Intra-assay variation

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]. 

The intra-assay variation for plasma was performed in a tenfold analysis and gave CVs of 0.8-4.1% with the exception of tryptophan, methionine, and the imino acids (6.7-8.9%). Urine behaved similarly to plasma in the sense of reproducibility, with CVs between 3.6 and 8.6% in 23 analyses over a 6-month period. Exceptions in this respect were the amino acids with a low ninhydrin color yield, such as / -alanine and sarcosine. 

Taylor and colleagues [98] at the Mayo Clinic published a method for the simultaneous analysis of urinary cortisol and cortisone. They used 2H4 cortisol as an internal standard and took a 0.5-ml urine sample. An API 2000 with Turboion-spray source was used in the positive-ion mode. Chromatography was conducted on a standard-bore C18 column with Q8 precolumn filter. MRM was conducted in the positive-ion mode monitoring m/z 363—>121 for cortisol, 367—>121 for 2TL, cortisol, and 361— -121 for cortisone. Cortisol and cortisone were separated and both were eluted within 2 min. Inter- and intra-assay variation for both compounds was < 9% for amounts above 2 pig/dl. The values obtained agree well with those of other studies, such as ours (Table 5.3.2) [62]. They found a range for cortisol for adult males of 4.2-60 pg/24 h and for adult females 3.0-43 pg/24 h. In summary, the 3-min run time of their method has allowed the Mayo group to completely transfer their cortisol and cortisone workload from RIA and HPLC to MS/MS. 

The analytical variation of the Cobas Integra ISE method in New Lab A was studied in the validation process by running the two-level system quality control samples forlO days, and Daytrol for8 days. The intra-assay variations (n = 20) of the two system controls were much better than those quoted by the manufacturer. The within-batch variation of the lower system control was verified as 0.43% compared to 0.81% given by the manufacturer. The higher level varied by 1.4% the manufacturer quoted a respective variation of 2.5%. 

Fig. 11. Narrow inter-assay and intra-assay variations are critical parameters for successful high-throughput screens using FLIPR technology. Some final advice...

Immunoassay is one of the most sensitive analytical techniques described for phytoestrogen measurements in human biological fluids. Detection limits for target analytes are below 1 nmol/L for most methods with an average recovery of 92% and inter-/intra-assay variation less than 10% for most methods (WiUdnson et al., 2002). Immunoassays have been validated against other analytical techniques and excellent correlation has been demonstrated. Immunoassay can be a powerful alternative to GC-MS- or HPLC-based methods for studying the bioavaUabflity and metabolic fate of dietary isoflavonoids because of its ease, relatively low cost, availability for... 

Hasegawa et al. [76] measured miconazole serum concentration by a high performance liquid chromatographic method. The authors assessed whether the internal standard method produced an intra-assay error and found that the method gave more precise and more reproducible results compared to the absorption calibration curve method. With 0.5 pg/mL of miconazole, the coefficient of variation produced by that method was 3.41%, whereas that of the absorption calibration curve method was 5.20%. The concentration of absorptions calibration curve method showed higher values than the internal standard method. This indicated that the internal standard method was far more precise in measuring the miconazole serum concentrations than the absorption calibration curve method. 

Rao et al.20 demonstrated a fluorescence polarization immunoassay for evaluating serum concentrations of tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and doxepin) with respect to nonresponse, compliance, therapeutic window, and influences of age, sex, substance abuse, and toxicity. Abbott Laboratories TDx/TDxFLx Toxicology Tricyclic Assay FPIA (fluorescence polarization immunoassay) was used. This assay of 50 /uL samples contained tricyclic antidepressant antibodies raised in rabbits and fluorescein-labeled tricyclic antidepressant as a tracer. The assay was calibrated with imipramine in the range of 75 to 1000 fig/L (268 to 3571 nmol/L). Intra-assay and inter-assay coefficients of variation for internal quality control samples from the manufacturer were 4.2 and 4.7%, respectively. The limits of detection were 72,71,64, and 72 nmol/L for amitriptyline, imipramine, clomipramine, and doxepin, respectively. This high-throughput immunoassay was easy to use although amitriptyline, dosulepine, desipramine, and nortriptyline showed cross-reactivities ranging from 74 to 100%. 

