LECTURE 13 QUALITY ASSURANCEMETHOD VALIDATION

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.

Quality Assurance vs. Quality Control

Quality Assurance

An overallmanagement plan to guarantee theintegrity of data(The “system”)

Quality Control

A series of analytical measurements usedto assess thequality of the analytical data(The “tools”)

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

QUALITY CONTROL MEASURES

Standards and Calibration Blanks Recovery Studies Precision and Accuracy Studies Method Detection Limits

STANDARDS AND CALIBRATION

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

CALIBRATION CURVES

Graphical representation of the relationship between:

• The analytical signal

• The concentration of the analyte

and

Calibration Curve for DDT

y = 9.3005x + 4.3313

0

100

200

300

400

500

0 10 20 30 40 50 60

Parts per trillion DDT

Peak

are

a x

106

R2 = 0.9989

CONTINUING CALIBRATION VERIFICATION

Many methods don’t require that daily calibration curves are prepared

• A “calibration verification” is analyzed with each batch of samples

SAMPLE BATCH

10 - 20 samples (method defined) or less Same matrix Same sample prep and analysis

• Contains a full set of QC samples

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.

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

PERFORMANCE TESTING

Blind samples submitted to laboratories

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?Labs must periodically analyze with acceptable results in order to maintain accreditation

BLANKS, BLANKS, BLANKS

Laboratory Reagent Blanks

Instrument Blanks

Field Reagent Blanks

Trip Blanks

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

INSTRUMENT BLANK

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

Field Reagent Blanks Prepared in the lab, taken to the field

• Opened at the sampling site, exposed to sampling equipment, returned to the lab.

RECOVERY STUDIES

Matrix Spikes

Laboratory Control Samples

Surrogates .

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

LABORATORY CONTROL SAMPLE

• Subjected to all sample prep and analytical procedures

• Analyte spiked into reagent water

LABORATORY CONTROL SAMPLE

Also known as:

Laboratory Fortified Blank (LFB) Quality Control Sample (QCS)

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

QUALITY CONTROL MEASURES

Standards and Calibration Blanks Recovery Studies Precision and Accuracy Studies Method Detection Limits

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

PRECISION AND ACCURACY

Required with a change in instrumentation or personnel

Specific to the analyst

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”

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)

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

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)

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 -

METHOD VALIDATION

Confimation by examination

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.

METHOD VALIDATIONUSE 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.

SPECIFICATIONSwill 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

METHOD VALIDATIONFOR REGULATORY SUBMISSION, THE FOLLOWING ARE REQUIRED:Method Specificity / Confirmation of identityLinearityAccuracyPrecisionRangeLimit of detection,Limit of quantification Rubostness

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.

SPECIFICITY

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

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 = 0.995 and above

Prepare independently at leas 6 samples + blank and measure linearity.

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 > 0.995Accuracy: % Recovery = 100.0 +/- 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.

ppb Pb+2 AAS Signal

0.02 0.20660.05 0.55820.1 1.08020.15 1.69670.25 2.75210.4 4.3822

Linearity Test:

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 ppbAAS Signal 1.0902 1.7002

1.0806 1.7232

1.0781 1.6996

Precision. An analyst in the laboratory as well as two ther analysts from different laboratories analyzed the same blank sample spiked with 0.125 ppm Pb+2 with the following results.

RUNS Analyst A B C1 0.122 0.120 0.132 0.124 0.119

2 0.128 0.120 0.119 0.126 0.126

3 0.128 0.129 0.127 0.120 0.122

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.

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

RANGE

RANGE is the concentration interval over which linearity, accuracy, and precision are all acceptable.

Correlation coefficient of R2 equal or greater than 0.995Interlab precision of +/-3%Spike recovery of 100 +/-2%

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 sampleLoQ = 2x to 3x of LoD

RUGGEDNESS / ROBUSTNESS

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

STANDARD ADDITION METHOD INTERNAL STANDARD METHOD