A representative Q-TOF mass analysis of meibum in positive-ion mode is shown in
Figure 2 . Based on total ion current, the most predominant peak at
m/
z 282.3 corresponds to the protonated fatty acid amide oleamide. In addition to oleamide, the following fatty acid amides were also observed and identified in positive-ion mode as protonated species, although at relatively low intensities relative to oleamide
(Fig. 3) : myristamide (
m/
z 228.3), palmitamide (
m/
z 256.3), linoleamide (
m/
z 280.3), stearamide (
m/
z 284.3), and erucamide (
m/
z 338.2). The chemical structures of oleamide and the additional fatty acid amides
(Fig. 1)were confirmed in meibum through accurate mass determination and collision-induced dissociation.
Theoretical and observed exact mass measurements
(Table 1)of the fatty acid amides show that
m/
z 282.2788 (protonated oleamide) demonstrated a mass accuracy of 3.2 ppm for the elemental composition of C
18H
35NOH
+. NaI clusters were used as an internal standard (lock mass for the TOF) to obtain the accurate mass for the peak observed at
m/
z 282.3 within 3 ppm of the theoretical molecular weight of oleamide (C
18H
35NO) confirming the molecular formula of oleamide observed in the meibum. The
m/
z 228.2344 peak (protonated myristamide) showed an error of 7.4 ppm, the
m/
z 256.2651 peak (protonated palmitamide) showed an error of 4.2 ppm, and
m/z 338.3414 (protonated erucamide) showed error of 2.6 ppm. The exact mass measurement for peak
m/
z 284.2954 (protonated stearamide) was determined at 1 ppm; however, the peak corresponding to stearamide showed very low abundance.
The collision-induced product ion mass spectrum of the oleamide standard observed at
m/
z 282.3 compared with the peak observed at
m/
z 282.3 in acidified meibum
(Fig. 4)demonstrated the structure of the protonated molecule, [C
18H
35NO+H]
+ at
m/
z 282.3 to be identical, further confirming the presence of oleamide in meibum. The fragmentation pattern is consistent with a carbon–carbon double bond. In contrast, the positive-ion mode ESI MS/MS of palmitamide standard and palmitamide in the meibum
(Fig. 5)showed a product ion spectrum consistent with a single-saturation carbon chain. Comparison of the MS/MS for the five identified fatty acid amides is shown in
Figure 6 . Although the
m/
z peak of 280.27 is consistent with the fatty acid amide linoleamide, we were unable to perform exact mass determination or collision-induced dissociation on the peak because of its very low abundance in meibum with the methods used.
The expected
m/
z for the protonated molecule of oleic acid would be
m/
z 283.3, and the carbon 13 peak observed with high-resolution mass spectrometry for oleamide is also expected to be found at
m/
z 283.3. To examine whether the peak at
m/
z 283.3 is fully the C
13 of oleamide or partially from the protonated form of oleic acid, we compared the theoretical isotope ratio of the oleamide carbon 12:carbon:13 peak to the measured peak in meibum and with oleamide standard. C
13 has a natural abundance of 1.1% ± 0.02% and as oleamide contains 18 carbons in the molecular formula, the theoretical isotope ratio for the oleamide carbon 12:carbon 13 peak should be 19.8% ± 0.4%. The percent C
12/C
13 ratio of the oleamide standard was measured as 19.4%, within the expected range of the theoretical calculated ratio.
Table 2summarizes the C
12/C
13 results of the oleamide observed in the meibum. The percent ratio of the C
12/C
13 peak of oleamide in meibum was measured four separate times, and it averaged 22.5%, which is slightly higher than predicted and observed with the standard. This indicates that oleic acid is probably present in the
m/
z 283 peak from meibum measured in positive-ion mode, but accounts for approximately only 2% to 3% of the
m/
z peak 283.3.
Negative ion mode electrospray mass spectrometry, which is more conducive to fatty acid analysis, confirmed the presence of oleic acid in meibum, and the major peaks
(Fig. 7)included the deprotonated molecules [M−H]
− of myristic acid at
m/
z 227.2, palmitic acid at
m/
z 255.2, stearic acid at
m/
z 283.3, and oleic acid at
m/
z 281.3 (which appeared to be a very minor peak). Accurate mass, theoretical mass, calculated error, and molecular formula of the fatty acids and fatty acid amides in the meibum can be seen in
Table 2 . As with linoleamide, the identification of erucic and linoleic acid was not confirmed due to large mass errors of low-abundance peaks, although
m/
z peaks within the appropriate regions were present.