sulfoxide; and (5 )and Aldicarb-sulfone. (5) Aldicarb- sulfone. Figure 5. Product ion spectra and proposed fragmentation pathway of three N-methyl amino formic Unexpectedly,acid 57oxime Da ester: was (1) lost Methomyl; in pirimicarb. (2) Oxamyl; ( Structurally3) Aldicarb; (4) Aldicarb-sulfoxide; speaking, it has and two(5) Aldicarb- methyl groups linked sulfone. to the aminoUnexpectedly, group and 57 itDa should was lost produce in pirimicarb. a loss of Structurally 72 Da. In fact,speaking, the mass it has spectrum two methyl of pirimicarb groups indicatedlinked anto the unexpected Unexpectedly,amino group loss 57 ofDaand 57was it Da lostshould rather in pirimicarb. produce than 72Structurally a Da, loss as of shownspeaking, 72 Da. in itIn Figurehas fact, two 6themethyl. This mass groups is becausespectrum of of the nitrogen-containingpirimicarblinked indicated to the pyrimidine aminoan unexpected group heterocyclic and it loss should of ring produce57 Da adjoined rathera loss of thethan 72 esterDa. 72 In Da, oxygenfact, as the shown mass that couldspectrumin Figure induce of 6. This a methyl is because ofpirimicarb the nitrogen-containing indicated an unexpected pyrimidine loss of 57 heterocyclic Da rather than ring 72 Da, adjoined as shown the in Figureester oxygen6. This is that could transfer reaction.because According of the nitrogen-containing to a literature pyrimidine survey heterocyclic [37–39 ring], the adjoined proton the ester is localized oxygen that at could the pyrimidine induce a methyl transfer reaction. According to a literature survey [37–39], the proton is localized at nitrogen ﬁrst;induce and a methyl then thetransfer charge-remote reaction. According become to a literature the reaction survey [37–39], center the proton that forming is localizedan at ion-neutral the pyrimidine nitrogen first; and then the charge-remote become the reaction center that forming an complex andthe induce pyrimidine an alkylnitrogen cation first; and transfer. then the charge-remote become the reaction center that forming an ion-neutralion-neutral complex complex and induce and induce an alkylan alkyl cation cation transfer.

H OH2 O N OH H OH2 N O N N N OH + N N N O N N O N N N N + N N O O N +N C N N N -57Da N O + C m/z 239.0 N -57Da m/z 182.2 N O 239.0 m/z8.0x10239.07 m/z 182.2

6.0x107 7 239.0 8.0x10 -57 Da

7 4.0x10 182.2 7 71.8

6.0x10 (cps) Intensity 7 2.0x10 -57 Da

7 84.7 137.0 195.1 4.0x10 0.0 182.2 5071.8 100 150 200 250

Intensity (cps) Intensity m/z (Da)

2.0x107 Figure 6. FigureProduct 6. Product ion spectra ion spectra and and probable probable fragmentation fragmentation routes routesof Pirimicarb. of Pirimicarb. 84.7 137.0 195.1 0.0 2.4. Method Validation 50 100 150 200 250 m/z (Da) 2.4.1. Matrix Effect Figure 6. Product ion spectra and probable fragmentation routes of Pirimicarb. As shown in Figure7, the complex composition of diverse matrices might have different effects on the accuracy of identiﬁcation and quantiﬁcation. It was necessary to correct the matrix effects in LC-MS detection. Eight distinct types of organic vegetable samples or pesticide-free samples were Molecules 2018, 23, x 10 of 16

2.4. Method Validation

2.4.1. Matrix Effect

Molecules As2018 shown, 23, 2496 in Figure 7, the complex composition of diverse matrices might have different effects11 of 16 on the accuracy of identification and quantification. It was necessary to correct the matrix effects in LC-MS detection. Eight distinct types of organic vegetable samples or pesticide-free samples were usedused as matrix-matchedas matrix-matched blank blank to to evaluate evaluate thethe matrixmatrix effects on on the the ionization ionization of of fifteen ﬁfteen carbamate carbamate residues.residues. The The evaluation evaluation of of matrix matrix effects effects was was calculatedcalculated using the the following following equation equation [40]: [40 ]:

