Detection and Limit of Quantification

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

Kuselman, I., and Sherman, F. (1999), Assessment of limits of detection and quantification using calculation of uncertainty in a new method for water determination, Accred. Qual. Assur., 4,124-128. 

The linear range was wider than two orders of magnitude. The limits of detection and quantification were suitable to satisfy current legislation. 

In order to achieve high throughput with ORAC determinations, Huang et al.464 have developed a robotic eight-channel liquid handling system coupled with a microplate fluorescence reader, which has improved efficiency at least 10-fold, compared with the discontinued COB AS FARAII analyser. The CV was 15% or less and the limits of detection and quantification were 5 and 6.25, uM, respectively. 

Mocak, J. Bond, A.M. Mitchell, S. Scollary, G. 17. A statistical overview of standard (lUPAC and ACS) and new procedures for determining the limits of detection and quantification Applied to voltammetric and stripping 18. techniques. Pure Appl. Chem. 1997, 69, 297-328. 

Results verification is totally different from results validation. Results validation (point 4.7.5. and 5.9. of NBN-EN-ISO-CEI 17025 standard) shows, each year, or when it is judged necessary, that a given laboratory has the capacity to apply a particular method, repetitively, in respect of obtained data during initial validation. Trueness and statistical dispersion of results are the basis of the definition of the uncertainty of the standard of measurement [16] and, in some cases, the basis for the definition of the limit of detection and quantification. Management of data from validation results, as control card, could permit the detection and control of eventual deviation. Validation of results is the internal quality control procedure which verifies the stability of performance of the methods for which accreditation is sought, in the limited-scope procedural context. 

Left-censored data are characteristic of many bioassays due to the inherent limitation of the presence of a lower limit of detection and quantification. An ad hoc approach to dealing with the left-censored values is to replace them with the Unfit of quantification (LOQ) or LOQ/2 values. Alternatively, one can borrow information from other variables related to the missing values and use MI to estimate the left-censored data. In addition, the left-censored mechanism can be incorporated directly into a parametric model, and a maximum likelihood (ML) approach can be used to estimate the parameters (21). 

A selective CZE microassay was developed for the determination of dexa-methasone phosphate and its major metabolite, dexamethasone, in tears (325). An internal standard, indoprofen, was used for quantitation. The limits of detection and quantification were 0.5 and 2.0 pg/mL, respectively. The quantitative method was essential for the in-vivo determination of the dexamethasone concentration-time profiles in tears after the application of the anti-inflammatory drug. Two examples of rapid and simple drug analysis in pharmaceutical formulations using capillary electrophoresis can be found in the methods described for the separation of naphazoline, dexamethasone, and benzalkonium in nose drops (326). 

The RNAA method was optimized for the determination of low-concentration I in human brain samples. The detection limit for I in human brain samples, defined by 3-Jn where N is the number of background counts under the 433 keV peak, was calculated (Currie, 1968). The quantification limit (1 O-v/TV ) would be about three times the corresponding value for the detection Emit. Detection and quantification concentrations were calculated from limits of detection and quantification defined by Currie (1968) (Table 69.3). The sensitivity of our method was found to be adequate for occurring elemental concentrations. The precision, in terms of relative SD, was 5% at about 50 ng g levels of I. 

Ding et al. described an automated on-line SPE-LC-MS/MS method for the determination of macrolide antibiotics, including erythromycin, roxithromycin, tylosin, and tilmicosin in environmental water samples. A Capcell Pak ME Ph-1 packed-column RAM was used as SPE column for the concentration of the analytes and clean-up of the sample. One millilitre of a water sample was injected into the conditioned SPE column, and the matrix was washed out with 3 ml high-purity water. By rotation of the switching valve (see Fig. 4.2), macrolides were eluted in the back-flush mode and transferred to the analytical column. The limits of detection and quantification obtained were 2-6 and 7-20 ng/1, respectively, which is suitable for trace analysis of macrolides. The intra- and inter-day precisions ranged within 2.9-12% and 3.3-8.9%, respectively. At the three fortification concentrations tested (20, 200, and 2000 ng/1), recoveries of macrolides ranged from 86.5% to 98.3%. 

The EC requirements do not include limits of detection and quantification or MU, but instead require the determination of other statistical indicators of result reliability, that is, the decision limit (CCa) and the detection capability (CCP) as discussed in Section 8.7.10. 

