1. INTRODUCTION:

The World Health Organization claims (WHO) data published on 2000, the leading cause of global death was due to cardiovascular ailments. Sixteen percentage of worldwide death resulted from ischemic heart diseases (IHD). From 2019 to 2020, there was 2 million increase in death observed for IHD and 8.9 millions total death worldwide was recorded. Hence, it is essential to manage both pre and post myocardial infracted patients with lifelong therapy by the use of multidrug regimens. In order to achieve better patient compliance, cost effectiveness and frequency of dosing, multicomponent formulation is considered as therapeutically best option. Literature survey revealed that statins and β-blockers showed physical, chemical and therapeutic compatibilities.

Hence, Pravastatin and Nebivolol are selected for multi component analysis in bulk form. Pravastatin sodium belongs to Statin category and Nebivolol Hydrochloride is selective β₁-blocker. Pravastatin sodium (Fig. 1) is a statin-class drug that reduces cholesterol production by inhibiting Hydroxy Methyl Glutaryl Coenzyme A. (HMG-COA). It's used to control abnormal cholesterol levels and lower the chance of atherosclerotic plugs forming in the coronary arteries and other blood vessels. Because of the inhibitory mechanism of action, cholesterol production is reduced, resulting in an increase in the number of LDL receptors on cell surfaces, as well as improved LDL metabolism and clearance via receptor-mediated metabolism.

Figure 1: Chemical Structure of Pravastatin sodium

Nebivolol (Fig. 2) is a β-blocker that is used to treat high blood pressure and heart failure. Nebivolol is a highly selective antagonist for β1-adrenergic receptors with a limited affinity for β2-adrenergic receptors. It binds to the hearts’s 1 adrenergic receptors and competes with them, decreasing cardiac contractility and rate. This lowers blood pressure and cardiac output at the same time. Additionally, nitric oxide (NO) generation is increased by nebivolol, which relaxes vascular smooth muscle and has a vasodilatory effect.

Figure 2: Chemical structure of Nebivolol HCl

According to a review of the literature, there are no effective methods for simultaneously estimating Pravastatin sodium and Nebivolol Hydrochloride in bulk form and formulation. Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) was used to establish easy, precise, accurate, and cost-effective procedures for estimating Pravastatin sodium and Nebivolol hydrochloride in bulk medication.

2. EXPERIMENTAL WORK^6-9

2.1 Chemicals and reagents:

HPLC grade Acetonitrile, o-phosphoric acid, HPLC grade methanol, water, pH buffer capsule 4.0, 7.0, 9.2, phosphate buffer, THF, Pravastatin sodium and Nebivolol hydrochloride.

2.2 Instrumentation:

HPLC (Shimadzu) prominent LC 20 AD, manual sampler, software LC solution and detector (UV-visible), column C-18, Thermo Scientific octadecylsilane Hypersil (ODS), ultrasonicater, vacuum filter, and analytical balance. UV –spectrophotometer Lab India 3000+ with UV win software was used to determine the lambda max and iso-absorptive point.

2.3 Selection of wavelength:

The iso-absorptive wavelength was determined by using double beam UV visible spectrometer. For wavelength selection, a 20 μg/mL concentration of Pravastatin sodium and a 20 μg/mL concentration of Nebivolol HCl were dissolved in 20:80 ratio of ACN to water, and overlain spectra were recorded. The results of the trials revealed 257 nm was most suitablewavelength for the analysis.

2.4 Selection of chromatographic condition:

A reversed phase isocratic high performance liquid chromatography with a mobile phase of acetonitrile and water in the ratio of 20:80 was used to achieve chromatographic separation at ambient temperature. At a rate of 1.0 mL/min, the mobile phase was pumped. The detector wavelength was set at 257 nm, with a 15-minute run time.

The effect of chromatographic factors like mobile phase pH and flow rate was investigated in order to optimize chromatographic conditions. The chromatographic responses were assessed and the resulting chromatograms were recorded.

2.5Analytical Method Validation:

A calibration curve was constructed with Pravastatin sodium concentrations ranging from 20 to 100 g/ml and Nebivolol hydrochloride concentrations ranging from 10 to 50 g/ml. The approach was proposed and validated in compliance with ICH guidelines. The linearity, accuracy, precision (intraday and interday precision and repeatability), and robustness of the parameters were evaluated, and the quantity recovery, percentage recovery, and mean recovery for each were calculated.

