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'Certificate of Analysis' on Active Pharmaceutical Ingredien
Transcript of 'Certificate of Analysis' on Active Pharmaceutical Ingredien
Certificate of Analysis
' on Active Pharmaceutical Ingredient: Deltamethrin
University of Sunderland
Injection: 20 uL
Reversed Phase HPLC assay
Reversed Phase Related substances test
Reversed Phase HPLC assay
Reversed Phase Related Substances test
Deltamethrin + Dry Methanol
Karl Fischer Device
Syringe + Sample
Agilent Technology 1200 series 1290 Infinity
Model No ADP410
Preparation of Sample
20 m x 0.18 mm x 1.0 μm
Gas Chromatography Instrument
1% w/v Solution of Deltamethrin
Angle of Rotation
Sample - 0.10
Reference - 0.20
[ ] = 1000
l x C
Infra Red Spectroscope Instrument
Completely Dry Sample
Comparison of Graphs - No Impurities!
Detector 5973 MSD
Run Time 40 min
HeadSpace G1888 Series
Company Perkin Elmer
l = path length in decimeter
A certificate of analysis provides the data which helps to assure the quality of active pharmaceutical ingredients (APIs) whereby the aim is for later implementation within therapeutic agents.
This investigation focused on following the mandatory steps for completion of a certificate of analysis on Deltamethrin; a pyrethroid ester insecticide which is commonly used for the control of malaria.
The experimental methods used to acquire the relevant data were divided between the group of students and involved analyzing Deltamethrin in terms of appearance, identity, polarimetry, the presence of residual solvents and potential related substances.
Upon completion of the investigation it was proven that through systematic and cooperative team work a range of valuable results could be obtained for forming the basis of an official certificate of analysis, which furthermore, provided the students with an experience which replicates the common workings of the pharmaceutical industry.
rotation in degrees
concentration in g/mL
Reversed phase HPLC assay
- Peak at 10.488 retention time was obtained for standard and 10.486 for sample
- Non-polar compounds had stronger attraction with non-polar column because of van der waals forces.
- Less attraction in polar mobile phase, they therefore spend more time in the solvent and column resulting in longer retention time.
Reversed phase related substances test
- Similar to Previous Test
- Related substances peaks in 1.709, 2.212 and 5.972 retention time, same as BP mentioned
Normal phase related substances tests on Regiscell and Chiralpak
- Impurity at 3.181 minutes was detected in sample but not standard
- Non-polar mobile phase was used.
- Polar compounds will stick longer to the column than non-polar compounds.
- The non-polar ones will pass more quickly through the column
- Hence, impurity should be more non-polar than Deltamethrin
GC residual solvents
- There was one peak at 2.133 in both sample and standard of GC
- No such peak in blank one
- That peak should be either impurity or Deltamethrin
- Since the operation temperature of GC is 50-250
- Therefore one of them may not have reached its B.P (B.P of Deltamethrin is 300)
Karl Fischer titration
- Test for whether Deltamethrin will absorb moisture or not
- No water was detectable by this method
- Deltamethrin is insoluble in water
- Absence of water = Absence of polar impurity
1) The non-polar impurity was not eluted before 20 minutes time or was not dissolved in the mobile phase.
2) Chromatogram of normal phase HPLC shows that there is an impurity at ~3 minutes
3) No evidence to show absence of impurities
4) No polar impurity can be found in Karl Fischer titration
5) Different angles of rotation for sample and standard can be found in polarimetry
- Ability of a substance to affect polarized light is related to its chemical structure
- Compounds are optically active, frequently used as an indication of purity
- Sample rotated by 50 degrees while the Standard rotated by 100 when official range = 55.5-58.5
- Since lack of amount of sample and standard (1% w/v instead of 4% w/v)
- Hence, the results obtained ≠ official range
- Impurity found in normal phase HPLC (eluted earlier, more non-polar than Deltamethrin)
- Therefore, the peaks of impurity may have overlapped with the peaks of Deltamethrin (non-polar bonds) resulting in the same IR spectrum
The purpose of this experiment was to gain experience of how a less than straight forward problem could be solved by working as a productive and cooperative team, allowing for the collection of the necessary data which is required for the construction of a ‘
certificate of analysis
Pyrethroid ester insecticide
High potency; mimic the actions of the lesser stable natural pyrethrins, isolated from chrysanthemum flowers
Used for the control of diseases spread by insects (e.g. Malaria)
Works against insects when they come in contact with Deltamethrin and ingest it.
Causes disruption to the nervous system by delaying Na ion channels in nerves from closing, causing a series of repetitive nerve signals causing death.
Relatively safe in humans due to physiological stability - can sometimes cause neurotoxicity if poorly monitored.
Methods and Results
Normal Phase Related Substances HPLC on ChiralPak Column
Mobile Phase: 85% Hexane + 15% IPA
10 mg in 10 ml of mobile phase
Normal Phase Related Substances HPLC on Regiscell Column
The department of Health (2013)
, 2013 British Pharmacopoeia (Veterinary)
, London: The Stationary Office.
Regis Technologies, INC. Regiscell Column, Available at: www.registech.com (Accessed: 5/3/2014).
ICH Center for Biologics Evaluation and Research (CBER), Guidance for Industry, 1996
ICH Center for Drug Evaluation and Research (CDER), Review for Guidance - Validation of Chromatographic Methods, 1994
Chiral Technologies Europe (2009) CHIRALPAK® & CHIRALCEL®, Available at: http://www.chiral.fr/coated_chiralpak_and_chiralcel.asp (Accessed: 5/3/2014).