This document compares the requirements for quantitative chemical single laboratory validation (SLV) from several guidelines, including AOAC, FDA-CVM, EU, harmonized, ISO/IEC, IUPAC, and EURACHEM. It finds that the 1999 harmonized guidelines developed by CODEX, ISO, AOAC, FAO, IAEA and IUPAC provide the most complete and relevant requirements. The key requirements across most guidelines are accuracy and precision testing over multiple days with 3-6 replicates each of blank, low, middle, and high spike levels. Method ruggedness and intermediate precision should also be evaluated.
This document compares the requirements for quantitative chemical single laboratory validation (SLV) from several guidelines, including AOAC, FDA-CVM, EU, harmonized, ISO/IEC, IUPAC, and EURACHEM. It finds that the 1999 harmonized guidelines developed by CODEX, ISO, AOAC, FAO, IAEA and IUPAC provide the most complete and relevant requirements. The key requirements across most guidelines are accuracy and precision testing over multiple days with 3-6 replicates each of blank, low, middle, and high spike levels. Method ruggedness and intermediate precision should also be evaluated.
This document compares the requirements for quantitative chemical single laboratory validation (SLV) from several guidelines, including AOAC, FDA-CVM, EU, harmonized, ISO/IEC, IUPAC, and EURACHEM. It finds that the 1999 harmonized guidelines developed by CODEX, ISO, AOAC, FAO, IAEA and IUPAC provide the most complete and relevant requirements. The key requirements across most guidelines are accuracy and precision testing over multiple days with 3-6 replicates each of blank, low, middle, and high spike levels. Method ruggedness and intermediate precision should also be evaluated.
This document compares the requirements for quantitative chemical single laboratory validation (SLV) from several guidelines, including AOAC, FDA-CVM, EU, harmonized, ISO/IEC, IUPAC, and EURACHEM. It finds that the 1999 harmonized guidelines developed by CODEX, ISO, AOAC, FAO, IAEA and IUPAC provide the most complete and relevant requirements. The key requirements across most guidelines are accuracy and precision testing over multiple days with 3-6 replicates each of blank, low, middle, and high spike levels. Method ruggedness and intermediate precision should also be evaluated.
COMPARISION of QUANTITATIVE CHEMICAL SINGLE LABORATORY VALIDATION (SLV) REQUIREMENTS
By Jo Marie Cook, Florida Department of Agriculture and Consumer Services
August 2009
Overview: I reviewed several different validation guidelines and summarized the requirements of each for validation of quantitative, multiresidue type chemical methods. I noted the minimum requirements for each. Even more replicates are necessary for validation of qualitative methods. The "Guidelines for SLV of Analytical Methods for Trace-Level Concentrations of Organic Chemicals",was the most complete and relevant guide. It was developed through a harmonization project between CODEX, ISO, AOAC, FAO, IAEA and IUPAC in 1999. I believe it remains the most important guide for trace level, multi-residue work. All the other guides are more general in nature. This guide also discusses extension of a method or performance verification by another laboratory. The only significant difference between this guide and others is that most guides recommend calibration for the range of concentrations expected. The CODEX and EU guides concentrated on ranges within 2 times the regulatory limit. Most guides mention the need for SLV's and the decline of collaborative studies due to time and cost. Despite frequent reference to "Fit for Purpose", the need for SLV's and the number of replicates is not significantly diminished for multiresidue methods. If anything, more detailed SLV's are important due to the scarcity of collaboratively studied methods. Some performance parameters needed to be measured only once such as selectivity, ruggedness and stability. Verification of method performance in other laboratories required validation parameters of accuracy and precision, similar to those in collaborative studies. However, most of the guides stressed the need to measure realistic repeatability (multiple days and sample types) and reproducibility (multiple analysts, instruments, reagents, etc). None of the guides mentioned interlab (3 - 5 lab) validations, presumably because this additional validation information is not statistically significant until 7 or more laboratories are involved. For most guides, the number of replicates needed for accuracy and repeatability was three blank matrices plus three fortified levels, 5-6 replicates, repeated on three different days. The AOAC generic protocol was less stringent than any of the other guides. Because certified reference materials are seldom available, trueness or accuracy must be determined by spiking multiple varieties of blank samples. Inefficiency of extraction can be the major source of bias. This makes it more important to evaluate method response with standard additions, comparison to alternate methods, proficiencies, evaluate ruggedness, stability etc. It is important to determine if the method will extract the analytes of interest in incurred samples in the same manner as demonstrated by fortified blank matrix recovery studies. Most guides mentioned using Youden pairs to determine ruggedness. Variables which contribute significant imprecision should be corrected by additional method development.
