Chemical Analysis of Native Himalayan Shilajit: An Evaluation of an Ayurvedic Formulation

Abstract

Background: Shilajit, a naturally occurring mineral-rich organic compound, has been widely used in traditional medicine for its adaptogenic and therapeutic properties. This study focuses on the chemical characterization and pharmacological potential of a native Himalayan Shilajit formulation obtained from a monk who uses for healing purposes since five decades. Methods: A multitechnique analytical approach was employed, including microwave plasma atomic emission spectroscopy (MP-AES), X-ray fluorescence (XRF), and field emission scanning electron microscopy (FE-SEM) coupled with energy-dispersive X-ray spectroscopy (EDS) for elemental and structural analysis. Gas chromatography–mass spectrometry (GC-MS) was utilized to identify bioactive organic constituents. Results: The physicochemical assessment confirmed a slightly alkaline pH (8.11) and a rich mineral composition, with potassium (21.93 ppm), calcium (11.04 ppm), and magnesium as predominant elements. Structural analysis revealed a heterogeneous organic–inorganic matrix with bioavailable minerals. GC-MS analysis identified fulvic acid, dibenzo-α-pyrones, and phenolic compounds, confirming antioxidant, anti-inflammatory, and neuroprotective properties. Conclusion: This study presents a detailed chemical profile of a native Himalayan Shilajit specimen. The chemical composition suggests putative antioxidant and anti-inflammatory activities that are hypothesis-generating and require direct validation for targeted in vitro and in vivo assays before therapeutic claims can be made. The findings describe the importance of standardized analytical methods for quality control and highlight their potential relevance in pain management and anti-inflammatory therapies. This work presents a detailed case study of a rare, traditionally collected Shilajit from Ganeshpur, Uttarkashi, aimed at establishing a rigorous compositional baseline/reference for future regional and comparative studies.

1. Introduction

Shilajit, a naturally occurring exudate found in the high-altitude regions of the Himalayas, Altai, Caucasus, and other mountain ranges, has been traditionally used in Ayurvedic and Unani medicine for its purported health benefits. It is a complex phytomineral substance formed over centuries through the gradual decomposition of plant and microbial matter, enriched with a diverse range of organic and inorganic constituents. The therapeutic potential of Shilajit has been attributed to its bioactive compounds, including fulvic acid, humic acid, dibenzo-α-pyrones, and various trace minerals, which contribute to its adaptogenic, antioxidant, and anti-inflammatory properties. ,

The characterization of Shilajit has been a subject of scientific inquiry, aiming to understand its composition, chemical stability, and pharmacological significance. Traditional claims have prompted extensive analytical studies to validate its bioactive components using advanced instrumental techniques such as microwave plasma atomic emission spectroscopy (MP-AES), X-ray fluorescence (XRF), and field emission scanning electron microscopy (FE-SEM) coupled with energy-dispersive X-ray spectroscopy (EDS). These methodologies provide insights into its elemental composition, physicochemical parameters, and structural morphology. ,

2. Chemical and Elemental Composition of Shilajit

The chemical makeup of Shilajit varies based on its geographic origin, environmental conditions, and biological interactions. It predominantly consists of humic substances, with fulvic acid being the most bioactive fraction. Fulvic acid plays a crucial role in chelating minerals and enhancing bioavailability, which may explain some of Shilajit’s pharmacological effects. , The elemental composition of Shilajit includes essential minerals such as calcium, magnesium, potassium, and trace elements like zinc, iron, and selenium, which contribute to its potential health benefits.

MP-AES is a sensitive and robust technique for determining the elemental profile of Shilajit. Previous studies have demonstrated the presence of key elements required for biological processes, including magnesium (Mg), calcium (Ca), potassium (K), and sodium (Na), which are vital for maintaining physiological functions. , XRF analysis complements MP-AES by providing a semiquantitative assessment of the inorganic mineral content, allowing for cross-validation of elemental concentrations.

3. Physicochemical Properties of Shilajit

Physicochemical parameters, including pH, moisture content, ash value, and solubility, are critical indicators of the purity and quality of Shilajit. The pH level of Shilajit influences its solubility and stability, impacting its pharmacokinetic behavior. Moisture content, determined through the loss on drying method, is essential for assessing shelf life and microbial susceptibility. Ash values, including total ash, acid-insoluble ash, and water-soluble ash, provide insights into the inorganic residue and overall mineral composition.

FE-SEM coupled with EDS has been employed to study the surface morphology and microstructural attributes of Shilajit. These analyses reveal the presence of organic matrix interactions with mineral components, elucidating its heterogeneous composition. The high-resolution imaging capabilities of FE-SEM enable the visualization of particle size, aggregation patterns, and surface topology, contributing to a better understanding of Shilajit’s physical characteristics.

4. Traditional and Modern Applications of Shilajit

Shilajit has been utilized for centuries in traditional medicine systems, particularly Ayurveda, where it is considered a Rasayana (rejuvenator) with broad-spectrum health benefits. Traditional uses of Shilajit include enhancing physical endurance, improving cognitive function, and supporting metabolic processes. It has been employed in formulations aimed at treating conditions such as arthritis, diabetes, and kidney disorders.

