Item 2. Verification and Validation of Analytical Methods
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This document discusses the validation and verification of analytical test methods used at the Environmental Analysis Laboratory at Southern Cross University. It provides details on key validation parameters including accuracy, precision, selectivity, linearity, matrix effects, sensitivity, spike recoveries, trueness, range, ruggedness, and measurement uncertainty. Validation data is presented for methods analyzing metals and salts in freshwater, effluent, and wastewater samples by ICP-MS. The methods were verified to meet performance criteria in these sample matrices.
Item 2. Verification and Validation of Analytical Methods
- 1. Verification and Validation of Methods Mr Graham Lancaster Manager/ Director Environmental Analysis Laboratory Southern Cross University, Lismore, NSW
- 2. • Verification is ensuring performance on unchanged standard procedures. • Validation is ensuring the same performance where modifications to the standard or alternative method have been implemented. • Only verified/ validated test methods should be approved for use in the laboratory. Verification/ Validation of methods
- 3. • Test methods selected by the laboratory should be those that have been published internationally or nationally, and are deemed acceptable to the relevant technical bodies, whenever such suitable methods are available (ie. Standard Methods). • Non-standard methods should be based on published methods, and modified only to improve laboratory practices (e.g. safe sample digestion) or efficacy of the method. Selection of methods
- 4. Regional reference for method verification
- 5. • Published and standard test methods need to be verified to ensure that the documented performance can be met. • Validation and verification of test methods should follow ‘NATA General Accreditation Guidance – Validation and verification of quantitative and qualitative test methods’. • The accuracy of the method should be determined using suitable certified reference materials. Validation/ Verification of methods
- 6. • Method verification studies are typically less extensive than those required for method validation. • If Methods have been previously validated via collaborative studies (ie. ISO, Aus Standards, or published in Green Book) then a reduced verification is typically required. • Validation is always a balance between costs, risks and technical possibilities. Validation/ Verification of methods
- 7. • The extent of validation required will depend on the status of the method under consideration and the needs relating to its intended application. • Verification under conditions of use is demonstrated by meeting system suitability specifications based on below: • blanks, or un-inoculated media (e.g. in microbiology), to assess contamination; • laboratory control samples (e.g. spiked samples for chemistry or positive culture controls for microbiology) to assess accuracy; HOW- Validation/ Verification
- 8. • duplicates to assess precision; • calibration check standards analysed periodically in the analytical batch for quantitative analyses; • monitoring quality control samples, usually through the use of control charts; and • Interlaboratory participation (ASPAC) in a performance testing program provided that the tested material is representative of the method in terms of matrix, analytical parameters, concentration level(s), etc. HOW- Continued…
- 9. Digest Method Kjeldahl Comparison Phosphorus (%w/w) 80 81 82 83 84 85 ASPAC Median of 40 labs 0.297 0.308 0.142 0.170 0.193 0.449 Kjeldahl 0.299 0.308 0.142 0.171 0.192 0.434
- 10. Digest Method Validation Trial Potassium % acid dry ASPAC digest ash Median ASPAC 104-07 1.04 1.04 0.98 ASPAC 11-08 0.71 0.68 0.68 ASPAC 12-08 2.45 2.33 2.43 ASPAC 13-08 1.05 1.02 1.03 ASPAC 14-08 1.89 1.73 1.76
- 11. Digest Method Validation Trial Calcium % acid dry Kjeldahl ASPAC digest ash ASPAC 104-07 0.26 0.24 0.25 0.25 ASPAC 11-08 0.84 0.83 0.77 0.83 ASPAC 12-08 1.61 1.63 1.62 1.65 ASPAC 13-08 0.21 0.14 0.14 0.15 ASPAC 14-08 1.43 1.38 1.45 1.37
- 12. Validation of Metals and Salts Analysis in Freshwater, Effluent and Wastewater by ICPMS Environmental Analysis Laboratory Southern Cross University, Lismore, NSW Validation Example
- 13. • Perkin Elmer 350D ICPMS with Flexar HPLC (speciation)
- 14. • Perkin Elmer Avio 500 with ESI FAST sampler
- 15. Steps of Method Validation Sensitivity Accuracy Precision Trueness Limit of Detection & Quantitation Range Selectivity Linearity Matrix Effects Ruggedness Measurement Uncertainty
- 16. The following validation data applies to the determination of 13 metals and 6 salts by ICPMS for the following waters: • Fresh River Water • Wastewater • Effluent The specific elements are: • Metals - Silver, Arsenic, Lead, Cadmium, Chromium, Copper, Manganese, Nickel, Selenium, Zinc, Mercury, Iron and Aluminium • Salts - Calcium, Magnesium, Potassium, Sodium, Sulfur and Phosphorus Validation specific details…
- 17. • Selectivity refers to the ability of a method to discriminate a particular analyte in a complex mixture without interference from other components. The selectivity for ICPMS analysis is based on the choice of isotope. The isotopes chosen are derived from Standard Methods for the Examination of Water & Wastewater, 23rd Ed, 2017 (Table 3125:I) and are the same isotopes as those recommended by the manufacturer. Three types of interferences are common to ICPMS. These are: Physical interferences are generally related to viscosity effects and aerosol transport from the nebulizer. Matrix interferences can be defined on how the matrix affects the energy of the plasma and what happens when the sample enters the vacuum system (space charge effect). Spectral interferences can be catergorised into isobaric (elemental) and polyatomic (molecular). 1. Selectivity
