(2024) 18:122
Hashmi and Alegete ﻿BMC Chemistry
https://doi.org/10.1186/s13065-024-01214-2

BMC Chemistry

RESEARCH

Open Access

QbD green analytical procedure
for the quantification of tolvaptan by utilizing
stability indicating UHPLC method
Shadab Anwar Hashmi2 and Pallavi Alegete1,2*Abstract
For the first time a new QbD-assisted green stability indicating ultra-high-performance liquid chromatography(UHPLC) method was developed and validated for quantifying Tolvaptan. The method is simple, quick, cost-effective,
and stable, and it was used to formulate a quality target product profile (QTPP) with strategically defined criticalanalytical attributes (CAAs) to meet specific criteria. Chromatographic separation was undertaken using a 10 cm longcolumn of ACE excel super C18 with an interior diameter of 2.1 mm and particle size of 1.7 µm. The analysis was performed under controlled conditions at 25 ℃ with the mobile phase flowing at a rate of 0.2 mL/min and detection
occurring at 220 nm. Injected 3 µL of standard by using an isocratic mobile phase system consisting of acetonitrile
and water in a 95:5 v/v ratio. The diluents, prepared by mixing acetonitrile with water at a 90:10 volumetric ratio,
were utilized. The analyte’s retention time was determined to be 1.63 min. The developed method provided reliable
results with accuracy exceeding 99% and a correlation coefficient exceeding 0.999 ranged between 10 and 150 µg/
mL across the range for LOQ—150% levels. Notably, during forced degradation testing, Tolvaptan exhibited susceptibility to acidic hydrolysis. The method effectively separated degradation products during stress testing, demonstrating its stability-indicating status. Environmental sustainability assessment of the developed method was conducted
through the investigation of various indicators of Complex GAPI, Analytical Eco scale and Analytical GREEness and it
was concluded the optimized method aligns with environmentally friendly practices.
Keywords Tolvaptan, Quality by Design, Design Expert, Green Analytical Tools
Introduction
Tolvaptan is a non-peptide antagonist that selectively
inhibits the arginine vasopressin 2 receptor. It was synthesized by Otsuka Pharmaceutical Co., Limited, basedin Tokyo, Japan [1] Tolvaptan received approval in Japanfor the management of excessive fluid accumulation*Correspondence:
Pallavi Alegete
pallavi@iict.res.in
1
Department of Analytical and Structural Chemistry, CSIR-Indian Instituteof Chemical Technology, Hyderabad, Telengana 500007, India
2
Academy of Scientific and Innovative Research (AcSIR),
Ghaziabad 201002, Uttar Pradesh, India

in patients with cirrhosis and heart failure when usual
diuretics like loop diuretics, and thiazides become ineffective [2]. In 2009, Tolvaptan received approval fromthe Food and Drug Administration (FDA) for the management of clinically significant euvolemic and hypervolemic hyponatremia, particularly in cases where serum
sodium levels are below 125 mEq/L. Tolvaptan is prescribed for hyponatremia associated with cirrhosis,
congestive heart failure, and the inappropriate antidiuretic hormone syndrome (SIADH). Additionally, it has
been authorized for the treatment of autosomal dominant polycystic kidney disease [3] and hepatic edema[4]. Traditional approaches to managing these patients
often result in worsening hyponatremia and other© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

electrolyte imbalances by promoting the loss of bothwater and electrolytes. However, the use of a specificvasopressin V2 receptor antagonist has the potential tocounteract the effects of elevated AVP levels, leading toa reduction in water retention without disrupting thebalance of electrolytes [5].
Tolvaptan is a racemic mixture of (±)-4’-[(7 chloro2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)
carbonyl]-o-tolu-m-toluidide, and is a crystalline powderthat appears milky, with a molecular weight of 448.94 g/
mol. Its empirical formula is ­C26H25ClN2O3. Tolvaptan isclassified as a BCS Class IV medication due to its poor
solubility (~ 0.1 mg/250 mL) [6]. It demonstrates solubility in ethanol and methanol, as well as high solubility
in benzyl alcohol, while remaining insoluble in water.
Tolvaptan undergoes primary metabolism in the liver
through the action of CYP3A4 and is later excreted in thefeces [7]. The structure of the Tolvaptan is illustrated inFig. 1.
In light of the growing imperative for greener analytical techniques, it is crucial to consider and justify the
key aspects when proposing or selecting an analyticalmethod for determining a specific analyte [8]. Green analytical chemistry is gaining prominence and its adoptionof environmentally friendly substances in diverse analytical approaches, especially in liquid chromatography, has
become increasingly essential. Green analytical chemistry does not revolve solely around the surveillance ofenvironmental pollutants,instead, it focuses on the transformation and environmentally friendly improvement ofmethodologies [9].
The evaluation of the environmental repercussions ofchemical processes poses a significant challenge within
the realm of green chemistry. Standardized measurement procedures empower us to compare the environmental impact of established solutions against newly
developed alternatives, making it easier to recognize
and opt for the most environmentally friendly choices.
Several green metric tools, such as Green AnalyticalFig. 1 Structure of Tolvaptan

Page 2 of 19

Procedure Index (GAPI), Complex GAPI, Analytical Eco-Scale (AES), National Environmental Methods
Index (NEMI), Analytical GREEnness (AGREE), Analytical Method GREEnness Score (AMGS), AGREEprep,
Analytical Method Volume Intensity (AMVI), Modified
NEMI, HPLC-EAT (Environmental Assessment Tool),and other metric tools [10] have been created and areused to evaluate the environmental impact of analyticalprocedures.
The analysis of Tolvaptan using various HPLC techniques have been widely reported in literature that has
been summarized in below Table 1. The reported methods were not suitable for high-throughput and cost-effective quality control analysis due to their lengthy runtime,
expensive organic modifiers, and high flow rate. However,there is a lack of research on environmentally friendly
HPLC methods that demonstrate stability with the QbD
approach for the estimation of Tolvaptan, as guided by
ICH guidelines. To the best of our knowledge, this is thefirst ever QbD assisted green analytical procedure for thequantification of Tolvaptan by utilizing stability indicating UHPLC method.
A green analytical method was initiated on the AES,
AGREE, AGREE prep and Complex GAPI tool for greenness assessment of the analytical procedure [11]. The
proposed method is eco-friendly and adheres to thegreen analytical chemistry (GAC) principle. It is necessary to prioritize the development and implementationof such environmentally friendly techniques to minimize the adverse impact of chemical processes on theenvironment.

