Field of the Invention
The present invention is directed to an analytical method for development and validation of novel continuous HPLC method for the determination of Prasugrel base from the formulation comprising Prasugrel hydrochloride.
Background of the Invention
Prasugrel HCl is chemically known as 2-Acetoxy-5-(α-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno [3,2-c] pyridine hydrochloride. Prasugrel is member of the thienopyridine class of ADP receptor inhibitors like clopidogrel. Prasugrel base is practically insoluble in water and becomes slightly soluble when pH decreases at 20°C (μg/mL) i.e. 59.8 (pH 3.57), 14.2 (pH 4.85), 4.01 (pH 6.00), 0.15 (pH7.12). Whereas Prasugrel hydrochloride is slightly soluble in pH 1-4, Very slightly soluble in pH 5 (i.e. 100 mg in 1000 mL water) and insoluble in pH 6-7.
Prasugrel is a prodrug, oxidation by intestinal and hepatic cytochrome P-450 enzymes convert Prasugrel into its active metabolite. Prasugrel has a rapid and almost complete absorption after oral ingestion of a loading dose. Its active form binds irreversibly to the adenosine diphosphate (ADP) P2Y12 receptor on platelets for their lifespan, thereby inhibiting their activation and decreasing subsequent platelet aggregation. Prasugrel has a greater antiplatelet effect than clopidogrel because it is metabolized more efficiently. Some of the differences in metabolism between clopidogrel and Prasugrel may be explained by genetic polymorphisms affecting the cytochrome P-450 system.
Regulatory authorities set stringent standards for a pharmaceutical product before it can be released into the market. The U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER) has promulgated guidelines recommending
that drug applicants identify organic impurities of 0.1% or greater in the active ingredient. “Guideline on Impurities in New Drug Substances,” 61 Fed. Reg. 371 (1996); “Guidance for Industry ANDAs: impurities in Drug Substances,” 64 Fed. Reg. 67917 (1999). In order to obtain marketing approval for a drug product, manufacturers must submit to the regulatory authority evidence that the product is acceptable for administration to humans. Such a submission must include, among other things, analytical data showing the impurity profile of the product to demonstrate that the impurities are either absent, or present in a negligible amount. Therefore, there is a need for analytical methods to detect impurities to identify and assay those impurities.
There are various patent and non-patent literature are known in the art, which disclose several liquid chromatography methods to determine Prasugrel hydrochloride.
PCT patent publication no. 2013/024399 A1 disclose an improved reversed-phase liquid chromatographic method for the quantitative determination of Prasugrel hydrochloride and a stability indicating analytical method using the samples generated from forced degradation studies.
Indian patent publication no. 2852/DEL/2012 disclose the assay method of Prasugrel hydrochloride for the determining the shelf-life of Prasugrel hydrochloride as well as dosage form.
Reddy, M. N. Chandra et al. Development and validation of a reverse-phase liquid chromatographic method for related substances of Prasugrel for 5 and 10mg tablets. International Journal of Pharmacy and Pharmaceutical Sciences (2014) 6 (1), 90-94 disclose a HPLC instrument and inert sustain C18, 75 x 4.6 mm, 3μm used for determination of Prasugrel and its related substances.
Liu, E. et al. Determination of the content of Prasugrel and its related substances. Chemistry & Bioengineering (2014) (07) disclose HPLC method for determination of the content of Prasugrel and its related substances by using Diamonsil C18column(150mm×4.6mm,5μm) column with the temperature of 30℃, mobile phase consisting of acetonitrile∶ phosphate buffer (pH value 5.0) in the volume ratio of 50∶50, the flow rate of 1.0mL·min-1, the detection wavelength of 219nm, the injection volume of 10μL.
