Using mass spectrometry (UHPLC-MS), individual samples were analysed to determine the outcomes of all the stress studies. All degradants formed over a time, and from each constraint, study conditions are outlined in the experimental section. Five substantial degradation products were identified, isolated, and characterized by UHPLC-MS, Prep-HPLC, HRMS, 2D-NMR and FTIR techniques. Most of these degradation products are formed by the rearrangement of six-member carbohydrate ring of API into five-member furan ring
Isolation of degradation products:
The degradation products formed as considerable percentage of > 5% as a result of degradation was observed under peroxide and acid stress conditions. Luna C18 column (150 mm × 25 mm, 5 µ) and acetonitrile ,0.1% formic acid in aqueous were used as a mobile phase for purification. Consecutive injections of crude sample solutions were injected, and the fractions collection was done based on UV response and later mass confirmed by LC-MS. Collected separately fractions of various degradation products and lyophilized to get a free solid.
Structural confirmation of degradation products:
To get the structural information of Ertugliflozin API, all analytical data was recorded for reference purposes. Under ESI-MS positive mode conditions, [M + H]+ and ammonia adduct [M + H + NH3]+ ions were detected as 437.1350 and 454.1617, respectively, which confirms the molecular formula for ertugliflozin is C22H25ClO7. The confirmed data of ertugliflozin is mentioned at HRMS, HRMS/MS spectrum (Fig-4, Table-1), IR (Table-4), and 2D-NMR (Fig-7 & S1). HRMS/MS proposed tentative mechanism for protonated fragmentation ions of ERG are shown in Fig-5. The structural conformation of ERG is done by HRMS and 2D-NMR. All the other degradant products are characterized based on the comparison of this structural elucidation data.
Degradation product (DP-1) characterization:
The Ertugliflozin treatment with acid resulted in degradation product-1 (DP-1). This degradation product was isolated, and its mass was confirmed as [M + H]+ 508.1724 (-1.7675 ppm error) in HRMS for the molecular formula C25H30ClN5O8 shown in Fig. 4. The HRMS spectrum confirms the presence of one chlorine atom in the structure. The complete structure of the degradation product was determined by NMR and HRMS studies. The sample was prepared in DMSO-d6 solvent and subjected to NMR analysis. Initially recorded 1H NMR and D2O exchange experiments. On comparison of 1H and D2O data confirm the presence of five exchangeable protons in the compound and in aromatic region missing of 1,4 disubstituted ring pattern and presence the pattern of 1,2,4 trisubstituted ring. The key difference from API compound is the presence of secondary amide (H32), methylene protons(H31) and acetyl group(H34). In 1H NMR, a total of 30 protons were present, the secondary amide is at 8.08 ppm, and aromatic protons were shown between 6.0 ppm to 8.0 ppm. Primary and secondary alcoholic protons are at 4.5 to 5.5 ppm, and aliphatic protons resonance between 1.0 to 4.5 ppm. N-methylene protons are showing at 4.15 ppm and acetyl protons are at 1.85 ppm. O-methylene is at 4.00 ppm, and aromatic methylene protons are at 3.97 ppm. In 13C NMR a total of 25 carbons were showing; most downfield carbon is acetamide carbonyl showing at 169.26 ppm, followed by O-ethyl group attached quaternary carbon is at 154.36 ppm. The remaining aromatic carbons were between 100 to 140 ppm, aliphatic carbons were showing at 10 to 90 ppm. In HSQC experiment confirmed as H31 methylene protons attached carbons is showing at 37.16 ppm, and N-acetyl protons attached carbon is showing at 22.47 ppm. In the gDQ-COSY experiment, H31 protons showed correlation with the H32 proton. In 1H-15N HSQC experiment H32 proton was confirmed as an amidic NH proton, and its nitrogen value is showing at 115 ppm. In 1H-13C HMBC experiment, H31, H32, and H34 protons showed connectivity to C33 carbon, and H31 protons showed connectivity to C9, C10 and C11 carbons. All the key information of 1D and 2D NMR data confirms that 1,4 disubstituted ring converted to benzylacetamide. Finally, it formed N-(5-(2-chloro-5-(2,3,4-trihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octan-5-yl)benzyl)-2-ethoxybenzyl)acetamide and the 2D NMR spectrum was depicted in Fig. 7 & S2. The chemical shifts were in Table-3 and the proposed degradation mechanism was shown in Fig. 6.
