1. INTRODUCTION:

Aspirin (Fig. 1) is chemically designated as 2-acetoxybenzoic acid. It is used as an analgesic, anti-inflammatory and antipyretic [1]. It inhibits cyclooxygenase, which is responsible for the synthesis of prostaglandins and thromboxane hence leading to inhibition of platelet aggregation. It has also been reported that low doses of ASP may be given immediately after a heart attack to reduce the risk of concurrent attack and death of cardiac tissues. Isosorbide 5-mononitrate (Fig. 2) chemically designated as 8-nitrooxy-2,6-dioxabicyclo[3.3.0] can be used in prophylactic treatment of angina pectoris [2] and to reduce the need for sublingual nitroglycerin.

The aforesaid combination is reported for a significant use in prophylaxis of angina and also in the patients needing secondary prevention for myocardial infarction. Moreover the combination also exhibits the ability of interfering with different regulatory pathways which in turn makes it more efficacious and safer therapeutic option and reduces the chances of GI side effects.

Figure.1

Figure.2

An extensive literature review did not revealed any stability indicating RP-HPLC method for simultaneous determination of both the drugs. However there is one HPLC method of determination of ASP and ISMN [3]. Few methods are available to determine both the drugs either alone or in combination with other drugs. For analysis of ASP by RP-HPLC method with UV detection individually [4, 5], by LC-Tandem MS [6] and HPTLC method [7] was reported. Second-derivative UV spectrophotometry determination of salicylic acid and acetylsalicylic acid in aspirin delayed-release tablet [8], Derivative-ratio spectrophotometric method for the determination of ternary mixture of aspirin, paracetamol and salicylic acid [9] HPLC for ISMN in bulk material and extended release formulations [10] by GC-MS [11] supercritical fluid chromatography [12] and Flow-through UV spectrophotometric determination [13] have been reported.

2. EXPERIMENTAL:

2.1. Chemicals, Reagents, Standard and Sample:

Reference standards of ASP and ISMN with stated purity of 99.98 and 99.56% respectively were obtained as a gift sample from RA Chem Pharma Limited (Hyderabad, India). The pharmaceutical dosage form used in this study was Aspitrate^®capsules labeled to contained 150 mg ASP and 30mg of ISMN (Aristo Pharmaceuticals Pvt. Ltd), PAR was obtained from Mankind Pharma Ltd (New Delhi, India), HPLC grade acetonitrile and methanol were obtained from Merck specialties private limited (Mumbai, India), HPLC grade water and analytical grade chemicals were obtained from Qualigen Fine chemicals, (Mumbai, India).

2.2. Instrumentation:

RP-HPLC method development and validation were done on Shimadzu (Japan) liquid chromatograph equipped with LC-20AD pump, LC 20A UV/Vis detector and rheodyne 7725i injector with 20µ L loop. For instrument control, data acquisition and processing, the chromatographic system was interfaced to LC solutions software. The chromatographic separation was performed using reverse phase phenomenex^®Luna 5µ C18 (2) 100A (250 × 4.60mm) column. Shimadzu electronic balance BL -220H (Shimadzu Corporation, Japan.), value 1 stage vaccum pump Model: VE115, fast clean ultrasonic cleaner.

2.3 OPTIMIZATION OF SEPARATION CONDITIONS:

2.3.1 Effect of ratio of mobile phase:

Once a reasonable chromatogram is obtained, optimization can be started. By changing the mobile phase composition slightly, the position of the peaks can be predicted within the range of investigated changes. An optimized chromatogram was the one in which all the peaks are symmetrical and well separated in less run time.

Mobile phase was selected on the basis of best separation, peak purity index, peak symmetry, theoretical plate etc. After number of trials 55:28:17% v/v/v ratio of water: methanol: acetonitrile was selected as a mobile phase.

2.3.2 Effect of flow rate:

By keeping all other parameters of mobile phase system constant, the chromatograms were recorded with different flow rates like 0.8, 1.0 and 1.2 ml/min. With flow rates of 0.8 and 1.2 ml/min, peaks were broad and were not symmetrical. But a flow rate of 1.0 ml/min gave good symmetrical peaks and hence selected for further studies.

2.4. Chromatographic conditions:

The column used for chromatographic separations was reverse phase phenomenex^®Luna 5µ C18 (2) 100A (250 × 4.60 mm) column. The eluents were monitored at 217 nm with a sensitivity of 0.001 AUFS and samples of 20 µl were injected. The chromatographic separation was accomplished using mobile phase consisting of water: methanol: acetonitrile (55:28:17% v/v/v), filtered through 0.45 µ m membrane filter using value 1 stage vaccum pump and deaerated in fast clean ultrasonic cleaner. Mobile phase was pumped in isocratic mode at a flow rate of 1 ml/minat ambient temperature.

