Polymeric ionic liquid open tubular capillary column for on-line in-tube SPME coupled with UHPLC-MS/MS to determine endocannabinoids in plasma samples

Highlights

• Polymeric ionic liquid monomers (PIL) and PIL cross-linker was synthesized.

• Selective PIL-based wall-coated open tubular capillary columns were synthesized.

• PIL-based wall-coated open tubular capillary was used as a sorbent phase.

• PIL capillary was used in on-line in-tube SPME/UHPLC-MS/MS method.

• The method was applied to determine endocannabinoids in plasma samples.

Abstract

This manuscript describes the development of wall-coated open tubular capillary column with polymeric ionic liquids (PILs) for on-line in-tube solid phase microextraction coupled with ultra high-performance liquid chromatography tandem mass spectrometry (in-tube SPME/UHPLC-MS/MS) to determine anandamide (AEA) and 2-arachidonoyl glycerol (2 A G) in plasma samples. Selective PILs were synthetized from the [VC₆IM][Cl], [VC₁₆IM][Br], and [(VIM)₂C₁₀]2 [Br] - ionic liquids - by in-situ thermal-initiated polymerization in a fused silica capillary column for in-tube SPME. The synthesis procedure was optimized, and the capillary columns were characterized using spectroscopic and chromatography techniques. The chemically bonded and cross-linked PIL-based sorbent phase (thickness coating: 1.7 μm) presented high chemical and mechanical stability. Among the sorbents evaluated, the PIL-based capillary, [VC₁₆IM][Br]/[(VIM)₂C₁₀]2 [Br] presented the best performance with a sorption capacity of 37,311 ng cm⁻³ and 48,307 ng cm⁻³ for AEA and 2 A G, respectively. This capillary was reused more than ninety times without significant changes in extraction efficiency. The in-tube SPME-UHPLC-MS/MS method presented a linear range from 0.1 ng mL⁻¹ to 100 ng mL⁻¹ for AEA, and from 0.05 ng mL⁻¹ to 100 ng mL⁻¹ for 2 A G, with coefficients of determination higher than 0.99, p-value for Lack-of-fit test higher than 0.05 (α of 0.05), precision with coefficient of variation (CV) values ranging from 1.6 to 14.0% and accuracy with relative standard deviation (RSD) values from −19.6% to 13.2%. This method was successfully applied to determine AEA and 2 A G in plasma patients with Parkinson's disease. The concentrations in these plasma samples ranged from 0.14 to 0.46 ng mL⁻¹ for AEA and from <0.05 ng mL⁻¹ to 0.51 ng mL⁻¹ for 2-AG.

Introduction

Arachidonoyl ethanolamide (AEA) and 2-Arachidonoyl glycerol (2 A G) are the main endogenous ligands for the cannabinoid receptors (ER). The biosynthesis of AEA and 2 A G endocannabinoids derive from N-acylphosphophatidylethanolamines and diacylglycerols substrates respectively by mediation of specific hydrolyses or a combination of acyltransferases and hydrolyses [[1], [2], [3]]. The endocannabinoid system exerts important modulatory functions in the central nervous system, including retrograde control of excitatory or inhibitory synapses. Such functions enables the central nervous system participate to and regulate vital neurobiological processes in healthy patients [4]. Studies involving neuropsychiatric patients have found a close relation between the endocannabinoid system and neurological disorders. For example, Parkinson's patients presented higher endocannabinoids (eCBs) concentration in plasma samples than control groups (healthy voluntaries) [[5], [6], [7]].

High performance liquid chromatography coupled with tandem mass spectrometry LC-MS/MS has been considered the primary technique to determine trace levels of eCBs in plasma samples [4,8]. Considering, the physicochemical properties of the eCBs, reversed-phase chromatography (hydrophobic interactions) has been the most commonly used mode to determine these compounds. One limitation associated with LC-MS/MS analysis is its susceptibility to the matrix effect. Therefore, a biological sample preparation step is required to reduce matrix interferences (mainly macromolecules), isolate and concentrate (enrichment) the eCBs.

In this context, sample preparation techniques including liquid-liquid extraction (LLE) [9], conventional solid phase extraction (SPE) [10], salting-out liquid-liquid extraction (SALLE) [11], and column switching systems [12,13] have been used to determine eCBs in plasma samples by LC–MS/MS.

