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)

1

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

4

Sulfonamides • Amphoteric with weakly basic and

acidic characteristics

pH < 2 Cationic

pH = 3-5 Neutral

pH >5 anionic

5

• 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.

7

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).

8

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

47

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

48

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)

11

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).

12

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

13

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

14

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).

16

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).

17

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)

18

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

19

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

26

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

20

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

21

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.

22

Acknowledgments

• To my advisors :-Professor M.M. Nindi &

Professor S. Dube

• To the University of South Africa (UNISA)

• To MOE , Ethiopia

• To ChromSAAMS

23

Thank You!

24