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Background: A requirement of current good manufacturing practices for dietary supplements is that manufacturers must identify their dietary ingredients. DNA-based methods can provide species-level authentication that may sometimes be difficult to achieve using conventional morphological and chemical analysis methods. However, because of varying levels of DNA degradation in botanical materials, many commercial tests fail to generate consistent test results across all types of botanical materials. AOAC published guidelines for validation of botanical identification methods and proposed probability of identification (POI) as a method performance parameter. However, few DNA-based botanical authentication methods in the literature follow these guidelines and evaluate POI. Objective: To provide a targeted PCR method validation example that follows AOAC guidelines for validation of botanical identification methods. Methods: Using Matricaria chamomilla (chamomile) as an example, we performed a single-laboratory validation for a targeted PCR method that aimed to identify both raw and processed chamomile materials. The performance parameters of the test were evaluated by carrying out an inclusivity/exclusivity study and a Specified Superior Test Material/Specified Inferior Test Material study to demonstrate that the method’s POI meets industry requirements. Results: The chamomile samples were identified by the method and achieved a POI greater than 0.9 with respect to all types of chamomile botanical materials. Conclusions: The method was validated for DNA-based identification of raw and processed chamomile materials, such as sterilized powders and extracts. Highlights: This work will provide insight for laboratories and manufacturers that aim to develop and validate DNA-based botanical identification methods.

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Background: The applications of deoxyribonucleic acid (DNA) barcoding methods have been extended from authenticating taxonomic provenance of animal products to identifying botanicals used as herbal medicine and in botanical dietary supplements. DNA barcoding methods for botanical identification must be adequately validated to meet regulatory compliance. Objective: The goal of this study is to provide a validation protocol for a two-tiered DNA barcoding method that aims to identify raw botanicals. Methods: A barcode database was computationally validated to define the barcode combinations that can unambiguously identify botanicals in the database. A maximum variation sampling technique was used to capture a wide range of perspectives relating to DNA barcode-based botanical identification, including plant parts and species distance, for the experimental validation. Twenty-two authenticated botanicals were purposively sampled from different plant parts—covering both closely related and distantly related species—to validate the two-tiered DNA barcoding method. The performance of the method was assessed on accuracy, precision, ruggedness, and uncertainty. Results: High accuracy (100%) and precision (1.0) were obtained from the validation samples. The method was also found to be rugged and have acceptable uncertainty. Conclusions: The method was validated and suitable for DNA-based identification of botanical raw materials listed in the current database. Highlights: This work will provide support guidance for manufacturers and regulatory policy makers to implement equivalent validated and compliant DNA-based testing in quality control processes to improve botanical raw material identification and authentication.