2.6 Preparation of standard stock solutions:

Pravastatin standard stock solution:

The HPLC grade sample solution was weighed and transported to a 100 mL volumetric flask, where it was dissolved in the standard Pravastatin sodium (ACN: Water in the ratio 20:80). The flask was shaken and sonicated for 30 minutes at 45°C, and the volume was filled to the desired amount with solvent. 10 mL of the solution was pipetted into a 100 mL volumetric flask, which was then filled with solvent to a capacity of 100 mL. The concentration of the prepared stock solution was 100 g/ml.

Nebivolol standard stock solution:

The HPLC grade sample solution was weighed and transported to a 100 mL volumetric flask, where it was dissolved in the standard Nebivolol hydrochloride (ACN: Water in the ratio 20:80). The flask was shaken and sonicated for 30 minutes at 45°C, and the volume was filled to the desired amount with solvent. 10 mL of the solution was pipetted into a 100 mL volumetric flask, which was then filled with solvent to a capacity of 100 mL. The concentration of the prepared stock solution was 100 g/ml.

Chromatographic conditions:

The mobile phase was made up of Acetonitrile: water (20:80) was used and absorbance was measured at 257 with the run time 15 min and the flow rate was set at 1.0 ml/min respectively.

Preparation of mobile phase:

The mobile phase was made by mixing HPLC grade acetonitrile and water in a 20:80 ratio, and the chromatographic conditions were set for drug separation at a wavelength of 257nm. Before using the mobile phase, degassing is performed.

3. RESULT AND DISCUSSION:

3.1: Overlay Spectra of Pravastatin sodium and Nebivolol HCl: An Overlay spectra of both drug were carried out in 20:80 ratio of ACN to water as solvent, the is absorptive point was recorded at 257nm.(Figure-3)

Figure 3: Overlain spectra of Pravastatin sodium and Nebivolol HCl

3.2 Characterization of drugs (Pravastatin sodium and Nebivolol HCl) by IR- spectroscopic method:

3.2.1 IR spectra of Pravastatin sodium:An IR spectrum of Pravastatin sodium (Fig: 4) was taken by KBr press pellet technique using IR Affinity-1 spectrophotometer of Shimadzu. The pellet was prepared with the help of dried KBr. The spectrum was obtained and interpretated result was shown in Table-1.

Fig. 4. IR spectra of Pravastatin sodium

Table 1: IR interpretation of Pravastatin Sodium

Functional group	Observed frequency
O-H stretch	3300.51
CH₃ stretch	2965.72
CH₂stretch	2880.94
C=C stretch	1577.69
CH₂ bending	1399
C=O stretch	1727.13
C-O stretch	1040.30
CH bending	824.77

3.2.2 IR spectra of Nebivolol HCl: Similarly for Nebivolol HCl IR spectrum (Fig: 5) has been obtained and data was interpretated and result was shown in Table-2.

Fig. 5. IR spectra of Nebivolol HCl

Table 2: IR interpretation of Nebivolol HCl

Functional group	Observed frequency
O-H Streching	3217.17
C=C Aromatic	1620.80, 1563.32
C-F Stretching	1074.79
C-H stretching	2901.06
C-N stretching	1258.71
C-O stretching	1074.79

3.3 Optimized Method development^10-11

The composition of the mobile phase was investigated and optimised. Separation was found to be adequate using acetonitrile and water pH 3 in a 20:80 % v/v ratio. UV detection was performed at 257 nm, which is the wavelength at which both drugs absorb the most. The isocratic mode was chosen since the retention time for both medicines was less than 6 minutes at a flow rate of 1ml/min. Pravastatin sodium and Nebivolol hydrochloride have retention times of 2.092 and 3.623 minutes, respectively. Figure 6 depicts the chromatogram of the optimised trial.

Figure 6: At wavelength 257 nm, optimised chromatogram of standard drug containing 20 g/ml Pravastatin sodium and 20 g/ml Nebivolol HCl.

3.4 Validation of the developed method:

3.4.1 Linearity curve for the Pravastatin sodium ^12-13

For Pravastatin sodium, 2, 4, 6, 8, and 10 mL of standard stock solution (100 g/mL) were pipetted out and transferred to separate 10 mL volumetric flasks, and the volume was made up to mark using solvent. These concentrations were 20, 40, 60, 80, and 100 µg/ml respectively.

