Quantitative Silylation Speciations of Primary Phenylalkyl Amines

Technical Note pubs.acs.org/ac

Quantitative Silylation Speciations of Primary Phenylalkyl Amines, Including Amphetamine and 3,4-Methylenedioxyamphetamine Prior to Their Analysis by GC/MS Borbála Molnár,†,§,⊥ Blanka Fodor,†,§ Imre Boldizsár,‡,∥ and Ibolya Molnár-Perl*,†,§ †

Institutes of Chemistry and ‡Biology and §Departments of Analytical Chemistry and ∥Plant Anatomy, L. Eötvös University, 1117, Pázmány Péter sétány 1/A-C, Budapest, Hungary ⊥ Doctoral School of Pharmaceutical Sciences, Semmelweis University, 1085, Ü llői út 26, Budapest, Hungary S Supporting Information *

ABSTRACT: A novel, quantitative trimethylsilylation approach derivatizing 11 primary phenylalkyl amines (PPAAs), including amphetamine (A) and 3,4-methylenedioxyamphetamine (MDA), was noted. Triggering the fully derivatized ditrimethylsilyl (diTMS) species with the N-methyl-N(trimethylsilyl)-trifluoroacetamide (MSTFA) reagent, a new principle was recognized followed by GC/MS. In the course of method optimization, the complementary impact of solvents (acetonitrile, ACN; ethyl acetate, ETAC; pyridine, PYR) and catalysts (trimethylchlorosilane, TMCS; trimethyliodosilane, TMIS) was studied: the role of solvent and catalyst proved to be equally crucial. Optimum, proportional, huge responses were obtained with the MSTFA/PYR = 2/1−9/1 (v/v) reagent applying catalysts; A and MDA needed the TMIS, while the rest of PPAAs provided the diTMS products also with TMCS. Similar to derivatives generated with hexamethyldisilazane and perfluorocarboxylic acid (HMDS and PFCA) (Molnár et al. Anal. Chem. 2015, 87, 848−852), the fully silylated PPAAs offer several advantages. Both of our methods save time and cost by allowing for direct injection of analytes into the column; this is in stark contrast with the requirement to evaporate acid anhydrides by nitrogen prior to their injection. Efficiences of the novel catalyzed trimethylsilylation (MSTFA) and our recently introduced (now, for A and MDA extended) acylation principle were contrasted. Catalyzed trimethylsilylation led to diTMS derivatives resulting in on average a 1.7 times larger response compared to the corresponding acylated species. Catalyzed trimethylsilylation of PPAAs, A, and MDA were characterized with retention, mass fragmentation, and analytical performance properties (R2, LOQ values). The practical utility of ditrimethylsilyation was shown by analyzing A in urine and mescaline (MSC) in cactus samples.

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Through our studies, in 2 weeks old MSTFA/PYR (2/1, v/v) derivatized standard solution, again to our delight, the A-diTMS and MDA-diTMS products were identified: surprisingly, for AdiTMS based on the NIST library. However, the source and preparation background of A-diTMS spectrum were not available. A-diTMS spectrum was found in the software of “Mass spectra of designer drugs”,10 while A-diTMS tentative formation was learned via an e-mail received from authors (Supporting Information).11 To investigate the characteristic properties of this novel trimethylsilylation process, in order to optimize it as quantitative method, a many-sided research approach was undertaken. Reagent composition, solvent, and catalyst were selected as well as time and temperature for optimum analytical conditions

ecently, in order to trialkylsilylate PPAAs with the HMDS and PFCA reagents, to our delight, a new derivatization reaction was recognized: instead of the expected trimethylsilyl derivatives, their trifluoroacylated products were obtained. The principle’s advantages and practical utility along with the comparison of traditional methods were shown.1 Now, continuing our interest in the classical trimethylsilylation, pioneered by Pierce,2 a novel, selective, and quantitative method was realized again, applying MSTFA derivatization. This experience opened the second new approach in PPAAs analysis, including A and MDA. In the course of these studies, along with a recent one,1 premises related PPAAs’ MSTFA derivatizations, prior to their GC/MS analysis, were summed up.3−9 To quantitate A, MDA, and MSC, the use of MSTFA was reported in solvent free medium.3−9 A and MSC were trimethylsilylated also in the catalyzed version (MSTFA containing 1% TMCS),9 leading to A-monoTMS,3−9 while MSC resulted in diTMS9 species. © XXXX American Chemical Society

