Method Development and Validation for Nutraceuticals

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1 White Paper Method Development and Validation for Nutraceuticals Maud Silvent Technical Specialist David Neville Technical Specialist

2 Method Development and Validation for Nutraceuticals Introduction Nutraceutical is a term formed from the amalgamation of the words pharmaceutical and nutrition by Dr Stephen Felice in Others have since tried to further define the term to distinguish and clarify the difference between functional foods, dietary supplements and nutraceuticals. There is now some consensus that a nutraceutical can be loosely defined as a functional food or supplement that aids in the prevention or alleviation of a disease state or disorder (except for anaemia), and not just supplement the diet. Therefore, a nutraceutical may be incorporated into a food product/matrix, or into a pill, capsule, tablet etc. There is no strict legal definition in the US, Canada or Europe to define nutraceutical, but other legal requirements do exist to cover dietary supplements and novel foods within these markets. Many of the compounds present in nutraceuticals that may possess beneficial health effects are derived from botanical sources, and will be considered to be natural in form. These products can be as diverse as proteins, peptides, lipids, flavonoids and isoflavanoids, polyphenols and tannins. Additionally, vitamins and minerals, sugar molecules such as glucosamine and chondroitin, may also be considered as nutraceuticals. Therefore, the range of nutraceuticals that require analysis is diverse, both in types of structures and matrices, and this is matched by the diversity of techniques and extraction conditions that need to be employed for accurate quantitation of the active nutraceutical of interest. It is also important to note that the purification, or not, of the nutraceutical from its natural matrix must also be considered as there may be contaminants/impurities/adulterants present that are deleterious to human health, or whose amounts are regulated by legislation (heavy metals, allergens, toxins). These must also be incorporated into a testing regime to ensure the safety of the product end-user. This article focuses on the analytical approaches that must be considered when performing a method development and validation exercise to ensure that your test method is fit-for-purpose, and meets the regulatory need of your nutraceutical of interest. The ultimate aim is to accurately quantify your nutraceutical of interest whether in final product matrix or as a raw material prior to incorporation into product. Due to the inherent complexity of many of the nutraceutical products that exist on the market, and to the complexity of the nutraceuticals themselves, there is no single analytical approach that can be undertaken. The extraction of the nutraceutical can be performed in a number of ways from simple liquid extraction into water or organic solvent, to more complex distillation procedures. This extraction is prior to final analytical procedures that may be HPLC- or GC-based with fluorescent, UV, mass spectrometric, refractive index, FID or other detection techniques. Additionally, ELISA, enzyme-based kits and chemical assays can be used. These assays are often separate from the type of testing regime that may be necessary to define the physicochemical properties of your product of interest. Therefore, the principles that must be followed to ensure the assay is accurate, precise, reproducible, and fit-for-purpose are outlined. Development of Assay At the outset of any method development and validation procedure it is important to understand the requirements of the assay, how the data will be used, who will perform the assay and where the assay will be employed. Factors such as in-line or near-line testing (quick and easy), experience of operator, QC/QA or NPD laboratory (robust), for regulatory submission, equipment capabilities, and method transfer will need to be considered. These findings will help in determining the best approach that can be taken to ensure that the method fulfils the business need. The experience of the laboratory concerned in analysing similar matrices, a comprehensive literature search to discover what is currently available (approved methods from AOAC, Pharmacopoeias, AACC and ISO, or published peer-reviewed articles) and the availability of appropriate standards can be investigated. Approved methods can form the basis of any new method that may be required, and are excellent starting points for development studies. As many of the analytical methods currently used are based on HPLC separation prior to detection by the most appropriate method for the nutraceutical of interest (UV, RI, Fluorescence, ELSD, MS etc.), this will be the focus of this article. However, the principles still apply to other types of analytical methods that Page 2

