Hexamethyldisilazane as an Acylation Generator for

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Hexamethyldisilazane as an Acylation Generator for Perfluorocarboxylic Acids in Quantitative Derivatization of Primary Phenylalkyl Amines Confirmed by GC-MS and Computations Borbála Molnár, Antal Csámpai, and Ibolya Molnár-Perl Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503786j • Publication Date (Web): 10 Dec 2014 Downloaded from http://pubs.acs.org on December 23, 2014

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Analytical Chemistry

Hexamethyldisilazane as an Acylation Generator for Perfluorocarboxylic Acids in Quantitative Derivatization of Primary Phenylalkyl Amines Confirmed by GC-MS and Computations Borbála Molnár1-3, Antal Csámpai2, Ibolya Molnár-Perl1* 1,2

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2

Institute of Chemistry , Departments of Analytical and Inorganic Chemistry, L. Eötvös University, 1117, Pázmány 3 Péter sétány 1/A, Budapest, Hungary; Doctoral School of Pharmaceutical Sciences , Semmelweis University, 1085, Üllői út 26, Budapest, Hungary ABSTRACT: A novel, selective acylation of primary phenylalkyl amines (PPAAs) using hexamethyldisilazane (HMDS) & perfluorocarboxylic acids (PFCAs) is noted. Couples, like HMDS & trifluoroacetic acid (TFA), or HMDS & pentafluoropropionic acid (PFPA), or HMDS & heptafluorobutyric acid (HFBA) trigger PPAAs’ quantitative acylation. Processes’ selectivity was characterized by applying all couples to derivatize benzyl, 2-phenylethyl, 3-phenylpropyl, 4phenylbutyl amines and their relevant substituted versions. Aliphatic amines were unreactive. Identification, quantification, proportionality and stoichiometry in derivatization processes were determined by gas chromatographymass spectrometry. Reaction conditions were optimized depending on reagents’ molar ratios, solvents and temperatures applied. The new acylation method, in comparison to the traditional ones - obtained with trifluoroacetic anhydride (TFAA), heptafluorobutyric anhydride (HFBAA) and N-methyl-bis(trifluoroacetamide) (MBTFA) – offers numerous advantages. Derivatives, provided by couples, can be directly injected onto the column, avoiding loss of species, saving time, work and cost in preparation process. Due to traditional reagents’ excess evaporation by nitrogen drying, the loss of trifluoroacylated species proved to be 65% or less. Regarding heptafluorobutyryl species, their losses varied between 25% and 5%. Unified huge responses, obtained with the HMDS & PFCA couples are attributable to their direct injection onto the column and to fragments sourced from the molecular ions and from their self chemical ionization ([M] , [M+147]+ i.e. [M+(CH3)2-Si=O-Si-(CH3)3]+). The reaction mechanism - due to HMDS symmetrical structure, acting HMDS as acylation generator for PFCAs - was confirmed by Density Functional Theory (DFT) computation.

Since the pioneer work of Pierce,1 analytical chemists both in academia and industry have used numerous silylating reagents, including HMDS, to prepare volatile products from organics with exchangeable proton.2-7 We preferred the use of HMDS because of its simplicity, cost affectivity in quantitation of active protons, being even in hindered positions.3-6 On course of trialkylsilylation optimization of 2-(3,4,5trimethoxyphenyl)ethylamine with the HMDS & TFA reagent, to our surprise, instead of the expected ditrimethylsilyl derivative,7 its trifluoroacylated product was obtained. This phenomenon proved to be a unique novelty in chemistry, especially in analytical chemistry. Namely, traditional acylation - one of the most widely used protocol8 derivatizing amines for GC analysis – are performed with TFAA,9-11 or HFBAA11-13 or MBTFA14 reagents, applying ethyl acetate (ETAC) as solvent (reagent/solvent=1/1, v/v). To investigate the characteristic properties of our novel acylation principle, a many-sided research was undertaken, along with computation. Reagent composition, solvent, time and temperature for optimum analytical conditions were optimized and controlled. In parallel with GC-

MS studies, DFT modeling was performed to highlight the mechanism and stoichiometry of principle. Analytical advantages of the novel method in comparison to traditional derivatizations9-14 (TFAA, HFBAA, MBTFA) were defined.

