Analytical Method Development and Validation for the Quantification of Acetone and Isopropyl Alcohol in the Tartaric Acid Base Pellets of Dipyridamole Modified Release Capsules by Using Headspace Gas Chromatographic Technique

A simple, sensitive, accurate, robust headspace gas chromatographic method was developed for the quantitative determination of acetone and isopropyl alcohol in tartaric acid-based pellets of dipyridamole modified release capsules. The residual solvents acetone and isopropyl alcohol were used in the manufacturing process of the tartaric acid-based pellets of dipyridamole modified release capsules by considering the solubility of the dipyridamole and excipients in the different manufacturing stages. The method was developed and optimized by using fused silica DB-624 (30 m × 0.32 mm × 1.8 µm) column with the flame ionization detector. The method validation was carried out with regard to the guidelines for validation of analytical procedures Q2 demanded by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). All the validation characteristics were meeting the acceptance criteria. Hence, the developed and validated method can be applied for the intended routine analysis.


Introduction
Dipyridamole (2,6-bis-(diethanolamino)-4,8-dipiperidino-(5,4-d)-pyrimidine) displays antithrombotic and antiaggregatory activity. e dipyridamole ( Figure 1) is used in combination with "blood thinners" such as warfarin to avoid clot formation after heart valve replacements. Clots are a serious complication that can cause strokes, heart attacks, or blocked blood vessels in the lungs (pulmonary embolisms). Dipyridamole is an antiplatelet drug. Dipyridamole is an odourless yellow crystalline powder, having a bitter taste.
Dipyridamole exhibits a relatively short biological halflife of less than one hour. erefore, extended release formulations of dipyridamole, which provide a continual administration of active ingredient over time, are preferred. Dipyridamole is soluble in acidic mediums with a pH below 4 and is practically insoluble in water. erefore, dipyridamole is readily absorbed in the more acidic regions of the upper gastrointestinal tract, but remains insoluble in the more basic regions of the intestine. To obtain a constant level of dipyridamole in the blood, it is advantageous to formulate a dipyridamole dosage form that releases dipyridamole at a controlled rate and at a de ned pH. Acidic components can be coadministered with dipyridamole to maintain a de ned pH level throughout administration. Dipyridamole can also be administered with other active ingredients, such as aspirin. Aspirin (acetylsalicylic acid) is an inhibitory substance which counteracts the aggregation of human blood platelets by inhibiting cyclooxygenase and thereby inhibiting the biosynthesis of the aggregation promoting thromboxane A2 [1,2]. e residual solvents present in tartaric acid-based pellets of dipyridamole modi ed release capsules are classi ed as class 3 solvents as per the ICH Q3C guidelines.
e dipyridamole in tartaric acid-based pellets of dipyridamole modi ed release 150 mg and 200 mg capsules are available in the market. Each capsule contains dipyridamole 200 mg and 150 mg respective dosage strength. e adults including the elders recommended dose is one capsule twice daily, usually one in the morning and one in the evening preferably with meals. e capsules should be swallowed whole without chewing as per eMC [3].
In the literature survey, quite a few GC method have been reported from the determination of the residual solvents in dipyridamole API [4], few liquid chromatographic methods have been reported for determination of dipyridamole in pharmaceutical preparation [4][5][6], and few methods have been reported for dipyridamole and its degradation product [7,8]. However, several methods were reported for determination of dipyridamole in combination with other drugs [9][10][11][12]. Estimation of dipyridamole and its metabolites in human plasma by liquid chromatographicmass spectroscopy and HPLC has been performed [13][14][15]. e aim of this study is to develop the simple and fast analytical method for estimation of residual solvents in the tartaric acid-based pellets of dipyridamole modi ed release capsules, and the method can be used for the routine analysis. e developed method was subjected for the analytical validation with respect to speci city, linearity, precision, accuracy, limit of detection (LOD), limit of quanti cation (LOQ), robustness, and ruggedness as per the ICH guidelines [16].

Chemicals and Reagents.
e GC grade N, N-dimethylsulfoxide, isopropyl alcohol, acetone, HPLC grade water, nitrogen gas, air, and hydrogen. e dipyridamole drug substance, placebo samples of dipyridamole modi ed release capsules, and samples of dipyridamole modi ed release capsules were supplied by Blue sh Pharmaceuticals Pvt. Ltd, Bangalore, India.

Equipment.
e analytical method was developed by using the Agilent 7890A coupled with G1888 network headspace sampler, analytical balance from Mettler Toledo, micropipette from Eppendorf, headspace crimp vials, and suitable glass apparatus for solution preparations.

Chromatographic Conditions.
e method was developed and validated by using fused silica DB-624 (30 m × 0.32 mm × 1.8 µm) column with the ame ionization detector (FID) and the chromatographic parameters are given in Table 1. e chromatographic retention time of acetone and isopropyl alcohol is given in Table 2.

Diluent Solution.
A mixture of N,N-dimethylsulfoxide and water was used as diluent.

