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Proceedings of 12th ISMAS Symposium cum
RSP-7
Workshop on Mass Spectrometry
LC-MS/MS Studies on Identification and Characterization of
Hydrolytic Products of Atorvastatin
Ravi P. Shah, Vijay Kumar and Saranjit Singh*
Department of Pharmaceutical Analysis National Institute of Pharmaceutical Education and Research (NIPER) Sector 67, S.A.S. Nagar 160062, Punjab Introduction
Atorvastatin (AT) is a drug of the class of lipid-lowering agents called statins. It acts by inhibiting the HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis in liver, thus reducing cholesterol content of hepatocytes [1]. AT is chemically [R-(R*, R*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4[(phenylamino)car-bonyl]-1H-pyrrole-1-heptanoic acid. Till date, only isolated studies exist, which provide structures of a few impurities/degradation products of the drug. These include diastereomer, tert butyl ester, lactone of AT [2] and oxo-products of drug and its lactone [3]. The methods of analysis for the drug include both LC and LC-MS techniques [4-6]. No study exists so far on the systematic forced decomposition behaviour of AT under hydrolytic stress conditions. Therefore, an endeavour of the present study was to: i) decompose the drug under hydrolytic conditions, ii) resolve the products on a HPLC column, iii) characterize the major products by LC-MS/MS studies, and iv) postulate the probable degradation pathways with the help of studies at different time points. Experimental
Materials and equipment
Pure AT was obtained as a gift sample from Dr Reddy’s Laboratories Ltd., India. HPLC grade acetonitrile was purchased from J.T. Baker (Mexico). Ultra pure water was obtained from a water purification unit (Elga Ltd., England). Buffer materials and all other chemicals were of analytical-reagent grade. The HPLC system from Shimadzu, Japan was used for LC studies, whereas LC-MS system consisted of Agilent HPLC and Bruker MicrOTOF-Q mass spectrometer. Stress decomposition studies
Acidic and alkaline hydrolytic decomposition studies were carried out using 0.2 N HCl, and 2 N NaOH, respectively. Neutral hydrolysis was performed in water. All the studies
were carried out at 80°C. The drug concentration was 0.25% w/v. Di-methyl sulfoxide
(DMSO) (50% v/v) was used for solubilization of drug in acidic, basic, neutral and oxidative
media. Samples were withdrawn at different time intervals and diluted ten times with
H2O:DMSO (50:50 v/v) before HPLC injection.

Results and Discussion
Development and optimization of LC method
The optimum resolution of drug and its hydrolytic products was influenced by pH of the aqueous phase, ratio of the organic modifier and the flow rate. Reasonable separations 12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa Proceedings of 12th ISMAS Symposium cum
RSP-7
Workshop on Mass Spectrometry
(resolution >1.0) and peak shapes were achieved on C-18 column (250 mm x 4.6 mm i.d., particle size 5 μm) using combination of ammonium acetate buffer (0.01 M, pH 3.0) and acetonitrile. A gradient run was employed wherein the solvents were run initially in a proportion of 54:46 for 22 min, the ratio was subsequently changed to 30:70 in 43 min and, then returned back to initial ratio. The flow rate, injection volume and detection wavelength were 1 mL min-1, 20 μL and 246 nm, respectively. Figure 1 clearly indicates that in acidic medium, the drug degraded within 1 h to major product III, and minor products V and VII. After 12 h, the decrease of III was accompanied by increase in VII and emergence of new peaks I, II and IV. The behaviour was similar after 24 h, with only difference being rise of a new small peak VI. No change in V was seen over the period. Fig. 1 Chromatograms showing AT and its degradation under different hydrolytic conditions, (X-axis shows time in min., whereas Y-axis shows absorbance in volts). Key: AT; atorvastatin, I-VII; hydrolytic Overall, the drug was degraded insignificantly (~ 2.5%) over 48 h in neutral and basic (Fig. 1) media, with formation of small quantities of II, III and/or VII. 12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa Proceedings of 12th ISMAS Symposium cum
RSP-7
Workshop on Mass Spectrometry
LC-MS/MS studies
The drug and diluted stressed samples were subjected to LC-MS studies using the same LC method. MS analysis was performed employing the APCI positive ions mode in mass range of 50-3000. High purity nitrogen was used as nebulizer and auxiliary gas. The mass parameters were optimized to the following values: hexapole Rf, 160.0 VPP; collision Rf, 150.0 VPP; pre pulse storage, 3.0 μs; collision energy, 10 eV/Z; quadrupole ion energy, 5.0 eV/Z; nebulizer, 1.4 bar; dry gas, 4.0 L min-1; dry temperature, 180°C; vaporizer temperature 375°C. Additionally, LC-MS/MS studies were carried out employing suitable collision energies. The LC-MS/MS chromatograms for the drug and hydrolytic products are displayed in Fig. 2. Fig. 2 LC-MS/MS line spectra of AT and its degradation products (I-VII). The m/z values of the drug