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Characterization of Triacylglycerides in Vegetable upon MALDI LTQ Orbitrap XL Chris Weise1, Tilman Achstetter1, Gerd Kloeck1, Tabiwang Arrey2, Thomas Moehring2, Martin Zeller2, Kerstin Strupat2 1Hochschule, Bremen, Germany; 2Thermo Fisher Scientific, Bremen, Germany

Results Results MS³ spectra of protonated or sodiated DAG show evidence of neutral loss of ∆m=56 TABLE 1: Sodiated TAG molecules observed upon FTMS detection of olive , Overview (C3H4O) and ∆m=74 (C3H6O2). The observed neutral losses in combination with , and linseed oil (only TAGs with CN57 are illustrated); + + Purpose: Characterization of triacylglycerides in vegetable oils. In FTMS full scan data, several compounds are identified upon MALDI. TAG ions are Tandem MS experiments of selected monoisotopic TAGs reveal a neutral loss of a fatty observed fragment ions at m/z 529 [M-RCOO-74] , m/z 265 [M-(RCOO)2-56] and alternative fatty acyl composition are illustrated in brackets. Ln: linolenic acid, + exclusively detected as [M+Na]+ adduct ions. Furthermore, the presence of acyl residue, e.g. ∆m=282.256 is indicative of an oleic acyl residue and equals to a m/z 321 [M+Na-(RCOO)2] are characteristic for MS³ spectra and fragmentation L: , O: , S: Methods: Analysis using Orbitrap-based instrumentation, regular MALDI sample pathway of this compound class (Figure 6 and 7). Sodiated fragment ion at m/z 625.51 n diacylglycerides (DAGs) and free fatty acids (FFA), both protonated ions, is observed in neutral loss of C18H34O2 (Figure 4). MS² spectra are dominated by sodiated and preparation and MS and MS approaches. reveals a corresponding fragmentation pathway (not illustrated here). Due to the type of composition FTMS full scan data (see Figure 2). Sodiated TAGs consisting of 57 carbon atoms protonated DAG ion signals. This is demonstrated in Figure 5, including possible m/z [M+Na]+ Result: Vegetable oils are distinguishable based on their triacylglyceride (TAG) (=CN57) display the most intense signal, followed by decreasing signal intensities for proposals about structural composition. In MS³ experiments, neutral losses occurrence of two or more fatty acyl residues in tandem MS and MS³ experiments, (non-stereospecific) distributions. different TAG clusters having CN55 and CN53 (inset in Figure 2). Based on the FTMS corresponding to a fatty acyl residue occur; furthermore, compound-specific fragment several fatty acyl compositions are determined (see Table 1). Alternative fatty acyl olive 901.72556 LLL full scan data, the structural properties, such as number of carbon atoms (CN), the ions, are observed. Literature suggests that these fragmentations occur with five- or compositions are assigned as well. Introduction elemental composition and the degree of saturation - illustrated through Ring Double six-membered transition states; these depend on the stereochemical position of the 903.74121 OLL Bond Equivalents (RDBE) - of the given TAGs are determined. Different vegetable oils fatty acyl residue on the glycerol backbone3. Upon the growth of the world’s population consumption of vegetable oils increased by 905.75686 OOL exhibit species-specific TAG patterns; i.e. the most abundant TAG molecule in , 4.5 % in 2011. The European Union (EU) is a leading consumer of vegetable oils. By m/z 907.772, is more saturated in hydrogens (RDBE 5.5) than most abundant TAG 907.77251 OOO 2020, the annual production of vegetable oils is expected to