Isoxsuprine Hydrochloride

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Keywords: UFLC, Lux Cellulose-4, Methyl Paraben, Enantiomers. 1. ... Analytically pure reference standard of ISX was kindly supplied by Jayco chemical ...

International Journal of Pharma Sciences Vol. 5, No. 2 (2015): 1019-1024 Research Article Open Access

ISSN: 2320-6810

Enantioselective Quantification of Beta-Adrenergic Agonist: Isoxsuprine Hydrochloride Sirisha Tadiboyina*, Gurupadayya Bannimath and Bharath Kumar Inturi Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS University, Mysuru-570015, Karnataka, India.

* Corresponding author: Sirisha Tadiboyina; e-mail: [email protected]

Received: 30 March 2015

Accepted: 16 April 2015

Online: 20 April 2015


Isoxsuprine hydrochloride, a beta-adrenergic agonist is a racemic mixture available in the market. As the drug consist of a single chiral center, two enantiomers were possible for it. The present work demonstrate the resolution of these two enantiomers on a polysaccharide based chiral column using ultra fast liquid chromatography attached with a diode-array detector (UFLC-DAD).The developed method was used to separate and quantify the isoxsuprine hydrochloride enantiomers in bulk and tableted formulations. The separation of the two enantiomers has been accomplished using cellulose tris (4-chloro-3-methylphenyl carbamate) stationary phase [Lux cellulose-4 column] with a mobile phase of 20mM ammonium bicarbonate buffer and methanol (60:40 v/v) at a flow rate of 1.0 mL/min. In order to achieve better resolution between the two enantiomers diethylamine (0.15%) was used as an organic modifier. The mobile phase variables which have an effect on the enantiomers separation like ionic strength, type and concentration of organic modifier were studied. Methyl paraben was used as an internal standard. The internal standard and the two enantiomers were eluted in the column at 4.5 min, 10.9 min and 13.6 min respectively. The method has been validated and found to be accurate and precise. Calibration curves were linear for both the enantiomers over the range of 1-11μg/mL(R2>0.999).A detection limit of 200ng/mL was found for each enantiomer. This chiral separation is used to quantitate the two isoxsuxprine enantiomers present in the bulk drug, tablet formulations and in pharmacological and toxicological studies.

Keywords: UFLC, Lux Cellulose-4, Methyl Paraben, Enantiomers. 1. INTRODUCTION

Isoxsuprine hydrochloride (ISX) is a β adrenergic agonist (Figure 1) mainly used to arrest premature labor. It also administered in the treatment of cerebral and peripheral vascular diseases [1]. Chemically known as 4-hydroxy-a-[1-[(1 -methyl-2-phenoxyethyl)-amino] propyl] phenol. ISX racemic drug is unstable in normal form hence it is marketed as a hydrochloride salt form.

Stereoselectivity plays a major role in the drug’s action. According to USFDA the single enantiomer drugs are safer, better tolerated and more efficient [2]. A search of literature survey indicated the pharmacological difference between (+) and (-) forms of ISX enantiomers with respect to binding affinity for alphaadrenoceptors [3].Hence there is a need for the separation of both the enantiomers. ISX is official in United State Pharmacopoeia (USP), it describes a simple spectrophotometric method for the assay of

bulk drug and column partition chromatographic method for the assay of tablets and ampoules [4]. To the extent of our knowledge the reported analytical methods that relate to ISX are three spectrophotometric methods by kinetic technique [5], charge transfer complexation [6] and through derivatisation [7]. One Flourimetric method [8] and a GC-MS method for the determination of ISX in biological fluids [9], simple HPLC method [10], stability indicating HPLC method [11] and a simultaneous estimation with ritodrine by HPLC [12]. A thorough literature survey reveals that there are methods for the resolution of ISX enantiomers by using derivatizing reagent [13]. Novel strong cation-exchange type chiral stationary phase [14] zwitter ionic chiral stationary phase [15]. Supercritical fluid chromatography [16] and capillary electrophoresis[17] which comprise of their own drawbacks (mentioned in the later part of this article). The proposed study is mainly designed to 1019

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overcome the drawbacks of existing methods, and

made an attempt to solve. OH






(a). Isoxsuprine Hydrochloride

(b). Methylparaben

Figure 1: Chemical structures of (a) Isoxsuprine hydrochloride and (b) Methyl paraben


2.1. Chemicals and reagents Analytically pure reference standard of ISX was kindly supplied by Jayco chemical industries. (Maharashtra, India).The formulation used in this study was the DUVADILAN tablet (Solvay pharma India Limited.) labelled to contain 10 mg of ISX, purchased from local pharmacy. Ammonium bicarbonate (HPLC grade, Sigma-aldrich, India), acetonitrile (HPLC grade, Merck Specialities Pvt. Ltd., Mumbai, India), and diethylamine (HPLC grade, Spectrochem Pvt. Ltd, Mumbai, India) were used in the analysis.