The linearity was obtained in a few conc. ranges. Table 3 depicts the linearity of Pravastatin sodium, and Figure 7 depicts the calibration plot.

Table 3: Pravastatin sodium Linearity curve at wavelength 257 nm.

Sr. No	Conc. (µg/mL)	Area (µ volt sec.)
1	20	1591623
2	40	3154587
3	60	4755761
4	80	6216789
5	100	7687225

Figure 7: Calibration plot of Pravastatin sodium at 257nm

3.4.2 Linearity curve for the Nebivolol HCl:

For Nebivolol HCl, 1, 2, 3, 4, and 5 mL of standard stock solution (100 µg/mL) were pipetted out and transferred to separate 10 mL volumetric flasks, and the volume was made up to mark using solvent. These concentrations were 10, 20, 30, 40, and 50 µg/ml respectively.

The linearity was obtained in a few conc. ranges. Table 4 depicts the linearity of Nebivolol hydrochloride, and Figure 8 depicts the calibration plot.

Table 4: Nebivolol hydrochloride linearity curve at wavelength 257 nm.

Sr. No	Conc. (µg/mL)	Area (µ volt sec.)
1	10	1145671
2	20	2067895
3	30	3245934
4	40	4187325
5	50	5074283

Figure 8: Linearity of Nebivolol hydrochloride at 257nm

3.4.3 Accuracy ^14-15

Accuracy is the proximity of the method's test results to the true value. Accuracy is frequently expressed as a percentage of assay recovery for a known amount of analyte added. Recovery studies were conducted by adding standard drug to the sample at three distinct levels of spiking, namely 80 %, 100%, and 120 % of the actual amount while accounting for the percentage purity of the additional bulk drug sample. Tables 5 and 6 indicate the accuracy of Pravastatin sodium and Nebivolol hydrochloride, respectively.

Table 7. Pravastatin sodium and Nebivolol hydrochloride Intraday Precision at 257 nm

Drug	Pravastatin sodium			Nebivolol hydrochloride
Concentration	40 µg/ml	60 µg/ml	80 µg/ml	20 µg/ml	30 µg/ml	40 µg/ml
Area	3154587	4755761	6216789	2067966	3245934	4187476
	3154615	4755779	6216852	2067895	3245887	4187325
	3154681	4755701	6216825	2067889	3245998	4187388
Mean	3154627	4755747	6216822	2067916	3245939	4187396
SD	48.263	40.841	31.607	42.829	55.717	75.844
%RSD	0.002	0.001	0.001	0.002	0.002	0.002

Table 8. Pravastatin sodium Interday Precision at 257nm

Day	Day 1			Day 2			Day 3
Concentration	40µg/ml	60µg/ml	80µg/ml	40µg/ml	60µg/ml	80µg/ml	40µg/ml	60µg/ml	80 µg/ml
Area (µ volt sec)	3154589 3154645 3154626	4755855 4755707 4755798	6216795 6216870 6216802	3154587 3154615 3154681	4755761 4755779 4755701	6216789 6216852 6216825	3154619 3154591 3154685	4755707 4755853 4755796	6216791 6216869 6216817
Mean	3154620	4755786	6216831	3154627	4755747	6216822	3154631	4755784	6216825
SD	28.478	74.648	57.553	48.263	40.841	31.607	48.263	74.648	39.716
%RSD	0.001	0.002	0.001	0.002	0.001	0.001	0.002	0.002	0.001

Table 9: Nebivolol hydrochloride Interday Precision at 257nm

Day	Day 1			Day 2			Day 3
Concentration	20 µg/ml	30 µg/ml	40 µg/ml	20 µg/ml	30 µg/ml	40 µg/ml	20 µg/ml	30 µg/ml	40 µg/ml
Area (µ volt sec)	2067882 2067959 2067885	3245945 3245891 3245978	4187481 4187330 4187393	2067891 2067923 2067886	3245932 3245885 3245996	4187332 4187415 4187345	2067966 2067895 2067889	3245934 3245887 3245998	4187476 4187325 4187388
Mean	2067908	3245938	4187401	2067900	3245937	4187364	2067916	3245939	4187396
SD	43.616	43.920	75.844	20.075	55.717	44.643	42.829	55.717	75.844
%RSD	0.002	0.001	0.002	0.001	0.002	0.001	0.002	0.002	0.002

Table 10: Pravastatin sodium repeatability at wavelength

Sr. No	Area (µ volt sec.)
1	2067899
2	2067954
3	2067531
4	2067824
5	2067913
6	2067289
Mean	2067735
S D	266.456
%RSD	0.013

Table 11: Nebivolol hydrochloride repeatability at wavelength 257nm257nm

Sr. No	Area (µ volt sec.)
1	3154872
2	3154209
3	3154967
4	3154223
5	3154498
6	3154785
Mean	3154592
S D	331.047
%RSD	0.010

3.4.6 Limit of detection (LOD)^20-21

The lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an accurate value is referred to as the LOD. The slope and the standard deviation of the response were used to determine LOD.

