Journal of Applied Pharmaceutical Science Vol. 7 (10), pp.

105-110, October, 2017

Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2017.71015
ISSN 2231-3354

Synthesis and establishment of Amlodipine impurity G reference

standard

Ly Dieu Ha1, Trang Thi Diem Nguyen2, Tri Minh Le1,2

1
Shool of Medicine, National University HCMC, Vietnam.
2
Faculty of Pharmacy, University of Medicine and Pharmacy at HCMC, Vietnam.

ARTICLE INFO ABSTRACT

Article history: Dimethyl-4-(2-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate which is impurity of

Received on: 07/05/2017 amlodipine besilate was synthesized. Hantzsch condensation of methyl acetoacetate with 2-chlorobenzadehyde
Accepted on: 07/07/2017 in 2-propanol at heated under reflux temperature at 85 oC for about 10 hours yielded the yellow powder. This
Available online: 30/10/2017 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-1,4-
Key words: dihydropyridine-3,5-dicarboxylate). The quality of the impurity was determined by high performance liquid
Hantzsch condensation, chromatography HPLC method and validated by guideline of The International Conference on Harmonization
amlodipine impurity G, 2003 Then this impurity was determined homogeneity and assigned value to establish reference standard as
WHO, ISO 13528. 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.

INTRODUCTION Chava et al., 2006 reported that 1,4-dihydropyridine

The regulatory agencies and pharmaceutical industries derivative were yielded during in the formative stage of the
are of increasing concern to the impurities affecting safety, amlodipine, one of degradative products was dimethyl-4-(2-
efficacy, and quality of pharmaceutical products. The impurities chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
were to be formed from starting with raw materials, intermediate dicarboxylate. Harishkumar et al., 2009 published the synthesis of
stages or impurity process. Quality and Safety of The diethyl-2,6-dimethyl-4-substituted phenyl-1,4-dihydropyridine-
International Conference on Harmonisation 2003, requires that 3,5-dicarboxylate by Knoevenagel and Hantzsch condensation.
impurities which are present in a drug substance although a Litvie and co-workers 2007 described Hantzsch condensation
quantity exceeding a certain limit, should be characterized, reaction of two equivalents of methyl-1,3-aminocrotonate with (m
identified and needs to be evaluated both qualitatively and and p)-methoxybenzaldehyde which resulted in formation of the
quantitatively. Amlodipine (RS)-3-ethyl-5-methyl-2-[(2-amino expected products 2,6-dimethyl-3,5-dimethoxycarbonyl-1-4-(m-
ethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine methoxyphenyl)-1,4-dihydropyridine and 2,6- dimethyl-3,5-
-3,5-dicarboxylate is a medication used to treat high blood dimethoxycarbonyl-1-4-(p-methoxyphenyl)-1,4-dihydropyridine.
pressure and coronary artery disease (Burges and Dodd, 1990). However, the synthesis of dimethyl-4-(2-chlorophenyl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (amlodipine
impurity G) has not been reported yet, while the monograph of
* Corresponding Author amlodipine salt in Pharmacopoeia (Bristist Pharmacopoeia, 2016
Ly Dieu Ha, Shool of Medicine, National University HCMC, Vietnam. or European Pharmacopoeia 8.8) required for controlling
Email: hadieuly @ gmail.com amlodipine impurity G as related substance.

© 2017 Ly Dieu Ha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
106 Ha et al. / Journal of Applied Pharmaceutical Science 7 (10); 2017: 105-110

