Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations

Highlights

• Nicotine and its metabolites were quantified in rabbit plasma by SPME-LC-MS/MS.

• A dual fiber extraction approach enabled analytes’ extraction at ppt levels.

• HLB/PAN biocompatible SPME fibers enhanced the recovery of polar analytes.

• The method performances complied to FDA criteria for bioclinical method validation.

• Nicotine release from nicotine-loaded smoking cessation formulations was evaluated.

Abstract

The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction–liquid chromatography–tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L ^− 1 and ranged between 98.3–112.2% for nicotine, 94.1–101.9% for cotinine, 94.7–107.0% for nornicotine, 81.1–107.2% for norcotinine and 94.3–115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L⁻¹. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.

Introduction

Smoking cessation often involves the application of nicotine replacement therapy, which is available as chewing gums, troches and transdermal patches. However, current transdermal patches are associated with side effects such as skin irritation and poor patient compliance [1]. Many strategies are currently being tested to improve the quality of these transdermal patches by overcoming irritation and prolonging the release of active ingredients. Encapsulation of nicotine into lipid nanoparticles could be an effective strategy. To test the efficacy of this new formulation, rabbits are often used as an animal model to monitor the levels of nicotine in the blood following transdermal application. Following permeation into the bloodstream, nicotine is readily metabolized into cotinine [2]. Plasma concentrations of cotinine are usually up to 10-fold higher than nicotine. Although pharmacologically inactive, cotinine is often regarded as a useful indicator of the nicotine metabolic pathway activity [2]. Thus, most studies focused on the assessment of nicotine delivery by transdermal route involve the determination of nicotine and cotinine in serum or plasma [3,4]. However, in vivo monitoring of the release of nicotine and its conversion into different metabolites for long periods in the same laboratory animal, has many experimental challenges. For instance, only limited quantity of blood can be withdrawn at each time point while the animal remains alive for the whole duration of the experiments. Thus, effective analytical protocols to measure targeted analytes at ultra-trace levels from minimal quantities of biofluids are required. In this context, it is critical to use a sample preparation/extraction technique able to, not only pre-concentrate the analytes of interest, but also minimize matrix co-extractants that can potentially affect the accuracy of the analysis with such limited sample sizes. Analysis of nicotine and its metabolites in biofluids has been extensively performed by liquid-liquid extraction (LLE) and protein precipitation (PP) [5], [6], [7], [8]. These strategies can provide limits of detection in the parts per billion level, however, they involve numerous steps (often not automated) to obtain clean extracts to be injected into the analytical system. Some of these steps involve multiple vortexing and centrifugation cycles, recovery of the supernatant with subsequent evaporation to dryness and reconstitution for pre-concentration. Moreover, considering the pKa of nicotine and its major metabolite, cotinine (8.58 and 4.79 respectively) several protocols involved re-extraction cycles with pH adjustments for neutralization of the analytes [9,10]. Other reported analytical methods, providing more relatively efficient and selective extraction of nicotine and its major metabolites from biofluids, compared to LLE, were based on SPE [11], and paper spray [12]. Solid Phase microextraction (SPME) was also employed as a convenient sampling/pre-concentration method for nicotine extraction from biofluids, reducing potential exposure to chemical toxicity (vs. LLE and SPE), lowering cost (vs. SPE and paper spray) and labor requirement (vs. LLE and SPE). Most of the SPME applications for analysis of nicotine and its metabolites involve head-space analysis [13], [14], [15], [16]. The use of HS-SPME was preferred mainly to avoid direct exposure to the extraction coating to complex biofluids and the occurrence of biofouling that would induce a drastic decline in the extraction ability of the SPME device. These methods, however, are limited by the poor volatility and high polarity of nicotine and derivatives that limit the recoveries obtainable due to a poor partition of the targeted analytes into the gas phase. However, in recent years, the development of biocompatible SPME coatings [17], [18], [19], [20], made overcoming such limitations possible by employing antifouling materials that ensure reusability of the sampling probes and minimize biases related to the attachment of unwanted matrix constituents on the coating surface, when the sampling device is directly immersed into the biofluid. The biocompatibility (or the matrix compatibility) of an SPME extraction phase is defined based on its antifouling properties. In fact, an extraction phase resistant to biofouling caused by the sample matrix components can be recognized as biocompatible . Moreover, a biocompatible SPME device is suitable for in vivo sampling when it is nontoxic and harmless to the living system studied [19].

To date, the most commonly used biocompatible SPME extraction phases are based on polydimethylsiloxane (PDMS) for gas-chromatography and polyacrylonitrile (PAN) for liquid-chromatography [19]. In this work, we developed and validated an analytical protocol, based on biocompatible SPME fibers and LC-MS/MS, to test the bioavailability, adsorption kinetics and metabolic interconversion of nicotine in rabbit plasma. Plasma samples were collected after transdermal application of a gel formulation for smoking cessation containing nicotine-stearic acid conjugate loaded into solid lipid nanoparticles. The slower release of nicotine by this formulation was initially tested through in vitro studies [21]. Further results were confirmed in rabbits using the sensitive analytical method described and developed in this work, for the ultra-trace analysis of nicotine and its metabolites from plasma specimens. We tested the performance of two SPME coatings, namely mixed-mode (MM)/PAN – constituted by sorptive silica particles functionalized with C₈ and benzene sulfonic acid moieties – and hydrophilic-lipophilic balance (HLB)/PAN, both for single and dual fiber extraction. Analysis of the extracts was performed with LC-MS/MS employing an efficient laminar flow ion flux through differential pressure regions of the mass spectrometer. Thus, there was no mass discrimination during ions transport, no optics surface charging and minimized ion beam drifting. The method was fully validated according to the FDA guidelines for bioanalytical method development and was successfully used to monitor the release of nicotine and its interconversion to secondary metabolites in rabbit plasma after transdermal administration of nicotine loaded smoking cessation formulations.