2.2. Instrumentation The ultra fast liquid chromatography (UFLC) used was Shimadzu Prominence LC-20AD equipped with a 1260 binary pump VL (35MPa), Prominence SIL-20ACHT Autosampler, and Prominence SPD-M20A Diode array detector. All weighing for analysis were performed on a Shimadzu electronic analytical balance AY-220 (Shimadzu).Water used for analysis was prepared from Millipak Express 20 filter unit.

2.3. Preparation of Standard solutions ISX reference standard (10mg) was accurately weighed, transferred to 100ml volumetric flask and dissolved in water by sonicating for 15 minutes, and then completed to volume with water(final concentration 0.1mg/ml). Also, an accurately weighed methyl para hydroxy benzoate reference internal standard(10mg) was transferred to 100ml volumetric flask and dissolved in water by sonicating for 15 minutes, then completed to volume with water(final concentration 0.1mg/ml). All the solutions were freshly prepared.

For construction of calibration graph, aliquot proportions (0.1-1.1 ml) of 0.1mg/ml ISX standard solution were taken into a series of 10ml volumetric flasks, 1 ml of methyl para hydroxy benzoate (0.1mg/ml) was added into each volumetric flask, and volume made up to 10ml with water to get 1, 3, 5, 7, 9 and 11 μg/ml of each enantiomer and 10μg/ml of methyl para hydroxy benzoate. All the dilutions were

filtered through a 0.45μm pore size membrane filter and degassed by an ultrasonicator.

2.4. Preparation of sample solution About 20 tablets were weighed and finely powdered. Powder weight equivalent to one tablet (claimed to contain 10mg of isoxsuprine hydrochloride) was transferred into a 100ml volumetric flask. Then extracted with water and sonicated for 30min and made up to volume with water. The solution was filtered by using 0.45μm millipore membrane filter. From the filtered solution 0.9ml of filtrate was pipetted into a 10ml volumetric flask and 1mL of (0.1mg/mL) internal standard was added and made up the volume with water. Concentration of isoxsuprine hydrochloride enantiomers were determined by peak area ratio of enantiomer to that of internal standard. Concentration of unknown sample was calculated from regression equation.

2.5. Chromatographic conditions The filtered dilutions were chromatographed by the set of conditions on Shimadzu LC-20AD Prominence series. The mixture of 20mM ammonium bicarbonate buffer and acetonitrile in the ratio of (60:40v/v) was used as mobile phase for the elution of the drug on Lux Cellulose-4 column (250 mm × 4.6 mm, 5μm) at 1.0 ml/min of flow rate. Methyl para hydroxy benzoate used as internal standard and it eluted at 4.5 min. ISX enantiomers were successfully eluted at 10.92 min and 13.6 min with a run time of 25 min and detection was performed by diode array detector at 220 nm.


3.1. Optimization of the method 3.1.1 Selection of column The separation of enantiomers was done by direct and indirect techniques [18]. The use of dervetising reagents to convert the racemic drug into diastereomers was indirect way of separating the enantiomers, but it is a tedious technique. Now a day’s most of the stereoselective estimation was done using the modern chiral stationary phases (CSP’S) [19]. Among the different CSP’S available in the market, cellulose based polysaccharide chiral stationary phase 1020

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was one of the best CSP for the separation of aromatic compounds with different functional groups. These immobilized polysaccharide columns have a wide range of applicability because of their diverse chiral recognition mechanisms [20]. Hence the immobilized cellulose-based polysaccharide column (Lux cellulose 4) was selected for the project. The CSP in Lux cellulose -4 is Cellulose tris (4-chloro-3methylphenylcarbamte) bonded to silica gel. The resolution of enantiomers may be ascribed due to hydrogen-bonding and dipole–dipole interactions [21]. When compared to the non-substituted cellulose derivative, the phenylcarbamates bearing electronwithdrawing substituents (chlorine) exhibit better chiral recognitions by affecting the polarity of the carbamate group by inductive effect.

3.1.2. Optimization of buffer and its ionic strength: Based on the pKa of drug molecule, basic buffer was used for screening. During that process a combination of ammonium bicarbonate buffer and methanol (60:40 v/v) showed good separation between the enantiomers. The ionic strength of the buffer was optimized [22] by preparing different concentrations (10-50Mm) of buffer. The retention of enantiomers was found to be indirectly proportional to buffer ionic strength (Figure 2). The concentration of buffer and the retention time of enantiomers were found to be viceversa, with 10mM ammonium bicarbonate buffer the enantiomers were eluted lately but when the concentration increases to 50mM the enantiomers were eluted early, the resolution between the enantiomers is poor. Hence from the study in order to achieve a better resolution and faster elution the buffer strength was optimized at 20mM concentration.

