آنالیز مستقیم کربوهیدرات ها در پلاسمای حیوانی به وسیله کروماتوگرافی یونی به همراه اسپکترومتری تراکمی (انبوه) و تشخیص آمپرسنجی ضربه ای برای استفاده به عنوان ابزار تشخیصی غیر تهاجمی Direct analysis of carbohydrates in animal plasma by ion chromatography coupled with mass spectrometry and pulsed amperometric detection for use as a non-invasive diagnostic tool

During the IC-ECD–MS/MS method development the methods were validated with basic validated parameters shown in Table 1. As can be seen in Table 1, there is no significant difference considering validated data that could prefer one of the methods. The advantages of particular method are considered during the practical use in determination of the authentic samples. Thus, the IC-ECD method is considered to be the most accurate routine method to qualify carbohydrates, whereas IC–MS/MS provides a better selectivity for problematic samples and is the tool of choice to elucidate unknown peaks. Electrochemical detector is not expensive and thus available to each laboratory. In MS detection the selected carbohydrates can be isomers yielding identical mass spectra. Verification of the identity of individual carbohydrates is feasible using a comparison of chromatographic retention times with reference or isotope labeled compounds. A very high specificity is obtained with single ion monitoring channels, which allows the determination of coeluting compounds and the elimination of background from the non-carbohydrate components of the sample matrix as well as the mobile phase. The characteristic multi-reaction monitoring (MRM) transitions of glucose (187.4/91.0) are used for monitoring carbohydrates in plasma samples. The results in Fig. 5 (Table 2) show that the concentration of glucose in plasma was increasing during the 40 day raising period. In the control group, the final concentration was 19.1% higher on day 40 than the concentration measured on day 16 counting from the first day of the raising period. In the test groups that were administrated the fortified fodder; the increase in the glucose level in plasma was smaller. In the case of administrated CoQ10, the final concentration was approximately 7% higher and in the case of fodder with a combination ofALAand CoQ10 the final concentration was only 2% higher than on day 16. The most rewarding result was obtained with pure ALA where the final plasma glucose concentration was 6% lower, thus showing that the glucose level decreased during the administration of fodder fortified with ALA (50 mg/kg). Our results are in accordance with the literature data and likewise show that CoQ10 and ALA food additives may reduce the plasma level of glucose and have beneficial effect in the case of diabetes, if taken as food additives. Tandem mass spectrometry represents an advanced quantitative analysis of complex mixtures performed by creating the fragments of a molecular ion by collisionally activated dissociation (CAD) and thus providing a fingerprint assay. Product ions are generated from the molecular ion by covalent bond cleavage within the carbohydrate backbone and/or losses of water. Mannitol and lactulose were measured in MRM mode. The parent ion for mannitol was 189.2 m/z (M+Li)+ and fragment ions were 171.3, 96.8 and 81.1 m/z. For lactulose, the selected parent ion was 349 m/z (M+Li)+ and fragment ions were 330.9, 187.0 and 97.2 m/z. A typical chromatogram with selected ion transitions of mannitol and lactulose is shown in Fig. 6. In MRM mode, the values of the L/M index could be measured in dog blood even 48 h after the peroral application of the non-steroidal anti-inflammatory drug meloxicam. After drug administration, higher L/M index values were found in plasma. Supplemented CoQ10 accelerated the recovery of the colon, which was observed through lower values of L/M index. The concentration of CoQ10 in plasma was measured with IC–MS/MS in MRM mode. The obtained results are shown in Table 3.