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The five batch analysis and its role in the registration process

Date:12-12-2022

Introduction


My name is Roberto Sardinha. I have a degree in Chemistry from the University of São Paulo (USP) in 2001. As soon I finished my degree, I had the opportunity to join a family-owned company that manufactures pesticides, and that’s when I got to know this interesting sector.


In 2004 I joined Bioagri Laboratories, which is currently Mérieux NutriSciences, where I had the opportunity to work on different studies, like animal metabolism and five batch, being a study director of five batch for about five years. After that I got different positions in the company but keeping close relationship with the progress of the five-batch analysis.


In this article I am going to discuss about the analysis of five batch and the importance of the study for the assessment of the product effects to the human health and to the environment.


The role of the five batch in the registration process


A simple way to describe about its importance is saying that through the knowledge of the composition of a chemical - with the appropriate knowledge of its components - it is possible to predict the product’s safety concerning the human health and the environment.


In the registration equivalent technical product process, the composition profile of the tested product is compared with the reference product. The reference product is the one with known composition profile and for which all toxicological and ecotoxicological evaluation tests has been carried out. Therefore, when the chemical composition of the tested product is equivalent to the composition of the reference product, it is considered that all toxicological and ecotoxicological knowledge of the reference product is also equivalent.


"A technical product may be considered equivalent whereupon: the maximum level of each non-relevant impurity is not increased by more than 50% in relation to the maximum level of the profile of the technical reference product or when the absolute maximum level is not increased above of 3 g / kg (whichever represents the highest incremental level), where there are no relevant new impurities and where the maximum level of relevant impurities is not increased." (Decree 4074.2002.)


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Chemistry Division


The five-batch study


Five-batch analysis comprises several different analyses performed on a representative five batches of a technical product produced in a specific manufacturing site and following a specific manufacturing process. As result, the five batch provides the full chemical composition of the tested product.


When I talk about full composition, I am talking about the identity and amount of the active ingredient and the impurities in significant levels (≥ 1g/Kg). It is also analyzed the impurities of toxicological concern with specifications published by different regulatory agencies, like FAO, EFSA and others.


The composition profile must reach at least 980 g/Kg identified and quantified.


The first and the most important step of the five-batch analysis is called screening analysis, followed by quantification and identification of the active ingredient, quantification, and identification of the significant and relevant impurities.


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Mass spectrometry lab


Screening analysis


The screening analysis aims to determine which are the impurities to be present in the composition profile, or as stated above, the significant impurities.


What are the possible impurities that may be present in the product?


As it is a technical pesticide product, with a known manufacturing process and known origin of the raw material used in its production, it is not a blind analysis with infinite possibilities. Considering its knowledge, the impurities can be:



So, the first step is to list the possible impurities considering the above list and to prepare the needed reference material and methods to investigate their presence on significant levels in the test samples.

The second step is done a scan analysis, which is able to detect non expected impurities and possible by products for which there are no reference material available.


Usually, this analysis employs chromatographic technique, which is developed according to the chemical characteristics of the active ingredient and the possible impurities. In most of the cases the system LC-DAD-MS is the best choice for this investigation.


Whereupon:


LC – Liquid Chromatography. Promotes the separation of impurities and active ingredient.


DAD – Diode Array Detector. Detects the signals of the substances after separation in the chromatographic column. The DAD can record a range of wavelengths, providing a chromatogram in the chosen wavelength and a 3D plot of the chromatogram showing the signals in the whole recorded range.


MS – Mass Spectrometry. Provides qualitative information as m/z of the peaks detected by the DAD, which refers to the impurity’s chemical identities.


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Figure 1 – Typical DAD chromatogram for screening analysis

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Figure 2 – Typical mass spectrum of impurity with fragmentation

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Figure 3 – 3D chromatogram


The result of the screening analysis is the list of impurities to be considered as significant, which will be fully characterized and quantified.


