Analytical Techniques for Simultaneous Estimation of Multi-Component Drug Formulations: A Comprehensive Review

Simultaneous estimation happens when one or more active pharmaceutical ingredients (APIs) within a given dosage type are determined by a single analytical technique without any form of separation beforehand [1]. This method is used very commonly in analysis of pharmaceuticals especially in combination doses where one drug is a fixed proportion of another and so forth.

There is a marked rise in the significance of simultaneous estimation as the combination drug therapy is now widely used [2]. Fixed-dose combinations (FDC) are commonly applied in the management of chronic conditions like hypertension, diabetes as well as infections to improve therapeutic effects, decrease adverse effects as well as enhance compliance [2]. There is therefore need to have accurate estimation of each component in order to achieve good therapeutic results.

Simultaneous estimation also plays a vital role in quality control of pharmaceutical formulations [3]. Id, purity, potency and stability of drug products need analytical methods to check the identity, purity, potency and stability. Techniques like HPLC, UV spectrophotometry and HPTLC are highly used because of their accuracy, reproducibility and ability to analyse a mixture of components [3].

Moreover, regulatory compliance requires validated analytical methods to be utilized [4]. Under the guidelines of the International Council for Harmonisation, analytical procedures should exhibit accuracy, precision, specificity, robustness and repeatability in simultaneous estimation of multiple components of drugs [4].

Although it has strengths, simultaneous estimation has a number of analytical complications. Overlapping spectra is one of the great problems especially in the spectrophotometric technique since the components absorb at the same wavelength hence difficult to distinguish [1]. The other very important concern is that of matrix interference, in which the excipients or impurities within the formulation itself interfere with analytical measurements, thus affecting accuracy [5]. Moreover, chemically related drugs, i.e. with identical physicochemical properties, e.g. polarity and solubility, may not separate under chromatography, and considerable method optimization is necessary [6].

In order to counter such shortcomings, new levels of analytical methods are required in large numbers [7]. Time-sensitive techniques, including UPLC, LC–MS/MS, and chemometric-assisted ones, offer high sensitivity, selectivity, and accuracy, allowing estimating drugs even in the complicated matrix and at low dosage levels with high accuracy [7].

This review will aim to give a full description of the methods of analysis in measuring multiple component drugs, such as spectroscopic, chromatographic as well as hyphenated methods. The methods development plans, validation needs, benefits, constraints and recent developments in the field are also discussed in the review.

1. ANALYTICAL TECHNIQUES CLASSIFICATION:

1.1 Spectroscopic Methods:

The most commonly applied methods of analysis to determine the compositions of multi-component drug formulations are spectroscopic because they are simple, fast and cost-effective. A foundation to these techniques is the interaction of electromagnetic radiation with matter, more specifically the absorption of UV-visible light by pharmaceutical molecules [8].

Figure 1: Block Diagram of a UV–Visible Spectrophotometer (Single Beam Type)

1.1.1 UV–Visible Spectrophotometry:

One of the most widely used methods of simultaneous estimations is the UV -Visible spectrophotometry. It is founded on the absorbance of drugs at certain wavelengths in the UV-visible range (200800 nm). To perform the multi-component analysis, there are methods like simultaneous equation method, absorbance ratio (Q-analysis), and multicomponent analysis that are commonly utilized [1].

The method is especially applicable when there are adequate differences in the absorption maxima ( 45 ) of the drugs. But in cases of strongly overlapping spectra, more sophisticated methods are needed.

1.1.2 Derivative Spectroscopy:

Derivative spectroscopy is a modern variation of the UV spectrophotometry that boosts the ability to distinguish between overlapping spectra. It entails transformation into first, second or higher derivatives of normal absorption spectra.

The zero-crossing method is the most popular and in zero-crossing, one component records zero absorbance, and the other is accurately determined [9]. The process enhances selectivity, but does not involve physical separation of components.

1.1.3 Ratio Spectra Methods:

Ratio spectra techniques entail the division of absorption spectrum of a mixture with the spectrum of a standard component (divisor). This method can be used to remove interference of intersecting spectra and is also able to accurately quantify each component [10].

Or, even better improvement can be done by employing ratio derivative method, a method which takes ratio spectra with derivative techniques to enhance the resolution and sensitivity.

