FAQ on calibration

Rationale
From experiences gained in inter-laboratory comparison studies, being it proficiency tests or method validation studies by collaborative trial, we know that the importance of instrument calibration and its effect on analysis results is frequently underestimated. Participants to some of the studies expressed their wish to get more guidance on instrument calibration; despite a number of international guidance documents is already available. This situation could be explained by the difficult language applied in some of the guides, respectively the lack of knowledge on where to find very practical and easily understandable guidance. The more than five million hits gained at the time of writing this text with one of the most prominent internet search engines for the term "instrument calibration" do not really contribute to improve the situation. Therefore we felt it necessary to prepare this document that aims to address some aspects of standard preparation and instrument calibration for the determination of polycyclic aromatic hydrocarbons (PAHs), respectively mycotoxins in food that seem to provide difficulties to some operators. Like in every question-and-answer scenario one will face the fact that either a question can be asked extremely specific, resulting in a similar specific answer or a question can be asked rather general and the answer thus might also be rather general. This might result in a dilemma, in which the answer to a question might not be the one of interest, because either it is too specific and not applicable to the exactly (slightly different) question one might have, or it is too general and does not touch specific aspect one had in mind. In other words: “If a model is simple, it likely will be wrong, if it is complex, it surely is impractical” Applying this to this guide, the compromise was to try to answer both to relevant general issues but also to a few specific ones that are sometimes encountered. The format of the guide was chosen on purpose, because as a frequently asked questions (FAQ) document it remains open to address and include any question regarding standard preparation and instrument calibration that might come up in future.

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FAQ on calibration Index Standard preparation .................................................................................................................. 3 1. Where do I get reference materials for PAH analysis from? ......................................... 3 2. Which level of purity of reference materials is acceptable? .......................................... 4 3. Which are the advantages of gravimetric standard preparation? ................................... 4 4. Which type of balance do I need for the preparation of calibration standards?............. 4 5. Is serial dilution of a standard solution for the preparation of calibration standards acceptable? ..................................................................................................................... 5 6. How shall I store PAH standard solutions?.................................................................... 6 7. Which containers shall I use for storage of standard solutions? .................................... 7 8. How shall I estimate the shelf life of my standard preparations? .................................. 7 9. Which type of volumetric glassware may I use for the preparation of calibration standards? ....................................................................................................................... 8 10. How do I verify the concentration of my standard preparations? .................................. 8 11. How many points do I need for a calibration curve? ..................................................... 9 12. How many replicates per calibration point?................................................................. 11 13. Why shall the concentration levels of the calibration standards be equidistant? ......... 12 14. Which range of concentration has the calibration to cover? ........................................ 14 15. Which type of internal standard shall I use? ................................................................ 15 16. When do I need to prepare matrix matched calibration standards? ............................. 16 17. How do I determine matrix effects? ............................................................................. 17 18. In which sequence shall I measure the calibration standards? ..................................... 18 Evaluation of calibration measurements .................................................................................. 18 19. How shall I test for linearity of the calibration?........................................................... 18 20. Does a correlation coefficient (r) of 0.99 indicate linearity of calibration? ................. 19 21. Which level of R² is sufficient?.................................................................................... 19 22. Which information can I get from the plot of residuals? ............................................. 20 23. What is the residual standard deviation?...................................................................... 20 24. May I force the calibration curve through the origin?.................................................. 20 25. What is homo- and heteroscedasticity? ........................................................................ 20 26. How do I test for homoscedasticity / heteroscedasticity? ............................................ 21 27. Linear regression or weighted linear regression – which shall I apply? ...................... 21 28. May I remove outliers? ................................................................................................ 21 29. How do I estimate confidence and prediction intervals? ............................................. 22 General ..................................................................................................................................... 23 30. Is there any internationally harmonised document on calibration?.............................. 23 31. Where can I get guidance on calibration? .................................................................... 23

