High-performance Thin Layer Chromatography (HPTLC) is one of the many sophisticated, flexible, robust, and cost-effective separation techniques employed in the discovery, development, and analysis of new drugs.
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Principles of HPTLC
HPTLC is an extension of thin-layer chromatography (TLC). Thin-layer chromatography involves the separation of different components in a sample depending upon the relative affinity of each compound towards the stationary phase or mobile phase, while HPTLC helps in better resolution of compounds with lower limits of detection and quantifies separated components with the use of an integrated software platform.
This technique uses relatively smaller plates for development compared to thin-layer chromatograms and thus the development time is usually 7-20 min. The short development time ensures that data that is produced by performing HPTLC is reliable, reproducible, and useful. However, it is necessary to choose a good solvent system that helps in moving all components of the test sample from the baseline of the stationary phase to obtain demarcated sample zones that can be quantified.
HPTLC is also used in combination with other powerful and advanced analytical techniques such as Fourier-transform infrared spectroscopy (FTIR), mass spectrometry (MS), and HPTLC-Scanning Diode Laser.
HPTLC in the Pharma Industry
HPTLC with densitometry is used for determination of the quantity and purity of active ingredients as well as preservatives in marketed formulations that may be synthetic or herbal. For instance, the technique is used in post-production quality control of batches of chemotherapeutic agents, and to regulate caffeine content in herbal formulations.
In many studies, HPTLC has also revealed its ability to analyze drugs without letting auxiliary substances interfere in the results. Over the years, researchers have developed accurate and simple HPTLC methods for the estimation of several active constituents such as acetaminophen, diclofenac, and famotidine, to name a few.
While Jha P. et al. developed a novel HPTLC method to determine the omeprazole content in capsules, the same method has been used for stability testing of different oral dosage forms.
Development and quantification of biomarkers
HPTLC methods have been beneficial in identifying and quantifying biomarkers not only for therapeutic purposes but also to quantify biomarkers that are indicative of exposure to environmental health hazards such as nicotine.
Testing of fruits, vegetables, vitamin supplements, as well as food items for the presence of pesticides, water, chemicals by HPTLC is accepted globally due to its ability to characterize small molecules.
A recent study by Morschheuser L. et al. demonstrated that HPTLC could be employed to estimate protein in combination with aptamers. HPTLC-apta staining opens up various possibilities such as the determination of lysozyme (an enzyme that produces severe allergic reactions in some individuals), in food and beverages.
HPTLC is used in bioavailability studies to study marketed formulations as well as novel therapeutic agents. For instance, Abdelwahab et al. demonstrated that HPTLC could be used to monitor drug-drug interactions as was seen when thalidomide and dexamethasone were administered simultaneously in rat plasma. The study used development methods that were validated by the FDA.
Drug durability is another aspect that is a source of concern for researchers. HPTLC plates with pre-coated silica gel 60F254 using a mobile phase mixture of methanol, ethyl acetate, and ammonia developed by Bober K. was successful in giving substantial information on the degradation products of diphenhydramine.
Alternative applications of HTPLC
Nutraceuticals and natural products
HPTLC helps in determining if the raw materials used to prepare the dietary supplements are of substandard quality. It is also useful in phytochemical analysis of the nutraceutical properties of various plant parts used in traditional systems of medicine.
This method has also been used in combination with diode array detectors (DAD) and post-chromatographic reactions with 1,1-Diphenyl 2-picryhydrazyl radical (DPPH) to establish the antioxidant activity of nutraceuticals.
Fingerprint analysis by HPTLC of naturally derived products is increasingly gaining popularity as the ideal method of screening for adulterants, right from the time of cultivation of raw materials until processing into the required therapeutic dosage form.
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Common adulterants in many food supplements and soft drinks such as sildenafil (a phosphodiesterase inhibitor) and its analogs, and sibutramine can be detected by employing HPTLC.
HPTLC has been used to investigate cases of chemical warfare, drug, and alcohol abuse as well as to pin down suspects involved in petty and heinous crimes. Quantities of picogram concentration can also be detected through HPTLC. HPTLC has been especially helpful in detecting cannabis, the most commonly abused drug for recreational purposes.
Kulkarni et al. have been successful in developing new chromogenic spray reagents such as saturated ammonium metavanadate and p-anisidine for a study on cannabis detection by isolating cannabinoids from urine samples.
Take home message
The pharmaceutical industry has witnessed a rise in the use of HPTLC alone or in combination with other techniques such as MS, FTIR for the analysis of bulk drugs and formulations. In addition, HPTLC is also being applied successfully in fields of biomedicine, biochemistry with a growing trend in its application in modern agriculture for estimating pesticide residues in fruits and vegetables.
- Kostik et al. (2015). Application of high performance thin layer chromatography with densitometry for determination of active ingredients and preservatives in various pharmaceutical marketed formulations. IOSR Journal of Pharmacy. http://eprints.ugd.edu.mk/13055/
- Abourashed EA et al. (2004). HPTLC determination of caffeine in stimulant herbal products and power drinks. Journal of Pharmaceutical and Biochemical Analysis. 36(3):617-20. https://doi.org/10.1016/j.jpba.2004.06.029.
- Attimarad M et al. (2011). High-performance thin layer chromatography: A powerful analytical technique in pharmaceutical drug discovery. Pharm Methods. 2(2):71-75. doi: 10.4103/2229-4708.84436
- El-Ahmady SH et al. (2016). Advances in Testing for Adulteration of Food Supplements. Woodhead Publishing Series in Food Science, Technology and Nutrition. https://doi.org/10.1016/B978-0-08-100220-9.00024-2
- Munoz K et al. (2011). Rapid HPTLC-based method for quality control: simultaneous chemical analysis and antioxidant activity determination in herbal, nutraceutical and functional foods. Procedia Food Science. 1; 960-64. https://www.sciencedirect.com/science/article/pii/S2211601X11001453
- Bober K. (2017). Application of HPTLC with Densitometry for Evaluation of the Impact of External Factors on Contents of Diphenhydramine in Its Solutions. Int J Anal Chem. 2017:4914292. doi: 10.1155/2017/4914292
- Abdelwahab S et al. (2019). Simultaneous Determination of Thalidomide and Dexamethasone in Rat Plasma by Validated HPLC and HPTLC With Pharmacokinetic Study. J Chromatogr Sci. Feb 1;57(2):130-138. DOI: 10.1093/chromsci/bmy094
- Kulkarni UK et al. (2018). High Performance Thin Layer Chromatographic Detection of Cannabis in Forensic Interest. Journal of Chemical and Pharmaceutical Research. 10(11):9-14. www.jocpr.com/.../...ection-of-cannabis-in-forensic-interest-9742.html
- Bazylak G et al. (2000). HPTLC screening assay for urinary cotinine as biomarker of environmental tobacco smoke exposure among male adolescents. Journal of Pharmaceutical and Biomedical Analysis. 24(1);113-123. DOI: 10.1016/S0731-7085(00)00402-7
- Morschheuser L et al. (2016). HPTLC-aptastaining - Innovative protein detection system for high-performance thin-layer chromatography. Sci Rep. 6:26665. doi: 10.1038/srep26665.