Intra-assay

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. 

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...

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. 

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)... 

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

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). 

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. 

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... 

Compound Intra-assay average Intra-assay CV (%) Inter-assay average Inter-assay CV(%)... 

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. 

Precision may be considered at three levels repeatability, intermediate precision, and reproducibility (2, 3). Repeatability expresses the precision obtained by repeatedly analyzing, in one laboratory on the same day by one operator using one piece of equipment, aliquots of a homogeneous sample, each of which has been independently prepared according to the method procedure. Repeatability is also termed intra-assay or within-day precision. It is assessed using a minimum of nine determinations. Repeatability can help in determining the sample preparation procedure, the number of replicate samples to be prepared, and the number of injections required for each sample in the final method setting. 

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. 

In PCR product, the untreated sample can be considered as a strand-specific intra-assay negative control. Electrochemical signals upon thermal denaturation undoubtedly designated the presence of a complementary sequence. Genosensors showed a different behaviour as underlined by the signal ratio between the denaturated and non-denaturated sample that highlighted the dissimilar efficiency of each probe. 

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. 

Imprecision source Intra-assay Intra-assay Inter-assay... 

An in vitro bioassay can be designed in several ways, but requires statistical validity. A one point assay is not valid. The bioassay should be designed to consider factors that introduce variability, and the analysis should test such variability. A measurement series of a test sample should be compared to an equivalent series of the reference material, carefully considering the comparisons between the linear portions of the dose-response curves (Mire-Sluis et al., 1996). To test validity of a bioassay inter- and intra-assay variability should be considered in both preparation, and in the case of multiwell plates, the variability between each plate. To reduce the positional effect in plate tests, it is advisable to distribute the points on the curves randomly and also to include a reference standard in each plate (Gaines-Das and Meager, 1995). One of the most widely used techniques to validate a bioassay s performance is to include internal duplicates. The data arising from the comparison can be important in assessing the test s variability. 

The calculated intra-assay accuracies ranged from... 

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. 

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