Analysis of benzo[c]phenanthridine alkaloids in Eschscholtzia californica cell culture using HPLC-DAD and HPLC-ESI-MS/MS

Figures & data

Fig. 1. Biosynthetic Pathway and Chemical Structures of the eight Target Benzo[c]phenanthridine Alkaloids in California Poppy.

Notes: Solid arrow stands for a single-step enzyme reaction and dotted arrows represent a multiple-step one (adapted from Ref.²⁰).

Fig. 2. HPLC-DAD Analysis Results of a Standard Sample (A) and a Real Sample Obtained from California Poppy Cell Culture (B).

Notes: 1 sanguinarine; 2 chelerythrine; 3 chelirubine; 4 macarpine; 5 dihydrochelerythrine; 6 dihydrochelirubine; 7 dihydrosanguinarine; 8 dihydromacarpine.

Table 1. Characteristics of the Alkaloids Determined by HPLC-DAD Analysis.

Compound	RT (min)	λmax (nm)	R^2	LOD (μg/mL)	LOQ (μg/mL)
Sanguinarine	17.09 ± 0.11	213.9, 273.9, 327.5, 472.0	0.9999	0.01	0.03
Chelerythrine	18.98 ± 0.09	213.6, 267.9, 316.7, 404.1	0.9978	0.61	2.03
Chelirubine	20.29 ± 0.07	234.8, 284.6, 353.8, 509.7	0.9965	0.76	2.55
Macarpine	22.59 ± 0.03	243.1, 283.4, 345.4	0.9963	0.79	2.64
Dihydrochelerythrine	42.60 ± 0.04	236.0, 281.0	0.9997	0.24	0.81
Dihydrochelirubine	43.18 ± 0.03	237.2, 278.6, 337.0	0.9932	0.11	3.59
Dihydrosanguinarine	43.83 ± 0.03	232.4, 283.4, 321.5	0.9955	0.09	2.90
Dihydromacarpine	44.16 ± 0.11	217.1, 283.4, 339.4	0.9981	0.06	2.05

Notes: RT: retention time (data represent the means and standard deviations of 9 samples); λ_max: characteristic wavelengths in DAD spectrum; R²: correlation coefficient of linear calibration curve; LOD: limit of detection; LOQ: limit of quantification.

Table 2. Operating Conditions of ESI-MS/MS for the Detection of Eight Target Alkaloids.

Compound	Observed mass (m/z)	Cone voltage (V)	Selected MRM mode (m/z)	Collision Energy (eV)
Sanguinarine	332.3	56	332.3 → 274.1	30
Chelerythrine	348.5	50	348.5 → 289.6	80
Chelirubine	362.4	60	362.4 → 317.4	80
Macarpine	392.5	60	392.5 → 347.3	80
Dihydrochelerythrine	350.5	48	350.5 → 303.7	78
Dihydrochelirubine	364.6	78	364.6 → 334.4	68
Dihydrosanguinarine	334.4	44	334.4 → 319.0	72
Dihydromacarpine	394.6	28	394.6 → 361.5	72

Fig. 3. MS/MS Spectrum of Eight Target Benzo[c]phenanthridine Alkaloids Analyzed by ESI-MS/MS.

Notes: (A) sanguinarine; (B) dihydrosanguinarine; (C) chelerythrine; (D) dihydrochelerythrine; (E) chelirubine; (F) dihydrochelirubine; (G) macarpine; (H) dihydromacarpine.

Fig. 4. MRM Chromatogram Analyzed by HPLC-ESI-MS/MS for a Real Sample Obtained from California Poppy Cell Culture.

Notes: 1 sanguinarine; 2 chelerythrine; 3 chelirubine; 4 macarpine; 5 dihydrochelerythrine; 6 dihydrochelirubine; 7 dihydrosanguinarine; 8 dihydromacarpine.

Fig. 5. Benzo[c]phenanthridine Alkaloid Profiles during the California Poppy Cell Culture.

Notes: (A) sanguinarine (●); chelerythrine (□); chelirubine (◆); macarpine (△), (B) dihydrosanguinarine (●); dihydrochelerythrine (□); dihydrochelirubine (◆); dihydromacarpine (△). Data represent the mean and standard deviation of duplicate samples, (C) cell growth profiles in gram dry cell weight (gDCW, ●) and in gram fresh cell weight (gFCW, ■).

Oldham JT, Hincapie M, Rejtar T, Wall PK, Carlson JE, Lee-Parsons CWT. Shotgun proteomic analysis of yeast-elicited California poppy (Eschscholzia californica) suspension cultures producing enhanced levels of benzophenanthridine alkaloids. J. Proteome Res. 2010;9:4337–4345.10.1021/pr1000412

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