Isolation and characterization of novel degradation products of valsartan by NMR and high resolution mass spectroscopy: Development and validation of valsartan by UPLC

Article abstract of DOI:10.14233/ajchem.2020.22526

Valsartan is used to treat high blood pressure, heart failure and diabetic kidney disease. It was subjected to forced degradation under acidic, basic and peroxide mediated oxidation. The forced degradation was performed according to ICH guidelines Q1A(R2)?Stability Testing of New Drug Substances and Products. The drug was inert under peroxide mediated hydrolysis, two degradants (DP-Val-1, DP-Val-2) were formed in base hydrolysis and one degradant (DP-Val-3) was formed in acid hydrolysis. These degradants were initially identified through LC-MS and isolated by mass mediated purification system. DP-Val-1 and DP-Val-3 are already reported but their structures were not confirmed by 2D NMR studies. DP-Val-2 is novel degradant and its structure has been elucidated by substantial analysis of high resolution mass spectrometry and 1D, 2D NMR spectroscopy. DP-Val-1 consists of a mixture of rotamers and this hypothesis was confirmed by variable temperature NMR (VT-NMR). A stability indicating RP-UPLC method was developed and validated for assay determination valsartan API drug. A valsartan UPLC method was validated on Acquity BEH C-18 2.1 × 100 mm, 1.7 μm column with shorter runtime of 6 min and the method was validated as per regulatory guidelines in terms of specificity, accuracy, linearity, precision, limit of detection and limit of quantitation.

Full text of DOI:10.14233/ajchem.2020.22526

Asian Journal of Chemistry; Vol. 32, No. 5 (2020), 1064-1068
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22526
Isolation and Characterization of Novel Degradation Products of Valsartan by NMR and
High Resolution Mass Spectroscopy: Development and Validation of Valsartan by UPLC
1,2
2
1
1
KASANI KUMARASWAMY , HIMA BINDU GANDHAM , RUDRAKSHULA J.D. PRASAD , B. AMARENDER REDDY ,
1
1,*
MURALIDHARAN KALIYAPERUMAL and CHIDANANDA SWAMY RUMALLA
1Department of Medicinal Chemistry, GVK Biosciences Pvt. Ltd., IDA Mallapur, Hyderabad-500076, India
2Department of Engineering Chemistry, Andhra University, Visakhapatnam-530003, India
*Corresponding author: E-mail: chidanand_swamy@yahoo.co.in
Received: 8 November 2019;
Accepted: 24 December 2019;
Published online: 29 April 2020;
AJC-19835
Valsartan is used to treat high blood pressure, heart failure and diabetic kidney disease. It was subjected to forced degradation under
acidic, basic and peroxide mediated oxidation. The forced degradation was performed according to ICH guidelines Q1A(R2)−Stability
Testing of New Drug Substances and Products. The drug was inert under peroxide mediated hydrolysis, two degradants (DP-Val-1, DP-
Val-2) were formed in base hydrolysis and one degradant (DP-Val-3) was formed in acid hydrolysis. These degradants were initially
identified through LC-MS and isolated by mass mediated purification system. DP-Val-1 and DP-Val-3 are already reported but their
structures were not confirmed by 2D NMR studies. DP-Val-2 is novel degradant and its structure has been elucidated by substantial
analysis of high resolution mass spectrometry and 1D, 2D NMR spectroscopy. DP-Val-1 consists of a mixture of rotamers and this
hypothesis was confirmed by variable temperature NMR (VT-NMR).A stability indicating RP-UPLC method was developed and validated
for assay determination valsartanAPI drug. A valsartan UPLC method was validated onAcquity BEH C-18 2.1 × 100 mm, 1.7 µm column
with shorter runtime of 6 min and the method was validated as per regulatory guidelines in terms of specificity, accuracy, linearity,
precision, limit of detection and limit of quantitation.
