Research Article - (2013) Volume 4, Issue 5
Keywords: Buflomedil; Degradation products; Thin-layer chromatography (TLC); HPLC
Instability of pharmaceuticals affects both the safety and efficacy of drug therapy. Therefore, appropriate analytical stability-indicating methods which allow accurate andprecise drug quantitation in the presence of its degradation products are needed in order to assess the stability of pharmaceuticals. Recently, the development of stability indicating methods has increased enormously, and is evenbeing extended to drug combinations.
Buflomedil HCl (Figure 1), 4-(1-pyrrolidinyl)-1-(2,4,6- trimethoxyphenyl)-1-butanone hydrochloride, is a vasoactive agent which increase the peripheral and cerebral blood flow in ischaemic tissues of patients with vascular diseases particularly at the microcirculatory level  It is rapidly absorbed from the gastrointestinal tract, reaching maximal plasma concentration within 1.5-4 h.
There have been few reports for its determination in formulation and in biological fluids using gas chromatography [2-4], highperformance liquid chromatography [5-7] and reversed-phase ion pair high-performance liquid chromatography [8,9] which mainly concerned with poising or toxicological cases. Therefore, only few methods were determined of BFM in presence of its degradation products.
The TLC-spectrodensitometry system: Camag TLC scanner 3 S/N 130319 operated with winCATS software, Linomat 5 autosampler (Switzerland), Camag micro syringe (100 μL) and TLC aluminum sheet (20×20 cm) precoated with silica gel 60 F254. (Merck KgaA, Darmstad, Germany) were used.
The HPLC system: Agilent 1200 series chromatographic system equipped with quaternary pump, microvacuum degasser, thermostatted column compartment and variable wavelength UV–VIS detector was used. Sample injections were made through an Agilent 1200 series autosampler. Data collection and processing were performed using Agilent ChemStation software, version A.10.01. Column Agilent Zorbax SB-C18 (150 mm×4.6 mm, 5 μm particle size i.d.) was from (Agilent Technologies, Polo Alto, CA, USA).
A “Jenway 3505” pH-meter (Jenway, UK), equipped with combined glass electrode was used for pH adjustment.
Chemicals and reagents
Pure sample: BFM was kindly supplied by Global Napi Pharmaceuticals (6th October City, Egypt). The purity of the sample was found to be 99.99% according to reported HPLC method .
Pharmaceutical dosage form: Loftyl® film coated tablet batch No.77721 manufactured by Kahira Pharm. & Chem. Ind. Co., under licence of Abbott Laboratories, Purchased from Egyptian market labeled to contain 150 mg Buflomedil/tablet.
Reagents: All chemicals used throughout the work were of analytical grade and solvents were of spectroscopic and HPLC grade: methanol, acetonitrile, triethylamin (s d fine-chem limited, Industerial state, Mumbai), 5N aqueous hydrochloric acid solution and 10N Sodium hydroxide, toluene, propanol, Butanol, Glacial acetic acid and ammonia solution 33% (El-Nasr Pharmaceutical Chemicals Co., Abu- Zabaal, Cairo, Egypt), doubly distilled deionized water (Otsuka, Cairo, Egypt).
• BFM stock standard solution: a stock solution containing 1 mgmL−1 in methanol was prepared by dissolving 100 mg of Buflomedil in sufficient amount of methanol and completing to 100 mL with the same solvent.
• BFM working standard solution: a volume of 20 mL of BFM stock standard solution (1 mgmL−1) were transferred into a 100 mL volumetric flask and the volume was completed to the mark with methanol to obtain 200 μgmL−1 final concentration.
• BFM acid induced : a stock solution of degraded BFM was prepared as described under degraded samples [2.3.1.]
• Laboratory prepared mixtures: For TLC, solutions containing different ratios of drugs and their degradation products in methanol were prepared from their working solutions. For HPLC, the solutions were prepared from the working solutions and diluted with the mobile phase.
Degraded samples: Degraded BFM sample was prepared by accurately weighing 50 mg of pure BFM, refluxed with 50 mL 5N HCl for 4 hours. The solution was then neutralized to pH 6.8-7.2 using 10N NaOH, Then evaporated under vacuum, extracted two times with 20 mL methanol, filtered with 50 mL volumetric flask and completed to volume with methanol to produce final concentration equivalent to 0.5 mgmL−1. Degradation was checked every half hour by using thin layer chromatography using toluene: propanol: triethylamine (50:50:5, v/v) . Complete degradation was confirmed by reported HPLC method using mobile phase constituted methanol: water: acetic acid (70:30:0.1, v/v/v) containing 5×10-3 M sodium dodecyl sulfate as a developing solvent and detection at 272 nm.
