Determination of co-eluted isomers in wine samples by application of MS/MS deconvolution analysis.

Introduction Wine is defined as an alcoholic beverage, which is produced by fermentation of fresh grapes or must and the organic acids of beverages are important in several respects. All organic acids have this quality to some degree, but some have their own characteristic flavour, taste or aroma. For example, citric acid has a fresh acid flavour different from that of malic acid, while succinic acid has an unusual salty, bitter taste in addition to its sourness [1]. Wine is also rich in phenolic compounds that are important components with health promoting properties and they also affect organoleptic characteristics, such as colour, astringency and aroma [2]. Recently, Ginjom et al. identify and quantify the individual phenolic compounds present in wine at different wine-making stages from crushing through to bottled and aged wine, produced in different Queensland (Australia) wineries [3]. Interestingly, they were not able to identify a compound (named U1) with a low molecular mass (m/z at 175) and relatively large eluting peak. In a recent paper, our research group have identified in wines two isomeric compounds, corresponding to U1, with molecular weight of 176 Da by using two LC-MS systems, i.e. ion trap (LC-IT) and LC-Q-Orbitrap. The two isomers were organic acids, i.e. 3-isopropylmalic acid (3-IPMA) never identify in wines, and 2-isopropylmalic acid (2-IPMA), never quantify in wines [4]. In this study, the quantification of these two compounds was performed by using LC-IT in ten among red and white wine samples and the average concentrations were determined at 1.78 mg L-1 (range 0.56-4.13 mg L-1) and 23.0 mg L-1 (range 6.7-41.6 mg L-1) of 3-IPMA and 2-IPMA, respectively. The LC-IT method used MS/MS analyses at 0.9 V excitation amplitude by extracting the product ions m/z 73 and m/z 115 for 3-IPMA and 2-IPMA respectively. Thus, the two compounds were separated by using extract ion chromatogram (EIC) of the two characteristics m/z ions. However, the different fragmentation yield of their product ions affects on the quantitation sensitivity. Therefore, to ensure the similar sensitivity between two isomers, their chromatographic separation is needed with increasing the analysis time. In order to ensure the requested specificity of the method, a different approach, based on a series of energy resolved MS/MS experiments, was carried out. By this approach a clear differentiation among all the isomers was obtained but, to emphasize such differences, it was necessary to develop a mathematical algorithm that distinguishes the MS/MS spectra of the isomers. This algorithm (LEDA) consists in the application of matrix of linear regression equations to different experimental data. In our case, the experimental data used were the abundance ratios of product vs. precursor ions selected during the MS/MS method set-up [5-7]. In this way, it was possible to resolve the MS/MS spectra assigning the correct signal to the isomer also for chromatographically unresolved peaks. Experimental The LC-MS/MS analysis was carried out using a Varian 1200L triple quadrupole system (Palo Alto, CA, USA) equipped by two Prostar 210 pumps, a Prostar 410 autosampler and an Elettrospray Source (ESI) operating in positive ions. The product ion scan spectra were acquired in the m/z range from 50 to 650, scan time 600 ms; argon was used as collisional gas and the collision energy (CE) was increased stepwise in the range 5 to 40 V. The obtained energy resolved tandem mass spectrometry (ERMS) spectra were employed to study the fragmentation of molecular species of studied isomers and build their breakdown curves. The chromatographic parameters employed to analyse the samples were tuned to minimize the run time. The column used was a Pursuit XRs C18 30 mm length, 2 mm internal diameter and 3 m particle size, at constant flow of 0.25 mL min-1, employing a binary mobile phases elution gradient by 10 min. total run time. The solvents used were 10 mM formic acid in water solution (solvent A) and 10 mM formic acid in acetonitrile (solvent B). The LEDA post-processing mathematical tool was used to guarantee the identification of the isomer present in analyzed samples without their chromatographic separation. The algorithm is based on the consideration that each MS/MS spectrum might be represented as the sum of contribution of each isomer present in the unresolved chromatographic peak. In order to obtain reliable data, the relative abundances of the different product ions were calculated with respect to the reference ion abundance, so that possible misleading results due to compound-dependent different product ion yields are avoided. For this purpose, the available signal of the precursor ion was acquired as reference ion (Ri) which allowed us to obtain the characteristic ratios among the selected product ions for isomer speciation. Therefore, knowing the characteristic abundance ratios of pure isomer, a deconvolution of these spectra is possible based on a series of linear regression equations as follows: (P_i⁄R_i )_(m )= ∑_(x=1)^n▒〖(P_i⁄R_i )_x*〖[%] 〗_x 〗 (1) Where: (Pi/Ri)m: is the abundance ratio between the product (Pi) vs reference ions (Ri) measured (m) in the sample; (Pi/Ri)x : are the characteristic abundance ratios between the product ion vs reference ion of pure isomers; [%]x: is the concentration (%) of isomers in the sample. Precision and accuracy of LEDA algorithm were evaluated by the LC-MS/MS analysis of standard mixtures prepared with different composition of the isomer pair. Then, the LC-MS/MS method with LEDA post-processing tool was apply to the determination of 2-IPMA and 3-IPMA in real samples of wine extracts prepared as reported by Ricciutelli et al [4]. Results The quantitative results obtained applying the LEDA approach on the abundance ratios of data collected from the MS/MS analysis of the standard mixtures are: accuracy 97.3 % and 96.3 % with a precision, expressed as RSD %, of 1,9 and 1,8 for 2-IPMA and 3-IPMA respectively. Also the application of LEDA on the wine samples shows a good correlation between the quantitative data obtained with the classical chromatography separation and linear equations deconvolution analysis of MS/MS spectra. Conclusions The proposed LC-MS/MS method coupled with mathematical algorithm (LEDA), applied to deconvolute the MS/MS spectra from unresolved chromatographic peak, was effective, allowing the determination of the isomers presents in wine samples. The obtained results confirm the ability of the LEDA approach to deconvolute a mixture of isomers from chromatographically unresolved peaks, allowing short analytic runs and high throughput in processed samples. It is to emphasize that, in the last decade, many investigation were carried out to develop ancillary MS technique able to give a different analytical dimension to the researcher in the field (e.g. ion mobility methods). In this paper we have shown that the energetic dimension of MS/MS experiments can be fruitfully employed to solve isomers characterization and quantification problems. Reference G.C. Whiting; Journal of the Institute of Brewing, 82 (1976), pp 84-92. J.A. Kennedy; Ciencia E Investigacion Agraria, 35 (2008), pp 107–120. I. Ginjom B. D’Arcy, N. Caffin, M. Gidley; Food Chemistry, 125 (2011), pp 823–834. M. Ricciutelli, S. Moretti, R. Galarini, G. Sagratini, S. Vittori, S. Lucarini, G. Caprioli; Food Chemistry, 294 (2019), pp. 390-396. M. Menicatti, L. Guandalini, S. Dei, E. Floriddia, E. Teodori, P. Traldi and G. Bartolucci; Rapid Commun. Mass Spectrom., 30 (2016), pp 423-432 M. Menicatti, L. Guandalini, S. Dei, E. Floriddia, E. Teodori, P. Traldi and G. Bartolucci; Eur J Mass Spectrom., 22 (2016), pp 235-243 M. Menicatti, M. Pallecchi, S. Bua, D. Vullo, L. Di Cesare Mannelli, C. Ghelardini, F. Carta, C. T. Supuran and G. Bartolucci; J Enzyme Inhib Med Chem., 33 (2018), pp 671-679