Development and Validation of an HPLC-UV Method for the Determination of Levofloxacin from a Prolonged–Release Mesh Implant in Rat Blood

1. Introduction

The creation of novel analytical approaches for detecting medicinal compounds in biological matrices represents a key area of pharmaceutical analysis. Precise measurement of active pharmaceutical ingredients depends on the establishment and validation of robust, dependable methods. Such methodologies are essential to guarantee the quality, safety, and therapeutic effectiveness of pharmaceutical products [1,2]. Levofloxacin (LVX) is a third-generation fluoroquinolone antibiotic with broad-spectrum activity against Gram-positive and Gram-negative bacteria. It is widely used in the treatment of respiratory, urinary tract, skin, and soft tissue infections due to its favorable pharmacokinetic properties, high bioavailability, and strong tissue penetration. Despite these advantages, systemic administration of LVX can be associated with adverse effects and suboptimal drug concentrations at the site of infection, particularly in chronic or localized infections that require sustained antimicrobial exposure [3,4].

Localized drug delivery systems have emerged as an effective strategy to overcome the limitations of conventional systemic therapy. Among these systems, implantable drug-loaded meshes have attracted increasing attention because they allow prolonged and controlled drug release directly at the target site, thereby reducing systemic exposure and improving therapeutic efficacy. Prolonged-release antibiotic implants are especially valuable in preventing and treating post-surgical infections and in managing chronic infections where long-term antimicrobial presence is required [5,6]. However, accurate evaluation of drug release behavior and systemic absorption from such implants necessitates sensitive and reliable analytical methods for drug quantification in biological matrices.

Several analytical methods have been reported for the determination of LVX in human plasma or serum using high-performance liquid chromatography (HPLC) with ultraviolet (UV) [7,8,9,10,11,12,13,14] or fluorescence detection (FLD) [15,16,17,18], as well as LC–MS/MS [19,20,21,22,23] techniques. HPLC coupled with UV detection remains one of the most widely used techniques for the quantitative determination of fluoroquinolone antibiotics in biological samples. HPLC-UV offers advantages such as robustness, cost-effectiveness, ease of operation, and adequate sensitivity for routine pharmacokinetic and bioavailability studies [24,25]. While LC–MS/MS methods provide high sensitivity, their high operational costs and limited accessibility restrict their routine use in many laboratories. Moreover, all of the reported HPLC-UV methods are optimized for conventional dosage forms or systemic administration and may not be directly applicable to studies involving prolonged-release implantable systems.

To date, limited attention has been given to the development and validation of analytical methods specifically tailored for the determination of LVX released from implantable mesh systems in small laboratory animals [26,27]. Rat models are commonly employed in preclinical studies to evaluate the pharmacokinetics, biocompatibility, and release characteristics of implantable drug delivery systems. Therefore, a validated, simple, and reliable HPLC-UV method for the quantification of LVX in rat blood is essential to support such investigations and ensure accurate interpretation of pharmacokinetic data.

The present study aimed to develop and validate a rapid, accurate, and precise HPLC-UV method for the determination of LVX in rat blood following administration of a prolonged-release LVX mesh implant. The method was validated in accordance with International Council for Harmonisation (ICH) Q2 (R2) guidelines, assessing parameters such as linearity, accuracy, precision, and robustness. The proposed method is intended to provide a practical analytical tool for routine analysis and preclinical evaluation of implant-based LVX delivery systems.

2. Materials and Methods

2.1. Chemicals and Reagents

LVX reference standard was obtained from KRKA (Slovenia; batch No. UG1456). Acetonitrile and methanol of gradient grade suitable for HPLC were purchased from Merck (Germany; batch Nos. I1167530 135 and I1154907 124, respectively). Orthophosphoric acid (85%, HPLC gradient grade) was also supplied by Merck (Germany; batch No. K52625773 029). Polypropylene hernia mesh implants loaded with LVX were used for in vivo experiments. A standard stock solution of LVX was prepared by accurately weighing 10 mg of LVX reference standard and transferring it into a 100 mL volumetric flask, 30 mL of methanol was added and shaken, then the volume of the solution was made up to the mark with solvent. Quality control (QC) solutions were then prepared by diluting the stock solution with mobile phase to the desired concentrations. The gelatin used in the research was obtained from Sigma-Aldrich) Germany), chitosan from Merck) Germany), citric acid from Sigma-Aldrich) Germany) and glycerin from Sigma-Aldrich) Germany). Ethyl alcohol meeting the requirements of the pharmacopoeia and having analytical purity was also used in the experimental studies. All reagents and materials used in the research process had high-quality indicators and were considered suitable for analytical purposes.

