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Quality problems in determination of organic compounds in environmental samples, such as PAHs and PCBs
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Trends in Analytical Chemistry, Vol. 29, No. 7, 2010
Quality problems in determination of organic compounds in environmental samples, such as PAHs and PCBs Piotr Konieczka, Lidia Wolska, Jacek Namies´nik The literature shows that most interlaboratory comparisons deal with determination of inorganic compounds or physico-chemical parameters. The determination of organic compounds, particularly in environmental samples, is still a challenging analytical problem, which is why there are few interlaboratory comparisons on this subject. The amount and the quality of information obtained from interlaboratory comparisons depend on both their organizer (who selects and prepares material for investigation, and provides statistics based on the results of measurements) and participants (who carry out measurements, determine the validation parameters of the relevant analytical procedure, and publish the results of analyses). We discuss the difficulties in determining organic compounds on the basis of our own investigations on polyaromatic hydrocarbons and polychlorinated biphenyls in environmental samples. We present means of overcoming these difficulties as conclusions emerging from the results of interlaboratory comparisons. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Analytical procedure; Environmental sample; Interlaboratory comparison; Organic compound; PAH; PCB; Polyaromatic hydrocarbon; Polychlorinated biphenyl; Sample preparation; Trace-organic constituent
1. Introduction Piotr Konieczka*, Lidia Wolska, Jacek Namies´nik Department of Analytical Chemistry, Chemical Faculty, Gdansk University of Technology ul. Narutowicza 11/12, 80-233 Gdan´sk, Poland
*
Corresponding author. Tel.: +485 83 47 21 10; Fax: +485 83 47 26 94; E-mail: [email protected]
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The overriding aim of every analysis is to acquire reliable results that supply the fullest possible information about the object of study on the basis of measurement results obtained during the assay of a sample. Results are obtained as a consequence of measurements made with the appropriate analytical procedures and measuring instruments. These must be properly prepared for the job in hand so that the final results are reliable. The determination of analytes at ever lower concentrations in samples with ever more complex matrices is one of the main lines of development in contemporary analytical chemistry. This is an exceedingly difficult, complicated task, so it presents a great challenge to analysts, who need to be aware of the problems surrounding quality assurance and quality control (QA/QC).
Assuring the appropriate quality of analytical measurement results involves monitoring the reliability of measurement apparatus and the range of applicability of analytical procedures. For this purpose, a system of QC of analytical measurements is applied, involving: assuring the metrological coherence of the results; estimating the uncertainties of the measurement results; validating the analytical procedures applied; utilizing (certified) reference materials; and, participating in different kinds of interlaboratory comparisons. Participation in interlaboratory comparisons provides an opportunity to compare oneÕs own results with those obtained by other laboratories and to demonstrate oneÕs competence, which is especially important in the case of accredited
0165-9936/$ - see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.03.012
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laboratories or those applying for accreditation. Moreover, such comparisons enable the participants to search for and to identify unexpected errors on the basis of comparisons with an external standard and with their own earlier results, and, if an error is discovered, to take steps to rectify it [1]. The procedure for determining specific compounds (particularly those occurring at trace and ultra-trace levels) in environmental samples involves the following steps [2]: (1) sample collection; (2) transport and storage of samples; (3) preparation of samples for analysis (e.g., filtration, mineralization, extraction, and fractionation); (4) final analyte determination (including the method of calibration); (5) calculation and interpretation of data; and, (6) QA/QC of analytical measurements and results. In view of the need to confirm the reliability of analytical results obtained by analytical laboratories, the results of tests and the analytical methods applied must be documented and verifiable. One of the most important ways of monitoring analytical results is to participate in interlaboratory comparisons, which facilitate the evaluation of the results of measurements performed on identical, or at least very similar, test samples at two or more laboratories under stipulated conditions. However, before embarking on an interlaboratory comparative study, the organizer should be absolutely clear as to the purpose, since this will determine how such a comparison will be prepared (the object of study, the type of samples to be assayed, sample preparation techniques, etc.) and how it is to be carried out. Interlaboratory comparisons are organized in order to [3]: (1) assess the reliability of determinations; (2) acquire experience; and, (3) improve the quality of analytical determinations. It is worth emphasizing that only a very few interlaboratory comparisons have been run on the determination of organic compounds in environmental samples, as opposed to the many given over to the determination of metals or inorganic ions. Undoubtedly, this is because of the far greater number of sources of errors that can be committed during the determination of the content of organics and the correspondingly greater difficulty of such analyses.
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analytical equipment (e.g., GC-MS, LC-MS, ICP-MS, and GC · GC), computers and the relevant software, are themselves sufficient to guarantee the high quality of the measurement and data-processing steps in the analytical procedure. However, analysts are coming to realize that it is collection, transport and storage of a sample, and its preparation for final determination that have the greatest influence on the quality of the information obtained from the measurement data [2]. Of especial interest to analysts are problems concerning the determination of polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in environmental samples. More than 30 years ago, the US Environmental Protection Agency (EPA) officially recognized these compounds as major environmental pollutants. Of the most toxic, 16 PAHs and seven PCBs were placed on the list of priority contaminants, and, as indicators of the level of environmental pollution, were designated for monitoring studies. The interest of the scientific community, greater environmental awareness, and increasing pressure on the part of society were all crucial factors causing the sudden increase in the need for information on levels of environmental pollution by PAHs and PCBs, among other compounds. Since that time, original papers on novel methodologies and equipment [4–7] and review papers [8–11] have appeared. Papers describing the procedures and the results of analytical tests for determining PAHs and PCBs in environmental samples (air, water, soil, bottom sediments, suspended particulate matter, organisms) have also been published [12–27]. But does this mean that the determination of PAHs and PCBs in environmental samples is now problem free? And do the results give reliable information about the environment? The results of numerous studies performed have demonstrated the possibilities and the limitations of contemporary analytical techniques in acquiring reliable information on the levels and forms of occurrence of PAHs and PCBs in the environment. Moreover, they have persuaded us to take a fresh look at the problems involved in their determination in environmental samples characterized by matrices of variable, highly diversified composition.
3. Implementation of interlaboratory comparisons 2. The problems involved The effect of a procedure on the final analytical result may be due to the way in which the successive steps are carried out, the materials from which the apparatus and accessories are made, and the content of the sample itself (matrix effects). It is generally believed that present levels of knowledge, together with the availability of state-of-the-art
Interlaboratory comparisons are undertakings in which tasks are performed by both the organizer and the individual participants. The tasks of the organizer include [2]: (1) selection and preparation of test samples of appropriate stability; (2) distribution of test samples, together with detailed instructions concerning measurement procedures and presentation of results; http://www.elsevier.com/locate/trac
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(3) statistical analysis and processing of results; and, (4) compilation of the final report. The tasks of the participants are to: (1) validate the analytical procedure used for the measurements; (2) perform measurements (calibration, determination of validation parameters, and estimation of uncertainty); (3) calculate measurement results; and, (4) send measurement results together with a detailed description of the applied analytical procedure to the organizer. The correct preparation and execution of each of these tasks will ensure optimal running of interlaboratory comparisons and will supply a full set of data regarding possible sources, types and magnitudes of error, which are the cause of the differences between actual and expected measurement results. There are three main types of proficiency testing (PT): (1) evaluating measurement procedures; (2) testing the competence of laboratories; and, (3) certification of materials. 3.1. Evaluating measurement procedures During this type of PT, all participants, in accordance with the same protocol and using the same procedure, perform tests to determine characteristic features in a batch of identical test samples. The results are used to estimate the characteristic parameters of the procedure, which are usually internal – interlaboratory precision, systematic error, analyte recovery, internal QC parameters, sensitivity, limit of detection (LOD), range of applicability. The role of the organizer in this type of study is to prepare the sample(s) in such a way that every step in the analytical procedure being tested can be monitored and that relevant conclusions can be drawn. 3.2. Testing the competence of laboratories One or more analyses are carried out by a group of laboratories on the basis of one or more homogeneous, stable test samples using a selected procedure or one routinely applied by all the laboratories participating in the interlaboratory comparison. The results are then compared with those of the other laboratories or against a known or guaranteed reference value. In this case, too, choice and appropriate preparation of test samples should enable the largest possible number of parameters of the procedure to be monitored by the participating laboratories. 3.3. Certification of materials As a result of the tests, a reference value is assigned to a parameter (e.g., concentration) or a physical property in the test material or test sample, usually of known uncertainty.
