Determination of 30 Pesticides Residues in Teas by GC-MS, GC-MS/MS and LC-MS/MS

Call for Voluntary Collaborators

Priority Project of AOAC INTERNATIONAL (2010)

High-Throughput Analytical Method for Determination of multi-Classes and multi-Kinds of 668 Residue Pesticides and Chemical Pollutants in Tea

by GC-MS,GC-MS/MS and LC-MS/MS

—Shrunken AOAC Collaborative Study Method

AOAC Study DirectorGuo-Fang Pang

Chinese Academy of Inspection and Quarantine,No 3 Gaobeidian North Rd. 100123,Chaoyang District, Beijing, China.

Email:, Fax:+86-335-5997608

1. Scope

This method is applicable for the qualitative and quantitative determination of multi-classes and multi-kinds of 668residue pesticides and chemical pollutants in green tea,black tea, puer tea and woolong tea by GC-MS, GC-MS/MS and LC-MS/MS. The limits of detection (LOD) for 490 pesticides determined by GC-MS were between 1.0-500 μg/kg, and for 448 pesticides determined by LC-MS/MS were between 0.03-4820 μg/kg. There are 482 pesticides with LOD≤100µg/kg for GC-MS method, accounting for 98% of the pesticides tested and there are 417 pesticides for LC-MS/MS, accounting for 93% of the pesticides analyzed; there are 264 pesticides with LOD≤10µg/kg for GC-MS method, accounting for 54% of the pesticides tested, 325 pesticides for LC-MS/MS method, making up 73% of the pesticides analyzed. There are 270 pesticides that can be analyzed by both GC-MS and LC-MS/MS. There are 264 pesticides with LOD≤100µg/kg for GC-MS method, accounting for 98% of the pesticides tested, 247 for LC-MS/MS, making up 91% of the pesticides analyzed; there are, however, 133 pesticides with LOD≤10µg/kg for GC-MS method, accounting for 49% of the pesticides tested, 200 pesticides for LC-MS/MS method, making up 74% of the pesticides analyzed.

2.Safety measures

Some organic reagents areused in this experiment, for example acetonitrile, toluene and n-hexane and so on. They have some potential harm to the human body. Laboratory assistantshould wear latex gloves and operation in the hood.

3. Principle

Test samples are extracted with acetonitrile using homogenizer, the extracts arecleaned up with Cleanert TPT cartridge or Envi-Carb/PSA cartridges.The pesticides are eluted with acetonitrile-toluene (3:1,V/V), after concentration,dryness and dissolution, the samples are analyzed by GC-MS, GC-MS/MS or LC-MS-MS, quantified with matrix-matched internal standard calibration curve.

4. Apparatus

4.1GC/MS system.—Model 7890A gas chromatograph connected to a Model 5975C mass selective detector with electron impact ion source(EI) and equipped with a Model 7683 autosamplerand equipped with Chemstation data processing software systems(Agilent Technologies, Wilmington, DE),with a DB-1701 capillary column (30 m×0.25 mm×0.25 μm, Agilent J&W Scientific, Folsom, CA) or equivalent.

4.2GC-MS/MS system.—Model 7890A gas chromatograph connected to a Model 7000B triple quadrupole mass spectrometerwith electron impact ion source (EI) and equipped with a Model 7693 autosampler and equipped with Mass Hunter data processing software systems (Agilent Technologies, Wilmington, DE), with a DB-1701 capillary column (30 m×0.25 mm×0.25 μm, Agilent J&W Scientific, Folsom, CA) or equivalent.

4.3LC-MS/MS system.—An Agilent Series 1200 HPLC system connected to an 6430 triple quadrupole mass spectrometer equipped with electrospray ionization (ESI)and equipped with a Model G1367D autosamplerandequipped with Mass Hunter data processing software systems(Agilent Technologies, Santa Clara, California, USA), witha Zorbax SB-C18, 2.1×100 mm, 3.5 μm (Agilent Technologies, Santa Clara, California, USA) or equivalent.

4.4Homogenizer.—Rotational speed higher than l3500 r/min, T-25B (Janke & Kunkel, Staufen, Germany) or equivalent.

4.5Rotary evaporator.—Buchi EL131 (Flawil, Switzerland) or equivalent.

4.6Centrifuge. —Centrifugal speed higher than 4200 r/min, Z320 (B. HermLe AG, Gosheim, Germany) or equivalent.

