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) 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
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)

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