Electromembrane extraction of phenytoin from biological fluids: A survey on the effects of molecularly imprinted polymer and carbon nanotubes on extraction efficiency

https://doi.org/10.1016/j.microc.2020.104800Get rights and content

Highlights

  • A simple, rapid, and efficient EME-HPLC-UV method has been developed.

  • The method was applied for the determination of phenytoin in biological fluids.

  • Effects of CNTs & MIP on EME efficiency were evaluated.

  • The method is environmentally friendly due to use of low volume of solvent (<30 µL).

  • Removing of further sample preparation steps has made the method so affordable.

Abstract

In the present study, an electrically-assisted microextraction method termed electromembrane extraction (EME) followed by a simple high performance liquid chromatography-ultraviolet detection was developed and validated for the determination of phenytoin in human biological samples. Main parameters influencing the electromembrane extraction were evaluated and optimized using the Box-Behnken experimental design. The membrane consisted of 1-octanol immobilized in the pores of a hollow fiber. As a driving force, a 40 V electric field was applied to facilitate the migration of analytes from the sample solution to an acceptor solution through a supported liquid membrane. The optimum donor and acceptor solutions pHs were achieved 10 and 13, respectively. The enrichment factor was > 82 within 15 min led to 55% absolute extraction recovery. In optimum conditions, the method provided the linearity in the range of 10–1000 ng/mL (R2 >0.999). The repeatability of the method was indicated as relative standard deviations (%RSD) between 3.6% and 8.9% (n = 3). The limits of detection and quantitation were 3.0 and 10.0 ng/mL, respectively. The sensitivity of HPLC-UV for determining phenytoin was enhanced by electromembrane extraction. Also, in this study, the effects of some nano-sorbents like carbon nanotubes and molecularly imprinted polymer on membrane performance and EME efficiency were evaluated. The EME technique can be introduced as a new approach for screening of molecularly imprinted polymers in their primary steps of characterization. Finally, the proposed EME-HPLC-UV method was applied for phenytoin determination in human plasma and urine samples with relative recoveries ranged between 88–92% indicating the reliability of the method.

Introduction

Drug substances determination and quantification are very important in pharmacological and toxicological sciences, pharmacokinetics, forensic studies, and clinical trials. In these kinds of investigations, samples are blood, saliva, spinal fluid, plasma, urine and other biological fluids. These biological fluids are the complex matrices consisting of hundreds/thousands of various components, and the target drug substance (analyte) is mostly in very low concentrations in these samples. Also, sample preparation and analyte extraction are necessary for analysing the analytes of interest in all the mentioned cases [1], [2], [3].

The chemical analysis steps will be influenced by the sample preparation procedure [4,5]. The main role in the sample preparation step is changing the real sample matrix to the appropriate sample for analysis. Meanwhile, a method that follows the following purposes with respect to the type of analyte can be more appropriate: removals of more probable interruptions, analyte concentration increasing, being robust and repeatable independent of sample matrix, using less amounts of sample, less organic solvent volume or not using organic solvent, specific and selective method for target analyte and also compatibility with the instruments [1], [2], [3], [4], [5], [6].

Liquid-liquid extraction and solid phase extraction methods are the most frequently used methods in chemical and pharmaceutical analysis, in order to extract and prepare the samples. These extraction methods disadvantages are considered as a long time sample preparation, organic solvent high consumption, highly-needed sample volume, need to other sample treatment steps, and difficulty in these methods automation. Nowadays, the analytical methods are more concerned for developing the method, which needs extractor (solvent) phase volume to be very lower or solvent-free/solvent less methods to be considered. Therefore, common traditional methods are being replaced with new miniaturized sample preparation methods that were called micro-extraction. Hollow fiber-liquid phase micro-extraction (HF-LPME) is one of the liquid phase microextraction methods that are extensively used, nowadays. Despite of this method simplicity and effectiveness, extraction may time-consuming [7,8]. Therefore, electro-membrane extraction (EME) method was introduced by Pedersen-Bjergaard and Rasmussen in 2006 in order to increase the rate and efficiency [9]. This method was actually the sub-set of the HF-LPME method. At first, a hollow fiber (usually made from polypropylene) was impregnated with a water-immiscible organic solvent which was named supported liquid membrane (SLM). The end of the hollow fiber (HF) was closed and after that its inside was filled by the aqueous solution with definite pH as the acceptor phase. After that, the mentioned HF was put inside the sample aqueous solution with definite pH as the donor phase, and at the final stage, two platinum electrodes were put inside the donor and acceptor phases with respect to the mentioned analyte charge. A magnetic stirrer was utilized to ensure uniformity throughout the container. The extraction was performed by applying electric potential difference (voltage) on two ends of the HF. The driving force achieved by the electric potential difference (voltage) was applied on the width of two donor and acceptor phases. In this method, extraction was performed with respect to active transport, while HF-LPME extraction method was based on passive diffusion. The molecules of analytes distributed between donor phase-SLM and acceptor phase-SLM interphases based on distribution coefficient. Applying the potential difference on both sides of donor and acceptor acts as a driving force, and also could significantly reduce the extraction time [10,11].

