Synthesis and establishment of Amlodipine impurity G reference standard

Article history: Received on: 07/05/2017 Accepted on: 07/07/2017 Available online: 30/10/2017 Dimethyl-4-(2-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate which is impurity of amlodipine besilate was synthesized. Hantzsch condensation of methyl acetoacetate with 2-chlorobenzadehyde in 2-propanol at heated under reflux temperature at 85 o C for about 10 hours yielded the yellow powder. This product was purified by column chromatography and the structure was identified by spectroscopic methods (IR, MS, NMR), that was characterized amlodipine impurity G (dimethyl -4-(2-chlorophenyl)-2,6-dimethyl-1,4dihydropyridine-3,5-dicarboxylate). The quality of the impurity was determined by high performance liquid chromatography HPLC method and validated by guideline of The International Conference on Harmonization 2003 Then this impurity was determined homogeneity and assigned value to establish reference standard as Appendix 3 of World Health Organization (WHO technical series 943, 2006) and Statistical methods for use in proficiency testing by inter-laboratory comparison, International Organization for Standardization (ISO 13528), 2005.

The paper reports a synthesis route and establishment of dimethyl-4-(2-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate as impurity reference substance of drug substance of amlodipine in order to quality control of the impurity before manufacturing process.

Equipments
The IR spectrum was recorded by using FT-IR Spectrophotometer Nicolet iS50, Thermo Scientific (KBr dishs).Mass spectrum was recorded on Shimadzu LC/MS IT-TOF.Melting point and purity was determined on Q20 DSC.NMR was performed by using Bruker Mercury 500 MHz spectrometer.TMS was used as an internal reference standard for the proton experiment (δ=0.00 ppm).The HPLC method was developed on Prominence Liquid Chromatography SPD20-A, Shimadzu, Japan.Gemini-NX C 18 (150×4,6 mm; 5 µm) column was used.

Method validation and establishment of impurity Reference standard
The purity of compound AmG2 was checked by TLC and high performance liquid chromatography equipped with Auto Sampler and PDA detector.Mobile phase: 2.3 g/L solution of amonium acetate -methanol (30:70 v/v), Column: Gemini-NX (octadecylsilyl silica gel for chromatography)-C 18 (150 x 4.6 mm, 5 m) or equivalent; Flow rate: 1.0 mL / min, Wavelength set up at 273 nm; Injection volume: 20 l; Column oven: Ambient; Run time: 3 times of RT of amlodipine impurity G (British Pharmacopoeia, 2016).
The analyse method of synthesized impurity and the various parameters for development and validation according to ICH guidelines (ICHQ2A) were performed.Selectivity and sensitivity was determined by analyzing control samples in replicates (n = 6) spiked with the analyte at the lowest level of the calibration standard.Accuracy of the samples was calculated using the calibration curve.
The establishing impurity reference standard was based on the homogeneity; assigned value from two Lab conformed to Good Laboratory Practice (GLP) or ISO/IEC 17025 main principles.

Synthesis of amlodipine impurity G (dimethyl-4-(2chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate).
The reaction Hantzsch condensation of o, m, pmethoxylbenzaldehyde isomers with methyl-3-aminocrotonate in propanol showed that the o-methoxylbenzaldehyde was compiled much slower during 48 hours, but the mainly obtained product was not 1,4-DHP as the hypothesis of mechanism involves to form derivative 1,4-DHPs of Litvie and co-workers had proved.Addition, Angeles et al (2001) reported that Hantzsch condensation of symmetric 1,4-DHPs are formed as impurites other than to yield 1,4-DHP ring, such as observation of reaction of 2-nitrobenzaldehyde with ammonium hydroxyde and acetoacetic acid ester by Hantzsch, isolated the different compounds including 1,2-dihydropyridine, cyclic amide and substituted hydroxamic acid without the 1,4-DHP product.In the case of our paper, the steric hindrance and electron drawing ability of the substituted 2-chloro on benzaldehyde of amlodipine impurity G which has not been mentioned, as expected reaction would be Michael addition of the second molecule of methyl acetoacetate rather than to proton transfer and dehydration lead to formation of the imino-Knoevenagel intermediate (Fig. 1).An interesting observation the spot trace of imino-knoevenagel intermediate was noted.
By TLC was detected only synthesized impurity in comparison with authentic sample of amlodipine impurity G EPCRS.Thus, the reaction of methyl acetoacetate with 2chlorobenzaldehyde as starting material in propanol gave the Hantzsch condensation was convenient for synthesis amlodipine impurity G which was higher yield than was expected (yield 78 %).

