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INTRODUCTION

GC-MS and LC-MS/MS are the two common tech-

niques for pesticide residue analysis. Due to the

wide range of physiochemical properties of pesti-

cides, two separate analyses by GC-MS and LC-MS/

MS are often required to cover multiple pesticides of

interest. In general, LC is favored for polar, less

volatile compounds, while GC is preferred for vola-

tile, thermally-stable ones, and also for pesticides

that do not ionize well in atmospheric pressure ioni-

zation. On the other hand, GC needs derivitization

and extensive sample clean-up, while LC-MS/MS is

prone to matrix co-eluting issues, either ion sup-

pression or isobaric interference from matrix.

UltraPerformance Convergence Chromatography

(UPC2®) provides an alternative approach for pes-

ticde residue analysis. UPC2 uses compressed carbon

dioxide, in supercritical state or sub-supercritical

state, as its primary mobile phase, sub-two micron

particles as the support of stationary phases, and

cutting-edge chromatography system as the separa-

tion platform. The non polar nature of the neat car-

bon dioxide can be altered with polar solvents, and

achieves separation for both highly nonpolar and po-

lar compounds with unique selectivity. In addition,

with the use of different stationary chemistries, its

selectivity can be altered dramatically. These render

UPC2 a potential powerful tool for the separation of

multiple pesticides with wide range of physiochemi-

cal properties. Recently, both GC and LC amenable

pesticides with wide range of polarity have been suc-

cessfully analyzed on a single SFC-MS/MS system in

a single injection (1).

The aim of this work is to demonstrate the capability

of UPC2 in simultaneous analysis of multiple pesti-

cides with wide range of properties, and to explore

the unique benefits of UPC2 in multiple pesticide

residue analysis.

ANALYSIS OF ANALYSIS OF MULTIPLE MULTIPLE PESTICIDES BY SFCPESTICIDES BY SFC--MS/MS WITH MS/MS WITH SUBSUB--2 MICRON PA2 MICRON PARTICLERTICLE COLUMNCOLUMNSS -- A FEASIBILITY STUA FEASIBILITY STUDY DY

Jinchuan Yang, Brian Tyler, Joseph P. Romano, Jennifer A. Burgess Waters corporation, 34 Maple St, Milford, MA 01757, U.S.A.

References

1. M. Ishibashi, T. Ando, M. Sakai, A. Matsubara, T.

Uchikata, E. Fukusaki, T. Bamba, J. Chromatogra. A,

1266 (2012) 143–148

RESULTS Optimization of the make-up solvent

The effects of make-up solvent flow rate, water

concentration, and acidicity or basicity of make-up

solvent on the MS peak area of 18 pesticides were

investigated using 100ppb pesticides mixture solvent

solution. The results of those investigation are shown

in Fig. 1 to Fig. 3.

Linearity and LOD

The linearity of the MS response was investigated

using both the spinach matched pesticide standard

solutions and the solvent standard solutions with

pesticide concentrations from 0.001ppb to 100ppb.

The LODs were also estimated during the linearity

study. The results are presented in Table 3.

Selectivity

The selectivity in UPC2 separation can be easily

changed by using different stationary phases. Fig. 4 is

a comparison of separation of 17 pesticides on two

different columns, HSS C18 SB column and BEH 2-EP

column.

Table 3. LODs and calibration curve linearity of

18 pesticides in solvent and in spinach extract

CONCLUSION UPC2 can simultaneously analyze residues of

pesticides of wide range of polarity and MW.

UPC2 offers a solution for a high-throughput

pesticide residue analysis since both GC

amenable and LC amenable pesticides can be

simultaneously analysis on a single system.

Coupling of Xevo TQ-S MS with UPC2 provides

excellent detection sensitivity that meet stringent

regulations.

The selectivity in UPC2 separation can be

conveniently changed with different stationary

phases using the same mobile phase, which

provides a solution to co-eluting issues.

Additional investigation is needed on the

reproducibility and robustness of this technique.

METHODS

Pesticides standards: Eighteen pesticides having different basic chemical structures, wide range of polarities, and molecular weights were chosen in this study. Table 1 lists their formula, MWs, log Pow, and common analysis techniques.

