Dispersive SPE Cleanup for Analysis of Veterinary Drugs in Animal Samples · PDF fileDuring Dispersive SPE Cleanup for Analysis of Veterinary Drugs in Animal Samples •Olga Shimelis,
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Using a New High Capacity Lipid Depletion Material in Comparison to a C18 Adsorbent
During Dispersive SPE Cleanup for Analysis of Veterinary Drugs in Animal Samples
www.sigma-aldrich.com
•Olga Shimelis, Michael Ye, and Xiaoning LuSupelco, Div. of Sigma-Aldrich, Bellefonte, PA 16823 USA
T411127
2
Introduction
In recent years the concept of using QuEChERS for sample cleanup has been successfully applied to foods other than fruits and vegetables such as breads, milk, and oils. In addition, the range of analyzed compounds has been broadened from pesticides to other types of contaminants (e.g., veterinary drugs). LC-MS methods have become main-stream for analysis.The SPE phases that were used as QuEChERS sorbents to date included primary-secondary amine (PSA) for the removal of acids, polar pigments and sugars; graphitized carbon black (GCB) for the removal of color pigments such as chlorophyll; and C18 for the removal of lipid and non-polar components. The C18 sorbent was, until recently, the only one that was available for the removal of fats and non-polar compounds from samples.In this work we evaluated the use of a new lipid-removal sorbent (Z-Sep+) vs. a DSC-18 for analysis of a number of veterinary drug residues in milk and kidney samples.
3
Experimental
Evaluation of SPE Sorbents for Fat Removal
A standard mix of oleins in acetonitrile (100-200 µg/mL each) was used as a test sample to evaluate the performance of different phases for the removal of fats.1 mL of the prepared solution was mixed with 25 mg of the different SPE sorbents, then centrifuged and the resulting sample was analyzed by LC-ELSD for the removal of oleins.
•HPLC Conditions:column: Ascentis® Express C18, 5 cm x 2.1 mm, 2.7 µm particles
flow rate: 0.5 mL/min.temp.: 30 oC
det.: ELSD (evap 30, neb 50, gas 1.50)injection: 3 µLgradient: 0 min. 100% methanol, 0-3 min. 0-100% isopropanol, 3-6 min. 100% isopropanol,
6-10 min. 100% methanol
4
Experimental (contd.)
Analyses of Veterinary Drugs
Compound ClassAbamectin B1a Avermectins (anthelmintics)Amoxicillin Antibiotics (beta-lactams)Chloramphenicol Antibiotics (phenicols)Ciprofloxacin Antibiotics (fluoquinolones)Furazolidone NitrofuranesLevamisole AnthelminticLincomycin Antibiotics (macrolides)Salbutamol Beta-blockersSulfanilamide Sulfonamides
Table 1. Classes for Veterinary Drug Compounds used in the Current Study
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MS Conditions
Compound Q1 Q3 ESIAbamectin B1a 890.9 567.7 +Amoxicillin 366.22 208.06 +Chloramphenicol 321.1 152.1 -Ciprofloxacine 332.2 288.2 +Furazolidone 226.2 122.1 +Levamisol 205.2 178.2 +Lincomycin 407.2 126.2 +Salbutamol 240.3 148.1 +Sulfanilamide 173.1 93.1 +A separate injection was done for analysis of Chloramphenicol.
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LC Conditions
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.
instrument: AB QTRAP 3200, Agilent 1100-1200 Stackcolumn: Ascentis Express RPA, 5 cm x 2.1 mm, 2.7 µm particles
mobile phase A: 5 mM, 10 mM ammonium acetate buffer, pH 7 mobile phase B: 5 mM acetonitrile
flow rate: 0.5 mL/min.temp.: 35 oC
injection: 5 µLgradient: Min. %B
0 21 25 608 100
10-14 2
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For chloramphenicol injection:
Same column and mobile phase as above was used with a different gradient: Min. %B• 0 20• 1 20• 5 50• 5-9 20
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Ciprofloxacine
0
50
100
150
200
250
300
0 2 4 6 8 10Tim e (m in)
MR
M 3
32.2
/288
.2
Abamectin
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12
Time (min)
MR
M 8
90.9
/567
.7
Amoxicillin
01020304050
60708090
100
0 2 4 6 8 10Time (min)
MR
M 3
66.2
/208
.1
Ch loram phenicol
0
50
100
150
200
250
300
0 2 4 6 8Tim e (m in)
MR
M 3
21.1
/152
.1
Furazolidone
0
100
200
300
400
500
600
700
800
900
0 2 4 6 8 10T ime (min)
MR
M 2
26.2
/122
.1
Lev am isol
0
200
400
600
800
1000
1200
0 2 4 6 8 10Tim e (m in)
MR
M 2
05.2
/178
.2
Figure 1. Extracted MRMs of Compounds
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Lincomycin
0
1000
2000
3000
4000
5000
6000
7000
8000
0 2 4 6 8 10T ime (min)
MR
M 4
07.2
/126
.2
S a lbu tam ol
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
0 2 4 6 8 1 0T im e (m in )
MR
M 2
40.3
/148
.1
Su lfan ilam id e
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
0 2 4 6 8 1 0T im e (m in)
MR
M 1
73.1
/93.