EXPERIMENTAL SECTION HMDS (99.9), TFA (99.5), PFPA (97), HFBA (99.5), TFAA (≥ 99), HFBAA (≥ 99), MBTFA (∼98), acetonitrile (ACN, ≥ 99.9), ETAC, ≥ 99.9), pyridine (PYR, ≥ 99.9), benzylamine (BA, ≥ 99.5), 2-phenylethylamine (2-PEA, ≥ 99), o-methoxybenzylamine (OMBA, 98), 3-phenylpropylamine (3PPA, ≥ 98), m-methoxybenzylamine (MMBA, 98), pmethoxybenzylamine (PMBA, 98), 2-(m-methoxyphenyl)ethylamine (2-MMPEA, 97), 4-phenylbutylamine (4-PBA, 98), 2-(p-methoxyphenyl)ethylamine (2-PMPEA ≥ 98), 2-(3,4-dimethoxyphenyl)ethylamine {2-(3,4DiM)PEA; homoveratrylamine, ≥ 98} and 2-(3,4,5trimethoxyphenyl)ethylamine, {2-(3,4,5-TriM)PEA; mescaline, 99} were of highest analytical grade available, used as received (percent purity in parentheses): all products of Sigma Aldrich, St Louis, Mo, USA. Model compounds, 10-12 mg/10 mL, weighed with ± 0.01

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mg uncertainty, were dissolved in distilled water, neutralized with hydrochloric acid and further diluted into a unified stock solution containing in 10-20 µL aliquots about 0.1-0.2 µg PPAA of each. Model solutions, in triplicate, were rotary evaporated to dryness at 30–40 °C. Residues, or freeze dried cactus tissues∗, weighed directly into the derivatization vessel (± 0.001 mg uncertainty), were treated with 70 µL HMDS, 30 µL TFA or 30 µL PFPA or 30 µL HFBA and 100 µL ETAC, heated in oven at 80 °C for 20 min. Thereafter acylated solutions were transferred into the autosampler vial and 1 µL (containing 500-1000 pg PPAA derivatives of each) was injected into the GC-MS system. ∗(Homogenized 2.95 g gave freeze dried 0.260 g.) Derivatizations with TFAA, HFBAA and MBTFA were performed as detailed above except dried residues were treated with 100 µL TFAA & 100 µL ETAC or 100 µL HFBAA & 100 µL ETAC or 100 µL MBTFA & 100 µL ETAC and heated in oven at 80 °C for 20 min. Thereafter TFAA and HFBAA derivatives were evaporated to dryness by using N2 at ambient temperature. The dry residues were redissolved in 200 µL ETAC. The apparatus consisted of a Varian 240 GC-MS/MS system (Varian, Walnut Creek, CA, USA). The analyses were carried out using a Varian CP-8400 Autosampler, and a Septum Programmable Injector (SPI). The column used was a product of SGE (Victoria, Australia); SGE forte capillary BPX5: 30 m x 0.25 mm; df = 0.25 µm. The temperatures of the transfer line, ion trap and manifold were, in order of listing 300 °C, 210 °C and 80 °C, respectively. Under optimized temperature programs injections were made at 280 °C, and held at 280 °C for 3 min, then cooled down to 100 °C (100 °C/min); the column temperature profile was: 100 °C, held for 1.00 min, then heated up to 145 °C for 10 °C/min, than heated up to 195 °C for 5°C/min with a 1.00 min hold and finally heated up to 280 °C for 50 °C/min with a 1.80 min hold at 280 °C (total elution time was 20.00 min. Helium (purity: 6.0, 99.9999%) was used. The column flow rate was 1 mL/min. The general MS parameters were: Fil/Mul delay: 3.00 min; electron energy: 70 eV. Statistical analysis was performed with Student's twotailed t-test, p < 0.05 was considered significant.

