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Dispersive Liquid Liquid Micro Extraction (DLLME ... · DAD1 E, Sig=360,4 Ref=360,100...
Transcript of Dispersive Liquid Liquid Micro Extraction (DLLME ... · DAD1 E, Sig=360,4 Ref=360,100...
Eskinder Teklu
Dispersive Liquid–Liquid Micro
Extraction (DLLME) Technique for The
Simultaneous Determination of 15
Sulfonamide Antibiotics in Egg
Samples Using High Performance
Liquid Chromatography (HPLC)
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Outlines
1. Introduction
2. Experimental
Standards and Instrumentation
Results and Discussion
3. Conclusion
4. Acknowledgment
2
Introduction
Veterinary drugs : different types of compounds
used in farm animals for
Therapeutic (treatment)
Prophylactic (prevention)
Sub-therapeutic (growth promoters)
3
Cont’d …
• Overused and misused :- Retain residue
Edible tissues (muscle, liver & kidney) and
Products (egg, milk & honey)
• Direct toxic effect
• Development of antibiotic resistant bacteria
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Sulfonamides • Amphoteric with weakly basic and
acidic characteristics
pH < 2 Cationic
pH = 3-5 Neutral
pH >5 anionic
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• MRL = 100 ppb (total ) ( EC ,WHO)
Cont’d…
• Efficient, reliable & environmentally friendly
analytical methods are needed for monitoring the
residue of these drugs
• Challenge :- ppb & multiple compounds
Dispersive Liquid - Liquid Microextraction technique
(DLLME).
• The method is rarely applied in biomatrices (Li et al.,
2013; Prosen , 2014; Yan et al., 2013).6
Experimental
Standards: 15 sulfonamides
Instruments
High performance liquid chromatographic (HPLC) system, Agilent
1260 series with diode array detector (DAD).
Chromatographic separations : Zorbax Eclipse C18 Plus column
(100 x 4.6 mm ID., 3.5 mm particle size.
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Optimization of chromatographic conditions
Fig . 1. Chromatogram of fifteen SAs Standards (300 ppb) SGD, SAM, SAA, SDZ, STZ, SPY, SMR,
SMT, SMM, SCP, SDX, SSO, SBZ SQZ and SSA Respectively.
Conditions; flow rate of 1.8 mL min−1, column temperature 40 °C, injection volume of 5 μL,
wavelengths of 265 nm & 360 nm. A binary mobile phase comprising of solvent A (0.1% FA
water) and solvent B Acetonitrile with a gradient program of 10 % B ( 0 – 1 min), 40 % ( 1- 4
min) and 60 % B (4- 6 min).
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min1 2 3 4 5
Norm.
-20
0
20
40
60
DAD1 D, Sig=265,4 Ref=360,100 (C:\CHEM32\1\DATA\ESK 2016-04-26 19-45-52\MIX.D)
0.7
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0.9
45
1.6
27
1.9
38
2.3
01
2.4
96
2.7
69
3.2
88
3.7
18
3.8
59
4.0
98
4.3
13
4.6
17
4.7
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DAD1 E, Sig=360,4 Ref=360,100 (C:\CHEM32\1\DATA\ESK 2016-04-26 19-45-52\MIX.D)
2.6
80
5.2
02
9
Homogenized
5 mL
Extraction solvent
1 ml DLLMEFilter
4 µm
Extract
HPLC (Recovery)
Spike
HPLC
Yolk & Albumen
Fig. 2. Sample pretreatment procedure
Optimization of Sample Pretreatment Procedure
Selection of organic solvent
Fig . 3. Blank egg spiked at 300 ppb of 15 SAs mixtures ; extraction solvents each 5 ml (n = 5)
10
-10
0
10
20
30
40
50
60
70
80
MeCN Aceton MeOH
% R
eco
very
Solvents
SGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
Optimization of acidic conditions that favor the dissolution of the SAs
Fig . 4. Blank egg matrix spiked at 300 ppb of 15 SAs mixtures; FA : MeCN (25 : 75 ,v/v) ; (n = 5)
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0
20
40
60
80
100
120
0,02% 0,05% 0,10% 0,20% 0,40%
% R
eco
very
% Formic acid v/vSGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
Selection of Optimum Formic Acid to Acetonitrile Ratio
Fig . 5. Blank egg matrix spiked at 300 ppb of 15 SAs mixture ; formic acid to
acetonitrile ratio (FA : MeCN) 5 / 95 - 45/ 55 v/v ; (n = 5).
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0
20
40
60
80
100
120
5\95 15/85 25/75 35/65 45/55
% R
eco
very
FA:MeCN ratioSGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
5 ml ultrapure
water ( pH =
3.5)
Vortex for
30 sec
Centrifuge
3 min
HPLC
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Fig 6. DLLME Procedure
Optimization of DLLME Conditions
Fig . 7. Conditions : - water volume (5.0 mL), dispersive solvent (1.0 mL MeCN : FA; extraction solvents (DCM, DCE & CF; 400 μL each) (n = 5).
Selection of extraction solvent
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0
10
20
30
40
50
60
70
80
90
DCM DCE CF
%E
xtr
acti
on
Eff
icie
ncy
Extraction Solvent
SGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
Effect of Sample pH
Fig .8. Conditions: water volume (5.0 mL).; dispersive solvent MeCN :FA (1mL ); extraction solvent (400 μL Dicloro methane) (n = 5).
15
-20
0
20
40
60
80
100
120
pH = 2.5 p H = 3.5 p H = 4.5 pH = 5.5 PH = 6.5
% E
xtr
act
ion
Eff
icie
ncy
pH
SGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
Effect of the volume of extraction Solvent
Fig .9. Conditions :- Water (5.0 mL, pH , 3.5) ; dispersive solvent (1.0 mL MeCN :FA), extraction
solvent (200µL - 1000µl, Dichloromethane) ) (n = 5).