3 may be used, such as wet chemistry, colourimetric or enzyme-based. Following the initial scoping phase, development becomes a matter of experimentation to find the optimal analytical parameters, such as ph, extraction solution, mobile phase, type of liquid chromatography column, and retention time, for the method. The optimisation of each parameter will require careful attention to detail. This will ensure that maximum recovery of nutraceutical and accurate quantitation of the analyte of interest are achieved. Adequate time must be taken for each separate phase as these conditions will be used to determine the final, optimised and streamlined procedure in the next stage of the method validation. Additionally, it is the optimisation of each stage (see Figure 1) that ensures that progress through method validation is as easy as possible. It is important that the source and quality of reagents used, the supplier of the HPLC system (and model number of component parts) used, and instrument settings are specified within the method to ensure that the method can be reproduced by other operators and laboratories. Figure 1 HPLC method development single considerations It is important to note that method development and validation can be undertaken to analyse a component, but within a number of different matrices. As the nutraceutical may be incorporated into different product matrices, a decision must be made whether the development and validation is for one or multiple matrices. It is often better to develop and validate for multiple matrices rather than go through an extension-of-scope exercise. This, of course, is highly dependent on the initial product development cycle, and the amount of analytical time available to perform the necessary experiments. Validation of Assay Validation is defined by the International Organization for Standardisation (ISO) as verification, where the specified requirements are adequate for an intended use. Additionally, the term verification is defined as the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled (ISO/IEC 17025:2005 cl ). The method validation starts only when the method parameters have been finalised as part of the method development. In majority of cases, a validation protocol will be written, and agreed upon by the relevant people (analyst, laboratory manager, QC/QA department, clients etc.), following the ICH guidelines that are routinely based on ISO guidelines. The acceptance criteria will be set using data generated during the method development stage but will also depend of the agreed compound specification tolerance. Additionally, as previously mentioned, the source and quality of reagents used, and the supplier of the HPLC system (and model number of component parts) used are defined. Final instrument settings that are optimal for the nutraceutical of interest (column temperature, eluent gradient conditions, and detector settings) are also defined, and used, as part of the validation exercise. It is important to note that not all laboratories are equipped with the same HPLC systems. Therefore, part of the validation may incorporate a study to test the equivalence of results using different HPLC systems (see reproducibility below). Why is it important to validate a method once it has been developed? The answer is to be confident in your results and to ensure the method is fit-for-purpose for that specific compound in that specific matrix. When would it be required? At any stage of discovery, development, and manufacture of pharmaceuticals or food products, but also to perform stability studies, and to submit a dossier for claim substantiation or novel food ingredient. For example, knowing the uncertainty of the method would give you an indication if a trend in stability results is genuine or not. The purpose of a method validation is to confirm that the method is specific, precise, accurate and robust. The level and extent of the validation will evolve with the Page 3

method reproducibility) Limit of detection (LOD) and limit of quantitation (LOQ) Solution stability Robustness Each parameter will give specific indication of the method performance, and each topic

