Comparison of Luke Method with Disposable Pipette Extraction of

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					                                                                                       Disposable Pipette Extraction (DPX)

           Comparison of Luke Method with
           Disposable Pipette Extraction of
                 Pesticide Residues
     William E. Brewer, Ph.D., University of South Carolina

              Sherry Garris, S.C. Department of Agriculture
                          Chanin Craft, SCDA
                          Hongxia Guan, USC
                       Lynn R. Musgrove, USC
                        Julie Richardson, USC
                         Whitney Carlson, USC
                                                                                          William E. Brewer, US patent 6,566,145 B2




     Comparison of Multiresidue Extraction Methods                             Comparison of Multiresidue Extraction Methods
                                                                                               DPX (sdvb)
                              LUKE METHOD                                  1.  Blend 100 g sample w/ 200 mL acetone (from Luke)
1.     Blend 100 g sample with 200 mL acetone and filter                   2.  Transfer 2.5 mL into test tube and add 1 mL satd. NaCl
2.     50 mL extracted with 100 mL CH2Cl2/pet ether                        3.  Condition DPX tip with 0.5 mL ethyl acetate (or methanol)
                                                                           4.  Draw solution into DPX tip with air to mix
3.     Repeat 2 times with 50 mL CH2Cl2 after addition of NaCl
                                                                           5.  Wait app. 10-20 seconds (layers may separate)
4.     Organic layer diluted to 240 mL with acetone                        6.  Draw in 0.5 mL ethyl acetate and mix with air
5.     2-120 mL portions split and concentrated—OC and OP                  7.  Allow layers to separate (10 sec), and dispense bottom layer
6.     OP: solvent exchanged to 5 mL acetone—GC-NPD                        8.  Dispense upper organic layer into clean test tube
7.     OC: florisil column cleanup (several steps!)                        9.  Elute from TOP with additional 0.5 mL ethyl acetate
                                                                           10. Pass organic eluent through Na2SO4 and/or florisil (ECD) and collect
8.     OC: solvent exchanged to 5 mL iso-octane—GC-ECD                         into graduated C-tube
                                                                           11. Dry to app. 0.5 mL with heat and gas flow (bottom layer—salt)
Note: 5:1 conc. factor or app. 1.7 g/mL sample equiv.                      12. Remove bottom layer and bring to 0.5 mL mark; transfer to GC vial

                                                                           Note: 5:1 conc. factor or app. 1.7 g/mL sample equiv.




                                                                                                     2007 Study
     Previous DPX Research v. this Research
                                                                           • DPX L/L methodology
             2005 Study                            2006 study
•    Analyzed acetone extract from    •   Analyzed methanol extract, so
                                                                           • Solid phase-liquid-liquid extraction
     the Luke extraction                  processed samples from scratch   • Permits the use of acetone solution
•    Took 5 mL acetone extract and    •   Took 2.5 mL methanol extract
     added 10 mL water                    and added 2.5 mL sat’d. NaCl     • Direct comparison with Luke Method
•    1 mL final volume                •   0.5 mL final volume              • Use NPD and ECD detectors with minimal
•    DPX took app. 20 min. to         •   DPX took app. 3 min. per           solvent evaporation
     process up to 12 samples             sample—processed 1 at a time
     simultaneously (lever press)     •   Much better recoveries for       • Very similar chromatograms between 2 methods
•    Great recoveries for OC, OP          fungicides and polar compounds
     and nonpolar compounds
                                                                           • Florisil cleanup tips (Pat. Pend.)
•    Poor recoveries for fungicides




                                                                                                                                                      1
             Sample Preparation--Luke                                                                      Sample Preparation--Luke




                                Movie—DPX L/L

                                                                                              DATE      SAMPLE                     COMPOUNDS        DET.   LUKE          DPX          NOTES/TOL.

                                                                                                        Preliminary study
                                                                                              3/15/2007 green bell peppers         monitor          NPD    1.00 ppm      0.91 ppm over 1 week later
                                                                                                                                   acephate         NPD    1.42 ppm      0.32 ppm
                                                                                                                                   oxamyl           NPD    1.01 ppm      0.21 ppm
                                                                                                                                   L. cyhalothrin   ECD    0.08 ppm      0.10 ppm
                                                                                                                                   cypermethrin     ECD    0.14 ppm      0.09 ppm

                                                                                                        sweet potatoes             dicloran         ECD    0.74 ppm 0.50 ppm over 1 week later

                                                                                                        lettuce (spike)            buprofezin       NPD    0.98 ppm 0.45 ppm           over 1 week later

                                                                                                        spinach                    cyfluthrin       ECD    0.60 ppm 0.66 ppm over 1 week later
                                                                                                                                   cypermethrin     ECD    1.02 ppm 1.00 ppm




