The use of trimethylsilyl cyanide derivatization for robust and broad-spectrum

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1 Analytical and Bioanalytical Chemistry Electronic Supplementary Material The use of trimethylsilyl cyanide derivatization for robust and broad-spectrum high-throughput gas chromatography-mass spectrometry based metabolomics Bekzod Khakimov, Mohammed Saddik Motawia, Søren Bak, Søren Balling Engelsen Additional safety consideration Most silylation reagents require careful handling and users must follow all safety instructions. According to European Regulation (EC) No 1272/2008, the silylation reagents TMSCN, MSTFA, as well as other reagents such as BSTFA, MTBSTFA, BSA, TMCS are all flammable volatile liquids and harmful in contact with skin, eye and inhalation. Safety data of all these chemicals suggest careful handling (avoid air, moisture, contact with skin, eye, and swallow) and storage (cool, well-ventilated place, under inert gas, protected from direct sunlight and water which causes their decomposition). Stability and reactivity data show that all these reagents require similar conditions to avoid possible danger and degradations: no heat, flame, sparks and water. Avoid strong acids, base, aldehydes, ketones. The difference in boiling points of TMSCN (115 C) and MSTFA (131 C) is not significant, while BSA (72 C) and TMCS (57 C) have relative lower boiling point that increases their volatility. As part of this study, we evaluated evaporation of silylation reagents TMSCN, MSTFA as well as methoximation reagents and silylation reagent (M-TMSCN, M-MSTFA) both, at room temperature and at the incubation temperature used during derivatization. 100 µl of reagents were sealed in GC- MS vials using the same magnetic-silicon septum caps that are used throughout the analysis and 1

2 penetrated four times by needles installed in the autosampler. The volume of the reagents, TMSCN and MSTFA did not change after 48h of incubation at both temperatures, while the volume of M- TMSCN and M-MSTFA was reduced by 10-20%. The volume reduction of M-TMSCN and M- MSTFA may rather be due to the readily volatile pyridine used in methoxiamination step. ur data documents, that when using appropriate needles (D: 0.5 mm) and GC-MS vial septum caps, the reagents do not evaporate out of the GC-MS encapsulated vials even after two days which ensure both a safeness of derivatization reactions and high reproducibility. Most GC-MS labs that perform high-throughput analysis utilize autosamplers that facilitates further derivatization accuracy and safer handling of reagents. All silylation reagents produce byproducts during the reaction: MSTFA (byproduct: N- Methyltrifluoroacetamide), BSTFA (byproduct: mono(trimethylsilyl)trifluoroacetamide and trifluoroacetamide), BSA (byproduct: N-trimethylsilyl-pivalimidic acid) and TMCS (byproduct: hydrochloric acid) fall into the similar categories of hazard classifications (according to European Regulation (EC) No 1272/2008) as the byproduct of TMCN, hydrogen cyanide (CN). Most silylation protocols use µl of silylation reagents for derivatization of dried complex extracts of plants and/or animal origin. In this study, according to the reaction stoichiometry, the amount of TMSCN used for derivatization of standard mixture was times more than the amount of the reagent needed for silylation of all available active protons in the standard mixture. Moreover, the byproduct, CN might be consumed during protonation of TMSCN to form TMSCN +, since the basicity of TMSCN is much greater than the basicity of CN. Therefore, the silylation reaction rate may even increase because TMSCN + is more electrophile than TMSCN and easily attracts nucleophile substrate [1]. In addition, we have estimated the amount of the byproduct, CN formed during a standard silylation reaction to evaluate the potential toxicity. Potential toxic concentration of CN in the air is 300 mg/m 3 [2], while the smallest size of most standard laboratories is 60 m 3. ne mole of TMSCN produces one mole of CN, thus mole (an amount that is used in this study) of TMSCN produces mole of CN, that is equivalent to X = g = mg. For example, if one hundred samples are derivatized simultaneously, a total of mg of CN will be formed. In an average laboratory of 60 m 3, this amount corresponds to a concentration of mg/m 3. This amount is 20 times less than the toxic amount of CN for human health, which is 300 mg/m 3. A CN concentration of mg/m 3 may form only if all TMSCN (40 µl) is used, in all 100 samples, and evaporated. Liberation of this amount of CN is 2