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Normally, laboratories of proven competence (reference laboratories) are chosen to participate in this type of study in order to test a material that is a candidate for a reference standard using a procedure facilitating the estimation of concentration (or another parameter) encumbered with the least error or the least known uncertainty. 3.4. Particular problems We now describe the particular problems that need to be taken into account during the organization of and participation in interlaboratory comparisons. The relevance of the conclusions regarding the assay of the test materials [i.e. analytical procedure, laboratory or certified reference material (CRM)] will depend on the way in which these problems are monitored within the PT framework. 3.4.1. Selectivity. Selectivity is a necessary condition for obtaining reliable measurement results. In the case of chromatographic analysis – the ‘‘classical’’ technique for determining organic compounds – selectivity is represented by the separation factor, the value of which is a function of the difference in retention times and chromatographic peak widths. The parameters affecting selectivity where chromatographic techniques are concerned are: (1) the type of column used; and, (2) the chromatographic separation parameters (e.g., temperature program, gas flow rate). With a suitable chromatographic column, the analyte signal can be distinguished from the interferent signal. If an inappropriate column is used, the materials to be assayed may well be determined in combination. In the case of PAHs, it happens that compounds (e.g., benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[j]fluoranthene) are determined as pairs of isomers (b+j or k+j) or even as the sum of all three, even by very experienced laboratories [18]. Again, PCBs are often determined as the sum of PCB 28 + PCB 31 or PCB 138 + PCB 162; moreover, the order of elution of the isomers PCB 28 and PCB 31 [28], PCB 138 and PCB 153 is frequently confused. Fig. 1 presents examples of chromatograms obtained during the determination of PCB analytes using two different columns [28]. The separation of PCB 31 from PCB 28 cannot be done on a column containing stationary phase DB17, but is possible with a DB1701 column. Organizers of interlaboratory comparisons should be aware of the errors that may be committed in connection with the difficulties in obtaining measurement selectivity. They should prepare the PT material in such a way that both the errors and their sources are identifiable. The simplest way of doing this is to make up a few
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RT: 17.07 - 34.72 18.29
07 - 34.72 18.29
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DB- 17
PCB 28
90
PCB 28
80
18.24 24.54
19.46
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PCB 101
60
PCB 153
25.49
22.68
20.83
26.05
PCB 105
50
18.24
27.53
40
19.46
21.99
18.78
22.81
30
30.23
26.92
PCB 170 31.88
29.08
23.77
31.72
20
32.63 32.82
10 0 100
18.28
PCB 28*
90
19.45
PCB 105*
80
18.78
26.91
25.46
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31.98
60
PCB 153*
22.66
50
PCB 101*
27.51
26.04
30.21 31.87
40
PCB 170*
30 20.34 20.97
20 10
30.37
18.08 22.51
33.50
28.51 29.89
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24
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RT: 17.60 - 35.40 19.45
PCB-28
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PCB-28
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19.36
80 70 60 50 40
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20.63
21.96
23.94 23.72
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27.34
PCB-101
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PCB-15329.31 28.66
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PCB PCB-170
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PCB-28*
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PCB-101*
PCB-153*
30 34.42
20
PCB-170* PCB
10 0 18
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26 28 Time (min)
30
32
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Figure 1. Some chromatograms obtained during the analysis of samples of appropriately prepared extracts for the determination of PCB analytes; separation was achieved using different types of chromatographic column [28].
solutions of scarcely separable analytes in widely differing concentrations.
3.4.2. Calibration. An optimally selected calibration technique is of the prime importance for decoding the
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output signal of the measuring instrument to obtain the analytical signal. As every analyst is well aware, calibration is integral to every analytical process [2]. After all, the fundamental analytical task is to determine the concentration of a particular substance (the analyte) in a sample. But this can be done with the aid of an analytical instrument only on the basis of measurement information supplied by that instrument (analytical signals) for the sample. Calibration is defined in various ways. For example, the IUPAC Gold Book defines calibration in analysis as: ‘‘The set of operations which establish, under specified conditions, the relationship between values indicated by the analytical instrument and the corresponding known values of an analyte’’ [29]. Calibration is also defined as ‘‘a model attempting to predict the value of an independent variable in the case when only the dependent variable is known’’ [30]. Generally speaking, ‘‘analytical calibration’’ is understood to mean the representation of the real (true, theoretical) dependence of the analytical signal on the analyte concentration in empirical form (the calibration curve), and then using that curve to determine the analyte concentration in the sample in order to obtain the ‘‘analytical result’’. The calibration curve is constructed under given analytical conditions using standard/reference samples. The calibration method generally depends on the following factors [2,3]: (1) the type of measuring instrument; (2) the number of samples;
(3)
the possibility of preparing samples over a wide range of analyte concentrations (in order to check the full measurement range of the monitoring/ measuring instrument); (4) the required accuracy of a measurement; (5) the composition of the sample matrix; and, (6) the possibility of the sample composition changing during the analytical process. A common error when constructing a calibration curve is to plot it for a very wide range of concentrations. It is well known that a calibration curve is linear only within a strictly defined interval of concentrations; the curve will certainly exhibit one set of characteristics for concentrations close to the LOD, another for higher concentrations, and yet another when levels approach the limit of the measurement range. The dependence of detector response on the concentration of the compound to be determined takes the shape of a sloping letter S. In the case of PAHs, it was found that the slope of the calibration curve covering the lower range of concentrations could be up to three times less than that of the curve relating to the higher range of concentrations in the samples investigated [31]. This must be taken into consideration when this step of the analytical procedure is carried out. As an example of the influence of the calibration step on the final analytical result, Table 1 ranks laboratories on the basis of relative error value. One of the ways of checking whether the calibration step (as an integral part of the final determination step)
Table 1. Ranking* of laboratories participated in interlaboratory comparison Lab code
lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab
3 23 19 15 7 8 16 13 1 22 11 6 2 17 9 20 21 5
PCB 52
118
138
153
180
23 8.5 26 35 11 12 24 62 21 57 51 97 68 141 63 211
19 14 30 42 39 27 6.1 48 104 56 62 249 32 1.4 45 27 584
4.6 29 6.0 0.30 7.7 33 0.90 20 68 35 74 48 33 90 330 58 50 33
8.1 21 7.5 5.3 23 47 81 24 5.3 61 72 156 17 92 69 275 465 74
9.6 11 27 15 18 7 28 14 28 57 85 13 23 88 74 27 47 0.16
EST, External standard method; IST, Internal standard method. * Compiled on the basis of the mean of relative error (in %) of the results and reference values for all analytes.
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Mean
Calibration
13 17 19 19 20 25 28 34 45 51 69 70 75 80 109 109 130 180
IST EST IST IST IST IST (C13) IST EST EST IST EST IST EST EST EST EST EST EST
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affects the final measurement result is to supply, along with the material to be tested during the interlaboratory comparison, a sample (samples) of a standard solution, the assay of which does not require a sample-preparation step. If the results for the standard solution are consistent with the reference value, then the calibration step can be regarded as reliable [19]. 3.4.3. Collection, transportation and storage. Most analysts would agree that collection, transportation and storage may be the source of the greatest errors in the determination of not only PAHs and PCBs, but also other compounds. The entire analytical procedure should be treated as an unbroken chain of operations and activities. If, as the saying goes, a chain is as strong as its weakest link, then these stages exert a decisive influence on the reliability of the measurement results obtained using a particular analytical procedure. This is frequently forgotten, attention being focused solely on the analytical techniques used to separate, to determine and to identify analytes in appropriately prepared extracts. Unfortunately, the effect of collection, transportation and storage on the final analytical result has not yet been taken into account in PT. 3.4.4. Sample preparation. If a suitable method is used to prepare a sample for testing in an interlaboratory comparison, it is possible to assess the efficiency of the sample-preparation step for final determination in a laboratoryÕs analytical procedure. The final extract obtained during the isolation step contains not only the analyte but also other compounds making up the matrix. This matrix effect on the final result of an assay can be divided into two types: (1) direct – due to the difficulty in obtaining and guaranteeing measurement selectivity; and, (2) indirect – when the matrix components react chemically and/or physically with analytes, they alter the real concentrations of the analytes (chemical reactions of sample components; adsorption, desorption, absorption). In order to verify the sample-preparation step, samples of material should be tested, the matrix composition of which corresponds to the matrix composition of samples routinely analyzed by laboratories. The samples to be analyzed can be divided in a general way according to whether they are in the solid, liquid or gaseous state. The vast majority of available reference materials have a solid matrix (e.g., soil, sediments, food, and tissue lyophilisates) Unfortunately, there are very few such materials with liquid or gaseous matrices. When determining the organic content of solid samples, it is important to monitor the isolation of the analyte from the matrix – this is given by the calculated recovery. That is why PT organizers are reminded to use
