INTRODUCTION:

The ADP receptor P2Y12 plays a critical role in plate let activation and aggregation.¹ It is required to amplify and sustain the initial response to a vascular damage thereby leading to stable thrombus formation. The P2Y12 receptor is a validated target for prevention of major adverse vascular events in patients with acute coronary syndromes (ACS, comprising unstable angina [UA], non-ST elevation myocardial infarction [MI] and ST elevation MI), as demonstrated by the thienopyridine class of drugs, including clopidogrel and prasugrel.² Thienopyridines are converted to active metabolites that irreversibly bind and inactivate the P2Y12 receptor for the life of the platelet.

Prasugrel, achieving more pronounced inhibition of platelet aggregation, showed superior efﬁcacy versus clopidogrel in moderate-to high-risk patients with ACS undergoing percutaneous coronary intervention (PCI), but the improved efﬁcacy was associated with an increased bleeding risk.³ Novel principles of P2Y12 antagonism may have the potential to improve efﬁcacy without adversely affecting safety. Therefore, a reversible and selective P2Y12 antagonist has the potential to provide an improved therapeutic window as compared with the thienopyridines.

Prasugrel hydrochloride is chemically known as 5-[(1RS)-2-cyclopropyl-1-(2fluorophenyl)-2-oxoethyl]-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate hydrochloride (Fig. 1) and molecular weight is 409.9. Prasugrel is an effective 3^rd generation oral thienopyridine which is class of anti platelet agents along with Clopidogrel and Ticlopidine.⁴ It blocks a specific receptor on the platelet surface, which may results in clogged arteries and may lead to heart attack.⁵ It is effectively preventing ischemic events in patients with acute coronary syndrome undergoing percutaneous coronary intervention, which increases in bleeding and improved net clinical outcome.⁶ It inhibits adenosine diphosphate–induced platelet aggregation more rapidly and consistently in healthy volunteers. Prasugrel is an orally bioavailable pro-drug metabolized to an active adenosine diphosphate (ADP) receptor antagonist, which is a potent inhibitor of platelet activation and aggregation mediated by the P2Y12 ADP receptor.

Figure 1: Chemical structures of Prasugrel hydrochloride and its impurities

Prasugrel inhibits adenosine diphosphate induced platelet aggregation more rapidly, more consistently, and to a greater extent than do standard and higher doses of clopidogrel in healthy volunteers and in patients with coronary artery.^7,8 Literature survey revealed that only a few analytical methods such as liquid chromatography – mass spectroscopic (LC-MS), high performance thin-layer chromatographic (HPTLC), Ultra violet spectroscopic (UV), and high performance liquid chromatographic (HPLC) methods have been reported.^9-11 The purpose of this study was to develop simple, rapid, precise, specific and accurate RP-HPLC method for the estimation of the drug in pure and in pharmaceutical dosage forms. The method was validated by evaluation of the linearity, precision, accuracy and as per ICH guidelines.¹² Gas chromatography is the analytical technique used for product identification (under very controlled conditions) and must be directly coupled to a mass spectrometer when information other than a comparative program is required, such as positive identification of peaks on the chromatogram.¹³ The basic principal of gas chromatography is that greater the affinity of the compound for the stationary phase, more the compound will be retained by the column and longer it will be before it is eluted and detected. To our knowledge, there is no GC-MS method for determination of Prasugrel hydrochloride in pharmaceutical preparations in literature. Therefore, we report GC-MS and HPLC methods for determination of Prasugrel hydrochloride in pharmaceutical preparations. The proposed methods in this study are accurate, sensitive, and precise and can be easily applied to different impurities [fluoro, bromo, methyl bromo, dibromo (Fig.1)] tablet as pharmaceutical preparation. The results obtained by the methods were statistically compared and there was no significant difference between two methods.

RESULTS AND DISCUSSION:

Development and optimization of method: The main objective of the chromatographic method development was focused on the separation of prasugrel peak from its related substances. The related substances are 1-(bromomethyl)-2-fluorobenzene (Fluoro imp), 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone (Bromo imp), 1-bromo-1-(2-fluorophenyl) propan-2-one (Methyl bromo imp), 1,1-dibromo-1-(2-fluorophenyl) propan-2-one. Prasugrel hydrochloride samples spiked with these impurities were injected in gradient mode using different combinations of the following chromatographic parameters:

i) Different stationary phases like C₈, C₁₈, cyano, phenyl etc., ii) Different mobile phases like phosphate, sulphates and acetate buffers with different pH (2.0-8.0). iii) Different organic modifiers like acetonitrile, methanol and ethanol