High extraction efficiency is readily attained using LLE and SALLE, which are simple techniques, but require extensive sample handling and exhibit poor selectivity. SPE-based sample preparation provides enhanced sample cleanup and enrichment of analytes at trace levels, but involves multiple steps and greater cost per sample [14].

On-line sample preparation approaches systems, such as column switching LC-MS/MS that use fully automated protocols not only shorten the total analysis time (high throughput) but also usually provide better accuracy and precision.

In column switching systems, for example, the in-tube SPME capillary is coupled to LC-MS/MS, the target fractions (after the trace enrichment and macromolecules exclusion) from the first column (capillary column with selective stationary phase) are transferred to the second column with different stationary phase for chromatographic separation [15].

Packed columns with different stationary phases have been evaluated for on-line trace enrichment of AEA and 2 A G from plasma samples. For example, a POROS™ R1 [poly (styrene-divinylbenzene)] column [30 mm × 2.1 mm-id (d_p = 20 μm)] [12], and a RP-8 ADS restricted access material column [25 mm × 4 mm-id (d_p = 25 μm)] [13]. Open tubular capillary columns with chemically bounded phases are an interesting alternative to packed columns. These capillary columns presented high stability, low back pressure, and frit-less (avoiding the adsorption of macromolecules from biological samples).

Polymeric ionic liquids (PILs) are a potential class of stationary phases for sample preparation, which can be engineered (structurally tuning) to exhibit desired selectivity toward specific groups of compounds [16]. For example, long aliphatic alkyl chains can enhance the PILs ability to undergo dispersion interactions with nonpolar analytes. Similarly, grafting a hydrogen-bond basic anion to the PIL favors the extraction of hydrogen-bond acids such as alcohols and organic acids [17]. Anderson and co-workers developed chemically bonded and highly crosslinked PIL-based sorbent phases for SPME [16,18]. These phases consisted of in-situ copolymerization of 1-vinyl-3-hexylimidazolium chloride ([VC₆IM][Cl]) or 1-vinyl-3-hexadecylimidazolium bromide ([VC₁₆IM][Br]) IL monomers and 1,12-di (3-vinylimidazolium)dodecane dibromide ([(VIM)₂C₁₂]2 [Br]) IL cross-linker [19], resulting in extremely robust SPME devices, which were successfully reused for several times for headspace and direct immersion extractions.

IL as well as PIL have been reported as a sorbent for in-tube SPME. Wang et al. [20] reported the synthesis of an IL-based monolith to determine organic acids in food samples. The authors modified the glycidyl methacrylate with 1-aminopropyl-3-methylimidazolium chloride and copolymerized with acrylamide and N,N′-methylenebisacrylamide to obtain the monolith. This is an interesting strategy to incorporate the IL into the capillary, however the presence of glycidyl methacrylate and N,N′-methylenebisacrylamide in the monolith structure might represent sites for secondary interaction with analytes or matrix endogenous components. Sun et al. [21] reported a PIL sorbent for fibers in-tube SPME. In this work the authors used cupper wires as a support to polymerize 1-dodecyl-3-vinylimidazolium bromide and 1,6-di (3-vinylimidazolium) hexane bibromide. These fibers were packed in an ILs-functionalized copper tube (fiber in-tube SPME) to determine five estrogens in domestic sewage samples. Although this capillary presented good extraction efficiency, the use of frits to retain the fibers inside the tube can adsorb macromolecules from biological samples. Moreover, the preparation of the fibers coating and the fiber-packed tube symbolize additional steps during the capillary synthesis compared to the open tube capillary columns.

In this study, selective PILs were synthetized from the [VC₆IM][Cl], [VC₁₆IM][Br], and [(VIM)₂C₁₀]2 [Br] ionic liquids by in-situ thermal-initiated polymerization in a fused silica capillary column for in-tube SPME. Among the stationary phases evaluated, the wall-coated open tubular PIL-based capillary, [VC₁₆IM][Br]/[(VIM)₂C₁₀]2 [Br] presented the best performance. This frit-less PIL capillary column was successfully used for in-tube SPME-UHPLC-MS/MS system to determine AEA and 2 A G in plasma samples from Parkinson's disease patients.