Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 69 No. 1 pp. 139ñ143, 2012 ISSN 0001-6837 Polish Pharmaceutical Society

DEVELOPMENT OF CHROMATOGRAPHIC METHOD FOR DETERMINATION OF DRUGS REDUCING CHOLESTEROL LEVEL

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

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

Keywords: hyperlipidemia, HPLC, GC, aryloxyalkylcarboxylic acids derivatives, 3-hydroxy-3-methylglutaryl- coenzyme A reductase inhibitors (HMG-CoA), fenofibrate, ciprofibrate, bezafibrate, gemfibrozil, atorvastatin calcium, fluvastatin sodium, rosuvastatin calcium, lovastatin, simvastatin

Hyperlipidemia (HLP) is a group of lipid aryloxyalkylcarboxylic acids. This class of drugs metabolism disorders with various pathogenesis. Its include new generation drugs: bezafibrate, ciprofi- characteristic feature is an increase in cholesterol brate, etofibrate, fenofibrate and gemfibrozil (1ñ4). level, especially the low density lipoprotein fraction The available literature of the last several years (LDL) level or triglyceride level in blood. An includes several publications describing separately increase in total and LDL cholesterol level is related an identification and determination of lovastatin to the increased risk of ischemic heart disease as derivatives and derivates of aryloxyalkylcarboxylic well as cerebral, coronary and peripheral circulation acids in pharmaceutical products and biological disorders. material. HPLC or UPLC methods with octadecyl The medications against hyperlipidemia and packed column with UV-VIS detector (11, 13ñ16) cholesterol level reduction are 3-hydroxy-3-methyl- and mass detector (12), the capillary electrophoresis glutaryl-coenzyme A reductase inhibitors (HMG- (9) and electrochemical methods ( 7) are used for CoA) also known as statins and aryloxyalkylcar- statins analysis. boxylic acid derivatives, also known as fibrates. The tests on fibrates relied mainly on the Statins inhibit biosynthesis of endogenous choles- HPLC method with Symmetry ODS or Hypersil terol at the mevalonic acid synthesis level and ODS columns using a spectrophotometric detector reduce of total cholesterol, LDL fraction and triglyc- (12) or mass spectrometry (10, 12), the method of erides levels in plasma and increases HDL fraction capillary electrophoresis (6) and gas chromatogra- level. A mother compound for a group of 3- phy with mass spectrometry detector (12). hydroxy-3-methylglutaryl-coenzyme A reductase In 2006, E. Kublin et al. have published a study inhibitors (HMG-CoA) is lovastatin. Currently, a on the determination of all statins by gas chro- semi-synthetic derivatives are available (simvastatin matography with FID detector (5). and pravastatin sodium), as well as synthetic com- In 2010, a study on determination of all drugs pounds ñ atorvastatin calcium, fluvastatin sodium (fibrate group) with a single column and a uniform and lately rosuvastatin calcium (1ñ4). methodology (HPLC with Symmetry C18 column The fibrates inhibit VLDL lipoprotein synthe- and spectrophotometric detector) has been pub- sis in liver and accelerate catabolism by increasing lished by the same authors (17). In the Department lipoprotein lipase activity. Also they improve HDL of Basic and Applied Pharmacy, National Medicines level and affect the return of cholesterol. A clofi- Institute, a determination method for all statin class brate is a mother compound of the derivatives of drugs by HPLC with Symmetry C18 column and

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

139 140 ELØBIETA KUBLIN et al.

Table 1. Retention time Rf for the tested compounds (HPLC). Table 2. Retention time Rf for the tested compounds (GC). CHEMICAL NAME RETENTION TIME CHEMICAL NAME RETENTION TIME [min] [min] ROSUVASTATIN CALCIUM 2.4 CIPROFIBRATE 1.6 BEZAFIBRATE 2.8 GEMFIBROZIL 2.9 ATORVASTATIN CALCIUM 3.3 FENOFIBRATE 7.1 FLUVASTATIN SODIUM 3.6 BEZAFIBRATE 7.4 CIPROFIBRATE 3.9 LOVASTATIN 9.6 GEMFIBROZIL 6.1 SIMVASTATIN 9.9 LOVASTATIN 7.5 ATORVASTATIN 11.4 CALCIUM SIMVASTATIN 9.8 FENOFIBRATE 13.3

Table 3. Statistical assessment of the results of simultaneous determination of active substances content in the drugs by HPLC.

