Elsevier

Talanta

Volume 221, 1 January 2021, 121626
Talanta

Large volume sample stacking of antiepileptic drugs in counter current electrophoresis performed in PAMAPTAC coated capillary

https://doi.org/10.1016/j.talanta.2020.121626Get rights and content

Highlights

  • PAMAPTAC coating reduces sorption of the analytes on the capillary wall.

  • Separation, sample stacking and forcing out of matrix controlled by high voltage.

  • Sample stacking with sensitivity enhancement factor of about 100.

  • Baseline separation of vigabatrine, pregabaline, and gabapentine in human serum.

  • LOQ for antiepileptics is about 2 · 10−8 mol/L in combination with conductometry.

Abstract

Electrophoretic stacking is developed for sensitive determination of three zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, in human serum. CE separation is performed in a 25 μm fused silica capillary covalently coated with the copolymer of acrylamide with 5% content of permanently charged 3-acrylamidopropyl trimethylammonium chloride (PAMAPTAC). In background electrolyte of 500 mM acetic acid, the 5% PAMAPTAC generates an anodic electro-osmotic flow with a magnitude of (−18.6 ± 0.5) · 10−9 m2V−1s−1, which acts against the direction of the electrophoretic migration of the analytes. A sample of the antiepileptic prepared in a 25% v/v infusion solution and 75% v/v acetonitrile is injected into the capillary in a large volume attaining a zone length of up to 270 mm. After turning on the separation voltage, the antiepileptics are isotachophoretically focussed behind the zone of Na+ ions with a sensitivity enhancement factor of 78. For the clinical determination of antiepileptics, the human serum is diluted with acetonitrile in a ratio of 1:3 v/v and a zone with a length of 90 mm is injected into the capillary. The method is linear in the 0.025–2.5 μg/mL concentration range; the attained limit of quantification is in the range 18.3–22.8 nmol/L; the within-day precision for the migration time is 0.8–1.2% and for the peak area 1.5–2.4%.

Introduction

The basic organic pharmaceutical type of amines, amino acids or nitrogenous heterocycles corresponds to a broad range of bioactive substances, such as antiepileptics - vigabatrin (VGB), pregabalin (PGB), gabapentin (GBP); antidiabetics (metformin and its derivatives) [1], anaesthetics (ketamine and its derivatives) [2], beta-lactam antibiotics [3] and many other compounds. Low-molecular basic pharmaceuticals constitute suitable analytes for capillary electrophoresis (CE), because they can be separated in acidic background electrolytes (BGE), where they migrate as cations through protonisation of the organically bonded nitrogen. The performance of CE in capillaries of small internal diameter (ID), typically ≤ 25 μm, leads to high separation efficiency [4]. This is an essential condition for achieving baseline separation of pharmaceuticals in complex clinical samples, such as blood, urine, tissue microdialysates, etc. Most of the mentioned low molecular pharmaceuticals neither absorb in the UV/VIS part of the spectrum nor provide a signal in fluorescence detection (FD). Thus, the use of these detectors requires derivatisation of the sample. This complication can be avoided by using contactless conductivity detectors (C4D) [5,6] as a universal detection technique [7,8].

The broader use of the CE/C4D methodology in combination with minimum sample pretreatment for clinical and toxicological purposes is prevented by the low sensitivity of this method, caused by the very small sample volume injected into the thin separation capillary. In standard CE separations, it is recommended that higher separation efficiency be achieved using an injected sample plug of only about 1% of the overall capillary length. However, it has been demonstrated that, if specific separation parameters are adjusted, the length of the sample zone injected into the capillary can be substantially increased without significant reduction of the separation efficiency. Thus, LOD can be lowered by several orders of magnitude from a level of about 10−5 mol/L to submicromolar or even nanomolar levels [9]. This technique is known as large volume sample stacking (LVSS) and its successful use is dependent on several preconditions [[10], [11], [12]]: i) reducing the conductivity of the clinical sample by dilution with water or adding an organic solvent with lower conductivity than the BGE, ii) injecting a long sample zone into the separation capillary, in the extreme case filling the whole capillary with the sample, iii) after turning on the high voltage, the analyte migrates faster in the sample zone with suppressed conductivity than in the BGE zone and focussed at the sample/BGE boundary, iv) after entering the BGE zone, the concentrated analyte separates in the capillary zone electrophoresis mode (CZE). The achievement of a high stacking enrichment factor is connected with the need to force the sample matrix out of the separation capillary during the focussing process by the simultaneous application of the hydrodynamic pressure or by electroosmotic flow (EOF).