4.7Nitrogen evaporator.—EVAP 112 (Organomation Associates, Inc. New Berlin, MA) or equivalent.

4.8Visiprep 5-port flask vacuum manifold.—RS-SUPELCO57101-U, (Sigma Aldrich Trading Co., Ltd), see Fig.1 or equivalent.

4.9Variable volume pipette: 10 μL, 200μL, 1 mL.

4.10Balance: Capable of accurately measuring weightsfrom 0.05 to100g within±0.01 g.

5. Reagents and materials

5.1Solvents. —Acetonitrile, toluene and n-hexane: HPLC grade.

5.2Acetonitrile-toluene (3:1,V/V).

5.3Anhydrous sodium sulfate. —analytically pure. Baked at 650 ℃ for 4h and kept in a desiccator.

5.4SPE cartridges. —Cleanert–TPT(2000mg,12mL,Agela,China)or Envi-Carb/PSA (500mg/500mg, 6mL, Supelco,USA)or equivalent.

5.5SPE Tube Adapter. —For 12 mL SPE Tubes(57267) , for 6 mL SPE Tubes(57020-U) (Sigma Aldrich Trading Co., Ltd) or equivalent.

5.6Disposable flow control valve liners for visiprepTM-DL(57059),(Sigma Aldrich Trading Co., Ltd), see Fig. 1.

5.7Pear-shaped flask. —80 mL (Z680346-1EA, Sigma Aldrich Trading Co., Ltd), see Fig. 1 or equivalent.

Fig 1. Solid phase Extraction Equipment

5.8Reservoir. —30 mL(A82030,Agela,China) , see Fig. 1 or equivalent.

5.9Centrifuge tube. —80mL.

5.10 Milli pore filter membrane (nylon) . —13 mm×0.2 μm.

5.11 Preparing three standard working solutions: The three standard stock solutions for collaborative study such as quality control standard stock solutions, pesticide mixed standard stock solutions and internal standard stocksolutions are provided by study director, volumes are all 1.0mL and they need diluting into standard working solutions before use. The concrete steps are as follows: erect any of ampoule bottles containd stock solutions till the solutions thoroughly flow down the inner walls of the bottle from the top, and carefully open the ampoule and transfer the solutions to a 10mL volumetric bottle. To ensure complete transfer, rinse the ampoule bottle with 3 ×1mL toluene and transfer the washings into the 10mL volumetric bottle. Then use toluene to dilute the solutions to the graduations, and mix homogeneously to form a corresponding standard working solution. The concentrations of quality control standard working solutions are shown in Table 1, while the concentrations of pesticide mixed standard working solutions are listed in Table 2. The concentration of heptachlor epoxide internal standard working solutions is35µg/mL, while the concentration of i chlorpyrifos methylnternal standard working solutions is40µg/mL, with all standard working solutions kept from exposureto light in a4°C fridge.

Table1. The concentration of quality control standard working solutions

GC-MS(GC-MS/MS) / LC-MS/MS
No. / Pesticides / Concentration/μg/mL / No. / Pesticides / Concentration/μg/mL
ISTD / heptachlor-epoxide / 2.0 / ISTD / chlorpyrifos-methyl / 2.0
1 / trifluralin / 10.0 / 1 / prometon / 2.0
2 / pyrimethanil / 5.0 / 2 / isoprocarb / 2.0
3 / propyzamide / 10.0 / 3 / furalaxyl / 2.0
4 / procymidone / 5.0 / 4 / Pirimiphos-methyl / 2.0
5 / quinoxyphen / 5.0 / 5 / picoxystrobin / 2.0
6 / bifenthrin / 5.0 / 6 / butralin / 2.0
7 / tebufenpyrad / 5.0 / 7 / phenothrin / 10.0

