Inter-assay

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%. 

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. 

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). 

Other aspects of sample preparation to consider, depending on the objective of the CZE method, are sample denaturation, reduction and/or derivatization, and the use of an IS. IS are widely used in CE separations to aid in normalization of slight inter-assay variability and in method troubleshooting. 

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... 

The LDH+ALT reactor provided a linear response from 0.1 to 50 pmol/L lactate, thereby increasing lactate conversion by 117-183% relative to LDH alone. The intra- and inter-assay CV were both less than 5%, and recoveries ranged from 93 to 106%. Even though roughly 100% of the LDH and ALT added bound to the support under the immobilization conditions used, the activities of the immobilized enzymes were ca. 3% of those of the free enzymes, which is consistent with previous results obtained by the same [67] and other authors [69,70]. Jointly immobilized LDH and ALT preserved ca. 50% of their original activity after 60-90 days of intermittent use. On the other hand, immobilized luciferase was less markedly inhibited than that in the free solution by substances present in the biological samples assayed [71]. 

Figure 3.29.A shows a flow-cell of 20 iL inner volume used to hold immobilized anti-mouse IgG bound to a rigid beaded support (activated Pierce trisacryl GF-2000). The cell was used to develop a two-site immunoassay for mouse IgG by consecutive injection of the sample, acridinium ester-labelled antibody and alkaline hydrogen peroxide to initiate the chemiluminescence, which started the reaction sequence shown in Fig. 3.29.B. Regenerating the sensor entailed subsequent injection of an acid solution, which resulted in a determination time of ca. 12 min (this varied as a fimction of the flow-rate used, which also determined the detection limit achieved, viz. 50 amol for an overall analysis time of 18 min) [218]. The sensor was used for at least one week with an inter-assay RSD of 5.9%. Attempts at automating the hydrodynamic system for use in routine analyses are currently under way.

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- 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... 

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

With known analyte concentrations, the processed data provide calibration points, and Immusoft comprises fitting procedures to deduce the corresponding calibration curve that can be stored in the program for further experiments. Different calibration curves can be stored depending for instance on the assay protocol, on the specific features of the chip used or on the medium in which the assay is performed. In most cases, the calibration is performed with six independent chips of eight channels and cumulated in order to get a stable batch calibration. Then the results can be referred to this internal batch calibration. For routine control, one calibration each week is recommended to be sure that the chemistry is still in the specifications (e.g. + 10% of inter-assay standard deviation). 

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. 

HPLC with column switching and mass spectrometry was applied to the online determination and resolution of the enantiomers of donepezil HC1 in plasma [38]. This system employs two avidin columns and fast atom bombardment-mass spectrometry (FAB-MS). A plasma sample was injected directly into an avidin trapping column (10 mm x 4.0 mm i.d.). The plasma protein was washed out from the trapping column immediately while donepezil HC1 was retained. After the column-switching procedure, donepezil HC1 was separated enantioselectivity in an avidin analytical column. The separated donepezil HC1 enantiomers were specifically detected by FAB-MS without interference from metabolites of donepezil HC1 and plasma constituents. The limit of quantification for each enantiomer of donepezil HC1 in plasma was 1.0 ng/ml and the intra-and inter-assay RSDs for the method were less than 5.2%. The assay was validated for enantioselective pharmacokinetic studies in the dog. 

In the field of environmental analyses, to achieve the required comparability and traceability of measurements, there is a clear need for matrix reference materials. These reference materials must present a sufficient matrix matching with real environmental samples, otherwise they are useless, due to a lack of corn-mutability, that is to say their ability to demonstrate inter-assay properties comparable to real samples. This matrix match can be considered as a more important property of the RM than the level of uncertainty of the measurand concentration [6, 7]. It has been estimated that only 10% of the required matrix RM in the field of environment are currently available [8]. 

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

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. 

The calculated intra- and inter-assay accuracies ranged from 101-107 %, calculated over all analytes and all three QC levels. 

Two methods are usually used for the evaluation of real-time PCR data, namely the standard curve method and the comparative threshold (Ct) method. The standard curve method relies on the use of purified cDNA pasmid standards, which will result in only a relative quantification. Other more specific standards can be used, e.g., in vitro transcribed RNA, which gives an absolute qantitation, however, this method is very labour intensive and not commonly used (Martell et al. 1999). The standard curve is included in each PCR run, and therefore provides a correction control for the PCR efficiency, making inter-assay comparisons easier. The comparative Ct method uses algorithms to calculate relative expression levels, compared to a calibrator (e.g., a control sample). A detailed description of the mathematics is given by Livak and Schmittgen (2001). After calculation the normalized expression... 

Kim et al. [48] developed a rapid, sensitive, and selective LC-ESI-MS/ MS method for fhe deferminafion of lornoxicam in human plasma. Lornoxicam and isoxicam (infernal standard) were extracted from human phasma wifh efhyl acefafe at acidic pH and analyzed on a Sunfire Cis column with the mobile phase of methanol-ammonium formate (10 mM, pH 3) (70 30). The analyte was detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in the multiple-reaction-monitoring mode. The standard curve was linear (r = 9998) over the concentration range of 0.5-500 ng/ml. The coefficient of variation and relative error for the intra- and inter-assay at four QC level were 0.7% fo -4.2% and —4.5% to 5%, respectively. The recoveries of... 

Two immunosensors developed by O Regan et al. [89,90] have demonstrated their usefulness for the early assessment of acute myocardial infarction (AMI). Human heart fatty-acid binding protein (H-FABP) is a biochemical marker for the early assessment of AMI. The authors constructed an amperometric immunosensor for the rapid detection of H-FABP in whole blood. The sensor is based on a one-step, direct sandwich assay in which the analyte and an alkaline phosphatase (AP) labelled antibody are simultaneously added to the immobilized primary antibody, using two distinct monoclonal mouse anti-human H-FABP antibodies. The substrate p-amino-phenyl phosphate is converted to p-aminophenol by AP, and the current generated by its subsequent oxidation at +300 mV vs. Ag/AgCl is measured. The total assay time is 50 min, and the standard curve was linear between 4 and 250 ng ml . The intra- and inter-assay coefficients of variation were below 9%. No cross-reactivity of the antibodies was found with other early cardiac markers, and endogenous substances in whole blood did not have an... 

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... 

---