Analytical blank, defined

Several terms have been used to define LOD and LOQ. Before we proceed to develop a uniform definition, it would be useful to define each of these terms. The most commonly used terms are limit of detection (LOD) and limit of quantification (LOQ). The 1975 International Union of Pure and Applied Chemistry (lUPAC) definition for LQD can be stated as, A number expressed in units of concentration (or amount) that describes the lowest concentration level (or amount) of the element that an analyst can determine to be statistically different from an analytical blank 1 This term, although appearing to be straightforward, is overly simplified. If leaves several questions unanswered, such as, what does the term statistically different mean, and what factors has the analyst considered in defining the blank Leaving these to the analyst s discretion may result in values varying between analysts to such an extent that the numbers would be meaningless for comparison purposes. 

The detection limit is defined as the concentration of the element that will yield a signal whose intensity is equal to two times the standard deviation of a series of at least 10 measurements of the analytical blank or of a very dilute solution (confidence level 95%). In practice, concentrations should be at least 10 times higher than the detection limit to give reliable measurements (cf. 21.5.3). 

The Union of Pure and Applied Chemistry (lUPAC) defines the Hmit of detection as the lowest concentration level than can be determined to be statistically significant from an analytical blank . The lowest net X-ray intensity N lo that still can be distinguished in a statistically significant manner from the average background level can be written as ... 

The IDL for a specific chemical analyte is defined to be the lowest possible concentration that can be reliably measured. Experimentally, if lower and lower concentrations of a given analyte are measured, the smallest concentration that is barely detectable constitutes a rough estimate of the IDL for that analyte using that particular instrument. For years, EPA has required laboratory analysts to first measure a blank a replicate number of times. The most recent guidelines appeared in 40 CFR (Code of Federal Regulations) Part 136, Appendix B (11). The steps that are recommended are listed as follows ... 

In our laboratory, saturation shake-flask solubility is routinely performed at pH 6.8 and pH 1.0 (for acids only) 0.5 mL of buffer is added to about 2 mg of sample in a small borosilicate vial (2 mL) and sonicated for 3 minutes using a sonicating bath. The suspension is then incubated at 25.0°C for 20 hours in a thermostated water bath at a shaking speed of200 cycles/min.The phases are separated by decantation/centrifugation.The supernatant is carefully collected and the solute quantified using the analytical method defined previously. Before each injection, a blank is performed to avoid phantom peaks due to carryover or impurities present in the system. After the centrifugation it is advisable to re-check the pH of the medium. 

A method s detection limit is the smallest amount or concentration of analyte that can be detected with statistical confidence. The International Union of Pure and Applied Chemistry (lUPAC) defines the detection limit as the smallest concentration or absolute amount of analyte that has a signal significantly larger than the signal arising from a reagent blank. Mathematically, the analyte s signal at the detection limit, (Sa)dl, is... 

Specificity is defined in Directive 96/46/EC as the ability of a method to distinguish between the analyte being measured and other substances. According to SANCO/825/00, blank values must be reported using representative matrices. They... 

Brandt [200] has extracted tri(nonylphenyl) phosphite (TNPP) from a styrene-butadiene polymer using iso-octane. Brown [211] has reported US extraction of acrylic acid monomer from polyacrylates. Ultrasonication was also shown to be a fast and efficient extraction method for organophosphate ester flame retardants and plasticisers [212]. Greenpeace [213] has recently reported the concentration of phthalate esters in 72 toys (mostly made in China) using shaking and sonication extraction methods. Extraction and analytical procedures were carefully quality controlled. QC procedures and acceptance criteria were based on USEPA method 606 for the analysis of phthalates in water samples [214]. Extraction efficiency was tested by spiking blank matrix and by standard addition to phthalate-containing samples. For removal of fatty acids from the surface of EVA pellets a lmin ultrasonic bath treatment in isopropanol is sufficient [215]. It has been noticed that the experimental ultrasonic extraction conditions are often ill defined and do not allow independent verification. 

For this reason, a number of analysts uses a further limit quantity, namely the limit of quantification, xLq, (limit of determination), from which on the analyte can be determined quantitatively with a certain given precision (Kaiser [1965, 1966] Long and Winefordner [1983] Currie [1992, 1995, 1997] IUPAC [1995] Ehrlich and Danzer [2006]). This limit is not a general one like the critical value and the detection limit which are defined on an objective basis. In contrast, the limit of quantification is a subjective measure depending on the precision, expressed by the reciprocal uncertainty xLq/AxLq = k, which is needed and set in advance. The limit of quantification can be estimated from blank measurements according to... 

The DL and QL for chromatographic analytical methods can be defined in terms of the signal-to-noise ratio, with values of 2 1-3 1 defining the DL and a value of 10 1 defining the QL. Alternatively, in terms of the ratio of the standard deviation of the blank response, the residual standard deviation of the calibration line, or the standard deviation of intercept (s) and slope (5) can be used [40, 42], where ... 

