Analysis of Difficult Matrices with a New QuEChERs Product

Analysis of Difficult Matrices with a
New QuEChERs Product
• E. Barrey, K. Stenerson, O.Shimelis
M.Ye, J. Claus, and D. Vitkuske
Supelco, Div. of Sigma-Aldrich
Bellefonte, PA 16823 USA
sigma-aldrich.com
T412162
Introduction
• QuEChERS stands for Quick, Easy, Cheap, Effective, Rugged, Safe
• The method is used for extraction and cleanup of samples for pesticide
analysis in fruits, vegetables, processed food and animal feeds
• The extraction step uses acetonitrile and a salting out effect (with MgSO4)
• The cleanup step is accomplished by using dispersive SPE
• Cleanup sorbents include:
– PSA – for removal of polar pigments, sugars and acids
– Graphitized carbon Black – for removal of chlorofill and carotenoids
– C18 – for removal of lipids and non-polar components
Reference: M. Anastassiades, S.J. Lehotay, et al., J. AOAC Int. 86, 412–431 (2003)
2
• Most fruits and vegetables contain less than 1% with the
exception of:
– Avocado, 10-15% fat
– Olives, 15% fat
– Nuts, ex: almonds – 49% fat
• Animal products contain varying amounts of fat:
– Beef Kidney, 5% fat
– Whole cow’s milk, 3-4% fat
– Salmon, 12% fat
• Researchers have used methods in addition to C18 dSPE to
improve the removal of fatty components prior to analysis:
– Freezing out the fats
– Using hexane for liquid-liquid extraction of fats
• Another way to remove fatty components – use of new sorbents
– Z-Sep and Z-Sep+
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What are Z-Sep/C18 and Z-Sep+ Particles?
• Supel™ QuE Z-Sep/C18
• Supel QuE Z-Sep+
• 50 mg of Discovery® DSC-18 and 20 mg
of Z-Sep (Proprietary HybridSPE®
zirconia-coated silica)
• Sold in 2 mL centrifuge tubes; 100
tubes/pack (catalog # 55284-U)
• Use for cleanup of LC-MS samples
ZrO
C18
ZrO
ZrO
ZrO
ZrO
ZrO
ZrO
+
• Sold in 12 mL centrifuge tubes, 500 mg
– 50 tubes/pack (catalog # 55296-U)
– Sample pack 5 tubes/pack
– Bulk, 20 g (catalog # 55299-U)
ZrO
C18
ZrO
• Dual Bonded C18 and Z-Sep
C18
C18
ZrO
C18
C18
C18
ZrO
C18
C18
C18
C18
ZrO
C18
4
Why Z-Sep/C18 and Z-Sep+?
Removal of Oleins from Solution by Using Various Sorbents
25 mg of sorbent was mixed with 1 mL solution of oleins (400 mg/mL total
concentration) in acetonitrile. The remaining mono-, di- and trioleins were
quantified by LC-ELSD and retention was calculated.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Z-Sep+
ZSep/C18
C18
PSA
NH2
alumina
LRA
Silica
5
Experimental - Veterinary Drugs in Kidney & Milk
Extraction & Cleanup
Place 1 g kidney sample into
50 mL centrifuge tube
Place 2 mL (g) milk sample into
50 mL centrifuge tube
Add 2 mL 50 mM phosphate buffer, mix
Add 8 mL acetonitrile, mix for 1 min
and centrifuge
Add 8 mL acetonitrile, mix
Keep sample at 60 oC for 45 min, mix periodically
Cool down in cold water, then centrifuge
Separate the supernatant into 15 mL centrifuge tube
Add 0.1 mL conc. formic acid and 500 mg Z-Sep+
Add 500 mg DSC-18
Shake for 1 min, centrifuge
Evaporate supernatant to 0.75 mL at 50 ºC. Add 0.15 mL acetonitrile, and adjust to
FV= 1 mL with water. Filter prior to LC-MS analysis
6
LC-MS Conditions
instrument: AB QTRAP 3200, Agilent 1100-1200 Stack
column: Ascentis® Express RPA, 5 cm x 2.1 mm, 2.7 µm
•mobile phase : (A) 5 mM 10 mM ammonium acetate buffer pH 7
(B) 5 mM acetonitrile
•
flow rate: 0.5 mL/min
•
temp.: 35 °C
•
det.: MS-MS via MRMs
•
injection 5 µL
Both C18 and RPA columns were tested for this separation. The RPA
column was used because it provided better retention for more polar
analytes, such as salbutamol and sulfanilamide.
