High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) as a tool in the determination of biomarkers of Passiflora-based herbal medicines.

High-resolution magic-angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) for the identification of the metabolic profile of plants under conditions very close to that in which the compounds are present in the matrix, the herbal medicines in this case. This enables selectivity in the determination of the active principle and other biomarkers present in the complex matrix, avoiding degradation products, which may occur in the extractive processes required in several analytical methods. In this study, HR-MAS analysis was applied in the quality control of seven Passiflora-based herbal medicines, using metabolic fingerprinting to confirm plant species and identify biomarkers. Vitexin and isovitexin were identified as major compounds in three of these herbal medicines (0.17 to 0.55%), while salicin was the majority in two others. On the other hand, no significant flavonoid contents were observed in the remaining two. In addition, it was possible to detect ethanol, a non-target compound, in all herbal medicines in concentrations varying between 0.009 and 0.342%. In this way, combined with chemometrics 1H HR-MAS NMR proved to be suitable for the qualitative and quantitative study of Passiflora biomarkers, using a minimal pre-treatment of the sample.

Important issues in plant tissues analyses by HR-MAS NMR.

INTRODUCTION: High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy enables the analysis of the metabolic profile of plant and animal tissues under close to natural conditions, as well as of other heterogeneous natural or synthetic materials. Neither sample pretreatment is required after fragmentation nor powdering of the sample before insertion into the rotor. However, the efficiency of the method depends strongly on the sample preparation, rotor insertion procedure, and analysis conditions. OBJECTIVE: To identify some of the variables that affect the spectral data and to propose solutions that minimise their impact on the quality of the analyses and results. METHODS: Dried plant tissues were powdered, weighed, and homogenised in a 50 µL rotor with an optimised volume of deuterated solvent and sample in order to prevent material from escaping during spacer insertion, avoiding variations in magnetic susceptibility. Factors affecting the quality of HR-MAS NMR analysis such as particle size, sample and solvent amounts, solvent polarity, swelling time, rotor manipulation and pulse sequence setting were evaluated. RESULTS: A strong correlation was observed between the signal area and the particle size of the powdered sample. The spectral profile varied depending on the deuterated solvent used. An incubation period was necessary to achieve adequate swelling of the sample and to ensure good data reproducibility. Proper sealing of the rotor, number of cycles and τ time on cpmgpr1d pulse sequence were found to affect the signal areas. CONCLUSION: The study highlights the need for standardised sample preparation and instrumental setup protocols in order to achieve high reproducibility and obtain reliable data from HR-MAS NMR analyses.