Measuring degradation

Question 1

Why is there is a need to measure degradation of drug products?

Answer 1

• 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

Question 2

Typical forced conditions

Answer 2

-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

Question 3

What is SIM?

Answer 3

Stability indicating method

Question 4

What does a good SIM do?

Answer 4

Ideally detect both API + degradation products simultaneously (but this is not always possible)

Question 5

What is an ideal candidate for SIM and used most widely?

Answer 5

Chromatography

Question 6

What is the most widely used chromatographic technique in stress testing studies?

Answer 6

HPLC

High pressure liquid chromatography

Question 7

What are the method requirements for API stress testing?

Answer 7

• Resolve API from degradation products
• Sensitive enough to detect small levels
• Quantify with high degree of accuracy and precision

Question 8

Describe HPLC instrumentation

Answer 8

Solvents→solvent mixing valve→pump→injection valve→column→detector

Question 9

in HPLC, what conditions is the pump under?

Answer 9

set a flow rate of 1mg/min to deliver mobile phase

Creates a back pressure typically ~1000 psi (maximum 5000 psi)

Question 10

Dimensions of pump in HPLC

Answer 10

Typically

<25cm x 0.5cm (or lower)

Question 11

Describe how sample injection in HPLC works

Answer 11

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.

Question 12

Describe the peaks coming out in reverse phase HPLC

Answer 12

polar peak comes out first
hydrophobic impurities come out later

because C8 or C18 akyl chains in system

Question 13

Describe the peaks coming out in normal HPLC

Answer 13

Hydrophobic impurities come out first
polar peak comes out later
therefore column must have polar properties

Question 14

In RP HPLC what is the mobile phase like?

Answer 14

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

Question 15

in RP HPLC, how to make peaks further apart?

Answer 15

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

Question 16

How to elute analyte?

Answer 16

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.

Question 17

How to speed up analysis and aid peak separation?

Answer 17

Gradient use

Question 18

How to know how well separated/resolved peaks are?

Answer 18

Resolution

Question 19

Area under peak is proportional to

Answer 19

amount of stuff

Question 20

RT=

Answer 20

retention time of an un-retained component

Question 21

W=

Answer 21

width at base of peak

width at half height

Question 22

Neff=

Answer 22

a measure of column efficiency

it reflects the number of plates thus the number of times analyte can partition between mobile and stationary phases

Question 23

accuracy and presion purposes

Answer 23

higher resolution

Question 24

gradient

what does steeper gradient mean?

Answer 24

solvent A→solvent B over time
steeper gradient (more rapid change in solvent composition) will elute analytes faster

Question 25

Isocratic

Answer 25

single composition of solvent
same constant composition
to resolve peaks - change composition to do this

Note isocratic gradient is not actually a gradient

Question 26

How to do quantative analysis of HPLC results?

Answer 26

-measure peak heights
-measure peak areas (preferred)
Both require: calibration and standards

Question 27

Quantative analysis of HPLC results

2 types

Answer 27

-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

Question 28

Describe external calibration method

Answer 28

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

Question 29

internal standard method

Answer 29

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.

Question 30

try the calculations and look over them - add to display book

Answer 30

done? - 5

not done - 1

Question 31

how are the degradation measured?

Answer 31

• 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)

Question 32

take home problems done?

Answer 32

yes=5

put in display book