Method validation
System suitability Reference solution: Dissolve 4 mg of amlodipine impurity G EPCRS in acetonitril and dilute to 20.0 mL with the mobile phase.Test solution: Dissolve 10.0 mg of the substance to be examined in acetonitril and dilute to 10.0 mL with the mobile phase.Dilute 2.0 mL of this solution to 10.0 mL with the mobile phase.
The % RSD of areas from repeated injects of samples not be more than 2.0 %.The SST is suitable for analysis (Table 1 &  Fig 3).

Specificity Study
The sample was analyzed in Specific conditions mobile phase Study.Chromatogram of mobile phase doesn't show interference at the retention time of amlodipine impurity G. Therefore this method is specific for determination of amlodipine impurity G.

Precision (Repeatability):
The same procedure is repeated to remaining six preparations about 200 μg/ml concentration.% RSD for the RT and area are tabulated as below in Table 3.

Intra-and inter-day precision
Determination of amlodipine impurity G concentration in samples (n = 6 at each concentration for intra-day and n = 6 days for interday precision).The % RSD of areas from six preparations precision level is not more than 2.0 %.These results indicate high precision of the proposed methods (table 4).

Accuracy
The accuracy of the test method was demonstrated by preparing recovery samples of at the level of 50 %, 100 % and 150 % of target concentration).The recovery of amlodipine impurity G by HPLC method estimated 99.0 % with RSD < 2 % which comply to accuracy method (table 5).

Robustness
The robustness of test method was demonstrated by carrying out Mobile phase variation ± 2.0 % (76: 24  64: 36), flow variation ± 10 % (0.9 mL to 1.1 mL/min) the results are tabulated as below in Table 6.Careful validation proved advantages of the method: high sensitivity, accuracy, selectivity and suitability for quality control laboratories.

Homogeneity test
For a homogeneity check, 12/100 vial of the samples of amlodipine impurity G packaged in glove-box with nitrogene were selected at random, and the contents of two test portions from each sample were determined.The data are shown in Table .7,together with sample averages and between-test-portion ranges.
The homogeneity was tested by Data analysis-tools of Microsoft excel 2010, Anova-single factor, comparison with values F and F crit, showed that F< F crit , so it may be concluded that the samples are adequately homogeneous in the batch.
For the stability check, three of the samples were tested by the same laboratory one month later, and gave an average result of X 1 = 99.23 %.The difference X 1 -X o = 0.04 %.
Standard deviation for method assessment has been set at 0.211 (Table 9).Assessment criterion for a stability check 0.3.s*= 0.3 * 0.211 = 0.063, so it has to be concluded that the samples are adequately stable.

Assigned value
After checking the suitability system of two lab (Table 8), the assigned value was determined.The result showed that there is no change the values of x* and s* from one iteration to the third modified data.So convergence may be assumed and the assigned value was 99.34 %, with the uncertainty p s U x / * 25 . 1  = 0.076.

CONCLUSION
In conclusion, a facile synthetic route to amlodipine impurity G has been developed with overall 78 % yield starting from 2-chlorobenzaldehyde, methyl acetoacetate and amonium salt in propanol and heated under reflux temperature at 85 o C for about 10 hours with stirring.The product was purified and structural elucidation by spectroscopic techniques to confirm its of dimethyl-4-(2-chlorophenyl)-2, 6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate.
The simple, accurate and precise RP-HPLC method for the determination of amlodipine impurity G as BP 2016 was verified.The establishment of authentic sample was carried out, it is concluded that the amlodipine impurity G (99.34 % as is) may be recommended as reference standard for routine and qualitycontrol analysis in the drug substances and formulations.
Financial support and sponsorship: Nil.

Table 1 :
The parameters of System Suitability Testing.

Table 2 :
Linearity Results of compound amlodipin impurity G Fig. 4: Linearity of amlodipine impurity G.

Table 3 :
Results of Repeatability of amlodipine impurity G by HPLC method.

Table 4 :
Results of Intra-and inter-day precision of amlodipine impurity G by HPLC method.

Table 5 :
Results of accuracy of amlodipine impurity G by HPLC method.

Table 7 :
Measurement results for homogeneity check.

Table 8 :
The parameter of SST from two Lab for assay of amlodipin impurity G.

Table 9 :
Determination of assigned value of amlodipine impurity G by two laboratories, together with values for the expanded uncertainties (U) of the results as reported by the laboratories.