Interference of oleamide with analytical and bioassay results

Abstract

During sample preparation and analysis, samples are coming in contact with different labware materials. By four unrelated analytical (phytochemical and pharmaceutical) case-studies and employing different analytical techniques, we demonstrated the potential misinterpretation of analytical results due to the use of contaminants-leaching labware during sample handling. Oleamide, a common polymer lubricant and a bioactive compound, was identified as a main analytical interference, leaching from different labware items into solvents, recognised as chemically compatible with the tested polymer material. Moreover, anti-inflammatory effect of oleamide at 100 μg mL^-1 and considerable pro-inflammatory effect of the plastic syringe extractables (containing oleamide) at the same level were shown in a TLR4-based bioassay. Taking these results into account, together with the fact that oleamide can be a compound of natural origin, we would like to notify the professional public regarding the possible erroneous oleamide-related analytical and bioassay results due to the use of oleamide-leaching labware. Researchers are alerted to double check the real source of oleamide (labware or natural extract), which will prevent further reporting of false results. Analysis of procedural blanks with de-novo developed UHPLC-ESI-MS method is, among some other strategies, proposed for detection of oleamide interference and avoidance of misleading results of certain analyses.

Figure 1

Chromatogram of extracts obtained after multiple extractions of labware: PVDF (d = 25 mm, 1), PVDF (d = 8 mm, 2), PVDF filter paper (3), H-PTFE (4), regenerated cellulose (5), plastic syringe (20 mL, 6), plastic syringe (5 mL, 7), plastic centrifuge vial (50 mL, 8), plastic Pasteur pipette (9), methanol treated with N₂ flow (10) and lipid standards mixture (11). HPTLC silica gel plate was developed according to the method for separation of lipid classes and documented under white light illumination after derivatisation with MoP. Legend: PL - phospholipids, AMPL - acetone-mobile polar lipids, ST - sterols, FA - fatty alcohols, FFA - free fatty acids, TAG - triacylglycerols, KE - ketones, WE - wax esters, HC - hydrocarbons.

Figure 2

DI-ESI-MS spectra in positive mode, corresponding to: eugenol ethanolic standard solution analysed after filtration (a), ethanol as blank, analysed without filtration (b) and eugenol ethanolic standard solution filtered using centrifuge tubes containing cellulose acetate membrane filter (c).

Figure 3

Chromatograms of: blank (90% acetone_(aq):1 M TEAA with pH 7; 85:15, v/v) without filtration (1), filtered blank (2), oleamide methanolic standard solution (3), and filtered Japanese knotweed leaves extract (4, 5). Solutions were applied as 8 mm bands on a pre-developed C₁₈ RP HPTLC plate and developed by 0.1% TBHQ in methanol:acetone (1:1, v/v). Plate was visualised under white light illumination without (track 5) and with post-chromatographic derivatisation with MoP (tracks 1–4).

Figure 4

TIC chromatograms of: filtered API solution (a), methanolic extract of 5 mL plastic syringe (b) and blank (c).