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3D Plot of screening chromatogram


Impurities characterization or identification


In some cases, the mass spectrum obtained in the screening analysis can clear the identity of the impurity. It happens when the impurity has a very clear and close difference comparing to the active ingredient. Anyway, for regulatory purposes, further data should be presented to confirm the identity of the impurity.


Once the chemical identity of the impurity is clear, it is possible to go to the reference material and proceed with the next steps.


However, usually, the mass spectrum is not enough to be sure about the molecular structure of the impurity. In these cases, the regular procedure is to isolate the impurity from the test sample in quantity and purity adequate to employ further spectrometric and spectroscopic techniques that clear its molecular structure with no doubts. Any possible doubt would provoke a requirement from the regulatory agencies.


The techniques used to complete the characterization can be: fragmentation in mass spectrometry, high resolution mass spectrometry (HRMS), Nuclear Magnetic Resonance (NMR) in different configurations (H1-NMR, C13-NMR, COSY, NOESY, and others), and other techniques, as required.


Finally, the identity of impurity is presented with at least its molecular structure, molecular formula, chemical name, and CAS number.


Quantification of significant impurities


The same chromatographic method developed for the screening analysis is usually employed for the quantification of impurities.


However, the method needs to be adjusted to be used for quantitative purpose and must be validated, and the reference material obtained for the characterization step can be used for quantification.


The method validation follows internationally recognized protocols, testing the parameters like linearity, precision, accuracy, limits of detection, and quantification.


The criteria for acceptance of validation results are determined by the protocols, and once everyone is reached, the method can be used.


The five samples are analyzed, and their amounts presented as % or g/Kg.


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Residue laboratory


Quantification and Identification of the active ingredient


As it is a known substance, the quantification and identification of the active ingredient is not as challenging as it is for the impurities.


The identification of the active ingredient must present at least two different techniques. Usually, it is employed MS and NMR in comparison with the reference material.


For quantification, the method for liquid chromatography is developed and validated, or in some cases, for gas chromatography, when the active ingredient is a regular organic compound.


When it comes to inorganics, the whole approach is different, and it is not being discussed in this article.


As with impurities, method validation follows internationally recognized protocols and results are expressed in terms of % or g/Kg.


Analysis of relevant impurities


Relevant impurities are substances with known toxicological concerns, and for which there are maximum limits established by different regulatory agencies, like FAO, EFSA, IUPAC, INC 02/2008 (Brazil), and others.


It is important to know these specifications before starting the study of the five batch, in order to plan and execute the analyses well.


Relevant impurities are developed aiming to reach the limit of quantification below the maximum established levels, and also analyzed by a method validated in accordance with international protocols.


Due to the low levels of some specifications, the analysis of relevant impurities requires sensitive and selective techniques and methods. The mass spectrometry is the best choice for most of the relevant impurities.


See some examples:

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Other analysis


As we saw in the discussion above, the main important steps of five batch analysis, with important complementary analyses to reach the mass balance, showing all the impurities at significant levels that contribute to reaching the minimum of 980 g/Kg as required by the regulatory agencies.


Once analyzed all the organic impurities and active ingredient, it is time to analyze the possible inorganic and solvents, according to the manufacturing process of the test product.


These substances employ different techniques, as shown below:



All the above analyses are performed by methods validated in accordance with international guidelines. Results are expressed in terms of % or g/Kg.

Results


As a result, of the entire study, a table is presented with the composition profile of the analyzed five batches, as follows:


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(1)ND = Not Detected (lower than the limit of detection)


Conclusion


The result of the five-batch study provides the entire composition profile according to the results obtained from the analysis of the five batches of the test product.


This result is used by the registrant to indicate the composition profile of the product, which comprises the minimum active ingredient content and the maximum content of the impurities.


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Test Facility Piracicaba


Mérieux NutriSciences – Bioagri Laboratórios Ltda.


 
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