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com).chemindustry. native and labelled) as well as PAH mixtures. LGC standards Important suppliers of reference materials for PAH in Europe (non-exhaustive list) ALFA Aesar. packaging size) can be found on the webpage www. SIGMA Aldrich.FAQ on calibration Standard preparation 1.chemexper. It contains single substance reference materials (neat and in solutions. Dr. A non-exhaustive list of suppliers.com A large collection of PAH reference substances.g.com chemexper. LGC. Chiron. Ehrenstorfer. among others different certified reference materials. Where do I get reference materials for PAH analysis from? A number of suppliers of chemicals have PAH standards in their assortment. and in solution): ChemIndustry: example of search for benzo[a]pyrene A similar searchable database which returns besides the name of different suppliers also some information on the product (e. The following link gives an example for suppliers of benzo[a]pyrene (neat. is included in the 2008/2009 catalogue of LGC. VWR certified reference materials (CRMs) for PAHs The Institute for Reference Materials and Measurements (IRMM). respectively links to other sources of information is given in the following: The International Society for Polycyclic Compounds (ISPAC) has on its website a list of suppliers of polycyclic aromatic hydrocarbons and heterocyclic aromatic compounds both neat and in solution: ISPAC Standards A searchable database on suppliers of different chemicals is on the homepage of Chemindustry (www. the National Institute of Standards and Technology (NIST) 3 .

will be fit for the purpose. care must be given that impurities do not interfere with the target analytes. Which level of purity of reference materials is acceptable? A purity of 100 % would be desirable.g. 3. Handling of low volumes of liquids is difficult due to the influence of many factors such as surface tension.1 mg) with a factor between 3000 and 5000. respectively 0. For gravimetric standard preparation it shall be noted that the uncertainty from weighing increases with decreasing amounts of weighed substance. Which type of balance do I need for the preparation of calibration standards? An analytical balance with a readability of 0.1 mg. This has consequences for the selection of the type of balance and the weighing procedure applied. The purity of the reference substances shall be considered in the calculation of the standard concentrations. Before starting with gravimetric standard preparation make sure that the balance is working properly. which results normally in smaller uncertainties. The uncertainty of the purity shall be included in the measurement uncertainty estimate. The US Pharmacopeia [1] defines the minimum permissible weight of a balance as a load that will give a relative uncertainty of less than 0. 0. 4 .FAQ on calibration 2. 4. Thermal equilibration might take a couple of hours especially in case of large solvent volumes. Hence the operator has to choose a reference material with a purity that is suitable for the particular task. by applying suitable check weights. and leads frequently to bias.1%. However. A prerequisite for gravimetric standard preparation is thermal equilibrium of the balance and all chemicals and consumables which are used for the standard preparation. As a rule of thumb the minimum weight can be estimated for a balance by multiplying the readability of the balance (e. which means that the uncertainty of weighing is at an acceptable level. but in reality most of the target PAHs (15+1 EU priority PAHs) are available on the market in purities of above 95%. Which are the advantages of gravimetric standard preparation? The weighing procedure is more precise than handling of volumes.01 mg for weighing of substances at levels as low as about 30 milligram.

care has to be given that the balance is calibrated and working according to the specifications. Even worse would be scheme B which includes a cascade of dilutions of the calibration standards. too low air humidity leads to electrostatic problems and might cause bias. Besides the risk of unidentified bias it provides high uncertainty of the concentration of standard CS5. Further information on the use of balances in standard preparation can be found in open source literature in e. According to the law of error propagation the uncertainty of CS5 is equal to the square root of the sum of uncertainties of the preparations S to CS5.g.g. 5 . which is prepared in six dilution steps. which is of course larger than the uncertainty of any other calibration standard shown in Figure 1. McDowall [2] 5. Is serial dilution of a standard solution for the preparation of calibration standards acceptable? No! Two aspects have to be taken into account in standard preparation by serial dilution. Also provisions on environmental conditions must be respected. Figure 1 A presents a standard preparation scheme where bias in the preparation of dilution 1 (D1) from the stock standard solution (S) cannot be identified from the measurement results of the calibration standards (CS1 to CS5). a paper published by Ch Burgess and R.001 weighed amount does not include the tare weight of the weighing vessel! Eq 1 It has to be noted that the minimum weight corresponds only to the amount of substance weighed and In any case.D.FAQ on calibration However the applicability of this rule of thumb depends on the precision of the balance and has to be evaluated experimentally according to Eq 1: 3 x Stdev of 10 measurements ≤ 0. e. The probably more important aspect is the lack of ability of identifying biased standard preparation.

