Lower limit of quantification

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. 

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 ... 

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... 

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... 

The mass-selective detector is more specific and allows a lower limit of quantification than the nitrogen-phosphorus detector (NPD). 

The terminal elimination half-life was 20 % lower in patients compared to healthy subjects. Probably the elimination half-life was not accurately determined in patients as XYZ456 concentrations were close to the lower limit of quantification. Thus, as the elimination has a biphasic profile, for patients this elimination half-life probably corresponds to a mix of ti/2,xi and ti/2,xz. 

Apart from the Chirasil-L-Val method, sarin enantiomers were also separated by a 2D-GC technique on chiral Cyclo-dex B material prior to NPD monitoring (Spruit et al., 2000, 2001). An additional GC-based approach allowed baseline separation of cyclosarin enantiomers on a GAMMA DEX column monitored by EI-MS (Reiter et al, 2007). VX enantiomers were chromatographed on a Chiracel OD column by LC coupled to an electrochemical detector yielding a lower limit of quantification of about lOng/ml blood (Van der Schans et al, 2003). To our knowledge no chiral HPLC-MS separation of OPCs has been described so far although mobile phases are compatible with MS detection. 

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