Received: July 10, 2015 Accepted: September 26, 2015

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DOI: 10.1021/acs.analchem.5b02599 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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RESULTS AND DISCUSSION Introductory experiences with our 2 weeks old MSTFA/PYR (2/ 1, v/v) derivatized standard solution called our attention to the diTMS transformation of PPAAs, including A and MDA, in PYR medium. This observation, meaning the existence of A-diTMS, was confirmed.11 Thus, further studies seemed to be promising to derivatize PPAAs both in solvent free and in TMCS catalyzed9 media as well as varying the MSTFA/PYR (v/v) reagent composition and the impact of TMCS and TMIS12 catalysts. These approaches were performed for the first time (Figures 1 and 2 and Table 1). Results, based on separate monoTMS/diTMS GC/MS evaluations (monoTMS responses were obtained for all PPAAs in MSTFA/ETAC = 2/1, v/v reagent composition; Supporting Information Figure S1), proved the crucial role of PYR and catalysts. Performing solvent free MSTFA derivatization resulted in the diTMS species (Figure 1, dotted green columns); except the cases of A and MDA, both eluted as the monoTMS derivatives (Figure 1, dotted red columns), in accordance with the literature.3−9 Applying PYR in the MSTFA/PYR = 2/1−9/1, v/v range (data in Figure 1) led to commensurable responses as obtained with the solvent free MSTFA (striped green columns, 2-PEA, OMBA, 2-(3,4-DiM)PEA, MSC) or to somewhat larger ones, consisting both of diTMS and monoTMS products (striped green + striped red columns, BA, MMBA, PMBA, 2-MMPEA, 2PMPEA). A and MDA provided monoTMS species (striped red columns) only. Using TMCS catalyst, varying reagent composition in 150 μL of total volume (MSTFA/PYR/TMCS = from 100/48/2 up to 100/40/10, v/v), resulted in considerably increased diTMS responses (Figure 1, plain green columns, Table 1, diTMS species). Reactivities of A and MDA, even under these conditions, proved to be insufficient. To obtain A-diTMS and MDA-diTMS species, additional studies are needed. Further derivatizations were performed with specially activated MSTFA (MSTFATMIS).12 We applied this reagent, at first, in the MSTFATMIS/PYR = 2/1 (v/v) medium leading to AdiTMS and MDA-diTMS in a quantitative/proportional manner (Figure 2, red peaks’ line, spectra 2A, 4A). In order to gain further insight into the ditrimethylsilylation process, parameters, affecting the reaction (solvent, reaction time, and temperature), had to be defined. Changing the proton acceptor PYR both for ETAC and ACN (MSTFATMIS/ETAC or ACN = 2/1, v/v), it turned out that applying ETAC primarily the monoTMS products were formed (Figure 2, green peaks’ line, spectra 1A, 2A, 3A, 4A), while ACN favors the diTMS production (Figure 2, blue peaks’ line, spectra 2A, 3A, 4A). Also exclusively the diTMS species were obtained in the solvent free medium (orange peaks’ line, spectra 2A, 4A), however, resulting in ∼1/10th of the responses compared to optimum conditions (Figure 2, red line, spectra 2A, 4A). Varying time (10, 20, 30, 60, 90 min) and temperature (70, 80, 90, 100 °C) of ditrimethylsilylations, in each case, led to optimum conditions (90 °C, 60 min). Concerning advantages of ditrimethylsilylation compared to monotrimethylsilylation methods,3−9 based on response relations, undoubtedly diTMS derivatization is to be preferred: Table 1, responses, IU/pg, A-diTMS vs A-monoTMS, 1.63 × 104 vs 0.64 × 104, and MDA-diTMS vs MDA-monoTMS, 2.03 × 104 vs 0.57 × 104. Turning to the derivatization reproducibility, characterized with relative standard deviation percentages, (RSD %, details in

were determined. In parallel with GC/MS studies, on the basic research level, the derivatization process’s stoichiometry, derivatives’ proportionality, stability, reproducibility, and practical utility were documented. Analytical advantages along with the recently published HMDS and PFCAs technique,1 extended now to the analysis of A and MDA, were compared. Proposal’s practical utility was demonstrated with the quantitation of A in urine and MSC in cactus samples.

