INTRODUCTION:

Pulmonary arterial hypertension (PAH) is a group of diseases characterised by an increase in pulmonary vascular resistance that leads to right ventricular failure and death. Ambrisentan (AMBR), an endothelin receptor antagonist selective for the endothelin type-A receptor, has been approved for the treatment of pulmonary hypertension^1-3.

Identification of an impurity exceeding the required controlled threshold is critical for the quality of an active pharmaceutical ingredient (API) and pharmaceutical product from a regulatory standpoint. The majority of impurities may originate from the API's synthetic process or from chemical degradation of API or products transported or stored in any given conditionPulmonary arterial hypertension (PAH) is a group of diseases characterised by an increase in pulmonary vascular resistance over time^4-6. Forced degradation studies of APIs using acid, base, oxidative, photolytic, and thermal stress are a common method of simulating impurities that may appear or increase during transportation or at the shelf to unacceptable levels^7-11. Although stress stability studies and characterization of AMBR degradation products have been reported in the literature, none of them attempted to isolate and characterise major degradation products through degradation^12-16. The primary goal of this study is to isolate and characterise the major degradation product by employing all modern sophisticated spectral techniques.

MATERIALS AND METHODS:

Chemicals and Reagents:

Pure Ambrisentan, HPLC Grade Acetonitrile, Formic acid and Milli-Q Water.

Instruments and parameters:

To attain an adequate separation of AMBR and its degradation product, a suitable UPLC method developed on Waters Acquity UPLC® (Waters, Milford, USA) system (consisted of a quaternary solvent manager, a sample manager, and a photodiode array detector) to monitor the percentage of degradation, the number of degradation products forming with respect to time and to check the purity of finally isolated degradation product. A gradient method was developed on Acquity CSH Phenyl hexyl column (100 × 2.1mm, 1.7μ) using 0.1% Formic acid (Solvent A) and Acetonitrile (Solvent B) as eluents (Fig. 3.a). The gradient solvent program set as follows: (Tmin / % Proportion of Solvent B): 0-1/20, 1-6/90, 6-7/90, 7-8.5/20, and 8.5-10/20. The column equilibrated with 10 column volumes of mobile phase at the initial gradient composition before sample injection. The flow rate is 0.3mL/min, the injection volume is 2.0 μL, the detection wavelength is 262 nm, and the column temperature is 25°C.

Mass Spectroscopy:

For MS/MS analysis, an Agilent 1290 series liquid chromatography instrument (Agilent Technologies, USA) attached to a quadrupole - time of flight (Q-TOF) mass spectrometer (Q-TOF LC/MS) 6540 series, Agilent Technologies, USA) was used. The data acquisition was under the control of Mass Hunter workstation software. Positive ionization mode in Electrospray ionization (ESI) optimized. The typical operating source conditions for mass (MS) scan in positive ESI mode optimized as follows: Fragmentor voltage is 150 V, the capillary at 4000 V, the skimmer at 65 V, Nitrogen as the drying gas (325°C, 8L/min), and nebulizing (35 psi) gas. For collision-induced dissociation (CID) experiments, keeping MS1 static, the precursor ion of interest was selected using the quadrupole analyzer, and the productions were analysed using a TOF analyzer. Ultra-high pure nitrogen gas as collision gas.

Nuclear magnetic resonance spectroscopy (NMR):

NMR spectra (1H, 13C and 2D-NMR) were recorded on 500MHz spectrometer (Bruker Avance 500 spectrometer). The studies wererecorded in CDCl3-d6 solvent. 1H chemical shifts values were reported on the δ scale in ppm relative to TMS (δ= 0 ppm), whereas 13C NMR in relative to CDCl3 (δ = 77.0 ppm). Multiplicities of NMR signals are labeled as s (singlet), m (multiplet, for unresolved lines), etc. The chemical shift values were assigned with the help of heteronuclear single quantum correlation spectroscopy (HSQC) and heteronuclear multiple bond correlation (HMBC) spectroscopies.