3 very unlikely, mainly due to the small amount of TMSCN that reacts (as mentioned earlier TMSCN is in excess for complete silylation, and accordingly 1/200 of used TMSCN will form CN), sample is moisture free and vials are tightly sealed with septum leads. Therefore, application of TMSCN in high-throughput GC-MS analysis is safe and may provide easy and powerful silylation. 1. M.V.Kashutina, S. L. Ioffe, V. A. Tartakovskii, Rassian Chem. Rev. 44 (1975) Environmental and ealth Effects. Cyanidecode.org. Retrieved on MultiPurpose Sampler (MPS) and Cooled Injection System (CIS) parameters GC-MS injection parameters of the left MPS equipped with 10 µl syringe were as follows: injection was performed in sandwich mode, top air volume 1.0 µl, air volume below 1.0 µl, air volume above 1.0 µl, injection volume 1.0 µl, injection speed 50.0 µl s -1, fill speed 0.2 µl s -1, viscosity delay 4 seconds, pre and post injection delay 2 seconds, vial penetration 30 mm, and injection penetration 40 mm. Parameters of the right MPS, equipped with 100 µl syringe were as follows: add volume 40 and/or 80 µl, add speed 5 µl s -1, viscosity delay 4 seconds, post add delay 2 seconds, eject speed 50 µl s -1, source vial penetration 30 mm and destination vial penetration 28 mm. Both syringes were pre and post washed two times with acetone followed by n-hexane. The agitator parameters for sample incubation were as follows: incubation time varied depending on the chosen silylation time, agitator speed was 750 rpm, agitator on time was 59 seconds, agitator off time was 1 second and agitator temperatures were 30 C and 37 C for methoxiamination and silylation, respectively. The CIS port parameters were as follows: initial temperature 40 C, equilibration time 1.0 min, initial time 0.5 min, heating rate 12.0 C s -1, end temperature 320 C and hold time 0.5 min. 3

4 Important considerations in automation of derivatization and GC-MS analysis igh-throughput GC-MS metabolomics requires the use of robots prior to reduce the level of experimental error. owever, automated and simultaneous derivatization of samples requires derivatization reagents to be present in the autosampler at all times throughout the analysis. Most of the commercially available derivatization reagents requires specific storing conditions prior to avoid degradation and reactivity lost. It is important to fulfill the storing conditions of the reagents recommended by the manufacturers when keeping them on the autosampler for a long time. Moisture and direct sunlight can easily cause degradation of the reagents, silyl-derivatives and/or alter the original content and in turn introduce a bias in the silylation of samples [13, 14]. It is also important to use an excess amount of reagent for complete silylation of metabolites and to suppress a little amount of water, which might be present in the reaction mixture. ydrolytic stability of the silyl-derivatives highly depends on the structural and steric features of the molecules. The general hydrolytic stability of silyl derivatives of the different classes of compounds decreases in the following order: alcohols > phenols >carboxylic acid > amines > amides [15]. Moreover, stability of trimethylsilyl derivatives depends on the type of the injection port used in the analysis. Despite most silyl-derivatives of metabolites are thermally stable, they may degrade in contact with stainless steel injection ports at high temperature, and therefore it is recommended to use glass injection ports (e.g. glass liners) for high-temperature GC injections [15]. Most high-throughput GC-MS metabolomic studies aimed to obtain a quantitative data. In order to compare GC-MS profiles of these samples, it is crucial to keep the derivatization reaction time (time interval between the addition of derivatization reagent into sample and GC-MS injection) constant over the entire analysis, as derivatization time differences alters the GC-MS profiles of the identical samples significantly, both qualitatively and quantitatively. Moreover, caution must be taken not to use polar solvents, with active protons (e.g. R, RC), throughout the derivatization (even for the injection needle wash), since they can easily react with the silylation reagent. Likewise, appropriate GC column with inert stationary phase (e.g. silicon-based columns) must be used for the analysis of trimethylsilylated samples. Injection of silylation reagent into the GC column with a polar stationary phase (e.g. polyethylene glycol based and free fatty acid based columns) will result in unreliable GC profiles with artifacts and column degradation products. 4