Keywords: Valsartan, Degradation products, UPLC method.
ional barrier is the activation energy required to covert one form
of rotamer to another form, intramolecular hydrogen bonding
and steric repulsion between two adjacent atoms influence the
stability of rotamers [5,6]. Variable temperature (VT) NMR is
preferred method to confirm the rotamers, where in NMR spectrum
multiple signals were observed at room temperatures while at
higher temperatures, spectrum simplifies as the equivalent peaks
are averaged out [7].
There are several reports are available on valsartan stress
degradation products characterisation on the basis of MS, MS/
MS, 1D NMR and its validation [8-10]. However degradation
product structures have not been confirmed on the basis of 2D-
NMR and VT-NMR studies. In the present study, two known
and one novel degradant viz. DP-Val-1; N-pentanoyl-N-(tetra-
zolo[1,5-f]phenanthridin-6-ylmethyl)valine, DP-Val-2; 2′-(2H-
tetrazol-5-yl)-[1,1′-biphenyl]-4-carboxylic acid and DP-Val-
3 ((2′-(2H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl)methyl)valine
INTRODUCTION
Valsartan is a non-peptide compound and contains a mono-
carboxylic acid, amide functional group and consisting of L-valine
in which amino hydrogens have been replaced by a pentanoyl
and a [2′-(1H-tetrazol-5-yl)biphenyl]-4-yl]methyl group and
chemically described as N-(1-oxopentyl)-N-[[2′-(1H-tetrazol-5-
yl) [1,1′-biphenyl]-4- yl]methyl]-L-valine. It is an angiotensin II
receptor blocker, a medication used to manage hypertension, heart
failure and decrease the incidence of developing adult-one set
diabetes [1,2]. Forced degradation or stress degradation is the
process to determine the stability of drug, formation of degradant
components, preliminary kinetics and generally 5-20 % of the
degradation of drug substance have been accepted for analytical
method validation [3,4].
Rotational isomerism is a form of conformational isomerism,
it can be interconverted by rotation about single bond. The rotat-
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Vol. 32, No. 5 (2020)
Isolation and Characterization of Novel Degradation Products of Valsartan by NMR and HRM Spectroscopy 1065
were identified and their structures have been confirmed by NMR
and HRMS.
used for peroxide mediated oxidation. The major degradants
were identified in acid and base hydrolysis.
Ultra performance liquid chromatography (UPLC) is a alter-
nate technology for the HPLC analysis and has been adopted in
laboratories around the world. The main advantage of UPLC
system to eliminate a significant time and cost, speed, resolution
and sensitivity. UPLC flow rate range 0.01-2 mL/min, back
pressure up to 18000 psi and the detector high sensitive than
HPLC. In the present work, UPLC analysis has been applied for
the method validation, assay determination of valsartan bulk
drug and reduced analysis time with good efficiency.
RESULTS AND DISCUSSION
The degradants were identified after 5 h of reflux conden-
sation, however, percentage formation was very less, thus refluxed
was continued to 24 h. The percentage of conversion was moni-
tored through LC-MS, where 1 mL of drug reaction mass was
diluted with mobile phase and injected 2 µL in the LC-MS
system. Two major degradants identified in base hydrolysis
and one degradant identified from acid hydrolysis (Fig. 1). The
identified degradants were taken for isolation.
EXPERIMENTAL
1.92
Valsartan was a gifted sample from an API unit situated in
Hyderabad, India. Chemicals and solvents used were of HPLC
grade, acetonitrile (Merck), methanol (Thermo-Fisher Scientific),
formic acid(Merck), trifluoroacetic acid (Merck), ammonium
bicarbonate (Sigma-Aldrich), DMSO-d6 (Cambridge isotope
Limited).
2.50
Peroxide mediated hydrolysis
2.25
2.00
1.75
1.50
1.25
Valsartan
1.00
High resolution mass spectrometry (HRMS): Accurate
mass was measured with Q-TOF micro mass instrument equipped
with micro-chanel plate detector and multimode ionization source
(ES+APCI). The optimum conditions were desolvation gas flow
700 L/h, capillary voltage 3500V, cone voltage 30V, MCP voltage
2700V, positive ionisation mode. Leucine Enkephalin (556.2771
Da) was used to calculate Lteff and the elemental compositions
were calculated with Masslynx(4.1) software.
