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Gladys Chia
Chew Yu Mei
Yvonne Teo
Cornelyus Ng
Lau Leslie
17 September 2008
5:33:00 PM
Topic: Re-establishment of Accuracy, Precision and Linearity
Hello :) I am back in school, continuing Alex's Project on Stability Testing of Metformin. This week I have been testing the method he has come up with for its accuracy, precision and linearity. Metformin standard solutions are injected into the High Performance Liquid Chromatography (HPLC) machine, to test for the stability of metformin at different concentrations.
Metformin Standard is used for calibration purposes at different concentrations; 50ppm, 100ppm, 150ppm, 200ppm and 250ppm. 3 replicates of each concentration is carried out starting from the lowest concentration. As for Accuracy, 3 replicates of 50ppm, 100ppm and 150ppm will be tested. Precision required 6 replicates of 100ppm. In all, it is a total of 30 injections. Linearity is achieved from the calibration plot.
Method 1. Weigh out 0.0125g of Metformin Standard in a weighing boat. 2. Mix it well with HPLC water and pour it into the 25ml volumetric flask, top up with HPLC water. 3. Use 1000ul pipette to pipette out amounts of Metformin Stock solution respectively into 10ml volumetric flasks; 50ppm-1ml, 100ppm-2ml, 150ppm-3ml, 200ppm-4ml, 250ppm-5ml. Top up the volumetric flasks with HPLC water.
20ul of the solutions will be injected each time and it runs for 5 mins before the chromatogram is printed out. Between intervals after the calibration and accuracy, the HPLC system has to be flushed by injecting DI water so as to remove the injected solution in the column.
Results
1. Calibration The average means of each concentration (50-250ppm) is calculated and a calibration plot is plotted using Windows Excel.
2. Accuracy The average means of each concentration (50-150ppm) is calculated and the 3 values of measured concentration of metformin is extrapolated from the calibration plot. Using the following formula to achieve accuracy results, which must be 100%±2%. ** % Recovery= Measured Concentration/True Concentration x 100% ** (eg: of True Concentration is 50ppm, Measured Concentration from the calibration plot is 48.59ppm)
3. Precision 6 determinations of 100pm is measured and the average mean is calculated. Standard Deviation (SD) of the 6 results is calculated as well and determined using the following formula. The result has to be less than 2%, in order to say that it is precise. **% Relative Standard Deviation= SD/Mean x 100%**
4. Linearity R2 has to be more than 0.99 to conclude that the calibration plot is linear.
The results have to be accurate, precise and linear in order to conclude that this is a well developed method.