References: AOAC: AOAC Guidelines for Single Laboratory Validation of Chemical Method for Dietary Supplements and Botanicals, http://www.aoac.org/dietsupp6/Dietary-Supplement-website/DSHomePage2.html, 2002-12-19 Single Laboratory Validation, Generic Protocol (Chemistry) - Draft 8, AOAC, A. Pohland, [email protected]
COMPARISION of QUANTITATIVE CHEMICAL SINGLE LABORATORY VALIDATION (SLV) REQUIREMENTS
By Jo Marie Cook, Florida Department of Agriculture and Consumer Services August 2009 Single Laboratory Validation Acceptance Criteria (Chemistry Methods) - Draft 8, AOAC, A. Pohland, [email protected] Protocols for AOAC Method Validation Programs - Rev 2, 3-17-2008, AOAC-OMB, G. Latimer, [email protected] FDA-CVM:
FDA Guideline 3 V: Guideline for Approval of a Method of Analysis for Residues, FDA, http://www.fda.gov/cvm/Guidance/1731.htm FDA Guideline 118, Mass Spectrometry for Confirmation of the Identity of Animal Drug Residues, http://www.fda.gov/cvm/Guidance/guide118.pdf Second Analyst Validation of Study 419.01 - Multiclass Determination and Confirmation of Antibiotic Residues in Honey using LC-MS_MS, Mayda Lopez, FDA CVM EU: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, Official Journal of the European Communities, HARMONIZED SLV: (AOAC, Food and Agriculture Organization of the United Nations, International Atomic Energy Agency, International Union of Pure and Applied Chemistry) Guidelines for Single-Laboratory Validation of Analytical Methods for Trace-Level concentrations of Organic Chemicals, AOAC/FAO/IAEA/IUPAC, 1999 http://www.iaea.org/trc/pest-qa_val2.htm ISO / IEC: (International Organization for Standardization & International Electrotechnical Commission) Guidelines for checking and validating test and calibration methods according to ISO/IEC 17025, A011, 1-9-2008, Ver. 4 IUPAC (International Union of Pure and Applied Chemistry: Harmonized Guidelines for Single-Laboratory Validation of Methods of Analysis, IUPAC Report, Thompson, Ellison, Wood, 11-4-1999 EURACHEM: The Fitness for Purpose Analytical Methods, A Laboratory Guide to Method Validation and Relate Topics, EURACHEM Working Group, David Holcombe, 1998, ISBN: 0-948926-120 GENERAL READING: Principles and Practices of Method Validation, edited by A Fajgelj and A. Ambrus, ISBN 0-85404-783-2, Royal Society of Chemistry 2000, www.rsc.org
COMPARISION of QUANTITATIVE CHEMICAL SINGLE LABORATORY VALIDATION (SLV) REQUIREMENTS
By Jo Marie Cook, Florida Department of Agriculture and Consumer Services August 2009 Performance Parameters
AOAC
FDA-CVM Guides 3.4, 145-chemistry, 118
EU
Harmonized Multiresidue SLV Guide
ISO/IEC 17025 A011
DEVELOPMENT and OPTIMIZATION - to be conducted for new methods and may/or may not be included in SLV's by additional laboratories Fit for Purpose Fit for Purpose Purpose Test "System" Selectivity or Specificity
Range of Blank matrices
3-4 MS fragments
Retention time GC/MS=2%, LC/MS=5% 3-4 MS fragments Full scan 20% match SIM = 10% match MS x MS = 20% match
Range of related compounds
(metabolites, derivatives,,,)
Blank spiked w/ interference
False Positive False Negative Quant. Enhancement or suppression Calibration Curves
required
EUROCHEM Guide
Fit for Purpose
Method, conc. Range, matrix, use Selectivity Index, matrix interference
Fit for Purpose
IP Points
20
5 representative commodities 5
Bias due to matrix suspected
to be significant
< 5%
zero < 10%
Zero + 3 levels times 2 10
LCL, AL, AL*2 Non-linear = 7 levels times 3 Matrix vs standards in solvent 16 Zero, LCL, AL times 5 2-3 * AL times 3
6 levels 0 - 150%, evenly distributed Run in duplicate or triplicate in random order Test matrix effect w/ std. add. Plot residuals Estimate, only a rough guide 3 times SD of matrix blank
3 times SD or average of blank
Detection Capability (LOQ)
10 time SD or average of blank
Y Y Y Y 5 levels including zero
At least 3 levels
3 times signal to noise
Calibration curve method
5 20 blanks, 3 times signal to noise Decision limit plus 1.64 times the SD of the reproducibility
1, 2, 3, 4 weeks or more Store at -20C or less 0, 1, 2, 3, 4 weeks (-20C)
Youden pairs
Youden pairs
Slope of calibration curve
Spike interferences Other methods
Zero + 6 levels Range of sample levels Linear level of instrument and method
Decision Limit (LD or LOD)
Sensitivity
Stability (light, temperature)
Analytes in standard solution Incurred residue or matrix fortified Optimization (re-extraction, sample preparation, alternate solvents, shake time/type, pH, detectors, cleanup, instrument, columns) Ruggedness (minor changes)
IUPAC
Youden pairs
16 Zero, LCL, AL times 5 2-3 * AL times 3
Required
Required
10% RSD or 2 times LOD Not recommended. State the uncertainty at concentration
Zero + 6 levels
10 blanks or low level spikes
Terms not generally accepted. "minimum detectable value" Blank + 3SD of lowest concentration 10 blanks or low level spikes Blanks + 10 SD May not be lower than the lowest quantitative measure.
