Method Development and Validation in Dietary Supplement Testing
In order to ensure dietary supplement safety and validity, analytical testing is used to verify ingredient identity,
purity, composition, and potency. These tests are far from random; they require careful selection and validation before being applied to consumer products. In many cases, the ingredients in a product are associated with standardized approaches available through organizations like USP or AOAC. In others, new methods (non-standard) need to be developed. The figure below walks through the stages of method validation and development, starting with using a standard method in the bottom circle.
Searching for a suitable method
When a testing lab receives a new product, they first search for a standardized method applicable to the ingredients and matrix. The ideal situation is when a method is available that matches the exact conditions of the product. Sometimes, there are methods for similar products, but they need to be expanded to include a new ingredient in its scope. Other times, a method exists but must be adjusted to work with a lab’s available equipment. In some cases, no method is available and a new one must be developed based on experience and expertise.
Method validation
No matter the case, methods must be validated prior to testing. Validation includes confirming and reporting the selectivity, sensitivity, range, accuracy, precision, and robustness of a method using regulated approaches. Even if a method is only slightly modified, it must be fully validated before use. Validation is done using blanks (including reagent, sample, and spiked blanks) and reference materials and standards. This allows scientists to confirm that the method can detect the compounds of interest (either ingredient or contaminant compounds) without interference from analytical factors.
Method development
In the case that no method exists and a new one must be developed, scientists use a combination of experience, expertise, published literature, and similar products to create a scientifically valid workflow. Usually, labs will try multiple instruments and methods to determine which is the most reliable and reproducible for a new ingredient. For example, UPLC, LCMS, and GCMS methods might be developed to quantify a compound’s concentration, but only the UPLC method has high accuracy. Unlike method validation, method development is not regulated and is done based on the lab’s equipment and capabilities.
Since method development is not regulated, it is crucial that proper validation is performed when an entirely new method is proposed. Validation can be done in a single laboratory, but ideally is done with multiple labs to ensure reproducibility. The figure below outlines the many steps involved in validating a method. This is used for newly developed methods and when changes are made to existing methods.
Validation vs verification
It should be noted that there is a difference between method validation and verification. Validation evaluates the analytical performance of a new or expanded method for a given sample. Verification confirms that a method works for a product and tests the proficiency of an existing method.
What does validation actually involve?
- System suitability: Making sure the instrument, reagents, and method parameters can actually measure the compound of interest. If the instrument detector cannot detect the compound, then it is not much use for quantification and identification! Suitability is tested using a reference standard for the compound of interest and confirming that it can be detected and matches expected outputs.
- Calibration curves: A solvent is spiked with various concentrations of a reference standard, and the measured concentration using the method is recorded and plotted. The linearity of the resulting curve is used to confirm that the compound can be measured properly.
- Specificity/selectivity: Specificity and selectivity are similar measurements, ultimately ensuring that any compound detected in the sample is actually there, not just something in the solvent or instrument interference. This requires a solvent blank, a sample, and a sample spiked with a reference standard. The method is specific and selective if the compound of interest is found in the samples, but not in the blank.
- Precision: Precision is analyzed by evaluating differences in results from the same sample measured multiple times using the same method. In other words, precision is a measurement of method error. Precise methods will have very little differences in the results from sample to sample.
- Accuracy: Accuracy is how close the measured concentration of a compound is to the expected concentration. Typically, samples are spiked with varying concentrations of a reference standard and the measured recovery is calculated. Accurate methods will have a high recovery percentage, or have a concentration value close to the initial spiked value.
The experts at Eurofins have the experience and expertise required for method validation, verification, and development with a broad range of analytical instruments and product matrixes.
Our Eurofins team of experts can help you with all of your botanical testing needs.
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