Optimization of silylation using N-methyl-N-(trimethylsilyl)-trifluoroacetamide, N,O-bis-(trimethylsilyl)-trifluoroacetamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide for the determination of the estrogens estrone and 17α-ethinylestradiol by gas chromatography–mass spectrometry

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Abstract

This paper reports an improved silylation procedure for simultaneous determination of the steroid hormones 17α-ethinylestradiol (EE2) and estrone (E1) using gas chromatography–mass spectrometry (GC–MS). This follows a re-assessment of some of the popular silylation procedures using N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), N-O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA), which lead to the formation of trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) derivatives. Silylation of EE2 using MSTFA or BSTFA + 1% TMCS in ethyl acetate, acetonitrile and dichloromethane solvents produced multiple peaks corresponding to TMS-E1, and 3-mono-TMS-EE2 and/or 3,17-di-TMS-EE2 in variable proportions depending on the solvent used. When pyridine or dimethyl formamide solvents were used in the silylation of EE2 under the same reaction conditions, only 3,17-di-TMS-EE2 derivative was formed. Derivatization using MTBSTFA reagents using ethyl acetate, acetonitrile, dichloromethane, pyridine and dimethyl formamide resulted in almost 100% conversion of mono-TBS-EE2 to the TBS-E1. Therefore, typical methods used in some previous GC–MS determinations of E1 and EE2 in environmental water and/or sediment samples are subject to speculation. However, we can confirm that any of the TMS reagents can be used with either pyridine or dimethyl formamide under suitable reaction conditions.

Introduction

Silylation is the most common and versatile method used to derivatize organic compounds containing active hydrogen atoms (e.g. –OH, double bondNH, –NH2, –SH, –COOH), which results in products with reduced polarity (less dipole–dipole interactions), enhanced volatility and increased thermal and catalytic stability necessary for optimal sensitivity and resolution of various components in mixtures by GC–MS analyses [1]. Another important advantage of silylation in GC–MS analysis is to enhance mass spectrometric properties by producing more favourable diagnostic fragmentation patterns [2].

Estrogenic hormones can be silylated using reagents such as N,O-bis-(trimethylsilyl)-acetamide (BSA), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA), or N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA), which lead to the formation of trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) derivatives, respectively. Catalysts such as trimethyl-chlorosilane (TMCS), together with trimethylsilyl-imidazole (TMSI) or tert-butyldimethylsilyl-chlorosilane (TBCS) and tert-butyldimethylsilyl-imidazole (TBSI), are usually added to enhance derivatization.

BSTFA and its by-products including mono-(trimethylsilyl)trifluoroacetamide and trifluoroacetamide have high volatility which results in little interference with early eluting peaks in chromatograms. While MSTFA has similar reaction properties to other TMS donors, it has particular advantage over BSTFA and other TMS reagents owing to the higher volatility of MSTFA itself and its by-product, N-methyltrifluoroacetamide.

Silylation reagents and the resulting derivatives are generally moisture sensitive, which requires them to be sealed to prevent deactivation by water vapour. TBS derivatives are approximately 104 times more stable to hydrolysis than TMS derivatives [2], [3], [4] in addition to enhanced specific mass spectrometric features. However, TMS reagents can be more effective in derivatizing sterically hindered functional groups. For instance, it has been shown that TBS reagents only react with the phenolic OH group at C3 position of 17α-ethinylestradiol (EE2), and do not react with the tertiary OH group at C17 position on the same molecule [5], [6], [7].

The effectiveness of a derivatization procedure can depend on a number of factors, including reaction conditions such as temperature and time, the nature of the derivatization reagent and the solvent. Strong silylation reagents should be used with caution because attack on unexpected active centers on molecules may give rise to non-uniform products [8]. Although silylation of estrogens can sometimes be carried out without additional solvent, some reagents (e.g. BSTFA) themselves act as solvent, the effectiveness of derivatization can depend on solvent choice. Some of the common solvents used in silylation using TMS or TBS reagents include ethyl acetate [1], [5], [9], acetonitrile [5], [7], [10], [11], pyridine [12], [13], [14], [15], [16] and dimethyl formamide [4], [17], [18]. These solvents are used with excess reagent which facilitates efficiency of the reaction as well as the prevention of the hydrolysis of the products because of exposure to moisture. The solvents used in silylation should be capable of dissolving the sample, or the derivatization products, and should be aprotic and not contain active hydrogen atoms [1].