Quality by design
The Quality by Design (QbD) [11] encourages proactive methodology to analytical quality, ensuring it’sbuilt into the process from the beginning, not justchecked at the end. This proactive approach ensuresthat quality is ensured into the very design of the analytical procedure, leading to more reliable and consistent outcomes ICH Topic Q8 [12] Pharmaceutical
Quality by Design (QbD) has undergone advancementswith the introduction of ICH Q10 (Pharmaceutical
Quality System), ICH Q9 (Quality Risk Management),and ICH Q8 (R2) (Pharmaceutical Development)
throughout the years [13–15]. One notable benefit of
employing the QbD technique lies in its adaptability
to conduct a qualification aligned with the distinct
Analytical Target Profile established for the method’s
intended utilization [16].
The QbD methodology offers a comprehensive framework for process optimization, enabling the identification of product features and the origins of inconsistencies[17]. It begins with setting the objective and then defining

Column: symmetry C18 (150 4.6 mm; 5 μm)
Mobile phase: Acetonitrile: methanol: buffer(680 mg potassium dihydrogen phosphatein 500 ml water, pH-3adjusted with orthophosphoric acid) in the ratio of 40:10:50 v/v
flow rate: 1.5 mL/min. Temp: room temp
Wavelength: 260 nm

Method Development and Validation of RPHPLC Method for the Estimation of Tolvaptan in Bulk and its Tablet Dosage Form

Development and Validation of RP-HPLC
Method For Estimation of Tolvaptan in
Bulk and its Pharmaceutical Formulation

Method Development and Validationof Tolvaptan in its API and Formulationby using PDA Detector-RP-HPLC

Analytical Method Development and Validation of Tolvaptan and its Related Substancesin Drug Product by RP-HPLC

An Eco-friendly RP-HPLC and UV-Method
Development and Validation for an Estimation of Tolvaptan in Bulk and Tablet Dosage
form Followed by Forced Degradation Studies

A New Approach for Analytical Method
Development and Validation for Quantification of Tolvaptan Using RP-HPLC in Bulk
and in its Tablet

Enantioselective Analysis of Tolvaptan in
Rat and Dog Sera by High Performance
Liquid Chromatography and Application to
Pharmacokinetic StudyA New Stability Indicating RP-HPLC Method
Development and Validation for the
Estimation of Tolvaptan with Forced Degradation Studies in Bulk and Tablet

1

2

3

4

5

6

7

8

4.536 min5.224 min7.419 min

3.4 min

4.7 minColumn: ODS 250 × 4.6 mm, 5 µm
Mobile phase: 60:40 (v/v) mixture of acetonitrile and 0.1% orthophosphoric acid buffer
Flow rate: 1.0 mL/min Temp: 30 °C
Wavelength: 270 nm

2.59 minColumn: CHIRALCEL OD-R (250 mm × 4.6 mm, 8.8 min
5 µm)
Mobile phase: Acetonitrile–water–acetic acid(55:45:1, v/v/v)
Flow rate: 1 mL/min Temp: 40 °C
Wavelength: 265 nm

Column: WatersC18 100 mm × 4.6 mm, 5 µm
Mobile phase: Methanol: Water (Upgrade)adjusted to pH-3 with GAA (60:40)
Flow rate: 0.7 mL/min Temp: ambient temp
Wavelength: 267 nm

Column: Sunsil C18 column(150 mm × 4.6 mm; 5 µm)
Mobile phase: Acetonitrile: Water [45:55]
Flow rate: 1 mL/min Temp: ambient temp
Wavelength: 266 nm

Chandmalin and Rao [29]Anusha and Kalaichelvi [28]Chakravarthy and Shankar [27]Boggula et al. [26]References

Both method development and validation
force degradation study were done in bulk
and tablets
Orthophosphoric acid buffer was usedin the mobile phase

Pharmacokinetic Study on rat and Dog
and sera was performed
Acetic acid was used in the mobile phase

Degradation studies were not conducted
Glacial Acetic acid was used for pH adjustment
Run time was more and the method
was not driven by QbD

Ganipisetty et al. [33]Furukawa et al. [32]Bhavyasri et al. [31]HPLC method with validation and stress stud- Patel et al. [30]ies were performed and reported
Run time is more and the method
is not driven by QbD

Tolvaptan and its Related impurities weredone in drug product and performed
method validation
Phosporic acid buffer

Conventional method with no forced degradation data
Phosphate related buffer

Development and validation were done
through HPLC and there was no forced degradation data performed
Forced degradation studies were not done

Method validation was performed but force
degradation study was not done
Phosporic acid buffer

Retention time Observation

Column: Inertsil ODS-3V (250 × 4.6 mm; 5 μm) 13.96 min
Mobile phase: Mobile phase: A: 0.1% ­H3PO4
Solution in 1000 mL H
­ 20
Mobile phase: B: Methanol: ACN (50:50)
Flow rate: 1 mL/min Temp: 35 °C
Wavelength: 254 nm