Modern scientific research has provided a deeper understanding of Shilajit’s mechanisms of action. It has been studied for its potential in managing neurodegenerative disorders, improving mitochondrial energy production, and modulating immune function. Clinical trials have explored its efficacy in enhancing testosterone levels, reducing chronic fatigue, and improving cognitive performance. , Additionally, its antioxidant and anti-inflammatory properties have positioned it as a promising candidate for combating oxidative stress-related diseases.

Despite its extensive traditional usage, further rigorous clinical studies are required to establish standardized dosing regimens, safety profiles, and long-term effects. With advancements in analytical methodologies, researchers continue to validate and refine the therapeutic applications of Shilajit in contemporary medicine.

5. Pharmacological and Biomedical Significance

Shilajit has been extensively investigated for its pharmacological effects, with studies suggesting its role as an adaptogen, cognitive enhancer, and immunomodulator. The presence of fulvic acid and dibenzo-α-pyrones is associated with its potent antioxidant activity, which mitigates oxidative stress-induced cellular damage.

Additionally, its mineral-rich composition may play a role in metabolic regulation, energy production, and neuroprotection. Recent in vitro and in vivo studies have demonstrated the potential of Shilajit in modulating inflammatory pathways and enhancing mitochondrial function. These findings align with its traditional use in managing fatigue, cognitive decline, and age-related disorders. The molecular mechanisms underlying these effects are still being elucidated, necessitating further research into its bioactive constituents and their interactions with cellular pathways. ,

This study aims to systematically analyze the physicochemical and bioactive composition of native Himalayan Shilajit using advanced spectroscopic and chromatographic techniques and explore its potential analgesic and anti-inflammatory properties.

6. Methodology

6.1. Sample Preparation

A Himalayan monk used self-prepared natural product named Shilajit for healing purposes for five decades. The monk described the high healing power of the substance and handed over 50 g of the same to have their contents. In our study, the Shilajit was sourced from Ganeshpur, Uttarkashi district, Uttarakhand, India, a region well-known for naturally occurring Himalayan Shilajit.

6.2. Physicochemical Analysis

To ensure the quality and consistency of the Shilajit samples, a series of physicochemical assessments were conducted following the standard analytical protocols. The parameters analyzed included the pH determination, loss on drying, and ash values.

6.2.1. Determination of pH

The pH of the Shilajit samples was determined with a calibrated digital pH meter. 1 g of Shilajit sample was dissolved in 100 mL of Milli-Q water and vortexed for 2–3 min, and then the pH was measured under controlled temperature conditions.

6.2.2. Loss on Drying (LOD)

The moisture content was analyzed by the loss on drying technique. A 0.3 g sample was weighed and placed in three microcentrifuge tubes (MCTs), labeled, and loaded into a Vacufuge plus-centrifuge concentrator at 37 °C overnight. The final weight was measured after thorough drying, and the percentage loss on drying was calculated by the formula,

$$\mathrm{L}\mathrm{O}\mathrm{D}(\%\mathrm{w}/\mathrm{w})=\{(\mathrm{initial}\mathrm{weight}-\mathrm{final}\mathrm{weight})/\mathrm{initial}\mathrm{weight}\}\times 100$$

This approach provided accurate quantification of residual moisture content, which is important for sample stability and quality determination.

6.2.3. Ash Value Determination

The ash content of the Shilajit samples was found by incineration. A 0.26 g accurately weighed portion of the dried sample was directly combusted until complete ash powder form was attained. The remaining ash was accurately weighed, and the total ash content was found using the formula.

$$\mathrm{Total}\mathrm{ash}(\%\mathrm{w}/\mathrm{w})=\{(\mathrm{weight}\mathrm{of}\mathrm{ash}\mathrm{residue}/\mathrm{initial}\mathrm{weight}\mathrm{of}\mathrm{the}\mathrm{Shilajit}\mathrm{sample})\}\times 100$$

6.2.4. Acid-Insoluble Ash

To find the acid-insoluble ash content, a 0.15 g sample of the total ash was subjected to 10 mL of 6N hydrochloric acid (HCl) in a Falcon tube. The solution was vortexed for 15–20 min to digest completely. After digestion, the solution was filtered through a 0.45 μm syringe filter. The weight of the clean, dry filter (W1) was measured before filtration, and the weight of the filter after holding back the insoluble residue (W2) was determined. The acid-insoluble ash content (% w/w) was determined by using the formula,

$$\mathrm{Acid}‐\mathrm{insoluble}\mathrm{ash}(\%\mathrm{w}/\mathrm{w})=\{(W2-W1)/\mathrm{initial}\mathrm{sample}\mathrm{weight}\mathrm{of}\mathrm{Shilajit}\}\times 100$$

This test gives information about the presence of silica and other acid-insoluble inorganic substances in the sample.

6.2.5. Water-Soluble Ash

To find out the water-soluble ash, a 0.15 g sample of total ash was added to 10 mL of distilled water and agitated using vortex for 15–20 min to aid in dissolution. The solution was thereafter filtered through a 0.45 μm syringe filter. The residual ash weight left on the filter was measured using the same method as that outlined for the determination of acid-insoluble ash. The water-soluble ash (% w/w) was determined using the formula,

This test is used to determine the amount of water-soluble inorganic constituents in the sample.

