Method for evaluating biodegradability of complex organic mixture

Abstract

The present invention relates to a method for assessing the biodegradability of complex organic mixtures, such as pharmaceutical compositions, whereby it is possible to understand the orientation of complex products that cannot be assessed by currently available standard rapid biodegradability tests (RBT).

Description

Technical Field

[0001] The present invention relates to a method for evaluating the biodegradability of complex organic mixtures (such as pharmaceutical compositions), thereby understanding the fate of complex products that cannot be evaluated by currently available standard Ready Biodegradability Tests (RBTs). Background Art

[0002] Biodegradation is the process by which organic matter is broken down by microorganisms into its simplest natural building blocks (e.g., CO2, H2O, NH3), which can be integrated into natural biogeochemical cycles. Human and industrial activities have resulted in the emergence of a range of new polluting compounds, which have had many adverse effects when released into the environment, prompting the development of many programs to try to remove these compounds.

[0003] Evaluation of the biodegradability of chemicals is one of the main issues in environmental risk assessment. Biodegradability tests are designed to evaluate the chemical under bulk conditions when it is the only carbon source for microfauna to survive.

[0004] Rapid biodegradability tests (RBTs) are the basis of a comprehensive testing strategy for the biodegradation of pure substances. They are a series of tests (from n°301A to n°301F and n°310) proposed by the Organization for Economic Cooperation and Development (OECD). Microorganisms and the test substances are usually cultured in a pH 7 buffered medium (called "mineral medium") containing N, P and trace elements. The kinetics of biodegradation are monitored over a period of at least 28 days by evaluating metabolic parameters such as oxygen consumption, carbon dioxide production or dissolved organic carbon consumption. RBT measures ultimate biodegradability or complete biodegradability, and if a chemical passes one of the RBTs, it can be classified as readily biodegradable (OECD, Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD, 2006).

[0005] Specific chemical analyses can be used to assess the primary biodegradation of the test substance and to determine the concentration of any newly formed intermediates.

[0006] This additional evaluation is mandatory only in the MITI method (301C), but it is optional for all other RBTs (OECD, Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD, 2006, OECD, Test No. 301: Ready Biodegradability, 1992.; OECD, Test No. 310 Ready Biodegradability-CO2 in sealed vessels (Headspace Test), 2014.).

[0007] The term primary biodegradation refers to changes in the structure of a substance caused by biological events, resulting in the loss of specific properties of the substance.

[0008] This can be calculated based on supplementary chemical analysis of the parent compound carried out at the beginning and end of the test (OECD 301, 310) (REGULATION (EC) No 440 / 2008 of 30 May 2008).

[0009] Substances that are not readily biodegradable are considered persistent unless their environmental degradability is demonstrated in more expensive and complex simulation tests (test methods n° 303, 306, 307, 308 and 309, OECD, 1992c, d, f, g, h). The type of simulation test to be performed depends on the potential receptor environment of concern (wastewater treatment plants, surface water, sediments, soil). Simulation tests are designed to evaluate the long-term chemical behavior in the environment. However, these tests are limited to very few pure substances because they are costly, technically complex and time-consuming (e.g. the use of several different tests and the use of 14C-labeled chemicals) (N. Nyholm, A. et al comparative study of test methods for assessment of the biodegradability of chemicals in seawater—Screening tests and simulation tests, Ecotoxicol. Environ. Saf., 1992, 23, 173190; JFEricson, Evaluation of the OECD 314BActivated Sludge Die-Away Test for Assessing the Biodegradation ofPharmaceuticals, Environ. Sci. Technol., 2010, 44, 375-381; F. Brillet, A. Maul, MJ Durand and T. Gérald, From laboratory to environmental conditions: a new approach for chemical's biodegradability assessment, Environ. Sci. Pollut. Res. 2016, 23, 18684-18693).

[0010] An in silico model has been developed (F. Pizzo, A. Lombardo, A. Manganaro and E. Benfenati, In silico models for predicting ready biodegradability under REACH: a comparative study, Sci. Total Environ., 2013, 463-464, 161–168) and is available under REACH.

[0011] However, predictions of overall persistence using multimedia fate models remain limited to available data and may not be appropriate for assessing environmental persistence as a function of chemical intrinsic properties and environmental conditions.

[0012] Although currently used biodegradability tests are cheap and easy to perform, they also have some limitations. For example, they are conducted under standardized conditions and cannot reflect highly variable environmental conditions (such as seasonality). In addition, even if risk assessment can be performed through conservative models, the concentrations of most chemicals tested are unlikely to occur in the environment.

[0013] There are additional criticisms of the microbial inoculum used in biodegradation testing. Prior to use, the inoculum source needs to be cleaned (to limit carbon contamination other than that of the test substance) or acclimatized (e.g. for 301C testing).

[0014] Furthermore, the final output may be affected by the total cell density, species diversity, the origin and history of the inoculum sample, the ratio between food and biomass, and the duration of the evaluation period (which has been limited to 28 days in standard experiments).

[0015] Another limitation of these tests is that they usually do not take into account the consequences of biodegradation, assuming complete degradation of the investigated molecule to its elemental bricks. Today, the development of modern reliable analytical techniques makes it possible to study the biodegradability of substances (or mixtures of substances such as in pharmaceutical, nutraceutical or cosmetic preparations) in a more accurate way, both at a qualitative and quantitative level, thus providing interesting and unprecedented complementary analyses to the primary biodegradation studies currently used.

[0016] We believe that the use of new technologies can enable a more accurate and realistic assessment of environmental risks and impacts, with a view to more sustainable development.

[0017] In addition, rapid biodegradability tests and simulation tests are usually for pure chemicals, and only a few cases of mixtures composed of structurally similar chemicals have been reported. An example is petroleum and surfactants. It is reported that the kinetics of biodegradation of the observed mixture is very different from the biodegradation kinetics of the isolated chemical entity, which is generally faster than the biodegradation kinetics of the mixture (European Chemicals Agency, 2017, Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7b: Endpoint specific guidance Draft Version 4.0 January 2017).

[0018] Clearly, moving away from reductionist analyses of single molecule biodegradation towards holistic analyses that consider the biodegradability of pools of chemical constituents could represent a powerful innovation with the potential to truly improve new reliable and sustainable designs. Summary of the invention

[0019] The authors of the present invention have performed RBT tests on complex mixtures of organic compounds and have observed that the biodegradability of a mixture of compounds cannot be considered as a simple sum of the biodegradability of each component. Furthermore, the authors have observed that currently available RBT techniques neither provide information on the type of degradation experienced by a complex mixture nor provide detailed information on the environmental impact of a complex mixture of organic compounds, including a pharmaceutical composition or a cosmetic composition or a food supplement composition or a medical device composition or a nutraceutical composition.

[0020] The authors of the present invention compared the biodegradation of compositions with similar medical effects but different components, such as two cough syrups and two compositions for reflux disease and functional dyspepsia.

[0021] The authors have demonstrated that, although the results provided by RBT indicate that the biodegradation of the cough syrups is substantially additive (see Figures 1 and 2 and Table 2 Experimental Section), a more detailed analysis using the methods provided in the specification and claims was able to demonstrate that the degradation products differed greatly between the syrups tested. Furthermore, testing of the gastric composition showed that the methods provided by the present invention provided more relevant information about the overall degradation of the tested composition.

[0022] The standard RBT provides no information about the mineralization of the tested compositions.

[0023] The authors of the present invention have developed a method which provides more relevant information on the mineralization of the tested mixtures and thus provides important information on the degradation products of said mixtures.

[0024] The method of the present invention comprises the RBT standard steps and additional steps, allowing the user to assess the degree of similarity between the degraded mixture and the environment (blank) in which the degradation is tested without the mixture. The more similar the composition is to the blank, the higher the degree of mineralization of the organic compounds of the mixture. Therefore, the method of the present invention provides relevant information about the degree of mineralization of the organic compound mixture.

[0025] Therefore, the object of the present invention is a method for evaluating the biodegradability of a mixture of organic compounds, comprising the following steps:

[0026] a) preparing at least one test flask or container containing a predetermined amount of a mixture of organic compounds of interest suspended in a suitable mineral medium and an inoculum, and at least one blank flask or container containing only said mineral medium and said inoculum, wherein said inoculum is obtained from activated sludge, effluent (non-chlorinated), surface water and soil, or a mixture thereof;

[0027] b) collecting a fingerprint of a sample from each flask or container prepared in a) by liquid chromatography-mass spectrometry at T0, T0 being the day of preparation in a), wherein each sample is filtered before collecting said fingerprint to eliminate microfauna therein;

[0028] c) collecting a blot of a sample from each test flask or container prepared in a) by liquid chromatography-mass spectrometry at Tn, where n is any integer greater than 0 and represents the number of days after said preparation in a), wherein each sample is filtered to eliminate microfauna therefrom before collecting said blot;

[0029] d) perform multivariate statistical analysis on the data obtained in b) and c);

[0030] e) comparing the multivariate statistical analysis results of each test sample and blank sample obtained in d), and evaluating the biodegradation of each test sample by evaluating the distance between the blank sample data obtained at Tn and the test sample data obtained at least at T0 and Tn.

[0031] Glossary

[0032] The term "mineral medium" has the meaning commonly used in the RBT standard test in the art (for example, as described in OECD, Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD, 2006; OECD, Test No. 311: Anaerobic Biodegradability of Organic Compounds in Digested Sludge: by Measurement of Gas Production, OECD, 2006; OECD, Test No. 310: Ready Biodegradability-CO2 in sealed vessels (Headspace Test), OECD, 2014; OECD, Test No. 301: Ready Biodegradability, 1992).

[0033] The term "mineralization" has the meaning commonly used in the art and refers to the decomposition of compounds in organic matter by which nutrients in these compounds are released in soluble inorganic form (minerals).

[0034] The pooled sample according to the present invention has a commonly used meaning in the art, and defines a quality control sample containing an equal amount of each test sample (ie, excluding a blank sample).

[0035] According to this specification, T0 is the day when the test starts, that is, the day when the preparation a) is performed.

[0036] According to the present specification, Tn is any day after T0, so T1 is the first day after the day the preparation described in a) is completed, T28 is the 28th day after the day the preparation described in a) is completed, and so on.

