Quantification, lower limit

The use of robotics can be adopted also in sample preparation steps, in particular on-line SPE [7], This necessity is particular evident when small quantity of starting materials is available and the target molecules are present at low concentration levels. With the advent of miniaturization and automated procedures for samples handling, treatments and analysis, the lost of analytes due to a laboratory steps can be reduced. The reduction of analyte losses and the possibility to analyze even a total sample (no loss) leads to lower limits of detection (and consequently lower limits of quantification). Smaller volumes bring to obtain adequate sensitivity and selectivity for a large variety of compounds. In addition, on-line SPE requires low solvent consumption without the need to remove all residual water from cartridges, since elution solvents are compatible with the separation methods. 

The assessment of DNA adducts may provide a sensitive indicatCH of previous exposure. The enzyme-linked immunosorbent assay (ELISA) has a lower limit of detection of about 0.08 femtomol per microgram of DNA (Perera et oL, 1982). This assay requires (1) the development of an antibody specific for a certain chemical metabolite bound covalently to DNA and (2) the isolation of DNA from some tissue sample, ag., skin biopsy, or lymphocytes of an exposed individual. It is anticipated that further refinement of such immunologic techniques may lower the threshold of sensitivity by one or two orders of magnitude. One such refined test is the ifitrasensitive ymatic radioimmunoassay (USE-RIA), purported to be about five times more sensitive than ELISA (Hsu et oL, 1981 Shamsuddin et oL, 1985 Harris et oL, 1985). Quantification by the development of monoclonal antibodies to aflatoxin Bj metabolites bound to DNA (Groopman et oL, 1982 Sizaret et oL, 1982) has now been reported. 

Lower limit of quantification (LLOQ) the lowest amount of an analyte in a sample that can be determined quantitatively with suitable precision and accuracy. 

For selectivity, there should be evidence that the substance being quantified is the intended analyte. Therefore, analyses of blank samples of the appropriate biological matrix (plasma, urine, or other matrix) should be obtained from at least six sources. Each blank sample should be tested for interference, and selectivity should be ensured at the lower limit of quantification (LLOQ). 

Lower Limit of Quantification. The lowest standard on the calibration curve should be accepted as the limit of quantification if the following conditions are met ... 

Actinides were determined at the ultratrace level in moss samples collected from the eastern Italian Alps (1500 m a.s.l.). The frozen samples were cut into 1-2 cm sections and analyzed separately to obtain the distribution curves of the vertical concentrations. For plutonium and americium isotope analysis, 1-2 g of the samples were ashed, leached, separated with respect to analytes and analyzed by alpha spectrometry and LA-ICP-MS after the plutonium or americium had been electroplated on a stainless steel disk.23 Estimated limits of quantification of LA-ICP-MS for actinide radionuclides deposited on stainless steel plates after chemical separation are summarized in Table 9.45. For most of the long-lived radionuclides in moss samples, lower limits of determination were found at the 10 15gg 1 concentration level compared to those of a - spectrometry 23... 

Lower Limit of Quantification (LLOQ) The lowest concentration of the analyte of interest in a matrix that can be quantitatively determined using the standard curve with acceptable precision and accuracy. The LLOQ is usually defined as the lowest concentration at which the assay imprecision does not exceed 20%. Upper Limit of Quantification (ULOQ) The highest concentration of an analyte in a matrix that can be quantitatively determined using the standard curve with an acceptable precision and accuracy. If the analyte concentrations in the postdose samples are higher than the ULOQ, then a dilution QC is needed to cover the highest anticipated dilution. 

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. 

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed [33] and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the IS. The collision-induced transition m/z 380 > 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 —> 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.1 to 20.0 ng/ml. The lower limit of quantification of the LC/MS/MS method was 0.1 ng/ml. The intra- and inter-day precisions were below 10.2% and the accuracy was between 2.3% and +2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to fit the donepezil plasma concentration-time curve. Maximum plasma concentration was... 

Macek et al. [120] developed a method to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The method is based on the protein precipitation with acetonitrile and a reversed-phase liquid chromatography performed on an octadecylsilica column (55 x 2 mm, 3 /im). The mobile phase consisted of methanol-10 mM ammonium acetate (60 40). Omeprazole and the internal standard, flunitra-zepam, elute at 0.80 0.1 min with a total rim time 1.35 min. Quantification was through positive-ion made and selected reaction monitoring mode at m/z 346.1 —> 197.9 for omeprazole and m/z 314 —> 268 for flunitrazepam, respectively. The lower limit of quantification was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. The method was applied to the analysis of samples from a pharmacokinetic study. 

Erturk et al. [40] determined vigabatrin in human plasma and urine by HPLC after derivatization with 4-chloro-7-nitrobenzofurazan with fluorescence detection at 520 nm with excitation at 460 nm. A column (20 cm x 3.9 mm) of Shim-Pack Cig (5 /tm) with a mixture of 10 mM phosphoric acid-acetonitrile (60 40) as a mobile phase (flow rate 1.0 ml/ min) was used. The assay was rectilinear over the concentration range of 2.0-20.0 fig/ml for plasma and 1.0-15.0 yg/ml for urine. The lower limit of detection and the lower limit of quantification for vigabatrin were 0.1 and 0.2 /ig/ml, respectively, in plasma and urine. Both the within-day and day-to-day reproducibilities and accuracies were less than 5.46% and 1.6%, respectively. 

The technique was further improved by employing a polymer coating on the polymeric fibers packed in a fused silica capillary. The coating material was based on GC stationary phases. The polymer-coated fiber-packed capillary was used as the sample loop of the LC injection valve for the extraction of phthalate esters from river water and wastewater.22 The coated-fiber extraction capillaries demonstrated a better extraction efficiency and lower limit of quantification (LOQ) than the uncoated-fiber capillaries. Also, the coated fibers were similarly packed in a PEEK tube, which was used as the injection loop or integrated in the rotor of an LC injection valve employed for the extraction of phthalates. The results clearly showed that an extraction with high selectivity could be established with an appropriate type of polymer coating.23... 

Microbial tests are substantially less certain than chemical ones, especially when they entail quantification. Microbial limits must be looked at with some skepticism the real value might be something quite different—either higher or lower—than the reported one. Consider any absolute number with some caution the answers are never as clear as they might seem with microbiology. When it comes to enumeration, microbiology can be less than a pure science. 

Ion suppression is so far mainly considered in the context of sensitivity and the lower limit of quantification of an assay. But it has to be emphasized that short term variations in ion yields—particularly due to matrix components—can compromise the accuracy of analyses Whenever the variation of ion yield has a differential impact on target analyte and internal standard, accuracy is compromised. This means that the reliability of LC-MS/MS analyses critically depends on (1) how similar the impact of ion suppression or ion enhancement on target analyte and internal standard compound is and on (2) how similar the matrices of calibrator samples and actual patients samples are with respect to the modulation of ionization efficacy. This problem can be of relevance for an entire measuring series—if systematic differences in the ionization modulation properties of calibration materials and actual patients samples are present—or it may non-systematically affect individual patients samples as well. 

LLE liquid-liquid extraction, SPE solid phase extraction, LPME liquid-phase microextraction, ESI electrospray ionization, APCI atmospheric pressure chemical ionization, SSI sonic spray ionization, Q quadrupole, QqQ triple quadrupole, TOF time-of-flight, IT ion trap, IS internal standard, CV coefficient of variation, MRE mean relative error, LOD limit of detection, LLOQ lower limit of quantification... 

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