Hexamethyldisilazane and perfluorocarboxylic acid couples achieve trialkylsilylation and acylation of active proton containing organics in a single step

A novel, multifunctional group mediated one-step derivatization (MGOD) method has been developed employing the couples of hexamethyldisilazane (HMDS) and perfluorocarboxylic acid (PFCA). 1 MGOD relies on the unique reactivity of HMDS and PFCA to provide simultaneous trialkylsilylation and acylation in a single step. While primary aliphatic amines and diamines are generally nonreactive (certainly secondary and tertiary amines are nonreactive either), MGOD allows for trialkylsilylation of amino acids (AAs), amino alcohols, amino sugars, 2,6-diaminoheptanedioic acid, di-and tripeptides, and in turn moderates the basicity of their free amino groups and facilitates subsequent acylation. The identification, quantification and stoichiometry of MGOD were characterized by selective mass fragmentations and monitoring by GC–MS. Reaction conditions were optimized and analytical performance characteristics were established with respect to a reproducibility of 5.6 RSD%, a linearity of R 2 = 0.996 and limit of quantitation values (LOQ) of 41–75 pg/μL. Derivatives were prepared without preliminary extraction steps, avoiding the use of organic solvents. The practical utility of MGOD was evidenced by derivatization of amino acids in a branched-chain amino acid (BCAA), nutritional supplement (recovery data of standard addition to BCAA varied between 92 and 110%.), in 100, 200 and 300 µL volumes of human urine demonstrating the proportionality of the method, and in peptide hydrolysates.


Introduction
Since the pioneering work of Pierce [1] numerous silylating reagents, including HMDS, have been applied in the preparation of volatile products from organics with exchangeable-proton-containing groups [2].In the case of AAs, silylation and the use of GC (instead of special apparatus requiring an amino acid analyzer) were preferred [3].However, after many efforts it has turned out that (i) silylation-based processes, when employed alone, fall short: unambiguous, stoichiometric derivatization resulting in a single reaction product could not be obtained [3]; and (ii) even performing one step alkyl-chloroformate derivatization needs compromise in terms of side products [4].Yet, the overall significance of the topic and the difficulties encountered with the HPLC techniques -attributable to ion suppression phenomenahave led to the introduction of new GC-MS based proposals [4][5][6][7][8][9][10][11].Mainly one-step trialkylsilylations [5][6][7][8][9][10][11] and two-steps derivatization [12] (1st step silylation, 2nd step acylation) have been suggested.As to the present study (based on our recent experiences [13][14]) we introduced the novel MGOD principle: we could expect the full derivatization of AAs (simultaneous trialkylsilylation and acylation), with the same HMDS & PFCA reagent, in a single step.Thus, we were delighted to see that our assumption in cases of AAs met the requirements: under well-defined conditions the N-acyl, O,S-TMS-TMS ester species are formed, in a stoichiometric manner.In addition, full derivatization has been confirmed with all those types of organics that are able to react with the HMDS & PFCA couples, manifesting trialkylsilylation and acylation in a single step Scheme 1).This phenomenon has proved to be a novelty in chemistry, especially in analytical chemistry.
In this study, our aims were to optimize the novel MGOD approach: (i) demonstrating the selective fragmentation patterns and the types of the multifunctional group containing organics monitored by GC-MS, (ii) varying reagent composition including solvent selection, time and https://doi.org/10.1016/j.microc.T temperature variation to define optimum analytical conditions, (iii) confirming stoichiometric behavior of the novel approach, in standard solutions, and (iv) highlighting practical utilization of the MGOD method, determining the free amino acid contents of a nutritional supplement, human urines and peptide hydrolyzates.

Sample preparation
Model compounds (0.1-1.0 mg/mL) and the corresponding amounts of BCAA samples and peptide hydrolyzates, weighted with analytical precision were dissolved in 2 M hydrochloric acid and further diluted into one or more unified stock solution containing aliquots, about 0.7-1.1 × 10 −3 g analyte in each.Model solution, various amounts (100, 200, 300 µL) of centrifuged urine, BCAA and peptide hydrolyzates were rotary evaporated to dryness at 30-40 °C.The residues were treated with 100 µL PYR or ETAC or ACN + 70 µL HMDS and 30 µL TFA or 30 μL PFPA or 30 µL HFBA and heated at 60, 70, 80 or 100 °C, for 10, 30 or 50 min.Thereafter, the differently diluted solutions were transferred into the autosampler vial and 1 µL injected into the GC-MS system.Note: it should be remarked that all derivatization tests were performed in parallel and injected three times of each.
Derivatization under optimum condition was performed with TFA as detailed above: dried residues were treated with 100 µL PYR, 70 µL HMDS and 30 µL TFA heated at 80 °C, for 30 min.

