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Silylation of acetaminophen by trifluoroacetamide-based silylation agents
Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 433–437
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Silylation of acetaminophen by trifluoroacetamide-based silylation agents Magda Caban ∗ , Piotr Stepnowski Institute for Environmental and Human Health Protection, Faculty of Chemistry, University of Gdansk, ul. Wita Stwosza 63, 80-308 Gda´ nsk, Poland
a r t i c l e
i n f o
Article history: Received 25 January 2018 Received in revised form 15 March 2018 Accepted 16 March 2018 Available online 17 March 2018 Keywords: Acetaminophen Silylation Mass spectra interpretation BSTFA Amide reaction
a b s t r a c t In the presented report, we have described the silylation reaction between the amide group in acetaminophen and a two most popular trifluoroacetamide-based silylation reagents – BSTFA and MSTFA. Both reagents had a amide groups on structures. An investigation was made through the performance of a set of experiments, GC–MS analysis, and a theoretical study, namely interpretation of mass spectra, presentation of the resonance states of all the involved compounds and SN 2 reaction schemes, which was found to be different when BSTFA and MSTFA was applied. The negligible effect of used solvent was also described. The fragmentation of TMS-derivatives (MS spectra) was presented and it has confirmed our previous investigations with silylation of pharmaceuticals, and a general rules of fragmentation patterns. Thanks to this the structure of di-O,O-TMS-acetaminophen was proven. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Silylation, as a type of derivatisation, aims to lower the polar character of the reacting compound. This reaction is performed by the replacement of labile hydrogen in the carboxyl, hydroxyl, amine or amide functional group into a non-polar fragment [1]. In most cases this group is an alkylsilyl substituent, like the trimethylsilyl (TMS) or tert-butyldimethylsilyl group. Silylation has great importance in analytical chemistry, mainly gas chromatography, because of the increased volatility and thermal stability of polar analytes [2,3]. Commercially available silylation reagents have differences in their reaction properties. Some of them are universal, but most react in a specific functional group [4]. The necessary information is easily available in the manufacturers’ folders. The effectiveness of silylation depends on various parameters, like reaction time, temperature, the presence of a catalyst and the solvent used. In our previous studies we investigated and optimized the derivatisation reaction in these silylation reagents for various groups of pharmaceuticals [5–8]. We observed that there is a need for the optimisation of silylation reaction conditions, especially when a mixture of pharmaceuticals is targeted. The N-based active groups in pharmaceuticals are generally less active for silylation compared to hydroxyl and
∗ Corresponding author. E-mail address: [email protected] (M. Caban). https://doi.org/10.1016/j.jpba.2018.03.037 0731-7085/© 2018 Elsevier B.V. All rights reserved.
carboxyl groups. All of already available silylation reagents OH and COOH groups (most silyl reagent, works with and dimethyl(3,3,3-trifluoropropyl)silyldiethylamine, which was developed in our laboratory [7,9]), while only the selected additionally with amine groups, for example BSTFA (N,Obis(trimethylsilyl)trifluoroacetamide) and MSTFA (N-methyl-N(trimethylsilyl)trifluoroacetamide). Thereby, special attention should be taken for acetaminophen derivatisation, while both hydroxyl and amide groups occur in their structure. One of the tested parameters was the solvent for silylation. In most protocols the silylation reagents are used alone, but the appropriate solvent can increase the reaction rate and speed. The role of the addition of a polar aprotic solvent is to increase the solubility of less soluble compounds, but also to increase reaction performance. The reason is that silylation is an SN 2 nucleophilic substitution reaction [10] and an aprotic polar solvent has the ability to stabilize the intermediate state (Fig. S1, Supplementary Information). The solvents of choice for silylation reagents are pyridine, DMSO, DMF, THF, or acetonitrile. It seems that pyridine has a great ability to support silylation. Pyridinium-like compounds, which act as catalytic Lewis bases, are believed to react with silyl chlorides to form silyl pyridinium ion pairs, whose subsequent reaction with the alcohol substrate yields silyl ether products [11]. Suppliers of silylation reagents often suggest the preparation of a mixture of the reagent with pyridinium and with the addition of trimethylchlorosilane (TMCS) as a catalyst. Pyridinium as a solvent for the silylation reaction is important not only for the efficiency of the reaction, but also the stability of the analytes. The ideal example is the case of the
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M. Caban, P. Stepnowski / Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 433–437
Fig. 1. The mass spectra of acetaminophen-mono-O-TMS and acetaminophen-di-O,O-TMS.
