Derivatization with dansyl chloride, as described below, was highly effective and was therefore used for all studies described for the LC-MS/MS method (31).
To derivatize the samples, we then added an equal volume of 1 g/L dansyl chloride in acetone, vortex-mixed each sample for 1 min, and incubated the samples in a heating block at 60[degrees]C for 3 min.
Derivatization with dansyl chloride overcame this problem without substantially increasing the complexity or time requirements of preanalytical sample preparation.
In summary, we have developed a highly sensitive method for the simultaneous measurement of E1 and E2 in human serum by LC-MS/MS after derivatization with dansyl chloride.
Gal-Hyl can be measured using an amino acid analyzer (12), but simpler methods have been developed for measurement of urinary Gal-Hyl, using HPLC after derivatization with dansyl chloride (13-16).
We used HPLC-grade acetonitrile (Rathburn Chemical), glass-distilled acetone (Rathburn), HPLC-grade isopropanol (Fluka), glacial acetic acid (BDH), anhydrous sodium acetate, 5-dimethylaminonaphthalene-l-sulfonyl chloride, dansyl chloride, sodium carbonate, and L-lysine (Sigma Chemical).
5 mol/L sodium carbonate solution in distilled water; 20 g/L dansyl chloride in acetone; 0.
7-mL polypropylene tubes, to which 50 [micro]L of sodium carbonate solution and 50 [micro]L of dansyl chloride solution were then added.