                                                                                              DATE       SAMPLE                    COMPOUNDS        DET.   LUKE          DPX          NOTES/TOL.
DATE      SAMPLE                 COMPOUNDS        DET.   LUKE      DPX          NOTES/TOL.
                                                                                                         Methanol conditioning**
3/20/2007 lettuce (spike)        acephate         NPD       0.69         0.09                   4/4/2007 roma tomatoes (spike)     chlorothalonil   ECD           0.32         1.03
                                 dursban          NPD       0.49         0.30                                                      allethrin        ECD           0.89         0.98
                                 thiabendazole    NPD       1.46         0.47                                                      captan           ECD           0.93         0.41
                                                                                                                                   bifenthrin       ECD           0.95         1.00
                                                                                                                                   L. cyhalothrin   ECD           0.93         1.04
3/22/2007 lettuce (spike)        acephate         NPD       0.59         0.25
                                                                                                         butter lettuce            L. cyhalothrin   ECD    0.04 ppm 0.04 ppm Tol: 2.0 ppm
                                 dursban          NPD       0.40         0.42
                                 thiabendazole    NPD       1.31         0.87                 4/11/2007 tomatoes (spike)           acephate         NPD           0.51         0.07
                                                                                                                                   chloropropham    NPD           1.14         0.85
                                 chlorothalonil   ECD       0.11         0.40                                                      dursban          NPD           0.99         0.98
                                 captan           ECD       0.34         0.16                                                      thiabendazole    NPD           1.15         0.60
                                 L. cyhalothrin   ECD       0.41         0.51                                                      phosmet          NPD           1.05         0.80
                                 dursban          ECD       0.37         0.41
                                                                                                         red apples                DPA              NPD    1.60 ppm 1.18 ppm Tol: 10.0 ppm

3/27/2007 cauliflower (spike)    dursban          NPD       0.94         0.78 2 extractions              potatoes                  chlorpropham     NPD    6.47 ppm 4.09 ppm Tol: 50 ppm
                                 fipronil         NPD       0.83         0.62 "
                                                                                                         pears                     thiabendazole    NPD    0.52 ppm 0.38 ppm Tol: 10 ppm




                                                                                                                                                                                                           2
        2/09/07 ECD 000005 spiked cucumbers                                                              3/15/07 NPD 000010 green bell peppers
        **early studies—sorbent needed cleaning
Response_

                                                                                                  Response_
                                    Signal: ECD000005.D\ECD1A.CH


   3.5e+07                                               allethrin                                      1300000
                                                                                                                                 acephate
                                                                                                                                                Signal: NPD000010.D\NPD2B.CH




       3e+07                                                                                            1200000



   2.5e+07
                                                                                                        1100000

                                                                                                        1000000
                                                                                                                                                         malathion




                                                                                                                       monitor
                                                                                                         900000
       2e+07                                                                                             800000

                                                                                                         700000
   1.5e+07
                                                                                                         600000

                                                                                                         500000
                                                                                                                                   oxamyl
       1e+07
                                                                                                         400000

                                                                                                         300000
   5000000
                                                                                                         200000

                                                                                                         100000
                   5.00   6.00   7.00   8.00   9.00   10.00 11.00 12.00 13.00 14.00 15.00
Time                                                                                                            3.00    4.00     5.00   6.00   7.00   8.00   9.00 10.00 11.00 12.00 13.00 14.00 15.00
                                                                                                  Time




                                                                                                          3/15/07 ECD 000023 sweet potatoes
         3/15/07 ECD 000018 green bell peppers
                                                                                                 Response_

Response_                                                                                                                                       Signal: ECD000023.D\ECD1A.CH
                                                                                                        4e+08

                                                                                                    3.8e+08
                                    Signal: ECD000018.D\ECD1A.CH
                                                                                                    3.6e+08

       3e+07                                                  L. cyhalothrin                        3.4e+08

                                                                                                    3.2e+08
                                                                                                                                                      dichloran
                                                                                                        3e+08
   2.5e+07                                                                                          2.8e+08

                                                                                                    2.6e+08

                                                                                                    2.4e+08
       2e+07
                                                                                                    2.2e+08

                                                                                                        2e+08

   1.5e+07                                                                        cypermethrin      1.8e+08

                                                                                                    1.6e+08

                                                                                                    1.4e+08

       1e+07                                                                                        1.2e+08

                                                                                                        1e+08

                                                                                                        8e+07
   5000000                                                                                              6e+07