RESULTS AND DISCUSSION Introductory, GC-MS derivatization study showed that the HMDS & TFA couple acylates PPAAs selectively: primary aliphatic amines do not react at all. In order to gain further insight into the selectivity and stoichiometry of this process, further studies were performed, to determine optimum molar ratio range applying [HMDS]/[TFA] from 0.44/1 up to 7.1/1. The impact of solvents (ETAC, ACN, PYR) was also investigated (Table 1). As optimum molar ratio, the [HMDS]/[PFCA] = 0.85/1 reagent proportion of HMDS/PFCA/ETAC = 7/3/10 (v/v/v) - as solvent ETAC was selected. Bold printed, blue values represent the optimum reagent composition, the mean of the optimum range. Varying time (10, 20, 30 min) and temperature (70 °C, 80 °C, 90 °C) of reactions, in each

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cases, lead to optimum conditions (80 °C, 20 min). Table 1. Efficiency of PPAAs Derivatization with the HMDS & TFA Couple Depends on the Molar Ratios ([HMDS]/[TFA]) and Solvents Applied: Ethyl Acetate (ETAC), Acetonitrile (ACN) and Pyridine (PYR), all in Proportions of (HMDS & TFA)/Solvent = 1/1 (v/v), Determined by GC-MS. 4

Iu/pg x 10 * (RSD%); [HMDS]/[TFA] varied ⇓ PPAAs

0.44/1

0.56/1 3.3/1 2.07/1 ETAC**

7.1/1

0.85/1 ACN PYR

BA

3.19 (1.53)

3.96 3.18 2.86 3.92 (4.76) (2.60) (0.77) (2.90)

-

2-PEA

3.59 (1.14)

4.81 3.41 3.06 4.68 (4.28) (2.20) (0.79) (3.30)

-

OMBA

3.48 (3.81)

4.99 4.07 3.82 5.00 0.77 (4.36) (3.93) (4.06) (3.28) (2.46)

MMBA

3.28 (0.22)

3.88 (3.64)

3.01 3.01 4.09 3.33 (3.31) (1.22) (3.56) (3.74)

PMBA

3.75 (2.47)

4.93 (4.81)

4.21 4.01 4.69 4.34 (1.91) (0.94) (3.12) (0.73)

2-MMPEA

2.78 (1.83)

3.70 3.25 2.82 3.70 3.73 (2.66) (2.76) (3.61) (4.12) (2.55)

2-PMPEA

3.15 (3.78)

4.72 (3.70)

3.89 3.59 4.60 4.73 (1.35) (1.86) (2.73) (2.49)

2-(3,4DiM)PEA

1.76 (1.33)

2.55 (4.81)

2.33 2.29 2.55 2.53 (1.90) (4.58) (1.77) (3.21)

2-(3,4,5TriM)PEA

1.61 (3.19)

2.26 2.09 2.14 2.13 2.19 (2.25) (0.84) (2.38) (1.15) (2.85)

Indications: * = average responses from 500 pg/injections and 1000 pg/injections of each; in parentheses: relative standard deviation percentages (RSD%); The bold printed, blue values are obtained from optimum range [HMDS]/[PFCA] = 0.56/1 - 2.07/1; ** = peak profiles and mass spectra are detailed in Supporting Information, Figs. S1a – S1e, values for the HMDS & TFA couple.