3.3 x σ

LOD = --------------------------

Where, σ = the standard deviation of the

Response

S = the calibration curve's slope

The slope and standard deviations of the linearity curve were determined for concentration ranges of 20-100 µg/mL for Pravastatin sodium and 10-50 µg/mL for Nebivolol HCl.

3.4.7 Limit of quantitation (LOQ)^22-23

It is the smallest amount of analyte in a sample that can be quantified with sufficient precision and accuracy. The slope and the standard deviation of the response were used to determine the LOQ. The LOQ was determined after obtaining the data from the linearity curve.

10 x σ

LOQ = --------------------------

Where, σ = the standard deviation of the response

S = the calibration curve's slope.The slope and standard deviations of the linearity curve were determined for concentration ranges of 20-100 µg/mL for Pravastatin Sodium and 10-50 µg/mL for Nebivolol HCl.

The LOD and LOQ for Pravastatin sodium is 5.611189 and 17.0036, respectively.

The LOD and LOQ for Nebivolol hydrochloride is 6.77271 and 20.52336, respectively.

3.4.8 Robustness^24-25

Robustness is a measure of a method's ability to stay unaffected by minor but deliberate changes in method parameters.

HPLC robustness was tested by varying the wavelength and flow rate. The robustness of a method was determined by changing the wavelength or the flow rate of a mobile phase. A 20 µg/mL injection of the stock solution was produced and recorded. Tables 12 and 13 show robustness data for Pravastatin sodium and Nebivolol hydrochloride, respectively.

Change in Wavelength:

Table 12: Pravastatin sodium and Nebivolol hydrochloride robustness at 257±2 nm wavelength.

Wavelength	Difference	Pravastatin sodium (min.)	Nebivolol HCl (min.)
255	-2	2.012	3.673
257	0	2.046	3.689
259	+2	2.058	3.652

Change in flow rate of mobile phase:

Table 13: Pravastatin sodium and Nebivolol hydrochloride robustness at 257±2 nm wavelengthby changing the flow rate.

Flow rate (ml/min.)	Difference	Pravastatin sodium (min.)	Nebivolol HCl (min.)
0.9	-0.1	1.956	3.541
1	0	1.943	3.650
1.1	+0.1	2.012	3.532

The overall validation parameter data are shown in Table 14. It complied with ICH guidelines.

Table 14: Summary of RP-HPLC validation parameters at 257nm wavelength.

Parameter	Pravastatin sodium	Nebivolol HCl
Linear range in (µg/mL)	20-100	10-50
Regression coefficient (R²)	0.9997	0.9981
%Accuracy	99.38	100.1
Repeatability (n=6)	%RSD NMT 2	%RSD NMT 2
Precision Interday precision Intraday precision	%RSD NMT 2	%RSD NMT 2
LOD	5.611189	6.77271
LOQ	17.0036	20.52336

4. DISCUSSION:

Reversed phase-high performance liquid chromatography (RP-HPLC)

