Validation of cuvette tests for drinking water analysis - HACH LANGE

PRACTICE REPORTLABORATORY ANALYSISCUVETTE TESTSDRINKING WATERValidation of cuvette tests fordrinking water analysisIn 2007 the IWW (Rheinisch-Westfälische Institut für Wasser Beratungs- undEntwicklungsgesellschaft mbH) was commissioned to carry out a study to“determine the formal permissibility of the use of cuvette tests for the analysisof drinking water in conformity with the 2001 Drinking Water Ordinance(Trinkwasserverordnung 2001).” The study concluded that: the precision,trueness and limit of detection of the studied LANGE cuvette testscorrespond to the requirements of the Drinking Water Ordinance(DWO) concerning analysis methods. They are therefore suitable inprinciple for official analyses of drinking water.Author:Ralf König- Diplom-Chemiker- Vertical Market ManagerHACH LANGE

2DRINKING WATER ANALYSIS_REQUIREMENTSDo your cuvette tests meet drinkingwater legislation?AccreditationIn principle, non-standardised methods canbe included in the accreditation, if they havebeen proven to be suitable for the user andhave been validated accordingly. The characteristics of the analysis methodmust satisfy the requirements specified inAnnex 5 of the Drinking Water Ordinance.As well as the basic proof of the suitabilityof the methods (e.g. through this study), alaboratory must also provide the simplifiedproof that it satisfies the requirements withregard to the characteristics. The test method must be approvedby the accreditation body as part of theaccreditation process (ISO 17025) and mustbe included in the certificate (or the annex tothe certificate). In this context, proof of the fitness ofthe method for the intended purpose mustbe provided (in accordance with section 5.4of ISO 17025). The necessary validation workmust be carried out by the laboratory and ifnecessary, the manufacturer of the method. The laboratory carries out the stipulatedinternal (control cards) and external qualityassurance (successful participation in roundrobin tests) for the method. Finally, compliance with the abovementioned requirements must be formallyconfirmed or recognised by the notificationbody in accordance with section 15 (5) of theDrinking Water Ordinance.If these criteria are satisfied, there is no legalimpediment to the inclusion of cuvette tests inthe laboratory’s internal “method portfolio”.BackgroundIn Germany, official analyses of drinkingwater are regulated by law. Theymay only be carried out by speciallyauthorised, notified laboratories, usingmethods that are specified in conformitywith the Drinking Water Ordinance. Asthe German Drinking Water Ordinanceof 2001 almost completely embodiesthe content of the European DrinkingWater Directive, most of its conditionsand requirements apply in similar formin the other Member States of the EU.In contrast to the situation with regardto wastewater, the drinking waterlegislation specifies no concretemethodological requirements. Inprinciple, any method that yieldssufficiently reliable results and whoseperformance characteristics conform tothose defined in Annex 5, section 15 ofthe Drinking Water Ordinance, can beused. The performance characteristics(PC) are trueness, precision and limitof detection, which are expressedas a percentage (in this case 10 %)of the limit value. Another, no lessimportant “authorisation criterion” isthe requirement that the alternativemethod must conform to the generallyacknowledged technical standards.Also, a laboratory that wants touse cuvette tests as alternatives tostandardised methods must possessa valid accreditation in conformity withDIN ISO 17025.Requirements concerningaccreditationIf no method is specified by the client,an analysis laboratory is obliged touse the most appropriate analysismethods [3]. These appropriatemethods must have been publishedeither in international, regional ornational standards or by reputabletechnical organisations, or in relevantscientific journals, or have been clearlydescribed by the manufacturer of themethods (e.g. cuvette tests).Methods developed or adopted by thelaboratory may also be used if theyare suitable for the intended use andappropriately validated. The client mustbe informed about the method chosen.Basic validation of the cuvettetestsA test method is validated byexamining its:- measurement uncertainty- limit of detection and limit ofquantification- selectivity- linearity- precision- trueness- ruggedness against externalinfluences (cross-sensitivities,influences in the sample matrix)Furthermore, the alternative methodshould give results that are sufficientlycomparable to those obtained usingthe corresponding standard method.www.hach-lange.com