7.5×10–1
5.0×10–1
2.5×10–1
0
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50
1.92
2.2
2.0
1.8
Valsartan
Base mediated hydrolysis
1.6
H-Class UPLC instrument: H-Class UPLC equipped
with quaternary solvent manager and 2996 PDA detector was
used for method validation. Method conditions were column:
Acquity UPLC BEH C−18 2.1 × 100 mm 1.7 µm, mobile phase:
(A) 0.05 % triflouroacetic acid; B: 0.05 % triflouroacetic acid
in acetonitrile with gradient time (%) of B 0/40, 3/98, 5/98, 6/
40; flow rate: 0.3 mL/min; column temp.: 30 ºC.
1.4
1.2
DP-Val-2
1.43
1.0
8.0×10–1
6.0×10–1
4.0×10–1
2.0×10–1
0
DP-Val-1
2.06
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50
1.92
LC-MS instrument:WatersSQD-2 massinstrumentcoupled
with UPLC, Column:Acquity BEH C-18 2.1 × 50 mm 1.7 µm,
mobile phaseA: 0.075 % formic acid; mobile phase b: 0.075 %
formic acid in acetonitrile, gradient time (%) of A 0/95, 2.5/2,
3.5/2, 4/95; column temp. 35 ºC, flow rate: 0.5 mL/min; cone
volatage: 30 V, capillary voltage 2800 V; Nebulization gas 750
L/h.
Mass mediated purification system: Preparative HPLC
coupled with 2545 binary gradient module, 2767 sample manager,
515 make up pump and SQ Detector-2. Column: Inertsil ODS-
3, 5 µm 20 × 250 mm, mobile phase A: 0.01 M ammonium
bicarbonate, mobile phase B: acetonitrile; gradient time (%) of
B 0/30, 11/90, 11.5/98, 13/98, 15/30; flow rate 19 mL/min,
detection at 248 nm.
NMR analysis: Valsartan degradation impurities were
recorded on Bruker 500 MHz resolution instrument with top
spin software, DMSO-d6 solvent was used for all the experiments
and TMS used as internal standard.
Stress methods: The stress conditions viz. acid, base
hydrolysis and oxidation were carried out as per ICH guideline,
A 0.5 N HCl was used for acid hydrolysis and refluxed for 5 h
and the formation of degradant percentage was very low and
the reflection is extended to 24 h. A 1N NaOH was used for base
catalyzed hydrolysis and refluxed for 24 h and 30 % H2O2 was
1.6
1.4
Acid mediated hydrolysis
1.2
1.0
8.0×10–1
6.0×10–1
4.0×10–1
2.0×10–1
0
Valsartan
DP-Val-3
1.27
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50
Time
Fig. 1. Degradation chromatogram of valsartan
Isolation of acid and base degradation products: The
degradation products were isolated through mass mediated
preparative HPLC. The degradants were separated through prep-
arative column, identified with mass and the resulting fractions
were collected separately and lyophilized. Identified degradant
products were assigned as DP-Val-1, DP-Val-2 and DP-Val-3
(Fig. 2).
Structure elucidation of DP-Val-1: The mass spectrum
of DP-Val-1 shows protonated molecular ion peak at 434.2195
[M+H]+ and protonated molecular formula C24H28N5O3 was
confirmed by HRMS spectrum (Fig. 3).

1066 Kumaraswamy et al.
Asian J. Chem.