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materials in which the analytes are bound to the matrix components in very much the same way as in real samples. That this condition is being satisfied is clear from the ever-widening diversity of newly-produced reference materials (e.g., river sediment, sewage sediment and marine sediment as materials with a sediment-type matrix). The tiny number of available liquid and gaseous reference materials compels comparison organizers to prepare such materials for PT precisely. All the isolation techniques used in this step, especially those based on solid-phase extraction or the use of membranes, need to take into account the adsorption of PAHs and PCBs on the walls of samplers, vessels and other equipment used for calculating the final analytical result. On the basis of test results, it was possible to calculate the extent of PAH adsorption on the walls of vessels – the more aromatic rings in the analyte molecule, the greater the adsorption. The proportions adsorbed of the total amount of PAH analyte introduced into the sample-preparation set-up were as follows [32]: (1) two- and three-ring compounds – <5% (2) four-ring compounds – 8–40% (3) five-ring compounds – 45–65% (4) six-ring compounds – 70%. In the case of PCBs, the level of adsorption increased with increasing numbers of chlorine atoms in the molecule. The proportions adsorbed of the total amount of PCB analyte introduced into the sample-preparation setup were as follows [32]: (1) 3 and 4 chlorine atoms – <1% (2) 5 chlorine atoms – 4% (3) 6 chlorine atoms – 4–8% (4) 7 chlorine atoms – 20% (5) 10 chlorine atoms – 60%. Only with awareness of the effect of adsorption on the total-analyte recovery from a sample can the isolation procedure be designed to ensure quantitative transfer of analytes from the matrix. The situation can be illustrated with an example of the rational preparation for interlaboratory comparisons of water samples containing PAHs and PCBs [33]. Each laboratory received two samples of water for analysis (A and B), prepared in the following way. A. A fixed volume (2 lL) of a reference solution of PAHs in methanol was added to a bottle containing 1 L of distilled water. The added compounds were adsorbed on the walls of the 1 L bottle. B. 15 mg of a certified reference material (bottom sediment, the addition of which was meant to simulate the presence of suspended particulate matter in water) were added to a bottle containing 1 L of distilled water. After the mixture had been stirred, a fixed volume (2 lL) of a reference solution of PAHs http://www.elsevier.com/locate/trac
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Table 2. Conclusions to be drawn depending on the proficiency testing (PT) results obtained (Y = Result consistent with reference value; N = Result inconsistent with reference value; CRM = Certified reference material, StSol = Standard solution, ExCRM = Extract from CRM) Material sent for testing CRM
StSol
ExCRM
Y N
Y N Y
N
Y
Y
Y
N
N
Y
N
N
Y
Y
Y
N
N
Y
N
N
Y
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Conclusion
Y
Y
Y
N
N
Y
N
N
Y
Y
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The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition. The analytical procedure does not yield reliable results for samples of a given matrix composition; it is hard to pinpoint which step(s) in the analytical procedure is/are responsible for the discrepancy between results. Only the calibration step of the analytical procedure has been carried out correctly, so no inference can be drawn about the reliability of the whole procedure. The calibration step of the analytical procedure has been carried out incorrectly; consequently, results are obtained that are inconsistent with the reference value. The measurement results obtained using the final determination step of the analytical procedure are consistent with the reference values for samples of a given matrix composition, but no inference can be drawn about the reliability of the whole procedure. The measurement results obtained using the final determination step of the analytical procedure are inconsistent with the reference values for samples of a given matrix composition; consequently, the results obtained with this procedure cannot be consistent with the reference value. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; the calibration step of the procedure has therefore been correctly performed. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; but the calibration step of the procedure has been carried out incorrectly, so the results are inconsistent with the reference value – a practically impossible situation. The analytical procedure does not yield reliable results for samples of a given matrix composition; all that can be said is that the calibration step of the procedure has been carried out correctly and that it is not responsible for the inconsistent results. The analytical procedure does not yield reliable results for samples of a given matrix composition; moreover, the calibration step of the procedure has been carried out incorrectly. It is this step that is primarily responsible for the inconsistent results. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; in addition, it confirms that the final determination step of the procedure as applied to such samples has been carried out correctly. The analytical procedure yields reliable results for samples of a given matrix composition; but the final determination step of the procedure as applied to such samples has been carried out incorrectly, so the results obtained are inconsistent with the reference value – a practically impossible situation. The analytical procedure does not yield reliable results for samples of a given matrix composition; all that can be said is that the final determination step of the procedure has been carried out correctly and that it is not responsible for the inconsistent results relating to samples of a given matrix composition. The analytical procedure does not yield reliable results for samples of a given matrix composition; moreover, the final determination step of the procedure has been carried out incorrectly, and it is this step that is primarily responsible for the inconsistent results relating to such samples. Both the calibration step and the final determination step of the analytical procedure for samples of a given matrix composition have been performed correctly, but no inference can be drawn about the reliability of the whole procedure. Only the calibration step of the analytical procedure has been performed correctly, so no inference can be drawn about the reliability of the whole procedure. The measurement results obtained using the final determination step of the procedure are inconsistent with the reference values for samples of a given matrix composition; consequently, the results obtained with this procedure cannot be consistent with the reference value. The calibration step of the analytical procedure has been carried out incorrectly; consequently, results are obtained that are inconsistent with the reference value. The consistency of the result for an extract sample is purely accidental; in combination with an incorrectly performed calibration step, such a situation is practically impossible. Both the calibration step of the analytical procedure and its final determination step have been carried out incorrectly; consequently, the results are inconsistent with the reference value. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; both the calibration step and the final determination step of the procedure have been carried out correctly, which means that the results are reliable. The final conclusion is that all the steps of the procedure have been performed correctly.
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Table 2. (continued) Material sent for testing CRM
StSol
ExCRM
Y
Y
N
Y
N
Y
Y
N
N
N
Y
Y
N
Y
N
N
N
Y
N
N
N
Conclusion
The analytical procedure yields reliable results for samples of a given matrix composition; moreover, the calibration step has been carried out correctly, so that measurement results are reliable. But the measurement results obtained using the final determination step of the procedure are inconsistent with the reference values for such samples – a practically impossible situation. The analytical procedure yields reliable results for samples of a given matrix composition; moreover, the final determination step has been carried out correctly, so that measurement results are reliable. But the measurement results obtained using the calibration step of the procedure are inconsistent with the reference values for such samples – a practically impossible situation. The analytical procedure yields reliable results for samples of a given matrix composition. But neither the calibration step nor the final determination step of the procedure have been carried out correctly; consequently, the results are inconsistent with the reference value – a practically impossible situation. The analytical procedure does not yield reliable results for samples of a given matrix composition; however, both the calibration step and the final determination step have been carried out correctly and yield reliable results; nevertheless, the conclusion to be drawn is that the results of the remaining steps of the procedure must be unreliable. The analytical procedure does not yield reliable results for samples of a given matrix composition; however, the calibration step has been carried out correctly and yields reliable results, but since the final determination step has been carried out incorrectly, it is responsible for the unreliability of the results of the remaining steps of the procedure. Conclusion: the matrix composition exerts a decisive influence on the consistency of the measurement results. The analytical procedure does not yield reliable results for samples of a given matrix composition; the calibration step has been carried out incorrectly, so the results obtained are inconsistent with the reference value. The consistency of the result for an extract sample is purely accidental; in combination with an incorrectly performed calibration step such a situation is practically impossible. The analytical procedure does not yield reliable results for samples of a given matrix composition; the calibration and final determination steps are definitely responsible for the unreliability of the whole procedure; however, it is difficult to define the possible effect of the remaining steps of the procedure.
in methanol was added. The suspension was invisible, due to the tiny quantity of sediment and the dark glass of the bottle, and fell to the bottom quite quickly. This time, the PAHs were adsorbed primarily on the surface of the suspended particles. Another way of defining and at the same time monitoring the influence of matrix composition on an assay result is to send out a sample of an extract of the test material together with the sample of the test material itself [18–20]. Any difference between the measurement results for the two samples will indicate a serious source of error at the sample-preparation step. Table 2 lists the conclusions to be drawn, depending on the results of the PT, the object of which was reference material and/or a standard solution and/or an extract of the reference material. Sample-preparation techniques exert a definite influence on the recovery of analyte from the matrix, and so also on the final result of the determination and its reliability. The method of preparing the samples in the above-mentioned aqueous solutions for analysis of organic compounds serves as an example. Samples A and B, prepared as stated above, were sent to three laboratories (1, 2 and 3) for analysis. Each laboratory used the individual isolation and enrichment steps in procedures for analyte extraction [33].
In Laboratory 3, suspended particulate matter was removed by filtration. In the case of sample B, this also removed the compounds adsorbed on the suspended particles. Hence, the low recovery of analytes, in particular of non-volatile PAHs. The isolation process used in Laboratory 2 involved the transfer of the suspended matter onto an extraction disc, which was then subjected to solvent extraction. This gave a high recovery of analytes, especially of nonvolatile PAHs. Such a high recovery was possible because the analytes introduced into the sample (dissolved in an organic solvent) were adsorbed on the surface of sediment particles, as a result of which ageing (penetration of the sediment and stronger bonding with it) was minimal. Quantitative desorption of PAHs occurring naturally in the suspension was impossible, given the conditions under which the isolations were carried out in these laboratories. Table 3 lists the results (recoveries) obtained by the participating laboratories. It is worth stressing that there was a seven-fold difference between the determinations of PAH levels in sample B obtained by the laboratories [33]. Special interlaboratory comparisons are also undertaken to assess a particular (often recommended) technique for isolating analytes from a matrix [21–25].