                                                                                                        4e+07

                                                                                                        2e+07
                   11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00
Time                                                                                                        4.00         5.00     6.00     7.00       8.00   9.00   10.00 11.00 12.00 13.00 14.00 15.00
                                                                                                 Time




                   3/15/07 NPD 000011 lettuce
                                                                                                                             3/15/07 ECD 000019 spinach
                                                                                                         Response_
       Response_

                                                                                                                                                  Signal: ECD000019.D\ECD1A.CH

          390000
                                         Signal:   NPD000011.D\NPD2B.CH

                                                                                                            1.8e+07                                                                      cypermethrin
          380000
                                                                                                            1.6e+07
          370000


          360000                                                                                            1.4e+07                                              cyfluthrin
          350000


          340000
                                                      buprofezin                                            1.2e+07

          330000
                                                                                                                1e+07
          320000


          310000                                                                                            8000000
          300000


          290000
                                                                                                            6000000

          280000
                                                                                                            4000000
          270000


          260000
                                                                                                            2000000
          250000
                                                                                                                   15.0015.5016.0016.5017.0017.5018.0018.5019.0019.5020.0020.5021.0021.50
          240000
                                                                                                         Time
          230000


          220000

                                           11.00
               5.00 6.00 7.00 8.00 9.0010.00       13.00
                                               12.00   14.00
                                                           15.00
                                                               16.00   18.00
                                                                   17.00   19.00
       Time




                                                                                                                                                                                                          3
                3/20/07 NPD 000001 lettuce                                                                                                        3/22/07 NPD 000014 lettuce

Response_                                                                                                                     Response_


                                                                                                                                                                           Signal: NPD000014.D\NPD2B.CH
                                                                                                                                 2600000
                                                       Signal: NPD000001.D\NPD2B.CH

   2000000               acephate                                                                                                2400000                    acephate
                                                                                                                                 2200000
   1800000
                                                                                                                                 2000000
                                                                                                                                                                                    dursban
   1600000
                                                              dursban                                                            1800000

                                                                                                                                 1600000
   1400000
                                                                                                                                 1400000
   1200000
                                                                                                                                 1200000

   1000000                                                                                                                                                                                           thiabendazole
       800000
                                                                               thiabendazole                                     1000000

                                                                                                                                     800000

                                                                                                                                     600000
       600000
                                                                                                                                     400000
       400000                                                                                                                        200000

       200000                                                                                                                                        5.00    6.00   7.00    8.00    9.00    10.00 11.00 12.00 13.00 14.00 15.00
                                                                                                                              Time

              4.00    5.00          6.00            7.00    8.00    9.00    10.00 11.00 12.00 13.00 14.00 15.00
Time




                      3/22/07 ECD 000026 lettuce                                                                                                  3/27/07 NPD 000003 cauliflower
   Response_                                                                                                                         Response_

                                                       Signal: ECD000026.D\ECD1A.CH
                                                                                                                                                                              Signal: NPD000003.D\NPD2B.CH
          2.8e+08

          2.6e+08                                    dursban                                                                                                                        dursban
                         chlorothanonil




                                                                                                                                        1600000
          2.4e+08

          2.2e+08                                                                                                                       1400000

              2e+08

          1.8e+08                                                                                           L. cyhalothrin              1200000


          1.6e+08                                                                                                                       1000000
          1.4e+08
                                                                                                                                            800000
          1.2e+08

              1e+08
                                                                                                                                            600000
                                                                                                                                                                                    fipronil
              8e+07
                                                                                                                                            400000
              6e+07

              4e+07                                                         captan                                                          200000
              2e+07


                        8.00                 9.00   10.00   11.00   12.00   13.00   14.00   15.00   16.00   17.00                                5.00       6.00    7.00     8.00    9.00    10.00   11.00   12.00   13.00   14.00
   Time                                                                                                                              Time




         Chlorothalonil spiked at 0.05 ppm (50 ppb)




               4/04/07 ECD 000040 roma tomatoes
                                                                                                                                             4/05/07 ECD 000034 butter lettuce

                                                                                                                              Response_
       Response_
                                                                                                                                                                           Signal: ECD000034.D\ECD1A.CH
                                                                                                                                 1.7e+07
                                                       Signal: ECD000040.D\ECD1A.CH
                                                                                                                                 1.6e+07

          4.5e+08                                     allethrin                                                                  1.5e+07
                             chorothalonil




              4e+08                                                                                                              1.4e+07

                                                                                                                                 1.3e+07
          3.5e+08

              3e+08
                                                                                                                                 1.2e+07

                                                                                                                                 1.1e+07
                                                                                                                                                                                                             L. cyhalothrin
                                                                                                                                     1e+07
          2.5e+08

              2e+08
                                                                            captan                           L. cyhalothrin      9000000