Blank Solution.
Transfer 5 mL of diluent into 20 mL headspace and crimp cap immediately.

Preparation of Standard Solution (Stock).
Weigh and transfer about 500 mg of isopropyl alcohol and 500 mg of acetone into 50 mL volumetric ask containing about 30 mL of diluent mix and make up to the mark with diluent.

Preparation of Standard Solution.
Pipette out 5 mL of the above standard stock solution into 100 mL volumetric ask and make up to the mark with diluent. Transfer 5 mL of above solution into 20 mL headspace vials, and crimp cap immediately.

Preparation of Sample Solution.
Open ve capsules and crush the pellets using mortar pestle. Weigh and transfer 500 mg of crushed powder into 20 mL headspace vial, add 5 mL of diluent, and crimp the cap immediately.

System Suitability Criteria.
e present relative standard deviation of standard peak area for six replicate injections should not be more than 10.

Method Development and Optimization.
e analytical method development was initiated by using the Agilent 7890A coupled with G1888 network headspace sampler, fused silica DB-624 (30 m × 0.32 mm × 1.8 µm) column with the ame ionization detector (FID), carrier gas as helium. e front injector conditions (injector temperature 140°C, carrier gas ow 2.0 mL/min, and split ration 5 : 1), front detector conditions (detector temperature 260°C, hydrogen ow 40 mL/minute, and air ow 300 mL/minute), oven conditions (40°C for 10 minutes and increasing the temperature to 250°C at the rate of 30°C/minute and hold for 10 minutes), headspace oven temperature 100°C, loop temperature 105°C, transfer line temperature 110°C. e dimethylsulfoxide used as diluent for solution preparations. Based on the above experiment, we found the very less resolution between acetone and isopropyl alcohol. e further experiments were conducted by altering the chromatographic conditions to achieve satisfactory resolution between acetone and isopropyl alcohol. e further experiment was conducted by using the above experiment chromatographic parameters and by changing the front injector condition and headspace sampler condition. Based on the above experiment, we found the very less resolution between acetone and isopropyl alcohol and found the placebo peak interference was observed at the retention time of acetone. e aim is to resolve the issue of placebo peak interference, needs to modify the diluent or headspace oven temperature conditions to nalize the method conditions. e further experiment was conducted by using the fused silica DB-624 (30 m × 0.32 mm × 1.8 µm) column with the ame ionization detector (FID), carrier gas as nitrogen. e front injector conditions (injector temperature 140°C, carrier gas ow 1.0 mL/min, and split ration 20 : 1), front detector conditions (detector temperature 260°C, hydrogen ow 30 mL/minute, and air ow 300 mL/minute), oven conditions (40°C for 10 minutes and increasing the temperature to 210°C at the rate of 35°C/minute and hold for 5 minutes), headspace oven temperature 80°C, loop temperature 90°C, transfer line temperature 100°C. e misture of N,N-dimethylsulfoxide and water used as diluent for solution preprations. Based on the above experiment, it was found that no placebo interference was observed at the retention time of acetone and isopropyl alcohol, and resolution was found satisfactory.
Based on the optimization of the trials, the abovementioned chromatographic conditions were nalized for the quanti cation of the acetone and isopropyl alcohol in tartaric acid-based pellets of dipyridamole modi ed release capsules. Hence, this method can be validated and introduced for the routine analysis.

Method Validation.
e developed analytical method for quanti cation of the residual solvents in the tartaric acidbased pellets of dipyridamole modi ed release capsules was validated as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) [15]. e validation parameters [17,18] speci city, estimation of limit of detection (LOD), and limit of quanti cation (LOQ), accuracy, precision, linearity, range, ruggedness, and robustness were examined [17,18].

System Suitability.
To check the system suitability criteria, the solutions were prepared and injected as per the test method. All the parameters were found well within the acceptance criteria (Table 3).

Limit of Detection and Limit of Quanti cation.
e limit of detection (LOD) and limit of quanti cation (LOQ) were established by the signal-to-noise ratio method by preparing the known concentrations of acetone and isopropyl alcohol and injected into gas chromatography headspace instrument as per the test method. e limit of detection and limit of quanti cation for each matrix were determined from the signal-to-noise ratio (S/N) method of 3 : 1 and 10 : 1 by injecting the standard solutions (Table 4). e LOD and LOQ were veri ed by analysis of spiked standard solutions prede ned acceptance criteria. e percent relative standard deviation for area response found for acetone at LOD concentration is 7.1 and LOQ is 3.5 and for isopropyl alcohol at LOD concentration is 5.2 and LOQ is 3.7.

Speci city.
Speci city was accomplished by injecting the samples as per the test method and as a part of the speci city study. Blank, acetone, isopropyl alcohol solvent, and placebo were prepared and injected as per test method. No peak interference at the retention time of acetone and isopropyl alcohol was observed. erefore, we conclude that this method is selective and suitable for the identi cation and quanti cation of the acetone and isopropyl alcohol in the dipyridamole modi ed release capsules. e chromatograms are given for the blank (Figure 2), placebo (Figure 3), acetone (Figure 4), isopropyl alcohol ( Figure 5), standard (Figure 6), as-such sample (Figure 7), and spiked blend solution (Figure 8).

