Inter-assay precision

Table 12.1 Accuracy (% deviation), intra-assay, and inter-assay precision (%CV) obtained in the validation of an assay for sirolimus in whole blood. Data from [16] ...

Bogusz et al. [68] reported the LC-APCI-MS analysis of sixteen phenethylamines after SPE. Subsequently, optimization of the mobile-phase composition for the LC-ESI-MS analysis of methylamphetamine and related compounds was described [69]. Methanol in the mobile phase resulted in a slightly higher abundance of the [M+H]. Formic acid was preferred over other additives. Microporous ultrafiltration was used in the sample pretreatment of urine samples. The intra-assay and inter-assay precision and accuracy in the 100-1000 ng/ml range were 2.3-6.3%, 0.8-5.9%, and 1.6-6%, respectively, for amphetamine in urine. 

Hoja et al. [74] reported a method for LSD and A-desmethyl-LSD in urine. The analytes were extracted from urine by means of an Extrelut-3 SPE cartridge. The eluate of the cartridge was evaporated to dryness and reconstituted in mobile phase prior to LC-ESl-MS analysis. LOQ were 0.05 and 0.10 ng/ml, with linearity up to 20 ng/ml. Intra-assay and inter-assay precision at 0.1 ng/ml were better than 9 % and 14%, respectively... 

Precision describes the variability in a set of measurements, i.e. how closely repeated measurements on a single sample agree. Precision can be calculated for samples within an individual itin (intra-assay precision) or across multiple runs (inter-assay precision). Precision is customarily expressed as the percentage coefficient of variation (%CV, or simply CV), which is the standard deviation expressed as a percentage of the mean ... 

However, the quantification of platinum compounds was achieved with good intra-and inter-assay precision (range 1-13%... 

Precision defines the scatter of repeated analysis, or the coefficient of variation of analytical results. Both intra-assay and inter-assay precision must be investigated. Intermediate precision describes the influence of different analysts, equipment, days and other intra-laboratory variabihty. Inter-laboratory comparison is also of interest in establishing the precision of the method. AU testing on accuracy and precision must be carried out by replicate analyses of a statistically relevant number of samples. Depending on the use of the method, it may be necessary to estabhsh both parameters over the measurable range, or in the case of content determination simply in the range of 80-120% of the nominal value. 

Inter-assay precision Run samples (s2 replicates) across >3 analytical runs. Samples used for precision testing may include commercial QC samples, but precision testing should also include species-specific samples of the relevant matrix (e.g., serum, plasma, urine) of the intended study samples Test >3 samples at low, medium, and high concentrations across the calibration curve range Calculate mean, SD, and CV% CV < 20%, CV < 25% at LLOQ... 

Inter-assay precision to establish reproducibility of an assay by comparing replicates across multiple runs... 

QC (see Section 41.5.9) samples that will be used to monitor the assay during routine use should also be subject to the intra- and inter-assay precision testing outlined earlier during the validation process. 

The 12o(-HSD, a NADP" dependent enzyme, was so coimmobilized with bioluminescent enzymes on Sepharose 4B. To improve sensitivity bacterial diaphorase was sometimes used instead NADPHrFMN oxidoreductase, since background is less. The diaphorase containing systems are very sensitive to differences in mixing and shaking. However with an injection method the inter -assay precision is good (8%) and the standard curve is linear from 0.4 to 200 mol/1. 

The ability to repeat the same method under different conditions, e.g. change of analyst, reagent, or equipment or on subsequent occasions, e.g. change of electrode, measurements recorded over several weeks or months, this is covered by the between batch precision or reproducibility, also known as inter-assay precision. 

Precision Multiple instruments, capillaries, runs, analysts, and days to calculate repeatability (intra-assay), intermediate precision (inter-assay), and reproducibility (overall) variability of method... 

Precision of the reduced CE-SDS method was studied via repeatability (intra-assay) and intermediate precision (inter-assay) studies. Figure 12 shows electropherogram of the intraassay repeatability data, and Table 4 demonstrates the precision of the assay (%RSDs of 0.3, 0.6, and 3.7% for the HC, LC, and non-main species, respectively). 

Method precision refers to the variability in measurement of the same sample. There are three main components of method precision repeatability (also known as system or intraassay precision), intermediate precision (also known as inter-assay or intra-laboratory precision), and reproducibility precision (also known as ruggedness, overall or inter-laboratory... 

In a protocol about collaborative studies [10] it is also considered what is called preliminary estimates of precision. Among these the protocol defines the total within-laboratory standard deviation . This includes both the within-run or intra-assay variation (= repeatability) and the between-run or inter-assay variation. The latter means that one has measured on different days and preferably has used different calibration curves. It can be considered as a within-laboratory reproducibility. These estimates can be determined prior to an interlaboratory method performance study. The total within-laboratory standard deviation may be estimated fi-om ruggedness trials [10]. 