Reddy, K. C. Sekhar et al. Development and validation of stability indicating reverse-phase HPLC method for the determination of Prasugrel hydrochloride and its related substances. International journal of pharmaceutical sciences and research (2014), 5 (3), 919-927 disclose RP-HPLC method for the determination of Prasugrel hydrochloride and its related substances by using Sunfire C18, 5μm (250mm x 4.6mm) column with a mobile phase A: 0.1% v/v orthophosphoric acid in water and mobile phase B: 0.1% v/v orthophosphoric acid in acetonitrile and the developed method was validated for specificity, forced degradation studies, sensitivity (LOD and LOQ), linearity, precision, accuracy, stability of standard and sample solutions and robustness.
Pulla R. P., Estimation of Prasugrel in tablet dosage form by RPHPLC. International Journal of Chemistry Research (2011), 2 (3), 34-36 disclose a simple, precise, rapid and accurate reverse phase HPLC method was developed for the estimation of Prasugrel in tablet dosage form by using an Xterra RP C18, 250x4.6 mm, column with 5 μm particle size and the mobile phase consisting of 0.03M K2HPO4 in water pH: 3.2 adjusted with Acetonitrile (25:75).
Kumar, S. A., Development and validation of RP-HPLC method for the estimation of Prasugrel in bulk as well in pharmaceutical dosages form. International Journal research of Pharmacy (2013), 4 (3), 254-260 disclose a simple, sensitive, precise, and
specific reverse phase HPLC method for the determination of Prasugrel in bulk and its tablet dosage forms by reverse phase chromatography on XTerra column C18 (4.6 x 150mm, 5 mm) with a mobile phase composed potassium dihydrogen phosphate and the pH has been adjusted to 3.0 by Orthophosphoric Acid & Acetonitrile in the ration of 40:60 v/v.
It was known in the art that Prasugrel hydrochloride was approved by USFDA in the dose strength of EQ 5mg & EQ 10mg base. In the approved product, it was very difficult to go at lower level by using XRD technique due to lower amount of analyte. The average weight of Prasugrel tablet is about 128 mg against FDA approved 5 mg Prasugrel tablet and 256 mg against FDA approved 10 mg Prasugrel tablet. While quantification of Prasugrel base from the formulation containing Prasugrel hydrochloride, due to high amount of excipient response of Prasugrel the characteristic peak was poor and cannot be detected at lower levels by using XRD technique.
Performing routine testing in a quality control laboratory with standardized methods requires analytical instrumentation with high reliability and ease-of-use, combined with optimal cost-of-ownership. Several attempts to determine the amount of Prasugrel base in a formulation containing Prasugrel hydrochloride by using liquid chromatography have been described previously. None of the prior art disclose the determination of Prasugrel base from the formulation containing Prasugrel hydrochloride by a continuous HPLC process involving removal of the salt and elution of the base during the process.
So, there still exists a need to develop an improved suitable single stability indicating Liquid Chromatography method that can be used to determine the quantity of Prasugrel base in the formulation containing Prasugrel hydrochloride which is not only industrially scalable but also method is short and cost-effective.
Summary of the Invention
One aspect of the present invention provides a continuous method for the quantitative determination of Prasugrel base from the formulation containing Prasugrel hydrochloride with the help of solvents comprising acetonitrile: water in ratio 90:10 v/v and 1% v/v of orthophosphoric acid.
Another aspect of the present invention provides a continuous method for quantification of Prasugrel base in a formulation containing Prasugrel hydrochloride by HPLC, wherein the HPLC method comprises the steps of:
(a) preparation of solution comprise the step of; (i) dissolve the Prasugrel hydrochloride into washing solvent to obtained residue, (ii) wash and process the residue obtained in step (i) multiple times with washing solvent to obtained precipitate, (iii) dissolve the precipitate obtained in step (ii) in diluent to obtained residue, (iv) wash and process the residue obtained in step (iii) multiple times with diluent to obtained final precipitate, (v) dissolve the precipitate obtained in step (iv) in diluent to obtained solution;
(b) inject the solution into an HPLC column;
(c) elute the solution with a mobile phase comprising buffer and a diluent; and
(d) measure the amounts of Prasugrel and each impurity with a detector.