A ME =AMatrix × 100% ME = A × 100% AS where AMatrix is the peak area of the standard solution with the matrix-matched blank and AS is the wherepeak A areaMatrix ofis the the standard peak area solution of the with standard initial solutionmobile phase. with As the shown matrix-matched in Table 3, the blank percentages and AS is of the peakthe area matrix ofthe effects standard of 15 carbamate solution withpesticides initial at mobile three different phase. Asconcentrations shown in Table (2, 20,3, and the percentages200 ng mL−1) of theranged matrix 95.4–111.2%effects of 15 carbamate(see Table 2 pesticides for details). at three Since different there was concentrations no obvious ion (2, 20,suppression and 200 ng or mL ion− 1) rangedenhancement 95.4–111.2% at the (see chosen Table levels2 for of details).quantification Since for there 15 carbamate was no obviouspesticides ion in eight suppression distinct types or ion enhancementof samples, atthe the matrix chosen effect levels can ofbe quantiﬁcationignored. for 15 carbamate pesticides in eight distinct types of samples, the matrix effect can be ignored.

9x105

8x105 7x105 6x105

5x105 Apple 4x105 Loofah

5 Chinese celery 3x10 Eggplant Intensity (cps) Intensity 2x105 Cowpea Mushroom 1x105 Tea 0 Pak choi 0 2 4 6 8 10 12 14 16 Time (min) FigureFigure 7. Typical7. Typical total total ion ion chromatograms chromatograms of ofﬁfteen fifteen carbamatescarbamates at at 2 2 μg/kgµg/kg spiked spiked in ineight eight matrices matrices (cleaning(cleaning by by 50 50 mg mg C18 C18 and and 150 150 mg mg PSA). PSA).

TableTable 3. 3.Matrix Matrix effects effects of of the the ﬁfteen fifteen carbamatescarbamates pesticides in in distinct distinct samples. samples.

MatrixMatrix Effects/% Effects/% (n = (3;n = 2, 3; 20, 2, 20020, ng200 mL ng− mL1) −1) CompoundCompound Pak ChoiPak Choi Chinese Chinese Celery Celery LoofahLoofah EggplantEggplant CowpeaCowpea AppleApple MushroomMushroom TeaTea Aldicarb-sulfoxide 98.6 98.5 96.3 98.8 96.0 95.4 96.6 97.6 Aldicarb-sulfoxide 98.6 98.5 96.3 98.8 96.0 95.4 96.6 97.6 Aldicarb-sulfone 97.8 96.5 98.3 99.1 95.7 96.2 98.4 96.9 Aldicarb-sulfone 97.8 96.5 98.3 99.1 95.7 96.2 98.4 96.9 PirimicarbPirimicarb 105.1105.1 102.0102.0 98.098.0 98.798.7 96.396.3 98.998.9 101.2101.2 99.299.2 Carbofuran-3-hydroxyCarbofuran-3-hydroxy 102.3 102.3 101.3 101.3 103.5 103.5 107.5 107.5 101.2 101.2 102.5 102.5 101.0 101.0 105.4 105.4 MethomylMethomyl 110.2 110.2 108.6 108.6 107.2 107.2 108.1 108.1 105.4 105.4 111.2 111.2 105.7 105.7 109.2 109.2 OxamylOxamyl 102.5 102.5 98.6 98.6 105.2 105.2 106.3 106.3 102.0 102.0 102.1 102.1 105.2 105.2 106.2 106.2 AldicarbAldicarb 107.5 107.5 102.1 102.1 100.2 100.2 101.3 101.3 100.8 100.8 103.2 103.2 100.5 100.5 102.3 102.3 Tsumacide 105.0 100.9 102.3 98.6 97.9 99.0 102.6 102.7 Tsumacide 105.0 100.9 102.3 98.6 97.9 99.0 102.6 102.7 Propoxur 102.3 105.4 102.7 104.9 101.8 102.4 103.1 101.5 CarbofuranPropoxur 102.5 102.3 105.9 105.4 106.2 102.7 103.1 104.9 98.7 101.8 99.2 102.4 97.4 103.1 98.5 101.5 CarbarylCarbofuran 102.3102.5 104.1105.9 106.5106.2 104.5103.1 101.798.7 102.499.2 106.997.4 107.498.5 IsoprocarbCarbaryl 102.3 102.3 104.6 104.1 102.5 106.5 104.8 104.5 101.2 101.7 107.5 102.4 108.4 106.9 101.8 107.4 MethiocarbIsoprocarb 105.9 102.3 104.1 104.6 102.4 102.5 106.2 104.8 107.1 101.2 105.1 107.5 106.3 108.4 105.1 101.8 FenobucarbMethiocarb 102.1105.9 105.2104.1 102.6102.4 107.8106.2 110.0107.1 108.4105.1 107.6106.3 106.8105.1 BanolFenobucarb 105.6 102.1 104.2 105.2 102.9 102.6 104.1 107.8 103.2 110.0 102.5 108.4 102.3 107.6 101.1 106.8 Banol 105.6 104.2 102.9 104.1 103.2 102.5 102.3 101.1 2.4.2. Linearity and Analytical Limits Satisfactory correlation coefficients (R > 0.996) were obtained for fifteen carbamate residues in matrix-matched blank over the concentration range of 0.05–200.0 ng mL−1. The analytical limits of the proposed method were measured by the limit of detection (LOD) and the limit of quantification (LOQ) using the following equations [41]: 3 LOD = C S S/N Molecules 2018, 23, 2496 12 of 16