[0037] According to the present specification, inoculum generally refers to microfauna collected from activated sludge, effluent (unchlorinated), surface water and soil or from a mixture thereof. The microfauna is generally mainly composed of cercozoa, metazoans, ciliated protozoa, amoebas; tardigrades and filamentous bacteria may sometimes appear. Generally speaking, the inoculum contains any species commonly found in microfauna communities in activated sludge, effluent (unchlorinated), surface water and soil.

[0038] According to the present specification, a test flask or container is a flask or container containing a mixture of organic components of interest, a suitable mineral medium and an inoculum, wherein the inoculum is obtained from activated sludge, effluent (non-chlorinated), surface water and soil, or a mixture thereof.

[0039] According to the present specification, a blank flask or container is a flask or container containing a suitable mineral medium and an inoculum obtained from activated sludge, effluent (non-chlorinated), surface water and soil, or a mixture thereof.

[0040] The terms targeted method, suspect screening and untargeted screening (or analysis) are used in this specification.

[0041] Three related methodological approaches can be used to stratify the chemical burden in samples from different sources, depending on the level of prior knowledge related to the compounds of interest under consideration, namely (i) targeted approaches for known compounds, (ii) suspect screening for known unknowns, and (iii) untargeted screening for unknown unknowns.

[0042] Suspects may be further "converted" into targets by collecting comprehensive mass spectral reference data that can unambiguously identify the suspect compounds (usually relying on the availability of reference standard compounds). The remaining signals in the sample are often referred to as "non-target" or "unknown unknown" signals, which cannot be easily identified and require further structural elucidation.

[0043] By definition, "targets" are compounds of known chemical name and structure for which quantitative targeted methods are available and for which guidelines already exist to harmonize method performance assessments (e.g. Commission Decision 2002 / 657 / EU on food). Targeted screening can also be performed using high-resolution instrumentation (e.g. quadrupole time-of-flight - qToF), which opens the door to simultaneous targeted, suspect and untargeted analysis.

[0044] A "suspect" is a compound known by chemical name and structure, which is expected ("suspected") to be present in the sample. The typical approach applied in this context is suspect screening, which aims to generate semi-quantitative data and helps to better elucidate the composition of complex mixtures by simultaneously generating data for multiple compounds in each individual sample. In most cases, analytical standards are not readily available, so the associated analytical methods are not validated and the identity of the compounds is uncertain.

[0045] The qualitative annotation step refers to the assignment of given compound identities to signals detected by the suspect method and relies on the existence of a compound list or the elaboration and implementation of a reference library to match the generated experimental data with structural descriptors indexed from a defined compound list.

[0046] Finally, “untargeted” analysis attempts to detect “unknown unknowns” compounds without any a priori criteria. In general, sample preparation and data acquisition are similar between suspect screening and untargeted analysis, while data analysis and data mining are different. Although these approaches are extremely challenging, they represent the most promising strategy to advance our knowledge of complex mixtures. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1. Biodegradation of different concentrations of mixtures A and B (panels A and B, respectively). All data are reported as the average of two or three replicates.

[0048] Figure 2. PCA models for 100 mg / L samples A and B (panels A and B, respectively).

[0049] Figure 3. PCA models for 1000 mg / L samples of Mixture A and B (panels A and B, respectively).

[0050] Figure 4. Clustering analysis of 100 mg / L mixture A and B samples (panels A and B, respectively).

[0051] Figure 5. Cluster analysis of 1000 mg / L Mixture A and B samples (panels A and B, respectively).

[0052] Figure 6 . Schematic diagram of the method described in the present invention.

[0053] Figure 7. PCA model for the ready biodegradability test OECD 301F mixtures C (50 mg / L) and D (75 mg / L) samples (panels A and B, respectively).

[0054] Figure 8. Cluster analysis of samples of mixtures C (50 mg / L) and D (75 mg / L) in the ready biodegradability test OECD 301F (panels A and B, respectively). DETAILED DESCRIPTION

[0055] The present invention provides for the first time a method for evaluating the biodegradability of complex mixtures of organic compounds.

[0056] The impact of pharmaceutical compositions in the form of complex mixtures of organic compounds (i.e. pharmaceutical compositions, for example, comprising plant extracts as active ingredients and / or comprising a plurality (wherein the number is at least two, but preferably at least 10, at least 100, at least 1000) of organic compounds) on the environment is increasingly relevant, whereas there is currently no method or standardized protocol for assessing the biodegradability of complex mixtures of organic compounds (also in the form of pharmaceutical compositions).

[0057] The currently available method, OECD Rapid Biodegradability Test (RBT) (OECD, Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD, 2006; OECD, Test No. 311: Anaerobic Biodegradability of Organic Compounds in Digested Sludge: by Measurement of Gas Production, OECD, 2006; OECD, Test No. 310: Ready Biodegradability-CO2 in sealed vessels (Headspace Test), OECD, 2014; OECD, Test No. 301: Ready Biodegradability, 1992.) is not suitable for evaluating the biodegradability of complex mixtures of organic compounds (e.g., pharmaceutical compositions, cosmetic compositions, etc.), nor is it suitable for providing information on the mineralization level of the mixture in a standard biodegradability assay.

[0058] The authors of the present invention analyzed four different compounds by RBT OECD N°301F (OECD, Test No.301: Ready Biodegradability, 1992; OECD, "Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD" 2006), and the results showed that using the method no information about the degradation output of the composition can be obtained. Therefore, the authors developed a method that allows technicians to easily assess the mineralization level of the analyzed mixture.

[0059] The products tested were mixtures A, B, C and D, the compositions of which are summarized in Table 1 in the examples below.

[0060] The object of the present invention is a method for evaluating the biodegradability of a mixture of organic compounds comprising the following steps:

[0061] a) preparing at least one test flask or container containing a predetermined amount of a mixture of organic compounds of interest suspended in a suitable mineral medium and an inoculum, and at least one blank flask or container containing only said mineral medium and said inoculum, wherein said inoculum is obtained from activated sludge, effluent (non-chlorinated), surface water and soil, or a mixture thereof;

[0062] b) collecting a blot of a sample from each flask or container prepared in a) by liquid chromatography-mass spectrometry at T0, T0 being the day of said preparation a), wherein each sample is filtered before collecting said blot to eliminate microfauna therein;

[0063] c) collecting a blot of a sample from each test flask or container prepared in a) by liquid chromatography-mass spectrometry at Tn, where n is any integer greater than 0 and represents the number of days after said preparation in a), wherein each sample is filtered to eliminate microfauna therefrom before collecting said blot;

[0064] d) perform multivariate statistical analysis on the data obtained in b) and c);

[0065] e) comparing the multivariate statistical analysis results of each test sample and blank sample obtained in d), and evaluating the biodegradation of each test sample by evaluating the distance between the blank sample data obtained at Tn and the test sample data obtained at least at T0 and Tn.

[0066] Furthermore, according to the present invention, step c) can be repeated at different Tn, and steps d) and e) are performed for each blot of the test sample and blank sample collected at the same Tn for each value of n.

[0067] According to the present invention, non-limiting examples of organic compound mixtures are represented by pharmaceutical compositions or cosmetic compositions or food supplement compositions or medical device compositions or nutraceutical compositions. According to one embodiment of the present invention, the organic compound mixture comprises at least one plant extract or at least a portion of a plant extract.

[0068] According to the present invention, the mineral medium is a medium described in the standard RBT test in the art, usually prepared from stock solutions of the mineral components potassium phosphate and sodium phosphate plus ammonium chloride, calcium chloride, magnesium sulfate and iron (III) chloride at appropriate concentrations.

[0069] Various stock solutions can be used, and can be appropriately mixed and diluted to prepare a suitable culture medium. Those skilled in the art can refer to any OECD RBT protocol, such as OECD, Test No. 301: Ready Biodegradability, 1992 and the Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD, 2006.

[0070] Various mineral media for bacterial growth are known in the art and are suitable for use in the practice of the present invention.Depending on the inoculum, the medium may be a basal minimal medium or a supplemented minimal medium (eg containing growth factors, etc.).

[0071] In any case, as in the RBT test, the only source of organic carbon in the culture medium of the methods of the present invention is provided by the mixture of organic compounds of interest (ie, the mixture whose degradation is analyzed by the methods of the present invention).

[0072] According to the invention, the inoculum is prepared following a standard RBT protocol, such as OECD 301 (A to F) or similar protocols.

[0073] The inoculum can come from a variety of sources: activated sludge, effluent (unchlorinated), surface water and soil, or mixtures thereof. For example, the activated sludge can be taken from a treatment plant or a laboratory-scale unit that primarily receives domestic sewage, or can be taken from a mixture of a variety of sources.

[0074] Those skilled in the art can easily prepare the inoculum by following OECD 301A-F and OECD 310. In one embodiment of the present invention, OECD 301F can be followed. The specific protocol for preparing a suitable inoculum can be found in the OECD GUIDELINE FOR TESTING OF CHEMICALS Adopted by the Council on 17 th July 1992 pages 48 to 51.

[0075] Without limiting the present invention, the inoculum can be prepared by collecting a fresh sample of activated sludge from an aeration tank of a sewage treatment plant or a laboratory-scale device that mainly treats domestic sewage, removing coarse particles if necessary, and then maintaining the sludge in an aerobic state. In the case where the sludge contains or is believed to contain inhibitors, it is preferably washed and resuspended for inoculation. Preferably, the main micro-animal component of the inoculum is determined before use, for example by optical microscopy. The inoculum comprises or consists of bacteria / micro-animals isolated from the above sources.

[0076] Alternatively, the inoculum may be derived from secondary effluent from a treatment plant or laboratory-scale facility that primarily receives domestic sewage and / or surface water (eg, rivers, lakes, ponds, etc.) If necessary, the inoculum may be concentrated.

[0077] According to the present invention, each mixture of interest will be suspended in an appropriate amount of mineral medium together with the inoculum as described above. Preferably, in step a) of the method described and claimed herein, at least two flasks or containers are prepared for each mixture of interest, and even more preferably, the test will be performed in triplicate and three flasks or containers will be prepared for each mixture of interest in step a). The flask or container containing the mixture of interest is also a test flask or test container as defined herein.