Duplicating scheme C with two independent stock standard solutions provides the highest level of information about the correctness of the calibration standards. 6 . 6. In practice the preparation of calibration standards needs thorough planning. The handling of low volumes or low masses shall be avoided as much as possible. Do not put PAH standard solutions in the freezer as the solubility of some PAHs might be affected at low temperatures.g. D1 dilution 1. By doing so. In case of PAHs limitations have to be encountered in the preparation of the stock standard solutions.FAQ on calibration Figure 1: Different schemes for the preparation of calibration standards. dibenzopyrenes) in the majority of organic solvents. How shall I store PAH standard solutions? PAH standard solutions shall be stored in amber glass ware in the dark due to potential degradation of PAHs by UV light. an error in the preparation of an intermediate dilution (D1 to D5) should be detectable in the measurement results of the calibration standards. Opened commercial standards and own standard preparations should be stored cooled to avoid solvent losses. which are caused by the low solubility of some PAHs (e. CS1 to CS5: calibration standard solutions) A B S D1 CS1 CS2 CS3 CS4 CS5 C The most appropriate of the three schemes is shown in Figure 1 C. Each arrow represents one dilution step (S: stock standard solution. The calibration standard solutions are prepared from independent dilutions of the stock standard solution. Room temperature (about 20 °C) is recommended for storage of PAH standard solutions by a number of suppliers.

independent. Usually laboratories use one standard preparation at a time as standard solutions are expensive. Therefore it might be worth to look for alternatives. It is also recommended to divide the stock standard solution preparations for storage into several units of small volume in order to conserve the composition of the parts of the preparations. How shall I estimate the shelf life of my standard preparations? The shelf-life of a product is the time that the average characteristic of the product remains within an approved specification. second. In addition the preparation of fresh standard solutions would make the determination of the shelf life superfluous. As a general rule. The tested standard solution must be independent from the standard solution that is used for instrument calibration in order to identify any changes. There is not any general guidance on this.FAQ on calibration 7. This sounds very well in theory. standard solution over the whole period of shelf life experiments. The question which has to be answered is how much may the change of the composition of the standard preparations contribute to the combined measurement uncertainty. which are at the time being not in use. More economically would be applying a single. loss of solvent etc. The time of the study has to cover at least this first estimate of the shelf life. The second problem consists of the identification of changes in practise. or information from literature. The first constraint is given by defining of the maximum tolerable change of concentration. At the beginning of such studies little knowledge of the stability of the standard solutions is available. However this does not provide the requested information because in case of significant differences it is not possible to trace back which of the two standard solutions has changed. 8. which might be caused by degradation of the analyte. A possibility could be to apply in the shelf life experiments a chemical as internal standard that is available in large amounts at low costs. An appropriate value has to be set on case by case basis. This chemical serves as reference point. and hence estimate its shelf life. Translated to standard preparation this means that the change of standard concentration respectively the associated uncertainty must not exceed certain predefined limits. The solution of the 7 . the headspace above the standard solution shall be as small as possible. Hence requesting the preparation of a fresh standard solution for each set of shelf life experiments would be illusionary. However. a relative change of the standard concentration of 1 % to 2 % could be acceptable. Which containers shall I use for storage of standard solutions? Amber glass ware with Teflon® lined closures should be used. and related to that to the set up of the experimental plan to proof the agreement with the predefined specifications. Hence the shelf life has to be estimated based on experiences made with similar substances. but causes several problems for the implementation into practise.

04 ml for a 25 ml flask). Hence the concentration of the standard preparation shall be evaluated against an independent standard preparation. 10. In the experiments relative response factors between the analyte and the reference chemical are determined. The integrity/stability of standard preparations has to be monitored over the whole shelf life of the standard preparation. The minimum requirement is to verify the concentration of a new standard preparation against the concentration of the preceding standard preparation. The maximum tolerances for different volumes are given in ISO standard 1042:1983 as well (for instance it is ± 0. 8 . e. Best practise in that respect would be verification against a solution with certified values for the analyte(s). More likely the concentration of a particular standard preparation can only be verified against other standard preparations. How do I verify the concentration of my standard preparations? The verification of the standard concentration is crucial for assuring the quality of analysis results. parallax error) of volumetric glass ware will contribute to the total uncertainty of the standard preparation probably to a larger extend than the tolerances according to ISO standard 1042:1983. Which type of volumetric glassware may I use for the preparation of calibration standards? The contribution of glassware tolerance to the global uncertainty of the method is very low but not negligible.FAQ on calibration reference point has to be prepared freshly for each set of shelf life experiments. Class A glassware according to ISO standard 1042:1983 shall be applied. 9. However it has to be pointed out that the handling (filling.g. the concentration of aflatoxin standard solutions in methanol/water can be verified by photometry. and any changes are monitored. Gravimetric standard preparation is considered superior to volumetric standard preparation with regard to precision. which lowers the risk of bias. For light sensitive substances the glass ware shall be produced from amber glass. emptying. Control charts shall be applied for this purpose. Such certified reference materials (CRMs) are frequently not available. The selection of a chemical serving as reference point depends on the properties of the analyte. Repeated measurements shall be performed at each control point in order to estimate the variability of the measurements. The low costs would allow using large quantities in the standard preparation. In a limited number of cases the concentration of standard preparations can be verified by application of reference methods. The shelf life of the standard preparation can be shortened or extended depending of the experimental results.

