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Development of a new, rapid and sensitive HPTLC method for estimation of Milnacipran in bulk, formulation and compatibility study
⁎Corresponding author. Tel.: +91 9314490535. singhvigautam@gmail.com (Gautam Singhvi),
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A simple, sensitive and rapid high performance thin layer chromatographic (HPTLC) method has been developed and validated for quantitative determination of Milnacipran Hydrochloride (MIL) in bulk and formulations. The chromatographic development was carried out on HPTLC plates precoated with silica gel 60 F254 using a mixture of acetonitrile, water and ammonia (6:0.6:1.6) (v/v/v) as mobile phase. Detection was carried out densitometrically at 220 nm. The Rf value of drug was found to be 0.63 ± 0.02. The method was validated as per ICH guideline with respect to linearity, accuracy, precision, robustness etc. The calibration curve was found to be linear over a range of 100–1000 ng μL−1 with a regression coefficient of 0.999. The accuracy was found to be very high (99.12–100.87%). %RSD values for intra-day and inter-day variation were not more than 1.43. The method has demonstrated high sensitivity and specificity. The method was applied for compatibility studies also. The method is new, simple and economic for routine estimation of MIL in bulk, preformulation studies and pharmaceutical formulation to help the industries as well as researchers for their sensitive determination of MIL rapidly at low cost in routine analysis.
Keywords
HPTLC
Milnacipran
Validation
Preformulation
Low cost
1 Introduction
Milnacipran Hydrochloride (MIL) is a serotonin–norepinephrine reuptake inhibitor (SNRI) used as an antidepressant synthesized, developed and marketed by Pierre Fabre Médicament (Boyer and Briley, 1998). It has been approved by the Food and Drug Administration (FDA) in January 2009 for the management of fibromyalgia syndrome, characterized by widespread pain and decreased physical function (Kranzler and Gendreau, 2010). Inhibition of both neurotransmitters simultaneously works synergistically to treat both depression and fibromyalgia. Unlike the tricyclic antidepressants (TCAs), MIL has no significant direct action on adrenergic, muscarinic, or histaminergic receptors – pharmacologic actions associated with many of the unpleasant side effects of TCAs (Puozzo et al., 1998, 2002). MIL (Fig. 1) is highly water soluble, leading to rapid and wide absorption, with maximum concentration observed within 2–4 h after dosing. The bioavailability of MIL is high (approximately 85–90%), and absorption is not affected by food intake. MIL undergoes minimal first-pass metabolism, with approximately 55% of the drug excreted unchanged in urine. Its relatively short half-life (approximately 6–8 h) is compatible with the recommended twice-daily dosing (Puozzo et al., 2002).
Chemical structure of Milnacipran.
Extensive literature survey revealed that there is no official high performance thin layer chromatography (HPTLC) method reported in major pharmacopeias like USP, EP, JP, BP and IP. There are very few analytical methods available for estimation of MIL alone. Some liquid chromatographic (LC) methods for determination of MIL combined with many other antidepressants in human plasma have already been published (Lacassie et al., 2000; Duverneuil et al., 2003). A micellar electro-kinetic capillary chromatographic method was developed for separation and determination of antidepressants and their metabolites in biological fluids (Labat et al., 2002). A liquid chromatography with spectrofluorimetric detection (Puozzo et al., 2004) and one GC–MS method is reported for determination of MIL in human plasma (Ucakturk and Safak, 2010). Recently, there are a few references for LC and UV spectroscopic methods that are published for determination of MIL in aqueous matrix and pharmaceuticals (Mehta and Khatri, 2010; Dias et al., 2010; Parejiya et al., 2011; Singhvi et al., 2013) but with sensitivity in microgram levels.
The above literature revealed that all available methods are sensitive up to microgram level only or need some complex reactions or require large amount of organic solvents or require sophisticated instrument or costly in determination of MIL in routine analysis.
Nowadays, HPTLC has become a routine analytical technique due to its advantages of reliability in quantitation of analytes at micro and even in nanogram levels and cost effectiveness. The major advantage of HPTLC is that several samples can be analyzed simultaneously using a small quantity of mobile phase unlike HPLC. This reduces the time and cost of analysis. In addition, it minimizes exposure risks and significantly reduces disposal problems of toxic organic effluents, thereby reducing possibilities of environment pollution. HPTLC also facilitates repeated detection (scanning) of the chromatogram with same or different parameters. Simultaneous assay of several components in a multi component formulation is possible (Kulkarni et al., (2000); Renger et al., 2011; Shewiyo et al., 2012).