10 LOQ = C S S/N where S/N is the average signal to noise ratio and CS is the concentration of the speciﬁc pesticide. The estimated values were tested with a suitable number and kind of samples containing the 15 carbamate pesticides at the corresponding concentrations. All related parameters of the proposed method are summarized in Table4. The LOD and LOQ were 0.2–2.0 µg kg−1 and 0.5–5.0 µg kg−1, respectively, which demonstrated a good analytical limit of this method for carbamate residues.

Table 4. Linear ranges, linear equations, correlation coefficients (R) limits of detection (LODs) and limits of quantification (LOQs) of the fifteen carbamate pesticides.

Linear Range LOD LOQ Compound Linear Equation R (1/X2) (ng mL−1) (µg kg−1) (µg kg−1) Aldicarb-sulfoxide 0.05–200 Y = 2.42 × 104X + 812.9 0.9963 0.2 0.5 Aldicarb-sulfone 0.50–200 Y = 1.44 × 104X + 10,476 0.9972 2.0 5.0 Pirimicarb 0.05–200 Y = 5.49 × 105X + 15,110.0 0.9968 0.2 0.5 Carbofuran-3-hydroxy 0.10–200 Y = 1.75 × 104X + 457.2 0.9964 0.3 1.0 Methomyl 0.05–200 Y = 2.91 × 104X + 102,771 0.9962 0.2 0.5 Oxamyl 0.05–200 Y = 8.38 × 104X + 2287.1 0.9965 0.2 0.5 Aldicarb 0.05–200 Y = 1.96 × 104X + 7283.2 0.9973 0.2 0.5 Tsumacide 0.05–200 Y = 6.64 × 104X + 3330.4 0.9967 0.2 0.5 Propoxur 0.05–200 Y = 1.27 × 105X + 3441.9 0.9963 0.2 0.5 Carbofuran 0.20–200 Y = 6.31 × 104X + 543.2 0.9982 0.3 1.0 Carbaryl 0.05–200 Y = 6.01 × 104X + 4445.6 0.9968 0.2 0.5 Isoprocarb 0.05–200 Y = 9.33 × 104X + 15,304 0.9957 0.2 0.5 Methiocarb 0.05–200 Y = 9.86 × 104X + 6565.0 0.9965 0.2 0.5 Fenobucarb 0.05–200 Y = 1.24 × 105X + 7436.7 0.9962 0.2 0.5 Banol 0.05–200 Y = 1.01 × 105X + 2436.7 0.9971 0.2 0.5 Y, peak area; X, mass concentration, ng mL−1; 1/X2, least square method.

2.4.3. Accuracy and Precision The accuracy and precision of the method were measured by the recoveries and coefficients of variation (CVs) for intra- and inter-day. Therefore, the standard mixtures solution of fifteen carbamate residues was spiked into distinct types of samples including greengrocery, Chinese celery, loofah, eggplant, cowpea, apple, mushroom and tea leaves, and 24 spiked samples (eight types at three concentrations of 2, 20, and 200 µg kg−1) were obtained. All spiked samples were detected three times a day on five different days following the process of Figure1. The results are shown in the Supplementary Materials. The recovery of fifteen carbamate residues (88.1–118.4%) fell within the recommended Eurachem guidelines of 80–120% [42]. The analysis precision, measured as the coefficient of variation percentage (% CV) of the recovery (3.2–9.8%), was well under the criteria of 10% [42].

