Practical and methodological issues related to high accuracy qNMR measurements in food

Practical and methodological issues related to high accuracy qNMR measurements in food

 

^*Bruno Carius Garrido¹, Lucas J. de Carvalho²

¹ Biotoxin Metrology, National Research Council, Halifax, Nova Scotia, Canada

*bruno.garrido@nrc-cnrc.gc.ca

² Organic Analysis Laboratory, Instituto Nacional de Metrologia, Qualidade e Tecnologia – Inmetro, Duque de Caxias, RJ, Brazil

 

Despite the growing interest in qNMR, many of its analytical aspects remain only partially studied or not studied at all. Metrological traceability of the results and calibration methods are often disregarded and some acquisition methods and pulse sequences such as solvent suppression are used indiscriminately without a deep understanding of its implications on the measurement results. The goal of this work is to discuss the use of the Pulse Length Based Concentration Determination (PULCON) for external calibration as well as solvent suppression techniques in the quantification of organic compounds while promoting a full validation of the methods involved and the assessment of parameters that might influence the measurement results.

To assess the accuracy of the PULCON calibration and the factors influencing it, solutions of two different certified reference materials (CRM) produced by Inmetro, namely dimethylsulfone (DMSO₂) and dimethylterephtalate (DMTP), were prepared in CDCl₃ and analysed. DMTP was used as the reference compound for calibration while DMSO₂ was measured as the analyte. Different solutions were prepared in the 0.04 – 20.8 mg/g range to assess the effect of different concentrations and concentration pairs (analyte/internal standard) in the results. The effect of solvent suppression was assessed in two different groups of solutions: organic solvents and D₂O. Firstly, different reference solutions were prepared in DMSO-d₆, CD₃CN and CDCl₃. Several pulse sequences were evaluated including simple pre-saturation, noesy pre-saturation, WET and excitation sculpting. To assess the effect of solvent suppression in D₂O, solutions containing analytes with different chemical shifts were assessed (maleic acid, glucose, lactose, salbutamol and vanillin). A Bruker avance III HD spectrometer operating at 11.74 T was used in the study with three different probes: an inverse probe (BBI), a direct smart probe probe (BBFO) and an inverse prodigy cryoprobe (CPP).

We built an updated model for evaluating the measurement uncertainties of the PULCON method considering all the relevant components for matrix measurements and validated the PULCON method using Mate infusions quantified by GC-FID using a standard addition calibration curve. This method was successfully applied to several different materials in our laboratory such as Cannabis and milk (quantification of lactose). We show that if instrumental parameters are not properly taken into account with this method, measurement errors can be as high as 71.5 %. However, when done properly, the method is capable of achieving 0.12 % relative errors.

Regarding solvent suppression, we show the performance of the qNMR method using different pulse sequences for different solvents and matrices and demonstrate that analogously, the use of some pulse sequences for some quantifications can lead to errors of up to almost 50 % while under optimized conditions, these values can range from less than 1 % to about 5 %.

 

Acknowledgments: FAPERJ for the financial support.