Column: Nucleosil-C18 (250 × 4.6 mm; 5 µm)
Mobile phase: Buffer of Sodium dihydrogen
phosphate: Acetonitrile (45:55%V/V) or 10:90
v/v
Flow rate: 1 mL/min Temp: ambient temp
Wavelength: 264 nm

Column: AMCHEMTEQ-USA-ACI C18(150 mm × 4.6 mm; 5 µm)
Mobile phase: Water: Acetonitrile (40:60)
Flow rate: 1 mL/min Temp: 25 °C
Wavelength: 254 nm

Chromatographic conditions

S. No. Titles

Table 1 Comparison of reported analytical methods for the estimation of Tolvaptan

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122
Page 3 of 19

Column: ODS-3v column, 150 × 4.6mm,
5.0 mmMobile phase: Orthophosphoric acidand acetonitrile as solvent in the ratioof 40:60 (v/v)
Flow rate: 1 mL/min Temp: 30 °C
Wavelength: 254 nm

Development and Validation of Novel Stability Indicating RP- HPLC Method for Quantification of Tolvaptan in Bulk and Pharmaceutical Dosage Form

Rapid RP-HPLC Method Development
and Validation of Tolvaptan in Bulk and Pharmaceutical Dosage Form for an InternalStandard

Application of Response Surface Methodology in Development and Optimizationof Stability Indicating RP-HPLC Method
for Determination of Tolvaptan in Bulk
and Formulation

A New Stability-Indicating and Validated RPHPLC Method for the Estimation of Tolvaptan
in Bulk and Pharmaceutical Dosage Forms

Quantification of Tolvaptan API by utilizing UHPLC method and stability indicating
approach with validation by optimizing
Design-Expert software

9

10

11

12

13

3.24 min3.68 min2.59 minColumn: ACE Excel super C18 100, 1.7µm × 2.1 mm
Mobile phase: acetonitrile and waterin the ratio of 95:5 V/V
Flow rate: 0.2 mL/min Temp: 25 °C
Wavelength: 220 nm

1.63 min

References

A QbD-assisted green stability-indicating
UHPLC method was optimized and achieved
reduced runtime
Forced degradation studies were performed,
and method optimization was achievedby using the quality design based centralcomposite design approach

Routine traditional method in bulk and pharmaceutical dosage forms
orthophosphoric acid buffer was usedin the mobile phase

Design of experiment was performed
with conventional method in bulk and formulation
Phosphate buffer was used
Run time was more & method not driven
by green analytical approaches

Internal standard was used with a conventional mobile phase and performed method
development and validation
orthophosphoric acid buffer was usedin the mobile phase

Current method

Bonthu et al. [37]Sutar and Magdum [36]Vijaya Sri et al. [35]Conventional method development and vali- Khaleela and Rahaman [34]dation were performed in bulk and pharmaceutical dosage forms
Orthophosphoric acid buffer was usedin the mobile phase

Retention time Observation

Column: C18 (100 mm × 4.6 mm I.D., 5 µm)
3.46 min
Mobile phase: orthophosphoric acid and acetonitrile in the ratio of 35:65 V/V
Flow rate: 1 mL/min Temp: 30 °C
Wavelength: 254 nm

Column: Kromasil C18 (250 mm × 4.6 mm,
5 µm)
Mobile phase: Acetonitrile and phosphatebuffer with pH 5.5 (70:30% V/V)
Flow rate: 1.0 mL/min Temp: 25 °C
Wavelength:

Column: Eclipse C18 column (100 mm × 4.6mm, 3.5 μm particle size)
Mobile phase: Methanol:0.2M phosphatebuffer (70:30v/v)
Flow rate: 1.0 mL/min Temp: 25 °C
Wavelength: 267 nm

Chromatographic conditions

S. No. Titles

Table 1 (continued)

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122
Page 4 of 19

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 5 of 19

Table 2 Different trials for method development for Tolvaptan
S. No. Chromatographic conditions

Observations

Reference figure

Figure 2

Different column
1

Column- Phenomenex Luna C 18 250 × 4.6 mm (10 µm)
M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 1 mL/
min Inj. Volume: 3 µL Time: 10 min Diluents: Acetonitrile
and water as 90:10

Peak shape is adequate but a short Retention Time required

2

Column: Phenomenex Luna C18 250 × 4.60 mm (5 µm)
M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 1 mL/
min Inj. Volume: 10 µL Time: 10 min Diluents: Acetonitrile
and water as 90:10

Peak splitting (peak shape was not adequate)

3

Column: Eclipse plus C18 100 × 4.60 mm (3.5 µm)
M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.5 mL/
min Inj. Volume- 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Peak shape was not adequate, tailing factor > 2

4

Column: ACE Excel C18 100 × 2.1 mm (2 µm)
M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow- 0.2
mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Peak shape not adequate, theoretical plate < 2000 main peakstarts with another peak, no clear separation

Different mobile phase with different flow rate
5

M.P: Acetonitrile and Methanol in 95:5
Theoretical plates are too low (lot lesser than 2000)
Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.1 mL/min
Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and wateras 90:10

6

M.P –Acetonitrile and Methanol in 95:5
Column- Ace Excel Super C18 Temp- 25 °C Flow-0.3 mL/
min Inj. Volume- 3 µL Time- 5 min Diluents- Acetonitrile
and water as 90:10

7

M.P: Acetonitrile and Methanol in 95:5
Theoretical plates are too low (less than 2000)
Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.5 mL/min
Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and wateras 90:108

M.P: 0.1%TFA in Acetonitrile and methanol in ratio 95:5
Peak not detected
Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.1 mL/min
Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and wateras 90:10

9

M.P: 0.1%TFA in Acetonitrile and methanol in ratio 95:5
Theoretical plates are too low
Column: Ace Excel Super C18 Temp: 25 °C Flow: 0.3 mL/min
Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile and wateras 90:10

