Analytical Method Development and Validation Harsh Review RGSCOP.pptx

Review on Analytical Method Development and validation by HPTLC
#HPTLC#Instrumental Method of Analysis

1. A REVIEW ON ANALYTICAL METHOD DEVELOPMENT AND VALIDATION BY HPTLC PRESENTED BY: MR. HARSH RANGRAO DESHMUKH (FINAL YEAR B. PHARMACY) GUIDED BY: PROF. PRAJAKTA G. THETE M.PHARM (QUALITY ASSURANCE) KCT’S R. G. SAPKAL COLLEGE OF PHARMACY, ANJANERI, NASHIK

2. CONTENT ❑ Abstract ❑ Introduction ❑ Rationale of Work ❑ Literature Review ❑ Applications of HPTLC ❑ Advantage and Disadvantage of HPTLC ❑ Summary And Conclusion ❑ Future Scope ❑ References

3. Abstract:- High Performance Thin Layer Chromatography (HPTLC) is a widely used analytical technique for the separation, identification, and quantification of components in complex mixtures. HPTLC is an enhanced and automated method of thin layer chromatography (TLC) that offers superior separation performance and detection limits and is frequently a great substitute for GC and HPLC. High Performance Thin Layer Chromatography (HPTLC) is the most powerful advanced form of Thin Layer Chromatography (TLC) and it consists of chromatographic layers of ulmost separation efficiency and the application of sophisticated instrumentation for all steps in the procedure include accurate sample application, standardized reproducible chromatogram development and software controlled evaluation. HPTLC is a concept that includes a widely standardized methodology based on scientific facts as well as the use of validated methods for qualitative and quantitative analysis. HPTLC meets all quality requirements for today’s analytical labs, to increase the resolution and to allow more accurate quantitative measurements [3]. 1

4. Keywords:- HPTLC, Analytical method development, validation, Mobile Phase, Stationary Phase, Chromatography, Instrumentation, Application. 2

5. ❑ Introduction:- HPTLC (High Performance thin layer chromatography) is the automated, sophisticated form and improved method of TLC. It is a powerful analytical method equally suitable for qualitative and quantitative analytical tasks. It is also known as planer or flat bed chromatography. HPTLC is conducted on TLC plates which are coated with purified silica gel with a particle range of 2-10um as opposed to 2-25um for standard commercial TLC plates. The narrow particle size range means that a greater number of theoretical plates are available for separation and thus the spots on the TLC plate remain tighter. These type of plates may be run in a standard type of TLC tank but optimal performance is obtained from horizontal development of the plates using  HPTLC. 3

6. Rationale of Work:- The Objective of this dissertation work is as follows:- Aim of the present work is to Develop some new analytical methods and validation parameter regarding HPTLC. To Develop rapid, sensitive and selective method. Economic and accurate method Method Validation according to ICH Guidelines. Accurate Quantification and Identification of various compounds in a mixture, ensuring precision. 4

7. Literature Review:- 1. Beddi Bhavya Shree et. al. (2017) This review paper highlights the development and validation of HPTLC-based analytical methods, with a practical evaluation. It meets standards while minimizing errors and investigations. This review article provides advice for selecting the optimal mobile phase, validating practices, and understanding analytical procedures. 2. R. N. Kulkarni et. al. (2021) In this review, we discuss the HPTLC is a popular method for identifying medicinal plants, screening lichen substances, quantifying active ingredients and herbal drugs, conducting phytochemical and biomedical analyses, and detecting adulterants in formulations. Using a little amount of solvent allows for greater sensitivity and faster sample processing. This is a sophisticated approach that utilizes thin layer chromatography's full potential. This paper provides an overview of HPTLC, including the instrument used, the technique, and its advantages over TLC. This page focuses on the use of HPTLC and includes examples of evaluated pharmaceuticals, drugs, and formulations. 5