2.2. Animal Experiments and İmplantation Procedure

A prospective laboratory trial has been conducted in our research. The applicability of the proposed method was tested on rat plasma samples by using a HPLC method. The macroscopic structure of the implant utilized in this study, as well as the critical steps of the surgical implantation procedure in the rat model, are illustrated in Figure 1. All stages of the research were considered and approved by the Ethical Committee Board of Azerbaijan Medical University (Protocol No. 28; 05.07.2023) as a research conducted in the Helsinki Declaration principles. The UNIFLEX surgical endoprosthetic mesh is a non-absorbable implant manufactured from biocompatible polypropylene and exhibits high mechanical strength. Its mesh-like, porous structure promotes effective integration with surrounding tissues by facilitating fibroblast migration and collagen deposition, thereby enabling reliable repair of tissue defects. The UF-2010 model is produced in a standard white color and features blue orientation marking lines that assist in accurate intraoperative placement (Figure 1a). The implant is sterilized using ethylene oxide and is intended for single use only.

To 10 g of gelatin, 100 mL of water at a temperature of 90–95 °C is added, and the mixture is stirred until complete dissolution is achieved. The resulting hot solution is then filtered through a cotton filter. After cooling the solution to 40–45 °C, 50 mL of 95% ethyl alcohol is added. As a result, a cloudy, milky suspension forms as a precipitate. The mixture is thoroughly stirred, the container is covered with cellophane, and the solution is left at room temperature for 12 hours.

The formed precipitate is separated from the liquid phase by filtration through a paper filter. To the obtained precipitate, 95% ethyl alcohol (approximately 50–75 mL) is added until complete precipitation of gelatin from the solution occurs. The container with the mixture is then placed in a refrigerator for 12–24 hours. After this period, the liquid phase is decanted, and the precipitate is dissolved in 50 mL of water by heating at 90–95 °C. The resulting hot solution is evaporated on a water bath until a thickened dry residue is obtained. The residue is dried in a thermostat at 40–45 °C, yielding approximately 5.25 g of dry gelatin.

To 5 g of purified gelatin, 20 mL of purified water is added, and complete dissolution is carried out in a water bath with heating. The solution is then cooled to 40–45 °C, after which 0.05–0.1 g of citric acid is added. Subsequently, 2 g of chitosan is introduced into the solution and stirred until complete dissolution is achieved. Thereafter, the following components are added sequentially: 0.5 g of LVX, 10 mL of glycerin, and 5 mL of propolis tincture. The mixture is thoroughly stirred until a homogeneous mass is obtained. Purified water is then added to adjust the final volume of the solution to 50 mL.

This volume is intended for application onto five mesh prostheses measuring 20 × 10 cm; thus, 10 mL of the prepared solution is used for each mesh to form a film. The mesh endoprosthesis is placed on a glass surface, and the prepared solution, preheated to 40–45 °C, is evenly applied in a thin layer using a spatula. The coated endoprosthesis is then placed in a thermostat and dried at 35–40 °C for approximately 12 hours. Upon solidification, an elastic film with a smooth surface and prolonged antibacterial activity is formed on the mesh endoprosthesis. Following the coating process, the sterile implant was surgically placed onto the abdominal wall defect in the rat model (Figure 1b) and secured in the final anatomical position (Figure 1c).

Prior to use, the endoprosthesis is packaged and sterilized with ethylene oxide. The components of the claimed composition were selected experimentally to achieve a positive effect corresponding to the stated objective. Since LVX is the principal therapeutic component of the proposed formulation, all specific implementation examples were carried out using a base amount of the active substance, namely 0.5 g of LVX. The quantitative composition limits enabling the achievement of the stated objective were determined.