silylation of 17␣-ethynylestradiol, which without the addition of pyridine into the sample can convert into estrone, because of the removal of the labile ethinyl group [7,12]. In one of our previous studies we investigated the influence of a solvent among other parameters for silylation of beta-blocker and beta-agonist pharmaceuticals [6]. BSTFA + 1% TMCS was mixed with dichloromethane, ethyl acetate, pyridine, toluene or hexane and the efficiency of the silylation of the target analytes was determined. The results showed that a mixture of the silylation reagent with ethyl acetate gave the highest efficiency, but generally the values were similar. In the present study, we tested the trifluoroacetamide-based reaction agents (BSTFA and MSTFA) for silylation of acetaminophen. These reagents are already proven to be suitable for the acetaminophen derivatisation. Nevertheless, we present a deep study of mass spectra and fragmentation of derivatives to check the differences between those two versatile reagents. 2. Materials and methods 2.1. Chemicals Acetaminophen (paracetamol, N-(4-hydroksyfenylo)acetamide, CAS 103-90-2, M = 151.17 g/mol) was purchased from SigmaAldrich (Germany). The stock solution of acetaminophen (1 mg/mL) was prepared in methanol, as well as a working solution (5 g/mL).
The derivatising reagents, BSTFA with 1% TMCS (trimethylchlorosilane) (hereafter BSTFA +1% TMCS) and MSTFA, were purchased from Sigma-Aldrich. BSTFA +1% TMCS was also purchased from Synthese Nord (Germany). The organic solvents (ethyl acetate, acetonitrile, acetone, methanol) were supplied by POCH (Polskie Odczynniki Chemiczne, Poland).
2.2. Methods The derivatisation protocol was the same for all experiments. Specifically, 100 L of working solution of acetaminophen was introduced into a 2 mL amber glass vial, and the solvent was evaporated in a nitrogen stream. 100 L of derivatisation reagent/mixture was introduced into the vial. After vortex mixing of the sample, the vial was heated for 30 min at 60 ◦ C in a heating block. The obtained samples were analyzed by GC/MS (Shimadzu GCMS-QP2010 SE). An Rtx-5 (30 m × 0.25 mm × 0.25 m; Restek) capillary column was used. The injector temperature was 300 ◦ C. The injection (1 L of the sample) was performed by the autosampler. The temperature program was as follows: starting temperature 120 ◦ C, 1 min in 120 ◦ C, then a rate of 12 ◦ C/min, to reach 270 ◦ C. The helium was kept in a constant pressure mode of 100 kPa. The EI ion source temperature was 200 ◦ C, while the interline temperature was 300 ◦ C. The mass spectrometer recorded the spectra in the m/z range of 45–650 and a speed of 3 scans per second. At least two repetitions
M. Caban, P. Stepnowski / Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 433–437
435
Fig. 2. The fragmentation patterns of mono- (m/z 223, Part A) and di-TMS (m/z 295, Parts B) derivatives of acetaminophen. The charges of the fragment were omitted on purpose to simplify the graph.
Table 1 The relative abundance of the acetaminophen derivatives depending on the used derivatisation mixture. No
Reagent
Acetaminophen-mono-O-TMS
Acetaminophen-di-O,O-TMS
1 2 3 4 5 6 7 8 9
BSTFA + 1%TMCS BSTFA + 1%TMCS/pyridine BSTFA + 1%TMCS/ethyl acetate BSTFA + 1%TMCS/pyridine/ethyl acetate BSTFA + 1%TMCS/acetone BSTFA + 1%TMCS/acetonitrile MSTFA/ethyl acetate Acetonitrile + 1%TMCS Pyridine + 1%TMCS
0 0 0 0 10% 10% 5% traces traces
100% 100% 100% 100% 90% 90% 95% 0 0
of each experiment were performed (with three GC–MS analysis per sample), with deviation of results not higher that 3%.