Other documents might lay down a different number of calibration levels. is usually applied for the estimation of linear calibration functions. How many points do I need for a calibration curve? Before answering to this question the purpose of the calibration experiment has to be defined. One has to distinguish between the calibration of a measurement system and the check of the validity of the calibration of a measurement system. The EURACHEM Guide "The Fitness for Purpose of Analytical Methods" specifies for that purpose at least six concentration levels plus blank. However it also says that the number of levels shall be increased for an initial assessment of the calibration function. The minimum number of calibration points/levels is defined by ISO standard 11095:1996 for the basic calibration method to three. Hence ISO regarded four calibration levels sufficient for the estimation of the calibration function. called in ISO standard 11095:1996 the "basic method". If the blank or zero sample does not produce any signal it can be excluded from the calibration experiments. The above mentioned IUPAC guide does not specify any concrete number of calibration levels. Commission Decision 2002/657/EC stipulates at least 5 concentration levels including zero for the construction of a calibration curve. Both topics are treated in depth by international standards such as ISO standard 11095:1996 and the IUPAC guideline "Guidelines for calibration in analytical chemistry". The linearity of the instrument response was probably tested for the analysis method that became an ISO standard. Less knowledge on the shape of the calibration functions requires performing of measurements on more concentration levels.FAQ on calibration Bracketing calibration as detailed in ISO standard 11095:1996 shall be preferably applied for the verification measurements. including blank. It encompasses the measurement of a certain number of reference materials (calibration standards) at different concentration levels. For example ISO standard 15302:2007 specifies four calibration levels. as this technique yields usually greater accuracy than linear calibration. ISO 8466-1:1990 demands even ten calibration levels. 9 . The inclusion of blank or zero levels into the calibration design is required. whereas the LGC/VAM guide "Preparation of Calibration Curves" defines seven calibration levels. as minimum requirement for an initial assessment of the calibration function. The first case. if the blank or zero sample produces a signal that is of the same nature as the signal produced by the analyte. 11. This initial assessment is equal to operations performed during method validation to assess the linear range of a measurement method. As can be seen the design of calibration experiments and the number of calibration levels depend very much of the purpose of the experiment and of existing knowledge.

9950x + -0.0357 5.8 9 1.9837x + 0.85 7.0813 8. Increasing the number of calibration levels and the number of replicate analyses per level reduces the width of confidence and prediction intervals. and at least one more calibration level is needed for the statistical assessment of the calibration model.05 1.85 5.05 3 3.8 0.9 5. The underlying data are displayed in Table 1.8 3.0178 7 1.8 0.1 5.05 2 2.05 8.0139 10 .8 0.9688x + 0.1 8.1 8.8 0.9871x + 0. Each calibration level was measured once.05 2 2.1 8.85 7. Figure 2 shows the confidence intervals for simulated calibration experiments performed at different numbers of calibration levels. However the return in terms of narrowing confidence intervals is diminishing with the number of calibration levels.1 5. Exceeding ten calibration levels does not provide any additional benefit.05 Number of calibration points 4 Response 1.9688x + 0.1 7.1396 8.8 0. Table 1: Data of simulated calibration experiments including different numbers of calibration levels 2 Level 1 2 3 4 5 6 7 8 9 Slope(x) + Intercept 1.FAQ on calibration In general three concentration levels are required to fit a non-linear function.

How many replicates per calibration point? The ISO standard 11095:1996 demands at least two replicate analyses per calibration level and recommends as many as possible. and to detect any shifts or errors. At least two replicate analyses are necessary to evaluate the calibration for constancy of the residual standard deviation. 12. 11 . This procedure is based on the information gained in an initial calibration experiment. At least two. preferably three calibration levels are used to monitor via control charts the validity of the calibration function. The check of the validity of a calibration system has to be clearly distinguished from the initial calibration. four (green dashed line). three (red dashed line). ISO standard 11095:1996 applies the term "Control method" for the check of the validity of a calibration system. seven (purple dashed line) and nine (grey dashed line) concentration levels (concentration points). The lines in the middle represent the calibration curves corresponding to the different scenarios.FAQ on calibration Figure 2: Confidence intervals for simulated calibration experiments at two (black dashed line). This information is needed to decide on which regression model is most appropriate (see below).