Another aspect of the present invention provides a continuous method for quantification of Prasugrel base in a formulation containing Prasugrel hydrochloride by HPLC, wherein the HPLC method comprises the steps of: (a) preparation of solution comprise the step of; (i) dissolve the Prasugrel hydrochloride into washing solvent to obtained residue, (ii) wash and process the residue obtained in step (i) multiple times with washing solvent to obtained precipitate, (iii) dissolve the precipitate obtained in step (ii) in diluent to obtained residue, (iv) wash and process the residue obtained in step (iii) multiple times with diluent to obtained final precipitate, (v) dissolve the
precipitate obtained in step (iv) in diluent to obtained solution; wherein the diluent comprising mixture of acetonitrile and water in ratio of 90:10 %v/v.
Another aspect of the present invention provides a continuous method for quantification of Prasugrel base in the formulation containing Prasugrel hydrochloride, wherein the process comprises eluting the sample solution with a mobile phase comprising buffer and a diluent, wherein the buffer is prepared by mixing water with 85% v/v of orthophosphoric acid and adjusting the pH of the mixture to about 2.9±0.05 with diluted potassium hydroxide.
Additional aspect and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not restrict the invention, as claimed.
Brief Description of the Figures
Figure 1: HPLC chromatogram of Blank
Figure 2: HPLC chromatogram of standard
Figure 3: HPLC Chromatogram of placebo
Figure 4: HPLC Un-spike test Chromatogram of Sample 5mg
Figure 5: HPLC Un-spike test chromatogram of Sample 10mg
Figure 6: HPLC spike test chromatogram of Sample 5mg
Figure 7: HPLC spike test chromatogram of Sample 10mg
Description of the Invention
Present invention was designed to develop and validate a new, fast and accurate reversed-phase high performance liquid chromatographic method for the estimation and quantification of Prasugrel base from the formulation comprising Prasugrel hydrochloride. The chromatographic separation has been achieved on silica column with mobile phase consisting mixture of acetonitrile and water (90:10 v/v) with pH 2.9±0.05 adjusted with orthophosphoric acid buffer at flow rate of 1.0ml/min and detector wavelength 235 nm.
While working on the present invention, Inventors have found that when Prasugrel hydrochloride tablet powder was dissolved and washed 4 to 8 times with washing solvent; further dissolved and washed 3 to 6 times with diluent, the obtained solution was injected into a HPLC column, the obtained solution is completely free of Prasugrel hydrochloride. The developed method was capable to detect and quantify the Prasugrel base at 1.48% & 4.94% of the specification level. The method was validated for its specificity, linearity, accuracy, precision, limit of detection and quantification according to ICH guidelines.
The term “limit of detection (LOD)” herein relates to the lowest concentration of analyze that can be clearly detected above the base line signal, is estimated is three times the signal to noise ratio.
The term “limit of quantization (LOQ)” herein relates to the lowest concentration of analyte that can be quantified with suitable precision and accuracy, is estimated as ten times the signal to noise ratio.
The term “gradient elution” herein relates to the change in the composition of the gradient eluent over a fixed period of time, stepwise or at a constant rate of change, as
the percentage of the first eluent is decreased while the percentage of the second eluent is increased.
The term “gradient eluent” herein relates to an eluent composed of varying concentrations of first and second eluents.
The term “multiple times” herein relates to about 3 to about 8 times.
The term “wash and process” herein relates to the process comprising dissolving the residue and/or precipitate in washing solvent and/or diluent, shaking, sonicating for appropriate time, centrifugation of mixture at appropriate RPM and discard the supernatant liquid.
The term “wash and/or washing” herein relates to dissolving and/or washing the mixture/ solution or precipitate in different solvents to remove impurity.