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Technical Note

EXPERIMENTAL SECTION

MSTFA (≥97.0), MSTFA activated I, catalog no. 50994, containing ammonium iodide and ethanethiol, forming in situ the MSTFA/TMIS = 1000/2 (v/v) reagent (further on MSTFATMIS), TMCS (≥99.0), HMDS (99.9), TFA (99.5), ACN (≥99.9), ETAC (≥99.9), PYR (≥99.9), benzylamine (BA, ≥ 99.5), 2-phenylethylamine (2-PEA, ≥ 99), D-amphetamine sulfate (A, ≥ 99), o-methoxybenzylamine (OMBA, 98), mmethoxybenzylamine (MMBA, 98), p-methoxybenzylamine (PMBA, 98), 2-(m-methoxyphenyl)ethylamine (2-MMPEA, 97), 2-(p-methoxyphenyl)ethylamine (2-PMPEA, ≥ 98), (±)-3,4-methylenedioxyamphetamine hydrochloride (MDA, ≥ 99), 2-(3,4-dimethoxyphenyl)ethylamine (2-(3,4-DiM)PEA, homoveratrylamine, ≥ 98), and 2-(3,4,5-trimethoxyphenyl)ethylamine (mescaline, MSC, 99) were of highest analytical grade, used as received (percent purity in parentheses): all products of Sigma-Aldrich, St. Louis, MO. Model compounds (10−12 mg/10 mL), weighed with ±0.01 mg uncertainty, were dissolved in distilled water, neutralized with hydrochloric acid, and further diluted into a unified stock solution providing finally in 1 μL of derivatized solution 25− 2000 pg of PPAAs, including A and MDA of each. Model solutions and/or cactus and urine extracts (detailed sample preparation in the Supporting Information), in triplicate, were rotary evaporated to dryness at 30−40 °C. Residues for trimethylsilylation were treated with (i) 40 μL of PYR, 100 μL of MSTFA, and 10 μL of TMCS; or (ii) 50 μL of PYR was mixed with 100 μL of MSTFATMIS; or (iii) 150 μL of MSTFA alone; then heated in an oven (90 °C, 60 min). Acylation were performed with 70 μL of HMDS, 30 μL of TFA, and 100 μL of ETAC, heated in an oven at 80 °C for 20 min. Thereafter derivatized solutions were transferred into the autosampler vial and 1 μL was injected into the GC/MS setup. The apparatus consisted of a Varian 240 GC/MS/MS system (Varian, Walnut Creek, CA). 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 × 0.25 mm; df = 0.25 mm. The temperatures of the transfer line, ion trap, and manifold were in order of listing 300, 210, 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 to 145 °C for 10 °C/min, then to 230 °C for 5 °C/min, and finally to 280 °C for 50 °C/min with a 1.00 min hold (total elution time were 24.50 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 two-tailed t-test, and p < 0.05 was considered significant. B


Technical Note


Figure 2. Peak profiles and mass spectra of A (spectra 1A, 2A) and MDA (spectra 3A, 4A), obtained with MSTFATMIS only (orange lines) or applying MSTFATMIS/solvents in 2/1 (v/v) ratios: ETAC (green lines), ACN (blue lines), PYR (red lines).

