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.

Identify low mass volatile organic compounds from cometary ice analogs using gas chromatography coupled to an Orbitrap mass spectrometer associated to electron and chemical ionizations.

Analysis of organic matter extracted from meteorites showed that solar system objects present an important molecular diversity. To improve our understanding of such organic matter, new analytical technologies must be developed. The present study displays the first experiments using a GC-FT-Orbitrap-MS to decipher the molecular diversity observed in experiments simulating the evolution of cometary ices. The proposed analytical strategy focuses on the analysis of 110 volatile organic compounds (VOC) with mainly 1 to 6 carbon atoms generated in such cometary ice analogs. Electron ionization (EI) and chemical ionization (CI) modes with methane (CH4) or ammonia (NH3) were optimized and compared. Those developments maximized the intensity of molecular, protonated or deprotonated ions, and improved high-resolution molecular formula unambiguous identification: EI mode is too energetic to provides there detection, while it is not the case in CI mode. Particularly, NH3 as a reagent gas improves amine identification in positive mode (PCI), and esters, alcohols, carbonyls, amides, carboxylic acids and nitriles in negative mode (NCI). The combination of both EI and CI mass spectrum analysis improves molecular identification, thanks to the detection of molecular, deprotonated or protonated ion of highest intensity and fragment formula assignments. The EI and NCI NH3 combination allows formula assignments up to 94% of our database with limit of detection up to 7 ppm. This procedure has been validated for untargeted GC-FT-Orbitrap-MS analysis of VOC coming from the processing of cometary ice analogs.

Application of prediction intervals to the interpretation of analytical methods robustness studies.

Since the emergence of the Analytical Quality by Design concept and the announcement of the future ICH Q14 "Analytical Procedure Development" guideline [1], a significant revival of the interest of the laboratories for the robustness study of their methods can be seen. Design of Experiments is a well-established tool used for this task, because of its ability to study the individual 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. Indeed, the role of this study is also to verify, regardless of the very significance of the factors, that the chosen variations of the operating parameters of the method do not modify the experimental variance measured under the operating conditions for which the parameters are strictly defined. We therefore propose, in addition to the analysis of the individuals effects of the factors, an original tool which makes it possible to verify that the studied factors, when they vary together, do not lead to a significant variation in the measured responses. This tool is based on the prediction interval of the matrix experimental results, for each of the studied responses. This concept of prediction interval is already used in the context of analytical methods accuracy study. In a robustness design of experiments, it can represent the interval in which should be found, with a given probability, the response value if complementary experiments are conducted. It therefore represents the variation induced simultaneously by the different factors and can be easily compared to a maximum acceptable deviation of the considered response. We will detail this proposal and illustrate it with an example from a robustness study carried out on a liquid chromatography assay method.

Biomimetic self-assembling acylphthalocyanines.

We synthesized a series of biomimetic self-assembling phthalocyanines equipped with carbonyl groups as recognition motifs, a central zinc atom and diverse solubilizing alkyl chains mimicking for the first time with these robust pigments the natural chlorosomal bacteriochlorophylls. Upon self-assembly a very broad and red-shifted Q-band absorption extending to over 900 nm is put into evidence.

DNA methylating and demethylating treatments modify phenotype and cell wall differentiation state in sugarbeet cell lines.

In plants organogenesis, cell differentiation and dedifferentiation are fundamental processes allowing high developmental plasticity. Such plasticity involved epigenetic mechanisms but limited knowledge is available concerning quantitative aspects. Three sugarbeet (Beta vulgaris L. altissima) cell lines originating from the same mother plant and exhibiting graduate states of morphogenesis were used to assess whether these differences could be related or not to changes in DNA methylation levels. Methylcytosine percentages from 18.3 to 28.8% and distinct levels of DNA methyltransferase (EC 2.1.1.37) activities were shown in the three cell lines. The lowest methylcytosine percentage was associated to organogenesis. In order to test the plasticity of these cell lines, various treatments causing DNA hypo or hypermethylation were performed at different times and concentrations. In this collection of treated lines with+/-10% of methylcytosine percentages, loss of organogenic properties and cell dedifferentiation were observed. As cell wall formation fits well with cell differentiation state, the lignification process was further investigated in treated and untreated lines as a biochemical marker of the phenotypic changes. For example, peroxidase specific activities (EC 1.11.1.7) varied from 0.7 to 0.02 pkat mg(-1) of protein in organogenic and dedifferentiated lines, respectively. A negative relationship between peroxidase activities, incorporation of cell wall-bound phenolic compounds as ferulate and sinapate derivatives and methylcytosine percentages was obtained. This is the first biochemical evidence that phenotypic changes in plant cells induced by DNA hypo- or hypermethylating treatments are correlated in a linear relationship to modifications of the cell wall differentiation state.

Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures.

Investigations have been made to develop an efficient protocol for micropropagation allowing to improve hypericin and pseudohypericin productions in Hypericum perforatum L. in vitro cultures. The role of growth regulator treatments has been particularly studied. Three in vitro culture lines with different morphological characteristics were obtained during H. perforatum micropropagation and referred to shoots, calli and plantlets according to their appearance. Multiplication and callogenesis from apical segments from sterile germinated seedlings were obtained on solid MS/B5 culture medium in the presence of N6-benzyladenine (BA) (0.1-5.0 mg/l BA). Regenerative potential of shoots was assessed on medium supplemented with auxins (0.05-1.0 mg/l), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The main goal of the research was to summarize the influence of plant growth regulators on hypericin and pseudohypericin productions in in vitro cultures of Hypericum. A rapid method for naphtodianthrone quantification was developed. The use of a reversed-phase high performance liquid chromatography (HPLC) method with fluorescence detection was used. Identification of the compounds was confirmed by electrospray ionization-mass spectrometry (ESI-MS) with electrospray in negative ion mode [M-H] . Calli, shoots and plantlets of H. perforatum produced hypericin and pseudohypericin. The concentration range of BA from 0.1 to 2.0 mg/l improved the production of hypericin (25-50 microg/g dry mass (DM)) and pseudohypericin (170-350 microg/g DM) in shoots. In callus cultures, BA (4.0-5.0 mg/l) did not changed hypericin contents (15-20 microg/g DM) but influenced pseudohypericin productions (120-180 microg/g DM). In the presence of auxins (IAA and IBA), Hypericum plantlets produced hypericin (30-100 microg/g DM) and pseudohypericin (120-400 microg/g DM). The presence of IAA did not influence naphtodianthrone productions in plantlets, but IBA decreased hypericin and pseudohypericin amounts in plantlets. The specific accumulation of the naphtodianthrones in in vitro cultures was influenced by phytohormonal supplementation of the medium. Results indicated that the production of hypericin and pseudohypericin could be increased by carefully adapted in vitro cultures. Hypericum in vitro cultures represent promising systems for hypericin and pseudohypericin productions.