COMPOUND/PRODUCT NO. OF TOTAL STANDARD CONFIDENCE RSD NAME SAMPLES AVERAGE X DEVIATIONS INTERVAL X ± ∆X (%) n (%) Pu = 95% (%) Atorvastatin calcium ñ tablets (Corator) 6 100.55 0.90 100.55 ± 0.86 0.90 Fenofibrate ñ capsules (Lipanthyl) 6 102.47 0.83 102.47 ± 0.79 0.81 Simvastatin ñ tablets (Zifam) 6 95.90 0.99 95.90 ± 0.94 1,03 Fenofibrate ñ capsules (Lipanthyl) 6 99.48 1,51 99.48 ± 1.44 1,52

Figure 1. Atorvastatin calcium/fenofibrate solution chromatogram Figure 2. Simvastatin/fenofibrate solution chromatogram (at 238 (at 246 nm). nm) Development of chromatographic method for determination of... 141 spectrophotometric detector was developed and is which could be applied to analyze the complete currently in print. class of 3-hydroxy-3-methylglutaryl-coenzyme A The available literature of the last several years reductase inhibitors (HMG-CoA) and aryloxyalkyl- includes a single publication on simultaneous deter- carboxylic acid derivatives in routine analysis and to mination of cholesterol level reducing drugs (3- assess its analytical and economical aspects. hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors ñ HMG-CoA) i.e., statins and ary- EXPERIMENTAL loxyalkylcarboxylic acid derivatives i.e., fibrates. The authors applied a HPLC test method with mass Reference materials sensitive detector (8). Rosuvastatin calcium (Zentiva), atorvastatin A combination of selected statin and fibrate calcium (Biocon Ltd.), simvastatin (Ph. Eur.), lovas- class drugs are applied in modern treating hypocho- tatin, pravastatin sodium, fluvastatin sodium fenofi- lesterolemia. The most common combination is brate CRS (Ph. Eur.), bezafibrate (KRKA), etofi- fenofibrate (100 mg to 267 mg/day) and simvastatin brate (Merz Co. GmbH), ciprofibrate (Sanofi (10ñ80 mg/day) or atorvastatin calcium (10ñ80 Chimie), gemfibrozil (Egis Ltd.), clofibrate (ICI). mg/day). A simultaneous determination of most commonly used drugs is presented in the study. Medical products A purpose of this study was to develop a sim- Corator ñ coated tablets 40 mg ple, sensitive and unified quantification method, (PrzedsiÍbiorstwo Farmaceutyczne LEK-AM), Zifam ñ coated tablets 20 mg (Polfa Warszawa S.A.), Lipanthyl ñ capsules 100 mg (Laboratoires Fourier).

Reagents and apparatus High purity HPLC reagents: acetonitrile, methanol (Rathburn), 85% orthophosphoric acid (AppliChem), Dionex liquid chromatograph with spectrophotometric detector and Agilent Technologies 6890N gas chromatogram with FID detector.

High performance liquid chromatography (HPLC)

Simultaneous quantification of atorvastatin and fenofibrate Drugs: Corator and Lipanthyl. The determina- tion was carried out at 246 nm for atorvastatin calci- Figure 3. Chromatogram of the mixture statins + fibrates (HPLC) um and at 286 nm for fenofibrate.

Figure 4. Chromatogram of the mixture statin/fibrate (GC) 142 ELØBIETA KUBLIN et al.

