Microextraction by Packed Molecularly Imprinted Polymer Combined Ultra-High-Performance Liquid Chromatography for the Determination of Levofloxacin in Human Plasma

Fluoroquinolones are considered as gold standard for the prevention of bacterial infections. To improve assessment of antibacterial efficacy, a novel method for determination of levofloxacin was developed and validated. Deep eutectic solvents (DESs) as only green solvent were used as a porogen for preparation of water-compatiblemolecularly imprinted polymers (MIPs) with a pseudotemplate.-e DESs-MIPs were characterized in detail, including scanning electron microscope, nitrogen sorption porosimetry, and Fourier transform-infrared spectra. Clearly, themaximum binding capacity of levofloxacin onDESs-MIPs in water andmethanol was 0.216 and 0.077μmol g, respectively.-eDESs-MIPs as adsorbingmaterials were applied inmicroextraction by packed sorbent (MEPS), and the DESs-MIPs-MEPS conditions were optimized. -e DESs-MIPs-MEPS coupled with ultra-high-performance liquid chromatography (UHPLC) was used to determine levofloxacin in human plasma.-emethod was found linear over 0.05–10μgmL with coefficient of correlation equal to 0.9988. -e limit of detection and limit of quantification were 0.012 and 0.04μgmL, respectively. At three spiked levels, the precision of proposed method was between 95.3% and 99.7% with intraday and interday relative standard deviations ≤8.9%. Finally, the developed method was used to examine levofloxacin from human plasma of 20 hospitalized patients after transrectal ultrasound-guided prostate biopsy, and the average concentration (±SD) of levofloxacin was 2.35± 0.99μgmL in plasma.


Introduction
Bacterial resistance of antimicrobial agents has become a serious public health problem in the worldwide context for several decades.New drug-resistant bacterial strains are emerging every day, resulting in wide-spread of antibiotic resistance.erefore, the World Health Organization (WHO) has proposed the possible strategic objectives for tackling antimicrobial drug resistance [1].One of them emphasizes strengthening research of new antibacterial agents, whereas another relies on the optimization of the usage of antimicrobial medicines.To tackle the spread of irrational use, accurate evaluation of prescribed antimicrobial agents is necessary [2].Levofloxacin, one of the commonly used third-generation quinines, has a broad spectrum of activity against Grampositive and Gram-negative bacteria [3,4].Due to its low probability of adverse effects and outstanding antibacterial activity with oral administration, levofloxacin has been widely applied to the treatment of infectious diseases, including acute exacerbation of chronic bronchitis and community-acquired pneumonia [5,6].
e mass spectrometry technique could create a low detection limit and high resolution, but it is not widely available for all laboratories, resulting from the high cost of the equipment.Anyhow, a valid sample pretreatment is necessary prior to chromatographic analysis.e main purpose of sample pretreatment is to eliminate interferences, enrich target analytes, and create a reproducible method [17].
Microextraction by packed sorbent (MEPS), initially introduced by Abdel-Rehim et al. [18], is a miniaturization of conventional solid-phase extraction (SPE) with the advantages of easy-to-automate, cost-effective, eco-friendly, and time efficient.us, the new sample pretreatment technology has been widely used in food, environmental water, and biological sample analyses [17,[19][20][21].However, the commercial MEPS sorbents, such as C2, C8, and C18, ionic exchange sorbent, and polystyrene polymer [19] lack selectivity, which usually results in decrease of separation and enrichment efficiency.erefore, selective sorbent, for example, molecularly imprinted polymers (MIPs), is developed to meet the requirement of selective extraction of target analytes from complex matrix samples [22].
MIPs are the synthetic materials with the advantage of high predetermined recognition ability to target molecules, and they combined with MEPS have been used for the selective extraction of drugs from human plasma and urine sample [23][24][25].In the preparation process of MIPs, porogen plays an important role, which should dissolve template and conduct synthetic reaction.Organic solvents (such as chloroform and toluene) are often employed for obtaining excellent imprinting efficiency.But the side effects of toxic reagents may involve serious risks to operators, as well as damage to the environment [26].
To overcome the toxicity of organic solvents, deep eutectic solvents (DESs) have emerged as a green solvent with the advantages of environmental friendliness, biodegradability, low cost, easy preparation and chemical inertness with water [26].
e DESs can be obtained simply by complexion of quaternary ammonium salts with hydrogen bond donors, which are able to form eutectic mixture.As a result, DESs as porogen have been successfully applied in the synthesis of MIPs, and DESs-MIPs have been used as SPE sorbents [27][28][29][30][31].However, the application of DESs-MIPs combined with MEPS (DESs-MIPs-MEPS) for extracting levofloxacin from biological samples has not been reported.
In this work, DESs were used as pure porogens for the preparation of DESs-MIPs, and the DESs-MIPs were synthesized using gatifloxacin as pseudotemplate, which could avoid the problem of "template leakage" [32].
e microstructure, chemical composition, and selectivity of DESs-MIPs were characterized in detail.DESs-MIPs-MEPS procedure followed by UHPLC was used for selective enrichment and determination of levofloxacin in human plasma.e scientific novelty of this work is the use of the MEPS technique with DESs-MIPs as packing sorbents.Stock solutions of levofloxacin and ciprofloxacin were prepared in methanol at a concentration of 100 μg mL −1 and stored at −20C o .e working solution of levofloxacin was freshly prepared by dilution of stock solution before use.Internal standard solution of ciprofloxacin was freshly prepared by mixing of stock solution and methanol to gain concentrations of 50 μg mL −1 .