2.5. Preparation of standard solutions:

Standard stock solutions of ASP (100 µg/ml), ISMN (100 µg/ml) and PAR (100 µg/ml) were prepared in mobile phase.

2.6. Preparation of calibration graph:

Stock solution was diluted with mobile phase to obtain a series of concentrations of ASP and ISMN ranging from 1 to 10 µg/ml and 1 to 10 µg/mlrespectively, each containing 5 µg/mlPAR as an IS. The solutions were analyzed in triplicate. The calibration graph was plotted by using peak area ratios of ASP and ISMN to IS peak area versus concentration of standard solution.

2.7. Preparation of capsules for assay:

Twenty capsules each containing 150mg of ASP and 30mg of ISMN were weighed and average weight was calculated. A quantity equivalent to 10 mg ASP and 2 mg of ISMN was weighed, transferred to a 100 mL volumetric flask, extracted with mobile phase, and made up with the same solvent. This solution was filtered through Whatman filter paper (0.45 µm).The clear solutions obtained were diluted with the same mobile phase to give the final concentration within the linearity range of both the drugs.

2.8. Forced degradation studies:

2.8.1. Acid degradation study:

Acid degradation was performed by subjecting the drug solution to accelerated degradation by refluxing with 0.01N HCl at 70°C. The sampling was done for every 30 min and the volume was made up with methanol. The forced degradation was performed in the dark to exclude the possible degradation effect of light. The resulting solution (5 µg/ml for ASP and 1 µg/ml for ISMN) were injected and chromatograms were recorded.

2.8.2. Base degradation study:

Alkali hydrolysis was performed by subjecting the drug solution to accelerated degradation by refluxing with 0.01N NaOH at 70°C. The sampling was done for every 30 min and the volume was made up with methanol. The chromatograms were recorded as described in the acid induced degradation study.

2.8.3. Hydrogen peroxide-induced (oxidation) degradation study:

Initial oxidation studies were performed in 3% H₂O₂at room temperature for 10 hrs, subsequently the drug was exposed to 6% H₂O₂at room temperature and analyzed periodically. The resultant solution was appropriately diluted and chromatograms were recorded as described in the acid induced degradation study.

2.8.4. Heat degradation study:

For dry heat degradation study, the standard drugs were powdered and kept in an oven at 60 ⁰C for 24 h. Dilutions were prepared in methanol appropriately and then analyzed under the optimized chromatographic conditions.

3. RESULTS:

3.1. RP-HPLC method optimization:

For the RP-HPLC, chromatographic conditions were optimized to get best resolution and peak shape. The selection of mobile phase was based on peak parameters; (symmetry, theoretical plates and capacity factor) ease of preparation and cost. Retention times for ASP is 2.05±0.056 min and ISMN is 4.27±0.016 min were obtained with good separation. It was obtained with reverse phase phenomenex^®Luna 5µ C18 (2) 100A (250 × 4.60 mm) column and mobile phase consisting of water: methanol: acetonitrile (55:28:17% v/v/v) at a flow rate of 1 ml/min. The optimum wavelength for detection and quantification was at 217 nm which was selected for further studies after several preliminary investigatory chromatographic runs. A typical chromatogram obtained from the analysis of drugs using the developed method is shown in Fig. 2. Under the described experimental conditions, all peaks were well defined and free from tailing.

3.2. Validation of the method:

The analytical method was validated with respect to parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), limit of quantitation (LOQ), robustness and ruggedness in compliance with ICH guidelines [14].

3.2.1 Linearity:

The linearity of an analytical procedure is the ability to obtain test results that are directly proportional to the concentration of an analyte in the sample. To check the linearity, standard calibration graph was plotted by using peak area ratios of ASP and ISMN versus concentration of standard solution. The curve showed good linearity over the concentration range of 1-10 µg mL^-1for ASP and 1-10 µg mL^-1for ISMN. The correlation c oeffic ie nt values were found to be 0.999 and 0.998 for ASP and ISMN, respectively. The slope and intercept values are shown in Table 1.

