industrial technology institute test method validation verification h.p.p.s.somasiri principal...
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Industrial Technology Institute 3 Basic Concepts in Method Validation Specificity Analyte Stability Analytical Range Calibration Range Recovery (Accuracy) Precision Limit of Detection (LOD) Limit of Quantification (LOQ) Measurement uncertaintyTRANSCRIPT
Industrial Technology Institute
Test Method Validation & Verification
H.P.P.S.SomasiriPrincipal Research Scientist / SDD-QAD /QM
Industrial Technology Institute
SLAB2015/11/10, BMICH
VER 6
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Introduction
An analytical method is the series of procedures from receipt of a sample to the production of the final report.
Validation is the process of verifying that a method is fit for purpose.
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Basic Concepts in Method ValidationSpecificityAnalyte StabilityAnalytical RangeCalibration RangeRecovery (Accuracy)PrecisionLimit of Detection (LOD)Limit of Quantification (LOQ)Measurement uncertainty
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Specificity
Shows that the detected signal is due to the analyte, not another compounds/ s.
Use matrix blanks, reagent blanks for conformation of specificity
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Example: Confirmation of Identity
Standard Non Polar Column
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Time (minutes)
Resp
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Sample Non Polar Column
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Time (minutes)
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Standard Polar Column
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Sample Polar Column
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Analyte StabilityAnalyte stability during sample storage, analyte
stability in sample processing, analyte stability in sample extract should be studied.
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Analytical RangeRange of the analyte that can be analysed with the
proposed selected method.
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Calibration Range If a linear calibration is used, the range in which the
response for the anayte shows a linear correlation with the analyte’s concentration should be determined.
This is known as linear dynamic range.
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Linear Dynamic RangeColour Vs time
y = 0.7518x2 + 0.9696x + 9.2679R2 = 0.9891
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0 1 2 3 4 5 6
Time
Col
our
Coconut oil
Palm Oil
Expon. (Coconut oil)
Poly. (Palm Oil)
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Recovery% Recover of the anlalyte on extraction, clean-up,
derivatization and measurement.Can be done with
- Certified Reference Material (CRM,s)- Standard Reference materials (SRM,s)- Interlaboratory comparisons- Spiking (@ 20 %, 50 % and 80 %)
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Accuracy (Trueness)
How close the result to the expected (assigned) value
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Example: AccuracySample Blank value
(mg/L)RM value (mg/L)
1 0.001 1.462 0.018 1.803 0.001 1.174 0.018 1.205 0.002 1.226 0.002 0.747 0.002 1.168 0.014 1.239 0.001 1.2010 0.002 1.29Mean value 0.004 1.25
RM assigned value: 1.22 ± 0.08 mg/L; Laboratory value for RM: 1.246 mg/L
Mean laboratory Value fromRM value certificate1.246 mg/L 1.22 mg/L
Bias
value
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Accuracy versus Precision
Accuracy (Trueness)
PrecisionAccuracy &
Precision
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Precision Is measured as “repeatability “ or reproducibility
Repeatability Measurement is repeated with a minimum of variations (same person, same laboratory, same equipment, over a short period of time.ReproducibilityMeasurement is repeated with as much variation as possible. (Different analyst, different laboratory, different equipment, over a long period of time).
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Limit of Detection (LOD) Point where the variability of the measurement is such that it is
impossible to distinguish between random fluctuation in the value measured and actual signal.
– LOD = YB + 3XSB
• YB = Mean value measured from the blank
• SB = Standard Deviation of the value measured from the blank
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Replicate (n) Instrument response
Calculated Concentration
(ppb)1 29550 0.1002 28653 0.0973 29401 0.0994 28532 0.0975 29712 0.1016 29983 0.1017 28167 0.095
Mean 0.099StdDev 0.0023 Times StdDev 0.007Detection Limit 0.106
TBT in Biota
Limit of Detection
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Limit of Quantification (LOQ) Point where the measured value could be quantified reliably
LOQ = YB + 10XSB
• YB = Mean value measured from the blank
• SB = Standard Deviation of the value measured from the blank
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Example:TBT in BiotaReplicate (n) Instrument
responseCalculated
Concentration (ppb)
1 29550 0.1002 28653 0.0973 29401 0.0994 28532 0.0975 29712 0.1016 29983 0.1017 28167 0.095
Mean 0.099StdDev 0.0023 Times StdDev 0.007Detection Limit 0.106
Limit of Quantitation
Blk + 5s 0.110 Blk + 6s 0.113Blk + 10s 0.122
Limit of Quantification (LOQ)
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Sensitivity- Cadmium Analysis by AAS
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0.00 0.20 0.40 0.60 0.80 1.00 1.20
Cd Concentration (mg/L)
Abs
orba
nce
slope = 0.241 ± 0.011
intercept = 0.009 ± 0.003
r-square = 0.99442 XX
xx
xx
Sensitivity
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Measurement Uncertainty
Uncertainty estimates should take account of:Overall long-term precisionBias and its uncertainty, including the
statistical uncertainty involved in the bias measurements, and the reference material or method uncertainty
Calibration uncertaintiesAny significant effects operating in addition to
those listed above
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Test Method Verification
Any standard method shall be verified before being used in the laboratory
Verify the RecoveryAccuracyPrecision (Repeatability and Reproducibility)LOD and LOQ
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