Reagents and solvents that have been used to prepare TMS derivatives of estrogens include BSTFA with pyridine [15], BSTFA with isooctane/acetone (99/1, v/v) [19], BSTFA + 1% TMCS with pyridine [16], [20], MSTFA with ethyl acetate [21], MSTFA + 1% TMCS with acetonitrile/pyridine (5/1, v/v) [22], BSA with dimethyl formamide [17]. TBS derivatives of estrogens have been prepared using MTBSTFA with ethyl acetate [5], acetonitrile [5], [7], dichloromethane [5] or hexane [5]. Jeannot et al. [19] used BSTFA without additional solvent to derivatize EE2, E1 amongst other estrogens.

In a recent investigation, Thorpe et al. [13] employed a two-stage derivatization procedure for silylation of the estrogens EE2 and 17β-estradiol (E2), by reacting them first with MTBSTFA at 120 °C for 30 min followed by BSTFA at 60 °C for 4 h in pyridine. This study indicated that the phenolic hydroxyl group is derivatized with MTBSTFA, but the sterically hindered hydroxyl group at the 17C position on the D-ring of EE2 molecule reacts only with BSTFA.

We recently reported [6] that EE2 is partially converted to E1 during derivatization using BSTFA and MTBSTFA with ethyl acetate as solvent and suggested that some of the current methods needed re-evaluation. Labadie and Budzinski [23] have developed a new silylation procedure for the determination of steroid hormones using MSTFA with ammonium iodide and mercaptoethanol. They confirmed that there was no breakdown of TMS-EE2 to TMS-E1, but suggested that the conversion of TMS and TBS-EE2 derivatives to the respective E1 derivatives, which we highlighted in our previous report [6], occurred during the derivatization procedure rather than in the gas chromatograph. In this current study, we investigate reaction conditions for the derivatization of EE2 and E1 using popular silylation reagents and solvents.

Section snippets

Chemicals

The following were supplied by Sigma–Aldrich (Australia): E1, EE2 and anthracene (purity >98%); derivatization grade MSTFA, MTBSTFA, TBCS, TBSI; and GC grade anhydrous methanol, acetone, ethyl acetate, acetonitrile, dichloromethane, pyridine and dimethyl formamide. Derivatization grade BSTFA, containing 1% TMCS, and TMSI were supplied by Alltech (Australia).

Reagents and solutions

BSTFA + 1% TMCS + 2% TMSI (v/v/w) was prepared by mixing an appropriate amount of TMSI with BTSFA containing 1% TMCS (v/v). Reagent mixtures of

TMS derivatization at 60 °C

Derivatization of E1 and EE2 with BSTFA and MSTFA was carried out at 60 °C using five different solvents: ethyl acetate, acetonitrile, dichloromethane, pyridine and dimethyl formamide. Fig. 1a displays a typical TIC of TMS-E1, while Fig. 1b shows its mass spectrum and the proposed fragmentation pattern. The TMS-E1 molecular ion, [M]+ m/z 342 was the base peak. Other major ions were m/z 327 [M-15]+ due to the loss of a methyl group from derivative, m/z 257 [M-85]+ and an ion with m/z 218.

The TICs

Breakdown of EE2 to E1

The multiple peaks observed in most TICs of EE2 derivatives indicate that there are several derivatization products under most reaction conditions. The fact that some peaks in the TICs of derivatized EE2 have identical retention times and mass spectra as the corresponding E1 derivative, indicate a tendency for the TMS and TBS derivatives of EE2 to break down to the corresponding E1 derivative. The conversion was near 100% for TBS-EE2, but generally less for TMS-EE2, for which the extent of

Conclusions

A number of questions on current silylation methods used in the determination of the estrogens EE2 and E1 have been answered in this study. Since our initial report of the problem of breakdown of TMS or TBS-EE2 derivatives to the corresponding E1 derivatives, we have discovered that reaction conditions, the choice of reagents and/or solvents all play a role in the effectiveness of the derivatization procedures.

We confirm that MTBSTFA is not suitable to derivatize EE2 in any of the solvents

Acknowledgement

A.S. is the holder of a La Trobe University Postgraduate Research Scholarship.

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