Table2. The concentration ofpesticide mixedstandard working solutions,

GC-MS(GC-MS/MS) / LC-MS/MS
No. / Pesticides / Concentration/μg/mL / No. / Pesticides / Concentration/μg/mL
1 / 2,4'-DDE / 40.0 / 1 / acetochlor / 4.0
2 / benalaxyl / 10.0 / 2 / benalaxyl / 2.0
3 / bifenthrin / 10.0 / 3 / bensulide / 6.0
4 / bromophos-ethyl / 10.0 / 4 / butralin / 2.0
5 / bromopropylate / 20.0 / 5 / clomazone / 2.0
6 / chlorfenapyr / 80.0 / 6 / diazinon / 2.0
7 / diflufenican / 10.0 / 7 / chlorpyrifos / 20
8 / dimethenamid / 4.0 / 8 / ethoprophos / 2.0
9 / fenchlorphos / 20.0 / 9 / fenazaquin / 2.0
10 / picoxystrobin / 20.0 / 10 / flutolanil / 2.0
11 / pirimicarb / 10.0 / 11 / indoxacarb / 2.0
12 / pirimiphos-methyl / 10.0 / 12 / imidacloprid / 4.5
13 / propyzamide / 10.0 / 13 / kresoxim-methyl / 20.0
14 / pyrimethanil / 10.0 / 14 / phenothrin / 10.0
15 / Quinoxyfen / 10.0 / 15 / picoxystrobin / 2.0
16 / tebufenpyrad / 10.0 / 16 / pirimiphos-methyl / 2.0
17 / tefluthrin / 10.0 / 17 / propoxur / 5.0
18 / tolclofos-methyl / 10.0 / 18 / tebufenpyrad / 2.0
19 / trifluralin / 20.0 / 19 / triadimefon / 2.0
20 / vinclozolin / 20.0 / 20 / trifloxystrobin / 2.0

5.12 Preparing matrix-matched internal standard solutions and establishing matrix-matched internal standardcalibration curve: Prepare5 portions of green tea andwoolong tea blank sample solutionaccording to operational procedures of this method 6.1,6.2.1 and 6.2.2, then add respectively20,50,100,200 and 300 µL pesticide mixed standard working solutions(see Table 2), and add 40μL heptachlor epoxide internal standard working solution(see 5.11)for GC-MS and GC-MS/MS, add 40μL chlorpyrifos methyl internal standardworking solution (see 5.11),for LC-MS/MS, and mix homogeneously. Evaporate to dryness under a stream of nitrogen in water-bath at 35℃,and then use 1.5mL hexane to dissolve the residue for GC-MS and GC-MS/MS, use 1.5mL acetonitrile-water (3:2,V/V) to dissolve residue for LC-MS/MS, and mix homogeneously with ultrasonic before being filtered through 0.2um filtering membrane to become5 point matrix-matched internal standard calibration mixed solutions, which is used for establishinga 5 point matrix-matched internal standard calibration curve. The matrix-matched internal standard calibration mixed solutions should be prepared for immediate use. The linear correlation coefficient (R2)of 20 pesticide matrix-matched internal standard calibration curve shall be greater than 0.995.

6. Determination

6.1 Extraction

Weigh 5 g sample (accurate to 0.01 g) into a 80mL centrifuge tube, add 15mL acetonitrile, and homogenize at 13500 r/min for 1 min, then centrifuge at 4200 r/min for 5 min. Transfer the supernatants to a pear-shaped flask. Then the sample isre-extracted in the same way and the supernatants combined, and concentrate the extract to ca 1 mLwith a rotary evaporator in a water-bath of 40 ℃for clean-up.

6.2 Clean-up

6.2.1Condition the cartridges

Before adding the concentrated extractsolution, anhydrous sodium sulfate (ca 2 cm) was placed on the top of the Cleanert-TPT cartridge which placed into the fixing bracket and connected a pear-shaped flask at the lower(see Fig.1). Activatethe cartridge with 10mL acetonitrile-toluent (3:1, V/V), and the effluent isdiscarded.

6.2.2Cleanup the sample extraction solution

by the time when rinsing liquid level has reached the top ofthe anhydrous sodium sulfate layer, transfer the concentrated extractsolution into Cleanert-TPT cartridge and collect the eluates with a new pear-shaped flask; wait until the concentrated liquid surface reaches the top of the anhydrous sodium sulfate layer before cleansing the concentrated solution bottle with 3×2 mL acetonitrile-tolene (3+1,V/V) and transferring the washings into the cartridge. Connect a 30mL reservoir onto the upper part of the cartridge (e.g. Fig.1), elute the cartridge with 25mL acetonitrile-tolene (3+1,V/V) and concentrate the eluates to ca 0.5mL by a rotary evaporator at40℃water-bath.