434.2195
100
435.2260
464.5866
486.6195
172.2909
217.2927
588.3235
298.2599
406.4803
572.6629
350.5700
285.7674
634.9454
m/z
0
180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640
Fig. 3. HRMS spectrum of DP-Val-1
7
6
1
3
HO
5
O
30
31
HN
30
5
31
N
N
N
2
15
21
18
15
21
18
4
32
N
1
4
6
N
N
29
28
29
HO
N
8
8
2
16
16
N
N
28
20
19
9
9
32
20
O
3
17
22
17
O
10
22
11
26
11
26
25
7
O
10
19
23
27
27
24
12
25
12
23
13
13
24
14
14
Valsartan
m.f.: C24H29N5O3
Exact mass: 435.2270
DP-Val-1
m.f.: C24H27N5O3
Exact mass: 433.2114
20
O
16
HN
15
N
6
5
24
HN
23
4
N
6
3
N
N
14
8
14
11
1
1
3
18
N
N
22
21
O
HO
9
19
13
N
2
H
7
4
5
12
17
12
25
13
2
OH
26
7
11
10
19
8
6
4
2
ppm
8
10
16
20 18
17
15
Fig. 4. COSY spectrum of DP-Val-1
9
DP -Val- 2
m.f.: C14H10N4O2
Exact mass: 266.0804
DP -Val-3
m.f.: C19H21N5O2
Exact mass: 351.1695
happened due to bond formation between C-20 (benzene ring)
and N-32 (tetrazole ring). HSQC analysis provided the infor-
mation that it had 7 aromatic methines, 2 aliphatic methines,
4 aliphatic methylene and 3 methyl groups.
Fig. 2. Proposed structures for valsartan degradation products
1H NMR spectrum of DP-Val-1 at room temperature reve-
Structure elucidation of DP-Val-2: A protonated mole-
cular ion peak of DP-Val-2 was observed at 267.0892 [M+H]+
and protonated molecular formula C14H11N4O2 with below 5
ppm error was confirmed by HRMS spectrum (Fig. 5).
1H NMR spectrum revealed that DP-Val-2 had 8 aromatic
protons, out of 4 protons having 1,4-disubstituted benzene
pattern and remaining 4 protons having 1,2-disubstituted benzene
pattern. In COSY experiment, H-2,6 (7.77 ppm) correlated
with H-3,5 (7.18 ppm), H-11 (7.63 ppm) correlated with H-10
(7.41 ppm), H-10 (7.41 ppm) correlated with H-9 (7.41 ppm)
and H-11 (7.63 ppm), H-9 (7.41 ppm) correlated with H-10
(7.41 ppm) and H-8 (7.35ppm) as shown in Fig. 6.
In HMBC experiment, H-2,6 (7.77 ppm) correlated with
18th position carbonyl carbon at 167.6 ppm, C-4 (146.4ppm)
and C-2,6 (128.5 ppm) (Fig. 7). This main key proton versus
carbon correlation in HMBC and proton versus proton corre-
lations in COSY supports the structure of DP-Val-2 as shown
in Fig. 2.
aled a duplication of signals, which suggested the presence of
two rotamers in solution, similarly variable temperature (VT)
1H NMR studies also supports this hypothesis. One set of 1H
NMR signals were observed at 100 ºC indicates that DP-Val-1
possesses rotamers and this was due to N-pentanoyl linkage
of DP-Val-1 and also exihibited 1,2,4-trisubstitution benzene
pattern protons instead of 1,4-disubstituted benzene protons
of valsartan drug. DP-1 had total seven aromatic protons and
nineteen aliphatic protons while 13C NMR data revealed that
DP-1 had 15 aromatic carbons and 9 aliphatic carbons. 13C
NMR data also showed two set of signals at room temperature
due to rotamers. In COSY experiment, H-21(8.37, 8.53 ppm)
correlated with H-17(7.64, 7.74 ppm). H-17(7.64, 7.74 ppm)
correlated with H-21(8.37, 8.53 ppm) and H-18(8.68, 8.81
ppm) (Fig. 4).