2.5. Sample Analyses After validation of the analyti cal methodology through above experimentation, it was used to detect numerous types of samples including apple, carrot, chili pepper, Chinese celery, coriander, cowpea, crown daisy, eggplant, garlic sprout, grape, ginger, leek, lettuce, loofah, mushroom, needle mushroom, orange, pakchoi, radish, shiitake, spinach and tea leaves. Carbofuran and its metabolite Carbofuran-3-hydroxy were the predominant detected residues of the ﬁfteen carbamates. Meanwhile, of the 26 samples (Table S3), eggplant contributed the detected residues of maximum frequency, with concentrations of carbofuran at 85.5, 77.4, 68.4, 35.6, 25.6, 17.3, 16.5 and 11.8 µg kg−1, respectively. Pokchoi, spinach, and Chinese celery all had considerable detection rate and concentration of carbofuran. Among all samples, although green tea had the second highest detection rate, the concentration of carbamate residues were localized at relative low concentration of 6.4, 1.5, 1.2, 0.8, 0.6, 0.6, 0.4, 0.3, 0.2 and 0.2 µg kg−1, respectively. This was mainly due to the further degradation of Molecules 2018, 23, 2496 13 of 16 carbamate residues in the drying process of tea manufacturing. Cowpea occasionally had the highest concentration of carbofuran at 180.2 µg kg−1.

3. Materials and Methods

3.1. Chemicals and Reagents Acetonitrile and Methanol were of HPLC grade and obtained from Merck (Darmstadt, Germany). Formic acid (≥98%) was of HPLC grade and purchased from Aladdin (Shanghai, China). High purity water was obtained using a Milli-Q water purification system from Millipore (Bedford, MA, USA). Cleanert® Pesticarb, Cleanert® PSA and Cleanert® C18 for QuEChERS were purchased from Agela Technologies (Beijing, China). Millipore filters of polytetrafluoroethylene (0.22 µm) were purchased from ANPEL Lab (Shanghai, China). Standards of fifteen carbamates were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany): Aldicarb-sulfoxide, Aldicarb-sulfone, Pirimicarb, Carbofuran-3-hydroxy, Methomyl, Oxamyl, Banol, Aldicarb, Metolcarb, Propoxur, Carbofuran, Carbaryl, Isoprocarb, Mercaptodimethur and Fenobucarb. Individual standard solutions were prepared in HPLC-grade methanol at a concentration of 1.0 mg/mL and stored at −28 ◦C temporarily. Samples were collected from local supermarkets, including vegetables (leeks, pakchoi, crowndaisy, coriander, spinach, lettuce, Chinese celery, garlic sprout, loofah, chili pepper, eggplants, cowpea, radish, and turnip), fruits (apples, kiwis, grapes, and oranges), tea (green tea, black tea, and flower tea), and edible fungus (shiitake, mushroom, and needle mushroom). Each fresh sample was crushed and mixed into homogenate, and stored in plastic bottles at −20 ◦C until analysis.

3.2. Instrumentation HPLC analysis was performed on a Shimadzu LC-30AD system (Shimadzu, Kyoto, Japan) which consist of two interconnected pump units: one with an integrated degasser and the other with a mixer, and is comprised of a UHPLC gradient system, a refrigerated autosampler, and a column oven compartment. Mass spectrometric detection was performed on an AB SCIEX QTRAP® 5500 (AB SCIEX instruments, Foster, CA, USA) in MRM mode and EPI mode. A Turbo V™ Ion Source (ESI) interface in positive ionization mode was used. Both the UHPLC and mass spectrometer were controlled remotely using Analyst® software v. 1.6.2 (AB SCIEX instruments, Foster City, CA, USA). A Waters BEH C18 column (1.7 µm 2.1 mm × 100 mm, Waters, Milford, MA, USA) was applied for analysis. The mass spectrometer was equipped with an electrospray ionization source and spectra were acquired in the positive ion multiple reaction monitoring (MRM) mode and enhanced product ion (EPI) scan modes. MS was optimized using a capillary voltage of 5.50 kV and desolvation temperature of 500 ◦C. The cone gas pressure and desolvation gas pressure were 50 psi. Nitrogen was used as the cone and collision gasses, respectively. The raw data were analyzed using an Analyst 1.6.2 workstation (AB SCIEX, Foster, CA, USA).