Instrumentation.
e morphology of the resultant polymers was measured with scanning electron microscope (SEM, Hitachi X-650, Tokyo, Japan).Brunauer-Emmett-Teller (BET) surface area and porosity of the polymers were measured on a V-Sorb 2800TP surface area and pore size analyzer (Gold APP Instruments, Beijing, China) at 77 K by nitrogen adsorption-desorption isotherms.Fourier transforminfrared (FT-IR) spectra of choline chloride and polymers were recorded on OPUS spectroscopy produced by Bruker Optics (Germany) in the 4000-400 cm −1 region.e chromatographic analysis was performed with an Acquity H-Class UHPLC system equipped with a binary high-pressure pump, an autosampler, a thermostated column compartment, and a diode array detector (Water, Milford, MA, USA).e chromatographic separation was performed with ACQUITY UPLC BEH C 18 column (2.1 mm × 100 mm I.D., 1.7 μm, Water, Milford, MA, USA).Methanol : pH 3 phosphoric acid solution (28 : 72, v/v) was used as the mobile phase with a detection wavelength of 293 nm.e flow rate and the injection volume were 0.3 mL min −1 and 1.0 μL, respectively.

Preparation of DESs-MIPs by Precipitation
Polymerization.For preparing DESs, choline chloride (0.1 mol) and ethylene glycol (0.2 mol) were mixed and heated at 80 °C until a homogeneous liquid formed.Prepolymerization mixture was prepared by mixing pseudotemplate (gatifloxacin), MAA, EDMA, AIBN, and DESs (Table 1) and placed in dark for 24 h.e mixture was sparged with nitrogen for 10 min, sealed, and polymerized in a 60 °C water bath for 24 h.e resultant polymer was crushed with a mortar and pestle, and the template was extracted with acetic acid/MeOH (1/9, v/v) using a Soxhlet apparatus.
e nonimprinted polymers (NIPs) without template (DESs-NIPs) were synthesized in the same manner.

Equilibrium Rebinding Experiments.
e dried particle (20.0 mg) was placed in 5 mL Ep tube containing 3 mL of levofloxacin solution (0.1 mmol/L).After incubation for 24 h at room temperature, the suspension was centrifuged and determined by UHPLC.e imprinting effect of the polymer was assessed with the imprinting factor (IF), which is defined as follows: 2 Journal of Chemistry where Q MIP and Q NIP are the amount of bound levofloxacin on the DESs-MIPs and DESs-NIPs, respectively.
To estimate the binding parameters of DESs-MIPs and DESs-NIPs, the data of static adsorption experiment were processed with the Langmuir model.e Langmuir model assumes that polymers possess only a single class of binding site and can be expressed as follow: where Q max is the apparent maximum number of binding site for levofloxacin, b is the association constant, and C e is the equilibrium template concentrations.

