Sulfonamides in Honey

Effective Detection Using Purospher® HPLC Columns

Sulfonamide antibiotics are commonly used in veterinary medicine such as for the prevention and treatment of bacterial diseases in bees. However, this can lead to sulfonamide residues in honey. This is of major concern because of possible allergic reactions in sensitive or sensitized people. Moreover, it can contribute to the development of antibiotic-resistant pathogenic bacteria. Due to this risk and the status of honey as a pure and natural product, the European Union forbids the use of sulfonamide antibiotics in agriculture. The limit of quantitation (LOQ) ranges between 20 and 50 ng/g, depending on the country. For effective detection of sulfonamides, Merck's Purospher® HPLC columns offer highly reliable results, with excellent separations and outstanding peak symmetry.

Application Example

Separation of sulfonamides in honey

Column	Purospher® STAR RP-18 endcapped (5μm) LiChroCART® 150-4,6 HPLC column
Precolumn	Purospher® STAR RP-18 endcapped (5μm) LiChroCART® 4-4
Oven temperature	45°C
Injection volume	50 μL
Flow Rate	0.7 mL /min
Mobile Phase A	0.020 M Acetate buffer pH 4.75 MeCN (98:2, v/v)
Mobile Phase B	0.020 M Acetate buffer pH 4.75 MeCN (68:32, v/v)
Detection	Fluorescence
Wavelength	EX 420 / EM 485
Sample Rate	200 ms
Time Constant	1 sec
Sample	Honey sample spiked with 20 ppb for all sulfonamides + PABA (3) and internal standard (9)
Peaks	1. sulfaguanidine, 2. sulfanilamide, (internal standard) 3. p-aminobenzoic acid 4. sulfacetamide 5. sulfadiazine 6. sulfathiazole 7. sulfapyridine 8. sulfamerazine 9. sulfamethizole 10. sulfamether 11. sulfamethazine 12. sulfamethoxypyridazine 13. sulfachloropyridazine 14. sulfadoxine

Gradient
Time (min)	Eluent A	Eluent B	Flow Rate (mL/min)
0.0	98	2	0.7
31	65	35	0.7
41	25	75	0.7
41.1	5	95	0.7
48	5	95	0.7
48.1	98	2	0.7
60.9	8	2	0.7

Post-column derivatization
The post-column reagent, a mixture of fluorescamine: 2-mercapto-ethanol : acetonitrile : phosphate buffer 0.021 M (0.276% sodium dihydrogen phosphate monohydrate in water, w/v, adjusted to pH 3.0 with ortho-phosphoric acid 85 %) (0.025 : 0.2 : 25 : 75, w/v/v/v) was stored in the dark at 4°C
Coil:	10 m x 0.25 mm i.d. thermostated within the column oven
Reaction pump flow:	0.2 mL /min

Specification of Purospher® RP-18 endcapped