HPTLC methods to assay active ingredients in pharmaceutical formulations: A review of the method development and validation steps

Abstract

High-performance thin-layer chromatography (HPTLC) is still increasingly finding its way in pharmaceutical analysis in some parts of the world. With the advancements in the stationary phases and the introduction of densitometers as detection equipment, the technique achieves for given applications a precision and trueness comparable to high-performance liquid chromatography (HPLC). In this review, the literature is surveyed for developed and validated HPTLC methods to assay active ingredients in pharmaceutical formulations published in the period 2005–2011. Procedures and approaches for method development, validation and quantitative assays are compared with the standard ways of conducting them. Applications of HPTLC in some other areas are also briefly highlighted.

Introduction

Though being an old technique, today in some parts of the world, Thin-layer chromatography (TLC) is still increasingly finding its way in the assay of active ingredient(s) in pharmaceutical analysis. This is due to the evolution of the instrumentation, automization and the development of new adsorbents and supports [1]. Moreover, TLC features in a broad range of applications, such as the analysis of herbal medicines, dietary supplements, biological and clinical samples, food and beverages, environmental pollutants and chemicals [2].

Like all chromatographic techniques, TLC is based on a multistage distribution process, which involves a suitable “adsorbent” (the stationary phase), solvents or solvent mixtures (the mobile phase or eluent), and the sample molecules. high-performance thin-layer chromatography (HPTLC) is an advanced form of instrumental TLC, which does not only include the use of high performance adsorbent layers (e.g. silica gel with refined uniform particles, approximately 5 μm in diameter, as compared to 12 μm in TLC), but also adopted instrumentation e.g. the development chambers. It usually also implies a standardized methodology for development, optimization, documentation and the use of validated methods. The HPTLC technique is applied in qualitative and quantitative separations of compounds in mixtures, where the quantitative mode operates in a more optimized way (standardized with a given procedure), hence, applicable in the assay of compounds in samples.

There are several advantages of using HPTLC for the analysis of compounds as compared to other techniques, like HPLC, spectrometric, titrations, etc. [3], [4]. Some of the advantages are:

• The separation process is easy to follow: especially with coloured compounds,

• Several samples can be separated in parallel on the same plate resulting in a high through-put, and a rapid low-cost analysis,

• Two-dimensional separations are easy to perform,

• Specific and sensitive colour reagents can be used to detect separated spots,

• HPTLC can combine and consequently use different modes of evaluation, allowing identification of compounds having different light-absorption characteristics or different colours,

• Contact detection allows radiolabelled compounds to be monitored and microbial activity in spots to be assessed,

• The TLC plates are disposable; therefore, neither regeneration nor essential clean-up are required, and

• Chromatographic development and detection of the separated spots on a plate are generally separate processes in time, therefore after separation, the plates can be stored for a long time, and detection performed at a later stage to obtain the analytical information.

Literature search reveals a large number of published papers that describe various usage of the (HP)TLC technique in many analytical fields. To emphasize on the importance, challenges and opportunities of the HPTLC technique, various older review papers are available in the literature [3], [5], [6]. In a recent (2011) review paper, Kaale et al. [7] reported the availability of a large number of publications on (HP)TLC methods that are developed, validated and applied in pharmaceutical analysis for the assay of active ingredient(s) and stability testing. The review in [7] gives a brief summary of the use of TLC in drug quality testing and therapeutic drug monitoring in resource-constrained settings in Africa and discusses the shortcomings associated with the use of TLC in Africa. It also gives a general overview of HPTLC methods and their application in drug testing in the above settings. In our review, we will focus on the quantitative mode of HPTLC as used in the assay of active ingredient(s) in pharmaceutical formulations. The different steps applicable to this technique during method development, validation and quantitative analysis will be discussed in relation to the practice observed in publications from 2005 to 2011. The paper is thus clearly different from [7].

Most of the reviewed publications were originating from Asia (mostly India), from some western European countries, and few, about twenty, were from Africa, where the technique has facilitated drug quality assurance, since the more sophisticated and costly analytical equipment, such as HPLC or liquid chromatography–mass spectrometry (LC–MS) is insufficiently available.

Given the context, one might expect to find many methods developed for quality assurance of drugs for diseases mostly affecting these areas, such as malaria, tuberculosis and HIV/AIDS. However, they were developed for many types of diseases. Table 1 shows pharmacological classes, which treat diseases, covered by some of the reviewed publications.