A new, simple, and rapid stereoselective normal phase-liquid chromatographic (NP-LC) method was developed to separate and quantify the solifenacin succinate and its three stereoisomers. The stereoisomeric separation was achieved on Chiralpak IC (
Solifenacin, a well-known muscaranic receptor antagonist belongs to the anticholinergics used for the treatment of patients with overactive bladder such as urinary urgency, urinary incontinence, and high urinary frequency. It acts as a selective antagonist to the M(3) receptor and works by relaxing the bladder muscles to prevent urgent, frequent, or uncontrolled urination. It is available in the market under the brand name Vesicare in the form of tablets for oral administration containing 5 or 10 mg of solifenacin succinate taken once daily [
Chemical structures of Solifenacin and its three stereoisomers and two precursors.
Development of an analytical method for the quantitative determination of stereoisomers in the drug substances/drug products having chiral centers is an important and challenging task during the drug synthesis and formulation research in pharmaceutical companies. It is highly essential to monitor and control other isomers to meet the stringent quality requirements. Although many analytical techniques like gas chromatography (GC), capillary electrophoresis (CE), and liquid chromatography (LC) can be employed for this purpose, liquid chromatography with a chiral stationary phase (CSP) is the most widely used technique [
Samples of solifenacin succinate, a mixture of all four stereoisomers of solifenacin and its three stereoisomers were received from synthetic laboratory, Megafine Pharma (P) Ltd., Nashik, India. HPLC grade n-Hexane, ethanol, and isopropyl alcohol were purchased from Qualigen fine chemicals, Mumbai, India. Diethylamine was purchased from Merck, Mumbai, India.
The LC system used for method development and method validation was Agilent 1200 series connected with a photodiode array detector (PDA) and variable wavelength detector (VWD). Chiral detection was performed using advanced laser polarimeter (PDR Chiral Inc.). The output signal was monitored and processed using EZChrome Elite software version 3.2.1. The chiral columns used during method development were Chiralcel OD-H, Chiralpak AD-H, Chiralpak-IA, Chiralpak-IB, and Chiralpak-IC (Daicel Chemical Industries, Japan) with 5
The chromatographic column used was a Chiralpak-IC, 250 mm length
A mixture of all four stereoisomers standard solution of 6
The selectivity of the developed method was checked by injecting solifenacin succinate spiked with all stereoisomers and its precursor at 0.15% level with respect to target analyte concentration.
The precision of the method was carried out by injecting six individual preparations of solifenacin succinate spiked with 0.15% of all the stereoisomers (Isomer-1 to Isomer-3) with respect to target analyte concentration (i.e., 1.0 mg
The intermediate precision of the method was verified by performing the analysis on different day using different instrument number in the same laboratory by a different analyst. The results of method precision and intermediate precision were expressed in terms of percentage of stereoisomers in six spiked test preparations. The RSD (%) for results of intermediate precision and results of method precision was calculated and compared with each other.
The LOD and LOQ of all the three stereoisomers were determined using calibration curve method according to ICH Q2R1 recommendations by establishing the lowest concentration that can be measured with precision. Precision study was also carried out at the LOQ level by injecting six individual preparations of all the stereoisomers and calculating RSD (%) of the area of each stereoisomer.
The linearity of an analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to the concentration of the analyte in the sample. Linearity test solutions were prepared by diluting the stereoisomers stock solution to the required concentrations. The solutions were prepared at six concentration levels from LOQ to 500% with respect to the stereoisomers specification level of 0.15% (i.e., LOQ. 0.075, 0.150. 0.225, 0.300, 0.600, and 0.750% related to solifenacin). The calibration curves for the stereoisomeric impurities were drawn by plotting the peak areas of stereoisomers versus its respective concentration. The correlation coefficient, intercept, and slope of the calibration curve were calculated and reported.
The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found. The recovery experiments were conducted to determine an accuracy of a developed HPLC method for the quantification of all three potential stereoisomeric impurities of solfenacin (Figure
To assess robustness of the method, the experimental conditions were deliberately altered, and resolution between the stereoisomers was evaluated. The effect of flow rate was studied at 0.9, 1.0, and 1.1 mL
To achieve the good separation between solifenacin succinate and its three stereoisomers, five chiral columns, namely Chiralpak AD-H, Chiralcel OD-H, Chiralpak IA, Chiralpak IB, and Chiralpak IC, were screened under normal phase conditions. Owing to the basic nature of the analyte, 0.1% DEA was added to the starting mobile phases n-Hexane/IPA (80/20, v/v) and n-Hexane/EtOH (80/20, v/v). A mixture of all four stereoisomers of solifenacin succinate was used during the method development. Indication of separation between all the four peaks was noticed on all the columns except Chiralpak IB column. Method optimization experiments were performed using Chiralpak IC column due to its high selectivity compared to other columns screened under this study. Chiralpak IC is the latest column in the immobilized polysaccharide category of columns. Its chiral selector tris (3, 5-dichlorophenylcarbamate) was immobilised on silica gel and hence compatible with all kinds of solvents.
On Chiralpak IC column, retention times were found to be longer in case of IPA compared to ethanol as a modifier in the mobile phase. Interestingly, change in elution order was observed with change in alcohol modifier in presence of constant chromatographic conditions (i.e., flow rate, column oven temperature
Chromatographic characteristics of system suitability solution.
