Analyzing Biomarkers in an Enhanced Polyherbal Formula with Antidiabetic Attributes through High-performance Thin-layer Chromatography and Liquid Chromatography–Mass Spectrometry/ Mass Spectrometry
Authors: Venkatesh Uppara , Ramana B. V. 
Published: January 2024

ABSTRACT

Context:
In this research, we subjected a polyherbal formulation (PHF) prepared in-house, previously evaluated for its antidiabetic effects in streptozotocin-induced diabetic rats, to further analytical characterization using high-performance thin-layer chromatography (HPTLC) and Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS).

Aim:
The aim of this research was to measure the active components within a newly developed in-house PHF with antidiabetic properties, prepared using dried leaves of Tinospora cordifolia, dried bark of Cinnamomum zeylanicum, dried seeds of Trigonella foenum, and dried seeds of Nigella sativa, employing HPTLC and LC-MS/MS techniques.

Materials and Methods:
Magnoflorine (MAG), cinnamaldehyde (CIN), trigonelline (TRI), and thymoquinone (THY) were identified as the active components of the ethanolic extract of the PHF. We utilized HPTLC and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the characterization of these constituents. The gradient mobile phase consisted of water, acetonitrile, and 0.1% trifluoroacetic acid. Chromatographic separation was achieved using an XBridge C18, 250 × 4.6, 3 mm analytical column with a flow rate set at 1.0 mL/min, maintained at ambient temperature employing a gradient system that spanned 30 min. Mass scanning was conducted in positive mode, with a scan speed ranging from 100 to 2000 amu/s, covering a mass range from 20 to 1974 Da. Electron spray ionization mode was utilized, with a source temperature maintained at 150°C and a desolvation temperature at 350°C. For HPTLC separation, precoated silica gel 60 GF254 plates were employed, employing a mobile phase consisting of toluene/chloroform/ethyl acetate/methanol/formic acid in a ratio of 2.5:2.5:2.5:2.0:0.5 (v/v). The plates were developed at room temperature, extending to a distance of 9.0 cm. Each individual component (MAG, CIN, TRI, and THY) was scanned and quantified at its respective maximum absorption wavelength (237 nm, 259 nm, 305 nm, and 338 nm).

Results:
Experimental parameters such as band size, chamber saturation duration, solvent front migration, slit width, and others were carefully examined to determine the optimized chromatographic conditions. LC-MS analysis of the alcoholic extract of the PHF successfully identified the presence of all five bioactive chemical constituents, namely, MAG, CIN, TRI, and THY. In addition, the drug samples exhibited satisfactory resolution with retention factor values of 0.27 ± 0.01, 0.64 ± 0.01, 0.55 ± 0.01, and 0.92 ± 0.01 for CIN, MAG, TRI, and THY respectively, using HPTLC.

Conclusion:
The integration of Ayurvedic formulations with contemporary high-throughput screening techniques holds great promise in discovering more potent and biocompatible drug leads. The results of this study offer compelling scientific evidence that strengthens the case for the therapeutic utility of the PHF, further underscoring its significance in the field of health care.