Received: 2 November 2023 Revised: 13 March 2024 Accepted: 22 April 2024

DOI: 10.1002/sscp.202300207

RESEARCH ARTICLE

Simultaneous estimation of mometasone furoate and

olopatadine hydrochloride by reversed-phase
high-performance liquid chromatography

Bhoomi Patel Ruchi Patel

Shree S. K. Patel College of

Pharmaceutical Education and Research, Abstract
Ganpat University, Mehsana, India The simultaneous determination of Mometasone furoate (MOM) and Olopata-
Correspondences
dine hydrochloride (OLO) in a combined dose has been developed and
Bhoomi Patel, Department of validated using a specific, precise, accurate, fast, reliable, and economical high-
Pharmaceutical chemistry and Quality performance liquid chromatography technique. Methanol:water:acetonitrile
assurance, Shree S. K. Patel College of
Pharmaceutical Education and Research, (20:25:55, v/v/v) was used as the mobile phase during the separation. The proce-
Ganpat University, Ganpat Vidyanagar, dure was performed using a low-pressure gradient elution on the reversed-phase
Mehsana, Gujarat, India.
Phenomenex C18 column. At 238 nm, separated components were densitomet-
Email: bhoomi16692@gmail.com
rically measured. The flow rate was fixed at 1.0 mL/min with a continuous run
up to 10 min, while the retention time was located near about 3.2 and 6.1 min
for OLO and MOM, respectively. The method was validated for Linearity and
range, precision, reproducibility, specificity, accuracy, limit of detection, limit of
quantification, and robustness as per the International Council for Harmoniza-
tion Q2(R1) guideline. MOM and OLO regression coefficients (r2 ) were found
to be 0.9994 and 0.9997, over the range of 2.5–15 and 33.25–199.5 µg/mL, respec-
tively. MOM and OLO percentage recoveries were measured to be 99.49 ± 0.14
and 99.98 ± 0.22, respectively. The method can be successfully applied for rou-
tine analysis of the quantitative determination of MOM and OLO in a combined
dose.

KEYWORDS
analytical method development, high-performance liquid chromatography, mometasone
furoate, olopatadine HCl, validation

1 INTRODUCTION etyl)−11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,11,12,14,15,
16 octahydrocyclopenta[a]phenanthren-17-yl] furan-
Mometasone furoate (MOM) Figure 1A, chemically 2-carboxylate topical glucocorticoid receptor agonist
[(8S,9R,10S,11S,13S,14S,16R,17R)−9-chloro-17-(2-chloroac- with dermatological properties [1]. It is effective for the
treatment of skin diseases like eczema, dermatitis, and
Article Related Abbreviations: ESI-MS/MS, electrospray psoriasis. Hence, MOM nasal spray is commonly used
ionization-tandem mass spectrometry; HPLC, high-performance liquid as an anti-allergic, anti-asthmatic, anti-inflammatory,
chromatography; HPTLC, high-performance thin-layer and dermatologic agent. Mometasone is official in Indian
chromatography; ICH, International Conference on Harmonization;
Pharmacopeia, British Pharmacopeia, United State
LOD, limit of detection; LOQ, limit of quantification; MOM,
Mometasone furoate; OLO, Olopatadine HCl; Rf , retention factor. Pharmacopeia, and European Pharmacopeia. From the

Sep Sci plus 2024;2300207. www.sscp-journal.com © 2024 Wiley-VCH GmbH. 1 of 13

https://doi.org/10.1002/sscp.202300207
2 of 13 PATEL and PATEL

As per the literature review, a specific HPTLC method

Validated for estimation of MOM and OLO methods is
available [51].
A detailed Literature review was done for methods
already submitted on MOM, OLO, and with other com-
binations but it was found no reversed-phase HPLC was
done for simultaneous estimation of MOM and OLO in
combination.
The present manuscript describes a novel, cost-effective,
and quick chromatographic separation method for the
simultaneous estimation of OLO and MOM in a combined
dose. In the present study, a simple, rapid, precise, robust,
and comparatively economical HPLC method was devel-
oped and validated for the simultaneous determination of
MOM and OLO. The described method is validated with
respect to the International Conference on Harmonization
(ICH) guideline Q2 R1 [52].