Preparation of standard solutions: Ca. 5 mg of mobile phase: ACN : water (70:30, v/v), at pH = 2.5; reference material (atorvastatin calcium) and 25 mg column temperature: 35OC, autosampler tempera- of reference material (fenofibrate) was transferred to ture: 15OC, mobile phase flow rate: 1.2 mL/min, a 100 mL flask, dissolved in methanol and diluted to injection volume: 10 µL, wavelength: 238 nm. volume with methanol. All tested compounds were separated (Table 1 Preparation of tested solutions: Ca. 220 mg of shows the retention times). Separation factor powdered Corator tablets (ca. 20 mg of active sub- between two adjacent peaks (fluvastatin and ciprofi- stance ñ atorvastatin calcium) and 250 mg of brate ñ peak 4 and 5, Fig. 3, is 2.30. Fig. 3 shows an Lipanthyl coated tablets (ca. 100 mg of active sub- example chromatogram. stance ñ fenofibrate) was transferred to a 50 mL A mixture of the following substances with flask, approx. 40 mL of methanol was added and methanol was used (GC method): atorvastatin calci- shaken by mechanical means for 30 min. The solu- um ñ 5.94 mg/mL, lovastatin ñ 1.56 mg/mL, sim- tion was diluted to volume with methanol and fil- vastatin ñ 1.48 mg/mL, fenofibrate ñ 1.42 mg/mL, tered. Two and a half mL of the solution was dilut- ciprofibrate ñ 5.96 mg/mL, bezafibrate ñ 4.88 ed to 20 mL with methanol. Figure 1 shows an mg/mL and gemfibrozil ñ 1.34 mg/mL. example chromatogram, Table 3 shows the results The following identification conditions were and statistical assessment. developed: column HP ñ 1; 30 m ◊ 0.25 mm ◊ 0.25 µm; detector temperature: 320OC, injection chamber Simultaneous quantification of simvastatin and temperature: 300OC, column temperature: program ñ fenofibrate initial temperature 190OC for 1 min; increment Drugs: Zifam and Lipanthyl. The determina- 10OC/1 min to final temperature 280OC, 2 min, gas tion was carried out at 238 nm for simvastatin and flow: 2.9 mL/min, injection volume: 1,0 µL, split: 286 nm for fenofibrate. 10:1. Preparation of standard solutions: Ca. 5 mg of All tested compounds were separated. Table 2 reference material (simvastatin) and 25 mg of refer- shows the retention times. Separation factor ence material (fenofibrate) was transferred to a 100 between two adjacent peaks (fenofibrate and bezafi- mL flask, dissolved in methanol and diluted to vol- brate ñ peak 3 and 4, Fig. 4) is 2.08. Figure 4 shows ume with methanol. an example chromatogram. Preparation of tested solutions: Ca. 210 mg of powdered Zifam tablets (approx. 20 mg of active DISCUSSION substance ñ simvastatin) and 250 mg of Lipanthyl capsules (approx. 100 mg of active substance ñ In conjunction with a small number of reports fenofibrate) was transferred to a 50 mL flask, on identification methods and simultaneous quan- approx. 40 mL of methanol was added and shaken tification of statins and fibrates, especially for drugs by mechanical means for 30 min. The solution was and biological material, a sensitive, accurate, unified diluted to volume with methanol and filtered. A vol- and easily available method for determination of ume of 2.5 mL of the solution was diluted to 20 mL content and purity is required. The quoted methods with methanol. Figure 2 shows an example chro- are usually used for the analysis of single 3- matogram, Table 3 shows the results and statistical hydroxy-3-methylglutaryl-coenzyme A reductase assessment. inhibitor (HMG-CoA) class and aryloxyalkylcar- boxylic acid derivative class compounds (maximum Identification of a mixture of aryloxyalkylcar- 2ñ3 compounds). boxylic acid derivatives (fibrates) and 3-hydroxy- For HPLC, a Symmetry C18 250 ◊ 4.6 mm, 5 3-methylglutaryl-coenzyme A reductase inhi- Ïm column was used for the qualitative and quanti- bitors (statins) tative assay of 3-hydroxy-3-methylglutaryl-coen- A mixture of the following substances in zyme A reductase inhibitors (HMG-CoA, statins) methanol was used for HPLC: atorvastatin calcium and aryloxyalkylcarboxylic acid derivatives ñ 49.2 µg/mL, lovastatin ñ 49.0 µg/mL, simvastatin (fibrates) with the mobile phase: ACN : water ñ 48.2 µg/mL, fluvastatin sodium ñ 50.2 µg/mL, (70:30, v/v), at pH = 2.5. rosuvastatin calcium ñ 47.5 µg/mL, fenofibrate ñ A new GC method for identification of ana- 206.3 µg/mL, ciprofibrate ñ 95.5 µg/mL, bezafi- lyzed fibrates and statins was developed. Column brateñ 48.2 µg/mL and gemfibrozil ñ 330.8 µg/mL. HP ñ 1; 30 m ◊ 0.25 mm ◊ 0.25 µm at initial tem- The following assay conditions were used: perature 190OC for 1 min; increment 10OC/ 1 min to Symmetry C18, 250 ◊ 4.6 mm, 5 µm column; final temp. 280OC, 2 min was used. Development of chromatographic method for determination of... 143

