Abstract
Methanol-based solutions are commonly used as extraction or dissolving solvents in analytical and bioanalytical methods for the analysis of ibuprofen (IBU). However, the potential for the formation of IBU alkyl esters in these solutions has not been addressed in these applications. The recent disruption in the supply of acetonitrile (ACN) triggered the need to identify alternative solvents for IBU analysis in pharmaceutical dosage forms. We observed that, in a mixture of methanol and water (70:30,v/v), more than 0.05% (wt/wt) of IBU methyl ester formed in 24 hours and 0.2% (wt/wt) formed in 3 days; whereas in a mixture of isopropyl alcohol and water (70:30, v/v), 0.1% (wt/wt) of IBU isopropyl ester formed in 3 days. This led to a systematic investigation into the formation of IBU methyl ester in aqueous methanol solutions at various pH. The IBU methyl ester was analyzed by gradient liquid chromatography (LC), and its identity was confirmed by LC, LC-MS and LC-PDA against an authentic standard. The ultimate dissolving solvent, consisting of methanol and 20 mM potassium phosphate buffer at pH 6.0 (70:30, v/v), resulted in an acceptable IBU solution stable up to 3 days.
Introduction
Ibuprofen, (±)-2-(p-isobutylphenyl) propionic acid (IBU), is commonly used as an analgesic and a non-steroidal antiinflammatory drug in a variety of pharmaceutical formulations. The use of methanol and/or alkyl alcohol based solvents has been reported, particularly for sample extraction and preparations in pharmaceutical and biological matrices. For example, publications [1-4] described using methanol for IBU stock and working standard and liposomal sample preparations, and an article [5] reported using isopropanol for IBU solutions.
The recent industry-wide acetonitrile (ACN) shortage has triggered a search for alternative solvents. Different organic aqueous solutions were tested for diluent equivalence in analysis of IBU. Methanol (MeOH), ethanol (EtOH), and isopropyl alcohol (IPA) were selected and evaluated for sample solubility and chromatographic properties. It was observed that a new peak formed at appreciable levels in all of the alcohol-containing diluents. More than 0.05% (wt/wt) of the degradation product formed in a solution of MeOH and water (70:30) in 24 hours. A literature search revealed no discussion of the formation of IBU alkyl esters during the use of aqueous alcohol mixtures as sample diluents in analytical methods. However, there have been reports of the chemical synthesis [6] and enantioselective hydrolysis [7] of these compounds. From an analytical standpoint, it is particularly important to consider and control IBU solution stability. This concern prompted a systematic investigation into the IBU solution stability of different pH.
IBU alkyl ester sample solutions were prepared by dissolving IBU reference standard in the corresponding alkyl alcohol solutions and stored at ambient laboratory conditions. The solutions were injected onto an HPLC system at the time of preparation, and the same solutions were re-injected every 24 hours for 3 days. The reaction products were quantitated against a 4% IBU standard solution (actual concentration: 0.2 mg/mL). IBU alkyl ester analysis employed a 21-minute gradient Liquid Chromatographic (LC) method, which was based upon the European Pharmacopoeia (EP) [8] and The United States Pharmacopoeia (USP) [9] compendia methods. The identity of IBU methyl ester was further confirmed by Liquid Chromatography-Photo Diode Array (LC-PDA), Liquid Chromatography-Mass Spectroscopy (LC-MS) and MS/MS against an authentic standard prepared in-house.
The work presented in this communication note provides detailed insight into the esterification behavior of IBU in alkyl alcohol solutions, and demonstrates that esterification can be controlled by using a buffered methanol solution at pH 6 and above.
Experimental
Sample, solvents and reagents
IBU reference substance used as a working reference standard was qualified using USP methods against a USP reference standard.
MeOH, IPA, and ACN were of HPLC grade, and formic acid was of 99% purity.
Anhydrous Alcohol containing 90% EtOH was of analytical grade.
ortho-phosphoric acid (85% v/v), KOH, KH2PO4, and K2HPO4 were of analytical grade, and used in the preparation of phosphate buffers at different pH levels.
Water used in this study was obtained using a water purification system.
