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.