1. INTRODUCTION:

In recent years, researchers have been using different techniques to create fingerprints of various sample types.¹^,² The industry often bases its compliance with QC criteria and current good manufacturing practises on pharmacopoeial standards.³^,⁴ Pharmacopoeial standards are required to evaluate and guarantee the quality of drugs distributed on the domestic pharmaceutical market. These standards enable regulators to perform quality control of drugs, drug substances, and excipients used in drug manufacturing and allow consumers to form an independent opinion about the drug quality.⁵ When compared to other techniques, chromatography tools continue to be superior for the analysis of compounds.⁶^,⁷

Most pharmacopoeal monographs list chromatography studies as one of the essential identity tests.⁸^,⁹ Various chromatographic is available to ensure the quality of drugs substances but High-performance thin layer chromatography (HPTLC) is highly feasible, reliable, straightforward, quick, and effective technique for qualitative and quantitative analysis of drug compounds.¹⁰ HPTLC is an analytical technique based on TLC, which has been improved to allow for quantitative analysis of the compounds. The application of HPTLC is widely regarded and approved, and in order to standardise the test procedures, numerous approaches are being developed. HPTLC is one of the chromatographic methods that are accessible and is used for constituent identification, impurity identification and quantification, and active substance quantification.¹¹ When it comes to analytical objectives, HPTLC is one of the best TLC techniques due to its improved accuracy, reproducibility, and ability to document the results when compared to normal TLC. As a result, HPTLC technologies are likewise the most suitable technique for the quality control of herbal drugs.¹²^,¹³ Medications derived from plants that are used to treat and enhance human health are known as herbal medicines.¹⁴ Herbal medications must undergo the same level of quality control and quality assurance as pharmaceuticals created using chemicals. But unhappily, the laws governing herbal medications are less strict than those governing modern drugs^.¹⁵^,¹⁶ This is leading to a decline in the quality standards of herbal products through intentional and occasionally unintentional adulteration, fake medications, drug substitution, and many other tactics that are likely to lower the quality of the herbal products that are marketed and consumed for a healthy lifestyle. Instead, it is negatively impacting everyone's health in the population as a whole in harmful ways. ¹⁷ Given the growing demand for herbal remedies, it is necessary to ensure their quality. Almost 80% of people rely on herbs for healing, prevention, and treatment.¹⁸^,¹⁹^,²⁰ The use of herbal remedies as dietary supplements for disease prevention or alternative/ complementary medicine (CAM) for disease treatment has grown in popularity in recent years.²¹ Numerous instruments and processes must be employed in order to check and guarantee that the essential quality is present in the herbal material and products. Rules and/or norms that is nearly as strict as those for synthetic pharmaceuticals must be set in order to carry out quality control testing of the herbs.²²^,²³^,²⁴ This will help to maintain the high standards of quality in the herbal pharmaceutical sector, which is a challenging task and a crucial demand in pharmaceutical research and quality control. The quality of the final formulations is ultimately influenced by a variety of physical, chemical, and geographic factors that affect the quality of these raw components.²⁵^,²⁶^,²⁷ Many chemical and phytochemical tests, analytical techniques, and hyphenated analytical techniques are used to evaluate the quality characteristics of the herbal ingredients in the herbal medications. ²⁸^,²⁹^,³⁰

The analysis and quality control of these products are progressing in an integrated and thorough way, taking into account the complex nature of herbal medicines. Like all other industrial products, pharmaceutical items are constantly subjected to extremely stringent and strictly regulated quality control and quality assurance examinations. The primary objective of these tests is to confirm and strengthen a pharmaceutical product's "Quality," "Purity," "Efficacy," and "Safety in Use." These four elements are necessary for any medicinal product. One of the primary barriers to the widespread adoption of plant-based pharmaceuticals is the inconsistent quality of the manufactured medications. High-performance thin layer chromatography (HPTLC) is one of the sophisticated instrumental methods for qualitative and quantitative analysis of plants and herbal remedies.³¹^,³² Procedures for developing analytical methods and validating them are essential to the search for new medications and pharmaceuticals.³³