role of TMCS (Figure 1, red and green striped versus green plain columns). Uncertainty in stability is associated with monoTMS formation (Figure 1, red striped columns, BA, MMBA, PMBA, 2MMPEA, 2-PMPEA). Contrasting our recent acylation1 and the just now developed ditrimethylsilyation principles, both are quantitative, reproducible, and comparable (Table 1). Characterizing acylation and ditrimethylsilylation analyses they are identical in any sense in their simplicity and advantages over classical derivatization for GC (direct injectability onto the column, time, work, and cost efficiency). As to their derivatization dependence, the specified response behavior, particularly in cases of A, MDA, and MSC, the use of catalyzed trimethylsilylation should be preferred, resulting in 1.9 (A), 2.7 (MDA), and 1.6 (MSC) greater responses, compared to their, by HMDS and TFA provided, acylated species. Regarding analytical performance characteristics, R2 and LOQ values both benefit ditrimethylsilylation (Table 1, data in 6th and 7th vertical columns). R2 varied between 0.9991 and 0.9999 for ditrimethylsilylation and 0.9986 and 0.9999 for acylation. LOQ values fall in the ranges of 4.8−16 ng/mL (average 7.2 ng/mL) for ditrimethylsilylation and 6.1−31 ng/mL (average 12.4 ng/mL) for acylation, respectively. Practical Utility of Proposal. The advantages of ditrimethylsilylation contrasting with the just now extended acylation principle, A from urine, MSC from cactus tissues, were summed up in parallel determinations (A, Figure 3; MSC, Figure 4). Linearity, proportionality, and reproducibility of this method is indicative of a 100% yield of analyte derivatization. This is evidenced by using model solutions (Table 1, R2, LOQ values) and natural matrixes, A in urine and MSC in cactus tissue up to 10 ng/injected amounts (A content in urine was in average 3.69 μg/ mL, with 6.7 RSD%; MSC content in cactus proved to be in average 0.54% (w/w), with 5.5 RSD %). Regarding MSC quantitation from four various amounts of tissue extract, now, from a different, albeit same, cactus type (Lophophora Willimamsii, Lem.), considerably less MSC content (0.54%) was obtained, in comparison with the earlier measured value (1.39%).1 However, the 0.54% falls also in the range reported by others (0.053−4.7%).13

Figure 1. Responses in integrator units (IU/pg × 104) for PPAAs, A, and MDA depending on reagent compositions: monoTMS (red) and diTMS (green) columns (Supporting Information, Figure S1, monoTMS derivatives).

Table 1, IU/pg values and Figure 1, uncertainty bars), in model solutions they varied between 0.10% and 5.1% (for diTMS derivatives, average 2.14 RSD %) and 0.56% and 3.12% (for acylated species, average 1.49 RSD %). The derivative composition and stability behavior confirm the unambiguous C


Technical Note


Table 1. Structures, Mass Fragmentation, and Analytical Performance Characteristics of PPAAs Derivatives; Comparison of Retention and Response Values as Di-TMS (Green Columns, Completed by Spectra 1A−4B) and as Acylated Species (Blue Columns), Obtained by GC/MSa

a

Indications, as in Figures 1 and 2, SFI = selective fragment ion; R2 = R squared value for linear regression in the standard curve, in the range of limit of quantitation (LOQ) and 400 ng/mL values. M+· = molecular ions; * = average responses obtained from three separate derivatizations, injected two times of each. Supporting Information, Figure S1, monoTMS and Figure S2, HMDS and TFA species of A and MDA. Response differences between acylated and ditrimethylsilylated species were significant, all at the level, p < 0.05

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Technical Note


of LOQ and 400 ng/mL concentrations) varied between 0.9991−0.9999 (ditrimethylsilylation) and 0.9986−0.9999 (acylation); LOQ proved to be in the ranges of 4.8−16 ng/mL (average 7.2 ng/mL) for ditrimethylsilylations and 6.1−31 ng/ mL (average 12.4 ng/mL) for acylations, respectively. Reliability and reproducibility of analyses were characterized with the relative standard deviation percentages confirming in average 2.14 RSD % (catalyzed ditrimethylsilylation) and 1.49 RSD % (HMDS and TFA acylation) (Table 1, IU/pg value, responses indicated by blue and green columns, one by one). The utility of our proposal was shown by contrasting acylation and ditrimethylsilylation processes in the quantitation of A from urine and MSC from cactus stem tissue.

