Fig. 1. Our strategy combining HPLC and LC/MS for the comprehensive quality control of traditional medicine.

Fig. 2. Chemical structures of bufadienolides isolated from ChanSu.

Fig. 3. Identification of bufadienolides in ChanSu by LC/MS analysis. From top to bottom, the chromatograms represent HPLC/UV profile monitored at 296 nm, LC/MS total ion current (TIC), and extracted ion chromatograms for different [M + H]⁺ ions. For peak identification: 1, ψ-bufarenogin; 2, gamabufotalin; 3, arenobufagin; 4, bufotalinin; 5, telocinobufagin; 6, desacetylcinobufagin; 7, bufotalin; 8, 19-oxo-cinobufagin; 9, cinobufotalin; 10, bufalin; 11, cinobufagin; 12, resibufogenin.

Fig. 4. Identification of gamabufotalin by HPLC/DAD/APCI-MS/MS: (a) online UV spectrum; (b) full scan mass spectrum, showing molecular adduct ions including [M + H]⁺ = m/z 403; [M + 18]⁺ = m/z 420; [M + 42]⁺ = m/z 444; [M + 60]⁺ = m/z 462; and [2M + H]⁺ = m/z 805; (c) MS/MS spectrum for the [M + H]⁺ ion at m/z 403.

Fig. 5. HPLC/UV quantitation of major bufadienolides in ChanSu crude drug: (a) chromatogram of eight bufadienolide standards; (b) chromatogram of ChanSu methanol extract: 1, gamabufotalin; 2, arenobufagin; 3, telocinobufagin; 4, bufotalin; 5, cinobufotalin; 6, bufalin; 7, cinobufagin; 8, resibufogenin.

¹³C NMR spectral data of bufadienolides (125 MHz, DMSO-d₆)

¹H NMR spectral data of bufadienolides (500 MHz, DMSO-d₆)

Note: Those peaks whose multiplicity was not designated are multiplets (m).

Cytotoxic activities of bufadienolides against human cancer cells (n = 3)

Regression equations of bufadienolides for HPLC/UV quantitation (n = 8)

Note: In the regression equation y=ax + b, x refers to the concentration of pure bufadienolides (μg/ml); y the peak area; r, the correlation coefficient; LOD, limit of detection; LOQ, limit of quantitation.

Recovery test for the determination of bufadienolides in ChanSu crude drug (n = 3)

Note: calculated amount = detected amount−original amount; recovery = calculated amount/added amount × 100%. The original amount was calculated according to the calibration.

Contents of eight major bufadienolides in ChanSu drug samples (mg/g)