CHANGZHOU PHARMACEUTICAL FACTORY

ESTABLISHED IN 1949

MOA of Ledipasvir-Co-Povidone
Appearance
Off-white to white powder

Identification
1)Accurately weigh suitable quantity of sample to be examined, then prepare test solution in ethanol to obtain
a solution contains 0.1mg of Ledipasvir-co-povidone per ml. Perform test by UV Spectrometer, exhibits
maximum at 207nm and 330nm(±2nm).
2) In the chromatogram of assay, the retention time of principal peak of test solution is similar to that of the
principle peak of system suitability solution.
Loss on drying : Not more than 3.0%
Test method(European Pharmacopoeia 2.2.32): determined on 1.0g by drying in vacuo at 105℃ for 1h.
Solubility:
Test method(European Pharmacopoeia 8.0,General notices):Freely soluble in Ethanol and dichloromethane,
practically insoluble in Water.
Heavy metals: NMT 0.002%
Carry out the limit test for heavy metals (The united states pharmacopeia,231, method ‖), using 1.0g of the
test sample, using the residue obtained in test for residue on ignition; not more than 0.002%.

Related substance: Liquid chromatography

Diluent: Acetonitrile
Test solution: Accurately weigh about 25mg of the sample to be examined, transfer into a 50-ml volumetric
flask, dissolve with diluent and dilute to volume, mix well.
Chromatographic System
Column: XBridge C18 (150mm×4.6mm×5μm)
Wavelength: 328nm Flow rate: 1.0ml/min
Column temperature: 35℃ Injection volume: 10µl
Mobile phase A:0.1%H3PO4
Mobile phase B: Acetonitrile
Eluted using mobile phase A and B with a gradient program as follows:
Time (min) Mobile phase A (%) Mobile phase B (%)
0.01 80 20
5.00 80 20

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 CHANGZHOU PHARMACEUTICAL FACTORY
ESTABLISHED IN 1949

30.00 10 90
40.00 10 90
40.01 80 20
50.00 80 20
Procedure: Separately inject 10µl of blank solution and test solution once into the chromatographic system,
record the chromatograms. Calculated the content of each impurity with formula as below:
Ai
imp%   100%
As
Ai: the peak area of impurity in test solution
As: the peak area of ledipasvir in test solution
Limits: LPS-8 not more than 0.15%; LPS-B not more than 0.15%; LPS-C not more than 0.15%; LPS-D not
more than 0.15%; LPS-D2 not more than 0.15%; LPS-One not more than 0.15%;any unknown impurity
should be not more than 0.10%; and the sum of the content of all impurities should be not more than 1.0%.

Assay: Liquid chromatography

Liquid chromatography as described in the test for Related substances.
Reference solution: Accurately weigh about 10mg of the reference standard, transfer into a 100-ml volumetric
flask, dissolve with diluent and dilute to volume, mix well.
Test solution: Accurately weigh about 10mg of the sample to be examined, transfer into a 100-ml volumetric
flask, dissolve with diluent and dilute to volume, mix well.
Procedure: Separately inject 10µl of blank solution once, reference solution for five times and test solution
twice into the chromatographic system, record the chromatograms. RSD of peak areas for five injections of
reference solution should be not more than 2.0%.
Calculated the assay with external standard method.
Limits:Ledipasvir:47.5%~55.0%

Residual solvents: Gas chromatography

Blank solution:DMSO
Reference stock solution A: Accurately weigh about 0.25g of isopropyl acetate, 0.25g of ethyl acetate, 0.25g
of acetone, 1.0g of ethanol, transfer to a 100-ml volumetric flask that contains appropriate volume of DMSO,
dilute to volume with DMSO and mix well.
Reference stock solution B: Accurately weigh about 0.44g of N,N-dimethyl formamide, 0.205g of acetonitrile,
0.050g of 1,2-dimethoxyethane, transfer to a 100-ml volumetric flask that contains appropriate volume of

NO.518 LAODONG EAST ROAD, CHANGZHOU, 213018 JIANGSU, CHINA

Tel:+86-51988813251 Fax:+86-519-87828412 E-mail: heyan1215@126.com Website: www.czpharma.com
 CHANGZHOU PHARMACEUTICAL FACTORY
ESTABLISHED IN 1949

DMSO, dilute to volume with DMSO and mix well. Further transfer accurately 1.0ml of above solution to a
10-ml volumetric flask, dilute to volume with DMSO and mix well.
Reference solution: Accurately transfer 1.0ml of reference stock solution A and 1.0ml of reference stock
solution B to the same 10-ml volumetric flask, dilute to volume with DMSO and mix well.
Test solution: Accurately weigh about 0.5g of sample to be examined, transfer to a 10-ml volumetric flask that
contains appropriate volume of DMSO, dissolve with DMSO and dilute to volume, mix well.
Chromatographic System:
Column: DB-624, 30m×0.53mm×3.0μm; Detector: FID
Injector temperature: 160℃ Detector temperature: 280℃
Carrier gas: Nitrogen gas The flow rate of carrier gas: 10KPa;
Hydrogen: about 35ml/min; Air: about 350ml/min.
Split ratio: 20:1 Injection volume: 1.0μl
Column temperature: The initial column temp is maintained at 40℃ for 5 minutes, then is rasied to 60℃ at
the rate of 5℃/min and maintained for 5 minutes, then is rasied to 200℃ at the rate of 5℃/min and
maintained for 5 minutes.
Procedure: Inject blank solution once and reference solution for fives times into the chromatographic system,
record the chromatograms, the resolution of adjacent peaks should be not less than 1.5, and RSD of peak areas
for replicate injection of reference solution should be not more than 10.0%. Then inject test solution once into
the chromatographic system, record the chromatogram. Calculation: Calculate the percent, in % of each
solvent in the portion of sample comparing to reference solution.
Limits: isopropyl acetate should be not more than 5000ppm; Ethyl acetate should be not more than 5000ppm;
N,N-dimethyl formamide should be not more than 880ppm; acetonitrile should be not more than 410ppm;
1,2-dimethoxyethane should be not more than 100ppm; acetone should be not more than 5000ppm; ethanol
should be not more than 20000ppm.

NO.518 LAODONG EAST ROAD, CHANGZHOU, 213018 JIANGSU, CHINA

Tel:+86-51988813251 Fax:+86-519-87828412 E-mail: heyan1215@126.com Website: www.czpharma.com