expand by 23 % to 507 compound in linseed oil, m/z 895.678 (RDBE 11.5, see Figure 3). Measurements with sesame 901.72556 LLL million tons, only 30 tons will be produced within EU. Therefore, import rates for electrospray ionization (ESI) MS of these vegetable oils (not shown here, described vegetable oil into the EU are predicted to rise up to 42 %, which will be 18 % of the 903.74121 OLL elsewhere) reveal significantly more complex and more difficult to interpret mass entire production worldwide1. These facts as well as excessive fluctuation of the price Figure 4: MALDI FTMS MS² Scan of TAG (M+Na)+ 57:3 (m/z 907.772) from olive oil Figure 6: MALDI ITMS MS³ spectrum (ITMS + p MALDI MS³ [email protected] spectra as a result of the Na+- and NH +- adduct ions of TAGs formed in ESI mode. 905.75686 OOL give rise to questions about product safety. Due to the current market situation, cases 4 using 25% Normalized Collision Energy for CID, isolation width 2 u, an average [email protected] [165-2000]) of TAG 57:3 (m/z 907.772)  DAG 39:2 (protonated of economic adulteration of vegetable oils with non-authentic and less expensive oil of nine scans, detected with resolving power 60,000 at m/z 400. DAG, m/z 603.5), average of 21 scans, resolving power 60,000 at m/z 400. 907.77251 OOO (SOL) 2 Figure 2: (a) MALDI FTMS Full Scan (FTMS + p MALDI Full MS [150-2000]) of olive adjuncts occur frequently . It emphasizes that economic adulteration is a widespread 909.78816 SOO (SSL) phenomenon with an accelerating trend causing serious economic and health care oil; an average of 25 scans detected with resolving power 60,000 at m/z 400, + 265.4 ITMS + p MALDI MS³ [email protected] mass range m/z 150-2000, (b) Inset of MALDI FTMS Scan of olive oil; abundance DAG (MDAG+Na) walnut 897.69426 LLnLn issue. The costs caused by economic adulteration were numbered with US $12 billion 625.51585 m/z [email protected] [165-2000] for economy and with US $290 billion for health care. Considering mentioned issues, of TAG containing CN 53, CN55 and CN57 is displayed. 899.70991 LLLn the development of a fast and comprehensive analysis brings valuable information TAG (M +Na)+ 901.72556 LLL (SLnLn) and contributes to quality control and consumer’s health. b) TAG FTMS + p MALDI MS² 907.77212 ∆m = 282 903.74121 OLL (SLLn) m/z [email protected] [250-2000] a) ∆m = 282.256 Methods C18H34O2 905.75686 OOL (SLL) C H O ∆m = 56 907.77251 OOO (SOL, SSLn) Sample Preparation 379.09244 18 34 2 895.67861 LnLnLn α-cyano-4-hydroxycinnamic acid matrix (c=3.5 g/L, dissolved in 84 % Acetonitrile, 13 % C H O linseed 247.3 3 4 Ethanol, 0.003 % TFA) and vegetable oil (1µL dissolved in 1mL 100 % chloroform) are DAG (M+H)+ 529.5 897.69426 LLnLn used. Finally 1µL of 1:10 premixed matrix/analyte molecule solution is spotted and 603.53393 321.3 ∆m = 74 899.70991 LLLn (OLnLn) allowed to dry onto the MALDI plate. ∆m = 304.238 C3H6O2 901.72556 LLL (OLLn) Mass Spectrometry C18H33O2Na 603.53448 907.77271 603.5 903.74121 OLL (OOLn) A Thermo Scientific MALDI LTQ Orbitrap XL mass spectrometer is used to analyze 905.75686 OOL TAGs from vegetable oils (linseed oil, olive oil, sesame oil, walnut oil). Fourier transform mass spectrometry (FTMS) full scan information (60,000 resolution @ m/z 300 400 500 600 700 800 900 200 250 300 350 400 450 500 550 600 907.77251 OOO 400) are obtained in positive ion mode. Additionally, tandem MS data (MSn using FTMS m/z m/z and ITMS) of selected triacylglycerides using collision induced