2 MATERIALS AND METHOD

2.1 Materials

Analytically pure standard OLO was procured as a gift

FIGURE 1 Structure of Mometasone furoate and Olopatadine sample from USV Pharma Pvt. Ltd. having 99.94% w/w of
HCl. purity and MOM was also procured as a gift sample from
Vadish Pharma Pvt. Ltd. Having 99.98% w/w of purity. AR-
grade methanol was procured from SD Fines Chemicals
literature review, it is observed that many analytical Ltd. HPLC-grade methanol, acetonitrile (ACN), and water
methods have been developed involving spectrophotomet- were procured from SD Fines Chemicals Ltd.
ric, high-performance liquid chromatography (HPLC),
high-performance thin-layer chromatography (HPTLC),
and stability-indicating methods for the estimation of 2.2 Instrument and chromatographic
MOM in pharmaceutical formulations. Table 1 shows the condition
literature survey of MOM.
Olopatadine HCl (OLO) Figure 1B, chemically 2- A quaternary low-pressure gradient pump configuration
[(11Z)−11-[3-(dimethylamino)propylidene]−6H-benzo[c]- Shimadzu LC-2030C Plus (Shimadzu) system including an
[1]benzoxepin-2-yl]aceticacid; HCl anti-histaminergic ultraviolet-visible lamp with the dual-wavelength detec-
agent with dual action H1 receptor antagonist and mast tor (SPD-M20A prominence), degasser unit, column oven,
cell stabilizer [27]. Hence, OLO nasal spray is commonly and auto-sampler was used. The system control software,
used as anti-allergic and anti-histaminic. Olopatadine is LC Solution was used for controlling system parameters,
official in Indian Pharmacopeia and United States Phar- data acquisition, and data processing to obtain output
macopeia. From the literature review, it is observed that chromatograms. For the preparation of standard solutions,
many analytical methods have been developed involving chemicals were weighed on Sensitive Sartorius (CP 224S)
spectrophotometric, HPLC, LC, HPTLC, Volttametry, analytical balance (Sartorius AG). A glass vacuum filtra-
stability-indicating HPLC method, and LC-electrospray tion assembly (Millipore) with a 0.22 um membrane filter
ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used to filter the mobile phase solution and an ultra-
and UPLC method for the estimation of OLO in pharma- sonic cleaner (Frontline FS 4) was used for degassing by
ceutical formulations. Table 2 shows the literature survey sonication.
of OLO. Stationary phase (column): Phenomenex C18 column
A combination of MOM and OLO is available in the mar- (250 × 4.6) mm, 5 µm particle size; mobile phase:
ket as a nasal spray is used to treat symptoms (e.g., stuffy or methanol:water:ACN (20:25:55, v/v/v); detection wave-
runny nose, itching, and sneezing) of seasonal (short-term) length: 238 nm; flow rate: 1 mL/min; injection volume:
allergic rhinitis (hay fever). 10 µL; temperature: 40◦ C; run time: 10 min.
PATEL and PATEL 3 of 13

TA B L E 1 Literature survey of Mometasone furoate.