1.1. Standardization of medicinal herbs and their products

It is challenging to standardise the plant based product because the phyto-constituents contained in herbal formulations varied based on the climate, makeup of the soil, and the location where it is grown. Due to their accessibility and lack of regulation as medications, many herbal cures could offer serious safety risks. ³⁴ WHO has produced a variety of technical guidelines and publications in order to guarantee the safety and calibre of medicinal plants and herbal products. These include Good Agricultural and Collection Practices (GACP) principles and procedures for quality control of medicinal plant resources.³⁵ Natural medicines are being contaminated and replaced due to the slow increase in deforestation areas. This adulteration and substitution compromise the medicine's safety and efficacy. Currently, large portions of the population use Asian herbal treatments. Asian herbal medications may include harmful heavy metals ³⁶^,³⁷ and unreported prescription drugs, according to evidence from many different countries, which could be extremely dangerous for your health. ³⁸^,³⁹Adulteration, substitution, and a lack of knowledgeable people are the key contributors to the paucity of true herbal remedies. Utilizing cutting-edge quality control techniques and proper standards is important to guarantee the quality of the medicinal herbal products. The identity, excellence, and purity of herbs and herbal products are verified using standards. ⁴⁰ Preliminary identification, physical characteristics, chemical characteristics, and biological characteristics all have an impact on the purity of herbs. The purity of the herbal products affects their quality and freshness.⁴¹ Quality control of herbals is much more important if we want to preserve the integrity of natural commodities and herbs. Purity of the material, assay of the active chemical constituent of larger relevance of the specific herb, and identification of compounds, adulterants, and replacements are all pharmacopeial components of quality control.⁴² The herbal medicines that contain dangerous contaminants and residues, microorganisms, heavy metals, and radio nuclides are examined to assure the purity of herbal ingredients. ⁴³ The process of standardisation involves comparing the qualitative and quantitative characteristics of herbs to recognised or controlled criteria and parameters.⁴⁴^,⁴⁵ Based on various significant evaluation characteristics, including organoleptic properties, ash values, moisture content, microbiological contamination, and chromatographic and spectroscopic evaluations, the WHO has established standards for standardisation methods and procedures for herbal medications. Pharmacognostical schemes and physicochemical studies are the two most crucial prerequisites for the authentication and standardisation of organoleptic testing.⁴⁶^,²⁶ Microscopical analysis, macroscopic (shape and markings), identifications (adulterants and genuine drug), physicochemical parameters (moisture content, acid insoluble ash, water soluble ash), pharmacognostical scheme, qualitative and quantitative, and other reported parameters play a significant role in the authentication of herbs and their formulations. Secondary metabolites such as alkaloids, tannins, glycosides, saponins, and flavonoids can be recognised and utilised to confirm parameters for standardisation.⁴⁷ Modern scientific methods for the analysis of herbal medicines are critically necessary if traditional herbs are to be generally acknowledged globally. A thorough and trustworthy pharmacognostical study can offer scientific foundations for the calibre of conventional herbs and ayurvedic items.⁴⁸

1.2. WHO Guidelines for Quality Control of Herbal Drugs

In an organised business where pharmaceutical companies make ayurvedic medicines, the majority of these regulations are followed. However, as most traditional Ayurvedic medicine producers work on a small scale, they do not employ such stringent tests.⁴⁹ They mostly rely on organoleptic properties and visual evaluation. The main obstacles are a shortage of technically skilled workers and a lack of funding for organised R and D and quality control testing. The WHO (World Health Organization) issued comprehensive recommendations for medicinal plant materials in 1992, and these standards are very useful in ensuring the homogeneity of herbal products.⁵⁰ Specified tests include those for determining foreign matter, macroscopic and microscopic, thin layer chromatography, ash values, extractable matter, specific tests, quality control tests in accordance with specifications, compliance with process and product parameters - process standardisation, microbiological tests to ensure complete absence of all pathogens, pesticide residues, arsenic and heavy metals, and radioactive contamination. In addition to the core identification standards, emphasis has been placed on rigorous microbiological testing, the absence of pesticide residue, and other aspects.⁵¹ Specific testing are indicated for certain elements that have a specific biological activity. Important chemical techniques, such as chromatography and spectroscopy, have been explicitly mentioned as having been used.⁵²