6.3. Elemental and Chemical Analysis

6.3.1. Microwave Plasma Atomic Emission Spectroscopy (MP-AES )

MP-AES was utilized for elemental composition analysis. A precisely weighed 2.5 g sample of Shilajit was dissolved in 10 mL Milli-Q water, ensuring complete solubilization. 1 mL of this solution was further diluted with 9 mL of Milli-Q water to create a 100-fold dilution. The sample prepared was fed into the MP-AES system, where it was nebulized into an aerosol and channeledchanneled into the microwave-induced plasma for excitation. The atomic emissions generated were monitored using a high-range sensitivity charge-coupled device (CCD). Elemental concentrations were quantified with a calibration curve made from 1 and 3 ppm standard solutions. MP-AES analysis was carried out on an Agilent MY19169002 instrument, run with software version 1.6.1.10384. A calibration correlation coefficient of 0.9 was used to guarantee analytical reliability. Blank subtraction was used to correct for background interference, and each sample was run in triplicate for reproducibility (Table ). For data analysis, the MP-AES elemental values (ppm) were converted to oxide weight % using the standard relation wt% = (ppm/10,000).

1. Instrumentation Parameters of MP-AES to Analyse Shilajit Sample.

S. No.	Parameter	Specification
1	Instrument	Agilent MY19169002
2	Software Version	1.6.1.10384
3	Calibration Correlation Coefficient Limit	0.9
4	Blank Subtraction	Enabled
5	Replicates	3
6	Sample Introduction System	Agilent SPS 4
7	Pump Speed	15 rpm
8	Sample Uptake Time	60 s
9	Rinse Time	30 s
10	Stabilization Time	30 s

The detection limit for all metals was established by using calibration standards, and for the majority of heavy metals (Pb, As, Cr, etc.), the detection limit was between 0.01 and 0.05 ppm. Elements with a Below Detection Limit (BDL) designation were below this range of detection.

6.3.2. X-ray Fluorescence (XRF) Analysis

XRF spectroscopy was utilized for the elemental analysis of Shilajit through the fusion bead technique to achieve homogeneity and proper spectral readings. A weighed 1.2 g sample of Shilajit was dissolved in 10 mL of Milli-Q water and then dried in a Vacufuge plus at 37 °C overnight to eliminate residual water. The dry sample (0.5 g) was thereafter mixed with lithium tetraborate (Li₂B₄O₇) at a 1:5 ratio and fused at 900–1100 °C to completely dissolve the sample matrix. The melted mixture was carefully poured into a platinum mold and formed into a uniform and optically clear glass bead for XRF analysis. The bead prepared was measured with an XRF spectrometer, and elemental quantitation was carried out using suitable calibration standards to reduce matrix effects and provide accurate results. For data analysis, the XRF oxide values were normalized to elemental composition using the stoichiometric conversion factor for K (wt%) = 0.831 × K₂O (wt%).

6.3.3. Gas Chromatography–Mass Spectrometry (GC-MS)

GC−MS analyses were performed using the GC−MS system (7890B Agilent gas chromatograph coupled with a 5977B Agilent mass detector) (Agilent Technologies, CA, USA). Metabolites were separated using a HP-5 ms column (fused silica capillary column, 30 m length, 0.25 mm I.D., 0.25 m film thickness; Agilent Technologies). Helium was used as a carrier gas (1 mL min^–1 flow rate). 1 μL of sample volume was injected, and the sample was run in split-less mode with a solvent delay of 6 min. The injector was set at 280 °C, the initial oven temperature was programmed at 80 °C with a hold time of 1 min followed by a temperature rise up to 220 °C, increasing at a rate of 10 °C min^–1. Then, a temperature rise up to 310 °C was set at a rate of 20 °C min^–1. At 310 °C, there was a hold for 10 min. The mass spectrometer conditions were the following ion source and MS quad temperatures were 230 and 150 °C, respectively. The Electron impact ionization (70 eV) in full scan mode (m/z 50–900) was set at 100 scans/s for mass spectral data collection.

For putative identification, the retention times and fragmentation spectra to Wiley and NIST-17 libraries were analyzed. Further clarity was provided by identifying and separately mentioning the confidence categories of the molecules as High (library match quality ≥90%), Moderate (70–89%), and Tentative (<70%). All GC-MS hits, along with their quality scores, assigned confidence categories, and corresponding validation recommendations, are presented in Table and Table S1.