Sr. Ref.
no. Drug Pharmacopeia Method and description no.
1. Mometasone furoate IP 2022 UV spectroscopy [2]
Solvent: Ethanol
Assessment: at 249 nm
2. Mometasone furoate aqueous IP 2022 HPLC [2]
nasal spray S phase: A column with porous silica bonded to
octadecylsilane packing (100 × 4.6 mm, 5 µm)
M phase: Water: Methanol (45:55, by volume)
Flow rate: 1 mL/min
Assessment: at 238 nm
3. Mometasone furoate cream IP 2022 HPLC [2]
S phase: A column with porous silica bonded to
octadecylsilane packing (100 × 4.6 mm, 5 µm)
M phase: Water: Methanol (45:55, by volume)
Flow rate: 1 mL/min
Assessment: at 238 nm
4. Mometasone furoate IP 2022 HPLC [2]
ointment S phase: A column with porous silica bonded to
octadecylsilane packing (100 × 4.6 mm, 5 µm)
M phase: Water:Methanol (45:55, by volume)
Flow rate: 1 mL/min
Assessment: at 238 nm
5. Mometasone furoate and BP 2020 Liquid Chromatography [3]
Mometasone furoate S phase: Column packed with end-capped octadecyl silyl
monohydrate silica gel (250 × 4.6 mm, 5 µm)
M phase: MeCN:Water (50:50, by volume)
Flow rate: 1 mL/min
Assessment: at 254 nm
6. Mometasone furoate USP 2020 Liquid Chromatography [4]
S phase: Column with packing L7 (250 × 4.6 mm, 5 µm)
M phase: Methanol:Water (65: 35, by volume)
Flow rate: 1.7 mL/min
Assessment: at 254 nm
7. Mometasone furoate cream, USP 2020 Liquid Chromatography [4]
Mometasone furoate S phase: Column with packing L60 (250 × 4.6 mm, 5 µm)
ointment, and Mometasone M phase: Gradient- MeCN:Water
furoate topical solution Flow rate: 2 mL/min
Assessment: at 254 nm
8. Mometasone furoate European Phar- Non-aqueous potentiometric titration [5]
macopeia Solvent: Anhydrous acetic acid Titrant: Perchloric acid
Sr. Ref.
No. Drug Method Description No.
1. Mometasone furoate and Absorbance Solvent: Methanol [6]
salicylic acid in topical correction Assessment: at 300 and 250 nm
formulation method
2. Mometasone furoate and RP-HPLC S phase: Thermo reversed phase C18 analytical column [7]
Formoterol fumarate (250 × 4.6 mm, 5 µm)
M phase: Methanol:Water (pH 3.5) (85:15, by volume)
Flow rate: 1 mL/min
Assessment: at 225 nm
3. Mometasone furoate, RP-HPLC S phase: Inertsil C18 column (250 × 4.6 mm, 5 µm) [8]
Hydroquinone M phase: MeCN:Methanol (90:10, by volume)
and tretinoin in topical Flow rate: 0.5 mL/min
formulation Assessment: at 266 nm
(Continues)
4 of 13 PATEL and PATEL

TA B L E 1 (Continued)
4. Mometasone furoate in nasal UV Solvent: Methanol [9]
spray spectrophotometry Assessment: at 220 and 310 nm
5. Mometasone furoate in bulk UV Solvent: Methanol [10]
and formulation spectrophotometry Assessment: at 246 nm
6. Mometasone and Stability S phase: Waters X Terra C18 column (250 × 4.6 mm, 5 µ) [11]
Ketoconazole in indicating M phase: Buffer (Triethyl amine in water, pH adjusted to
formulation RP-HPLC 6.5 with glacial acetic acid):MeCN (40:60, by volume)
Flow rate: 1.5 mL/min
Assessment: at 250 nm
7. Mometasone furoate and HPLC S phase: Reversed phase C18 analytical column (250 × [12]
Formoterol fumarate in 4.6 mm, 5 µm)
rotacaps M phase: Methanol:Water (pH 3.5) (85:15, by volume)
Flow rate: 1 mL/min
Assessment: at 225 nm
8. Terbinafine HCl and Absorbance Solvent: Methanol [13]
Mometasone furoate correction Assessment: at 282 and 248 nm
informulation method
9. Mometasone furoate and HPLC S phase: C18 column (250 × 4.6 mm, 5 µm) [14]
Formoterol fumarate in M phase: Sodium dihydrogen orthophosphate buffer:MeCN
metered dose inhaler (50:50, by volume)
Flow rate: 1 mL/min
Assessment: at 220 nm
10. Mometasone furoate and Stability S phase: Silica gel G60 F254 [15]
Salicylic acid indicating M phase: Hexane:Chloroform:Methanol:MeCN (6:6:1:0.3, by
TLC volume)
densitometry Assessment: at 250 nm
11. Mometasone Intrinsic S phase: Silica gel G60 F254 [16]
furoate in the presence of stability M phase: Toluene:Ethyl acetate:Methanol:Ammonia
Salicylic acid by HPTLC (6.4:1.5:2.0:0.1, by volume)
method Assessment: at 250 nm
12. Formoterol fumarate and Stability S phase: Inertsil C8 column (250 × 4.6 mm, 5 µm) [17]
Mometasone furoate in indicating LC M phase: Ammonium acetate buffer solution (0.05 M, pH
respicaps method 5.0): MeCN (30:70, by volume)
Flow rate: 0.8 mL/min
Assessment: at 247 nm
13. Formoterol fumarate and Stability S phase: Enamal C18 column (250 × 4.6 mm, 5 µm) [18]
Mometasone furoate in indicating the M phase: MeCN:0.05 M orthophosphoric acid:Methanol
combined formulation HPLC (60:30:10, by volume)
method Flow rate: 1 mL/min
Assessment: at 248 nm
14. Terbinafine HCl and HPTLC S phase: Silica gel G60 F254 [19]
Mometasone furoate in M phase: Toluene:Ethyl acetate: Glacial acetic acid (8:4:0.1,
combined by volume)
formulation Assessment: at 248 nm
15. Mometasone furoate and RP-HPLC S phase: Thermo reversed phase C18 analytical column [20]
Formoterol fumarate (250 × 4.6 mm, 5 µm)
M phase: Methanol:Water (pH 3.5) (85:15, by volume)
Flow rate: 1 mL/min
Assessment: at 225 nm
16. Mometasone furoate in HPLC S phase: C18 column (250 × 4.6 mm, 5 µm) [21]
human plasma M phase: Methanol:Water (88:12, by volume)
Flow rate: 1 mL/min
Assessment: at 248 nm
(Continues)
PATEL and PATEL 5 of 13