Method Precision (Repeatability).
e method precision or intraassay precision was performed by preparing the six replicate test preparations (n � 6) of dipyridamole 200 mg modi ed release capsules by spiking acetone and isopropyl alcohol speci cation level (Table 5 and Figure 9) analyzed as per the test method. e concentration in parts per million was calculated and found to be within the acceptance criteria. e relative standard deviations obtained for acetone were 1.0% and isopropyl alcohol 1.4%. e graphs for acetone and isopropyl alcohol are shown in Figure 9.

Accuracy.
Accuracy of the proposed analytical procedure was evaluated from the assay results of the acetone and isopropyl alcohol as per the test method. A series of sample solutions were prepared in triplicate (six replicate test preparations for LOQ and about 200% levels) by spiking the acetone and isopropyl alcohol in placebo except LOQ level in the range of about 25%, 50%, 100%, and 150% of speci cation level and injected into HPLC system and analyzed as per the test method. e concentrations of acetone are 7.5 µg/mL, 1300 µg/mL, 2738 µg/mL, 5050 µg/mL, and 7694 µg/mL and of isopropyl alcohol are 28 µg/mL, 1304 µg/mL, 2745 µg/mL, 5063 µg/mL, and 7715 µg/mL. Individual % recovery, mean % recovery, % RSD, and squared correlation coe cient for linearity of the test method were calculated, and the results were found to be within the acceptance criteria (Table 6). e linearity graphs from accuracy results for acetone and isopropyl alcohol are shown in Figures 10 and 11, respectively.

Linearity.
e linearity was studied by analyzing the standard solutions. A series of solutions of acetone and   ≤10.0 0.9 e present relative standard deviation of isopropyl alcohol peak area for six replicate injections.

Journal of Analytical Methods in Chemistry
150% of speci cation level and injected into HPLC system and analyzed as per the test method. e concentrations of acetone are 7 µg/mL, 1260 µg/mL, 3024 µg/mL, 5040 µg/mL, and 7560 µg/mL and of isopropyl alcohol are 27 µg/mL, 1261 µg/mL, 3027 µg/mL, 5045 µg/mL, and 7567 µg/mL. e square of correlation coe cient, slope, and % y-intercept at 100% level, intercept, and residual sum of   squares were calculated, and the results were found to be within the acceptance criteria (Table 7). e linearity graphs from accuracy results for acetone and isopropyl alcohol are shown in Figures 12 and 13, respectively.

Ruggedness (Intermediate Precision).
Intermediate precision was performed by preparing the six replicate test preparations (n � 6) of dipyridamole 200 mg    modi ed release capsules by spiking acetone and isopropyl alcohol at speci cation level and analyzed as per the test method by using di erent Headspace gas chromatography system, di erent column of same make by di erent analyst on di erent day. e concentration in parts per million was calculated and found to be within the acceptance criteria. e overall concentration in parts per million for replicate preparations (n � 12) of method precision and intermediate precision was calculated and found to be within the acceptance criteria (Table 8). e relative standard deviations obtained for acetone was 1.4% and isopropyl alcohol was 3.1%.

Solution Stability.
e solution stability of acetone and isopropyl alcohol was determined by keeping sample solution and standard solutions at room temperature for 1 day and 2 day and measured against freshly prepared standard solution.
e standard solution and sample solutions were found stable for 2 days at room temperature.

3.2.9.
Robustness. Robustness of the proposed method was performed by keeping the chromatographic conditions constant with the following deliberate variations: (i) Change in carrier gas ow rate (ii) Change in column oven temperature (iii) Change in headspace sampler vial equilibration time (iv) Change in headspace vial oven temperature e standard solution was injected six times in replicate for each abovementioned change.
e system suitability parameters like % relative standard deviation for area response and % relative standard deviation for retention time    were recorded for acetone and isopropyl alcohol and found well within the acceptance criteria. e results are given in Table 9, and the graphs for acetone and isopropyl alcohol are shown in Figures 14 and 15, respectively.

Application of the Proposed Method.
e developed analytical method was applied to the analysis of real samples from the manufacturing unit. All the analytical validation parameters could be con rmed, and the method was proven   to be suitable for routine analysis regarding rapid and accurate results.

Conclusions
A simple, sensitive, accurate, robust headspace gas chromatographic method was developed for the quantitative determination of residual solvents in tartaric acid-based pellets of dipyridamole modi ed release capsules. e proposed method was validated and found to be precise, accurate, linear, robust, and rugged, and all the validation parameter results were found satisfactory. e described method is suitable for routine analysis of production samples at laboratories.

Conflicts of Interest
e authors declare that there are no con icts of interest regarding the publication of this paper.