The electromembrane extraction method is significantly mentioned for its simplicity, quickness, proper sample clean up, very low consumption of sample and solvent, analyte enrichment, method flexibility, and no requirement to other sample preparation steps [12]. Its reported applications included drug substances [9,[13], [14], [15], [16], [17], [18], [19], [20]], chemicals and dyes [21,22], peptides and amino acids [23], [24], [25], plant hormones [26], heavy metals [27], minerals and anions [28,29] extraction from various foods, environmental samples and also biological fluids.

In addition to this simple method various methods have been invented for EME. These inventions in the method performing included a drop to drop method, on-microchip extraction, pulsed EME, simultaneous extraction of acidic and basic compounds, combination with electro-assisted liquid-liquid microextraction, and carbon nanotubes application in membrane structure [10], [11], [12], [13], [14], [15], [16], [17].

Carbon nanotubes (CNTs) have been applied in various extraction methods as sorbent like solid phase extraction, solid phase micro-extraction (SPME), and HF-LPME [30]. Moreover, several reports were accomplished on their applications in EME method. In those investigations, using carbon nanotubes has increased extraction efficiency due to the high surface area and their adsorption capacity [31,32]. Molecularly imprinted polymers (MIP) with many applications are considered as the porous sorbents made in molecular imprinting method. Also, molecular imprinting is a method for preparing a polymer with selective recognition sites and porosity making. In other words, the selective recognition sites by molecular imprinting make 3D permanent paths and memories for transferring molecules into the polymeric structure [33,34]. In addition, the most important characteristics of molecularly imprinted polymers are their high affinity to the target molecule and their selective absorption capacity [33], [34], [35]. Accordingly, the main applications of molecularly imprinted polymers in field of chemical analysis included using them as the sorbent in SPE, chromatography column, and electrochemical sensors.

In this research, an EME-HPLC-UV method has been developed and validated for phenytoin (PT) determination in human biological fluids. Also, the effects of important factors like organic solvent composition, pH of donor and acceptor solutions, extraction time and voltage on extraction efficiency have been examined. The appropriate conditions optimization for EME method was performed using Box-Behnken experimental design approach. In addition, some nano-sorbents like carbon nanotubes (CNT/EME) and molecularly imprinted polymer (MIP/EME) and their effects on membrane performance and extraction efficiency were also evaluated. At the Final stage, the developed EME-HPLC-UV method were used for drug determination in human plasma and urine samples.

Phenytoin (PT) (5, 5-diphenylimidazolidine-2, 4-dione) is an antiepileptic drug belonged to the class of hydantoin that is frequently utilized for treating seizure and epilepsy. In addition, phenytoin is used in order to prevent the caused seizure by head trauma and brain surgeries. The phenytoin chemical structure and physiochemical characteristics are indicated in Fig. 1. A proper analytical method development for phenytoin determination and quantification in biological fluids is important, because of its pharmacokinetic properties and the extensive prescription for treating the epilepsy, narrow therapeutic index, and also the importance of therapeutic drug monitoring [36].

Several analytical methods have been reported for the phenytoin determination in biological fluids including HPLC [37], [38], [39], [40], [41], LC–MS and LC–MS/MS [42], GC and GC–MS [43,44], immunoassays [45] and capillary electrophoresis [46].

There is no report in literature about using EME method for phenytoin extraction from biological samples. Therefore, an EME method development, and after that, a simple HPLC-UV analysis will be useful for the phenytoin quantitative analysis in biological samples. The proper speed and simplicity, consumption of low volume of organic solvent (<30 µL), proper analyte enrichment and preconcentration, sample clean up, and removing the further sample preparation steps are recognized as the proposed EME method advantages.

