Development of a Method Based on DLLME and UFLC-DAD for the Determination of Antibiotics in Honey Samples and the Study of Their Degradation Kinetics

Santana, Ana C. B. A.; Santana, Marta V. A.; Pereira, Pedro A. P.

doi:10.21577/0103-5053.20180028

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Journal of the Brazilian Chemical Society

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Articles • J. Braz. Chem. Soc. 29 (7) • July 2018 • https://doi.org/10.21577/0103-5053.20180028 copy

Development of a Method Based on DLLME and UFLC-DAD for the Determination of Antibiotics in Honey Samples and the Study of Their Degradation Kinetics

Authorship SCIMAGO INSTITUTIONS RANKINGS

In this work, a method was developed, based on dispersive liquid-liquid microextraction (DLLME) and analysis by ultra-fast liquid chromatography coupled to diode array detector (UFLC-DAD), to simultaneously determine ten antibiotics of two different classes in honey, as well as to evaluate their degradation kinetics in this matrix. The extraction was optimized by a Box-Behnken design and the method provided good linearity and precision, limits of detection (LODs) in the range of 3.1-6.8 µg kg^-1 and recoveries between 82.9 and 105.7%. The decay velocities along the storage period have followed a first order model for nine antibiotics, while one has fitted a zero order model. In the analysis of 33 samples, sulfadimethoxine and tetracycline were found in four samples of the same year and type of blooming, freshly produced in different places, at concentrations, respectively, below and 79% higher than the established by the Brazilian legislation. This suggests an application of those drugs to the beehives, probably shortly before the samples were taken.

Keywords:
honey; sulfonamide and tetracycline residues; degradation kinetics; DLLME; UFLC-DAD

Factor	Low (-1)	Mean (0)	High (+1)
ESV / µL	100	150	200
DSV / µL	500	750	1000
SO / g	0.3	0.55	0.8
pH	3.0	4.0	5.0

Factor	SS (× 10⁹)	d_f	MS	F	p
(1) ESV	0.4647829	2	232391457	93.3987	0.0106
(2) DSV	0.1927039	2	96351928	38.7241	0.0252
(3) pH	0.0684321	2	34216034	13.7515	0.0678
(4) SO	0.0870157	2	273100332	17.4859	0.0541
1 × 2	1.092401	4	19778941	109.7597	0.0090
1 × 3	0.0791158	4	19778941	7.9492	0.1148
1 × 4	0.0011247	2	562330	0.2260	0.8156
2 × 3	0.0294013	3	9800429	3.9388	0.2091
2 × 4	0.105500	2	52750154	21.2004	0.0450
3 × 4	0.0003003	1	300304	0.1207	0.7614
Pure error	0.0049763	2	2488166	-	-
Total SS	2.639755	26	-	-	-

Compound	Regression equation	R²	Linear range / (µg kg^-1)	LOD / (µg kg^-1)	LOQ / (µg kg^-1)
SDZ	y = 236.42x - 64.894	0.9982	10-100	3.2	10.0
STZ	y = 239.16x - 356.19	0.9973	10-100	3.2	10.0
SMRZ	y = 284.43x - 299.51	0.9996	10-100	3.5	10.0
SMTZ	y = 240.46x - 250.68	0.9978	10-100	3.5	10.0
SMX	y = 318.53x - 132.6	0.9981	10-100	3.1	10.0
SDM	y = 227.19x + 588.87	0.9982	10-100	3.5	10.0
OTC	y = 110.38x - 242.63	0.9984	20-200	6.8	20.0
TC	y = 103.79x - 355.75	0.9988	20-200	6.5	20.0
CTC	y = 104.36x - 120.8	0.9903	20-200	6.3	20.0
DXC	y = 100.82x - 168.97	0.9876	20-200	6.3	20.0

Compound	40 µg kg^-1			60 µg kg^-1			100 µg kg^-1
Compound	R_m / %	RSD_r / %	RSD_R / %	R_m / %	RSD_r / %	RSD_R / %	R_m / %	RSD_r / %	RSD_R / %
OTC	92.8	6.3	6.8	95.4	4.5	6.0	94.6	5.4	4.8
TC	91.4	11.5	13.2	97.9	6.6	7.1	101.9	3.6	5.4
CTC	83.5	5.3	14.7	96.8	5.3	7.9	101.3	3.1	5.3
DXC	83.3	9.5	13.7	89.9	8.7	8.6	91.4	7.8	7.8

Compound	20 µg kg^-1			40 µg kg^-1			60 µg kg^-1
Compound	R_m / %	RSD_r / %	RSD_R / %	R_m / %	RSD_r / %	RSD_R / %	R_m / %	RSD_r / %	RSD_R / %
SDZ	98.3	4.0	10.2	104.9	3.1	7.3	105.6	5.2	7.9
STZ	87.4	6.9	6.3	101.8	3.8	7.1	99.6	4.4	6.5
SMRZ	93.8	4.6	13.3	99.1	6.7	6.1	94.9	2.4	6.5
SMTZ	98.6	8.8	7.8	105.0	4.6	7.4	104.7	4.4	6.5
SMX	87.8	3.1	7.9	82.9	3.6	7.0	95.3	5.8	8.8
SDM	95.8	4.2	16.1	99.9	3.0	6.5	105.7	4.9	8.1

