Abstract
In this study, we report the systematic approach for characterization of two major degradant impurities, which are not listed in any compendia and were formed during the stability studies of Dihydroergotamine mesylate injection (DHE). An ion-pair UPLC chromatographic method was developed to quantify the related substances present in the DHE injection drug product. The same was used to monitor the impurity profiling during its stability. The two unknown impurities were observed at RRT about 0.08 (Impurity-1) and RRT about 0.80 (Impurity-5) and found to be significantly increasing on stability. Forced degradation studies revealed the nature of the impurity and conditions required for enriching them. A Mass compatible HPLC method was developed to quantify only these two impurities using 25% ammonia and formic acid in water. Their mass numbers were identified using LC MS/MS with triple quadruple mass spectrometer coupled with a HPLC. These two impurities were then isolated from enriched products using preparative HPLC. These impurities were then characterized using Mass and NMR analysis along with Q-TOF elemental analysis.
Introduction
Dihydroergotamine (DHE), a classified alkaloid under ergot alkaloids, exhibits contraction of blood vessels around the brain [1]. It also impacts blood flow patterns that are associated with evident types of headaches, consequently used in the treatment of migraine. The therapeutic activity of DHE in migraine is generally attributed to the agonist effect at 5-HT1D receptors. Two current theories have been proposed to explain the efficacy of 5-HT1D receptor agonists in migraine. One theory suggests that activation of 5-HT1D receptors located on intracranial blood vessels, including those on arterio-venous anastomoses, leads to vasoconstriction, which correlates with the relief of migraine headache [2, 3]. The alternative hypothesis suggests that activation of 5-HT1D receptors on sensory nerve endings of the trigeminal system results in the inhibition of pro-inflammatory neuropeptide release.
Commercially available Dihydroergotamine is ergotamine hydrogenated in the 9, 10 positions as the mesylate salt and is official in both US and European Pharmacopeia. It is chemically ergotaman-3′,6′,18-trione,9,10-dihydro-12′-hydroxy-2′-methyl-5´-(phenylmethyl)-(5′α)-, monomethanesulfonate. As shown in Fig. 1 Structure of Dihydroergotamine. Its molecular weight is 679.80 and its empirical formula is C33H37N5O5⋅CH4O3S. Five related alkaloids or related substances were reported in official pharmacopeia, which are by-products during the process of fermentation. Detailed structures along with its name and nature are tabulated in Table 1.
List of European pharmacopeia officially listed specified impurities
| Impurity Name | Chemical structure | Category |
| Impurity-A (Ergotamine) |  | By-product from fermentation |
| Impurity-B (Dihydroergostine) |  | By-product from fermentation |
| Impurity-C (8-Hydroxy dihydroergotamine) |  | By-product from fermentation |
| Impurity-D (2epi-dihydroergotamine) |  | By-product from fermentation/Degradant |
| Impurity-E (dihydroergocristine) |  | By-product from fermentation/Degradant |
Dihydroergotamine mesylate is commercially available in injection and inhalation forms [4]. Injection form is supplied in sterile ampoules for I.V., I.M., or subcutaneous administration containing per mL dihydroergotamine mesylate 1 mg, 94% w/w ethanol 6.2% volume, glycerine 15% by weight, and qs with water.
Dihydroergotamine mesylate injection [5] when loaded on stability as per ICH guidelines Q1, significant increase of unspecified impurities were observed. Two major impurities observed were at RRT∼0.08 and RRT∼0.80. Below Table 2 is the compilation of impurities for two different formulations with different processes.
Compilation of % w/w of major degradant impurities in the two formulations
| %w/w Impurity RRT∼0.08 | %w/w Impurity RRT∼0.80 | |||
| 25 °C/60%RH-6M | 40 °C/75%RH-6M | 25 °C/60%RH-6M | 40 °C/75%RH-6M | |
| Formulation-1 | 1.42 | 1.09 | 7.85 | 6.32 |
| Formulation-2 | 1.23 | 0.87 | 6.88 | 6.12 |
Degradation studies were carried out to study the stability of Dihydroergotamine injection at different stress conditions. The aforementioned impurities were monitored in all stress conditions. It revealed that Impurity at RRT 0.08 is a base degradant and impurity at RRT 0.80 is an acid, photo, thermal, and oxidative degradation product [6, 7]. As shown in Fig. 2, two impurities of concern at RRT∼0.08 and RRT∼0.80 as Impurity-1 and Impurity-5 are depicted as in Table 3, the compilation of major degradants in various stress conditions.
