LC-MS/MS and NMR Characterization of impurities in Epalrestat
Maruthi R, Chandan R.S*, Anand Kumar Tengli
Department of Pharmaceutical Chemistry, JSS College of Pharmacy,
JSS Academy of Higher Education and Research, Mysuru - 570 015, (KA), India.
*Corresponding Author E-mail: rschandan@jssuni.edu.in
ABSTRACT:
For the identification and characterization of main impurities in Epalrestat, a responsive and rapid analysis of LC-MS/MS and NMR was created. Epalrestat is used in diabetic neuropathy therapy, which in patients with diabetes mellitus is one of the most severe long-term complications. Epalrestat is a derivative of carboxylic acid and a non-competitive and reversible inhibitor of the reductase of the aldose. It reduces intercellular sorbitol deposition that is thought to be causing diabetic neuropathy, retinopathy, and nephropathy. Aldose reductase is the key enzyme in the polyol pathway, the enhanced activity of which is the basis for diabetic neuropathy this enzyme is targeted by the aldose reductase inhibitors (ARI). Epalrestat is the only commercially available API. It is easily absorbed in the neutral tissues and has minimal side effects inhibiting the enzyme. At 5.1 RT in Epalrestat, one of the primary impurities was observed in the analytical HPLC process. Preparative HPLC technique was used to further characterize the impurity. To predict the structure, isolated impurity was subjected to amass and NMR analysis of LC-MS/MS was performed using a reverse-phase column of C18 and with a mobile step consisting of methanol and ammonium type in a ratio of 70:30 to pH 4.6. The injection volume is 10 μL, and the binary gradient system was used to isolate. The flux rate was maintained at 1.0 ml/min. The temperature of the column was held at 45oC. For this study, the MS conditions were adopted with a scan range of m/z= 50 to 500 with a dwell time of 3 seconds.
KEYWORDS: LC-MS/MS, NMR, HPLC, Epalrestat, Diabetic.
INTRODUCTION:
Figure. 1: Structure of Epalrestat
EXPERIMENTAL:
Chemicals and Reagents:
Epalrestat (99% pure) samples provide by TCI Chemicals Pvt. Ltd. HPLC grade Acetonitrile was procured from Merck. Analytical reagent grade Ammonium acetate was purchased from Loba Chemie.
Instrumentation:
Shimadzu LC-20AD with PDA detector were used for chromatographic separations. LC solution software was used for data analysis. Shimadzu electronic weighing balance was used for weighing samples and Mark ultra sonicator was used for sonication of mobile phases and samples.
LC-MS/MS was performed by using Shimadzu LC-MS/MS 8030 system with triple Quadra pole mass spectrometry. NMR was performed on FT NMR spectrometer system – 400MHz (Make: Agilent USA model: 400 MRDD2).
RESULTS AND DISCUSSION:
LC-MS/MS study of Epalrestat key impurity:
The LC-MS spectrum of Epalrestat key impurity (Figure. 2) showed peak at RT 5.1 minutes displayed the protonated molecular ion (M++H) at m/z = 319, this is corresponding to the molecular formula C15H13NO3S2. And there Fragmentation pathway. [5-9]
(Figure. 2, 3 and 4).
Figure. 2: Epalrestat Pure Drug MS/MS spectra
Figure. 3: Epalrestat impurity MS/MS spectra
Table 1: m/z Values of Ionization in LC-MS/MS
|
Peak |
m/z values |
|
M+ peak |
319 |
|
274 |
|
|
100 |
|
|
58 |
Fig 3: Fragmentation Pathway
NMR Study of Epalrestat key impurity:
The NMR data (1H, 13C) of Epalrestat impurity was noted using CDCl3 as solvent at 400MHz for H1 and 100MHz for 13C on Agilent FT-NMR 400MHz spectrometer.
Fig 4: 13C NMR of Epalrestat
The 13C NMR spectrum (Fig 4) of Epalrestat impurity has showed R-CH2-R peaks at 16.1ppm and 40.1ppm. The peak at 193.6 is due to R-CH=O and the peak at 40.144ppm is due to C-N. The additional peaks from 100 to 150ppm are due to C=C.
Fig 5:1H NMR of Epalrestat
The 1H NMR spectrum (Fig 5) of Epalrestat impurity has showed aromatic peaks in between 6.5 and 7.5ppm. Peaks from 4.5 to 6.5 are due to C=C bonds and the peak at 2.2ppm and 2.4ppm are due to R-OOR
Negative Ionization
Epalrestat Impurity (Mol weight: 319.22g/mol; (M+H))
Fig 6: Epalrestat impurity MS/MS Spectra
Fig 7:1H NMR Spectrum of Epalrestat impurity in CDCl3.
The 1H NMR spectrum (fig. 7) of Epalrestat impurity has showed aromatic peaks in between 6.5 and 7.5ppm. Peaks from 4.5 to 6.5 are due to C=C bonds and the peak at 2.2ppm and 2.4ppm are due to R-OOR.
Fig 8: 13C NMR Spectrum of Epalrestat Impurity in CDCL3.
Fig 9: Structure for Epalrestat Impurity
The 13C NMR spectrum (Fig. 8) of Epalrestat impurity has showed R-CH2-R peaks at 16.1ppm and 40.1ppm. The peak at 193.6 is due to R-CH=O and the peak at 40.144ppm is due to C-N. The additional peaks from 100 to 150ppm are due to C=C.
CONCLUSION:
A rapid and sensitive LC-MS/MS characterization method for predicting the structure of key impurity was developed. The predicted structure was based on mass and NMR data.
ACKNOWLEDGEMENT:
We would like to thank JSS Academy of Higher Education and Research for their facilities in successfully completion our research work.
CONFLICTS OF INTEREST:
The authors affirm no conflicts of interest.
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Received on 22.01.2020 Modified on 18.04.2020
Accepted on 14.06.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(1):11-13.
DOI: 10.5958/0974-360X.2021.00003.2