The experimental data in Tables 1, 2 clearly demonstrate that HMDS together with PFCAs behave as an acylation reagent in proton donor solvents. Therefore, we also provided computational evidence (DFT modeling at B3LYP/6-31 G(d)15,16 level: Scheme 1, Supporting Information, Tables S1 - S14). The special interaction of HMDS A with PFCAs B, the formation/transformation intermediaries C - G, result in stable endproducts J - K, including the acylated amine J. In this process, HMDS, due to its symmetrical structure, triggers to form cationic intermediate, C, which getting stabilized by two electrons’ delocalization, exemplifying the Olah theory.17 Ring opening C with perfluorocarboxylate D provides activated ester E. Again, E with amine F affords carbinol I and acylated amine J via the proton transfer between ions G and H. In the last step I is converted irreversibly into silane species K by half equivalent of A, necessary for the overall process completion (Scheme 1). This exceptional reactivity

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Scheme 1. Acylation Route of PPAAs via the Reaction with the HMDS & PFCA Couples

of HMDS is also in line both with our data and the literature8, confirming that reagents - BSA, BSTFA, MSTFA, MTBSTFA, up to now also HMDS - are silylating, exclusively. Summing up mechanism from stoichiometrical point of view it is clear that calculated molar ratio of [HMDS]/[PFCA] = 1.5/1 supports our experimental proof, being compatible with the wide optimum molar ratio ranges of [HMDS]/[PFCA] = 0.56/1 - 2.07/1, due to the huge excess of both parts of the couple (Table 1.: bold printed blue response values). As to the similar efficacy of solvents ETAC and ACN, this was also supported by the reaction mechanism attributable to the fact that both solvents do have an acidic character in contrary to PYR’s basic one; PYR is a competitor of HMDS to react with the proton (H+), which has a crucial role in the primary generation of cyclic cationic intermediate C with a hydrogen in a bridged position (Scheme 1.). The scope and validity of the novel acylation process was extended, by replacing the HMDS & TFA both with the HMDS & PFPA and the HMDS & HFBA couples (Table 2.). To our delight, the corresponding pentafluoropropionyl and heptaflurobutyryl species were formed, quantitatively and proportionally, proving the selective acylation of PPAAs, equally (Table 2., values in lines TFA, PFPA and HFBA; peak profiles and mass spectra are detailed in Supporting Information, Figs. S1a - S1e). As an example, the last vertical column in Table 2. shows the peak profiles from BA up to 2-PMPEA and the fragmentation for BA derivatives (spectra 1A, 2A, 3A). The joint evaluation of the HMDS & PFCA acylated products’ fragmentation patterns along with their products’ efficiencies reveals unique properties and advantages of the new acylation principle. a) Acylated species obtained with the HMDS & PFCA couples demonstrated unified huge responses (Table 2.), expressed as integrator units/pg values (Iu/pg) attributable to their molecular ions ([M] ) and/or to their characteristic fragments formed by self chemical ionization {[M+147]+ i.e. [M+(CH3)2-Si=O-Si-(CH3)3]+, Table 2., Supporting Information, Figs. S1a - S1e}. b) Responses proved to be independent on the PFCA part of couples (Table 2., average responses, values in the 9th vertical column). These concordant response values call attention to the special properties of the new

