FusionChrom Analytical Method Development And Method Validation.
Method development and validation
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Transcript of Method development and validation
Method development and validation of Drug substance(BSN)
for Assay & Related Substances using HPLCPresented ByAbdul DiwkarMSc BT-13006
GuideMr.Avinash Jagdale
Introduction
BSN STRUCTURE
• 4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]benzene-1-sulfonamide monohydrate
• ASSAY• RELATED SUBSTANCES
Manpreet kaur et al(2012)
OBJECTIVES• Method development and validation of drug substance(BSN)for ASSAY using high performance liquid chromatography
• Method development and validation of drug substance(BSN)for RELATED SUBSTANCES using High Performance Liquid Chromatography
Materials and MethodsColumn use
FOR ASSAY FOR RELATED SUBSTANCES
Column nameInertsil ODS 3V Zodiac C18 Inertsil ODS-3 Ascentis express
C18mode of chromatography Reversed Phase Reversed Phase Reversed Phase Reversed Phase
operating pH 2-10 2-10 2-9 2-10
particle size 5um 5um 5um 2.7um
• CHROMATOGRAPHIC CONDITIONS: For Related Substances
• Buffer :10ml of triethylamine dissolve in 10 litre water adjust pH 3.02 with orthophosphoric acidMobile phase Buffer: Acetonitrile (60:40)
Column temperature 40° c
Flow rate 2.0ml/min
Injection volume 10 μl
Wavelength 220nm
• CHROMATOGRAPHIC CONDITIONS: For Assay• Buffer:-2ml of triethylamine dissolve in 2 litre water
adjust pH 3.01 with orthophosphoric acidMobile phase Buffer: Acetonitrile (45:55)
Column temperature 40° c
Flow rate 2.0ml/min
Injection volume 20 μl
Wavelength 205nm
Review Of Literature• ET-1 is a potent vasoconstrictor that is overexpressed in the
plasma and the lungs of patients with PAH(Giaid et al ).• Some research work on animal has shown that by blocking
ETB receptors, ET-1 vasoconstrictive activity is enhanced (via the ETA receptor), due to inhibition of the transient ETB induced vasodilatation and ET-1 clearance. (Black et al )
• Reverse phase-high performance liquid chromatography (RPHPLC) is a simple, rapid, sensitive and precise method for the determination of BSN and method developed on Agilent XDB C18 column (150 mm × 4.6 mm, i.d., 5 μ) for Assay (R. Kalaichelvi and E. Jayachandran)
Results
• Related substances • Trial 1:-The experimental trials was carried out as
given in USP.PH of a buffer set 3.02 instead of 2.5.and mobile phase composition changes to 60:40 (buffer: Acetonitrile) instead of 55:45.
Blank Standard Impurity B
Impurity E Impurity A Impurity DObservation: - Impurity B eluting at the retention time of BSN. Impurity C is not eluted till 30minSample Retention
TimeImpurity E 1.378Impurity D 1.620Impurity B 4.621BSN 4.654Impurity A 12.651Impurity C Not Eluted
Trial 2• Gradient program setMobile phase A: BufferMobile phase B: AcetonitrileGradient program
Time(minute)
%A %B
0.0 90 10
20.0 90 10
30.0 10 90
40.0 10 90
50.0 90 10
60.0 90 10
ObservationBase line obtained was uneven. Impurity B eluting at the retention time of BSN.