4 nutraceutical development phase. Typical method parameters recommended by FDA, USP, and ICH are as follows: Specificity, Linearity range Accuracy Method precision (incorporating method repeatability, intermediate precision and method reproducibility) Limit of detection (LOD) and limit of quantitation (LOQ) Solution stability Robustness Each parameter will give specific indication of the method performance, and each topic is covered in greater detail below. Specificity The specificity for the compound(s) of interest needs to be evaluated to understand the ability of the method parameters to separate any potential interferences coming from the sample matrix like other excipients, potential degradation products and the mobile phase. Ideally a placebo sample with the same ingredients/excipients but without the compound(s) of interest will be available to analyse to determine the specificity of the method. A forced degradation study can also be performed to artificially stress the product and obtain potential degradation compounds that could interfere with the analysis of the compound(s) of interest. Linearity The linearity will assess the proportional range of the method response, i.e. the concentration range over which the method can accurately determine the compound concentration. A minimum of five standard concentration levels, covering the concentration range in the final dilution of the sample matrix to be analysed, is recommended for investigation. In the case of a chromatography technique, the different standard solutions will be injected and analysed in ascending and descending order to assess if the method is affected by sample carryover into the next injection/analysis. It is also at this stage that the standardisation parameters (use of internal or external standards for accurate quantitation of analyte) are investigated. Accuracy The accuracy is the capability of an analytical method to determine the correct measurement, or exactness between a measured value and a theoretical value. The best approach is to work with certified reference materials (CRMs). However, those are not always available for the compound(s) of interest or for the specific matrix. Working with CRMs would increase the confidence in accessing the efficiency of the extraction procedure of the compound(s) of interest from the sample matrix. A second approach would be to spike the sample extraction solution with a known concentration of the compound(s) of interest and check how much is recovered. This approach will not investigate the efficiency of the extraction procedure on a sample embedded in a matrix, but only how the sample matrix may affect the spiked sample extraction procedures. Investigation of different levels of spiking is the best approach when CRMs are not available. It is important that the level of spiking should cover the concentration of the compound(s) of interest in your sample matrix over time. Figure 2 Precision and accuracy Precision The method repeatability is the ability of an analytical method to produce consistent results. The precision of a method is determined by its repeatability, intermediate precision, and reproducibility. A simplified diagram to illustrate accuracy and precision is shown in Figure 2. The repeatability will be measured to define the consistency of results when analysed by the same analyst, using the same instrument under the same analytical conditions. In most cases, it is assessed by preparing and analysing the same sample six to ten times. The percentage standard deviation will be assessed and compared to the acceptance criteria laid out in the validation protocol. The intermediate precision will be measured to define the consistency of results when samples are prepared and analysed by a second analyst using the same analytical conditions. Where possible, it is also best practice to perform the new analysis using a second column of the same specification as the first, and a second HPLC system (may or may not be the same manufacturer/model of HPLC system). This will evaluate the intra-laboratory precision. The percentage standard deviation will be assessed and compared to the acceptance criteria laid out in the validation protocol. The reproducibility evaluates the consistency of results between different laboratories or interlaboratory precision. It is worthwhile to note Page 4

The repeatability, intermediate precision, and reproducibility are used to determine the method uncertainty or relative uncertainty.

5 that The International Conference for Harmonisation (ICH) addresses ruggedness as intermediate precision and reproducibility. The percentage standard deviation will be determined and compared to the acceptance criteria laid out in the validation protocol. The repeatability, intermediate precision, and reproducibility are used to determine the method uncertainty or relative uncertainty. Limit of Detection (LOD) and Limit of Quantitation (LOQ) The limit of detection (LOD) is commonly defined as the lowest concentration of the compound(s) of interest that can be detected under given method conditions using the defined instrument parameters. In chromatographic analysis a signal-to-noise (S/N) approach may be taken, where there is a detected baseline noise in the method. The LOD may be calculated as being of three times the signal-to-noise (S/N) ratio. This is, in essence, an instrumental detection limit. It is important to note that though the compound can be detected, it may not necessarily be within the linearity of the assay. Therefore, the parameter limit of quantitation must be defined. Figure 3 The relationship between LOQ, LOD and linear range of assay The limit of quantitation (LOQ) is the lowest concentration of the compound(s) of interest that can be accurately and precisely quantified under given method and conditions. The LOQ may be calculated as being the sample concentration that gives a ten times the signalto-noise ratio, and lies within the linear range of the assay. The relationship between LOQ, LOD and the linear range of the assay are illustrated in Figure 3. Solution Stability There may be times when samples cannot be immediately analysed, or there is a time delay between sample preparation and actual HPLC analysis, or prepared samples need to be reanalysed. Different conditions of temperature and times-of-storage of prepared samples and standards are assessed to determine if these are detrimental to sample quantitation. Robustness The robustness will assess the method ability to obtain consistent results when small variations in the analytical parameters are applied. The parameters under scrutiny could be column temperature, mobile phase concentration, flow rate, ph value, sample and standard solution stability, mobile phase stability, any other parameters specific to a particular technique that could change over time and could potentially affect the results. Control Once the method has been validated, it may be useful to perform continuous quality control on the assay to ensure that it is performing within specifications. Control (or Shewhart) charts are useful for large volume or continuous work. They require starting with at least values to calculate a mean and a standard deviation (SD), which form the basis for control values equivalent to the mean ± 2 * SD (warning limits) and the mean ± 3 * SD (rejection limits). At least one replicate test portion of a stable in-house reference material and a blank are run with every batch of test samples. The calculated mean and standard deviations (or range of replicates) of the controls are continuously assessed. These values are used for ongoing determination of warning and rejection limits. The analytical process is in control if not more than 5% of the values fall in the warning zone. Any value falling above the rejection limit or two consecutive values in the warning region requires investigation and corrective action. Additionally, trends in the values obtained can indicate that further investigation of the analytical method may be required. Challenges A major challenge for nutraceutical analysis in both method development and validation is the availability of standard or certified reference materials. In the case of a compound where no previous method exists, it can be difficult to obtain a reference material, or considerable cost can be incurred in synthesising reference material. Additionally, because the compounds of interest may be endogenous to a matrix Page 5

