HPLC-grade acetonitrile and methylene chloride were obtained from EM Science, and dansyl chloride, estriol, estrone sulfate, and ethinylestradiol were obtained from Sigma-Aldrich.
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.
Sample preparation was modified by preliminary ultrafiltration of samples, an increase in the volume of sample used in the dansylation procedure, and an increase in the concentration of dansyl chloride to avoid the production of mono-dansyaated Gal-Hyl.