3. Results and discussion The acetaminophen has two hydrogens which can be potentially substituted by trimethylsilyl (TMS) groups from the silylating reagent, with two probable derivatives, acetaminophen-mono-OTMS and acetaminophen-di-N,O-TMS at first glance. In view of the gas chromatography analysis, the total derivatisation of analytes is recommended, which means that all the labile hydrogens in analyte structures are replaced by TMS groups. This makes the analytes as non-polar as possible, which causes adsorption in the injector or column less probable and increases the resolution of capillary columns. The mono-TMS derivative retention time in tested conditions was 8.56 min, while the di-TMS derivative had a lower retention time of 7.50 min, despite the fact that the first one have greater molecular mass. This is a prove of the stronger interferences of polar compounds with capillary columns. The mass spectra of both acetaminophen derivatives are presented in Fig. 1. The m/z of 73 resulted from the elimination of TMS groups. Molecular ions are present. Acetaminophen-mono-OTMS (M+ ·; = 223 m/z) is substituted in the phenolic group as the more reactive group with the silylation reagent [2]. The structure of acetaminophen-di-TMS (M+ ·; = 295 m/z) needs to be confirmed by mass spectra interpretation, while in the literature two structures were proposed, differentiated by the position of a second TMS group. In one report it was proposed that hydrogen attached to the nitrogen of amide group is replaced by TMS [13–15]. In another report TMS was proposed to be attached to oxygen of the acetamide group [16]. This can be solved by the analysis of fragmentation patterns obtained in a mass spectrum. In Fig. 2 the location of charges were omitted to simplified the graphs and from the reason that the accurate location of
charge is not known. Fig. 2 Part A, presents the fragmentation of acetaminophen-mono-O-TMS. The loss of mass 15 and 73 is very characteristic for TMS derivatives [6]. The loss of mass 15 results in a presence of ions 280 m/z (acetaminophen-di-TMS, Figs. 1 and 2 Part B) and ion 208 m/z (acetaminophen-mono-TMS, Figs. 1 and 2 Part A). What is also noticeable is that the primary fragmentations occur mainly in the beta position to the aromatic ring. This prerequisite is useful to identify the structure of acetaminophen-di-TMS. Fig. S2 (Supplementary Information) presents the structure of the acetaminophen-di-N,O-TMS. To produce an ion with the m/z of 206, the 89 mass needs to be lost. This mass belongs to the fragment −O-Si(CH3 )3 . To lose this fragment the presented structure needs to break the bond in the alpha position to the aromatic ring, but as was mentioned, beta-fragmentation is preferred. This is the reason why in fact Part B of Fig. 2 presents the appropriate structure of acetaminophen-di-O,O-TMS (instead acetaminophen-di-N,O-TMS) and its fragmentation pattern. Table 1 presents the relative abundances of acetaminophenmono-O-TMS and acetaminophen-di-O,O-TMS depending on the utilized reagent and the supplier of the reagent. BSTFA + 1%TMCS alone gives a single di-TMS derivative of acetaminophen. A mixture of BSTFA + 1%TMCS and pyridine gives the same result. The addition of acetone and acetonitrile to the silylation reagent makes derivatisation not complete (in the same specific condition for or mentioned reaction), and the mono-TMS and di-TMS derivatives coexist in the chromatogram with a ratio of areas 1–9, respectively. To check if TMCS has the possibility to react with acetaminophen, mixtures of TMCS with pyridine or acetonitrile were tested, but give only traces of acetaminophen-mono-TMS. Thereby, the previously mentioned catalytic role of pyrydinium for alcohol silylation was not a case of acetaminophen silylation (or change of reaction conditions is needed) [11]. MSTFA gives generally the same results as BSTFA. This was also proven in our previous study, when we performed an optimiza-
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Fig. 3. The resonance of electron and tautomerisation in A. carbonyl groups of acetaminophen, B. BSTFA and C. MSTFA structures.
Fig. 4. The possible reactions between acetaminophen-mono-O-TMS and BSTFA and MSTFA.
tion of silylation using BSTFA and MSTFA at various temperatures and times for selected pharmaceuticals, and acetaminophen among others [5]. Information concerning the acetaminophen-di-TMS structure can be helpful to investigate the mechanism of the silylation of amides. Generally, the amide reaction occurs via an attack on the carbonyl (as nucleophiles), breaking the carbonyl double bond and forming a tetrahedral intermediate. Resonance delocalisation of nitrogen non-bonded electrons into the adjacent carbonyl (C O) group, and amide-iminol tautomerisation [17], increase the nucleophile character of oxygen. Thereby the attack of the silylation reagent (electrophile) in the case of the amide is directed into the carbonyl. The resonance of the non-bonded