Further, stretching frequency at 3415 cm− 1 in FT-IR spectra specifies the presence of NH and OH groups, and 1645 cm− 1 stretching frequency directs the presence of amide keto groups. The stretching frequency at 811 cm− 1 leads to the existence of the C-Cl group. A significant FT-IR spectrum established the existence of keto, amide, alcohol, and chloride functional groups, as presented in Table 4. For DP-1, HRMS/MS spectrum and proposed tentative mechanism for protonated fragmentation ions were shown in Fig-4 & 5.
Degradation product (DP-2) characterisation:
The DP-2 compound was formed in acid hydrolysis of the ERG compound. To acquire structural information, DP-2 HRMS analysis was performed and 371.1037 [M + H]+ was achieved as a protonated molecule with − 2.0463 ppm error for the calculated molecular formula C21H19ClO4 shown in Fig. 4. The HRMS spectrum confirms the presence of one chlorine atom in the structure, and the mass of the degradation product has 66 units less than parent ertugliflozin. To obtain the meticulous structure of the degradation product 1D and 2D NMR experiments have been performed. The 1H NMR data of DP-2 showed extreme differences from API compound. Here we could not see the bicyclic ring protons and noticed the primary alcohol proton at 5.56 ppm as a triplet, it was exchanged in D2O exchange. The methylene protons show as a doublet at 4.51 ppm, furan ring protons showing at 6.59 and 7.28 ppm with J value 3.2 Hz. In aromatic region 1,4 disubstituted ring, 1,2,4 trisubstituted pattern protons were present like parent compound at 6–8 ppm, and in aliphatic region O-ethoxy protons, methylene protons were shown between 1.0-4.5 ppm region. In 13C NMR, a total of 19 carbons were shown due to symmetry C9-C13 and C10-C12 carbons showing each as one signal. Most downfield carbon is a ketone, and it is resonating at 180.11 ppm. Quaternary carbons of furan ring are showing at 150.15, 161.7 ppm, and O-ethoxy attached aryl carbon is at 157.05 ppm; remaining aryl carbons are showing at 110 to 140 ppm. In the aliphatic region 4 carbons were showing, key methylene carbon is at 55.94 ppm, by HSQC experiment confirmed the same. Other key information from HSQC experiment is identifying the furan ring protonated carbons. From COSY experiment, primary alcohol proton H26 showed a correlation with H25 methylene protons, and furan ring protons H21, H22 are shown correlation with each other. From g-HMBC experiment confirms H25 protons showing connectivity to C22 and C23 carbons and H1, H3 protons show connectivity to C18 carbon. Based on NMR data concluded that bicyclic ring has been rearranged and formed 2,5 substituted ring furan rings as structure depicted in Fig. 7 & S3 and formed (4-chloro-3-(4-ethoxybenzyl)phenyl)(5-(hydroxymethyl)furan-2-yl)methanone. The chemical shifts were in Table-3 and proposed degradation mechanism was shown in Fig. 6.
Further, stretching frequency at 3481 cm− 1 in FTIR spectra confirms the presence of OH groups, and 1637 cm− 1 stretching frequency specifies the presence of keto groups. The stretching frequency at 756 cm− 1 directs the presence of the C-Cl group. FT-IR significant frequencies established the existence of alcohol, keto, and chloro functional groups, as depicted in Table 4. For DP-2, HRMS/MS spectrum and proposed tentative mechanism for protonated fragmentation ions were shown in Fig-4 & 5.