Table 1. ^{Summary of validation parameters for the
proposed method:}

^P^ara^m^e^t^e^r	_ASP	_I_S_MN
_L_i_nearity	_{1 – 10}µg/ml	_{1 – 10}µg/ml
Intercept (c)	4985	376.4
Slope (m)	50695	9161.3
Correlation coefficient	0.9997		0.998
LOD	10 ng/ml		4 ng/ml
_L_O_Q	200 ng/ml		500 ng/ml

3.2.2 Precision:

The precision of analytical procedure determines the closeness of agreement between series of measurement obtained from the multiple sampling of same homogeneous sample under prescribed condition. The Precision of the method was studied in terms of intraday and interday precision of sample injections (5 µg mL^-1). Intraday precision was investigated by injecting six replicate samples of each of the sample on the same day. The % RSD obtained for ASP and ISMN were found to be 0.341 and 0.534 respectively.

Interday precision was assessed by analysis of the 6 solutions on three consecutive days. The % RSD obtained for ASP and ISMN were found to be 0.452 and 0.925 respectively. The results are given in Table 2.

Table 2. Precision studies

Precision	ASP (5 µg/ml)	ISMN (5 µg/ml)
_I_{nterday precision (n=3),% *RSD}	0.452	0.925
_I_{ntraday precision (n=6), % *RSD}	_0.341	_0.534

* Mean of six observations

3.2.3. Accuracy:

To study the accuracy of the methods, recovery studies were carried out by spiking of standard drug solution to preanalyzed sample at three different levels: 80, 100, and 120%. The resultant solutions were then reanalyzed by the proposed methods. Weight equivalent to 10 mg and 2 mg of ASP and ISMN respectively was taken and dissolved in 100 ml of mobile phase. To the above solution 8 mg of standard ISMN was added to make the concentration equivalent to 100 µgmL^-1 for both the drugs (Standard addition method). It was done by mixing known quantity of standard drugs with the analyzed sample formulation and the contents were reanalyzed by the proposed method. At each level of the amount, six determinations were performed. The mean recoveries obtained were in the range of 98-101% for both the drugs. The results are given in Table 3.

3.2.4. Specificity:

3.2.4.1 Placebo interface:

To establish the interface of tablet excipients (placebo), a study has been conducted and assay was performed on placebo (lactose monohydrate, starch, purified talc and aluminum stearate) in triplicate equivalent to the weights of placebo in the portion of test method. At the retention times of ASP and ISMN, there were no additional peaks observed in the chromatogram, this indicates that the excipients employed in the formulation do not interfere in the estimation of ASP and ISMN. (Fig. 4)

3.2.5. LOD and LOQ:

Limit of detection (LOD) and Limit of quantitation (LOQ) values were determined by the signal-to-noise (S/N) ratio. LOD is the concentration of the analyte that give signal-to-noise (S/N) ratio of 3:1 at which analyte can be readily detected. LOQ is the concentration of the analyte that give a signal-to-noise (S/N) ratio of 10:1 at which analyte can be readily quantified with accuracy and precision. The limits of quantification (LOQ) for ASP and ISMN was found to be 200 ng mL^-1and 500 ng mL^-1and limit of detection (LOD) for ASP and ISMN was found to be 10 ng mL^-1and 4pg mL^-1respectively, Fig 3.

3.2.6. Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Robustness of the method was investigated under a variety of conditions including changes in composition of mobile phase and flow rate. % RSD of assay was calculated for each condition. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust Table 4 and 5.

Figure.3 A typical chromatogram of Standard ASP (7 µg/ml), ISMN (7 µg/ml) and PAR (5 µg/ml) measured at 217 nm

Figure.4 A typical chromatogram of Blank

_{Table 3. Accuracy (Recovery studies)}

Table 4. Robustness studies

_P_ara_m_e_t_er_s	_Rt₍_mi_n)
Flow rate (ml/min)	ASP	ISMN
0.9	2.326	4.685
1.0	2.056	4.276
1.1	_1.854	_3.562

Table 5. Robustness studies

_P_ara_m_e_t_er_s	_Rt₍_mi_n)
Mobile phase Composition %v/v/v (Water: Methanol: Acetonitrile)	ASP	ISMN
53:30:17	_2.142	_4.524
_55:25:20	_2.075	_4.582
_52:28:20	_2.152	_3.562

3.2.7. Ruggedness:

The ruggedness of the method was assessed by comparison of the intra-day and inter-day assay results for ASP and ISMN that has been performed by two analysts. The % RSD values for assays performed in the different laboratory by two analysts did not exceed 2, indicating the ruggedness of the method Table 6.

Table 6. Ruggedness studies:

Drugs Peak area	% RSD
_{Analyst 1 Analyst 2}
_{ASP 441348 442348}	_0.7456
ISMN 79525 79425	0.9687

* mean of six observations

3.3. Forced degradation studies:

Analysis of samples obtained upon stress testing of ASP and ISMN under different conditions using the optimized HPLC conditions suggested the following degradation behavior:

3.3.1. Acid-induced degradation study:

It was found that around 40% of the ASP degraded upon heating in 0.01N HCl for 3 h at 70°C, forming degradation products with Rt of 2.63 and 3.06 min (Figure). The rate of hydrolysis in acid was faster when compared to that in alkali. For ISMN, additional peaks were observed with Rt of 5.42, 6.45 and 7.4 respectively (Fig. 5a).

