agilent 6470 triple quadrupole lc/ms system and emr-lipid ...emr vs quechers workflow 400 µl...
TRANSCRIPT
Agilent 6470 Triple QuadrupoleLC/MS System and EMR-Lipid
–The Combination for Confident
Pesticide Quantitation
Christoph Mueller
Agilent Technologies
LCMS Application Scientist
EMEAI Food Market Program Manager
Agenda
• 6470 QQQ innovations and quantitation performance
• Experimental• Sample Preparation• LC-QQQ method
• Evaluation of Matrix Removal
• Evaluation of Pesticide Recovery
6470 Innovations
Agilent 1290 Infinity II
High Speed Pump
Multisampler
Multicolumn Thermostat
Upgradeable to 6495 QQQ with iFunnel Technology
Experimental
• Samples• Avocado (high lipid content)• Spinach (low lipid content)
• Pesticides• Test mix from Agilent Pesticides tMRM Application Kit (G1733BA) with 250
pesticides• + some herbizides for negative ion performance evaluation• Spiked to samples before extraction to asses recovery 10 and 100 ng/g• Spiked to extracts after extraction 10 and 100 ng/g
• Extraction• EN QuEChERS extraction salt + PSA/C18 dSPE (for high lipid content)• EN QuEChERS extraction salt + EMR lipid dSPE + EMR lipid polish salts
EMR vs QuEChERS workflow
400 µL extract + 600 µL water in autosampler vial. (Spinach: 200 µL extract + 800 µL water)
Vortex 1 min, centrifuge at 5,000 rpm for 5 min.
Transfer 6 mL to Bond Elut EN dispersive SPE 15 mL tube (PSA/C18)
Centrifuge (5000 rpm, 5 min)
Add Bond Elut EN extraction salts, shake 1 min
5g Avocado (10 g spinach) + STD, 5 mL water, 10 mL ACN (1% acetic acid) in 50 mL centrifuge tube, shake 1 min
200 µL extract + 800 µL water in autosampler vial.
Vortex 1 min, centrifuge at 5,000 rpm for 5 min.
Transfer 5 mL of supernatant to a EMR—Lipid polish tube.
Vortex 1 min, centrifuge at 5,000 rpm for 5 min.
Add 5 mL water, then 5 mL of ACN extract to EMR-Lipid dSPE 15 mL tube
Centrifuge (5000 rpm, 5 min)
Add Bond Elut EN extraction salts, shake for 2 min
10 g Avocado (10 g spinach) + STD, 10 mL ACN (1% acetic acid) in 50 mL centrifuge tube.
PSA/C18 EMR
UHPLC and QQQ ParametersUHPLC column Agilent Zorbax Eclipse Plus C18 RRHD
2.1 x 150 mm, 1.8 µm
Mobile Phase A: water + 5 mM ammonium acetate + 0.1% acetic acidB: methanol + 5 mM ammonium acetate + 0.1% acetic acid
Gradient program
Min.031720
%B5
30100100
Flow rate 0.4 mL/min
Dynamic MRM acquisition with fast polarity switching
(for matrix evaluation: precursor ion scanand MS scan)
AVOCADO MATRIX
Evaluation of Matrix Removal
6x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (** -> **) Avocado EN acid 0.d
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Precursor Ion Scan for Product Ion m/z 184 – Phosphatidylcholin- Phospholipids = Group of compounds known for causing ion supression- Q1 Scan – Collision C. – Q2 fixed at m/z 184
6x10
0.2
0.225
0.25
0.275
0.3
0.325
0.35
0.375
0.4
0.425
0.45
0.475
0.5
0.525
0.55
0.575
0.6
0.625
0.65
0.675
0.7
0.725
0.75
0.775
0.8
0.825
0.85
0.875
0.9
0.925
0.95
0.975
+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (** -> **) Avocado EN acid 0.d
Counts vs. Acquisition Time (min)15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2 20.4 20.6 20.8 21 21.2 21.4 21.6 21.8
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
+ESI EIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (778.6000, 780.0000, 782.8000, 784.8000 ... -> 184.0000) Avocado EN acid 0.d