electrons occurs not only in the acetaminophen structure, but also in the BSTFA and MSTFA (Fig. 3, Parts A–C respectively). In a case of acetaminophen we omitted the further delocalization to aromatic ring and hydroxyl group. The mentioned delocalization will now be discussed as something which could have an impact on acetaminophen silylation via the SN 2 reaction. In the silylation reaction the nucleophile is the analyte compound (specific atom or groups of atoms), while the electrophile is the silylation reagent. Oxygen in the acetaminophen structure has properties of the nucleophile. MSTFA has one TMS group connected to nitrogen with an electrophile character, created by the fact that there is a resonance between this nitrogen and the oxygen in the amide group (Fig. 3, Part C). In the case of BSTFA,
the nitrogen atom has no opportunity to be electrophile (Fig. 3, Part B), thereby from the two obtained TMS groups, only the one which is connected to oxygen is reactive in silylation. What is more, there is a tautomerisation opportunity for BSTFA, but N,Nbis(trimethylsilyl)trifluoroacetamide is much less abundant [18]. This makes BSTFA the only silylating reagent, presented already in 1968 [19], from the popular ones which is a donor of a TMS group from the side of oxygen, not nitrogen. The possible reactions between acetaminophen-mono-O-TMS and BSTFA and MSTFA are presented in Fig. 4, Parts A and B, respectively. The intermediate state in the first case has an O(␦+). . . Si. . . O(␦-) fragment, while the second reaction has an O(␦+). . . Si. . . N(␦-) fragment. Of course, the TMCS is presented in this reaction, which can be also active in reaction. The experiments with TMCS with two solvents (pyridine, acetonitrile) shows no reactivity of this silyl compound. 4. Conclusions Although the reaction between BSTFA and MSTFA occur by the different scheme, the effect was noticed to be the same, namely conversion of acetaminophen into di-O,O-TMS-derivative. The trifluoroacetamide-based silylation reagent are powerful chemicals for silylation of polar compound which possess both hydroxyl and amides groups, what was also proven for levetiracetam and lamotrigine [20]. Nevertheless, we proven here that the TMS group is transferred into oxygen atom of amide group, neither to nitrogen
M. Caban, P. Stepnowski / Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 433–437
atom. Actually, we present the reaction between two amides, while one of them is more nucleophile, second more electrophile. This study can be helpful to confirm the structure of TMS derivatives in a future study of derivatisation via silylation of compounds reach in polar moieties (for example the metabolites and transformation products). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jpba.2018.03.037. References [1] D.R. Knapp, Handbook of Analytical Derivatization Reactions, John Wiley, New York, 1979. [2] N.D. Danielson, P.A. Gallagher, J.J. Bao, Chemical reagents and derivatization procedures in drug analysis, Encycl. Anal. Chem. (2008) 7042–7076. [3] R.A. Meyers, Chemical reagents and derivatization procedures in drug analysis, in: Encycl Anal. Chem. Appl. Theory Instrum, John Wiley and Sons, Tarzana, CA, USA, 2011. [4] N.G. Todua, A.I. Mikaia, Mass spectrometry of analytical derivatives. 2. Ortho and para effects in electron ionization mass spectra of derivatives of hydroxyl, mercapto and amino benzoic acids, Mass Spektrom. 13 (2016) 83–94. [5] N. Migowska, M. Caban, P. Stepnowski, J. Kumirska, Simultaneous analysis of non-steroidal anti-inflammatory drugs and estrogenic hormones in water and wastewater samples using gas chromatography-mass spectrometry and gas chromatography with electron capture detection, Sci. Total Environ. 441 (2012) 77–88. [6] M. Caban, P. Stepnowski, M. Kwiatkowski, N. Migowska, J. Kumirska, Determination of -blockers and -agonists using gas chromatography and gas chromatography-mass spectrometry–a comparative study of the derivatization step, J. Chromatogr. A 1218 (2011) 8110–8122. [7] M. Caban, M. Czerwicka, P. Łukaszewicz, N. Migowska, P. Stepnowski, M. Kwiatkowski, et al., A new silylation reagent dimethyl(3,3,3-trifluoropropyl)silyldiethylamine for the analysis of estrogenic compounds by gas chromatography-mass spectrometry, J. Chromatogr. A 1301 (2013) 215–224. [8] J. Kumirska, A. Plenis, P. Łukaszewicz, M. Caban, N. Migowska, A. ´ et al., Chemometric optimization of derivatization reactions Białk-Bielinska, prior to gas chromatography-mass spectrometry analysis, J. Chromatogr. A 1296 (2013) 164–178.