2. High-Performance Thin-Layer Chromatography (HPTLC)

Due to its simplicity, adaptability, affordability, and sophistication, the HPTLC technique is frequently a superior alternative to other chromatography techniques. It is an automated form of thin layer chromatography with superior and advanced separation efficiency and detection limits.⁵³^,⁵⁴^,⁵⁵ High-performance thin-layer chromatography (HPTLC) gained popularity with the development of high-performance adsorbent layers and sophisticated instruments for sample application, chromatogram creation, derivatization, and chromatogram evaluation.⁵⁶ The planar or flat-bed chromatography are other names for high-performance thin-layer chromatography. The three primary industrial uses of HPTLC technology are in the fields of clinical, pharmaceutical, and food testing. ⁸ The two most active research areas at the moment are the evaluation of pharmacological bulk compounds and dosage forms, as well as natural phytochemical medications.⁵⁷ The majority of material classes, including alkaloids, glycosides, carbohydrates, terpenoids, phenols, and steroids can be analysed using HPTLC.⁵⁸ Because of its simplicity, low cost, great sensitivity, and quick separation, it is an extensively used analytical tool. ⁵⁹^,⁶⁰ (Figure -1).

Figure -01: Graphical representation of HPTLC analysis and Mass spectroscopy (HPTLC-MS)

2.1. High-Performance Thin-Layer Chromatography (HPTLC) Principle

The HPTLC operates using the same principles as TLC, such as the adsorption principle of separation. The capillary action allows the solvent or mobile phase to flow. The analytes migrate toward the stationary phase in accordance with their affinities (adsorbent). The component with stronger affinity moves more slowly in the direction of stationary phase. A component with low affinity moves quickly in the direction of the stationary phase. The components are then separated on a chromatographic plate.⁶¹^,⁶² Prior to beginning HPTLC, determine the analytical goal, which could be quantification, qualitative identification, separation of two components from multicomponent mixtures, or analysis time optimization.⁶² The HPTLC method requires fundamental understanding of the nature of the sample, specifically its structure, polarity, volatility, stability, and solubility characteristic, the process of developing a method takes lots of trial and error.⁶³ The most challenging issue is typically deciding with what kind of mobile phase to begin. It is simple to choose a stationary phase; simply start with silica gel as it is practical and nearly suitable for all types of medications. Three-level strategies are used to optimise the mobile phase involves identifying those solvents with an average ability to separate sample, utilising hexane or water to change the solvent's potency, tested at the third level as mixes rather than neat solvents, which can be further optimised by the usage of modifier like acids or bases.⁶⁴^,⁶⁵ Fluorescence mode or absorbance mode are used to find analytes. However, if the analytes cannot be detected accurately, then modifications in stationary phase or mobile phase, as well as pre- or post-chromatographic derivatization, are required. Only after obtaining a decent chromatogram, which can be accomplished by making a little adjustment to the mobile-phase composition, can optimization begin. This results in a chromatogram that is reasonable and has all the desired peaks in symmetry and clearly separated^.⁶⁶^,⁶⁷

2.2. HPTLC analysis of herbal drugs

2.2.1. Drug identification and Authentication

Drug identification and authentification are the fundamental requirements for every type of quality testing of formulations as well as raw ingredients.⁶⁸^,⁶⁹ If any prescription formulation has been taken before the verification of the raw ingredients, there is a higher chance that different drugs or species have been used. The effects on bioactivity or introduction of dangers in adulterated ⁷⁰^,⁷¹, faked, or contaminated herbal problems.,⁷²,⁷³ items pose health. Effective identification techniques and dependable procedures for the detection of foreign material are necessary for the purity and quality of drugs in order to recognise and lessen the risk of these issues. ⁷⁴ Depending on the variety, these markers ⁷⁵ may be species- or family-specific; they can be used to detect the presence of a single herbal compound or to calculate its percentage in a mixture. Several taxonomic, chemical, proteomic, and genomic markers ⁷⁶^,⁷⁷ can be used to validate and identify the constituents of herbal medicines. ⁷⁸ These methods include molecular marker analysis, such as protein or DNA analysis, ⁷⁹ analysis or profiling of secondary metabolites by high-performance thin-layer chromatography (HPTLC)⁸⁰,⁸¹ high-performance liquid chromatography (HPLC)⁸²^,⁸³^,⁸⁴^,⁸⁵ gas chromatography (GC)⁸⁶^,⁸⁷^,⁸⁸^, and capillary electrophoresis (CE) all of which can be combined with mass spectrometry. ⁸⁹^,⁹⁰^,⁹¹