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Table 3. PAH analyte recoveries obtained by the participating laboratories for samples A and B [32] Compound
Percentage of PAH levels determined in samples A and B with respect to reference values [%] Laboratory 1 (LLE)
Laboratory 2 (SPE disc for accelerated extraction)
Laboratory 3 (SPE columns)
Sample
A
B
A
B
A
B
Naphthalene Anthracene Fluoranthene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[g,h,i]perylene Benzo[k]fluoranthene Indeno[l,2,3-cd]pyrene
85 105 95 59 60 42 59 34
87 105 94 59 61 40 61 34
44 68 66 58 63 51 59 41
16 60 114 88 91 104 86 87
35 54 60 52 62 42 55 40
31 40 58 21 27 17 22 12
Both the recovery and the method of its determination affect the final result of a determination. The recovery value must be used to correct the obtained measurement value. Like the result of every analytical measurement, the recovery is encumbered with uncertainty. Hence, the value of this uncertainty must be taken into account as a component of the combined uncertainty of the final determination. 3.4.5. The uncertainty of a measurement result. Uncertainty is a fundamental property of every measurement. It is always present at every step of every measurement procedure. Hence, it is not a property that causes additional problems during the measurement process [34– 37]. The major sources of uncertainty during the testing of samples using an appropriate analytical procedure include the following [1,3,38]: (1) erroneous or imprecise definition of the magnitude to be determined; (2) a non-representative sample; (3) incorrect application of the analytical methodology; (4) systematic human errors in reading analogue signals; (5) ignorance or lack of awareness of the effect of all external conditions on the result of an analytical measurement; (6) the uncertainty inherent in the calibration of the monitoring/measurement apparatus; (7) the resolution of the measurement apparatus; (8) the uncertainty inherent in the application of standards and/or reference materials; (9) the uncertainty of parameters determined during separate measurements and applied in calculating the final result of the determination (e.g., physico-chemical constants); 714
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(10)
approximations and assumptions relating to the use of a particular measurement apparatus and applied during the performance of a measurement; and, (11) fluctuations of apparently identical external conditions occurring during repeated measurements. Every analytical result is the consequence of a measurement performed. The overriding aim of the analyst is to obtain an analytical result reflecting as closely as possible the expected (real, true) value. The certainty of an analytical result therefore depends on the uncertainties inherent in every step of an analytical procedure. The parameter determining the final uncertainty of a result is the one with the greatest uncertainty. Hence, the sources and the types of uncertainty must be defined for every single step of an analytical procedure – for every measured magnitude, in fact [1,38]. Determining the uncertainty of a measurement enhances its reliability, renders the results obtained during interlaboratory comparisons comparable, and helps to establish the significance of the difference between a measurement result and a reference value [38]. One way of discovering problems relating to the construction of the uncertainty budget is for intercomparison organizers to insist that the participating laboratories state the uncertainty values for every step of the analytical procedure that they apply during PT. Comparison of uncertainty values will then show up the main sources of uncertainty and give an idea of the accuracy of its estimation. 3.4.6. Data processing and interpretation of results. The presentation of measurement results and their interpretation may be an additional source of information as far as the reliability of results is concerned. During the preparation of an interlaboratory study, the organizer can impose a particular way of presenting
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Table 4. Dubious situations resulting from disagreement between uncertainty (U), limit of detection (LOD) and standard deviation (SD) values delivered by PT participants as a final result of PT Data delivered by PT participants Analyte
Unit
Results 1
2
3
PCB 28
ng/g
18.0
14.3
22.8
Anthracene
ng/L
33.0
33.2
29.9
4
35.5
Conclusions
Mean
SD
LOD
18.4
4.3
2.0
10%
32.9
2.3
15
30%
5
the results, which can considerably improve the quality of data interpretation For example, by stipulating the number of replicate measurements for particular analytes, the uncertainty inherent in the repeatability of results can be estimated. Again, the demand that the LOD be stated yields information on the quality of measurement results depending on the ratio of the given LOD to the concentration level determined. It is therefore very important to prepare (e.g., in the form of a results sheet) an appropriate way of data transmission that includes all the information needed for
STEP OF THE ANALYTICAL PROCEDURE
SAMPLING
SAMPLE PREPARATION
FINAL DETERMINATION
DATA TREATMENT
U
Standard uncertainty due to repeatability calculated is about 13%, so expanded uncertainty should be no less than 26%. It is underestimated. Standard uncertainty due to LOD value is about 45%, so expanded uncertainty should be no less than this value. It is underestimated. Additionally, if LOD is equal to 15 ng/L, the limit of quantification (LOQ) should be equal 45 ng/L. Reliability of results below LOQ is really poor.
correct, reliable data treatment. This information should include the following: (1) sample-preparation method; (2) enrichment method; (3) calibration method; (4) calibration-curve parameters; (5) numbers of standards and their ranges used during calibration; (6) raw results (all repetitions); (7) mean, standard deviation, LOD, uncertainty values; (8) how the uncertainty budget is estimated; and, (9) all graphs (calibration curve, chromatograms).
THE WAY OF CONTROL
NO
− test samples with different matrix composition YES − standard solution − extract from test sample − standard solution YES − extract from test sample − data presentation YES − uncertainty estimation − validation parameters calculation
Figure 2. Ways of interaction, monitoring and obtaining additional information from interlaboratory comparisons on the steps in an analytical procedure.
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Table 4 presents equivocal situations resulting from discrepancies between LODs, uncertainty and standard deviation. These discrepancies would be shown up on an appropriately designed results sheet sent to participants. However, if appropriate (often very specific) processing techniques are applied to the results, additional information becomes available regarding, say, laboratory competence or the potential of a tested analytical procedure [21,22,26,27]. 4. Conclusions The principal objective of interlaboratory comparisons is to obtain additional information on the object of comparison (analytical method, reference material, or laboratory). But this additional information can be reliable and exhaustive only when both the organizer and the participant of the comparison present the results of the analyses in an optimal manner. On the one hand, this way of organizing and carrying out such comparisons yields the fullest possible information on possible errors, and how to eliminate or at least prevent them. On the other hand, reliable measurements result in subsequent comparisons of the same type. As far as determining the content of organic compounds is concerned, the matrix composition of the sample under scrutiny has a significant influence on the reliability of the measurement result. This influence is far greater than in determining, say, metal ions or physico-chemical parameters (e.g., pH or conductivity). That is why, when an interlaboratory-comparison organizer is preparing appropriate samples for analysis during the comparison, it is imperative to define the matrix composition and to take into account its effect on the result of determination. In this context, the following parameters are crucial – how analytes are bound to the matrix components, analyte bioavailability, biodegradation, volatility and stability. Attention needs to be paid to these questions, both during the organization of the comparison and while it is taking place. The statistical processing of the measurement results also needs to be looked at. It goes without saying that the statistician who evaluates the results of analyses must have at least a rudimentary knowledge of analytical chemistry. At the comparison stage, analyst and statistician must therefore work in close cooperation. The possible sources of ‘‘interference’’ in the execution of interlaboratory comparisons, outlined in this article, are the main points of which the organizers and participants of such studies should be aware. By way of summary, Fig. 2 presents ways of interacting, monitoring and obtaining additional information from interlaboratory comparisons regarding the steps of an analytical procedure.
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References [1] I.R. Juniper, Accred Qual. Assur. 4 (1999) 336. [2] P. Konieczka, J. Namies´nik, Quality Assurance and Quality Control in the Analytical Chemical Laboratory: A Practical Approach, CRC Press/Francis and Taylor, Boca Raton, FL, USA, 2009. [3] P. Konieczka, Crit. Rev. Anal. Chem. 37 (2007) 173. [4] L.A. Berrueta, B. Gallo, F. Vicente, Chromatographia 40 (1995) 474. [5] R.A. Doong, S.M. Chang, Y.C. Sun, J. Chromatogr., A 879 (2000) 177. [6] L. Wolska, M. Rawa-Adkonis, M. Gdaniec, J. Namies´nik, Ecol. Chem. Eng. 12 (2005) 611. [7] V. Lepane, J. Chromatogr., A 845 (1999) 329. [8] M. Lee, M.V. Novotny, K.D. Bartle, Analytical Chemistry of Polycyclic Aromatic Compounds, Academic Press, Boca Raton, FL, USA, 1981. [9] M. Rawa-Adkonis, L. Wolska, J. Namies´nik, Crit. Rev. Anal. Chem. 32 (2006) 63. [10] A. Kot, B. Zabiegała, J. Namies´nik, Trends Anal. Chem. 19 (2000) 445. [11] J.H. Hermans, F. Smedes, J.W. Hofstraad, W.P. Coflnoz, Environ. Sci. Technol. 26 (1992) 2028. [12] J.L. Zhou, K. Maskaoiu, Environ. Pollut. 121 (2003) 269. [13] V. Pino, J.H. Ayala, A.M. Afonso, V. Gonzalez, J. Chromatogr., A 949 (2002) 291. [14] A.J. King, J.W. Readman, J.L. Zhou, Mar. Pollut. Bull. 48 (2004) 229. [15] X. Jiang, D. Martens, K.W. Schramm, A. Kettrup, S.F. Xu, L.S. Wang, Chemosphere 41 (2000) 901. [16] G. Witt, Mar. Pollut. Bull. 31 (1995) 237. [17] M.S. Garcia-Falcon, C. Perez-Lamela, J. Simal-Gandara, Anal. Chim. Acta 508 (2004) 177. [18] S. Balzamo, P. de Zorzi, S. Barbizzi, E. Calabretta, M. Potalivo, S. Rosamilia, Accred. Qual. Assur. 14 (2009) 487. [19] M. Ricci, O. Bercaru, R. Morabito, C. Brunori, I. Ipolyi, C. Pellegrino, A. Sahuquillo, F. Ulberth, Trends Anal. Chem. 26 (2007) 818. [20] M. Gluschke, Accred. Qual. Assur. 11 (2006) 470. [21] W. Kandler, R. Schuhmacher, S. Roch, P. Schubert-Ullrich, R. Krska, Accred. Qual. Assur. 9 (2004) 82. [22] S.A. Senseman, T.C. Mueller, M.B. Riley, R.D. Wauchope, C. Clegg, R.W. Young, L.M. Southwick, H.A. Moye, J.A. Dumas, R.W. Mersie, J.D. Mattice, R.B. Leidy, J. Agric. Food Chem. 51 (2003) 3748. [23] K. Hartonen, S. Bøwadt, H.P. Dybdahl, K. Nylund, S. Sporring, H. Lund, F. Oreld, J. Chromatogr., A 958 (2002) 239. [24] G. Asmund, M. Cleemann, Sci. Total Environ. 245 (2000) 203. [25] A. Jaouen-Madoulet, A. Abarnou, A.M. Le Guellec, V. Loizeau, F. Leboulenger, J. Chromatogr., A 886 (2000) 153. [26] P. Konieczka, T.P.J. Linsinger, B. Zygmunt, J. Namies´nik, Accred. Qual. Assur. 10 (2005) 241. [27] P. Konieczka, T.P.J. Linsinger, J. Namies´nik, Accred. Qual. Assur. 11 (2006) 584. [28] P. Konieczka, J. Namies´nik, Chem. Anal. (Warsaw) 53 (2008) 785. [29] A.D. McNaught, A. Wilkinson (Compilers), IUPAC Compendium of Chemical Terminology, 2nd Edition (The ‘‘Gold Book’’), Blackwell Scientific Publications, Oxford, UK, 1997. XML on-line corrected version: http://goldbook.iupac.org (2006-), created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8 (doi:10.1351/goldbook). [30] Joint Committee for Guides in Metrology (JCGM), International Vocabulary of Metrology – Basic And General Concepts and Associated Terms (VIM), JCGM 200:2008, (http://www.bipm.org/ utils/common/documents/jcgm/JCGM_200_2008.pdf).