6.2.3Addinginternal standardand filter

For GC-MS and GC-MS/MS, add 40μL heptachlor epoxide internal standard working solution(see 5.11) into the above-mentioned concentrated cleanup solution; for LC-MS/MS, add 40μL chlorpyrifos methyl internal standardworkingsolution(see 5.11), evaporate to dryness under a stream of nitrogen at 35℃in water-bath, while for GC-MS and GC-MS/MS, use 1.5mL hexane to dissolve the residue; for LC-MS/MS, use 1.5mL acetonitrile-water (3:2,V/V) to dissolve residueand mix homogeneously with ultrasonic before being filtered through a 0.2μm filtering membrane, which is then submitted for analysis.

Study Director reminds each collaborator: when conditioning SPE cartridges and cleaning up the test sample extracted solutionsnever allow the SPE cartriges to dry to prevent any inlet of air, which will affect cartridge efficiencies.

6.3 Determination

6.3.1 GC/MS analyticalconditions

(a)Column: DB-1701 capillary column (30m×0.25mm×0.25μm), or equivalent;

(b)Column temperature: 40℃ hold 1 min , at 30℃/min to 130℃, at 5℃/min to 250℃, at 10℃/min to 300 ℃, hold 5 min;

(c)Carrier gas: Helium, purity ≥99.999%, flow rate: 1.2mL/min;

(d)Injection port temperature: 290℃;

(e)Injection volume: 1μL;

(f)Injection mode: Splitless, purge on after 1.5 min;

(g)Ionization mode: EI;

(h)Ion source polarity: positive ion;

(i)Ionization voltage: 70 eV;

(j)Ion source temperature: 230℃;

(k)GC-MS interface temperature: 280℃;

(l)Solvent delay: 14 min;

(m)Ion monitoring mode: selected ion monitoring(SIM), each compound selects onequantifying ion and twoqualifying ions. All of the detected ions aremonitored basedon the programmed time and the sequence of peaks. The retention times, quantifying ions, qualifying ions and the ion aboundances for each compound of 20 pesticides and internal standard are listed in Table 3. SIM acquisition of monitored ions for GC-MS areshown in Table 4.

Table 3. Retention times, quantifying ions, qualifying ions, ion aboundances, LODs and LOQs for 20 pesticidesby GC-MS

No. / Pesticides / Retention
time,min / Quantifying
ion / Qualifying
ion 1 / Qualifying
ion 2 / LOQ,
μg /kg / LOD,
μg /kg
ISTD / heptachlor-epoxide / 22.15 / 353(100) / 355(79) / 351(52)
1 / trifluralin / 15.35 / 306(100) / 264(72) / 335(7) / 20 / 10
2 / tefluthrin / 17.42 / 177(100) / 197(26) / 161(5) / 10 / 5
3 / pyrimethanil / 17.45 / 198(100) / 199(45) / 200(5) / 10 / 5
4 / propyzamide / 18.73 / 173(100) / 255(23) / 240(9) / 10 / 5
5 / pirimicarb / 18.83 / 166(100) / 238(23) / 138(8) / 20 / 10
6 / dimethenamid / 19.57 / 154(100) / 230(43) / 203(21) / 10 / 5
7 / tolclofos-methyl / 19.77 / 265(100) / 267(36) / 250(10) / 10 / 5
8 / fenchlorphos / 19.80 / 285(100) / 287(69) / 270(6) / 40 / 20
9 / vinclozolin / 20.27 / 285(100) / 212(109) / 198(96) / 10 / 5
10 / pirimiphos-methyl / 20.28 / 290(100) / 276(86) / 305(74) / 20 / 10
11 / 2,4'-DDE / 22.84 / 246(100) / 318(34) / 176(26) / 25 / 12.5
12 / bromophos-ethyl / 23.12 / 359(100) / 303(77) / 357(74) / 10 / 5
13 / Picoxystrobin / 24.53 / 335(100) / 303(43) / 367(9) / 20 / 10
14 / chlorfenapyr / 27.08 / 247(100) / 328(54) / 408(51) / 200 / 100
15 / quinoxyphen / 27.13 / 237(100) / 272(37) / 307(29) / 10 / 5
16 / benalaxyl / 27.48 / 148(100) / 206(32) / 325(8) / 10 / 5
17 / bifenthrin / 28.61 / 181(100) / 166(32) / 165(35) / 10 / 5
18 / diflufenican / 28.73 / 266(100) / 394(25) / 267(14) / 10 / 5
19 / tebufenpyrad / 29.19 / 318(100) / 333(78) / 276(44) / 10 / 5
20 / bromopropylate / 29.39 / 341(100) / 183(54) / 339(51) / 20 / 10