Construction of TLC calibration curves: Aliquots (1-10 mL) of BFM standard solution (200 μgmL-1) equivalent to 200-2000 μg of BFM were accurately transferred into a series of 10 mL volumetric flasks and the volumes were completed to the mark with methanol to give final concentrations of 20-200 μgmL-1 of BFM. Aliquots (10 μL) of each concentration equivalent to 0.2-2 μg of BFM were applied to the TLC plates, the plates were developed to 13 cm using the developing mobile phase [Butanol: ammonia: triethylamine (8:0.5:0.5, v/v/v)]. The plates were then removed, air dried, visualized under UV lamp at 254 nm, and scanned at 272 nm. The calibration curve representing the relationship between the relative peak area (calculated following the external standard tech nique using an external standard of 1 μg/band of BFM) and the corresponding concentration was plotted and the regression equation was calculated.
Construction of HPLC Calibration Curves: Aliquots of BFM were accurately transferred from its working standard solutions (200 μgmL-1) equivalent to 10-200 μg into separate series of 10 mL volumetric flasks; the volumes were completed to the mark with the mobile phase of methanol: water: acetonitrile: triethylamine (50:30:20:0.4, v/v/v/v, pH 6.5). A 20-μL aliquot of eachsolution was injected onto a ZorbaxSB-C18 column (150 mm×4.6 mm, 5 μm particle size i.d.); using the mobile phase, at flow rate 0.7 mLmin-1 and detection at 272 nm. The calibration curves were constructed by plotting the peak ratio, using 20 μgmL-1 of BFM as the external standard, and the corresponding concentration in micrograms per milliliter and the regression equation were computed.
Assay of laboratory-prepared mixtures: The peak areas or peak area ratios of the laboratory-prepared mixture were scanned and processed as described for the calibration for each of the proposed TLC or HPLC methods, respectively. The concentration of Buflomedil in each mixture was calculated using the specified regression equations.
Application to pharmaceutical formulations: Ten Loftyl® tablets [labeled to contain 150 mg BFM] were accurately weighted and finely powdered. An accurate weight of the powdered tablet equivalent to 100 mg BFM were sonicated with 60 mL methanol for 20 min, filtered into a 100 mL volumetric flask and completed to volume with methanol. The obtained solution labeled to contain 1 mgmL−1. A volume of 20 mL of previous stock solution were transferred into a 100 mL volumetric flask and the volume was completed to the mark with distilled water to obtain 200 μgmL−1 final concentration. For TLC, 10 μL was applied onto TLC plates, whereas for HPLC analysis, the last solution was further diluted by transferring 0.5-10 mL aliquots of it to 10-mL volumetric flasks and the volumes were completed with the HPLC mobile phase. The general procedures described above for each method were followed to determine the concentration of Buflomedil in the prepared dosage form solutions.
The development of analytical methods for the determination of compounds in the presence of their degradation products without previous chemical separation is always a matter of interest. A stability indicating method has been studied and validated for the determination of Buflomedil in presence of its degradation products , acid degradation of BFM was carried out by refluxing with 5N HCl to give two main degradation products which are 1,3,5 Trimethoxybenzene and butane- pyrrolidinium salt , (Figure 2).
Although stability-indicating methods have been extended to include determination of BFM in presence of its degradation products, our review of the literature indicated that these methods are much sensitive and no such TLC-densitometric method has been described for determination of BFM in presence of its degradation products.
In this work, a TLC densitometric method was used for the determination of BFM by separation from their degradation products, depending on the difference in Rf values. Complete separation of BFM from its degradation products was achieved using Butanol: ammonia: triethylamine (8:0.5:0.5, v/v/v) as the mobile phase. Densitometric scanning was performed at 272 nm with accepted results (Figure 3a and b).
Good chromatographic separation of BFM from its degradation products could be achieved by using a Zorbax SB-C18 (150 mm×4.6 mm, 5 μm particle size i.d.) column, a mobile phase consisting of methanol: water: acetonitrile: triethylamine (50:30:20:0.4, v/v/v/v, pH 6.5), UV detection at 272 nm, and a flow rate of 0.7 mL min-1 (Figure 4). Several trials were done to reach the optimum chromatographic separation, and the suggested chromatographic system allows complete baseline separation in reasonable time [11,12].
