DEVELOPMENT AND VALIDATION OF A CHROMATOGRAPHIC METHOD

1. REFERENCE STANDARDS:

• 1.a. Established source and known grade (DMF - Drug Master File

or COA - Certificate of Analysis)
• 1.b. Percentage of purity from assay will be taken into account in the calculations.
• 1.c. Amount of residual compounds (GC, heavy metals, inorganic salts, water,

residual solvents, weight loss) should be known.

2. CHOICE OF STANDARDIZATION: EXTERNAL OR INTERNAL

• 2.a. Simple formations and sample preparation: external standard
• 2.b. Gas chromatography, bio-studies or complex medium and sample

preparation: internal standard.

3. SELECTION AND DEVELOPMENT OF THE CHROMATOGRAPHIC METHOD:

• 3.a. The chromatographic method from the literature (Pharmacopoeia) must be the first

choice, with freedom of coloumn choice (according to the Lx tables in the USP).
Peak shape should be examined in this step already (see 4.a.).
• 3.b. The method should be adjusted to be stability indicating and selective, if this is

impossible, a new method should be developed and validated to be stability indicating.
• 3.c. Potential impurities and degradation products should be used with the sample medium.
• 3.d. Peak purity of the active substance is checked (by phodo-diode array detector to verify

that the method is selective, and a single component peak is quantified.
• 3.e. The analysis time range should be kept practical, no more than 40 min, i.e., the retention

parameter should be kept k(first) >= 2 and k(last) <= 40 (flow-rate, column length and solvent
composition can be adjusted to speed up the separation).
• 3.f. Final method is defined by the following parameters:
• Column type: geometry and brand
• Mobile phase
• Flow rate and pressure
• Temperature
• Sample volume
• Detection
• Sample preparation(ID)

4. COLUMN AND INSTRUMENT PERFORMANCE:

SYSTEM SUITABILITY TEST

The following parameters must be checked, preferably during method development:
• 4.a. Number of theoretical plates, N>=2000. [16(tR/W) ^2]
• 4.b. Tailing factor 0.5 <= T =< 2.

If these requirements cannot be fulfilled, the column must be replaced. This test is done
during the method development (3.a.).
• 4.c. Precision RSD <= 1-2% in 10 injections of the standard. If RSD > 1%, the

autosampler should be checked, or a leak might be detected in the instrument.
• 4.d. Resolution, Rs >= 2 between adjacent peaks; [(tR2-tR1)/0.5(w1+w2)]
Note:
This step should be measured as part of adjustment of the method to be stability indicating and selective.
Sections 1 and 2 verify that the chromatographic system and column follow the requirements
of the analysis. The next steps aim to establish the method of analysis.
 

1. METHOD VALIDATION

• 5.a. Linearity: the range of linearity is checked by injections of 5-6 concentrations of the

reference standards (in triplicates) below and above the expected concentration of the
samples (20%-120%). The slope and intercept are determined. r >= 0.999, and the
range of linearity (in conc. terms) is established.
• 5.b. Accuracy is measured as the (amount measured)/(amount claimed) in the following

procedures:
• In drug formulations: a standard is added to the placebo (80%, 100%, 120%), and the

recovery is measured
• Recovery from sample preparation procedures (such as dilution, extraction or

filtration) should be established.
• Solution stability of the samples (A fresh sample is compared to one which was left in

solution for the entire duration of the test procedure).
• 5.c. Intermediate precision or ruggedness: 5 measurements of the first step of accuracy under two

different occasions (two columns, or two operators).
• 5.d. Limit of detection (LOD) and limit of quantification (LOQ):
• The intercept from the linearity test (in section 5.a) can be used for LOQ

measurements (intercept x 5 or 10).
• Analysis of repeatability at the quantification limit, using both sample and reference

standard. Concentrations that yield 10-15% RSD will be acceptable as LOQ concentrations.
• 5.e. Robustness of the chromatographic method: variables in this test can be the

following: column batch #, solvent strength in the mobile phase, temperature, flow rate,
pH of the mobile phase, ionic strength in the mobile phase, sample diluent,
injection volume, wavelength of detection. The parameter measured: response,
retention time, selectivity and/or resolution.

Robustness of the sample preparation procedure: variables in this test can be the
following: duration of extraction, extraction medium, filtration type, temperatures.
Parameter measured: accuracy.
 
 
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