Hence more than five replicate analyses per calibration level do not provide big additional benefit. corresponding to the highest concentration level and one concentration level at the lower end of the concentration range. As a result the calculated slope and intercept might be influenced disproportionally by one data point. The signal value of this data point was changed from about 800 to 600. 13. one at a time. If you deviate from this calibration procedure. As a consequence. NOTE: A very important thing to consider is that all performance data associated with a standard method is based on the calibration procedure mentioned therein. both the slope and the intercept of the calibration curve change significantly. The respective data points are indicated by bold dots. Why shall the concentration levels of the calibration standards be equidistant? The reason is that the higher the concentration of a respective calibration standard. The effect is demonstrated based on a simulated calibration experiment. as the number of calibration levels. In Figure 3 each calibration level corresponds in concentration to the double of the next lower concentration level. the data point at the upper end of the calibration range gets higher weight. In each of the experiments one data point got a relative offset of -20 %. were manipulated. as mentioned before. The offset of the red dot (●) at the higher level has a much bigger influence on the 12 .g. the law of diminishing return. and calibration curves were determined by linear regression. Since the residual (absolute signal value) caused by the relative offset (20 %) is much higher at the upper end of the calibration range than at the lower end. it is your responsibility to demonstrate that the modified calibration procedure will give equivalent results. which aims to minimise the sum of the squared residuals. the more it is weighted for the calculation of the calibration curve (this is called leverage). pipetting mistakes. Figure 3: Simulated calibration experiments with a relative offset of -20% of one data point in each experiment. The contrary is the case if the data for the highest concentration level would be biased. Two data points of the example. The effect of the offset of the data point at the lower end of the calibration range (green dot) on the regression curve is marginal.FAQ on calibration Increasing the number of replicate analyses follows. This effect is based on the principle of the applied regression method. Such relative offsets are caused in practise by e.

despite they are frequently found in practise. The difference in effect on the regression curve of one biased calibration point is displayed in Figure 4 both for a set of six equidistant concentration levels and a set of six unevenly distributed concentration levels (multiplication factor = 2). Leverage 800 600 Signal 400 200 0 0 20 40 60 80 Analyte concentration It has to be stressed that the application of calibration designs based on standard concentrations that correspond to multiples of the next lower concentration is strongly discouraged. The offset of the data point at the highest concentration level has less influence on the regression curve in the calibration with equidistant concentration levels than with unevenly distributed concentration levels.FAQ on calibration resulting red calibration curve (—) than the offset of the green dot (●) on the resulting green calibration curve (—). Figure 4: Effect of one biased calibration point (at concentration level 80. offset of signal = -20%) on the regression curve of calibration experiments with equidistant (pink) and unevenly distributed (blue) analyte concentration levels 800 700 600 Signal 500 400 300 200 100 0 0 20 40 60 80 100 Analyte concentration Unevenly distributed Equidistant Linear regression Linear regression 13 .

where shown by experiments to be appropriate. and to re-analyse it. but is per se not applicable for all analysis methods due to the alteration of matrix effects.FAQ on calibration 14. As rule of thumb the ratio between the concentrations of the highest and lowest concentration levels shall not exceed a factor between 10 and 20. 14 . a dilution can be made. provided the interest concerns only this small working range. However.g. In that respect caution has to be given to “simply” diluting the test sample extract to bring it to a concentration level that is covered by the instrument calibration. The upper range of concentration that a calibration experiment may span is not defined. As a result it can be very narrow in concentration (e. This might be possible in many cases. Which range of concentration has the calibration to cover? The calibration shall cover at least the content/concentration range in which you will need to report results. The calibration standards have to be at concentration levels corresponding to the concentration levels of the ready to measure/inject sample. However factors such as homo-/heteroscedasticity (see below) shall be taken into account in the design of the experiments. Occasionally the analyte content of test samples will exceed this concentration range the instrument is calibrated for. The calibration range defines also the working range of the analysis method. around a legislative limit).