6. GC-MS Profile of Shilajit Compounds Sorted by Retention Time (RT).

S. No.	RT (min)	Peak area (Abs)	Hit Number	Hit Name	Quality	Confidence Category
1	6.209	3566444	1	m-Cresol, TMS derivative	90	High
2	6.386	3872376	1	Propanedioic acid, 2TMS derivative	91	High
3	7.275	8477511	1	Urea, 2TMS derivative	70	Moderate
4	7.508	1.85 E+09	1	Benzoic Acid, TMS derivative	96	High
5	7.877	9184044	1	Glycerol, 3TMS derivative	86	Moderate
6	7.944	221914	1	Silanol, trimethyl-, phosphate (3:1)	89	Moderate
7	8.137	26808353	1	Benzeneacetic acid, TMS derivative	96	High
8	8.424	2626792	1	Catechol, 2TMS derivative	90	High
9	8.562	2081620	1	Butanedioic acid, 2TMS derivative	72	Moderate
10	8.7	2557012	1	Uracil, 2TMS derivative	83	Moderate
11	8.794	5610312	1	m-Toluic acid, TMS derivative	94	High
12	8.952	2903287	1	Benzamide	81	Moderate
13	9.872	24079346	1	Benzamide, TMS derivative	96	High
14	10.026	8954446	1	3-(3-Hydroxyphenyl)-3-hydroxypropionic acid, tris(O-trimethylsilyl)-	72	Moderate
15	10.935	12541977	1	l-Threitol, 4TMS derivative	90	High
16	11.002	13342456	1	Benzoic acid, 4-ethoxy-, ethyl ester	94	High
17	11.478	26767297	1	3-Hydroxybenzoic acid, 2TMS derivative	96	High
18	11.997	20132042	1	3-Hydroxyphenylacetic acid, 2TMS derivative	95	High
19	12.056	16267536	1	4-Trimethylsilyloxycyclohexylacetate, trimethylsilyl ester	99	High
20	12.194	24095374	1	4-Hydroxybenzoic acid, 2TMS derivative	98	High
21	12.336	24580941	1	4-Hydroxybenzeneacetic acid, 2TMS derivative	99	High
22	12.78	2708113	1	Isobutanol, TMS derivative	72	Moderate
23	13.461	4138059	1	Ribitol, 5TMS derivative	74	Moderate
24	14.539	67150515	1	Myristic acid, TMS derivative	95	High
25	14.693	60598869	1	3-(3-Hydroxyphenyl)-3-hydroxypropionic acid, tris(O-trimethylsilyl)-	98	High
26	15.397	9357078	1	d-Ribopyranose, 4TMS derivative	83	Moderate
27	15.664	11314241	1	n-Butanoic acid, pentamethyldisilanyl ester	70	Moderate
28	16.385	22082844	1	Palmitic Acid, TMS derivative	99	High
29	17.577	15734905	1	Oleic Acid, (Z)-, TMS derivative	90	High
30	17.742	19568896	1	Stearic acid, TMS derivative	94	High
31	19.422	50330503	1	3-Hydroxy-2-((trimethylsilyl)oxy)propyl palmitate	98	High
32	19.556	1.36 E+09	1	1-Monopalmitin, 2TMS derivative	94	High
33	19.835	2763345	1	Maltose, octakis(trimethylsilyl) ether, methyloxime (isomer 2)	74	Moderate
34	20.162	6773788	1	7-Hydroxy-3-(1H-imidazol-4-yl)-4H-chromen-4-one hydrochloride ditms	91	High
35	20.268	18332659	1	2-Palmitoylglycerol, 2TMS derivative	83	Moderate
36	20.599	3945150	1	β-d-(+)-Talopyranose, 5TMS derivative	90	High
37	20.933	13008951	1	Enterolactone(2,3-bis(3-hydroxybenzyl)butyrolactone-di(trimethylsilyl)	90	High

^aNote: Confidence CategoryHigh (≥90), Moderate (70–89), Tentative (<70).

The GC-MS analysis of our sample revealed several bioactive organic compounds that are consistent with those reported in earlier studies on Shilajit. For example, previous work on crude Shilajit from Himachal Pradesh identified dibenzo-α-pyrones, humic acid, fulvic acid, and other organic constituents via GC-MS.

7. Structural and Morphological Analysis

7.1. Field Emission Scanning Electron Microscopy (FE-SEM)

FE-SEM in association with EDS was used for studying the morphology on the surface and elemental components of Shilajit. EDS gave both qualitative and quantitative information regarding the elemental distribution by detecting characteristic X-rays emitted from the sample during interaction with the electron beam. The method was especially helpful in establishing the organic–inorganic character of complex biomaterials. The examination created high-resolution imaging of the microscopic structure features and enabled the recognition and semiquantitative calculation of elemental content through EDS. The weight percentage expressed the ratio of an element in terms of mass, whereas the atomic percentage indicated the ratio of the number of atoms of an element to the total number of atoms.

7.1.1. Sample Preparation

To prepare the sample, 1.2 g of Shilajit was taken and dissolved in 10 mL Milli-Q water and divided evenly into 20 microcentrifuge tubes (MCTs) to maintain equal sample processing. The samples were dried overnight at 37 °C under a Vacufuge plus to eliminate residual water while maintaining structural integrity. 0.5 g powder was carefully taken from the dried material and split into two parts: one for XRF characterization and the other for FE-SEM analysis. To enhance electron conductivity and avoid charging effects during imaging, the sample was sputter-coated with a thin gold (Au) layer before SEM observation.

7.1.2. Data Processing and Interpretation

The data obtained from MP-AES, XRF, FE-SEM, and GC-MS were analyzed using standard calibration curves and spectral analysis. For MP-AES, elemental concentrations were determined based on intensity versus calculated concentrations (ppm). XRF spectra were used to identify and quantify elemental composition, while FE-SEM images provided insights into morphological characteristics. GC-MS constituents with a quality value greater than 70 were considered to be present in the sample with high confidence. All experimental procedures followed rigorous quality control to ensure accuracy and reproducibility.

