Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 67 No. 5 pp. 455ñ461, 2010 ISSN 0001-6837 Polish Pharmaceutical Society

ANALYSIS

METHODS OF CHROMATOGRAPHIC DETERMINATION OF MEDICINES DECREASING THE LEVEL OF CHOLESTEROL

ELØBIETA KUBLIN1, BARBARA KACZMARSKA-GRACZYK1, EWA MALANOWICZ1 and ALEKSANDER P. MAZUREK1,2

1Department of Basic and Applied Pharmacy, National Medicines Institute, 30/34 Che≥mska St., 00-725 Warszawa, Poland 2Department of Drug Chemistry, Medical University of Warsaw, 1 Banacha St., 02- 097 Warszawa, Poland

Abstract: With reference to common application of HPLC to routine analytical tests on medicinal products decreasing the level of cholesterol, including three compounds from this group ñ , and , we developed a new method for determining two other compounds ñ and . The developed HPLC method may be used for identification and qualitative determination of selected com- pounds ñ derivatives of aryloxyalkylcarboxylic acids as well as it may be used for simultaneous separation and determination of all compounds from the group of using one column and the same methodology. The results and statistical data indicate good sensitivity and precision. The RSD value presented is equivalent to the newly developed method of determinination of ciprofibrate and gemfibrozil in the substances and medicinal products ñ capsules and coated tablets.

Keywords: hyperlipidemia, HPLC, derivatives of aryloxyalkylcarboxylic acids, bezafibrate, ciprofibrate, , gemfibrozil, etofibrate,

Hyperlipidemia (HLP) is a group of disorders The selected medicines applied in the treat- in the lipid balance of various pathogenesis, which ment of hyperlipidemia, particularily leading to a demonstrate an increase in the cholesterol concen- decrease in the level of cholesterol, have been apart tration, mostly the level of lipoprotein fractions of from , the derivatives of aryloxyalkyl-car- low density (LDL) and/or the concentration of boxylic acids ñ so called fibrates. triglycerides in blood. The increase in the total cho- Fibrates inhibit the synthesis of lipoproteins lesterol and LDL concentration is closely connected VLDL in the liver and they accelerate catabolism by with an increase in the risk of appearance of the car- an increase in the activity of lipoprotein lipase. They diac ischemia and disorders in the cerebral, coronary increase the removal of fractions of the cholesterol and peripheral circulation. In May 2001, the LDL from the organism and they change their struc- National Cholesterol Education Program issued the ture by means of increasing their sizes and decreas- third edition of the guidelines of Adult Treatment ing density. Moreover, the compounds from this Panel (ATP III). The most important parameter of group influence the increase of the HDL fraction the lipid profile is the cholesterol concentration LDL and the reverse transportation of cholesterol. The ñ the proper level shall be below 130 mg/dL, specific mechanism of action of fibrates relies on though, the optimum concentration LDL has been their interaction with nuclear receptors, so called assumed as below 100 mg/dL. The level for the cho- PPAR (peroxisome proliferator activated receptors), lesterol HDL has been also increased ñ the correct which are crucial transmitters of stimuli for the number shall be at the level of more than 40 mg/dL genes controlling the metabolism of lipids. As a for men and more than 50 mg/dL for women and the result of their action, there is a decrease in the level risk factor ñ the concentration below 40 mg/dL. The of triglycerides by 20ñ50%, the increase of choles- third most important parameter is the concentration terol HDL by 10ñ15% and a decrease in the LDL of triglycerides ñ the correct number shall constitute fraction. Apart from the significant influence on the the concentration not exceeding 150 mg/dL. profile of lipids and lipoproteins, this group of com-

* Corresponding author: e-mail: [email protected]

455 456 ELØBIETA KUBLIN et al. pounds demonstrate other mechanisms of activity method (12) and the method of capillary elec- that favorably influence the prevention of athero- trophoresis (13). sclerosis (1, 2). The part of the studies describes the determi- The mother compound of the group of deriva- nation of compounds from this group and their tives of aryloxyalkyl carboxylic acids is clofibrate metabolites in the biological material, mainly in i.e., the ethyl ester of 2-(p-chlorophenoxy)-2- blood, plasma and urine. The authors applied the methylpropanoic acid introduced to health care in method of HPLC in ODS columns along with the 1963. This group also encompasses the medicines of use of the spectrofluorimetric detector (3) and the later generation: bezafibrate, ciprofibrate, etofibrate, fluorescent method (9). fenofibrate and gemfebrozil. Chemical formulas and There are also studies on determination of names are presented in Figure 1. fibrates by means of gas chromatography using MS In the available literature, there are some pub- detector (7, 14, 15). All the cited methods are main- lications discussing the identification and determi- ly used for testing single compounds. nation of derivatives of aryloxyalkylcarboxylic Because there is a limited number of informa- acids in pharmaceutical preparations and biological tion on the methods of identification and quantita- material. The tests on fibrates in substances and tive tests on the whole group of fibrates, particular- tablets or capsules relied mainly on the HPLC ly in pharmaceutical products and biological materi- method with Symmetry ODS columns or Hypersil al, it was justified to develop a sensitive, precise, ODS using a spectrophotometric detector (3) or unified and easily accessible method of determina- mass spectrometry (4ñ7), spectrofluorimetric meth- tion of the content and purity of the aforementioned ods (8, 9), electrochemical methods (10, 11), TLC compounds.

