Detection of Adulterated Naodesheng Tablet (Naodesheng Pian) via In-Depth Chemical Analysis and Subsequent Reconstruction of Its Pharmacopoeia Q-Markers

Abstract

Naodesheng Tablet (Naodesheng Pian), a traditional Chinese medicine formula for stroke treatment, is made up of five herbal medicines, i.e., Sanqi, Gegen, Honghua, Shanzha, and Chuanxiong. However, the current Pharmacopoeia quality-marker (Q-marker) system cannot detect possible adulteration. Our study tried to use a new strategy, i.e., standards-library-dependent ultra-high-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS/MS) putative identification, to reconstruct the Q-marker system. Through the strategy, 30 isomers were successfully differentiated (such as 2'-hydroxygenistein, luteolin, and kaempferol; ginsenoside Rg2 and ginsenoside Rg3; ginsenoside Rf and ginsenoside Rg1). In particular, 11 compounds were unexpectedly found in Naodesheng, including 2'-hydroxygenistein, 7,4'-dihydroxyflavone, pectolinarigenin, 7-methoxy-4'-hydroxyisoflavone, scoparone, matrine, 3,3',4',5,6,7,8-heptamethoxyflavone, 5-hydroxyflavone, diosgenin, chloesteryl acetate, and (+)-4-cholesten-3-one. In total, 68 compounds were putatively identified and fully elucidated for their MS spectra. Subsequently, relevant compounds were further investigated using UV-vis scanning experiments, semi-quantitative analysis, and quantum chemical calculation. Finally, five adulterated Naodesheng Tablets were used for validation experiments. The experiment successfully detected five adulterated ones via a lower-version LC-MS analysis. On this basis, three new candidates (hydroxy safflor yellow A (HSYA), citric acid, and levistilide A), along with puerarin and notoginsenoside R1, are re-nominated as the Q-markers for LC-MS analysis. The LC-MS analysis of puerarin, notoginsenoside R1, HSYA, citric acid, and levistilide A can clearly detect adulteration regarding all five herbal medicines mentioned above. Therefore, the reconstructed Q-markers are described as a "perfect" quality control system to detect adulteration in Naodesheng and will offer a valuable recommendation for the Pharmacopoeia Commission.

Keywords: Naodesheng Pian; UHPLC-Q-Exactive-Orbitrap MS/MS; counterfeiting recognition; quality control.

Figure 1

The TIC diagrams of Naodesheng Tablet in the UHPLC-Q-Orbitrap MS identification under negative mode (A) and positive mode (B). The positive mode was the supplement for the negative mode.

Figure 2

Structures of identified bioactive compounds from Naodesheng Tablet (A) for isomers; (B) for non-isomeric compounds. The chiral atoms in all sugar residue groups have been marked in their absolute configurations to avoid possible misreading. D-glucose is expressed as the Fischer project formula. The wave line in HSYA (7) indicates uncertain stereo configuration. The red tick √ means the old Q-markers.

Figure 3

The UV-vis spectra of 7 compounds (2, 7, 12, 36, 39, 50, and 65).

Figure 4

The results of the adulteration detection validation experiment of CNT 1~CNT 5. (A) Naodesheng Tablet and CNT 1 by extraction of C₂₁H₁₉O₆ (puerarin [ M − H], m/z 415); (B) Naodesheng Tablet and CNT 2 by extraction of C₄₇H₇₉O₁₈ (notoginsenoside R1 [ M − H], m/z 931); (C) Naodesheng Tablet and CNT 3 by extraction of C₂₇H₃₁O₁₆ (HSYA [ M − H], m/z 611); (D) Naodesheng Tablet and CNT 4 by extraction of C₆H₇O₇ (citric acid [ M − H], m/z 191); (E) Naodesheng Tablet and CNT 5 by extraction of C₂₄H₂₉O₄ (levistilide A [ M + H], m/z 381). The analytic technology was UHPLC-ESI-Q-TOF-MS. (A–D) Under the negative model; (E) under the positive model.

Figure 5

The preparation of the lyophilized aqueous extract of Naodesheng Tablet.