Table 1. List of pesticides in this study

Spinach matrix extract: Waters DisQuE™ kit was used and AOAC QuEChERS protocol was followed in the preparation of spinach extract. Details of the

procedure can be found in DisQuE Care and Use Manual (Waters Lit. 715001888). Standard solutions: Spinach matrix matched pesticide standard solutions were prepared by mixing 800µL acetone water (3/1 v/v) mixture, 100µL matrix extract, and 100µL pesticide standard stock solution at certain concentrations. The pesticide standard stock solutions were prepared by serial dilution of 1mg/mL pesticide solutions with acetone water (3/1) mixture to certain concentrations. Pesticide standard solvent solutions were prepared by mixing 100µL pesticide stock solution with 800µL acetone water (3/1) mixture and 100µL MeCN with 1% acetic acid. UPC2-MS/MS Analysis: Waters UPC2 system was coupled with Xevo TQ-S MS. A splitter for MS was used to incorporate make-up solvent and split eluent

to MS probe before the pressure regulator. MassLynx 4.1 was used for instrument control and for data process.

UPC2 Conditions: Column: ACQUITY® UPC2 BEH-2EP, 3.0x100mm, 1.7µm Column Temp: 65oC Sample Temp: 4oC Injection vol.: 2µL Flow rate: 1.5mL/min Back pressure: 1500 psi Mobile phase A: CO2 Mobile phase B: MeOH with 10mM ammonium formate Gradient: Initial hold at 100%CO2 for 2 minutes, linear gradient from 0% to 10% modifier B in 5 minute, then a second gradient from 10% to 45% modifier B in 2 minute, and hold at 45% B for 1 minute before return to initial condition. Make-up solvent: MeOH + 1%H2O + 0.1% NH4OH Make-up flow rate: 0.2mL/min

Xevo® TQ-S MS Conditions:

Capillary voltage, 3000 V; source temperature, 150 ◦C; desolvation temperature, 600 ◦C; cone gas flow rate, 150 L/h; desolvation gas flow rate, 1000 L/h; collision gas flow rate, 0.15 mL/min. The dwell time: auto (6 to 34 ms). MRM transitions were optimized by combining the infusion of pesticide standard solutions with the make-up solvent at 200µL/min. Table 2 shows

the optimized MRM transition parameters for the 18 pesticides.

Table 2. MRM Transition parameters for the 18

pesticides.

Figure 2. Effects of addition of trace water in make-up solvent

on peak area. Data shown are normalized to the peak areas

obtained without any water added to make-up solvent (methanol). All other conditions are the same.

Figure 1. Effect of make-up solvent flow rate on peak areas of

18 pesticide standards. All data are normalized to the peak ar-eas obtained at 0.5mL/min make-up flow rate. All other condi-

tions are the same.

Figure 3. Effects of addition of acid (0.1% acetic acid) or base

(0.1% ammonium hydroxide) in make-up solvent on peak area. Data shown are normalized to the peak areas obtained

without any acid or base (methanol with 1% water). All other

conditions are the same.

No. Pesticide Formula MW

Monoisotopic

MW log Pow

Analysis

Technique1

1 Diquat dibromide C12H12Br2N2 344 341.9367 -4.6 IC or IP-LC

2 Fosetyl C6H18O9P3Al 354 353.9979 -2.7 IC

3 Maleic hydrazide C4H4N2O2 112 112.0273 -1.96 LC

4 Methamidophos C2H8NO2PS 141 141.0013 -0.8 GC

5 Methomyl C5H10N2O2S 162 162.0463 0.09 LC

6 Acetamiprid C10H11ClN4 223 222.0672 0.8 LC, GC

7 Carbendazim C9H9N3O2 191 191.0695 1.51 LC

8 Azinphos-methyl C10H12N3O3PS2 317 317 2.75 GC

9 Phosmet C11H12NO4PS2 317 317 2.78 GC

10 Thifluzamide C13H6Br2F6N2O2S 528 525.8421 3.56 GC

11 Tralomethrin C22H19Br4NO3 665 660.8098 5.05 GC

12 Amitraz C19H23N3 293 293.2 5.5 LC, GC

13 Emamectin benzoate(B1a) C49H77NO13 888 887.5395 5.76 LC

14 Chlorfluazuron C20H9Cl3F5N3O3 541 538.963 5.8 LC

15 Acequinocyl C24H32O4 385 384.2301 6.2 LC

16 Pyridaben C19H25ClN2OS 365 364.1376 6.37 GC

17 Cypermethrin C22H19Cl2NO3 416 415.0742 6.6 GC, LC

18 Etofenprox C25H28O3 377 376.2038 7.05 LC

Note: 1. Analysis technique that is commonly used for pesticide is listed.