Figure.5 (a) A typical chromatogram of ASP, ISMN and degradation products in the acid induced degradation study

3.3.2. Alkali-induced degradation study:

In alkali induced degradation study, it was observed that around 20% of ASP and ISMN degraded upon heating in 0.01N NaOH for 9 h at 70°C ASP and ISMN (figure 5b)

Figure.5 (b) A typical chromatogram of ASP, ISMN and degradation products in the alkali induced degradation study

3.3.3. Hydrogen peroxide-induced (oxidation) degradation study:

In oxidative conditions, 20% degradation of the ASP and ISMN was obtained after exposure to 6% H₂O₂ for 48 h (Figure 5c).

Figure.5 (c) A typical chromatogram of ASP, ISMN and degradation products in the Hydrogen peroxide-induced (oxidation) degradation study

3.3.4. Heat degradation study:

In the heat degradation study, ASP and ISMN showed additional peaks at Rt of 2.42, 3.20 (about 30% degradation) and 3.96, 6.8 (about 15% degradation), respectively. (Figure 5d)

Figure.5 (d) A typical chromatogram of ASP, ISMN and degradation products in the heat degradation study

In all the degradation studies, except that of acid induced degradation and heat degradation, there was no formation of degradants when compared to the standard solution of the drug. This indicated that probably the drug degraded to low molecular weight nonchromophoric compounds. Singh and Bakshi in their article on stress testing of drugs substances and products recommended a target degradation of 20–80% for establishing the stability-indicating nature of the assay method, and even the intermediate degradation products should not interfere with any stage of drug analysis. The drugs showed extensive degradation in acid hydrolytic conditions [15].

3.3 Analysis of marketed formulation:

The proposed procedures were successfully applied for the analysis of ASP and ISMN in formulation and the drug contents in each sample were calculated by comparing with the appropriate standard solution of the drug. No interference due to excipients was detected in the chromatograms produced. The results of analysis are summarized in Table 7.

_T_a_{ble 7. Analysis of marketed formulation}

_Drug

^L^a^b^e^l^e^d

^Amount

^(mg/capsule)

_{Total % Label %
RSD}

_{Amount Claim}

_Found

^(mg/capsule)

_ASP

₁₅₀

_{149.84 99.98 0.059}

ISMN

₃₀

_{29.54 98.46 0.60}

Table 8. Stability studies for the developed method

Degradation condition	Number of degradation products (Rt)
Acid-Induced	5 (2.63, 3.06, 5.42, 6.45, 7.01)
Base- Induced	3 (1.52, 2.63, 3.42)
Oxidative- Induced	1 (2.647)
Heat	4 (2.42, 3.20, 3.96, 6.8)

4. DISCUSSION:

The results show that within the concentration range tested, there was an excellent correlation between peak area and concentration. Linearity was established using least squares linear regression analysis of the calibration curve. Precision of the developed method was studied under intraday precision and inter day precision of injection. Low % RSD values show that the developed method is precise. The recovery values between prescribed limit of 98-101% shows that the method is free from interference of excipients present in formulation.

In our study separation of ASP and ISMN occurred within the run time of only 5 minutes, in contrast to the previous method where separation of ASP and ISMN occurred by using a mixture of water: methanol (water pH adjusted to 3.4 using dilute orthophosphoric acid) with an analysis time of 10 min. In our study PAR is used as an Internal Standard which accommodates itself with in the retention time of ASP and ISMN. Previous reported method employed chlorzoxazone as I.S which elutes at a run time of 10 min which is far away from the retention time of analytes of interest, leading to wastage of costly solvents and time. In our study buffer was not used, which will protect the column from accumulation of inorganic salts by preventing back pressure during re-conditioning step.

5. CONCLUSION:

The validated RP‐HPLC method employed here proved to be simple, fast, precise and accurate for the estimation of ASP and ISMN in pharmaceutical dosage form without any interference from the excipients. The good recoveries were obtained in all cases as well as the reliable agreement with the reported procedure proved that the proposed method could be applied efficiently for determination of ASP and ISMN in oral dosage form with satisfactory precision. The method is sensitive for quantitative detection of the analytes in pure drug and its pharmaceutical dosage form and thus can be used for routine analysis.