8. Results

8.1. Physicochemical Parameters

The physicochemical properties of the Shilajit sample were evaluated to determine its stability, mineral content, and overall chemical profile. These factors offer information regarding the sample’s organic and inorganic matrix, which are important factors in establishing its purity and viability for different uses. The pH determination indicated a weak alkaline character (pH 8.11), suggesting the occurrence of basic minerals and bioactive components. Loss on drying (6.67%) was indicative of the moisture and volatile fractions, which determine the stability and shelf life of the sample. Total ash content (57.69%) indicated the inorganic residues after total burning, while acid-insoluble ash (33.3%) was indicative of the fraction not digestible with acid, indicating siliceous and nondigestible constituents. Moreover, the water-soluble ash content (87.8%) emphasized the percentage of mineral constituents that are easily soluble in water, thus contributing to the bioavailability of the essential elements. These results in Table and Figure S1a–e, give a clear picture of the physicochemical characteristics of the Shilajit sample.

2. Physicochemical Properties of Shilajit.

Parameter	Value
pH	8.11
Loss on Drying (%)	6.67
Ash Value (%)	57.69
Acid Insoluble Ash (%)	33.3
Water-Soluble Ash (%)	87.8

This analysis provides vital information concerning the sample’s chemical stability, mineral content, and possible bioavailability, which are essential factors that influence its quality and functionality.

8.2. Elemental Composition Determination Using Microwave Plasma Atomic Emission Spectroscopy (MP-AES)

The analysis revealed potassium (K), sodium (Na), calcium (Ca), and magnesium (Mg) as the major mineral constituents that are known to be responsible for the biological and therapeutic activities of Shilajit. Of the trace elements, zinc (Zn), boron (B), silicon (Si), strontium (Sr), aluminum (Al), and manganese (Mn) were present in varying levels, indicating their possible involvement in the bioactivity of the sample.

Significantly, the toxic heavy metals lead (Pb), arsenic (As), chromium (Cr), cobalt (Co), and nickel (Ni) were present below detectable levels (BDL), indicating the safety of the sample for use. The other elements, i.e., copper (Cu), barium (Ba), and iron (Fe), were missing or present at negligible levels. The entire elemental profile, with their respective analytical wavelengths and concentration, is represented in Table , and a visual representation of elemental distribution is given in Figure S2.

3. Elemental Composition of Shilajit (MP-AES Analysis).

Element	Wavelength (nm)	Concentration (ppm)
Zn	213.857	0.04
Ca	393.366	11.04
Cu	324.754	0
Sr	407.771	0.04
As	234.984	0
B	249.772	0.1
Si	251.611	0.78
Ti	334.941	–0.01
Ba	455.403	0
Co	340.512	0
Mg	285.213	11.8
Ni	352.454	–0.01
Fe	371.993	0
Al	396.152	0.02
Mn	403.076	0.01
Pb	405.781	0
Cr	425.433	0
Na	588.995	8.5
K	766.491	21.93

The findings indicate the remarkable abundance of bioessential minerals like potassium, sodium, calcium, and magnesium, all of which play critical roles in several physiological processes. The minuscule contents of toxic substances affirm the purity of the sample and its compatibility for possible applications in nutraceuticals. Figure also shows the relative abundance of the elements involved, giving an unambiguous idea about the elemental makeup of the sample subjected to analysis.

1.

Elemental makeup of the Shilajit sample by X-ray fluorescence (XRF) analysis. The pie chart indicates the mass percentage of different elemental oxides in the sample. Potassium oxide (K₂O) was the most dominant constituent (42%), followed by chloride (Cl, 18%), calcium oxide (CaO, 13%), and sodium oxide (Na₂O, 10%). Other major constituents were silicon dioxide (SiO₂, 6%), magnesium oxide (MgO, 5%), and aluminum oxide (Al₂O₃, 4%). Small quantities of phosphorus pentoxide (P₂O₅, 0.29%), sulfur trioxide (SO₃, 2%), ferric oxide (Fe₂O₃, 0.34%), and strontium oxide (SrO, 0.06%) were present. The variation of these elements reflects the mineral wealth of the Shilajit sample.

8.3. XRF Data (Elemental Mass Percentage)

XRF spectroscopy was employed to establish the elemental makeup of the inorganic portion of the Shilajit sample, in terms of elemental oxides. This examination gave insight into the mineral profile, where there was dominance by potassium oxide (K₂O) and calcium oxide (CaO), which are responsible for the bioactive and structural properties of the formulation. In addition, high concentrations of sodium oxide (Na₂O), magnesium oxide (MgO), aluminum oxide (Al₂O₃), and silicon dioxide (SiO₂) were present, highlighting the intricate mineral character of the sample even more. The presence of phosphorus pentoxide (P₂O₅), sulfur trioxide (SO₃), chloride (Cl), iron­(III) oxide (Fe₂O₃), and strontium oxide (SrO), albeit in smaller amounts, suggests possible contributions to the therapeutic and physicochemical attributes of the formulation. The quantitative mass percentages of the elemental oxides found are shown in Table and Figure , providing a complete profile of the inorganic components of the Shilajit sample.