6 (such as a plant extract), the level of analyte often varies, depending on the agronomic conditions. Therefore, developing and optimizing methods for endogenous compounds usually requires a great deal of experimentation for the extraction and isolation phase of the method. Ensuring that all of the analytes of interest have been extracted from the matrix is critical for the analysis of complex matrices, such as processed foods. There is less difficulty where the compound of interest is embedded within clean matrices (tablet, capsule etc.) with known excipients. Considerable expense can be incurred when developing and validating new methods. This expense may be warranted where it can be foreseen that the method will be used for multiple samples over an extended time period. Both the business need, and return-oninvestment (ROI), must be taken into consideration when assessing the feasibility of developing and validating any new method. It is also worth noting that as instrumental technology and detection methods evolve, new methods may be necessary to meet the demands of speed and efficiency. Additionally, regulatory requirements may force the adaptation of new methods that are more sensitive, both in terms of compound determination and identification of impurities/adulterants within the sample matrices. Conclusion Method development and validation is necessary to ensure that the analytical procedures for a nutraceutical are fit-forpurpose, and can accurately quantify the compound of interest. The process can be time consuming and expensive, both in terms of man-hour and equipment/consumables costs. The work performed is carefully planned and must be documented at all stages to ensure that due process is followed. Once the method is developed, a validation protocol is agreed and, deviations from this procedure can occur unless agreed upon and approved by the relevant authorities. This does give some scope for limited further method development, but ideally this would be minimal. The purpose of the development phase is to try and ensure that the validation is performed using as close to as final a method as is possible. The validated final method can be specific for single or multiple matrices, but each matrix must be tested as part of the method development and validation exercise. Before the method can be put into routine use, the validation report must be issued and approved. Subsequently, the method can be transferred to the laboratory where appropriate training of personnel can be undertaken. When the required level of proficiency is attained, the method can be placed into routine use. David Neville Technical Specialist David is a Senior Associate Principal Scientist at Reading Scientific Services Ltd (RSSL). His expertise is in the analysis of sugars, carbohydrates, bioactive molecules and proteins in both food and biological matrices, and has published widely on these topics. Additionally, he has developed and validated a number of methods for detection of bioactive compounds within varied food matrices. Maud Silvent Technical Specialist Maud is a Senior Scientist at Reading Scientific Services Ltd (RSSL). Her expertise is in ion chromatographybased analysis of sugars in varied food matrices. Many of the analyses are novel and require, mostly performing method development and method validation. For further information, please contact Customer Services on enquiries@rssl.com Page 6

Analytical method validation. Presented by Debbie Parker 4 July, 2016

Analytical method validation. Presented by Debbie Parker 4 July, 2016 Analytical method validation Presented by Debbie Parker 4 July, 2016 Introduction This session will cover: Guidance and references The types of test methods Validation requirements Summary Slide 2 PharmOut