Standardization of polyherbal formulations is essential in order to assess the quality of drugs, based on the concentration of their active principles (markers or standards). As Mentat tablet (MT), a herbal formulation consist of natural multi-ingredient formula with active constituents responsible for treatment of neurological disorder and improvement of mental health, therefore it is worthwhile to take it as a model for standardization of polyherbal formulation. In this work, an ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for simultaneous quantification of 28 major bioactive constituents. Multiple-reaction monitoring scanning was employed for quantification in positive and negative mode. The analysis was accomplished on Waters AQUITY UPLC BEH C18 column with linear gradient elution of acetonitrile 0.1% formic acid with water 0.1% formic acid solutions at a flow rate of 0.3 mL/min. The comparative analysis of samples was carried out by hierarchical cluster analysis and principle component analysis. The proposed method was applied to analyze 4 different batches of marketed formulation with acceptable linearity (r2, 0.9984-0.9999), precision, stability and recovery (RSD ≤ 3.74 %), evaluated under optimum conditions. The developed method is capable of controlling quality of polyherbal formulations having similar markers and herbs.

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Herbal medicines (HMs) refer to one herb or complex mixtures, which usually contains hundreds of chemically different components. Their curative effects are principally based on the synergic effect of their multi-targeting and multi-ingredient preparations [1, 2]. Consequently, quality control becomes a troublesome issue for crude drugs and their medical preparations. Therefore, the method that employs pharmacologically active components to evaluate the quality and authenticity of the complex preparations is confronted with severe challenges and better analytical strategies to assure their efficacy, safety, and consistency are in great demand [3].
Moreover, the chemical compounds in the poly herbs in HMs products may vary depending on harvest seasons, plant origins, drying processes and other factors. Thus, it seems to be necessary to determine most of the phytochemical constituents of herbal products in order to ensure the reliability and repeatability of pharmacological and clinical research, to perceive their bioactivities and eventual side effects of active compounds and to enhance product quality control [4, 5].
Currently, selection of a single or a few specific components from a certain herbal medicine as markers for quality assessment is a widely applied strategy. However, it cannot afford sufficient quantitative information for the other medicinal compositions and cannot accurately reflect the quality of HMs products. All the HMs compositions play important roles in the therapeutic effects. Therefore, selecting multiple constituents from different medicinal herbs as evaluation markers has been gradually applied for the quality control of HMs [6, 7].
Mentat tablets (MT, commercial product) is a polyherbal medication with each tablet composed of extracts (136 mg of Bacopa monnieri, 70 mg of Centella asiatica, 52 mg of Withania somnifera, 52 mg of Evolvulus alsinoides, 52 mg of Nardostachys jatamansi, 50 mg of Valeriana wallichii, 50 mg of Embelica ribes, 50 mg of Prunus amygdalus, 42 mg of Acorus calamus, 36 mg of Tinospora cordifolia, 36 mg of Terminalia chebula, 36 mg of Emblica officinalis, 32 mg of Oroxylum indicum, 32 mg of Celastrus paniculatus) and powders (80 mg of Bacopa monnieri, 18 mg of Withania somnifera, 18 mg of Mucuna pruriens, 18 mg of Elettaria cardamomum, 18 mg of Terminalia arjuna, 18 mg of Anethum sowa, 18 mg of Ipomoea digitata, 14 mg of Zingiber officinale, 14 mg of Terminalia belerica, 14 mg of Myristica fragrans, 10 mg of Syzygium aromaticum). MT is a unique all-natural multi-ingredient formula that promotes brain health. It improves the mental quotient, memory span, concentration ability, stress threshold and exhibit significant anti-parkinsonian activity. MT also offers protection against convulsions, which is beneficial in insomnia with its sedative and tranquilizing effects [8-10]. There are various pharmaceutical manufacturers that produce herbal formulations with similar herbs combinations to treat various neurological disorders. Chemically, bacosides the saponin mixture of Bacopa monnieri and triterpenoid glycosides in Centella asiatica have been considered as the key active components. [11-14]. Phytochemical investigations show important classes of bioactive constituents in selected plants as in combination of MT that are responsible for the bioactivity [15-20].
Literature survey reveals that, analytical methods including thin layer chromatography (TLC) [21], high performance thin layer chromatography (HPTLC) [22, 23], liquid chromatography (LC) [6, 24, 25], liquid chromatography coupled with mass spectrometry (LC-MS) [26-29], nuclear magnetic resonance (NMR) [30] have been developed for the quantitative analysis of the bioactive constituents in HMs to assess the quality of the complex preparations. Natural alteration in preparation processes and climate affects the safety and batch-to-batch uniformity of HMs products. Highly sensitive analytical methods are thus required to identify ingredients and evaluate lot-to-lot consistence.
To the best of our knowledge, there is no method have been reported for the simultaneous estimation of selected 28 multi-markers in herbals by UPLC-ESI-MS/MSand no such formulation approach has been explored on this important drug combination for quality consistency evaluation of this herbal preparation. Compared to conventional TLC, HPTLC, HPLC–UV method which will take a longer analysis time, UPLC-ESI-MS/MS method in multiple reaction monitoring (MRM), tandem MS scan mode offers detection effectively with more powerful approach, to rapidly quantify multi-ingredients in complex sample matrices due to its rapid separation power, low detection limit, high sensitivity, selectivity and specificity.
The methods used or reported in literature only contained one or two compounds, without consideration of other active ingredients. This paper describes for the first time a simple, accurate and reliable UPLC-ESI-MS/MS method for the simultaneous determination of 28 bioactive multiple components from different polyherbs including: bacoside A (mixture of bacoside A3, bacopaside II, bacopaside X and bacopasaponin C), withanolide-A, withaferin-A, asiaticoside, madecassoside, jatrorrhizine, palmatine, magnoflorine, curcumin, gallic acid, protocatechuic acid, ferulic acid, caffeic acid, ellagic acid, rosamarinic acid, ursolic acid, catechin, apigenin, luteolin, quercetin, rutin, kaempferol-3-O-rutinoside, corilagin, chrysin and chlorogenic acid with single runtime of 10 min.
The results indicated that the developed method is fast, sensitive and convenient to show the real quality of the polyherbal preparations. Therefore, this proposed method could be reliable and feasible for quality assessment of MT and all other herbal formulations manufactured by various pharmaceutical manufacturers with similar herbs combinations containing either these 28 markers or less. The method was also applied to analyze the multiple components in MT different batches from same manufacturer. Our results will facilitate the comprehensive quality control of MT and other similar related preparations. The quantitative results were further analyzed by multivariate statistical analysis i.e., hierarchical cluster analysis (HCA) and principle component analysis (PCA) to provide more information about the chemical differences and batch-to-batch uniformity.