Novel Chiral LC Methods for the Enantiomeric Separation of Bicalutamide and Thalidomide on Amylose Based Immobilized CSP

Home , Thalidomide

Current Pharmaceutical Analysis, 2011, 7, 47-53 47 Novel Chiral LC Methods for the Enantiomeric Separation of Bicalutamide and Thalidomide on Amylose Based Immobilized CSP

I.V. Soma Raju1,*, P. Raghuram2,* and J. Sriramulu2

1Invagen Pharmaceuticals Inc. New York, NY, USA 2Department of Chemistry, Sri Krishna Devaraya University, Anantapur-515003, India

Abstract: Fast chiral liquid chromatographic methods are developed for the separation of bicalutamide and thalidomide enantiomers in bulk drug samples in an elution time of about 15 min. The chromatographic separation was carried out on various solvents using an amylose 3,5-dimethylphenylcarbamate immobilized onto silica gel (Chiralpak-IA). The resolution (Rs) between the enantiomers is found to be greater than 1.5 in the developed method. Chiralpak-IA column played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic effi- ciency. The column’s compatability to non standard chromatographic solvents is checked and found suitable. Keywords: Bicalutamide, Thalidomide, Chiralpak-IA, Enantiomeric separation.

INTRODUCTION widely used is liquid chromatography (LC) employing a chiral stationary phase (CSP) [5-7]. Bicalutamide (Fig. 1.1) is an oral non-steroidal anti- androgen used in the treatment of prostate cancer and hirsu- O O tism [1]. And thalidomide (Fig. 1.2) is a sedative-hypnotic, HN and multiple myeloma medication [2]. These two active O N pharmaceutical substances are racemic mixtures. N O Fig. (1.2). Thalidomide: (RS)-2-(2,6-dioxopiperidin-3-yl)-1H- F isoindole-1,3(2H)-dione F F O Polysaccharide derived chiral stationary phases (CSPs) HN have been recognized as the most powerful packing materials for the chromatographic separation of enantiomers in OH analytical and preparative applications due to their broad O SO application field and their remarkable loading capacity [8- 17]. Due to their coated nature, these CSPs can only be used with a limited range of solvents such as the polar solvents (e.g. acetonitrile, alcohols) or non-polar solvents (e.g. al- kanes) in combination with some polar components as modi- F fiers (mainly alcohols). Solvents with intermediate polarities, Fig. (1). Structure and label of bicalutamide, thalidomide and trans- such as methyl t-butyl ether, ethyl acetate, tetrahydrofuran, Stilbene Oxide. acetone, 1,4-dioxane and chlorinated solvents can partially or Fig. (1.1). Bicalutamide:N-[4-cyano-3-(trifluoromethyl)phenyl]-3- totally dissolve the above CSPs. Therefore, they must be [(4-fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide excluded in the optimization of chromatographic parameters Enantiomers of racemic drugs often differ in pharma- on these CSPs. Immobilization of a polysaccharide deriva- cokinetic behavior or pharmacological action [3]. The devel- tive on the support is an evolutionary strategy to make a CSP opment of analytical methods for the quantitative analysis of compatible with the whole range of organic solvents, which chiral materials and for the assessment of enantiomeric pu- will consequently extend its application scope [18]. rity is extremely challenging due to the fact that enantiomers Chiralpak-IA (amylose 3,5-dimethylphenylcarbamate posses virtually identical properties [4]. Although many ana- immobilized onto silica gel) has been extensively investi- lytical techniques can be employed to achieve this, the most gated and proved to be versatile in enantiomeric separation and excellent in solvent compatibility. In the present work,

*Address correspondence to these authors at the Invagen Pharmaceuticals the key role of mobile phase in the enantiomeric selectivity Inc. New York, NY, USA; Tel: 917 434 0797; Fax: 631 231 4288; shown by this immobilized CSP is checked. A structured E-mails: [email protected] approach for solvent compatibility is followed [19] and also Department of Chemistry, Sri Krishna Devaraya University, Anantapur- the use of non standard solvents is verified as stated by 515003, India; E-mail: [email protected]