These correlations in COSY and H-21 (8.37, 8.53 ppm),
H-17 (7.64, 7.74 ppm, δ, 8 Hz), 18(8.68, 8.81 ppm, δ, 8 Hz)
protons chemical shift values, multiplicity and coupling constant
values supported that DP-Val-1 had 1,2,4-trisubstituted benzene
pattern. It is because of cyclic six membered ring formation
Structure elucidation of DP-Val-3: The mass spectrum
of DP-Val-3 shows protonated molecular ion peak at 352.1774
267.0892
100
268.0982
260.3504
283.3553
167.2147
244.1138
350.4620 369.3628
339.3016
332.4613
308.3763
176.2511
214.4846
200.0480
m/z
0
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380
Fig. 5. HRMS spectrum of DP-Val-2

Vol. 32, No. 5 (2020)
Isolation and Characterization of Novel Degradation Products of Valsartan by NMR and HRM Spectroscopy 1067
ppm
8
6
4
2
ppm
8
6
4
2
Fig. 9. HMBC spectrum of DP-Val-3
Fig. 6. COSY spectrum of DP-Val-2
accuracy of the method. Accuracy was proved by spiking 10
% of standard solution to 50, 100 and 150 % of the sample.
Valsartan standard solution (0.3 mg/mL) was injected in
the UPLC system for system suitability test, the retention time
of the valsartan was 2.73 min, while USP tailing and plate
count values were 1.12 and 50437, respectively. Intra-day and
inter-day precession methods was checked with six repeated
concentration preparations and the results are shown in Table-1.
TABLE-1
VALIDATION PARAMETERS OF VALSARTAN
Intraday method precision (n = 6, % of RSD)
Interday method precision (n = 6, % of RSD)
LOD-LOQ
Limit of detection (mg/mL)
Limit of quantification (mg/mL)
Precision at LOQ (% R.S.D.)
Linearity
Calibration range (mg/mL)
Calibration points
Correlation coefficient
0.3
0.2
ppm
8
6
4
2
0.0009
0.003
0.7
Fig. 7. HMBC spectrum of DP-Val-2
[M+H]+ and protonated molecular formula C19H22N5O2 was
confirmed by HRMS spectrum with below 1 ppm error (Fig. 8).
1H NMR analysis shows that DP-Val-3 had 8 aromatic
protons and 10 aliphatic protons. 13C NMR spectra also revealed
14 aromatic carbons and 5 aliphatic carbons. In HSQC experi-
ment, 8 aromatic methine protons, 2 aliphatic methine protons,
1 methylene group and 2 methyl groups in DP-Val-3 were
observed, while in HMBC experiment, H-8 (3.80, 3.95 ppm)
correlated with C-3 (66.3 ppm), C-9 (133.6 ppm) and C-10,14
(128.5ppm) (Fig. 9). These main key protons versus carbon
correlation in HMBC supports the structure of DP-Val-3 as
shown in Fig. 2.
Method development and validation: UPLC method with
6 min run time method was developed and validates as per
regulatory guidelines in terms of precession (intra & inter-day),
limit of detection and quantitation. Linearity was performed
with 25, 50, 75, 100, 125 and 150 % of the sample, accuracy
and the recovery experiments were conducted to determine the
0.075-0.450
6
0.999
The detection limit and quantitation were 0.0009 mg/mL
(S/N 4.84) and 0.003 mg/mL (S/N 16.64), respectively and
the valsartan linearity was demonstrated with the concentration
ranging 0.075-0.450 mg/mL, while the correlation coefficient
was greater than 0.999. The accuracy and recovery of assay of
valsartan was 99.24 % and the results are shown in Table-2.
TABLE-2
ASSAY RECOVERY OF VALSARTAN
Amount added
(µg/mL)
Amount recovered
(µg/mL)
Level (%)
Recovery (%)
50
100
150
160.08
200.10
300.05
158.14
200.41
296.45
98.78
100.15
98.80
352.1774
100
353.1894
207.1043
130.5292
703.7441
235.2574
306.3963
0
m/z
125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700
Fig. 8. HRMS spectrum of DP-Val-3

1068 Kumaraswamy et al.
Asian J. Chem.
Method robustness was checked by changing the organic
solvent composition ( 0.2 mL/min), pH ( 0.2), column temp.
( 5 ºC) and different systems. No illustrious changes were
observed and the stability of valsartan drug solution was checked
at precise temperature (2-8 ºC) for the period of 40 days,
mobile phase stability was checked after 2, 4 and 7 days with
valsartan drug solution and no significant changes were observed.
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CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
https://doi.org/10.1016/j.jpba.2010.05.022