With the objective of developing an advanced method for rapid separation with shorter runtime, a simple, precise, and accurate stability-indicating isocratic RP-LC method coupled with PDA detector was developed for the quantitative determination of flupirtine maleate in bulk and in capsule dosage form. Good resolution between the peaks for degradation products and the analyte was achieved on a Waters Agilent XDB C18 (
Flupirtine maleate is a nonopioid centrally acting, structurally dissimilar from other analgesics. It is amino pyridine derivative [
Structure of flupirtine maleate.
Flupirtine is used for the treatment of acute and chronic pain, that is, for painful increased muscle tone of the posture and motor muscles, primary headache, tumor pain, dysmenorrhea, and pain after orthopedic operations and injuries. Some neuroprotective effects due to NMDA receptor antagonistic properties of flupirtine may also be used in the treatment of Creutzfeldt-Jakob disease, Alzheimer’s disease, and multiple sclerosis [
Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light which enables recommendation of storage conditions, retest periods, and establishing shelf life. An extensive literature survey revealed the availability of few analytical methods which includes spectrophotometric, chromatographic and bioanalytical methods [
Authentic sample of flupirtine maleate was kindly supplied by Spectrum Pharma Research Solutions (Andhra Pradesh, India). Flupirtine maleate capsules (Retense-Sun pharmaceuticals Ltd.) containing 100 mg flupirtine maleate as per label claim were purchased from local pharmacy. HPLC grade water, methanol, acetonitrile, and triethylamine were procured from S.D. Fine-chem Ltd. (New Delhi, India). High purity water was prepared by using Millipore Milli-Q plus water purification system, Bedford, MA, USA.
The HPLC consists of a Waters 2695 series with photodiode array detector and Empower 2 software. The chromatographic separation was performed using Agilent XDB C18, 5
A stock solution of flupirtine (1 mg/mL) was prepared by accurately weighing 100 mg of flupirtine in 100 mL volumetric flask then it was dissolved in methanol and was made up to the volume with diluent (methanol). Aliquots of the standard stock solutions of flupirtine were transferred using A-grade bulb pipettes to 10 mL volumetric flask and solutions were made up to the volume with the diluent to give the final concentrations of 20, 50, 70, 100, 120, and 150
To determine the content of flupirtine maleate in capsules (label claim: 100 mg flupirtine maleate), 20 capsules were opened and the contents were weighed and mixed. An aliquot of powder equivalent to the weight of one capsule was accurately weighed and transferred to 100 mL volumetric flask and was dissolved in diluent and made up to the volume with diluent. The volumetric flask was sonicated for 30 minutes to affect complete dissolution. The solutions were filtered through a 0.45
Linearity was established by least squares linear regression analysis of the calibration curve. The linearity of response for flupirtine assay method was determined by preparing and injecting solutions with concentrations of about 20, 50, 70, 100, 120, and 150
Precision was measured in terms of repeatability of application and measurement. The system precision was carried out using six replicate injections of standard concentration. The method precision was carried out using six replicate injections of sample concentration and the %RSD was calculated.
Accuracy of the method was determined by standard addition method. A known amount of standard drug was added to the fixed amount of preanalyzed sample solution. The standard addition method was performed at 50%, 100%, and 150% levels of sample solution. The resulting solutions were analyzed in triplicate at each level as per the ICH guidelines.
Limit of detection is defined as lowest concentration of analyte that can be detected, but not necessarily quantified, by the analytical method. Limit of detection is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.
Limit of quantification is the concentration that can be quantitated reliably with a specified level
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters and provides an indication of its reliability during normal usage. Robustness
Specificity is the ability of the analytical method to measure the analyte free from interference due to other components. Specificity was determined by comparing test results obtained from analysis of sample solution containing ingredients with the test results obtained from standard drug.
To determine whether the analytical method and assay were stability-indicating, flupirtine standard drug was stressed under various conditions to conduct forced degradation studies. Intentional degradation was attempted to stress conditions of photolytic degradation, acid hydrolysis (using 1N HCl), base hydrolysis (1N NaOH), oxidative degradation (20% H2O2), and thermal treatment (heated at 100°C for 5 hrs) to evaluate the ability of the proposed method to separate flupirtine from its degradation products.