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[9] M. Caban, K. Mioduszewska, P. Łukaszewicz, N. Migowska, P. Stepnowski, M. Kwiatkowski, et al., A new silylating reagent—dimethyl(3,3,3-trifluoropropyl)silyldiethylamine—for the derivatisation of non-steroidal anti-inflammatory drugs prior to gas chromatography-mass spectrometry analysis, J. Chromatogr. A 1346 (2014) 107–116. [10] M.V. Kashutina, S.L. Ioffe, V.A. Tartakovskii, Silylation of Organic Compounds Silylation of Organic Compounds, 733 (1975). [11] P. Patschinski, C. Zhang, H. Zipse, The lewis base-catalyzed silylation of alcohols—a mechanistic analysis, J. Org. Chem. 79 (2014) 8348–8357. [12] A. Shareef, M.J. Angove, J.D. Wells, 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 17alpha-ethinylestra, J. Chromatogr. A 1108 (2006) 121–128. ˛ ˛ ˛ ´ Comparison of the efficiency [13] S. Kurkiewicz, A. Dzierz˙ ega-L ecznar, K. Stepie n, of silylating agents in GC/MS analysis of non-steroidal anti-inflammatory ˛ Nauk. 7 (2010) 28–32. drugs, Farmakol. Przeglad [14] J.F. Klebe, H. Finkbeiner, D.M. White, Silylations with bis(trimethylsilyl) acetamide, a highly reactive silyl donor, J.Am. Chem. Soc. 339 (1966) 3390–3395. [15] I. Dalmázio, T.M.A. Alves, R. Augusti, An appraisal on the degradation of paracetamol by TiO 2/UV system in aqueous medium. Product identification by gas chromatography-mass spectrometry (GC–MS), J. Braz. Chem. Soc. 19 (2008) 81–88. [16] K. Hoffmann, T.A. Baillie, The use of alkoxycarbonyl derivatives for the mass spectral analysis of drug-thioether metabolites. Studies with the cysteine, mercapturic acid and glutathione conjugates of acetaminophen, Biomed. Environ. Mass Spectrom. 15 (1988) 637–647. [17] D.P. Fairlie, T.C. Woon, A.C. Willis, Amide-iminol tautomerism effect of metalation, Inorg. Chem. 572 (1994) 6425–6428. [18] H. Adolfsson, D.J. Ager, J.M. Aizpurua, T. Aoyama, K.M. Baines, Science of Synthesis: Houben-Weyl Methods of Molecular Transformations, vol. 4, Thieme, 2014. [19] D.L. Stalling, C.W. Gehrke, R.W. Zumwalt, A new silylation reagent for amino acids bis(trimethylsilyl)trifluoroacetamide (BSTFA), Biochem. Biophys. Res. Commun. 3 (1968) 278–283. [20] P. Nikolaou, I. Papoutsis, A. Dona, C. Spiliopoulou, S. Athanaselis, Development and validation of a GC/MS method for the simultaneous determination of levetiracetam and lamotrigine in whole blood, J. Pharm. Biomed. Anal. 102 (2014) 25–32.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Silylation of acetaminophen by trifluoroacetamide-based silylation agents Magda Caban ∗ , Piotr Stepnowski Institute for Environmental and Human Health Protection, Faculty of Chemistry, University of Gdansk, ul. Wita Stwosza 63, 80-308 Gda´ nsk, Poland
a r t i c l e
i n f o
Article history: Received 25 January 2018 Received in revised form 15 March 2018 Accepted 16 March 2018 Available online 17 March 2018 Keywords: Acetaminophen Silylation Mass spectra interpretation BSTFA Amide reaction
a b s t r a c t In the presented report, we have described the silylation reaction between the amide group in acetaminophen and a two most popular trifluoroacetamide-based silylation reagents – BSTFA and MSTFA. Both reagents had a amide groups on structures. An investigation was made through the performance of a set of experiments, GC–MS analysis, and a theoretical study, namely interpretation of mass spectra, presentation of the resonance states of all the involved compounds and SN 2 reaction schemes, which was found to be different when BSTFA and MSTFA was applied. The negligible effect of used solvent was also described. The fragmentation of TMS-derivatives (MS spectra) was presented and it has confirmed our previous investigations with silylation of pharmaceuticals, and a general rules of fragmentation patterns. Thanks to this the structure of di-O,O-TMS-acetaminophen was proven. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Silylation, as a type of derivatisation, aims to lower the polar character of the reacting compound. This reaction is performed by the replacement of labile hydrogen in the carboxyl, hydroxyl, amine or amide functional group into a non-polar fragment [1]. In most cases this group is an alkylsilyl substituent, like the trimethylsilyl (TMS) or tert-butyldimethylsilyl group. Silylation has great importance in analytical chemistry, mainly gas chromatography, because of the increased volatility and thermal stability of polar analytes [2,3]. Commercially available silylation reagents have differences in their reaction properties. Some of them are universal, but most react in a specific functional group [4]. The necessary information is easily available in the manufacturers’ folders. The effectiveness of silylation depends on various parameters, like reaction time, temperature, the presence of a catalyst and the solvent used. In our previous studies we investigated and optimized the derivatisation reaction in these silylation reagents for various groups of pharmaceuticals [5–8]. We observed that there is a need for the optimisation of silylation reaction conditions, especially when a mixture of pharmaceuticals is targeted. The N-based active groups in pharmaceuticals are generally less active for silylation compared to hydroxyl and
∗ Corresponding author. E-mail address: [email protected] (M. Caban). https://doi.org/10.1016/j.jpba.2018.03.037 0731-7085/© 2018 Elsevier B.V. All rights reserved.
