A Sensitive Liquid Chromatography-tandem Mass Spectrometry Method for the Estimation of Cilostazol in Bulk and in a Pharmaceutical Formulation

 

Vishnu. K, Narenderan. S. T, Vishnupriya. S, Babu. B*, Meyyanathan. S. N

Department of Pharmaceutical Analysis, JSS College of Pharmacy

(JSS Academy of Higher Education and Research, Mysuru) Udhagamandalam, India.

 

ABSTRACT:

The present study reports on a liquid chromatography-tandem mass spectroscopy method for the analysis of Cilostazol drug substance in its formulation. The materials utilize a Zorbax SB C18 analytical column with electrospray ionization and a triple quadrupole mass detector in multiple reaction monitoring (MRM) mode, resulting in a short analysis of 1.5 min runtime and the analyte elute at 0.76 min. Change from SIM to MRM mode resulted in increased sensitivity and low quantitation limit which in turn produces a detection limit of 1 ng/ml. The obtained method was validated in a range of 5-100 ng/ml with a correlation coefficient of 0.9991. The percentage recovery of the present method was found to be in the range of 94% to 100%. The developed method was validated as per the ICH guidelines.  

 

 

INTRODUCTION:

Cilostazol is chemically 6- [4- (1- cyclohexyl- 1H- tetrazol- 5-yl) butoxyl]- 3, 4- dihydro- 2 (1H)- quinolinone (Fig. 1). It is cyclic adenosine monophosphate phosphodiesterase III-inhibitors, inhibiting phosphodiesterase activity and suppressing cAMP degradation with a resultant increase in cAMP in platelets and blood vessel leading to inhibition of platelet aggregation and vasodilation. Therefore, Cilostazol is used for the treatment of intermittent claudication resulting from peripheral arterial diseases¹. Based on the literature survey, it was found that Cilostazol has been analyzed by spectrophotometry², HPLC in pharmaceutical formulation alone and in combination with other drugs^3,4.

 

Fig. 1: Structure of Cilostazol

 

To our knowledge, there is no LC-MS/MS method developed for the estimation of Cilostazol in API and formulation. Hence, the aim of the current study is to develop a sensitive method for the estimation of Cilostazol in the pharmaceutical formulations. The developed method was validated as per ICH guidelines.

 

MATERIAL AND METHODS:

Chemicals:

The Cilostazol drug substances were kindly provided as a gift sample from Ranbaxy Pvt Ltd. Methanol of LC-MS grade by Sigma Aldrich, ammonium acetate and acetic acid by Rankem Fine Chemical Limited and Water of LC-MS/MS grade from Milli-Q RO system (Millipore, Bedford, USA) were used.

 

LC-MS/MS instrumentation:

LC system coupled with a tandem mass tandem quadrupole mass spectrometer (Shimadzu 8030, Tokyo Japan) equipped with electrospray ionization (ESI) interface, LC-20AD pump, SPD-M20 PDA detector, CTO-20AC column oven, CBM-20 alite controller and SIL-20AC autosampler was used. The data were recorded using Lab solution data station software.

 

 

 

Operating condition LC-MS/M^5-7

Zorbax SB C₁₈ column (50 mm x 4.6, 5µm) was used as a stationary phase with a flow rate of 0.8ml/min, with a detection range of 190-400nm. Eluent A consists of 10mM ammonium for mate pH adjusted to 3.5 using 1% formic acid and Eluent B consist of methanol in the ratio (5:95). The column temperature was kept at 25°C and injection volume of 10µl. Electrospray positive mode was used with MRM monitoring for m/z 370.05>125.10 and 370.05>288 transition for Cilostazol with collision energy -24 and -18, respectively.

 

Selection of a mass range:

A 1000 ng/ml of Cilostazol was injected in the mass spectrometer directly and the operation conditions were optimized. Obtained transitions 370>125.10 and 370.05.288 were used to monitor the Gemfibrozil (Fig. 2).

 

 

Fig. 2: Mass scan spectra of Cilostazol

 

Solution preparation:

Preparation of working standard solution:

1mg/ml of Cilostazol stock solution was prepared by dissolving 10 mg of drug in 10ml of methanol. Further dilutions were made with methanol to produce the working concentration of 1000 ng/ml.

