A8231 A8232

Standards and Reagents

Standards Reagents

A.8.2.4. Standard and Reagents Solutions Preparation

A.8.2.4.1. Metformin Stock Standard Solution A.8.2.4.1.1. Metformin Stock Standard Solution. A.8.2.4.1.2. Metformin Purity Check Solution.

A.8.2.4.2. Internal Standard Solutions A.8.2.4.2.1. Is Stock Solution. A.8.2.4.2.2. Is Working Solution. A.8.2.4.2.3. Phenformin Purity Check Solution.

A.8.2.4.3. Identification Solution

A.8.2.4.4. Preparation of Standard Calibration Curve Samples

A.8.2.4.5. Preparation of Quality Control Samples

A.8.2.4.5.1. Quality Control Samples For Regular Run. A.8.2.4.5.2. Quality Control Samples for Dilution Integrity.

A.8.2.5. Description of Method

A.8.2.5.1. Sample Preparation A.8.2.5.2. Chromatographic Conditions A.8.2.5.3. Standardization and Calculation

A.8.2.6. Method Development

A.8.2.7. Method Validation

A.8.2.7.1. Specificity/Selectivity

A.8.2.7.2. Linearity

A.8.2.7.3. Accuracy and Precision A.8.2.7.3.1. Intra-Day Accuracy and Precision. A.8.2.7.3.2. Inter-Day Accuracy and Precision.

A.8.2.7.4.1. Absolute Analytical Recovery.

A.8.2.7.4.2. Relative Analytical Recovery.

A.8.2.7.5. Sensitivity

A.8.2.7.6. Stability

A.8.2.7.6.1. Stability in Biological Plasma Samples. A.8.2.7.6.1.1. Short- Term Stability-. A.8.2.7.6.1.2. Freeze and Thaw Stability. A.8.2.7.6.1.3. Long-Term Stability-. A.8.2.7.6.2. Post-Preparative Stability. A.8.2.7.6.2.1. Autosampler Stability-. A.8.2.7.6.2.2. Dry Extract Stability. A.8.2.7.6.3. Stock Solution Stability.

A.8.2.7.7. Dilution Integrity A.8.3. Data and Results

A.8.3.1. Specificity/Selectivity

A.8.3.2. Linearity

A.8.3.3. Accuracy and Precision

A.8.3.3.1. Intra-Day Accuracy and Precision A.8.3.3.2. Inter-Day Accuracy and Precision

A.8.3.4. Recovery

A.8.3.4.1. Absolute Analytical Recovery A.8.3.4.2. Relative Analytical Recovery

A.8.3.5. Sensitivity

A.8.3.6. Stability

A.8.3.6.1. Stability in Biological Plasma Samples

A.8.3.6.1.1. Short-Term Stability. A.8.3.6.1.2. Freeze and Thaw Stability. A.8.3.6.1.3. Long-Term Stability. A.8.3.6.2. Post-Preparative Stability A.8.3.6.3. Stock Solution Stability

A.8.3.7. Dilution Integrity

A.8.4. Conclusions

The developed method of analysis provided a sensitive and specific assay for metformin in human plasma. It was shown that this method is suitable for the analysis of metformin in the biological plasma samples. For the bioequivalence study, the following recommendations were implemented:

■ Clinical samples collection, handling, processing, and running should take into consideration the stability conditions furnished by the stability tests in this validation study.

■ A standard calibration curve including blank sample and standard zero sample should be generated for each analytical run and should be used to determine the sample concentrations in the unknown clinical samples.

■ For each run, QC samples at each of the low, medium, and high concentrations should be included.

■ QC samples should be analyzed together with the unknown clinical samples and should be allocated judiciously taking into consideration the estimated drug level throughout the batch, in order to detect any analytical drift.

■ Clinical plasma samples of volunteers in all periods should be analyzed with their own calibration curve and QC samples as one batch in a single analytical run.

■ No determinations should be done below the LLOQ or above the ULOQ of the standard calibration curve. Alternatively, appropriate dilution should be intended for samples of concentration above the ULOQ (Figs. A1-A7).

Peak: Metformin—ISTD

Peak: Metformin—ISTD

Amount ratio (ng/mL) FIGURE A.1 A representative standard calibration curve.

International pharmaceutical research center

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