Trends in Analytical Chemistry, Vol. 29, No. 7, 2010 [31] L. Wolska, M. Gdaniec-Pietryka, P. Konieczka, J. Namies´nik, Talanta 78 (2009) 730. [32] L. Wolska, The analytical result and quality of environmental information, Zeszyty Naukowe Politechniki Gdan´skiej, Chemia 57 (2008) 1 (in Polish). [33] L. Wolska, Anal. Bioanal. Chem. 391 (2008) 2647.
Trends [34] A. Populaire, E. Campos Gimenez, Accred. Qual. Assur. 10 (2005) 485. [35] S. Roy, A.-M. Fouillac, Trends Anal. Chem. 23 (2004) 185. [36] V.R. Meyer, J. Chromatogr., A 1158 (2007) 15. [37] R. Kadis, Accred. Qual. Assur. 3 (1998) 237. [38] P. Konieczka, J. Namies´nik, J. Chromatogr., A 1217 (2010) 882.
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Quality problems in determination of organic compounds in environmental samples, such as PAHs and PCBs Piotr Konieczka, Lidia Wolska, Jacek Namies´nik The literature shows that most interlaboratory comparisons deal with determination of inorganic compounds or physico-chemical parameters. The determination of organic compounds, particularly in environmental samples, is still a challenging analytical problem, which is why there are few interlaboratory comparisons on this subject. The amount and the quality of information obtained from interlaboratory comparisons depend on both their organizer (who selects and prepares material for investigation, and provides statistics based on the results of measurements) and participants (who carry out measurements, determine the validation parameters of the relevant analytical procedure, and publish the results of analyses). We discuss the difficulties in determining organic compounds on the basis of our own investigations on polyaromatic hydrocarbons and polychlorinated biphenyls in environmental samples. We present means of overcoming these difficulties as conclusions emerging from the results of interlaboratory comparisons. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Analytical procedure; Environmental sample; Interlaboratory comparison; Organic compound; PAH; PCB; Polyaromatic hydrocarbon; Polychlorinated biphenyl; Sample preparation; Trace-organic constituent
1. Introduction Piotr Konieczka*, Lidia Wolska, Jacek Namies´nik Department of Analytical Chemistry, Chemical Faculty, Gdansk University of Technology ul. Narutowicza 11/12, 80-233 Gdan´sk, Poland
*
Corresponding author. Tel.: +485 83 47 21 10; Fax: +485 83 47 26 94; E-mail: [email protected]
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The overriding aim of every analysis is to acquire reliable results that supply the fullest possible information about the object of study on the basis of measurement results obtained during the assay of a sample. Results are obtained as a consequence of measurements made with the appropriate analytical procedures and measuring instruments. These must be properly prepared for the job in hand so that the final results are reliable. The determination of analytes at ever lower concentrations in samples with ever more complex matrices is one of the main lines of development in contemporary analytical chemistry. This is an exceedingly difficult, complicated task, so it presents a great challenge to analysts, who need to be aware of the problems surrounding quality assurance and quality control (QA/QC).
Assuring the appropriate quality of analytical measurement results involves monitoring the reliability of measurement apparatus and the range of applicability of analytical procedures. For this purpose, a system of QC of analytical measurements is applied, involving: assuring the metrological coherence of the results; estimating the uncertainties of the measurement results; validating the analytical procedures applied; utilizing (certified) reference materials; and, participating in different kinds of interlaboratory comparisons. Participation in interlaboratory comparisons provides an opportunity to compare oneÕs own results with those obtained by other laboratories and to demonstrate oneÕs competence, which is especially important in the case of accredited
0165-9936/$ - see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.03.012
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laboratories or those applying for accreditation. Moreover, such comparisons enable the participants to search for and to identify unexpected errors on the basis of comparisons with an external standard and with their own earlier results, and, if an error is discovered, to take steps to rectify it [1]. The procedure for determining specific compounds (particularly those occurring at trace and ultra-trace levels) in environmental samples involves the following steps [2]: (1) sample collection; (2) transport and storage of samples; (3) preparation of samples for analysis (e.g., filtration, mineralization, extraction, and fractionation); (4) final analyte determination (including the method of calibration); (5) calculation and interpretation of data; and, (6) QA/QC of analytical measurements and results. In view of the need to confirm the reliability of analytical results obtained by analytical laboratories, the results of tests and the analytical methods applied must be documented and verifiable. One of the most important ways of monitoring analytical results is to participate in interlaboratory comparisons, which facilitate the evaluation of the results of measurements performed on identical, or at least very similar, test samples at two or more laboratories under stipulated conditions. However, before embarking on an interlaboratory comparative study, the organizer should be absolutely clear as to the purpose, since this will determine how such a comparison will be prepared (the object of study, the type of samples to be assayed, sample preparation techniques, etc.) and how it is to be carried out. Interlaboratory comparisons are organized in order to [3]: (1) assess the reliability of determinations; (2) acquire experience; and, (3) improve the quality of analytical determinations. It is worth emphasizing that only a very few interlaboratory comparisons have been run on the determination of organic compounds in environmental samples, as opposed to the many given over to the determination of metals or inorganic ions. Undoubtedly, this is because of the far greater number of sources of errors that can be committed during the determination of the content of organics and the correspondingly greater difficulty of such analyses.
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analytical equipment (e.g., GC-MS, LC-MS, ICP-MS, and GC · GC), computers and the relevant software, are themselves sufficient to guarantee the high quality of the measurement and data-processing steps in the analytical procedure. However, analysts are coming to realize that it is collection, transport and storage of a sample, and its preparation for final determination that have the greatest influence on the quality of the information obtained from the measurement data [2]. Of especial interest to analysts are problems concerning the determination of polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in environmental samples. More than 30 years ago, the US Environmental Protection Agency (EPA) officially recognized these compounds as major environmental pollutants. Of the most toxic, 16 PAHs and seven PCBs were placed on the list of priority contaminants, and, as indicators of the level of environmental pollution, were designated for monitoring studies. The interest of the scientific community, greater environmental awareness, and increasing pressure on the part of society were all crucial factors causing the sudden increase in the need for information on levels of environmental pollution by PAHs and PCBs, among other compounds. Since that time, original papers on novel methodologies and equipment [4–7] and review papers [8–11] have appeared. Papers describing the procedures and the results of analytical tests for determining PAHs and PCBs in environmental samples (air, water, soil, bottom sediments, suspended particulate matter, organisms) have also been published [12–27]. But does this mean that the determination of PAHs and PCBs in environmental samples is now problem free? And do the results give reliable information about the environment? The results of numerous studies performed have demonstrated the possibilities and the limitations of contemporary analytical techniques in acquiring reliable information on the levels and forms of occurrence of PAHs and PCBs in the environment. Moreover, they have persuaded us to take a fresh look at the problems involved in their determination in environmental samples characterized by matrices of variable, highly diversified composition.