Linearity and ranges: Under the previously described experimental conditions, linear relationships were obtained by plotting the drug concentrations against peak areas for each drug, for both chromatographic methods. The corresponding concentration ranges, calibration equations, LOD and LOQ and other statistical parameters are listed in Table 1.
|Range||0.2-2 µg/band||10-100 µg mL-1|
|SE of slope||0.005389||0.000142|
|SE of intercept||0.007163||0.016|
|Correlation coefficient (r)||0.9999||1|
|Accuracy(Mean ± SD)||100.11 ± 0.516||100.44 ± 0.722|
|LOD*||0.0340 (μg/band)||1.5928 (μgmL-1)|
|LOQ*||0.1031 (μg/band)||4.8269 (μgmL-1)|
|Precision (RSD %)
|Specificity||100.53 ± 0.534||99.89 ± 0.978|
*LOD: Limit of detection, LOQ: Limit of quantitation
*LOD = (SD of the response/ slope) × 3.3, LOQ = (SD of the response/ slope) × 10
aThe intraday of samples of BFM (0.1,1and1.4μg/band) for TLC and (40, 80 and 160 μgm-1) for HPLC
bThe inter-day of samples of BFM (0.1,1and1.4μg/band) for TLC and (40, 80 and 160 μgm-1) for HPLC
Table 1: Results of regression and validation parameters of the proposed chromatographic methods for determination of Buflomedilin presence of its acid induced degradation products.
Accuracy: The accuracy of the investigated methods was validated by analyzing pure samples of both BFM and its degradation products. The concentrations of the BFM was calculated from the calculated regression equation. Good results are shown in Table 1.
Precision: Precision was evaluated by calculating intra- and inter-day precision by repeating the assay of three different concentrations three times in the same day and assaying the same samples in triplicate on three successive days, using the developed chromatographic methods and calculating the recovery % and RSD%. Results in Table 1 indicate satisfactory precision of the proposed methods.
Specificity: Specificity was ascertained by analyzing different mixtures containing the BFM and its degradation products in different ratios. Other parameters such as resolution, capacity factor and selectivity for the separated spots and peaks were then calculated, Table 1.
Application to commercial tablets: The suggested methods were successfully applied for determination of Buflomedil inLoftyl® tablets. The results shown in Table 2 were satisfactory and with good agreement with the labeled amounts. Applying the standard addition technique, no interference due to excipients was observed as shown from the results in Table 2.
|Loftyl® film coated tablet 150mg/Tablet B.N. 77721(mean ± SD)||98.87 ± 0.828||101.13 ± 0.255|
|Standard addition(mean ± SD)||100.01 ± 0.909||100.30 ± 0.472|
Table 2: Determination of Buflomedil in Loftyl®Tablets by the proposed chromatographic methods.
In order to validate the suggested chromatographic methods, an overall system suitability testing was done to determine ifthe operating systems are performing properly. Good results were obtained and shown in Table 3 and 4.
|Retention factor (Rf)||0.56 ± 0.02||0.04 ± 0.01|
|Resolution (Rs)||3.4||Rs >2|
|Tailing factor (T)||1.06||1.07||T <2|
|Capacity factor (K')||5.1||3||K' >2|
|Selectivity factor (α)||1.7||α >1|
Table 3: Statistical analysis of parameters required for system suitability testing of TLC method.
|Parameter||BFM||BFM Deg.||Reference value|
|Resolution (Rs)||4.69||Rs >0.8|
|Tailing factor (T)||1.33||1.67||T = 1*|
|Capacity factor (K')||6.52||3.23||K' >2|
|Selectivity factor (α)||2.02||α >1|
|Column efficiency (N)||3599||2906||N >2000|
|Height equivalent to theoretical plate (HETP)||0.016||0.019||AS HETP increase the column efficiency decrease|
*For a typical symmetrical peak
Table 4: Statistical analysis of parameters required for system suitability for HPLC.
When results obtained by applying the proposed methods for analysis of pure BFM compared to those obtained by applying the reported method , they showed no significant difference regarding accuracy and precision, and results were given in Table 5.
|Parameter||TLC||HPLC||**Reported HPLC method|
|Student's t||0.3377 (1.8124)*||0.01797(2.201)*|
|F||3.104 (5.0503)*||2.673 (4.3874)*|
*The values in parentheses are the corresponding tabulated values at p=0.05
**HPLC method (C-18, using methanol: water: acetic acid (pH=4) in the ratio of 70:30:0.1, v/v/v) containing 5×10-3 M sodium dodecyl sulfate at flow rate of and 0.7 mL/min and detection is 275 nm)
Table 5: Statistical comparison of the results obtained by the proposed methods and the reported one for Determination of BFM in presence of its acid induced degradation products.
In order to compare the ability of the proposed methods to determine pure BFM, the obtained results were subjected to statistical analysis using one-way ANOVA test, there was no significant difference between all of the proposed methods (Table 6).
The proposed methods are simple and rapid methods of their analysis especially in quality control laboratories. The suggested chromatographic methods provide simple, accurate, and reproducible stability-indicating methods for the quantitative analysis in presence of degradation products. The developed TLC method is highly sensitive than the reported method  and has the advantages of short run time, large sample capacity, and use of minimal volume of solvents. The HPLC method gives a good resolution between the drug and its degradation product with suitable analysis time. It is highly specific. The applied methods could be useful for stability investigation of the active drug and checking the extent of degradation in pharmaceutical formulations.