Component | System suitability parameter | ||
Theoretical plate | Asymmetry | Resolution | |
Isomer-1 | 9320 | 1.17 | — |
Isomer-2 | 8627 | 1.18 | 3.2 |
Isomer-3 | 7900 | 1.18 | 6.4 |
Solifenacin | 7415 | 1.15 | 5.7 |
(a) Isomer elution order using n-Hexane : IPA : DEA (70 : 30 : 0.1) mobile phase. (b) Isomer elution order using n-Hexane : EtOH : DEA (85 : 15 : 0.1) mobile phase. (c) Typical chromatogram representing isomeric separation of Solifenacin Succinate using n-Hexane : EtOH : IPA : DEA (60 : 15 : 25 : 0.1) mobile phase. (All trials have been conducted at column temperature
Further, the effect of temperature on the resolution between all the stereoisomers was studied. Column temperature was maintained at 10, 20, 30, and
Effect of column temperature on resolution between stereoisomers.
The selectivity of method was checked by injecting the test sample spiked with each individual stereoisomer and its precursor at 0.15% level of analyte concentration. An excellent separation between all the peaks was observed (Figure
Method selectivity as illustrated by a typical chromatogram of solifenacin spiked with its precursor and stereoisomers at 0.15% level. (Chromatographic conditions- Chiralpak-IC, 250 mm
The RSD (%) of content of isomer-1 to isomer-3 in method precision study was within 1.9% and in intermediate precision study was within 3.8%. The results of method precision and intermediate precision were compared with each other. The overall RSD (
Precision, linearity, and accuracy analysis results of solifenacin succinate.
Validation components | Isomer-1 | Isomer-2 | Isomer-3 |
---|---|---|---|
%RSD in MP ( | 1.35 | 1.89 | 1.26 |
%RSD in IP ( | 3.42 | 3.80 | 3.27 |
Mean | 0.147 | 0.158 | 0.156 |
0.004 | 0.004 | 0.005 | |
Overall %RSD ( | 2.72 | 2.53 | 3.21 |
Correlation coefficient ( | 0.99955 | 0.99968 | 0.99947 |
Slope | 34545.27 | 33742.05 | 33984.35 |
Intercept | 592.75 | 1884.67 | 562.54 |
LOQ | |||
50% | |||
100% | |||
150% |
The LODs of isomer-1, isomer-2 and isomer-3 were 0.003, 0.002, and 0.004%, respectively (of analyte concentration i.e., 1.0 mg
Linear calibration plots were drawn for each stereoisomer (isomer-1, isomer-2, and isomer-3) ranging from LOQ to 500% of stereoisomer specification (NMT 0.15%), and the correlation coefficient obtained was greater than 0.999. These results demonstrate that an excellent correlation existed between the peak area and concentration of isomer-1 to isomer-3. The correlation coefficient, intercept, and slope for each stereoisomer is shown in Table
The percentage recovery of stereoisomers (isomer-1 to isomer-3) in bulk drugs samples ranged from 93.3% to 104.6%. The results of accuracy are reported in Table
Typical chromatogram of solifenacin test sample, (a) unspiked and (b) spiked with stereoisomers at 0.15% level.(Chromatographic conditions- Chiralpak-IC, 250 mm
Unspike test sample
Spike test sample
In all the deliberate varied chromatographic conditions (flow rate, column temperature, and composition), the resolution between successive stereoisomers was greater than 3.0, illustrating the robustness of the developed method.
The solution stability of solifenacin succinate and its three stereoisomers was assessed by keeping the spiked sample solutions in a tightly capped volumetric flask at room temperature on a laboratory bench for 48 h, calculating the amount of isomer-1, isomer-2, and isomer-3 at every 6 h intervals and comparing with freshly prepared solution. The solution stability experiment data confirms that sample solutions prepared in the mobile phase were stable up to 48 h at room temperature (
The analysis results of the commercial formulation sample and bulk drug sample are shown in Table
Results (%) of formulation tablet analysis and bulk drug batches sample analysis.
Sample source | Isomer-1 | Isomer-2 | Isomer-3 |
---|---|---|---|
Formulation-1 | Not detected | 0.10 | Not detected |
Formulation-2 | Not detected | 0.05 | Not detected |
B.NO.SOL/A107/I/03 | Not detected | 0.01 | Not detected |
B.NO.SOL/A107/I/04 | Not detected | 0.01 | Not detected |
B.NO.SOL/A107/I/05 | Not detected | 0.01 | Not detected |
The method presented in this communication describes the development of a rapid, simple, and accurate HPLC method that separates all four stereoisomers (isomer-1, isomer-2, isomer-3, and solifenacin) with good resolution. The developed method was validated to ensure the compliance in accordance with ICH guideline. The developed method will be useful for monitoring the quantitative determination of stereoisomers in the bulk and formulation samples.
The authors wish to thank the management of Megafine Pharma for supporting this paper. They also thank colleagues in the Research and Development department, Megafine Pharma (P) Ltd., for their cooperation in carrying out this work. Further, the authors wish to acknowledge the contribution of Mr. K. Vijaya Bhaskar, Research Associate of Daicel Chiral Technologies (India) Pvt Ltd., for his valuable experimental work.