3. Implementation of interlaboratory comparisons 2. The problems involved The effect of a procedure on the final analytical result may be due to the way in which the successive steps are carried out, the materials from which the apparatus and accessories are made, and the content of the sample itself (matrix effects). It is generally believed that present levels of knowledge, together with the availability of state-of-the-art
Interlaboratory comparisons are undertakings in which tasks are performed by both the organizer and the individual participants. The tasks of the organizer include [2]: (1) selection and preparation of test samples of appropriate stability; (2) distribution of test samples, together with detailed instructions concerning measurement procedures and presentation of results; http://www.elsevier.com/locate/trac
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(3) statistical analysis and processing of results; and, (4) compilation of the final report. The tasks of the participants are to: (1) validate the analytical procedure used for the measurements; (2) perform measurements (calibration, determination of validation parameters, and estimation of uncertainty); (3) calculate measurement results; and, (4) send measurement results together with a detailed description of the applied analytical procedure to the organizer. The correct preparation and execution of each of these tasks will ensure optimal running of interlaboratory comparisons and will supply a full set of data regarding possible sources, types and magnitudes of error, which are the cause of the differences between actual and expected measurement results. There are three main types of proficiency testing (PT): (1) evaluating measurement procedures; (2) testing the competence of laboratories; and, (3) certification of materials. 3.1. Evaluating measurement procedures During this type of PT, all participants, in accordance with the same protocol and using the same procedure, perform tests to determine characteristic features in a batch of identical test samples. The results are used to estimate the characteristic parameters of the procedure, which are usually internal – interlaboratory precision, systematic error, analyte recovery, internal QC parameters, sensitivity, limit of detection (LOD), range of applicability. The role of the organizer in this type of study is to prepare the sample(s) in such a way that every step in the analytical procedure being tested can be monitored and that relevant conclusions can be drawn. 3.2. Testing the competence of laboratories One or more analyses are carried out by a group of laboratories on the basis of one or more homogeneous, stable test samples using a selected procedure or one routinely applied by all the laboratories participating in the interlaboratory comparison. The results are then compared with those of the other laboratories or against a known or guaranteed reference value. In this case, too, choice and appropriate preparation of test samples should enable the largest possible number of parameters of the procedure to be monitored by the participating laboratories. 3.3. Certification of materials As a result of the tests, a reference value is assigned to a parameter (e.g., concentration) or a physical property in the test material or test sample, usually of known uncertainty.
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Normally, laboratories of proven competence (reference laboratories) are chosen to participate in this type of study in order to test a material that is a candidate for a reference standard using a procedure facilitating the estimation of concentration (or another parameter) encumbered with the least error or the least known uncertainty. 3.4. Particular problems We now describe the particular problems that need to be taken into account during the organization of and participation in interlaboratory comparisons. The relevance of the conclusions regarding the assay of the test materials [i.e. analytical procedure, laboratory or certified reference material (CRM)] will depend on the way in which these problems are monitored within the PT framework. 3.4.1. Selectivity. Selectivity is a necessary condition for obtaining reliable measurement results. In the case of chromatographic analysis – the ‘‘classical’’ technique for determining organic compounds – selectivity is represented by the separation factor, the value of which is a function of the difference in retention times and chromatographic peak widths. The parameters affecting selectivity where chromatographic techniques are concerned are: (1) the type of column used; and, (2) the chromatographic separation parameters (e.g., temperature program, gas flow rate). With a suitable chromatographic column, the analyte signal can be distinguished from the interferent signal. If an inappropriate column is used, the materials to be assayed may well be determined in combination. In the case of PAHs, it happens that compounds (e.g., benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[j]fluoranthene) are determined as pairs of isomers (b+j or k+j) or even as the sum of all three, even by very experienced laboratories [18]. Again, PCBs are often determined as the sum of PCB 28 + PCB 31 or PCB 138 + PCB 162; moreover, the order of elution of the isomers PCB 28 and PCB 31 [28], PCB 138 and PCB 153 is frequently confused. Fig. 1 presents examples of chromatograms obtained during the determination of PCB analytes using two different columns [28]. The separation of PCB 31 from PCB 28 cannot be done on a column containing stationary phase DB17, but is possible with a DB1701 column. Organizers of interlaboratory comparisons should be aware of the errors that may be committed in connection with the difficulties in obtaining measurement selectivity. They should prepare the PT material in such a way that both the errors and their sources are identifiable. The simplest way of doing this is to make up a few
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RT: 17.07 - 34.72 18.29
07 - 34.72 18.29
100
DB- 17
PCB 28
90
PCB 28
80
18.24 24.54
19.46
70
PCB 101
60
PCB 153
25.49
22.68
20.83
26.05
PCB 105
50
18.24
27.53
40
19.46
21.99
18.78
22.81
30
30.23
26.92
PCB 170 31.88
29.08
23.77
31.72
20
32.63 32.82
10 0 100
18.28
PCB 28*
90
19.45
PCB 105*
80
18.78
26.91
25.46
70
31.98
60
PCB 153*
22.66
50
PCB 101*
27.51
26.04
30.21 31.87
40
PCB 170*
30 20.34 20.97
20 10
30.37
18.08 22.51
33.50
28.51 29.89
24.28
0 18
20
22
24
26
28
30
32
34
Time (min)
- 35.40 19.45
DB- 1701
RT: 17.60 - 35.40 19.45
PCB-28
100
PCB-28
90
19.36
19.36
80 70 60 50 40
18.03
20.63
21.96
23.94 23.72
30
27.34
PCB-101
31.78 32.66
PCB-15329.31 28.66
18.93
29.44
20
34.46
10
8.03
20.63
18.93
2
PCB PCB-170
0 100
19.44
PCB-28*
90 80
26.78
70 60
27.33
50 40
19 44
29.29
31.76 32.64
23.93
PCB-101*
PCB-153*
30 34.42
20
PCB-170* PCB
10 0 18
20
22
24
26 28 Time (min)
30
32
34
Figure 1. Some chromatograms obtained during the analysis of samples of appropriately prepared extracts for the determination of PCB analytes; separation was achieved using different types of chromatographic column [28].
solutions of scarcely separable analytes in widely differing concentrations.
3.4.2. Calibration. An optimally selected calibration technique is of the prime importance for decoding the
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output signal of the measuring instrument to obtain the analytical signal. As every analyst is well aware, calibration is integral to every analytical process [2]. After all, the fundamental analytical task is to determine the concentration of a particular substance (the analyte) in a sample. But this can be done with the aid of an analytical instrument only on the basis of measurement information supplied by that instrument (analytical signals) for the sample. Calibration is defined in various ways. For example, the IUPAC Gold Book defines calibration in analysis as: ‘‘The set of operations which establish, under specified conditions, the relationship between values indicated by the analytical instrument and the corresponding known values of an analyte’’ [29]. Calibration is also defined as ‘‘a model attempting to predict the value of an independent variable in the case when only the dependent variable is known’’ [30]. Generally speaking, ‘‘analytical calibration’’ is understood to mean the representation of the real (true, theoretical) dependence of the analytical signal on the analyte concentration in empirical form (the calibration curve), and then using that curve to determine the analyte concentration in the sample in order to obtain the ‘‘analytical result’’. The calibration curve is constructed under given analytical conditions using standard/reference samples. The calibration method generally depends on the following factors [2,3]: (1) the type of measuring instrument; (2) the number of samples;
(3)
the possibility of preparing samples over a wide range of analyte concentrations (in order to check the full measurement range of the monitoring/ measuring instrument); (4) the required accuracy of a measurement; (5) the composition of the sample matrix; and, (6) the possibility of the sample composition changing during the analytical process. A common error when constructing a calibration curve is to plot it for a very wide range of concentrations. It is well known that a calibration curve is linear only within a strictly defined interval of concentrations; the curve will certainly exhibit one set of characteristics for concentrations close to the LOD, another for higher concentrations, and yet another when levels approach the limit of the measurement range. The dependence of detector response on the concentration of the compound to be determined takes the shape of a sloping letter S. In the case of PAHs, it was found that the slope of the calibration curve covering the lower range of concentrations could be up to three times less than that of the curve relating to the higher range of concentrations in the samples investigated [31]. This must be taken into consideration when this step of the analytical procedure is carried out. As an example of the influence of the calibration step on the final analytical result, Table 1 ranks laboratories on the basis of relative error value. One of the ways of checking whether the calibration step (as an integral part of the final determination step)
Table 1. Ranking* of laboratories participated in interlaboratory comparison Lab code
lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab lab
3 23 19 15 7 8 16 13 1 22 11 6 2 17 9 20 21 5
PCB 52
118
138
153
180
23 8.5 26 35 11 12 24 62 21 57 51 97 68 141 63 211
19 14 30 42 39 27 6.1 48 104 56 62 249 32 1.4 45 27 584
4.6 29 6.0 0.30 7.7 33 0.90 20 68 35 74 48 33 90 330 58 50 33
8.1 21 7.5 5.3 23 47 81 24 5.3 61 72 156 17 92 69 275 465 74
9.6 11 27 15 18 7 28 14 28 57 85 13 23 88 74 27 47 0.16
EST, External standard method; IST, Internal standard method. * Compiled on the basis of the mean of relative error (in %) of the results and reference values for all analytes.