[0078] According to the present invention, the method may also include step f), from T0 to T28 (T28 is the 28th day after the preparation), by means of one or more calibrated continuous reader probes, or by arranging an appropriate number of containers for each predetermined measurement for discontinuous reading, measuring oxygen consumption and / or carbon dioxide production and / or dissolved organic carbon consumption.

[0079] According to OECD GUIDELINE FOR TESTING OF CHEMICALS Adopted by the Councilon17 th Any of the RBT standard methods described in July 1992 pages 48 to 51 may measure oxygen consumption, and / or carbon dioxide production and / or dissolved organic carbon consumption at will by one or more calibrated continuous reader probes or by discontinuous readings arranged for an appropriate number of containers for each predetermined measurement. Preferably, the measurement in f) is a manometric respirometry method, in which O2 consumption and / or CO2 production are measured continuously. For example, oxygen consumption can be determined by measuring the amount of oxygen required to maintain a constant gas volume in the respirometer bottle (the amount of oxygen produced by electrolysis), or can be determined by changes in volume or pressure (or a combination of both) in the instrument. The released carbon dioxide is absorbed by potassium hydroxide solution or other suitable absorbent. During the biodegradation of the test mixture, the amount of oxygen absorbed by the microbial population (corrected by the absorption of a blank inoculum run in parallel) can be expressed as a percentage of ThOD or COD.

[0080] In fact, the method according to the invention provides in step f) the necessary data for carrying out the RBT standard test.

[0081] According to the present invention, before the LC-MS acquisition, the test samples collected in c) are preferably organized in a random sample queue, and the sample queue also includes one or more mixed samples and one or more blank samples. The analysis blank samples and mixed samples can be collected at the beginning, middle and end of the sample sequence.

[0082] Although the method can be performed by collecting blots of the test sample and blank sample at a single Tn, the blots of the test sample and blank sample can be collected at different days after the preparation a), and thus can be performed at Tn for different values ​​of n.

[0083] In this case, the analysis of step d) and the comparison of step e) are performed for each blank sample and test sample for which said blot was collected on the same day (same Tn).

[0084] In this specification and claims, Tn refers to the date on which the sample was collected and the blot was acquired.

[0085] As defined in the glossary, a mixed sample according to the present invention is a quality control sample comprising equal amounts of each of the test samples (ie excluding one or more blank samples).

[0086] Before taking the blots [in b) and c)], each test sample is filtered to eliminate microfauna in the sample. This allows the biodegradation to stop at the desired time Tn. Such filtration can be performed using any filter known to those skilled in the art that is suitable for removing contained microfauna from a liquid sample. Non-limiting examples of suitable filters are filters with a pore size of 0.45 mm or less, preferably ≤ 0.25 mm (e.g. 0.22 mm or less).

[0087] Liquid chromatography-mass spectrometry can be performed using any standard procedure known to those skilled in the art. Preferably, the liquid chromatography-mass spectrometry of the method is performed by UHPLC-qToF, i.e., UHPLC / qToF-MS (ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry). Commercially available reagents can be used.

[0088] According to the present invention, the multivariate statistical analysis is performed by processing data obtained from liquid chromatography-mass spectrometry and analyzing the complex blots obtained therefrom.

[0089] Preferably, an unsupervised, untargeted analysis is performed, so the algorithm used reads patterns from unlabeled data by an expert and does not identify compounds from the blot.

[0090] Multivariate data analysis (MVDA) facilitates the understanding and interpretation of blot data by providing a holistic view of the associations between compounds and corresponding metabolites.

[0091] There are many kinds of multivariate statistical tests. Specifically, these can be divided into:

[0092] - Unsupervised methods, such as principal component analysis (PCA), hierarchical clustering analysis (HCA) - dendrogram, heatmap;

[0093] -Supervised methods, such as Partial Least Squares (PLS), Partial Least Squares-Discriminant Analysis (PLS-DA), Orthogonal Partial Least Squares (OPLS), and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA).

[0094] Among unsupervised algorithms, PCA is one of the most widely used multivariate techniques in exploratory analysis (Worley B, Powers R, "Multivariate Analysis in Metabolomics." Current Metabolomics 1 (2013): 92-107). If we have a high-dimensional data set, such as dozens or hundreds of compounds or peaks per sample, we would like to find the combination that best explains the total variation in the original data set, thereby showing the "natural" trend or pattern and clusters between the samples according to the experimental conditions. PCA is one of the most powerful methods for this type of dimensionality reduction. Although the number of PCs is equal to the number of variables, only a limited number of PCs are explained. In addition, if the first few PCs can explain most of the variation in the data, we can use two-dimensional or three-dimensional graphs (called scores or loadings) to visualize the data (Joliffe, Ian. Principal component Analysis. John Wiley & Sons, 2005).

[0095] As an unsupervised method, PCA is often used in metabolomics studies to highlight experimental differences in sample groupings.

[0096] Cluster analysis aims to identify how many groups there are in the original data set. All clustering algorithms group observations so that samples in the same group or cluster are more similar to each other than to samples in other groups. Hierarchical clustering creates a hierarchy and uses a cluster graph to represent the hierarchy, or in other words, the clusters are organized into a hierarchical tree.

[0097] The data can be processed by performing peak peaking and peak alignment, setting appropriate mass tolerance, retention time tolerance and signal filtering threshold.

[0098] According to the present invention, the confidence level can be set between X% and Y%, preferably at 95%. The goodness of fit can be measured by R 2 The X algorithm is used to evaluate the prediction quality, and the Q 2 Evaluate the algorithm.

[0099] Observational diagnosis can be achieved by Hotelling T 2 and DModX tests were performed.

[0100] Specifically, the mass tolerance can be set to 10 ppm, the retention time tolerance can be set to 10 seconds, and the signal filter threshold can be set to 1000 counts. The mass tolerance can also be set to 5 ppm.

[0101] Variables with missing value frequencies and coefficients of variation exceeding 20% ​​can be removed and data imputed. In addition, filters can be applied to remove those variables that are present in the analytical blank samples.

[0102] The median of the parallel experiments can be applied to eliminate the influence of random noise, and the reference mixed sample can be normalized using PQN (Probabilistic Quotient Normalization).

[0103] The processed data can be saved in CSV format, and the data matrix in CSV format can be reprocessed by the SIMCA program (version 16.0.2.10561, January 22, 2020). Subsequently, the data matrix can be mean-centered and Pareto scaling can be applied before data analysis.

[0104] The processed data providing a complex profile of the samples may then be analyzed by PCA (Principal Component Analysis) and / or hierarchical cluster analysis.

[0105] The confidence level for both analyses is preferably about 95%.

[0106] Through the above steps, in the R 2 , Q 2 、Hotelling T 2After the classical test of , dMoDX, the clean data matrix can be used to build statistical models. The covariance matrix and the standardized principal component scores are selected by unsupervised methods for principal component analysis (PCA) calculation. After applying Ward's method to form hierarchical clusters, the distances between the groups are determined using Euclidean distances through CA, and the analysis results of the clusters are presented as cluster diagrams.

[0107] Step e) of the method according to the invention is a step that provides useful information about the mineralization of the mixture of interest. In fact, the PCA and / or HCA of the sample is carried out some time after the preparation of the test sample in a) (for example 28 days, T28, in compliance with the RBT criterion) and / or at an earlier stage (one or more other dates Tn) and compared with the PCA and / or HCA results of the blank sample, allowing the user of the method to assess whether biodegradation has occurred, or whether biodegradation has not occurred or is incomplete, confirming or not confirming the RBT result when the RBT step (step f) is also carried out. In fact, both PCA and HCA make it possible to visualize the distance between the test sample and the blank sample in the form of points or clusters.

[0108] Specifically, when performing multivariate statistical analysis by PCA, the user of the method will evaluate in step e) that when the PCA results of the test sample at Tn are close to the PCA results of the blank sample at the same Tn in 2D space, biodegradation has occurred, and when the PCA results of the test sample at Tn are not close to the PCA results of the blank sample at the same Tn in 2D space, biodegradation has not occurred or only partial biodegradation has occurred.

[0109] According to the present specification, the PCA result is considered to be "close" when the points of the test sample and the points of the blank sample are both within a 2x3 grid (2 on the X-axis and 3 on the Y-axis) rectangle of the PCA grid, preferably within a 2x2 grid of the PCA grid, more preferably within a 1x1 grid rectangle of the PCA grid, and even more preferably within the same grid of the PCA grid.

[0110] Figure 2b , Figure 3b and Figure 7b A clear example of a “close” point (i.e. a biodegradable mixture of interest) can be seen in Figure 2a , Figure 3a and Figure 7a Clear examples of "no-close" points can be seen.

[0111] If the distance between samples is observed on the X-axis, the variation is considered to be more correlated because this describes PC1 (PC1 is the first principal component of PCA), which contains the largest percentage of variance in the model. The Y-axis distance describing PC2 (PC2 is the second principal component of PCA) will indicate less difference between samples because PC2 describes a lower percentage of variance.

[0112] When multivariate statistical analysis is performed by HCA, the user of the method will assess in step e) that when the HCA results of the test sample at Tn form a cluster with the HCA results of the blank sample at the same Tn, biodegradation has occurred, and when the HCA results of the test sample at Tn do not form a cluster with the HCA results of the blank sample at the same Tn, no biodegradation has occurred or only partial biodegradation has occurred.

[0113] Generally speaking, the T0 test sample remained separated from the other samples in both analyses. However, when the test sample at Tn was close to the T0 sample in PCA and formed a cluster with the T0 sample in HCA, it meant that biodegradation had not yet occurred.

[0114] In a preferred embodiment, one of the Tn's is T28.

[0115] If necessary, in particular when no biodegradation or incomplete degradation is assessed in e), a targeted qualitative analysis is performed on one or more of the test samples to identify undegraded components or complex degradation intermediates in the sample.

[0116] According to the invention, the method can be advantageously applied to mixtures of organic compounds, such as pharmaceutical compositions, cosmetic compositions, food supplement compositions, medical device compositions or nutraceutical compositions. The medical device composition is a composition according to any of the categories described in Directive 2017 / 745 / EEC on medical devices, which consist of a substance or combination of substances that is absorbed by the human body or dispersed locally in the human body.

