Reference standards handling

Two different and possibly complementary approaches have been explored. One utilizes a panel of quantifiable internal reference standards (QIRS), which are common proteins present widely in tissues in relatively consistent amounts.11,22 In this instance because the reference proteins are intrinsic to the tissue they are necessarily subjected to identical fixation and processing, and incur no additional handling or cost, other than synchronous performance of a second IHC assay (stain), such that the intensity of reaction for the QIRS and the test analyte can be compared by IA, allowing calculation of the amount of test analyte (protein) present on a formulaic standard curve basis. The other approach seeks to identify external reference materials and to introduce these into each step of tissue preparation for cases where IHC studies are anticipated in this instance the logistical issues of production, distribution, and inclusion of the reference standard into all phases of tissue processing also must be considered, along with attendant costs. 

The laboratory shall have procedures for safe handling, transport, storage and ttse of reference standards and reference materials in order to prevent contamination or deterioration and in order to protect their integrity. 

Maintenance records of reference standards, stock standard solutions, and working standards are not complete, and a method of handling these standards is not properly documented. Storage of stock and working standards, labeling of all standards, and a plan for disposal are not well defined. 

Moxalactam disodium reference standards are equilibrated under controlled humidity conditions to raise the moisture content to a level where the material can be handled by ordinary means. This is accomplished by spreading a thin layer of material in a wide mouth weighing bottle which is placed in a 42% relative humidity chamber for about 16 hours before use. The 42% relative humidity chamber is prepared by storing a saturated aqueous solution of potassium carbonate in a desiccator or closed chamber held at room temperature. After a 16 hour equilibration period, the water content of the standard is determined by Karl Fischer titration. The activity of the standard is then corrected for the water content of the equilibrated reference material. Experience indicates that the water content of a properly equilibrated reference material should be in the range of 10 to 12%. 

To serve its intended purpose, each reference standard must be properly stored, handled, and used. Generally, reference standards should be stored in their original containers away from heat and protected from light. Follow any special instructions accompanying the containers. 

Since the aim of the protocol is to ensure the mitigation of problems, the essential elements of the protocol consists of sections that include (a) an Introduction, (b) treatment and disposition of data, (c) types of methods being transferred, (d) materials, reference standards, and reagents being used, (e) recommended type of equipment, (f) sample handling, (g) predetermined acceptance criteria, and (h) an Acknowledgment section. An example of a typical table of contents (TOC) of an analytical methods transfer protocol is discussed in Table 16-2. 

Reference standards are labeled such that their identity, potency, correct use, and correct storage can be assured at all times. Reference standard labeling must also ensure that laboratory personnel handling the material are properly informed of safety risks associated with potentially harmful chemicals. 

In most forms of quantitative spectroscopy, the sample and standard should be handled identically. In this IR asTsay6, one accurately weighs about 130 mg. of sample and of USP Propoxyphene Hydrochloride reference standard transfer both (quantitatively) to 125 ml. separatory funnels, containing 25 ml. of water. Add 0.U ml. of sodium hydroxide solution (l in 2) and 50 ml. of chloroform. Extract for 3 minutes and then allow the layers to separate. Drain the organic phase through anhydrous sodium sulfate into a 250 ml. beaker. Repeat the extraction with 3-50 ml. portions of chloroform and pool them in the 250 ml. beaker. Evaporate (on steam bath with air) to a small volume then transfer (quantitatively) to a 50 ml volumetric flask, dilute to volume with chloroform and mix. Using a suitable infrared spectrophotometer and 1 mm. cells, read the sample and standard at the maximum (about 5-80 a) using chloroform as the blank. The calculation is then samp. Abs. 

The sample should be as pure as possible, but standards have been used where the major constituent is approximately 85% of the mixture, as long as the remainder is made up of known compounds. Suggestions to use mixtures as reference standards have been made to reduce the number of assays run and the number of samples handled. This is clearly the case for a racemic mixture in an achiral HPLC assay, where both enantiomers have the same retention and detector response. It can also be beneficial to use known mixtures of diastereomers as a standard, provided there is sound reasoning that they give equivalent response to the method of detection. 

A reference standard log is typically maintain to provide an inventory of the standard, indicating who, when, and how much standard was removed. Care should be exercised when handling to avoid contamination of the standard. Furthermore, observe safety cautions when handling potent drug substance materials. 

Once a well-characterized, pure reference standard of known concentration is available, one must be aware of the fact that the actual preparation of the assay calibrators can also influence the PK standard curve. The physical treatment and handling of concentrated protein solutions can be very challenging. Some reference standards require specific handling techniques to prevent aggregation or denaturation of the protein. The effects of vortexing can be disastrous as can the process used to thaw frozen reference standard or calibrator solutions. Details such as the dilution pattern that is used in spiking the calibration solutions can contribute to assay bias and between-laboratory discrepancies. 

Samples of reference substances specified in the current instructions for testing should be obtained. Reference standards and any secondary standards prepared from them should be dated and be stored, handled and used so as not to prejudice their quality. 

A broad range of technical requirements is important in a laboratory quality system. These include aspects such as the selection of appropriately qualified and experienced personnel sampling, sample handling and preparation laboratory accommodation and environmental conditions equipment and reagents calibration reference standards and reference materials traceability (of standards and of samples) the selection or development, validation, and control of methods estimation of the uncertainty of measurements reporting of results and quality control and proficiency testing. 

USP publishes the list of official reference standards in a number of venues. The most accurate information regarding the availability of USP RS can be confirmed on line at USP s website www.usp.org and is updated every 24 hours. Users of USP RSs should note that the storage and handling instructions printed on the RS label take precedence over the storage and handling instructions that might be listed elsewhere on USP s website or in the official USP publications. 

Reference standard Selection of a standard, absolute spin concentration, storage, and handling of the standard... 

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