Standards, controls, and samples (250 fiL each) were treated with 500 fiL acetonitrile-acetic acid (99 1 v/v) containing IS (2.50 jUg/mL), vortexed for 10 sec, incubated for 5 min, and centrifuged at 15,000 g for 5 min. The supernatants (1650 //L) were loaded onto a polypropylene 96-well plate containing 900 fxL HPLC water under low vacuum. The SPE plates were conditioned with 500 fxL methanol followed by 300 jx. acetonitrile-water-acetic acid (30 69.5 0.5 v/v/v) (solvent A), washed with 1000 /xL solvent A, dried under full vacuum for 10 min, wiped dry with paper, eluted with 500 jxL methanol-trifluoroacetic acid (99.9 0.1 v/v) (solvent B) and then with 400 //L solvent B for 2 min, evaporated to dryness at 65°C under a gentle air stream, reconstituted with 200 /xL methanol-hydrochloric acid (0.1 M) (70 30 v/v) and assayed. The injection volume was 50 i L. Figure 11.3 shows chromatograms of blank plasma and spiked plasma with lumefantrine. A calibration curve was constructed in a concentration range of 25 to 20,000 ng/mL. Intra-assay and interassay coefficients of variation were below 5.2 and 4.0%, respectively. The limit of detection was 10 ng/mL. The limit of quantification was 25 ng/mL. 

A calibration curve was constructed over a concentration range of 1 to 50 fig/mL with a correlation coefficient of 0.998. Intra-assay and inter-assay coefficients of variation were less than 7.9 and 9.5%, respectively. Mean absolute recoveries at 10 and 20 fig/mL were 72 and 76%, respectively. Limit of detection was 0.3 fig/mL. Limit of quantification was 1.0 fig/mL. 

Linear 1/y2 regression analyses of the ratio of the peak area of lercanidipine to the concentration compared with the ratio of the IS were constructed over the range of 0.05 to 30.00 ng/mL. Correlation coefficients exceeded 0.995. Intra-assay and inter-assay coefficients of variation were less than 7.3 and 6.1%, respectively. The limit of detection was calculated to be 0.02 ng/mL, and the limit of quantitation was 0.05 ng/mL. 

The hydrogel coatings achieved the lowest limits of detection (LTD) 1300 to 1600 amoles/spot, but exhibited significant assay variation (22% intra-slide to 37% inter-slide CV). LLD levels of surface-modified polysfyrene slides (Maxisorb black. Nunc) equaled 1500 amole/spof af 15 to 32% CV, while reflective (mirror-like) slides coated with 3-aminopropyltriethoxysi-lane (Amersham) showed the lowest variahon with CVs at 11 to 14%. 

All calculations were made on three concentration levels. The intra-assay coefficient of variation (CV) for plasma ranged from 3.8 to 7.6%, the latter at a level of... 

The intra- and inter-assay variation is determined in tenfold analyses of a pool plasma sample. Table 3.4.2 shows the results of these measurements. The linearity of this method should be assessed for all analytes. Pristanic acid and the C26 0 fatty acid were linear up to 16 pmol/1, phytanic acid to 100 pmol/l and the C22 0 and C24 0 fatty acid to 200 pmol/1. The lower detection limit for all analytes was at a level of less than 0.01 pmol/l, for the lower reporting levels (LOQ), an analysis of a blank solution was taken into account. The blank levels of the analytes phytanic acid, pristanic acid, and fatty acids C22 0, C24 0 and C26 0 were 0.04, 0.01, 0.41, 0.68 and... 

Repeatability (intra-assay or within-day precision) is obtained when the analysis is carried out in one laboratory by one operator, using one piece of equipment over a relatively short time span. It reflects the variation in replicate procedures performed within a short time period, with the same operational conditions. 