However, when the LOQ of a method is significantly lower than the actual concentrations monitored for compliance with a MRL, it may be more appropriate to carry out the validation experiments based on a lowest calibrated level (LCL), typically 0.5 x the MRL. For use in a regulatory program, the limits of detection and quantification are important parameters when the method will be applied to estimate exposure to residues, where there may be an interest in monitoring residues at concentrations below the MRL, or when conducting residue analyses for substances that do not have ADIs or MRLs. For monitoring compliance with a MRL, it is important that an LCL be included in the analysis that adequately demonstrates that the MRL concentration may be reliably determined. The LCL of a method used to support a MRL should not be less than the LOQ. 

As noted previously, Decision 2002/657/EC does not specify a requirement for the validation of analytical methods to include limits of detection and quantification, but instead includes the decision limit and the detection capability as required performance criteria. The alpha... 

Dr. Taylor continues his general advice "Later speakers will discuss how measurement variability quantitatively defines the limits of detection and quantification. Due to the nature of measurement, each laboratory (analyst) will have somewhat different measurement uncertainties, and hence different limits of detection. Published values of LCD s (Limits Of Detection), MDL s (Method Detection Limits), or what-have-you for methodology are typical, at best hence they have no predictable quantitative relation to those obtained by any laboratory or analyst. Each must evaluate them for itself and will make somewhat different decisions concerning precision and detection when analyzing the same samples. Each has the professional obligation to obtain all information necessary to support the quality of its data, which must be technically sound and defensible. 

Zorn, M. E., R. D. Gibbons, and W. C. Sonzogni. 1997. Weighted least squares approach to calculating limits of detection and quantification by modeling variability as a function of concentration. Anal. Chem. 69 (15), 3069-3075. 

Eurachem guide [285], which discusses when, why, and how methods should be validated. However, for the pharmaceutical industry, the main reference source is the ICH Guidelines [286], which provides recommendations on the various characteristics to be tested for the most common types of analytical procedures developed in a pharmaceutical laboratory. The main characteristics of any analytical method to be tested are specificity, linearity, accuracy, precision, solution stability, limits of detection and quantification, and robustness. Specific aspects should be considered for a CE method including method transfer between instrument manufacturers, reagent purity and source, electrolyte stability, capillary treatment and variations in new capillaries, and buffer depletion. Fabre and Altria [284] discuss CE method validation in more detail and include a number of examples of validated CE methods for pharmaceutical analysis. Included in Table 4.3 are a number of validated pharmaceutical assay methods. 

A chemical marker for the off-flavor of wine, in bottles with cork stoppers, has been proposed as 2,4,6-trichloroanisole (2,4,6-TCA). In one approach, the analyte was extracted from liquid sample using a single drop of an imidazolium-based ionic liquid. The ionic liquid was heated, releasing the 2,4,6-TCA into an IMS in negative polarity. The limits of detection and quantification were 0.2 and 0.66 ng/L, respectively, and precision was reported for 10 ng/L as 1.4% (repeatability, n = 5) and 2.2% (reproducibility, n = 5 during 3 days). 

A related field of research that has found certain apphcations in pharmaceutical analysis is flash photolysis. This technique has been adapted with UV absorbance, fluorescence, and electrochemical detection of photolysis products separated by LC to enhance limits of detection and quantification. Products of these reactions have also been further investigated following LC separation. 

The most important parameters in method validation for bioanalysis applications are linearity, precision, accuracy, selectivity, and limits of detection and quantification. In... 

The sensitivity of the method is evaluated by the limit of detection and quantification. In general, there are no specific criteria for the limit of detection, but for bioanalysis purposes, the limit of quantification is established for the concentration with precision and accuracy lower than 20%. Limits of detection and quantification obtained in CE with UV detection are generally higher than in HPLC, owing to the small optical path in the detection window and injected volumes. As discussed previously, by using preconcentration procedures (off-line sample preparation and/or stacking sample injection techniques) and a more sensitive detection system, particularly LIE and MS detection, suitable detection or quantification limits could be obtained for the apphcation of the methods to the analysis of real samples. 

Analyte enrichment (preconcentration) in order to improve the method sensitivity (reduction of the limits of detection and quantification). 

Because of the pervasive and pernicious occurrence of matrix effects, it is usually advisable to build a routine check on the extent of these effects into any method that has been shown to be subject to them, e.g. the ME/RE procedure (Matuszewski 2003) described in Section 5.3.6a. A particularly deceptive cause of ionization suppression that is not really a matrix effect is the mutual interference of an analyte and its co-eluting SIS (Liang 2003 Sojo 2003), discussed in some detail in Section 5.3.6a. While any level of suppression (or enhancement) of ionization efficiency is undesirable, the mutual suppression of analyte and an isotope-labeled SIS appear to be equal, with minimal effect on the validity of the quantitative analysis, although it may adversely affect limits of detection and quantification as a result of the... 

---