1573-4129/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd. 48 Current Pharmaceutical Analysis, 2011, Vol. 7, No. 1 Raju et al. manufacturer [20]. These two APIs are selected as they be- 1.2.2. Chromatographic Conditions long to similar therapeutic category. The chromatographic conditions are optimized using a Few analytical methods had been reported in the litera- Chiralpak-IA column. The column temperature is main- ture for enantiomeric separation of bicalutamide and tha- tained at 25°C and the detection is monitored at a wave- lidomide by HPLC but all these methods are developed on length bicalutamide 270 nm and thalidomide 210 nm (Table cellulose based (Chiralpak AD-H and Chiralcel OD-H) and 1). The injection volume is 20 μL. Methanol is used as diluent. whelk O1 columns [21-25]. So far, to our present knowledge 1.2.3. Preparation of Sample Solution no chiral HPLC methods are reported in the literature for the enantiomeric separation of bicalutamide and thalidomide on Stock solutions of bicalutamide and thalidomide (1000 Chiralpak-IA [immobilized chiral stationary phase]. This g mL-1) are prepared individually by dissolving appropriate paper deals with method development for the enantiomeric amount of the substances in the diluent. The analyte concen- separation on immobilized stationary phases for bicalu- tration of 200 g mL-1 for bicalutamide and 50 g mL-1 for tamide and thalidomide active pharmaceutical ingredients. thalidomide. Working solutions of bicalutamide and tha- Compatibility of dichloromethane, ethyl acetate and tetrahy- lidomide are prepared in diluents. drofuran with Chiralpak-IA is checked. 1.3. Method Development and Optimization 1. EXPERIMENTAL DESIGN 1.3.1. Introduction 1.1. Chemicals In principle all miscible solvents can be used with an immobilized CSP either in their pure form or as mobile Samples of bicalutamide and thalidomide are obtained phase components. The exhaustive investigation of various from Hetero Labs Ltd., Hyderabad, India. HPLC grade n- solvents for their general behavior in enantioselective separa- hexane, dichloromethane, isopropyl alcohol, methanol, etha- tions is undoubtedly essential in the mobile phase selection. nol, acetonitrile, tetrahydrofuran, ethyl acetate and methyl t- A large series of organic solvents have been investigated. It butyl ether are purchased from Merck, Darmstadt, Germany. is found that, among the usual solvents for chromatography, Trans-stilbene oxide is purchased from Fluka, Germany. methyl t-butyl ether, CH2Cl2, tetrahydrofuran, ethyl acetate, together with the standard solvents are those with the highest 1.2. Procedure potential in terms of enantioselectivity on Chiralpak® IA 1.2.1. Equipment [19]. Although this CSP can afford more or less comparable separations with standard mobile phases as Chiralpak® AD, The LC System, used for method development, and its real interest lies in the use of non-standard ones. method verification are Shimadzu 2010 & Agilent 1100 Model Systems with variable UV detector. The output signal The method development is done with standard solvents is monitored and processed using LCsolution & chemstation like normal phase solvents, i.e polar solvents and reverse software on pentium computer. phase solvents and then with non standard solvents. The chiral column used in method development Chiral- 1.3.2. Optimization Strategy (Performance Control Analysis) pak -IA is manufactured by Daicel Chemical Industries Ltd; The performance of the column is checked by its ability Japan having 5 m particle size in 250 x 4.6 mm dimension. to separation of enantiomers in trans-stilbene oxide (Fig. 1.3)

Table 1. Optimized Conditions for the Analysis of Bicalutamide and Thalidomide Enantiomers

Compound Bicalutamide Thalidomide

Wave length 270 nm 210 nm

Flow 1.0 mL/min

Temperature 25°C

Diluent Methanol

Mobile phase - 1 Methanol: Hexanes: Tetrahydrofuran (30:50:20 v/v)

Mobile phase - 2 Dichloromethane: Methanol: n-Hexane (40:20:40 v/v )

Mobile phase - 3 Ethyl Acetate: Methanol: n-Hexane (30:55:15v/v )