Figure 2: Effect of ionic strength on stereoisomer retention.

3.1.3. Effect of % organic modifier: For the separation of most of the analytes containing basic and acidic functional groups the addition of additives plays an important role. Triethylamine (TEA) and Diethyl amine (DEA) are basic modifiers which improve the peak shape and reduce the peak tailing. When compared to triethylamine (TEA), diethylamaine (DEA) showed better resolution between the enantiomers. Hence in the present work inorder to

improve the peak shape and reduce the tailing, DEA was used as organic modifier (Figure 3). At 0.02% DEA the enantiomers were eluted early having poor resolution, as the concentration increase to 0.2% the enantiomers were eluted lately but with better resolution. Hence from the study in order to achieve a better resolution and faster elution the % DEA was optimized at 0.15%.


Sirisha Tadiboyina et al. / Int J Pharma Sci. 2015, 5(2): 1019-1024

Figure 3: Effect of % organic modifier on stereoisomer retention.

Figure 4: Chromatogram of (A) blank solution, (B) Resolution of ISX enantiomers from bulk drug and (C) pharmaceutical dosage form (Duvadilan 10mg TABLET). Table 1: Chromatographic data for Isoxsuprine hydrochloride enantioseperation on Lux Cellulose-4 chiral column

S.No 1

Chromatographic parameter Capacity factor of first eluted enantiomer (K1)


Capacity factor of second eluted enantiomer (K2)



Resolution factor





Value obtained 1.42

Selectivity factor


Theoretical plates


The chromatographic parameters like capacity factor (K’) (gives information about the interaction of enantiomers), Selectivity factor (α) (defined as a ratio of capacity factors) and Resolution factor (Rs) (degree of separation between two enantiomers on a chromatography system) were analysed for ISX enantiomers (Figure 4) and listed in Table 1. 3.2. Method validation The method was validated according to the international conference on Harmonization guidelines for validation of analytical procedure [23]. 3.2.1. Linearity The calibration curve was obtained with six concentrations of the standard solution 1-11µg/mL.


The solutions were prepared in triplicate. The linearity was evaluated by the least square regression method. Results were summarized in Table 2.

3.2.2. Precision: The precision of the assay was determined by repeatability (intra-day) and intermediate precision (Inter-day). Intra-day precision was evaluated by assaying sample, at the same concentration on the same day. Six sample solutions of three different concentrations (3, 5 and 7 µg/mL) were prepared and assayed. The intermediate precision (inter-day) was studied by comparing the assays on different days (3 days). The results were summarized in Table 3.

Table 2: Linearity data of Enantiomer-1 and Enantiomer-2.


Beer’s range (ppm) Slope (m) Y Intercept Correlation coefficient (r2) LOD (ppm) LOQ (ppm)

Enantiomer-1 1-11ppm 0.319 0.458 0.999 0.091 0.278


1-11ppm 0.328 0.431 0.999 0.0812 0.246


Sirisha Tadiboyina et al. / Int J Pharma Sci. 2015, 5(2): 1019-1024

Table 3: Intra –Day and Inter-Day Accuracy and Precision Data of UFLC method for Isoxsuprine hydrochloride.

Theoretical concentration (µg/mL) Enantiomer 1 3 5 7 Enantiomer 2 3 5 7

Intra-Daya Accuracy (%) 100.41 99.82 99.54

1.003 0.293 0.595

99.8 99.45 99.63

1.524 0.319 0.119

3.2.3 Accuracy: The accuracy of an analytical method is determined by how close the test results obtained by application of the analytical procedure to an analyte of known purity or by recovery studies (where a known amount of standard is spiked in the placebo). In this study, a number of different solutions were prepared with a known added amount of drug substance and injected in triplicate. Percent recovery was calculated as shown in


Inter-Daya Accuracy (%)

Precision (RSD %)

100.52 101.29 101.6 101.7 100.1 101.64

pH of the mobile phase (8.0±0.2) Flow rate (1.0±0.2 mL/min) Column temperature (300C ± 50C)

1 11 1 11 1 11

3.2.5 Solution stability and mobile phase stability: The sample and standard solutions were injected at 0 hr (comparison sample), 24hr and after 48 hr (stability sample) at ambient room temperature (30̊ C). The %RSD for 0 hr, 24hr and 48 hr for sample and standard solutions were