1.1.4 Chemometric-Assisted Methods:

Chemometric based techniques involve the combination of statistical and mathematical resources and spectroscopic data to solve multi-component mixtures. Popular methods in simultaneous estimation include Principal Component Analysis (PCA), as well as Partial Least Squares (PLS) regression [5].

They are quite useful with very overlapping spectra, and multifaceted drug matrices, where they substantially increase the accuracy and predictability.

Benefits of Spectroscopic Techniques:

Spectroscopic techniques offer several advantages in pharmaceutical analysis. They are generally cost-effective, as they require relatively inexpensive instrumentation compared to chromatographic methods [11]. These techniques are also simple, rapid, and involve minimal sample preparation, resulting in reduced analysis time. Furthermore, spectroscopic methods are highly suitable for routine quality control analysis due to their efficiency, reliability, and ease of operation.

1.2 Chromatographic Methods:

The use of chromatographic methods has been regarded as the gold standard of the simultaneous determination of multi-component drug preparations because of their high selectivity, sensitivity, and reproducibility. Such techniques allow the isolation of the components by disparities in physicochemical characteristics like polarity, molecular weight, and contact with stationary and mobile phases [8].

1.2.1 High-Performance Liquid Chromatography (HPLC):

The most common methods have been High-Performance Liquid Chromatography (HPLC) which is predominantly employed in the simultaneous estimation of drugs in combined dosage product. Of its kinds, the most frequently used one is Reverse Phase HPLC (RP-HPLC) because of its versatility and its ability to work with a large variety of pharmaceutical compounds [3].

RP-HPLC (most common):

RP-HPLC uses a non-polar stationary phase (e.g. C18 column) and a rather polar mobile phase. It can be used to analyse moderately polar to non-polar drugs and offers high separation efficiency [3].

Gradient vs Isocratic Techniques:

• Isocratic elution: Employs fixed composition of the mobile phase; simple and repeatable but need not be able to separate complex mixtures.
• Gradient elution: During analysis, the composition of the mobile phase is varied; enhances separation of compounds possessing different polarities, and reduces the time used in analysis [12].

Applications in Combination Drugs:

HPLC finds a wide application in the analysis of fixed-dose combinations of antihypertensive, antidiabetic, antiviral drugs, and others. It is also used in the study of stability, testing of dissolution and in bioanalytical testing [6].

1.2.2 Ultra-Performance Liquid Chromatography (UPLC):

Ultra-Performance Liquid Chromatography (UPLC) is a more advanced version of HPLC, as it uses sub-2 µm columns made with particle sizes and higher pressure of operation.

1. Faster analysis: UPLC uses a lot of less time than traditional HPLC does, which results in high sample throughput [13].
2. Higher resolution: It offers a better resolution of peaks, sensitivity and accuracy which makes it much more applicable to the analysis of complex multi-component drugs [13].

UPLC finds more and more applications in drug industry in the context of high throughput analysis and method development.

1.2.3 High-Performance Thin Layer Chromatography (HPTLC):

High-Performance Thin Layer Chromatography (HPTLC) is an advanced version of the thin-layer chromatography with better resolving and quantifying performance.

HPTLC has the advantage of analysing a large number of samples in a single plate, and is also a cost-efficient and efficient method of conducting routine quality control [14]. It is commonly applied in herbal preparations, stability, and in screening of pharmaceutical mixes.

The Chromatographic Methods have the following advantages.

• High precision and accuracy: give reproducible and reliable findings in the analysis of multiple components [8].
• Great selectivity and sensitivity.
• Applicable to complicated mixtures and analysis at the trace level.

Shortcomings of Chromatographic Techniques.

• High cost: It needs costly instrumentation and maintenance.
• Harder operation: Requires qualified employees to develop and optimize the methods.
• Slow preparation of samples in certain instances.

1.3 Hyphenated Techniques:

The combination of the separation ability of chromatograph systems and the detection way of spectroscopic systems is a big step forward in the analysis of pharmaceuticals where hyphenated methods of analysis are utilized. The approaches are especially useful in the simultaneous determination of multi-component drug preparations, in particular, in cases when high selectivity and sensitivity are needed. The most popular qualitative/quantitative methods used in analysing the pharmaceutical compounds are Liquid Chromatography Mass Spectrometry (LC-MS/MS) and Gas Chromatography Mass Spectrometry(GC-MS) [7].