[0117] In any part of the present description and claims, the term "comprising" may be replaced by the term "consisting of".

[0118] The examples reported below are intended to better illustrate the embodiments disclosed in this description and should in no way be considered as limiting the preceding description and subsequent claims.

[0119] Example

[0120] Table 1 - Test mixtures

[0121]

[0122] a The reporting method is given on the package insert.

[0123] Two commercially available cough syrups (one containing synthetic ingredients [A] and one containing only natural ingredients [B]) were selected as real examples of widely distributed pharmaceutical ingredient formulations. Their biodegradation was evaluated using UHPLC-qToF "All Ions MS / MS" acquisition technology and compared with that of pure non-naturally occurring API (Active Pharmaceutical Ingredient) bromhexine. The chemical compositions of mixtures A and B declared in the package inserts of the analyzed drug mixtures are summarized in Table 1 above.

[0124] Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-qToF) is a state-of-the-art spectrometer characterized by ultra-high performance chromatographic separation of molecular species and has been successfully adopted due to its rapid speed, high-resolution separation, high-resolution mass spectrometry, and high sensitivity.

[0125] Even though high-resolution mass spectrometry and other spectroscopic methods have been previously reported for studying the environmental impact of pharmaceutical and cosmetic formulations, this is the first time that a UHPLC-qToF “All Ions MS / MS” approach has been used to achieve metabolite profiling by untargeted data processing of a drug formulation tested for rapid biodegradability and by performing targeted analysis to more deeply investigate the rapid biodegradation output.

[0126] Mixture A and B

[0127] Biodegradation

[0128] Pure glucose, magnesium sulfate heptahydrate ACS Reag. Ph. Eur and Reag. USP for analysis, sodium acetate anhydrous USP, 99% phosphoric acid, 99+% anhydrous potassium hydrogen phosphate for analysis, 99% anhydrous potassium dihydrogen phosphate for analysis, pure iron (III) chloride hexahydrate, 98+% disodium hydrogen phosphate dihydrate, 99% anhydrous pure calcium chloride were purchased from Carlo Erba Reagents srl (Cornaredo, Milano, Italy). 99% extra pure ammonium chloride and 85% extra pure potassium hydroxide were purchased from Italchimica SPA (Pontecchio Polesine, Rovigo, Italy). Buffered peptone water (ISO) was purchased from ThermoFisher.

[0129] Purified water was prepared by an Arium Mini water purification system (from Sartorius Goettingen, Germany).

[0130] Sample preparation

[0131] The samples were processed according to the procedure reported in Respirometry n° 301F (OECD). In order to enable correct recording by the BOD sensor and to have sufficient material for instrumental analysis, few adjustments were planned, as shown in Table S4.

[0132] Table S4. Experimental parameters for the breathing test

[0133]

[0134] Sodium acetate was used as reference substance. The test was carried out at a constant temperature of 22°C.

[0135] Inoculum preparation.

[0136] Five activated sludge samples and five river water samples were collected from different places and mixed in equal volumes to prepare the inoculum. The inoculum was oxygenated, stirred, and glucose, peptone, and dipotassium hydrogen phosphate were added, and the values ​​of redox potential, oxygen consumption, and total dry matter were monitored every day. When used, the dry matter was determined at 100°C to measure the same amount (30 mg / ml) in the container containing the test substance.

[0137] Inoculum composition

[0138] The inoculum was obtained by mixing equal amounts of activated sludge from eight different areas and river water from two different rivers. The combined inoculum was oxygenated, stirred, and glucose, peptone, and monopotassium orthophosphate were added. Oxygen, redox, and total suspended solids values ​​were monitored daily.

[0139] Before using the inoculum, the total dry matter was determined. The composition of the microfauna was determined by light microscopic analysis and consisted of the following species: Aspidisca cicada, Zoothamnium, Rotiferi, Euglyphia.

[0140] Instrument parameters

[0141] Biological oxygen demand is continuously monitored by a BOD EVO sensor system 6, which consists of six BOD EVO sensors, a six-position stirring base, a dark glass bottle, a subcap for absorbing carbon dioxide and a magnetic stirring bar. The BOD EVO sensors transmit the data wirelessly to a dedicated BODSoft TMSoftware (VELP Scientifica, Usmate, Monza-Brianza, Italy). All experiments were performed at controlled temperatures in a refrigerated thermostat equipped with an automatic temperature regulation system and forced air circulation to ensure stability and uniformity of the internal temperature (VELP Scientifica, Usmate, Monza Brianza, Italy). The temperature of the thermostat was monitored by a data logger with an external probe in the range of -40 to +80°C (XS Instruments, Carpi, Modena, Italy).

[0142] UHPLC ESI-qToF Method

[0143] All solvents were of high purity analytical grade and used without further purification. ULC / MS grade anhydrous methanol was purchased from Biosolve (Dieuze, France). Ultra-high purified water was purchased from Prepared in ultrapure water system (ELGA, UK). LCMS 98%-100% formic acid and dimethyl sulfoxide ≥99% were purchased from Sigma-Aldrich (St. Louis, MO). Mixture A was purchased (purchased from a local pharmacy, batch number 181092), and mixture B was produced by Aboca (batch number 19C2076); bromhexine hydrochloride, sucralose, maltitol, and ambroxol were purchased from SigmaAldrich (St. Louis, MO). High-purity reference standards used to build internal databases and calibration curves were purchased from Extrasynthese (Genay, France), Sigma-Aldrich (St. Louis, MO), PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany), and ChromaDex (Irvine, CA). High-purity reference standard stock solutions were prepared at 500 ppm in methanol, water / methanol (80:20, v / v), or methanol / dimethyl sulfoxide (80:20, v / v). Working solutions were prepared by diluting an appropriate volume of the stock solution with water / methanol (50:50, v / v). The internal standard sulfadimethoxine-d6 was purchased from Sigma-Aldrich (St. Louis, MO). The internal standard stock solution was prepared at 5 ppm in methanol. All stock solutions were stored in glass vials at -80°C.

[0144] Targeted approach

[0145] Sample preparation: The samples were filtered on 0.20 μm Millipore cellulose acetate syringe filters and used for acquisition of chromatographic profiles according to the following conditions without any dilution.

[0146] Instrument parameters. The instrument platform used consisted of a UHPLC series 1290 coupled with a high-resolution (2 GHz) quadrupole time-of-flight (qToF) mass spectrometer series 6545 (Agilent Technologies, Santa Clara, CA). The UHPLC was equipped with a binary pump, autosampler, multicolumn thermostat, isocratic pump, and solvent cabinet. The autosampler was maintained at 15°C. The analytical column was C18 VanGuard TM Pre-column (5x2.1mm, 1.6μm) protected C18 (100 x 2.1 mm, 1.6 μm), both columns were provided by Waters (Milford, MA) and thermostated at 40° C. HCO2H aqueous solution (0.1%, v / v) was used as mobile phase A, HCO2H (0.1%, v / v, in MeOH) was used as mobile phase B, and elution gradient analysis was performed according to the following gradient table (Table S5).

[0147] Table S5. UHPLC ESI-qToF method: elution gradient

[0148]

[0149] Flow rate: 0.3 mL min -1 The injection volume for 1000 mg / L samples was 3 μL, while the injection volume for 100 mg / L samples was 10 μL.

[0150] The UHPLC system was coupled to a qToF mass spectrometer equipped with a Dual AJS ESI source operating in negative and positive ionization modes with a scan range of 50 to 1700 m / z. The optimized instrument parameters are reported in Table S6 below.

[0151] Table S6. UHPLC ESI-qToF method: MS parameters

[0152]

[0153] By using N2 three different collision energy values ​​(20eV, 30eV and 40eV), each analytical standard reference compound was collected in data-dependent mode (AutoMS / MS, Agilent Technologies), thereby building an internal database. In full ion mode, the collection of metabolite traces of the studied samples was carried out using a collision energy value of 30eV. In both acquisition methods, a reference mass solution containing purine and six (1H, 1H, 3H-tetrafluoropropoxy) phosphazines was directly injected into the ESI source by an isocratic pump and ionized with the sample solution to perform mass calibration, thereby obtaining accurate mass time-of-flight data. MassHunter software version B.07 (Agilent Technologies, Santa Clara, CA) was used for data acquisition and processing.

[0154] Software. MassHunter software version B.07 (Agilent Technologies, Santa Clara, CA) was used for data acquisition and processing.

[0155] The accurate masses of various molecular species were calculated by the isotope distribution calculator, MassHunter Data Analysis core version 8.0.8208.0 (Agilent Technologies, Santa Clara, CA) data analysis tool. Data analysis. Detailed description by the "Qualitative Analysis" program and the "Find-by-Formula" algorithm. The data file was loaded into "Qualitative Analysis" and then "Find-by-Formula" was run on the low channel against the MS / MS library. Find-by-Formula returns possible precursor formulas found in the library and their product ions. Using the product ion list, "Qualitative Analysis" extracts the EIC from the high channel and aligns it with the EIC of the precursor.

[0156] The co-eluting fractions were calculated and compounds passing the threshold (user set to 70) were retained. Qualitative Analysis returned a total score formed by the mass score, isotopic abundance score, isotopic spacing score and retention time score, with a user set threshold of 70. The EIC was obtained by setting the m / z spread to a symmetrical 2.5 ppm.

[0157] Non-targeted approach

[0158] Sample preparation: Samples were prepared as described for the targeted UHPLC-qToF method.

[0159] Multivariate statistical analysis

[0160] Data collection: Prior to UHPLC-qToF acquisition, two syrup samples at T0 and T28 and the blank sample consisting of mineral medium and inoculum were organized into a random sample queue. The analytical blank and mixed samples were collected at the beginning, middle and end of the sample sequence. Each sample was collected in triplicate.

[0161] The instrument parameters were the same as used in the targeted UHPLC-qToF method, positive ions for mixture A and negative ions for mixture B.

[0162] Data processing : Peak quantification and peak alignment were performed using the software R [R version 3.5.2 (2018-12-20), 2018 The R Foundation for Statistical Computing]. The mass tolerance was set to 10 ppm, the retention time tolerance was set to 10 seconds, and the signal filtering threshold was set to 1000 counts.

