Mr. Ramakant Yadav, an application specialist under the guidance of Akshay Charegaonkar (Managing Director), works at Anchrom Enterprises Pvt Ltd, Mumbai, India. The company specializes in instrumental Planar Chromatography and is renowned for its expertise in developing novel, quantitative, and regulatory-compliant analytical methods for a wide range of products, including pharmaceutical formulations, APIs, herbal products, food items, organic intermediates, and dyes. Mr. Yadav finds HPTLC advantageous due to its rapidity, ease of use, cost-effectiveness, and data outputs such as plate images both pre- and post-chromatographic derivatization, along with the ability to evaluate data through image, profile, and spectrum comparisons.
Introduction
Crocus sativus L. commonly known as saffron, is a perennial stemless herb that is widely cultivated in Iran, India and Greece. It is obtained by drying the stigma of C. sativus L., which belongs to the Iridaceae family. Saffron plays a pivotal role in modern and traditional medicine, it is utilized for the prevention and treatment of various diseases and has anti-hypertensive, antioxidant, antidepressant, and anti-inflammatory activity. This precious spice holds a broad spectrum of applications in the food and cosmetic industries, serving as both a flavoring and coloring agent. The quality of saffron is affected by various factors, such as cultivation regions, climate, drying process, and storage conditions. However, the high value of this product makes it very susceptible to economic adulteration, which involves the mixing of low-quality spices with saffron, the addition of plant materials, and the use of natural or artificial colorants to imitate the color of saffron.
HPTLC is widely implemented in the food industry as a convenient and low-cost approach for separations of different chemical components, such as adulterants and contaminants. It is well suited for adulteration studies, because it is inexpensive and time-saving. By HPTLC, 15-20 samples can be detected simultaneously on one plate in about 20-30 minutes. The solvent consumption is only about 20 mL for those 20 samples and little waste is produced. Hence a method was developed for comparison of marketed saffron samples with pure saffron sample (BRM) to find possible adulterants.
Standard solutions
100 mg of Crocus sativus L. (Saffron BRM) is dissolved in 10 mL of 70 % ethanol.
Sample preparation
100 mg of Crocus sativus L. (Saffron) marketed formulations are dissolved in 10 mL of 70 % ethanol. The samples are vortexed thoroughly and centrifuged at 3000 rpm for 5 min. After centrifugation, the supernatant was collected and used for the application.
Chromatogram layer
HPTLC plates silica gel 60 F254 (Merck), 20 x 10 cm are used.
Sample application
2.0 and 5.0 µL of sample solutions and 2.0 and 5.0 µL of standard solutions are applied as bands with the Automatic TLC Sampler (ATS 4), 15 tracks, band length 8.0 mm, distance from the left edge 20.0 mm, track distance 11.4 mm, distance from the lower edge 8.0 mm.
Chromatography
Plates are developed in the ADC 2 with chamber saturation (with filter paper) for 20 min and after activation at 33 % relative humidity for 10 min using a saturated solution of magnesium chloride, development with ethyl acetate – methanol – water 18:4:3 (V/V) to the migration distance of 70 mm (from the lower edge), followed by drying for 5 min.
Post-chromatographic derivatization
The plate is pre-heated at 105 °C for 3 min using the TLC Plate Heater and is then sprayed with 3 mL of natural product A reagent (1 g of 2-aminoetheyl diphenylborinate in 200 mL of ethyl acetate) using the Derivatizer.
Documentation
Images of the plate are captured with the TLC Visualizer in UV 254 nm, UV 366 nm, and white light.
Results and discussion
The analysis conducted on saffron samples involved a comparison between saffron (BRM) and saffron available in the market (branded and non-branded). Upon developing the chromatographic plate, it was observed that the fingerprints of all the saffron samples from the market were identical to that of the saffron (BRM). Notably, no adulterants were detected in any of the saffron samples, as there were no discernible colored bands observed apart from the characteristic fingerprint pattern.
Further investigation involved taking spectra of the major bands detected in all the saffron samples for the purpose of comparison. The results of this spectral analysis revealed that all the spectra from the different saffron samples, including both BRM and market-sourced saffron, matched identically. This suggests consistency and purity among the saffron samples, reinforcing their authenticity and quality.
HPTLC fingerprints: White light (A), UV 254 nm (B) & UV 366 nm (C) prior to derivatization, and white light after derivatization (D) and UV 366 nm after derivatization (E); Tracks 1 & 7: Saffron (BRM), tracks 2 & 8: marketed branded sample 1, tracks 3 & 9: marketed branded sample 2, tracks 4 & 10: marketed branded sample 3, tracks 5 & 11: marketed non-branded sample 1, and tracks 6 & 12: marketed non-branded sample 2
Left: Stack and flip view of saffron BRM and marketed saffron samples; middle: spectrum comparison of the two prominent zones found at RF 0.22 and 0.43 in both the marketed saffron samples and the BRM; right: HPTLC peak profiles for saffron BRM and marketed saffron samples
Literature
[1] American Herbal Pharmacopoeia
Further information is available on request from the authors.
Contact
Mr. Akshay Charegaonkar, A-101, Shree Aniket Apartments, Navghar Road, Mulund East, Mumbai, Maharashtra 400081, India, hptlc@anchrom.in, www.anchrom.in