- 18. • The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. Linearity is determined by the software after calibration and is checked by comparing the instrument response (y as counts) with the concentration (x as mg/L) of the calibration standard for which the linear model y = Ax + b is used. • Routine metals analysis is achieved with linearity using 0.01, 0.1 and 1mg/L calibration standards. • Salts (Ca, Mg, K, Na) calibration standards are 20 and 200mg/L, while S and P are 10 and 100mgL. • Tables 2-7 Appendix 1 details the instrument response and calibration coefficient for each calibration standard, and the results of blanks and standards when analysed as samples during the validation work. 2. Linearity
- 19. Table 2- Counts and Linearity data 1/2/2011
- 20. • Matrix effects are often caused by the alteration of ionization efficiency of target analytes in the presence of co-eluting compounds in the same matrix. Matrix effects may be identified by the use of standard additions and internal standards. These effects can be corrected for by use of internal standards. • The methods routinely used by EAL utilize Scandium, Rhodium and Terbium as internal standards which have been prepared from 1000ppm stock solution. • Table 8 lists the concentration of each internal standard and the elements they are linked to. • An internal standard response of 75-125% is often acceptable. • If the response is outside this range samples are to be diluted by a factor of five and re-analysed. 3. Matrix Effects
- 21. Table 8: Internal standards and concentrations routinely utilized by EAL Internal Standard Elements Concentration Scandium Ca Mg K Na P Al Cr Fe Mn Ni Cu Zn 500ppb Rhodium As Se Ag Cd 50ppb Terbium Hg Pb 20ppb
- 22. • Sensitivity is often defined as the lowest analyte concentration that can be measured with acceptable accuracy and precision. Instrument Detection Limit (IDL) – is the constituent that produces a signal greater than three standard deviations of the mean noise level. It can be determined from seven replicate analysis of a blank sample. Method Detection Level (MDL) or Level of Detection (LOD) – the constituent concentration that can be measured and reported with 99% confidence that the analyte concentration is greater than zero. It can be estimated by analyzing seven blank samples which have been spiked with the analyte concentration within 2-5 times the IDL. The standard deviation of the analyte is multiplied by three to give the MDL. Practical Quantitation Limit (PQL) or Limit of Reporting (LOR) – is determined by multiplying the MDL by 5 and rounding to the nearest decimal unit. Table 9 Appendix 1 contains IDL, MDL and PQL obtained after following the above procedure. 4. Sensitivity
- 23. Table 9: Detection Limits
- 24. • Accuracy is the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value, and the value found. • Accuracy was determined by analysis of National Measurement Institute (Proficiency Study AQA 09-18 Samples 1 and 2), Certified Reference Materials (CRM’s) CWW-TM-A and CWW-TM-B and WaterChek Round 109 and 110 proficiency testing samples, the results of which were compared to the known or certified value. 5. Accuracy and Precision
- 25. • Precision of a method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. • The metals CRM’s and NMI samples were analysed over three (3) different days (1/2/2011, 21/2/2011 and 7/3/2011) with each sample matrix analysed on seven (7) consecutive occasions. Tables 10 – 33 Appendix 1 contain the accuracy and precision data for the matrices analysed. 5. Accuracy and Precision
- 26. Accuracy and Precision Table 10: AQA 09-18 S1 - Freshwater 1/2/2011
- 27. • Spike recovery criteria is typically (at least) 50% for many validation guidelines however obviously 100% is ideal. • Each matrix (except Minerals 1 and 2) was spiked with a multi-element metals standard at three different concentrations over the working range of the calibration. The spike concentration for routine metals was 0.005, 0.200 and 0.800mg/L. Spiked recovery data is provided in Tables 34 – 56. 6. Spike Recoveries
- 28. Spike Recoveries Table 34: Spike recoveries AQA 09-18 S1 Freshwater – 1/2/2011
- 29. • Trueness refers to 'the closeness of agreement between the average value obtained from a large series of test results and an accepted value • Subjective validation is a cognitive bias by which people will consider a statement or another piece of information to be correct if it has any personal meaning or significance to them. • Trueness/Bias can be calculated by summing the difference between the analytical result and certified result for each analyte. • An estimate of the average bias can be obtained by comparing test results, generated in different runs over several days, with the known value. 7. Trueness/Bias
- 30. See sensitivity and refer to Table 9. 8. Limit of Detection and Limit of Quantitation
- 31. • Range as an interval from the upper to the lower concentration of the analyte in the sample. • See linearity and Tables 2-7. 9. Range
- 32. • Ruggedness is a measure of reproducibility of test results under the variation in conditions normally expected from laboratory to laboratory & from analyst to analyst. • Ruggedness can be determined from assessment of CRM results over a period of time where the analyses are performed by different operators under different environmental conditions and where instrument performance maybe affected by the condition of the torch and sample introduction system. • Table 65 contains results for seven (7) groundwater samples analysed by EAL and mgt Labmark Melbourne (NATA Accreditation number 1261). Results for these samples compare exceptionally well. 10. Ruggedness
- 33. Table 65: Comparison data for samples analysed by mgt Labmark and EAL – November 2010
- 34. • Measurement uncertainty is a property of measurement result, not of the method, equipment or laboratory and therefore it is to be expected that it is assessed only once the result is obtained. • Measurement Uncertainty (MU) has been calculated using EAL’s Quality procedure QWORK 17.1 ‘Estimating Uncertainty of Measurement’. • This method has been adapted from EURACHEM/CITAC Guide, 2000 and provides an estimate of the combined uncertainty of measurement. 11. Measurement Uncertainty
- 35. Thanks for your attention • Reference documents