[0163] Variables with missing value frequencies and coefficients of variation exceeding 20% ​​were removed and data imputed. In addition, filters were applied to remove those variables present in the analytical blank samples.

[0164] The median of the parallel experiments was applied to eliminate the influence of random noise, and data were normalized by PQN (probability entropy normalization) for the reference mixed sample.

[0165] The processed data were saved in CSV format and the data matrix in CSV format was reprocessed by the SIMCA program (version 16.0.2.10561 on January 22, 2020). Subsequently, the data matrix could be mean-centered and Pareto scaling could be applied before data analysis.

[0166] Statistical Models Performance evaluation was performed and a 95% confidence level was used.

[0167] The goodness of fit and prediction were evaluated by the following R 2 X and Q 2 (Table S7) for evaluation.

[0168] Table S7.Q 2 and R 2 X-statistical model performance evaluation

[0169]

[0170] Computational diagnostic tool Hotelling T 2and DModX to ensure that the model is not deformed, so there are neither strong nor weak outliers. The model is robust because the observed values ​​are below the calculated critical values ​​and there are no outliers.

[0171] PCA analysis : After data processing, in order to evaluate the complex imprints obtained, multivariate principal component analysis (PCA) was used. The PCA model was constructed using the first two principal components providing a 2D model (confidence level 95%).

[0172] Cluster analysis : Cluster analysis was performed using a hierarchical algorithm according to an unsupervised approach (confidence level 95%). Distance indices were determined using Ward linkage (JH Ward, Hierarchical Grouping to Optimize an Objective Function, J. Am. Stat. Assoc., 1953, 58, 236-244) and Euclidean distance.

[0173] Semi-quantitative determination.

[0174] -Relative ratio (RR) calculation.

[0175] The relative ratio (RR) was calculated by the ratio of the area counts of the compound to the area counts of the internal standard (sulfadimethoxine-d6).

[0176] - Calculation of percentage reduction

[0177] The percent reduction of a compound is the ratio of the RR at T28 to the RR at T0, expressed as a percentage.

[0178] Results and discussion

[0179] UHPLC chromatographic conditions were optimized to achieve the best ionization and to elute all species of interest within 21 minutes. Pure water and methanol (both acidified with formic acid) were used as mobile phases, following a linear gradient composition suitable for eluting both hydrophilic and lipophilic compounds within a time frame compatible with UHPLC analysis.

[0180] By using UHPLC-qToF "all ion MS / MS" (S. Naz, H. Gallart-Ayala, SN Reinke, C. Mathon, R. Blankley, R. Chaleckis, CE Wheelock; Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition. Analytical Chemistry 2017; 89: 7933-7942) acquisition technology, high-resolution mass spectrometry (HRMS) data can be acquired using different conditions: (1) low collision energy values ​​(2) high energy values. Low energy spectra mainly show only the molecular (or precursor) ions of the compound, while high energy spectra provide the precursor plus its fragment ions.

[0181] Utilizing All Ions MS / MS data, targeted qualitative screening analyses can be performed with confidence, as well as structural elucidation and fragmentation pattern identification of compounds present in complex products during biodegradability studies.

[0182] The large number of signals obtained from UHPLC-qToF analysis can be used to perform untargeted studies without structural identification. Untargeted analysis provides information about the physicochemical changes of samples during RBT by observing the grouping routes of samples in statistical models through principal component analysis (PCA) and cluster analysis (CA).

[0183] The breathalyzer test n° 301F (according to OECD Guidelines OECD, Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD, 2006) was chosen as the method for biodegradability assessment.

[0184] The tests were carried out at the two concentrations required by the OECD 301F method (50 mg / L and 100 mg / L), in addition, we tested an out-of-range concentration (1000 mg / L) in order to better identify all metabolites formed during the test. Samples were analyzed by both untargeted and targeted methods at the beginning of the rapid biodegradation test and after 28 days.

[0185] At a concentration of 50 mg / L, the RBT of mixture A passed the ten-day window criterion and achieved 81% biodegradation after 28 days of incubation, while mixture B passed the ten-day window criterion and achieved 74% biodegradation on day 28. The biodegradation curves are shown in Figure 1.

[0186] The test was repeated at concentrations of 100 mg / L and 1000 mg / L. The results show that mixture A is easily biodegradable again at 100 mg / L (86% at 100 mg / L BD28 days), but not easily biodegradable at 1000 mg / L (6% at 1000 mg / L BD28 days), while mixture B is easily biodegradable under both conditions (77% at 100 mg / L:BD28 days, 75% at 1000 mg / L BD28 days). Table 2 and Figure 1 summarize these results.

[0187] Table 2: Results of the rapid biodegradability test (OECD 301F) of mixture A and mixture B

[0188]

[0189] 1 The passing level for rapid biodegradability by respirometry is 60% ThOD removal. These passing values ​​must be achieved within a 10-day window within 28 days of testing. The 10-day window begins when the extent of biodegradation reaches 10% ThOD and must end before day 28 of the test. Chemicals that reach the passing level after 28 days are not considered to be readily biodegradable.

[0190] Even though 1000 mg / L was not the concentration considered in the validation method, it is interesting to observe the different behaviors of the analyzed samples. In the case of mixture A, the microfauna of the inoculum did not seem to be able to fully activate the biodegradation, probably because it was affected by the experimental conditions. We believe that this can be attributed to both the high concentration of substrate and / or some initially formed metabolites. No similar effect was observed in the case of mixture B, suggesting that different mixtures can have different dose-dependent effects on the experimental microenvironment. In order to better study this phenomenon, the spectral semi-quantitative evaluation of the residual main components of mixture A at 50 mg / L, 100 mg / L and 1000 mg / L was performed after 28 days under RBT conditions: sucralose 1, maltitol 2 and bromhexine 3 (see Table S1 below).

[0191] Table S1: Semi-quantitative evaluation of sucralose 1, maltitol 2 and bromhexine 3 by estimating the relative ratio RR and calculating the reduction percentage. RR is the ratio of the area counts of the compound to the area counts of the internal standard (sulfadimethoxine-d6). The reduction level at the end of the experiment is the ratio of the RR at T20 to the RR at T0, expressed as a percentage.

[0192]

[0193] Sucralose was completely non-degradable and its original concentration was detected in all samples (see Table S1). In contrast, the degradation of bromhexine 3 was hardly affected by the sample concentration, with only 6%, 12%, and 12% of the parent compound present at the end of the experiment in the 50 mg / L, 100 mg / L, and 1000 mg / L samples, respectively. Maltitol 2 was completely biodegraded in the 50 mg / L and 100 mg / L samples, but 62% of the original concentration was detected in the 1000 mg / L sample, which largely led to the failure of the RBT at this concentration.

[0194] In addition, the composition of 100 mg / L and 1000 mg / L mixtures (A and B) before and after 28 days was studied by UHPLC-qToF analysis under RBT conditions following both untargeted and targeted approaches.

[0195] The UHPLC-qToF method used to acquire the data is characterized by chromatographic separation in a reversed-phase UHPLC column followed by detection by a qToF mass spectrometer. All eluting species were selectively characterized by their retention time and high-resolution mass. Therefore, after acquiring the chemical blot, the data were aligned. Variables with missing value frequencies and coefficients of variation exceeding 20% ​​were removed, noise was subtracted, and the corresponding raw data matrix was normalized and scaled.

[0196] In the case of R 2 2 X, Q 2 (Model Diagnostic Test), Hotelling T 2 After classical tests such as DModX (observational diagnostic test), the obtained data matrix is ​​used to build a statistical model. The covariance matrix and standardized principal components are selected for unsupervised principal component analysis (PCA) calculation.

[0197] In the experiments at 100 mg / L (Figure 2) and 1000 mg / L (Figure 3), it was found that the first two components of 2D-PCA explained more than 90% of the total model variance for mixture A (PC1 74.9%, PC2 15.8% 100 mg / L; PC1 57.5%, PC2 33.2% 1000 mg / L) and mixture B (PC1 91.4%, PC2 2.2% 100 mg / L; PC1 96.1%, PC2 2.1% 1000 mg / L).

[0198] The graph demonstrates that for mixture A, the observed values ​​(scores) at 28 days are quite different from those observed for the mineral medium, whereas for mixture B, the biodegradation appears to be higher, but still not completely consistent with the mineral medium (compare Figure 2a / 2b and Figure 3a / 3b).

[0199] Another method for qualitative evaluation of the metabolite traces obtained by UHPLC-qToF is to use unsupervised "cluster analysis" (CA) for statistical interpretation to obtain natural hierarchical groupings. By CA, after applying the Ward connection method (H. Ward, Hierarchical Grouping to Optimize an Objective Function, J. Am. Stat. Assoc., 1953, 58, 236-244) as a standard method for forming hierarchical clusters, the distances between groups were determined using Euclidean distances, and the results of the cluster analysis were presented in the form of cluster diagrams (Figures 4 and 5). The clusters generated by mixture A (100 mg / L) are basically two: one formed by the sample collected after 28 days under the biodegradation test conditions, and the other formed by the sample collected at the beginning of the study and the mineral medium ( Figure 4a For mixture B (100 mg / L), similarly, two clusters were identified: one formed by the sample collected at the beginning of the test, and the other formed by the sample collected 28 days later and the mineral medium, ( Figure 4b ) confirmed the observations of PCA. Similarly, untargeted analysis of samples with a concentration of 1000 mg / L showed that the derivative mixture produced by the degradation of mixture B was closer to the derivative mixture produced by the mineral medium than the molecular imprint of the mixture obtained by the biodegradation of A (compare Figure 4a / 4b and Figure 5a / 5b). However, in both cases, mineralisation does not appear to have been fully realised.

[0200] These data clearly show that the biodegradation output results obtained after 28 days of RBD testing, considering the mixture of compounds, are strongly influenced by the chemical composition of the starting feedstock, which produces a new mixture of compounds that is not completely consistent with the compound mixture of the mineral medium, which should be completely degraded into basic bricks such as carbon dioxide, water, ammonia and sulfide.

[0201] Furthermore, these results suggest that untargeted analysis by UHPLC-qToF offers an attractive approach for evaluating the actual biodegradation of chemical mixtures, overcoming some of the limitations in sensitivity exhibited by currently used RBTs.