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Comparison of PC-(18:n/18:n) between EMR-Lipid and PSA/C18Increasing level of saturation of fatty acid Increasing level of removal
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
+ESI EIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (778.6000, 780.0000, 782.8000, 784.8000 ... -> 184.0000) Avocado EN acid 0.d
Counts vs. Acquisition Time (min)18.3 18.4 18.5 18.6 18.7 18.8 18.9 19 19.1 19.2 19.3 19.4 19.5 19.6 19.7 19.8 19.9 20 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9 21 21.1 21.2 21.3 21.4 21.5 21.6 21.7 21.8 21.9
LLPCPC(18:2/18:2)
OLPCPC(18:1/18:2)
OOPCPC(18:1/18:1)
Lyso-PC-(C18:n/0:0)
Significantly smaller than PC, but FA saturation has the same effect on level of removal6x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
+ESI EIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (518.5000, 520.5000, 522.5000 -> 184.0000) Avocado EMR 0-r002.d
Counts vs. Acquisition Time (min)15.7 15.8 15.9 16 16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 17 17.1 17.2 17.3
Lyso-PC-(C18:3/0:0)Lyso-PC-(C18:2/0:0)
Lyso-PC-(C18:1/0:0)
Avocado EMR 10 [Fenpyroximat]
16.25 16.3 16.35 16.4 16.45 16.5
4x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
422.1 -> 366.2 Area=83779
422.1 -> 135.1 Area=20859
Avocado EN acid 10 [Fenpyroximat]
16.25 16.3 16.35 16.4 16.45 16.5
4x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
422.1 -> 366.2 Area=94675
422.1 -> 135.1 Area=22753
Assessment of Total Extracted MatrixMS Scan, High Temperature and Extended Chromatography
7x10
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
3.25
3.5
3.75
4
4.25
4.5
4.75
5
5.25
5.5
5.75
6
6.25
6.5
6.75
7
+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Avocado EN acid 0.d
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Diacylglycerols(+ some MGDG)
Triacylglycerols???
SPINACH MATRIX
Evaluation of Matrix Removal
Precursor Ion Scan for Product Ion m/z 184 – Phosphatidylcholin- Group of compounds known for causing ion supression- Q1 Scan – Collision C. – Q2 fixed at m/z 184
6x10
0
0.5
1
1.5
2
2.5
+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (** -> **) Avocado EN acid 0.d
6x10
0
0.5
1
1.5
2
2.5
+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (** -> **) Spinach EN 0.d
6x10
0
0.5
1
1.5
2
2.5
+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 [email protected] (** -> **) Spinach EMR 0.d
Counts vs. Acquisition Time (min)14.8 15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2 20.4 20.6 20.8 21 21.2 21.4 21.6 21.8
Avocado PSA/C18
Spinach PSA/C18
Spinach EMR-Lipid
Assessment of Total Extracted MatrixMS Scan, High Temperature and Extended Chromatography
7x10
0
1
2
3
4
5
6
+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Avocado EN acid 0.d
7x10
0
1
2
3
4
5
6
+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Spinach EN 0.d
7x10
0
1
2
3
4
5
6
+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Spinach EMR 0.d
Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Avocado PSA/C18
Spinach PSA/C18
Spinach EMR-Lipid
PESTICIDE RESULTS
Excellent performance at default MRL (10µg/kg)
Pirimicarb
Signal Qualityin region with large max. number of concurrent MRMs
Uniconazole-P Fipronil Diflubenzuron Flusilazole
Fenamiphos Picoxystrobin Bupirimate Fenoxycarb
Recovery of Pesticides from Avocado MatrixEMR Lipid vs QuEChERS• 256 pesticides spiked
• Reference for recovery is solvent calibration, which results in greater deviation from 70-120% range.
