Quantification of Antibiotics in Multicomponent Drug Formulations using UV-Vis Spectrometer with PLS and MCR-ALS

Abstract

This study explores the potential of spectroscopic analysis combined with Partial Least Squares Regression (PLS) and Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) for the simultaneous quantification of antibiotics in multicomponent drug formulations, specifically clofazimine (CLZ) and dapsone (DAP). The analysis also evaluated the in vitro release profile of the drugs in a fixed-dose combination tablet. High-Performance Liquid Chromatography with Photodiode Array Detection (HPLC-PDA) was used as a reference analytical technique to validate and compare the chemometric models. Both PLS and MCR-ALS models demonstrated high accuracy, with MCR-ALS showing superior predictive capability for CLZ, while both models presented similar performance for DAP quantification. Notably, the results from both models were consistent with the dissolution profile, indicating no statistically significant differences between the spectroscopic and chromatographic quantification methods. Furthermore, the dissolution profile confirmed the immediate release of both active pharmaceutical ingredients (APIs), with no statistically significant differences between the spectroscopic and chromatographic quantification methods. This study highlights the efficiency and versatility of chemometric techniques as an alternative to conventional methods in the quality assessment of anti-leprosy medications.

Highlights

• Simultaneous quantification of antibiotics in fixed-dose combination tablets.
• Spectroscopy integrated with PLS and MCR-ALS for drug quality control.
• MCR-ALS demonstrates superior accuracy with recovery rates near 100%.
• Chemometric effectively analyze in vitro drug release profiles.
• Robust alternatives to conventional methods for pharmaceutical quality assessment.

Introduction

Leprosy, or Hansen’s disease, is a chronic infectious condition caused by Mycobacterium leprae, primarily affecting the skin, peripheral nerves, and mucous membranes [1]. Currently, the specific treatment for people with leprosy is multidrug therapy standardized by the World Health Organization (WHO), known as MDT, which follows a standard regimen according to the patient’s operational classification as Multibacillary (MB) or Paucibacillary (PB) [2]. The drugs used for treatment are rifampicin (RMP), dapsone (DAP), and clofazimine (CLZ), administered in combination [2], [3]. Treatment for MB leprosy in adults lasts 12 months, comprising three drugs: RMP (600 mg, administered monthly under the supervision of a healthcare professional), CLZ (300 mg, administered monthly, and 50 mg in a daily dose), and DAP (100 mg in a daily dose, used without direct supervision). The treatment regimen for PB leprosy in adults lasts 6 months and uses two drugs: RMP (600 mg, administered monthly under supervision) and DAP (100 mg daily dose, also used without direct supervision) [1], [2]. Thus, the standardization of MDT, in terms of time and therapeutic agents, plays an essential role in improving treatment adherence and efficacy.

Although antimicrobial therapy has proven effective in curing leprosy, there are reports of several cases of resistance to these drugs [4]. These resistance mechanisms can arise from various factors, with one of the primary causes being poor patient adherence to treatment due to its long duration and the need for strict daily compliance [5]. Faced with these challenges, there is an urgent need to look for alternatives to improve the availability and quality of therapies, including the development of combined fixed formulations that can guarantee greater practicality and safety in administration, and encourage local production [6], [7]. Implementing these strategies can significantly enhance leprosy management and strengthen the autonomy of the Brazilian health system. [8].

As for the analytical methodologies used to determine CLZ and DAP in combination, few publications are available, most of which are based on chromatographic separation techniques [2], [9], [10], [11]. Although chromatographic analytical techniques are widely used due to their high precision and efficiency, they have significant limitations, such as the high consumption of organic solvents, making them environmentally unsustainable, as well as requiring highly qualified operators to carry them out [12]. On the other hand, spectroscopic methods, such as UV-Vis spectroscopy, stand out for offering several advantages, including simplicity of handling, lower cost instrumentation, shorter analysis times, and the possibility of use by professionals with a lower level of qualification [13]. However, one of the main challenges of UV-Vis spectroscopy is its low analytical selectivity [14], especially in complex mixtures where spectral overlap occurs. To overcome this limitation, the use of chemometric tools, such as multivariate calibration models, has proved highly effective. These techniques, based on the use of many spectral variables, make it possible to overcome the overlap of signals between active pharmaceutical ingredients (APIs) and excipients, enabling both qualitative and quantitative analyses with greater precision [15], [16]. In addition, these methods offer a greener profile, significantly reducing the use of chemicals, energy, and waste generation, contributing to more sustainable laboratory practices [17].

Multivariate calibration models for quantifying APIs in mixtures include Partial Least Squares (PLS) and Multivariate Curve Resolution with Alternating Least Squares (MCR-ALS) [14], [18], [19]. Although they work in different ways, both chemometric models have advantages and limitations that make them suitable for different applications. PLS regression correlates spectral information with a dependent variable, such as the concentration of the API present in the tablet. This chemometric model builds a predictive model capable of estimating the concentration of APIs in new samples from the corresponding spectra, with high precision and efficiency [20], [21]. MCR-ALS, in turn, decomposes data matrices, such as UV-Vis spectra, to identify the pure components (APIs) in mixtures containing interferents, such as excipients. It allows relative concentrations and pure spectra of the components to be recovered by iteratively adjusting the model parameters until the experimental data is reproduced with good accuracy [19]. To guarantee physically meaningful solutions, restrictions such as non-negativity, unimodality and closure are applied, reducing spurious solutions and increasing the reliability of the results [22], [23].

This study explored the application of two chemometric approaches, MCR-ALS with correlation constraint and PLS regression, for determining the content of CLZ and DAP in a combined fixed-dose solid formulation, using UV-Vis spectroscopy. The methodology was structured in three main stages. In the first stage, a 52 factorial design was carried out for the construction of synthetic mixtures of CLZ and DAP, followed by the division of the data into calibration and test sets. The second stage focused on the development of the combined fixed-dose tablet and the subsequent determination of the contents of both drugs. In the third stage, the dissolution profiles of the developed tablets were constructed, assessing their pharmaceutical performance. The chemometric models developed were compared to the reference method, High Performance Liquid Chromatography with Photodiode Array Detector (HPLC-PDA), allowing the quantification performance of CLZ and DAP to be assessed and the applicability of the proposed methodologies to be validated.

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Materials

Clofazimine (CAS 2030-63-9, C27H22Cl2N4, 473.396 g.mol-1, purity 98%, batch E/1233/G22001, BRMAQ) and Dapsone (CAS 80-08- 0, C12H12N2O2S, 248.302 g.mol-1, purity > 97 %, batch 20220502, BRMAQ). β-cyclodextrin (≥97%, Sigma-Aldrich), SEPITRAP 4000 (Castor oil 65%, SEPPIC), SEPITRAP 80 (Polysorbate 80 55%, SEPPIC), Microcrystalline cellulose 102 (Sigma Aldrich), Sodium amide glycolate (Sigma-Aldrich), Colloidal Silicon Dioxide (SigmaAldrich), Sodium Stearyl Fumarate (Sigma-Aldrich)

Hilthon A. Ramos, Igor Eduardo Silva Arruda, Lucas José de Alencar Danda, Rafaella F. Sales, Julia M. Fernandes, Monica Felts de La Roca Soares, Jose M. Amigo, M. Fernanda Pimentel, José Lamartine Soares Sobrinho, Quantification of Antibiotics in Multicomponent Drug Formulations using UV-Vis Spectrometer with PLS and MCR-ALS, Chemometrics and Intelligent Laboratory Systems, 2025, 105354, ISSN 0169-7439, https://doi.org/10.1016/j.chemolab.2025.105354.

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