Table 4. SIM acquisition of 20 pesticide ions by GC-MS

Group / Start time/min / Monitored Ions, m/z / Dwell time,ms
1 / 14.85 / 306,264,335 / 80
2 / 16.85 / 177,197,161,198,199,200 / 80
3 / 17.97 / 173,255,240,166,238,138 / 80
4 / 19.43 / 154,230,203,285,287,270,134,197,210 / 40
5 / 20.00 / 285,212,198,290,276,305, / 40
6 / 21.77 / 353,355,351, 246,318,176 / 40
7 / 22.93 / 359,303,357 / 160
8 / 24.00 / 335,303,367 / 53
9 / 25.87 / 237,272,307,247,328,408,148,206,325, / 40
10 / 28.49 / 181,166,165,266,394,267,318,333,276,341,183,339 / 40

6.3.2 GC-MS/MS analytical conditions

For GC-MS/MS, excluding the ion monitoring mode is multi reaction monitor (MRM), other operating conditions arethe same with GC-MS. Each compound ismonitored by one Quantifying precursor/product ion transition, and one Qualifying precursor/product ion transition. The monitored ion transitions and the collision energiesof the 20 pesticides and the internal standard see Table 5. MRM acquisition of monitored ion transitions for GC-MS/MS areshown in Table 6.

Table 5. Retention times, monitored ion transitions, collision energies, LODsand LOQs for20 pesticides by GC-MS/MS

Table 6. MRM acquisition of 20 pesticide ions by GC-MS/MS

Group / Start time/min / monitored ion transitions, m/z / Dwell time,ms
1 / 14.76 / 306/264,306/206 / 50
2 / 15.87 / 177/127,177/101,200/199,183/102 / 50
3 / 18.06 / 173/145,173/109,238/166,238/96 / 50
4 / 19.26 / 230/154,230/111,287/272,287/242,267/252,267/93 / 25
5 / 20.07 / 290/233,290/125,285/212,285/178 / 50
6 / 21.87 / 353/282,353/263 / 33
7 / 22.6 / 359/331,359/303,318/248,318/246 / 50
8 / 23.59 / 335/303,335/173 / 50
9 / 26.71 / 148/105,148/79,408/363,408/59 / 33
10 / 27.88 / 266/246,266/218,181/166,181/165 / 33
11 / 28.96 / 341/185,341/183,333/276,333/171 / 50

6.3.3 LC-MS/MS analytical conditions

(a)Column: ZORBAX SB-C18, 3.5 μm, 100 mm ×2.1 mm or equivalent

(b)Mobile phase program and the flow rate: refer to table 7.

Table 7. Mobile phase program and the flow rate

Step / Time/min / Flow rate/(μL/min) / Mobile phase A
(0.1%Formic acid Water)/％ / Mobile phase B
(Acetonitrile)/％
0 / 0.00 / 400 / 99.0 / 1.0
1 / 3.00 / 400 / 70.0 / 30.0
2 / 6.00 / 400 / 60.0 / 40.0
3 / 9.00 / 400 / 60.0 / 40.0
4 / 15.00 / 400 / 40.0 / 60.0
5 / 19.00 / 400 / 1.0 / 99.0
6 / 23.00 / 400 / 1.0 / 99.0
7 / 23.01 / 400 / 99.0 / 1.0

(d) Injection volume: 10µL

(e) Ionization mode: ESI

(f) Ion source polarity: positive ion

(g) Nebulizer gas: nitrogen gas

(h) Nebulizer gas pressure: 0.28Mpa

(i) Ion spray voltage: 4000v

(j)Dry gas temperature: 350℃

(k) Dry gas flow rate: 10 L/min

(l) Monitored ion transitions, collision energiesand fragmentations of20 pesticides and the internal standard areshown in Table 8.

(m) MRM acquisition of monitored ion transitions for LC-MS/MS areshown in Table 9.