8. 3.Salim Bala Muhammad et.al . (2020) This review will generally focus on developing a sensitive, specific, and reliable HPTLC method, optimize chromatographic conditions for separation and detection, Ensure accurate and precise quantitation of analytes , Minimize interference from matrix components and establish a robust and reproducible method. In addition, the major goal of this review is to discuss specific validation criteria and the benefits of method validation, such as ensuring dependable and correct outcomes. Complies with regulatory regulations. improves technique credibility. 4. Miss Payal Badhe et.al . (2024) Studied that High-Performance Thin Layer Chromatography (HPTLC) is a highly effective alternative to GC
and HPLC. This automated technology improves separation performance and detection limitations. HPTLC
can be used to research biological materials and phytochemicals, assess herbal remedies and active
components, fingerprint formulations, and detect adulterants. HPTLC can identify forensically important
compounds. Using a small volume of solvent allows for faster and more sensitive testing of multiple samples.
This is a complex instrumentation approach that makes use of all thin-layer chromatography capabilities. 6

9. Principle:- HPTLC involves the similar theoretical principle of TLC i.e. the principle of separation is adsorption An analyte migrates up or across a layer of stationary phase (most commonly silica gel), under the influence of a mobile phase, which moves through the stationary phase by capillary action. The distance moved by the analyte is determined by its relative affinity for the stationary vs the mobile phase. Mobile phase flow by capillary effect and component move according to their affinities towards the adosrbant . The component with higher affinity towards the adsorbant travels slowly. The component with lower affinity towards the stationary phase travels faster. Finally the components are separated on a chromatographic plate according to their affinity and separation also based on their solubility in mobile phase. 7

10. Difference Between TLC & HPTLC :- Features TLC HPTLC Technique Manual Instrumental Plates Lab Made/Pre-coated Pre-coated Plate Height 30 μ m 12 μ m Layer of sorbent 250μm 100μm 250μm 100μm Stationary phase Silica gel, Alumina & Kiesulguhr (diatomaceous earth) Wide choice of stationary phases like silica gel for normal phase and C8, C18 for reversed phase modes Analysis time 20-200min 1-3min Separations 10 - 15 cm 3 - 5 cm Mean particle size 10-12 μ m 5-6 μ m Efficiency Less High due to smaller particle size Sample holder Capillary/pipette Syringe

11. Features TLC HPTLC Sample spotting Manual spotting Auto sampler Size of sample Uncontrolled/Solvent dependent Controlled Solvent independent Shape of sample Circular (2-4 nm dia ) Rectangular (6mm L X 1mm W) Sample tracks per plate ≤ 10 ≤ 36 (72) Vol. range 1 to 10 μL 0.1 to 500 μL Development chamber More amount New type that requires less amount of mobile phase Wavelength range 254 or 366 nm, visible 190 or 800 nm, Monochromatic Detection Limits (Absorption) 1-5 pg 100-500 pg

12. Features TLC HPTLC Detection limits (Fluorescence) 50-100 pg 5-10 pg PC connectivity, Method storage, Validation No Yes Quantitative analysis No Yes Scanning No Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer Analysis judgment By analyst By machine

13. Basic Drug Selecition Criteria For New Analytical Method Development:- An analytical method for quantifying the medication in body fluids may not be accessible. Analytical procedures for a medication when combined with other substances may be unavailable. The currently used analytical approach may necessitate the use of costly reagents and solvents. It may also include extraction and separation methods, which may not be reliable. Analytical Method Development (AMD):- Analytical technique development yields official test methods. Quality control laboratories employed these approaches to ensure the efficacy, purity, safety, and performance of drug goods. Regulatory authorities place high priority on analytical procedures in production. Modern pharmaceutical analysis needs the following requirements:- The analysis ought to take a minimum time and should be economical. The accuracy of the analysis should accept the Pharmacopoeial guidelines. The chosen method should be precise and selective. Analytic methods can be spectral, chromatographic, electrochemical, hyphenated, or various. Analytical technique development involves selecting an accurate assay procedure to identify the composition of a formulation. 9