Forced degradation in acidic media was performed by taking 100 mg of flupirtine pure drug into 100 mL volumetric flask followed by the addition of 1N HCl and 70 mL diluent and the mixture was heated under reflux for 2 hrs at 60°C, and the volume was made up to the mark with diluent and filtered. The resultant solution was diluted to obtain 100
Oxidative degradation was performed by taking 100 mg of flupirtine pure drug in 100 mL volumetric flask then 1 mL of 20% H2O2 and 70 mL of diluent were added and the mixture was heated under reflux for 2 hrs at 60°C, and the volume was made up to the mark with diluent. Appropriate aliquot was taken from the above solution and diluted with diluent to obtain a final concentration of 100
Photostability was performed by exposing the pure drug to
The standard drug was placed in an oven at 100°C for 5 hrs to study dry heat degradation. For HPLC study, the resultant solution was diluted to 100
The main target of the chromatographic method is to achieve the separation of flupirtine maleate along with the degradation products. The maximum absorption wavelength of the reference drug substance and the forcibly degraded drug solution is 344 nm; hence this wavelength was selected as the detection wavelength for LC analysis. Pure drug along with its degraded products was injected and run in different solvent systems. Initially acetonitrile and water in different ratios were tried. Since acetonitrile
Calibration curves were obtained for flupirtine from which the linear regression equation was computed and found to be
Linearity regression data of flupirtine maleate (
Parameter | Flupirtine maleate |
---|---|
Linearity | 20–150 |
Correlation coefficient | 0.9996 |
Slope | 30296 |
Intercept | 15084 |
System precision was carried out by injecting standard flupirtine maleate six times and the method precision was carried out by injecting the sample flupirtine maleate for six times. The %RSD for repeatability of both standard and sample solutions was found to be 0.1 and 0.56, respectively. This shows that precision of the method is satisfactory as % relative standard deviation is not more than 2.0%. The results are depicted in Tables
System precision of flupirtine maleate (
S. no. | Area of flupirtine maleate |
|
---|---|---|
(1) | 1703662 | 3.107 |
(2) | 1705406 | 3.132 |
(3) | 1708279 | 3.137 |
(4) | 1706769 | 3.142 |
(5) | 1705713 | 3.159 |
(6) | 1704132 | 3.163 |
Mean | 1705660 | 3.14 |
S.D | 1702 | 0.0202 |
%RSD | 0.1 | 0.64 |
Method precision of flupirtine maleate (
S. no. | Area of flupirtine maleate |
|
---|---|---|
(1) | 1729347 | 3.067 |
(2) | 1707363 | 3.085 |
(3) | 1734438 | 3.089 |
(4) | 1721039 | 3.160 |
(5) | 1707799 | 3.165 |
(6) | 1724954 | 3.165 |
Mean | 1720823 | 3.121 |
S.D | 11188 | 0.0461 |
%RSD | 0.65 | 1.47 |
The accuracy of the method was established by recovery studies. The recovery of flupirtine maleate by proposed method is satisfactory and means recovery was found to be in the range of 99.44–100.28%. The results are shown in Table
Recovery data of flupirtine maleate (
Concentration level (%) | Spiked concentration ( |
Concentration obtained ( |
% Recovery | Mean % recovery |
---|---|---|---|---|
50 | 50 | 49.7 | 99.40 | 99.83 |
50 | 50.04 | 100.09 | ||
50 | 50.00 | 100.01 | ||
| ||||
100 | 100 | 99.95 | 99.95 | 100.03 |
100 | 99.98 | 99.98 | ||
100 | 100.17 | 100.17 | ||
| ||||
150 | 150 | 150.49 | 100.33 | 100.28 |
150 | 150.65 | 100.43 | ||
150 | 150.13 | 100.09 |
LOD and LOQ were determined by the standard deviation method and were found to be 1.64 and 4.97
Under all the deliberately altered chromatographic conditions (flow rate, mobile phase, and temperature), all peaks were adequately resolved and elution orders remained unchanged which indicate that the method is robust. The results are summarized in Table
Robustness of the method (
S. No | Parameter | Modification | Retention time (min) | %RSD |
---|---|---|---|---|
(1) | Flow rate | 0.9 mL/min | 3.16 | 0.86 |
1.1 mL/min | 2.84 | 0.97 | ||
(2) | Mobile phase | −5% | 2.97 | 0.72 |
+5% | 3.05 | 0.89 | ||
(3) | Temperature | −5°C | 3.11 | 0.40 |
+5°C | 3.14 | 0.48 |
The method was declared specific, as there were no interfering peaks at the retention time of flupirtine and the flupirtine peak was well resolved from the peaks of all possible degradation products. A typical chromatogram of standard and sample flupirtine for specificity study is shown in Figures
Typical chromatogram of flupirtine maleate standard.
Sample chromatogram of flupirtine maleate.
The results of stress testing studies indicated a high degree of selectivity of the method. Typical chromatograms obtained from stressed samples are shown in Figures
Summary of forced degradation results.
S. No | Stress condition |
|
Peak area | % |
% |
---|---|---|---|---|---|
(1) | Acid degradation | 3.574 | 1669932 | 97.21 | 2.79 |
(2) | Base degradation | 3.606 | 1648315 | 95.95 | 4.05 |
(3) | Oxidative degradation | 3.571 | 1597637 | 93.00 | 7.0 |
(4) | Photolytic degradation | 3.640 | 1546934 | 90.05 | 9.95 |
(5) | Thermal degradation | 3.533 | 1629303 | 94.84 | 5.16 |
Determination of flupirtine maleate in capsule dosage form.
Parameter | Value |
---|---|
Label claim mg/capsule | 100 mg |
Drug content % ± SD*1 |
|
%RSD*2 | 0.86 |
Typical chromatogram obtained after acidic degradation of flupirtine maleate (peak 1 degradation product showing
Chromatogram of alkali stressed sample of flupirtine maleate showing degradation peaks at 10.061 and 14.346 min, respectively.
Chromatogram of oxidative stressed sample of flupirtine maleate. (peaks 1, 2, and 3 are degradation products having
Typical chromatogram of photolytic stressed sample showing degradation peak at 16.119 min.
Typical chromatogram for thermal stressed degradation of flupirtine. 1, degradation product
The developed HPLC technique is precise, specific, accurate, and stability-indicating. Statistical analysis proves that the method is reproducible and selective for the analysis of flupirtine in pharmaceutical dosage form. The method can be used to determine the purity of the drug available from various sources. As the method separates the drug from its degradation products, it can be employed as stability-indicating.
The authors are thankful for Spectrum Pharma Research Solutions, Andhra Pradesh, India, for providing the gift sample of flupirtine maleate.