Specimen chromatogram depicting two impurities of concern at RRT∼0.08 and RRT∼0.80 as Impurity-1 and Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Compilation of % w/w of major degradant impurities at various stressed conditions
| Stress Condition | %w/w Impurity RRT∼0.08 | %w/w Impurity RRT∼0.80 |
| 1N HCl–60C; 30Min | 0.14 | 4.57 |
| 1N NaOH–60C; 5Min | 2.41 | 0.06 |
| Photo Stress; 3Hrs | 0.09 | 5.43 |
| Oxidative stress; 30% H2O2; 1 Day | ND | 7.12 |
| Thermal-60 °C; 7 Days | 0.39 | 7.46 |
The current research focus is on enriching, isolation, and separation of these two unspecified impurities followed by their structural characterizations.
Materials and methods
Materials
The samples required for study are Dihydroergotamine drug substances and Dihydroergotamine Injection USP 1 mg mL−1 were formulated at Dr. Reddys IPDO, Innovation Plaza (Dr. Reddys Limited, Hyderabad, India).
Impurities enrichment and isolation process were done at Dr. Reddy's IPDO facility. The Reagents used for analysis i.e., 1-Heptane sulfonic acid sodium salt (AR grade), Potassium dihydrogen phosphate (AR grade), orthophosphoric acid (88% w/w ACS grade), Acetonitrile (Merck-HPLC grade), Hydrochloric acid 36% (AR grade), and Water (Milli-Q grade).
Instrumentation and methodologies
Analytical UPLC chromatographic conditions
Chromatographic separation was performed on Water Acquity UPLC system configured separation module equipped with photodiode array detector (PDA) with Empower pro-data handling system [waters corporation, MILFORD, MA01757, USA]. The analysis was carried out on Acquity UPLC BEH shield RP18, 100 mm × 2.1 mm, 1.7 µm Particle size column. The separation of specified and unspecified impurities was achieved by gradient elution of mobile phase A, buffer pH of 2.5, and mobile phase B, which is a mixture of mobile phase A and acetonitrile with a flow rate of 0.3 mL min−1 and column temperature of 50 °C. Gradient program involves mobile Phase A with 75% till 25 min, then linearly changes to 55% within 55 min, and 50% in 10 min.
Enrichment and isolation of impurities (degradation impurity-1 and -5) by preparative HPLC
Agilent 1,200 series preparative (PS) liquid Chromatograph configured with UV-VIS detector and fraction collector was used for purification. A Phenomenex Gemini NX C18 250 mm × 30 mm, 5 µm Particle size column was employed for isolation of degradation impurities. A mixture of Milli Q water, 25% ammonia water, and 98% formic acid (1,000:10:5 v/v/v) with a pH of 8.50 is used as mobile phase A and a mixture of acetonitrile and mobile phase A as mobile phase B. The chromatographic conditions are tabulated in Table 4.
Gradient program
| Time (minutes) | % Mobile phase-A | % Mobile phase-B |
| 0 | 60 | 40 |
| 12 | 50 | 50 |
| 20 | 15 | 85 |
| 24 | 15 | 85 |
| 25 | 60 | 40 |
| 31 | 60 | 40 |
For Impurity-1 (at RRT∼0.08): Refluxed Dihydroergotamine sample with 1N HCl for about 5 h to yield 40%w/w of Impurity-1 when injected in UPLC methodology.
For Impurity-5 (at RRT∼0.80): Subjected Dihydroergotamine sample to 1.2 million lux hours, 200-W hour/square meter for about 5 days. About 20%w/w of Impurity-5 was observed in the stressed sample when injected in UPLC methodology.
The above solutions were loaded into a preparative LC as per above methodology. Fractions with about 95% purity are pooled and subjected to rotavapor to remove the solvent. The resultant mixture was lyophilized separately using a freeze dryer (Virtis advantage 2 XL) to yield impurity-1 and impurity-5.
LC-MS/MS conditions
LC-MS/MS analysis was carried out on Applied biosystems triple quadruple mass spectrometer (AB SCEIX QTRAP 4500) coupled with a Shimadzu UHPLC with LC-30 AD pumps, SIL-30AC auto-injector, CTO-20AC column oven, and SPD-M20A detector. Analyst software version 1.6.2 was used for data processing. The turbo ion spray voltage was maintained at 5,500 mV, the temperature was set at 550 °C, scan rate was 200 Da/S with unit resolution. High pure nitrogen gas is used as auxiliary gas and curtain gas. Zero air is used as the nebulizer gas. The LCMS data was acquired by selecting Q1 MS Q1 with a scan range from m/z 100–1000 Da in 0.1 amu steps with 2.0s dwell time. The analysis of the samples was carried out by using Develosil UG-5, 250 × 4.6 mm, 5 µm Particle size column. A mixture of Milli Q water, 25% ammonia water, and 98% formic acid (1,000:10:5 v/v/v) with a pH of 8.50 is used as mobile phase A and a mixture of acetonitrile and mobile phase A as mobile phase B [8]. UV detection was carried out at 280 nm, the flow rate was 1.5 mL min−1 and data acquisition time was 31 min.