Instrumentation
The apparatus consisted of a Varian 240 GC-MS/MS system (Varian, equipped with a Varian CP-8400 Autosampler, and with Programmable Injector (SPI).Walnut Creek, CA, USA).The column used was a product of Agilent (Santa Clara, CA, USA); Agilent J & W GC column, HP 5MS: 30 m × 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 gradient conditions, the optimized temperature programs, different for the SPI and for the column, were as follows: injections were made at 280 °C, and held at 280 °C for 3 min, then cooled down to 100 °C (100 °C/min), column temperature starts at 100 °C, held for 1.00 min, then heated up to 145 °C for 10 °C/min, then 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 2.00 min hold at 280 °C (total elution time was 20.20 min).Helium (purity: 6.0, which means He of 99.9999%) was used as a carrier gas, with the column flow rate fixed at 1 mL/min.The general MS parameters were: Fil/Mul delay: 2.00 min; mass defect: 0 mmu/100 µ; filament current: 25 µA; Target TIC: 20,000 counts; Prescan Ion Time: 1500 µs; Scan mode: Fast; Scan Time: 0.17 s/scan; Multiplier offset: Autotune + 100 V; electron energy: 70 eV.
Investigations performed as a function of the reaction time (10, 20, 30 and 50 min) and temperatures (60, 70, 80 and 100 °C) (detailed data not shown), revealed that quantitative MGODs need at least 20 min reaction time and 70 °C temperature, and do not change under any fortified conditions: neither at extended reaction time nor at increased temperature.Thus, remaining on the safe side, as optimum, 30 min reaction time and 80 °C reaction temperature were selected.

Fragmentation pattern analysis
The fragmentation process of fully derivatized species in all cases investigated, led to characteristic high masses (Table 1, Table S1, Fig. 1 (2) selective, decomposition products of the molecular ions obtained by the loss of one methyl group ([M-15] + ), (3) specific reactions of the molecular ions with one TMS radical, manifesting 'self-chemical ionization' by the species [M+TMS] + or [M-15+TMS] + .These fragmentation phenomena are characteristic to the ion trap detector, independently of the reagent partners (the type of PFCA, AA, amino alcohol, peptide, etc.) used (Table 1, Table S1, Figs. 1 and 2, Fig. S1).
Further details demonstrate the peak profiles and mass spectra of the N-TFA, O,S-TMS-TMS ester derivatives of selected AAs (Fig. 1) and of those of amino alcohols, amino-glucose, 2,6-diaminoheptanedioic acid and peptides derivatives (Fig. S1, Supplementary Materials).
The above listed fragmentations all, are supporting the unambiguous correlations and proofs provided by the novel MGOD process.Unfortunately, reproducible derivatives from arginine have not been obtained.

MGOD' s selectivity
In order to gain further insight into the understanding of MGOD, it should be emphasized that primary aliphatic mono-and diamines do not react at all.This is of primary importance and serves as completing evidence in understanding the peculiarity of the MGOD process (Scheme 1.); full derivatization (i) takes place exclusively of those analytes that have active proton containing functional groups (Scheme 1, A species); (ii) The process very likely starts with trialkylsilylation, resulting in a decrease in the basicity of the analyte's amino group (Scheme 1, B species), initializing the immediate availability for acylation (Scheme 1, C species).

Solvent and reagent selection for MGOD
When varying the solvent, PYR was preferred, prior to separation by GC (reaction mixtures containing ETAC and ACN formed two phases).
Regarding the reagent's PFCA compounds TFA proved to be the best selection in terms of the retention and response properties of the derivatives (Table 1, Table S1 and Table S2).Since, comparing retention and response values, it can be stated that of the tested 19 AAs (i) elution times of the N-TFA derivatives proved to be the shortest (Table 1, Table S1), and that (ii) the N-TFA derivatives provided considerably greater response values in the overwhelming number of AA derivatives (13 out of 19), contrasting with the corresponding N-TFA, N-PFP and N-HFBspecies (Table S2).
Concerning the optimum molar ratio range of the HMDS & TFA couple, it has been repeatedly shown [12] that the range of [HMDS]/

Practical utility of the method
An unlimited number of applications of this new approach can be expected.Derivatives were prepared from the evaporated dry species, without preliminary extraction steps.Subsequent to MGOD, derivatization products were directly injected onto the column, thus, it is avoiding all the disadvantages associated with the use of organic solvents, and satisfying the requirements of green chemistry.The practical utility of the proposal is shown a) by the quantitation (including recovery studies with standard additions) of the nutrition supplement's BCAA (Fig. 3a, b), b) by the determination of various amounts of females' urine quantitating their free AAs (Table 2., Fig. 4.) and c) by measuring the composition of peptide hydrolysates (Fig. S2.).
It is important to underline that the presentation of practical utility of the novel MGOD was combined with our direct sample preparation working strategy [13][14][15].