TA B L E 1 (Continued)
17. Mometasone furoate, HPTLC S phase: Silica gel G60 F254 [22]
Miconazole M phase: Toluene:Ethyl
nitrate, and Nadifloxacin in acetate:Ethanol:Formic acid,
cream (10: 3: 2: 0.5 by volume)
formulation Assessment: at 235 nm
18. Eberconazole nitrate, RP-HPLC S phase: Waters Xterra C18 (150 mm X 4.6 mm, 5 µ) [23]
Mometasone furoate and M phase: Water:Methanol (35:65, by volume)
Methyl paraben Flow rate: 1.5 mL/min
in formulation Assessment: at 235 nm
19. Mometasone furoate and HPTLC S phase: Silica gel G60 F254 [24]
Nadifloxacin in M phase: Dichloroethane:Diethyl ether:Ammonia:
formulation Methanol:Ethyl acetate (6:3:0.2:1.75:3.5, by volume)
Assessment: at 254 nm
20. Fusidic acid and Mometasone RP-HPLC S phase: C18 (150 × 4.6 mm, 5 µm) [25]
furoate in combination M phase: 10 mM ammonium formate in water pH 5.5:
Methanol (65:35, by volume)
Flow rate: 1 mL/min
Assessment: at 240 nm
21. Mometasone furoate and HPLC S phase: Hypersil BDS, C18 column (150 × 4.6 mm, 5 µm) [26]
Eberconazole in cream M phase: Methanol:MeCN (15:85, by volume)
Flow rate: 1 mL/min
Assessment: at 240 nm