the concentration of the internal standard added to the sample shall preferably be in the middle of the range of expected analyte concentrations There are different options for the choice of internal standards. if the analysis method comprises chromatography with optical detection (such as fluorescence or UV-absorption) the chosen internal standard has to behave chemically and physically very similar to the analyte (e. the internal standard must not be found in the sample itself. If the chosen internal standard has a much different retention time and therefore most likely a rather different chemical behaviour (e. The applicability of the different possibilities depends on the purpose of the internal standard and the applied detection system. For example. The same holds true for deuterium substituted PAHs. in terms of polarity) it is likely that it also behaves different from the analyte during extraction or clean-up. Often analogues of the actual analyte are taken for this purpose. close structural analogues of the analyte are preferably used.FAQ on calibration 15. Another option is provided by the application of fluorinated analogues of the target PAHs. In the field of mycotoxins aflatoxicol (a metabolite of aflatoxins) is used as internal standard for the determination of aflatoxins. in extraction and clean up steps). As a result. squaric acid for the determination of moniliformin (for HPLC-FL methods) or de-epoxy deoxynivalenol (DOM-1) for GC methods. which show in HPLC also slightly different retention characteristics compared to the native compounds.g. because chemical properties are very similar and chromatographic separation can easily be achieved. Examples are the use of verrucarol for the determination of fusarium toxins (for GC methods). Structural isomers of target analytes (e. otherwise the interpretation of the internal standard data can be jeopardized. Also structurally similar substances have been proposed when a derivatisation is required and the analogue (internal standard) must react in the same manner as the analyte.g. 15 . but must be chromatographically resolved from the analyte. Which type of internal standard shall I use? The most important properties of a suitable internal standard are: • • • the internal standard must behave the same or at least very similar to the analyte in question.g. benzo[b]chrysene) are applied for the determination of PAHs in food by high performance liquid chromatography with fluorescence detection (HPLC-FLD).

The basic assumption with this technique is that relative responses between the analyte and the labelled analogue stay constant.g. Also fluorescence detection can be subject to matrix influences (e. fluorescence quenching). e. Isotope dilution with isotope labelled analogues of the target analyte is frequently applied to compensate for matrix effects. might lead to problems in distinguishing between the mass spectrometric signals of the native compound and the labelled analogue. The possibility of deuterium-hydrogen exchange cannot be excluded with deuterated compounds. C13 labelled compounds do not provide such problems. different retention characteristics compared to the native compounds. However the costs for this kind of labelled substances are substantially higher than for deuterated substances and the availability is limited. Also the loss of deuterium atoms in chemical reactions of the analyte. where the matrix (even after clean-up procedures) has an influence on the signal obtained for the analyte during measurement. which might provide problems when it comes about compensation of matrix effects in mass spectrometry. When do I need to prepare matrix matched calibration standards? A matrix matched calibration is needed in those cases. Many analysis systems are sensitive to matrix effects. Perdeuterated substances as commercialised for some PAHs have. However care must be taken. such as derivatisation reactions. that the matrix used to prepare the calibrant is sufficiently well matched to the matrix of the sample. The differently labelled substances might show significant physico-chemical differences. This phenomenon is encountered in the determination of acrylamide by GC-MS after chlorination and consecutive dehydrochlorination. which makes them unsuitable for quantitative analysis. This offers the detection of both substances (the analyte and the labelled internal standard) with the same or very similar retention time. as mentioned above. 16 . LC-MS or GC-MS.g.FAQ on calibration In the case of chromatography coupled to mass selective detection the substances of choice are isotope labelled analogues of the analyte. The hydrogen isotope clusters of some fragment ions of the labelled and native acrylamide overlap partially. which is necessary for compensating for matrix effects. 16. The choice between deuterated and C13-labelled substances needs to take into account different facts.

To answer this question. The number of degrees of freedom is for each calibration experiment N-2 (with N=number of data). Before proceeding it must be guaranteed that the precision of the two calibration curves is comparable. the question has to be answered whether the deviations from the ideal values (slope=1. Otherwise any significant difference between the calibration curves might be hidden by the different level of precision. If the confidence intervals include for the slope the value one. than it can be concluded that there is not any statistical difference between the calibration with calibration standards in solvent solution and the matrix matched calibration. Ignoring these facts would lead in the earlier case to constant bias and in the latter case to proportional bias.FAQ on calibration 17. intercept=0) are significant or just random. and the corresponding concentration values (x-values) are calculated. Given that no significant differences of the residual standard deviations were identified. This is accomplished by testing the residual standard deviations of the two calibration curves for significant differences (with an F-test) at the 99% confidence level. An intercept different from zero indicates concentration independent bias. Matrix matched calibrations are an alternative to isotope dilution to compensate for matrix effects when using mass spectrometry for measurement. A slope different from one indicates potential concentration proportional signal enhancement respectively signal suppression. and for the intercept the value zero. However as the regression is based on a particular data set. The procedure to identify matrix effects is the same as to estimate a recovery function. This regression curves contains the information on matrix effects. These values are called in the following "apparent concentration values". In the first step a calibration curve is constructed by linear regression with the calibration standards in solvent solution. In the next step another calibration curve is constructed from the measurement data of the matrix matched calibration standards. Matrix effects are encountered when the intercepts and/or the slopes of the regression curves for the two sets of calibration solutions are significantly different from each other. How do I determine matrix effects? Matrix effects can be identified from calibration curves obtained with matrix matched calibration standards and calibration solutions in solvent. the confidence intervals (95% confidence level) of the regression parameter have to be determined. 17 . In the next step a linear regression is performed on the concentration data of the calibration standard solutions in solvent (x-values) and the apparent concentration values (used as signal data – y-values). the measurement data (y-values) of the matrix matched calibration are applied to the calibration function gained with the calibration standards in solvent. as a consequence of the variability in the limited number of data points.