The paper reports a synthesis route and establishment of time: 3 times of RT of amlodipine impurity G (British
dimethyl-4-(2-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine- Pharmacopoeia, 2016).
3,5-dicarboxylate as impurity reference substance of drug The analyse method of synthesized impurity and the
substance of amlodipine in order to quality control of the impurity various parameters for development and validation according to
before manufacturing process. ICH guidelines (ICHQ2A) were performed. Selectivity and
sensitivity was determined by analyzing control samples in
MATERIALS AND METHODS replicates (n = 6) spiked with the analyte at the lowest level of the
calibration standard. Accuracy of the samples was calculated using
Materials
the calibration curve.
Chemicals (AR- Analytical Grade reagents) methyl
The establishing impurity reference standard was based
acetoacetate, 2-propanol 2-chlorobenzaldehyde, ammonium
on the homogeneity; assigned value from two Lab conformed to
acetate were purchased from Merck Chemicals. Acetonitrile
Good Laboratory Practice (GLP) or ISO/IEC 17025 main
(HPLC grade), methanol (HPLC grade) were purchased from JT.
principles.
Baker. For column chromatography silica gel (Sigma-Aldrich, 63-
200 m) was used. Thin layer chromatography was carried out
RESULTS AND DISCUSSION
using silica gel 60 F254 aluminum sheets from Merck.
Synthesis of amlodipine impurity G (dimethyl-4-(2-
Equipments chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
The IR spectrum was recorded by using FT-IR dicarboxylate).
Spectrophotometer Nicolet iS50, Thermo Scientific (KBr dishs). The reaction Hantzsch condensation of o, m, p-
Mass spectrum was recorded on Shimadzu LC/MS IT-TOF. methoxylbenzaldehyde isomers with methyl-3-aminocrotonate in
Melting point and purity was determined on Q20 DSC. NMR was propanol showed that the o-methoxylbenzaldehyde was compiled
performed by using Bruker Mercury 500 MHz spectrometer. TMS much slower during 48 hours, but the mainly obtained product was
was used as an internal reference standard for the proton not 1,4-DHP as the hypothesis of mechanism involves to form
experiment (δ=0.00 ppm). The HPLC method was developed on derivative 1,4-DHPs of Litvie and co-workers had proved.
Prominence Liquid Chromatography SPD20-A, Shimadzu, Japan. Addition, Angeles et al (2001) reported that Hantzsch
Gemini-NX C18 (150×4,6 mm; 5 µm) column was used. condensation of symmetric 1,4-DHPs are formed as impurites
other than to yield 1,4-DHP ring, such as observation of reaction
Methods of 2-nitrobenzaldehyde with ammonium hydroxyde and
Synthesis of amlodipine impurity G (dimethyl-4-(2- acetoacetic acid ester by Hantzsch, isolated the different
chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5- compounds including 1,2-dihydropyridine, cyclic amide and
dicarboxylate). substituted hydroxamic acid without the 1,4-DHP product. In the
Dissolve 3.9 g (0.05 mol) ammonium acetate in 20 ml 2- case of our paper, the steric hindrance and electron drawing ability
propanol. Add gradually drop by drop in round bottom flask 12.9 of the substituted 2-chloro on benzaldehyde of amlodipine
ml (0.1 mol, 13.13 g) methyl acetoacetate and 5.7 ml (0.05 mol, impurity G which has not been mentioned, as expected reaction
7.1 g) 2-chlorobenzaldehyde. Stir vigorously and headed under would be Michael addition of the second molecule of methyl
reflux temperature at 85 oC for about 10 hours with stirring. The acetoacetate rather than to proton transfer and dehydration lead to
precipitate was filtered and washed with cool methanol (3 x 30mL) formation of the imino-Knoevenagel intermediate (Fig.1). An
and dried in the vacuum at 40 oC to constant weight. The residue interesting observation the spot trace of imino-knoevenagel
transfered to chromatography column (10 x 30 cm, silica gel 40-60 intermediate was noted.
m), 1- 99 % CH2Cl2-EtOAc and 5- 95 % CH2Cl2 - EtOAc as By TLC was detected only synthesized impurity in
eluant to give two fraction AmG1-2. The AmG2 was evaporated to comparison with authentic sample of amlodipine impurity G
dryness and recrystallized from cool methanol to obtain while EPCRS. Thus, the reaction of methyl acetoacetate with 2-
powder (15,04 g). chlorobenzaldehyde as starting material in propanol gave the
Hantzsch condensation was convenient for synthesis amlodipine
Method validation and establishment of impurity Reference impurity G which was higher yield than was expected (yield 78
standard %).
The purity of compound AmG2 was checked by TLC AmG2 compound was obtained as while off powder, Rf
and high performance liquid chromatography equipped with Auto (CH2Cl2 – EtOAc, 98:2) = 0,313; mp 194.10 oC, and purity test on
Sampler and PDA detector. Mobile phase: 2.3 g/L solution of DSC Q20 and HPLC was about 99.85 %. The IR absorption bands
amonium acetate - methanol (30:70 v/v), Column: Gemini-NX IR max(cm-1): 3306, 2947, 1702, 1651, 1493, 1231, 1020, 1118
(octadecylsilyl silica gel for chromatography)-C18 (150 x 4.6 mm, and 755 cm-1. HR-MS revealed the molecular formula to be
5 m) or equivalent; Flow rate: 1.0 mL / min, Wavelength set up C28H27ClN2O7 m/z [M-H]-: 334.0829 (Cal. 335.0814).
at 273 nm; Injection volume: 20 l; Column oven: Ambient; Run
 Ha et al. / Journal of Applied Pharmaceutical Science 7 (10); 2017: 105-110 107

Fig. 1: The pathway of synthesis of amlodipine impurity G.

Fig. 2: Structure of amlodipine impurity G.

