1. INTRODUCTION:
Gabapentin (GBP), 1-(aminomethyl) cyclo-hexaneacetic acid, is chemically unique cyclohexane derivative of gabba amino butyric acid (GABA) that was synthesized to cross blood brain barrier, and mimic the inhibitory effects of this neurotransmitter on the CNS. Gabapentin is effective as adjunctive therapy for patients with partial and secondarily generalized tonic-clonic seizures.1,2 It is official in United State Pharmacopoeia 30.3 Mecobalamin (MCB), (1R, 2R, 4S, 7S)-7-{[(2S)-3- hydroxy-2-phenylpropanol]oxy}-9,9-dimethyl-3-oxa-9-azonia tricycle [3.3.1.02,4] nonane, is a supplement for vitamin, used in treatment of Vitamin B12 deficiency of dietary origin.1,4 It is official in Japanese pharmacopoeia.5 Alpha lipoic acid (ALP), (R)-5-(1, 2-dithiolan-3-yl) pentanoic acid, is antioxidant, and used in treatment of diabetes and HIV. It also has been used for cancer, liver ailments, and various other conditions.1,4 It is official in United State Pharmacopoeia 30.3 Combination of Gabapentin, Mecobalamin and Alpha lipoic acid treats both the problems associated with all types of neuropathy i.e., neuralgia (neuronal pain) and neuron degeneration. Gabapentin is proved to be very effective and well tolerated in the treatment of neuropathic pain. Alpha lipoic acid is a universal antioxidant which prevents oxidative damage of neurons. Mecobalamin increases myelin sheath formation thereby regenerates neuron. Literature survey reveals many reported methods for the analysis of GBP by ultra-violet (UV),6,7 high-performance liquid chromatography (HPLC)8-11 and high-performance thin-layer chromatography (HPTLC).12 Various methods have been reported for determination of MCB by UV,13-17 HPLC5,17,18 and HPTLC.12 Estimation of ALP by UV,19,20 HPLC3,21,22 and GC,21 either individually or in combination with other drugs are reported. To the best of our knowledge, there is no analytical method reported for simultaneous determination of ternary mixture containing GBP, MCB and ALP. Therefore, an attempt has been made to develop a simple, accurate, rapid and reproducible ratio spectra derivative spectroscopic method for simultaneous determination of GBP, MCB and ALP in tablet dosage form and validate it, in accordance with ICH guidelines.23
2. MATERIALS AND METHODS:
Pharmaceutical grade of GBP (Zydus Research Center, Ahmedabad, Gujarat, India), ALP (Centurion Laboratories, Vadodara, Gujarat, India) and MCB (Centurion Laboratories, Vadodara, Gujarat, India) were kindly supplied as gift samples, certified to contain >99% (w/w) on dried basis. Commercially available trigabantin 100 (Sun Pharma, Sikkim) tablets claimed to contain 100mg Gabapentin, 0.5mg Mecobalamin and 100mg Alpha lipoic acid have been utilized in the present work. Methanol of Analytical grade was purchased from Merck Chemicals, India and Rankem Chemicals, India. Sartolon Polyamide, 0.20mm pore size membrane filter, Sartorius AG. 37070 Goettingen, Germany, and 0.45 mmpore size, 47mm Ø, Sartolon Polyamide, Sartorious AG, Germany.