3All measurements were carried outwith a HACH LANGE DR 5000 UV-VISspectrophotometer (see Fig. 1).The basic calibration was carried outusing calibration solutions preparedby fortifying matrix-free solutions(ultrapure water) with the analytesof interest [1]. Certified referencematerials were used to fortify theanalytes. The limit of quantificationwas determined by measuring tencalibration solutions with equidistantconcentration levels starting from thelowest concentration of the measuringrange of the cuvette test over 10 days.The total measuring range wasvalidated by measuring from two tofive additional calibration solutions upto the highest concentration of themeasuring range.For the validation of the cuvettetests, the basic calibration data wereevaluated and used to calculate thefollowing method specifications. Calibration function andperformance characteristics,- Gradient (b, measure of sensitivity),- Intercept- Standard deviation of the residuals(s y , scatter of the measured valuesaround the line of regression)- Method standard deviation (s x0 ,absolute measure of the precisionof the calibration)- Method variation coefficient (V x0 ,relative method standard deviation,relative measure of the precision ofthe calibration) Verification of the linearity and Determination of the limits ofdetection and quantification inaccordance with DIN 32645.In addition, the homogeneity of thevariances over the measuring range wasexamined.The performance characteristics werecalculated with “Software for thestatistical control of analytical data– SQS” (Version 1.51, Perkin Elmer,Rodgau-Jügesheim).Fig. 1: DR 5000 spectrophotometer withtouchscreen, rotational barcode measurementand automatic error detection for reliableevaluation of all LANGE cuvette testsNo. Parameter LANGE cuvette test Standard Measuring range of DWO limit value PC (%) PC (mg/l)cuvette test1 Aluminium LCK301DIN ISO 10566 0.02–0.5 mg/l 0.2 mg/l 10 0.02(Chromazurol S) (Pyrocatechol violet)2 Ammonium LCK304DIN 38406-50.02–2.5 mg/l 0.5 mg/l 10 0.05(Indophenol blue) (Indophenol blue)3 Chlorine, free LCK310DIN EN ISO 7393-2 0.05–2 mg/l min. 0.1 mg/l, - -(after disinfection) (DPD)(DPD)max. 0.3 mg/l4 Chlorine, total LCK310DIN EN ISO 7393-2 0.05–2 mg/l - - -(DPD)(DPD)5 Iron, trace LCK521DIN 38406 E1-1 0.01–1 mg/l 0.2 mg/l 10 0.02(1,10-Phenanthroline) (1,10-Phenanthroline)6 Manganese, trace LCW532DIN 38406 E20.005–0.5 mg/l 0.05 mg/l 10 0.005(PAN indicator)(Formaldoxim)7 Nitrate LCK339DIN38405 D9-1 1–60 mg/l 50 mg/l 10 5(2,6-Dimethylphenol) (2,6-Dimethylphenol)8 Nitrite, trace LCK541(Diazotisation)DIN EN 26777(Diazotisation)0.005–0.1 mg/l 0.5 mg/l, or 0.1 mg/lat water treatmentplant outlet10 0.019 Ortho-phosphate LCK349(Molybdenum blue)DIN EN ISO 6878(Molybdenum blue)0.15–4.5 mg/l 6.7 mg/l - -Table 1: Methodology, limit values and performance characteristics (PC) of the selected analysis parameters in conformity with the 2001 DrinkingWater Ordinance (DWO)

4DRINKING WATER ANALYSIS_VALIDATIONValidation of cuvette tests: precisionFig. 2: 5 cm rectangular cuvette for the precisedetermination of trace amounts of iron,manganese and nitriteV x0 (%)32,521,510,50LCK301 AluminiumMethod variation coefficients of cuvette testsPrecisionEvaluating the precision ofthe method using the methodvariation coefficientThe method variation coefficient(relative method standard deviation,V x0 ) is used to evaluate the precisionof a calibration. The method variationcoefficient is a measure of the qualityof a test method. Furthermore, itenables analysis methods with differentmeasuring ranges to be compared. The method variationcoefficients of the examinedcuvette tests (Fig. 3) are allbelow 3 %. The methodstherefore exhibit good to verygood precision.Especially noteworthy are the cuvettetests for ammonium, iron, nitrate andphosphate, whose method variationcoefficients are clearly below 1 %,indicating that they are very preciseindeed.2,2 2,21 2,131,84LCK304 Ammonium0,42LCK521 Iron, trace0,701,19LCW532 Manganese,traceLCK339 NitrateLCK541 Nitrite, traceLCK349 ortho-PhosphateLCK310 Chlorine, freeLCK310 Chlorine, totalFig. 