A reliable validated high-performance liquid chromatography-photodiode array detection method for quantification of terpenes in Copaifera pubiflora, Copaifera trapezifolia, and Copaifera langsdorffii oleoresins

Abstract The Copaifera oleoresins are widely used in folk medicine to treat various diseases. The goal of this study was to develop a validated reverse-phase high-performance liquid chromatography method with photodiode array detection (RP-HPLC-PDA) to quantify eight terpenes: ent-hardwickiic acid, ent-copalic acid, ent-7α-acetoxy hardwickiic acid, ent-16-hydroxy-3,13-clerodadiene-15,18-dioic acid, ent-5,13-labdadiene-15-oic acid, junenol, ent-kaurenoic acid, and 13E-ent-labda-7,13-dien-15-oic acid in the oleoresins of Copaifera pubiflora L. (OCP), Copaifera trapezifolia L. (OCT) and Copaifera langsdorffii L. (OCL). The linearity of the method was confirmed in the range of 20.00–500 µg.mL−1 (r2 > 0.999). The limit of quantification was between 1,05 and 16.89 µg.mL−1. Precision and accuracy ranges were found to be %RSD <0.2 and 96% to 110%, respectively. Based on the obtained results, the developed analytical method is rapid, precise, accurate, and sensitive for quantifying these terpenes in Copaifera's oleoresins. Graphical Abstract


Introduction
The Copaifera L. genus occurs in South America, Africa, and Asia.It comprises approximately 70 species of large trees, of which sixteen of them are endemic to Brazil.
Thus, quality control studies of copaiba oil are indispensable for the safe use of these oleoresins (Carneiro et al. 2017).The lack of characterising the Copaifera oleoresins from different species of copaiba at quality control constitutes an obstacle for registering and exporting products containing Copaifera oleoresins (Da Silva et al. 2017).Therefore, the isolation of main constituents and validation of an analytical method that fulfills all the analytical applications' requirements can ensure the reliability and safety of Copaifera oleoresins.
The analytical methods for quality control must be validated to provide reliable data in compliance with international regulatory guidelines (ANVISA RE899 2003; Souza et al., 2013).Therefore, we are reporting the isolation and identification of Copaifera oleoresins main constituents, the development and validation of an analytical method to quantify the main compounds in crude Copaifera oleoresins obtained from Copaifera pubiflora L. (OCP), Copaifera trapezifolia L. (OCT) and Copaifera langsdorffii L. (OCL).
The natural Copaifera oleoresins chemical compositions vary, depending on the collection site and its different species (Langenheim 1994;Barbosa et al. 2012).Their exploitation presents management characteristics that define the application possibilities and establish the quality for the market.One of them refers to the possible mixture of oleoresins from different Copaifera species and even oleoresins collected from specimens of different ages and locations, which causes variability in the chemical composition of commercialised oleoresins (Veiga et al. 1997).Thus, it is necessary to establish controlled procedures from the oleoresin collection to the commercialisation of standardised Copaifera oleoresins.Being one of the most critical aspects of quality control, constituting one of the focuses on the pharmaceutical and cosmetic industries (Barbosa et al. 2012).This is mandatory for the final products' quality, efficacy, and safety assurances.
Thereby, this situation reflects the difficulty in carrying out a chemical control of the product, being essential to establish techniques that allow rigorous quality control for the quantitative chemical standardisation of the Copaifera oleoresins commercial products.So, our research group has worked on pharmacological monitoring of qualitatively and quantitatively characterised Copaifera oleoresins fractions, and their isolated compounds, as agents responsible for several biological activities (Carneiro et al. 2017;Da Silva et al. 2017;Carneiro et al. 2020).
Therefore, facing the regulatory pressure to develop products within sanitary standards, efforts have been undertaken to standardise Copaifera oleoresins considering their volatile and non-volatile constituents.Also, the biological activities of oleoresins fractions and isolated compounds have been undertaken to determine the role of the chemical composition on the reported biological activities.For example, there is evidence of the correlation between anti-inflammatory activity and Copaifera oleoresins with higher diterpenic acid levels in their composition (Tappin et al., 2004).Another example is the diterpene ent-copalic acid as the chemical marker of the Copaifera genus, once this metabolite has been found in oleoresins of all species of this genus (Carneiro et al. 2017).This compound can serve as a quality marker both qualitatively and quantitatively.It is pointed out that the developed method is reliable, of low cost, versatile, and simple to be used routinely to analyse crude Copaifera oleoresins and their final products (Carneiro et al. 2017;Da Silva et al. 2017).
The quality control involves the assessment of the presence and quantification of the major metabolites and optimising several analytical parameters, from sample preparation, chromatographic separation, detection, and quantification, which are intrinsically related to the metabolites of interest.Thus, makes it possible to differentiate samples of oleoresins and indicate their quality through the presence of the major metabolites.