2.2.2. Standard Solution preparation

All the solvents used for preparation of standard solutions should be HPLC Grade. The solubility of pharmaceuticals was taken into consideration when choosing the solvent for standard solutions. The stock solution were prepared by taking 10 ml volumetric flasks, by taking 10 mg of standard drug and added 5 ml of solvent, sonicated for 10 minutes and make up the volume upto the mark with suitable solvent. This stock will be used for the HPTLC analysis. ⁹²

2.2.3. Sample preparation

Solubility is the fundamental problem with chromatographic analysis. If the incorrect solvent is utilised for the type of sample, the analysis's results will suffer.⁹³ To obtain greater separation and TLC development resolution, the solvent selected for the sample must be compatible with the solvent system.⁹⁴^,⁹⁵To much concentrated solution should not be used, it affect the travelling of bands and separation. Volatile and non-polar solvents must be used on the plate.⁹⁶ Polar solvents are commonly used to dissolve components for reversed phase chromatography. The commonly used solvents in HPTLC development are water, methanol, ethanol, acetic acid, formic acid, acetonitrile ethyl acetate, toluene, chloroform, hexan, Pet. ether. ⁹⁷^,⁹⁸

2.2.4. Selection Chromatographic of Layers

The HPTLC layer is available as very tiny particle size pre-coats of silica gel, which is a common adsorbent.⁹⁹ Other substances that are utilised as adsorbents include Alumina, powdered cellulose, Kiesulguhr G, Polyamide powder, Fuller earth, Magnesol, and Sephadex.¹⁰⁰ The type of sample to be analysed will determine which stationary phase should be used. HPTLC can be regarded as the most advanced form of modern TLC. It uses HPTLC plates featuring small particles with a narrow size distribution. As a result, homogenous layers with a smooth surface can be obtained.¹⁰¹ HPTLC uses smaller plates (10 × 10 or 10 × 20 cm) with significantly decreased development distance (typically 6 cm) and analysis time (7–20 min). HPTLC plates provide improved resolution, higher detection sensitivity, and improved in situ quantification and are used for industrial pharmaceutical densitometry quantitative analysis.¹⁰² Normal phase adsorption TLC on silica gel with a less polar mobile phase, such as chloroform– methanol, has been used for more than 90% of reported analysis of pharmaceuticals and drugs. Lipophilic C-18, C-8, C-2; phenyl chemically-modified silica gel phases; and hydrocarbon- impregnated silica gel plates developed with a more polar aqueous mobile phase, such as methanol–water or dioxane–water, are used for reversed-phase TLC.¹⁰³ HPTLC plates need to be stored under appropriate conditions. Before use, plates should be inspected under white and UV light to detect damage and impurities in the adsorbent. It is advisable to prewash the plates to improve the reproducibility and robustness of the results.¹⁰⁴ Microparticulate sorbents with particle diameters between 10 and 30 mm are the stationary phases used in TLC. In terms of band spreading (efficiency) and resolution, chromatographic performance is improved by a smaller mean particle size and a narrower size range.¹⁰⁵ To create thin-layer chromatography plates, rectangular plastic, aluminum, or glass sheets are coated with adherent, uniform layers of sorbents that are about 250 mm thick. An insoluble fluorescent reagent may be included in commercially produced plates to help with the detection of solute spots.¹⁰⁶ These plates are available in several sizes ranging from 5 cm to 20 cm square. Silica and powdered cellulose are the two most frequently used sorbents, and adsorption and partition are the corresponding sorption mechanisms. ¹⁰⁷^,¹⁰⁸

