Intra-assay controls

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. 

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. 

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. 

The quality control samples are used to control for intra-assay variability. 

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%. 

The analytical part of this study confirmed the adequate imprecision of holoTC determination, with mean intra- and inter-assay coefficients of variation (CVs) ranging from 2.9% to 4.1% on assay controls and from 6.0% to 7.7% on our local pooled control serum, good holoTC linearity from 8.8 to 143.3 pmol/L (r = 0.99), good recovery in spiked specimens (mean 95%, interval 90 100%), mean recovery determined by dilution (100%, range 93-111 %) and detection limit (0.07 pmol/L). 

Each test must be repeated at least three times and each microplate has it own control with quadruple wells for each concentration. The intra-assay... 

A selective, sensitive, and rapid hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry was developed for the determination of donepezil in human plasma [32], Donepezil was twice extracted from human plasma using methyl-ferf-butyl ether at basic pH. The analytes were separated on an Atlantis HILIC Silica column with the mobile phase of acetonitrile ammonium formate (50 mM, pH 4.0) (85 15, v/v) and detected by tandem mass spectrometry in the selective reaction monitoring mode. The calibration curve was linear (r = 0.9994) over the concentration range of 0.10-50.0 ng/ ml and the lower limit of quantification was 0.1 ng/ml using 200 /d plasma sample. The CV and relative error for intra- and inter-assay at four quality control levels were 2.7% to 10.5% and —10.0% to 0.0%, respectively. There was no matrix effect for donepezil and cisapride. The present method was successfully applied to the pharmacokinetic study of donepezil after oral dose of donepezil hydrochloride (10 mg tablet) to male healthy volunteers. 

As lymphocytes, monocytes, and macrophages are the primary targets for viral infection, the penetration of antiviral agents is important. Lymphocytes and monocytes are indicated as peripheral blood mononuclear cells (PBMC). The preparation of control PBMC from blood was reported in considerable detail by Jemal et al. [38], In addition, a validated assay for the determination of ATA in PBMC was developed. The determination of protease inhibitors in hmnan PBMC was reported by several groups [39-40]. After LLE, the analytes were analysed by LC-MS. Both methods enable the intra-cellular determination of the analytes and can be applied for TDM and pharmacokinetic studies. 

Calcitonin acts on receptors in bone osteoclasts with a resulting reduction of bone resorption, and it also acts on the renal tubular reabsorption of phosphate. The phosphaturic effects are accompanied by diuresis and increased excretion of other electrolytes. Calcitonin and parathyroid hormone act as a dual negative feedback mechanism in controlling calcium in intra- and extracellular fluids. The range of calcitonin assays suitable for laboratory animals is limited, but the hormone can be measured by two-site radioimmunometric assays (Moukhtar et al. 2005). 

Assay validation characterizes the assay performance so that the significance of the measured assay values obtained is readily understood [75]. Test methods should be validated when important decisions are to be based on the data generated [30]. Thus, the extent of method validation depends on the stage of clinical supply manufacture. The key elements of assay validation for a method are to establish reliability, the intra- and interlaboratory test variation, and relevance, the meaning of the results for a specific purpose [19]. The robustness of an analytical procedure (according to the ICH-Validation of Analytical Methods, 1993) is its measured capacity to be unaffected by small variations in controlled parameters and reliability under normal usage [35]. 

Fig. 3 (continued) assayed by TUNEL. Numerous TUNEL-positive cells were observed after treatment with gpl 20, while almost no apoptotic cells were seen in cultures that were not incubated with gpl 20 (p<0.001 serum-free media (control) vs. 200 ng/ml gpl 20 in serum-free media for 24 h). Almost all occludin-positive cells were TUNEL positive for 200 ng/ml gpl 20. Control with buffer only and no enzyme showed no TUNEL staining (not shown), (b) After intra-caudate-putamen (CP) injection of HIV-1 500 ng gpl 20, fewer occludin-positive structures were seen, (c) Decrease in the number of claudin-5 (a tight junction protein)-positive structures parallels the leakage of Evans blue (EB) in CP previously injected with HIV-1 gpl 20. (d) Reduction in the number of laminin (a basement membrane proteinj-positive structures observed after intra-CP injection of HIV-1 gpl 20, suggesting a degradation of the basement membrane protein (modified from [105])... 

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