The objective of the present study was to develop and validate a new, simple, accurate, specific and reproducible HPTLC method for determination of MIL in bulk, preformulation studies and pharmaceutical formulation to help the industries as well as researchers for their sensitive determination of MIL rapidly at low cost in routine analysis.
2 Experimental
2.1 Reagents and chemicals
Milnacipran Hydrochloride was obtained as a gift sample from Torrent Pharmaceuticals Limited (Ahmedabad, India). All chemicals used were of analytical grade and solvents were of HPLC grade. Methanol, acetonitrile, triethylamine, ammonia and orthophosphoric acid were procured from Merck (India). Ultrapure water (Milli-Q Plus, Millipore®, India) was used throughout the analysis.
2.2 Instrumentation and chromatographic conditions
Chromatography was performed on 5 cm × 5 cm aluminum foil plates precoated with 0.2 mm layers of silica gel 60 F254 (E. Merck, Germany). Before use, the plates were prewashed with methanol and water mixture then dried in the current of dry air and activated at 120 °C for 5 min. Samples were applied as bands 6 mm wide, 15 mm apart, by use of a CAMAG (Switzerland) Linomat 5 applicator with a CAMAG microliter syringe. A constant application rate of 150 nL s−1 was used. Different mobile phases containing water, chloroform, methanol, acetonitrile, acetone, ethyl acetate, dichloro methane, ammonia, triethylamine in different proportions were examined. Of these, the mixture of acetonitrile, water and ammonia (6:0.6:1.6) (v/v/v) was found to be most suitable for the studies. The Retardation Factor (Rf) value of MIL was found to be 0.63 ± 0.02 in optimized mobile phase. The densitogram obtained from a standard solution of MIL is shown in Fig. 2.
Chromatogram of standard Milnacipran: peak of 500 ng (Rf = 0.63).
Linear ascending development was performed in a twin-trough glass chamber previously saturated with mobile phase vapor for 1.5 min at room temperature (RT, 25 ± 2 °C) and relative humidity 60 ± 5%. The development distance was approximately 40 mm. After development, the plates were dried in an oven at 120 °C. Densitometric scanning, at 220 nm, was performed with a CAMAG TLC scanner 3 in absorbance mode. The source of radiation was a deutrium lamp emitting a continuous UV spectrum in the range of 190–400 nm.
2.3 Calibration curve and linearity
A stock solution containing 100 μg/mL of MIL was prepared by dissolving an accurately weighed 10 mg portion of the drug in methanol in a 10 mL volumetric flask. Different volumes of stock solution (0.1, 0.3, 0.4, 0.6, 0.8 and 1 μL) were spotted on an HPTLC plate in triplicate to obtain concentrations of 100, 300, 400, 600, 800 and 1000 ng/band, respectively. The data of peak area versus drug concentration were treated by linear least-squares regression. Three quality control level, Lower quality control samples (LQC) = 200 ng/band, medium quality control samples (MQC) = 500 ng/band, and higher quality control samples (HQC) = 900 ng/band were determined for various validation studies.
3 Method validation
The analytical method was validated for specificity, sensitivity, accuracy, precision, robustness, and ruggedness in accordance with ICH guidelines (ICH: Q2(R1)).
3.1 Specificity and sensitivity
The specificity of the developed method was established analyzing the sample solutions containing MIL standard and marketed tablets in relation to interferences from formulation ingredients. The spot for MIL in the sample was confirmed by comparing Rf values of the spot with that of the standard.
The sensitivity of measurement was estimated in terms of the limit of quantification (LOQ) and the limit of detection (LOD). The LOQ and LOD were calculated by the use of equations LOD = 3 × N/B and LOQ = 10 × N/B where N is the standard deviation of the peak area of the drug (n = 3), taken as a measure of noise and B is the slope of the corresponding calibration plot.
3.2 Accuracy
To check the accuracy of the method, recovery measurements were performed by the addition of a standard drug solution at three different levels (50%, 100% and 150%) to a pre-analyzed sample solution (200 ng/band so that after addition of standards, samples would be in the linear range). Three replicate estimations were carried out for each concentration level.
3.3 Precision
Precision of the method, determined from replicate analysis of the sample, was expressed as repeatability (intra-batch) and intermediate precision (inter-batch). The intra-day and inter-day precision studies were carried out by estimating the responses of three quality control (QC) standards in triplicates under same experimental conditions three times on the same day and on three different days. From the results obtained, the precision was expressed as percentage relative standard deviations (%RSD) from mean intra and inter-day assays.
