Fusaric acid as a novel proton-affinitive derivatizing reagent for highly sensitive quantification of hydroxysteroids by LC-ESI-MS/MS

Abstract

A highly sensitive derivatization method for liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry of dehydroepiandrosterone (DHEA), testosterone (T), pregnenolone (P5), and 17␣-OH-pregnenolone (17-OHP5) was developed based on the use of fusaric acid as a reagent. DHEA, P5, and 17-OHP5 were rapidly and quantitatively converted to the 3-fusarate esters by treatment with fusaric acid and 2-methyl-6-nitrobenzoic anhydride. The positive ESI-mass spectra of the fusarate esters of each steroid were dominated by the appearance of [M ϩ H] ϩ as base peaks. The fusarate derivatization of these steroids showed 17.6-fold (DHEA), 11.9-fold (P5), 3.3-fold (17-OHP5), and 1.8-fold (T) higher sensitivity to those of the corresponding picolinate derivatives in LC-selected reaction monitoring. (J Am Soc Mass Spectrom 2010, 21, 249 -253) © 2010 American Society for Mass Spectrometry I n the recent development of analytical methodology for hydroxysteroids that were generally insensitive to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), two types of derivatization designs have been proposed to provide high sensitivity to such kind of steroid molecules. The introduction of permanently charged moieties into steroid molecules was proven to be a promising method [1][2][3][4]. The use of 1-methylpyridinium for hydroxy group [2] and 2-hydrazino-1-methylpyridine for carbonyl group [4] as derivatizing reagents showed higher sensitivity in analyses of testosterone (T), 5␣-dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) by LC-ESI-MS/MS. However, the application of these permanently charged derivatives to steroids possessing more than two hydroxy and/or carbonyl groups in one molecule seemed to be inconvenient and failed to obtain simple ESI-mass spectra with the appearance of protonated molecule ([M ϩ H] ϩ ) due to the formation of multiple ions of [M Ϫ H] -and [M] 2ϩ [4]. The introduction of high proton-affinitive moiety into steroid molecules was the alternative choice to obtain simple ESI mass spectra characterized by the formation of [M ϩ H] ϩ with high intensity [5][6][7][8]. The use of dimethylaminoaphthalene-sulfonate [5,6], pyridine-sulfonate [7], pyridine-dicarboxylate [8] derivatives for steroidal hydroxyl group, and hydroxime [9, 10] and 2-pyridine-hydrazone [11] derivatives for steroidal ketones have been investigated. However, the introduction of sulfonate moieties was limited to the phenolic hydroxy group of estrogens. Derivatization of T and DHT with pyridine-2,3-dicarboxylic anhydride resulted in the formation of the mixture consisted of two regio-isomers of half esters in their chromatograms [8]. Generation of syn-and ant-isomeric peaks might also be inevitable in the analyses of T and DHT using hydroxime and pyridine-hydrazone derivatizations [9,11].Recently, we have demonstrated that the introduction of picolinate moiety into hydroxysteroids provided some advantages to LC-ESI-MS/MS analyses of these compounds in a positive mode, involv...

Derivatization of Secondary Aliphatic Alcohols to Picolinates – A New Option for HPLC Analysis with Chiral Stationary Phase

“…On the other hands, several hydroxy steroids have been changed to picolinates for LC-ESI-MS spectroscopy. [10][11][12] However, these studies have aimed to develop high sensitivity method, which was different from our study.…”

A sensitive approach for simultaneous quantification of carbonyl and hydroxyl steroids using 96-well SPE plates based on stable isotope coded-derivatization-UPLC-MRM: method development and application

“…Papers in the literatures report that reagents for the derivatization of hydroxyl groups were mainly picolinic acid, 18 fusaric acid, 19 2-uoro-1-methylpyridinium p-toluenesulfonate, 20,21 isonicotinoyl azide, 22 and DMBA.…”

A Rapid Method with UPLC for the Determination of Fusaric Acid in Fusarium Strains and Commercial Food and Feed Products

“…Several methods are in use for the detection of FA in agricultural commodities and cell culture, involving highperformance liquid chromatography (HPLC) coupled with UV or ESI-MS/MS [19][20][21]. In addition, GC-MS methods and several qualitative methods such as the use of luminescent bacterium assay have been developed [22].…”

“…In addition, GC-MS methods and several qualitative methods such as the use of luminescent bacterium assay have been developed [22]. However, these methods involving HPLC in FA analysis require at least 20 min for completion [21], and it is also less sensitive than UPLC (requires about 20 ll injection volume while UPLC only needs 1 ll). The GC-MS method has almost never been used since it was reported in 1995, and the major disadvantage is its complex extraction and analysis process [14].…”