Figure 3

Retention Time is too highFigure 4

Different temperature and flow rate
10

Column: ACE Excel Super C18 100 × 2.1 mm (2 µm)
M.P: Acetonitrile and water (95:5) Temp: 45 °C Flow: 0.5 mL/
min Inj. Volume- 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Theoretical plates and resolution are too low

11

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)
M.P: Acetonitrile and water ( 95:5) Temp: 45 °C Flow: 0.3 mL/
min Inj. Volume: 3 µL Time- 5 min Diluents- Acetonitrile
and water as 90:10

Theoretical plate and resolution are too low

12

Column- ACE Excel Super C18 100 × 2.1 mm (2µm)
M.P –Acetonitrile and water ( 95:5) Temp: 45 °C Flow: 0.2
mL/min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Theoretical plates and resolution are good

13

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)
M.P: Acetonitrile and water (95:5) Temp: 45 °C Flow: 0.1 mL/
min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Theoretical plates and resolution are good but RT is not adequate

Figure 5

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 6 of 19

Table 2 (continued)
S. No. Chromatographic conditions

Observations

Reference figure

14

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)
M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.5 mL/
min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Theoretical plates and resolution are too low

Figure 6

15

Column: ACE Excel Super C18 100 × 2.1 mm (2 µm)
M.P:Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.3 mL/
min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Theoretical plates are less than 2000 but resolution is good

16

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)
M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.2 mL/
min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Theoretical plates and resolution are good and can be
adopted in the present method

17

Column: ACE Excel Super C18 100 × 2.1 mm (2µm)
M.P: Acetonitrile and water (95:5) Temp: 25 °C Flow: 0.1 mL/
min Inj. Volume: 3 µL Time: 5 min Diluents: Acetonitrile
and water as 90:10

Theoretical plates and resolution are good but RT is not adequate

the Quality Target Product Profile (QTPP), encompassing essential requirements for safety, efficacy, and quality [12]. These prerequisites form the basis for productdesign and must be consistently replicated to achieve
the aimed benefits. Recent developments in combining
green analytical chemistry with the Quality-by-Design
approach were discussed [18, 19].
Employing risk-assessment tools, further investigation
into CPPs and CQAs is carried out through a Design ofExperiment (DOE) study. DOE is a statistical techniquethat allows for the systematic investigation of systems
and processes to comprehend the primary and interactive impacts of diverse CPPs. Additionally, it offers flexibility by forecasting the magnitude of these interactions[20].
The Design of Experiments (DoE) methodology
involves the organized variation in controlled input

Fig. 2 Fishbone diagram

factors to assess their impact on output responses. Thissystematic variation facilitates the identification of crucial input factors, the determination of optimal settings
for these factors resulting in optimized output responses,
and the elucidation of interactions among the input
factors. The choice of an optimal experimental design
should take into account various factors, including welldefined objectives, the number of input factors andinteractions under examination, statistical validity andefficiency inherent in each design. In this paper we used
Design-Expert software for doing the DoE and further
analysis was conducted to assess robust chromatographic
conditions.
To the best of our knowledge for the first time we havedeveloped and validated a QbD assisted stability indicating green UHPLC method that leads to the developmentof more resilient methods, generating consistent, reliable,

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 7 of 19

Table 3 Design summary layout
Std

Run

Factor 1

Factor 2

Factor 3

Response 1

Response 2

Response 3

A:Flow rate

B:Temperature

C:Mobile phasecomposition

Retention time

Tailing Factor

Theoretical plates

mL/min

C

mL

Min

13

1

0.2

25

93

1.61

1.1

2298

18

2

0.2

25

95

1.63

1.2

2210

5

3

0.1

20

97

1.71

1.3

2273

3

4

0.1

30

93

1.71

1.2

2154

14

5

0.2

25

97

1.61

1.2

2269

9

6

0.1

25

95

1.71

1.3

2167

16

7

0.2

25

95

1.62

1.1

2202

15

8

0.2

25

95

1.61

1.2

2260

7

9

0.1

30

97

1.71

1.3

2156

4

10

0.3

30

93

1.51

1.1

2212

19

11

0.2

25

95

1.61

1.2

2243

1

12

0.1

20

93

1.7

1.1

2145

6

13

0.3

20

97

1.5

1.3

2234

11

14

0.2

20

95

1.61

1.2

2164

8

15

0.3

30

97

1.51

1.3

2251

17

16

0.2

25

95

1.63

1.2

2267

12

17

0.2

30

95

1.59

1.1

2121

2

18

0.3

20

93

1.54

1.1

2138

10

19

0.3

25

95

1.58

1.3

2278

20

20

0.2

25

95

1.63

1.2

2277

and high-quality data. Consequently, it reduces the likelihood of regular incidents in routine environments, saving both time and resources by minimizing the need forinvestigations.

Materials and methods

Analytical solutions
Preparation of mobile phase

A combination of acetonitrile and water, stored in two
separate 1-L bottles. The ratio of acetonitrile to waterwas
95:5% of the total flow.

Chemicals and standards

Diluents

Tolvaptan (API) with a purity of 99% was attained as a giftsample from Extrovis, located in Hyderabad, Telangana,India. Consistently throughout the study, we employed
HPLC-grade acetonitrile procured from E. Merck (India)
Ltd. in Mumbai. To achieve HPLC-grade water, we utilized a Milli-Q Plus water purification system sourced
from Millipore in Milford, MA, USA.

Diluents were made by mixing acetonitrile and waterwith a volumetric ratio of 90:10.