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Mean
Calibration
13 17 19 19 20 25 28 34 45 51 69 70 75 80 109 109 130 180
IST EST IST IST IST IST (C13) IST EST EST IST EST IST EST EST EST EST EST EST
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affects the final measurement result is to supply, along with the material to be tested during the interlaboratory comparison, a sample (samples) of a standard solution, the assay of which does not require a sample-preparation step. If the results for the standard solution are consistent with the reference value, then the calibration step can be regarded as reliable [19]. 3.4.3. Collection, transportation and storage. Most analysts would agree that collection, transportation and storage may be the source of the greatest errors in the determination of not only PAHs and PCBs, but also other compounds. The entire analytical procedure should be treated as an unbroken chain of operations and activities. If, as the saying goes, a chain is as strong as its weakest link, then these stages exert a decisive influence on the reliability of the measurement results obtained using a particular analytical procedure. This is frequently forgotten, attention being focused solely on the analytical techniques used to separate, to determine and to identify analytes in appropriately prepared extracts. Unfortunately, the effect of collection, transportation and storage on the final analytical result has not yet been taken into account in PT. 3.4.4. Sample preparation. If a suitable method is used to prepare a sample for testing in an interlaboratory comparison, it is possible to assess the efficiency of the sample-preparation step for final determination in a laboratoryÕs analytical procedure. The final extract obtained during the isolation step contains not only the analyte but also other compounds making up the matrix. This matrix effect on the final result of an assay can be divided into two types: (1) direct – due to the difficulty in obtaining and guaranteeing measurement selectivity; and, (2) indirect – when the matrix components react chemically and/or physically with analytes, they alter the real concentrations of the analytes (chemical reactions of sample components; adsorption, desorption, absorption). In order to verify the sample-preparation step, samples of material should be tested, the matrix composition of which corresponds to the matrix composition of samples routinely analyzed by laboratories. The samples to be analyzed can be divided in a general way according to whether they are in the solid, liquid or gaseous state. The vast majority of available reference materials have a solid matrix (e.g., soil, sediments, food, and tissue lyophilisates) Unfortunately, there are very few such materials with liquid or gaseous matrices. When determining the organic content of solid samples, it is important to monitor the isolation of the analyte from the matrix – this is given by the calculated recovery. That is why PT organizers are reminded to use
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materials in which the analytes are bound to the matrix components in very much the same way as in real samples. That this condition is being satisfied is clear from the ever-widening diversity of newly-produced reference materials (e.g., river sediment, sewage sediment and marine sediment as materials with a sediment-type matrix). The tiny number of available liquid and gaseous reference materials compels comparison organizers to prepare such materials for PT precisely. All the isolation techniques used in this step, especially those based on solid-phase extraction or the use of membranes, need to take into account the adsorption of PAHs and PCBs on the walls of samplers, vessels and other equipment used for calculating the final analytical result. On the basis of test results, it was possible to calculate the extent of PAH adsorption on the walls of vessels – the more aromatic rings in the analyte molecule, the greater the adsorption. The proportions adsorbed of the total amount of PAH analyte introduced into the sample-preparation set-up were as follows [32]: (1) two- and three-ring compounds – <5% (2) four-ring compounds – 8–40% (3) five-ring compounds – 45–65% (4) six-ring compounds – 70%. In the case of PCBs, the level of adsorption increased with increasing numbers of chlorine atoms in the molecule. The proportions adsorbed of the total amount of PCB analyte introduced into the sample-preparation setup were as follows [32]: (1) 3 and 4 chlorine atoms – <1% (2) 5 chlorine atoms – 4% (3) 6 chlorine atoms – 4–8% (4) 7 chlorine atoms – 20% (5) 10 chlorine atoms – 60%. Only with awareness of the effect of adsorption on the total-analyte recovery from a sample can the isolation procedure be designed to ensure quantitative transfer of analytes from the matrix. The situation can be illustrated with an example of the rational preparation for interlaboratory comparisons of water samples containing PAHs and PCBs [33]. Each laboratory received two samples of water for analysis (A and B), prepared in the following way. A. A fixed volume (2 lL) of a reference solution of PAHs in methanol was added to a bottle containing 1 L of distilled water. The added compounds were adsorbed on the walls of the 1 L bottle. B. 15 mg of a certified reference material (bottom sediment, the addition of which was meant to simulate the presence of suspended particulate matter in water) were added to a bottle containing 1 L of distilled water. After the mixture had been stirred, a fixed volume (2 lL) of a reference solution of PAHs http://www.elsevier.com/locate/trac
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Table 2. Conclusions to be drawn depending on the proficiency testing (PT) results obtained (Y = Result consistent with reference value; N = Result inconsistent with reference value; CRM = Certified reference material, StSol = Standard solution, ExCRM = Extract from CRM) Material sent for testing CRM
StSol
ExCRM
Y N
Y N Y
N
Y
Y
Y
N
N
Y
N
N
Y
Y
Y
N
N
Y
N
N
Y
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Conclusion
Y
Y
Y
N
N
Y
N
N
Y
Y
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The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition. The analytical procedure does not yield reliable results for samples of a given matrix composition; it is hard to pinpoint which step(s) in the analytical procedure is/are responsible for the discrepancy between results. Only the calibration step of the analytical procedure has been carried out correctly, so no inference can be drawn about the reliability of the whole procedure. The calibration step of the analytical procedure has been carried out incorrectly; consequently, results are obtained that are inconsistent with the reference value. The measurement results obtained using the final determination step of the analytical procedure are consistent with the reference values for samples of a given matrix composition, but no inference can be drawn about the reliability of the whole procedure. The measurement results obtained using the final determination step of the analytical procedure are inconsistent with the reference values for samples of a given matrix composition; consequently, the results obtained with this procedure cannot be consistent with the reference value. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; the calibration step of the procedure has therefore been correctly performed. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; but the calibration step of the procedure has been carried out incorrectly, so the results are inconsistent with the reference value – a practically impossible situation. The analytical procedure does not yield reliable results for samples of a given matrix composition; all that can be said is that the calibration step of the procedure has been carried out correctly and that it is not responsible for the inconsistent results. The analytical procedure does not yield reliable results for samples of a given matrix composition; moreover, the calibration step of the procedure has been carried out incorrectly. It is this step that is primarily responsible for the inconsistent results. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; in addition, it confirms that the final determination step of the procedure as applied to such samples has been carried out correctly. The analytical procedure yields reliable results for samples of a given matrix composition; but the final determination step of the procedure as applied to such samples has been carried out incorrectly, so the results obtained are inconsistent with the reference value – a practically impossible situation. The analytical procedure does not yield reliable results for samples of a given matrix composition; all that can be said is that the final determination step of the procedure has been carried out correctly and that it is not responsible for the inconsistent results relating to samples of a given matrix composition. The analytical procedure does not yield reliable results for samples of a given matrix composition; moreover, the final determination step of the procedure has been carried out incorrectly, and it is this step that is primarily responsible for the inconsistent results relating to such samples. Both the calibration step and the final determination step of the analytical procedure for samples of a given matrix composition have been performed correctly, but no inference can be drawn about the reliability of the whole procedure. Only the calibration step of the analytical procedure has been performed correctly, so no inference can be drawn about the reliability of the whole procedure. The measurement results obtained using the final determination step of the procedure are inconsistent with the reference values for samples of a given matrix composition; consequently, the results obtained with this procedure cannot be consistent with the reference value. The calibration step of the analytical procedure has been carried out incorrectly; consequently, results are obtained that are inconsistent with the reference value. The consistency of the result for an extract sample is purely accidental; in combination with an incorrectly performed calibration step, such a situation is practically impossible. Both the calibration step of the analytical procedure and its final determination step have been carried out incorrectly; consequently, the results are inconsistent with the reference value. The analytical procedure (all its steps) yields reliable results for samples of a given matrix composition; both the calibration step and the final determination step of the procedure have been carried out correctly, which means that the results are reliable. The final conclusion is that all the steps of the procedure have been performed correctly.
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Table 2. (continued) Material sent for testing CRM
StSol
ExCRM
Y
Y
N
Y
N
Y
Y
N
N
N
Y
Y
N
Y
N
N
N
Y
N
N
N
Conclusion
The analytical procedure yields reliable results for samples of a given matrix composition; moreover, the calibration step has been carried out correctly, so that measurement results are reliable. But the measurement results obtained using the final determination step of the procedure are inconsistent with the reference values for such samples – a practically impossible situation. The analytical procedure yields reliable results for samples of a given matrix composition; moreover, the final determination step has been carried out correctly, so that measurement results are reliable. But the measurement results obtained using the calibration step of the procedure are inconsistent with the reference values for such samples – a practically impossible situation. The analytical procedure yields reliable results for samples of a given matrix composition. But neither the calibration step nor the final determination step of the procedure have been carried out correctly; consequently, the results are inconsistent with the reference value – a practically impossible situation. The analytical procedure does not yield reliable results for samples of a given matrix composition; however, both the calibration step and the final determination step have been carried out correctly and yield reliable results; nevertheless, the conclusion to be drawn is that the results of the remaining steps of the procedure must be unreliable. The analytical procedure does not yield reliable results for samples of a given matrix composition; however, the calibration step has been carried out correctly and yields reliable results, but since the final determination step has been carried out incorrectly, it is responsible for the unreliability of the results of the remaining steps of the procedure. Conclusion: the matrix composition exerts a decisive influence on the consistency of the measurement results. The analytical procedure does not yield reliable results for samples of a given matrix composition; the calibration step has been carried out incorrectly, so the results obtained are inconsistent with the reference value. The consistency of the result for an extract sample is purely accidental; in combination with an incorrectly performed calibration step such a situation is practically impossible. The analytical procedure does not yield reliable results for samples of a given matrix composition; the calibration and final determination steps are definitely responsible for the unreliability of the whole procedure; however, it is difficult to define the possible effect of the remaining steps of the procedure.
in methanol was added. The suspension was invisible, due to the tiny quantity of sediment and the dark glass of the bottle, and fell to the bottom quite quickly. This time, the PAHs were adsorbed primarily on the surface of the suspended particles. Another way of defining and at the same time monitoring the influence of matrix composition on an assay result is to send out a sample of an extract of the test material together with the sample of the test material itself [18–20]. Any difference between the measurement results for the two samples will indicate a serious source of error at the sample-preparation step. Table 2 lists the conclusions to be drawn, depending on the results of the PT, the object of which was reference material and/or a standard solution and/or an extract of the reference material. Sample-preparation techniques exert a definite influence on the recovery of analyte from the matrix, and so also on the final result of the determination and its reliability. The method of preparing the samples in the above-mentioned aqueous solutions for analysis of organic compounds serves as an example. Samples A and B, prepared as stated above, were sent to three laboratories (1, 2 and 3) for analysis. Each laboratory used the individual isolation and enrichment steps in procedures for analyte extraction [33].
