Quantitation limit of detection

The UV-vis spectrophotometric methods require validation of the method for the analysis of pharmaceutical compounds. Once the method is developed, data regarding precision, accuracy, linear response behavior, limit of quantitation, limit of detection, selectivity, and ruggedness are generated. These terms are very well-defined in many compendial monographs and in ICH, USP, and FDA guidelines. These are not addressed further. However, it should be emphasized that without appropriate validation data and reasonable understanding of how the method results will be affected by minor day-to-day variation of experimental parameters, routine generation of acceptable data may be difficult. 

There are no official guidelines on the sequence of validation experiments and the optimal sequence can depend on the method itself. A potentially useful sequence for a liquid chromatographic method is 1) Selectivity of standards (optimizing separation and detection of standard mixtures) 2) precision of retention times and peak areas 3) linearity, limit of quantitation, limit of detection, range 4) selectivity with real samples 5) trueness or accuracy, at different concentrations 6) ruggedness. 

The following analytical performance parameters were included into the validation process selectivity stability during chromatograidiic development and in solution spot stability prior to the run and after development linearity and range precision reproducibility limit of quantitation limit of detection accuracy. The definitions used for the performance parameters the methods applied to determine them and the acceptance criteria were also described. Therefore, these papers can be recommended to be used by practicising chromatographers. 

An analytical method vahdation study should include demonstration of the accuracy, precision, specificity, limits of detection and quantitation, linearity, range, and interferences. Additionally, peak resolution, peak tailing, and analyte recovery are important, especially in the case of chromatographic methods (37,38). 

The method limit of quantitation and limit of detection must be determined as well as the limit of linearity. The limit of quantitation is defined as the level at which the measurement is quantitatively meaningful the limit of detection is the level at which the measurement is larger than the uncertainty and the limit of linearity is the upper level of the measurement rehabihty (39). These limits are determined by plotting concentration vs response. 

Method Transfer. Method transfer involves the implementation of a method developed at another laboratory. Typically the method is prepared in an analytical R D department and then transferred to quahty control at the plant. Method transfer demonstrates that the test method, as mn at the plant, provides results equivalent to that reported in R D. A vaUdated method containing documentation eases the transfer process by providing the recipient lab with detailed method instmctions, accuracy and precision, limits of detection, quantitation, and linearity. 

In hplc, detection and quantitation have been limited by availabiHty of detectors. Using a uv detector set at 254 nm, the lower limit of detection is 3.5 X 10 g/mL for a compound such as phenanthrene. A fluorescence detector can increase the detectabiHty to 8 x 10 g/mL. The same order of detectabiHty can be achieved using amperometric, electron-capture, or photoioni2ation detectors. 

A multiresidue analytical method based on sohd-phase extraction enrichment combined with ce has been reported to isolate, recover, and quantitate three sulfonylurea herbicides (chlorsulfuron, chlorimuron, and metasulfuron) from soil samples (105). Optimi2ation for ce separation was achieved using an overlapping resolution map scheme. The recovery of each herbicide was >80% and the limit of detection was 10 ppb (see Soil chemistry of pesticides). 

Although the most sensitive line for cadmium in the arc or spark spectmm is at 228.8 nm, the line at 326.1 nm is more convenient to use for spectroscopic detection. The limit of detection at this wavelength amounts to 0.001% cadmium with ordinary techniques and 0.00001% using specialized methods. Determination in concentrations up to 10% is accompHshed by solubilization of the sample followed by atomic absorption measurement. The range can be extended to still higher cadmium levels provided that a relative error of 0.5% is acceptable. Another quantitative analysis method is by titration at pH 10 with a standard solution of ethylenediarninetetraacetic acid (EDTA) and Eriochrome Black T indicator. Zinc interferes and therefore must first be removed. 

Limit of Detection (LOD) The minimum concentration of a substance being measured that, in a given matrix and with a specific method, has a 99% probability of being identified, qualitatively or quantitatively measured, and reported to be greater than zero. 

Accuracy and precision Specificity Limit of detection Limit of quantitation Linearity and range... 

The interface should provide quantitative information with a reproducibility better than 10% with low limits of detection and have a linear response over a wide range of sample sizes (low picograms to p,g). 