14. General Methodology For HPTLC Method Development:- Selection of chromatographic plates (HPTLC plates) Pre-washing of the plates/Layer Pre-washing Activation of the HPTLC plates/Pre-Coated Plates Sample Preparation and application Selection of Mobile Phase (solvent system) Pre-conditioning (chamber saturation) Application of sample. Chromatographic development and Drying Detection of spots. Scanning & documentation. 10

15. Applicator Developer Derivatizer Scanner Visualizer Software

16. 11 1) Selection of chromatographic plates:- Hand made plates which are made up of cellulose and other materials which are not used much now a day. Pre-coated plates- The plates with different support materials and sorbent layers with different format and thickness are used for qualitative and quantitative analysis. Support materials used in plates:- Glass Polyester/ polyethylyne Aluminium Sorbents used in plates- Silica gel 60F, Aluminium oxide, Cellulose, silica gel chemically modified – a)amino group(NH2), b) CN group Smaller particle size of silica helps in greater resolution and sensitivity.

17. 2) Pre-washing of the plates/Layer Pre-washing:- 12 It is purification step. The main purpose of the pre-washing is to remove impurities which include water vapours and other volatile substances from the atmosphere when they get exposed in the lab environment. In case of silica 60F (most widely used sorbent) the major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using Methanol: water (9:1), this is the major advantage of the step of pre-washing. Some common methods are:- -Ascending -Dipping -Continuous Solvents used for pre-washing- Methanol Chloroform:Methanol (1:1) Choloroform : Methanol: Ammonia (90:10:1)

18. 3) Activation of the HPTLC plates/Pre-Coated Plates:- Freshly opened box of HPTLC plates doesn't need activation. If plates exposed to high humidity or kept in hand for longer time then activation is required and it's activation results by removing moisture. The plates are activated by placing in an oven at 110- 120° C for 30 min, this step will removes water that has been physically absorbed on surface at solvent layer. Activation at higher temp and for longer time is avoided which leads to very active layer and there is risk of sample being decomposed.

19. 4) Sample Preparation and application:- Sample preparation- It's important to prepare proper sample for successful separation. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones. It needs a high concentrated solution, as very less amount of sample need to be applied. After that dry the plates and store in dust free atmosphere. Solvents used are- • Methanol, • Chloroform: Methanol (1:1), • Ethyl acetate: Methanol (1:1), • Chloroform: Methanol: Ammonia (90:10:1)

20. Sample application:- Usual concentration range is 0.1-1µg / µl, above this causes poor separation and volume recommended for HPTLC-0.5-5µl. The size of sample spot applied must not exceed 1mm in diameter. Problem from overloading can be overcome by applying the sample as band. Some applicators used for application of sample- Selection of applicator to be used depends on- -Sample volume -No. of sample to be applied Capillary tubes Micro bulb pipettes Micro syringes Automatic sample applicator. • The major criteria is that they shouldn't damage the surface while applying sample. • The sample are completely transfer to the layer. • Advantage of application of sample as bands are- Better seperation due to rectengular shape. Response of densitometer is higher in case of band because of uniform distribution of sample in band.

21. 5) Selection of mobile phase (solvent system):- Chemical properties of analytes and sorbent layer factors should be considered while selection of mobile phase. Various components of Mobile Phase should be measured separately and then placed in mixing vessel. The less amount of mobile phase is required then TLC. This prevents contamination of solvents and also error arising from volumes expansion or contraction on mixing. Multi component mobile phase once used not recommonded for further use due to diffirent evaporation and adsorption by layer.

22. 6) Pre-conditioning (chamber saturation):- • Un- saturated chamber causes high Rf values. • Saturated chamber by lining with filter paper for 30min prior to development-uniform distribution of solvent vapours -less solvent for the sample to travel-lower RF values • For low polarity mobile phase there is no need of saturation. However saturation is needed for highly polar mobile phase. • Chamber saturation influence separation profile.