BCAA samples
In the context of this study all response values were calculated from AA/μL injection: 1 μL aliquots of 200 μL derivatized solutions (diluted 20x) contained 12.0-13.0ng BCAA sample, in total.
On one hand, proportionality proof was provided by the quantitation of various amounts of the BCAA samples (Fig. 3a).Val, Leu, Ile, Asp and Orn contents were expressed in ng/injected AAs and characterized with the relative standard deviation percentages (RSD%, in parentheses) varying between 1.63 and 6.1 RSD%.
On the other hand, common analytical performance characteristics were tested via the standard addition of the initially present AAs (Val, Leu, Ile, Asp and Orn) completed by the addition of an 'external standard' Tyr (Fig. 3b).
Fig. 3b reveals the linearity, recovery and repeatability values of the free AA contents of BCAA samples.Excellent linearities of 0.996-0.999and recoveries of 92-110% were confirmed.Intersection values of horizontal axes correspond to the initial AA contents of the BCAA samples in ng/injection; i.e.Val (1.42 ng), Leu (3.72 ng), Ile (1.59 ng), Asp (0.45 ng) and Orn (0.36 ng) were obtained in accordance with the nominal AA contents within less than 7.0%.Repeatability of parallel tests, (RSD percentages in parentheses) varied between 2.98 RSD% and 6.5 RSD%.Regarding Tyr, responses (3.64 RSD%) proved to be in excellent accordance with those obtained from model solution determined in parallel.

Conclusion
A selective and quantitative novel analytical process has been presented, providing full derivatization of amino acids, amino alcohols, amino sugars, amino carboxylic acids and di-and tripeptides, without extraction, in a single step.
The recognition of this novel method can be attributed to the unique reactivity of the hexamethyldisilazane & perfluorocarboxylic acid couples in all those cases when the analyte possesses active proton containing function.In these cases, trialkylsilylation and the immediate initialized acylation proceed in a single step.The crucial point of this approach, its selectivity, needs to be underlined: aliphatic monoamines and diamines do not react at all, evidencing the need for the introductory trialkylsilylation of the analyte leading to an increased concentration of the deprotonated amino entity, which is thereby immediately available for acylation.
As to the reagent's PFCA content TFA proved to be the best selection: in terms of their derivatives' and response properties.Comparing responses and reproducibility values, out of the tested 19 AAs N-TFA derivatives' elution times proved to be the shortest and N-TFA derivatives provided considerably higher response values in the overwhelming part of AA derivatives (in 13 out of 19), contrasting the corresponding N-TFA, N-PFP and N-HFB-species.
Analytical performance properties (averages of several model solutions) obtained under this study, provided an average reproducibility of 5.6 RSD%; linearity (in the range of 41-5370 pg/injection) R 2 0.996; LOQ (in the range of 41-75 pg/μL values) of 58 pg/μL; and recoveries between 92 and 110%, calculated from standard additions of AAs.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Fig. 1 .
Fig. 1.Peak profile and characteristic mass spectra of representative N-TFA, O,S-TMS-TMS ester derivatives of AAs.Detailed data in Table1.

Fig. 3 .
Fig. 3. (a, b) Analytical performance characteristics of the MGOD method.(a) Proportionality proof of the AA contents of various amounts of BCAA samples obtained with the MGOD method.Analyses were performed with 0.45-9.03ng injected AAs (bold printed data).In parentheses relative standard deviation percentages (RSD%).Samples were derivatized in parallels and injected three times of each.(b) Analytical performance characteristics obtained via standard addition of the initial constituents (Val, Leu, Ile, Asp, Orn) of the BCAA sample, completing with Tyr as external standard.All response values are related to ng AA/μL injection: aliquots of 200 μL derivatized solutions, diluted 20 x, containing 12.0-13.0ng BCAA sample in 1 μL injected solution.