Author- Pankaj Gothi, COO- Laboratory Division, SWA Environmental Private Limited

What is method validation & why it’s necessary to validate the methods?

Method validation is the process of determining whether a given testing method can consistently deliver reliable, accurate, and reproducible results across multiple trials. This involves evaluating both the repeatability within a laboratory and the reproducibility across different laboratories, ensuring that the method stands up to statistical scrutiny.

As we know, there are always two types of methods whenever you adopt one— the method is either standard or non-standard.

• If the method adopted for testing is standard, then it’s already believed  to be extensively validated by intra-laboratory studies and statistically significant samples, along with measurement uncertainty
• But if the methods are non-standard, developed in-house, or modified from a standard, then the method requires validation.

Method Validation for Pesticides in Gas Chromatograph

First, you need to get the resource requirements according to the method, like: Reference materials, Glassware, Instruments, Reagents, Analytes, Instrument variability etc.

Following are key parameters to check for method validation in Gas Chromatography:

1. Specificity/Selectivity:
   Ability of the method to distinguish the target analyte from other components in the sample. Ensures no interference from other substances.
2. Accuracy & Recovery:
   Measures how close the results are to the true value.Usually expressed as percentage recovery of known added standards, like verifying against reference materials.
3. Precision:
   Precision means consistency of method results when measuring. It includes:

•                                   Repeatability: Consistency of results under similar conditions (same analyst, instrument)
•                                   Reproducibility: Consistency under different conditions (different labs, analysts, instruments)

Calculate Standard Deviation (SD) and Relative Standard Deviation (RSD) from these.

4. Linearity:
   Assesses if the method provides results proportional to analyte concentration within a specific range.
   Determined by plotting a calibration curve and assessing the correlation coefficient (R²).
5. Range:
   Range is derived from linearity — the minimum and maximum concentrations that the method can measure reliably under acceptable criteria.
6. Limit of Detection (LOD):
   People often confuse LOD and MDL — they are different.

•                                 LOD is the smallest concentration that can be detected but not necessarily quantified.
•                          It means you can detect the analyte but cannot reliably quantify it.

The Minimum Detection Limit (MDL) is always lower than LOD. When your results approach LOD, measurement uncertainty increases. If analyte is detected but not quantified, you can only report that it is present.

7. Limit of Quantification (LOQ):
   LOQ is the lowest concentration you can quantitatively measure with acceptable accuracy and precision.
   It is higher than LOD.

Note: LOQ is not a “maximum detection limit” — that term is incorrect technically.

8. Robustness:
   After method development, robustness must be tested to ensure small variations (temperature, pH, humidity) don’t affect results.
9. Ruggedness:
   Validated methods must be rugged — meaning the method works reproducibly under normal lab conditions, across different labs, instruments, and analysts.
10. Measurement Uncertainty:
    Degree of confidence in measurement results, including all errors and uncertainties in the process.
11. Stability:
    Method stability checks whether the analyte or parameter remains stable during storage, preparation, and analysis.

A detailed step by step worksheet for METHOD VALIDATION for pesticide residue analysis by Gas Chromatography is provided her with — necessary for labs aiming to ensure their methods are reliable, reproducible, and compliant with international accreditation standards like ISO/IEC 17025 & NABL.

As we discussed the method validation, with the example of Organochlorines Pesticides in Water, the same can be applied to other parameters in water

Method Validation for  Organochlorine Pesticides (OCPs)
Discipline Matrix		Water/Wastewater			Date of Analysis:
Parameter Name		p, p’- DDT			QM/Verified by:			Urv Patel
Method Reference:		EPA 8081 B: 2007			TM/Supervised by:			Pankaj Gothi
Instrument Details		Gas Chromatography -1110			Analyst:			Rekha Karavadra
1	Sample Preparation			EPA 8081 B: 2007
2	Stock Standard Details			20 ppm
3	Working Range			1.0, 1.5, 3.0, 5.0, 10.0 ppm
4	Calibration Graph Details			Attached
5	Performance Characteristics
	5.1	Linearity R2 Value				0.997
		SD/LOD/LOQ of Linearity	SD (Standard Deviation)			0.188
			LOD (Limit of Detection)			0.619
			LOQ (Limit of Quantification)			1.877
	5.2	Accuracy % /Recovery (RSD%)				4.573
	5.3	Precision and Repeatability (RSD%)				3.408
		SD/LOD/LOQ of Repeatability of lowest Std	SD (Standard Deviation)			0.034
			LOD (Limit of Detection)			0.112
			LOQ (Limit of Quantification)			0.341
Validation						p, p’- DDT
1	Linearity
	Acceptance Criteria:		R2 ≥0.995	R2 Value			0.9974
	Accepted or Rejected:		Accepted
	Amount in ppm	Calculated amount in ppm
	1	0.778
	1.5	1.4617
	3	3.2722
	5	5.0834
	10	9.9045
	Calibration Curve & Regression Analysis	y = 1x – 0
	Slope (m) =	1.0000
	Intercept (c) =	0.0000
III	Limit of Detection & Quantification as per calibration graph
	Sr No	Standard Concentration  in ppm	Observed Concentration in ppm	Predicted Concentration	Residual Concentration (observed-predicted)		Std. Dev. of Residual Concentration
	1	1	0.7780	1.000037124	-0.2220		0.1877
	2	1.5	1.4617	1.500036842	-0.0383
	3	3	3.2722	3.000035996	0.2722
	4	5	5.0834	5.000034868	0.0834
	5	10	9.9045	10.00003205	-0.0955
	SD	√[  ∑X-Xavg.)2 / (n-1) ]		0.1877
	LOD PPM	3.3SD		0.619
	LOQ PPM	10SD		1.877
II	Accuracy: % Recovery
	Sample No.	Concentration in units	Observed Value Against STD	Result (X) in Units	% recovery % R	RSD %	Acceptance Criteria		Accepted or Rejected
	1	1.00	0.952	0.95214	952	4.573	Recovery between  75  – 125 %		Accepted
	2		1.000	0.99977	1000
	3		1.043	1.04344	1043
	Average		X or X avg.	0.998	998
			SD	0.046
III	Precision and Repeatability:
	Acceptance Criteria:		RSD < 20%		Accepted or Rejected:			Accepted
	Sample No.	True Concentration in ppm	Observed Concentration in ppm	Area in (mv*min)	Peak Height in (mv)		RSD %   ( Area)	RSD % (Peak Height)	RSD % (Observed conc.)
	1	1.00	0.987	13.832	122.427		3.41	7.41	3.408
	2	1.00	0.952	13.349	124.479
	3	1.00	1.017	14.257	136.928
	4	1.00	1.043	14.629	146.688
	5	1.00	1.000	14.01675	132.6305
	Average		0.9998	14.017	132.631
	SD		0.0341	0.4776	9.8316
2	Limit of Detection, Quantification of Repeatability of Lowest Standard
	Sr No	Standard Concentration  in ppm	Observed Concentration in ppm	Predicted Concentration	Residual Concentration (observed-predicted)		Std. Dev. of Residual Conc
	1	1.000	0.987	1.000037124	-0.0134		0.0341
	2	1.000	0.952	1.000037124	-0.0479
	3	1.000	1.017	1.000037124	0.0169
	4	1.000	1.043	1.000037124	0.0434
	5	1.000	1.000	1.000037124	-0.0003
	SD		√[  ∑X-Xavg.)2 / (n-1) ]		0.0341
	LOD PPM		3.3SD		0.112
	LOQ PPM		10SD		0.341