BEZAFIBRATE C19H20ClNO4 GEMFIBROZIL C15H22O3 2-(4-{2-[(4-chlorobenzoyl)amino]ethyl}-phenoxy)-2-methyl- 5-(2,5-dimethylphenoxy)-2,2-dimethyl-pentanoic acid propanoic acid

ETOFIBRATE C18H18ClNO5 2-[(pyridin-3-yl)carbonyloxy]ethyl 2-(4-chlorophenoxy)-2-

FENOFIBRATE C20H23ClO4 methylpropanoate propan-2-yl 2-{4-[(4-chlorophenyl)carbonyl]-phenoxy}-2- methylpropanoate

CIPROFIBRATE C13H14Cl2O3 CLOFIBRATE C12H15ClO3 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methylpropanoic acid ethyl 2-(4-chlorophenoxy)-2-methylpropanoate

Figure 1. Chemical formulas and names of fibrate compounds Methods of chromatographic determination of medicines decreasing the level of cholesterol 457

With reference to common application of the tional compounds ñ ciprofibrate and gemfibrozil and HPLC method to routine analytical tests on medicinal further, to develop another simple, sensitive and uni- products including three compounds from this group ñ fied method of identification and quantitative determi- fenofibrate, bezafibrate and etofibrate, it was decided nation, which might be applied to test the whole group to develop a new method for determining the two addi- of the derivatives of aryloxyalkylcarboxylic acids in

Figure 2. Regression curves for gemfibrozil (A) and ciprofibrate (B) ñ standard sample 458 ELØBIETA KUBLIN et al.

Figure 3. Chromatogram of standard sample of ciprofibrate

Table 1. Retention time (Rt) for the tested compounds the routine analysis and their assessment from the ana- lytical and economical point of view. COMPOUND Rt [min] BEZAFIBRATE 2.9 EXPERIMENTAL CIPROFIBRATE 4.0 ETOFIBRATE 4.3 Reference materials GEMFIBROZIL 6.3 Bezafibrate (KRKA), etofibrate (Merz Co. CLOFIBRATE 7.5 GmbH), fenofibrate (Laboratoires Fournier), FENOFIBRATE 13.6 ciprofibrate (Sanofi Chimie), gemfibrozil (Egis Ltd.), clofibrate (ICI).

Table 2. Statistical assessment of the results of determining the contents of active substances in the substances/products. NAME OF NUMBER AVERAGE FROM STANDARD CONFIDENCE COMPOUND/ OF TESTS ALL MEASUREMENTS DEVIATION INTERVAL X ± ∆X RSD PRODUCT n X (w %) S Pu = 95% (w %) % Gemfibrozil substance 6 99.59 0.17 99.59 ± 0.16 0.17 Gemfibrozil in tablets (Gemfibral) 6 103.27 0.51 103.27 ± 0.16 0.16 Ciprofibrat substance 6 99.26 0.13 99.26 ± 0.13 0.14 Ciprofibrat in capsules (Lipanor) 6 103.88 0.80 103.88 ± 1.12 0.77 Methods of chromatographic determination of medicines decreasing the level of cholesterol 459

Figure 4. Chromatogram of standard sample of gemfibrozil

Tested substances and medicinal products flow rate: 1.2 mL/min, injection volume: 10 µL, Ciprofibrate (Laboratories Winthrop), (gemfi- detection wavelength: 233 and 274 nm for ciprofi- brozil ñ Egis), Lipanor, capsules 100 mg (Sanofi brate and gemfibrozil, respectively. Aventis), Gemfibral, coated tablets 300 mg The analyte solutions have been prepared in (Polpharma S.A.). methanol at a concentration of 1.0 mg/mL for both ciprofibrate and gemfibrozil. The respective reten- Reagents and apparatus tion times were: 4.0 min for ciprofibrate and 6.3 min Reagents of high purity for HPLC: acetonitrile, for gemfibrozil. methanol (Lab-Scan), ortho-phosphoric acid 85% p.a. (AppliChem) and liquid chromatograph Standard curves (Dionex) with a spectrophotometric detector. Standard solutions of ciprofibrate and gem- brozil were prepared in methanol at selected con- HPLC method centrations (0.049 ñ 198.0 µg/mL and 0.198 ñ 197.9 µg/mL, respectively). Determinations have been Determination of ciprofibrate and gemfibrozil performed under the conditions described above and After testing the series of columns, the standard curves were constructed (Fig. 2). Symmetry C18 column, 250 mm ◊ 4.6 mm, 5 µm and a spectrophotometric detector were chosen for Quantitative determination of ciprofibrate and the test. The following determination conditions gembrozil have been worked out: mobile phase: acetonitrile : Preparation of standard solutions: In 100 mL water (70 : 30, v/v) at pH 2.5, column temperature: flasks 5 mg reference material of ciprofibrate and 35OC, auto sampler temperature: 25OC, mobile phase gembrozil were dissolved and filled up with 460 ELØBIETA KUBLIN et al.