7
Results
Recoveries of veterinary drugs spiked into the matrix samples at the
indicated levels (n=3).
Spike level
(µg/kg)
Recoveries milk
(%RSD)
Recoveries kidney
(%RSD)
milk
kidney
Z-Sep+
C18
Z-Sep+
C18
Abamectin
25
50
61%(10)
3%(100)
44%(8)
4%(86)
Amoxicillin
15
30
31%(7)
40%(16)
63%(3)
19%(98)
1.25
2.5
106%(4)
100%(6)
95%(3)
110%(6)
5
10
22%(26)
73%(5)
57%(22)
54%(22)
12.5
25
98%(13)
90%(3)
73%(15)
70%(21)
Levamisol
5
10
98%(7)
95%(9)
82%(4)
64%(6)
Lincomycin
5
10
63%(11)
75%(9)
128%(11)
132%(12)
Salbutamol
5
10
37%(17)
80%(13)
79%(9)
88%(9)
Sulfanilamide
20
40
62%(6)
77%(8)
58%(5)
47%(9)
Compounds
Chloramphenicol
Ciprofloxacine
Furazolidone
Quantified against calibration curve standards made in solvent.
8
Discussion
• In milk, C18 cleanup yielded better recoveries for most drugs. In beef
kidney, Z-Sep+ generally yielded better recoveries.
• Abamectin recovery was poor in both matrices with C18 cleanup.
Detection was probably affected by ion supression due to coeluting
phospholipids. Recovery was improved with Z-Sep+ cleanup.
• Z-Sep+ cleanup removed more color from beef kidney extracts than C18
(not shown).
• Z-Sep+ cleanup required the use of formic acid to avoid retention of
more acidic and chelating compounds (e.g.ciprofloxacine).
9
Experimental - Pesticides in Olives
Extraction & Cleanup
Weigh 10 g olive sample into 50 mL centrifuge tube
Add 10 mL acetonitrile, mix
Add citrate extraction tube (55227-U) and shake for 1 min
Centrifuge at 3200 rpm for 5 min
Aliquot 0.7 mL of supernatant to QuEChERS tubes:
PSA (55287-U), PSA/C18 (55288-U), Z-Sep/C18 (55284-U)
Shake for 1 min and centrifuge at 5000 rpm for 5 min
Dilute as necessary and analyze
10
LC-MS Conditions
instrument: AB QTRAP 3200, Agilent 1100
column: Ascentis Express C18, 5 cm x 2.1 mm , 2.7 µm (53822-U)
mobile phase: (A) 10 mM ammonium acetate buffer pH 7, (B) 5 mM ammonium
acetate in acetonitrile
•
temp.: 30 °C
•
det.: MS-MS via MRMs
•
injection: 5 µL
•
sample: olives spiked with a mixture of 38 pesticides at 50 ppb, most amenable
to LC-MS
11
Results
Recoveries of selected pesticides spiked into the matrix samples at 50 ppb (n=3).
Quantified against calibration curve standards made in solvent.
12
Discussion
• Pesticide recoveries were better using Z-Sep/C18 cleanup than PSA/C18
or PSA alone.
• For fenhexamid, anilazine, and sethoxydim, ion suppression was not
observed for the Z-Sep/C18 mix but was evident for PSA and PSA/C18.
13
Experimental - Pesticides in Avocado
Extraction & Cleanup
Place 3 g homogenized avocado sample into
50 mL centrifuge tube (55248-U)
Add 25 mL acetonitrile, mix for 1 min
Add Supel QuE Acetate tube (55234-U),
mix for 1 min
Centrifuge samples for 5 min
Add the following cleanup sorbents to 3 mL extract:
Supel QuE Z-Sep+ (55296-U), PSA/C18 mix, or
PSA/Z-Sep+ mix
Separate 1 mL supernatant
Proceed with GC-MS analysis
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GC-MS Conditions
instrument: Agilent 7890/5975 GC/MS
GC column: (1) SLB®-5ms 20 m x 0.18 mm I.D., 0.18 µm
(2) SLB-5ms 20 m x 0.18 mm I.D., 0.36 µm
oven: 70 °C (2 min.), 15 °C/min. to 325 °C (5 min.)
inj.: programmed, (1) 75 °C (0.89 min.), 600 °C/min. to 325 °C (5 min.)