Instrumentation

The method development and validation were doneusing an Agilent UHPLC system (Infinity 1220 LC)
coupled with a Diode Array Detector (DAD). Information gathering and subsequent analysis were conductedwith Open LAB software and ACE Excel super C18(100 mm × 2.1 mm, 1.7 μm) column was utilized foranalysis.

Stock solution preparation

About precisely weighing, 10 mg of Tolvaptan API was
added to a 10 mL volumetric flask. The API was then dissolved in 100% acetonitrile to obtain a concentration of1000 µg/mL.
Blank standard

Diluent was taken as blank.
Standard solution preparation.

Standard preparation of 100 µg/mL solution was madethrough taking 1 mL of the stock solution and 9 mL
diluent was added to it. Different concentration of test
solution was made from the stock solution by dilution

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 8 of 19

calculation to get the desired concentration for specificconcentration for method validation parameters.
Operating chromatographic conditions

The isocratic mobile phase comprising Acetonitrile:
Water [95:5 v/v], was passed through the column with aconsistent flowing rate of 0.2 mL/min at room temperature of 25 °C with an injection volume of 3 µL for a totalof 5 min. The stationary phase employed was the ACE
Excel Super C18 column (100 mm × 2.1 mm) and 1.7 µm
size of particles of silicones. The other experimental conditions before the operating chromatographic conditions
were given in Table 2. The UV–visible detector remained
set at a detection wavelength of 220 nm.
Quality by design (QbD) execution

The practice of Quality by Design (QbD) principles inthe development of analytical techniques has gainedwidespread popularity as a well-established practice toachieve enhanced robustness and improved method performance. The implementation of Analytical Quality by
Design (AQbD) methodology enables a comprehensive
understanding of essential variability sources based on
scientific principles and risk assessment. This involves
identifying Critical Method Parameters (CMPs)/Critical
Process Parameters (CPPs) (buffer pH and mobile phaseratio) through risk assessment and factor screening thisCMPs further evaluation affecting the critical analytical attributes (CAAs)/CQA (peak response, peak tailing,
theoretical plates number and retention time) Once CPPs
and CQAs are identified, a risk assessment is performed
to evaluate the potential implications of CPP variations
Table 4 ANOVA fit statistics of proposed method

on CQAs by DoE. Variable parameters are designed asa fishbone diagram in Fig. 2 which depicts all the factorsthat influenced on further research and investigation.
The analysis of optimization data was conducted by statease Design-Expert software. Response surface factor(RSF) was used for experimental design it contains Central Composite Design (CCD), [11, 21]. used three factors
around three centre points for 20 runs. The repetitionof center points were done to ascertain repeatability,
enhance the robustness of the experimental design, andassess pure error. The values were presented in Table 3
A comprehensive statistical analysis incorporating prediction equations, ANOVA, actual vs. predicated plots,
lack-of-fit analysis, 3D plots were employed to thoroughly evaluate each (CQA)/CAA and define the experiment of design.
Validation parameters

The established method underwent validation for linearity, accuracy, precision, specificity, robustness, limit
of detection (LOD) and limit of quantification (LOQ), as
per the “ICH Q2 (R1)”.
Linearity

Linearity in the validation parameter indicates the capability, within a specified range, to produce test resultsthat are directly proportional to the concentration of theanalyte in the sample.
Calibration solutions for the assay method were
equipped at six concentrations between 10 µg/mL and150 µg/mL. HPLC analysis confirmed linearity of themethod, with peak areas plotted against drug concentrations and analyzed using least square regression.
Accuracy

Retention time Tailing factor Theoretical plates
Std. Dev

0.01580.0430

35.97

Mean

1.62

1.20

2215.95

C.V. %

0.9788

3.59

1.62

R2

0.97120.8456

0.7848Adjusted ­R20.94520.70670.5911

Predicted ­R20.7561

0.0946− 0.2803

Adeq. precision

20.19018.40166.8243Lack of fit(p-values)

0.07130.41540.2872

Accuracy can be described as proximity of measured values to the actual or accepted values. This involves quantifying the quantity of the standard recovered involved
conducting triplicate measurements at three distinct
concentrations (50%, 100%, and 150%). The calculation
includes determining the mean percentage recoveries at
all levels, and the % RSD (relative standard deviation) isthen computed.

(See figure on next page.)
Fig. 3 2D-contours (1-2-3) and 3D-response surface(4-5-6) plots showing the influence of CMPs, i.e., flow rate (A), temperature (B) mobile phase
concentration (C) on retention time as the CAA. b 2D-contours (1–2-3) and 3D-response surface (4-5-6) plots showing the influence of CMPs, i.e.,
flow rate (A), temperature (B) mobile phase concentration (C) on tailing factor as the CAA. c 2D-contours(1–2-3) and 3D-response surface(4-5-6)
plots showing the influence of CMPs, i.e., flow rate (A), temperature (B) mobile phase concentration (C) on theoretical plate as the CAA. e
perturbation plot showing deviation from reference point with 1. Retention time 2. Tailing factor 3. Theoretical plateHashmi and Alegete ﻿BMC ChemistryFig. 3 (See legend on previous page.)(2024) 18:122

Page 9 of 19

Hashmi and Alegete ﻿BMC ChemistryFig. 3 continued(2024) 18:122

Page 10 of 19

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 11 of 19

Fig. 4 Different Column trials. A Phenomenex Luna C 18 250 × 4.6 mm (10 µm). B Phenomenex Luna C18 250 × 4.60 mm (5µm). C Eclipse plus C18
100 × 4.60 mm (3.5µm). D ACE Excel C18 100 × 2.1 mm (2µm)

Fig. 5 Different mobile phase and flow rate trials. A Mobile phase ACN: MeOH (95:5) and flow rate 0.5 mL/min. B Mobile phase ACN: MeOH (95:5)
and Flow rate 0.3 mL/min. C Mobile phase ACN: MeOH (95:5) and flow rate 0.1mL/min