[0202] These untargeted analyses provided an overall overview of the mixture behavior and suggest that further investigations using targeted approaches should be conducted with the goal of understanding the fate of each specific compound present in the complex mixture after 28 days of RBT.

[0203] The same UHPLC-qToF technique as used for the untargeted analysis was used to characterize some of the compounds present in mixtures A and B. With this approach, pure reference samples of selected compounds such as sucralose, bromhexine, and ambroxol (as known metabolites) were analyzed to correctly identify them in mixture A. The MS and MS / MS experimental data for sucralose and bromhexine were consistent with those reported in the literature. Preliminary identification of the compounds present in mixture B was achieved by using the Personal Compound Database and Library (PCDL) of natural compounds present in the laboratory.

[0204] In the starting mixtures A and B, based on high resolution mass, retention time, MS / MS fragmentation pattern, isotopic profile and isotopic abundance, Figure 6 Compounds 1–6 reported in were unambiguously identified.

[0205]

[0206] UHPLC-qToF analysis was used to detect bromhexine (3) and its metabolites in 1000 mg / L mixture A after 28 days under RBT conditions. Bromhexine (3) had a typical MS / MS fragmentation characterized by the cleavage of the benzylamino bond, resulting in two different fragments, namely: an aromatic fragment carrying a bromine atom, measured at m / z 263.8843; and a cyclohexane moiety bonded to the amino group, measured at m / z 114.1277.

[0207]

[0208] Molecular ion m / z 377.0048[(M+H+2) + ] and the corresponding fragment ions m / z 114.1280 and m / z 263.8843 (acceptable differences less than 5 ppm were observed compared to the calculated m / z) clearly indicated that bromhexine and the same related metabolites were still present after 28 days.

[0209] Specifically, the extracted ion at m / z 263.8841 indicated the presence of several bromhexine metabolites that share the common characteristic of bromoaryl fragments. Some of these were assigned different confidence levels. For example, the structure of Ambroxol 7 could be identified (level 1), with full overlap between the experimental data and the pure reference standard data identified by the characteristic fragmentation reported below:

[0210]

[0211] As mentioned above, semi-quantitative analysis of the spectral data revealed that 12% of the initial bromhexine was not degraded after 28 days, with metabolites 8 and 9 (above) having the highest levels, which may be because the N-demethylation of bromhexine and the hydroxylation of the cyclohexyl ring are reactions that are kinetically favorable to the metabolism of RBT micro-animals.

[0212] Similarly, a targeted analysis of sucralose was performed on mixture A. After 28 days, the search for the molecular ion m / z 419.0038 [(M+Na) + ], its presence was detected without any significant difference in concentration. This result was further confirmed by its fragment EIC at m / z 238.9848, demonstrating that the presence of related metabolites with common fragments was not detected in this case.

[0213] The biodegradability of the mixture B was investigated in a targeted manner, focusing on the fate of sucrose (4), acteoside (5), and grindelic acid (6) identified as selected representative compounds in the mixture at T0 of the RBT.

[0214] The experimental MS / MS fragmentation patterns of actinomycin (5) and oxalic acid (6) are shown below:

[0215]

[0216] Actinomycin showed a peak corresponding to m / z 161.0244 [caffeic acid-H2O-H - ], resulting in the loss of the caffeoyl moiety of -] obtained anion m / z 461.1664. Glycol oxalic acid (6) produced a fragment with mass 205.1596, assuming spiro-tetrahydrofuran ring opening and further anion rearrangement, which has not been reported in the literature. The proposed elemental composition of the fragment is C 14 H 21 O, the calculated monoisotopic accurate mass is m / z 205.1596, which corresponds to the m / z recorded for the fragmentation of pure oxalic acid standard and oxalic acid in the sample.

[0217] After 28 days of RBT, it was not possible to detect the presence of compounds 4, 5 and 6, nor their direct derivatives.

[0218] Finally, in order to compare the fate of incompletely degraded APIs as pure chemical entities or as part of pharmaceutical formulations, pure bromhexine (3) was subjected to RBT at 0.7 mg / L (corresponding to its concentration in 1000 mg / L mixture A). After 28 days, evaluation of the relative area % (Table S1) analysis showed that only 46% of the pure compound was degraded (compared to 88% observed in the mixture A). This result is not easy to explain and can be tentatively attributed to the higher activity of the microfauna in the presence of other readily available energy sources. However, it clearly shows that the degradation profile of a specific molecule can be very different when obtained in pure form or as part of a complex chemical mixture, suggesting that mixtures, similar to complex systems, can exploit some emerging, not completely predictable properties. In conclusion, based on the results collected in this manuscript, we suggest that modern technologies (such as UHPLC-qToF) should be considered to more accurately and reliably evaluate biodegradation and, therefore, the environmental impact of pharmaceutical and cosmetic formulations. We demonstrate here that, using an untargeted approach, two different pharmaceutical formulations could be considered readily biodegradable according to currently used analytical protocols, but were in fact not fully mineralized, regardless of whether the synthetic or natural components were considered, even though, as expected, the natural component was much more degraded to mineral media. Incomplete mineralization of the formulations yielded new mixtures of derivatives that may merit detailed analysis using high-performance methods, such as those based on mass spectrometry. Furthermore, we observed that the biodegradability of a mixture of compounds cannot be considered as a simple sum of the biodegradability of each component. In this direction, the new technology offers a holistic approach that simultaneously considers the fate of chemical systems that are bound to biodegrade, while providing very rational targeted analyses that are able to elucidate most components of the final chemical footprint.

[0219] For these reasons, we believe that the use of these modern technologies and the development of new protocols and regulations based on them will improve the ability to predict, control and prevent the environmental impacts of chemical mixtures, such as those found in the pharmaceutical, cosmetic and personal care product markets.

[0220] The results reported in this paper suggest a new approach to evaluating biodegradability that is more based on systems thinking, a paradigm that must always be considered when talking about environmental and biological systems.

[0221] Mixture C and D

[0222] Two commercially available complex formulated products commonly used to treat reflux disease and functional dyspepsia, one containing synthetic ingredients (mixture C) and one containing only natural ingredients (mixture D), were selected as real examples of large-scale distribution formulations of pharmaceutical ingredients to study the transformation products during rapid biodegradability testing.

[0223] The ingredients of both products are described in the package insert and are summarized in Table 1 above, namely "Mixture C" based on omeprazole. According to the latest report of the Italian Medicines Agency, omeprazole is currently one of the most widely present active pharmaceutical ingredients in medicines used to treat gastric diseases in the world and in Italy.

[0224] Rapid biodegradation test

[0225] The respirometry test OECD n°301F [OECD, Revised Introduction to the OECD Guidelines for Testing of Chemicals, Section 3, OECD 2006, OECD, Test No.301: Ready Biodegradability, 1992.] was chosen as the method for the evaluation of the ready biodegradability of our two complex formulations. Each experiment was set up according to the OECD guidelines, so inocula obtained from equal amounts of activated sludge and river water were used and their macroscopic composition was characterized (e.g. as defined in the experimental section). The inoculum was not pre-adapted to the mixture tested, so the results can be considered representative and robust. The oxygen consumption was recorded by a calibrated continuous reader probe. In order to ensure the reproducibility of the results for each product, three parallel tests were performed. At a concentration of 50 mg / L, "Mixture C" turned out to be not readily biodegradable, since the biodegradation rate was 26% after elimination of nitrification on the 28th day. "Mixture D" tested at the same concentration turned out to be readily biodegradable as it passed the ten-day window criterion, achieving 91% biodegradation at day 28 (Table 3).

[0226]

[0227] For "Mixture D", the test was repeated at a concentration of 75 mg / L, confirming the results obtained at 50 mg / L.

[0228] The transformation products of 50 mg / L “Mixture C” and 75 mg / L “Mixture D” after 28 days were studied by UHPLC-qToF analysis under OECD 301F test conditions using an untargeted and suspect screening approach.

[0229] Sample preparation

[0230] The workflow for targeted suspect screening and untargeted analysis involves multiple steps from sample preparation and data acquisition to data mining, expert review, and data interpretation. The first sample preparation step is critical and a compromise must be found between selectivity and sensitivity.

[0231] The concept of cleaning to remove the most abundant interferences is based on procedures used for targeted methods, whereby low-concentration analytes are enriched by purification. However, this also introduces new problems, especially when the nature of the compounds to be detected is not fully known in advance.

[0232] In the context of our studies, sample handling was minimized to minimize the selective treatment of the various transformation compounds. Therefore, the test mixture only had to undergo a filtration step, which has the advantage of maintaining the integrity of the sample, thus limiting the variations associated with sample preparation.

[0233] UHPLC-ESI-qToF analysis

[0234] The UHPLC-qToF method used to acquire the blot data is characterized by chromatographic separation in a reversed-phase UHPLC C18 column followed by qToF mass spectrometer detection. An ESI source was used to generate various ion molecular species. Positive ion mode was used to analyze "Mixture C" due to the presence of the nitrogen-containing compound "omeprazole". In the case of "Mixture D", negative ion mode ionization was used because it is rich in phenolic compounds.

[0235] The mobile phase consists of water / methanol, according to a general elution gradient from high percentage water to high percentage organic solvent, which satisfies the separation of most analytes and limits matrix effects. The column is finally flushed (e.g., with 99% methanol) to avoid carry-over between injections. Formic acid is added to the mobile phase as a phase modifier to stabilize the pH, enhance peak shape and promote ionization.

[0236] To ensure reproducibility and achieve a better level of confidence in the results produced, each sample was collected in triplicate, and pooled samples were collected at the beginning, middle, and end of the analytical period.

[0237] Data processing

[0238] The post-acquisition data processing step consists of converting the raw instrument data into organized tabular files containing peak lists for subsequent annotation and statistical analysis (for targeted suspect screening and untargeted analysis, respectively). This part fundamentally depends on the availability and performance of bioinformatics tools. In our case, to extract information from the raw data, Mass Profiler Professional (from Agilent), R packages (from Rfoundation), and Simca (from Umetrics / Sartorius) were used.