DESs-MIPs-MEPS Conditions.
For the DESs-MIPs-MEPS procedure, the polyethylene frit was inserted into the insulin syringe (1 mL), and then 4.0 mg of DESs-MIPs was packed.After that the second polyethylene frit was inserted into the syringe on the top.Prior to each MEPS, the DESs-MIPs-MEPS syringe was activated with 1.0 mL of methanol and 1.0 mL of water.In a typical process, 400 μL of the sample solution (water-methanol, 25 : 75, v/v) plus 10 μL of internal standard was aspirated 20 times of draw-eject cycles at a speed of 40 μL s −1 .en, the DESs-MIPs-MEPS syringe was washed with 200 μL of water-methanol (50 : 50, v/v) and eluted with 400 μL of acetonitrile/ammonia (95/5, v/v).e eluate was evaporated to dryness with nitrogen gas and quantified to 50 μL of volume with the mobile phase.Finally, the treated sample was injected into UHPLC for quantitative analysis.
2.6.Sample Pretreatment.Plasma samples were collected using the standard venipuncture technique from healthy volunteers and hospitalized patients with transrectal ultrasound-guided prostate biopsy (12 h after oral administration of levofloxacin at a dose of 500 mg, n � 20), who had signed the informed consent for the usage of their plasma.is study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of e Second Hospital of Tianjin Medical University.100 μL of plasma was added into 300 μL of methanol and 10 μL of internal standard, vortexed for 5 min, and then centrifuged at 12 000 rpm for 5 min.e supernatant was transferred and filtered through a 0.45 μm syringe filter before DESs-MIPs-MEPS.

Preparation and Characterization of DESs-MIPs.
In traditional MIPs studies, porogen is typically aprotic and weakly polar volatile organic compound, which will promote the formation of template-monomer complex.DESs consist of charged species and exhibit intraliquid structural properties.
e resulted polymers were characterized by scanning electron microscopy (SEM), nitrogen adsorption-desorption experiments, and Fourier transform-infrared (FT-IR) spectra.
e SEM images (Figures 1(a) and 1(b)) showed that the polymers based on DESs possessed rough and irregular morphology, and a lot of small cavities which could be due to the DESs.But the polymers based on methanol were agglomerated by smaller microglobules with a particle size of near 2-3 μm (Figures 1(c) and 1(d)).
e FT-IR spectra of choline chloride and polymers (Figure 2) provided information to confirm the successful synthesis of DESs-MIPs.Choline chloride had the characteristic peaks of ] C-O (primary alcohol) near 1011 cm −1 and ] C-N (tertiary amine) near 1086 cm −1 .ese characteristic peaks did not appear in the spectra of DESs-MIPs and DESs-NIPs, which indicates that choline chloride is not involved in polymerization and acted just as porogen.e characteristic bands of-COO-(1258 and 1156 cm −1 ) and C�O (1730 cm −1 ) in the spectrum of DESs-MIPs and DESs-NIPs demonstrated the existence of poly(EDMA).e significant peak at 3540 cm −1 was assigned to O-H stretching from MAA. ere are no obvious differences between the spectra of DESs-MIPs and DESs-NIPs, which indicates that pseudotemplate has been washed out completely after elution.
e BET surface areas of DESs-MIPs and DESs-NIPs were 111.2 and 120.9 m 2 /g, which are far higher than those of Me-MIP (6.9 m 2 /g) and Me-MIP (10.6 m 2 /g).In addition, the pore size of DES-based polymer is also far higher than that of methanol-based polymers (Table 2). is was similar to the SEM result, and this indicated that DESs as porogen could change the morphology and improve the surface area and adsorption site of the resultant DESs-MIPs.

Binding Characteristic and Selectivity.
To demonstrate the superior property, the selectivity of resultant polymers was compared in different solvents.As shown in Figure 3, the binding capacity of levofloxacin adsorbed by DESs-MIPs was found to be significantly higher than that of DESs-NIPs, methanol-MIPs, and methanol-NIPs, as well as the binding capacity in water solution was significantly higher than that in methanol, dimethyl sulfoxide, and dimethyl formamide.ese results indicated great water compatibility of DESs-MIPs, which resulted from the "solvent effects" that MIPs prepared in polar porogen show better recognition in similarly polar solvent [32].Figure 4 shows the static adsorption curves in water and methanol.e binding capacities of the DESs-MIPs were much higher than that of DESs-NIPs, methanol-MIPs, and methanol-NIPs at various concentrations.e Q max and b   were calculated from the Langmuir model and are listed in Table 3.Clearly, in water, Q max on DESs-MIPs (0.216 mmol/ g) was considerably higher than that on DESs-NIPs (0.119 mmol/g), methanol-MIPs (0.097 mmol/g), and methanol-NIPs (0.062 mmol/g).In methanol, the DESs-MIPs likewise possessed higher Q max .
e IF of DESs-MIPs was 1.8 in water, while 1.2 in methanol.is result once again indicated the water compatibility of DESs-MIPs.

