Assays precision

Precision is a measure of random error and is defined as the agreanent among replicate measurements of a defined sample. It is generally expressed as the percentage coefficient of variation (%CV). %CV is calculated as 

TABLE 41.1 Summary of Validation Procedures and Performance Goals for Quantitative Assays 

Validation Parameter Design/Plan Target Performance Goals and Acceptance Criteria  

Calibration cnrve Perform duplicate analysis of standards across analytical runs CV S 20%, CV 25% at LLOQ, and recovery 

LLOQ Refer to calibration curve accuracy and precision data The lowest standard concentration that demonstrates acceptable accuracy and precision CV 25% and recovery of 80-120% 

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A study was conducted to measure the concentration of D-fenfluramine HCl (desired product) and L-fenfluramine HCl (enantiomeric impurity) in the final pharmaceutical product, in the possible presence of its isomeric variants (57). Sensitivity, stabiUty, and specificity were enhanced by derivatizing the analyte with 3,5-dinitrophenylisocyanate using a Pirkle chiral recognition approach. Analysis of the caUbration curve data and quaUty assurance samples showed an overall assay precision of 1.78 and 2.52%, for D-fenfluramine HCl and L-fenfluramine, with an overall intra-assay precision of 4.75 and 3.67%, respectively. The minimum quantitation limit was 50 ng/mL, having a minimum signal-to-noise ratio of 10, with relative standard deviations of 2.39 and 3.62% for D-fenfluramine and L-fenfluramine. 

Essential features of an automated method are the specificity, ie, the assay should be free from interference by other semm or urine constituents, and the sensitivity, ie, the detector response for typical sample concentration of the species measured should be large enough compared to the noise level to ensure assay precision. Also important are the speed, ie, the reaction should occur within a convenient time interval (for fast analysis rates), and adequate range, the result for most samples should fall within the allowable range of the assay. 

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

Table 5.20 Intra-assay precision and accuracy of the LC-ToF-MS determination of Idoxifene. Reprinted from J. Chromatogr, B, 757, Comparison between liquid chromatography-time-of-flight mass spectrometry and selected-reaction monitoring liquid chromatography-mass spectrometry for quantitative determination of Idoxifene in human plasma , Zhang, H. and Henion, J., 151-159, Copyright (2001), with permission from Elsevier Science...

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

Nominal concentration (jUg/ml or pg/g) Intra-assay precision (CV%) ( =10) Intra-assay precision (CV%) (n=6) Intra-assay accuracy (%> ( =10) Intra-assay accuracy (%) ( =6) Recovery (%) (mean SD)... 

This chapter describes how to be more successful in HPLC operation by summarizing a series of standard operating procedures representing the best practices of experienced HPLC analysts. It offers some brief guidelines on maintenance/troubleshooting and on means of enhancing HPLC assay precision. 

Repeatability (intra-assay precision) Precision under the same operating conditions over a short interval of time (e.g., 10 subsequent injections) " ... 

The authors determined specificity using the known hydrolytic degradation products. The precision of spiked samples of these degradation products were determined and found to be acceptable (99.9 0.4%). Accuracy of the method was determined using spiked recoveries of piroxicam benzoate, and the recoveries were acceptable (99.1-100.5%). Assay precision n = 6, RSD = 0.4%) was in accord with recommended criteria [7]. Within-day precision was performed on two instruments on two separate days, and the overall intermediate precision was 1.0%. The method was linear over the expected analyte concentration range giving a regression line of 1 = 0.999. The detection (DL) and quantification levels (QL) were assessed, and the latter was determined as 0.185 pg/ml (ca. 0.04%). 

This differs from simple within-assay precision in that it might be considered to compare the sum total of the operations carried out in an assay of a particular sample from the beginning, carried out by a single operator, with the same sum total of all critical operations, carried out by the same operator, probably in sequence with the initial assay and certainly within the same day. 

Intra-assay precision is evaluated by analyzing aliquots of a homogeneous material several times by one person on one day with the same equipment. Each analysis is independent, so the intra-assay precision is telling us how reproducible the analytical method can be. Intraassay variability is greater than instrument variability, because more steps are involved. Examples of specifications might be that instrument precision is <1 % and intra-assay precision is 2%. 

Define the following terms instrument precision, injection precision, intra-assay precision, ruggedness, intermediate precision, and interlaboratory precision. 

Like in the case of immunoassays, the MIP-ILAs will be governed by the Law of Mass Action when working at equilibrium so that the reagents are under equilibrium conditions and subject to temperature fluctuations. Shaking can also affect the local concentration of reagents and the reaction rate these factors must be controlled to improve the assay precision. 

Viral detection assays based on infectivity suffer from significant variability, which necessitates the use of statistical evaluation. Polymerase chain reaction-based assays are currently being developed and validated for viral clearance. With PCR assays, there is a potential to distinguish between inactivation and physical removal, perform mass balance studies, evaluate more than one vims at a time for a given process step, reduce the time for completing clearance studies, and accurately quantitate the amount of vims bound to such surfaces as chromatography resins. Table 5 compares the assay precision between an infectivity assay and a quantitative PCR assay. 

Dilution Integrity To check dilution integrity, a QC sample prepared at a concentration greater than the ULOQ is analyzed using dilution in blank matrix. Acceptable assay precision and accuracy are required. 

Inter- and Intra-Assay Precision Intraassay precision and accuracy are assessed within one batch (QCs, standards, etc.), whereas interassay precision and accuracy are assessed using separate batches. 

The assay precision data is obtained from the 100% level of assay accuracy. (See Sec. 4.1). 

The assay precision data are obtained from the 0.5-ppm level of accuracy. (See item 4.)... 

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