(2) 60 °C (0.28 min.), 600 °C/min. to 325°C (5 min.)
carrier gas: helium, 1 mL/min. constant flow
injection: (1) 25 µL LVI, PTV solvent vent, variable injection speed
(2) 10 µL LVI, PTV solvent vent, rapid injection speed
split vent flow: 100 mL/min, (5 psi) until 0.28 min., 60 mL/min. at 2.78 min.
liner: 4 mm ID focus liner w/taper
MS conditions: SIM
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Results
Total residue remaining and GC-MS background after cleanup
Avocado (15% fats)
From 1.44 g avocado
Residue remaining after
cleanup of avocado
extracts
A total of 200 mg of sorbent or sorbent mix per mL of extract used in cleanup
Z-Sep+ cleanup
12
14
16
18
20
22
24
Time (min)
C18/PSA cleanup
GC-MS Analysis of avocado
extracts (scan mode) in the
same y-scale
16
12
14
16
18
Time (min)
20
22
24
Recoveries and (RSDs) of pesticides from avocado using different
cleanup methods.
140
120
C18/PSA
Z-Sep(+)/PSA
Z-Sep+
100
80
60
40
20
Deltamethrin
Cypermethrin
Cyfluthrin isomers
Coumaphos
Methoxychlor
4,4'-DDT
Endosulfan I
Chloropyrifos
Metolachlor
Malathion
Heptachlor
gamma-BHC
Qunitozene
Simazine
Dimethoate
0
Hexachlorobenzene
Z-Sep+
20 ng/g
98 (1)
92 (2)
58 (1)
97 (7)
92 (2)
86 (1)
91 (1)
78 (1)
104 (2)
94 (0.4)
93 (1)
79 (2)
78 (6)
97 (6)
121 (5)
133 (10)
114 (2)
124 (2)
alpha-BHC
Z-Sep+/PSA
20 ng/g
87 (17)
84 (14)
39 (15)
91 (18)
85 (12)
71 (7)
78 (10)
78 (12)
89 (8)
86 (6)
102 (5)
94 (6)
69 (2)
82 (4)
95 (5)
88 (11)
87 (31)
42 (87)
Trifluralin
Spike level
Trifluralin
α-BHC
Hexachlorobenzene
Dimethoate
Simazine
Qunitozene
γ-BHC
Heptachlor
Malathion
Metolachlor
Chloropyrifos
Endosulfan I
4,4'-DDT
Methoxychlor
Coumaphos
Cyfluthrin isomers
Cypermethrin isomers
Deltamethrin
C18/PSA
20 ng/g
71 (5)
91 (17)
44 (10)
0
74 (7)
62 (15)
75 (13)
58 (4)
74 (14)
74 (7)
72 (10)
68 (9)
60 (56)
42 (27)
68 (10)
0
matrix
matrix
Best recoveries and least matrix
interference with Z-Sep+ only cleanup.
C18/PSA samples analyzed using GC/MS conditions (1)
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Discussion
• Using Z-Sep+ and Z-Sep+/PSA improved cleanup vs. C18/PSA, as
evidenced by GC-MS scan data and the measured total extractables
(not shown).
• Z-Sep+ removed more residue than other cleanups. The addition of PSA
did not remove any additional residue. Z-Sep+ can be used as a single
cleanup sorbent for this application.
• Permethrin pesticides could not be seen in the C18 cleaned extract due to
the presence of high amounts of sample matrix.
• Lower recoveries were observed for hexachlorobenzene, deltamethrin and
other lipophilic pesticides using C18/PSA cleanup.
• For selected pesticides, the method sensitivity decreased as more
injections into the GC system were performed (data not shown).
Modifications to the GC-MS systems may be necessary for running fatty
samples, such as installation of back-flashing capability.
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Conclusions
• Zirconia-based Z-Sep and Z-Sep+ sorbents retain more oleins than
traditional cleanup sorbents.
• In beef kidney and avocado, Z-Sep+ cleanup removed more background
than PSA/C-18.
• In beef kidney and avocado, extracts cleaned with Z-Sep+ exhibited
comparable or better analyte recoveries than PSA/C-18.
• In olives, Z-Sep/C18 cleaned extracts exhibited better pesticide recoveries
than PSA and PSA/C18. This is possibly due to ion supression effects
from the presence of phospholipids in the PSA and PSA/C18 cleaned
extracts.
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Trademarks
Ascentis, Discovery, HybridSPE, and SLB are registered trademarks of
Sigma-Aldrich Co. LLC.
Supel is a trademark of Sigma-Aldrich Co. LLC.
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