3: Method variation coefficients (V x0 ) of the LANGE cuvette tests (basic calibration)0,510,74Analysis precision at the limitvalue (≤10 %)In analytical chemistry, the precision ofa method depends on the concentrationof the analytes. It decreases as thelower limit of the measuring range isapproached. Under the drinking waterlegislation, the results of authorisedmeasurement methods must notdeviate by more than 10 % from thelimit values specified in the legislation.The performance characteristicsspecified for precision in the DrinkingWater Ordinance relate to theconcentration of the parameter at thelimit value. This is intended to ensurethat the analysis method employedis at least suitable for detecting theconcentration of the analyte reliably atthe limit value.The analytical precision of the cuvettetests was calculated with “Software forthe statistical control of analytical data– SQS” (Version 1.51, Perkin Elmer,Rodgau-Jügesheim) and the data ofthe basic validation. All validated cuvettetests for which performancecharacteristics are specified inthe Drinking Water Ordinance(aluminium, ammonium, iron,manganese, nitrite and nitrate)have a high level of analyticalprecision at the limit value. Theyall give results that do not exceedthe maximum deviation of 10 %.In the case of ammonium, aluminium,iron and nitrate, the scatter ofthe results is less than 3 %. TheDrinking Water Ordinance defines noperformance characteristics for theparameters chlorine (free/total) andortho-phosphate.www.hach-lange.com

5Precision under repeatabilityconditionsAnother quality objective of routineanalysis is to ensure the constantprecision and trueness of the analysisresults over a long period of time. Theprecision of the method was examinedand assessed over a period of 2 to3 weeks by carrying out systematicallyrepeated control analyses (ten in total,with double determination) in fortified realdrinking water samples and on differentmeasurement days,. For this purposethe within series standard deviation(double determination per measurementday) and the between series standarddeviation were calculated, as was thetotal standard deviation in accordancewith Funk et al. [5]. Under repeated conditions,all the examined cuvette testsexhibit good to very goodprecision.The analysis results were not found tovary as a function of time.Ruggedness: The influence ofdifferent reagent batches onprecisionRuggedness is a measure of therelative insensitiveness of an analysismethod to changes in the generalanalytical conditions. The ruggednessof the cuvette tests was examinedusing reagents from differentproduction batches. To do this, tripledeterminations of a standard solution ina matrix-free solution (ultrapure water)were carried out under repeatabilityconditions with cuvette tests from twodifferent production batches.The mean values of the two measurementseries (in each case N=3) withdifferent reagent batches differ onlyslightly. For all examined cuvette tests,the relative standard deviation over allmeasured values (N=6), as a measureof scatter, is good to very good. For allmethods, the relative standard deviationis below 3 %. The reagent batch has nodetectable influence on precision.TruenessDetermination of the recoveryratesTrueness is a measure of the deviationof the measured value (or the meanof several measured values) from thecorrect (true) value due to a systematicerror.To test the trueness of the cuvettetests, the analytes were fortified withcertified reference materials to createreal drinking water matrices [1]. Thefortified test solutions were analysedsix times under repeatability conditionsusing the cuvette tests. The truenesswas determined via the recoveryrate after the measured values hadbeen checked for outliers (Grubbsmethod, P=99 %). The recoveryrate corresponds to the mean of themeasured values (N=6) expressed as apercentage of the target concentrationof the fortified analytes.Fig. 4: Cuvette test LCK310 Chlorine (free andtotal)Fig. 5: Cuvette test LCK349 ortho-Phosphate