Fig. 6 Different mobile phase and flow rate trials. A Mobile phase 0.1%TFA in ACN: MeOH (95:5) and flow rate 0.3 mL/min. B Mobile phase 0.1%TFA
in ACN: MeOH (95:5) and flow rate 0.1 mL/min

Precision

Precision measures the degree of reproducibility of
the analytical results. It indicates how consistently themethod can produce the same results when repeatedunder the same conditions. Precision was determinedat two levels: system precision and method precision.
System precision involved five injections from the same
standard solution. Assessment of method precision

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 12 of 19

Fig. 7 Different flow rate and temperature trials at 45 °C. A Flow rate 0.5 mL/min and Temperature 45 °C. B Flow rate 0.5mL/min and temperature
45 °C. C Flow rate 0.5mL/min and temperature 45 °C. D Flow rate 0.5 mL/min and Temperature 45 °C. E Blank flow rate 0.5 mL/min and temperature
45 °C

Fig. 8 Different flow rate and temperature Trials at 25 °C. A Flow rate 0.5 mL/min and Temperature 25 °C. B Flow rate 0.5 mL/min and Temperature
25 °C. C Flow rate 0.5 mL/min and Temperature 25 °C. D Flow rate 0.5 mL/min and Temperature 25 °C. E Blank flow rate 0.5 mL/min and Temperature
25 °C

involved injecting six samples of the standard solution ata consistent concentration. The resulting peak responses
were utilized to determine the average area, standard
deviation and % RSD for both conditions.
Limit of detection (LOD) and limit of quantification (LOQ)

limited to ± 0.1 mL/min, mobile phase composition wereconsidered as ± 1% v/v, and temperature changes were
controlled with ± 5 °C. Duplicate injections of sample
were executed and calculations were made for the average peak response, standard deviation and % RSD.

The lowest concentration of the analyte that can be accurately detected is known as LOD with a specified degreeof confidence. It indicates the sensitivity of the method.
The lowest amount of analyte that can be precisely andaccurately quantified with a certain level of confidence is
known as the limit of quantification. The LOD and LOQ
were evaluated from the calibration curves by applying
statistical calculations.

Solution stability

Robustness

Stability indicating stress study

Robustness defines the method’s ability to withstandminor variations in conditions, such as column temperature, ratio of mobile phase, and flow rate. Flow rate were

The evaluation of the intrinsic stability of the drug molecule is facilitated through the utilization of stress studies.
Both FDA and ICH guidelines require the incorporation

Solution stability pertains to the ability of the solutions tomaintain their concentration, purity, and analyte integrity over a period of time under defined storage conditions. Standard solution was analyzed of day one and day
seven to check the solution stability using the validated
HPLC method and percentage change in peak area wascalculated for above period of time.

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 13 of 19

Fig. 9 UV spectra of Tolvaptan

Fig. 10 Calibration curveof stress studies to understand and examine alterations in
the characteristics of both a drug substance and a drug
product across different timeframes and under diverse
environmental conditions [22, 23]. The ICH guidelines
mandate the incorporation of stress studies involving
altered conditions, encompassing oxidation, acidic, basepH, exposure to light, dry heat, and hydrolysis.

Oxidation Hydrogen peroxide was used for oxidationof the solution. An electron transfer pathway is involvedin the drug’s oxidative breakdown. For oxidation 10 mg
API was combined with 5 mL of acetonitrile (ACN), followed by the addition of 5 mL of 6% H
­ 2O2 solution, constituting the degradation solution and yielding a stock
solution of 1000 µg/mL. This solution was refluxed for
6 h at 60 °C, Subsequently, a 100 µL aliquot of the solu-

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 14 of 19

Table 5 Validation results of proposed UHPLC method for theestimation of Tolvaptan
Parameters

TLP

Acceptable criteria
0.999

Linearity
Concentration range (µg/mL)

10–150 µg/mL

Slope

49.22

Intercept

75.48

Correlation coefficient (r)0.99916
98–102% ± 2.0%

Accuracy (% recovery)
50% mean ± RSD

99.964 ± 0.397472

100% mean ± RSD

99.775 ± 0.590492

150% mean ± RSD

100.0793 ± 0.822959

Precision

RSD (%)

Method precision

0.4681

System precision

0.0482LoD

0.00606 mg/mL

LoQ

0.0183677 mg/mL

Robustness

RSD (%)

Flow 0.1 (mL/min)

0.0251

Flow 0.3 (mL/min)

0.4941

Mobile Phase_ACN:H2O(94:6)

0.1072

Mobile Phase_ACN:H2O(96:4)

0.0819Temperature 30 °C

0.0461

Temperature 20°C

0.2719

Solution stability (5 days)

0.31

NMT 2.0%

NMT 2.0%

NMT 2.0%

tion was withdrawn and diluted to 1 mL with a dilution
solution. This sample was then injected for HPLC analysis
for to detect degraded products and separate them fromthe original peak.
Acidic hydrolysis Acidic hydrolysis involves catalyzing
the molecule’s ionizable functional groups. Acid degradation, induced by either HCl or H
­ 2SO4 at varying concentrations ranging from 0.1 N to 5 N, is a common method.
In this experiment, 0.2 N HCl was employed as the degradation solution. For acidic hydrolysis, 10 mg of Active
Pharmaceutical Ingredient (API) was combined with 5
mL of acetonitrile (ACN), followed by the addition of 5
mL of 0.2 N HCl, resulting in a stock solution of 1000 µg/
mL. The mixture underwent reflux for 6 hours at 60 °C.
Subsequently, a 100 µL aliquot of the solution was withdrawn and diluted to 1 mL with a dilution solution. This
sample was then injected for High-Performance Liquid
Chromatography (HPLC) analysis to detect degradedproducts and separate them from the original peak.