The limit of detection is the smallest amount of an analyte that is required for reliable determination, identification or quantitation. More mathematically, it may be defined as that amount of analyte which produces a signal greater than the standard deviation of the background noise by a defined factor. Strictly for quantitative purposes, this should be referred to as the limit of determination . The factor used depends upon the task being carried out and for quantitative purposes a higher value is used than for identification. Typical values are 3 for identification and 5 or 10 for quantitation. 

The limit of detection (LOD) (see Figure 2.6) is defined as the smallest quantity of an analyte that can be reliably detected. This is a subjective definition and to introduce some objectivity it is considered to be that amount of analyte which produces a signal that exceeds the noise by a certain factor. The factor used, usually between 2 and 10 [11], depends upon the analysis being carried out. Higher values are used for quantitative measurements in which the analyst is concerned with the ability to determine the analyte accurately and precisely. 

Signal-to-noise ratio The ratio of the intensity of the analytical signal to that of the noise. This is used in determining the limits of detection and quantitation. 

It is appropriate at this juncture to illustrate the power of chemiluminescence in an analytical assay by comparing the limits of sensitivity of the fluorescence-based and the chemllumlnescence-based detection for analytes in a biological matrix. The quantitation of norepinephrine and dopamine in urine samples will serve as an illustrative example. Dopamine, norepinephrine, and 3,4-dihydroxybenzy-lamine (an internal standard) were derivatized with NDA/CN, and chemiluminescence was used to monitor the chromatography and determine a calibration curve (Figure 15). The limits of detection were determined to be less than 1 fmol injected. A typical chromatogram is shown in Figure 16. 

Figure 2.14. The definition of the limits of detection, LOD, respectively quantitation, LOQ (schematic).

Conclusions the residual standard deviation is somewhat improved by the weighting scheme note that the coefficient of determination gives no clue as to the improvements discussed in the following. In this specific case, weighting improves the relative confidence interval associated with the slope b. However, because the smallest absolute standard deviations. v(v) are found near the origin, the center of mass Xmean/ymean moves toward the origin and the estimated limits of detection resp. quantitation, LOD resp. 

Situation and Criteria A method was to be developed to determine trace amounts of cyanide (CN ) in waste water. The nature of the task means precision is not so much of an issue as are the limits of detection and quantitation (LOD, LOQ), and flexibility and ease of use. The responsible chemist expected cyanide levels below 2 ppm. 

Legend No number of measurement. Cone concentration in fig, CN"/100 ml Absorb absorbance [AU] slope slope of regression line t CV intercept see slope res. std. dev. residual standard deviation Srts -n number of points in regression LOD limit of detection LOQ limit of quantitation measurements using a 2-fold higher sample amount and 5-cm cuvettes—i.e., measured absorption 0. .. 0.501 was divided by 10. 

Figure 4.31. Key statistical indicators for validation experiments. The individual data files are marked in the first panels with the numbers 1, 2, and 3, and are in the same sequence for all groups. The lin/lin respectively log/log evaluation formats are indicated by the letters a and b. Limits of detection/quantitation cannot be calculated for the log/log format. The slopes, in percent of the average, are very similar for all three laboratories. The precision of the slopes is given as 100 t CW b)/b in [%]. The residual standard deviation follows a similar pattern as does the precision of the slope b. The LOD conforms nicely with the evaluation as required by the FDA. The calibration-design sensitive LOQ puts an upper bound on the estimates. The XI5% analysis can be high, particularly if the intercept should be negative.

This example assumes that RIA was chosen. The principle behind RIA is the competition between the analyte A and a radioactively tagged control C (e.g., a /-marked ester of the species in question) for the binding site of an antibody specifically induced and harvested for this purpose. The calibration function takes on the shape of a logistic curve that extends over about three orders of magnitude. (Cf. Fig. 4.38a.) The limit of detection is near the B/Bo = 1 point (arrow ) in the upper left corner, where the antibody s binding sites are fully sequestered by C the nearly linear center portion is preferrably used for quantitation. 

Determine the limit of detection LOD and limit of quantitation LOQ according to the interpolation at level y = a + CL of the regression line and its lower CL this is sensitive to the calibration-point pattern ... 

LOD) calculate and display the limits of detection and quantitation LOD, LOQ. [Note This form of calculating the LOD or LOQ was chosen because the results are influenced not only by the noise on the baseline, but also by the calibration design from the educational point of view this is more important than the consideration whether any agency has officially adopted this or that LOD-model. For a comparison, see Figs. 2.14, 2.15, and 4.31]. 