2.3 Preparation of solutions bilized in 50 mL of methanol with sonication at a slightly

rising temperature to solubilize the substance as precisely
2.3.1 Standard stock solution as possible. The volume was raised to mark height with
solvent.
MOM (5 mg) and OLO (66.5 mg) standard materials were
carefully weighed and placed in volumetric flasks (100 mL)
separately, where they were solubilized in methanol. The 2.4 Validation parameters
flasks were stirred, and the volumes were filled to the
marked height with methanol to produce a solution having 2.4.1 Linearity
50 µg/mL for MOM and 665 µg/mL for OLO concentra-
tions, accordingly. A series of standard solutions of 33.25–199.5 µg/mL
of OLO and 2.5–15 µg/mL of MOM were prepared.
Three injections of each concentration were analyzed to
2.3.2 Combined mixture determine the linearity. To determine the coefficient of
determination (R2 ), a calibration plot with concentra-
OLO standard powder (665 mg) and MOM (50 mg) tion (µg/mL) versus peak area of OLO and MOM was
standard powder, thoroughly mixed, were added to a constructed.
285 mg combination of various excipients to make a
final quantity of 1000 mg synthetic mixture. Active
pharmaceutical ingredients and additives standard pow- 2.4.2 Precision
ders were correctly homogenized to ensure consistent
dispersion. Method precision
The repeatability of the chromatograph was determined by
continuous observing and noting the response of solutions
2.3.3 Sample solution (n = 6) for MOM (5 µg/mL) and OLO (66.5 µg/mL) without
modifying the parameter of the designed approach were
The combined mixture (100 mg), equivalent to 5 mg MOM used to verify the instrument’s precision. The findings
and 66.5 mg OLO, was accurately weighed and put in a were expressed as percentage relative standard deviation
volumetric flask of 100 mL, where the mixture was solu- (% RSD).
6 of 13 PATEL and PATEL

TA B L E 2 Literature survey of Olopatadine hydrochloride.

Sr. Ref.
no. Drug Pharmacopeia Method & Description no.
1. Olopatadine HCl, IP 2022 HPLC [28]
Olopatadine HCl S phase: A column with porous silica bonded to
Ophthalmic solution, and octadecylsilane packing (150 × 4.6 mm, 5 µm)
Olopatadine HCl tablets M phase: Buffer:MeCN (72:28, by volume)
Flow rate: 1 mL/min
Assessment: at 299 nm
2. Olopatadine HCl and USP 2020 HPLC [29]
Olopatadine HCl S phase: Column with packing L7 (150 × 4.6 mm, 5 µm)
ophthalmic solution M phase: Buffer:MeCN (72:28, by volume)
Flow rate: 1 mL/min
Assessment: at 299 nm
Sr. Ref.
No Drug Method Description no.
1. Olopatadine HCl in bulk UV spectroscopy Solvent: Methanol and Water (50:50, by volume) [30]
and formulations Assessment: at 299 nm
2. First order derivative Solvent: Methanol and Water (50:50, by volume)
Assessment: at 289 nm
3. Second order Solvent: Methanol and Water (50:50)
derivative Assessment: at 267 nm
4. Olopatadine HCl in eye UV spectroscopy Solvent: Distilled water [31]
drops and tablets Assessment: at 301 nm
5. First order derivative Solvent: Distilled water
Assessment: at 262 nm
6. Olopatadine in bulk and UV spectroscopy Solvent: Methanol and 0.1 N HCl (50:50, by volume) [32]
pharmaceutical Assessment: at 206 nm
formulation
7. Olopatadine and its LC-ESI-MS-MS S phase: Develosil ODS HG-5 column (150 × 4.6 mm, 5 µ) [33]
metabolites in human M phase: 10 mmol/L acetic acid: Methanol (55:45, by
plasma volume)
Flow rate: 0.1 mL/min
Assessment: at m/z 179 and 247
8. Olopatadine HCl in bulk Area under curve Solvent: Distilled water [34]
and pharmaceutical Assessment: at 220–230 nm
formulation
9. Second order Solvent: Distilled water [35]
derivative Assessment: at 224 nm
10. Olopatadine HCl in human Liquid S phase: Altima amino (250 × 4.6 mm, 5 µm) [36]
sera chromatography M phase: MeCN:Water:Ammonium acetate (30:55:15, by
volume)
Flow rate: 1 mL/min
Assessment: at 257 nm
11. Olopatadine HPTLC S phase: Silica gel G60 F254 [37]
M phase: Methanol:Water:Glacial acetic acid (8:2:0.2, by
volume)
Assessment: at 247 nm
12. HPLC S phase: Inertsil ODS (150 × 4.6 mm, 5 µm)
M phase: MeCN:Methanol:Water (10:20:70, by volume)
Flow rate: 1.2 mL/min
Assessment: at 254 nm
(Continues)
PATEL and PATEL 7 of 13