Mobile phase - 4 Methyl t-butyl ether: n-Hexane: Methanol (50:10:40 v/v)

Novel Chiral LC Methods for the Enantiomeric Separation Current Pharmaceutical Analysis, 2011, Vol. 7, No. 1 49

2.2. Multi Wavelength Analysis The multi wavelength analysis is done to analyze bicalutamide and thalidomide together and is found suitable. O Wavelength program is as given below. Chromatogram is Fig. (1.3). Trans-Stilbene Oxide. enclosed for mobile phase combination 1. (Figs. 2 and 3). with a criterion of resolution not less than 2.0 and tailing factor between 0.8 to 2 and theoretical plates not less than Time(min) Wavelength(nm) 4000 for the enantiomeric peaks. Performance is measured 0 270 before and after the experiments and at regular intervals. The set criterion for performance control was met before and 5.0 210 after the experiments. 1.3.3. Selection of Mobile Phase Samples of bicalutamide and thalidomide analysed in 4 optimized mobile phases resulted in 50 : 50 enantiomers as The investigation of above listed solvents for their gen- expected theoretically meeting system suitability criterion. eral behavior in enantio selective separations is made in the The column is found working satisfactorily after analysis mobile phase selection [19]. The method development is using standard solvents and non standard chromatographic done with standard solvents i.e normal phase solvents [polar solvents. solvents] and reverse phase solvents and then with non standard solvents. The target is to achieve resolution between 2.3. Analytical Method Validation two enantiomers of not less than 1.5 with and tailing factor 2.3.1. Limit of Quantification (LOQ) between 0.8 to 2.0 and theoretical plates not less than 4000. The quantitation limit of an individual analytical proce- 1.3.4. Using Polar and Non Polar Solvents dure is the lowest amount of analyte in a sample which can The trials are unsuccessful in n-hexane combination with be quantitatively determined with suitable precision and ac- -1 ethanol and IPA in which either peaks are not eluted or reso- curacy. LOQ was found to be 0.1g mL for bicalutamide -1 lution is not achieved. In ethanol combination bicalutamide and 0.05 g mL for thalidomide with signal to niose ratio enantiomers separated but with distorted peaks while tha- above 10. lidomide no peaks are eluted. 2.3.2. Precision 1.3.5. Method Trials in Non Standard Solvents Bicalutamide and thalidomide samples are injected in replicates [n=3] and %RSD is less than 1% for the enanti- Tetrahydrofuran combination with methanol and hexanes omer peak areas. resulted in satisfactory resolution more than 1.5 between the two target compounds enantiomers and theoretical plates 2.3.3. Linearity and Range more than 4000 and tailing factor between 0.8 and 2.0. The To establish linearity of the method, calibration solutions combination of the three solvents dichloromethane, methanol were prepared from stock solution at six concentration lev- and n-hexane is successful in achieving satisfactory resolu- els. Concentration levels ranging from LOQ to 150% (LOQ, tion and other chromatographic parameters. The combination 50, 80,100,120 and 150%) were prepared. Average peak area of ethyl acetate with methanol and n-hexane yielded satisfac- at each concentration level was subjected to linear regression tory resolution and other chromatographic parameters. The analysis with the least square method. Calibration equation combination of the three solvents methyl t-butyl ether, n- obtained from regression analysis was used to calculate the hexane and methanol is also successful. Methanol has played corresponding predicted responses. The residuals and sum of a decisive role in achieving the separation and resolution in the residual squares were calculated from the corresponding all the four mobile phase combinations. predicted responses. The correlation coefficient was more than 0.995 demonstrating a linear relationship between con- 2. RESULTS AND DISCUSSION centration and response within the specified range. 2.3.4. Robustness 2.1. Optimized Mobile Phases The robustness of an analytical procedure is a measure of The following four mobile phases are optimized for the its capacity to remain unaffected by small, but deliberate analysis of bicalutamide and thalidomide enantiomers in variations in method parameters and provides an indication which resolution is above 1.5, Theoretical plates were more of its reliability during normal usage. Flow rate and tempera- than 5000 and tailing factor is between 0.8 and 2.0 (Table ture were changed by 10% in all the 4 mobile phases and 2.1-2.4, respectively). found satisfactory resolution demonstrating the method’s 1. Methanol: Hexanes: THF (30:50:20 v/v) robustness (Fig. 4). 2. MDC: Methanol: n-Hexane (40:20:40 v/v) CONCLUSION 3. Ethyl Acetate: Methanol: n-Hexane (30:55:15v/v) Four novel chiral LC methods are described for the enantiomeric separation of bicalutamide and thalidomide. Chiral- 4. MTBE: n-Hexane: Methanol (50:10:40 v/v). 50 Current Pharmaceutical Analysis, 2011, Vol. 7, No. 1 Raju et al.