Figure 5. Chromatogram of the mixture of fibrates.

methanol yielding concentrations of 0.05 mg/ mL. containing the following concentrations: ciprofi- Preparation of tested solutions: a) To 230 mg of cap- brate ñ 0.15 mg/mL, etofibrate ñ 0.05 mg/mL, sules Lapanor (equivalent to ca. 50 mg of the active bezafibrate ñ 0.015 mg/mL, fenofibrate ñ 0.01 substance of ciprofibrate) 40 mL of methanol was mg/mL, clofibrate ñ 0.30 mg/mL and gemfibrozil ñ added and the mixture was shaken for 30 min. The 0.25 mg/mL. Retention time values are presented in solution was filled up to 50 mL with methanol and Table 1. filtered. One mL of the solution was diluted with 20 mL of methanol and used for tests. b) To 350 mg of RESULTS AND DISCUSSION powdered tablet mass of Gemfibral (equivalent to ca. 250 mg of the active substance ñ gemfibrozil ) 80 The linearity of determinations within the test- mL of methanol was added and the mixture was ed ranges of concentration were found for both shaken for 30 min.. The solution was filled up to 100 ciprofibrate and gembrosil (Fig. 2). The following mL with methanol and filtered. One mL of the solu- values for determination limits (0.049 µg/mL and tion was transferred to 50-mL flask and filled up 0.198 µg/mL) and detection limits (0.0198 µg/mL with methanol. This solution was used for tests. and 0.099 µg/mL) were found for ciprofibrate and gemfibrozil, respectively. Chromatograms of stan- Determination of the mixture of fibrates dard samples of ciprofibrate and gemfibrozil are Simultaneous testing of six fibrates used in presented in Figures 3 and 4. The chromatograms of therapy by HPLC method under the conditions substances and medicinal products of these fibrates described above was accomplished for the solution were identical, respectively. The results and statisti- Methods of chromatographic determination of medicines decreasing the level of cholesterol 461 cal data presented in Table 2 indicate good sensitiv- 5. Kushnir M.M., Rockwood A.L., Nelson G.J., ity and precision of the method. Yue B.F., Urry F.M.: Clin. Biochem. 38, 319 The chromatographic conditions established, (2005). proved to be also favorable for separation of six 6. Kim S.-Ch., Carlton K.: Trends Anal. Chem. investigated fibrates (Fig. 5). 24, 635 (2005). The developed HPLC method may be success- 7. Fatta D., Nikolaou A., Achilles A., Merc S.: fully used for identification and determination of Trends Anal. Chem. 26, 513 (2007). fibrates ñ derivatives of aryloxyalkylcarboxylic 8. Tang B., Jia B. X. , Cui G. W., Ding Y.: Anal. acids, as substances and in medicinal products. Chim. Acta 516, 221 ( 2004). 9. Kuroda N., Sugihara S., Sugihara Y., Wada M., Kishikawa N., Ohba Y., Nagashima K.: J. REFERENCES Chromatogr. A 1066, 119 (2005). 10. Yardimici C., Ozaltin N.: Anal. Bioanal. Chem. 1. Chemistry of medicines (Polish). Zejc A., 378, 495 (2004). Gorczyca M. Eds.: pp. 421ñ424, PZWL, 11. Xu M. T., Song J. F., Guo W., Li N.: Warszawa 2002. Electroanalysis (N. Y.) 16, 1038 (2004). 2. Chemistry of medicines (Polish). Zajπc M., 12. Misztal G., Komsta L.: J. Planar Chromatogr. Pawe≥czyk E. Eds., pp. 403ñ405, PoznaÒ 18, 188 (2005). University of Medical Sciences, PoznaÒ 2000. 13. Komsta £., Misztal G., Majchrak E., Haurze A.: 3. Chen R.R., Zelesky T., Ilasi N., Sekulic S.S.: J. J. Pharm. Biomed. Anal. 41, 408 (2006). Pharm. Biomed Anal. 37, 239 (2005). 14. Weigel S., Kallenborn R., H¸hnerfuss H.: J. 4. Quintana J. B., Reemtsma T.: Rapid Commun. Chromatogr. A 1023, 183 (2004). Mass Spectrom. 18, 765 (2004). 15. Soliman M.A., Pedersen J.A., Suffet I.H.: J. Chromatogr. A 1029, 223 (2004).

Received: 04. 08. 2009