Basic hydrolysis The basic hydrolysis entails the catalytic
breakdown of ionizable functional groups within the molecule. Alkali degradation induced by NaOH of different
concentration from 0.1 N to 5 N. Here 0.2 N HCl was used
as a degradation solution. Basic hydrolysis was conducted
as follows: 10 mg of API was combined with 5 mL of acetonitrile (ACN), followed by the addition of 5 mL of 0.2 N
NaOH, resulting in a stock solution of 1000 µg/mL. The
solution was then refluxed for 24 hours at 60°C. Afterward, a 100 µL aliquot of the solution was withdrawn anddiluted to 1 mL with a dilution solution. Subsequently, thissample was injected for High-Performance Liquid Chromatography (HPLC) analysis to detect degraded products
and separate them from the original peak.
Thermal degradation Sample of solid drug was exposedto dry heat for accelerated degradation, here 10 mg of API
was kept in hot air oven for 5 days. A 100 µg/mL solution
was prepared using a 1000 µg/mL stock sample and thisresulting solution was utilized for the subsequent HPLC
analysis.
Photolysis Photo stability studies aim to produce primary degradants of the drug substance by subjecting it to
UV or fluorescent conditions. The ICH guidelines specify
conditions for photo stability testing [24] recommending
exposure to light at least 1.2 million lux hours and 200 W
hours per square meter for drug substance and solid/liquid drug product samples. The commonly acknowledged
wavelength range for initiating photolytic degradation is300–800 nm. Photo oxidation, induced by light stress conditions through a free radical mechanism, was also considered [25]. 10 mg of API was exposed to direct sun lightfor 3 days. Subsequently, a 1000 µg/mL stock solution was
prepared from it, and a 100 µg/mL sample was drawn foranalysis.

Results and discussion
Method optimization

The objective of this study was to develop a QbD assisted
quick and environmentally friendly stability-indicating
UHPLC method for the estimation of Tolvaptan. From
the analytical QbD approach, CMPs have cause-andeffect relationship possessing the capacity to impact thechosen CAAs. The most vital CMPs are mobile phasecomposition, Diluents, flow rate, column oven temperature and length of the column; three pivotal CAAs arepeak tailing, retention time, and theoretical plates. The
Design of Experiments (DoE) methodology utilized apinpoint central process and established a design spaceHashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 15 of 19

Fig. 11 a Robustness flow rate. (A) Flow rate 0.2 mL/min (B) Flow rate 0.1 mL/min. (C) Flow rate 0.3 mL/min. b Robustness mobile phase. (A) Mobile
phase ACN: ­H2O (95:5 v/v) (B) Mobile phase ACN: ­H2O (94:6 v/v) (C) Mobile phase ACN: ­H2O (96:4 v/v). c Robustness temperature. (A) Temperature
25 °C (B) Temperature 30 °C. C Temperature 20 °C

Table 6 Summary of force degradation studies
NameTime (h)Assay (%w/w) in
Mass balance
degradation sample (assay + total
impurity)

RemarkPeak purity

Acid Degradation (0.2 N HCL)

6h

98.43

99.99

Two degradation peaks peak 1
and peak2 were observed

Pass
Pass

Base Degradation (0.2N NaOH)

24 h

99.94

99.98

No degradation was observed

Oxidation ­(H2O2 30%)

6h

99.72

100.1

No degradation was observed

Pass

Thermal (105 °C)

5 days

99.53

100.1

One degradation peak 1 was observed

Pass

Photolysis (sunlight)

3 days

99.82

100.3

One degradation peak 1 was observed

PassHashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 16 of 19

Fig. 12 Force degradation study. (A) Blank. (B) Standard. (C) Acid 6 h. (D) Base 24 h. (E) 30% ­H2O2. (F) Thermal 5 days. (G) Photolytic 3 days

rooted in statistical significance. Validation of the method
ensured a reliable confirmation of quality results. In thecurrent study, CCD was created from Design-Expert
software, using three factors such as flow rate, column
temperature and acetonitrile composition in the mobile
phase. A total of 20 experiments of face centered CCD
were created with 8 factorial, 6 axial, and 6 center points[21]. The chosen factors, responses and experimental

values are tabulated in Table 3. Subsequently, the experimental results from this data underwent statistical evaluation using the Design-Expert software. The CAAs ofeach response were analyzed using various statisticaltechniques such as prediction equations, ANOVA, actualvs. predicted plot, lack of fit, contour plot. Understanding the performance of the technique across various
experimental scenarios offered a fundamental grasp ofHashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Page 17 of 19

Fig. 13 Green assessment tools. a Complex Gapi. b AGREE tool. c AGREE prep

Table 7 Analytical eco-scale penalty points
Principle/reagents/chemicals

Penalty points

Water

0

Acetonitrile

12

UHPLC

1

Occupational hazard

3

Sample storage (RT)0

Sonicator

1

Waste

5

Waste management3

Sum of total penalty points

25

Analytical eco-scale75

the method control approach. The involvement of thetest quadratic polynomial models was assessed using
ANOVA, and the corresponding results were showcasedin Table 4.
The adjusted (Adj. ­R2) and predicted (Pred. ­R2) were
utilized to assess the polynomial regression equations. The ­R2 values for the determination coefficients
approached 1, indicating that the data closely adhered
to the regression statistics line with an accuracy exceeding 99%. Extension studies were conducted to study
the 3D response plots with 2D contour. The impact offactors such as the mobile phase composition, temperature and flow rate of the LC system on responses
like retention time, theoretical plates, and tailing factor was unveiled through 3D response surface plots.
Figure 3a–e displays representative graphs of these
responses, demonstrating the intricate interaction
between factors A and B and their interdependence,
while factor C remained constant. The 3D surface plots
illustrated a decrease in retention time with increasing