[0239] Untargeted analysis

[0240] Basically, the purpose of performing untargeted non-analysis is to detect any signal present in the chemical footprint generated by the rapid biodegradation test and to visualize the test results after unsupervised multivariate statistical analysis.

[0241] Therefore, peak extraction, peak alignment, and peak integration were performed to align the common peaks found in different samples and report their intensities or areas, respectively. Variables with missing value frequencies and coefficients of variation exceeding 20% ​​were removed, noise was subtracted, and the corresponding raw data matrices were normalized and scaled.

[0242] The resulting peak list contains a series of features labeled with retention time and HR-MS. The classical test R was performed at a confidence level of 95%. 2 X, Q 2 (Model Diagnostic Test), Hotelling T 2 After DModX (observation diagnostic test), the data matrix thus obtained was used to build a statistical model. The covariance matrix and standardized principal components were selected for unsupervised principal component analysis (PCA) calculations. It can be observed that in the 2D plot (Figure 7), it was found that the first two components of 2D-PCA explained more than 95% of the total variance of the "Mixture C" (PC1 70.7%, PC2 26.9%) and "Mixture D" (PC1 98.3%, PC2 0.6%) models.

[0243] The graph demonstrates that for "Mixture C" the observed values ​​(scores) at day 28 differ greatly from those observed for the mineral medium, whereas in the case of "Mixture D" the biodegradation appears to be completely consistent with that of the mineral medium.

[0244] Another method for qualitative evaluation of the blots obtained by UHPLC-qToF is to use unsupervised "cluster analysis" (CA) for statistical processing to obtain natural hierarchical groupings. With CA, after applying the Ward connection method [H. Ward, Hierarchical Grouping to Optimize an Objective Function, J. Am. Stat. Assoc., 1953, 58, 236-244] as a standard method for forming hierarchical clusters, the distances between groups are determined using Euclidean distances, and the results of the cluster analysis are presented as cluster diagrams.

[0245] "Mixture C" produced essentially three clusters: one formed by the sample collected after 28 days under the biodegradation test conditions, another formed by the sample collected at the beginning of the study, and another formed by the mineral medium (Figure 3). For "Mixture D", two clusters were identified: one formed by the sample collected at the beginning of the test, and another formed by the sample collected after 28 days and the mineral medium (confirming the PCA observations).

[0246] Targeted suspected substance screening analysis

[0247] Using targeted suspect screening analysis, annotations of suspect compounds with varying confidence levels were obtained depending on the type and extent of structural information collected and provided through the analytical workflow.

[0248] In this example, raw data collected at the beginning of the RBT and at day 28 were background subtracted and matched to data present in two compound libraries:

[0249] - "Internal PCDL" experimental database containing 1000 natural compounds characterized by their retention times, accurate masses and accurate MS / MS fragments

[0250] -Metlin_Metabolites_AM_PCDL, a database containing accurate masses and accurate MS / MS fragments of various compounds and metabolites.

[0251] The two libraries were integrated into Mass Hunter Qualitative Analysis (Agilent) and used to identify natural compounds and omeprazole TP using the "Find by Formula" mining algorithm. Obviously, "Metlin PCDL" is targeted to "Mixture C" and "Internal PCDL" is targeted to "Mixture D".

[0252] At the beginning of the test, some typical compounds such as sucrose, liquiritin and apigenin were detected in the mixture of "Mixture C", while omeprazole was detected in the mixture of "Mixture D". The presence of these compounds is consistent with the composition of these two drugs. Then, their high-resolution mass, retention time, high-resolution MS / MS fragmentation pattern, isotopic spacing and isotopic abundance are consistent with the data of the verified reference standard.

[0253] On day 28, the same search was performed on both libraries. Evaluation of the data showed that omeprazole was still detectable in "Mixture C", while sucrose, liquiritin and apigenin were no longer detectable in "Mixture D". The data for omeprazole were consistent with the known data.

[0254] Semi-quantitative determination

[0255] According to the semi-quantitative determination by area % evaluation, among all the omeprazole TPs in t28, the most abundant one was omeprazole TP_12 (omeprazole TP_12 5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole (ufenprazole), followed by TP 5, TP7 (omeprazole TP_5 4-methoxy-3,5-dimethyl-picolinic acid (CAS n°138569-60-5; omeprazole TP_7 5-methoxy-2-(4-methoxy-3,5-dimethyl-pyridin-1(2H)-yl)-1H-benzo[d]imidazole) and others.

[0256] It can be inferred that a series of omeprazole transformation compounds were formed after 28 days, indicating that the metabolic rate was kinetically unfavorable for the microfauna in the OECD 301F rapid biodegradability test.

[0257] We tested the rapid biodegradability of omeprazole-based “Mixture C” and “Mixture D” using the respiromanometric test (OECD 301F).

[0258] "Mixture C" turned out to be not readily biodegradable, whereas "Mixture D" turned out to be readily biodegradable.

[0259] Samples from a rapid biodegradability test were studied using UHPLC-qToF “All Ions MS / MS” technology for untargeted and suspect screening analysis to follow the behavior of two antacid drugs.

[0260] Significant changes were observed in the samples from the start of the pneumomanometric test to day 28, due to the consumption of the product by the microfauna. The incomplete mineralization observed for "Mixture C" was also confirmed by untargeted analysis and suspect screening studies. On the other hand, for "Mixture D", the selected compounds were not detected, which were well metabolized by the microfauna.

[0261] In conclusion, these data highlight that the use of UHPLC-qToF “All Ions MS / MS” methods, which enable the use of a variety of analytical approaches, has great potential in the future of biodegradability studies, allowing the evaluation of the behavior of compounds even when they are present in complex mixtures.

[0262] Materials and methods

[0263] Biodegradation

[0264] Magnesium sulfate heptahydrate ACS Reag. Ph. Eur and Reag. USP for analysis, sodium acetate anhydrous USP, 99% phosphoric acid, 99+% anhydrous potassium hydrogen phosphate for analysis, 99% anhydrous potassium dihydrogen phosphate for analysis, pure iron (III) chloride hexahydrate, 98+% disodium hydrogen phosphate dihydrate, 99% pure anhydrous calcium chloride were purchased from Carlo Erba Reagents srl (Cornaredo, Milano, Italy). 99% extra pure ammonium chloride and 85% extra pure potassium hydroxide were purchased from Italchimica SPA (Pontecchio Polesine, Rovigo, Italy). Purified water was prepared by an Arium Mini water purification system (from Sartorius Goettingen, Germany).

[0265] Sample preparation: The samples were processed according to the procedure reported in Respirometry n°301F (OECD). In order to enable correct recording by the BOD sensor and to have enough material for the planned instrumental analysis in a 1 L container, 0.4 L of mineral medium and 50 mg / L or 75 mg / L of sample were added. Sodium acetate was used as reference substance. The test was carried out at a constant temperature of 22°C.

[0266] Inoculum preparation. Five activated sludge samples and five river water samples were collected from different places and mixed in equal volumes to prepare the inoculum. The inoculum was aerated, stirred, and glucose, peptone, dipotassium hydrogen phosphate were added, and the values ​​of redox potential, oxygen consumption and total dry matter were monitored daily. At the time of use, the dry matter was determined at 100°C to measure the same amount (30 mg / ml) in the container containing the test substance.

[0267] Inoculum composition. The inoculum was obtained by mixing equal amounts of activated sludge from eight different areas and river water from two different rivers. The combined inoculum was oxygenated, stirred, and glucose, peptone, and monopotassium orthophosphate were added. Oxygen consumption, redox potential, and total suspended solids values ​​were monitored daily.

[0268] Before using the inoculum, the total dry matter was determined.

[0269] The composition of the microfauna was determined by light microscopy analysis and consisted of the following species: Litonotus Fasciola, Aspidisca costata, Vorticella acquadulcis, Vorticella convallaria, Amoebas, Rotifers, Tardigrades.

[0270] Instrument parameters. Biological oxygen demand is continuously monitored by a BOD EVO sensor system 6 consisting of six BOD EVO sensors, a six-position stirring base, a dark glass bottle, a sub-cap for absorbing carbon dioxide and a magnetic stirring bar. The BOD EVO sensors transmit data wirelessly to a dedicated BODSoft TM Software (VELP Scientifica, Usmate, Monza Brianza, Italy). All experiments were performed at controlled temperature in a refrigerated thermostat equipped with an automatic temperature regulation system and forced air circulation to ensure stability and uniformity of the internal temperature (VELP Scientifica, Usmate, Monza Brianza, Italy). The temperature of the thermostat was monitored by a data logger with an external probe in the range of -40 to +80 °C (XS Instruments, Carpi, Modena, Italy).

[0271] UHPLC ESI-qToF Method

[0272] All solvents were of high purity analytical grade and used without further purification. ULC / MS grade anhydrous methanol was purchased from Biosolve (Dieuze, France). Ultra-high purified water was purchased from Prepared in ultrapure water system (ELGA, UK). For LC-MS Formic acid (98%-100%) and dimethyl sulfoxide (≥99%) were purchased from Sigma-Aldrich (St. Louis, MO).

[0273] "Mixture C" containing omeprazole was purchased (purchased from a local pharmacy, batch number LC52792), and "Mixture D" was produced by Aboca (batch number 21D1848); omeprazole was purchased from Sigma-Aldrich (St. Louis, MO).

[0274] High purity reference standards used to build the internal database and calibration curves were purchased from Extrasynthese (Genay, France), Sigma-Aldrich (St. Louis, MO), PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany), and ChromaDex (Irvine, CA). High purity reference standard stock solutions were prepared at 500 ppm in methanol, water / methanol (80:20, v / v), or methanol / dimethyl sulfoxide (80:20, v / v). Working solutions were prepared by diluting an appropriate volume of stock solution with water / methanol (50:50, v / v). Internal standard sulfadimethoxine-d6 was purchased from Sigma-Aldrich (St. Louis, MO). Internal standard stock solutions were prepared at 5 mg / L in methanol. All stock solutions were stored in glass vials at -80 °C.

[0275] Prior to collecting the blot, each blotted sample was filtered to eliminate microfauna and thereby substantially stop the biodegradation process at the desired time t.

[0276] Targeted Suspect Screening Method

[0277] Sample preparation: The samples were filtered on 0.20 μm Millipore cellulose acetate syringe filters and used for acquisition of chromatograms without any dilution according to the following conditions.