In one embodiment of present invention provides a continuous method for quantification of Prasugrel base in a formulation containing Prasugrel hydrochloride by HPLC, wherein the HPLC method comprises the steps of: (a) preparation of solution comprise the step of; (i) dissolve the Prasugrel hydrochloride into washing solvent to obtained residue, (ii) wash and process the residue obtained in step (i) multiple times with washing solvent to obtained precipitate, (iii) dissolve the precipitate obtained in step (ii) in diluent to obtained residue, (iv) wash and process the residue obtained in step (iii) multiple times with diluent to obtained final precipitate, (v) dissolve the precipitate obtained in step (iv) in diluent to obtained solution; wherein the wash and process step comprising sonication, centrifugation, washing and filtration.
Another embodiment of present invention, the washing solvent comprising mixture of orthophosphoric acid and water.
Another embodiment of present invention, the concentration of orthophosphoric acid is 1% v/v. The 1% orthophosphoric acid was prepared by taking 1ml of 85% v/v orthophosphoric acid in 100ml volumetric flask and make the volume 100ml with water.
Another embodiment of present invention, the washing solvent comprising mixture of orthophosphoric acid and water, wherein the washing solvent was prepared by taking 3ml of 1% orthophosphoric acid in 1000ml volumetric flask and make the volume 1000ml with water.
Another embodiment of present invention, the diluent comprising mixture of acetonitrile and water in a ratio of 90:10 v/v.
Another embodiment of present invention provides a Mobile Phase A, wherein the Mobile Phase A is buffer. The buffer was prepared by taking 3.0ml of 85% orthophosphoric acid into 1000ml water and adjust the pH to 2.9±0.05 with dilute potassium hydroxide solution.
Another embodiment of present invention provides a dilute potassium hydroxide (1%) solution, wherein the potassium hydroxide solution was prepared by taking 1gm of potassium hydroxide in 100ml volumetric flask, dissolved in water and make up the volume with water to 100ml.
Another embodiment of present invention provides a Mobile Phase B. Mobile phase B may include the organic solvent selected from the group comprising methanol, acetonitrile, propanol or isopropanol, water or mixtures thereof. The Mobile Phase B comprises mixture of acetonitrile and water in ratio of 90:10 v/v.
Another embodiment of present invention, the HPLC column is a Inertsil ODS-3 250mm x 4.6mm column with 5μ particles.
Another embodiment of present invention, the filtration was carried out through 0.45μ nylon filter paper.
Another embodiment of present invention, the column temperature is about 30°C to about 35°C.
Another embodiment of present invention, the separation was carried out at 30°C column temperature at a flow rate of 1.0ml/min.
Another embodiment of present invention, the detection wavelength is 235nm.
Another embodiment of present invention, the retention time in the column is 35min.
Another embodiment of present invention, the instrument used in the quantification of drug is HPLC equipped with UV/ PDA detector.
Another embodiment of present invention, quantification may be performed in isocratic or gradient mode. In isocratic mode, the mobile phase composition is constant throughout. A gradient mode was carried out by a gradual change over a period in the percentage of the two or more solvents making up the mobile phase. The change in solvent is controlled by a mixer which mixes the solvents to produce the mobile phase prior to its passing through the column.
Another embodiment of present invention, quantification was performed in gradient mode.
Another embodiment of present invention, the limit of detection was found to be about 1.48%.
Another embodiment of present invention, the limit of quantification was found to be about 4.94%.
Another embodiment of present invention, the Prasugrel hydrochloride tablet powder was dissolved in washing solvent with 4 to 8 washes and further dissolved in diluent with 3 to 6 washes, the Prasugrel hydrochloride was dissolved and elute from the sample. The residue was left with Prasugrel base along with other inactive ingredients. To overcome impact of placebo Prasugrel base, standard sample shall be prepared by spiking in placebo with subsequent washings with water and sonication.
While the invention has been described in term of its specific embodiments, certain modification and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the invention.