2.2.5. Selection of mobile phase

The adsorbent substance utilised as the stationary phase, along with the physical and chemical characteristics of the analyte, are used to determine the mobile phase.¹⁰⁹ To guarantee correct mixing of the contents, separate, exact volumetric measurements of each component of the mobile phase must be made in suitable volumetric glasses and shaken. The mobile phase of the appropriate solvents must be selective via one’s own experience, literature or trial and error methods.¹¹⁰ Mobile phases are typically made up of nonpolar and polar solvent with or without an acid or basic modifier to improve resolution. They are less polar than the silica gel layer (normal-phase TLC). In reversed-phase TLC, the mobile phase, which is frequently a mixture of methanol, acetonitrile, or tetrahydrofuran with water, is less polar than the stationary phase.¹¹¹^,¹¹² The mobile phases used in TLC are extremely diverse, and they are frequently chosen empirically. Combinations of two or more solvents are frequently used because the eluting power, or strength, of the solvents can be easily changed to optimize a separation by changing the solute distribution ratios^.¹¹³^,¹¹⁰ In order to increase resolution, mobile phase eluting power should be changed such that solute Rf values fall between 0.2 and 0.8; For separations on silica gel and other polar adsorbents, solute migration rates and, consequently, their Rf values, are governed by the overall polarity of the mobile phase; Rf values will be significantly increased by small additions of a slightly polar solvent, such as diethyl ether, to a nonpolar solvent, such as methylbenzene.¹¹⁴ The best way to separate polar and ionic solutes is to mix a polar organic solvent, like n-butanol, with water; by adding small amounts of ethanoic acid or ammonia to the water, you can make basic and acidic solutes more soluble, respectively. ¹¹⁵

2.2.6. Pre Washing

Pre-washing the TLC/HPTLC layer is occasionally required to get rid of contaminants that usually come from the binder. Typically, this only becomes an issue if the detection method uses fluorescence quenching or the detection reagent is sensitive to the contaminants.¹¹⁶ To water vapour or volatile impurities, the plates must be cleaned. It may be clean with a suitable solvent such as methanol.¹¹⁷ Silica Gel 60F is widely used sorbent, the major disadvantages of this sorbent is that it contains iron impurity. ¹¹⁸ This iron impurity is removed by using Methanol: Water (9:1), this is the main advantage of this process. In order to prevent contamination, plates are typically handled only at the upper edge. Unless chromatography creates impurity fronts due to plate contamination, plates are typically utilised without preparation. For reproducibility studies and quantitative analysis, layers are frequently prewashed with 20 ml of methanol ¹¹⁹, ¹²⁰ or mixture of methanol and ethyl acetate, Chloroform and methanol (1 :1 ), Chloroform, methanol and Ammonia (90:10:1 ) or even the method's mobile phase may also be used. ¹²¹^,¹²²

2.2.7. Pre-Conditioning (Chamber Saturation):

Saturation is necessary for highly polar mobile phases although there is no need for saturation for low polarity mobile phases. Un-saturation of chamber cause high Rf value and bending or tailing effect.¹²³ Saturation of chamber by lining with filter paper for 20 min prior to development facilitate the uniform distribution of solvent vapour leads to better separation and lower Rf value.¹²⁴^,¹²⁵

2.2.8. Conditioning (activation of plate )

The presence of moisture significantly affects the travelling of band and separation on TLC plate. Moisture containing plates were observed fronting and tailing effect, it disturbs the uniform development of bands. So conditioning of plate is must be done before using of plates. Freshly opened box does not need activation; plate exposed to high humidity should be adjusted for laboratory conditions. Plates are placed in an oven at 120 ° C for 15 to 20 minutes to perform conditioning.¹²⁶

2.2.9. Sample Application:

The sample spot's or band's diameter cannot be larger than 1 mm. In HPTLC, there are several techniques for spotting samples. One allows for the application of small amounts of samples to the HPTLC plate using a Hamilton micro-syringe.¹²⁷ It has been demonstrated that applying confined, homogeneous bands of specified length produces better chromatographic outcomes than spot application. Modern HPTLC procedures are available with these automated application devices, while using HPTLC system selection of the sampling speed is key factor which effect the fine and uniform application of band.²⁵