6. AUTHORS CONTRIBUTIONS:

S Hasan Amrohi participated in the collection of literature review, study design, drafting the protocol and in the development and implementation of the work. Mahesh Nasare collected the literature review and carried out the optimization of chromatographic conditions. Afra Nazneen carried out method validation and stability testing. Satish J participated in the study design.

7. ACKNOWLEDGEMENT:

The authors extend their appreciation to the School of Pharmacy, Anurag Group of Institutions for providing excellent research facilities and RA Chem Pharma Limited (Hyderabad, India) for supplying pure samples of ASP and ISMN. We would like to convey special thanks to Dr. Ranjitha (IICT, Hyderabad) and Mr. Akif ul Haque for reviewing the text.

8. REFERENCES:

1. Aspirin drug review from www.drugbank.ca/drugs/DB00945

2. Isosorbide 5-mononitrate drug review from www.medicinenet.com (assessed March 3^rd2009).

3. Gandhimathi, M., et al. 2003. Simultaneous determination of aspirin and isosorbide 5-mononitrate has been developed in pharmaceutical formulation by reversed phase high pressure liquid chromatography. J. Pharm. Biomed. Anal. 32, 1145-1148.

4. Suresh Kumar, S., et al. 2010. Analytical method development and validation for aspirin. Int. J ChemTech. Res. 2, 389-399.

5. Ismail., et al. 2008. RP-HPLC method for the simultaneous determination of aspirin, atorvastatin and pioglitazone in capsule dosage form. Asian. J. Research Chem. 1, 40-42.

6. Bae, SK., et al. 2008. Determination of acetylsalicylic acid and its major metabolite, salicylic acid, in human plasma using liquid chromatography-tandem mass spectrometry: application to pharmacokinetic study of Astrix in Korean healthy volunteers. Biomed. Chromatogr. 22, 590-5.

7. Purushotam Sinha, K., Mrinalini Damle, c., Bothara, K.G., 2009. A validated stability indicating HPTLC method for determination of aspirin and clopidogrel bisulphate in combined dosage form. Eurasian. J. Anal. Chem. 4, 152-160.

8. Zenon kokat, Kinga burda., 1998. Simultaneous determination of salicylic acid and acetylsalicylic acid in aspirin delayed-release tablet formulations by second-derivative UV spectrophotometry. J. Pharm. Biomed. Anal. 18, 871-875.

9. Fawzi El-Yazbi, A., Hassan Hammud, A., Sulaf Assi, A., 2007. Derivative-ratio spectrophotometric method for the determination of ternary mixture of aspirin, paracetamol and salicylic acid. Spectrochimica Acta Part A. 68, 275–278.

10. Rajan Verma, K., Sanjay Garg., 2002. A validated high performance liquid chromatographic method for the analysis of isosorbide mononitrate in bulk material and extended release formulations. J. Pharm. Biomed. Anal. 30, 583-591.

11. Piergiorgio Zuccaro., et al. 1990. Determination of isosorbide-5-mononitrate in human serum by gas chromatography-mass spectrometry. J. Chromatogr. 525, 447-453.

12. Olle Gyllenhaal., Johan Hulthe., 2002. Direct injection of aqueous samples in packed column supercritical fluid chromatography of isosorbide-5-mononitrate from drug release testing. J. Pharm. Biomed. Anal. 29, 381–386.

13. Antonio Ruiz-Medina., et al. 2001. Flow-through UV spectrophotometric sensor for determination of (acetyl) salicylic acid in pharmaceutical preparations. Int. J. Pharm. 216, 95-104.

14. ICH Harmonized Tripartite Guideline: Validation of Analytical Procedures: Text and Methodology Q2 (R1) (November 2005) International Conference on Harmonization, Geneva, Switzerland.

15. Singh, S., Bakshi, M., 2000. Pharm. Technol. Online. 24, 1–14.

Received on 29.10.2013 Modified on 22.11.2013

Asian J. Research Chem 7(1): January 2014; Page 41-47

_Drug	_Level_(%)	_{Amount Taken}(µg/ml)	Amount added (µg/ml)	_{Total Amount Found}(µg/ml)	_{% Recovery}	_{% RSD}
_ASP	₅₀	₄	₂	_5.96	_99.33	_0.9185
	₁₀₀	₄	₄	_7.90	_98.75	_0.4523
	₁₅₀	₄	₆	_9.92	_99.2	_0.9658
_ISMN	₅₀	₄	₂	_5.89	_98.16	_0.1221
	₁₀₀	₄	₄	_8.12	_100.01	_0.9632
	₁₅₀	₄	₆	_10.03	_100.3	_1.2405