Measuring degradation Flashcards
Why is there is a need to measure degradation of drug products?
- requirement in international guidelines for marketing pharmaceutical products e.g. ICH, FDA
- evaluates likely degradation products and pathways
- by separately stressing API, formulation, formulation minus API (placebo) we can discover the origin of degradation products of the finished product apart from manufacturing process or raw material impurities
- helps with dosage form design and storage
Typical forced conditions
-Acid hydrolysis 0.1M HCl
-Base hydrolysis 0.1M NaOH
-Temperature 50-60°C
-Humidity 70-80%
-Oxidation (3-30% peroxide)
-Light irridation
Aim to produce 10-20% degradation
What is SIM?
Stability indicating method
What does a good SIM do?
Ideally detect both API + degradation products simultaneously (but this is not always possible)
What is an ideal candidate for SIM and used most widely?
Chromatography
What is the most widely used chromatographic technique in stress testing studies?
HPLC
High pressure liquid chromatography
What are the method requirements for API stress testing?
- Resolve API from degradation products
- Sensitive enough to detect small levels
- Quantify with high degree of accuracy and precision
Describe HPLC instrumentation
Solvents→solvent mixing valve→pump→injection valve→column→detector
in HPLC, what conditions is the pump under?
set a flow rate of 1mg/min to deliver mobile phase
Creates a back pressure typically ~1000 psi (maximum 5000 psi)
Dimensions of pump in HPLC
Typically
<25cm x 0.5cm (or lower)
Describe how sample injection in HPLC works
Loading Loop
-in the load position, mobile phase flows from the HPLC pump to the column without entering the sample loop. Therefore, you can load the sample, from the syringe into the loop without it going to the column. Any excess sample flows out as waste
Injecting sample
Rotate to inject position allows the mobile phase to flow in from the HPLC pump, through the sample loop and out to the column. Thus, the sample enters the column.
Describe the peaks coming out in reverse phase HPLC
polar peak comes out first
hydrophobic impurities come out later
because C8 or C18 akyl chains in system
Describe the peaks coming out in normal HPLC
Hydrophobic impurities come out first
polar peak comes out later
therefore column must have polar properties
In RP HPLC what is the mobile phase like?
Polar
usually water mixed with an organic solvent such as methanol, aceteonitrile or isopropanol
analytes are eluted with most polar ones first
Retention time increased by using more polar solvent or decreased by using more organic modifier
alternatively, retention time can be increased by using less polar stationary phase, e.g. C18 instead of C4
in RP HPLC, how to make peaks further apart?
Change ratio of methanol (organic modifier) and water e.g. 90% methanol 10% water - quite together 50% of each - further apart 80% water 20% methanol - furthest apart
How to elute analyte?
When using a gradient the composition changes with time usually starting with most polar (e.g. Water). Organic solvent (organic modifier) is then mixed in increasing amounts as time increases. When the composition reaches a certain critical value the analyte is eluted.
How to speed up analysis and aid peak separation?
Gradient use
How to know how well separated/resolved peaks are?
Resolution
Area under peak is proportional to
amount of stuff
RT=
retention time of an un-retained component
W=
width at base of peak
width at half height
Neff=
a measure of column efficiency
it reflects the number of plates thus the number of times analyte can partition between mobile and stationary phases
accuracy and presion purposes
higher resolution
gradient
what does steeper gradient mean?
solvent A→solvent B over time steeper gradient (more rapid change in solvent composition) will elute analytes faster
Isocratic
single composition of solvent
same constant composition
to resolve peaks - change composition to do this
Note isocratic gradient is not actually a gradient
How to do quantative analysis of HPLC results?
-measure peak heights
-measure peak areas (preferred)
Both require: calibration and standards
Quantative analysis of HPLC results
2 types
-External standard method
Concentration calculated from ratio of sample peak areas to the corresponding standard peak areas
-Internal standard method
More precise method. Less chance of error from reproducibility and sample preparation concerns
Also often used in pharmacokinetics when trace levels of drugs or metabolites are present in a biological matrix
Describe external calibration method
The area under the peak is proportional to the amount of X which has passed the detector, and this area can be calculated automatically by the computer linked to the display.
a sample with a peak area of 200,000 has twice the amount of that with a peak area of 100,000 for the analyte.
A standard curve can be obtained from known concentrations of standards
equation of straight line y=mx+c
internal standard method
inject your sample and standard at the same time. Can’t be same as will give one peak. Need to be apart but not too far away
Internal standard :related to analyte but with distinct retention time that is close to analyte. More reliable method as external factors (temperature) this affects both samples
Analysis
Add standard to analysis mixture. Relative areas are compared and used to calculate conc. Preferably sample preparation should be same for both sample and standard. Although in some assays the standard can be added after extraction.
Response factors are used to calculate unknown concentration by comparing sample solution with a calibration solution. The internal calibrant concentration is kept the same in both solutions.
try the calculations and look over them - add to display book
done? - 5
not done - 1
how are the degradation measured?
- measured in same way as API using the external or internal calibrants
- placebo samples are important to distinguish background peaks
- amounts are small so better detectors and thin bore capillary tubes can be used e.g. LC-MS (liquid chromatography - mass spectrometry)
take home problems done?
yes=5
put in display book
