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Graduation Project

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Hala Kh

on 6 March 2013

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Transcript of Graduation Project

German Jordanian University
School of Applied Medical Sciences
Chemical-Pharmaceutical Engineering Department Outline
Revalidation for the process used for the characterization and quantification of AMT (4- Aminomethyltrioxsalen-Hydrochloride) using HPLC Introduction Introduction
Objectives
Experimental
Methodology
Representative graphs
Conclusion
Future work and recommendation Roche diagnostics, Penzberg, Germany Life cycle of an analytical method The validation method of an analytical process aims to obtain experimentally justified evidence that this process is able to give results characterized by the required accuracy and precision. AMT: 4'-AMINOMETHYLTRIOXSALEN HYDROCHLORIDE


C15H16ClNO3
Molecular weight= 293.75 gram/mole
light-sensitive
Stored at temperature between 2-8 °C Importance of the analysis of AMT AMT reacts with nucleic acids under UV radiation to inhabit the function of some nucleic acids in samples

Oligonucleotides also react with AMT. The resulting product may contain only a specific amount of AMT that is sit by the producer. Previous work Figure 1: Calibration curve of AMT Standards using self-prepared stock solution Table 1: Calibration details of AMT standards using self-prepared stock solution Figure 2: Chromatogram of (H2O) using self-prepared stock solution Figure 3: Zoomed chromatogram of an AMT sample using self-prepared stock solution Table 2: Calculated amount of an AMT sample using self-prepared stock solution Objectives Writing a validation plan a new test-prescription according to Roche's quality specifications Main objective: In order to achieve this objective further experiments where done to:

1.Choose the appropriate stock solution
2.Test the effect of changing the gradient on the peak’s shape
3.Test each validation parameter and specify it’s range Experimental 1.Reverse Phase Chromatography
2.HPLC setup
3.Material used Reverse Phase Chromatography Hydrophobic stationary phase (non-polar)

Separation based on polarity

Stainless steal column with pores to increase the surface area available for binding HPLC setup Elution flow rate = 0.8 ml/min
Pressure = 160 Bar
Column temperature = Room temperature
Injection volume = 10 µMicroliters
Sample temperature = 4 °C, protected from light
Detector: UV detector at 260 nm Material used 1 M Triethylammoniumacetate produced by Calbiochem
1 M Triethylammoniumacetate produced by Fluka
Triethylamine
Acetic acid
Distilled water
Acitonitrile
AMT calibrator powder sample
AMT liquid sample Methodology 1.Linearity
2.Accuracy
3.Specificity
4.Suitability range
5.Precision (repeatability, intermediate precision)
Choosing the appropriate stock solution(s) Eluents prepared by the Calbiochem stock solution Figure 4: Overlay between AMT sample and H2O sample using Calbiochem stock solution and two columns Eluents prepared by the Fluka stock solution Figure 5: Chromatogram of (H2O) using Fluka stock solution Figure 6: Zoomed chromatogram of the resulting signal for AMT standard with d.f 1:25 using Fluka stock solution Eluents prepared by the self-prepared stock solution Figure 7: Chromatogram of (H2O) using self-prepared stock solution Figure 8: Zoom chromatogram of a resulting signal of AMT sample using self-prepared stock solution Figure 12: Calibration curve of AMT standards using self-prepared stock solution after forcing the curve from zero Table 10: Calibration details of AMT standards using self-prepared stock solution and column P592 after forcing the curve from zero Table 9: Calculated amount of AMT sample using self-prepared stock solution without forcing the curve from zero Testing the accuracy Figure 13: An example for a resulting signal of AMT sample using the stainless steel capillary (recovery=100%) Figure 14: Chromatogram shows the resulting signal for the same AMT sample using Table 12: Recovery results using column P593 and a capillary column Effect of changing the gradient on the peak’s shape Table 3: Gradient program for analyzing AMT samples Table 4: Gradient program for analyzing AMT samples after omitting the 55 B% Figure 9: Overlay between AMT sample and (H2O) sample after omitting step 55 B% from the gradient program Table 5: Short gradient program for analyzing AMT samples with omitting 55 B% Figure 10: Overlay between AMT sample and (H2O) sample after shortening the gradient program System suitability test (SST) Table 13: Retention time precision of column P593 Repeatability (intra assay) and intermediate precision (inter assay) Table 14: Standard deviation and RSD for the results of Fluka stock solution and self-prepared stock solution Conclusion

The mobile phase prepared from the Fluka stock solution and the self-prepared stock solution have given a very stable and under-peak-free signals and also a specified and accurate separation of the AMT peak

A five-point calibration curve instead of a three-point calibration curve has shown better results for the quantity of an AMT sample

Neither changing the gradient program nor shortening it has given a better peak's shape Ranges of the specificity, accuracy and precision are specified

Recovery is a measure of accuracy and it is around 96%

The system suitability test represented by the retention time precision has a CV less than or equal to≤ 0.5 for the four columns. Future work and recommendation Any incoming AMT sample will now be analyzed by colleagues using this revalidated process to continue proving its accuracy

The method can also be applied using a core shell column. This column is supposed to give the same signals but in a shorter time with a relatively low back pressure

The procedure of the characterization and quantification of AMT can be transferred to an UHPLC (RPC) Testing the range for the linearity of the calibration curve Table 6: Previous calibration curve dilutions Table 7: Calibration curve dilutions used for revalidation Figure11: Calibration curve of AMT standards using self-prepared stock solution Table 8: Calibration details of AMT standards using self-prepared stock solution Testing the specificity Figure 15: Spectra of an AMT sample tested for specificity Questions Table 11: Calculated amount of AMT sample using self-prepared stock solution and column P592 after forcing the curve from zero Aya Fahed Al-Bitar
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