Table 2.1. Results in the Optimized Conditions for the Analysis for Mobile Phase-1

Compound Bicalutamide Thalidomide

Peak 1 Peak 2 Peak 1 Peak-2 RT 5.0 5.6 7.5 10.5 % of Each component 49.86 50.14 49.85 50.15 % RSD for area 0.07 0.12 0.15 0.18

Resolution 2.5 6.7

Mean Tailing factor 1.2 1.2 0.9 0.9 Theoretical plates 7286 5759 7709 5874

Table 2.2. Results in the Optimized Conditions for the Analysis for Mobile Phase-2

Compound Bicalutamide Thalidomide

Peak 1 Peak 2 Peak 1 Peak-2 RT 3.4 3.7 6.7 7.2 % of Each component 48.83 51.17 49.44 50.56 % RSD for area 0.37 0.17 0.12 0.87 Resolution 1.7 1.5 Mean Tailing factor 1.1 1.3 1.2 1.1 Theoretical plates 7876 7364 9699 9944

Table 2.3. Results in the Optimized Conditions for the Analysis for Mobile Phase-3

Compound Bicalutamide Thalidomide

Peak 1 Peak 2 Peak 1 Peak 2 RT 3.3 3.6 5.9 9.1

% of Each component 49.76 50.24 49.83 50.17

% RSD for area 0.16 0.20 0.90 1.08

Resolution 1.6 10.1

Mean Tailing factor 1.3 1.3 1.1 1.1

Theoretical plates 6392 7416 8648 8819

Table 2.4. Results in the Optimized Conditions for the Analysis for Mobile Phase-4

Compound Bicalutamide Thalidomide

Peak 1 Peak 2 Peak 1 Peak 2 RT 3.6 4.2 8.3 11.9

% of Each component 49.78 50.22 49.94 50.06

% RSD for area 0.86 0.68 0.05 0.01

Resolution 3.5 9.4

Mean Tailing factor 1.2 1.2 1.1 1.1

Theoretical plates 7080 8082 11266 11044 Novel Chiral LC Methods for the Enantiomeric Separation Current Pharmaceutical Analysis, 2011, Vol. 7, No. 1 51

mV mV Det.A Ch1 500 500 Det.A Ch1 Bicalutamide MP-1 Thalidomide MP-1 Isomer-1 / 4.978 Isomer-2 / 5.644 Isomer-1 / 7.492

250 250 Isomer-2 / 10.451

0 0 0.0 2.5 5.0 7.5 10.0 12.5 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min min mV Det.A Ch1 mV Det.A Ch1 300 Bicalutamide MP-2 50 Thalidomide MP-2 Isomer-1 / 3.422 Isomer-2 / 3.726

Isomer-1 / 6.740 Isomer-2 / 7.169 200

100

0 0 0 1 2 3 4 5 6 7 0.0 2.5 5.0 7.5 min min mV mV100 Det.A Ch1 Det.A Ch1 Bicalutamide MP-3 75 Thalidomide MP-3 1000 Isomer-1 / 3.280 Isomer-2 / 3.620

50 Isomer-1 / 6.500

Isomer-2 / 9.559 500 25

0 0

0.0 2.5 5.0 7.5 10.0 12.5 0.0 2.5 5.0 7.5 min min mV mV Det.A Ch1 Det.A Ch1 Thalidomide MP-4 1000 300 Bicalutamide MP-4

Isomer-1 / 8.269 750 Isomer-1 / 3.562 Isomer-2 / 4.176 Isomer-2 / 11.862 200

500 100 250 0 0 0.0 2.5 5.0 7.5 10.0 12.5 0 1 2 3 4 5 6 7 min min

Fig. (2). Bicalutamide and thalidomide chromatograms in optimized conditions.