Fourier transformed infrared spectroscopy (FTIR):

The FTIR spectra were recorded in the range of 4000–400 cm−1 using PerkinElmer Spectrum RX I spectrophotometer (Waltham, MA, USA). The sample pellet was prepared using dried potassium bromide (KBr) powder into a mortar with 1% (w/w) of the sample.

Preparative LC conditions:

A semi-preparative chromatograph from Waters (Milford, MA, USA) equipped with 515 HPLC pump and 2489 UV visible detector was used. μBondapak from Waters (Milford, MA, USA) C18 Prep column (125˚A, 300 × 7.8mm, 10μ) was employed for isolation. The mobile phase consisted of 0.1% formic acid and Acetonitrile (40:60% v/v), flow rate was 4mL/min.

Sample preparation:

Degradation reaction and isolation of degradation product:

For isolation of major degradation product about 1 g of AMBR was dissolved in 30mL of diluent (70:30 H2O: ACN) and refluxed for 30 min at 80°C, resulted in the formation of one DP (5%). When the hydrolysis was extended to 6 h the amount of degradation product was increased to 20.74%, without the formation of other degradation product Fractions containing > 99% of DP were pooled together and concentrated on rotavapor to remove the solvents. The degradation product was obtained as a light brown colour powder.

RESULTS AND DISCUSSION:

Characterization of degradation product:

LC-MS/MS studies of DP:

The results of LC–MS analysis indicated the presence of protonated ions [M + H]+ of degradation product (Fig. 1) and AMBR at m/z 303.1500 and 379.1650 Da, respectively. This shows that there is mass difference of 76 Da between DP and AMBR. To investigate the structural changes of DP, MS/MS of both protonated DP and AMBR were examined. All possible structures were proposed for product ions of DP and AMBR were shown in Table 1. The MS/MS spectrum (Fig. 2) of the protonated drug shows the product ions at m/z 303, m/z 179, m/z 125 and m/z 107. These peaks are indicative of the presence of 2,2-diphenylethen-1-ylium moiety and 4,6- dimethylpyrimidin-2-ol moieties in the drug. Thus, the MS/MS data provided the evidence for the proposed structure of DP The HRMS data of all the product ions are given in Table 1.

Fig. 1. LC-ESI-MS spectra of [M+H] + Ion of DP.

Table 1: HRMS Data of product ions of protonated AMBR and DP

AMBR and DP	Proposed formula	Calculated mass	Observed mass	Error	Nitrogen rule
	C22H23N2O4+	379.1652	379.1661	-2.37	Even
	C21H19N2O3+	347.1390	347.1399	-0.29	Even
	C21H17N2O2+	329.1285	329.1295	-2.73	Even
AMBR	C20H19N2O+	303.1492	303.1499	-1.98	Even
	C20H17N2O+	301.1335	301.1341	-1.99	Even
	C14H11+	179.0855	179.0854	0.56	Even
	C14H10•+	178.0777	178.0779	-0.56	Even
	C6H9N2O+	125.0709	125.0710	-0.80	Even
	C6H7N2+	107.0604	107.0604	0.00	Even
	C5H8N+	82.0651	82.0654	-3.66	Odd
	C20H19N2O+	303.1492	303.1500	-2.64	Even
	C14H11+	179.0855	179.0860	-2.79	Even
DP	C14H10•+	178.0777	178.0778	-0.56	Even
	C6H9N2O+	125.0709	125.0707	1.60	Even
	C6H7N2+	107.0604	107.0608	-6.54	Even
	C5H8N+	82.0651	82.0649	2.44	Odd