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0
20
40
60
80
100
120
200 µL 400 µL 600 µL 800 µL 1000 µL
% E
xtr
acti
on
Eff
icie
ncy
Extraction Solvent Volume
SGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
Effect of Dispersive Solvent Volume
Fig . 10. Conditions : - Water (5.0 mL, pH 3.5); extraction solvent (400 µl, Dichloromethane) ;
dispersive solvent (0.5 – 2 mL MeCN : FA ), (n = 5).
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0
10
20
30
40
50
60
70
80
90
100
0.5 mL 1 mL 1.5 mL 2 mL
% E
xtr
acti
on
Eff
icie
ncy
Volume of Dispersive Solvent
SGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
Effect of Centrifuge Time
Fig . 11. Conditions :- Water volume (5.0 mL, pH 3.5); extraction solvent (400 µl, Dichloromethane)
; dispersive solvent (1mL MeCN :FA ), centrifuge time ( 3 min - 10
min); (n = 5)
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0
20
40
60
80
100
120
1 min 3 min 5 min 7 min 10 min
% E
xtr
acti
on
Eff
icie
ncy
Centrifuge Time
SGD SAM SAA SDZ STZ SPY SMR SMT SMM SCP SMX SSO SBZ SQZ SSA
Method Validation
Selectivity/Specificity
Fig. 12 . a & b Blank egg sample before enrichment and after enrichment proceduresrespectively
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min1 2 3 4 5
mAU
0
1
2
3
4
DAD1 D, Sig=265,4 Ref=360,100 (C:\CHEM32\1\DATA\ESK 2016-06-28 13-21-36\blank1.D)
min1 2 3 4 5
mAU
0
0.5
1
1.5
2
DAD1 D, Sig=265,4 Ref=360,100 (C:\CHEM32\1\DATA\ESK 2016-06-28 17-08-00\blank1.D)
5.0
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a b
Compounds Regression equation
(n = 6) Linear Range
(µg/L)
R2
LOD (µg/L) LOQ (µg/L)
SGDY = 0.005x + 0.03 6.4 - 100
0.995
4.3 6.4
SAMY =0.001x + 0.042 16 – 100
0.991
6.7 15.9
SAAY = 0.003x + 0.010
50 - 1000 0.994
6.4 10.2
SDZ Y = 0.010x + 0.189 17 - 1000 0.9987.2 16.4
STZ Y =0.001x + 0.305 16 - 1000 0.9977.4 15.7
SPYY = 0.009x + 0.068
14 - 700 0.993
6.9 13.2
SMR Y = 0.075x + 0.404 6 - 10000.994 4.5 5.4
SMT Y = 0.005x + 0.283 10 - 10000.998
5.9 9.2
SMM Y= 0.030x + 0.0.279 11 - 7000.997
5.4 10.5
SCP Y= 034x + 0.588 7 - 1000 0.990 4.7 7.0
SMX Y = 0.34x + 0.525 8 - 1000 0.995
5.1 7.8
SSO Y =0.003x + 0.272 18 - 1000 0.997
8 17.6
SBZ Y = 0.010x + 0.514 14 - 1000 0.994 6.3 13.9
SQZ Y =0.013x + 0.598 13 - 5000.995 6.7 12.4
SSA Y=0.0004x + 0.004 9 - 500 0.996
5 8.3
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CompoundsPrecision
Intra – day (n =6) % RSD Inter – day (n = 6) % RSD
50 (µg/L) 100 (µg/L) 500 (µg/L) 50 (µg/L) 100 (µg/L) 500 (µg/L)
SGD 10.9 10.3 9.5 11.3 12.9 9.5
SAM 16.2 22.3 9.8 11.3 15.2 14.6
SAA 13.1 9.2 12.3 14.4 9.3 10.4
SDZ 15.3 13.2 13.8 14.3 10.4 6.9
STZ 15.8 12.4 15.1 14.3 8.2 7.3
SPY 6.3 10.7 9.7 14.5 11.5 4.8
SMR 14.4 11.8 9.8 14 9.2 5.5
SMT 11.8 15.4 13.7 13 10.4 15.3
SMM 15.4 10.6 10.1 12.7 15.8 10.2
SCP 14.9 15.9 9.3 13.7 11.8 16.8
SMX 16.4 14.7 10.8 11.5 7.8 12.6
SSO 15.6 17.5 14.7 12.1 13.5 4.4
SBZ 14.7 16.5 12.1 9.3 6.4 8
SQZ 12.5 14.8 9.7 12.8 14 11
SSA 9.9 13 14.1 13.7 16.8 7.2
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Conclusions
Results obtained in
Linear range (6.4 - 1000 µg/L),
Regression coefficient (0.993 - 0.998),
LOD ( 4.3 - 7.4 µg/Kg),
LOQ ( 5.4 - 17.6 µg/Kg),
Precision (%RSD) at (50%, 100% & 500% of target level)
•Intra-day ( 6.3 - 15.8, 9.2 - 17.5 & 9.3 -15.1 , respectively)
•Inter-day (9.3 - 14.5, 6.4 -16.8 & 4.8 - 16.8 , respectively)
indicated that the method is promising for the determination of
target compounds in egg matrix.
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Acknowledgments
• To my advisors :-Professor M.M. Nindi &
Professor S. Dube
• To the University of South Africa (UNISA)
• To MOE , Ethiopia
• To ChromSAAMS
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Thank You!
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