Time-resolved And Chemometrics-assisted Fluorescence For Qualitative Identification And Quantitative Analysis Of The Active Constituents In Traditional Chinese Medicines

Traditional Chinese medicine(TCM)plays an essential role in preventing and healing human diseases in several Asian countries and has earned more and more reputation all over the world due to its good therapeutic performance and low toxicity,especially for treating chronic diseases.Usually,most TCMs comprise complex mixtures of different phytochemical constituents but only a few components are bioactive.The contents of the bioactive components of herbal medicines are highly variable depending on geographical origins,climate,cultivation,and the growth stage when harvested,thereby,altering its therapeutic effects.Consequently,it is essential to identify and measure the bioactive constituents of medicinal plants to ensure the reliability and repeatability of clinical research.Up to now,there have several analytical methods for the qualitative and quantitative analysis of the analytes in TCM samples,mainly including LC–MS.Although the analytical performance of these methods is widely accepted,the analysis of targets tends to be laborious,relatively expensive and time-consuming.For these reasons,the development of simpler and greener methods,i.e.without separations and minimizing the use of organic solvents,is of great interest,for the determination of the target compounds.Fluorescence spectroscopy is already known to be very useful for developing greener methodologies.The main advantages of molecular fluorescence spectroscopyare its sensitivity and selectivity,in addition to its ease of use,instrumental versatility,speed of analysis and its non-destructive character.Nevertheless,natural medicines are multi-componentsystems with mostly unknown components,serious spectra overlap will also occur between the analytes of interest and potential interferents.These spectra overlap will lead to heavy collinearity of the measurement data.One approach to improve the analytical selectivity in the complex samples would be the use of fluorescence excitation–emission measurements(EEMs),in conjunction with different chemometric algorithms to build a second-order calibration.These methods permit the determination of the compounds of interest,without the use of chromatography,in a sample with overlapping spectral interferences that are not included in the calibration set(known as the second-order advantage).The alternative method to measure fluorescence,time-resolved fluorescence decay patterns are additional effective means of fluorophore separation,as spectrally overlapping signals can often be segregated by distinct fluorescencelifetimes.The use of time-resolved fluorescence techniques could assist develop a non-destructive methodology to determine chemical composition.So far,qualitative identification of the active components in traditional Chinese medicines by separating fluorescence lifetimes has not been investigated yet.The aim of this work is to developanew,fast and reliable method based on time-resolved(lifetimes)fluorescence and excitation–emission matrix fluorescence combined with second-order algorithms,to identify and determinethe active components of herbal medicines.Chapter 1.OverviewThis overview covers current chemometric methodologies using second-order advantage to solve problems of analyzing highly complex matrices.Among the existing algorithms,it focuses on those most frequently used e.g.,the standard for second-order approaches to data analysis,PARAFAC(parallel factor analysis),and MCR-ALS(multivariate curve resolution alternating least squares),as well as the most recently implemented ATLD(alternating trilinear decomposition)and its variants,and U-PLS/RBL and N-PLS/RBL(unfolded partial least-squares and multi-dimensional partial least-squares,both combined with residual bilinearization).A series of examples is discussed in detail.Emphasis is directed toward the most popular multi-way data,i.e.,second-order or matrix data of EEMs and time-resolved emission spectra(TRES).Chapter 2.A comparison of several second-order algorithms for simultaneous determination of neomangiferin and mangiferin with severe spectral overlapping in Anemarrhenae RhizomaThis work presents a greener approach for simultaneous determination of neomangiferin and mangiferin,the major bioactive constituents with severe spectral overlapping in Anemarrhenae Rhizoma,combining the sensitivity of molecular fluorescence and the selectivity of chemometric multivariate calibration algorithms.In this study,we compared the analytical performance of two group chemometric algorithms including PARAFAC,ATLD,SWATLD(self-weighted alternating trilinear decomposition)and APTLD(alternating penalized trilinear decomposition),and PLS-based methods such as U-PLS/RBL and N-PLS/RBL.The statistical parameters for the validation set of the second-order calibration were evaluated through the relative error of prediction(REP%),the average recovery(Rec%),and the root mean square error ofprediction(RMSEP).Prediction results for the validation set by trilinear algorithms showed that the values were satisfactory for neomangiferin,and higher and not acceptable values for mangiferin,while U-PLS and N-PLS predictions were very successful for two analytes.Therefore,U-and N-PLS/RBL were chosen to determine neomangiferin and mangiferin in more complex real samples simultaneously,and U-PLS/RBL algorithm