Time-resolved fluorescence and chemometrics-assisted excitation-emission fluorescence for qualitative and quantitative analysis of scopoletin and scopolin in Erycibe obtusifolia Benth.

This paper presents a new strategy for qualitative identification of scopoletin and scopolin in Erycibe obtusifolia Benth using time-resolved (lifetimes) fluorescence and quantitative analysis with chemometrics-assisted excitation-emission matrix (EEM) fluorescence. Due to the significant spectral overlapping among analytes and interference, the use of the more selective time-resolved fluorescence is proposed for qualitative identification in quality control of traditional Chinese medicine (TCM) for the first time. Using the strategy of combining EEM fluorescence with second-order calibration method, i.e. parallel factor analysis (PARAFAC), the simultaneous quantification of scopoletin and scopolin in the complex system of Erycibe obtusifolia Benth was achieved successfully. The predicted concentrations were compared with the values obtained using high performance liquid chromatography-coupled to fluorimetric detector (HPLC-FLD), and no significant differences between them were observed. Therefore, the proposed methods using time-resolved fluorescence for qualitative analysis and EEMs coupled with second-order calibration for quantitative analysis in TCM are comparable and provide a suitable alternative to the chromatography-based method.

Simultaneous determination of α-asarone and ß-asarone in Acorus tatarinowii using excitation-emission matrix fluorescence coupled with chemometrics methods.

A chemometrics-assisted excitation-emission matrix (EEM) fluorescence method was proposed for simultaneous determination of α-asarone and ß-asarone in Acorus tatarinowii. Using the strategy of combining EEM data with chemometrics methods, the simultaneous determination of α-asarone and ß-asarone in the complex Traditional Chinese medicine system was achieved successfully, even in the presence of unexpected interferents. The physical or chemical separation step was avoided due to the use of "mathematical separation". Six second-order calibration methods were used including parallel factor analysis (PARAFAC), alternating trilinear decomposition (ATLD), alternating penalty trilinear decomposition (APTLD), self-weighted alternating trilinear decomposition (SWATLD), the unfolded partial least-squares (U-PLS) and multidimensional partial least-squares (N-PLS) with residual bilinearization (RBL). In addition, HPLC method was developed to further validate the presented strategy. Consequently, for the validation samples, the analytical results obtained by six second-order calibration methods were almost accurate. But for the Acorus tatarinowii samples, the results indicated a slightly better predictive ability of N-PLS/RBL procedure over other methods.

[Fluorimetric analysis of camptothecin in Chinese herbal medicine common Camptotheca fruit].

Three-dimensional (3D) fluorescence spectra and thin layer fluorescence chromatogram of common Camptotheca fruit (CCF) crude drug, camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) have been studied, and a novel fluorimetric method for determination of CPT in CCF crude drug has been established. In 3D fluorescence spectra, CPT presented 3 fluorescence peaks with excitation wavelengths lambdaex of 215, 255 and 365 nm, separately, and all peaks with emission wavelength lambdaem of 430 nm. HCPT presented 4 fluorescence peaks with lambdaex of 220, 265, 325 and 375 nm, separately, and all peaks with lambdaem of 555 nm. The fluorescence of CPT is much stronger than that of HCPT. Comparison of 3D fluorescence spectra and analysis of thin layer fluorescence chromatogram revealed that the main fluorescent component of CCF is CPT. HCPT and other components basically do not interfere with the fluorescence of CPT. Under the condition of pH 3.0-6.7, CCF aqueous solution can produce strong and steady fluorescence. Using methanol as solvent, the extracting solution of CCF was prepared, and diluted properly with water, then measured fluorescence intensity at lambdaex/lambdaem = 365/430 nm to determine the content of CPT. A linear calibration curve covered the concentration range 0.002 35-0.235 microg x mL(-1). The regression equation was IF = 9 + 30,844 c, with correlation coefficient r = 0.999 (n=9). The method has been applied to the analysis of CPT in a CCF sample, with a result of 0.127% and a spiked recovery rate of 102%. The reliability of the method has been verified by a thin layer chromatography-fluorescence scanning method. Experimental results demonstrated that this method can be used for quality evaluation of CCF crude drug.

[Fluorescence spectra and absorption spectra of carvacrol].

Fluorescence spectra and absorption spectra of carvacrol, an active component of Chinese herbal medicines, have been studied. The ionization constant and fluorescence quantum yield of carvacrol were measured according to spectral data. Under the condition of pH < 2.0, fluorescence intensity of carvacrol increases with the increase in pH value. In the range of pH 2.0-8.0, carvacrol gives a strong and steady fluorescence with maximum excitation wavelength 278 nm and emission wavelength 306 nm. When pH > 8.0, the fluorescence intensity decreases with the increase in pH value. Ionization constant of carvacrol was measured to be pK(a) = 10.44 +/- 0.06 using a pH-absorbance method; and pK(a) = 10.40 +/- 0.04 using a pH-fluorescence method. Fluorescence intensity of carvacrol was remarkably enhanced when methanol was added into its aqueous solution. Using L-tryptophane as a reference, the fluorescence quantum yield of carvacrol aqueous solution was measured to be 0.121 at excitation wavelength 278 nm; while in a solution containing 80% methanol, the quantum yield was measured to be 0.324.

[Fluorescence spectra of Bletilla striata aqueous extraction].

Three-dimensional fluorescence spectrum and ultraviolet absorption spectrum of Bletilla striata extraction drawn by boiling water were reported. Three fluorescence peaks, located at lambda(ex)/lambda(em) = 227/299 nm, 271/299 nm and 258/ 389 nm respectively, were observed in the three-dimensional fluorescence spectrum. Among them, the two former peaks having the same emission wavelengths were produced by one fluorescent component, while the latter peak was produced by another. Three-dimensional fluorescence spectrum is a characteristic mark of Bletilla striata, therefore it can be used in qualitative identification. The distance between emission wavelengths of the two components was 90 nm, so the fluorescence intensity of two components could be measured separately without pre-separation. For dilute solutions of Bletilla striata aqueous extraction, good linear relationships between fluorescence intensity and concentration of two components were obtained, thereby the quantitative determination of the two components may be carried out. In the range of pH 1.5 to pH 12.5, the fluorescence intensity of the two components changes with the increase in pH, indicating dissociable protons exist in the molecular structure of the two components.

[Fluorescence spectra of Dichroa febrifuga aqueous extraction].

Fluorescence spectra of chang shan (Dichroa febrifuga Lour) aqueous extraction were studied. In the three-dimensional fluorescence contour spectrum, three fluorescence peaks of quinazoline alkaloids, which are the active components of chang shan, were observed. The excitation wavelengths of the peaks were 235, 270 and 320 nm, respectively, and the emission wavelength of all the peaks was 430 nm. Three-dimensional fluorescence contour spectrum is the very image of fingerprint, suitable for qualitative identification of traditional Chinese medicine. In the range of pH 3 to pH 6, the fluorescence spectrum of chang shan aqueous extractions changes with the variation in pH value. The reason for this spectral change might be the protonation of N-1 in quinazolone ring of beta-dichroine (febrifugine) molecule. There is an excellent linear relationship between the fluorescence intensity and the concentration of chang shan under nearly neutral conditions, thereby a quantitative method for the determination of quinazoline alkaloids may be established.