Assay Development and Validation. Reproducibility of this ELISA assay was determined based on a set of clomazone standards that were run on different plates on the same day (intra-assay) and on different days (inter-assay). The intra-assay coefficient of variation of the standards changed from 1.5 at the highest clomazone concentration (250 ppb) to 22 at the lowest concentration of 1.4 ppb. The coefficient of variation(CV) at clomazone rate of 12.5 ppb was 10. Similar values were obtained for the inter-assay variability, with the CV of the 1.4 ppb concentration being 22.5, and the CV for the 250 ppb concentration being 2.7. The CV for the 10 ppb concentration of clomazone was about 5 between tests. Analysis of the data for this range of clomazone concentrations indicates that there is good correlation (r =0.97) between the log of the concentration of clomazone and percent inhibition in the assay when the linear regression equation was used. Based on these results, the limit of the test s sensitivity was defined as 2 ppb (10 ppb in soil) and the limit of detection was set at 1 ppb. 

The intra-assay imprecision (SDs) of the HCV Versant bDNA and Amplicor RT-PCR assays ranges from 0.05 to 0.31ogio the Versant bDNA assay is more precise. The biological variation of HCV viral load ranges from 0.5 logio... 

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 ... 

Using the proposed BL-EIA, the measurable range for Angiotensin I and Endothelin-1 were 7.81 - 1000 pg/mL and 15.63 - 1000 pg/mL, respectively. The intra-assay coefficients of variation of Angiotensin I and Endothelin-1 at each standard point were below 9.7% and 11%, respectively. (Fig. 2)... 

In the proposed assay, there was no interference in the measurement of the aequorin activity by biotinylated luciferase. The sensitivity of aequorin was 7.58xl0 0 mol/assay (blank + 3SD, Fig. 2, left), the calibration linearity of the assay ranged from 1.21 X lO mol/assay to 1.94 x lO mol/assay and the intra-assay coefficients of variation (CV) for 8 replicates with each standard point were from 1.0 to 3.2%. These results are similar to the results from the separate assay. 

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. 

In the simultaneous luminescent assay of Aq, b-Luc and HRP, the sensitivity of Aq was 2.8 xlO"20 mol/assay (blank + 3SD), which is similar to the result of the individual assay for Aq. The intra-assay coefficients of variation (CV) of 8 replicates for each standard point of Aq were from 2.2 to 4.2%. The detection limit of b-Luc (blank + 3SD) was 6.5 x 10 19 mol/assay. The intra-assay CVs of 8 replicates with each standard point for b-Luc were from 2,6 to 4.9%. We added benzalkonium chloride to luminescent reagent of HRP for inactivation of b-Luc, because the luminescence of b-Luc interfered with the measurement of HRP. The luminescence of b-Luc decayed after 6,5 min. The detection limit of HRP (blank+3SD) was 1.9 x 1017 mol/assay. The intra-assay CVs of 8 replicates with each standard point for HRP were from 2.4 to 7.9%. The CV values were obtained in the presence of all three enzymes and were similar to individual assay. The measurements of the three enzymes were completed in 13 minutes with a single assay medium at room temperature. 

In addition, a standard solution of 640 ng/ml 6-oxo-PGF assayed in 8 consecutive assays gave a mean assay (+ s.e.m.) of 638 + 31 ng/ml, with an interassay coefficient of variation of 13.5%. The intra-assay coefficient of variation was 12.4%, though both these coefficients of variation... 

A similar HPLC method with a postcolumn derivatization system was used for the analysis of total thiamine in human whole blood as well as in serum, cerebrospinal fluid, and milk (26). The HPLC system consisted of a pBondapak column and the mobile phase was a mixture of methanol-50 mM sodium citrate buffer pH 4.0 (45 55, v/v) plus 10 mM sodium 1-octanesulfonate. Two milliliters of blood was needed. The minimum detectable amount was 60 fmol of thiamine. The intra-assay and inter-assay coefficients of variation were 2.3% and 3.9%, respectively. The recovery of TPP added to blood samples was 98.7%. 

The ID-GC-MS-SIM method of Adlecreutz and coworkers (1993) is perhaps the most frequently used in clinical and medical studies, although intra- and total-assay variabilities of all analytes are considered to be high, probably reflecting low-analyte concentrations in biological fluids (Wilkinson et al., 2002). Other GC-MS methods report lower inter-assay variation of 6%-ll% isoflavones in serum. The latest method of measuring urinary excretion of isoflavones (daidzein, genistein, equol. 

---