P003 - Horizon Technology

Abstract
Pesticide Residues in Wine - Methods
Pesticides are used on agricultural commodities such as table grapes and wine grapes to protect
against insects, fungi, mold and other agents that may affect crop yield, cosmetic appearance, and
flavor properties. Wine, being a major agricultural commodity, makes it especially important to
know the types and quantities of these pesticides that are present in the product as these affect
the overall purity, quality and safety to consumers. As a result, many analytical methodologies
have been created to monitor these compounds in this food matrix, as dictated by the FDA. Given
the number of samples that must be tested in any given year, it is necessary to develop fast,
automated SPE methodology for the extraction of these compounds from wine, in addition to
post-extraction treatment of extracts prior to GC/MS analysis.
The established Alcohol and Tobacco Tax and Trade Bureau method developed to manually
extract and then analyze for 20 organochlorine pesticides in wine was optimized for an automated
solid phase extraction system using Amino Propyl and HLB cartridges as well as DryDisk
membrane technology for drying extracts. The process of method optimization was developed
into a flow chart to identify the method steps where optimization was performed and report on
the results. The sample preparation optimization flow chart established a scientific process to
achieve efficiency of the extraction while demonstrating good recoveries of these 20
organochlorine pesticides. This optimized method demonstrates an improvement to existing
manual extraction methodology, and also models an extraction scheme that can be used for other
organochlorine pesticides as well as organophosphorous pesticides and antifungal compounds.
Experimental & Objectives
The SPE process and evaporation of extracts was
automated using the cartridge SmartPrep® Extractor
and XcelVap® Evaporation System from
Horizon Technology.


Results & Conclusions
Modified TTB Method Steps
TTB Method Steps
1. Mix 20 mL wine with 20 mL DI Water (40 mL)
2. Spike sample - 50 µl of pesticide mix (50 µg/mL)
3. Condition Oasis HLB cartridge with 10 mL of:
i. 50:50 Ethyl Acetate:Hexane
ii. Methanol
iii. DI Water
4. Load sample at 5 mL/min
5. Air dry for 10 mins (4 PSI)
6. Elute cartridge with 5 mL of
i. 80:20 Ethyl Acetate:Hexane
ii. 50:50 Ethyl Acetate:Hexane
iii. 50:50 Ethyl Acetate:Hexane
7. Load aminopropyl cartridge with 1/3 column volume of
magnesium sulfate (drying agent)
8. Condition aminopropyl cartridge with 5 mL 50:50 Ethyl
Acetate:Hexane
9. Load and collect extract
10. Evaporate with XcelVap to 1.0 mL @ 60°C using nitrogen
pressure profile of 4-24 PSIG
11. Analyze by GC/MS
1. Mix 20 mL wine with 20 mL DI Water (40 mL)
2. Spike sample - 50 µl of pesticide mix (50 µg/mL)
3. Condition Oasis HLB cartridge with 10 mL of:
i. 50:50 Ethyl Acetate:Hexane
ii. Methanol
iii. DI Water
4. Load sample by gravity (initial vacuum pull)
5. Air dry for 15 mins
6. Elute cartridge with 5 mL of
i. 80:20 Ethyl Acetate:Hexane
ii. 50:50 Ethyl Acetate:Hexane
iii. 20:80 Ethyl Acetate:Hexane
7. Load aminopropyl cartridge with 1/3 column volume of
magnesium sulfate (drying agent)
8. Condition aminopropyl cartridge with 5 mL 50:50 Ethyl
Acetate:Hexane
9. Load and collect extract
10. Evaporate with Nitrogen to 0.1 mL
11. Add 1 mL 0.1% corn oil/EtoAc
12. Analyze by GC/MS
Solid Phase Extraction Method Optimization
Develop an automated solid phase extraction (SPE) method for the extraction of 20
organochlorine pesticides using an established regulated manual SPE method for wine
testing
Demonstrate comparability of automation and manual SPE methodologies through good
recoveries of water and wine samples

