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
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 For instance, only limited quantity of blood can be withdrawn at each time point while the animal remains alive for the whole duration of the experiments.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.
Section snippets
Materials
The reference materials nicotine, cotinine, anabasine, nornicotine were purchased from Sigma Aldrich (TX, USA) and norcotinine was purchased from Toronto Research Chemicals (North York, Ontario, Canada). The deuterated standards nicotine-D3 and cotinine-D3 were acquired from Cerilliant-Sigma Aldrich (Austin, TX, USA). The physical-chemical properties and molecular structure of the targeted analytes are summarized in Table S1. The pH 10 buffer consisting of disodium tetraborate, sodium
Method optimization
Considering the ionizable nature of nicotine and its metabolites, pH adjustment of the sample matrix is critical to guarantee adequate recovery of the analytes onto SPME devices. The adjustment of matrix pH was carried out in order to promote the neutral form of the analytes and, consequently, favor their partition onto the extraction phase rather than in the plasma samples, which is an aqueous-based media. Considering the pKa values of analytes, we decided to adjust the pH of the plasma to 10,
Conclusions
In this work, biocompatible SPME fibers, HLB/PAN, were used for the simultaneous extraction of nicotine and 4 of its metabolites from rabbit plasma treated with different smoking cessation formulations. Analysis of the extracts by UPLC-MS/MS utilizing a laminar flow ion guide enabled the achievement of LOQs at parts per trillion levels for all the analytes, (except nornicotine) and linear dynamic ranges within three orders of magnitude of concentration in the plasma samples.
Figures of merit of
CRediT authorship contribution statement
Nipunika H. Godage: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft. Erasmus Cudjoe: Formal analysis, Methodology, Validation. Rabin Neupane: Investigation, Resources. Sai HS. Boddu: Investigation, Resources. Pradeep Kumar Bolla: Investigation, Resources. Jwala Renukuntla: Investigation, Resources. Emanuela Gionfriddo: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors are grateful to the University of Toledo for funding. The authors would like to express their gratitude to Perkin Elmer Inc., and in particular to Dr. Frank Kero for enabling the use of the LX 50 UPLC and the QSight 220 Mass Spectrometer in their laboratory at the University of Toledo. The authors would also like to acknowledge Prof. Jose O. Rivera and Paso del Norte Health Foundation grant awarded to The University of Texas at El Paso, for supporting this work, and Amit Dahal for
References (27)
- et al.
Determination of nicotine and cotinine in rat plasma by liquid chromatography-tandem mass spectrometry
J. Chromatogr. A.
(2003) Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods
J. Pharm. Biomed. Anal.
(2004)- et al.
Simultaneous and sensitive measurement of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human plasma by liquid chromatography-tandem mass spectrometry
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2009) - et al.
A high area, porous and resistant platinized stainless steel fiber coated by nanostructured polypyrrole for direct HS-SPME of nicotine in biological samples prior to GC-FID quantification
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2017) - et al.
A critical outlook on recent developments and applications of matrix compatible coatings for solid phase microextraction
TrAC-Trends Anal. Chem.
(2019) - et al.
Simple, fast and sensitive LC–MS/MS analysis for the simultaneous quantification of nicotine and 10 of its major metabolites
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2014) - et al.
A fully validated LC–MS/MS method for simultaneous determination of nicotine and its metabolite cotinine in human serum and its application to a pharmacokinetic study after using nicotine transdermal delivery systems with standard heat application in adult smokers
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2016) - et al.
A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2010) - et al.
Challenges and opportunities in dermal/transdermal delivery
Ther. Deliv.
(2010) - et al.
Nicotine chemistry, metabolism, kinetics and biomarkers.
Handb. Exp. Pharmacol.
(2009)
Nicotine replacement therapy: an overview.
Int. J. Health Sci.
Pharmacology of nicotine: addiction, smoking-induced disease, and therapeutics
Annu. Rev. Pharmacol. Toxicol.
A simple, fast, and sensitive method for the measurement of serum nicotine, cotinine, and nornicotine by LC-MS/MS
J. Sep. Sci.
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