Sample Retention Time
Impurity E 24.051
Impurity D 24.741
Impurity B 26.210
BSN 26.185
Impurity A 27.334
Impurity C 28.700
Trial 3• Gradient program changed to separate impurity B
Time(minute)
%A %B
0.01 90 1012.0 90 1015.0 10 9020.0 10 9030.0 90 10
Trial 11• From all the trials taken till now satisfactory results not
achieved hence decided to change the PH of the buffer from 3.0 to 2.5 and also change the diluent from Buffer: ACN to Buffer: Methanol: ACN (300:200:500) Time(minute)
%A %B
0.00 70 30
17.0 70 30
22.0 30 70
25.0 30 70
27.0 70 30
30.0 70 30
• Observation: -Peak shape of BSN found satisfactory. Resolution between impurity B and BSN peak is 2.6.Noise observed at retention time of impurity A & impurity C Sample Retention Time
Impurity E 2.052
Impurity D 2.420
Impurity B 12.955
BSN 14.393
Impurity A 20.544
Impurity C 22.545
Trial 12• To reduce the noise decided to change the gradient
program. Rest all the parameters same as trial 11. Time(minute)
%A %B
0.00 70 30
17.0 70 30
22.0 40 60
25.0 40 60
27.0 70 30
30.0 70 30
• Observation: -Peak shape of BSN found satisfactory. Resolution between impurity B and BSN peak is 2.5.Base line at retention time of impurity A & impurity C found satisfactory. Sample Retention Time
Impurity E 1.723
Impurity D 2.058
Impurity B 12.442
BSN 13.880
Impurity A 21.109
Impurity C 24.179
Linearity• linearity of an analytical procedure as its ability (within
a given range) to obtain test results that are directly proportional to the concentration (amount) of analyte in the sample.
• All the parameters kept constant as in trial no 12 to confirm method which is developed for related substances is proper.
• Observation:-linearity of the related substances performed and results found within the range as shown in the picture given below.
Name Concentration % Retention Time
Lin level 1
50 1.721
Lin level 2
80 1.720
Lin level 3
100 1.722
Lin level 4
120 1.720
Lin level 5
150 1.719
Assay Trial 1• The experimental trials was carried out as given in USP.PH of a
buffer set 3.01 instead of 2.5.and mobile phase composition remain same .Rest all the chromatographic parameters set as follows
• CHROMATOGRAPHIC CONDITIONS: Mobile phase : Buffer: Acetonitrile (45:55) Column temperature : 40° c Column : Inertsil ODS-3V Flow rate : 2.0ml/min Injection volume : 20 μl Wavelength : 205nm
• Observation: -Baseline found satisfactory .Retention time of a sample is 4.892
Trial 2• All chromatographic conditions are same as Trial 1
except column and flow rate decreases to 1.5 to reduce runtime of the sample
• Observation: -Baseline found satisfactory .Retention time of a sample is 3.341
Trial 3• Mobile phase composition changes 45:55 to
40:60.Wavelength changes to270nm column temperature changes to 35°c to reduce runtime of the sample
• Observation: -Baseline found satisfactory .Retention time of a sample is 2.456
Linearity and Accuracy of Assay
• Experimental design same as trial 3. • Observation:-linearity of the Assay performed and
results found within the range as given below. Name Concentration % Retention TimeLin level 1 50 2.456Lin level 2 75 2.455Lin level 3 100 2.455Lin level 4 125 2.456Lin level 5 150 2.456
Linearity
• Accuracy:-accuracy of an analytical procedure as the closeness of agreement between the conventional true value or an accepted reference value and the value found
• Experimental design same as trial 3• Observation:-Accuracy of the Assay performed and
results found within the range as given below. According to USP % recovery should be from 98% to 102%.
Name % Recovery %RSD
Accuracy level 1 101.1
Accuracy level 2 100.7 0.8
Accuracy level 3 100.7
Conclusion• The Method for Assay and Related Substance using HPLC
given in the U.S.Pharmacopeial was modified to suite the laboratory condition and availability of instruments and reagents. The new developed method was validated and results of validation test were found within the acceptable range given by U.S.Pharmacopeial. Hence the method was set as a final developed method for analysis of DRUG (BSN).
• The Method so developed is now ready to be transferred to the production unit where large number of DRUG (BSN) formulation can be analyzed using this set method within short period of time & Impurities can be monitored properly.
Bibliography• http://pubchem.ncbi.nlm.nih.gov/ • http://www.drugbank.ca/drugs/DB00559 • http://pubchem.ncbi.nlm.nih.gov/image/imagefly.cgi?
cid=104865&width=800&height=800 • http://www.drugbank.ca/drugs/DB00559 • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3588285/pdf/e-69-00o12.pdf • http://www.patient.co.uk/pdf/28696.pdf • http://www.medscape.com/viewarticle/755543_6 • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2605321/pdf/vhrm-4-0943.pdf 12.12 • http://www.hindawi.com/journals/cri/2011/929876/ • http://www.pharmacelsus.de/assay_development/ • http://www.sigmaaldrich.com/