IBU methyl ester reference standard was synthesized in-house and its 1H NMR spectrum was in agreement with the reference standard.
Sample solutions
All IBU sample solutions were prepared at a concentration of 5 mg/mL using IBU working standard, except in the LC-MS experiment where the sample concentration and injection volume were scaled appropriately to obtain detectable signal and sensitivity. The sample solution was prepared at about 40 mg/mL for the LC-MS experiment for identification purposes.
The IBU methyl ester reference standard was used as an absolute standard for identification purposes. It was prepared by dissolving 50 mg of IBU reference standard in 10 mL of anhydrous MeOH and a few drops of sulfuric acid was added as a catalyst. The mixture was kept at room temperature in a water bath shaker at 150 rpm for 48 hours. The solution was extracted with methylene chloride and the solvent was removed under reduced pressure. The resulting compound was re-dissolved and diluted to 1 mg/mL in MeOH for LC/MS analysis.
Apparatus and equipment
HPLC analysis was performed on a LC system with a dual wavelength Absorbance Detector. A 150 x 4.6 mm, 5 μm C18 column was used as stationary phase.
LC-MS measurements were performed on a Mass Spectrometer coupled to an HPLC system. The LC component consisted of a degasser, gradient pumps, an autosampler, a column thermostat, and a photo-diode array detector. The MS was equipped with an electrospray source and operated at positive ionization mode. The whole LC-MS system was operated by software.
NMR spectrometer equipped with a 5 mm TXI (1H, 13C) probe was used for acquisition of the 1H NMR spectrum for the IBU methyl ester standard. The temperature was set to 298 K. Chloroform-d1 was used as the solvent.
Methods
HPLC
The HPLC conditions were adapted from the IBU monographs of the EP and USP. The method was modified for determination of IBU methyl ester to ensure that there would not be any coelution with other known related substances. A C18 column was used for separation and the injection volume was 20 μL. The column temperature was maintained at 35 °C during the analysis. The mobile phases were: A, 34% ACN in water with 0.1% formic acid; B, 100% ACN. Gradient elution profile: 100% A at 0 min, 100% B at 15 min, 100% A at 16 min, and end at 21 min. The flow rate was set at 1.5 mL/min. UV detection was performed at 215 nm. A Chromatography Data System was utilized for data acquisition.
HPLC/MS
The HPLC method for LC-MS analyses were performed under the same experimental conditions described in the HPLC section, except the injection volume was scaled in order to increase sensitivity. Besides the common MS parameters used, the spray voltage was 4.92 kV, capillary temperature was 274.88 °C and the capillary voltage was 35.95 V. The electron energy of MS/MS measurements was set at 27%. Full scan mode (m/z) was 60-600 in MS and 60-320 in MS/MS. The output flow from the column was split in a ratio of 1:2, where 0.5 mL/min entered the MS detector and 1 mL/min was passed to waste.
Results and Discussion
Characterization of the in-house reference material
The characterization of the in-house reference material was established by 1H-NMR and mass spectrometry. The σ values, intensities, multiplicities and coupling constants of the signals matched with those in the standard spectrum.
IBU methyl ester
IBU sample solutions were prepared at a concentration of 5 mg/mL in the respective dissolving solvents listed in Table 1. 50 mg of IBU working standard was accurately weighed and transferred into a 10-mL volumetric flask. It was then dissolved and diluted to volume with the dissolving solvent. The sample solutions were analyzed by LC at the time of preparation, and kept at ambient laboratory conditions. The aged solutions were re-injected every 24 hours for 3 days.
The investigation on IBU methyl ester formation will be discussed, as it is representative of the other IBU alkyl esters, which may be examined in a similar approach.
The weight percent (w/w%) of IBU ester for the aged solutions was calculated against the 4% IBU reference standard solution; a plot of methyl ester yield on the 3-day old solutions versus the pH values is shown in Figure 1.
The trend demonstrates that solution pH influences ester formation. Decreasing pH accelerated ester formation, and the short- and straight-chain alkyl esters formed faster than the longeror branched-chain alkyl esters. Although the ester may hydrolyze in acidic media, the presence of an excess of alcohol ensured that the ester formation process was dominant in the equilibrium reaction. Figure 2 is an overlay of the sample solutions of different pH, and Figure 3 represents the overlaid chromatogram of the IBU methanol solution and the in-house standard.