1
H-NMR (500 MHz, CDCl3) H 2.31 (6H, s, CH3), 3,61 acetonitril and dilute to 10.0 mL with the mobile phase. Dilute 2.0
(6H, s, COOCH3), 5.40 (1H, s, CH), 5.67 (1H, s, NH), 7.03 (1H, mL of this solution to 10.0 mL with the mobile phase.
dt, arom., J=7.5 &1.0 Hz, 7.12 (1H, t, arom., J =7.5 Hz), 7.24 The % RSD of areas from repeated injects of samples not
(1H, d, arom., J =7.5 Hz), 7.36 (1H, d, arom., J=7.5 Hz); 13C be more than 2.0 %. The SST is suitable for analysis (Table 1 &
NMR (125 MHz, CDCl3): C 19.4, 37.3; 50.8, 104.0, 126.9, 127.3, Fig 3).
129.3, 131.2, 144.0, 145.9, 168.0.
Table 1: The parameters of System Suitability Testing.
S (µV x AS N
Method validation and establishment of impurity Reference No Injects RT (min)
second) asymetric Theory places
standard 1 3.872 6023226 1,272 3521
2 3.879 6033593 1.269 3530
Method validation 3 3.881 6028266 1.273 3532
System suitability 4 3.883 6037178 1.274 3544
5 3.877 6030399 1.271 3526
Reference solution: Dissolve 4 mg of amlodipine impurity G 6 3.875 6032021 1.272 3523
EPCRS in acetonitril and dilute to 20.0 mL with the mobile phase. Average 3.878 6030781 1.272 3529
Test solution: Dissolve 10.0 mg of the substance to be examined in RSD 0.104 0.079 0.114 0.233
108 Ha et al. / Journal of Applied Pharmaceutical Science 7 (10); 2017: 105-110

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

Reference solution Synthesis of amlodipine impurity G
amlodipine impurity G
EPCRS
Area of Area of
Concentrat Concen
amlodipine amlodipine Content
ion tration
impurity G impurity G %
(µg/ml) (µg/ml)
peak peak
202.0 6059302 205.4 6062882 99.34
6061920 213.8 6314527 99.40
6011289 229.0 6780092 99.65
Fig. 3: HPLC Chromatography of amlodipine impurity G. 6014870 199.2 5887082 99.46
6041440 204.8 6027511 99.05
6036902 206.6 6095237 99.29
Specificity Study
Number of samples 6
The sample was analyzed in Specific conditions mobile
Average mean (%) 99.37
phase Study. Chromatogram of mobile phase doesn’t show SD 0.20
interference at the retention time of amlodipine impurity G. RSD (%) 0.20
Therefore this method is specific for determination of amlodipine
impurity G.
Table 4: Results of Intra- and inter-day precision of amlodipine impurity G by
HPLC method.
Linearity study
Intra-day 1 Intra-day 2
Preparation of sample concentrations were about 100 Number of samples
Content (%) Content (%)
µg/mL, 160 µg/mL, 200 µg/mL, 240 µg/mL, 300 µg/mL 1 99.34 99.00
respectively. Correlation coefficient should not be less than 0.999 2 99.40 99.26
3 99.65 99.15
(Table 2 & Fig 4) 4 99.46 99.41
The calibration curves were linear over the 5 99.05 99.25
range of 100.3 to 307.6 μg/mL resulted in the regression equation 6 99.29 99.60
Average 99.37 99.28
ŷ = 30110 x + 15733 (r2 > 0.9999). The LOD and LOQ of the SD 0.20 0.21
calibration graph were 0.97 μg/ml and 3.23 μg/mL, respectively. % RSD 0.20 0.21

Table 2: Linearity Results of compound amlodipin impurity G Table 5: Results of accuracy of amlodipine impurity G by HPLC method.
Concentration 100.3 162.4 203.9 245.4 307.6 Concentration
Area of
(µg/ml) from %
% Level amlodipine Concentration
Average Areas 30259 491302 61644 74045 927072 regression Recovery
concentration impurity (g/ml)
14 1 05 04 7 equation ± SD
G peak
(g/ml)
3103426 104.2 102.5
98.6 ±
50 2995989 100.5 99.0
0.287
3048975 101.8 100.7
Area Linearity of amlodipine impurity G
6194965 205.8 205.2
10000000 99.8 ±
100 5986070 198.9 198.3
9000000 0.114
y = 30110x + 15734 6094431 202.1 201.9
8000000
R² = 1 9249577 305.3 306.7
7000000 150 8934276 301.6 296.2
99.0 ±
6000000 1.125
9101464 306.8 301.8
5000000
4000000
Intra- and inter-day precision
3000000
2000000 Determination of amlodipine impurity G concentration in
1000000 samples (n = 6 at each concentration for intra-day and n = 6 days
0 for interday precision). The % RSD of areas from six preparations
0 50 100 150 200 250 300 350 precision level is not more than 2.0 %. These results indicate high
Concentratio precision of the proposed methods (table 4).