2.3. Biological Samples

Blood samples were collected from experimental rats following surgical implantation of a polypropylene mesh loaded with LVX. To evaluate the long-term release profile of LVX from the implanted mesh, blood samples were obtained on days 1, 3, 5, 9, and 14 after implantation. All samples were handled and stored under appropriate conditions until analysis. Rat blood samples used for analysis were provided by the laboratory of the Teaching Surgery Clinic of Azerbaijan Medical University. For preparation of the biological samples, 2.0 mL of whole blood was transferred into a centrifuge tube and centrifuged at 4000 rpm for 5 minutes at 4 °C. Following centrifugation, 1.0 mL of the separated plasma was collected and diluted to 2.0 mL with methanol for protein precipitation. The mixture was vortex-mixed and centrifuged again under the same conditions. Subsequently, 1.0 mL of the clear supernatant was transferred to a clean tube, diluted to 2.0 mL with the mobile phase, thoroughly mixed, and filtered through a 0.45 μm PTFE (polytetrafluoroethylene) membrane filter prior to HPLC analysis.

2.4. Chromatographic Conditions

Chromatographic analysis was performed using an Agilent 1260 HPLC (Agilent Technologies, USA) equipped with a UV detector. Data acquisition and peak integration were performed by OpenLab CDS software (version: 2.5.0.842). Separation was achieved on a Zorbax SB-C18 column (4.6 × 250 mm, 5 μm particle size). The mobile phase consisted of acetonitrile and 0.1% (v/v) orthophosphoric acid in water in an 80:20 ratio and was delivered at a flow rate of 1.4 mL/min. The mobile phase was prepared by mixing 800 mL of acetonitrile with 200 mL of deionized water adjusted to pH 3.5 in a 1000 mL volumetric flask. The mixture was degassed using an ultrasonic bath prior to use. The column temperature was maintained at 25 °C. The injection volume was 20 μL, and detection was carried out at a wavelength of 296 nm. The total chromatographic run time was 6 minutes. All solvents used were of HPLC-grade purity. Statistical evaluation of the analytical results was performed using Student’s t-test.

3. Results and Discussion

In accordance with the study objective, a reliable HPLC–UV method was developed and optimized for the determination of LVX in rat blood samples following implantation of a prolonged-release mesh formulation. Method development focused on achieving adequate chromatographic resolution, sensitivity, and reproducibility while maintaining a simple and rapid analytical procedure suitable for biological samples.

3.1. Optimization of Chromatographic Conditions

Key chromatographic parameters, including stationary phase, mobile phase composition, flow rate, injection volume, column temperature, and detection wavelength, were systematically investigated. Optimal separation and peak symmetry for LVX were achieved using a Zorbax SB-C18 column (4.6 × 250 mm), with a mobile phase consisting of acetonitrile and water (80:20, v/v) adjusted to pH 3.5 with orthophosphoric acid. The optimized conditions included a flow rate of 1.4 mL/min, column temperature of 25 °C, injection volume of 20 μL, and UV detection at 296 nm. Under these conditions, LVX was well resolved from endogenous matrix components with a total run time of 6 minutes. Figure 2 shows chromatograms of drug-free plasma of a rat (a), the LVX standard solution (b), and biological samples obtained after implantation of the prolonged-release formulation (c). No significant interfering peaks were observed at the retention time of LVX, confirming the selectivity of the method.

3.2. Method Validation

Validation of the developed HPLC–UV method was carried out in accordance with applicable analytical regulatory guidelines, especially the ICH Q2(R2) guidelines, including assessment of accuracy, linearity, precision, sensitivity, selectivity and stability.

Selectivity: Six blank plasma samples were analyzed to evaluate potential interferences by comparing the retention time values with those obtained from plasma samples spiked with LVX. As shown in Figure 2a, there is no interfering peak from the endogenous compounds.

Stability: The stability of LVX was investigated throughout different storage conditions. The stability results of LVX are summarized in Table 1. As shown, after 30 days of storage at room temperature (25 °C), the assay value of LVX decreased from 100.0% to 98.9%, corresponding to a 1.1% change from day 0. These minor variations are considered within the acceptable range for pharmaceutical stability.

Accuracy

Method accuracy was evaluated using QC samples prepared by spiking known amounts of LVX into the mobile phase. Working solutions at concentrations of 2, 4, and 8 μg/mL were analyzed in triplicate. As summarized in Table 2, recoveries ranged from 99.24% to 99.97%, with a mean recovery of 99.68%. The low standard deviation (0.26%) and narrow confidence interval (99.48–99.88%, p = 95%) demonstrate the high accuracy of the method across the studied concentration range. These results meet the acceptance criteria specified in the ICH Q2(R2) guidelines.