A simultaneous quantification of commonly 4. Bon-Ba≥aziÒska M.: Medicine after graduation used drugs by HPLC method was carried out due to (Polish), Vol. 12, 111 (2003). the common combination of selected fibrates 5. Kublin E., Kaczmarska-Graczyk B., Mazurek (fenofibrate) and statins (simvastatin and atorvas- A.P.: Acta Pol. Pharm. Drug Res. 62, 404 tatin calcium) in modern treating hypocholes- (2006). terolemia. 6. Komsta £., Misztal G., Majchrak E., Haurze A.: The results and statistical data presented in J. Pharm. Biomed. Anal. 41, 408 (2006). Table 3 show high sensitivity and precision of a 7. Komorsky-LovriÊ ä., NigoviÊ B.: J. method. RSD values are valid for the developed Electroanal. Chem. 593, 125 (2006). method of simultaneous determination of fenofi- 8. Hernando M.D., Aq¸era A., Fern·ndez-Alba brate/simvastatin and fenofibrate/atrovastatin calci- A.R.: Anal. Bioanal. Chem. 387, 1269 (2007). um in tablets and capsules. 9. Li M., Fan L.-Y., Zhang W., Sun J., Cao Ch.- X.: J. Pharm. Biomed. Anal. 43, 387 (2007). CONCLUSIONS 10. Yuan H., Wang F., Tu J., Peng W., Li H.: J. Pharm. Biomed. Anal. 46, 808 (2008). The presented HPLC method can be used for 11. Kadav A.A., Vora D.N.: J. Pharm. Biomed. simultaneous separation and determination of all Anal. 48, 120 (2008). analyzed 3-hydroxy-3-methylglutaryl-coenzyme A 12. Lambropoulou D.A., Hernando M.D., reductase inhibitors (HMG-CoA, statins) and ary- Konstantinou E.M., Thurman E.M., Ferrer I., loxyalkylcarboxylic acid derivatives (fibrates) with Albanis T.A., Fern·ndez-Alba A.R.: J. a single column and a single run. The GC method Chromatogr. A 1183, 38 (2008). allows for fast identification of analyzed drugs from 13. Sarr F.S., AndrÈ C., Guillaume Y.C: J. both classes of compounds. Chromatogr. B 868, 20 (2008). 14. Iriarte G., Gonzales O., FerreirÛs N., Maguregui REFERENCES M. I., Alonso R. M., JimÈnez R. M.: J. Chromatogr. B 877, 3045 (2009). 1. Therapy and drugs (Polish), K≥osiewicz- 15. Ali H., Nazzal S.: J. Pharm. Biomed. Anal. 49, Latoszek L. Ed., Center of Scientific 950 (2009). Information Polfa, Warszawa 6, 1999. 16. Hefnawy M., Al-Omar M., Julkhuf S.: J. Pharm. 2. Chemistry of medicines (Polish), Zejc A., Biomed. Anal. 50, 527 (2009). Gorczyca M. Eds.: p. 408, PZWL, Warszawa 17. Kublin E., Kaczmarska-Graczyk B., 1998. Malanowicz E., Mazurek A.P.: Acta Pol. 3. Chemistry of medicines (Polish), Zajπc M., Pharm. Drug Res. 67, 455 (2010). Pawe≥czyk E. Eds.: p. 403, PoznaÒ University of Medical Sciences, PoznaÒ 2000. Received: 29. 12. 2010