at the beginning and at the end of the sample sequence. Evaluation of calibration measurements 19. The decision on whether to measure the calibration standards at the beginning of a sample sequence. Linearity may also mean the estimated parameters are linear which would also be true for a parabola.FAQ on calibration 18. or randomly distributed over the sample sequence depends of the stability of the measurement system. In which sequence shall I measure the calibration standards? Generally the sequence in which the calibration standards are measured should be random. However the design of the measurement sequence has to be modified if any instrument drift is expected. How shall I test for linearity of the calibration? For the purposes of this document linearity means the calibration can be best described by a straight line. something that is not a straight line at all. A straight line can be described by Eq 5: y nk = β0 + β1 x n + ε nk with β0 β1 ynk xn εnk = intercept = line slope = the kth measured response of calibration level n = the concentration of the analyte in calibration level n = the residual for the kth measurement of calibration level n. If the measurement system is stable throughout the whole sample sequence then all approaches will give equal results. Such modifications could exist of repeated analyses of the calibration solutions during the measurement sequence or the inclusion of an increased number of quality control samples in the measurement sequence. ISO standard 11095:1996 specifies as general requirements that "the measurements from which the calibration function was calculated are representative of the normal conditions under which the measurement system operates" and "that the measurement system is in a state of control". Eq 5 The residual is the difference between the measured response and the response value calculated from the calibration function: ) ε nk = y nk − y n 18 .

and a value of 0 indicates that y can not be predicted by x. which is markedly not a straight line. Does a correlation coefficient (r) of 0.99.98 while for others 0.FAQ on calibration Plotting all ε nk over y n (residuals over fitted) results in the so called residual plot. 21. may have a correlation coefficient of 0. is the lack-of-fit test. This plot is a very valuable diagnostic tool. Another. If the lack-of-fit test is not significant then a straight line function describes the calibration data appropriately. Which level of R² is sufficient? One can not define a sufficient level of R2! The closer R2 to 1 the better the quality of the predictions made through the calibration. If the points are evenly distributed around a horizontal line trough zero the straight line function will be appropriate (see Figures 5 & 6). But certain calibration problems my never get beyond R2 = 0.998 is a sign of an error. And the r-value may improve by adding a quadratic term to one's calibration function which then is certainly not linear in our sense of the word. An r-value of 1 indicates that y can be completely predicted by x. more complex approach. Replicate measurements at each ) 100 0 2000 4000 6000 Fitted values 8000 10000 Residuals -400 -300 -200 -100 0 100 0 -200 Residuals -100 0 200 300 2000 4000 6000 Fitted values 8000 10000 Figure 5: straight line appropriate Figure 6: straight line inappropriate calibration level are a prerequisite for a lack-of-fit test. 20. 19 .99 indicate linearity of calibration? No! The correlation coefficient is a measure of how much of the variability of y can be predicted by x. A parabola.

But it can also be used to check for homo. then the intercept term is dropped from the calibration function and the calibration curve is assumed to originate at x = 0 and y = 0.FAQ on calibration 22. May I force the calibration curve through the origin? If the test of significance shows that the estimated intercept is not different from zero. 25. Which information can I get from the plot of residuals? The plot of residuals (see point 19) can show whether the assumption of linearity is met. The smaller the residual standard deviation. 24. 23. If the data's variability changes from one end of the range to the other the data is called to be heteroscedastic. What is homo. 20 . Otherwise the intercept term must be kept and the calibration curve is assumed to originate at x = 0 and y = intercept.and heteroscedasticity? Homoscedasticity is the term for calibration data having about equal variability over the whole calibration range. It is used to calculate significance of the intercept and the slope. What is the residual standard deviation? The residual standard deviation is a measure of the goodness-of-fit of the calibration. the closer are the measured data point to the calculated calibration curve.or heteroscedasticity of the calibration data and it is an indicator of the residual variability.