6DRINKING WATER ANALYSIS_VALIDATIONValidation of cuvette tests: truenessThe recovery rates of the examinedcuvette tests lie between 92.5 and102 %.over the total range of matrices wereachieved with the cuvette tests for ironand ortho-phosphate (96.9–98.6 %).Fig. 6: Ideal for determining low concentrationsof manganese – LCW532LCK/Parameter Recovery rate (%)LCK301 Aluminium 91.8LCK304 Ammonium 98.2LCK310 Chlorine, free 94.9LCK310 Chlorine, total 91.5LCK521 Iron, trace 99.2LCW532 Manganese, trace 98.0LCK339 Nitrate 100.8LCK541 Nitrite, trace 91.2LCK349 ortho-Phosphate 97.6Table 2: Recovery rate of the LANGE cuvettetests in ultrapure water They therefore meet therequirements of the DrinkingWater Ordinance with regardto the required trueness (10 %deviation relative to the limitvalue).Recovery rates in differentmatricesOne of the key quality criteria of a testmethod is its suitability for use with realsamples. Matrix effects and individualprocess steps can express themselvesin increased imprecision and/or asconstant or proportional systematicdeviation of the analysis results fromthe “true” values. Calculation of therecovery function in a special drinkingwater matrix is a suitable way todetermine a matrix effect. As a matrix,real drinking water was fortified withhypochlorite (0.8 mg/l free Cl 2 ), calcium(365 mg/l CaCO 3 = 20°dH), iron(2 mg/l Fe) and manganese (1 mg/l Mn)standard solutions.For each cuvette test, a recoveryfunction was generated over thetotal measuring range using the fourdrinking water matrices.The recovery rates of all examinedcuvette tests were between 90 and100 % and meet the requirementsof the Drinking Water Ordinance of2001 with regard to the performancecharacteristic “trueness”.The aluminium cuvette test, whichgave a recovery rate of only 85.1 %in the manganese matrix, formed anexception. The highest recovery rates The recovery rates for allmethods are good to very good,depending on the matrix.Checking the limit ofquantification (in accordancewith DIN 32645)Annex 5 of the Drinking WaterOrdinance defines performancecharacteristics for the limit of detectionas a percentage of the limit value.The limit of detection is the lowestconcentration of an analyte that canbe determined qualitatively, while thelimit of quantification is the lowestconcentration of an analyte thatcan be determined quantitatively.The limit of quantification is thelowest concentration at which themeasurement satisfies a specifiedprecision requirement.As drinking water analyses involvedetermining the concentration of theanalytes quantitatively, the limit ofquantification rather than the limit ofdetection was used for the purposesof this validation, to assess theperformance of the test method.The limit of quantification wasdetermined in accordance withDIN 32645 (calibration linemethod) by measuring 10 standardcalibration solutions with equidistantconcentrations, starting at the lowestconcentration of the measuring rangeof the HACH LANGE test method, overa period of ten days.www.hach-lange.com

7 The cuvette tests fordetermining aluminium,ammonium, iron (trace),manganese (trace), nitrateand nitrite (trace) satisfy theperformance characteristicsspecification for the limit ofdetection.For the parameters “chlorine” (freeand total) and “ortho-phosphate”,no performance characteristicsare specified in the Drinking WaterOrdinance of 2001. The limits ofquantification of these methodsare in the range achieved using thecorresponding standard photometricmethods.Comparison of the cuvettetests with standard photometricmethodsThis study is based on a comparisonof the performance characteristicsof both methods (cuvette tests v.standard photometric methods).The performance characteristics ofthe photometric test methods weredetermined separately within theframework of the study [1].In addition, for the purpose ofassessing the comparability of analysisresults from real samples, 6-foldanalyses of fortified drinking watermatrix samples were carried out underrepeatability conditions using thecuvette tests and the standardisedphotometric methods. All the examinedstandard photometric methods andall the examined cuvette tests gaverecovery rates in the drinking watermatrix, as a measure of trueness,between 91.0 and 103.8 %. For allthe examined methods, the withinNo. Examined cuvette test Measuring rangeof cuvette test inmg/lseries relative standard deviation underrepeatability conditions, as a measureof precision, was in the range between0.29 