carboxyl groups. All of already available silylation reagents OH and COOH groups (most silyl reagent, works with and dimethyl(3,3,3-trifluoropropyl)silyldiethylamine, which was developed in our laboratory [7,9]), while only the selected additionally with amine groups, for example BSTFA (N,Obis(trimethylsilyl)trifluoroacetamide) and MSTFA (N-methyl-N(trimethylsilyl)trifluoroacetamide). Thereby, special attention should be taken for acetaminophen derivatisation, while both hydroxyl and amide groups occur in their structure. One of the tested parameters was the solvent for silylation. In most protocols the silylation reagents are used alone, but the appropriate solvent can increase the reaction rate and speed. The role of the addition of a polar aprotic solvent is to increase the solubility of less soluble compounds, but also to increase reaction performance. The reason is that silylation is an SN 2 nucleophilic substitution reaction [10] and an aprotic polar solvent has the ability to stabilize the intermediate state (Fig. S1, Supplementary Information). The solvents of choice for silylation reagents are pyridine, DMSO, DMF, THF, or acetonitrile. It seems that pyridine has a great ability to support silylation. Pyridinium-like compounds, which act as catalytic Lewis bases, are believed to react with silyl chlorides to form silyl pyridinium ion pairs, whose subsequent reaction with the alcohol substrate yields silyl ether products [11]. Suppliers of silylation reagents often suggest the preparation of a mixture of the reagent with pyridinium and with the addition of trimethylchlorosilane (TMCS) as a catalyst. Pyridinium as a solvent for the silylation reaction is important not only for the efficiency of the reaction, but also the stability of the analytes. The ideal example is the case of the
434
M. Caban, P. Stepnowski / Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 433–437
Fig. 1. The mass spectra of acetaminophen-mono-O-TMS and acetaminophen-di-O,O-TMS.
silylation of 17␣-ethynylestradiol, which without the addition of pyridine into the sample can convert into estrone, because of the removal of the labile ethinyl group [7,12]. In one of our previous studies we investigated the influence of a solvent among other parameters for silylation of beta-blocker and beta-agonist pharmaceuticals [6]. BSTFA + 1% TMCS was mixed with dichloromethane, ethyl acetate, pyridine, toluene or hexane and the efficiency of the silylation of the target analytes was determined. The results showed that a mixture of the silylation reagent with ethyl acetate gave the highest efficiency, but generally the values were similar. In the present study, we tested the trifluoroacetamide-based reaction agents (BSTFA and MSTFA) for silylation of acetaminophen. These reagents are already proven to be suitable for the acetaminophen derivatisation. Nevertheless, we present a deep study of mass spectra and fragmentation of derivatives to check the differences between those two versatile reagents. 2. Materials and methods 2.1. Chemicals Acetaminophen (paracetamol, N-(4-hydroksyfenylo)acetamide, CAS 103-90-2, M = 151.17 g/mol) was purchased from SigmaAldrich (Germany). The stock solution of acetaminophen (1 mg/mL) was prepared in methanol, as well as a working solution (5 g/mL).
The derivatising reagents, BSTFA with 1% TMCS (trimethylchlorosilane) (hereafter BSTFA +1% TMCS) and MSTFA, were purchased from Sigma-Aldrich. BSTFA +1% TMCS was also purchased from Synthese Nord (Germany). The organic solvents (ethyl acetate, acetonitrile, acetone, methanol) were supplied by POCH (Polskie Odczynniki Chemiczne, Poland).
2.2. Methods The derivatisation protocol was the same for all experiments. Specifically, 100 L of working solution of acetaminophen was introduced into a 2 mL amber glass vial, and the solvent was evaporated in a nitrogen stream. 100 L of derivatisation reagent/mixture was introduced into the vial. After vortex mixing of the sample, the vial was heated for 30 min at 60 ◦ C in a heating block. The obtained samples were analyzed by GC/MS (Shimadzu GCMS-QP2010 SE). An Rtx-5 (30 m × 0.25 mm × 0.25 m; Restek) capillary column was used. The injector temperature was 300 ◦ C. The injection (1 L of the sample) was performed by the autosampler. The temperature program was as follows: starting temperature 120 ◦ C, 1 min in 120 ◦ C, then a rate of 12 ◦ C/min, to reach 270 ◦ C. The helium was kept in a constant pressure mode of 100 kPa. The EI ion source temperature was 200 ◦ C, while the interline temperature was 300 ◦ C. The mass spectrometer recorded the spectra in the m/z range of 45–650 and a speed of 3 scans per second. At least two repetitions
M. Caban, P. Stepnowski / Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 433–437
435
Fig. 2. The fragmentation patterns of mono- (m/z 223, Part A) and di-TMS (m/z 295, Parts B) derivatives of acetaminophen. The charges of the fragment were omitted on purpose to simplify the graph.
Table 1 The relative abundance of the acetaminophen derivatives depending on the used derivatisation mixture. No
Reagent
Acetaminophen-mono-O-TMS
Acetaminophen-di-O,O-TMS
1 2 3 4 5 6 7 8 9
BSTFA + 1%TMCS BSTFA + 1%TMCS/pyridine BSTFA + 1%TMCS/ethyl acetate BSTFA + 1%TMCS/pyridine/ethyl acetate BSTFA + 1%TMCS/acetone BSTFA + 1%TMCS/acetonitrile MSTFA/ethyl acetate Acetonitrile + 1%TMCS Pyridine + 1%TMCS
0 0 0 0 10% 10% 5% traces traces
100% 100% 100% 100% 90% 90% 95% 0 0
of each experiment were performed (with three GC–MS analysis per sample), with deviation of results not higher that 3%.