Preparation of Placebo solution: 100mg of Prasugrel Hydrochloride placebo was weight and transferred into a 50-mL centrifuge tube. 40 mL of washing solvent was added into the centrifuge tube and shake for 2 minutes and sonicate 30 min at about 20°C with intermittent shaking to obtained solution. Centrifuge this solution for 5 minutes at 8000 rpm and allow to settle the particle. Discard the supernatant liquid carefully to obtained residue. This process was repeated for 6 times to obtained final residue.
Further, the obtained residue was transferred in centrifuge tube and dissolved in 20ml diluent and shake for 2 minutes. Transfer the content to 100 mL volumetric flask. Repeat this procedure four times and transfer the content to same volumetric flask. Sonicate the content for 5 minutes with occasional swirling at about 20°C and keep the
volumetric flask to attain the room temperature. Makeup the volume of volumetric flask 100ml with diluent and mix well to obtained solution. Filter this solution through 0.45μ Nylon filter and after discarding first 5 mL of filtrate collect the sufficient final solution in HPLC vial.
Preparation of Standard solution: Weight about 100mg placebo and 4mg of Prasugrel Base and transferred into a 50ml centrifuge tube. 40 mL of washing solvent was added into the centrifuge tube and shake for 2 minutes and sonicate 30 min at about 20°C with intermittent shaking to obtained solution. Centrifuge for this solution for 5 minutes at 8000 rpm and allow to settle the particle. Discard the supernatant liquid carefully to obtained residue. This process was repeated for 6 times to obtained final residue.
Further, the obtained residue was transferred into centrifuge tube and dissolved in 20ml diluent and shake for 2 minutes. Transfer the content to 100 mL volumetric flask. Repeat this procedure four times and transfer the content to same volumetric flask. Sonicate the content for 5 minutes with occasional swirling at about 20°C and keep the volumetric flask to attain the room temperature. Make up to volume of volumetric flask 100ml with diluent and mix well to obtained solution. Filter this solution through 0.45μ Nylon filter and after discarding first 5 mL of filtrate collect the sufficient final solution in HPLC vial.
Preparation of Sample Solution for 5mg Prasugrel Hydrochloride Tablet: 20 tablets were crushed and transfer 100mg tablet powder into a 50ml centrifuge tube. 40 mL of washing solvent was added into the centrifuge tube, shake for 2 minutes and sonicate 30 min at about 20°C with intermittent shaking to obtain solution. Centrifuge of this solution for 5 minutes at 8000 rpm and allow to settle particle. Discard the
supernatant liquid carefully to obtain residue. This process was repeated for 6 times to obtained final residue.
Further, the obtained residue was transferred in to centrifuge tube and dissolved in 20ml diluent and shake for 2 minutes. Transfer the content to 100 mL volumetric flask. Repeat this procedure four times and transfer the content to same volumetric flask. Sonicate the content for 5 minutes with occasional swirling at about 20°C and keep the volumetric flask to attain the room temperature. Make up to volume of volumetric flask 100ml with diluent and mix well to obtain solution. Filter this solution through 0.45μ Nylon filter and after discarding first 5 mL of filtrate collect the sufficient solution in HPLC vial.
Preparation of Sample Solution for 10mg Prasugrel Hydrochloride Tablet: 20 tablets were crushed and transfer 100mg tablet powder into a 50ml centrifuge tube. 40 mL of washing solvent was added into the centrifuge tube, shake for 2 minutes and sonicate 30 min at about 20°C with intermittent shaking to obtain solution. Centrifuge of this solution for 5 minutes at 8000 rpm and allow to settle particle. Discard the supernatant liquid carefully to obtain residue. This process was repeated for 6 times to obtained final residue.
Further, the obtained residue was transferred in to centrifuge tube and dissolved in 20ml diluent and shake for 2 minutes. Transfer the content to 100 mL volumetric flask. Repeat this procedure four times and transfer the content to same volumetric flask. Sonicate the content for 5 minutes with occasional swirling at about 20°C and keep the volumetric flask to attain the room temperature. Make up to volume of volumetric flask 100ml with diluent and mix well to obtain solution. Filter this solution through 0.45μ Nylon filter and after discarding first 5 mL of filtrate collect the sufficient solution in HPLC vial.