23. 7. Application of sample:- The sample spot applied must not be larger than 1mm in diameter. There are several strategies for sample spotting, one of which is self-loading capillary, which allows for a smaller volume of samples to be put to the plate. Surface with platinum-iridium tubing bonded to the end of a piece of glass tubing. LINOMAT 5: SAMPLE APPLICATOR:- Quantitative Analysis This analysis is most frequently employed. The typical application volume is 6–20ul. The typical band size is 6 mm. For aqueous samples 8 mm. Distance from side: 12-15 mm; distance from bottom: 8 mm. In-situ clean – up:- Used for separating components from samples. Samples were applied 110 mm from the bottom, developed in ether, plate dried, sliced 120 mm from the bottom, rotated 180˚, and developed with the appropriate mobile phase. Micro - prep isolation:- To isolate components on a milligram scale and identify them using spectroscopy. The usual band length is 190mm. The volume applied ranged from 2 to 10 ul . Chromatography requires several comparable plates.

24. 8) Chromatographic development and drying:- Plates are spotted with sample and air dried and placed in the developing chambers. The different methods used for development of chambers are like- Ascending Descending Horizontal Autometic multiple development, Circular, anti-circular device and multiple developments are some other methods. After development, remove the plate and mobile phase is removed from the plate to avoid contamination of lab atmosphere. Dry in vacuum Desiccators with protection from heat and light. CAMAG Automatic Developing Chamber

25. 9) Detection of spots:- Immediately after development is done, the plates are taken from the chamber and dried with a drier to remove any free mobile phase. In general, the detection can be made using iodine vapor in an iodine chamber. 10) Scanning & Documentation:- Scanning- • The scanner converts band into peak and peak hieght or area is related to the concentration of substance on spot/band. • The peak height and area under spot are measured by instrument and recorded. Documentation- • Documentation is important because labeling every single chromatogram can avoid mistake in respect of order of application. Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded. The HPTLC equipment is connected to a computer and data recording and storage devices. The development of HPTLC plates is scanned at certain UV wavelengths by the instruments, and the discovered spots are displayed on computers as peaks.

26. Schematic Procedure for HPTLC Method Development:-

27. Instrumentation of HPTLC:-

28. Validation of HPTLC Methods:- Method validation ensures the reliability, accuracy, and reproducibility of the analytical method. The key validation parameters include: Specificity: The method should distinguish the analyte from other components in the sample. Linearity: The method’s ability to produce results proportional to the concentration of the analyte across a given range. Precision: This includes repeatability and intermediate precision (intra-day and inter-day variations). Accuracy: The closeness of the measured value to the true value. Limit of Detection (LOD) and Limit of Quantification (LOQ): The lowest concentration of analyte that can be detected and quantified with acceptable accuracy and precision. Robustness: The method’s capacity to remain unaffected by small, deliberate variations in method parameters. Ruggedness: The method's reproducibility under varied conditions, such as different laboratories or analysts. 15

29. Some commonly used software in HPTLC include: VisionCATS : A popular software for HPTLC data analysis, peak detection, and quantitation. Camag TLC Scanner: Software for controlling the Camag TLC Scanner and analyzing HPTLC plates. WinCATS : A software for HPTLC data analysis, peak detection, and quantitation, similar to VisionCATS . TLC Analyzer: A software for analyzing and documenting HPTLC plates. HPTLC Explorer: A software for data analysis, peak detection, and quantitation, specifically designed for HPTLC. Empower: A chromatography data system (CDS) software that can be used for HPTLC data analysis. OpenChrom : An open-source software for chromatography data analysis, including HPTLC. TLC Studio: A software for analyzing and documenting HPTLC plates. 16

30. Common HPTLC Problems:- Method Development Issues- • Poor separation of analytes • Inconsistent Rf values • Tailoring or fronting of peaks • Insufficient resolution 2. Validation Issues • Lack of specificity • Inadequate linearity • Poor accuracy • Insufficient precision • Inadequate robustness 3. Quantitation Issues • Inaccurate calibration • Incorrect integration parameters • Insufficient sensitivity • Interference from impurities • Inadequate sampling 5. Specific HPTLC Challenges • Sample preparation issues (e.g., extraction, derivatization) • Plate-to-plate variability • Mobile phase optimization • Detection wavelength selection 17