Satisfactory separations were achieved on Sunfire C₁₈ 250 mm x 4.6 mm, 5µ (Make: Waters) column with 0.1% OPA in water and 0.1% OPA in acetonitrile as mobile phases A and B, column oven temperature as 45 °C, flow rate 1.0 ml/min with injection volume 20 µl. The choice of monitoring UV wave length 220 nm was prasugrel and most of its related substances show maximum UV absorbance at 220 nm. The gradient elution given as follows. Time (min)/ A (v/v): B(v/v); T0.01/85:15, T17/80:20, T29/65:35, T45/25:75. For the synchronous determination of the pharmaceutical types of prasugrel as well as Fluoro imp in a specific, simple and exact inverse phase fluid chromatographic method has been created according to ICH rules, since the simultaneous determination of Prasugrel as well as bromo imp has not had the consolidated approach available.

Specificity:

The blank and standard solutions were injected and found that there is no interference. The peak of Prasugrel hydrochloride and its impurities retention time were found at 3.28 min and 6.61 min. The HPLC chromatograms of Blank, Prasugrel hydrochloride standard, bromo impurity standard and Prasugrel hydrochloride and bromo impurity sample were shown in Fig. 2, Fig. 3

Figure 2: Blank Chromatogram of Prasugrel hydrochloride

Figure 3: Chromatograms of mixture of Prasugrel hydrochloride (1) and 1-bromo-1-(2-fluoro phenyl) propan-2-one (2)

Linearity:

The peak areas were observed from 8 to 48 μg/mL for Prasugrel hydrochloride and 25 to 150 μg/mL for 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone respectively, which were displayed in the Table 1. Linearity between 8 and 48 μg/mL was acquired for both prasugrel hydrochloride and fluoro impurity. Calibration graph was plotted for concentration and absorbance. The correlation coefficient (r) and equation of calibration curve obtained were 0.9994 and y = 40137 x + 3923 for Prasugrel hydrochloride. The correlation coefficient (r) and equation of calibration curve obtained were 0.9993 and y = 15360x + 1737 for 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone. The results were shown in Fig. 4 and Fig. 5.

Table 1: Linearity Data Prasugrel hydrochloride, 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone

S. No.	Prasugrel hydrochloride			2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone
S. No.	Conc. (µg/mL)	Rt (min)	Area	Conc. (µg/mL)	Rt (min)	Area
1	8	2.304	311978	25	2.789	365409
2	16	2.307	652642	50	2.796	787688
3	24	2.307	976516	75	2.801	1183751
4	32	2.309	1318948	100	2.807	1532622
5	40	2.308	1582766	125	2.807	1882622
6	48	2.306	1927579	150	2.805	2324326
	r=0.9994			r=0.9993
	y=40137x+3923			Y=15360x+1737

Figure 4: Graph represent Linearity of Prasugrel hydrochloride

Figure 5: Graph represent Linearity of 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone

This was performed by preparing six standard solutions of the same concentration level (100 %) and infused into the HPLC as indicated by the method. The % RSD was found to be < 0.69 and < 0.77 for Prasugrel hydrochloride and 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone was shown in Table 2.

Table 2: Precision Details Prasugrel hydrochloride and 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone

S. No	Prasugrel hydrochloride			1-(bromomethyl)-2-fluorobenzene
S. No	Conc. (µg/mL)	Rt (min)	Area	Conc. (µg/mL)	Rt (min)	Area
1	32	2.323	1325737	100	2.719	1582794
2	32	2.326	1333591	100	2.72	1586610
3	32	2.328	1327928	100	2.723	1581968
4	32	2.333	1322519	100	2.732	1572724
5	32	2.335	1315862	100	2.733	1564518
6	32	2.337	1308550	100	2.747	1599699
Mean			1322365			1581386
SD			8961			12038
%RSD			0.68			0.76

Table 5: Robustness Details with Differentiation in Rate of Flow of Prasugrel hydrochloride, and 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone

S. No		Prasugrel hydrochloride			2-bromo-1-cyclopropyl-2-(2-fluoro phenyl)ethanone
	Rate of Flow (mL/min)	Peak Area (Mean)*	Std. Dev.	RSD (%)	Average Peak Area*	SD	%RSD
1	0.9 mL/min	1336364	9066	0.68	1596606	12580	0.79
2	1.0 mL/min	1329442	6762	0.51	1601908	14006	0.87
3	1.1 mL/min	1326214	9672	0.73	1596866	7328	0.46

