научная статья по теме DEVELOPMENT AND VALIDATION OF UHPLC–DAD METHOD FOR THE DETERMINATION OF CHOLESTERYL-HEXAHYDROPHTHALOYL-5-FLUOROURACIL IN LIPID NANOEMULSION Химия

Текст научной статьи на тему «DEVELOPMENT AND VALIDATION OF UHPLC–DAD METHOD FOR THE DETERMINATION OF CHOLESTERYL-HEXAHYDROPHTHALOYL-5-FLUOROURACIL IN LIPID NANOEMULSION»

ОРИГИНАЛЬНЫЕ СТАТЬИ

УДК 543

DEVELOPMENT AND VALIDATION OF UHPLC—DAD METHOD FOR THE DETERMINATION OF CHOLESTERYL-HEXAHYDROPHTHALOYL-5-FLUOROURACIL IN LIPID NANOEMULSION

© 2015 Fars K. Alanazi*, Nazrul Haq**, ***, Awwad A. Radwan*, Ibrahim A. Alsarra**, ***, Faiyaz Shakeel**, ***, 1

*Kayyali Chair for Pharmaceutical Industry, Department of Pharmaceutics, College of Pharmacy, King Saud University

P.O. Box 2457, Riyadh 11451, Saudi Arabia **Center of Excellence in Biotechnology Research (CEBR), King Saud University P.O. Box 2460, Riyadh 11451, Saudi Arabia ***Department of Pharmaceutics, College of Pharmacy, King Saud University P.O. Box 2457, Riyadh 11451, Saudi Arabia

1 E-mail: faiyazs@fastmail.fm Received 06.08.2013; in final form 07.06.2014

An ultra HPLC—diode array detector (UHPLC—DAD) method was developed and validated for rapid determination of cholesteryl-hexahydrophthaloyl-5-fluorouracil (CH5F) conjugate in standard drug, lipid na-noemulsion and dissolution samples. The chromatographic identification of this conjugate was achieved on Hypersil GOLD 50 x 2.1 mm reversed phase Ci8 column having a 1.9 p.m packing as a stationary phase using methanol—water (80 : 20, v/v) as a mobile phase, at a flow rate of 0.4 mL/min with DAD detection at 276 nm. The proposed UHPLC—DAD method is linear in the concentration range of 1—50 p.g/mL with correlation coefficient of 0.998. The proposed method is precise, accurate, robust, sensitive and specific for analysis of the conjugate. High assay value (98.7%) of CH5F conjugate in lipid nanoemulsion was obtained by the proposed method. Forced degradation studies indicated that conjugate was sufficiently stable under oxidative stress conditions degraded under acid, base and thermal stress conditions. The proposed UHPLC—DAD method successfully and resolved drug conjugate peak in the presence of its degradation products which established stability and indicating property of the method. Results of the present study indicated that the proposed UHPLC—DAD method can be successfully used for routine determination of CH5F conjugate in standard drug and pharmaceutical formulations.

Keywords: UHPLC—DAD, cholesteryl-hexahydrophthaloyl-5-fluorouracil, lipid nanoemulsion, dissolution.

DOI: 10.7868/S0044450215050059

5-Fluorouracil (5-FU) is a very common anticancer drug belonging to antimetabolite class that is recommended clinically for the management and treatment of various kinds of tumors like colorectal, breast and ovarian tumor [1, 2]. It is administered via intravenous injection in which 5-FU plasma concentration could not be maintained for longer duration of time due to rapid metabolism [3, 4]. Prodrug/conjugation approach showed potential for enhancing therapeutic efficacy and reducing adverse effects of several antitumor drugs including 5-FU [1, 5—9].

Literature survey revealed that various liquid chromatographic methods such as HPLC and gas chroma-tography have been reported for the quantification of 5-FU in standard drug samples, pharmaceutical dosage forms and biological fluids [10—19]. As 5-FU produces various adverse effects on oral and parenteral

therapy, cholesteryl-hexahydrophthaloyl conjugate of 5-FU was developed in present study in order to eliminate its adverse effects and enhance therapeutic efficacy. In the present study, cholesteryl-hexahydrophth-aloyl conjugate of 5-FU (cholesteryl-hexahydrophth-aloyl 5-fluorouracil, CH5F, with molecular weight of 652.88) was synthesized. In the last few years, UHPLC is frequently used for rapid quantification of drugs which offered several advantages such as high sensitivity, improved resolution, high analytical speed, short analysis and run time over conventional liquid chromatographic techniques [20—24]. However, no UHPLC methods have been reported for quantification of 5-FU or any of its conjugate/derivative or prodrug so far. Therefore, the aim of present study was to develop and validate a rapid, facile, precise, accurate and robust stability-indicating UHPLC method cou-

pled with diode array detector for rapid quantification of newly synthesized conjugate CH5F in standard drug, lipid nanoemulsion and in vitro dissolution samples utilizing isocratic elution, taking into considerations a variety of International Conference on Harmonization recommended test conditions [25]. The proposed UHPLC—DAD method is completely novel for CH5F conjugate. The proposed method could be utilized for routine analysis of CH5F conjugate in its pharmaceutical formulations and for prediction of its shelf life.

