Characterization of Degradation Products Resulted from Acidic Hydrolysis of Lisinopril Under Drastic Conditions

Objective: Characterization and structure elucidation of the degradation products which resulted from the acidic hydrolysis of lisinopril under drastic condition. Method: Ultra-performance liquid chromatography coupled with mass/mass spectroscopy (LC-MS/MS) with simple and sensitive method was used to detect the formed degradation products. The fragmentation patterns of the degradation products were investigated. Results: The validation of analytical method was satisfied to the recommend criteria of international conference of harmonization. Four degradation products were formed due to the acidic hydrolysis of lisinopril. Conclusion: Two degradation products were described in E. Ph. (8) as impurities. While both of 6-amino-2-((1-carboxy-3-phenylpropyl)amino) hexanoic acid (m/z 309) and 1-(2,6-diaminohexanoyl)pyrrolidine-2-carboxylic acid (m/z 244) were formed and identified.


INTRODUCTION
Lisinopril,2S)-1-[(2S)-6-amino-2-{[(1S)-1carboxy-3-phenylpropyl] amino}hexanoyl] pyrrolidine-2 carbo-xylic acid is an angiotensin converting enzyme (ACE) inhibitor, used for the treatment of hypertension, heart failure, and acute myocardial infarction [1][2] .A wide variety of separation and detection techniques have been applied to the analysis of lisinopril, such as gas chromatography coupled with mass spectrometry (GC/MS) [3][4] , high performance liquid chromatography (HPLC) [5][6] , and high performance liquid chromatography coupled with mass spectrometry (HPLC/MS) [7][8] .The degradation profile of a drug substance is critical to its safety assessment and formulation process in addition to dosage form administration.For safety reasons, the degradation products or impurities of a drug that exceed 0.1% must be identified prior to clinical trials 9 .This paper presents a modified validated method for determination of lisinopril with high resolving from the formed degradation products or impurities by LC-MS/MS in addition to identification of the four formed degradation products by employing LC-MS/MS analysis technique and studying the fragmentation behavior of the resulted product ions to elucidate their chemical structures.

Materials
Lisinopril certified reference standard (CRS), USA Pharmacopeia, USA.Lisinopril raw material was purchased from Sigma aldrish, Germany.Formic acid was from Sharlu, Spain; Acetonitrile and methanol HPLC grade were brought from J. T. Baker, USA.All other reagents were of analytical grade.Distilled

Mass spectroscopy conditions
LC-MS/MS separation was performed on 40° C with injection volume of 2µl on C18 (1.7µm 2.1X 100 mm) end capped (BEH).Mobile phase flow rate was 0.2 ml/min.The optimum TQ mass spectrometer parameters were equipped as following: the ionization process was done by ESI in positive ion mode, Source temperature was at 120°C, drying nitrogen was used as dissolving gas at flow rate of 700 l/h and dissolving temperature was at 400°C, Capillary voltage was at 3Kv and cone voltage at 40 Kv, dwell time per transition was 0.112 (sec), and ion transition for screening degradation products fragments was at m/z 1000.The selected collision energy value for every degradation product was applied to screen its fragment ions pattern with accepted relative abundance values for molecular characterization.

Preparation of mobile phase
The mobile phase was prepared according to Zhou et al., 10 with a slight modification in its composition (aqueous to organic ratio) to resolve lisinopril and degradation products.It consists of an acidified aqueous solution with formic acid (pH 2.9): methanol: acetonitrile in a ratio of (75: 15: 10, v/v), respectively.The mixture was stirred continuously for 30.0 min to ensure complete equilibrium.The final mobile phase solution was filtered on 0.2µm millipore filtration system and degassed on ultra-sonicator path for 20.0 min to get rid of air bubbles.

Preparation of serial standard solutions
A stock solution was prepared by dissolving a respective weight of Lisinopril CRS in mobile phases to obtain a concentration of 5µg/ml.Serial concentrations of 50, 100, 150, 200, 250,300 and 350 ng/ml were prepared from the stock solution to construct a calibration curve.

