Analysis of anti-neoplastic drug in bacterial ghost matrix, w/o/w double nanoemulsion and w/o nanoemulsion by a validated ‘green’ liquid chromatographic method

Highlights

• Environmentally benign RP-HPLC method has been applied for the assay of 5-FU.

• The developed method was applied for the analysis of 5-FU in bacterial ghost matrix.

• It was also applied for analysis of 5-FU in injection, w/o and w/o/w nanoemulsion.

• Proposed method was found to be suitable for analysis of 5-FU in all samples.

Abstract

The objective of the present investigation was to develop and validate a ‘green’ reversed phase high-performance liquid chromatography (RP-HPLC) method for rapid analysis of a cytotoxic drug 5-fluorouracil (5-FU) in bulk drug, marketed injection, water-in-oil (w/o) nanoemulsion, double water-in-oil-in-water (w/o/w) nanoemulsion and bacterial ghost (BG) matrix. The chromatography study was carried out at room temperature (25±1 °C) using an HPLC system with the help of ultraviolet (UV)–visible detector. The chromatographic performance was achieved with a Nucleodur 150 mm×4.6 mm RP C8 column filled with 5 µm filler as a static phase. The mobile phase consisted of ethyl acetate: methanol (7:3% v/v) which was delivered at a flow rate of 1.0 mL min⁻¹ and the drug was detected in UV mode at 254 nm. The developed method was validated in terms of linearity (r²=0.998), accuracy (98.19–102.09%), precision (% RSD=0.58–1.17), robustness (% RSD=0.12–0.53) and sensitivity with satisfactory results. The efficiency of the method was demonstrated by the assay of the drug in marketed injection, w/o nanoemulsion, w/o/w nanoemulsion and BG with satisfactory results. The successful resolution of the drug along with its degradation products clearly established the stability-indicating nature of the proposed method. Overall, these results suggested that the proposed analytical method could be effectively applied to the routine analysis of 5-FU in bulk drug, various pharmaceutical dosage forms and BG.

Introduction

5-Fluorouracil (5-FU) is one of the major antineoplastic drugs used in the treatment of various solid tumors associated with several parts of the body [1], [2]. The literature survey demonstrated that various analytical methods such as thin layer chromatography (TLC), gas chromatography (GC), isotachophoresis, capillary electrophoresis or high-performance liquid chromatography (HPLC) have been reported for the quantification of 5-FU in plasma, bulk drugs, pharmaceutical dosage forms and biological fluids [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. Radioactivity, flame ionization, fluorescence, ultraviolet (UV) absorption or mass spectrometry (MS) techniques have been used as detection techniques for the quantification of 5-FU [15], [16], [17], [18], [19], [20], [21]. Among the various techniques available for the analysis of 5-FU, none of them discussed the ‘green’ as well as stability indicating property of the method. Moreover, not a single technique described the application of the analytical technique for the quantification of 5-FU in bacterial ghost (BG) matrix. It has also been observed that none of the previously reported methods has been validated for BG matrix despite these techniques address a variety of validation parameters in a specific biological matrix. BGs represent empty non-denaturized bacterial cell envelopes derived from Gram-negative bacteria by protein-E-mediated lysis. BGs can be utilized as the carrier for proteins/antigens, nucleic acids, drugs and soluble compounds for various medicinal and pharmaceutical applications [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35].

In recent years, overall sustainability of the pharmaceutical and healthcare sectors have become an issue. Regarding environmental aspects, the keyword is “green chemistry” which is being applied to drug design, formulation development, manufacturing and packaging, environmental risk assessment for new pharmaceuticals and minimizing greenhouse gas emissions in the distribution chain, etc. [36]. Due to this fact, the chemicals/solvents used for analytical studies cause greater toxicity to the environment than the species being determined in some conditions. But so far, only a little attention has been paid to the other features related to operational safety in addition to the environmental impact of analytical methodologies [37]. With this background, Green Analytical Chemistry (GAC-an aspect of green chemistry which focuses on diminishing the environmental influence of analytical methodologies) began as an investigation for realistic substitutes to the off-line management of discharged by-products and remains with a view to substituting contaminating procedures with the environment friendly ones [38]. The concept of GAC is spreading in analytical laboratories and the need to make analytical chemistry greener is becoming more widely accepted. In the past decade, the scientific reports found in the literature related to clean analytical chemistry or environmentally-benign analytical methods have been considerably increased [39].

To our surprise, despite several favorable features such as non-toxic, non-volatility, non-inflammable, non-aggressiveness, high biodegradability and cost effective, the full potential of environmentally benign solvents (and their combinations) as an eluent for TLC/HPLC analysis of drugs has not been fully utilized [36], [37], [38], [39].

With this background, the purpose of the current study was to develop and validate an environmental friendly, economical, fast, simple, precise, accurate, robust and stability-indicating reverse phase HPLC (RP-HPLC) method combined with UV detection for analyzing 5-FU using isocratic elution, taking into considerations a variety of International Conference on Harmonization (ICH)-recommended test conditions and its application in determination of this drug in marketed injection, in-house developed formulations i.e. water-in-oil (w/o) nanoemulsion, water-in-oil-in-water (w/o/w) nanoemulsion and BG matrix.