Test procedure
The specificity of the assay method will be investigated by injecting of the extracted placebo to demonstrate the absence of interference with the elution of the analyte.
Documentation
Print chromatograms.
Acceptance criteria
The excipient compounds must not interfere with the analysis of the targeted analyte.
Linearity
Test procedure
Standard solutions will be prepared at six concentrations, typically 25, 50, 75, 100, 150, and 200% of the target concentration. Three individually prepared replicates at each concentration will be analyzed. The method of standard preparation and the number of injections will be the same as used in the final procedure.
Documentation
Record results on a data sheet. Calculate the mean, standard deviation, and Relative Standard Deviation (RSD) for each concentration. Plot concentration (xaxis) versus mean response (yaxis) for each concentration. Calculate the regression equation and coefficient of determination (r2). Record these calculations on the datasheet.
Acceptance criteria
The correlation coefficient for six concentration levels will be ≥ 0.999 for the range of 80 to 120% of the target concentration. The yintercept must be ≤ 2% of the target concentration response. A plot of response factor versus concentration must show all values within 2.5% of the target level response factor, for concentrations between 80 and 120% of the target concentration.
Concentration
(mg/ml)

Concentration
as % of Analyte Target

Peak Area (mean
of three Injections)

Peak Area
RSD (%)

5

25



10

50



15

75



20

100



30

150



40

200



Equation for regression
line =

Correlation coefficient
(r^{2}) =

Range
Test procedure
The data obtained during the linearity and accuracy studies will be used to assess the range of the method.
The precision data used for this assessment is the precision of the three replicate samples analyzed at each level in the accuracy studies.
Documentation
Record the range on the datasheet.
Acceptance criteria
The acceptable range will be defined as the concentration interval over which linearity and accuracy are obtained per the above criteria, and in addition, that yields a precision of ≤ 3% RSD.
Accuracy
Test procedure
Spiked samples will be prepared at three concentrations over the range of 50 to 150% of the target concentration.
Three individually prepared replicates at each concentration will be analyzed. When it is impossible or difficult to prepare known placebos, use a low concentration of a known standard.
Documentation
For each sample, report the theoretical value, assay value, and percent recovery. Calculate the mean, standard deviation, RSD, and percent recovery for all samples.
Record results on the datasheet.
Acceptance criteria
The mean recovery will be within 90 to 110% of the theoretical value for nonregulated products. For the U.S. pharmaceutical industry, 100 ± 2% is typical for an assay of an active ingredient in a drug product over the range of 80 to 120% of the target concentration.
Lower percent recoveries may be acceptable based on the needs of the methods.
Sample

Percent of
Nominal (mean of three injections)

Amount of Standard
(mg)

Recovery (%)

Spiked

Found

1

75




2

100




3

150




Mean





SD





RSD%





Precision
Repeatability
Test procedure
One sample solution containing the target level of the analyte will be prepared. Ten replicates will be made from this sample solution according to the final method procedure.
Record the retention time, peak area, and peak height on the datasheet. Calculate the mean, standard deviation, and RSD.
Acceptance criteria
The typical RSD should be 1% for drug substances and drug products and ± 2% for bulk drugs and finished products.
Injection No.

Retention Time
(min)

Peak Area

Peak Height

Replicate 1




Replicate 2




Replicate 3




Replicate 4




Replicate 5




Replicate 6




Replicate 7




Replicate 8




Replicate 9




Replicate 10




Mean




SD




RSD%




Intermediate Precision
Test procedure
Intermediate precision (withinlaboratory variation) will be demonstrated by two analysts, using two HPLC systems on different days and evaluating the relative percent purity data across the two HPLC systems at three
concentration levels (50%, 100%, 150%) that cover the analyte assay method range 80 to 120%.
Documentation
Record the relative % purity (% area) of each concentration on the datasheet.
Calculate the operators' and instruments' mean, standard deviation, and RSD.
Acceptance criteria
The assay results obtained by two operators using two instruments on different days should have a statistical RSD ≤ 2%.

Relative %
Purity (% area)

Instrument
1

Instrument
2

Sample

S1
(50%)

S2 (100%)

S3
(150%)

S1
(50%)

S2
(100%)

S3
(150%)

Operator 1,
Day 1







Operator 1,
Day 2







Operator 2,
Day 1







Operator 2,
Day 2







Mean (Instrument)







Mean (Operators)







RSD%

S1 + S1

S2 + S2

S3 + S3




Instruments



Operators



Limit of Detection
Test procedure
The lowest concentration of the standard solution will be determined by sequentially diluting the sample. Six replicates will be made from this sample solution.
Documentation
Print the chromatogram and record the lowest detectable concentration and RSD on the datasheet.
Acceptance criteria
The ICH references a signaltonoise ratio of 3:1. Some analysts calculate the standard deviation of the signal (or response) of a number of blank samples and then multiply this number by two to estimate the signal at the limit of detection.
Record sample data results:
(e.g., concentration, S/N ratio, RSD%)

Limit of Quantitation
Test procedure
Establish the lowest concentration at which an analyte in the sample matrix can be determined with the accuracy and precision required for the method in question. This value may be the lowest concentration in the standard curve. Make six replicates from this solution.
Documentation
Print the chromatogram and record the lowest quantified concentration and RSD on the datasheet. Provide data that demonstrate the accuracy and precision required in the acceptance criteria.
Acceptance criteria
The limit of quantitation for chromatographic methods has been described as the concentration that gives a signaltonoise ratio (a peak with height at least ten times as high as the baseline noise level) of 10:1.
Record sample data results: (e.g., concentration, S/N ratio, RSD%)

System Suitability
Test procedure
System suitability tests will be performed on both HPLC systems to determine the accuracy and precision of the system by injecting six injections of a solution containing analyte at 100% of test concentration. The following parameters will be determined: plate count, tailing factors, resolution, and reproducibility (% RSD of retention time, peak area, and height for six injections).
Documentation
Print the chromatogram and record the data on the datasheet
Acceptance criteria
Retention factor (k): the peak of interest should be well resolved from other peaks and the void volume; generally k should be ≥ 2.0.
Resolution (Rs): Rs should be ≥ 2 between the peak of interest and the closest eluted peak, which is potentially interfering (impurity, excipient, and degradation product).
Reproducibility: RSD for peak area, height, and retention time will be 1% for six injections.
Tailing factor (T): T should be 2.
Theoretical plates (N): ≥ 2000.
System Suitability
Parameter

Acceptance
Criteria

Results

Criteria Met/
Not Met

HPLC 1

HPLC 2

Injection
precision for retention time (min)

RSD ≤ 1%




Injection
precision for
peak
area (n = 6)

RSD ≤ 1%




Injection
precision for
peak
height

RSD ≤ 1%




Resolution
(Rs)

Rs = ≥ 2.0




USP
tailing factor (T)

T = ≤ 2.0




Capacity
factor (k)

K = ≥ 2.0




Theoretical
plates (N)

N = ≥ 2000




Robustness
Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters.
Robustness provides some indication of the reliability of an analytical method during normal usage. Parameters, which will be investigated, are the percent organic content in the mobile phase or gradient ramp, pH of the mobile phase, buffer concentration, temperature, and injection volume. These parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment.
The chromatography obtained for a sample containing representative impurities, when using a modified parameter(s), will be compared to the chromatography obtained using the target parameters. The effects of the following changes in chromatographic conditions will be determined:
methanol content in the mobile phase adjusted by ±2%, mobile phase pH adjusted by ± 0.1 pH units, column temperature adjusted by ± 5°C. If these changes are within the limits that produce acceptable chromatography, they will be incorporated into the method procedure.
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