Shilajit is also known as Shilajeet, Shilajatu (in Sanskrit which means derived from rocks), mumie (juice of rock) or mummiyo. It is a Herbo mineraloid, the word ‘shilajit’ is made up of two words, Shila and jeet. ‘Shila’ means mountain and ‘jeet’ means to win. In summers, due to strong heat it seeps out from the layers of rocks on the mountain facing at high altitudes between 1000 m to 5000 m [1]. Like other herbs, it cannot be directly harvested from plants, and it takes years to form. It is a blackish-brown resinous exudate which is the result of slow decomposition of plant and microbial metabolites, and it takes years to form under the layer of rocks [2]. In traditional systems of medicine, shilajit is known to have an important contribution in human health. According to ayurveda, it has rejuvenating property and promotes healthy ageing [3]. It is considered as the natural remedy for over 3000 years in folk medicine for all types of diseases.

Shilajit, a traditional medicinal substance, embodies chemical complexity, comprising phytochemicals like fulvic acid, humic acid, and bioactive markers such as hippuric acid and urolithin A. The phyto-complexity of shilajit refers to the combination of organic compounds and minerals, which further leads to produce synergistic effect. Fulvic acid, a natural chelator, often helps in transport and absorption of essential minerals to the cells, thus contributing to its rejuvenating properties. Dibenzo-α-pyrones including urolithin A, has anti-inflammatory and antioxidant property which helps in mitochondria function. Hippuric acid is an essential organic compound which helps in the detoxification process of the body. This research aims to unravel these complexities using hyphenated methods, with special focus on markers pivotal to its therapeutic efficacy. Notably, this study refrains from elaborating on Shilajit varieties unrelated to its primary analysis, centering only on direct relevant classifications.

Some scientific research revealed that shilajit is a result of biological process. It might be formed from the long humification of latex or resin-producing plants, such as Euphorbia royleana and Trifolium repens, which grow near shilajit-bearing rocks [4]. Furthermore, claims have been put forth regarding bryophytes, as a source for shilajit, which includes species of mosses like Minium, Barbula and species of liverworts like Pellia, Asterella, Marchantia [2]. Meanwhile, some studies revealed that shilajit is formed through geological process which may have originated from vegetative fossils that have been compressed under layers of rocks for several years which transformed due to the high temperature and pressure [5]. The Charka Samhita describes four different varieties of shilajit, namely svarna (gold colored with red hue), rajat (silver or white), tamra (blue colored), and lauha shilajit (iron-containing blackish brown colored) [2]. In this study, lauha shilajit variety has been used.

As per literature review, quantification of Urolithins in plasma samples were investigated by chromatographic and spectroscopic methods [6,7]. In the past few years, many analytical as well as pharmacological studies have been reported on humic substances like Fulvic acid and Humic acid [8,9]. The elemental composition of Fulvic acid extract was studied by researchers through FT-ICR mass spectrometry [10]. Furthermore, HPLC method was developed for extracts of Mongolian shilajit, and isolated compounds were evaluated for free radical scavenging activity [11]. To date, no analytical method utilizing HPLC or LC-MS has been reported for the identification and quantification of hippuric acid and benzoic acid in shilajit. This study primarily aimed to quantify hippuric acid (Fig. 1A), benzoic acid (Fig. 1B), and fulvic acid from shilajit. Novel RP-HPLC method was developed, capable of accurately quantifying hippuric acid and benzoic acid. In contrast to it, fulvic acid was quantified using gravimetry, as it is a complex mixture of low-molecular-weight organic acids with highly variable composition and lacks well-defined, distinct peaks in chromatographic analysis. In addition to it, confirmation of markers based on mass to charge ratio (m/z) and fragment ion analysis was done by using LC-MS. There have been reports that the chemical constituents present in shilajit are rich in humic substances and urolithin A, which attribute to major physiological and pharmacological aspects [13]. Around 20–40 % inorganic substances as minerals are present in crude shilajit [14,2]. Geographical and environmental factors are known to affect the composition of natural materials. Some studies revealed that natural color of raw shilajit may vary with the amount of minerals present in it. One literature had reported the elemental composition of shilajit by using EDX, LIBS, ICP techniques [15]. Shilajit is known to be rich in wide range of minerals and trace elements. In this study, we primarily aimed to quantify and compare the mineral composition of selected samples, providing a basis for future work exploring such regional variations. To investigate that, 14 elements were quantified using inductively coupled plasma mass spectrometry (ICP-MS). Development of HPLC method is crucial for standardization, purification and quality control of shilajit pharmaceutical product. Techniques including HPLC, liquid chromatography-tandem mass spectrometry LC-MS/MS, and ICP-MS support high-precision profiling, guaranteeing safety assessment and reliability. ICP-MS technique will help researchers to detect the heavy metal content and its compliance with standard WHO guidelines. The therapeutic potential of Shilajit in pharmaceutical applications can be explored with the help of these studies.