Interpreting Metabolites and Biosynthetic Pathways of Musk with GC–MS

Limited evidence regarding the function and pathways of musk is available from databases due to the complexity, variability, and derivativity of chemical composition. Researchers harvested musk samples from six male forest musk deer at three different stages during maturation, and a gas chromatography and mass spectrometry (GC–MS) approach was utilized to explore the chemical composition. A paper based on this research was published in Frontiers in Pharmacology (1).

A small ruminant found in south-central China and northern Vietnam, forest musk deer (Moschus berezovskii Flerov) has been listed as an endangered species by the International Union for Conservation of Nature (IUCN) due to loss of habitat and poaching of the animal for its musk sac (2,3). Existing studies have revealed that natural musk is an excellent source of traditional Chinese medicine (TCM) pharmaceutical preparations, including bioactive components such as muscone (4). The high demand for musk in traditional Chinese medicine and perfumery has led to unsustainable harvesting, making its chemical characterization crucial for conservation efforts (while deep brown mature musk has unique and intense scents, attracting females during the breeding season from the end of October to February, immature musk is white and not intensely scented, as two phases are required for musk maturation [5–8]).An understanding of the composition of musk can inform the development of synthetic substitutes, which would reduce reliance on wild populations; with the improvement of analytical methods, measurement of metabolites would become more accessible and widespread (9,10). To date, studies on the composition of natural musk have primarily focused on characterizing contents and their quantities. However, relatively few comprehensive analyses of the pathways involved in the biosynthesis of metabolites in natural musk have been undertaken, thus motivating the researchers to conduct this analysis in their research (1).

Samples of musk taken from six healthy 3-year-old male forest musk deer were obtained in 2-mL EP tubes and underwent extraction with 0.48 mL of extraction liquid (3:1 methanol– chloroform). Samples were homogenized in a ball mill for 4 min at 45 Hz, then ultrasound-treated for 5 min in ice water. The resulting homogenate was centrifuged for 15 min at 13,000 rpm at 4 °C, and the supernatant (0.4 mL) was transferred to a fresh 2-mL GC–MS glass vial. GC–MS analysis was then performed (1).

The researchers believe that mapping the metabolites to multiple databases and defining them systematically, the determination of macrocyclic as organic oxygen compounds, the unambiguous categorization of lipids, and the construction of a deciphered biosynthetic pathway of muscone and steroids according to the compound-pathway-enzyme-reaction network analysis deepens the understanding of natural musk composition and offers new theoretical insights to develop and utilize musk. In addition, their efforts reduce the demand for wild forest musk deer resources. However, while their metabolomic data suggest involvement of muscone/steroid biosynthesis pathways, they believe that experimental validation (such as enzyme activity assays and isotope tracing) is required to confirm these mechanistic links. Future studies, therefore, should integrate multiomics approaches to dissect the genetic and enzymatic basis of musk maturation (1).

A solitary musk deer stands proudly among the forest grass. © iti - stock.adobe.com

References

1. Jie, H.; Li, F.; Liu, Q.; et al. Elucidating Metabolites and Biosynthetic Pathways During Musk Maturation: Insights from Forest Musk Deer. Front. Pharmacol. 2025, 16, 1503138. DOI: 10.3389/fphar.2025.1503138

2. Yang, Q.; Meng, X.; Xia, L.; et al. Conservation Status and Causes of Decline of Musk Deer (Moschus spp.) in China. Biol. Conserv. 2003, 109 (3), 333–342. DOI: 10.1016/S0006-3207(02)00159-3

3. Shusheng, G.; Shila, M. Decline of Musk Deer in China and Prospects for Management. Environ. Conserv. 2000, 27 (4), 323–325. DOI: 10.1017/S0376892900000369

4. Wang, J.; Xing, H,; Qin, X.; et al. Pharmacological Effects and Mechanisms of Muscone. J. Ethnopharmacol. 2020, 262, 113120. DOI: 10.1016/j.jep.2020.113120

5. Shen, H.; Liu, Z. The Musk Deer in China (in Chinese); Shanghai Scientific and Technical Publishers, 2007.

6. Qi, W. H.; Li, J.; Zhang, X. Y.; et al. The Reproductive Performance of Female Forest Musk Deer (Moschus berezovskii) in Captivity. Theriogenology 2011, 76 (5), 874–81. DOI: 10.1016/j.theriogenology.2011.04.018

7. Hawkins, T. H. Musk and the Musk Deer. Nature 1950, 166 (4215), 262. DOI: 10.1038/166262a0

8. Wu, J. Y.; Wang, W. The Musk Deer of China (in Chinese); China Forestry Publishing House, 2006.

9. Wang, S.; Shang, Y.; Liang, C.; et al. Binary Eluent Based Vortex-Assisted Matrix Solid-Phase Dispersion for the Extraction and Determination of Multicomponent from Musk by Gas Chromatography-Mass Spectrometry. J. Anal. Methods Chem. 2021, 2021, 9913055. DOI: 10.1155/2021/9913055

10. Jin, C.; Yan, C.; Luo, Y.; et al. Fast and Direct Quantification of Underivatized Muscone by Ultra Performance Liquid Chromatography Coupled with Evaporative Light Scattering Detection. J. Sep. Sci. 2013, 36 (11),1762–1767. DOI: 10.1002/jssc.201200946