EXPERIMENTAL

Materials. 5-FU was procured as a gift sample from Alfa Aesar (Ward Hill, MA). CH5F conjugate was synthesized and characterized in our laboratory (structure is presented below).

™yn

O

Structure of CH5F conjugate (mol. wt. 652.88).

Hydrochloric acid (HCl), sodium hydroxide (NaOH), hydrogen peroxide (H2O2) and UHPLC grade methanol were purchased from BDH Laboratory supplies (Liverpool, UK). Phosphatidylcholine, glycerol trioleate (triolein), cholesteryloleate and cholesterol were purchased from Sigma Aldrich (St. Louis, MO). Ultrapure water was procured from ELGA water purification unit (Wycombe, Bucks, UK). All other chemicals and reagents used were of analytical grade.

Instrumentation and chromatographic conditions.

Chromatographic separation of CH5F conjugate was performed at room temperature (22 ± 1°C), with Thermo Scientific UHPLC system (Thermo Scientific, Germany) equipped with a 3000 LC pump, 3000 autosampler, binary pumps, a programmable DAD detector, ultimate 3000 column oven, ultimate 3000 controller and an inline vacuum degasser. The software used in the system was Chromeleon, version 6.8. Chromatography was performed on a Thermo Hyper-sil GOLD 50 x 2.1 mm reversed phase C18 column (Thermo Scientific, Germany) having a 1.9 ^m packing as a stationary phase. The mobile phase consisted of methanol—water (80 : 20, v/v). The elution was performed at a flow rate of 0.4 mL/min with DAD detection at 276 nm. Samples (1 ^L) were injected using an ultimate 3000 series Thermo auto sampler.

Preparation of CH5F conjugate stock solution. Calibration curve for CH5F conjugate was plotted in the concentration range of 1 to 50 ^g/mL. Stock solution of 100 ^g/mL was prepared. Serial dilutions from this

stock solution were made by diluting the required ali-quots with mobile phase to get concentration in the range of 1 to 50 ^g/mL.

Method development. Various mixed organic/aqueous-organic solvent systems as mobile phase were tried for the development of suitable UHPLC—DAD method for the quantification of CH5F conjugate in its standard drug compound. The selection of the solvent system was decided on the basis of the sensitivity of the assay, suitability for stability studies, time required for the analysis, peak parameter, ease of preparation and cost effectiveness of solvents. Based on above criteri-ons, various mobile phases such as methanol—water, methanol—phosphate buffer, acetonitrile—phosphate buffer, methanol—sodium percholate buffer and acetonitrile—sodium percholate buffer at different proportions were tried. Out of tried mobile phases for UHPLC quantification, a combination of methanol— water (80 : 20, v/v) was selected as final eluent for further studies.

Validation studies. The proposed UHPLC—DAD method was validated for the linearity, accuracy, precision, sensitivity, robustness and specificity [26].

Freshly prepared solutions in the concentration range of 1—50 ^g/mL were used for construction of calibration curves. The mobile phase consisting of methanol—water (80 : 20, v/v) was delivered at 0.4 mL/min for column equilibration; the baseline was monitored continuously during this process. The chromatographic detection was performed at 276 nm. The prepared dilute solutions were injected in triplicates and peak areas were recorded using UHPLC system for each solution, and concentration was plotted against peak area.

Accuracy of the proposed method was determined by previously reported standard addition method. The standard CH5F conjugate solution (10 ^g/mL) was spiked with 0, 50, 100 and 150% extra CH5F conjugate standard solution and reanalyzed by the proposed UHPLC—DAD method. Each experiment was performed in triplicate. Percent recovery (%), percent relative standard deviation (RSD, %), and standard error for each concentration were calculated.

Precision of the proposed UHPLC—DAD method was determined at two levels i.e. repeatability (intra-day precision) and inter-day (intermediate) precision. Intra-day precision of the proposed UHPLC—DAD method was carried out by quantification of four different concentrations of CH5FU conjugate (10, 15, 20, and 25 ^g/mL) in triplicate on the same day. However, intermediate precision of the proposed UHPLC—DAD method was determined by repeating the studies on three different days.

Limits of detection (LOD) and quantification (LOQ) of the proposed UHPLC—DAD method were determined by signal to noise ratio (S/N ratio) method as reported previously using following equations:

LOD = 3.3S/N and LOQ = 10S/N.

The robustness of the proposed UHPLC—DAD method was determined to evaluate the effect of deliberate variation of chromatographic conditions on the determination of CH5F conjugate. The target concentration (10 ^g/mL) of CH5F conjugate was selected for this purpose. Robustness of the proposed UHPLC—DAD method was determined by changing the mobile phase flow rate from 0.4 to 0.45 and 0.35 mL/min, wavelength of detection from 276 to 280 and 272 nm and the concentration of methanol in mobile phase from 80 to 85 and 75%.

Forced degradation studies. Forced degradation studies were performed to determine the stability-indicating property and specificity of the proposed UHPLC—DAD method. These studies were performed at various stress conditions such as acid stress, base stress, oxidative stress and thermal stress conditions.

For acid and base-induced degradation, the target

Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.

Показать целиком