Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb Extractive silylation method for high throughput GC analysis of flaxseed cyanogenic glycosides Durre Shahwar a , Lester W. Young a , Youn Young Shim a,b,c , Martin J.T. Reaney a,b,c, ⁎ a Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada b Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan University, Guangzhou, Guangdong 510632, China c Prairie Tide Diversified Inc., Saskatoon, Saskatchewan S7J 0R1, Canada ARTICLE INFO Keywords: Cyanogenic glycosides Linum usitatissimum L. GC-FID LC-MS/MS ABSTRACT The reported methods for the estimation of CG are indirect, long and tedious uses solvent extraction which results in lower recoveries due to several co-eluted components. The present study aims to develop and validate a high throughput method for the analysis of cyanogenic glycosides (CG) in flaxseed using extractive silylation. The experimental procedure comprised of preparation of trimethylsilyl (TMS) derivatives of CG, their quanti- tation through gas chromatography with flame ionization detector (GC-FID) and further characterization by LC- MS/MS. Different validation parameters determined in the experiment include the relative standard deviation both inter-day and intraday less than 5%, recovery in the range of 79.9–112.7%, limit of detection 4.72–6.43 μg/ mL and limit of quantitation 14.31–19.50 μg/mL. Combinations of silylation reagent were screened in a central composite experimental design in order to examine their effect on the extraction recovery. Finally, the developed method was applied successfully to quantify CG in various flaxseed cultivars. Advantages of the extractive si- lylation are simple preparation, short reaction times and the combination of extraction and silylation in one step which indicate that the method has the potential to sensitively and accurately determine CG where large numbers of samples are being routinely analysed. 1. Introduction Cyanogenic glycosides (CG), minor components found in the seed of mature flaxseed (Linum usitatissimum L.), are the major source of flax- seed toxicity due to the release of hydrogen cyanide (HCN) when hy- drolysed [1,2]. Once released the HCN could be toxic. There is some evidence suggesting that acute cyanide poisoning may result from dietary exposure to elevated levels of some CG [3,4] though this has not been associated with flaxseed. In the mature flaxseed, CGs constitute approximately 0.1% of the dry seed weight but can reach as much as 5% of dry weight in seedlings. The total amount of CGs in flaxseed from Canadian cultivars typically ranges between 365 and 550 mg/100 g seed [5–8]. These latter concentrations are not likely to prove toxic when whole flaxseed or flaxseed products are consumed by human or used in animal feed mixtures. Nevertheless, jurisdictions such as Japan restrict the import of flaxseed due to its CG content. A rapid, cost-ef- fective, high throughput analytical method to determine CG con- centrations in flaxseed will be beneficial for analysis of process mate- rials, enforcing regulations and selection of plant breeding materials. Researchers have dedicated considerable efforts into the development of reliable methods for detecting and determining the concentration of cyanogenic compounds in an array of matrices. Both colorimetric and chromatographic techniques have been developed [9–11]. Colorimetric methods may detect cyanide evolution following either enzymatic or chemical hydrolysis of CGs [12–14]. Advantages of colorimetric techniques include more rapid chemical hydrolysis at elevated temperatures and higher throughput, however, greater in- accuracies are observed than with other methods [15]. Although en- zymatic hydrolysis of CGs followed by colorimetric determination is as sensitive as high-performance liquid chromatography (HPLC) detection of these compounds, these methods cannot be applied for identification and quantification of the specific CG because estimation based on total cyanide evolution rather than detection of its source. Moreover, enzy- matic hydrolysis needs separate ß-glucosidases for different CG and variability in the concentrations of these enzymes in seeds likely in- creases assay variability [16,17]. More direct analysis of CG is accomplished by separation-based analytical techniques such as HPLC and gas chromatography (GC) which produce acceptable results due to their high resolving power and automation [6,8,18]. Due to the polar nature of CG, HPLC and GC https://doi.org/10.1016/j.jchromb.2019.121816 Received 16 May 2019; Received in revised form 23 September 2019; Accepted 24 September 2019 ⁎ Corresponding author at: Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada. E-mail address: martin.reaney@usask.ca (M.J.T. Reaney). Journal of Chromatography B 1132 (2019) 121816 Available online 21 October 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved. T