In Laboratory 3, suspended particulate matter was removed by filtration. In the case of sample B, this also removed the compounds adsorbed on the suspended particles. Hence, the low recovery of analytes, in particular of non-volatile PAHs. The isolation process used in Laboratory 2 involved the transfer of the suspended matter onto an extraction disc, which was then subjected to solvent extraction. This gave a high recovery of analytes, especially of nonvolatile PAHs. Such a high recovery was possible because the analytes introduced into the sample (dissolved in an organic solvent) were adsorbed on the surface of sediment particles, as a result of which ageing (penetration of the sediment and stronger bonding with it) was minimal. Quantitative desorption of PAHs occurring naturally in the suspension was impossible, given the conditions under which the isolations were carried out in these laboratories. Table 3 lists the results (recoveries) obtained by the participating laboratories. It is worth stressing that there was a seven-fold difference between the determinations of PAH levels in sample B obtained by the laboratories [33]. Special interlaboratory comparisons are also undertaken to assess a particular (often recommended) technique for isolating analytes from a matrix [21–25].
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Table 3. PAH analyte recoveries obtained by the participating laboratories for samples A and B [32] Compound
Percentage of PAH levels determined in samples A and B with respect to reference values [%] Laboratory 1 (LLE)
Laboratory 2 (SPE disc for accelerated extraction)
Laboratory 3 (SPE columns)
Sample
A
B
A
B
A
B
Naphthalene Anthracene Fluoranthene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[g,h,i]perylene Benzo[k]fluoranthene Indeno[l,2,3-cd]pyrene
85 105 95 59 60 42 59 34
87 105 94 59 61 40 61 34
44 68 66 58 63 51 59 41
16 60 114 88 91 104 86 87
35 54 60 52 62 42 55 40
31 40 58 21 27 17 22 12
Both the recovery and the method of its determination affect the final result of a determination. The recovery value must be used to correct the obtained measurement value. Like the result of every analytical measurement, the recovery is encumbered with uncertainty. Hence, the value of this uncertainty must be taken into account as a component of the combined uncertainty of the final determination. 3.4.5. The uncertainty of a measurement result. Uncertainty is a fundamental property of every measurement. It is always present at every step of every measurement procedure. Hence, it is not a property that causes additional problems during the measurement process [34– 37]. The major sources of uncertainty during the testing of samples using an appropriate analytical procedure include the following [1,3,38]: (1) erroneous or imprecise definition of the magnitude to be determined; (2) a non-representative sample; (3) incorrect application of the analytical methodology; (4) systematic human errors in reading analogue signals; (5) ignorance or lack of awareness of the effect of all external conditions on the result of an analytical measurement; (6) the uncertainty inherent in the calibration of the monitoring/measurement apparatus; (7) the resolution of the measurement apparatus; (8) the uncertainty inherent in the application of standards and/or reference materials; (9) the uncertainty of parameters determined during separate measurements and applied in calculating the final result of the determination (e.g., physico-chemical constants); 714
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(10)
approximations and assumptions relating to the use of a particular measurement apparatus and applied during the performance of a measurement; and, (11) fluctuations of apparently identical external conditions occurring during repeated measurements. Every analytical result is the consequence of a measurement performed. The overriding aim of the analyst is to obtain an analytical result reflecting as closely as possible the expected (real, true) value. The certainty of an analytical result therefore depends on the uncertainties inherent in every step of an analytical procedure. The parameter determining the final uncertainty of a result is the one with the greatest uncertainty. Hence, the sources and the types of uncertainty must be defined for every single step of an analytical procedure – for every measured magnitude, in fact [1,38]. Determining the uncertainty of a measurement enhances its reliability, renders the results obtained during interlaboratory comparisons comparable, and helps to establish the significance of the difference between a measurement result and a reference value [38]. One way of discovering problems relating to the construction of the uncertainty budget is for intercomparison organizers to insist that the participating laboratories state the uncertainty values for every step of the analytical procedure that they apply during PT. Comparison of uncertainty values will then show up the main sources of uncertainty and give an idea of the accuracy of its estimation. 3.4.6. Data processing and interpretation of results. The presentation of measurement results and their interpretation may be an additional source of information as far as the reliability of results is concerned. During the preparation of an interlaboratory study, the organizer can impose a particular way of presenting
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Table 4. Dubious situations resulting from disagreement between uncertainty (U), limit of detection (LOD) and standard deviation (SD) values delivered by PT participants as a final result of PT Data delivered by PT participants Analyte
Unit
Results 1
2
3
PCB 28
ng/g
18.0
14.3
22.8
Anthracene
ng/L
33.0
33.2
29.9
4
35.5
Conclusions
Mean
SD
LOD
18.4
4.3
2.0
10%
32.9
2.3
15
30%
5
the results, which can considerably improve the quality of data interpretation For example, by stipulating the number of replicate measurements for particular analytes, the uncertainty inherent in the repeatability of results can be estimated. Again, the demand that the LOD be stated yields information on the quality of measurement results depending on the ratio of the given LOD to the concentration level determined. It is therefore very important to prepare (e.g., in the form of a results sheet) an appropriate way of data transmission that includes all the information needed for
STEP OF THE ANALYTICAL PROCEDURE
SAMPLING
SAMPLE PREPARATION
FINAL DETERMINATION
DATA TREATMENT
U
Standard uncertainty due to repeatability calculated is about 13%, so expanded uncertainty should be no less than 26%. It is underestimated. Standard uncertainty due to LOD value is about 45%, so expanded uncertainty should be no less than this value. It is underestimated. Additionally, if LOD is equal to 15 ng/L, the limit of quantification (LOQ) should be equal 45 ng/L. Reliability of results below LOQ is really poor.
correct, reliable data treatment. This information should include the following: (1) sample-preparation method; (2) enrichment method; (3) calibration method; (4) calibration-curve parameters; (5) numbers of standards and their ranges used during calibration; (6) raw results (all repetitions); (7) mean, standard deviation, LOD, uncertainty values; (8) how the uncertainty budget is estimated; and, (9) all graphs (calibration curve, chromatograms).
THE WAY OF CONTROL
NO
− test samples with different matrix composition YES − standard solution − extract from test sample − standard solution YES − extract from test sample − data presentation YES − uncertainty estimation − validation parameters calculation
Figure 2. Ways of interaction, monitoring and obtaining additional information from interlaboratory comparisons on the steps in an analytical procedure.
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Table 4 presents equivocal situations resulting from discrepancies between LODs, uncertainty and standard deviation. These discrepancies would be shown up on an appropriately designed results sheet sent to participants. However, if appropriate (often very specific) processing techniques are applied to the results, additional information becomes available regarding, say, laboratory competence or the potential of a tested analytical procedure [21,22,26,27]. 4. Conclusions The principal objective of interlaboratory comparisons is to obtain additional information on the object of comparison (analytical method, reference material, or laboratory). But this additional information can be reliable and exhaustive only when both the organizer and the participant of the comparison present the results of the analyses in an optimal manner. On the one hand, this way of organizing and carrying out such comparisons yields the fullest possible information on possible errors, and how to eliminate or at least prevent them. On the other hand, reliable measurements result in subsequent comparisons of the same type. As far as determining the content of organic compounds is concerned, the matrix composition of the sample under scrutiny has a significant influence on the reliability of the measurement result. This influence is far greater than in determining, say, metal ions or physico-chemical parameters (e.g., pH or conductivity). That is why, when an interlaboratory-comparison organizer is preparing appropriate samples for analysis during the comparison, it is imperative to define the matrix composition and to take into account its effect on the result of determination. In this context, the following parameters are crucial – how analytes are bound to the matrix components, analyte bioavailability, biodegradation, volatility and stability. Attention needs to be paid to these questions, both during the organization of the comparison and while it is taking place. The statistical processing of the measurement results also needs to be looked at. It goes without saying that the statistician who evaluates the results of analyses must have at least a rudimentary knowledge of analytical chemistry. At the comparison stage, analyst and statistician must therefore work in close cooperation. The possible sources of ‘‘interference’’ in the execution of interlaboratory comparisons, outlined in this article, are the main points of which the organizers and participants of such studies should be aware. By way of summary, Fig. 2 presents ways of interacting, monitoring and obtaining additional information from interlaboratory comparisons regarding the steps of an analytical procedure.
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