R1 =2.0 R2 = 6.6

Fig. (3). Typical wavelength program chromatograms of bicalutamide and thalidomide Mobile phase: Methanol: Hexanes: Tetrahydrofuran (30:50:20v/v). 52 Current Pharmaceutical Analysis, 2011, Vol. 7, No. 1 Raju et al.

Bica ‐ Bicalutamide 7080 6392 1.2 8082 Tha ‐Thalidomide 7876 1.3 7416 1.2 7286 11266 1.1 3.5 7364 1.3 1.6 1.2 1.1 11044 5759 1.3 1.7 9699 1.1 Bica ‐1 8648 1.1 1.2 2.5 9.4 7709 8819 1.2 9944 1.1 Bica‐2 10.1 0.9 1.1 5874 1.5 Tha ‐1 0.9 6.7

Tha ‐2

MP ‐ Mobile Phase Rs‐ Resolution T‐ Tailing Factor N‐ Theorotical Plates

Fig. (4). Comparison of performance parameters across mobile phases. pak-IA is found to be selective for bicalutamide and tha- [9] Subramanian, G. A Practical Approach to Chiral Separations by lidomide enantiomeric separation. The methods are showing Liquid Chromatography. VCH Verlagsgesellschaf mbH, Wein- heim, Germany, 1994. satisfactory data for all the parameters tested. The developed [10] Ahuja, S. Chiral Separations, ACS, Washington DC, 1997, Chap- methods can be used for the separation and quantitative de- ter 10, pp. 271. termination of enantiomers in active pharmaceutical ingredi- [11] Yashima, E.; Okamoto, Y. Chiral discrimination on polysaccha- ent samples of bicalutamide and thalidomide. rides derivatives. Bull. Chem. Soc. Jpn., 1995, 68, 3289-3307. [12] Yashima, E. Polysaccharide-based chiral stationary phases for Bicalutamide and thalidomide samples analysed in 4 op- high-performance liquid chromatographic enantioseparation. J. timized mobile phases resulting 50 : 50 enantiomers as ex- Chromatogr. A, 2001, 906,105-125. [13] Tachibana, K.; Ohnishi, A. Reversed-phase liquid chromatographic pected theoretically. The resolution between the two enanti- separation of enantiomers on polysaccharide type chiral stationary omers in 4 optimized mobile phases is above 1.5, tailing fac- phases. J. Chromatogr. A, 2001, 906, 127-154. tor in all cases is between 0.80 and 1.5 and theoretical plates [14] Aboul-Enein, H.Y. High-performance liquid chromatographic of the two enantiomeric peaks for the three compounds is enantioseparation of drugs containing multiple chiral centers on polysaccharide-type chiral stationary phases. J.Chromatogr. A, more than 5000. 2001, 906, 185-193. [15] Okamoto, Y.; Kawashima, M.; Yamamoto, K.; Hatada, K. Useful ACKNOWLEDGEMENTS chiral packing materials for high-performance liquid chromatographic resolution. Cellulose triacetate and tribenzoate coated on The authors would like to thank the Hetero Labs Khazi- macroporous silica gel. Chem. Lett., 1984, 13(5), 739-742. pally for donating the samples and supporting this work. [16] Okamoto,Y.; Kawashima, M.; Hatada, K. Useful chiral packing materials for high-performance liquid chromatographic resolution of enantiomers: phenylcarbamates of polysaccharides coated on sil- REFERENCES ica gel. J. Am. Chem. Soc., 1984, 106(18), 5357-5359. [1] Schellhammer, P.F. An evaluation of bicalutamide in the treatment [17] Okamoto, Y.; Aburatani, R.; Fukumoto, T.; Hatada, K. Useful of prostate cancer. Expert Opin. Pharmacother., 2002, 