*n=3 (Average of 3 Determinations)

Table 6: Robustness Details with Modification in Composition of Mobile Phase of Prasugrel hydrochloride, 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone

S. No		Prasugrel hydrochloride			2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone
S. No	Mobile Phase Variation (% v/v/v)	Average Peak Area*	SD	% RSD	Average Peak Area*	SD	% RSD
1	M.P-1-(BUFFER: ACN:51:49)	1322739	7875	0.60	1601123	15335	0.96
2	M.P-2 (BUFFER: ACN:50:50)	1326995	8770	0.66	1600378	16623	1.04
3	M.P-3 (BUFFER: ACN:49:51)	1336451	10751	0.80	1607058	6638	0.41

*n=3 (Average of 3 Determinations)

Table 7: Prasugrel/bromo impurity Ruggedness Data Change Day

S. No.	Precision (Inter-Day)
	Prasugrel			Fluoro impurity
		Area of Peak			Area of Peak
	conc. (µg/mL)	First Day	Second Day	conc. (µg/mL)	First Day	Second Day
1	32	1336934	1335638	100	1599202	1635929
2	32	1327283	1342833	100	1610292	1594633
3	32	1339474	1329383	100	1603848	1614182
4	32	1345849	1291132	100	1624122	1622901
5	32	1323938	1310293	100	1585393	1600322
6	32	1312384	1334842	100	1613033	1633932
Mean		1330977	1324020		1605982	1616983
SD		12147	19518		13204	17124
%RSD		0.91	1.47		0.82	1.06

Table 8: Prasugrel /bromo impurity Ruggedness Data Change Instrument

No	Instrument to Instrument
	Prasugrel hydrochloride			2-bromo-1-cyclopropyl-2-(2-fluorophenyl)ethanone
		Area of Peak			Area of Peak
	conc. (µg/mL)	Instrument 1	Instrument 2	conc. (µg/mL)	Instrument 1	Instrument 2
1	32	1334847	1336282	100	1584283	1593832
2	32	1335838	1332837	100	1598822	1612922
3	32	1331934	1327927	100	1612838	1581973
4	32	1322931	1331983	100	1623848	1600283
5	32	1332482	1327282	100	1591344	1622833
6	32	1343013	1330484	100	1613939	1595752
Mean		1333508	460183		1604179	1601266
SD		6526	3337		15142	14559
%RSD		0.49	0.73		0.94	0.91

GC-MS parameters:

Instrumentation:

Gas Chromatograph: Agilent Technologies, 7890A Network GC system or its equivalent.

Mass Spectroscopy: Agilent Technologies, 7000 GC-MS Triple Quad detector or 5975C inert XL EI/CI MSD with Triple-Axis detector or its equivalent. Data handling system: Agilent, Mass Hunter or MSD chemstation software or its equivalent. Column: DB-5 capillary column with 30 meters length, 0.32 mm internal diameter and 1.0 µm film thickness or its equivalent.

Auto sampler parameters:

Solvent A-Pre washes (Methanol):6; Solvent B- Pre washes (Methanol): 6; Sample washes 6; Sample pumps:6; Solvent A- Post washes (Methanol):6; Solvent A- Post washes (Methanol):6

GC Parameters: Carrier Gas: Helium; Flow: 1.5 mL/min; Injector temperature: 250 ˚C, 260 ˚C; Injection volume: 1.0 µL; Split mode: (5:1); Oven temperature -1:100 ˚C, 150 ˚C; Hold time^-1: 0.0 min; Rate-1: 15, 25˚C/min; Oven temperature-2: 220 ˚C, 280 ˚C; Hold time-2: 0.0, 7.0 min; Total run time: 15 min.

MS Parameters: Ionization Mode: EI; Scan type: MRM (Multiple reaction monitoring); Channels: Fluoro impurity (Precursor Ion-110), (Product Ion-84), Bromo impurity, Dibromo impurity, Methyl bromo Impurity (Precursor Ion-187), (Product Ion-108). Collision energy 20.0 V, Collision gas (N2): 1.50 mL/min, Quench Gas (He): 2.25 mL/min, MS1/MS2 resolution: Wide, MS Source temp: 230 ˚C, MS Quad temp: 150 ˚C, Aux-2 temp: 280 ˚C, Dwell (msec): 6-12 cycles/s, Solvent delay: 1.2-2 min, MS off: 11.0 min.