3. Results and discussion The acetaminophen has two hydrogens which can be potentially substituted by trimethylsilyl (TMS) groups from the silylating reagent, with two probable derivatives, acetaminophen-mono-OTMS and acetaminophen-di-N,O-TMS at first glance. In view of the gas chromatography analysis, the total derivatisation of analytes is recommended, which means that all the labile hydrogens in analyte structures are replaced by TMS groups. This makes the analytes as non-polar as possible, which causes adsorption in the injector or column less probable and increases the resolution of capillary columns. The mono-TMS derivative retention time in tested conditions was 8.56 min, while the di-TMS derivative had a lower retention time of 7.50 min, despite the fact that the first one have greater molecular mass. This is a prove of the stronger interferences of polar compounds with capillary columns. The mass spectra of both acetaminophen derivatives are presented in Fig. 1. The m/z of 73 resulted from the elimination of TMS groups. Molecular ions are present. Acetaminophen-mono-OTMS (M+ ·; = 223 m/z) is substituted in the phenolic group as the more reactive group with the silylation reagent [2]. The structure of acetaminophen-di-TMS (M+ ·; = 295 m/z) needs to be confirmed by mass spectra interpretation, while in the literature two structures were proposed, differentiated by the position of a second TMS group. In one report it was proposed that hydrogen attached to the nitrogen of amide group is replaced by TMS [13–15]. In another report TMS was proposed to be attached to oxygen of the acetamide group [16]. This can be solved by the analysis of fragmentation patterns obtained in a mass spectrum. In Fig. 2 the location of charges were omitted to simplified the graphs and from the reason that the accurate location of
charge is not known. Fig. 2 Part A, presents the fragmentation of acetaminophen-mono-O-TMS. The loss of mass 15 and 73 is very characteristic for TMS derivatives [6]. The loss of mass 15 results in a presence of ions 280 m/z (acetaminophen-di-TMS, Figs. 1 and 2 Part B) and ion 208 m/z (acetaminophen-mono-TMS, Figs. 1 and 2 Part A). What is also noticeable is that the primary fragmentations occur mainly in the beta position to the aromatic ring. This prerequisite is useful to identify the structure of acetaminophen-di-TMS. Fig. S2 (Supplementary Information) presents the structure of the acetaminophen-di-N,O-TMS. To produce an ion with the m/z of 206, the 89 mass needs to be lost. This mass belongs to the fragment −O-Si(CH3 )3 . To lose this fragment the presented structure needs to break the bond in the alpha position to the aromatic ring, but as was mentioned, beta-fragmentation is preferred. This is the reason why in fact Part B of Fig. 2 presents the appropriate structure of acetaminophen-di-O,O-TMS (instead acetaminophen-di-N,O-TMS) and its fragmentation pattern. Table 1 presents the relative abundances of acetaminophenmono-O-TMS and acetaminophen-di-O,O-TMS depending on the utilized reagent and the supplier of the reagent. BSTFA + 1%TMCS alone gives a single di-TMS derivative of acetaminophen. A mixture of BSTFA + 1%TMCS and pyridine gives the same result. The addition of acetone and acetonitrile to the silylation reagent makes derivatisation not complete (in the same specific condition for or mentioned reaction), and the mono-TMS and di-TMS derivatives coexist in the chromatogram with a ratio of areas 1–9, respectively. To check if TMCS has the possibility to react with acetaminophen, mixtures of TMCS with pyridine or acetonitrile were tested, but give only traces of acetaminophen-mono-TMS. Thereby, the previously mentioned catalytic role of pyrydinium for alcohol silylation was not a case of acetaminophen silylation (or change of reaction conditions is needed) [11]. MSTFA gives generally the same results as BSTFA. This was also proven in our previous study, when we performed an optimiza-
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Fig. 3. The resonance of electron and tautomerisation in A. carbonyl groups of acetaminophen, B. BSTFA and C. MSTFA structures.