3(9), 1313- chiral stationary phases for HPLC. amylose Tris(3,5-dimethyl- 1328. phenylcarbamate) and Tris(3,5-dichlorophenylcarbamate) sup- [2] Desikan, R.; Munsi N.; Zeldis, J. Activity of thalidomide (THAL) ported on silica gel. Chem. Lett., 1987, 16(9), 1857-1860. in multiple myeloma (MM) confirmed in 180 patients with ad- [18] Ali, I.; Aboul-Enein, H.Y. Immobilized polysaccharide CSPs: an advancement in enantiomeric separations. Curr. Pharm. Anal., vanced disease. Blood, 1999, 94(1), 603a, Abstract. 2007, 3(1), 71-82. [3] Sahajwalla, C.G. New Drug Development, Marcel Dekker. Inc., [19] Zhang, T.; Franco, C.K.P.; Ohnishi, A.; Kagamihara, Y.; Kurosawa, New York, 2004, pp. 421-426. H. Solvent versatility of immobilized 3,5-dimethylphenylcarbamate [4] Beesley, T.E.; Scott, R.P.W. Chiral Chromatography. John Wiley of amylose in enantiomeric separations by HPLC. J. Chromatogr. & Sons, Ltd., 1998, pp. 23-26. A, 2005, 1075(1-2), 65-75. [5] Subramanian, G. A Practical Approach to Chiral Separations by [20] Chiral technologies inc. Available at: [http://www.chiraltech.com/ Liquid Chromatography, VCH Publishers. Weinheim, Germany, col_csp.asp] [http://www.chiraltech.com/faqs.asp]. [Accessed: June 1994. 22, 2009] [6] Aboul-Enein, H.Y.; Wainer, I.W. The Impact of Stereochemistry in [21] Rao, R.N.; Raju, A.N.; Nagaraju, D. An improved and validated Drug Development and Use. Wiley, New York, 1997. LC method for resolution of bicalutamide enantiomers using amy- [7] Allenmark, S. Chromatographic Enantioseparation: Methods and lose tris-(3,5-dimethylphenylcarbamate) as a chiral stationary nd Applications, 2 ed., Ellis Horwood, New York, 1991. phase. J. Pharm. Biomed. Anal., 2006, 42(3), 347- 353. [8] Okamoto, Y.; Kaida, Y. Resolution by high-performance liquid [22] Török, R.; Bor, A.; Orosz, G.; Lukács, F.; Armstrong, D.W.; Péter, chromatography using polysaccharide carbamates and benzoates as A. High-performance liquid chromatographic enantioseparation of chiral stationary phases. J. Chromatogr. A, 1994, 666, 403-419. Novel Chiral LC Methods for the Enantiomeric Separation Current Pharmaceutical Analysis, 2011, Vol. 7, No. 1 53

bicalutamide and its related compounds. J. Chromatogr. A, 2005, common-size columns, capillary liquid chromatography and 1098(1-2), 75-81. nonaqueous capillary electro chromatography. J. Chromatogr. A, [23] Murphy-Poulton, S.F.; Boyle, F.; Gu, X.Q.; Mather, L.E. Thalido- 2000, 876(1-2), 157-167. mide enantiomers: Determination in biological samples by HPLC [25] Welch, C.J.; Szczerba, T.; Perrin, S.R. Some recent high- and vancomycin CSP. J. Chromatogr. B, 2006, 831(1-2), 48-56. performance liquid chromatography separations of the enantiomers [24] Meyring, M.; Chankvetadze, B.; Blaschke, G. Simultaneous sepa- of pharmaceuticals and other compounds using the Whelk-O 1 ration and enantioseparation of thalidomide and its hydroxylated chiral stationary phase. J. Chromatogr. A, 1997, 758(1), 93-98. metabolites using high-performance liquid chromatography in

Received: 31 March, 2010 Revised: 14 June, 2010 Accepted: 06 July, 2010