J Am Oil Chem Soc (2015) 92:1533–1547 DOI 10.1007/s11746-015-2735-z 1 3 ORIGINAL PAPER The Updated Bottom Up Solution Applied to Atmospheric Pressure Photoionization and Electrospray Ionization Mass Spectrometry William Craig Byrdwell 1 Received: 8 July 2015 / Revised: 25 September 2015 / Accepted: 5 October 2015 / Published online: 28 October 2015 © AOCS (outside the USA) 2015 by APPI-MS of vitamin D in the gelcaps gave values of 42.90 ± 0.83 μg, or 1716 ± 33 international units, in good agreement with APCI-MS. Keywords APPI-MS · ESI-MS · Triacylglycerols · Regioisomers · Lipidomics Introduction Human metabolism of triacylglycerols (TAG) is regiospe- cific, meaning that fatty acids are preferentially metabo- lized from specific locations on the three carbon atoms of the glycerol backbone, designated by the stereospecific numbering system (sn) as sn-1, sn-2, and sn-3. Fatty acids (FA), or more precisely fatty acyl chains, from the outer positions, sn-1 and sn-3 are preferentially removed from TAG, leaving 2-monoacylglycerols (MAG). TAG synthesis (catabolism) in plants and other animals similarly displays regiospecific trends. In cocoa butter and vegetable oils (VO), the monounsaturated FA (MUFA) oleic acid, 18:1, and the polyunsaturated FA (PUFA) linoleic acid, 18:2, are mainly esterified at the sn-2 position [1–3], while satu- rated FA (SFA) occur preferentially at either the sn-1 posi- tion [2] or at the sn-1 and sn-3 positions [1, 3]. Conversely, in fats of animal origin, specifically bovine milk and pork fat (lard), SFA are esterified mainly at the sn-2 position, although in beef fat (tallow), SFA are esterified predomi- nantly at the sn-1 and sn-3 positions [1]. The stereospecific forms of isobaric TAG are regioisomers. The metabolism and catabolism of TAG regioisomers has direct implica- tions for the structures of lipids into which FA are incorpo- rated throughout the human body. Regioisomerism of TAG is one trait of interest in full structural characterization of TAG. Other, even more Abstract The Updated Bottom Up Solution (UBUS) was recently applied to atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) of triacylg- lycerols (TAG). This report demonstrates that the UBUS applies equally well to atmospheric pressure photoioniza- tion (APPI) MS and to electrospray ionization (ESI) MS. Critical Ratio 1 (CR1), the [MH] + /Σ[DAG] + or [MNH 4 ] + / Σ[DAG] + ratio, does not exhibit the same strongly sigmoi- dal shape as it does by APCI-MS. CR1 varies more widely for APPI-MS than by APCI-MS, having a maximum value of 11.8, indicating a much greater effect of unsatu- ration on ion ratios in APPI-MS than APCI-MS. Critical Ratio 2, the [AA] + /[AB] + ratio for Type II TAG or [AC] + / ([AB] + +[BC] + ) ratio for Type III TAG, allows quantifica- tion of regioisomers of TAG, and shows good agreement for APPI-MS to regioisomer quantification determined by APCI-MS. Critical Ratio 3, the [BC] + /[AB] + ratio for Type III TAG, reveals new trends relating the degree of unsatu- ration by APPI-MS, and shows that structural assignments made by ESI-MS are in good agreement to APCI-MS data. In addition to providing valuable structural informa- tion, the Critical Ratios also constitute a reduced data set that allows APPI-MS or ESI-MS mass spectra to be recon- structed when processed through the UBUS. Quantification Electronic supplementary material The online version of this article (doi:10.1007/s11746-015-2735-z) contains supplementary material, which is available to authorized users. * William Craig Byrdwell C.Byrdwell@ars.usda.gov 1 Food Composition and Methods Development Lab, Beltsville Human Nutrition Research Center, U.S.D.A., Agricultural Research Service, 10300 Baltimore Ave., Beltsville, MD 20705, USA