Nuclear magnetic resonance (NMR)
1D (1H NMR, 13C NMR) and 2D (DEPT (135), 1H–1H COSY, HMBC, HSQC, and NOESY) NMR experiments were performed on a Bruker Advance III HD Cryo-600MHz NMR spectrometer (Bruker AG Industries, Faelladen, Switzerland), using deuterated Dimethyl sulfoxide (DMSO-d6) as solvent and TMS as internal standard at 25 °C for 1H NMR and 13C NMR spectra. The operating frequencies for 1H NMR and 13C NMR were 600.1337058 and 150.9178988Hz and the number of scans was 64 and 2,048 respectively.
Mass spectrometry
Mass and MS/MS spectrums were recorded on AB SCIEX QTRAP 4500 mass spectrometer equipped with an ESI source [9]. Source temperature as 500 °C, ion spray voltage as 5,500v (IS), Nitrogen gas was used as curtain gas, nebulizer gas (GS1) and collision gas, zero air used as heater gas (GS2), Decluttering potential were 60v (DP), collision energy was 18v (CE) and CXP was 8v. Detection of ions was performed in electrospray ionization, positive ion mode by direct infusion of the sample solution into the source [10].
Elemental composition by Q-TOF
Elemental composition was performed on SYNAPT G2. Si Q-TOF (Water solutions) equipped with an interface ESI source and Masslynks (version 4.1) was operation and processing software. The capillary voltage was 1,000v, cone voltage 25v, source temperature 120 °C, and desolvation temperature 300 °C. Samples solution was directly infused using Hamilton syringe, recorded mass spectra and elemental composition was deduced from mass data by applying the DBE: min = −100.0 max = 100 and tolerance is 10.0 PPM.
Results and discussion
Prediction for degradation impurity at RRT∼0.08 (Impurity-1)
Acid Degradation sample is run in LC-MS/MS method. The electron spray ionization (ESI) mass spectrum of Dihydroergotamine and degradation Impurity-1 (RRT∼0.08) showed protonated molecular ion peaks (M+H)+ at m/z 271.0 in positive ion mode [11]. Refer Fig. 3a for LC-MS/MS data.
A) LC-MS/MS data for Impurity-1; B) LC-MS/MS data for Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
The possible prediction for the structure with a mass of 270amu is by cleavage of NH- bond resulting in two fragments. Refer Figure 4 for possible degradation pathway.
Possible degradation pathway of Impurity-1
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Prediction for degradation impurity at RRT∼0.80 (Impurity-5)
Photo stress Degradation sample is run in LC-MS/MS method. The electron spray ionization (ESI) mass spectrum of Dihydroergotamine and degradation Impurity-5 (RRT∼0.80) showed protonated molecular ion peaks (M+H) + at m/z 584.2 and 588.2 respectively in positive ion mode. Refer Fig. 3b for LC-MS/MS data. Also extracted the UV spectra (as shown in Fig. 5) for the impurity-5 from PDA detector and compared it with the UV spectra of DHE [12].
The mass spectral data (m/z 588, M+H) displayed 4 amu more than Dihydroergotamine (m/z 584, M+H). The UV showed a 14 nm shift in the impurity (234 nm) when compared with those of Dihydroergotamine (220 nm).
Chromatographic purity of isolated impurities
Isolated and purified individual impurity-1 and impurity-5 of concentration 1 mg mL−1 along with dihydroergotamine were injected injected in UPLC methodology to identify the relative retention times of isolated impurities and their chromatographic purity [13]. It was identified that the enriched and isolated impurities were Impurity-1 at RRT∼0.08 and Impurity-5 at RRT∼0.80. The Chromatographic purity of both the impurities was found to be more than 98% pure. Refer Fig. 6 for chromatographic purity chromatograms [14].
For chromatographic purity for Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Structural characterisation of Impurity-1
Mass study
The ESI mass spectrum of Dihydroergotamine Impurity-1 (RRT = 0.08). was recorded in +ve polarity mode on the SYNAPT G2-Si TOF system. Refer Fig. 7 for fragmentation spectra.