Degradation product (DP-3) characterization:
Acid hydrolysis of ERG compound resulted in the DP-3 compound. To acquire structural information_ of DP-3, HRMS analysis was executed and observed protonated molecule [M + H]+ as 413.1139 with − 2.8246 ppm error, which confirms the calculated molecular formula C23H21ClO5 shown in Fig. 4. The mass of the degradation product has less by 24 units than that of ERG, and to know the exact structure. Additionally, NMR experiments were recorded. DP-3 compound 1D NMR is almost like the DP-2, observed little difference, instead of alcoholic proton seen acetyl group. In 1H NMR, a total 21 number of protons are present, with those 9 protons from the aromatic region and furan ring protons shown at 6.79 and 7.32 ppm with J = 3,6Hz. Aliphatic region, a total 12 number of protons were present, in that 3 methylene groups are showing between 3.5 to 5.5 ppm, and acetyl group protons were showing at 2.07 ppm. In 13C NMR number of carbon count is 21, in those 16 carbons from aromatic and 5 carbons from aliphatic region. The most downfield carbon is ketone at 180.22 ppm, followed by O-acetyl is at 169.86 ppm, and O-ethoxy attached aryl carbon at 157.05 ppm. The furan ring quaternary carbons are at 150.92 and 154.92 ppm, protonated carbons are at 122.12 and 112.85 ppm, remaining carbons were shown between 110 to 140 ppm, aliphatic carbons were shown at 10 to 65 ppm. To identify protonated carbons from total number of carbons, conducted HSQC experiment and it confirms that O-methylene carbons C25 is 57.47 ppm and C15 is 62.86ppm and furan ring protonated carbons were at 122.12 and 112.85 ppm. In COSY experiment H21, H22 protons show correlations. To check 2J and 3J correlations DP-3 compound HMBC experiment helped a lot, here the key correlations were H25, H28 protons are showing connectivity to C27 carbon, H25 showed connectivity to C22, C23 carbons also and H1, H3 protons showing connectivity to C18 carbon along with these remaining connectivity’s are fitting to the structure. The structure of degradation product-3 was (5-(4-chloro-3-(4-ethoxybenzyl)benzoyl)furan-2-yl)methyl acetate, and the 2D NMR spectrum was depicted in Fig. 8 & S4. The chemical shifts were in Table 3 and the proposed degradation mechanism was shown in Fig. 6.
Further confirmation done with FT-IR and 1744 & 1645 cm− 1 stretching frequency indicates the presence of keto groups, and signals at 1349,1039 cm− 1 confirm the presence of C-O groups. No signal in the region of 3400 to 3000 cm-1 confirms that OH & NH protons are not present in the structure. The stretching frequency at 756 cm− 1 directs the presence of the C-Cl group. Significant FT-IR spectrum confirmed the presence of keto, and chloro functional groups, as tabulated in Table 4. For DP-3, HRMS/MS spectrum and proposed tentative mechanism for protonated fragmentation ions are shown in Fig-4 & 5.
Degradation Product (DP-4) Characterisation:
DP-4 resulted in treating ERG with acid. After isolation, HRMS was recorded to know its mass. For structural information of DP-4, HRMS analysis was performed and obtained the protonated molecule [M + H]+ 389.0694 with − 2.9529 ppm error for the molecular formula C21H18Cl2O3 shown in Fig. 4 and the HRMS spectrum having the dichloro pattern which conforms the compound having two chlorine atoms. It has 48 units less in comparison with the parent ERG compound. NMR studies deduced the precise structure of the DP-4. The sample was prepared in DMSO-d6 solvent and the required 1D and 2D NMR analyses were conducted to find out the structure of DP-4. In 1H NMR data shows 18 protons, and a minor difference was observed between 1H NMR data of DP-2 and DP-4 i.e., missing of the alcoholic proton, in DP-4, H25 methylene is showing as a singlet at 4.93 ppm. The furan ring protons were shown at 6,82 and 7.31 ppm with J = 3.6Hz. Two strong doublets H9, H13, and H10, H12 are showing at 7.15, 6.86ppm, H1, H2, and H3 protons are between 7.5 to 7.9 ppm, and aliphatic protons are showing between 1 to 5 ppm. In 13C NMR number of carbon count is 19, due to symmetric nature in 1,4-disubstituted ring instead of 6 observed 4 signals. Most downfield carbon is ketone is resonating at 180.21 ppm, rest of the carbons showing at 110 to 160 ppm. In aliphatic region four carbons were showing, the key methylene carbon at C25 is at 36.88 ppm, O-ethoxy carbons are at 14.63 and 62.86 ppm, methylene carbon C7 is at 37.37 ppm. By HSQC experiment validated the protonated carbons, furan ring protonated carbon shifts showing at 122.26 and 112.62 ppm. In HMBC experiments observed, two important connectivity’s in that H25 proton showing 2J correlation with C23 carbon and 3J correlation with C22 carbon. the second important connectivity is H1, H3 protons showing connectivity C18 carbon. All important information of NMR data confirms that bicyclic ring has been rearranged and 1,5substituted furan ring has been formed. As a result, the structure forming (4-chloro-3-(4-ethoxybenzyl)phenyl)(5-(chloromethyl)furan-2-yl)methanone and the 2D NMR spectrum was depicted in Fig. 8 & S5. The chemical shifts were in Table 3 and the proposed degradation mechanism was shown in Fig. 6.
