Chemometric approaches in calibration experiments of trilinolenoylglycerol by liquid chromatography ion-trap mass spectrometry

Abstract

The article presents a comprehensive account of the application of chemometric approaches to determine the factors that influence the tri-␣-linolenoylglycerol (TALG) ammoniated adduct signal m/z 890.6 in an ion-trap mass spectrometer coupled to a liquid chromatograph and the estimation of different sources of errors involved in TALG calibration experiments. It was found that by using experimental design, the influence of the nebulizer pressure on the analytical signal is less pronounced than the influence of the drying gas flow and the chromatographic flow rate. The results revealed that without using tandem mass or atmospheric pressure chemical ionization, it is possible to fragment the TALG backbone and obtain the free ␣-linolenic acid by varying the drying gas flow and using an electrospray interface. The error decomposition approach revealed that the preparation error was 26 times higher than the instrumental error. [4,5]. Quantification of TAG using coupled techniques such as LCMS depends mainly on two aspects, the sample preparation technique and the instrumental condition settings. Well-established protocols and books [6 -8] have been devoted to the former and nowadays many research laboratories around the world invest time and resources to investigate new, rapid, and simpler techniques. Works regarding the latter aspect are not widespread in the literature, perhaps because variation of the instrumental conditions can cause drastic alterations to the results obtained with a tested and optimized method [9]. Continuity of results within long periods of time (years) is of paramount importance in clinical studies [10], impairing in some degree the interest in the exploration of new or existing methodologies aiming at optimizing the setting of the LCMS instrumental conditions used in the quantitative analysis of TAG. Superb LCMS instruments equipped with automatic optimization devices could be another reason for the limited number of publications on this field. It has been reported that the sole chromatographic system is influenced by more than 50 factors [11], hence the complexity of chromatography coupled to mass spectrometry overwhelms even the most sophisticated software intended for automatic optimization and method development. The classic one-at-the-time methodology for optimization has been widely used in TAG quantification studies. One of the drawbacks of this method is that it is a tedious procedure and the true optimum can be missed if there are important interactions between the factors. The optimization of a LCMS method is prone to difficulties resulting from the inherent high number of instrumental factors involved and their interactions. The determination of the local response surface and exploitation of the local factor dependence is essential. The discovery and application of methods to that effect are extremely important. This study was undertaken, first to estimate the optimal combination of liquid chromatography-ion-trap-mass spectrometry (LCITMS) experimental variables, specifically nebu...

Unbound DHA causes a high blank value in β‐oxidation assay: a concern for in vitro studies

“…With filtration, the insoluble products were retained on cellulose membrane filters (0.45 mm diameter pore) in a sampling manifold system (Millipore, USA), and the radioactivity of 0.5 ml of the clarified liquid was measured as above. As shown in Table 1, the filtration method allowed removing more BSA-bound [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C] FA than centrifugation, so filtration method was applied in all the other experiments presented in the study. In order to evaluate the effect of bio- logical extracts on the radioactive survival in control assay, 100 ml of rat liver homogenate (1/40, w/v) was added into the same buffered medium as described above, with the immediate following injection of the same volume of perchloric acid.…”

“…i) Oxidation of [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]FA bound to BSA. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]FA oxidation by liver homogenates was performed at 377C using two media.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]FA oxidation by liver homogenates was performed at 377C using two media. The first medium contained 13.3 mM HEPES, 83.3 mM KCl, 16.7 mM MgCl 2 , 0.7 mM EDTA, 1.0 mM L-carnitine, 0.4 mM CoASH, 2.0 mM ATP, 0.5 mM L-malate, and 25 mM cytochrome c, which allowed the measurement of both mitochondrial and peroxisomal FA oxidation activities.…”

“…However, few publications addressed this issue. In order to identify the radioactive survivals and their impact on the final results, we compared the radioactive background of different 14 C labeled FAs with a range of albumin from different species in the experiment of b-oxidation using rat liver homogenate. LCMS/MS was used to identify the products qualitatively and quantitatively.…”

“…In this study, seven [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C] FAs were mixed with albumin and assayed for b-oxidation in rat liver homogenates. In the process of identifying the radioactive background of control assay by LCMS/MS, the results indicated different binding capacity of FAs to albumin.…”