(1) Background: Ganoderic acids (GAs) are specific triterpenes of Ganoderma lucidum. The HPLC fingerprint profile of GAs of the fruiting body is well known, but their mycelial fingerprinting remains unclear. (2) Methods: An ethanol extract of the mycelium of G. lucidum (YK-01) was further purified via preparative HPLC. The triterpenoid compositions for four strains of G. lucidum and one strain of G. formosanum (purple lingzhi) were analyzed using HPLC. (3) Results: Nineteen lanostane triterpenes, including five new triterpenes, GA-TP (1), ganodermic acid J_c (GmA-J_c) (2), GmA-J_d (3), GA-TQ₁ (4), and ganoderal B₁ (5), and fourteen known triterpenes 6–19 were isolated from the ethanol extract. Their structures were identified by mass and extensive NMR spectroscopy. A green chemical HPLC analytical method was developed using ethanol and acetic acid as a mobile phase, and all isolated compounds can be well separated. These triterpenes comprise a unique HPLC chromatograph of the G. lucidum mycelium. All four G. lucidum strains showed the same HPLC chromatographic pattern, whereas G. formosanum displayed a different pattern. Quantitation methods for ganoderic acid T (10) and S (12) were also validated. (4) Conclusions: The triterpenoid HPLC analytical method can be used to identify the G. lucidum species and to determine the contents of GA-T and GA-S.

(1) Background: ganoderic acids (GAs) are specific triterpenes of Ganoderma lucidum. The HPLC fingerprint profile of GAs of the fruiting body has been well-known, but the mycelial fingerprinting remains unclear. (2) Methods: the ethanol extract of the mycelium of G. lucidum (YK-01) was further purified by preparative HPLC. The HPLC spectra for four strains of G. lucidum and one strain of G. formosanum (purple lingzhi) were investigated. (3) Results: nineteen lanostane triterpenes, including five new triterpenes, GA-TP (1), ganodermic acid Jc (GmA-Jc) (2), GmA-Jd (3), GA-TQ1 (4), and ganoderal B1 (5), and fourteen known triterpenes 6-19 were isolated from the ethanol extract. A green chemical HPLC analytical method was developed by using ethanol and acetic acid as mobile phase. These triterpenes comprise of a unique HPLC triterpenoid spectral pattern of G. lucidum mycelium. All four G. lucidum strains showed the same HPLC spectral pattern, whereas G. formosanum displayed a different pattern. Quantitation methods for ganoderic acid T (10) and S (12) were also validated. (4) Conclusions: the triterpenoid HPLC analytical method can be used to identify G. lucidum species, and to determine the contents of GA-T and GA-S.