Analytical method
The chromatograms for OCP, OCL, and OCT showed a complex chemical profile.The developed analytical studies allowed the identification of prominent chromatographic peaks (Figure S2).It does not distinguish between copalic acid (2 b) and its positional isomer (8 b) in the standard analytical solutions.It is noteworthy that the sum of 1 b, 3 b, and 8 b corresponds to approximately 60% of OCP.Compound 1 b is the main compound representing the oleoresins OCP and OCT (Table S2).They can be used as a chemical marker for the quality control of these oleoresins.Because most of the analytical methods commonly used for quality control cannot distinguish copalic acid (2 b) and its isomer (8 b), it is necessary to establish new diterpenes that can be used as chemical markers for different species of Copaifera.The content of the identified diterpenes was considered low for OCL (17.8%).
The analytical curve was constructed by correlating the concentration (x-axis) versus the ratio between the peak area displayed by compounds 1 b-8b and the peak area of the internal standard (PI) that was constant (y-axis).The analytical curves were linear over the proposed concentration range (20.0-500.0mg.mL À1 ).They presented a linear correlation coefficient (r) ranging between 0.9989 and 0.9994.From the (r) values, the determination coefficient (r 2 ) ranged between 0.9978 and 0.9998.Thus, it was possible to assure the linearity of the method.The limits of detection and quantification values (LOD and LOQ, respectively) indicated that the proposed method detects low concentrations down to 0.3474 mg.mL À1 (Table S3).
The intra-day and inter-day precision were determined by quintuplicate analysis of the compounds 1 b-8b.The relative standards deviation (RSD%) was lower than 2.0%, denoting that method is precise (Table S4).The method's accuracy was assessed by adding concentrations at high, medium, and low levels for the compounds 1 b-8b, allowing to calculate the method's accuracy.The developed method displayed good accuracy, exhibiting good recovery (between 96.00 and 110.00).It can also be observed that the values of relative standard deviation (RSD%) were less than 2.0%, as well as for precision (Table S5) (Baccarin et al., 2011).
The method's robustness was analysed by examining the sources that are subject to variations.The rrt (relative retention time) and concentration were considered to calculate effects (Ex) in robustness, which were converted to RSD% and analysed (Table S6).The Copaifera pubiflora oleoresin (OCP) was chosen to assess the robustness of the methodology.The SRD% was lower than 20% when all factors were considered, except for the concentration-response wavelength detection (k).It can be explained by the characteristic profile of the ultraviolet-visible spectrum of such terpenes.Slight variations in the wavelength elicit changes in the absorbance of these compounds.The results obtained allowed us to consider that the developed method is adequate for the oleoresins quality control.

Conclusion
The obtained results allowed the identification and quantification of eight terpenes.The developed method is an important tool for the quality control of oleoresins from Copaifera pubiflora, Copaifera trapezifolia, and Copaifera langsdorffii.The development of the methodology described here proved to be an excellent strategy of practical and useful application in the daily life of analysts, as it is an easily accessible commercial herbal medicine.The methodology provided quick identifications and reliability, allowing the characterisation of chemical constituents and differentiation between the different Copaifera oleoresins.The developed analytical method meets the parameters of the Brazilian Regulatory Agency.