g. bad chromatography. injection error. from tight at one end to spread out at the opposite end (see Figures 7 & 8). etc.or heteroscedasticity. May I remove outliers? If an outlying value can be traced back to a failure in the system (e. In the case of homoscedasticity the residuals are more or less all within a band parallel to the xaxis. 200 100 Residuals -100 0 -200 -300 0 2000 4000 6000 Fitted values 8000 10000 -1000 0 -500 Residuals 0 500 1000 1500 2000 4000 6000 Fitted values 8000 10000 Figure 7: homoscedastic data Figure 8: heteroscedastic data 27. If such a retrace does not come up with any failure then the outlying value should be considered as a real but rare incident and kept in the data set.FAQ on calibration 26. In the case of heteroscedasticity the residuals assume a fan shape. Therefore weighted linear regression should be used in such case.) then it is permissible to remove it or better yet to repeat the measurement in question. Linear regression or weighted linear regression – which shall I apply? For homoscedastic data ordinary linear regression is appropriate. 21 . pipetting error. either can be determined from the residual plot. How do I test for homoscedasticity / heteroscedasticity? Whether one is dealing with homo. 28. But if the data is heteroscedastic ordinary linear regression will result in inflated estimates of the residual standard deviation.

defines the region in which with a certain probability (usually 95%) the regression line would be found if the calibration were repeated under similar conditions. More important for the task of calibration is the prediction band which is wider than the confidence band.FAQ on calibration They are estimated based on the estimates of intercept ( β 0 ). and residual standard deviation ( σ ) according to Eq 6. If a weighted least squares approach has to be used because of heteroscedasticity the weighted equivalents of all the estimates are used in Eq 6 and 7. 22 . slope ( β 1 ). As such the confidence band is of minor interest. (x P − x ) 1 y P = β 0 + β 1 x P ± t p .n − 2σ 1 + + n ∑ ( x n − x )2 ) ) ) 2 Eq 7 The subscript C (Confidence) was replaced by the subscript P (Prediction). Otherwise the same definitions as above are true. n − 2σ + n ∑ ( x n − x )2 with Eq 6 yC xC ) = upper or lower bound of the confidence interval for xC = value of x for which to compute the confidence interval = estimate of the intercept = estimate of the slope = estimate of the residual standard deviation β0 ) β1 σ n ) t p .n − 2 = Student’s t for probability p and n-2 degrees of freedom = number of observations = average of all x-values of the calibration = individual x-values of the calibration x xn The confidence interval or in the case of regression analysis better confidence band. How do I estimate confidence and prediction intervals? ) ) ) ) ) ( x C − x )2 ) 1 y C = β 0 + β 1 x C ± t p . 29. The above formulas are for the ordinary least squares approach. The projection of the outer bounds of this prediction band onto the y-axis defines the range of values which could reasonably be expected if one were too predict a new y for a new x.

Where can I get guidance on calibration? LGC Best practice guide for calibration design LGC Document "Preparation of calibration curves – a guide to best practice. Almansa-López. Switzerland. Chem. 1996 31. Is there any internationally harmonised document on calibration? ILAC/OIML Guide on Calibration ILAC Guide G24:2007 / OIML D 10:2007 "Guidelines for the determination of calibration intervals of measuring instruments". IUPAC Recommendations 1998 K. Pure&Appl. ILAC. L. Currie (1998).Chem. (2003) L. Fundamentals and single component calibration". 20: 620-636 23 .. 2007.M.FAQ on calibration General 30. ISO. E. II. Switzerland. L. Gámiz-Gracia. A methodological approach". ISO. (2003) "Calibration in chemical measurement processes. Trends in Anal. 1997 ISO Standard ISO 8466-1:1990 "Water quality – Calibration and evaluation of analytical methods and estimation of performance characteristics.. Cuardos-Rodriguez. ISO. "Guidelines for Calibration in Analytical Chemistry – Part 1. Danzer.A. Part 1: Statistical evaluation of the linear calibration function". 1990 ISO Standard ISO 11095:1996 "Linear calibration using reference materials". Bosque-Sendra. Australia. Switzerland. Silverwater.M. 70: 993-1014 ISO Guide ISO Guide 32:1997 "Calibration in analytical chemistry and use of certified reference materials". Geneva. Geneva. Geneva. J.

LCGC Europe. A question of balance? Part 2: Putting principles into practice. 2005 2 Ch.FAQ on calibration 1 United States Pharmacopeia. 24 . Rockville. USA. McDowall. Maryland. 19/3 (2006). Burgess and R. 28th Edition.D. Chapter 41.