3. SELECTION OF SUITABLE WAVELENGTHS FOR ANALYSIS:
Solutions containing appropriate concentration of GBP,
MCB, ALP and mixture of GBP+MCB+ALP in methanol (glassware’s protected with
aluminium foil and keep all glassware below 25ºC) were scanned using UV-visible
spectrophotometer in “Spectrum mode” in the range of 800-200 nm and their
spectra were stored in computer. Using UV Probe software spectrum of mixture was
divided by spectrum of GBP (100μg/ml) and MCB (0.5μg/ml); GBP (100μg/ml)
and ALP (100μg/ml); MCB (0.5μg/ml) and ALP (100μg/ml) to get
ratio spectrum of ALP, MCB and GBP respectively. Ratio spectra of the drugs
were smoothed (
λ=10) and converted to first order derivative spectra
((λ=10) using UV Probe software. First order ratio
derivative spectra of the drugs were overlaid. From the overlain ratio
derivative spectra of GBP, MCB and ALP, 731.10nm, 768.53nm and 242.21nm were selected
as suitable analytical wavelengths for analysis of GBP, MCB and ALP
respectively (Fig. 1)
4. PREPARATION OF CALIBRATION CURVES:
Spectra of prepared standard binary mixture containing
100 + 0.5+ 100, 200 + 1.0 + 200, 300 +1.5+300, 400 + 2.0 + 400, 500 + 2.5 + 500μg/ml
of GBP + MCB + ALP recorded in spectroscopic condition. For ratio spectra of
GBP, standard spectra of the drugs mixture were divided by spectra of 0.5μg/ml
MCB and 100μg/ml ALP. Ratio spectra of GBP were smoothed ((∆λ=
10) and converted to first order derivative spectra ((∆λ= 10, SF = 10).
For ratio spectra of MCB standard spectra of the drugs mixture were divided by
spectra of 100mg/ml GBP and 100 mg/ml ALP. Ratio spectra of MCB were smoothed ((∆λ=
10) and converted to first order derivative spectra ((∆λ= 10, SF =
10). For ratio spectra of ALP, standard spectra of the drugs mixture were
divided by spectra of 0.5mg/ml MCB and 100 mg/ml GBP. Ratio spectra of ALP were
smoothed ((λ= 10) and converted to first order derivative
spectra ((λ= 10, SF = 1). Amplitudes (dA/dl) of obtained
ratio derivative spectra of the drugs were measured at selected wavelengths. Standard
calibration curves of dA/dl against Concentration were plotted.
5. VALIDATION OF METHOD:
Validation of developed method was carried out according to ICH Guideline for Validation of Analytical Procedures Q2 (R1) by linearity, limit of detection (LOD) and limit of quantitation (LOQ), accuracy, Precision, robustness and specificity.
5.1. Robustness:
Solution containing mixture of 300
g/ml of GBP, 1.5μg/ml of MCB and 300μg/ml
ALP was prepared and analyzed as per proposed method with small but deliberate
change in spectroscopic condition such as scanning speed, filter variability (0.25μm
and 0.45μm) and methanol from different manufacturers. The mean amplitude
(dA/dl) with its standard deviation and % relative standard deviation was
computed at each level.
5.2. Specificity:
Specificity of an analytical method was assessed by, defining its ability to measure accurately and specifically the analyte of interest without interferences from blank: Solution containing 300μg/ml GBP, 1.5μg/ml MCB, 300μg/ml ALP, mixture of 300μg/ml GBP, 1.5μg/ml MCB and 300μg/ml ALP were prepared and analyzed as per the proposed method.
5.3. Stability:
Solution containing mixture of 300μg/ml of GBP, 1.5μg/ml of MCB and 300μg/ml ALP was prepared. Prepared solution is analyzed after 24 h for stability of drugs in 0.1 N HCl, 0.1 N NaOH, light, thermal and hydrogen peroxide.
6. ANALYSIS OF PHARMACEUTICAL FORMULATION:
Twenty tablets were weighed accurately and their average weight was determined. The tablets were crushed to fine powder and from the triturate, tablet powder equivalent to 25mg of GBP, 0.125mg MCB and 25mg of ALP were weighed and transferred to 25ml volumetric flask. To this flask, 15ml methanol was added and the flask was sonicated for 5 min. The volume was adjusted up to the mark with methanol. The solution was then filtered through membrane filter paper (0.25μm). Filtrate contained mixture of 1000μg/ml GBP, 5μg/ ml MCB and 1000μg/ml ALP. The filtrate solution was suitably diluted with methanol to get a final concentration of 300mg/ml of GBP, 1.5μg/ml of MCB and 300μg/ml of ALP. For Ratio spectrum of GBP, MCB and ALP, spectrum of the mixture was divided by standard spectrum of MCB (0.5μg/ml) and ALP (100μg/ml); GBP (100μg/ml) and ALP (100μg/ml); and MCB (0.5 μg/ml) and GBP (100μg/ml) respectively. Obtained ratio spectra were smoothed (Dl ¼ 10) and converted to first order derivative spectrum (Dl ¼ 10, SF ¼ 10 for GBP and MCB; Dl ¼ 10, SF ¼ 1 for ALP). Amplitude (dA/dl) of GBP, MCB and ALP were measured at 731.10nm, 768.53nm and 242.21nm respectively. Concentrations of GBP, MCB and ALP were computed by putting value of their amplitudes in respective standard regression equation obtained from calibration curve. The analysis procedure was repeated six times with tablet formulation.