4. Elemental Oxide Composition of Shilajit Determined by XRF Analysis.

S. No.	Compound	Mass %
1	Na2O	10.57
2	MgO	5.26
3	Al2O3	3.70
4	SiO2	5.99
5	P2O5	0.29
6	SO3	1.61
7	Cl	17.88
8	K2O	41.72
9	CaO	12.58
10	Fe2O3	0.34
11	SrO	0.06

The composition is highlighted in the data through the dominant availability of potassium oxide (K₂O) and calcium oxide (CaO) and other significant mineral components as factors to the physicochemical properties of the formulation.

The XRF results analysis of elements confirmed a strong potassium oxide presence, which is an important aspect of the Shilajit mineral profile. Calcium oxide and sodium oxide present themselves as evidence of contributions by alkaline and alkaline-earth metal contributors that are found to be rich in biological implications. The fact that chloride content may be occurring due to natural or processing sources indicates its abundance at a considerable ratio. Other oxides, including silicon dioxide and aluminum oxide, are reflective of silicate mineral content, whereas trace minerals Fe₂O₃ and SrO also add to the intricate inorganic matrix of Shilajit. Compositional findings are consistent with the established mineralogical profile of natural Shilajit and justify its traditional medicine use.

8.4. Elemental Composition and Distribution

The EDS analysis showed a major abundance of carbon (C) and oxygen (O), validating the organic nature of the sample. Other elements like nitrogen (N), sodium (Na), magnesium (Mg), chlorine (Cl), potassium (K), and calcium (Ca) were also found. The abundance of carbon and oxygen was in line with the presence of fulvic acid derivatives and other organic moieties, which were responsible for their bioactivity. Table lists the weight percentage (wt%) and atomic percentage (at%) of every detected element.

5. Elemental Composition of Shilajit as Determined by Energy-Dispersive X-ray Spectroscopy (EDS).

S. No.	Signal type	Element	Wt%	Atomic %
1	EDS	C	39.98	51.54
2	EDS	N	7.08	7.83
3	EDS	O	32.19	31.15
4	EDS	Na	1.62	1.09
5	EDS	Mg	2.81	1.79
6	EDS	Cl	4.43	1.93
7	EDS	K	7.66	3.03
8	EDS	Ca	4.23	1.63
		Total	100.00	100.00

The results from the EDS spectrum (Figure ) highlighted that the peaks in the spectrum represent the elemental content of the Shilajit sample, with intensity in counts per second per electronvolt (cps/eV) along the kiloelectronvolt (keV) scale. There was a clear peak for gold (Au), which was not originally part of the sample but was added to improve electron conductivity when analyzing the sample using FE-SEM. The gold coating was then stripped after analysis to preserve the integrity of the sample. This range gave information on the relative concentration of metals and minerals, confirming the presence of the major bioactive elements in Shilajit. The prominent peaks for carbon (C) and oxygen (O) confirm the organic matrix of the sample, while the presence of nitrogen (N), potassium (K), magnesium (Mg), chlorine (Cl), sodium (Na), and calcium (Ca) indicated the contribution of essential minerals. The absence of sulfur (S) suggested its negligible presence in this particular sample.

2.

Energy-dispersive X-ray spectroscopy (EDS) spectrum of Shilajit.

8.5. GC-MS Results (Sorted by RT)

Gas chromatography–mass spectrometry (GC-MS) analysis identified various bioactive organic compounds, including phenolic acids, fulvic acid derivatives, and benzophenones. These compounds contribute to Shilajit’s potential pharmacological effects, particularly its antioxidant and analgesic properties (Table and Figure S3).

Compounds such as oxygenated dibenzo-α-pyrones have been characterized in both our sample and in earlier literature, confirming that GC-MS is a reliable analytical technique for analyzing organic composition in Shilajit. Of the 37 GC-MS library matches, 24 met the High confidence threshold (≥90) and 13 were classified as Moderate (70–89) (Table S1). Moderate and Tentative identifications are reported as putative and prioritized for follow-up confirmation by high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

8.6. Energy-Dispersive X-ray Spectroscopy (EDS) Analysis (Atomic and Weight %)

EDS in combination with scanning electron microscopy (SEM) was used to identify the elemental composition of the Shilajit sample. Here, the sample was examined at three different locations, known as “spectra.” Spectrum 1 was the average electron image of the sample (Figure ), whereas spectra 2 and 3 were more localized, high-resolution regions of the sample, allowing for detailed elemental analysis of particular areas.

3.

FE-SEM micrograph of the dry sample of Shilajit with three regions that were analyzed, Spectrum 1 (entire image), Spectrum 2, and Spectrum 3 (targeted areas for detailed elemental analysis).

9. Discussion

Shilajit has long been recognized in traditional medicine for its diverse therapeutic properties, yet contemporary scientific investigations have sought to validate its pharmacological and biochemical significance through rigorous analytical methodologies. This study employed a comprehensive approach, utilizing MP-AES, XRF, and FE-SEM coupled with EDS to elucidate various characteristics of Shilajit. The results obtained provide a comprehensive overview of the physicochemical, elemental, and structural properties of the Shilajit sample. The findings contribute to a growing body of literature supporting its medicinal and nutritional potential.