1
Standard injection at the following concentrations: ciprofloxacin, levamisol, lincomycin, salbutamol at 3 ng/mL, chloramphenicol at 0.75 ng/mL, furazolidone at 7.5 ng/mL, amoxicillin at 9 ng/mL, sulfanilamide at 12 ng/mL, andabamectin at 15 ng/mL.
Figure 1. Extracted MRMs of Compounds (contd.)
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Extraction and Cleanup MethodPlace 1 g kidney sample into
50 mL centrifuge tube
Add 2 mL 50 mM phosphate buffer, mix
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+ (55296-U) Add 500 mg
DSC-18
Shake for 1 min., centrifuge and proceed to evaporation
Place 2 mL (g) milk sample into 50 mL centrifuge tube
Add 8 mL acetonitrile, mix for 1 min. and centrifuge
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• Evaporate at 50 oC to 0.75 mL. • Add 0.15 mL acetonitrile and water to total volume of 1.0 mL. • Filter the sample prior to LC-MS analysis using 0.45 µm filter.
Results
A new sorbent (Z-Sep+) displayed a better capacity for removal of mono-, di- and tri-oleins from standard solutions in acetonitrile and acetonitrile:water. The retention is not dependent on the %water, like that for C18 sorbent (Figures, 2A, 2B).
Extraction and Cleanup Method (contd.)
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Figure 2. Removal of (A) Monoolein (B) Diolein and (C) Triolein from Solution by Different Sorbents
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Z-Sep+ C18 PSA NH2 alumina LRA Silica
100% acetonitrile
90% acetonitrile
75% acetonitrile
MonooleinA
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Figure 2. Removal of (A) Monoolein (B) Diolein and (C) Triolein from Solution by Different Sorbents (contd.)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Z-Sep+ C18 PSA NH2 alumina LRA Silica
in 100% acetonitrilein 90% acetonitrilein 75% acetonitrile
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Z-Sep+ C18 PSA NH2 alumina LRA Silica
in 100% acetonitrile
in 90% acetonitrile
Diolein
Triolein
B
C
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Table 2. Recoveries of Drugs Spiked into the Blank Extract. The difference from 100% is due to the presence of Ionization Effects
61%77%89%77%Sulfanilamide
96%93%58%39%Salbutamol
167%150%86%62%Lincomycin
77%106%93%101%Levamisol
74%80%97%113%Furazolidone
64%112%119%102%Ciprofloxacine
98%108%94%106%Chloramphenicol
41%120%116%102%Amoxicillin
9%10%10%85%Abamectin
C18 BlankZ-Sep+ BlankC18 BlankZ-Sep+ BlankCompounds
kidneymilk
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Table 3. Recoveries of Veterinary Drugs Spiked into the Matrix Samples at the indicated Levels (n=3). The Calibration Curve Standards were made in Solvent
47%(9)58%(5)77%(8)62%(6)4020Sulfanilamide
88%(9)79%(9)80%(13)37%(17)105Salbutamol
132%(12)128%(11)75%(9)63%(11)105Lincomycin
64%(6)82%(4)95%(9)98%(7)105Levamisol
70%(21)73%(15)90%(3)98%(13)2512.5Furazolidone
54%(22)57%(22)73%(5)22%(26)105Ciprofloxacine
110%(6)95%(3)100%(6)106%(4)2.51.25Chloramphenicol
19%(98)63%(3)40%(16)31%(7)3015Amoxicillin
4%(86)44%(8)3%(100)61%(10)5025Abamectin
C18Z-Sep+C18Z-Sep+kidneymilkCompounds
Recoveries kidney (%RSD)
Recoveries milk (%RSD)
Spike level(µg/kg)
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Discussion
Addition of 1% formic acid to the extract was necessary to get better recoveries for some compounds during cleanup using the new lipid removal material.The removal of phospholipids (phosphatidylcholine – PC) from milk samples was better when using Z-Sep+ in comparison to using C18. The PC from milk samples is shown in Figure 3. The recoveries of drug compounds from milk did not follow the trend for PC removal. The cleanup using C18 gave better recoveries from milk samples except for abamectin. Abamectin could not be recovered using C18 cleanup (due to ion suppression) but was better recovered using the new lipid removal material. Abamectin is the late-eluting compound and, possibly, the phospholipids contribute strongly to the ionization supression.An additional benefit for using the new lipid-removal phase is the more efficient color removal which is shown in Figure 4 for kidney samples. Also, the recoveries of drugs from kidney samples were better when using Z-Sep+
during cleanup (for amoxicillin, sulfonilamide and levamisol).