Preparation of sample solutions
Serial concentrations of sample solutions were skipped with 50 ng/ml of lisinopril standard solution to obtain concentrations of 150, 200 and 250 ng/ml.

Acidic hydrolysis of lisinopril
Lisinopril solutions of 50 µg/ml concentration were exposed to acidic hydrolytic degradation under the effect of 0.1 N HCl for 4 h at a temperature of 90°C.At the end of the experimental period, the solutions were diluted with the mobile phase and filtered through 0.2µm membrane to be injected into LC-MS/MS.

Validation of the analytical method
Validation of the analytical method was performed according to the international conference of harmonization guidelines (ICH) 11 for terms of linearity, accuracy, range, precision, specificity, sensitivity, and robustness and governed by United States pharmacopeia [USP 38 NF 29] 12 criteria for chromatographic separation.

Identification of the degradation products using LC-MS/MS
LC-MS/MS was employed for characterization of the degradation products 13 resulting from exposure of lisinopril to drastic hydrolysis conditions.First; the study was based on injecting lisinopril standard solution at concentration 200 ng/ml into LC-MS/MS system for determining the relative standard deviation percentage %RSD to confirm the precision and integrity of LC-MS/MS system conditions.Second; the study was extended to determine and follow the fragmentation pattern pathway of the degradation products which formed under the effect of acidic hydrolysis conditions of 0.1N HCl at temperature 90 °C for 4 h on lisinopril molecule.Three replicates were obtained from the acidic hydrolysis of lisinopril and diluted with the mobile phase, filtered through 0.2 µm membrane filter and injected in duplicates into the LC-MS/MS system.
Ion transition of screen spectrum was applied to m/z 1000 to detect the main separated degradation products and subsequently followed by running the spectrum to m/z 600 for every degradation product.The collision energy in a range of 10-35v at a fixed cone value was screened for every degradation product to select the appropriate value, which enables the detection of the fragment ions with accepted relative abundance value for degradation products characterization.

