The molecular structure of C16:1isop can be viewed in figure 4.4. As its name and structure
suggest C16:1isop is the precursor of C16:1synth where an isopropylidene protective group is
present on the glycerol backbone.
M m O O O OCH3
Figure 4.4: The molecular structure of C16:1isop. It’s formula is C23H44O4 (M) and the for-
mula for just the alkyl chain without the methoxyl group is C16H30 (m).
Presented in figure 4.5 is the ammonium adduct MSMS spectrum of C16:1isop and table 4.3
Figure 4.5: MSMS spectrum of C16:1isop[M+ NH4]+(marked with diamond). Formulas for
the major fragments can be viewed in table 4.3.
Table 4.3: The major signals in figure 4.5. All fragments have the charge of+1 and figure 4.4 states the formulas for M and m.
Mass [m/z] Formula Gain/loss 402.4 C23H48O4N M+NH4
385.3 C23H45O4 M+H
353.3 C22H41O3 M−CH3O
221.2 C16H29 m−H
The [m−H]+fragment seen for C16:1synth is also observed for C16:1isop, indicating the frag-
ment containing only the alkyl group. Additionally, the [M−CH3O]+observed for C16:1isop
can only be due to a loss of the methoxyl group as there are protective groups on the glycerol oxygens. Unfortunately, the intensity of the alkyl chain fragmentation is not high enough to yield beneficial information.
4.3 Summary
The MSMS spectrum of C16:1synth shows a range of fragments involving both the alkyl
chain and the oxygen containing glycerol backbone as well as the methoxyl group. The alkyl chain fragments into clusters with varying degree of unsaturation which can be repre- sented by CNH2N−3 and CNH2N−1. There are fragments, at 103 and 117 m/z, where parts of
the chain have fallen off along with either part of the glycerol or the methoxyl group. Further experiments are needed in order to unambiguously determine which of the oxygen contain- ing groups are participating in the mass loss. From the MSMS spectrum of C16:1isop it can
be concluded that the [M−CH3O]+signal is the result of the methoxyl group fragmenting.
In the next chapter, the HPLC-MS and MSMS data of the MAGs from the shark liver oil mixture are presented and analyzed.
5 NH
4
HPLC-MS/MSMS data of MAGs in
mixture
First a comprehensive analysis of the HPLC-MS data is presented. Second, the [M+ NH4]+ MSMS spectra of the standard (C16:1synth) and the sample (C16:1) are compared. Third, in
section 5.3, the MSMS spectra of [M+ NH4]+, [M+ H]+and [M−CH3O]+ are displayed in order to determine whether [M+ H]+and [M−CH3O]+take part in the major fragmentation process. Fourth, [M+ NH4]+ MSMS spectra are presented where different amount of col- lision energy has been applied to observe the resulting various degree of fragmentation. In sections 5.5 and 5.6 the [M+ NH4]+MSMS spectra are shown, major fragments presented and discussed.
5.1 HPLC-MS data
In figure 5.1 the EIC of [M+ NH4]+for all of the major compounds present in the mixture is depicted. Table 5.1 shows the corresponding chromatographic data.
Inspection of the MS spectra revealed that the ammonium ions were spontaneously frag- menting during the analysis. This is a known problem in LC-MS analysis called in-source fragmentation. EIC of the most abundant fragment ions, [M+H]+, [M−CH3O]+and [m−H]+ along with [M+ NH4]+, is shown in figure 5.2. Accurate mass values were acquired for all ions and are listed in table A.1 in appendix A.
C18:3 C22:6 C16:1 C16:0 C18:1 A C18:1 B C18:0
Figure 5.1: EIC of [M+ NH4]+ for each of the major compounds present in the mixture. Chromatographic data are listed in table 5.1
Visual observation of figure 5.1 concludes that C18:3 has by far the lowest intensity while C16:1 and C16:0 exhibit the highest.
In order to provide some level of quantitation, area fractions (AF) and specific ion area fractions (AFion) have been calculated. These quantities are defined in the introduction in
Table 5.1: HPLC-MS data for the EIC of [M+ NH4]+run depicted in figure 5.1. Shows i.a. run time (RT) and area fractions (AF) of the signals.
Compound RT [min] Formula Mass [m/z] AF [%] AFM+NH4 [%] C18:3 10.1 C22H44O4N 386.3 0.88 1.8 C22:6 10.5 C26H46O4N 436.3 5.2 11 C16:1 11.3 C20H44O4N 362.3 13 27 C16:0 12.7 C20H46O4N 364.3 12 24 C18:1 A 13.2 C22H48O4N 390.4 4.4 9.0 C18:1 B 14.2 C22H48O4N 390.4 7.9 16 C18:0 16.2 C22H50O4N 392.4 5.4 11
Judging by the AFs, both AF and AFM+NH4, the C16:1 and C16:0 are the most abundant followed by C18:1 B, C18:0, C22:6 and C18:1 A with C18:3 trailing far behind. However this abundance order changes when comparing different ions as figure 5.2 displays.