ESI (MS-MS) (+ve) Mass spectrum of DHE Impurity-1 (RRT = 0.08)
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
The ESI +ve ionization mass spectrum of Dihydroergotamine Impurity-1 (RRT = 0.08) displayed the protonated molecular ion at m/z: 270 indicating that the structural formula is C16H19N3O.
The ESI +ve ionization mass spectrum of Dihydroergotamine Impurity-5 (RRT = 0.80) displayed m/z = 586 (There is a difference of 4 Amu's with dihydroergotamine). The High resolution mass (-ve) spectral data (-ve) of the impurity showing m/z = 586.2675,C32H36N5O6 [M−H] with 1.5 ppm error, as shown in Fig. 8.
ESI (MS-MS) (-ve) mass spectrum of DHE Impurity-5 (RRT = 0.80)
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
FT-IR study
Fourier Transform Infra-Red (FT-IR) of Dihydroergotamine Impurity-1 (RRT = 0.08) was recorded on Perkin-Elmer FT-IR spectrophotometer. Triturate 3–4 mg of the substance being examined with 300–400 mg of dried KBr. Then carefully placed the disc obtained in a pellet holder and transfer the sample into the beam of the FT-IR spectrophotometer. Record the infra-red spectrum of the sample preparation over the range from 4,000 cm to 1 to 400cm−1. The following characteristic bands observed for specific functional groups are shown in Fig. 9 and tabulated in Table 5.
ATR-FTIR spectra for Impurity-1
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Characteristic bands observed for specific functional groups
| Wavenumber (cm−1) | Assignment | Mode of vibration |
| 3398.9 | –NH | Stretching |
| 2950.9 | Sp3–C–H | Stretching |
| 1,664 | C=O (amide) | Stretching |
| 1036.8 | C–O of carboxylic group | Stretching |
| 749.2 | C–H | Bending |
NMR study
All NMR experiments (One-dimensional (1H, 13C{1H}, DEPT135) and various twodimensional 2D NMR experiments such as 1H–1H gDQFCOSY, 1H–1H, ROESY, 1H-13CgHSQC, 1H-13CgHMBC, and 1H–15N gHSQC) were performed on Bruker Advance 600 MHz NMR spectrometer (600 and 150 MHz resonance frequency for 1H and 13C, respectively). The samples were dissolved in deuterated dimethylsulphoxide (DMSO-d6). The entire NMR measurements were carried out at 298 K. 1H and 13C chemical shift values were reported regarding DMSO-d6 solvent peak at 2.50 and 39.51 ppm, respectively. For Dihydroergotamine Impurity-1, two exchangeable signals were observed at 7.40 and 6.80 ppm, which did not show correlations in the 1H–13C HSQC spectrum. These two signals showed HMBC correlations to C17 (carbonyl) and C12, indicating that these two protons are possible from the CONH2 group. FT-IR spectrum also shows one transmittance band at 1,664.0 cm−1 corresponds to an amide carbonyl stretching frequency, not carboxylic acid. In addition, these two signals have shown correlations in the 1H–15N HSQC spectrum indicating that there are two protons attached to one nitrogen atom along with another NH proton at position 5. This suggested confirming the presence of one NH and one NH2 group in the molecule. The presence of nOe cross-correlations between H1/H10, H3/H5, H6/H16, H12/H18, and H13/H18 along with the HMBC correlations between H18/C17 and H18/C12 confirm the structure (as shown in Figure A Fig. 4), the correct structure formula is C16H19N3O, name is (6aR,9R,10aR)-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo [4,3-fg]quinoline-9-carboxamide as shown in Figure B in Figs 9 and 10.
Nmr spectras for dihydroergotamine Impurity-1
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
For Dihydroergotamine Impurity-5, the ESI mass spectrum (Fig. 5) has been recorded on SYNAPT G2-SI Q-TOF Q-LCMS/MS system. The sample is introduced into the system through HPLC by bypassing the column. In Impurity-5, the disappearance of an aromatic methane carbon at 119 ppm (positon-9) and also the singlet exchangeable proton∼11.00 pm (positon-8). The appearance of a new carbonyl at 199 ppm (positon-11). CH2 signal at 23.8 ppm (position 12) in API got deshielded and shifted to downfield about 44.3 ppm in impurity and it is showing HMBC with Carbonyl carbon at 198.1 ppm (position 11). Aromatic CH proton at 6.51 ppm (position 4) showing HMBC with Carbonyl carbon at 198.1 ppm (position 11). The chemical shift assignments of Dihydroergotamine Impurity-1 (RRT = 0.08) was tabulated in the Table 6 (Fig. 11).