TA B L E 2 (Continued)
13. Olopatadine HCl in pure HPLC S phase: Agilent Eclipse XDB-C18 (150 × 4.6 mm, 5 µm), [38]
forms and eye drops M phase: Phosphate buffer and MeCN (20:80)
Flow rate: 1 mL/min
Assessment: at 270 nm
14. Olopatadine HCl and Spectrophotometry Solvent: Methyl orange in 0.1 M NaOH [39]
Ketorolac Assessment: at 443 nm
15. Spectrophotometry Solvent: KMnO4 in 1 M
NaOH
Assessment: at 605 nm
16. Olopatadine HCl in Simultaneous Solvent: Methanol [40]
pharmaceutical equation method Assessment: at 320.0 and 206.0 nm
preparation
17. Olopatadine HCl in Spectrophotometry Solvent: Chloroform [41]
pharmaceutical Assessment: at 425 nm
formulation
18. Olopatadine HCl and First order derivative Solvent: Methanol [42]
Ambroxol HCl in their Assessment: at 299.20 and
synthetic 307.40 nm
mixture
19. Olopatadine in bulk and RP-HPLC S phase: C8 column (250 × 4.6 mm, 5 µm) [43]
formulations M phase: Methanol:MeCN (80:20, by volume)
Flow rate: 1 mL/min
Assessment: at 246 nm
20. Olopatadine HCl in tablets HPTLC S phase: Silica gel G60 F254 [44]
M phase: Methanol:MeCN:Triethylamine (8:2:0.2, by
volume)
Assessment: at 247 nm
21. Olopatadine HCl in tablets Stability indicating S phase: C8 column (250 × 4.6 mm, 5 µm) [45]
RP-HPLC M phase: MeCN:Methanol (55:45, by volume)
Flow rate: 1 mL/min
Assessment: at 252 nm
22. Olopatadine HCl in bulk Differential pulse Electrode: Mercury drop [46]
and voltammetry electrode and Ag/AgCl/KCl
formulations electrode
23. Olopatadine HCl HPTLC S phase: Silica gel G60 F254, (100 × 100 mm) [47]
M phase: Chloroform:Ethyl acetate:Methanol:Triethylamine
(6:4.5:2.5:0.8, by volume)
Assessment: at 254 nm
24. Olopatadine HCl Area under curve Solvent: Methanol [48]
method Assessment: at 275–320 nm
25. Olopatadine HCl and UV spectroscopy Solvent: Methanol [49]
Montelukast in pure and Assessment: at 220 nm
pharmaceutical
formulation
RP-HPLC S phase: Inertsil ODS C18 (250 × 4.6 mm, 5 µm) [50]
M phase: Methanol:MeCN (60:40, by volume)
Flow rate: 1.2 mL/min
Assessment: at 254 nm

Intermediate precision and OLO solutions (2.5, 5, and 7.5 µg/mL and 33.25,
The designed approach’s intra-day and inter-day 66.5, and 99.75 µg/mL) three times on the same day
variability were assessed by evaluating the rele- and different days. The outcomes are expressed as
vant findings for varying concentrations of MOM % RSD.
8 of 13 PATEL and PATEL

2.4.3 Limit of detection and limit of tion’s stability of MOM and OLO. In 24 h of storage,
quantification stability was assessed in the time interval. Methanol was
used to make both solutions. The observed results were
The proposed method’s limit of detection (LOD) and limit compared to a freshly made solution. By matching the peak
of quantification (LOQ) were obtained by solving the area, the solution’s stability was assessed.
below formulas [52].

A. LOD = 3.3 X σ/S 2.6 Combined mixture analysis

B. LOQ = 10 X σ/S
A combined mixture of OLO and MOM was prepared
where σ = the SD of the responses according to the abovementioned procedure. The response
S = slope of the calibration chart of the sample solution was measured at 238 nm under the
chromatographic condition mentioned above for the quan-
titation of OLO and MOM. The amounts of the OLO and
2.4.4 Accuracy MOM present in the sample solution were determined by
fitting the responses into the regression equation for OLO
The accuracy was determined by calculating MOM and and MOM.
OLO recoveries independently using the standard addi-
tion method. Known amounts of MOM and OLO standard
solutions were transferred to prequantified MOM and OLO 3 RESULTS AND DISCUSSION
sample solutions at 50%, 100%, and 150%, respectively.
3.1 Method development