The method was developed for the simultaneous quantification of Pravastatin sodium and Nebivolol HCl using RP-HPLC. All parameters were validated in accordance with ICH recommendations.Pravastatin sodium with Nebivolol HCl achieved method linearity at 257 nm. Pravastatin sodium and Nebivolol HCl had R2 values of 0.9997 and 0.9981, respectively. The R2 value was between 0.995 and 0.999, indicating acceptable linearity.For method accuracy, the percent drug recovery was determined for both medications at wavelength 257 nm at concentrations of 80, 100, and 120 %, and the average recovery was found to be 99.38 % for Pravastatin sodium and 100.1 % for Nebivolol HCl. As for the percentage of drug recovery, it should be kept within limitations, i.e. 100 ± 2%.Interday precision was calculated by making dilutions of 40, 60, and 80 µg/ml conc. for Pravastatin sodium and 20, 30, 40 µg/ml conc. for Nebivolol HCl and responses were obtained at wavelength 257nm. At wavelength 257 nm, on the 1st day, the % RSD was 0.002, 0.001, and 0.001. At wavelength 257 nm on the second day, the % RSD was determined to be 0.001, 0.002, and 0.001. At wavelength 257 nm on the third day, the % RSD was 0.002, 0.002, and 0.001. The % RSD for Pravastatin sodium was reported to be 0.010% at wavelength 257 nm for repeatability. The % RSD for Nebivolol HCl was discovered to be 0.013%, which is within the allowable range. It demonstrates that the approach meets the repeatability criterion. The LOD and LOQ for Pravastatin sodium were calculated using the linearity curve at wavelength 257, and the LOD was found to be 5.611 µg/ml and the LOQ was found to be 17.00 µg/ml. At wavelength 257 nm, LOD for Nebivolol HCl was 6.772 µg/ml and LOQ was 20.52 µg/ml.The approach was proven to be robust when the wavelength was changed to ± 2 mL and the flow rate was changed to ± 0.1 mL/min.

5. CONCLUSION:

Pravastatin sodium and Nebivolol Hydrochloride were successfully separated using the improved RP-HPLC technique. The approach used a 250 x 4.6 mm ODS C-18 (Shim-pack) column with a particle size of 5 m and a mobile phase composition of ACN: Water in a 20:80 ratio. The flow rate was set to 1 ml/min, and the wavelength was measured at 257 nm. Pravastatin sodium and Nebivolol Hydrochloride had retention times of 2.092 and 3.623min, respectively. The % RSD for intraday and interday precision was less than 2. Recovery experiments were used to determine the method's accuracy, which was found to be between 98 % - 102 %.The data for validation parameters is in accordance with the ICH requirements.In the future, the optimised method for the bulk drug can be used for the routine determination of Pravastatin sodium and Nebivolol HCl in bulk drug and formulation.

6. ACKNOWLEDGEMENT:

The author wishes to acknowledge Department of Pharmaceutical Analysis and Quality Assurance, Laureate Institute of Pharmacy, Jawalamukhi, Himachal Pradesh (176031), for providing necessary facilities in this research work.

7. REFERENCES:

1. https://www.betterhealth.vic.gov.au/health/ConditionsAndTreatments/circulatory-system

2. https://go.drugbank.com/drugs/DB00175

3. https://en.wikipedia.org/wiki/Pravastatin

4. https://go.drugbank.com/drugs/DB04861

5. https://en.wikipedia.org/wiki/Nebivolol

6. Beckett AH, Stenlake JB. Text book of practical pharmaceutical chemistry. Part II, CBS, Publishers and Distributors, New Delhi. Pg No158-164. 2005.

7. Lloyed R, Joseph J et al. Practical HPLC method development. 2nd ed. Jhon Willy and Sons, New York. Pg No653. 2004.

8. Hema, Swati Reddy G. A review on new analytical method development and validation by RP-HPLC. Int Res J Pharm Biosci. Pg No41-50. 2017.

9. Ravisankar P, Navya CN, Pravallika D, Sri DN. A review on step by step analytical method validation. ISOR J Pharm. Pg No 7-19. 2015 doi.org/10.22159/ijap.2018v10i6.28279

10. ICH, Q2 Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization of Technical Requirements for Registrations of Pharmaceuticals for Human Use, ICH harmonized Tripartite Guideline, Nov 2005.

11. https://www.ich.org/page/quality-guidlines

12. Shaikh HA, Jain V. Novel Reverse-Phase High-Performance Liquid Chromatography method development and validation for estimation of telmisartan and nebivolol hydrochloride in pharmaceutical dosage form. Asian J Pharm Clin Res. 2018;11(9):431-5doi.org/10.22159/ajpcr.2018.v11i9.27016.

13. Doshi DB, Bhatt PA. Development and Validation of UV Spectrophotometric Methods for the Estimation of Pravastatin Sodium in Bulk and Tablet Dosage Form. Research Journal of Pharmacy and Technology. 2010;3(3):869-71.

14. Seetharaman R, Lakshmi KS. Development and Validation of a Reverse Phase Ultra Performance Liquid Chromatographic Method for Simultaneous Estimation of Nebivolol and Valsartan in Pharmaceutical Capsule Formulation. Journal of Chromatography and Separation Techniques. 2014;5(3):1doi: 10.4172/2157-7064.1000229

15. Patel SR, Patel SM, Patel JI, Patel PU. Simultaneous determination of nebivolol and amlodipine in combined dosage form by derivative spectrophotometry. Research Journal of Pharmacy and Technology. 2011;4(1):109-12.