Mobile phase A: Buffer
Mobile phase B: Acetonitrile and water in the ratio (90:10 v/v)
Gradient programmed:
Time (Min.)
Mobile Phase-A (%)
Mobile Phase-B (%)
0.00
60
40
15.0
60
40
20.0
35
65
28.0
35
65
28.5
60
40
35.0
60
40
Separately injected equal volume of Diluent (blank), 1st & 2nd standard (6 replicates) and sample solution (2 replicates) in column and response was recorded. The results of the response are disclosed below in Table.
Table 1: Solubility Profile of Prasugrel base and Prasugrel HCl
Weight of Prasugrel Base at LOQ
0.2mg
Weight of Prasugrel Base at 150%
6.0mg
Weight of Prasugrel HCl at LOQ
0.22mg (Equivalent to 0.2mg of Prasugrel)
Weight of Prasugrel HCl at 150%
6.6mg (Equivalent to 6.0mg of Prasugrel)
Washing Solvent
0.003% Orthophosphoric acid
Diluent
Acetonitrile: Water (90: 10 v/v)
Table 2: Determination of PrasugrelHydrochloride after wash by washing solvent with Placebo
No. of Washing
Level
Weight of standard
Peak of Prasugrel HCl
Injection 1
Injection 2
Mean Response
1
Higher (150%)
6.6268 mg
603167
582730
592948
2
357042
357884
357463
3
165360
163990
164675
4
73619
72603
73111
5
35680
35179
35429
6
ND
ND
ND
Final Dilution
ND
ND
ND
1
Lower (LOQ)
0.2202 mg
27695
27869
27782
2
17376
17042
17209
3
3566
3417
3491
4
1600
1559
1579
5
ND
ND
ND
6
ND
ND
ND
Final Dilution
ND
ND
ND
Table 3: Determination of Prasugrel Base after wash by washing solvent with Placebo
Level
Peak of Prasugrel Base
No. of Washing
Weight of standard
Injection 1
Injection 2
Mean Response
1
Higher (150%)
6.1505 mg
ND
ND
ND
2
ND
ND
ND
3
ND
ND
ND
4
ND
ND
ND
5
ND
ND
ND
6
ND
ND
ND
Final Dilution
689948
689813
689880
1
Lower (LOQ)
0.2149 mg
ND
ND
ND
2
ND
ND
ND
3
ND
ND
ND
4
ND
ND
ND
5
ND
ND
ND
6
ND
ND
ND
Final Dilution
17074
17072
17073
It was evident from the results disclosed in Table 2 & Table 3, that Prasugrel base will not dissolve in washing solvent (0.003% orthophosphoric acid) and selectively Prasugrel hydrochloride will be washed out after sequential washings (about 5 to 6 times) with washing solvent (0.003% orthophosphoric acid). Further, it was evident that proposed method is capable to determine the Prasugrel base accurately up to LOQ level without any loss of Prasugrel base in Prasugrel HCl tablets (5 mg & 10 mg) in presence of Prasugrel hydrochloride.
Evaluation of System Suitability: Relative standard deviation for six replicate injections of standard preparation is not more than 2.0%. From first injection theoretical plates is not less than 2000 and tailing factor is not more than 2.0.
The % correlation between 1st standard solution and 2nd standard solution should be in the range of 95.0% and 105.0%. Correlation was calculated by formula:
AS2 WS1
Correlation = --------------x------------------x 100
AS1 WS2
Where:
AS2 = Area of Prasugrel base peak from two replicates of 2nd standard preparation.
AS1 = Area of Prasugrel base peak from six replicates of standard preparation.
WS1 = Weight of Prasugrel base standard in mg taken for preparation of 1st standard.