3.3. Sample Extraction Five grams of the homogenized sample was weighed in 50 mL Corning extraction tubes, and 5 mL of acetonitrile was added. The tubes were vibrated vigorously for 20 min in batches by a Multi Reax Vortexer (Heidolph, Schwabach, Germany). After the vortex, 1.0 g of sodium chloride and 2.0 g of sodium sulfate were separately added to each tube for the elimination of moisture, and the tubes were immediately manually shaken vigorously for 1 min to prevent sodium sulfate agglomeration. The process was followed by centrifugation at 8000 rpm for 3 min at 4 ◦C in a Sigma 2–16 K. centrifuge (Sartorius AG, Göttingen, Germany). The supernatant was collected in 10 mL extraction tubes and subjected to QuEChERS dispersive SPE cleaning. The dSPE agent contained 100 mg of primary secondary amine (PSA), 50 mg of C18EC, and 200 mg of magnesium sulfate. The tubes were vibrated for 30 s and centrifuged in the Sigma 2–16 K centrifuge at 13,000 rpm for 2 min at 20 ◦C. The supernatants Molecules 2018, 23, 2496 14 of 16 were collected and ﬁltered through 0.22 µm Anpel Syringe Hydrophilic PTFE ﬁlters (Anpel, Shanghai, China) before LC-MS/MS analysis. Organic vegetables were cultivated by Ying Zhou’s mother from her garden. The samples were homogenized with an Ultra-Turrax T 25 homogeniser (IKA® Werke, Staufen, Germany) and extracted using the QuEChERS method.

4. Conclusions In this paper, a quick and credible method to quantify and confirm fifteen carbamate pesticide residues by modified QuEChERS sample pretreatment combined ultra-high performance liquid chromatography–quadrupole-linear ion trap mass spectrometry was systematically developed. For modified QuEChERS part, the composition of the cleaning agents was optimized to adapt the general samples. For the instrumental analysis, based on traditional conformation criteria including selected ion pairs, retention time and relative intensities between transitions from MRM scan mode, the presence of carbamate pesticides in doubtful samples was further confirmed by the matching of carbamate pesticide spectra via EPI scan mode. Then, the fragmentation routes of all the carbamates were preliminarily explained based on the mass spectra that the carbamate could generate a stable characteristic fragment ion by a neutral loss of CH3NCO (−57 Da). The linearity, matrix effect, analysis limits, accuracy and repeatability were validated in diverse samples. All fifteen carbamates have good correlation coefficients above 0.996 at the range of 0.05–200 ng mL−1. The recoveries of intra- and inter-day experiments were in the range of 80–120% at three concentrations with coefficients of variation all better than 10%. Moreover, the whole process of one dozen samples from pretreatment to final report did not exceed 2 h, which is shorter and exacter than traditional methods. Finally, the method was applied to carbamate residues detection in 26 kinds of samples from local markets, in which carbamates were extensively detected but all below the standard of maximum residue limit.

Supplementary Materials: The following are available online, Figure S1: Recovery tests of different proportions of PSA, GCB, and C18 in multi-matrices, Table S1: Intra-day accuracy and precision (n = 3), Table S2: Inter-day accuracy and precision (n = 15), Table S3: The maximum residues of 15 carbamate pesticide residues in 26 different samples (n = 28). Author Contributions: Y.Z. conceived and designed the study; M.G., J.G., S.L. and W.G. performed the experiments; and Y.Z. analyzed the data and wrote the paper. Funding: This research received no external funding and the APC was funded by [Jiaxing Center for Disease Control and Prevention]. Acknowledgments: This work was supported by all colleagues of Jiaxing Center for Disease Control and Prevention. Special thanks are given to Ying Zhou’s mother, Yafen Wang, for the organic vegetables and fruits from her garden. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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