Liquid Chromatography Mass Spectrometry (LC-MS/MS) combines both liquid chromatography and tandem mass spectrometry, enabling effective separation and accurate detection of the analytes in terms of their mass to charge (m/z) ratios. The method is very appropriate in the characterization of non-volatile and thermally sensitive and polar molecules in pharmaceutical formulations [15]. LCMS/MS has outstanding sensitivity, selectivity, which can be used to detect the drug traces at trace levels in complex biological and pharmaceutical matrices. It finds wide applications in bioanalysis, pharmacokinetic analysis, profiling impurities, and testing stability of combination drug products. Moreover, the tandem mass spectrometry also gives structural details in the form of fragmentation pattern, allowing effective identification and verification of analytes [16].

Another potent hyphenated method is Gas Chromatography-Mass Spectrometry (GC-MS) that is mainly applied to volatile as well as semi-volatile compounds. Under GC-MS, the analytes are first of all separated in the gas chromatographic column followed by their detection by the mass spectrometry that provides qualitative and quantitative data [17]. IRGC–MS is universally used in impurity analysis, residual solvent determination as well as in forensic and environmental investigations, even though the technique has been restricted in use in pharmaceutical formulations to volatile or dramatizable compounds. The method is highly resolution and reproducible, hence can be utilized in simultaneous estimation of complementary elements of drugs.

Hyphenated techniques are important because they offer high sensitivity and structural identification. The methods are capable of analysing analytes in very low concentrations even in complex matrices which can be difficult to analyse using the conventional methods. Also, the mass spectrometry structural elucidation ability enables verification of drug identity and identification of degradation products, as well as characterization of impurities [18]. Although highly advantageous, the techniques entail costly instrumentation, highly trained staff, as well as complicated methodology development. This notwithstanding, the use of hyphenated methods cannot be abolished as an analysis tool in highly sophisticated pharmaceutical studies owing to their excellent analytical capability.

1.4 Electrochemical Methods:

Due to its simplicity, sensitivity, and low cost, electrochemical analyses have received growing popularity in the analysis of drug formulations that contain multiple components of the drug itself, as well as its impurities and reactions with other drugs in pharmaceutical analysis. The principles of these methods rest on the principle of measuring electrical signals including current or potential produced by redox reactions of analytes at the electrode surface. Electroanalysis techniques can also be applied specifically to the analysis of electroactive drugs and have been effectively used in quality control, bioanalysis and pharmaceutical studies [19].

One of the most popular electrochemical methods is voltammetry where the current is captured as a change with respect to an applied potential. Techniques like cyclic voltammetry, differential pulse voltammetry, and square wave voltammetry are regularly used in the analysis of pharmaceuticals. Voltametric techniques are sensitive and can measure several different analytes at the same time with different levels of redox or reduction potentials. These are particularly beneficial in trace-level analysis, and the electrochemical behaviour of drugs. Alongside, it utilizes modified electrodes to improve the selectivity and sensitivity of complex matrices, i.e. carbon nanotube or polymer-coated electrodes [20].

Another electrochemical method that is worth mentioning is Potentiometry which is aimed at determining the potential difference between two electrodes without causing any major current between them. It is much more frequently applied with ion-selective electrodes (ISEs) in the determination of particular ions or drug constituents in a mixture. Potentiometric techniques are less expensive, quick and easy and can be used in routine analysis. Though they are usually less sensitive than voltametric methods, they are extremely convenient in the simultaneous determination of drugs with different ionic properties [8].

Generally, the electrochemical techniques offer a number of benefits such as little sample preparation, quick analysis, and capability to identify the analytes in low concentration. Their use is however confined to electroactive substances and may be interfered with by other redox active species consequently affecting accuracy. Nevertheless, to overcome these shortcomings, electrochemical methods still remain useful in the simultaneous determination of multi-component drugs especially where resources are limited and on-site determination.