EMR-Lipid PSA/C18
Lower Recovery with EMR-Lipid vs. PSA/C18
Lower Recovery in PSA/C18 vs. EMR-Lipid
Better Recovery with PSA/C18
Avocado EMR 10 [Propamocarb]
3.2 3.25 3.3 3.35 3.4 3.45
4x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
189.1 -> 102.1 Area=112744
189.1 -> 144.0 Area=7224
Avocado EN acid 10 [Propamocarb]
3.2 3.25 3.3 3.35 3.4 3.45
4x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
189.1 -> 102.1 Area=203910
189.1 -> 144.0 Area=12887
Avocado EMR 10 [Oxamyl]
3.8 3.85 3.9 3.95 4 4.05 4.1
4x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
237.0 -> 72.1 Area=63456
237.0 -> 90.1 Area=12043
Avocado EN acid 10 [Oxamyl]
3.8 3.85 3.9 3.95 4 4.05 4.1
4x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
237.0 -> 72.1 Area=157305
237.0 -> 90.1 Area=29811
Avocado EMR 10 [Bifenazate]
12.65 12.7 12.75 12.8 12.85 12.9 12.95 13
4x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
301.1 -> 198.2 Area=20500
301.1 -> 170.1 Area=10103
Avocado EN acid 10 [Bifenazate]
12.65 12.7 12.75 12.8 12.85 12.9 12.95 13
4x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
301.1 -> 198.2 Area=107094
301.1 -> 170.1 Area=54042
Propamocarb Oxamyl Bifenazate
EMR
PSA/C18
Better Recovery with PSA/C18
Avocado EMR 10 [Spirotetramat]
12.95 13 13.05 13.1 13.15 13.2
4x10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
374.1 -> 330.3 Area=17378
374.1 -> 302.2 Area=14487
Avocado EN acid 10 [Spirotetramat]
12.95 13 13.05 13.1 13.15 13.2
4x10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
374.1 -> 330.3 Area=55790
374.1 -> 302.2 Area=46358
Avocado EMR 10 [Tribenuron-methyl]
10.4 10.45 10.5 10.55 10.6 10.65
3x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
396.0 -> 155.1 Area=18365
396.0 -> 181.1 Area=10656
Avocado EN acid 10 [Tribenuron-methyl]
10.4 10.45 10.5 10.55 10.6 10.65
3x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
396.0 -> 155.1 Area=30147
396.0 -> 181.1 Area=18051
Tribenuron-methyl Spirotetramat
EMR
PSA/C18
Better Recovery with EMR-Lipid
Avocado EMR 10 [Quinmerac]
5.1 5.15 5.2 5.25 5.3 5.35
4x10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
222.0 -> 204.0 Area=60598
222.0 -> 141.1 Area=17360
Avocado EN acid 10 [Quinmerac]
5.1 5.15 5.2 5.25 5.3 5.35
4x10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
222.0 -> 204.0 Area=6033
222.0 -> 141.1 Area=1759
Avocado EMR 10 [Triclopyr]
9.35 9.4 9.45 9.5 9.55 9.6 9.65 9.7 9.75
2x10
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
253.9 -> 195.9 Area=938
255.9 -> 197.9 Area=957
Avocado EN acid 10 [Triclopyr]
9.35 9.4 9.45 9.5 9.55 9.6 9.65 9.7 9.75
2x10
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
253.9 -> 195.9 Area=267
255.9 -> 197.9 Area=158
Avocado EMR 10 [Dichlorprop]
9.9 9.95 10 10.05 10.1 10.15 10.2
2x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
233.0 -> 161.0 Area=3332
235.0 -> 163.0 Area=2090
Avocado EN acid 10 [Dichlorprop]
9.9 9.95 10 10.05 10.1 10.15 10.2
2x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
233.0 -> 161.0 Area=906
235.0 -> 163.0 Area=623
Quinmerac Triclopyr Dichlorprop
EMR
PSA/C18
Better Recovery with EMR-Lipid
Avocado EMR 10 [2,4,5-T]
10.2 10.25 10.3 10.35 10.4 10.45
2x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
252.9 -> 194.9 Area=2463
252.9 -> 158.9 Area=498
254.9 -> 196.9 Area=2423
Avocado EN acid 10 [2,4,5-T]
10.2 10.25 10.3 10.35 10.4 10.45
2x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
252.9 -> 194.9 Area=548
252.9 -> 158.9 Area=140
254.9 -> 196.9 Area=571
Avocado EMR 10 [Bispyribac]
11.3 11.35 11.4 11.45 11.5 11.55 11.6
3x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
431.0 -> 275.1 Area=37806
431.0 -> 413.1 Area=11233
Avocado EN acid 10 [Bispyribac]
11.3 11.35 11.4 11.45 11.5 11.55 11.6
3x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
431.0 -> 275.1 Area=14581
431.0 -> 413.1 Area=5362
Avocado EMR 10 [Moxidectin]
17.3 17.35 17.4 17.45 17.5 17.55
3x10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
*640.4 -> 622.2 Area=1103
640.4 -> 528.2 Area=29
Avocado EN acid 10 [Moxidectin]
17.3 17.35 17.4 17.45 17.5 17.55
3x10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
*640.4 -> 622.2 Area=880
640.4 -> 528.2 Area=35
2,4,5-T Bispyribac Moxidectin
EMR
PSA/C18
Summary
• EMR-lipid clean-up dramatically reduces lipid contentinjected onto colum, while maintaining analyte recovery aswith other dSPE sorbents.
• EMR-lipid clean-up improves robustness of the method andlongevity of column and mass spectrometer.
• Productivity gains through less maintenance downtime.
• Sensitive and robust 6470 QQQ is perfect match for theanalysis of diluted extracts after EMR-lipid clean-up.
• Thank You!