Sulfonamides in Honey


Effective Detection Using Purospher® HPLC Columns

Detection of sulfonamides 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, MilliporeSigma'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

Sorbent characteristics Purity C18-silica with special modification and deactivation of the surface
Metal content Na, Ca, Mg, Al: 1 ppm; Fe 3 ppm
Particle shape Spherical
Particle size 5 μm
Pore size 90 Å (9 nm)
Pore volume 1.05 mL/g
Spec. surface area 480 m²/g
Carbon load 18.0 % C
pH range pH 2 - 8
Efficiency 80.000 N/m
Shipping eluent Acetonitrile/Water
 
Purospher® HPLC columns

Purospher® HPLC Columns
Excellent separations, symmetrical peaks

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