NMR structural characterization studies

1H NMR chemical shifts (δ) with coupling constants (J) and 13C NMR chemical shift values are presented for both DP and AMBR in Table 2. 1H and 13C NMR (Fig.3.i) of DP revealed 18 protons and 20 carbons while AMBR contained 21 protons and 22 carbons in DEPT135 and 13C experiments verified that DP has 2-CH3 (primary), 12-CH (tertiary) and 6-C (quaternary) carbons, while AMBR has 3-CH3 (primary), 12-CH (tertiary) and 7-C (quaternary) carbons. The experimentally obtained number of carbons precisely matched with the theoretically calculated number of carbons. The single bond carbon-proton heteronuclear correlation experiments are performed to confirm the rearrangement of correlation of CH (2) of AMBR to CH (1) and loss of correlation of CH3 (19) of AMBR in DP. Remaining moiety of AMBR molecule HSQC peaks are identical to DP HSQCspectrum. The observation of 1H, 13C, DEPT135 and HSQC NMR spectra of DP and AMBR indicates that loss of carboxylic acid and methoxy groups followed by double bond formation between second and third carbon atoms of AMBR resulted in the formation of DP. The observation of long-range carbon-proton correlations from HMBC experiment (Fig. 3) is consistent with the proposed structure. HMBC connections such as H1/C2, H1/C3, H1/C9, H1/C16, H19/C22, 23, H19/C18,20, H22,23/C18,20 and H22,23/C19 are showing notable correlations, further confirms the proposed chemical structure.

Fig. 2. LC-ESI-MS spectra of [M+H]+ Ion of AMBR

Fig. 3. HSQC spectra of AMBR in CDCl3

Table 2: 1H NMR chemical shifts (δ in ppm), coupling constants (J in Hz) and 13C NMR chemical shifts (δ in ppm) for AMBR and DP.

AMBR					DP
Atom number	1H	ppm/J	13C	DEPT	Atom number	1H	ppm/J	13C	DEPT
1	-	-	169.80	-	1	1H	7.82/s	135.77	CH
2	1H	6.35/s	77.31	CH	2	-		125.65	-
3	-	-	84.52	-	3	-		136.93	-
4, 5	-	-	-	-	4, 8, 10, 14	4H	7.18-7.25/m	127.10	CH (4)
6	-	-	138.53	-	5, 7, 11, 13	4H	7.25-7.29/m	128.25	CH (4)
7, 11, 13, 17	4H	7.23-7.27/m	127.94	CH (4)	6, 12	2H	7.35-7.36/m	130.15	CH (2)
8,10, 16, 14	4H	7.29-7.34/m	128.46	CH (4)	9	-		139.91	-
9, 15	2H	7.48-7.50/m	127.90	CH (2)	15	-		-	-
12	-	-	139.49	-	16	-		163.14	-
18	-	-	-	-	17	-		-	-
19	3H	3.30/s	53.21	CH3	18, 20	-		169.63	-
20	-	-	-	-	19	1H	6.69/s	115.55	CH
21	-	-	163.37	-	21	-		-	-
22	-	-	-	-	22, 23	6H	2.36/s	23.79	CH3 (2)
23, 25	-	-	169.44	-
24	1H	6.67/s	115.09	CH
26	-	-	-	-
27, 28	6H	2.35/s	23.75	CH3 (2)

IR Structural characterization studies:

IR spectra of DP and AMBR were compared to predict major differences in functional groups and found significant similarities. Overlaid IR spectra of DP and AMBR shows loss of C=O stretching (1752 cm-1), C-O ether stretching (1042 cm-1), O-H stretching (carboxylic acid, 2965 cm-1) in DP, which evidence the loss of carboxylic acid and methoxy functional groups. This was further corroborating the proposed chemical structure of DP by mass and NMR (Fig. 4).

Fig. 4. Overlaid IR spectrum of DP and AMBR

CONCLUSIONS:

Degradation of Ambrisentan drug substance has been performed under neutral hydrolytic conditions, achieved 20% of degradation. Isolated the degradation product of AMBR has been carried out using preparative HPLC and maximum purity of the DP identified through liquid chromatography. Discussed and elucidated the structure of degradation product through sophisticated spectroscopic techniques of HRMS, multidimensional NMR and FTIR analytical techniques. Based on this data it concluded that the degradation product is 2- (2,2-diphenylvinyloxy)-4,6-dimethylpyrimidine.

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Received on 19.06.2022 Modified on 28.08.2022

Research J. Pharm. and Tech 2023; 16(9):4151-4154.

DOI: 10.52711/0974-360X.2023.00680