acylation process, meaning excellent selectivity, reliability and reproducibility of derivatives. This phenomenon could be best characterized with the relative standard deviation percentages (RSD%) of responses (Table 2., values in 9th vertical column): varying between 0.56% (2PMPEA) and 2.01% (3-PPA). c) Contrasting the response values with the counterpart products - derivatized with TFAA, HFBAA and MBTFA reagents - it turned out that all three classical acylation processes resulted in lower responses (regarding as the basis of comparison, - as 100% - the average values for the PFCAs’ derivatives in Table 2.). The most commonly used TFAA derivatives show up with the lowest responses due to derivatives’ loss under TFAA excess elimination by N2 gas. Differences in responses, obtained with the TFAA and the HMDS & PFCA couples, served for calculation: significant differences were found, all proved to be at the level p < 0.05. Exclusive benefit of the HMDS containing reagents can be attributed to the ions of [M+147]+. d) Setting the elution profiles and mass spectra of the HMDS & PFCAs derived species (Supporting Information, Figs. S1a - S1e) - against those obtained via the traditional acylation processes (Supporting Information, Figs. S2a – S2d) - strict correlations of retention times and considerable differences in favor of the HMDS & PFCA couples are shown. The novel acylation process, resulted in fragments - sourced from self chemical ionization of molecular ions - benefit of HMDS, exclusively. Practical utility of the method. We can foresee numerous useful applications of our principle, which may include the analysis of amphetamines and cardiotonic amines. As a proof of principle we measured mescaline in freeze-dried cactus stem (Lophophora Williamsii), Figure 1. Supporting the utility of our method, we directly derivatized mescaline with the HMDS & TFA reagent, omitting the 24 h Soxhlet extraction before chromatography.18 Various amounts of freeze dried tissues were determined (a, 0.454 mg; b, 0.200 mg; c, 0.100 mg), in comparison to mescaline standard (d, 0.800 µg mescaline) and blank (e, reagent only). Via the novel method comparable mescaline contents were: a, 1.34%; b, 1.41%; c, 1.43%. Reproducibility and reliability proved to be: a, 4.70 RSD%; b, 2.69 RSD%; c, 3.48 RSD%.

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Table 2. Chemical Properties, Structures and Fragmentation Patterns of Primary Phenylalkyl Amines (PPAAs) Obtained with the HMDS & TFA, HMDS & PFPA and HMDS & HFBA Couples, Applying ETAC Under Optimum Conditions, Determined as Acylated Derivatives by GC-MS, Based on Their Selective Fragment Ions.

414

HFBA 6.40

317

464

TFA 7.22

233

380

PFPA 7.07

283

430

HFBA 7.54

333

480

TFA 7.70

231

378

PFPA 7.61

281

428

HFBA 8.13

331

478

TFA 7.84

233

380

PFPA 7.74

283

430

HFBA 8.28

333

480

TFA 8.17

233

380

PFPA 8.06

283

430

HFBA 8.63

333

480

TFA 9.21

247

394

PFPA 9.10

297

444

HFBA 9.65

347

494

245

392

295

442

HPBA 9.87

345

492

TFA 9.47

247

394

PFPA 9.36

297

444

HPBA 9.92

347

494

2-(3,4DiM)PEA 181.23

TFA 12.79

277

424

PFPA 12.55

327

474

HPBA 13.05

377

524

2-(3,4,5TriM)PEA

TFA 15.23

307

454

PFPA 14.94 357

504

211.26

HPBA 15.39 407

554

HMDS & PFPA

{91} 91

4.99

91

134, {104}, 136, {121},

HMDS &TFA

4.73

1.86 4.94 (2.13) (0.91) {38} 4.94 4.90

3.88

4.93

91

2.09 2.13

2.09 (2.01)

HMDS & HFBA

2.05 3.85 3.79

1.89 3.87 (4.23) (1.19) {49}

1.73 4.92 (3.71) (0.65) {35} 4.88

4.94 3.77

91

TFA 9.43 PFPA 9.29

2.56 4.77 (0.31) (0.85) {54} 4.76

2-PMPEA

267

PMBA

PFPA 5.92

2-MMPEA

4.81

OMBA

4.01

364

MMBA

450

217

2-PEA

303

TFA 5.93

1.70 3.97 (1.46) (1.02) {43}

BA

HFBA 5.36

3.98

(RSD%) TFAA {yields, %}

400

averages

one by one

other ions

253

104, {91}

2-PMPEA 151.21

PFPA 4.92

3.93

3.20

91

4-PBA 149.23

350

4.67

3.74 3.67

1.75 3.73 (3.65) (1.36) {47}

3.17 3.16 (1.55)