Validation of the analytical method
Table 1 summarized the results of the validation of the analytical method of lisinopril analysis  1 at a range between 50-350 ng/mL with mean recovery 99.93% of SD ± 0.42 for the spiked samples.The precision of the method evaluated by means of the relative standard deviation percentage (%RSD) of intraday ruggedness between two analysts at three different times per day (morning, noon, evening) was 0.29 and % RSD inter-day ruggedness between two analysts at three successive days was 0.74.The sensitivity of the method was represented by limit of detection 9.38 ng/ml and limit of quantitation 28.43ng/ml.The method is robust and can tolerate different labs variations according to robustness studies results.Specificity of the method to separate lisinopril from its degradation products was achieved with resolving of lisinopril from its degradation products with resolution factor of 3.28.System suitability of the validated method was accepted as described in Table 2. Studying the fragmentation pattern of lisinopril and its degradation products LC-MS/MS could identify the degradation products of lisinopril by studying its fragmentation patterns represented by the mass spectrum [14][15] .First, protonated lisinopril [M+1] (m/z 406 + ) fragments under collision induced by dissociation energy (CID) of 27.21v showed maximum relative abundance ion products at (m/z 246 + ) and (m/z 84 + ) as shown in Table 3 and Figure 3.The spectrum of protonated lisinopril (m/z 406 + ) shows a product ion at [406 + →360 + ] resulting from elimination of formic acid in a single step with a larger possibility than consecutive loss of water then carbon monoxide as there was no signal detected for water loss [406 + →388 + ].The signal of (m/z 309 + ) resulted from elimination of dihydropyrrole and carbon monoxide and migration of hydroxyl function group 16,17 , it can't be obtained the elimination process produced two molecules of dihydropyrrole and carbon monoxide or one neutral compound of dihydropyrrole carbaldehyde as there was no signals for their precursors.4.  MS/MS spectrum of the product ion (m/z 309 + ) was represented by Figure 9 and studied in Figure 10.The protonated degradation product (m/z 309 + ) resulted from hydrolysis of lisinopril and removal of pyrrolidine ring and carbon monoxide with migration of hydroxyl group to form with carbonyl group a carboxylic group 16,17 .
The fragmentation pattern of (m/z 309 + ) under CID energy of 28.4v shows peaks at (m/z 246 + ) due to elimination of formic acid with the amino group and cyclization of the lysine residue to from a tetrahydropyridine ring, (m/z 200 + ) due to elimination of formic acid, ( m/z 142 + ) is the same pathway for formation of the product ion of (m/z 246 + ) with elimination of the styrene 19 , and (m/z 84 + ) due to elimination of CH2COOH group from product ion of ( m/z 142 + ) or elimination of the but-3-en-1-ylbenzene group from the product ion of (m/z 201 + ) as presented in Table 5.     Figure 13 shows the fragmentation ion produced from the protonated degradation product (m/z 180 + ) by MS/MS.The protonated degradation product (m/z 180 + ) resulted from hydrolysis of lisinopril to give 2-amino-4-phenylbutonic acid due to removing 6aminohexanoyl pyrrolidine-2-carboxylic acid.The fragmentation pattern of (m/z 180 + ) under CID energy of 42.86vshows peaks at (m/z 162 + ) due to elimination of a water molecule, (m/z 134 + ) due to elimination of formic acid, (m/z 105 + ) due to elimination of 2-aminoacetic acid, and (m/z 72 + ) due to elimination of water and toluene molecules as represented in Table 7 and Figure 14.From the previous studying of the fragmentation pattern pathways and schematic analysis of the degradation products resulted from the acidic hydrolysis of lisinopril, it was found that four main degradation compounds were formed as summarized in the Table 8.Both degradation products of m/z (388 + and 180 + ) were described in Eur.Ph. (8 th ) as lisinopril impurities 10 .

Figure 1 .
Figure 1.Linear calibration curve of lisinopril concentrations and their corresponding peak areas.

Figure 2 .
Figure 2. Separation of lisinopril (200ng/ml) after applying modified chromatographic conditions for LC-MS/MS analysis at m/z 406 + in positive ion mode; it shows retention time of lisinopril at 6.61.

Figure 3 .
Figure 3. Product ion collusion induced dissociation (CID) mass spectrum for [M+1] ion (m/z 406 + ) of lisinopril.The formed degradation products as a result of acidic hydrolysis of lisinopril had been separated and detected by LC-MS/MS as shown in Figures4 and 5respectively.It was observed that the product ions of lisinopril as well as each formed degradation products appeared at mass peaks of m/z [M+1] of 406 + , 388 + , 309 + , 244 + and 180 + .

Figure 4 .
Figure 4. Separation of lisinopril and the resulted degradation products showed the main peak of lisinopril at 6.63 min and the peaks of degradation products at 4.79, 6.27, 9.34 and 10.31 min.

Figure 4 .
Figure 4. Possible schematic mechanism of the fragmentation pattern pathway of the product ion (m/z 406 + ) from lisinopril.

Figure 7
Figure7shows the fragmentation pattern of the protonated degradation product (m/z 388 + ) which has been investigated in Figure8and revealed that the product ion (m/z 388 + ) resulted from the elimination of a water molecule from lisinopril and formation of

Figure 8 .
Figure 8. Possible schematic mechanism of fragmentation pattern pathway of the product ion (m/z 388 + ).

Figure 14 .
Figure 14.Possible schematic mechanism of fragmentation pattern pathway of the product ion (m/z 180 + ).

Table 5 . Summary of chemical formulae, relative abundance, measured and exact mass of major ions observed in the product ion MS/MS spectrum of [M+ H] (m/z 309 + ). Chemical formulae Relative Abundance Measured mass m/z [H+1]
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