5 Table S1. Derivatization products of the metabolites of standard mixture, in the same order as Table 1 Entry Substrate Derivatized product 1 Valine NSiMe 3 2 Benzoic acid Entry Substrate Derivatized product 13 Phenylalanine N 2 14 Phenyllactic acid 3 Serine Me 3 Si N ydroybenzoic acid Me 3 Si C C 4 Threonine 16 Vanillin-MEX N 2 C Glycine Succinic acid C N Me 3 Si SiMe 3 Me 3 Si ydroxy-3-methoxybenzoic acid (trans)-ribose C 3 Me 3 Si N Me 3 Si C 3 7 Serine Me 3 Si C NSiMe 3 19 Ribitol Me 3 Si Me 3 Si 8 Threonine C 9 Aspartic acid NSiMe 3 Me 3 Si N Vanillic acid 2-ydroxycinnamic acid C ydroxyaceto -phenone 11 Malic acid Me 3 Si 22 (trans)-glucose Me 3 Si Me3 Si Me 3 Si N C 3 C 23 p-coumaric acid Me 3 Si 12 Vanillin C 3 24 (cis)-glucose Me 3 Si Me 3 Si Me 3 Si Me 3 Si N C 3 5

6 Entry Substrate Derivatized product Entry Substrate Derivatized product 25 Palmitic acid C 3 (C 2 ) Cholestrol 26 (trans)- Caffieic acid Me 3 Si Me 3 Si 27 (trans)-glucose- 6-phosphate-MEX P Me 3 Si Me 3 Si N C Amyrin Me 3 Si 28 (cis)-glucose- 6-phosphate-MEX P Me 3 Si N Me 3 Si Me 3 Si C Amyrin Me 3 Si 29 Sucrose Me 3 Si 37 Lupeol Me 3 Si 30 (trans)-maltose- MEX SiMe Me 3 3 Si Me 3 Si N C 3 38 leanolic acid Me 3 Si 39 Betulinic acid 31 Naringenin Me 3 Si Me 3 Si 40 Epoy- -amyrin Me 3 Si 32 Catechin Me 3 Si 41 ederagenin Me 3 Si 6

7 Fig. S1. (a) PCA scores plot of the replicate data matrix (16 x 41), with 16 samples, 4 replicates per method and abundances of 41 metabolites listed in Table 1. (b) ANVA analysis of the four derivatization methods (TMSCN, M-TMSCN, MSTFA and M-MSTFA) based on their scores on PC1, (c) ANVA analysis of the four derivatization methods based on their scores on PC2 7

8 Fig. S2. Comparison of total ion current chromatograms of trimethylsilyl derivative of 2,6-diphenyl-phenol at four different silylation time points, (a) 5 seconds, (b) 30 seconds, (c) 1 minute and (d) 5 minutes with two silylation reagents, TMSCN and MSTFA. *Silylation time refers to the incubation time of sample in agitator after addition of the reagent, excluding 35 seconds of robot operation time for washing needle, delays and injection 8

9 Fig. S3. Scores (a) and loadings (b) plots of PCA model developed on a matrix containing PARAFAC2 scores (relative abundances of all peaks including MEX-TMS derivatives of carbohydrates) of TMSderivatives of standard mixture detected by four derivatization methods, at 12 different silylation time points (including four replicates at the optimal silylation times) 9

10 Fig. S4. Relative peak abundances of trimethylsilyl derivatives of caffeic, malic, p-coumaric and phenyllactic acids in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and ln(min) scale of silylation time was used for better visualization 10

11 Fig. S5. Relative peak abundance of trimethylsilyl derivative of 2-ydroxy-3-methoxybenzoic acid in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 11

12 Fig. S6. Relative peak abundance of trimethylsilyl derivative of Naringenin in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization.. 12

13 Fig. S7. Relative peak abundance of trimethylsilyl derivative of Catechin in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 13

14 Fig. S8. Relative peak abundance of trimethylsilyl derivative of Succinic acid in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 14

15 Fig. S9. Relative peak abundance of trimethylsilyl derivative of 2-ydroxycinnamic acid in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 15

16 Fig. S10. Relative peak abundances of trimethylsilyl derivatives of serine in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 16

17 Fig. S11. Relative peak abundances of trimethylsilyl derivatives of threonine in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 17

18 Fig. S12. Relative peak abundances of trimethylsilyl derivative of glucose in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 18

19 Fig. S13. Relative peak abundances of trimethylsilyl derivative of glucose-6-phosphate in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 19

20 Fig. S14. Relative peak abundance of trimethylsilyl derivative of sucrose in four different derivatization methods (TMSCN, M-TMSCN, MSTFA, M-MSTFA) over silylation time range of 5 minutes to 30 hours. *Relative peak abundances of TMS-derivatives were calculated using PARAFAC2 modeling and logarithmic scale of silylation time was used for better visualization 20