Application of prediction intervals to the interpretation of the robustness study of a UHPLC method for the separation of cannabinoids.

Design of Experiments (DoE) is a well-established tool used for analytical methods robustness studies, because of its ability to assess the effect of a great number of factors in a minimal number of experiments. However, when assessing the robustness of an analytical method the analysis of the individual effect of each factor is not sufficient on its own. Some factors may not influence the robustness of the method, but their effect combined with the effects of other factors may have a significant contribution on the robustness of the method, which is not given by conventional analysis of DoE results. The aim of this work is to propose, in addition to the analysis of the individual effects of the factors, to estimate the joint effect of the factors by means of the matrix experimental results prediction interval. This prediction interval is the interval in which, with a given probability, should fall the next results, therefore it is an interesting tool to estimate the variation limits of the method results during routine use. We also propose the use of two other prediction intervals which can help to analyze the DoE results and give a conclusion on the method robustness. The first one is based on the DoE experimental error information, and it gives an estimation of the experimental error component impact on the factors joint effect. The second one is based on the factors non-significance limits, and it provides the information regarding the factors impact on the responses in the case where the conditions are, by definition, robust. We applied these proposals to the robustness study of a UHPLC method for the separation of phytocannabinoids and we could demonstrate that, in addition to the calculated effects values and robustness information, the use of the prediction intervals information provided additional information that allowed a better interpretation of the method performance parameters.

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis.

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides. Average tailing factor were quite comparable across all chromatographic modes, while the best peak capacity values were obtained under mixed-mode LC conditions. Selectivity for each chromatographic mode was also evaluated for six closely related peptides having minor modifications on their structures. The LC-based chromatographic modes confirmed their superior selectivity over UHPSFC. By contrast, when analyzing short peptides (di- or tripetides), UHPSFC was the only technique allowing to simultaneously separate highly polar and less polar peptides within the same run confirming its unique versatility. In addition, the sensitivity of each chromatographic approach was accessed by for two representative peptides by both UV and MS detection. With UV detection, limit of detection (LOD) values were comparable among the different chromatographic modes, ranging from 0.5 to 2 µg mL-1. However, major differences were found when employing MS detection (LOD values ranged from 0.05 to 5 µg mL-1). The best results were obtained under HILIC conditions, followed by SFC, and finally mixed-mode LC and RPLC modes.

New perspective for the in-field analysis of cannabis samples using handheld near-infrared spectroscopy: A case study focusing on the determination of Δ9-tetrahydrocannabinol.

The aim of the present study was to explore the feasibility of applying near-infrared (NIR) spectroscopy for the quantitative analysis of Δ9-tetrahydrocannabinol (THC) in cannabis products using handheld devices. A preliminary study was conducted on different physical forms (entire, ground and sieved) of cannabis inflorescences in order to evaluate the impact of sample homogeneity on THC content predictions. Since entire cannabis inflorescences represent the most common types of samples found in both the pharmaceutical and illicit markets, they have been considered priority analytical targets. Two handheld NIR spectrophotometers (a low-cost device and a mid-cost device) were used to perform the analyses and their predictive performance was compared. Six partial least square (PLS) models based on reference data obtained by UHPLC-UV were built. The importance of the technical features of the spectrophotometer for quantitative applications was highlighted. The mid-cost system outperformed the low-cost system in terms of predictive performance, especially when analyzing entire cannabis inflorescences. In contrast, for the more homogeneous forms, the results were comparable. The mid-cost system was selected as the best-suited spectrophotometer for this application. The number of cannabis inflorescence samples was augmented with new real samples, and a chemometric model based on machine learning ensemble algorithms was developed to predict the concentration of THC in those samples. Good predictive performance was obtained with a root mean squared error of prediction of 1.75 % (w/w). The Bland-Altman method was then used to compare the NIR predictions to the quantitative results obtained by UHPLC-UV and to evaluate the degree of accordance between the two analytical techniques. Each result fell within the established limits of agreement, demonstrating the feasibility of this chemometric model for analytical purposes. Finally, resin samples were investigated by both NIR devices. Two PLS models were built by using a sample set of 45 samples. When the analytical performances were compared, the mid-cost spectrophotometer significantly outperformed the low-cost device for prediction accuracy and reproducibility.

The analysis of cannabinoids in cannabis samples by supercritical fluid chromatography and ultra-high-performance liquid chromatography: A comparison study.

The aim of this work was to develop a supercritical fluid chromatographic method to study the applicability of this emerging technique to cannabinoid analysis and showcase its advantages. During method development, the authors focused on nine phyto-cannabinoids to assess the selectivity needed to potentially perform the quantitation of each cannabinoid. After method development, robustness studies were carried out on this method to gain more information about its qualitative behavior (in terms of critical resolutions) when varying some crucial parameters (concentration of additive, column temperature, starting gradient conditions and column batch). Once the robustness was evaluated and the parameters most affecting the selected responses were individuated, the SFC method was applied for a simulated routine use to generate quantitative results concerning the concentrations of the main cannabinoids in real cannabis samples. The samples were also analyzed by means of an ultra-high-performance liquid chromatographic method currently used in the laboratory for the same objective. Finally, the results obtained with both analytical methods were compared to evaluate their accordance. The Bland-Altman method was applied as a statistical strategy to evaluate the degree of accordance between the results generated and display the data in a difference plot. The ultra-high performance supercritical fluid chromatography quantitative results were in accordance with the ultra-high performance liquid chromatography results, demonstrating the applicability of this technique for cannabinoid analysis.

Implementation of a generic liquid chromatographic method development workflow: Application to the analysis of phytocannabinoids and Cannabis sativa extracts.

A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativa extracts. Our method development procedure consists in four steps: At the end, this method development workflow was performed in less than 4 days and minimizes the costs of the method development in liquid chromatography.