31. Advantages of HPTLC:- Speed: Fast analysis time, typically 10-30 minutes. Simplicity: Easy to perform, minimal sample preparation. Cost-effective: Low operating costs, minimal equipment requirements. High sensitivity: Detection limits in nanogram range. Multi-component analysis: Can separate and detect multiple components simultaneously. Flexibility: Can be used for various sample types (e.g., plant extracts, biological fluids). Repeatability: High precision and reproducibility. Non-destructive: Samples can be recovered after analysis. 18

32. Disadvantages of HPTLC:- Limited resolution: Compared to other chromatographic techniques (e.g., HPLC, GC). Qualitative results: Primarily used for identification and quantification, not structural elucidation. Operator-dependent: Results can vary depending on user expertise. Limited automation: Manual steps involved in sample application, development, and detection. Standardization: Requires careful standardization of experimental conditions. Detection limits: May not detect very low concentrations of analytes. Plate-to-plate variability: Can occur due to differences in TLC plates. Limited dynamic range: May not be suitable for samples with very high or low concentrations. 19

33. Applications of HPTLC:- HPTLC is extensively used across various fields:- Pharmaceutical analysis: Identification, quantification, and purity testing of drugs and pharmaceutical ingredients. Food safety and quality control: Detection of contaminants, adulterants, and residues in food products. Environmental monitoring: Analysis of water and air samples for pollutants and toxins. Forensic science: Investigation of crimes by analyzing evidence such as ink, paint, and biological fluids. Plant and animal research: Separation and identification of bioactive compounds, metabolites, and biomarkers. Cosmetic and personal care product testing: Analysis of ingredients, stability, and quality control. 19

34. 7. Herbal medicine and dietary supplements: Standardization and quality control of botanical extracts and products. 8. Toxicology: Detection and quantification of toxins, poisons, and drugs in biological samples. 9. Clinical research: Analysis of biological samples for disease diagnosis, treatment, and monitoring. 10. Quality control in industries: Monitoring of raw materials, intermediates, and final products in various industries. 11.Industrial Application: Process development and optimization, In-process check, validation etc. 12. Forensic: Poisoning investigation, Finger print analysis. 20

35. Summary & Conclusion:- The creation and validation of analytical methods by HPTLC include the establishment of a dependable and repeatable technique for the separation, identification, and quantification of analytes. Optimization of the stationary phase, instrumental parameters, and mobile phase are all included in the process. The performance attributes of the method, including specificity, linearity, accuracy, precision, detection limit, quantitation limit, and robustness, are evaluated during validation. For the quality and dependability of analytical data in a variety of domains, such as pharmaceuticals, food safety, and environmental monitoring, HPTLC technique development and validation are essential. HPTLC's affordability, speed, and ease of use make it a useful analytical tool for method development and validation. For the analysis of complicated samples, a well-designed and tested HPTLC method can yield precise, dependable, and accurate results. Validation assures that the procedure satisfies legal requirements and is appropriate for its intended use. 21

36. Future Scope:- Advancements in instrumentation Enhanced Detection systems Application in new areas:- *Pharmaceutical Industry *Food and agriculture *Environmental Monitoring Regulatory Acceptance *Standardization and validation * Inclusion in Pharmacopeias Integration with Digital Technologies * Data Management and analysis * Remote Monitoring and control Green Chemistry and Sustainability * Eco-friendly Solvents * Waste Reduction Education and Training Applications:- * Enhanced Training Tools * Increased Adoption in Academia 8. Pharmacogenomics and metabolomics 22