Table 8. Retention times, monitored ion transitions, collisionenergies, Fragmentations, LODs and LOQs for 20 pesticides by LC-MS/MS

No. / Pesticides / Retention
time,
min / Quantifying
precursor/
product ion
transition / Qualifying
precursor/
product ion
transition / Collision
energy,
V / Fragmentation
/V / LOQ,
μg /kg / LOD,
μg /kg
ISTD / chlorpyrifos-methyl / 13.1 / 322.0/125.0 / 322.0/290.0 / 15;15 / 80
1 / propoxur / 5.8 / 210.1/111.0 / 210.1 / 168.1 / 10;5 / 80 / 12.20 / 6.10
2 / clomazone / 7.3 / 240.1 / 125.0 / 240.1 / 89.1 / 20;50 / 100 / 0.20 / 0.10
3 / triadimefon / 9.9 / 294.2/69.0 / 294.2 / 197.1 / 20;15 / 100 / 3.90 / 1.95
4 / ethoprophos / 8.5 / 243.1/173.0 / 243.1 /215.0 / 10;10 / 120 / 1.40 / 0.70
5 / acetochlor / 9.6 / 270.2 / 224.0 / 270.2 / 148.2 / 5;20 / 80 / 23.70 / 11.85
6 / flutolanil / 11.1 / 324.2 / 262.1 / 324.2 / 282.1 / 20;10 / 120 / 0.60 / 0.30
7 / chlorpyrifos / 18.3 / 350.0/198.0 / 350.0/79.0 / 20;35 / 100 / 22.00 / 11.00
8 / imidacloprid / 4.73 / 256.1 / 209.1 / 256.1 / 175.1 / 10;10 / 80 / 53.80 / 26.90
9 / kresoxim-methyl / 12.2 / 314.1 / 267 / 314.1 / 206.0 / 5;5 / 80 / 50.30 / 25.15
10 / picoxystrobin / 13.3 / 368.1 / 145.0 / 368.1/205.0 / 20;5 / 80 / 4.20 / 2.10
11 / pirimiphos methyl / 10.7 / 306.2/164.0 / 306.2 / 108.1 / 20;30 / 120 / 0.10 / 0.05
12 / diazinon / 12.0 / 305.0 / 169.1 / 305.0 / 153.2 / 20;20 / 160 / 0.40 / 0.20
13 / bensulide / 13.5 / 398.0 / 158.1 / 398.0 / 314.0 / 20;5 / 80 / 17.10 / 8.55
14 / tebufenpyrad / 14.0 / 334.3/147.0 / 334.3 / 117.1 / 25;40 / 160 / 0.10 / 0.05
15 / indoxacarb / 15.0 / 528.0 / 150.0 / 528.0 / 218.0 / 20;20 / 120 / 3.80 / 1.90
16 / trifloxystrobin / 15.1 / 409.3/186.1 / 409.3 / 206.2 / 15;10 / 120 / 1.00 / 0.50
17 / pyriproxyfen / 14.7 / 322.1 / 96.0 / 322.1/227.1 / 15;10 / 120 / 0.20 / 0.10
18 / butralin / 15.9 / 296.1 / 240.1 / 296.1 / 222.1 / 10;20 / 100 / 1.00 / 0.50
19 / fenazaquin / 16.4 / 307.2/57.1 / 307.2 / 161.2 / 20;15 / 120 / 0.20 / 0.10
20 / phenothrin / 17.4 / 351.1 / 183.2 / 351.1/237.0 / 15;5 / 100 / 169.60 / 84.80

Table 9. MRM acquisition of 20 pesticide ions by LC-MS/MS

Group / Start time
/min / Monitored ion transitions, m/z / Dwell time,ms
1 / 0 / 256.1/209.1, 256.1/175.1, 210.1/111.0, 210.1/168.1, 240.1/125.0, 240.1/89.1, 243.1/173.0, 243.1/215.0, 270.2/224.0, 270.2/148.2, 294.2/69.0, 294.2/197.1, 306.2/164.0, 306.2/108.1, 324.2/262.1, 324.2/282.1, 305.0/169.1, 305.0/153.2, 314.1/267.0, 314.1/206.0, / 10
2 / 12.5 / 322.0/125.0, 322.0/290.0, 368.1 / 145.0, 368.1/205.0, 398.0 / 158.1, 398.0 / 314.0 , 334.3/147.0, 334.3 / 117.1, 528.0 / 150.0, 528.0 / 218.0, 409.3/186.1, 409.3 / 206.2, 322.1 / 96.0, 322.1/227.1, 296.1 / 240.1, 296.1 / 222.1, 307.2/57.1, 307.2 / 161.2, 351.1 / 183.2, 351.1/237.0, 350.0/198, 350.0/79.0 / 13

Study Director reminds each collaborator: the analytical parameters covered in this method are all from Agilent instruments. The preliminary selected results of analytical parameters of instruments from the other three companies, Waters, ThermoFisher and Applied Biosystems are tabulated in Annex A for collaborators’ reference only. When using the analytical parameters of these instruments and the monitoring ion of 20 target pesticides, collaborators shall have to pass the tea fortified test confirmation and practice examination as well as the acceptance criteria in terms of examination results in the AOAC Pre-collaborative study stage before adopting the analytical parameters in the official collaborative study, while in the meantime these analytical parameters used shall be entered in Excel worksheet 1 of Annex B.