1.5 Emerging Techniques:

New methods of analysis are increasingly becoming relevant in simultaneous estimation of multi-component drug formulation since they are increasingly efficient, sustainable, and capable of providing better data analysis. The methods help to overcome the shortcomings of traditional analytical techniques by increasing sensitivity, decreasing analysis time and minimizing environmental impact. The major focused methods are capillary electrophoresis and green analytical chemistry techniques, nano-analytical and artificial intelligence (AI)-supported chemometric methods.

Capillary electrophoresis (CE) is a very effective separation method that relies on the varying rates of movement of chargeable analytes under a given electric field in a thin capillary. Its benefits include a high resolution, quick analysis, and computerized less use of solvents. Some of the methods that have been effectively used to estimate simultaneous multiple pharmaceutical drugs encompass use of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) [21,24]. The topical research has shown that the application of the CE-based techniques is effective in terms of analysing a complex of drugs with enhanced selectivity and efficiency [24].

Increasing concerns about the environment and regulation have attracted great interest to green analytical chemistry approaches. These approaches are aimed at minimizing hazardous solvent utilization, generation of waste and enhancing the energy utilization. The use of greener solvents and miniaturized methods has been commonly proclaimed in pharmaceutical analysis [22,25]. The most recent studies emphasise the creation of green RP-HPLC techniques to estimate drugs simultaneously with proving their environmentally-friendly nature and analytical efficiency [25].

The nano-analytical methodology includes the use of nanomaterials like nanoparticles, nano sensors and nanostructured electrodes to improve the performance of the analytical process. These materials possess a high level of surface area and enhanced selectivity and good reactivity. An analytical application based on nanotechnology have been effectively used to detect drugs at trace levels in complex matrices and are currently under investigation to determine multi-component drugs [23].

Through artificial intelligence (AI) and chemometric analysis, pharmaceutical studies are undergoing change making it possible to analyse the data through sophisticated data processing, multivariate calibration and predictive modelling. Principal Component Analysis (PCA) and Partial Least Squares (PLS) regression are more commonly used methods to overcome redundancy in spectral data and enhance accuracy [5]. The latest development on analytical method development, such as RP-HPLC and hyphenated method, has integrated chemometric tools to increase the robustness and the reliability of the methods to estimate multiple drugs simultaneously [26 -28].

In general, new methods of analysis are highly beneficial particularly in terms of sensitivity, environmental friendliness and efficiency in the analysis process. Their broad use can also be restricted, though, by special equipment, technical skills and increased costs of operation. These innovative methods will be of vital importance in the future of pharmaceutical analysis and at the same time estimation of multi-component formulations of drugs despite the limitations.

2. METHOD DEVELOPMENT CONSIDERATIONS:

A well-established and sound analytical technique to use in simultaneous estimation of multi-component drug forms has to be optimized carefully by designing several important parameters. Applicability Proper development of methods makes the results of the analysis accurate, precise, selective and reproducible, especially in complex pharmaceutical mixtures [3].

The choice of solvent or mobile phase, particularly during chromatographic methodologies, can be considered one of the most vital issues in the development of the method. The type of the solvent to be used is determined by the solubility, polarity, and stability of the analytes on chemical grounds. Mobile phases used in High-Performance Liquid Chromatography (HPLC) can be mixtures of water with organic solvents (including methanol or acetonitrile) with or without buffering to adjust pH and enhance peak shape. The mobile phase greatly affects retention time, resolution, and peak symmetry and its optimization is important in the successful separation of multiple components [12].

A selection of the detection wavelength is another crucial parameter especially in UV Visible Spectrophotometry and HPLC/UV Detection. The wavelength is usually determined using the maximum absorbance ( λmax ) of the analytes so as to achieve maximum sensitivity and accuracy. When the absorption maxima of some analysed components are not at the same wavelength, in compromise wavelength, or multiple wavelength estimation a trade-off wavelength or wavelengths can be employed. The correct choice of wavelength will aid in the reduction of interference and specificity of the method [8].

Optimization of pH is also highly important in the development of methods because it influences the ionization state, solubility, and stability of drug molecules. Chromatographic methods The pH affects how analytes interact with the stationary phase, hence, affecting the retention time and the resolution in chromatographic methods. Supplier buffer solutions are commonly used to ensure consistent pH and increase reproducibility. Close attention to pH when working with ionizable compounds is especially significant to reach an ideal separation and peak shapes [12].