Fig. 4. The possible reactions between acetaminophen-mono-O-TMS and BSTFA and MSTFA.
tion of silylation using BSTFA and MSTFA at various temperatures and times for selected pharmaceuticals, and acetaminophen among others [5]. Information concerning the acetaminophen-di-TMS structure can be helpful to investigate the mechanism of the silylation of amides. Generally, the amide reaction occurs via an attack on the carbonyl (as nucleophiles), breaking the carbonyl double bond and forming a tetrahedral intermediate. Resonance delocalisation of nitrogen non-bonded electrons into the adjacent carbonyl (C O) group, and amide-iminol tautomerisation [17], increase the nucleophile character of oxygen. Thereby the attack of the silylation reagent (electrophile) in the case of the amide is directed into the carbonyl. The resonance of the non-bonded electrons occurs not only in the acetaminophen structure, but also in the BSTFA and MSTFA (Fig. 3, Parts A–C respectively). In a case of acetaminophen we omitted the further delocalization to aromatic ring and hydroxyl group. The mentioned delocalization will now be discussed as something which could have an impact on acetaminophen silylation via the SN 2 reaction. In the silylation reaction the nucleophile is the analyte compound (specific atom or groups of atoms), while the electrophile is the silylation reagent. Oxygen in the acetaminophen structure has properties of the nucleophile. MSTFA has one TMS group connected to nitrogen with an electrophile character, created by the fact that there is a resonance between this nitrogen and the oxygen in the amide group (Fig. 3, Part C). In the case of BSTFA,
the nitrogen atom has no opportunity to be electrophile (Fig. 3, Part B), thereby from the two obtained TMS groups, only the one which is connected to oxygen is reactive in silylation. What is more, there is a tautomerisation opportunity for BSTFA, but N,Nbis(trimethylsilyl)trifluoroacetamide is much less abundant [18]. This makes BSTFA the only silylating reagent, presented already in 1968 [19], from the popular ones which is a donor of a TMS group from the side of oxygen, not nitrogen. The possible reactions between acetaminophen-mono-O-TMS and BSTFA and MSTFA are presented in Fig. 4, Parts A and B, respectively. The intermediate state in the first case has an O(␦+). . . Si. . . O(␦-) fragment, while the second reaction has an O(␦+). . . Si. . . N(␦-) fragment. Of course, the TMCS is presented in this reaction, which can be also active in reaction. The experiments with TMCS with two solvents (pyridine, acetonitrile) shows no reactivity of this silyl compound. 4. Conclusions Although the reaction between BSTFA and MSTFA occur by the different scheme, the effect was noticed to be the same, namely conversion of acetaminophen into di-O,O-TMS-derivative. The trifluoroacetamide-based silylation reagent are powerful chemicals for silylation of polar compound which possess both hydroxyl and amides groups, what was also proven for levetiracetam and lamotrigine [20]. Nevertheless, we proven here that the TMS group is transferred into oxygen atom of amide group, neither to nitrogen
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atom. Actually, we present the reaction between two amides, while one of them is more nucleophile, second more electrophile. This study can be helpful to confirm the structure of TMS derivatives in a future study of derivatisation via silylation of compounds reach in polar moieties (for example the metabolites and transformation products). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jpba.2018.03.037. References [1] D.R. Knapp, Handbook of Analytical Derivatization Reactions, John Wiley, New York, 1979. [2] N.D. Danielson, P.A. Gallagher, J.J. Bao, Chemical reagents and derivatization procedures in drug analysis, Encycl. Anal. Chem. (2008) 7042–7076. [3] R.A. Meyers, Chemical reagents and derivatization procedures in drug analysis, in: Encycl Anal. Chem. Appl. Theory Instrum, John Wiley and Sons, Tarzana, CA, USA, 2011. [4] N.G. Todua, A.I. Mikaia, Mass spectrometry of analytical derivatives. 2. Ortho and para effects in electron ionization mass spectra of derivatives of hydroxyl, mercapto and amino benzoic acids, Mass Spektrom. 13 (2016) 83–94. [5] N. Migowska, M. Caban, P. Stepnowski, J. Kumirska, Simultaneous analysis of non-steroidal anti-inflammatory drugs and estrogenic hormones in water and wastewater samples using gas chromatography-mass spectrometry and gas chromatography with electron capture detection, Sci. Total Environ. 441 (2012) 77–88. [6] M. Caban, P. Stepnowski, M. Kwiatkowski, N. Migowska, J. Kumirska, Determination of -blockers and -agonists using gas chromatography and gas chromatography-mass spectrometry–a comparative study of the derivatization step, J. Chromatogr. A 1218 (2011) 8110–8122. [7] M. Caban, M. Czerwicka, P. Łukaszewicz, N. Migowska, P. Stepnowski, M. Kwiatkowski, et al., A new silylation reagent dimethyl(3,3,3-trifluoropropyl)silyldiethylamine for the analysis of estrogenic compounds by gas chromatography-mass spectrometry, J. Chromatogr. A 1301 (2013) 215–224. [8] J. Kumirska, A. Plenis, P. Łukaszewicz, M. Caban, N. Migowska, A. ´ et al., Chemometric optimization of derivatization reactions Białk-Bielinska, prior to gas chromatography-mass spectrometry analysis, J. Chromatogr. A 1296 (2013) 164–178.
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