One of the major challenges that one encounters during the lipidomic analysis of complex mixtures is contamination of the samples with synthetic compounds. The samples may be inadvertently contaminated on extracting, concentrating, drying, and storing the samples. The main sources of contamination are impurities in organic solvents, laboratory plastic ware, and vacuum grease and oil. Organic solvents, especially the aggressive ones such as chloroform, dichloromethane, and acetic acid, tend to dissolve many types of plastic and should never be used with anything but glass, fluoropolymers (e.g., Teflon; DuPont, Wilmington, DE), stainless steel, and noble metals.
19 Alcohols (methanol, ethanol, propanol) and alkanes (e.g., hexane, heptane, octane) are less aggressive, though their contact with plastic ware, even that approved for use with the above solvents, should be kept to minimum or avoided altogether. A frequent mistake is the use of Eppendorf tubes for handling and storing samples.
19 Even a brief exposure of the tube to a chloroformic solution of a sample will present a serious risk of contaminating the samples with plasticizers and other extractives. As an example, a partial HPLC MS spectrum of a sample whose solution in the CM solvent was briefly kept in a polyethylene container is presented in
Figure 11 . Two prominent HPLC peaks were detected with the major MS signals with
m/z values, respectively, of 663 and 637. Previously, ion
m/z 663 was reported in samples of human meibum
16 but was not assigned to any particular compound. The authors of the publication
16 used Eppendorf tubes for storing and handling the lipid samples. No HPLC analysis or structural elucidation of the ion(s) was performed at the time. In our experiments, the RT of peak
m/z 663 was approximately 4 minutes. Assuming that the detected ion was an (M+H)
+ adduct, its molecular mass of 662 was close to that of a WE (C
45H
90O
2) and a DAG (C
43H
84O
5 − H
2O). However, rushing to this conclusion would have been a mistake because subsequent fragmentation of ion
m/z 663 in sequential MS
n experiments gave us the following chain of product ions differing by 56 amu: 663 (MS1) → 607 (MS2) → 551 (MS3) → 495 (MS4) → 439 (MS5) → 383 (MS6) → 327 (MS7) (not shown). Note that the first four ions (663, 607, 551, 495) were also easily detectable in a simple MS1 experiment
(Fig. 11A) . These transformations were indicative of a sequential loss of several (up to six)
t-butyl groups and were formerly described for oxidized form of Irgafos 168 (C
42H
63O
4P,
Fig. 11A , inset)—a polymer additive present in polyethylene and polypropylene—and its derivatives.
20 In the negative ion mode, the compound gave two prominent signals,
m/z 407 and 473. These ions were also distinctive characteristics of Irgafos.
20 In a similar experiment, an apparently related, more polar compound with
m/z 637 and its related ion
m/z 581
(Fig. 11B)with an RT of approximately 25 minutes fragmented, releasing five
t-butyl groups: 637 (MS1) → 581 (MS2) → 525 (MS3) → 469 (MS4) → 413 (MS5) → 367 (MS6) (not shown). The
m/z value, a loss of only five
t-butyl groups and much higher polarity, were indicative of a compound related to Irgafos 168 in which one of the
t-butyl groups could have been replaced with a methoxy group (proposed molecular formula,C
39H
58O
5P). These results emphasize the necessity of exercising the utmost care while handling and analyzing samples and the importance of proper laboratory techniques.