and 2.6 %, and can therefore bedescribed as very good to good Thestandard photometric test method fornitrate is an exception, as the methodvariation coefficient of 4.9 % liesclearly below the 3 % limit which all theother values comply. It is noticeablethat the precision of the cuvette testsfor manganese and nitrate is betterthan that of the standard photometricmethod. In general, the limits ofquantification of the cuvettetests are of the same orderof magnitude as those of thestandardised photometricmethods.Limit ofquantification(mg/l)Limit ofdetection(%)1 LCK301 Aluminium 0.02–0.5 0.017 8.5 102 LCK304 Ammonium 0.02–2.5 0.0086 1.7 103 LCK310 Chlorine, free 0.05–2 0.016 16 -4 LCK310 Chlorine, total 0.05–2 0.022 22 -5 LCK521 Iron, trace 0.01–1 0.01 5 106 LCW532 Manganese, trace 0.005–0.5 0.004 8 107 LCK339 Nitrate 1–60 0.44 0.88 108 LCK541 Nitrite, trace 0.005–0.1 0.0063 6.3 109 LCK349 ortho-Phosphate 0.15–4.5 0.045 0.67 -PC of theDWO limit ofdetection (%)Table 3: Limits of detection of the examined LANGE cuvette tests in comparison with the specifiedPCs of the Drinking Water OrdinanceFig. 7: Cuvette test LCK521 Iron, trace

DRINKING WATER ANALYSIS_RESUMEEFinal assessment of the validationIn a validation study by the IWW, ninephotometric LANGE cuvette testswere experimentally validated andtheir performance characteristics wereexamined and assessed against therequirements of the 2001 DrinkingWater Ordinance. The cuvette tests for thedetermination of aluminium,ammonium, iron (trace),manganese (trace), nitrateand nitrite (trace) satisfy therequirements of the 2001Drinking Water Ordinance withregard to the performancecharacteristics trueness,precision and limit of detection.The performance characteristicsare all below 10 %. On the basisof the determined performancecharacteristics, these cuvette tests arebasically suitable for use in the analysisof drinking and raw water.The 2001 Drinking Water Ordinancespecifies no performance characteristicsfor three of the examined HACH LANGEcuvette tests: chlorine (free), chlorine(total) and ortho-phosphate.The performance characteristics for theortho-phosphate cuvette test (trueness,precision and limit of quantification) areall below 10 %. This cuvette test wouldtherefore also comply with the threeperformance characteristics and fulfilthe criteria.In the case of the cuvette tests forchlorine (free) and chlorine (total), theLANGE cuvette testMeasurementprinciple similar tothat of standardTruenessdeviation≤10 %Precisiondeviation≤10 %LCK301 Aluminium No Yes Yes YesLCK304 Ammonium Yes Yes Yes YesLCK521 Iron, trace Yes Yes Yes YesLCW532 Manganese, trace No Yes Yes YesLCK339 Nitrate Yes Yes Yes YesLCK541 Nitrite, trace Yes Yes Yes YesTable 4: Performance characteristics of the LANGE cuvette testsFig. 8: Cuvette test LCK339 Nitrateprecision and limit of quantification areabove 10 %, and the trueness is below10 %.This result shows that the performancecharacteristics are mostly of the orderof magnitude of those for standardphotometric methods. The reasonfor this is the limited stability of thestandard control solutions, which haveto be prepared freshly, and is thereforeindependent of the analysis method.Limit ofquantificationdeviation ≤10 %Literature[1] Prüfung und Bewertung ausgewählterHACH LANGE–Prüfverfahren für dieUntersuchung von Trinkwasser gemäßTrinkwasserverordnung 2001, 2008, IWWRheinisch-Westfälisches Institut für WasserBeratungs- und EntwicklungsgesellschaftmbH, Moritzstraße 26, 45476 Mülheim ander Ruhr[2] Verordnung über die Qualität vonWasser für den menschlichen Gebrauch(Trinkwasserverordnung – TrinkwV 2001)vom 21. Mai 2001; BGBl., Teil I, Nr. 24 vom28.05.2001, S. 959 - 980[3] DIN EN ISO/IEC 17025: 2005-08; AllgemeineAnforderungen an die Kompetenz vonPrüf- und Kalibrierlaboratorien (ISO/IEC17025:2005); Deutsche und EnglischeFassung EN ISO/IEC 17025:2005[4] Liste der Aufbereitungsstoffe undDesinfektionsverfahren gemäß § 11Trinkwasserverordnung 2001, UmweltBundesamt 2004[5] Qualitätssicherung in der AnalytischenChemie, Funk W., Dammann V., DonnevertG, 2005; Wiley-VCH, WeinheimDOC042.52.20035.Apr09HACH LANGE MAROC SARLAUVilla 14 – Rue 2 Casa PlaisanceQuartier Racine ExtensionMA-Casablanca 20000Tél. +212 (0)522 97 95 75Fax +212 (0)522 36 89 34info-maroc@hach-lange.comwww.hach-lange.maTél. +212 (0)522 97 95 75