1. LECTURE 13 QUALITY ASSURANCE METHOD VALIDATION

2. QUALITY ASSURANCE
A system of activities whose purpose is to provide to the producer or the user of a product or service the assurance that it meets the needs of the user.

3. Quality Assurance vs. Quality Control
An overall
management plan to
guarantee the
integrity of data
(The “system”)
A series of
analytical
measurements used
to assess the
quality of the
analytical data
(The “tools”)

4. QUALITY CONTROL CHARTS
- Visual representation of actual manufacturing data with respect to the target levels.

5. QUALITY CONTROL MEASURES
Standards and Calibration
Blanks
Recovery Studies
Precision and Accuracy Studies
Method Detection Limits

6. STANDARDS AND CALIBRATION
Prepared vs. Purchased Standard
Signals: Peak Area, Beer’s Law
Calibration Curves
Continuing Calibration Checks
Internal Standards
Performance Testing.

7. The concentration of the analyte
CALIBRATION CURVES
Graphical representation of the relationship between:
The concentration of the analyte
and
The analytical signal

9. CONTINUING CALIBRATION VERIFICATION
Many methods don’t require that daily calibration curves are prepared
A “calibration verification” is
analyzed with each batch of samples

10. SAMPLE BATCH 10 - 20 samples (method defined) or less Same matrix
Same sample prep and analysis
Contains a full set of
QC samples

11. INTERNAL STANDARDS
A compound chemically similar to the analyte
Not expected to be present in the sample
Cannot interfere in the analysis
Added to the calibration standards and to the samples in identical amounts.

12. INTERNAL STANDARDS Refines the calibration process
Analytical signals for calibration standards are compared to those for internal standards
Eliminates differences in random and systematic errors between samples and standards

13. Blind samples submitted to laboratories
PERFORMANCE TESTING
Blind samples submitted to laboratories ? ? ?
Labs must periodically analyze with acceptable results in order to maintain accreditation

14. BLANKS, BLANKS, BLANKS
Laboratory Reagent Blanks
Instrument Blanks
Field Reagent Blanks
Trip Blanks

15. LABORATORY REAGENT BLANKS
Contains every reagent used in the analysis
Is subjected to all analytical procedures
Must give signal below detection limit
Most methods require one with every batch

16. INSTRUMENT BLANK
A clean sample (e.g., distilled water) processed through the instrumental steps of the measurement process; used to determine instrument contamination.

17. Prepared in the lab, taken to the field
Field Reagent Blanks
Prepared in the lab, taken to the field
Opened at the sampling site, exposed to sampling equipment, returned to the lab.

18. RECOVERY STUDIES
Matrix Spikes
Laboratory Control Samples
Surrogates .

19. MATRIX SPIKES Sample spiked with a known amount of analyte
Subjected to all sample prep and analytical procedures
Determines the effect of the matrix on analyte recovery
Normally one per batch

20. LABORATORY CONTROL SAMPLE
Analyte spiked into reagent water
Subjected to all sample prep and
analytical procedures

21. LABORATORY CONTROL SAMPLE
Also known as:
Laboratory Fortified Blank (LFB)
Quality Control Sample (QCS)

22. SURROGATES Similar to an internal standard
Added to all analytical samples, and to all QC samples to monitor method performance, usually during sample prep
Methods often have specific surrogate recovery criteria
Most common in Organic methods

23. QUALITY CONTROL MEASURES
Standards and Calibration
Blanks
Recovery Studies
Precision and Accuracy Studies
Method Detection Limits

24. PRECISION AND ACCURACY
Required for initial certification and annually thereafter
A series of four laboratory control samples
Must meet accuracy (recovery) and precision (standard deviation) requirements, often in method

25. PRECISION AND ACCURACY
Required with a change in instrumentation or personnel
Specific to the analyst

26. METHOD DETECTION LIMIT
“The minimum concentration of a substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero”

27. METHOD DETECTION LIMIT
MDLs are determined according to ules from regulating body
Seven replicate laboratory control samples, analyzed for precision
Multiply standard deviation by 3.14 (Student’s t- value)

28. METHOD DETECTION LIMIT
Must be performed initially for certification
Must meet criteria specified in method
Must be performed with change in instrumentation or test method

29. QUALITY ASSESSMENTS
The overall system of activities whose purpose is to provide assurance that the overall control job is being done effectively. Two types: - Evaluation of the precision and accuracy of methods of measurements - Evaluation of the quality of manufactured goods sold for public consumption. (control charts)

30. SPECIFICATIONS
State how good the numbers should be and what precautions are required in the analytical procedure. Quality Asssurance begins with sampling. False Positive - False Negative -

31. METHOD VALIDATION
Confimation by examination

32. WHEN SHOULD METHODS BE VALIDATED?
A new method developed for a particular problem
Established methods revised for a particular problem
Established methods have changed with time
Established method used in different laboratory with different analysts at different times.
Establish the equivalence between two methods.