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. 

IA results are inherently less precise than those of chromatographic assays [40]. Owing to the greater inter-assay imprecision, more validation mns should be used (e. g., at least six batches) to achieve an adequate level of confidence in the estimates of assay performance [41]. Acceptance criteria should be defined a priori for the method validation they should be more lenient than those of chromatographic methods. 

Finally a validation step for the overall MISPE procedure is mandatory to allow the use of the method itself in place of the regulatory methods. The issues to be considered here are the accuracy and precision of the method based on the MISPE protocol, its limit of quantification, selectivity and ruggedness. In particular the inter- and intra-assay precision need to be checked with real samples and certified reference materials and methods [23,24]. 

The inter- and intra-assay precision (% C.V.) of this method were reported to be less than 8% across the range of the limits of quantification (0.05-10 ng/ml). The accuracy (% bias) for all spiked control concentrations did not exceed 4%. Same-day turnaround of results for over 100 samples was possible and was used to support an acute dose tolerance and pharmacokinetic study that involved the analysis of 1600 samples. 

Precision Instrument repeatability - ten replicate injections. RSD<1% Intra-assay precision. Multiple analysis of aliquots of a sample during one day. RSD < 2% Intermediate precision. Multiple analysts, instruments, days in one laboratory. Reproducibility by inter-laboratory studies to detect bias. 

Figure 1.1 Examples of (a) Levey-Jennings and (b) cumulative sum (cusum) control charts using inter-assay quality control data from an alprazolam GC-MS assay. Since the cusum chart persents the cumulative sum of deviations from the mean, it is more sensitive to small biases that develop over time, whereas Levey-Jennings charts are most useful for detecting changes in the precision of the assay...

If known pharmacological agents are available, then the screening assay is validated across multiple assays. This is more difficult if the target of interest is novel and no pharmacological tools are available. It requires rigorous attention to detail to achieve inter-laboratory precision even down to the way the compounds are solubilized and diluted, the types of equipment, and the detection technology. 

Values of 20% (25% at lower limit of quantification [LLOQ]) are recommended as default acceptance criteria for accuracy and inter-batch precision for ligand binding in practical use. Precision and accuracy should be established by analyzing four sets of QC samples at LLOQ, low, medium, and high levels in duplicate in six different batches during method development. In addition, a second proposed criterion for method acceptance takes into account the sum of inter-batch precision and the absolute value of accuracy be <30%. During practical application of such assays, the 4-6-30-rule may be applied - that is, for each batch four out of six QC samples must be within 30% of nominal concentration, but the two failed QC samples may not be at the same level. 

Standard curves were constructed by plotting the change in absorbance with time (dA/dt) versus concentration of ATI or ATF. Standards for the assay were prepared by dilution of stock solutions of ATI or ATF to the appropriate concentrations (0,25,50,100,175, and 250ng/mL for ATI 0,5,10,20,50, and lOOng/mL for ATF) with PBS (pH 7.4), with the addition of 1% (v/v) blank rat plasma. Assays were validated with respect to precision and accuracy, by analysis of QC samples at 25,100,250ng/ml for ATI and 5,20, lOOng/ml for ATF, respectively. Intra-assay and inter-assay variability were determined through the analysis of QC samples. 

Accuracy and precision are determined using a minimum of five to six determinations per concentration and are assessed for intra-assay (intra-batch or intra-run) and inter-assay (inter-batch or inter-run) conditions. Accuracy should be within 20 (30)% of the target concentration and precision should not exceed 20 (30)% of CV [37, 38]. 

There are also studies that comp)are sp>ectrophotometric and RP-HPLC determinations of iodine concentrations in urine (Bier et al., 1998). In the first one ammonium persulfate was used as an oxidant in the modified ceiic arsenite method. With the use of this sensitive method iodine concentrations can be determined in very small sp>ecitnens (50 pL). A Technicon Autoanalyzer II and a paired-ion-RP HPLC were the basic analytical equipment. The authors found that the precision of this optimized ammonium j ersulfate method yielded inter assay CVs of <10% for urinary iodine concentrations >10 pg/dL The detection limit was 0.0029 pg iodine. There was a high correlation between all three methods (r > 0.94 in any case) and the interpretation of the results was consistent. The authors suggested that the manual ammonium persulfate method could be performed in any routine clinical laboratory for urinary iodine analysis. Another benefit of the described methods is a p>ossibility to process a large number of samples with high accuracy and ininimal technician s time. 

While a substitute matrix can be used to prepare standard cahbrators for a drug compound that exists endogenously, VS/QCs should be prepared in the authentic matrix, regardless. VS data are used during method validation to characterize the intra- and inter-mn accuracy/precision and stability. QC data are used for assay performance monitoring and to accept or reject a run during in-study validation. 

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