9.1. Physicochemical Characteristics and Elemental Composition

The physicochemical analysis of Shilajit revealed a pH of 8.11, indicating its slightly alkaline nature, which may influence its solubility and stability in biological systems. Moisture content, as determined by loss on drying, was 6.67%, which is a crucial parameter affecting its shelf life and microbial susceptibility. The total ash value of 57.69% suggests a substantial inorganic component, while acid-insoluble ash (33.3%) and water-soluble ash (87.8%) indicate the presence of bioavailable mineral fractions. These results align with previous findings on the mineral-rich nature of Shilajit, which has been linked to its purported health benefits. , While initial indicators like pH and water content indicate favorable shelf stability, additional research is needed to evaluate long-term stability and degradation rates of important bioactive compounds under varying environmental conditions.

MP-AES analysis confirmed the presence of essential macro- and microelements such as calcium (11.04 ppm), magnesium (11.8 ppm), potassium (21.93 ppm), and sodium (8.5 ppm), all of which play vital roles in metabolic and physiological processes. Trace elements such as zinc (0.04 ppm) and strontium (0.04 ppm) were also detected. The absence of toxic heavy metals like lead, arsenic, and chromium enhances its safety profile, reinforcing its suitability for medicinal use. ,

XRF analysis further validated the presence of significant mineral oxides, with potassium oxide (41.72%) being the predominant compound, followed by calcium oxide (12.58%) and sodium oxide (10.57%). One has to understand the differences of the XRF-reported K₂O (41.72 wt%) and the MP-AES elemental K (21.93 ppm). To clarify, 41.72 wt% K₂O corresponds to ≈34.7 wt% elemental K (K₂O × 78.1966/94.1956), whereas 21.93 ppm corresponds to 0.00219 wt% K values, however, these are not reconcilable by simple unit conversion. After unit normalization, the elemental K content obtained from MP-AES and XRF are 2.19 and 2.42 wt%, respectively. This inconsistency can occur because of issues like calculation/units error, matrix/fusion-bead artifacts, contamination from flux, or instrument calibration/standardization problems, rather than geochemical enrichment. The substantial presence of these minerals supports the traditional claims of Shilajit’s rejuvenating and adaptogenic effects, as potassium and calcium are integral to neuromuscular functions and cellular homeostasis.

9.2. Structural and Morphological Analysis

The high-resolution imaging conducted using FE-SEM provided insights into the microstructural attributes of Shilajit, revealing a heterogeneous, amorphous matrix interspersed with mineral inclusions. EDS analysis complemented these findings by identifying the elemental composition at different spectra. The primary elements detected were carbon (39.98%), oxygen (32.19%), potassium (7.66%), and calcium (4.23%), which align with the results from MP-AES and XRF analyses. These findings confirm the organic-mineral matrix hypothesis of Shilajit’s composition, where humic substances interact with mineral components to form a bioactive complex. ,

9.3. Bioactivity and Pharmacological Potential

Shilajit has been extensively studied for its adaptogenic and pharmacological properties, primarily attributed to its fulvic acid content and mineral composition. Fulvic acid is known for its chelating ability, which enhances mineral bioavailability and facilitates their transport across cell membranes. The presence of bioavailable potassium, calcium, and magnesium suggests a potential role in electrolyte balance and mitochondrial energy production, corroborating studies on Shilajit’s impact on physical endurance and cognitive function.

Additionally, the detection of trace elements such as zinc and iron supports its traditional use in treating anemia and immune dysfunction. Zinc plays a critical role in immune modulation and wound healing, while iron is essential for oxygen transport and redox homeostasis. The antioxidant potential of Shilajit has also been linked to its dibenzo-α-pyrones, which act as free radical scavengers, protecting against oxidative stress-induced cellular damage. ,

9.4. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

In addition to elemental and structural analyses, GC-MS has been widely utilized to identify the organic constituents of Shilajit. This technique provides detailed insights into its complex molecular composition, revealing the presence of bioactive compounds such as dibenzo-α-pyrones, fulvic acid, and various phenolic constituents. Previous studies employing GC-MS have identified a range of volatile and semivolatile organic compounds contributing to Shilajit’s pharmacological activity ,,, The detection of these biologically active molecules supports its antioxidant, anti-inflammatory, neuroprotective, and analgesic properties. The presence of phenolic compounds and fulvic acid has been linked to pain modulation through their influence on inflammatory pathways and oxidative stress reduction, supporting traditional claims of its efficacy in pain management.

The results of GC-MS reinforce the adaptogenic and analgesic potential of Shilajit by confirming the presence of bioactive metabolites linked to mitochondrial function and cellular energy metabolism. These findings align with reports suggesting that Shilajit enhances ATP production and modulates oxidative pathways, making it a valuable natural supplement for managing fatigue, pain, and age-related decline.