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Figure 3. Comparison of Phosphatidylcholine in Milk Samples Cleaned Using Z-Sep+ or C18 dSPE. 2 Ions-M/Z 104 and 184 - were monitored
0.0E+00
2.0E+04
4.0E+04
6.0E+04
8.0E+04
1.0E+05
1.2E+05
1.4E+05
1.6E+05
0 2 4 6 8 10 12 14Time (min)
TIC
M/Z
104
Z-Sep+
C18
0.0E+00
2.0E+04
4.0E+04
6.0E+04
8.0E+04
1.0E+05
1.2E+05
1.4E+05
0 2 4 6 8 10 12 14Time (min)
TIC
M/Z
184
Z-Sep+
C18
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Conclusions
• When evaluated, the new material Z-Sep+ as a QuEChERS cleanup reagent compared to a C18:
• 1. Is better for removal of especially fatty compounds that contain single fatty acid chains.
• 2. Both positive and negative comparisons to the C18 sorbent were made when analyzing kidney and milk samples for monitored veterinary drugs:
• The new sorbent is better for removing color compounds.• The new sorbent requires addition of formic acid during the cleanup step to avoid
retaining more acidic and chelating compounds (e.g.ciprofloxacine).• The final recoveries from milk were better for this method when using C18 sorbent
with exception of abamectin.• The final recoveries from kidney were better for this method when using the new
sorbent with exception of salbutamol.
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References
1. Kaufmann, A., Butcher, P., Maden, K., Widmer, M. (2008). Quantitativemultiresidue method for a bout 100 veterinary drugs in different meat matrices by sub 2-μm particulate high-performance liquid chromatography couple to time of flight mass spectrometry. Journal of Chromatography A, 1194, 66-79.
2. Kinsella, B., Lehotay, S., Mastovska, K., Lightfield, A., Furey, A., Danaher, M. (2009). New method for the analysis of flukicide and other anthelminticresidues in bovine milk and liver using liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta, 637, 196-207.
3. Martos, P., Jayasundara, F., Dolbeer, J., Jin, W., Spilsbury, L., Mitchell, M., Varilla, C., Shurmer, B., (2010). Multiclass, Multiresidue Drug Analysis, Including Aminoglycosides, in Animal Tissue Using Liquid Chromatography Couple to Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry, 58, 5932-5944.
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4. Mastovska, K., Lightfield, A. (2008). Streamlining methodology for themultiresidue analysis of beta-lactam antibiotics in bovine kidney using liquid chromatography-tandem mass spectrometry. Journal of Chromatography A. 1202, 118-123.
5. Samanidou, V., Nisyriou, S. (2008). Multi-residue methods for confirmatory determination of antibiotics in milk. Journal of Separation Science, 31, 2068-2090.
6. Turnipseed, S., Anderson, W., Karbiwnyk, C., Madson, M., Miller, K. (2008). Multi-class, multi-residue liquid chromatography/tandem mass spectrometry screening and confirmation methods for drug residues in milk. Rapid Communications in Mass Spectrometry, 22, 1467-1480.
7. Whelan, A., Kinsella, B., Furey, A., Moloney, M., Cantwell, H., Lehotay, S., Danaher, M. (2010). Determination of anthelmintic drug residues in milk using ultra high performance liquid chromatography-tandem mass spectrometry with rapid polarity switching. Journal of Chromatography A, 1217, 4612-4622.
References (contd.)
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