Fluorous derivatization combined with liquid chromatography/tandem mass spectrometry: a method for the selective and sensitive determination of sialic acids in biological samples

Abstract

We have developed a novel method for selective and sensitive analysis of sialic acids (N-acetylneuraminic, N-glycolylneuraminic, and 2-keto-3-deoxy-D-glycero-D-galactonononic acid) utilizing liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with a fluorous derivatization technique. In this method, the carboxylic groups in the sialic acids are derivatized via amidation with heptadecafluoroundecylamine, a commercially available perfluoroalkylamine reagent. This reaction proceeds rapidly and readily at room temperature in the presence of a condensation reagent. Subsequently, the derivatives are retained specifically on an LC column with a perfluoroalkyl stationary phase by means of a fluorophilic or 'fluorous' interaction, and detected by positive electrospray ionization MS/MS. The detection limits of the examined sialic acids are in the range of 60-750 amol on column. We show that the proposed method can be used to analyze trace amounts of sialic acids in biological samples.

A new strategy for ionization enhancement by derivatization for mass spectrometry

“…MS is well suited for the detection because of its high sensitivity and selectivity. However, carboxylic acids tend to be poorly ionized by the ionization sources used in MS. To enhance the detection of carboxylic acids in ESI-MS/MS, several chemical derivatization procedures suitable for the positive ion detection have been developed [71][72][73][74][75][76]. For example, Cartwright et al reported that tris(2,4,6-trimethoxyphenyl)phosphonium propylamine (TMPP) bromide was used as the derivatization reagent for the LC-ESI-MS/MS analysis of carboxylic acids in pharmaceutical products [75].…”

Multi-Perfluoroalkyl Derivatization of Polyamines for Selective Liquid Chromatography-Tandem Mass Spectrometric Analysis Utilizing Fluorous Affinity

“…The mono-fluorous derivatives were obtained by derivatization of monoamines [n-butylamine (C 4 ), nhexylamine (C 6 ), and n-octylamine (C 8 )] with STFN using the procedure described for polyamines in section 2.2. On the other hand, to prepare the corresponding non-fluorous dialkyl-to tetraalkylated derivatives, the polyamines were derivatized with n-nonanoic acid (which has the same carbon number as STFN) in the presence of the condensation reagent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methylmorpholinium chloride (DMT-MM) with previously reported procedure [20]. These mono-fluorous and non-fluorous derivatives were analysed by selected ion monitoring mode in the positive ESI mode.…”

“…Utilizing such fluorous affinity, we reported that accurate and precise LCtandem mass spectrometric (MS/MS) analysis of biogenic compounds could be performed following derivatization with a fluorous-tag reagent [20][21][22]. Because the obtained perfluoroalkyl (fluorous) derivatives could be completely separated from non-fluorous species, such as endogenous components in the biological matrix, on the fluorous LC column, this method did not require further sample pretreatment to eliminate matrix-induced effects in MS/MS detection.…”

“…Although the fluorophilicity of mono-fluorous derivatives with STFN may be insufficient, the multi-fluorous polyamine derivatives corresponding to the diperfluoroalkylated to tetraperfluoroalkylated forms could be selectively retained on the fluorous LC column because of their adequate fluorophilicity. Furthermore, the fluorous derivatives could be detected with higher sensitivity using MS/MS owing to an improved ionization response in ESI [20][21][22]. After optimization of the analytical conditions, including the derivatization, the feasibility of the proposed method was demonstrated by analysing human plasma samples deproteinized with ultrafiltration.…”

Development of Analytical Methods Utilizing Selectivity of Fluorous Affinity and Their Applications

“…Many researchers have used stable isotope-labeled internal standards to overcome matrix effects, although the use of such internal standards, especially for deuterized ones, is sometimes limited because of unexpected behavior, such as different recoveries or retention times, during pretreatment or LC separation [13,14]. On the other hand, our developed separation-oriented derivatization method using fluorous affinity is useful for eliminating matrix effects because non-fluorous matrix components are formerly eluted and clearly separated from the fluorous derivatives by fluorous LC [15,16]. We applied this method to the accurate analysis of trace amounts of biogenic primary amines [DA, NE, 3-methoxytyramine (3-MT), normetanephrine (NM), tyramine (Tyr), 5-HT, tryptamine (Tryp), 5-methoxytryptamine (5-MT), and histamine (Hist)] in human plasma samples [17].…”