dissociation (CID) 200 400 600 800 1000 1200 m/z 1400 experiments are collected in order to reveal the structural compositions of TAGs. The workflow is outlined in Figure 1. Conclusion Data Analysis . MALDI, in combination with high-resolution, accurate mass detection, is a fast, Figure 3: Insets into MALDI FTMS Full Scan information of various vegetable reliable, valuable tool to distinguish vegetable oils qualitatively. MS and MSn data are post-processed using the QualBrowser feature of Thermo oils (inset into Full Scan MS data around m/z 900), average of 25 scans each. Scientific Xcalibur software version 2.1. a) olive oil, b) sesame oil, c) walnut oil, d) linseed oil. Detected with resolving . Full Scan MS and MSn are the key for sum formula and structural composition Figure 5: Same as Figure 4, MALDI FTMS MS² Scan of TAG 57:3 (m/z 907.772) Figure 7: Same as Figure 6, MALDI ITMS MS³ spectrum of TAG 57:3 (m/z power 60,000 at m/z 400 in the Orbitrap™ detector. proposals of endogenous TAGs. from Olive Oil using CID, including structural proposals with R = C H . 907.772)  DAG 39:2 (protonated DAG, m/z 603.5), including structural C H O Na 17 33 C57 H102 O6 Na 57 104 6 proposals with R = C17H33. . In spite of the general simplicity of ESI mode, MALDI proves to be more beneficial, 905.75708 907.77271 as only Na+- adduct ions are formed. This facilitates mass spectral interpretation of Figure 1: Workflow applied to characterize TAGs in various vegetable oils a) 6.5 RDBE 5.5 RDBE TAGs by MALDI over ESI. C57 H98 O6 Na C57 H100 O6 Na 901.72528 903.74133 O + + + Olive 8.5 RDBE 7.5 RDBE [M-(RCOO)2-56] [M-(RCOO)2] [M-RCOO-74] . Results of this study contribute to investigations of economic adulteration of Vegetable Oils (1 µL) dissolved in Chloroform (1 mL) CHCA Na+ Na+ m/z 265 m/z 321 m/z 529 vegetable oils. (c=3.5g/L)* O C R O 905.75714 903.74164 CH O + R 2 R O O O CH2 C C + + 7.5 RDBE 6.5 RDBE 907.77319 b) 901.72589 + References 5.5 RDBE C C O R [M+Na-RCOO] O HC 8.5 RDBE R C Sesame m/z 625.51585 O HC 1. OECD-FAO Agricultural Outlook 2011-2020 – Chapter 5 Oilseeds and oilseed O C CH2 olive sesame walnut linseed RCH2 products, 107-118 R O C O C 2. Ehling, S. [2011] - Consumer Product Fraud: Deterrence and Detection, C57 H96 O6 Na 901.72546 Walnut C O c) 8.5 RDBE H2C presentation, Annual Convention of Ozark Food Processors Association (GMA) 899.70990 903.74188 R C57 H94 O6 Na CH2 OR 7.5 RDBE R matrix - vegetable oil solution (10:1 (v/v)) 897.69427 905.75812 907.77478 3. Griffiths , W (2003) - Tandem Mass Spectrometry in the study of fatty acids, bile 9.5 RDBE O CH 6.5 RDBE 5.5 RDBE 2 C 247.3 10.5 RDBE R CH + acids and steroids, Mass Spectrometry Reviews, 22:81-152 C O [M+Na] 529.5 O 321.3 895.67908 m/z 907.77212 C H O Na 899.71063 + d) 57 92 6 Linseed O CH2 603.5 1.0 µL spotted onto MALDI sample plate & subjected to mass analysis 11.5 RDBE 897.69580 9.5 RDBE 901.72675 903.74194 [M-RCOO]+ 10.5 RDBE 8.5 RDBE 7.5 RDBE 905.75714 907.77338 see Figure 5 6.5 RDBE 5.5 RDBE m/z 603.53393

300 400 500 600 700 800 900 200 250 300 350 400 450 500 550 600 n 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 m/z All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Photographs of olives, sesame Full MS & MS data acquisition * dissolved in 84% ACN, m/z , walnuts and linseeds were made by KS. This information is not intended to encourage use of these products in 13% EtOH, 0.003% TFA) m/z any manners that might infringe the intellectual property rights of others.