                                                                                                                                 8000000

          1.5e+08                                                                                                                7000000

                                                                                                                                 6000000
              1e+08
                                                                                       bifenthrin                                5000000

              5e+07                                                                                                              4000000

                                                                                                                                 3000000

                        8.00                 9.00   10.00   11.00   12.00   13.00   14.00   15.00   16.00   17.00                2000000
       Time
                                                                                                                                            5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.0015.0016.0017.0018.0019.00
                                                                                                                              Time




         Chlorothalonil spiked at 0.05 ppm (50 ppb)
                                                                                                                               Incurred L cyhalothrin at 0.04 ppm (40 ppb)




                                                                                                                                                                                                                                     4
                                CONCLUSIONS                                                                                           Movie—DPX (polar/nonpolar)

• DPX L/L Method is a viable alternative to the Luke
  Method
• Great recoveries for nonpolar compounds and
  pyrethroids in particular
• Better recoveries* for chlorothalonil
• Fair recoveries* for polar compounds (e.g.,
  thiabendazole), and poor recoveries for acephate
• Surprisingly very “CLEAN” chromatograms
• Tomato extracts run on ECD “without” florisil cleanup
• *New: DPX-RP tips with DPX L/L provide great
  recoveries of chlorothalonil, thiabendazole and captan*




                 DPX cleanup tip (Pat. Pend.)                                                                                             DPX Quechers Tip
                                                                                                                       GC/MS chromatograms of 1 ppm OP mix in acetonitrile extract
   GC/MS chromatograms of 0.8 ppm OP mix in acetonitrile extract
                                                                                                                                           of muffin mix
                 Abundance


                                                         T C
                                                          I :   G041807a02 D d
                                                                          . \ a a m
                                                                               t . s
                                                                                                                          Abundance

                                                                                                F.A.: 3 x 107 counts
                      2800000


                      2600000




Corn muffin           2


                      2


                      2
                       4


                       2


                       0
                        0


                        0


                        0
                         0


                         0


                         0
                          0


                          0


                          0
                           0


                           0


                           0
                            0


                            0


                            0

                                                                                                                             1100000
                                                                                                                                                        TIC: G071807a05.D\data.ms



mix—no cleanup
                      1800000


                      1600000


                      1400000


                      1200000                                                                                                1000000
                      1000000


                       800000


                       600000                                                                                                 900000
                       400000


                       200000


                             4 0
                              . 0   5 0
                                     . 0    .
                                           6 00   7 0
                                                   . 0      8 0
                                                             . 0       .
                                                                      9 00   10 0
                                                                               . 0     11 0
                                                                                         . 0   12 0
                                                                                                 . 0   1 .
                                                                                                        3 00                  800000
                  i
                 T m - >
                    e -




                                                                                                                              700000
                 Abundance


                                                     TIC: G041607a03.D\data.ms
                     2000000                                                                                                  600000
                     1800000




With cleanup         1600000


                     1400000
                                                                                                                              500000

                     1200000
                                                                                                                              400000
tip—poly-amino       1000000


                      800000
                                                                                                                              300000
                      600000


                      400000


                      200000
                                                                                                                              200000

                                    5.00   6.00   7.00     8.00      9.00    10.00   11.00     12.00   13.00
                 Time-->
                                                                                                                              100000
                 Abunda c
                       n e


                                                         TIC: G041607a02.D\data.ms

                      1000000
                                                                                                                                        5.00   6.00   7.00   8.00   9.00   10.00   11.00   12.00   13.00
                       900000                                                                                             Time-->

Neat standard          8


                       7


                       6
                        0


                        0


                        0
                         0


                         0


                         0
                          0


                          0


                          0
                           0


                           0


                           0
                            0


                            0


                            0


                       500000


                       400000


                       300000


                       200000


                       100000



                                    5.00   6.00   7.00      8.00     9.00    10.00   11.00     12.00   13.00
                 T  e
                  im -->




        Very high recoveries with almost complete loss of fatty acid peaks




                  Future Research of DPX                                                                                                       Acknowledgments

• DPX clean up tips (remove fatty acids--PA, PSA)                                                                      • Dept. of Chemistry and Biochemistry, University of South
                                                                                                                         Carolina, Columbia, SC
• Mixed sorbent of polar and sdvb will provide
                                                                                                                       • SC Department of Agriculture, Columbia, SC
  multi-residue analysis—use 2 separate elutions
                                                                                                                       • DPX Labs, LLC, Columbia, SC
• Fully automated extraction following initial                                                                         • GERSTEL
  sample preparation (blending of sample to
  obtain representative sample)—GERSTEL
• Gerstel automation of Quechers using DPX-Q




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