Experimental section:

Chemicals, Equipment and Conditions: The investigated samples, Prasugrel hydrochloride reference sample, Prasugrel hydrochloride drug substance and its related substances: 1,1-dibromo-1-(2-fluorophenyl) propan-2-one, 1-(bromomethyl)-2-fluorobenzene, 2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone, 1-bromo-1-(2-fluorophenyl) propan-2-one (25 ppm) (limit) were gifted from Mylan Laboratories Ltd., Hyderabad. A gas chromatograph 7000 (Agilent) equipped with an electronically controlled splitless injection port and coupled with a triple quadrupole inert mass selective detector (5975C, Agilent) with electron impact ionization chamber and 7890A Network GC system or its equivalents was used for the GC-MS analysis. GC separation was performed on a DB-624 capillary column (60 m× 0.25 mm × 1.4 𝜇m) (J and W Scientific). Helium was the carrier gas with a constant pressure of 25.6 psi. About 1𝜇L of the sample solution was injected in splitless mode at 280 ˚C. The initial temperature of the oven was 150 ˚C and ramped with a rate of 10 ˚C per minute until achieving 250 ˚C. The temperature was held at 280 ˚C for not less than 11 min. Mass spectrometric parameters were set with electron impact ionization energy of 69.9 eV, ion source temperature of 230 ˚C, and MS quadrupole temperature of 150 ˚C. Mass parameters used as Dwell (msec); 6-12 cycles/s, Solvent delay 1.2-2 min, and Collision energy 20.0 V, Collision gas (N₂) 1.50 mL/min, Quench Gas (He) 2.25 mL/min. The MS system was routinely set in selective ion monitoring (SIM) mode. Analytical reagent (AR grade) Orthophosphoric acid and acetonitrile (HPLC grade) were procured from Merck (India) limited and pure milli-Q water was used with the help of millipore purification system (Millipore®, Milford, MA, USA).

Preparation of 4% Sodium chloride solution: weight and transfer about 20 grams of sodium chloride into a 500 mL volumetric flask, dissolve and dilute to volume with Milli-Q-Water.

Diluents: Methanol, Dichloromethane (MDC)

Standard stock solution preparation: Accurately weight and transfer about 25 mg of Fluoro imp, Methyl bromo imp, Bromo imp and Dibromo imp standards into a 25 mL volumetric flask. Dissolve 2.5 mL of above solutions to 25 mL with methanol individually. Further dilute 1.25 mL of above solution to 50 mL with methanol.

Standard solution preparation: Transfer about 5.0 mL of water (Milli-Q) and 5.0 mL of sodium chloride solution into 100 mL separating funnel, to this add 1 mL standard stock solution, then add 5.0 mL of MDC and shake vigoursly for about 1 min. Allow the layers to separate and collect the organic layer into a 20 mL vial or flask. Further, add another 5.0 mL of MDC, shake well for about 1 min. Allow layers to separate, collect and transfer the organic layer (MDC) into the same 20 mL vial of flask. Remove the moisture from MDC layer by adding about 500 mg of sodium sulphate. Transfer this solution to a 100 mL volumetric flask and make up the volume to mark with MDC and inject this solution.

Sample preparation:

Transfer about 5.0 mL of water (Milli-Q) into a 100 mL separating funnel, to this transfer about 100 mg of Prasugrel Hydrochloride, and shake well until the sample dissolves, then and 5.0 mL of 4% sodium chloride solution and shake well, then add 5.0 mL of MDC and shake vigoursly for about 1 min. Allow the layers to separate, collect the organic layer into a 50 mL vial or flask. Further, add another 5.0 mL of MDC, shake well for about 1 min. Allow layers to separate, collect and transfer the organic layer (MDC) into the same 20 mL vial or flask. Remove the moisture from MDC layer by adding about 500 mg of sodium sulphate. Transfer this solution to a 10 ml volumetric flask and make up the volume to the mark with MDC and inject this solution.

Blank Preparation:

Follow the procedure given in sample preparation without Prasugrel HCl sample. Evaluation of system suitability: Inject blank followed by standard preparation six times into the gas chromatograph with mass spectroscopy and record the chromatograms. The % relative standard deviation for the peak area of dibromo impurity should be not more than 15.0

Procedure: Inject blank followed by sample preparation into the gas chromatograph with mass spectroscopy. Record the chromatograms and note down the peak area response of dibromo impurity in the sample solution based on the retention time obtained in standard preparation chromatogram. The retention time of Fluoro imp, Methyl bromo imp, bromo imp and dibromo impurity is about 4.7, 7.0, 8.9 and 3.4 min respectively.

