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,N-diisopropylethylamine 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.

Figure 1

Reaction scheme for dansylation of unactivated alcohols.

Figure 2

Structures of the lipid compounds and internal standard.

Figure 3

LC-MS analysis of dansylated alcohols (5 μM each lipid compound and 0.1 μM internal standard, 10-μL injection) using MRM in the ESI positive mode. The t_R values (circled with broken ovals) indicate the dansylated derivatives of the selected compounds: 6β-OH testosterone (−2), deoxycholic acid (−2), and hydrocortisone (−3) indicate that more than one hydroxy group was dansylated, i.e. the di-, di-, and tri-dansylated derivatives, respectively.

Figure 4

Stability of the dansylated derivatives. Dansylation incubations were carried out by adding 5 nmol of each lipid compound and 0.1 nmol of internal standard. The resulting dansylation mixture (10 μL) was injected and analyzed by LC-MS/MS in the ESI positive mode. For the compounds containing more than one hydroxy group, the stability of the mono-dansylated derivative was measured.

Figure 5

DoGEX results of the reaction mixture of P450 7A1 and human liver extract after dansylation. An isotopic ratio range of 0.95-1.9 was selected in the automated search for the m/z 636 and 638 doublet at t_R 9.3 min, corresponding to the mono-dansylated 7α-OH cholesterol, with the green band corresponding to a doublet with this t_R.

Figure 6

Standard curves for LC-MS analysis of dansylated testosterone with (■) and without (●) human liver extract using MRM in the ESI positive mode. Different amounts of testosterone (ranging from 0.1 nmol to 100 nmol, with 0.1 nmol of internal standard) were added for the dansylation incubations, respectively, and 10 μL of each resulting dansylation mixture was injected.