WS2 = Weight of Prasugrel base standard in mg taken for preparation of 2nd standard.
Calculation: Percentage Assay of Prasugrel Base from the Prasugrel hydrochloride formulation was calculated by formula:
AT WS 100 P Average weight (mg)
%= --------- x ---------x----------x---------x----------------------------- x 100
AS 100 WT 100 LC (mg)
Where:
AT: Peak area of Prasugrel Base in sample solution.
AS: Average peak area of Prasugrel Base from six replicate injections of standard solution.
WT: Weight of sample (mg)
WS: Standard weight of RS\WS (mg)
P: Potency of WS\RS Prasugrel base
LC: Label Claim
The process of present invention was found linear, accurate, precise, robust and rugged between LOQ (4.94%) to 150% of label claim of Prasugrel in Prasugrel hydrochloride tablets.
Method validation: The method was validated for specificity, accuracy (recovery study), precision (repeatability & ruggedness), and robustness according to ICH guidelines by following procedure.
Specificity: The specificity of the method was done to check for the interference of impurities and of matrix in the analysis. For this first a blank solution (without any sample), then a solution with matrix and then drug solution of 20) μg/mL were injected into the column, under optimized chromatographic conditions. No interference due to impurities and matrix was observed which showed the method to be specific.
Robustness: It was determined by making slight changes in chromatographic conditions like change in the detection wavelength, change in the flow rate. It was observed that there were no marked changes in the chromatograms that suggest that the developed method is robust.
Accuracy: It was determined by carrying out recovery studies at different spiked levels (70%, 80%, 90% and 100%). The amount recovered, and the values of percent recovery were calculated. The high recovery showed that there is no interference from excipients.
Precision: It was checked by injecting replicate injections of the combined solution and then variability of the method was studied by analyzing the solution on the same day. The results were found to be within the specified range.
Ruggedness: It was done by performing the same procedure by two different analysts on two different days. The results were found to be within the specified range.
It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the invention. Various modifications and embodiments can be made without departing from the scope and spirit of the invention, which is defined by the following claims only.

We Claim:
1. A continuous method for quantification of Prasugrel base in a formulation containing Prasugrel hydrochloride by HPLC, wherein the HPLC method comprises the steps of: (a) preparation of solution comprise the step of; (i) dissolve the Prasugrel hydrochloride into washing solvent to obtained residue, (ii) wash and process the residue obtained in step (i) multiple times with washing solvent to obtained precipitate, (iii) dissolve the precipitate obtained in step (ii) in diluent to obtained residue, (iv) wash and process the residue obtained in step (iii) multiple times with diluent to obtained final precipitate, (v) dissolve the precipitate obtained in step (iv) in diluent to obtained solution; (b) inject the solution into an HPLC column; (c) elute the solution with a mobile phase comprising buffer and a diluent; and (d) measure the amounts of Prasugrel and each impurity with a detector.
2. The continuous method according to claim 1, wherein the wash and process step comprising sonication, centrifugation, washing and filtration.
3. The continuous method according to claim 1, wherein the washing solvent comprising mixture of orthophosphoric acid and water.
4. The continuous method according to claim 3, wherein the concentration of orthophosphoric acid is 1% v/v.
5. The continuous method according to claim 1, wherein the diluent comprising mixture of acetonitrile and water.
6. The continuous method according to claim 5, wherein the acetonitrile and water are present in ratio of 90:10 v/v.
7. The continuous method according to claim 1, wherein the HPLC column is a Inertsil ODS-3 250mm x 4.6mm column with 5μ particles.
8. The continuous method according to claim 1, wherein the buffer is prepared by mixing water with 85% v/v of orthophosphoric acid and adjusting the pH of the mixture to about 2.9±0.05 with diluted potassium hydroxide.
9. The continuous method according to claim 2, wherein the solution mixture is filtered through 0.45μ nylon filter paper.
10. The continuous method according to claim 1, wherein the column temperature is about 30°C to about 35°C.