Compound

Ionization

mode

Parent

ion [m/z]

Cone

Voltage [V]

Daughter

ion [m/z]

Collision

Energy [eV]

Daughter

ion [m/z]

Collision

Energy [eV]

1 Diquat Dibromide ESI+ 183 25 157 18 168.2 15

2 Fosetyl Alumium ESI- 109.1 20 81 10 63 15

3 Maleic Hydrazine ESI+ 113 20 85 10 67 15

4 Methamidaphos ESI+ 142 30 93.9 12 124.9 9

5 Methomyl ESI+ 185.1 20 64 8 128 6

6 Acetamiprid ESI+ 223 20 125.9 20 56 12

7 Carbendazim ESI+ 192 20 159.9 15 131.9 27

8 Azinphos-methyl ESI+ 339.9 15 132 14 159.9 10

9 Phosmet ESI+ 317.9 18 160 10 132.9 35

10 Thifluzamide ESI+ 528.7 20 488.7 25 167.9 24

11 Tralomethrin ESI+ 682.6 25 440.6 12 276.7 30

12 Amitraz ESI+ 294.1 15 163.1 13 253.1 13

13 Emamectin Benzoate ESI+ 886.3 50 158 30 82 80

14 Chlorfluazuron ESI+ 539.8 40 382.9 25 158 15

15 Acequinocyl ESI+ 357 20 329.1 15 203 15

16 Pyridaben ESI+ 365 20 309 8 147 20

17 Cypermethrin ESI+ 432.9 20 190.9 10 127 30

18 Etofenprox ESI+ 394.1 25 359 8 177 13

MRM transition 1 MRM transition 2

Pesticide RT (min) LOD (ppb) Linear Range (ppb) R2 LOD (ppb) Linear Range (ppb) R2

1 Diquat Dibromide 9.220 0.500 1 - 100 0.996 0.500 1 - 100 0.996

2 Fosetyl Alumium 8.540 2.500 2.5 - 100 0.989 5.000 10 - 100 0.976

3 Maleic Hydrazine 8.100 5.000 10 - 100 0.997 10.000 25 - 100 0.996

4 Methamidaphos 5.180 0.025 0.100 - 100 0.998 0.050 0.050 - 100 0.997

5 Methomyl 5.470 2.500 5 - 100 0.991 2.500 5 - 100 0.990

6 Acetamiprid 7.990 0.025 0.050 - 100 0.996 0.050 0.050 - 100 0.997

7 Carbendazim 5.430 0.100 0.250 - 100 0.998 0.100 0.250 - 100 0.999

8 Azinphos-methyl 5.700 0.050 0.100 - 100 0.998 0.050 0.100 - 100 0.997

9 Phosmet 5.420 0.100 0.100 - 100 0.998 0.100 0.100 - 100 0.998

10 Thifluzamide 6.930 0.005 0.010 - 100 0.997 0.025 0.025 - 100 0.998

11 Tralomethrin 6.55, 6.99, 7.44 0.500 1 - 100 0.995 0.500 1 - 100 0.997

12 Amitraz1 4.250 - - - - - -

13 Emamectin Benzoate 8.490 0.050 0.100 - 100 0.993 0.050 0.100 - 100 0.991

14 Chlorfluazuron 7.860 0.025 0.050 - 100 0.995 0.025 0.050 - 100 0.998

15 Acequinocyl 6.260 0.010 0.100 - 100 0.995 0.050 0.050 - 100 0.998

16 Pyridaben 5.220 0.025 0.100 - 100 0.997 0.025 0.100 - 100 0.997

17 Cypermethrin 4.86,5.06 0.100 0.250 - 100 0.997 N/A2 6.8 - 1042 0.998

18 Etofenprox 4.870 0.050 0.100 - 100 0.998 0.050 0.100 - 100 0.9981: Data not available due to experimental error

2: 43ppb residue cypermethrin was found in blank spinach extract

Solvent Spinach

Figure 4. Comparison of separation of pesticides in UPC2 on dif-ferent columns (HSS C18 SB column vs BEH 2-EP column). The same mobile phase elution gradient was used for both columns.