16. Raju NA, Begum S. Simultaneous RP-HPLC method for the estimation of the emtricitabine, tenofovir disoproxil fumerate and efavirenz in tablet dosage forms. Research Journal of Pharmacy and Technology. 2008;1(4):522-5doi: 10.4103/2229-4708.103876.

17. Vasanthan K, Vijayageetha R, Arcot AS. Method Development and Validation of RP-HPLC Method for Estimation of Nateglinide in Bulk Drug and Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 2010;3(3):804-6

18. Dinda SC, Desireddy RB, Kumar PJ, Chand AS, Rao PS, Kumar BA. Development and validation of RP-HPLC method for qunatitative analysis of Amlodipine besylate in pure and Pharmaceutical formulations. Research Journal of Pharmacy and Technology. 2013;6(2):204-7.Maslarska V. Development, optimization and validation of HPLC method for determination of Pravastatin Sodium in Tablets. Int. J. Adv. Pharmacy, Biol. Chem..2014;3:230-4.

19. Maslarska V. Development, optimization and validation of HPLC method for determination of Pravastatin Sodium in Tablets. Int. J. Adv. Pharmacy, Biol. Chem..2014;3:230-4.

20. Jacob J, Vishal JM. Development and Validation of Analytical Methods for Pravastatin. Research Journal of Pharmacy and Technology. 2013;6(10):1116-20.

21. Dey S, Sarkar S, Malakar J, Ghosh A, Gangopadhyay A, Mazumder B. Spectrophotometric method for simultaneous determination of atenolol and atorvastatin in tablet dosage forms. International Journal of Pharmaceutical and Biomedical Research. 2012; 3:40-3.

22. Raj ND, Kumaravel S, Murugan R, Narayanan SS, Vijayalakshmi R. Reverse phase HPLC method for determination of pravastatin in tablet dosage forms. Int J Res Pharm Sci. 2010;1:187-9.

23. Rao AL, Rajeswari KR, Sankar GG. Spectrophotometric method for the determination of nebivolol hydrochloride in bulk and pharmaceutical formulations. E-Journal of Chemistry. 2010;7doi:10.1155/2010/902697.

24. Meyyanathan SN, Rajan S, Muralidharan S, Birajdar AS, Suresh B. A validated RP-HPLC method for simultaneous estimation of nebivolol and hydrochlorothiazide in tablets. Indian journal of pharmaceutical sciences. 2008;70(5):687doi:10.4103/0250-474X.45420.

25. Pramod K, Amar Deep A, Pooja K, Mahendra Singh A. Method Development and Validation for Multicomponent Analysis of Rosuvastatin Calcium and Losartan Potassium in Bulk Drug by RP-HPLC. Journal of Drug Delivery and Therapeutics.2020;10(5-s):76-84doi https://doi.org/10.22270/jddt.v10i5-s.4341

Received on 11.01.2022 Modified on 03.07.2022

Research J. Pharm. and Tech 2023; 16(5):2477-2484.

DOI: 10.52711/0974-360X.2023.00408

Sr. No.	Recovery Sample			Fortified Sample			% Recovery
	Conc. (μg/ml)	Peak Area	Mean Peak Area	Conc. (μg/ml)	Peak Area	Mean Peak Area
1	20	1292563	1292545	20+40	3854770	3854764	99.41
		1292523			3854742
		1292551			3854781
2	40	2534589	2534586	40+40	5045878	5045881	99.54
		2534583			5045884
		2534587			5045882
3	60	4145638	4145636	60+40	6854803	6854798	99.20
		4145632			6854792
		4145640			6854801

Sr. No.	Recovery Sample			Fortified Sample			% Recovery
	Conc. (μg/ml)	Peak Area	Mean Peak Area	Conc. (μg/ml)	Peak Area	Mean Peak Area
1	10	1145682	1145678	10+20	3445981	3445985	100.2
		1145674			3445989
		1145680			3445987
2	20	2075889	2075885	20+20	4186329	4186325	100.8
		2075880			4186327
		2075887			4186320
3	30	3245936	3245934	30+20	5374285	5374283	99.34
		3245929			5374287
		3245938			5374278