CALCULATION:

Dibromo impurity (ppm) = AT X CS X P X 10000

AS CT

Where,

AT: Area counts of dibromo impurity peak in the chromatogram of the sample preparation.

AS: Average area counts of dibromo impurity peak in the chromatogram of the standard preparation.

CS: Concentration of dibromo impurity in the standard preparation (mg/mL).

CT: Concentration of sample preparation (mg/mL).

P: Purity / potency of dibromo impurity standard (%).

Based on the available maximum daily dose (60 mg/day) of Prasugrel HCl and per the threshold of toxicological concern (TTC) consideration, the toxicity or genotoxic limit is set as 25 ppm. The analysis was carried out on a stainless-steel column 250 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5 µm diameter [Sunfire C18, 250 mm × 4.6 mm, 5 µ (Make: Waters)] maintained at temperature 45 °C. Mobile phase A was prepared by mixing 1 ml of orthophosphoric acid in 1000 ml of water and mobile phase B was prepared by mixing 1 ml of orthophosphoric acid in 1000ml of acetonitrile. Diluent was prepared by mixing water and acetonitrile in the ratio of 60:40%v/v. Flow rate was kept as 1.0 mL/min, injection volume was 20µl, chromatographic data acquisition time was 55 min and UV detection was carried out at 220 nm. Retention time of prasugrel is about 22 min. The pump was in gradient mode and the program was as follows: Time (min)/ A (v/v): B(v/v); T0.01/85:15, T17/80:20, T29/65:35, T45/25:75, T55/10:90, T57/85:15, T65/85:15.

Preparation of solutions:

(a) System suitability solution: Prepare 1 mg/mL solution using Prasugrel hydrochloride enriched with 3-Fluoro prasugrel reference sample with diluent. Filter through 0.45 µ or finer porosity membrane filter. (b) Standard solution: Prepare 1.5 µg/mL using Prasugrel hydrochloride reference sample with diluent. Filter through 0.45 µ or finer porosity membrane filter. (c) Sample solution: Prepare 1 mg/mL using Prasugrel hydrochloride sample with diluent. Filter through 0.45 µ or finer porosity membrane filter.

Suitability requirements: The column efficiency as determined from the Prasugrel peak is not less than 20000 USP plate counts, USP tailing for the same peak is not more than 2.0 and USP resolution between Prasugrel and 3-Fluoro Prasugrel peak is not less than 2.5 obtained from system suitability solution. Relative standard deviation (RSD) for peak areas of prasugrel obtained from six injections of the standard solution is not more than 5.0%.

CONCLUSIONS:

The GC-MS, HPLC method developed for the determination of Prasugrel hydrochloride and its related substances is specific, sensitive, linear, precise, accurate and rugged. The method was fully validated, showing satisfactory data for all the method validation parameters tested. The developed method is stability indicating and can be very useful for quality monitoring of regular production samples and can also be employed to check the quality during stability studies.

ACKNOWLEDGEMENT:

One of the Author (EM) is thankful to our Research Supervisor Pilli VVN Kishore for providing us required facilities and motivation for completion of the research work. We also extend our gratitude towards Department of Sciences and Humanities, VFSTR University.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interests

REFERENCES:

1. Andre, P.; Delaney, S. M.; LaRocca, T.; Vincent, D.; DeGuzman, F.; Jurek, M.; Koller, B.; Phillips, D. R.; Conley, P. B. J. Clin. Invest. 2003; 112, 398;

2. (a) Antman, E. M.; Anbe, D. T.; Armstrong, P. W.; Bates, E. R.; Green, L. A.; Hand, M.; Hochman, J. S.; Krumholz, H. M.; Kushner, F. G.; Lamas, G. A., et al. Circulation 2004, 110, 82; (b) Smith, S. C., Jr.; Feldman, T. E.; Hirshfeld, J. W., Jr., et al. Circulation 2006, 113, 166;

3. Wiviott, S. D.; Braunwald, E.; McCabe, C. H., et al. N. Engl. J. Med. 2001, 2007, 357.

4. http://en.wikipedia.org/wiki/Prasugrel

5. http://www.rxlist.com/effient-drug.html

6. Baker W L and White C M, Role of Prasugrel, a Novel P2Y12 Receptor Antagonist, in the Management of Acute Coronary Syndromes, American Journal of Cardiovascular Drugs, 9 (2009) 4, 213-229.