 

Preparation of sample solution:

Twenty tablets were taken, weighed accurately and powdered. From the powdered sample, the amount equivalent to 100 mg of Cilostazol was accurately weighed and transferred to 100 ml volumetric flask. About 50 ml of methanol was added and the solution was sonicated for about 30 min and then the volume was made up to 100 ml using methanol. Further, the solution was shaken thoroughly and filtered through a syringe filter and the quality control samples of low-quality control (LQC) 5 ng/ml, medium quality control (MQC) 30 ng/ml and high-quality control (HQC) 85 ng/ml.

 

 

 

VALIDATION STUDY:

The method validation was performed in terms of specificity, precision, linearity, accuracy, limit of detection (LOD) and limit of quantification (LOQ) as per the ICH guidelines⁸. For specificity, the quality control (QC) samples were analyzed for any interferences in the region of the chromatogram where Cilostazol was expected to elute. The calibration graph was constructed by injecting the standard drug in the concentration range from 5-100 ng/ml into the LC-MS/MS system. The LOD and LOQ were determined as the lowest concentration giving a response of 3 and 10 times, respectively.

 

RESULTS AND DISCUSSIONS:

Specificity:

To determine that the excipients interfering standards were injected and no peaks were eluted along with the retention time of Cilostazol (Fig. 3). Hence, the developed method results showed that it was selective for the determination of Cilostazol in the formulation.

 

Fig. 3: LC-MS/MS chromatogram of blank and Cilostazol standard

 

Linearity:

The evaluation of the method to be linear was by six determinations at six concentration levels with a range of 5-100 ng/ml for Cilostazol and the standard deviation (SD) were found to be within the limits. The peak areas were used for the calculation of the calibration function by least square linear regression. A calibration curve was found to be linear with a mean regression of equation, y = 8539.1x - 9185.2, r² = 0.9991 respectively.

 

Accuracy and precision:

The precision of the method was determined by the intra-day and inter-day precision studies at three different concentrations and they were found to be within the limits (Table 1). The accuracy of the method was carried out for three quality control (LQC, MQC, and HQC) samples by standard addition method, and the accuracy was found to be 94 to 100% (Table 2). Application of the developed method was intended for the estimation of a commercial formulation of Cilostazol (Fig. 4).

 

Table 1: Accuracy and precision results of Cilostazol.

Sample (ng/ml)	Amount found (ng/ml) ± SD	Intra-day		Inter-day
		Accuracy (% N)	Precision (% RSD)	Accuracy (% N)	Precision (% RSD)
5	4.81 ± 0.04	96.20	0.95	94.86	4.42
30	29.26 ± 0.15	97.55	0.52	95.00	0.49
85	84.33 ± 0.05	99.21	0.06	97.40	0.84

SD, Standard deviation; RSD, Relative Standard Deviation.

 

Table 2: Recovery results for Cilostazol in the formulation.

Formulation	Label claim	Assay QC level (ng/ml)	Amount Found ± SD	Recovery %
F1	50 mg	5	4.79 ± 045	95.93
		30	29.16 ± 0.15	97.22
		85	84.12 ± 0.10	98.94

F₁ – Cilodoc Brand

 

 

Fig. 4: LC-MS/MS chromatogram of Cilostazol from the formulation

 

Limit of detection and limit of quantification:

The lowest limit detected for the method for Cilostazol was at 1 ng/ml based on the signal-to-noise ratio 3:1. Due to the increase in the sensitivity of the method, quantification was done at 5 ng/ml for Cilostazol.

 

Robustness:

The robustness of the method was determined by alteration of the chromatographic conditions and the results obtained were found to be within the limits proving that the developed method was found to be robust (Table 3).

 

Table 3: System suitability results for Cilostazol. 

S. No	Parameters	Obtained values
1	Retention time (min)	0.76 min
2	Theoretical Plate (N)	5621
3	Tailing Factor	1.2
4	Asymmetric Factor	1.4
5	Correlation coefficient	0.9991
6	LOD (ng/ml)	1
7	LOQ (ng/ml)	5

 

 

CONCLUSION:

In summary, a sensitive method was described for the quantification of Cilostazol in pharmaceutical formulations using LC-MS/MS in positive ionization mode. The developed method was shown to be sensitive, precise, accurate and validated as per ICH guidelines. The developed method can be successfully applied for the estimation of Cilostazol in the commercial formulation and in bulk drug.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 14.11.2018           Modified on 18.12.2018

Accepted on 21.01.2019         © RJPT All right reserved