[0278] Instrument parameters. The instrument platform used consisted of a UHPLC series 1290 combined with a high-resolution (2 GHz) quadrupole time-of-flight (qToF) mass spectrometer series 6545 (Agilent Technologies, Santa Clara, CA). The UHPLC was equipped with a binary pump, autosampler, multicolumn thermostat, isocratic pump, and solvent cabinet. The autosampler was maintained at 15°C.

[0279] For “Mixture C”, the analytical column was ACQUITY UPLC BEH C18 VanGuard TM ACQUITY UPLC protected by a pre-column (5x2.1mm, 1.6μm) C18 (100 x 2.1 mm, 1.6 μm), both columns were provided by Waters (Milford, MA) and thermostated at 40°C.

[0280] For "Mixture D", the analytical column is C18 VanGuard TM Pre-column (5x 2.1mm, 1.6μm) protected C18 (100 x 2.1 mm, 1.6 μm), both columns were provided by Waters (Milford, MA) and thermostated at 40°C.

[0281] According to the following gradient (Table 7), HCO2H aqueous solution (0.1%, v / v) was used as mobile phase A, HCO2H (0.1%, v / v, in MeOH) was used as mobile phase B, and the elution gradient analysis was performed with a flow rate of 0.3 mL min -1 The injection volume for 1000 mg / L samples was 3 μL, while the injection volume for 100 mg / L samples was 10 μL.

[0282] Table 4. Elution gradient

[0283]

[0284] The UHPLC system was coupled to a qToF mass spectrometer equipped with a dual AJS ESI source operating in positive ionization mode for "Mixture C" and in negative ionization mode for "Mixture D" with a scan range of 50 m / z to 1700 m / z. The optimized instrument parameters are shown in Table 5.

[0285] Table 5. MS parameters

[0286]

[0287] Internal PCDL construction was performed by acquiring each analytical standard reference compound in data-dependent mode (AutoMS / MS, Agilent Technologies) using three different collision energy values ​​of N2 (20 eV, 30 eV and 40 eV). In contrast, the sample traces studied were acquired in all-ion mode

[15]

[16] using a collision energy value of 30 eV. In both acquisition methods, a reference mass solution containing purine and hexa(1H,1H,3H-tetrafluoropropoxy)phosphazine was directly injected into the ESI source by an isocratic pump and ionized together with the sample solution for mass calibration, thereby obtaining accurate mass time-of-flight data.

[0288] software.MassHunter software version B.07 (Agilent Technologies, Santa Clara, CA) was used for data acquisition and processing. The accurate masses of various molecular species were calculated by the isotope distribution calculator - MassHunterData Analysis core version 8.0.8208.0 (Agilent Technologies, Santa Clara, CA) tool. The Metlin library used was Metlin_Metabolites_AM_PCDL, version 7.0.

[0289] Data analysis. Elaboration using the Qualitative Analysis program and the Find by Formula algorithm. Load the data file into Qualitative Analysis and run Find by Formula on the low channel against the MS / MS library. Find by Formula returns possible precursor formulas found in the library and their product ions. Using the product ion list, Qualitative Analysis extracts the EIC from the high channel and aligns it with the EIC of the precursor. The co-eluting fraction is calculated and compounds that pass a threshold (set by the user to 70) are retained. Qualitative Analysis returns a total score formed by the mass score, isotopic abundance score, isotopic spacing score, and retention time score, with a user-set threshold of 70. The EIC is obtained by setting the m / z spread to 2.5 ppm, symmetric.

[0290] Non-targeted approach

[0291] Sample preparation : Prepare samples as described for the targeted suspect screening method.

[0292] Multivariate statistical analysis

[0293] Data Collection . Prior to UHPLC-qToF acquisition, samples of mixtures Ct T0 and T28 and the blank samples consisting of mineral medium and inoculum were organized into random sample queues. Analytical blank samples and mixed samples were collected at the beginning, middle and end of the sample sequence. Each sample was collected in triplicate. The instrument parameters were the same as those used for suspect screening, positive ions for "Product" A and negative ions for "Mixture D".

[0294] Data processing.Peak quantification and peak alignment were obtained using the software R [R version 3.5.2 (2018-12-20), Copyright (C) 2018 The R Foundation for Statistical Computing]. The mass tolerance was set to 10 ppm and the retention time tolerance was set to 10 seconds. The signal filtering threshold was set to 1000 counts. Variables with missing value frequencies and coefficients of variation exceeding 20% ​​were removed and data interpolation was performed. In addition, filters were applied to remove those variables present in the analytical blank samples.

[0295] The median of the replicates was applied to eliminate the effect of random noise, while data normalization was performed by PQN (probabilistic entropy normalization) centering and scaling to a reference mixed sample.

[0296] The processed data were saved in CSV format, and the data matrix in CSV format was reprocessed using the SIMCA program (version 16.0.2.10561, January 22, 2020, Umetrics / Sartorius). Subsequently, the data matrix could be mean-centered and Pareto scaling could be applied before data analysis.

[0297] Statistical model performance evaluation was performed and a 95% confidence level was used. Goodness of fit and goodness of prediction were measured by Q 2 X and R 2 X is evaluated.

[0298] <![CDATA[Q 2 ]]> <![CDATA[R 2 ]]> "Mixture C"_50mg / L (omeprazole) 0.948 0.985 "Mixture D"_75mg / L 0.795 0.989

[0299] Computational diagnostic tool Hotelling T 2 and dModX to ensure that the model is not deformed, so there are neither strong nor weak outliers. The model is robust because the observed values ​​are below the calculated critical values ​​and there are no outliers.

[0300] PCA analysis : After data processing, in order to evaluate the complex imprints obtained, multivariate principal component analysis (PCA) was used. The PCA model was constructed using the first two principal components providing a 2D model (confidence level 95%).

[0301] Cluster analysis : Cluster analysis was performed using a hierarchical algorithm according to an unsupervised approach (confidence level 95%). Distance indices were determined using Ward linkage and Euclidean distance.

[0302] Semi-quantitative determination. At T28, the area counts for omeprazole and the area counts for the identified compounds were divided by the area counts for the internal standard (sulfadimethoxine-d6). All correction values ​​were then added together to give the total area. The area percentage for each compound at T28 was calculated by dividing each correction area by the total area and multiplying by 100.

Claims

1. A method for evaluating the biodegradability of a mixture of organic compounds, comprising the following steps: a) preparing at least one test flask or container containing a predetermined amount of a mixture of organic compounds of interest suspended in a suitable mineral medium and an inoculum, and at least one blank flask or container containing only said mineral medium and said inoculum, wherein said inoculum is obtained from activated sludge, effluent (non-chlorinated), surface water and soil, or a mixture thereof; b) collecting a sample blot from each flask or container prepared in a) by liquid chromatography-mass spectrometry at T0, T0 being the day of said preparation a), wherein each sample is filtered before collecting said blot, to eliminate the micro-fauna therein; c) collecting a sample blot from each test flask or container prepared in a) by liquid chromatography-mass spectrometry at Tn, where n is any integer greater than 0 and represents the number of days after said preparation in a), wherein each sample is filtered to eliminate microfauna therefrom before collecting said blot; d) Perform multivariate statistical analysis on the data obtained in b) and c); e) comparing the multivariate statistical analysis results of each test sample and blank sample obtained in d), and evaluating the biodegradation of each test sample by evaluating the distance between the blank sample data obtained at Tn and the test sample data obtained at least at T0 and Tn.

2. The method according to claim 1, wherein said step c) is repeated at different Tn, and said steps d) and e) are performed for each blot of said test sample and blank sample collected at the same Tn for each value of n.

3. The method according to claim 1 or 2, wherein one of said Tn is T28, and T28 is the 28th day after said preparation a).

4. The method according to any one of claims 1 to 3, further comprising the following steps: f) From T0 to T28, the oxygen consumption, and / or the carbon dioxide production and / or the dissolved organic carbon consumption are measured by one or more calibrated continuous reader probes or by discrete readings arranged with an appropriate number of containers for each predetermined measurement.

5. The method according to any one of claims 1 to 4, wherein the mineral medium is prepared from stock solutions of the mineral components potassium phosphate and sodium phosphate plus ammonium chloride, calcium chloride, magnesium sulfate and iron (III) chloride at appropriate concentrations.

6. The method according to any one of claims 1 to 5, wherein the culture medium is further supplemented with suitable growth factors.

7. The method according to any one of claims 1 to 6, wherein the test samples are organized in a random sample queue before being collected by liquid chromatography-mass spectrometry, wherein the sample queue also contains one or more mixed samples and one or more blank samples.

8. The method according to any one of claims 1 to 7, wherein the liquid chromatography-mass spectrometry technique is performed by UHPLC-qToF.

9. The method according to any one of claims 1 to 8, wherein the multivariate statistical analysis is an untargeted unsupervised analysis.

10. The method according to any one of claims 1 to 9, wherein the multivariate statistical analysis is performed by PCA (Principal Component Analysis) and / or HCA (Hierarchical Cluster Analysis).

11. The method according to claim 10, wherein the multivariate statistical analysis is performed by PCA, and in step e), when the PCA result of the test sample at Tn is close to the 2D space of the PCA result of the blank sample at the same Tn, the test sample is considered to be biodegradable, and when the PCA result of the test sample at Tn is not close to the 2D space of the PCA result of the blank sample at the same Tn, the test sample is considered to be not biodegradable or only partially biodegradable; and / or Wherein the multivariate statistical analysis is performed by HCA, and in step e), when the HCA result of the test sample at Tn forms a cluster with the HCA result of the blank sample at the same Tn, the test sample is considered to be biodegradable, and when the HCA result of the test sample at Tn does not form a cluster with the HCA result of the blank sample at the same Tn, the test sample is considered to be non-biodegradable or only partially biodegradable.

12. The method according to any one of claims 1 to 11, wherein a targeted qualitative analysis is performed on one or more of the test samples.

13. The method according to any one of claims 1 to 12, wherein the organic compound mixture is a pharmaceutical composition or a cosmetic composition or a food supplement composition or a medical device composition or a nutraceutical composition.

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