7. Williard H H, Merit L L, Dean F A and Settle F A. Instrumental methods of analysis, C.B.S. Publishers, New Delhi, 7th Edn, 2002, 1036-1058.

8. Srikanth I, Sharmila P, Vijaya bharathi K, Raju M, Lakshma M, Nagarjuna K A. Validated Reverse Phase HPLC Method for the Estimation of Prasugrel Hydrochloride in Pharmaceutical Dosage Forms, J Inno trends Pharma Sci, 2(5), 2011, 140-148.

9. Usha Rani G, Chandrasekhar B, Devanna N. Analytical Method Development and Validation of Prasugrel in Bulk and its Pharmaceutical Formulation using HPLC, J pp Pharma Sci, 01(07), 2011, 172-175.

10. Borole T C, Mehendre R, Damle M C, Bothara K G. Development and Validation of Stability indicating HPTLC Method for Determination of Prasugrel, J Che, Pharma Res, 2(4), 2010, 907- 913.

11. Kishore R, Venkateswara R A, Lavanya P, PaniKumar A D, Rama Krishna, Subba Reddy P V. Development of Validated RP-HPLC Method for the Estimation of Prasugrel HCl in Pure and Pharmaceutical Formulations, Pharmacy Res, 4(9), 2011, 3105-3110.

12. Pulla R P, Sastry B S, Prasad Y R, Raju N A. Estimation of Prasugrel in Tablet Dosage Form by RP-HPLC, Int J Chem Res, 2(3), 2011, 34-36.

13. R.L. Grob, E.F. Barry, Modern Practice of Gas Chromatography, 4^th ed., Wiley Interscience- John Wiley and Sons, New Jersey, 2004.

Received on 17.04.2024 Modified on 03.05.2024

Asian J. Research Chem. 2024; 17(2):103-110.

DOI: 10.52711/0974-4150.2024.00021

No		Prasugrel hydrochloride			2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone
No	Conc	Added Amount (µg/mL)	Amount Available (µg/mL)	*%Recovery (Mean) + %RSD**	Added Amount (µg/mL)	Amount Available (µg/mL)	*%Recovery (Mean) + %RSD**
1(n=6)	50%	8.00	8.01	100.06 + 0.19	25.00	24.89	99.56+ 1.22
2(n=6)	100%	16.00	16.06	100.40 + 0.84	50.00	50.41	100.81 + 0.71
3(n=6)	150%	24.00	23.81	99.23 + 0.98	100.00	74.61	99.48 + 0.97

S. No	Parameter	Prasugrel	Fluoro impurity
1	LOD	0.990	2.586
2	LOQ	2.999	7.837

S. No.	Sample		Prasugrel hydrochloride		Bromo impurity
S. No.	Sample	Label	Amount Found	%Purity + RSD*	Amount Found	%Purity + RSD*
1	CELAR	8mg/ 25 mg	8.02	99.81 + 1.07	24.92	99.29 + 0.60

	Prasugrel hydrochloride		2-bromo-1-cyclopropyl-2-(2-fluorophenyl) ethanone
	%Assay	Degraded	%Assay	Degraded
Acid	95.07	4.93	94.83	5.17
Alkaline	96.74	3.26	95.73	4.27
Oxidation	100.92	0.92	96.94	3.06
Dry Heat	97.79	2.21	98.15	1.85
Photo Stability	99.01	0.99	98.77	1.23
Neutral	99.41	0.59	99.57	0.43

Impurities	Structure	MF	M.Wt	Nature	Miscible
5-(2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c] pyridin-2-yl acetate hydrochloride		C₂₀H₂₁ClFNO₃S	409.90		methanol/ MDC
1-(bromomethyl)-2-fluorobenzene		C₇H₆BrF	189.02	Colourless liquid	methanol/ MDC
2-bromo-1-cyclo propyl-2-(2-fluorophenyl) ethanone		C₁₁H₁₀BrFO	257.10	Dark Brown liquid	Methanol/ MDC
1-bromo-1-(2-fluoro phenyl) propan-2-one		C₉H₈BrFO	231.06	Dark Brown liquid	Methanol/ MDC
1,1-dibromo-1-(2-fluorophenyl) propan-2-one		C₉H₇Br₂FO	310	Dark Brown liquid	Methanol/ MDC