C18:3 C22:6 C16:1 C16:0 C18:1 A C18:1 B C18:0
Figure 5.2: EIC of [M+ NH4]+(brown line), [M+ H]+(green line), [M−CH3O]+(blue) and [m−H]+ (pink) for each of the compounds present in the sample. Ion specific area fraction and AF are listed in tables 5.2 and 5.3
For the saturated MAGs, C16:0 and C18:0, the ammonium (brown line) and hydrogen (green line) adducts are most abundant with [M−CH3O]+ (blue line) small and [m−H]+(pink line) visually nonexistent.
The monounsaturated counterparts show variations where C16:1 and C18:1 B are similar where [M+ NH4]+> [M + H]+> [M−CH3O]+> [m−H]+. C18:1 A behaved completely dif- ferently with similar intensities of [M+ NH4]+and [M+ H]+but [M−CH3O]+and [m−H]+ visually almost nonexistent. Thus in addition to varying retention times, dissimilar abun- dance of these four major ions points to further evidence of different position of the double bond between C18:1 A and B.
When focusing on the polyunsaturates, C18:3 and C22:6, there are also different quanti- ties. C22:6 has similar ratios as C16:1 and C18:1 B but with higher quantities of [m−H]+. C18:3 displays however ratios unlike the others as [m−H]+> > [M + NH4]+ = [M + H]+> [M−CH3O]+. The potential explanations behind the unique behavior of C18:3 could stem from the double bond position in the alkyl chain, but the age of the sample could also be a factor as it was almost 7 months old at the time of measurement.
Due to the great width of the peaks, visual comparison and analysis only lends certain amount of information. Therefore ion specific area fractions and AF for all four ions have been com- piled and are presented in tables 5.2 and 5.3.
Table 5.2: Comparison of fragment specific area fractions for [M + NH4]+, [M + H]+, [M−CH3O]+and [m−H]+run shown in figure 5.2.
Compound AFM+NH4 [%] AFM+H[%] AFM−CH3O[%] AFm−H [%] C18:3 1.8 2.9 4.4 28 C22:6 11 9.9 6.8 35 C16:1 27 21 39 22 C18:1 A 9.0 14 2.0 5.1 C18:1 B 16 14 20 7.8 C16:0 24 24 21 2.1 C18:0 11 15 7.9 0.50
The major ratios between the compounds, according to AFM+H, are similar to AFM+NH4 with the exception of C18:1 A and B, where according to the AFM+H their ratio is 1:1 while
AFM+NH4 indicates a ratio of almost 1:2.
When comparing the AFM−CH3O the compounds line up in the following abundance order C16:1 > C16:0 > C18:1 B > C18:0 > C22:6 > C18:3 > C18:1 A. Thus judging from AFM−CH3O the amount of C18:1 A is lower than that of C18:3 which contrasts visual quan- tifications.
If the compounds are set up in order of abundance according to [m−H]+ data the result is C22:6 > C18:3 > C16:1 > > C18:1 B > C18:1 A > C16:0 > C18:0. This order is in stark contrast of what was presented in the start of this section.
Additionally, by comparing the AF within each compound there are great variations. It is clear that any attempt at relative quantification is going to be futile due to the varying de- gree of in-source fragmentation where both saturation and position of double bonds in the alkyl chain seem to play a role. One would either have to optimize the MS method to mini- mize in-source fragmentation or study this effect further by analyzing standards with known structures where this effect can be quantified for each compound and linear calibration would enable absolute quantification. That work could be justified by the possibility of developing a new method of analyzing these type of compounds on MS instruments that do not produce high resolution spectra but could base their analysis on varying degree of fragmentation.
Not surprisingly, the varying abundance trend can also be seen in the AF percentages shown in table 5.3. However, the AF of [m−H]+ and [M−CH3O]+ account for less than 8 % and 10 % of the total sum of the area fractions. Thus in general the [M+ NH4]+and [M+ H]+ dominate with their share of AF little below 50 % and 33 %, respectively.
Table 5.3: Comparison of the area fractions for the 4 major ions present in NH4-HPLC-MS
run shown in figure 5.2.
Area fraction (AF) [%]
Compound [M+ NH4]+ [M+ H]+ [M−CH3O]+ [m−H]+ C18:3 0.88 0.98 0.42 2.1 C22:6 5.2 3.3 0.65 2.7 C16:1 13 6.9 3.7 1.7 C16:0 12 7.9 1.9 0.16 C18:1 A 4.4 4.7 0.19 0.40 C18:1 B 7.9 4.7 1.9 0.61 C18:0 5.4 4.9 0.75 0.04 Sum 48.78 33.38 9.51 7.71