Linearity

Linearity was assessed by analyzing standard LVX solutions over the concentration range of 1–8 μg/mL. A calibration curve was constructed by plotting peak area versus concentration. As shown in Table 3 and Figure 3, the method exhibited excellent linearity with a correlation coefficient (R²) of 0.999. The regression equation was y = 72409.63x + 10.474, indicating a strong proportional relationship between detector response and analyte concentration within the tested range.

Precision

Method precision was evaluated in terms of both intra-day and inter-day repeatability using six replicate analyses of samples containing 2, 4 and 8 μg/mL of LVX. The results are presented in Table 4 and Table 5. Inter-day precision showed a mean recovery of 99.85% with a mean coefficient of variation (CV) of 0.24%, while intra-day precision yielded a mean recovery of 99.84% with a mean CV of 0.21%. These low variability values indicate excellent repeatability and robustness of the analytical method for routine application.

Sensitivity

The limits of detection (LOD) and quantification (LOQ) were calculated based on the standard deviation of the intercept and the slope of the calibration curve, in accordance with ICH recommendations. The LOD and LOQ were determined to be 0.3 μg/mL and 1.0 μg/mL, respectively, demonstrating adequate sensitivity for monitoring LVX concentrations in biological samples following prolonged release.

3.3. Application to Biological Samples

The validated method was successfully applied to the analysis of blood samples collected from six rats implanted with LVX-loaded mesh devices. The method enabled reliable quantification of LVX over the studied time points (Table 6), supporting its suitability for evaluating long-term drug release and systemic exposure from implantable formulations. The chromatograms obtained from the analysis of rat samples are shown in Figure 4.

Comparison of the Developed HPLC-UV Method with Previously Reported Methods for LVX Quantification

The HPLC-UV method developed in the present study demonstrates analytical performance characteristics well suited to its intended preclinical application, occupying a distinct niche within the extensive literature on levofloxacin quantification. As summarized in Table 7, while fluorescence and mass spectrometric methods achieve superior sensitivity with LOQs as low as 2.11 ng/mL in human milk and 100 ng/mL in serum, our method provides adequate sensitivity (LOQ 1.0 μg/mL) for monitoring therapeutic concentrations following prolonged-release mesh implantation in rats, where sustained drug levels above the MIC are anticipated. The linear range of 1–8 μg/mL is comparable to established HPLC-UV methods for human plasma (0.25–15 μg/mL), and the validation parameters, including mean recovery of 99.68% and intra-/inter-day CV <0.3%, compare favorably with published assays reporting recoveries of 87–99% and acceptable CV <5–15%. Unlike the majority of reported methods optimized for human plasma [28,29,30,31,32,33,34,35], serum, specialized clinical matrices [36], pharmaceutical formulations [37,38,39,40], or environmental samples [41], our method is specifically validated in rat blood, the most relevant preclinical model for evaluating implantable drug delivery systems. Furthermore, while Jiang et al. [42] employed HPLC for in vitro release testing from composite scaffolds, our study extends application to in vivo quantification over 14 days post-implantation, addressing a recognized methodological gap. The simple protein precipitation sample preparation and 6-minute run time confer practical advantages for routine preclinical analyses compared to more complex solid phase extraction, liquid-liquid extraction techniques. Thus, the developed method provides a fit-for-purpose, cost-effective, and accessible analytical tool that complements existing high-sensitivity clinical therapeutic drug monitoring platforms and pharmaceutical quality control assays by specifically addressing the requirements of preclinical implantable device research.

4. Conclusion

A selective, accurate, and precise HPLC-UV method has been successfully developed and validated for the determination of levofloxacin in rat blood. The method fulfills all critical validation parameters, including linearity, accuracy, precision, and sensitivity as per ICH guidelines, making it suitable for routine bioanalysis. Its successful application to plasma samples from rats implanted with a LVX-loaded polypropylene mesh demonstrates its practical utility in preclinical pharmacokinetic studies. This method effectively quantifies the sustained systemic exposure of LVX from a localized delivery system, addressing a gap in analytical approaches tailored for implantable drug delivery evaluations. The proposed HPLC-UV assay provides a cost-effective, accessible, and reliable analytical solution to support the development and optimization of prolonged-release antibiotic implants, facilitating accurate assessment of their in vivo release profiles and therapeutic potential.