7. RESULT AND DISCUSSION:
Excellent linearity was obtained for all the three drugs in the range of 100e500mg/ml for GBP and ALP; and 0.5e2.5 mg/ml MCB. Linearity of GBP, MCB and ALP were shown in Figs. 2e4 respectively. The correlation coefficients (r2) were found to be greater than 0.998 (n ¼ 6) in all instances. LOD and LOQ were found to be 3.09mg/ml and 9.37mg/ml for GBP; 0.03 mg/ml and 0.10 mg/ml for MCB; and 4.79mg/ml and 14.52 mg/ml for ALP (Table 1). The proposed method afforded high recoveries for GBP, MCB and ALP tablets. Results obtained from recovery studies shown in Table 2 indicate that this assay procedure can be used for routine quality control analysis of this ternary mixture in tablets. Precision of the analytical method was found to be reliable based on % RSD (<2%) corresponding to the peak areas. The % RSD values were less than 2, for intraday and inter-day precision. Hence, the method was found to be precise for all the three drugs. In all deliberately varied conditions for robustness study, the % RSD of GBP, MCB and ALP were found to be well within the acceptable limit (<1.5%) for robustness study (Table 3). The validated method was used in the analysis of marketed conventional tablet trigabantin 100 with a label claim: 100 mg GBP, 500 mg MCB and 100 mg ALP per tablet. The results for the drugs assay shown in Table 4 indicate a good agreement with the label claims. The spectrum of blank does not show any interference at the detection of GBP, MCB and ALP as it can be seen from the respective spectra (Fig. 5). The results of stability study of drugs shown in Table 5.
8. CONCLUSION:
The developed Ratio spectra derivative spectroscopic method is simple, accurate and precise for the simultaneous determination of GBP, MCB and ALP from tablets. It was successfully validated in terms of linearity, range, accuracy, precision, LOD, LOQ and robustness in accordance with ICH Guidelines. Thus, the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs in combination.
Fig. 1: Overlaid ratio derivative spectra of Gabapentin, Mecobalamin and Alpha lipoic acid in methanol.
Fig. 2: Linearity of ratio derivative spectra of Gabapentin (100-500 μg/ml) at 731.10 nm.
Fig. 3: Linearity of ratio derivative spectra of Mecobalamin (0.5-2.5 μg/ml) at 768.53 nm.
Fig. 4: Linearity of ratio derivative spectra of Alpha lipoic acid (100-500 μg/ml) at 242.21 nm.
Fig. 5: Spectrum of blank (placebo).
Table 1: Linearity, range, LOD and LOQ parameter for the simultaneous estimation of GBP, MCB and ALP (N=6).
|
Parameter |
GBP |
MCB |
ALP |
|
Range |
100-500 μg/ml |
0.5-2.5 μg/ml |
100-500 μg/ml |
|
Linearity Equation |
Y=0.0015x + 0.1433 |
Y= -0.2306x- 0.1196 |
Y=0.0031x –––– 0.0043 |
|
R2 |
0.9996 |
0.9998 |
0.9999 |
|
% RSD |
0.6827- 1.0350 |
0.3573-1.4616 |
0.1509-1.2399 |
|
LOD |
3.09 |
0.03 |
4.79 |
|
LOQ |
9.37 |
0.10 |
14.52 |
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Received on 13.12.2019 Modified on 18.01.2020 Accepted on 10.02.2020 ©A&V Publications All right reserved Research J. Science and Tech. 2020; 12(1):74-78. DOI: 10.5958/2349-2988.2020.00010.8 |
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