33. METHOD VALIDATION USE OBJECTIVES
States the pupose for which the results will be used.
Example:
Analytical data and results will be used to determine whether the new antibacterial ingredient will be effective in removing 99.9% of the gram negative bacteria.

34. SPECIFICATIONS
will detail on how the numbers need to be and what precautions are required in the analytical procedures.
Specifications might include:
Sampling requirements calibration checks
Accuracy and precision quality control samples
Rate of false results
Selectivity
Sensitivity
Acceptable blank values
Recovery of fortification

35. METHOD VALIDATION
FOR REGULATORY SUBMISSION, THE FOLLOWING ARE REQUIRED: Method Specificity / Confirmation of identity Linearity Accuracy Precision Range Limit of detection, Limit of quantification Rubostness

36. CONFIRMATION OF IDENTITY
It is necessary to establish that the signal being produced at the measurement stage, which has been attributed to the analyte, is only due to the analyte and not from the presence of something chemically or physically similar.

37. SPECIFICITY
Specificity is the ability of an analytical method to distinguish the analyte from everything else that might be in the sample.

38. LINEARITY
LINEARITY measure how well a calibration curve follows a straight line. A common measure of linearity is R2, square of the correlation coefficient. Good linear fit when R2 = and above Prepare independently at leas 6 samples + blank and measure linearity.

39. SAMPLE PROBLEM:
An AAS method to analyze Pb+2 in water has just been developed and is in the process of being validated. The validation protocol states the following criteria to be met (in addition to proof of identity and setting dynamic range): Determine whether the new method is validated. Linearity: R2 > Accuracy: % Recovery = /- 5% Precision: Intermediate Precision, STD < 0.5% Intralab Precision, STD < 2.0% The following validation experiments were done: Linearity Test. Blank samples were spiked with several known amounts of Pb+2. The following data were collected.

40. Linearity Test:
ppb Pb+2
AAS Signal
0.02
0.2066
0.05
0.5582
0.1
1.0802
0.15
1.6967
0.25
2.7521
0.4
4.3822

41. RECOVERY TEST. Blank samples were spiked with 0. 10 and 0
RECOVERY TEST. Blank samples were spiked with 0.10 and 0.15 ppb of Pb+2 and analyzed using the method. The following data were collected.
Spiked sample
0.10 ppb
0.15 ppb
AAS Signal
1.0902
1.7002
1.0806
1.7232
1.0781
1.6996

42. Precision. An analyst in the laboratory as well as two ther analysts from different laboratories analyzed the same blank sample spiked with ppm Pb+2 with the following results.
RUNS
Analyst A B C 1
0.122
0.120
0.132
0.124
0.119 2
0.128
0.126 3
0.129
0.127

43. ACCURACY
ACCURACY is “nearness to the truth”. Analyze blank , sample and a certified reference material (CRM) and compare the means. Do a recovery test. Analyze a blank sample spiked with 0.5, 1.0, 1.5X level of your analyte. And analyze for % Recovery.

44. PRECISION
PRECISION is how well replicate measurements agree with one another, usually express as standard deviation. Instrument precision – reproducibility observed when same quantity of a sample is analyzed using a particular instrument repeatedly (>10 times) Intra-assay precision – analyzing aliquots of a homogeneous material several times by one analyst on one day using same equipment. Inter-lab precision (reproducibility) aliquots of the same sample are analyzed by different analyst in different labs using different (?) instrument

45. RANGE
RANGE is the concentration interval over which linearity, accuracy, and precision are all acceptable. Correlation coefficient of R2 equal or greater than Interlab precision of +/-3% Spike recovery of 100 +/-2%

46. LIMITS OF DETECTION AND QUANTITATION
The detection limit is the smallest quantity of analyte that is “significantly different” from the blank. LoD: Measure blank samples at least 7 times and get the mean. Do similar analyses with 7 samples with analyte and calculate the standard deviation. LoD = blank mean + 3S LoQ = 6x to 10x the SD of blank sample LoQ = 2x to 3x of LoD

47. RUGGEDNESS / ROBUSTNESS
Robustness is the ability of an analytical method to be an affected by small deliberate changes in operating parameters.

48. STANDARD ADDITION METHOD
INTERNAL STANDARD METHOD