37. References:- P.D. Sethi (HPTLC) High performance thin layer chromatography, First edition, CBS Publisher and Distrributers . Reich, E. and Schhibli A.(2007)High performance liquid chromatography for analysis of medicinal plant, Thieme . Sherma J. Review of HPTLC in Drug Analysis: 1996-2009. JAOAC Int.2010; 93:754-64.• Arup U, Ekman S, Lindblom L, Mattsson JE.High performance Thin Layer Chromatography (HPTLC), 1993; 25:61-71. Pharmaceutical Analysis vol-II by Dr.A.V.Kastur Dr. K.R.Mahadik Nirali Publishers page no.28-30. Patel, R. B., Patel, M. R. and Patel, B. G. 2011. Experimental aspects and implementation of HPTLC. In “High performance thin layer chromatography (HPTLC) page no. 41-54., Srivastava, M. M. ed. Springer Berlin Heidelberg, New York, USA. Sherma , J. 2008. Modern thin layer chromatography. J. AOAC Int. 91: 1142-1144. Ravali R., Phaneendra M., BhanuJyothi K., RamyaSanthoshi L., Sushma K. Recent Trends in Analytical Techniques for the Development of Pharmaceutical Drugs. J. Bioanal . Biomed.2011: 1-7. Gocan S. Stationary phases for thin-layer chromatography. J. Chromatogram Sci. 2002; 40: 538–549. Indian Pharmacopoeia, Volume-1. published by The Indian Pharmacopoeia Commission,Central Indian pharmacopoeia Laboratory Govt. Of India, Ministry of Health & Family Welfare Sector-23, Raj Nagar, Ghaziabad-201 00, 2007 Patil R, Deshmukh T, Patil V, Khandelwal K. Review on analytical method development and validation. Res Rev J Pharm Anal 2014;3:1-10. Chauhan A, Mittu B, Chauhan P. Analytical method development and validation: a concise review. J Anal Bioanal  Tech 2015;6:1. ICH Harmonised Tripartite Guideline. 2005. Validation of analytical procedures: text and methodology, Q2 (R1) Geneva, http://www.ich.org. (Accessed on 01/04/2012) 23

38. 13) Camag planar chromatography catalog ; CAMAG Scientific Inc.: Wilmington, NC, 2010/11, http://www.camag.com. (Accessed on 01/04/2012) 14) Camag parameters for planar chromatography – useful hints, CAMAG, Muttenz , Switzerland, http://www.camag.com. (Accessed on 01/04/2012) 15) Koll, K.,Reich , E. and Blatter, A. et al. 2003. Validation of standardized high-performance thin-layer chromatographic methods for quality control and stability testing of herbals. J. AOAC Int. 86: 909-915. 16) Sherma , J. 2010. Review of HPTLC in drug analysis: 1996-2009. J. AOAC Int. 93: 754-764. 17) International Journal of Research in Pharmacy and Pharmaceutical Sciences ISSN: 2455-698X; Impact Factor: RJIF 5.22 Received: 28-02-2019; Accepted: 09-04-2019 www.pharmacyjournal.in Volume 4; Issue 3; May 2019; Page No. 83-88 18) International Journal of Scientific Research in Science and Technology www.ijsrst.com Publication Issue :Volume 11, Issue 1 January-February-2024 Page No. 445-455 19 ) Experimental Phytopharmacognosy A Comprehensive Guide By Dr. S.S. Khadabadi , Dr. S.L. Deore , Dr. B.A. Baviskar , Nirali Prakashan, Page No. 2.2 -2.7 20) Journal Of Chromatography library-Volume-9 HPTLC High performance thin-layer chromatography edited by A. Zatkis  and R.E. Kaiser 21) Monograph on High Performance Thin Layer Chromatography of Selected Medicinal Plants by Dr. Alok Sharma & Prof. (Dr.) G.D. Gupta, Nirali Prakashan 22) Sethi’s HPTLC High Performance Thin Layer Chromatography Quantitative Analysis of Pharmaceutical Formulations Vol.3 P.D.Sethi 23) Instrumental methods of chemical analysis Vol.2: Chromatography & Miscellaneous Methods by B.K. Sharma Page No. 241-267 24