Investigation of DESs-MIPs-MEPS Conditions.
To enhance the extraction efficiency of DESs-MIPs-MEPS, the relevant conditions were investigated, including the pH of loading sample, volume of loading sample, time of draweject cycles, washing solution, and elution solution.
e influence of the pH of the loading sample was studied over the range 2.0-12.0(adjusted with 1 M of hydrochloric acid and sodium hydroxide).300 μL of sample containing 0.5 μg mL −1 of levofloxacin was loaded.shown in Figure 5, the recoveries of levofloxacin increased from pH 2.0 to 6.0, remained at the best value with the pH values from 6.0 to 8.0, and decreased from pH 8.0 to 12.0.At a low pH (pH � 2.0), most of these carboxyl groups from the imprinted cavity in polymer skeleton were in the neutral form, whereas levofloxacin was positively charged and interacted mainly with ionized carboxyl groups.
is would explain the very low recoveries of levofloxacin.At the pH range 6.0-8.0,most levofloxacins were present in the neutral form, which was beneficial to electrostatic interactions between levofloxacin and DESs-MIPs.At higher a pH (pH � 12.0), the deprotonation of carboxyl groups was enhanced and the protonation of levofloxacin was decreased.is would dramatically decrease ionic interaction between levofloxacin and DESs-MIPs.
erefore, the optimal pH of loading sample was between 6.0 and 8.0, which included the pH of human plasma.
In MEPS, only a few milligrams of sorbent were packed as absorbent material.is resulted in that the extraction capacity was less than the traditional SPE.Fortunately, the increase of draw-eject cycles could enhance the binding amount of target molecules.erefore, the impact of sample volume and draw-eject cycles on the recovery of levofloxacin was optimized when the concentration of levofloxacin was 0.5 μg mL −1 .As shown in Figure 6(a), the recovery of levofloxacin increased with increase of draw-eject cycles and reached the maximum value after 20 of draw-eject cycles.When the time of draw-eject cycles was 20, the recovery of levofloxacin varied from 99.4% to 99.6% with the sample volume increasing from 300 μL to 400 μL and slightly    Journal of Chemistry decreased to 94.3% with 500 μL of sample volume.erefore, 400 μL of sample volume and 20 of draw-eject cycles were selected for further MEPS.
e aim of washing is to remove nonspecifically interfering substances.Figure 6(b) exhibits the effect of washing solution on the recovery of levofloxacin, including water, acetonitrile, methanol, water-acetonitrile (50 : 50, v/v), and water-methanol (50 : 50, v/v).300 μL of sample containing 0.5 μg mL −1 of levofloxacin was drawn into and expelled from the syringe for 20 cycles.No significant differences of recovery were observed when the volume of washing solution was at 100 μL.Further increase of volume led to a slight decrease in recovery.At different volumes of the washing solution, the highest recovery of levofloxacin was observed with water-methanol (50 : 50, v/v) as the washing solution.To achieve excellent recovery and remove as much interfering substances as possible, 200 μL of watermethanol (50 : 50, v/v) was employed as the washing solution for further experiments.
To optimize the smallest volume of eluent that can sufficiently wash the levofloxacin adsorbed on DESs-MIPs, methanol and acetonitrile containing aqueous ammonia solution (3%, 5% and 7%, v/v) were evaluated for eluting levofloxacin, as shown in Figure 6(c).Acetonitrile-ammonia offered the acceptable recovery of levofloxacin.e recovery of levofloxacin increased with the volume of eluent

6
Journal of Chemistry increasing from 100 μL to 400 μL and then barely changed from 400 μL to 500 μL, when the content of ammonia ranging was at 5% and 7%.erefore, 400 μL of acetonitrile-ammonia (95 : 5, v/v) was selected for elution in subsequent experiments.