The limit of detection (LoD) has already been mentioned in Section 4.3.1. This is the minimum concentration of analyte that can be detected with statistical confidence, based on the concept of an adequately low risk of failure to detect a determinand. Only one value is indicated in Figure 4.9 but there are many ways of estimating the value of the LoD and the choice depends on how well the level needs to be defined. It is determined by repeat analysis of a blank test portion or a test portion containing a very small amount of analyte. A measured signal of three times the standard deviation of the blank signal (3sbi) is unlikely to happen by chance and is commonly taken as an approximate estimation of the LoD. This approach is usually adequate if all of the analytical results are well above this value. The value of Sbi used should be the standard deviation of the results obtained from a large number of batches of blank or low-level spike solutions. In addition, the approximation only applies to results that are normally distributed and are quoted with a level of confidence of 95%. 

The ability to make analytical measurements depends intimately on the availability of well-defined standards and calibrants. Many measurements of analytes in seawater (such as dissolved organic carbon and dissolved organic nitrogen) cannot be compared among laboratories because of the lack of appropriate reference materials and blanks for instrument calibration and testing. Intercomparison exercises are critical (NRC, 1993, p. 75). 

The LOD must be assessed for limit tests. In some cases, the LOD should also be defined for quantitative impurity/purity tests. The LOD is generally expressed as either the minimum level at which the analyte can be reliably detected or as a set amount above the SD from the repeated analysis of suitable sample, such as a blank or negative control sample. 

It is not possible to prescribe specific pretreatment procedures here because these can only be decided upon when the system and the purpose of the experiments has been properly defined. However, a wealth of information exist in various biochemical reference books on how to isolate various biological compounds. The recommended techniques and methods could be used as part of the trace element speciation protocol often after slight modification, taking into consideration the following points First, the trace element blank levels have to be low, less than 10% of the total concentration in the sample. Second, the regents used should not interfere with subsequent analytical determinations. Third, the experimental conditions should not deviate markedly from those found in vivo, especially the pH and ionic strength of the medium. 

For qualitative methods, the LOD is defined as the threshold concentration at which the test becomes unreliable. A series of blank samples, spiked with different concentrations of the analyte, are each analyzed at least 10 times. The threshold, or cutoff, concentration is determined visually based on a response curve, plotting the percentage of positive results versus the concentration. In this respect, the LOD is also defined as the concentration at which 95% of the experiments give a clearly positive signal [15]. 

Two aspects are important for IQC (1) the analysis of control materials such as reference materials or spiked samples to monitor trueness and (2) replication of analysis to monitor precision. Of high value in IQC are also blank samples and blind samples. Both IQC aspects form a part of statistical control, a tool for monitoring the accuracy of an analytical system. In a control chart, such as a Shewhart control chart, measured values of repeated analyses of a reference material are plotted against the run number. Based on the data in a control chart, a method is defined either as an analytical system under control or as an analytical system out of control. This interpretation is possible by drawing horizontal lines on the chart x(mean value), x + s (SD) and x - s, x + 2s (upper warning limit) and x-2s (lower warning limit), and x + 3s (upper action or control limit) and x- 3s (lower action or control limit). An analytical system is under control if no more than 5% of the measured values exceed the warning limits [2,6, 85]. 

A literature survey shows that the concentration of the analyte found when analyzing a spiked blank sample matrix is often expressed as the percentage of the known or true drug concentration and is called recovery. By this definition, recovery is the same as accuracy, which is why accuracy is reported as recovery in many scientific reports. The difference is that the recovery, as defined, should be close to 100%, while the accuracy close to 0%. 

Both the limit of detection and the limit of quantification have been also defined as ratios of the analyte signal to the background signal (S/N). Thus, an S/N ratio of 3 has been used to define the detection limit, whereas a S/N ratio of 10 has been used to define the limit of quantification. Determination of the signal-to-noise ratios is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples. 

Both the detection limit and the limit of quantification, as defined, are often not very stable characteristics of an analytical method, because the blank signal and the signal generated by the very low concentrations of the analyte are frequently dependent on certain analytical parameters, including the purity of reagents, sample matrices, environmental conditions, instrumentation, and the analysts themselves. Sensitivity is a measure of the ability of an analytical method to discriminate between small differences in analyte concentration. It is defined as the analyte signal per unit concentration of the analyte. Despite the apparent simplicity of the sensitivity concept, a degree of confusion surrounds its use. This confusion stems from the perception that the sensitivity of a method is the same as the limit of detection. 

In order to conduct these analyses, the detection limit of the instrument must be known. The detection limit is defined (in ppm or ppb) as the concentration of analyte that allows a detectable signal to be measured with certainty - for example, three times the standard deviation of the background signal or the blank. If the volume of solution needed to obtain these results is known, the preceding values can be transformed to the absolute quantities or mole fractions (pico-mole, femtomole, etc.) that are needed to obtain the signal. In general, these values are excessively small because current instruments use excessively small volumes. 

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