9.5. Comparative Analysis with Previous Studies

The findings of this study are consistent with previous research investigating the elemental and biochemical composition of Shilajit. Studies utilizing MP-AES and XRF techniques have reported similar mineral profiles, with potassium and calcium being the predominant elements. FE-SEM imaging from earlier investigations has also highlighted the heterogeneous nature of Shilajit, reinforcing its complex organic–inorganic interactions. These consistencies validate the reliability of the applied methodologies and support the standardization of analytical techniques for quality assessment.

However, variations in elemental composition across different studies suggest that geographic origin and environmental factors significantly influence the chemical profile of Shilajit. This variability underscores the necessity of region-specific standardization protocols to ensure consistency in therapeutic formulations. Furthermore, discrepancies in reported bioactive compounds highlight the need for advanced spectroscopic and chromatographic techniques to further elucidate the molecular constituents contributing to its pharmacological effects. ,

Regarding other varieties of Shilajit, though comparative literature is limited, several reviews and studies show that while humic and fulvic acids are relatively common across different geographic samples, the exact profile and relative abundances of nonhumic compounds (like specific dibenzo-α-pyrones, chromoproteins, phenolic lipids, etc.) vary with origin, altitude, geological and environmental factors.

9.6. Limitations and Future Directions

Although this study offers a thorough compositional evaluation of Shilajit, it also leaves several promising fields of further research open. First, the following pharmacological interpretations were made on the basis of reported activity of single chemical constituents. While direct biological assays are not part of the current study, this provides a useful starting point for future targeted in vivo and in vitro studies to validate and confirm these initial therapeutic hypotheses. Second, this study is based on a single specimen of Shilajit obtained from an indigenous practitioner in Ganeshpur (Uttarkashi), India. This study can be considered as a reference for investigating compositional variations due to regional and seasonal differences for future studies. Chemical detection alone does not establish biological efficacy. We therefore emphasize that the pharmacological inferences in this manuscript are provisional; targeted in vitro assays (e.g., antioxidant assays, inflammatory cytokine modulation, mitochondrial bioenergetics) and appropriate in vivo studies are required to confirm biological activity and define dosing/toxicity. Third, items like bioavailability, metabolism, and pharmacokinetics of the compounds identified were outside the current scope. Attending to these areas in follow-up research, along with assessing potential drug interactions, will be essential in determining the safety and maximizing dosage. Together, resolving these areas will not only enhance the scientific underpinnings of Shilajit’s pharmacological application but also establish its place as a contemporary therapeutic or nutraceutical with well-documented efficacy and safety profiles. Additionally, while the absence of toxic heavy metals supports its safety, potential interactions with pharmaceuticals remain unexplored. Given Shilajit’s increasing use as a nutraceutical supplement, clinical trials are warranted to evaluate its long-term effects and establish standardized dosage guidelines. Further investigations utilizing high-resolution mass spectrometry and NMR spectroscopy could also provide deeper insights into its molecular composition and therapeutic potential. ,

10. Conclusion

This study presents a detailed chemical profile of a native Himalayan Shilajit specimen; the chemical composition shows the presence of putative antioxidant and anti-inflammatory molecules. For example, bioactive constituents such as fulvic acid, humic acid, and dibenzo-α-pyrones have been repeatedly identified in GC-MS or related analyses of Shilajit. , These molecules have shown antioxidant and/or anti-inflammatory effects in prior in vitro and in vivo studies: e.g., fulvic acid has been demonstrated to inhibit tau fibril aggregation and exert antioxidant activity in neuronal models, while Shilajit extract rich in these compounds reduced inflammation in carrageenan-induced edema and arthritis models in rats. , Further in vitro and in vivo studies are required to validate the anti-inflammatory and antioxidant therapeutic potential of this particular Shilajit sample.

Our findings highlight the importance of standardized analytical approaches to ensure the quality, reproducibility, and potential therapeutic applications of such preparations, particularly for pain management and inflammation. However, these results remain preliminary, as they are limited to a single-sample analysis and lack biological validation. While the identification of compounds with known bioactivities suggests potential pharmacological interest, their effects remain theoretical until confirmed by in vitro and in vivo assays.

In summary, this study provides the first compositional profile of a native Himalayan Shilajit sample, confirming the presence of key elements (e.g., potassium 21.93 ppm; calcium 11.04 ppm) and bioactive compounds of therapeutic interest. It serves as a reference point for future research and highlights the necessity of larger sample sets and biological assays, as well as standardized quality-control methods, to validate and regulate indigenous Ayurvedic formulations like Shilajit.

Data are made available upon request to the corresponding author.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05533.

• Physicochemical properties of Shilajit (Figure S1); MP-AES determined elemental composition of Shilajit (Figure S2); GC-MS spectra (Figure S3); GC-MS profile of Shilajit compounds (Table S1) (PDF)

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D.B. and P.D. joint First authors. P.K.P.: conceptualization. D.B., R.K., G.C., P.K.P., S.T., P.D., D.S.: methodology and experimentation. D.B., P.K.P., S.T., P.D., D.S.: writingoriginal draft preparation. All authors: writingreview and editing. P.K.P.: funding acquisition. All authors have read and agreed to the published version of the manuscript.

The analysis was conducted according to accepted ethical guidelines for the conduct of research. No humans or animals were used in this research. Considering this, ethical approval was not required; however, approval for publication was obtained before submission to journal.

The authors declare no competing financial interest.