FTIR spectrum indicates the presence of signal 1649 cm− 1 indicates the presence of the keto group, and signals at 1305 and 1246 cm− 1 indicate the presence of C-O groups. The stretching frequency at 841,703 cm− 1 directs the presence of the C-Cl group. No single in the region of 3400 to 3000 cm− 1 confirms that OH & NH protons are not present in the structure. FT-IR signals in spectrum conforms the structure includes of Chloro, and keto functional groups, as shown in Table 4. For DP-4, the HRMS/MS spectrum and proposed tentative mechanism for protonated fragmentation ions are shown in Fig-4 & 5.
Degradation product (DP-5) characterization:
Degradation product-5 was formed by treating the API compound with Hydrogen peroxide. For DP-5 structural information, HRMS analysis was performed and observed the protonated molecule [M + H]+ of 409.1037 with − 2.9086 ppm error for molecular formula C20H21ClO7 shown in Fig. 4. From the mass data, it is understood that 28 units were less than the parent API compound and had one chlorine atom in the structure. NMR studies have done the complete characterization of DP-5. We have recorded obligatory NMR experiments for a sample dissolved in DMSO-d6. Primary experiments of NMR 1H and D2O saying that, in 1H-NMR data showing 21 protons, in D2O exchange experiment 5 protons were exchanged and showing 16 protons. Hence confirmed the presence of five labile protons in the compound. The major difference from DP-5 compound to the parent compound is missing of O-ethoxy group and the presence of phenolic OH proton at 9.24ppm; remaining 4 labile protons are showing between 4.5 to 5.5 ppm (1o and 2o alcoholic). In aromatic region 1,4 disubstituted ring protons are at 6.66 and 6.97 ppm, chloro substituted ring protons are at 7.1 to 7.5 ppm, aliphatic methine and methylene protons are showing between 3 to 4 ppm. In 13C NMR 7 carbons were showing in aliphatic region and 11 signals were showing in aromatic region. More de-shielding carbon is phenolic OH located quaternary carbon, remain aromatic signals were shown between 100 ppm to 140ppm. In aliphatic region O- attached methylene and methine carbon were showing between 55 to 90ppm, aromatic methylene carbons are at 37.70 ppm. in HSQC experiment confirmed the shifts of methylene and methine carbons. In COSY experiment H24 proton showed connectivity to H19, H23 shows with H20, the H22 proton is with H21 proton, and H28 shows correlation with H25 protons. These connectivity’s are helped to identify the methine and methylene protons. In HMBC experiment, few important 3J correlation are observed H1,H3 and H27 protons are showing connectivity to C16 carbon. Also, H7 protons showing 3J connectivity to C3, C5 and C9, C13; 2J connectivity to C4, C8 carbons. NMR and mass data confirmed that ethoxy group leaved from the parent and formed phenolic OH on 1,4 disubstituted ring. The structure of the DP-5 has been elucidated and the NMR spectrum portrayed in Fig. 8 & S6. The chemical shifts were in Table 3 and the proposed degradation mechanism was shown in Fig-6. The FT-IR analysis also confirms the presence of OH group at 3443 cm− 1 stretching frequency. Stretching frequency at 755 cm− 1 directs the presence of the C-Cl group. No signal in the region of 1750 − 1600 cm− 1 confirms the absence of the keto group in the structure. Central frequencies established the presence of alcohol, Chloride functional groups, and data is tabulated in Table 4. For DP-5, HRMS/MS spectrum and proposed tentative mechanism for protonated fragmentation ions are captured in Fig-4 & 5.