Ure mass spectrometry of pentafluorobenzyl esters is reported to become about ten femtomoles (three,000 femtograms) (33). Therefore, our method is about 10-fold much more sensitive than this earlier method of FA analysis.To gauge the increase in FA detection sensitivity, we analyzed a variety of amounts of d8-20:four AMPP amide in constructive ion mode and a variety of amounts of d8-20:4 absolutely free acid in damaging ion mode. For the latter we monitored a significant higher mass solution ion due to the loss of CO2. We also tuned the instrument to optimize the cone voltage and collision energy for this transition in negative ion mode. Results are shown in Fig. two. Injection of 0.78 pg of d8-20:four AMPP amide offers rise to a peak area of 22,800 in positive ion mode versus 17,100 for 50 ng of d8-20:4 totally free acid in negative ion mode. Thus, the enhance in sensitivity for the AMPP derivatization method is 64,000-fold.MF59 Next we analyzed the FAs present in mouse serum, and the benefits are summarized in Table 2. Intra-assay coefficients of variation determined by five injections in the similar sample have been ordinarily much less than 4 . Inter-assay coefficients of variations depending on injections of six independent extractions of your same serum were normally less than 6 .NMDA Hence, the technique is highly reproducible. For these research we made use of the m/z 239 production ion. As noted above, this really is present in all the FAs, but its use is sufficient in the case of mouse serum. Additional analytical specificity may be obtained by monitoring analytespecific precursor ions, including these noted above for the 18:1 species. It need to be described that accurate quantification of the absolute level of any particular FA species calls for a chemically identical isotopic substituted internal common. Only within this way can one account for differences in ionizationTABLE 2.Calculated concentrations (pg/ l) and coefficients of variation ( ) for LC/ESI-MS/MS analysis of FA AMPP amides in commercial mouse serumFA (pg/ l) CV Intra-sample (ten l of Serum)a CV Inter-sample (10 l of Serum)bLauric Myristoleic Myristic Palmitoleic Palmitic Linolenic Linoleic Oleic Stearic Eicosapentaenoic Arachidonic 3-Arachidonic Eicosatrienoic Eicosadienoic Eicosenoic Acid Arachidic Docosahexaenoic Docosapentaenoic Docosatetraenoic Docosatrienoic Docosadienoic Erucic Behenic Nervonic Lignoceric143.two 12.9 299.4 295.eight 1286.9 185.2 462.0 2066.1 1820.four 72.0 873.0 24.2 147.0 53.9 98.7 21.4 605.6 148.2 66.9 14.5 0.eight eight.3 7.6 20.six 18.3.1 1.2 two.2 0.8 1.2 1.6 0.six 1.1 1.8 0.9 1.7 four.8 two.4 1.7 1.4 two.9 4.four three.9 three.5 two.4 three.1 three.0 1.7 3.5 1.7.six five.8 three.0 2.6 five.three 2.7 3.two four.3 six.1 3.7 five.two 5.9 1.5 3.8 two.four 8.1 six.9 7.9 9.0 two.7 two.7 10.4 five.3 six.0 7.A trace level of 12:1 fatty acid was observed but variability was higher as a consequence of its low level. CV, percent coefficient of variation.PMID:24187611 a Coefficient of variation for the evaluation with the very same sample of extracted and derivatized serum injected five occasions onto the LC/ESI-MS/MS. b Coefficient of variation for the evaluation of six independently extracted and derivatized serum samples.Journal of Lipid Analysis Volume 54,efficiencies inside the mass spectrometer supply and in differences in precursor-to-product ion generation for the unique FA molecular species. Also, if a deuterated FA is employed as in internal normal, deuterium ought to not be present at a web-site that results in an isotope impact on the volume of solution ion generated. An additional solution is usually to use a restricted quantity of heavy atom substituted FA internal standards and to determine the relative MS/MS signal i.