showed the best performance.The predicted concentrations by proposed methods were satisfactorily compared with those obtained using high performance liquid chromatography with ultraviolet detection.Chapter 3.Chemometrics-assisted excitation–emission fluorescence for simultaneous determination of esculin,esculetin and fraxin in Cortex FraxiniA novel method for the direct determination,i.e.,without chromatographic separation,of esculin,esculetin and fraxin with strong spectral overlapping in Cortex Fraxini samples has been developed on the basis of the collection of EEM and data processing with second-order chemometric methods.Excitation–emission matrices of the calibration set,validation set and ten different Cortex Fraxini samples were obtained for the analysis,recording emission between 420 and 550 nm,exciting between 300 and 410 nm.We compared the analytical performance of two groups of chemometric algorithms including trilinear algorithms(PARAFAC,ATLD)and PLS-based methods(U-PLS/RBL,N-PLS/RBL).The statistical parameters for the validation set of the second calibration were evaluated through the quality of the recovered spectral profiles,expressed by the correlation coefficients(Rem and Rex)in the emission and excitation modes with the related reference spectra,the correlation coefficients between predicted concentrations and nominal values,the relative error of prediction(REP%),the average recovery(Rec%),and the root mean square error ofprediction(RMSEP).It is important to note that,being resolution methods,two trilinear algorithms could achieve the correct spectral resolution of three compounds present in the validation samples,without any a priori information given to the analysis.The results demonstrate that U-and N-PLS/RBL yield the optimal results with relative error of prediction(REP%)less than or equal to 3.2% for esculin and esculetin,and 5.5% for fraxin in the validation set.This fact can be justified by the use of latent variables,which are flexible enough to overcome the problem of the high degree of spectral similarity among the analytes.Therefore,U-PLS/RBL was chosen to determine the analytes in more complex real samples,and this methodology was validated by an optimized liquid chromatography coupled to UV detector.The simplicity and the environmentally friendly nature of the experimental procedure,associated with the excellent analytical figures of merit,provide a valuable alternative for the quantification of the active components in herbal medicines.Chapter 4.Time-resolved fluorescence and chemometrics-assisted time-resolved emission spectra(TRES)for qualitative identification and quantitative analysis of the bioactive constituents in TCMsThis paper presents a new strategy for qualitative identification of scopoletin and scopolin in Erycibe obtusifolia Benth using time-resolved(lifetimes)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.A FLS-980 fluorescence spectrometer(Edinburgh)was used to record the time resolved fluorescence.Measurements of the fluorescence lifetimes and time-resolved emission spectra(TRES)were performed with standard time-correlated single-photon counting(TCSPC).The decay profile intensity was fitted to a one,two,three and four exponential decay using deconvolution with the instrument response function via the software FLS-980 provided by Edinburgh.The reliability of fitting was checked by numerical value of reduced chi-square(?2),and also by visual inspection of residual distribution in the whole fitting range.All data of both pure analytes were well fit by single exponential decay.The measured lifetimes are 4.91±0.03 ns for scopoletin and 2.14±0.03 ns for scopolin.The fluorescence lifetimes of the analytes showed no dependence on the emission wavelength and sample concentration.The time-resolved fluorescence decay of Erycibe obtusifolia Benth extract solution was obtained,which was excited at 358.2 nm and the fluorescence intensity decay registered at 430 nm,where both species emit.The values of the reduced-?2 for one lifetime decay was 9.55,for two lifetimes decay was 1.22,and for three lifetimes decay was 1.19.We decided to use two lifetimes(two exponential decay fitting)because it presented an even residual distribution.The values of the lifetimes resolved by two exponential decay fitting function were 4.88±0.04 and 2.17±0.04 ns respectively,which were well consistent with their actual ones of scopoletin and scopolin,implying that the qualitative discrimination of chemical composition in TCM was achieved successfully.When using a three component fitting,the results showed that in addition to two analytes,another component with a longer lifetime around 9.1±2.6 ns(but lower signal,about 2% of total intensity)existed in the TCM matrix,which resulted in the slight decrease of ?2.Using the strategy of combining TRES with second-order calibration methods including PARAFAC,APTLD and SWATLD,the quantification of scopoletin in the complex systems of Feng Liao Xing Yao Jiu and Feng Tong Yao Jiu was achieved successfully.The predicted concentrations were compared with the values obtained using high performance liquid chromatography-coupled to UV detector,and no significant differences between them were observed.Therefore,the proposed methods using time-resolved fluorescence for qualitative analysis,and TRES coupled with second-order calibration for quantitative analysis in TCMs are comparable and provide a suitable alternative to the chromatography-based method.