3.10 4.72 4.71

2.21 4.70 (3.71) (0.56) {47}

2.55

91

2-MMPEA 151.21

203

1.10 2.50 (2.86) (1.89) {44} 2.48

2.46 2.26

91

PMBA 137.18

TFA 4.95

162, {117}, 105, 91

MMBA 137.18

[M+ + 147]

164, 136, {121}, 91

3-PPA 135.21

[M]

136, {121},

OMBA 137.18

peak profiles from BA up to 2-PMPEA; spectra 1A, 2A, 3A for BA derivatives

134, {121},

2-PEA 121.18

4

Iu/pg × 10 ∗

176, 132,

107.15

min

{134}, 121,

BA

structures

tR,

164, {151},

MW, mol

selective fragment ions, m/z

194, {181},

PPAAs

derivatives of HMDS &

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2.18 2.19

1.14 2.21 (4.74) (1.97) {52} +

+

Indications, as in Table 1., as well as [M] = molecular ion; [M+147] = [M+(CH3)2-Si=O-Si-(CH3)3] ; in square braces = {abundant m/z values}; ∗ = average response values obtained from 500 pg or 1000 pg/injections of each; peak profiles and mass spectra, as example, are in the last vertical column for BA derivatives, for all others are demonstrated in Supporting Information, in Figs. 1a – S1e; Differences between the TFAA and the HMDS & TFA couples provided responses served for calculation; significant differences were found, all, at the level p < 0.05.

The mescaline content of the Lophophora Williamsii

cactus stem samples (1.39% in average) falls in the range

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reported by others (0.053% and 4.7%).18

being compatible with the wide optimum molar ratio ranges of [HMDS]/[PFCA] = 0.56/1 - 2.07/1, determined by GC-MS.

ASSOCIATED CONTENT Supporting Information Figs. S1a-1e and S2a-2d show GC-MS elution profiles and mass spectra of PPAA derivatives Tables S1- S14 give DFT calculation details. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding author *E-mail: [email protected]

Author Contributions Figure 1. Peak profiles (a - e) and mass spectra (1A, 1B) of mescaline from HMDS & TFA direct derivatizations: freezedried cactus (a, 1A; b, c); standard (d, 1B); blank (e)

The manuscript was written through contributions of all authors; all have given approval to manuscript’s final version.

Notes

CONCLUSION A new, selective way of acylation was demonstrated recognizing the unique potential of HMDS facilitating PFCA-mediated acylation reactions of PPAAs. Novel acylation reagents, like HMDS & TFA, HMDS & PFPA and HMDS & HFBA couples were introduced for the quantitative and selective derivatization of PPAAs. Aliphatic amines are unreactive. The optimum condition and analytical advantages of the novel principle are compiled in Tables 1. and 2. and in Supporting Information, Figs. S1a - S1e. Comparison of species derived by the new method with those performed via TFAA, MBTFA and HFBAA derivatizations (Supporting Figs. S2a - S2d) – are highlighted: retention properties of derivatives obtained via the novel and traditional ways were identical, however differences in their responses, proved to be in favor of derivatives, prepared with the couples. Advantages, indicating the superiority of the new acylation principle are as follows: the couples provided species can be directly injected onto the column, avoiding loss of species, saving time, work and cost of preparation process. The couples provided derivatives resulted in concordant high responses, independent on the PFCA part of the couple (Table 2., average responses, values in the 9th vertical column). Concordant response values call attention to the unique properties of the new acylation process, meaning excellent selectivity, reliability and reproducibility of derivatives. This phenomenon was characterized with the RSD% of derivatizations varying between 0.56% (2-PMPEA) and 2.01% (3-PPA). Statistical significance between the traditional and new method was calculated using Student's t-test. It was found to be significant at p < 0.05. The evidence of mechanism due to the symmetrical structure of HMDS was confirmed. Evaluating the mechanism from stoichiometrical point of view it turned out that calculated molar ratio of [HMDS]/[PFCA] = 1.5/1 supports our experimental proof,

The authors declare no competing financial interest

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