Antibiotic	Extraction technique	Solvent type (volume)	Sample amount / g	Extraction time / min	LOQ / (µg kg^-1)	Reference
Sulfonamide	LLE	acetonitrile (10 mL) and dichloromethane (2.5 mL)	5.0	60	N.D.	Verzegnassiet al.¹⁷17 Verzegnassi, L.; Savoy-Perroud, M.; J. Chromatogr. 2002, 977, 77.
Tetracycline	SPE	10% (v/v) methanol in ethyl acetate (5 mL)	3.0	10	15-30^a a Limits of detection	Viñaset al.²2 Viñas, P.; Balsalobre, N.; López-Erroz, C.; Hernández-Córdoba, M.; J. Chromatogr. A 2004, 1022, 125.
Tetracycline	SPE online	methanol (3 mL)	5.0	n.d.	23-40	Liet al.⁶6 Li, J.; Chen, L.; Wang, X.; Jin, H.; Ding, L.; Zhang, K.; Zhang, H.; Talanta 2008, 75, 1245.
Sulfonamide	SPE	acetonitrile (10 mL)	5.0	60	1.5-15	Sheridanet al.⁷7 Sheridan, R.; Policastro, B.; Thomas, S.; Rice, D.; J. Agric. Food Chem. 2008, 56, 3509.
Tetracycline	SPE	ethyl acetate:methanol 75:25 v/v (3 mL)	3.0	13	25	Pereset al.¹³13 Peres, G. T.; Rath, S.; Reyes, F. G. R.; Food Control 2010, 21, 620.
Chloramphenicol and thiamphenicol^b b two analytes;	DLLME	1,1,2,2-tetrachloroethane (30 µL) and acetonitrile (100 µL)	1.0	2	0.1-0.6^a a Limits of detection	Chenet al.⁵⁶56 Chen, H.; Chen, H.; Ying, J.; Huang, J.; Liao, L.; Anal. Chim. Acta 2009, 632, 80.
Sulfonamide and tetracycline^c c ten analytes. LOQ: limit of quantification; n.d.: not declared; LLE: liquid-liquid extraction; SPE: solid-phase extraction; DLLME: dispersive liquid-liquid microextraction.	DLLME	chloroform (200 µL) and acetonitrile (500 µL)	1.0	10	10-20	this method

Antibiotic	C₀ / (µg kg^-1)	Decay equation (ln C = -kt + ln C₀)	R²	t_1/2 / day	t₉₀ / day	t_max / day	Degradation^a a Estimated degradation of the antibiotic at tmax. C0: initial concentration; R2: correlation coefficient; t1/2: time calculated for a decay of 50% of the antibiotic, relative to its initial concentration; t90: time calculated for a decay of 90% of the antibiotic, relative to its initial concentration; tmax: maximum time for which the antibiotic was detected in the samples; SDZ: sulfadiazine; STZ: sulfathiazole; SMRZ: sulfamerazine; SMX: sulfamethoxazole; SDM: sulfadimethoxine; OTC: oxytetracycline; TC: tetracycline; CTC: chlortetracycline; DXC: doxycycline. / %
SDZ	40	ln C = -0.0649t + 3.7273	0.9434	11	35	20	72.7
	60	ln C = -0.0417t + 3.9661	0.9774	17	55	40	81.1
STZ	40	ln C = -0.1364t + 3.6794	0.9274	5	17	10	74.4
	60	ln C = -0.0414t + 3.8264	0.9132	17	56	40	80.9
SMRZ	40	ln C = -0.0286t + 3.4607	0.9455	20	67	35	63.2
	60	ln C = -0.0342t + 4.0777	0.9047	24	81	60	87.1
SMX	40	ln C = -0.0494t + 3.8607	0.9167	14	47	35	70.9
	60	ln C = -0.0431t + 4.1120	0.9771	16	53	40	82.1
SDM	40	ln C = -0.0474t + 3.6432	0.8854	14	48	20	61.2
	60	ln C = -0.0381t + 4.3387	0.9158	19	62	35	81.4
OTC	60	ln C = -0.0417t + 4.1622	0.9384	17	55	25	64.7
	100	ln C = -0.0382t + 4.4107	0.9660	18	60	45	82.1
TC	60	ln C = -0.0234t + 4.1620	0.9096	30	98	35	65.1
	100	ln C = -0.0112t + 4.5605	0.9815	62	206	120	73.9
CTC	60	ln C = -0.0485t + 4.0721	0.8693	14	47	20	62.1
	100	ln C = -0.0117t + 4.2968	0.9069	60	197	90	65.1
DXC	60	ln C = -0.0280t + 4.1799	0.9068	25	82	30	56.8
	100	ln C = -0.0164t + 4.5895	0.9280	42	140	90	77.1

Antibiotic	C₀ / (µg kg^-1)	Decay equation (C = -kt + C₀)	R²	t_1/2 / day	t₉₀ / day	t_max / day	Degradation^a a Estimated degradation of the antibiotic at tmax. C0: initial concentration; R2: correlation coefficient; t1/2: time calculated for a decay of 50% of the antibiotic, relative to its initial concentration; t90: time calculated for a decay of 90% of the antibiotic, relative to its initial concentration; tmáx: maximum time for which the antibiotic was detected in the samples; SMTZ: sulfamethazine. / %
SMTZ	20	C = -0.2659t + 21.977	0.9392	38	68	45	59.8
	40	C = -0.2928t + 41.247	0.9207	68	123	90	65.9
	60	C = -0.3074t + 60.649	0.9567	98	166	120	61.5

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