>5 each Analyte/commodity & standard solutions
Youden pairs
COMPARISION of QUANTITATIVE CHEMICAL SINGLE LABORATORY VALIDATION (SLV) REQUIREMENTS
By Jo Marie Cook, Florida Department of Agriculture and Consumer Services August 2009 Comparison to established method VALIDATION - basic measurements common to all levels of validation. Some SLV's may require some or all of the tests above. 2 - 3 or more as needed 5 During development - 20 Representative Sample Types to represent the range of composition Trueness (accuracy or bias) CVM Certified Ref. Material or see Recovery 3 levels (upper, lower, 5 levels including zero Calibration Curves middle) (Dietary Supl = 0, 5-7 points) Recovery 2 9 - 10 20 18 range of expected 0.5, 1, 2 LOQ times 5 1, 1.5, 2.0 LOD times 6 3,4 Analyst performing validation concentrations 5 incurred or 0.5, 1, 1.5 Permitted Level should not be aware of 0 * 3, 15%, 30%, 130% times 6 concentration / composition times 3 times 3 days If possible Incurred residues At least 10 <20% 18 Repeatability (% CV) At least 2 different Perform the required recovery Recovery on 3 different days sample types experiments over 3 days (Dietary Supl = 3 Blinded samples matrices) Times 2, At least 2 days 15- 20 18 Within-Lab Reproducibility or Each condition, Recovery on 3 different days Intermediate Precision (%CV) concentrations that differ (different reagents, analysts, by order of magnitude rooms, instruments, days) times 5 Measurement Uncertainty 6
Ruggedness (major changes)
Required
10
Required Determine range
Zero, LCL, AL times 5
2-3 * AL times 3
Required
? 2 levels, min and max
18 Blanks + 3 levels times 6
Required
Duplicates times several days,
range of concentration, different matrix Horwitz is incorrect < 120 ppb
10
Representative analytes and matrices
Zero, LCL, AL times 5
2-3 * AL times 3 Representative analytes and matrices
Required
Inter lab study
Notes: 1. Experiments may be combined to determine different parameters 2. Multiple analytes can be determined at the same time as long as they do not interfere 3. LOD = minimum required quantitative performance limit of the method 4. Recovery is measured by method of standard additions (100 (conc. 2 -conc 1)/concentration added to #2) 5.Concentration at y intercept plus 2.33 times the SD or the reproducibility of the intercept or 3 times signal to noise 6. Bias, as measured by recovery, is not a component of uncertainty. Bias (a constant) should be removed by subtraction before calculating MU standard deviations (AOAC Dietary Supplement Guidelines for SLV) 7. See FDA Guidance for Industry #118 8. AOAC general guidelines required fewer replicates and levels as recommended by the dietary supplement guide, which was more consistent with other guides. 9. The range of concentrations recommended for recovery and repeatability was narrow. Other guidelines recommended measurements across broader ranges. 10. per the CODEX guidelines: AL = Action Level or Acceptable Level (regulatory limit) and LCL = lowest calibration level
Analytical Purity Method Development and Validation by Gas Chromatography of L-Valine Methyl Ester Hydrochloride For Production of Anti-Hypertensive Drugs
Analytical Purity Method Development and Validation by Gas Chromatography of L-Valine Methyl Ester Hydrochloride For Production of Anti-Hypertensive Drugs
