DEVELOPMENT AND VALIDATION OF LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRIC METHOD FOR THE QUANTIFICATION OF CIPROFIBRATE FROM HUMAN PLASMA

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About Authors:
Emanual Michael Patelia*, Rakesh Thakur, Jayesh Patel
Department of Pharmaceutical analysis and chemistry (Gujarat technical university)
Department of Pharmacology (University of Bedfordshire)
ricky.emanual@gmail.com

Abstract:
To develop and validate liquid chromatography-tandem mass spectrometric method for the quantification of ciprofibrate from human plasma. Ciprofibrate and furosemide (IS) were extracted from human plasma using Oasis HLB 1cc 30 mg solid phase extraction cartridge. The chromatographic separation was performed on ACE C18, 50×4.6 mm, 5μ column. The mobile phase consisted of 0.001% ammonia in methanol-acetonitrile-water (70:20:10, v/v/v) was delivered at rate of 1 mL/min. Detection and quantitation were performed by a triple quadrupole equipped with electrospray ionization and multiple reaction monitoring in negative ionization mode (API 3200). The most intense [M-H]- transition for ciprofibrate at m/z 287.0→85.0 and for IS at m/z 328.9.0→204.9 were used for quantification. The developed method was successfully applied for bioequivalence study of ciprofibrate. The method was found to linear over the range of 25-30000 ng/mL (r>0.998). The lower limit of quantitation (LLOQ) was 25 ng/mL. The extraction recovery was above 90%. The accuracy was found to 101.26%-106.44%. The intra and inter-day precision expressed as % CV were 1.15% and 5.25%, respectively. The stability testing was also investigated and it was found that both drug and IS were quite stable. A simple, rapid, sensitive, accurate and precise LC-ESI/MS/MS method has been developed for the quantification of ciprofibrate from human plasma using SPE method. The method exhibited good linear response over the selected concentration range 25-30000 ng/mL. Selectivity and sensitivity were sufficient for detecting and quantifying ciprofibrate in human plasma. These features coupled with a short run time at 1.8 min compared to reported methods, facilitated a high analysis throughput, with the ability to quantify a larger number of clinical samples in a shorter time frame.

REFERENCE ID: PHARMATUTOR-ART-1942

Introduction:
Ciprofibrate is isobutyric acid used to reduce high levels of lipids such as triglycerides in the blood.

It is also used to reduce the likelihood of cardiovascular disease events [1-3]. Ciprofibrate is primarily indicated in conditions like Hypercholesterolaemia, Hyperlipidemia and Hypertriglyceridaemia. In patients with low HDL and high risk of atheromatous disease [4-6]. High-performance liquid chromatographies coupled with electrospray tandem mass spectrometry have developed for quantification of Ciprofibrate in human plasma for pharmacokinetic studies [7]. High-performance liquid chromatography has developed for determination of ciprofibrate in human plasma [8]. Stability-Indicating HPLC has developed for the determination of ciprofibrate in Bulk drug and Pharmaceutical dosage form [9]. High-performance liquid chromatography have developed for the determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma [10]. Spectrophotometric method have developed for the determination of Ciprofibrate content in tablets [11]. Method has developed for achiral and chiral determination of Ciprofibrate and its glucuronide in human urine by capillary electrophoresis [12]. Densitometry and videodensitometric TLC method have developed method for the determination of bezafibrate and ciprofibrate in pharmaceutical formulations [13]. The method official is in British Pharmacopeia 2011 [14]. All reported methods have long run time. Hence, it felt necessary to develop and validate a rapid and selective method that can be successfully applied to a bioequivalence study. In the present paper we would like to present a simple and high-throughput protein precipitation method for quantification of Ciprofibrate using Glimipiride as an internal standard with LC-MS/MS detection. The application of this validated method in analyzing samples from a bioequivalence study involving Ciprofibrate is also presented.

Experimental conditions:

Chemicals and Reagents
The reference standard of Ciprofibrate was provided by accutest pharmaceuticals Ltd. (Mumbai, India). The reference standard Furosemide was obtained from Derivados Quimicos, India. Purity of both the standards was higher than 100.1 %. The Ciprofibrate API, were obtained from Derivados Quimicos, India. High-purity water was prepared in-house using a Milli-Q A10-gradient water purification system (Millipore, Bangalore, India). LC-grade methanol and acetonitrile were purchased from J.T. Baker Inc, and sigma, germany. (Phillipsburg, NJ, USA). Ortho-phosphoric Acid and Sodium hydroxide were procured from AR Grade, Rankem Fine Chemical Ltd., New Delhi, India. Ammonia was procured form SD fine chemicals Mumbai.Drug-free (blank) human plasma containing heparin was obtained by enrolling healthy volunteers and taking their consent before bleeding. The plasma thus obtained was stored at −20°C prior to use.

Calibration Curve and Quality Control Samples
Two separate stock solutions of Ciprofibrate were prepared for bulk spiking of calibration curve and quality control samples for the method validation exercise as well as the subject sample analysis. The stock solutions of Ciprofibrate and Furosemide were prepared in 0.001% ammonia in methanol-acetonitrile-water (70:20:10, v/v/v) at free base concentration of 100 μg/mL and 1000 μg/mL. Primary dilutions and working standard solutions were prepared from stock solutions using Methanol:Water (80:20 v/v) solvent mixture. These working standard solutions were used to prepare the calibration curve and quality control samples. Blank human plasma was screened prior to spiking to ensure that it was free of endogenous interference at retention times of Ciprofibrate and internal standard Furosemide. An eight-point standard curve and four quality control samples were prepared by spiking the blank plasma with an appropriate amount of Ciprofibrate. Calibration samples were made at concentrations of 500, 1000, 2000, 10000, 20000, 100000, 200000, 400000 and 600000 ng/mL, and quality control samples were made at concentrations of 1000, 2000, 10000, 20000, 100000, 200000, 400000 and 600000  ng/mL for Ciprofibrate.

Liquid Chromatography and Mass Spectrometric Conditions
The chromatographic separation was performed onACE C18 column with 50x4.6 mm i.d., 5 μ particle size. The mobile phase consisted of 0.001% ammonia in methanol:acetonitrile:water (70:20:10, %v/v/v) (pH: 6.5) was delivered at rate of 1.0 mL/min with Splitter. Detection and quantitation were performed by a triple quadrupole equipped with electrospray ionization and multiple reaction monitoring in negative ionization mode. The tuning was performed with ion-spray voltage -4500 V and temperature was 550 °C. The most intense [M-H]- transition for Ciprofibrate at m/z 287.0 85.0 and for IS at m/z 328.9 204.9 were used for quantification. The developed method was successfully applied for bioequivalence study of Ciprofibrate. The method was found to linear over the range of 25-30000 ng/mL (r≥0.992). The lower limit of quantitation (LLOQ) was 25 ng/mL. Selectivity and sensitivity were sufficient for detecting and quantifying Ciprofibrate in human plasma. These features coupled with a short run time compared to reported methods, facilitated a high analysis throughput, with the ability to quantify a larger number of clinical samples in a shorter time frame.


Figure 1: Product ion mass spectrum of Ciprofibrate


Figure 2: Product ion mass spectrum of glimepride.

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