Subsequently, the IBU methanol sample solution was analyzed by LC-MS and LC-PDA-MS. Because of the lower sensitivity of IBU molecule, a concentration of 40 mg/mL was used to achieve the desired mass intensity. The extracted ion scans of IBU methyl ester from standard and sample solutions are presented in Figures 4 and 5, which gives molecular weight plus hydrogen peak of 221 m/z. The identical UV spectra were also obtained and presents in Figure 6.
Additionally, a two-stage mass analysis (MS/MS) allowed even more certainty for IBU methyl ester identification. Both Full Scan and SRM (Selective Reaction Monitoring) were conducted for the sample and standard solutions. The typical MS/MS product “daughter” ions of m/z 161, losing methyl ester moiety, and 91, were observed.
Conclusion
A study was conducted to investigate the formation of IBU methyl ester in alcohol based solutions of different pH, particularly at low pH. The results presented in this paper may be of importance for further consideration of IBU solution stability when utilizing alkyl alcohol based solutions for the analysis of IBU drug substance and products. By controlling the pH of the solution above 6, IBU solution stability can be prolonged.
Acknowledgement
The authors would like to acknowledge Hugh Ta for facilitating LC-MS work. We also thank David Rogers for proofreading the draft.
References
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Linzhe Liu is currently a Senior Scientist of Global R&D Operations at McNeilConsumer Healthcare, PA. She received her B.Sc. in Pharmaceutical Chemistry from Beijing University in China, and Maters in Pharmaceutical Sciences from the Catholic University of Leuven in Leuven, Belgium. Prior to joining McNeil, she worked as a Senior Analytical Chemist in Eli Lilly and Company. Her research expertise is in analytical method development and analytical technology applications to support the research and development of drug APIs and drug products. Readers may email her directly at: [email protected]
Steve Martellucci is a Principal Scientist at McNeil Consumer Healthcare responsible for specialty analytical testing in the R&D laboratories. He holds a BS degree from Penn State University. Prior to McNeil, he worked as an analytical chemist in drug discovery research for Johnson Matthey.
Dr. Kang Ping Xiao is a Principal Scientist leading an analytical team in the New Product Development department at McNeil Consumer Healthcare. He received his B.S. in Chemistry from Wuhan University, China, in 1990, and a Ph.D. in Analytical Chemistry from the University of Tokyo, Japan, in 1999. During his Ph.D. study, Dr. Xiao was awarded a young research scientist fellowship of Japan Society for the Promotion of Science from 1998 – 1999. He then completed postdoctoral fellowship at Michigan State University and The University of Michigan from 1999 to 2003. Dr. Xiao started his industrial career at Schering-Plough where he had developed a comprehensive guidance on the strategies to efficiently develop analytical methods for challenging pharmaceutical molecules. From 2007, Dr. Xiao has been working with formulation chemists at McNeil to direct pre-formulation and formulation studies for new products from the chemistry point of view.
Kenneth Day currently holds the position of Senior Scientist in Global R&D Operations at McNeil Consumer Healthcare, PA, where he supports the preformulation and development of new OTC drug products. He received his B.S. in Chemistry from Wayne State University in 2001 and M.S. in Organic Chemistry from the University of Illinois Urbana-Champaign in 2005. Prior to joining McNeil he has held various positions in analytical research and development within Discovery and Pharmaceutical Sciences at Pfizer Global R&D, supporting drug development from discovery through Phase III.
Dr Tony Osei is an R&D manager at McNeil Consumer Healthcare, Fort Washington PA. He received his B.Sc. in Chemistry from Kwame Nkrumah University of Science & Technology in Ghana and a PhD in Analytical Chemistry from University of Kansas. After a postdoctoral appointment in the Pharmaceutical Chemistry Dept at the same University, he joined Oread Labs in Lawrence, Kansas. For the past 15 years, Tony has been involved in analytical chemistry and formulation research at McNeil Consumer Healthcare.