Lower limits of quantitations

Accuracy, precision, and linearity of standards — The linear dynamic range was established as 10.22 pg/mL to 2.037 ng/mL with coefficient of determination (r2) below 0.996190 when using 1/x2 weighing for three consecutive accuracy and precision runs. The lower limit of quantitation (LLOQ) was accurate (inter-run mean bias = 1.1%) and precise (inter-run mean CV = 14.1%). Three levels of QCs were prepared. The inter-run mean bias varied from -4.3 to 1.0% and the inter-run mean CV varied from 4.6 to 6.6% for all QC levels. 

Christiaens et al. (2004) measured cyproterone acetate (CPA), a drug used to treat prostate carcinoma, in human plasma. A LiChrosphere RP-4 (25 x 2 mm inner diameter, 25 /an, Merck KGaA) cartridge was coupled with an OminiSpher C18 column (100 x 2 mm inner diameter, 3 /an) and 30 jiL plasma was injected directly. The lower limit of quantitation (LOQ) was 300 pg/mL. Recovery was 100%. 

Koal et al. (2004) measured four immunosuppressants (cyclosporine A, tacrolimus, sirolimus, and everolimus) in whole blood samples from transplant recipients. The samples were treated first with a protein precipitation step. The supernatant was extracted with a Poros Rl/20 perfusion column (30 x 2.1 mm, 20 tm, Applied Biosystems, Darmstadt, Germany) online. A Luna phenyl hexyl column (2 x 50 mm, Phenomenex, Schaffenburg, Germany) was used for separation. The total run time was 2.5 min. The lower limit of quantitation was 10 ng/mL for cyclosporine A and 1 ng/mL for the other three analytes. 

Other applications include bioequivalent measurements of bromazepam, an anticonvulsant, in human plasma. The lower limit of quantitation (LLOQ) was 1 ng/mL (Gongalves et al. 2005). Kuhlenbeck et al. (2005) studied antitussive agents (dextromethorphan, dextrophan, and guaifenesin) in human plasma LLOQ values were 0.05, 0.05, and 5 ng/mL, respectively. Other compounds studied were nucleoside reverse transcriptase inhibitors, zidovudine (AZT) and lamivudine (3TC) (de Cassia et al. 2004) and stavudine (Raices et al. 2003) in human plasma, and paclitaxel, an anticancer agent, in human serum (Schellen et al. 2000). 

As a generic method, the SPE cartridge was conditioned with 1.5 mL methanol (5.0 mL/min) and 1.5 mL water (5.0 mL/min), after which 100 /tL of spiked plasma was injected and washed with 3.0 mL water (2.0 mL/min). After switching online, the analytes were flushed and eluted with a fast gradient of mobile phase A (5 95 v/v acetonitrile water, 0.1% formic acid, and 10 mM ammonium acetate) and B (95 5 v/v acetonitrile water, 0.1% formic acid, and 10 mM ammonium acetate). The lower limit of quantitation was 0.2 to 2 ng/mL and linear range was 2 to 4 orders. Carry-over was 0.02 to 0.1 %. 

The recent trend of decreasing available sample volumes and requiring lower limits of quantitation (LLOQs) means better sample preparation procedures are under consideration. Further improvements MS sensitivity will eventually impact sample preparation strategies and sample throughput. 

Injector temperature, °C Column temperature, C Detector temperature, °C Lower limit of quantitation (ppm)b... 

The lower limit of detection given in Equation 5-5 is 3s/m, where s is the standard deviation of a low-concentration sample and m is the slope of the calibration curve. The standard deviation is a measure of the noise (random variation) in a blank or a small signal. When the signal is 3 times as great as the noise, it is readily detectable, but still too small for accurate measurement. A signal that is 10 times as great as the noise is defined as the lower limit of quantitation, or the smallest amount that can be measured with reasonable accuracy. 

The lower limit of quantitation is 0.5 ng/L (parts per trillion). To measure such small quantities requires extraordinary care at every stage of analysis to prevent contamination. Mercury amalgam fillings in a worker s teeth can contaminate samples exposed to exhaled breath. 

Quality Control (QC) QC samples are used to check the performance of the bioanalytical method as well as to assess the precision and accuracy of the results of postdose samples. QC samples are prepared by spiking the analyte of interest and the IS into a blank/control matrix and processing similar to the postdose samples. QC samples cover the low (3 x LLOQ LLOQ = lower limit of quantitation), medium, and high (70-85% of ULOQ ULOQ = upper limit of quantitation) concentration ranges of the standard curve and are spaced across the standard curve and the postdose sample batch. 

Although the lower limit of quantitation is established during assay validation and prior to microdosing, assay sensitivity remains an uncertainty until the actual analysis of the microdose samples as well. There is always the danger that plasma exposures from the microdose are lower than predicted and as a result plasma concentrations from some or all of the time points cannot be detected by the LC-MS/MS method. Reduction of this risk is achieved by collaborative communication between the bioanalytical chemist and the project team. Conservative estimates on bioavailability and clearance can be used to establish the necessary limit of detection needed to determine plasma concentrations for all time points. Updates on the progress of the assay development allow the team to decide if the achievable limit of detection will enable the determination of plasma concentrations from enough time points to make a go-no go decision. Of course, sensitivity is not an issue with AMS, which practically ensures that plasma concentrations will be determined, possibly for several days, enabling the observation of complex PK and clearance from deep compartments. 

Second, an internal standard must have adequate purity. Preferably, the contribution of an internal standard to any analyte should be less than 20 % of the corresponding lower limit of quantitation (LLOQ) of the analyte. Otherwise, the significant amount of analyte from the added internal standard can bias the reported signal to noise (S/N) ratio at the LLOQ and cause larger variability at low concentrations. The interference of an internal standard to other cointemal standards in a multianalyte method is rare, but it should be also evaluated. Though there are no reported criteria for this, it should be at least less than 15 % of the concentration of a cointemal standard in a multianalyte method. In addition, an internal standard should not correspond to any in vivo metabolic products of the analyte (e.g., hydrox-ylated metabolite, N-dealkylation metabolite). 

Typically, six to eight different nonzero concentrations that cover the dynamic range of the assay should be used to define the curve. Two STDs should be at concentrations lower than the Low QC sample and two STDs should be at concentrations above the High QC sample. Five levels of analytical QC, i.e LLOQ (Lower Limit of Quantitation), Low, geometric Medium (optional), Medium and High QC, are recommended. In general, the QC concentrations should differ from standard concentrations. The corresponding concentration distributions are LLOQ=same con-... 

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