Important issues in plant tissues analyses by HR‐MAS NMR

• Published in: Phytochemical analysis 2019-01, Vol.30 (1), p.5-13
• Main Authors: Flores, Igor S., Martinelli, Bruno C.B., Pinto, Vinicius S., Queiroz, Luiz H.K., Lião, Luciano M.
• Format: Article
• Language: English
• Subjects: analyses protocols; Animal tissues; Chemistry Techniques, Analytical - methods; Chemistry Techniques, Analytical - standards; Deuteration; Deuterium - chemistry; Eugenia - chemistry; Eugenia - metabolism; HR‐MAS NMR; Insertion; Magnetic permeability; Magnetic Resonance Spectroscopy - methods; Magnetic susceptibility; Malpighiaceae - chemistry; Malpighiaceae - metabolism; Metabolome; NMR; NMR spectroscopy; Nuclear magnetic resonance; Particle Size; Passiflora - chemistry; Passiflora - metabolism; phytochemical analysis; phytotherapeutic; Plant Extracts - chemistry; Plant Leaves - chemistry; Plant Leaves - metabolism; Plant tissues; Polarity; Powdering; Pretreatment; Reference Standards; Reproducibility; Reproducibility of Results; Sample preparation; Solvents; Solvents - chemistry; Spectroscopy; Spinning (materials); Swelling; Tissue analysis
• ISSN: 0958-0344; 1099-1565
• DOI: 10.1002/pca.2785

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. HR‐MAS NMR is a useful tool for plant tissues analysis, since it permits chemical insights directly of samples in their natural swollen state, avoiding extractions and purifications procedures, and thus drastically minimises the use of solvents. In this work, several issues on sample preparation and instrumental setup are examined, in order to assure reproducibility and reliable data are addressed. It was found that different aspects of sample preparation, rotor insertion procedure and spectra acquisition should be carefully observed to obtain reliable data by this methodology.