The assistance of hydrodynamic pressure has practical applications only in capillaries with ID of ≥50 μm, which have low hydrodynamic resistance, and its importance decreases for lower capillary ID [13,14]. In contrast, the application of counter-current EOF is useful in low ID capillaries and is employed especially in the separation of anionic analytes, where the counter-current EOF is generated by the dissociation of the silanol groups of the fused silica capillary or PDMS microchip [12,15,16].

This work describes a detailed study of LVSS in PAMAPTAC covalently coated capillaries with isotachophoretic focussing of the analytes behind the zone of Na+ ions, which are the most abundant cations in human serum. PAMAPTAC covalent coating of fused silica capillaries generates an anodic EOF, whose magnitude is controlled by the percentage content of highly basic 3-acrylamidopropyl trimethylammonium chloride (APTAC) in the polymeric mixture of neutral polyacrylamide (PAM). PAMAPTAC covalent coating with tuneable EOF was recently employed for the CE separation of ketamine and its derivatives [17] and separation of branched chain amino acids in the counter-current electrophoresis regime [18]. Here the PAMAPTAC coating is employed for sensitive CE determination of zwitterion antiepileptics (VGB, PGB, GBP) in human serum for therapeutic drug monitoring. VGB, known under the commercial name Sabril, is a structural analogue of gamma-aminobutyric acid (GABA) and inhibits its degradation through inhibition of the enzyme GABA-transaminase. It is used in the treatment of resistant epilepsy, complex partial seizures, secondary generalized seizures, and for monotherapy use in infantile spasms in the West syndrome [19,20]. PGB with the commercial name Lyrica suppresses neuropathic pain and anxiety disorders and it is used in epilepsy as an add-on therapy for partial seizures [[21], [22], [23]]. PGB was approved as a drug in the U.S.A. in 2004 and has been available as a generic drug in many countries since 2019. PGB is gradually replacing the older antiepileptic GBP known under the name Neurontin, which was already approved in 1993 and has been available as a generic drug since 2004. GBP is also used primarily for suppressing partial seizures, neuropathic pain and restless leg syndrome [24]. A number of methods have been developed for the simultaneous determination of VGB, PGB, and GBP in clinical samples or for checking the purity of pharmaceutical products, based on GC/MS following sample derivatisation [25,26], reverse-phase HPLC [27,28] or HILIC [[29], [30], [31]] with MS, HPLC/FD after sample derivatisation [32,33], CE with laser induced fluorescence (LIF) after derivatisation with 5-(4,6-dichlorotriazinyl)amino fluorescein [34], simple spectrofluorometry after derivatisation with fluorescamine [35], and sensors based on voltammetry [36] or potentiometry [37]. A detailed summary of the analytical procedures can be found in the review article [38].

Section snippets

Chemicals

Gabapentin (pharmaceutical secondary standard, traceable to USP) is purchased from (Fluka, St. Louis, MO, U.S.A.); rac-pregabalin (98%) from (Toronto Research Chemicals, Toronto, Canada) and vigabatrin (98%) from (Tocris Bioscience, Bristol, UK). All other chemicals are of analytical grade purity: NaOH is purchased from (Fluka, Buchs, Swiss); acetonitrile (ACN), HCl (37%) and acetic acid (AcOH, 99.8%) from (Sigma-Aldrich, Steinheim, Germany), infusion solution for intravenous application

Conventional CE separation in coated capillaries

The structures of VGB, PGB and GBP contain a functional group with a pKa value of about 4.0 and amino group with a pKa value of about 10; for the exact values, see Fig. 1. The dissociation of the carboxyl group is suppressed in acidic BGE with pH < 4.0, while the amino group is completely protonated and the antiepileptics are present as their cations. Aqueous AcOH solutions are tested for the CE separation, where the antiepileptics migrate as cations in the normal electrophoretic mode with the

Conclusion

The charged polymeric coating of fused silica capillaries plays a substantial role in reducing sorption of the analytes on the inner walls of the separation capillary and adjusting EOF according to the requirements of the specific application. Thus, baseline separation of the analytes can be achieved even in complex matrices, such as clinical samples. The adjustable EOF can also be beneficial in the LVSS technique, where a long sample zone is injected into the capillary. Na+, which naturally

Author contributions

Petr Tůma: conceptualization, methodology, validation, formal analysis, investigation, writing - original draft, writing - review & editing, supervision; Tomáš Hložek: conceptualization, methodology, formal analysis; Blanka Sommerová: formal analysis, project administration; Dušan Koval: conceptualization, methodology.

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.

Acknowledgement

Financial support from the Grant Agency of the Czech Republic, Grant No. 18-04902S, and the Charles University, Project 260531/SVV/2020, and Project GAUK 11920, are gratefully acknowledged.

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