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ASSOCIATED CONTENT

* Supporting Information

Figure 3. Peak profiles of acylated (a) and ditrimethylsilylated (b) derivatives from the same urine extract, corresponding to 3.36−3.88 ng (red lines) and 1.82−1.94 ng (green lines) A, 1 ng standard (orange lines) and blank (blue lines); sample preparation in the Supporting Information. Mass spectra obtained from urine in Figure S3, as acylated and as ditrimethylsilylated derivatives.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02599. Source and background of A-diTMS spectrum; detailed sample preparation; GC/MS peak profiles and mass spectra for PPAAs as monoTMS and as, by HMDS and TFA, acylated A and MDA; and spectra of A from urine and MSC from cactus samples (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

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

The authors declare no competing financial interest.

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REFERENCES

(1) Molnár, B.; Csámpai, A.; Molnár-Perl, I. Anal. Chem. 2015, 87, 848−852. (2) Pierce, A. E. Silylation of Organic Compounds; Pierce Chemical Company: Rockford, IL, 1968. (3) Toennes, S. W.; Steinmeyer, S.; Maurer, H.-J.; Moeller, M. R.; Kauert, G. F. J. Anal. Toxicol. 2005, 29, 22−27. (4) Pujadas, M.; Pichini, S.; Civit, E.; Santamariña, E.; Perez, K.; de la Torre, R. J. Pharm. Biomed. Anal. 2007, 44, 594−601. (5) Joya, X.; Pujadas, M.; Falcón, M.; Civit, E.; Garcia-Algar, O.; Vall, O.; Pichini, S.; Luna, A.; de la Torre, R. Forensic Sci. Int. 2010, 196, 38− 42. (6) González-Mariño, I.; Quintana, J. B.; Rodríguez, I.; Cela, R. J. Chromatogr. A 2010, 1217, 1748−1760. (7) Wan Raihana, W. A.; Gan, S. H.; Tan, S. C. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2011, 879, 8−16. (8) Peliçaõ , F. S.; Peres, M. D.; Pissinate, J. F.; De Martinis, B. S. J. Anal. Toxicol. 2014, 38, 341−348. (9) Liu, R. H.; Canfield, D. V.; Wang, S.-M. Quantitaion and Mass Spectromtric Data of Drugs and Isotopically Labeled Analogs; CRC Press: Boca Raton, FL, 2009; p 16, 52, 245. (10) Rösner, P. Mass Spectra of Designer Drugs [CD-ROM]; WileyVCH: Weinheim, Germany, 2015. (11) Rö sner, P. University of Kiel, Kiel, Germany. Personal communication, 2015. (12) Derivatization of Drug Substances with MSTFA; Analytix Notes 7, 2005; p 6. (13) Ogunbodede, O.; McCombs, D.; Trout, K.; Daley, P.; Terry, M. J. Ethnopharmacol. 2010, 131, 356−362.

Figure 4. Peak profiles of MSC derivatives, from the same extract, as acylated (a) and ditrimethylsilylated (b) species (corresponding to found MSC ng/crude tissue ng as follows: 0.84−0.86/151 (blue), 1.61− 1.70/302 (orange), 3.87−4.00/755 (green), and 7.29−8.12/1510 (red)), indicating in average 0.54% MSC content (5.5 RSD %); sample preparation in the Supporting Information. Mass spectra obtained from cactus in Figure S4, as acylated and as ditrimethylsilylated derivatives.

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CONCLUSION A novel approach was introduced for the quantitative and selective ditrimethylsilylation of PPAAs, including A and MDA, applying MSTFA in PYR medium, using catalysts (TMCS, TMIS). The optimum condition and analytical advantages of the new principle are compiled and contrasted with the recently recognized conditions1 in the present paper for the A and MDA extended acylation method (Table 1, Figures 1−4, and Supporting Information, Figures S1 and S2). Advantages, indicating the superiority of our new acylation and ditrimethylsilylation principles are comparable: both approaches provide directly injectable species onto the column, avoiding loss of derivatives, saving time, work, and cost of preparation process. Comparing responses of our two recent proposals it turned out that ditrimethylsilylation, on average, resulted in 1.7 times larger responses compared to acylation. Analytical performance characteristics were highlighted: R2 (in the range E


Quantitative Silylation Speciations of Primary Phenylalkyl Amines

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