3.4 Robustness and ruggedness
Robustness of the method was performed by spotting 900 ng of drug making small deliberate changes in various chromatographic conditions. The composition of the mobile phase, amount of mobile phase (10 ± 2 mL) and duration of saturation (20 ± 10 min) were varied. Time from application of MIL to the TLC plate to development of the plate and time from development of plate to scanning were also varied (20 ± 10 min).
Ruggedness of the method was performed by spotting 900 ng of drug by two different analysts maintaining same experimental and environmental conditions.
3.5 Stability studies
To test the stability of the drugs on the TLC plates, the freshly prepared solutions of the analyte were applied to the plates and developed plates were scanned at different intervals of 2, 6, 24, 48 and 72 h.
3.6 Method application
3.6.1 Analysis of MIL in marketed and in-house tablet dosage form
To determine the MIL content of marketed capsule dosage form and in-house tablets, powder equivalent to 10 mg of MIL was transferred to a 100 mL volumetric flask, extracted with methanol, sonicated for 15 min, and diluted to volume with same solvent. The resulting solution was filtered through a 0.45 μm filter (Millifilter; Milford, MA; USA). The solution (5 μL, 500 ng MIL) was applied in triplicate on an HPTLC plate for quantification using the proposed method.
3.6.2 Application in drug-excipient compatibility study
The developed method was implemented for compatibility studies of MIL with various excipients for formulation design. Drug and excipients were stored in various storage conditions as per ICH guideline (ICH, Q1A(R2)). Samples were analyzed for assay at various time points (initial, 1st month, 2nd month and after 3rd month).
4 Results and discussion
4.1 Calibration curve and linearity
A calibration curve was constructed by plotting peak area against concentration of Milnacipran (ng per spot). The results of regression analysis are shown in Table 1. They confirm the linearity of the standard curves over the range studied (100–1000 ng/mL). Linear regression of concentration versus peak area plots resulted in an average coefficient of determination (r2) greater than 0.999. The average equation for calibration curves was y = 3.271 x + 1933. The 3-D chromatographs of all calibration concentrations and QC samples (LQC, MQC and HQC) are shown in Figs. 3 and 4, respectively.
Parameter
Value
Linearity range (ng/mL)
100–1000 ng
Correlation coefficient (r2)
0.999
Slope (m) ± %RSD
3.271 ± 0.92
Intercept (c) ± %RSD
1933 ± 1.05
Rf
0.63 ± 0.02
LOD (ng/mL)
25.14
LOQ (ng/mL) ± %RSD
76.20 ± 0.95
Ruggedness (%RSD)
1.25
3D graph of calibration linearity graph: 100–1000 ng of milncipran with Rf at 0.63 ± 0.02, showing the peak linearity.
3D chromatographs of quality control concentrations (LQC: 200 ng, MQC:500 ng and HQC: 900 ng).
4.2 Specificity and sensitivity
The peak purity of MIL was assessed by comparing the spectra at three different levels, that is, peak start (S), peak apex (M), and peak end (E) positions of the spot and the results obtained as r2 (S, M) = 0.997 and r2 (M, E) = 0.998. Good correlation was obtained between standard and sample spectra of MIL.
Limit of Detection and Limit of quantification were found to be 25.14 ng and 76.20 ng, respectively. Hence the method was found to be highly sensitive for determination of MIL.
4.3 Accuracy
The developed method showed high and consistent recoveries at all studied levels. The results obtained from recovery studies are presented in Table 2. The mean % recovery ranged from 99.12% to 100.87%. Additionally, the obtained recoveries were found to be normally distributed with low %RSD (⩽1.261) at all concentration levels.
Spiked conc. (%)
Theoretical content (ng/band)
% Recovery ± S.D
RSD (%)
% Bias
50
300
99.12 ± 3.75
1.261
0.88
100
400
100.87 ± 3.62
0.897
−0.87
150
500
100.53 ± 1.25
0.249
−0.53
4.4 Precision
%RSD values for intra-day variation were not more than 1.43 and for inter-day variation were not more than 1.076. Low %RSD values indicated the good repeatability and intermediate precision of the method as shown in Table 3.
Concentration (ng/band)
Intraday
Interday
% Recovery (±SD)
%RSD
% Recovery (±SD)
%RSD
LQC (200)
99.84 ± 2.86
1.43
99.89 ± 2.15
1.076
MQC(500)
100.10 ± 0.93
0.18
100.14 ± 1.04
0.207
HQC(900)
99.93 ± 0.57
0.06
99.94 ± 0.85
0.095
4.5 Robustness and ruggedness
The results obtained in the new conditions were in accordance with the original results as shown in Table 4, though the Rf varied very slightly (0.63 ± 0.3). The %RSD values for peak area was less than 1.0 indicating the highly robust nature of the developed method. The %RSD was found to be less than 2% indicating the method was rugged when estimation was done by two different analysts (Table 4).