2.2.10. Chromatographic Development:

The linear development method in high-performance thin-layer chromatography is the most common technique here the plate is positioned vertically in an appropriate container with a solvent or mobile phase.¹²⁸ The mobile phase is generally fed by capillary action and both sides may produce chromatograms. Different type of developing tank is used for the development of plates but Twine-Trough Chamber (TTC) has many advantages over others. The TTC chamber's design enables development with just small amounts of mobile phase and simple pre-equilibration of the layer using mobile phase vapours or another conditioning liquid.⁶³^,¹²⁹ The stationary and mobile phases that are present during development, there is also a gas phase that can have a big impact on the separation's outcome. The kind, dimension, and saturation state of the chamber during development all affect this gas phase. Because unsaturated chambers frequently result in bending solvent fonts and poor reproducibility, they should be avoided. (Figure -2).

Figure -02 : Illustration of HPTLC Method Development and Validation

2.2.11. Detection of band and Scanning

The HPTLC instrument has attached to computer and data recording devices visualize and scanner. The development of band is viewed as peaks at wavelengths of selected UV regions. The height and the area of the peaks are determined by the instrument and recorded as a percentage. Fluorescence compound are visualized and scanned at 366 nm light. Band which are not visible at 254 nm and 366 nm are derivatized with suitable reagent or chemical like iodine, Dragendrof reagent, Anisaldehyde –Sulphuric acid reagent, 5% methanol-Sulphuric acid reagent, Vanilline-Suplphuric acid reagent and with other reagent, it depend on category of compound, ¹³⁰

2.3. HPTLC: Validation Process

Validation of the developed HPTLC method was carried out as per the International Conference on Harmonization (ICH) guidelines Q2 (R1)¹³¹^,¹³² for specificity, sensitivity, accuracy, inter-day and intra-day assay precision, repeatability, and robustness, limit of detection and limit of quantification.¹²¹^,¹³³

After performing all the experiments described in the validation protocol, obtained data are evaluated and compared with the acceptance criteria. If all criteria are met, the method can be regarded as valid. This approach is widely accepted for validation of qualitative HPTLC methods for identification during routine use. The validation protocol is a key instrument for structuring, regulating and documenting the validation processes, depending on the quality management system. The following elements must be included:

2.3.1. Specificity:

The capacity of a method to separate an analyte from all other compounds present or anticipated to be present in test samples is known as specificity. The method is particular since it was discovered that the peak of the standard was unaffected by other substances in the formulation. The overlay spectrum of the standard and samples was found to be similar or to have overlapped.¹³⁴ Three different sample types must be simultaneously chromatographed as part of the HPTLC analytical procedure: sample type 1 is the analyte in its purest form, or its reference standard; sample type 2 is the analyte mixed with all likely impurities; and sample type 3 is the representative blank mixed with all likely impurities. Degradation by products, reagents, intermediates, excipients, side products, and analyte isomers are examples of likely contaminants. The mixtures can be made by adding likely contaminants to test samples (API chemicals or finished goods) or placebos. By comparing the spectra at the peak start, peak apex, and peak end positions of the spot, one may determine the peak purity of a standard.¹³⁵

2.3.2. Linearity:

The capacity of an analytical process to produce results that are, within a certain range, directly proportional to the analyte concentration in the sample is known as linearity. Using the test procedure, responses from samples with various analyte concentrations are produced. ¹³⁶ Where multiple (i.e., 3) answers are received at each analyte level, it is generally recommended to employ at least five distinct concentrations. Plotting the method response (y-axis) as a function of the analyte concentration (x-axis) allows for later analysis using linear regression techniques, where slope, intercept, and correlation coefficient are presented. The concentration range must encompass both the highest and lowest levels expected during a study. By plotting the peak area of the marker compound against the concentration, several concentrations of the standard marker are used to create the calibration curves.¹³⁷^,¹³³ The conventional correlation coefficient should be more than 0.99, demonstrating strong linearity between concentration and peak area.¹³⁸,¹³⁹ A correlation coefficient greater than 0.999 is typically regarded as satisfactory for chromatographic procedures because it indicates that the data match the regression line well.¹³⁴

