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Eight human plasma preparation protocols were evaluated for their suitability for metabolomic studies by ultra-high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry: organic solvent protein precipitation (PPT) with either methanol or acetonitrile in 2:1 and 3:1 (v/v) ratios with plasma; solid-phase extraction (SPE) using C18 or HybridSPE cartridges; and a combination of PPT and SPE C18 cartridges and microextraction by packed sorbent. A study design in which the order of injection of the samples was not randomized is presented. The analyses were conducted in a BEH C18 column (1.7 μm, 2.1 mm × 100 mm) using a linear gradient from 100% water to 100% methanol, both with 0.1% formic acid, in 21 min. The most reproducible protocol considering both the univariate and the multivariate analysis results was PPT with acetonitrile in a 2:1 (v/v) ratio with plasma, offering a mean coefficient of variation of the area of all the detected features of 0.15 and one of the best clusterings in the principal component analysis plots. On the other hand, the highest number of extracted features was achieved using methanol in a 2:1 (v/v) ratio with plasma as the PPT solvent, closely followed by the same protocol with acetonitrile in a 2:1 (v/v) ratio with plasma, which offered only 1.2% fewer repeatable features. In terms of concentration of remaining protein, protocols based on PPT with acetonitrile provided cleaner extracts than protocols based on PPT with methanol. Finally, pairwise comparison showed that the use of PPT- and SPE-based protocols offers a different coverage of the metabolome.

In this study, a selective and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method requiring low sample volume (≤100 μL) was developed and validated for the quantitative determination of the opioid drug fentanyl in plasma and cerebrospinal fluid (CSF). A protein precipitation extraction with acetonitrile was used for plasma samples whereas CSF samples were injected directly on the HPLC column. Fentanyl and (13) C6 -fentanyl (Internal Standard) were analyzed in an electrospray ionization source in positive mode, with multiple reaction monitoring (MRM) of the transitions m/z 337.0/188.0 and m/z 337.0/105.0 for quantification and confirmation of fentanyl, and m/z 343.0/188.0 for (13) C6 -fentanyl. The respective lowest limits of quantification for plasma and CSF were 0.2 and 0.25 ng/mL. Intra- and inter-assay precision and accuracy did not exceed 15%, in accordance with bioanalytical validation guidelines. The described analytical method was proven to be robust and was successfully applied to the determination of fentanyl in plasma and CSF samples from a pharmacokinetic and pharmacodynamic study in newborn piglets receiving intravenous fentanyl (5 µg/kg bolus immediately followed by a 90-min infusion of 3 µg/kg/h).

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