Section snippets

Chemicals and materials

Phenytoin (PT) powder (99%) was kindly supplied by Alhavi Pharmaceutical Company (Tehran, Iran). Phenobarbital powder (99%) was supplied from Nantong Pharmaceutical Company (China). Methanol, ethanol, ammonium acetate, glacial acetic acid, hydrochloric acid (HCl), sodium hydroxide (NaOH), nitric acid, sulfuric acid, 2-ethylhexanol, 1-heptanol, 1-octanol, cetyl trimethylammonium bromide (CTAB), toluene, acrylamide, ethylene glycol dimethacrylate (EGDMA), azobisisobutyronitrile (AIBN) and Triton

SLM composition

The effect of various solvents as SLM was examined on phenytoin extraction in similar conditions. Moreover, some solvents combination effect was evaluated as SLM. Test results are indicated in Fig. 3. The maximum extraction was observed for 1-octanol as SLM amongst various solvents. Comparing the statistical results (p value < 0.05) demonstrated the significant difference between the maximum points obtained by 1-octanol from the other points. Also, extraction system was very unstable and high

Optimization of EME of phenytoin

In this research, several factors effect has been examined on EME of phenytoin, in order to obtain the optimum extraction conditions. Also, the SLM composition effect was examined by one factor at a time (OFAT) method. The other parameters like voltage, pH of the donor solution, and extraction time were optimized by the use of Box-Behnken experimental design.

The maximum extraction was observed by 1-octanol amongst various solvents. By comparing the statistical results, the significant

Conclusion

An efficient EME-HPLC-UV method has been developed and validated for the phenytoin determination in biological fluids. Also, effective parameters including SLM composition, applied voltage, donor and acceptor solution pH, and extraction time were evaluated and optimized. In addition, the effects of some solid nano-sorbents like carbon nanotubes and molecularly imprinted polymer on electromembrane extraction efficiency were completely explained. The proposed EME-HPLC-UV method can be applied in

Declaration of Competing Interest

There is no conflict to declare.

Acknowledgements

The authors would like to acknowledge financial and instrumental supports from Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (Grant No. 32968).

References (54)

  • C. Huang et al.

    Electromembrane extraction

    TrAC Trend. Anal. Chem.

    (2017)
  • Z. Aladaghlo et al.

    Application of electromembrane extraction followed by corona discharge ion mobility spectrometry analysis as a fast and sensitive technique for determination of tricyclic antidepressants in urine samples

    Microchem. J.

    (2016)
  • T. Rahmani et al.

    Study on electrical current variations in electromembrane extraction process: relation between extraction recovery and magnitude of electrical current

    Anal. Chim. Acta

    (2016)
  • M. Balchen et al.

    Electromembrane extraction of peptides

    J. Chromatogr. A

    (2008)
  • L. Strieglerová et al.

    Electromembrane extraction of amino acids from body fluids followed by capillary electrophoresis with capacitively coupled contactless conductivity detection

    J. Chromatogr. A

    (2011)
  • Z. Tahmasebi et al.

    Ultra-trace determination of Cr (VI) ions in real water samples after electromembrane extraction through novel nanostructured polyaniline reinforced hollow fibers followed by electrothermal atomic absorption spectrometry

    Microchem. J.

    (2018)
  • T.Y. Tan et al.

    Electro membrane extraction of biological anions with ion chromatographic analysis

    Anal. Chim. Acta

    (2012)
  • X. Liang et al.

    Carbon-based sorbents: carbon nanotubes

    J. Chromatogr. A

    (2014)
  • K.S. Hasheminasab et al.

    A new method for the enhancement of electromembrane extraction efficiency using carbon nanotube reinforced hollow fiber for the determination of acidic drugs in spiked plasma, urine, breast milk and wastewater samples

    J. Chromatogr. A

    (2013)
  • K.S. Hasheminasab et al.

    Development and application of carbon nanotubes assisted electromembrane extraction (CNTs/EME) for the determination of buprenorphine as a model of basic drugs from urine samples

    Anal. Chim. Acta

    (2013)
  • P.K. Owens et al.

    Molecular imprinting for bio-and pharmaceutical analysis

    TrAC Trend. Anal. Chem.

    (1999)
  • L. Wan et al.

    Core–shell molecularly imprinted particles

    TrAC Trend. Anal. Chem.

    (2017)
  • A. Beltran et al.

    useful sorbents for selective extractions

    TrAC Trend. Anal. Chem.

    (2010)
  • R.H.C. Queiroz et al.

    Quantification of carbamazepine, carbamazepine-10, 11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography

    J. Pharma. Biomed. Anal.

    (2008)
  • R. Shimoyama et al.

    Monitoring of phenytoin in human breast milk, maternal plasma and cord blood plasma by solid-phase extraction and liquid chromatography

    J. Pharma. Biomed. Anal.

    (1998)
  • S. Bardin et al.

    Determination of free levels of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry

    J. Pharma. Biomed. Anal.

    (2000)
  • M. Balchen et al.

    Electrokinetic migration of acidic drugs across a supported liquid membrane

    J. Chromatogr. A

    (2007)
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