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


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


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/

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