Another critical aspect is the choice of column, especially in chromatography. The factors of column length, particle size, and stationary phase composition (e.g., C18, C8, phenyl columns) play an important role in the efficiency of the separation and the length of the analysis. The most common one is the reverse-phase column more so the C18 type, as it can be widely employed in pharmaceutical analysis. Appropriate choices of column guarantee proper resolution of components and enhance the level of method robustness [6].

Lastly, the processes of sample preparation are vital in achieving sound and reproducible findings. Removal of impurities and sample preparation before analysis is commonly done using techniques like filtration, dilution, extraction and sonication. More sophisticated sample preparation techniques may be necessary in a complex matrix, extraction of the sample in either solid phase by solid-phase extraction (SPE) or liquid phase by liquid-liquid extraction (LLE) may be necessary to remove the interfering substances. Effective sample preparation also increases sensitivity of the method, lessens effects of matrices, and also improves the overall analytical performance [7].

3. METHOD VALIDATION (According to the ICH guidelines):

3.1 Accuracy:

Accuracy can be defined as the proximity of the experimental value as determined by the method of analysis with the true value. It is normally assessed by recovery tests, in which concentrations of the analyte that are known to be present are put on the sample matrix at various levels (e.g., 80%, 100 and 120 percent). The degrees of the results are indicated as percentage recovery and a range between 98-102 is usually acceptable in terms of pharmaceutical analysis [4,3].

3.2 Precision:

Precision specifies how accurately an analytical method will repeat, under typical operating conditions. It is presented as percent relative standard deviation of replicates, that is, percentage RSD. Precision is considered on various levels, such as repeatability (within the day only) and intermediate (within the day, but across analysts, or instruments). The acceptable limit of the %RSD is usually less than 2% and this is an indicator of good method precision [4,8].

3.3 Linearity:

Linearity This is a property of an analysis strategy, such as that a result calculated is proportional to the concentration of analyte in a given range. It is established by making calibration standards at varying concentrations and then a calibration curve of response against concentration is plotted. Linearity is indicated by an expression of the correlation coefficient (R 2 ) and it must be typically above 0.999 to have a good analytical performance [4,3].

3.4 Limit of Detection (LOD):

Limit of Detection (LOD) may be described as the lowest measurement level of the analyte which is measurable but not quantifiable using the presented experimental conditions. It is normally determined by the signal-noise ratio (usually 3:1) or other statistical features of the response and slope of the calibration curve. LOD stands out especially in the detection of trace levels of drugs or impurities [4].

3.5 Limit of Quantification (LOQ):

Limit of Quantification (LOQ) The lowest concentration of analyte that can be accurately and precisely determined. It has typically been computed based on a signal-noise ratio of 10:1 or in a statistically equivalent way to LOD. LOQ makes sure that the analysis procedure has the ability to quantify low levels of the analytes in pharmaceutical formulations reliably [4].

3.6 Robustness:

Robustness is the property of an analysis technique that allows it to be stable to small, intentional fluctuations in the parameters of the analysis technique. These changes can be variations in pH, mobile phase composition, flow rate, temperature or detection wavelength. A sound method has the ability to give dependable and steady findings when utilized in different circumstances, which suggests that it can be utilized in everyday analysis [3].

3.7 Specificity:

Specificity This is the characteristic of the analytical method to measure the analyte with the rest of the components including impurities, degradation products and excipients. It assures that the signal to be analysed is caused by the signal of interest only, and no other substances. This parameter is especially important when it comes to simultaneous estimation of multi-component drug formulations [8].

4. TECHNIQUES COMPARATIVE EVALUATION:

An analytical approach that compares the overall performance, applicability and limitations of commonly used methods of analytical techniques in the simultaneous estimation of multi-component drug formulations should be done to establish their relative performance. Methods used include UV- visible spectrophotometry, High-performance liquid chromatography (HPLC) and Liquid chromatography-mass spectrometry (LC-MS), which have a great difference in respect of sensitivity, cost, accuracy and use. All spectrophotometric techniques are typically appropriate when it comes to routine quality control because of their simplicity and low cost, but chromatographic and hyphenated methods are more sensitive and accurate with complex pharmaceutical analysis [8,3,7].

Table 1: Compare and Contrast Analysis of Analysis Methods.