Chemical shift assignments of dihydroergotamine Impurity-1 (RRT = 0.08)
| Position1 | 1H ( | J (Hz) | 13C ( | DEPT |
| 1 | 6.79 | d, J = 8.0 | 112 | CH |
| 2 | 7.01 | t, J = 8.0 | 122 | CH |
| 3 | 7.12 | d, J = 8.0 | 108.7 | CH |
| 4 | - | - | 133.1 | C |
| 5 | 10.61 | s | - | - |
| 6 | 6.96 | s | 118.5 | CH |
| 7 | - | - | 110.1 | C |
| 8 | - | - | 125.9 | C |
| 9 | - | - | 132.5 | C |
| 10 | 2.77 | m | 39.6 | CH |
| 11, 11′ | 2.67, 1.38 | m | 30.8 | CH2 |
| 12 | 2.64 | m | 41.7 | CH |
| 13 | 2.15 | t, J = 11.2, | 59.3 | CH2 |
| 13′ | 2.98 | m | - | - |
| 14 | - | - | 66.7 | CH |
| 15 | 1.95 | m | 26.5 | CH2 |
| 16, | 3.30 | dd, J = 4.0, 14.7 | 175.33 | C |
| 16′ | 2.49 | m | - | - |
| 17 | - | - | 42.7 | CH3 |
| 18 | 7.40, 6.80 | s | - | - |
| 19 | 2.34 |
s = singlet, d = doublet, dd = doublet of doublet, m = multiplet
Nmr spectras for dihydroergotamine Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Based on the diagnostic cross-peaks observed in HMBC & NOESY (shown by arrows in the structure) from Figs 12, 13 and 14 and salient features of NMR data and from the Mass data, a new ring-opened structure of indole moiety for the impurity has been elucidated and the structure has been elucidated as shown below in Fig. 15.
gCOSY NMR spectrum of dihydroergotamine Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
NOESY NMR spectrum of dihydroergotamine Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
gHSQC NMR spectrum of dihydroergotamine Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Characteristic nOe correlations (dotted arrows) and HMBC correlations (solid arrows) are used to determine the correct structure of Dihydroergotamine Impurity-1 (RRT = 0.08)
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Cross-peaks observation in HMBC & NOESY
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Conclusion
Impurities at RRT 0.08 and 0.80 (impurity-1 and impurity-5 respectively) in DHE injection which were found to be increasing above ICH threshold were identified for its mass number as m/z 271 and 588 M+H by LC-MS fragmentation respectively (Fig. 16). These two degradation impurities were found to be increasing in acidic environment and are then enriched, isolated, and characterized for their structure. Characteristic nOe correlations and HMBC correlations used to determine the correct structure of Dihydroergotamine Impurity-1 (RRT = 0.08) and Dihydroergotamine Impurity-5 (RRT = 0.80) exhibited no structural alerts for genotoxic nature. These impurities thus can be classified as identified and specified impurities as per ICH Q3B (R2). The proposed structures for Dihydroergotamine Impurity-1 and Imputiy-5 are presented in Fig. 17a and b respectively.
A) Proposed structure of dihydroergotamine Impurity-1; B) Proposed structure of dihydroergotamine Impurity-5
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01095
Declarations
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Competing interests
The authors have no relevant financial or non-financial interests to disclose.
Conflict of interest section
The authors declare that no potential conflicts of interest for this article's research, authorship, and/or publication.
Author contributions
Basappa Praveen: The acquisition, analysis, interpretation of data for the work and drafting of the work.
Dr. Dama Venugopala Rao: Assisting in data interpretation, prediction of structures and technical assistance in carrying the work.
Dr. M.S Uma Shankar: The conception, design of the work and final approval of the version to be published.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Abbreviation:
| ICH | International Council for Harmonisation |
| RRT | Relative Retention Time |
| NMR | Nuclear Magnetic Resonance |
| HSQC | Heteronuclear Single Quantum Coherence |
| HMBC | Heteronuclear Multiple Bond Correlation |
| NOESY | Nuclear Overhauser Effect Spectroscopy |
| ROESY | Rotating Frame Overhause Effect Spectroscopy |
| FTIR | Fourier-Transform Infrared Spectroscopy |
| ESI | Electrospray Ionization |
| Q-TOF | Quadrupole Time-of-Flight |
| amu | atomic mass unit |
Acknowledgements
Authors acknowledge technical insights of P. Rudra Mohan Reddy for his technical insights and suggestions during execution of the research.
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