Sub-stocks

Sub-stocks are also used for multi-analyte assays where a mixture of analytes in a single solution is prepared for subsequent preparation of QCs, calibration solutions or spiking solutions used for the preparation of matrix matched calibrators. In this case, individual stock solutions are combined to make one or more sub-stock solutions containing all of the analytes in one solution. This practice will reduce the number of steps that would be required versus making an individual spiking solution for each analyte. However, for methods that required a different LLOQ for each analyte, the sub-stocks and spiking solutions need to be prepared at the corresponding appropriate concentration for each analyte. 

At the onset of method development the appropriate concentrations of the sub-stock and spiking solutions may not be known, but estimates can be based upon the initial method development scheme and by considering what types of spiking solutions will be required for early sensitivity and recovery assessments. Also, to determine the required concentrations for these solutions, consideration must be given to the type of solvent and the volume of solution that will be used for any subsequent dilutions or for spiking the analyte into the control matrix (Section 9.5.6c). 

Table 9.6 An example of a preparation scheme for a calibration curve in solution prepared by direct dilution from a stock solution using three sub-stocks each of which was ...

The chemical stability of an analyte in a given solution that is stored under specific conditions for given time intervals is an important validation parameter that was discussed in general terms in Section 9.4.4f. An extreme form of instability is the propensity of the analyte to explode or be set afire, and such properties should be available in the MSDS information or, if the analyte is a new compound, the chemists associated with its synthesis or isolation will have discovered such properties before the analyst A crucial special case is the stability of the analyte (and thus of the analytical standard) in solution, as in stock, sub-stock and spiking solutions. The stabihty of analyte or of an analog internal standard in solution should be determined by comparing stored stock solution(s) to freshly made-up stock solution(s). For a stable isotope-labeled internal standard, stability data for the corresponding analyte are often used to estabhsh stahUity for such an SIS. 

Stability should be clearly established for the anticipated duration of storage and use by comparing peak areas for stored solutions against those for solutions made from fresh weighings of analytical standard at selected time intervals such as two weeks, one month, three months etc. As a practical consideration, it is often desirable to establish a full year of stock stability under appropriate storage conditions. If sub-stocks or spiking solutions are used and made in different solvents, then similar stability studies would be required for these but, for reasons of practicality, probably for shorter periods. 

Documentation of preparation of stock and sub-stock solutions must start with receipt of the reference standard, its Certificate of Analysis, assigned purity (and possibly chiral purity) and its history of storage and use after receipt (Section 9.4.4). Procedures for preparation and subsequent dilution of stock solutions are described in Section 9.5.4 and stability testing for these solutions in Section 10.4.Ih. Some of the relevant documentation might be included in general laboratory SOPs but full documentation of all study-specific procedures and data regarding preparation, storage and validation of stock solutions is required. 

Generalized requirements for validation of the stability of stock and sub-stock solutions were described in Section 10.2.7. The FDA guidance document (FDA 2001) does not, however, recommend any exact procedures about how this should be done Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. The procedure should also include an evaluation of analyte stability in stock solution . Also the following The stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least six hours. If the stock solutions... 

Transfer a 0.20 mL aliquot of blank serum or blood to an appropriately labeled 12x75 mm tube. Add 2 pL of the rodenticide anticoagulants mixed sub-stock standard. This 10 ng/mL calibrator serves as the reporting limit check for the assay. 

Laxative mixed sub-stock standard To a 10 mL volumetric flask, add 100 pL of each stock standard for desacetylbisacodyl, aloe-emodin, emodin, rhein, and phenolphthalein. Fill to volume with methanol. The final concentration of each compound in the mixed sub-stock is 1.0 pg/mL. Store this solution in a Teflon-lined capped amber vial at -10°C. 

Laxative working standard To a 13 x 100 mm tube containing 1 mL of blank urine, add 10 tL of laxative mixed sub-stock standard. Vortex to mix. Prepare fresh for each run duplicate single point calibrators are run at the beginning and end of each batch. 

QC sub-stock solutions To independent 10 mL volumetric flasks, add 150 pL (high QC) or 50 pL (low QC) of each QC stock solution. Bring to volume with methanol. Concentrations are high QC sub-stock, 1.5 pg/mL low QC sub-stock,... 

Working QC Add 10 pL of the high and low QC sub-stocks to separate 13x100 mm tubes containing 1.0 mL of blank urine. Prepare fresh for each run. Concentrations are high control, 15 ng/mL low control, 5 ng/mL. 

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