Parameters
% Mean recovery ± SD
% Bias
% RSD
Mobile phase composition
(6.1:0.6:1.6)
99.25 ± 1.35
0.75
0.767
(6:0.5:1.6)
98.47 ± 3.21
1.53
0.362
Mobile phase volume
100.72 ± 1.24
−0.72
0.137
Chamber saturation time
99.85 ± 1.57
0.15
0.175
Time of drug application to TLC plate development
99.68 ± 0.58
0.32
0.065
Time of TLC plate development to scanning
100.15 ± 2.87
−0.15
0.320
Change of analyst
99.85 ± 4.45
0.15
0.495
Stability up to 72 h
98.75 ± 2.93
1.25
0.330
4.6 Stability studies
There was no significant deviation in peak area (RSD < 1.5%) observed on analysis up to 72 h. No decomposition of the drug was observed during chromatogram development. These observations suggest that the drug is stable under the typical processing and storage conditions of the analytical procedure.
4.7 Method application
4.7.1 Analysis of MIL in marketed and in-house formulations
Drug bands at Rf 0.65 corresponding to MIL were observed in chromatograms obtained from tablet extracts. There was no interference from excipients present in the tablets. The assay of marketed capsules and in-house tablets was found to be 99.68% (SD ± 1.35) and 100.18 (SD ± 1.65), respectively. The good performance of the method indicated the suitability of this method for routine analysis of MIL determination in pharmaceutical dosage forms.
4.7.2 Application in drug-excipient compatibility study
Compatibility study of MIL with various excipients was analyzed with the developed method. Compatibility data after 3rd month are shown in Table 5. The assay values of MIL with all excipients were found to be 99.12–100.62 and 98.14–99.72 at long term and accelerate conditions respectively. The Rf values for all samples at both conditions were found to be 0.63 ± 0.03. The % RSD for all samples were less that 2%, indicated that the developed and validated HPTLC method can be used effectively for preformulation studies and routine analysis of Milnacipran.
Sample⁎
Long term 25 °C/60% RH
Accelerate condition 40°/75% RH
Assay⁎⁎ ± SD
Assay⁎⁎ ± SD
Pure drug
99.25 ± 1.50
99.05 ± 1.38
D + Micro crystalline cellulose
99.12 ± 2.34
98.25 ± 0.96
D + Dibasic calcium phosphate
100.40 ± 0.80
99.12 ± 1.50
D + Poly vinylpyrrolidone
99.05 ± 1.15
98.17 ± 0.75
D + HPMC 15 K
100.62 ± 1.15
99.72 ± 2.72
D + Magnesium stearate
100.03 ± 2.34
98.14 ± 1.25
D + Talc
100.62 ± 1.85
99.45 ± 0.93
4.8 Advantages of developed method
Extensive literature survey revealed that there is no official HPTLC method reported in major pharmacopeias like USP, EP, JP, BP and IP for determination of MIL in bulk and pharmaceutical formulation. There are few UV-spectroscopic methods and HPLC methods reported in journal publications but they have certain limitations like derivatization of samples or require large amount of sample and/or organic solvents or sensitive to microgram level. Few methods like gas chromatography–mass spectroscopy, micellar electrokinetic capillary chromatography, etc. are reported but these methods are not preferred for routine analysis of MIL in bulk, preformulation studies, formulations and stability studies because of high cost of analysis and skilled requirement for sample treatment.
The above literature revealed that all available methods are sensitive for microgram level only or need some complex reactions or require sophisticated instrument or costly in determination of MIL. There is a need of a new, simple, rapid, highly sensitive and economic HPTLC method for determination of Milnacipran.
It can be seen that this simple and reproducible HPTLC method has enough sensitivity to evaluate preformulation samples, stability samples and pharmaceutical dosage form of MIL at low cost. Thus this presented work will meet the industrial and research laboratories’ requirements.
5 Conclusion
A new, simple, and sensitive HPTLC method has been successfully developed and validated for determination of MIL in bulk and pharmaceutical dosage form. The method was found to be accurate, precise, and reproducible with good stability under various processing and storage conditions. In addition, the method was successfully employed for compatibility testing of MIL with various excipients for formulation design. Thus this developed and validated method will help the industries as well as researchers for their sensitive determination of MIL rapidly at low cost.
Acknowledgments
The authors are very thankful to Department of Science and Technology (DST), Govt of Rajasthan, India for financial support of this project. Authors are also thankful to Torrent Pharmaceuticals Limited (Ahmedabad, India) for providing with the gift sample of Milnacipran.
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