2.3.3. Precision

By examining the standard solution over the complete calibration range for three distinct days, the inter-day precision (RSD) was ascertained. By analysing the standard solution over the complete calibration range three times on the same day, the intra-day precision (RSD) was ascertained.¹⁴⁰ By examining sample solutions at three levels covering low, medium, and higher concentrations over a period of seven days (n = 5), interday precision was calculated. The mean value and %RSD (relative SD) percentage were calculated using the peak areas obtained. Precision reveals the presence of random error. Relative standard deviation (RSD) or coefficient of variation (COV) should be used to express the results.¹²³ When it comes to replication, precession is seen in terms of precision under the same circumstances, the same analyst, the same apparatus, a brief window of time, and similar chemicals being used on the same sample; based on seven measurements of the same site, the RSD for the peak area measurement should not be more than 1%; peak position: depending on repositioning the instrument seven times after each measurement, the RSD should not be more than 2%; Equal volume should be applied in seven areas when applying the sample, and the RSD shouldn't be more than 3% whether utilising the same sample with different reagents from different sources and in different labs, on different days, or with different analytes within the reproducibility of the laboratory, RSD shouldn't be more than 10%. ¹⁴¹

2.3.4. Accuracy

The degree of agreement between the value acknowledged as conventional true value or a recognised reference value and the value discovered is expressed as the analytical procedure's accuracy. The conventional addition method is used to evaluate the method's accuracy at three concentration levels (80%, 100%, and 120%).¹⁴² An analysis' accuracy is determined by the amount of systematic error present. This was done to verify that the standard was recovered at various formulation levels. It is described as the degree of agreement between the mean analytical value produced by repeatedly performing the test procedure and the real value. If the difference between the true value and mean measured value does not exceed the RSD values achieved for repeatability of the procedure, the accuracy is considered satisfactory.¹⁴³ This metric is crucial for pharmaceutical dosage forms since it tells us how well the analyte was recovered from sample preparation and how the matrix affected it. If the recovery rate is discovered to be 100%, it suggests that the suggested analytical procedure is devoid of constant and proportionate systematic error. It is necessary to have a blank matrix and known contaminants on hand to check the method's accuracy.¹⁴⁴^,¹³²

2.3.5. Robustness

To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done.¹⁴⁵^,¹⁴⁶ By introducing small changes in the mobile phase composition, mobile phase volume, duration of chamber saturation with mobile phase¹⁴⁶, time from spotting to development (five minutes, 20 minutes, and one hour), and time from development to scanning (five minutes, 20 minutes, and one hour), the effects on R_f value and peak area of drugs were examined. ¹⁴⁷^,¹⁴⁸. The %R.S.D. of peak area was calculated after the robustness of the procedure was tested in triplicate at a concentration level of 2 g/spot ¹⁴⁹.

2.3.6. Ruggedness

It expresses the precision within laboratories variations like different days, different analyst, and different equipment. Ruggedness of the method was assessed by spiking the standard 6 times in two different days with different analyst. This is one of the most important parameters for validation of HPTLC method. Experiments are usually recommended to evaluate ruggedness of a HPTLC method like sample preparation: shaking time, temperature, number of extractions; sample application, separation, chromatographic conditions and development distance; spot visualization: post chromatographic derivatization, spraying, dipping, reaction temperature and time; quantitative evaluation: drying of plates, detection and wavelength. ¹⁴³ Once the analytical method is developed, it should be performed independently by three analysts well conversant with practical aspects of the technique, analyzing the same sample under same experimental conditions to check reproducibility of the method.¹²⁵

2.3.7. Limit of Detection and Limit of Quantification

The lowest limit of detection, or LOD, is the level at which a drug may be found in a sample and identified or analysed. ¹²³ The smallest amount of analyte that can be identified is 10% or less of the Individual Impurity limit. The amount of analyte that must be applied must be increased if this is not achievable. Based on the signal to noise ratio, the limit of detection (LOD can be calculated. The absolute SD values of the 15 noise peak areas' means are calculated. LOD is the quantity of applied sample that results in a peak area with a value equal to the sum of the mean blank area and the three standard deviations.¹²⁶

LOD = 3.3 x σ / S

The lowest limit of quantification, or LOQ, is the value at which the amount of a substance can be quantified or detected that is how much quantity of drug quantity is present in the sample.¹⁵⁰^,³³ By lowering the recognised concentration, LOD and LOQ are empirically verified.