flow rate, while changes in mobile phase composition and column temperature had negligible effects
on retention time. The tailing factor was significantly
influenced by flow rate and mobile phase composition,with the maximum tailing factor observed at low andhigh flow rates. However, the maximum tailing factor
was observed under center point conditions of column
temperature (25°C) and mobile phase composition(95%). The column theoretical plates were minimallyaffected by flow rate, with maximum values observedat the center point temperature condition (25°C), butvarying at low and high points of organic compositionin the mobile phase and reaching minimum at center
point conditions. An increase in flow rate led to adecrease in theoretical plates, while the opposite trend
was observed for temperature. Additionally, slightvariations in retention time occurred with changes intemperature, but a significant decrease in temperature
resulted in an increase in theoretical plates.
Optimized chromatographic conditions

Different Column trials were reported in Fig. 4, various
mobile phases and flow rate trials were shown in Figs. 5,
6, different flow rate and temperature trials were shownin Figs. 7, 8 and the optimized conditions involved utilizing the ACE Excel Super C18 column (100 mm, 1.7 µm,
2.1 mm) as the stationary phase. A detection wavelength of 220 nm was chosen for the UV–Visible detector because at this wavelength, there was the highest
absorption as shown in Fig. 9. The isocratic mobile phase
comprised acetonitrile: Water [95:5], passed through thecolumn at a consistent flow rate of 0.2 mL/min at 25 °C
with an injection volume of 3 µL for 5 min as total run
time.

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

Method validation.

Linearity: Linearity was confirmed by construction ofcalibration curves. The calibration curve was displayed inFig. 10, R2 for tolvaptan within the concentration range
exceeded 0.99. Linearity results were shown in Table 5.
LOD and LOQ: The calculation determination of LOD
and LOQ from the calibration curve was successfully
achieved, and the results were presented in Table 5.
Accuracy: The recovery percentage was calculated foreach triplicate and % RSD was calculated, yielding values consistently below 2%. The results for accuracy were
given in the Table 5.
Precision: Different type of precision were calculated
at different concentrations and the %RSD was calculated which was consistently falling below the acceptable
threshold of 2% as outlined in the guidelines provided inTable 5.
Robustness: Different conditions of robustness were
evaluated by making small changes from the original
condition, and the resulting %RSD values were presented
in Table 5. Chromatograms illustrating these conditions
were depicted in Fig. 11.
Solution stability: The percentage change in area wascalculated indicating he values within the acceptable
range of not more than 2% and no degradation peak was
detected as shown in Table 5
Force degradation study: The study results of tolvaptan
degradation studies were given in the Table 6. Degradation percentage of tolvaptan was noticed in acidic hydrolysis, and excellent separation was attained between the
degradants, in acidic condition. Figure 12 shows the force
degradation chromatogram.

Method greenness assessment
The proposed methodology underwent a comprehensive
evaluation using AGREE prep and AGREE, two greenness assessment tools. The outcome of the assessments
were analyzed by inputting the procedure followed during the experiments into the software, and the results
were visually displayed in Fig. 13. Additionally, we evaluated the suggested approach using the complex GAPI,
which is a tool that assigns a score to a technique basedon its adherence to certain criteria. The score was represented by a pentagram in a pictogram, with colors of
the pentagrams indicating the degree of adherence tospecific criteria. Predominantly green and yellow pentagrams in the pictogram suggested that the suggested
approach is eco-friendly. Furthermore, we used the EAS
score, a comprehensive assessment tool that considers
six parameters. After applying penalties as per the tool’s
guidelines, our technique scored an impressive 75 points.
Analytical eco scale penalty points were summarized inTable 7 [11]. In summary, the suggested methodology is

Page 18 of 19

both rapid and environmentally friendly, as evidenced
by its high scores on complex GAPI, EAS AGREE and
AGREE prep tools.

Conclusion
A simple and rapid QbD assisted green stability-indicating chromatographic UHPLC method was developed for Tolvaptan. Leveraging AQbD and the Central
Composite Experimental Design lead to a notableenhancement in the performance and robustness of themethod, ensuring successful separation and estimationof Tolvaptan. The number of experimental runs werereduced by adopting Design-Expert software and validation was carried out in accordance with guidelines of
ICH Q2 R (1). The validation of the method covered allparameters, and their adherence to acceptable ranges
confirmed the linearity, precision, robustness, and sensitivity of the proposed method results. In the pursuitof a sustainable approach, the AES, Complex GAPI,AGREE tool and AGREE were employed for the green
assessment, revealing that the presented method’s
greenness profile stands out as exceptionally superior.
This underscores the method’s commitment to environmentally conscious practices, making it a compelling
choice for estimation of Tolvaptan.
Acknowledgements
The authors are thankful to Dr D. Srinivasa Reddy, Director, CSIR-Indian Institute of Chemical Technology, Hyderabad, Telengana, India for his encouragement and support.
Author contributions
Shadab Anwar Hashmi: Formal analysis and Validation; Pallavi Alegete:
Conceptualization, Methodology, Funding acquisition, Supervision, Writing—
Review and Editing.
Funding
Dr. Pallavi Alegete is thankful for CSIR-IICT for providing the start-up research
grant (MLP-0101) to carry out the present research work. The manuscript bears
CSIR IICT acknowledgement number: IICT/Pubs./2023/439.
Data availability
The datasets used and/or analysed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors give consent for the publication of the manuscript in the Journal of
BMC Chemistry.
Competing interests
The authors declare that they have no competing interests.
Received: 6 March 2024 Accepted: 21 May 2024

Hashmi and Alegete ﻿BMC Chemistry(2024) 18:122

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