Fig. 4: Linearity of amlodipine impurity G. Accuracy

The accuracy of the test method was demonstrated by
Precision (Repeatability): preparing recovery samples of at the level of 50 %, 100 % and 150
The same procedure is repeated to remaining six % of target concentration). The recovery of amlodipine impurity G
preparations about 200 μg/ml concentration. % RSD for the RT by HPLC method estimated 99.0 % with RSD < 2 % which
and area are tabulated as below in Table 3. comply to accuracy method (table 5).
 Ha et al. / Journal of Applied Pharmaceutical Science 7 (10); 2017: 105-110 109

Robustness Table 9: Determination of assigned value of amlodipine impurity G by two

laboratories, together with values for the expanded uncertainties (U) of the
The robustness of test method was demonstrated by results as reported by the laboratories.
carrying out Mobile phase variation ± 2.0 % (76: 24  64: 36),
Iteration x*o x*1 x*2 x*3
flow variation ± 10 % (0.9 mL to 1.1 mL/min) the results are x* - 1.5.s* - 99.04 99.02 99.02
tabulated as below in Table 6. Careful validation proved x* + 1.5.s* - 99.61 99.65 99.65
advantages of the method: high sensitivity, accuracy, selectivity Average 99.343
and suitability for quality control laboratories. Standard 0.194
deviation
New x* 99.325 99.337 99.337 99.337
Table 6: Robustness Study. New s* 0.193 0.211 0.211 0.211
Parameters Flow rate (mL/min) Mobile phase (v/v)
0.9 1.1 76: 24 64: 36
RT 4.14 3.48 5.12 3.02
Area of amlodipine 6162882 ± 5897082 ± 6262582 ± 5597102 ± Assigned value
impurity G peak 0.14* 0.31* 0.22* 0.61*
% Content 99.05 99.31 99.11 99.41 After checking the suitability system of two lab (Table
*SD of n=3. 8), the assigned value was determined. The result showed that
there is no change the values of x* and s* from one iteration to
Establishment of impurity Reference standard the third modified data. So convergence may be assumed and the
Homogeneity test assigned value was 99.34 %, with the uncertainty
For a homogeneity check, 12/100 vial of the samples of U x  1.25s * / p = 0.076.
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 CONCLUSION
with sample averages and between-test-portion ranges. In conclusion, a facile synthetic route to amlodipine
The homogeneity was tested by Data analysis-tools of impurity G has been developed with overall 78 % yield starting
Microsoft excel 2010, Anova-single factor, comparison with from 2-chlorobenzaldehyde, methyl acetoacetate and amonium salt
values F and Fcrit, showed that F< Fcrit, so it may be concluded that in propanol and heated under reflux temperature at 85 oC for about
the samples are adequately homogeneous in the batch. 10 hours with stirring. The product was purified and structural
For the stability check, three of the samples were tested elucidation by spectroscopic techniques to confirm its of dimethyl-
by the same laboratory one month later, and gave an average result 4-(2-chlorophenyl)-2, 6-dimethyl-1,4- dihydropyridine-3,5-
of X1 = 99.23 %. The difference X1 – Xo = 0.04 %. dicarboxylate.
Standard deviation for method assessment has been set at The simple, accurate and precise RP-HPLC method for
0.211 (Table 9). Assessment criterion for a stability check 0.3.s* = the determination of amlodipine impurity G as BP 2016 was
0.3* 0.211 = 0.063, so it has to be concluded that the samples are verified. The establishment of authentic sample was carried out, it
adequately stable. is concluded that the amlodipine impurity G (99.34 % as is) may
be recommended as reference standard for routine and quality-
Table 7: Measurement results for homogeneity check.
control analysis in the drug substances and formulations.
Test Test
Sample
portion portion 2 Descriptive Statistics
number
1 (%) (%)) Financial support and sponsorship: Nil.
1 99.28 99.03
2 99.36 99.44
3 99.30 99.39
Conflict of Interests: There are no conflicts of interest.
Sample average = 99.27%;
4 99.10 99.39
Standard deviation of sample
5 99.66 98.92
averages SD= 0.22
6 98.76 99.48
RSD = 0.22 % REFERENCES
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How to cite this article:

Ha LD, Nguyen LTD, Le TM. Synthesis and establishment of
Amlodipine impurity G reference standard. J App Pharm Sci, 2017;
7 (10): 105-110.