LOQ = 10 x σ / S

Where σ = Standard deviation, S = Slope of intercept

3. Hyphenated Technique

The possibility of combining two or more instrumental analysis techniques to exploit the maximal possible information from a single run is referred to as hyphenation.¹⁵¹ The coupling aims to produce an information-rich detection for both identification and quantification in comparison to a single analytical approach. When analysing a range of biological, chemical, and toxicological compounds, hyphenated analytical methods combine chromatographic and spectroscopic or spectrometric approach. The separated mixture components from the chromatographic technique will next enter the spectroscopic technique through an interphase.¹⁵²

3.1. High-Performance Thin-Laye Chromatography– Mass Spectrometry (HPTLC–MS)

The process of chromatography is used to separate mixtures of volatile and non-volatile compounds. This chromatography is carried out on a sheet of aluminium foil, glass, or plastic that has been lightly covered with an adsorbent substance, typically silica gel, cellulose, or aluminium oxide. An analytical method called mass spectrometry (MS) creates spectra of the masses of the atoms or drug molecules that make up a sample of material.⁹¹,¹⁵³ Chemical substances are ionised by mass spectrometry to produce charged molecules or molecular fragments. Then, using an electric or magnetic field or often by accelerating them, these ions are separated according to their mass-to-charge ratio. Ion fragments with an identical mass-to-charge ratio will deflect equally. An electron multiplier, a device that can detect charged particles, is used to find the ions. The molecules or atoms in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.¹⁵⁴ Because of mass spectrometry's great sensitivity, quick analysis, and capacity for structural characterisation, the direct coupling of HPTLC with MS by TLC/MS Interface can be bring together. The compounds separated in the form of HPTLC/TLC zone on both glass plates and aluminum foils take out directly from the plate using a suitable solvent delivered by the HPLC pump transferring them into a mass spectrometer for identification or structure elucidation. HPTLC-MS is a flexible approach that can be used to separate and identify pharmaceuticals and phytopharmaceuticals.¹⁵⁵

3.2. High-PerformanceThin-Layer Chromatography–Fourier Transform Infrared Spectroscopy (HPTLC–FTIR)

High-performance thin-layer chromatography (HPTLC) and spectrometric methods are increasingly being combined. After the separation of samples with HPTLC, it is necessary to use detection methods that enable sample identification and determination.¹⁵⁶^,¹⁵⁷ Infrared (IR) is one of the spectroscopic methods that has been integrated with HPTLC for the characterisation and identification of unknown sample. The main benefits of IR spectroscopy's is that little to no sample preparation is needed before using it, and results can be obtained quickly and without the use of reagents.¹⁵⁸ HPTLC and FTIR spectroscopy are used in the direct hyphenated HPTLC-FTIR methodology, an online coupling method for planar chromatography.¹⁵⁹ The direct on-line coupled HPTLC-FTIR has a number of advantages over earlier hyphenated techniques (HPTLC-Raman spectroscopy, HPTLC-PA, and HPTLC-MS), including simplicity of use and on-line coupling's improved operating characteristics. In contemporary laboratories, the HPTLC-FTIR linked technique is extensively used for both qualitative and quantitative examination. This technique has shown potential in a number of analytical fields, including drug, forensic, food, environmental, and biological analysis, among others. The hyphenated HPTLC-FTIR methodology will be created in the future to fully realise the promise of this method.¹⁶⁰

4. CONCLUSION:

The HPTLC techniques are most suitable for the determination and quantification of therapeutic phytochemicals present in herbal drugs. The method validated necessary for the accurate determination of phytocompounds, if validation has not be done for the any methods obtained result will be incorrect. The method for analysis of compound should be simple, precise, and reproducible; hence, it can be adopted for the routine analy- sis, standardization and quality control of herbal drugs. The hyphenation of HPTLC make it more sophisticated and reliable, also minimized the analysis time and process.

5. CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 05.07.2023 Modified on 08.08.2023

Research J. Pharm. and Tech 2023; 16(8):3964-3976.

DOI: 10.52711/0974-360X.2023.00652