GC/MS has been employed by Demeter et al. (1978) to quantitatively detect low-ppb levels of a- and P-endosulfan in human serum, urine, and liver. This technique could not separate a- and P-isomers, and limited sensitivity confined its use to toxicological analysis following exposures to high levels of endosulfan. More recently, Le Bel and Williams (1986) and Williams et al. (1988) employed GC/MS to confirm qualitatively the presence of a-endosulfan in adipose tissue previously analyzed quantitatively by GC/ECD. These studies indicate that GC/MS is not as sensitive as GC/ECD. Mariani et al. (1995) have used GC in conjunction with negative ion chemical ionization mass spectrometry to determine alpha- and beta-endosulfan in plasma and brain samples with limits of detection reported to be 5 ppb in each matrix. Details of commonly used analytical methods for several types of biological media are presented in Table 6-1. 

Under the experimental conditions used the method gave a limit of detection of 5 ng. Recoveries of W-nitrosoglyphosate from the fortified soil samples (10 g) at the 1 and 5 ppm levels were nearly quantitative (6). 

The data are also represented in Fig. 39.5a and have been replotted semi-logarithmically in Fig. 39.5b. Least squares linear regression of log Cp with respect to time t has been performed on the first nine data points. The last three points have been discarded as the corresponding concentration values are assumed to be close to the quantitation limit of the detection system and, hence, are endowed with a large relative error. We obtained the values of 1.701 and 0.005117 for the intercept log B and slope Sp, respectively. From these we derive the following pharmacokinetic quantities ... 

The semilogarithmic plot of the data is given in Fig. 39.8a. On this plot we can readily identify the linear 3-phase of the plasma concentrations between 30 and 240 minutes. The last three points (at 360, 480 and 720 minutes) have been discarded because the corresponding plasma values are supposed to be close to the quantitation limit of the detection system. 

Recoveries, limit of quantitation, and limit of detection Calculation of residues Important points... 

Liquid chromatography/mass spectrometry Lower limit of detection Limit of detection Limit of quantitation Florseshoe crab hemocyanin Liquid scintillation counting Matrix-assisted laser desorption/ ionization mass spectrometry m -Maleimidobenzoy 1-A -Hydroxysuccinimide 1 -Cyclohexyl-3-(2-Morptiolino-ethyl)carbodiimide rnetlio-/ -Toluenesulfonate (same as CDI)... 

The limit of determination [or limit of quantitation (LOQ)] is defined in Directive 96/46/EC as the lowest concentration tested at which an acceptable mean recovery (normally 70-110%) and acceptable relative standard deviation (normally <20%) are obtained. The specific requirements for LOQ in crops, food, feed, soil, drinking and surface water, air, body fluids, and tissues are described in Section 4. Because the abbreviation LOD usually means limit of detection rather than limit of determination, the authors prefer not to use this abbreviation here in order to avoid confusion, and LOQ is used throughout. According to Directive 96/46/EC no data with regard to the limit of detection must be given. 

The required limit of quantitation (LOQ) and limit of detection (LOD) have been extended to the parts per billion range as the European Community (EC) baby food -related guideline and the US consumer basket requirements became effective. 

Adequate sensitivity should be demonstrated and estimates of the limit of detection (LOD) and the limit of quantitation (LOQ) should be provided. The slope of the calibration line may indicate the ability of the method to distinguish the tme analyte concentration. The LOD of a method is the lowest analyte concentration that produces a reproducible response detectable above the noise level of the system. The LOQ is the lowest level of analyte that can be accurately and precisely measured. For a regulatory method, quantitation is limited by the lowest calibration standard. The techniques for these estimations should be described. 

Residue study protocols typically either include quality specifications for analytical procedures or refer to a written analytical method that includes such specifications. The protocol for an LSMBS should also include analytical quality specifications, either directly or by reference to a method. Analytical specifications usually include minimum and maximum recovery of analyte from fortified control samples, minimum number of such fortifications per set of samples, minimum linearity in calibration, minimum stability of response to injection of calibration solutions, and limits of quantitation and of detection. 

Similar considerations were taken into account throughout the process of designing the study and committing the design to a protocol. In addition to analytical quality specifications, decisions were made regarding definitions of limits of detection and quantitation, levels of apparent residues at which confirmation was required, and how such confirmation would be achieved. All of these decisions were based on fulfilling the objectives of the study while operating within unavoidable time and resource constraints. 

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