Dansylation of Unactivated Alcohols for Improved Mass Spectral Sensitivity and Application to Analysis of Cytochrome P450 Oxidation Products in Tissue Extracts

Abstract

Chemical derivatization is useful for improving the ionization characteristics of poorly or nonionizable analytes in liquid chromatography-mass spectrometry (LC-MS). Dansyl chloride has been widely used as a derivatizing reagent for fluorescence detection and for facilitating the MS detection of phenols and amines, but not for general alcohols. A new dansylation method for improving the mass spectral sensitivity of unactivated alcohols was developed. The dansylated derivative was formed after incubation of the test compound cholesterol and excess dansyl chloride in CH 2 Cl 2 in the presence of 4-(dimethylamino)-pyridine (DMAP) plus N,Ndiisopropylethylamine at 65 °C for 1 h, with an overall yield of 96%. The versatility of dansylation was investigated by utilizing representative lipid compounds (containing different numbers of hydroxy groups) for dansylation. All dansylated derivatives of the selected compounds were detected by LC-MS/MS in the electrospray ionization (ESI) positive ion mode. Validation of the method was established in terms of the sensitivity, stability, and repeatability of dansylation. The method was then applied to characterizing the P450 7A1 oxidation product (dansylated 7α-hydroxycholesterol) in human liver extracts using an LC-MS metabolomics/isotopic labeling approach (Tang, Z.; Guengerich, F. P. Anal. Chem. 2009, 81, 3071-3078). The dansylated derivative of the product was identified, with the signal increased by 10 3 -fold compared with a previous method (derivatization with succinic anhydride and ESI negative ion MS). Quantitation of testosterone in human liver extracts was also done as an example of the application of the dansylation method. Thus, dansylation is a potential method of modifying many alcohols for detection by fluorescence and LC-MS analysis.

Inhibition of Human Cytochrome P450 3A4 by Cholesterol

“…Following centrifugation (2000 ϫ g, 5 min), 1.4 ml of the organic layer was transferred and taken to dryness under an N 2 stream. A general method for derivatization with dansyl chloride was used with slight modification (32). Briefly, the samples were dissolved in 200 l of CH 2 Cl 2 containing 2 mg of dansyl chloride, 0.5 mg of 4,4-dimethylaminopyridine, and 2 l of triethylamine and incubated at 65°C for 1 h. The samples were dried under an N 2 stream and then dissolved in 100 l of CH 3 CN for analysis.…”

Oxidation of Dihydrotestosterone by Human Cytochromes P450 19A1 and 3A4

“…Dansylation-The dansylation reactions were performed as previously described (34). Briefly, samples were dissolved in 200 l of CH 2 Cl 2 containing 2 mg of dansyl chloride, 1 mg of 4,4-dimethylaminopyridine, and 20 l of triethylamine and incubated at 65°C for 1 h. The samples were dried under an N 2 stream and then dissolved in 50 l of CH 3 CN for analysis.…”

“…Concentrations of the oxidation product 19-hydroxydihydrotestosterone could be quantitated, based on the synthetic material and fragmentation of the dansyl group (34), and the values of multiple analyses with several samples of these products were ϳ40-fold less than dihydrotestosterone (determined as 2,4-DNPH derivative) in both plasma and urine ( Table 1). The range of concentrations was expected to be variable due to variations in individual urinary volumes; the concentrations varied 2-fold.…”

Derivatization in liquid chromatography for mass spectrometric detection

“…Dns-Cl reacted these compounds at 60ºC for 1 h in the presence of 4-(dimethylamino)-pyridine and N,Ndiisopropylethylamine. The generated derivatives were sensitively analyzed by LC/ESI-MS/MS (11). DnsCl is widely used for the analysis of the compounds having hydroxyl group (10)(11)(12)(13)(14)(15)(16)(17)(18)(19).…”

“…The generated derivatives were sensitively analyzed by LC/ESI-MS/MS (11). DnsCl is widely used for the analysis of the compounds having hydroxyl group (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Similarly, picolinic acid (20-26), fusaric acid (27), dimethylglycine (28), isonicotinoyl azide (29,30), and N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS) (31) were used for the derivatization of alcohols and phenols.…”