6.3.4 Qualitative determination

If the retention time of monitored peaks are the same with the peaks of the pesticide standard, and the ion abundance are within the expected limits (see Table 10), the sample is confirmed to contain this pesticide. If the results are still not be confirmed, the sample should be re-injected with acquisition in scan mode (sufficient sensitivity) or with additional confirmatory ions or using other instruments that have higher sensitivity.

Table 10.Maximum permitted tolerances for relative ion intensities using a range of mass spectrometric techniques

Relative intensity
(% of base peak) / EI-GC-MS(relative) / GC-MS/MS,LC-MS/MS(relative)
> 50 % / ± 10 % / ± 20 %
> 20 % to 50 % / ± 15 % / ± 25 %
> 10 % to 20 % / ± 20 % / ± 30 %
≤ 10 % / ± 50 % / ± 50 %

6.3.5 Quantitative determination

Using instrument data processing software of GC-MS, GC-MS/MS, LC-MS/MS to establish least 5 piontmatrix-matchedinternal standard calibration curve, and calculate the contents ofpesticides in the samples. The analytical results from the practice examination samples shall be entered in Excel Workshet of Tables 2 and 3of Annex B; the analytical results from collaborative study samples shall be entered inExcel worksheet of Tables 4 and 5 of Annex B.

Study Director reminds each collaborator: our past within-lab study has discovered that for green tea and woolong tea there are 12.5%-28.4% pesticides automatic integration errors for GC-MS and 4.0%-9.7% for GC-MS/MS, which need correcting manually. The frequencies of integration error occurrence are closlyrelated to the extent of contamination of the instrument.

So, in order to ensure the accuracy of qualitative and quantitative results, the following key points should be paid more attention:

(a) Before determination, check the sensitivity and stability of instrument come up to the requirements using quality control standard working solution(see section 5.11).

(b) After the determination of a series of the samples, check the sensitivity and stability of instrument again using quality control standard working solution(see section 5.11), for seeing that they are the same.

(c) The retention time and ion aboundance should be verified, exactly as the qualitative and quantitative requirements in the method, for ensuring all of the targetedpesticides are in the time window and can be identifiedaccurately.

(d) The integration of each pesticide should be checked. For the pesticide which has wrong integration, perform a manual operation from one peak valley to another peak valley.

7.Blank test

For the purpose of preventing the interference from the reagents, blank test of the whole process of the method shall be done to confirm there is no existence of any interferencefrom the reagents. The operation of the blank test is the same as the described in the Section 6 of method but with the omission of sample addition.

Study Director reminds:collaboratorshould providethe total ion chromatogram (TIC)of the blank test sample to the Study Director.

8.References

(1)

(2)Document No. SANCO/10684/ 2009“Method Validation and Quality Central Procedures for Pesticide Residues Analysis in Food and Feed

(3)AOAC Guidelines for single laboratory validation of chemical methods for dietary supplements and botanicals

(4)Youden W.J.& Steiner E.H.(1975) Statistical Manual of the AOAC, Arlington, VA.USA

(5)Standardization Administration of P.R.China (2008) GB/T 23204-2008 , Determination of 519 pesticides and related chemicals residues in tea—GC-MS method

(6)Standardization Administration of P.R.China (2008) GB/T 23205-2008, Determination of 448 pesticides and related chemicals residues in tea—LC-MS-MS method

(7)High-Throughput Analytical Techniques for determination of 653 multi-Classes and multi-Kinds of Residue Pesticides and Chemical Pollutants in Tea. Part I: The Tentative Probe into the Analytical Techniques of Multiresidues in Tea (J.AOAC Int. Manuscript ID-10-0008R.1)

(8)High-Throughput Analytical Techniques for determination of 653 multi-Classes and multi-Kinds of Residue Pesticides and Chemical Pollutants in Tea. Part II: Comparative Study of Extraction Efficiencies of the Three Sample Preparation Techniques (J.AOAC Int. Manuscript ID-10-0215R.1)