Show improvement or optimization to existing extraction methodology

Establish an efficient workflow for transitioning from manual to automated SPE
SPE - Manual vs. Automated SmartPrep Extractor
Wine sample preparation was performed manually with a vacuum manifold and automated using the SmartPrep Extractor.
Modifications to the TTB method steps were performed to optimize recovery and precision between replicates.
Manual Manifold
SmartPrep
Manual Manifold
SmartPrep
Compounds
Water
Water
Wine (Moscato)
Wine (Moscato)
a-HCH
b-HCH
g-HCH
d-HCH
Heptachlor
Aldrin
Heptachlor epoxide (B)
t-Chlordane
c-Chlordane
Endosulfan I
4,4`-DDE
Dieldrin
Endrin
Endosulfan II
4,4`-DDD
Endrin Aldehyde
Endosulfan Sulfate
4,4`-DDT
Endrin Ketone
Methoxychlor
87
99
85
90
83
78
83
80
75
83
75
84
85
84
80
87
83
72
85
83
STDEV
6
93
92
90
94
88
85
87
85
84
87
89
87
97
88
75
90
86
92
85
76
STDEV
5
101
111
98
108
50
42
80
48
47
72
42
73
61
96
40
23
98
52
114
66
STDEV
28
85
87
84
90
44
37
67
41
42
61
36
58
70
78
37
53
86
46
83
58
STDEV
20
Statistics
MEAN
83
%CV
7
MEAN
88
%CV
6
MEAN
71
%CV
39
MEAN
62
%CV
31
Automated SPE - Moscato vs. Chardonnay Wines
SmartPrep
Compounds
Wine (Moscato)
Wine (Chardonnay)
a-HCH
b-HCH
g-HCH
d-HCH
Heptachlor
Aldrin
Heptachlor epoxide (B)
t-Chlordane
c-Chlordane
Endosulfan I
4,4`-DDE
Dieldrin
Endrin
Endosulfan II
4,4`-DDD
Endrin Aldehyde
Endosulfan Sulfate
4,4`-DDT
Endrin Ketone
Methoxychlor
85
87
84
90
44
37
67
41
42
61
36
58
70
78
37
53
86
46
83
58
94
98
91
101
55
48
80
55
55
72
50
75
64
95
49
18
90
60
104
68
Cartridges:
 Waters Oasis® HLB | 6 cc, 200 mg
 Supelco Aminpropyl LC-NH2 | 3 cc, 500 mg






50:50 Ethyl Acetate:Hexane
80:20 Ethyl Acetate:Hexane
Methanol
Deionized Water
Pesticide Spike, Restek #32415
Magnesium Sulfate
MANUAL SPE
 BAREFOOT® Moscato (ALC. 9%)
 Livingston® Cellars Chardonnay (ALC. 12%)
Solvents & Standards:
GENERAL
OPTIMIZATION
Wine Samples:
AUTOMATED SPE
Aldrin | α-BHC | β-BHC | δ-BHC | γ-BHC (Lindane) | cis-Chlordane | trans-Chlordane
4,4′-DDD | 4,4′-DDE | 4,4′-DDT (50-29-3) | Dieldrin | Endosulfan I | Endosulfan II
Endosulfan sulfate | Endrin | Endrin aldehyde | Endrin ketone | Heptachlor
Heptachlor epoxide (isomer B) | Methoxychlor
LOAD
Compounds of Interest:
Index A | Cartridge Drying Times
Index B | Speed of Drying
Index C | Collection Fractions
Index D | Elution Volumes
Index E | Elution Flow Rate
Index F | Elution Soak Times
Index G | Sample Loading Flow Rate
Index H | Re-extraction for Breakthrough
Index I | Sample Line Rinsing
Index J | Sample Evaporation Test
Index K | Manual vs. Automation Point of Loss
Index L | DryDisk Test
Index M | Adjusting vacuum flow rates
This application demonstrated comparable recoveries of the 20 target compounds using automated SmartPrep Extractor SPE and
manual vacuum SPE. The water extraction yielded a mean of 83% recovery manually compared to 88% with the automated
SmartPrep Extractor. The wine extraction obtained good recoveries for various compounds (HCH, Endosulfan Sulfate, Endrin
Ketone); however, many other target compounds suffered loss of recovery. This was apparent in a similar pattern for both
manual and automated SPE results, and is suspected to be caused by a matrix effect. It is possible that wine containing a higher
sugar content (Moscato, in this case) may hinder recoveries by approximately 10%. Further SPE optimization is required for
optimal recoveries. Overall, efficient workflow from manual to automated SPE was established using the SmartPrep Extractor
and XcelVap. Automated SPE included the ability to perform the bottle rinsing process to maximize recovery. Using the nitrogen
pressure profile programming on the XcelVap provided consistent sample eluent evaporation.
To obtain final optimized method, please contact Horizon Technology, Inc. for more details
P003

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