and degradation products (I-VII) were 559, 541, 541, 541, 571, 523 and 523, respectively. The exact m/z value of V was not distinct. The MS/MS analysis of signal corresponding to parent drug at m/z 559 resulted in four major fragments at m/z 466, 440, 399 and 280 (Fig. 2). The figure also shows that the degradation products, except V, followed a very similar fragmentation pattern to the drug. Combining the information obtained from chromatographic profiles (Fig. 1) with MS/MS data (Fig. 2), the molecular structures were rationalized for various degradation products, except V. A general fragmentation pattern for the drug and the degradation products, except V is proposed in scheme 1. It is postulated that fragment of m/z 466 results from elimination of Ph-NH2, whereas the fragment of m/z 440 is formed by elimination of Ph-N=C=O from AT. The latter involves H-transfer from amide nitrogen to 4C of pyrrole. This transfer, via a four membered cyclic structure, is possible due to electronegative character of 4C owing to its sp2 hybridisation and aromaticity. 4C withdraws the amide proton, followed by formation of another bond between carbonyl carbon and amide nitrogen. Finally, heterolytic cleavage of 4C and carbonyl carbon bond releases Ph-N=C=O as a neutral molecule. Formation of fragment with m/z 399 is explained through heterolytic cleavage of pyrrole nitrogen and 1′C through four membered transition state involving hydrogen of 2′C. 12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa Proceedings of 12th ISMAS Symposium cum
RSP-7
Workshop on Mass Spectrometry
AT, I-IV, VI and VII
Table 1: Structures, name, elemental formulae and molecular masses of AT and its hydrolytic degradation products with their fragmentation patterns as per scheme 1. Mol. Formula and m/z values of fragments Diastereomers of Dehydrated atorvastatins 12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa Proceedings of 12th ISMAS Symposium cum
RSP-7
Workshop on Mass Spectrometry
It results in formation of another bond between 1′C and 2′C and finally heterolytic cleavage of N and 1′C bond. The structural details with fragmentation patterns are given in Table 1. Structures were further verified by degradation pathway. Postulated degradation pathway based on LC-MS/MS data
The kinetics of inter-conversion and the equilibrium between AT and the lactone at different pH have been reported [7]. AT and its distereoisomer (I) are reversibly converted to lactone III and its distereoisomer (II), with concomitant formation of VI and VII with molecular weight less by 18 amu. It suggests loss of water from II/III in acidic medium. This can be attributed to the instability of secondary alcohols in acidic medium at elevated temperature [8]. Formation of VII, the major degradation product, is followed by appearance of IV, which is 18 amu heavier than the former. This suggests the addition of H2O molecule to VII. Lactones are well known to undergo hydrolysis in acidic medium. Protonation of carbonyl oxygen results in cleavage of lactonic C-O bond forming a carbocation. A double bond in conjugation with carbocationic centre stabilizes the carbocation by allylic resonance stabilization and +ve charge can be localised on 3′C or 5′C. Hence, a nucleophile like H2O can attack on either of two cationic centres. However steric-hinderence due to OH of –COOH makes the attack of H2O unfavourable at 5′C resulting in formation of 3′-hydroxy derivatives. Conclusion
Atorvastatin undergoes severe hydrolytic degradation in acidic condition at 80°C after 24 h. However, under neutral and alkaline conditions, it undergoes only minor degradation. As MS/MS fragmentation patterns were same for AT/I, II/III and VI/VII, it could be concluded that diastereomers are formed from atorvastatin, atorvastatin lactone and dehydrated atorvastatin lactone, respectively. It is further suggested that IV represents any of the two diasteremer of dehydrated atorvastatin or both co-eluting diastereomers. The structure of V could not be elucidated from MS behaviour. References
1. Y. Shitara, Y. Sugiyama, Pharmacol. and Ther. 122 (2006) 71.
2. A. Mohammadia, N. Rezanour, M. Ansari Dogahehc, F. Ghorbani Bidkorbeh, M.
Hashemb, R.B. Walker, J. Chromatogr. B 846 (2007) 215-221.
3. M. DellaGreca, F. Cermola, M. Iesce, S. Montanaro, L. Previtera, F. Temussi, Tetrahedron 62 (2006) 7390-7395.
4. S. Erturk, A. Onal, S. MugeCetin, J. Chromatogr. B 793 (2003) 193-205.
5. W. Bullen, R. Miller, R. Hayes, J. Am. Soc. Mass Spectrom. 10 (1999) 55-66.
6. V. Borek-Dohalsky, J. Huclova, B. Barrett, B. Nemee, I. Ulc, I. Jelinek, Anal. Bioanal.
Chem. 386 (2006) 275-285.
7. A. Kearney, L. Crawford, S. Mehta, G. Radebaugh, Pharm. Res. 10 (1993) 1461-1465.
8. J. March, Advanced Organic Chemistry, A Wiley-Interscience Publication, Singapore,
Ravi P. Shah
Department of Pharmaceutical Analysis
National Institute of Pharmaceutical Education and Research (NIPER)
Sector 67, S.A.S. Nagar 160062, Punjab
12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa

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