Method Validation.
e DESs-MIPs-MEPS coupled with UHPLC was validated in accordance with specificity, linearity, sensitivity, accuracy, and precision.e specificity of the proposed method was characterized as noninterference at retention time of levofloxacin, which derived from the endogenous plasma.e specificity was evaluated through analysis of blank samples (n � 15).No interfering peaks from endogenous compounds were observed at the retention time of levofloxacin (2.13 min), and the DESs-MIPs-MEPS could remove potential interferences.Typical chromatogram for blank plasma is shown in Figure 7. Linear calibration curve of levofloxacin was constructed by plotting the peak areas versus nominal concentrations over the range from 0.05 to 10 μg mL −1 for plasma.e calibration curve was prepared at seven concentrations, which covered the range of expected concentrations in the tested plasma.An excellent linearity was obtained with a correlation coefficient equal to 0.9988.e limit of detection (LOD) and the limit of quantification (LOQ) determined as signal-to-noise ration of 3 : 1 and 10 : 1 were 0.012 and 0.04 μg mL −1 , respectively.e level of sensitivity could meet the requirement of trace residue detection in the target biological sample.
e precision and accuracy of the proposed method were determined by using spiked samples at three concentrations, including 0.05, 0.5, and 4.0 μg mL −1 for the blank plasma.e intraday and interday accuracies were tested by analysis of five sets on the same day and five separate days, respectively.No levofloxacin was determined in the blank plasma.As shown in Table 4, the precision values ranged from 95.3% to 99.7%, with intraday precision less than 5.1% and interday precision less than 8.9%.In addition, the sensitivity and the selectivity of the UHPLC method without DESs-MIPs-MEPS were performed.
e sensitivity of the UHPLC method reduced to an LOD of 0.024 μg mL −1 and an LOQ of 0.08 μg mL −1 , respectively.e selectivity of the UHPLC method was also evaluated through analysis of blank samples.As shown in Figure 8, no interfering peaks were observed at the retention time of levofloxacin (2.13 min).However, the interfering peaks at 2.64 min could affect quantification of ciprofloxacin (internal standard at 2.71 min). is results in the difficulty of selecting appropriate internal standard because the internal standard must exist in biopharmaceutical analysis.ese results indicated that the developed method based on DESs-MIPs-MEPS coupled with UHPLC could be used for the analysis of levofloxacin in plasma samples because the DESs-MIPs-MEPS could selectively enrich and separate levofloxacin and ciprofloxacin (internal standard) from complex biological samples.

Real Sample Analysis.
To verify the practical applicability of the developed method, 20 plasma samples obtained from hospitalized patients after transrectal ultrasound-guided prostate biopsy, who had been taking oral levofloxacin at a dose of 500 mg for 12 hours, were analyzed by the DESs-MIPs-MEPS-UHPLC method.e mean ± SD (n � 20) concentration of levofloxacin was 2.35 ± 0.99 μg mL −1 in plasma.Figure 7 showed the chromatographic separation of levofloxacin.e average concentration of levofloxacin obtained from plasma samples in this work agreed with that in the previous report [2,33].

Conclusion
In this work, DESs were selected as porogen for successful preparation of pseudotemplate DESs-MIPs.
e pseudotemplate DESs-MIPs possesses high adsorption capacity for levofloxacin in water and methanol.e DESs-MIPs used   for MEPS could avoid the problem of "template leakage" during application.e optimized DESs-MIPs-MEPS followed by UHPLC analysis was successfully applied to the determination of levofloxacin in plasma sample and provided excellent recoveries and reproducibility.Result of practical application demonstrated that the proposed DESs-MIPs-MEPS-UHPLC method was efficient, economic, and eco-friendly for the determination of levofloxacin in the biological sample.

Figure 5 :
Figure 5: Effect of pH on the recovery of levofloxacin.

Figure 6 :
Figure 6: Effect of sample volume and draw-eject cycles (a) washing solution (b), and eluent solution (c) on the recovery of levofloxacin.

Figure 7 :
Figure 7: Representative chromatograms of patient plasma sample (a) and blank plasma (b).

Table 1 :
Recipes of resultant polymers.

Table 2
a BET surface area.b BJH adsorption cumulative surface area.c Single-point adsorption total pore volume.d Total adsorption average pore with (4 V/A by BET).