Reference standards quantity required

Reference Standard. One of the limitations of the external standard method is that a well-characterized reference standard is essential. In addition, each analysis requires accurate weighings of small quantities (e.g., 10 mg) of reference standard. Therefore, weighing error can affect method precision and accuracy. 

The design of comparative assays requires that the standard preparation and preparations with unknown activity be compared within tests. Thus, the reference standard must be available in sufficient quantity for distribution to testing laboratories and must have a certified level of activity that remains stable for a period of years. 

The Consultative Committee for Amount of Substance (CCQM) has set up a definition of primary methods [1, 2] and has selected some methods with the potential of being primary , from the viewpoint of the end user. From the point of view of metrology, methods used for linking the chemical measurements with the SI system at the highest level should not refer to other amount of substance standards. This requirement excludes methods which are relative in their principle. Some other methods identified as having the potential to be primary yield information expressed as amount fraction. This is essential for evaluation of purity, but in order to convert it to a value useful for transfer of the unit, additional information on the identity (molar mass) and content of the impurities is required. This additional information is needed to convert the result into amount content or similar quantities. 

Identify all samples being set aside for FDA validation. The samples should include drug substance, drug product, major impurities, and degradation products being controlled for, references standard, and internal standard (the latter is not required if commercially available but is recommended to facilitate FDA laboratory work). If appropriate, blanks and any other materials not commercially available but specified in the analytical procedures should be provided. The samples are to be maintained by the sponsor until the FDA s reviewing chemist provides instructions as to where they should be forwarded. The total quantities and the manner of their subdivision (e.g., 400 tablets, 4 x 100 tablets/ bottle) should be indicated. The amounts provided should be adequate to permit at least three separate determinations, excluding sterility, by two different laboratories. 

How much material will be required and for what purpose If material is needed just for structural identification, < 100 mg may suffice however, if a reference standard is needed, this may require gram quantities. 

Because reference standard material will eventually be consumed, replacement material must be isolated or synthesized. Due to this process, the method of preparing the standard should be reproducible for the future. Laboratory synthesis of a reference standard is preferred. The synthetic route does not have to be extremely efficient, since only 100-200 g will be required over several years. Preparative chromatography to acquire gram quantities can be practical where the separation is clean and the target compound is present above 3% in a concentrated mixture. 

Any discussion of the method development/validation process should consider obtaining a sufficient quantity of a qualified reference standard and appropriate samples to support the development and validation campaign. In most cases, the limit of the accuracy of the analytical test will be related to the correctness of the standard s assay. There is little consensus on the common requirements of reference standards as outlined by the regulatory authority typical practice favors an approach of thorough analytical characterization of the standard supported by adequate documentation. 

The ultimate goal of an assay or an analytical procedure is to measure accurately a quantity or a concentration of an analyte, or to measure a specific activity, as in some assays for biomarkers. However, many activity assays, such as cell-based and enzyme activity assays, may not be very sensitive, may lack precision, and/or do not include the use of definitive reference standards. Assays based on measurements of physicochemical (such as chromatographic methods) or biochemical (such as ligand-binding assays) attributes of the analyte assume that these quantifiable characteristics are reflective of the quantities, concentration, or biological activity of the analyte. For the purpose of bioanalytical method validation, we will follow the recently proposed classifications for assay data by Lee et al. [4,5]. These classifications, as summarized below, provide a clear distinction with respect to analytical validation practices and requirements. 

For all analytical testing, standard substances are required as reference material. These standards exhibit defined quality, and serve for all types of identity, purity, degradation product and potency assays as reference quantity. In QC, reference and working standards are used. Reference standards are highly purified and extensively characterized by all appropriate physico-chemical, biochemical and immunochemical methods. Working standards are used in daily practice, and are calibrated against reference standard for routine use. 

Pyrrolizidine alkaloids poison animals grazing on toxic wild plants and those fed contaminated feed, causing economic losses. They poison humans through deliberate consumption of certain foods and herbal medicines and through consumption of food contaminated by wild plants, such as via transport of the toxins by bees into honey. Analytical methods are required for different purposes - to detect the presence of pyrrolizidine alkaloids, to quantify the total level of the toxins, or to measure the quantity of individual compounds. The task is made more challenging by the variety of PAs, their widespread nature and their different forms. Analytical methods are based on color reactions, enzyme linked immunosorbent assays (ELlSAs), spectroscopy, and the full range of chromatographic techniques. A lack of reference standards and... 

With most non-isothemial calorimeters, it is necessary to relate the temperature rise to the quantity of energy released in the process by determining the calorimeter constant, which is the amount of energy required to increase the temperature of the calorimeter by one degree. This value can be detemiined by electrical calibration using a resistance heater or by measurements on well-defined reference materials [1], For example, in bomb calorimetry, the calorimeter constant is often detemiined from the temperature rise that occurs when a known mass of a highly pure standard sample of, for example, benzoic acid is burnt in oxygen. 

In titrimetry certain chemicals are used frequently in defined concentrations as reference solutions. Such substances are referred to as primary standards or secondary standards. A primary standard is a compound of sufficient purity from which a standard solution can be prepared by direct weighing of a quantity of it, followed by dilution to give a defined volume of solution. The solution produced is then a primary standard solution. A primary standard should satisfy the following requirements. 

To find a numerical value for AHi, we need to know ArH° at one temperature, while evaluation of I requires ArG° at one temperature. The usual choice is to obtain ArH° and ArG° at T = 298.15 K from standard molar enthalpies of formation and standard molar Gibbs free energies of formation. Earlier in this chapter we referred to examples of these quantities. It is now time to define AfH° and AfG° explicitly and describe methods for their measurement. 

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