2.4.5 Specificity The prime objective of the method development was the
simultaneous analysis of OLO and MOM in a single
Prepared blank solution and standard solution run under run. The optimization of the stationary phase and mobile
the above chromatographic condition. phase was done simultaneously. Stationary phases such as
The peak due to OLO and MOM should be well resolved Phenomenex C-18 column (250 x 4.6 mm, 5 µm) and Phe-
from any other peak due to a blank solution. nomenex C-8 column (250 x 4.6 mm, 5 µm) were tested
with different mobile phases containing their organic mod-
ifier (methanol, ACN, and water). Using the Phenomenex
2.4.6 Robustness C-18 column, resolution values between OLO and MOM
peaks were found greater than 2.0 (Rs≥2.0). The sep-
Deliberate variation in chromatographic conditions is aration performance of the Phenomenex C-18 column
made to assess the sustainability of the analytical method was better than that of the other columns in terms of
to remain unaffected under such conditions. In this study, analysis time, tailing factor, good theoretical plates, and
the robustness of the developed analytical method follow- peak efficiency. As a consequence, the Phenomenex C-
ing conditions was applied: 18 column was used for further method development and
optimization.
I. Modification in mobile phase ratio ± 2 For selection and optimization of the mobile phase,
II. Modification in column temperature ± 5.0◦ C the various mobile phase compositions containing water
III. Modification in flow rate ± 0.2 mL/min and methanol (50:50, v/v), water and ACN (60:40, v/v),
IV. Modification in wavelengths ± 2 nm methanol and ACN (30:70, V/V) were tried in different
ratios, but the resolution, peak shape, theoretical plates
Responses such as peak area, tailing factor, and theoreti- were not found to be satisfactory. Finally, the mobile phase
cal plates were analyzed in three consecutive injections for containing water: methanol: ACN in the ratio (25:20:55,
each condition, and % RSD was calculated. v/v/v) was found to give the best resolution, improved sep-
aration, and peak symmetry for both drugs (Figure 2A–C).
Other parameters such as run time, flow rate, and col-
2.5 Solution stability umn temperature were finalized during development. The
column oven temperature was maintained at 40◦ C and elu-
The standard and sample solutions were kept in firmly ent with a flow rate of 1.0 mL/min was monitored at 238 nm
closed containers at working temperature to test the solu- with a UV detector.
PATEL and PATEL 9 of 13

F I G U R E 2 Chromatogram of Blank solution, sample solution, and standard solution mometasone furoate (MOM) (2.5 µg/mL) and
olopatadine HCl (OLO) (33.25 µg/mL) at 238 nm.

So with this condition, it was found the good separa- Beer’s law. The high correlation coefficient (r2 = 0.9994
tion of main peaks and tailing factor was less than 2.0 and and 0.9997) for MOM and OLO throughout this concentra-
the resolution was more than 2.0. Finally, the proposed tion range verified the calibration chart’s linearity. Table 4
method was subjected to method validation according to contains a list of all regression parameters.
the ICH guidelines.
3.2.3 Precision
3.2 Method validation
Method precision
3.2.1 System suitability parameter Table 4 shows the findings of the repeatability study. The
precision of the approach was determined by spotting and
Parameters such as retention time, theoretical plate, tail-
ing factor, and resolutions were determined. The system
suitability parameters are shown in Table 3. TA B L E 3 System suitability parameters.
PARAMETERS OLOa (n = 6) MOMb (n = 6)
3.2.2 Linearity Retention time (Rt) (min) 3.2 6.1
Resolution (R) 2.22 2.50
The method’s linearity was tested at different concentra- Theoretical plates (N) 6759 3687
tion standards, in the 2.5–15 µg/mL range for MOM and
Tailing factor 1.13 0.96
33.25–199.5 µg/mL for OLO. Over this concentration range,
a
OLO : Olopatadine HCl, MOM.
the calibration plot was linear, and the drug followed b
: Mometasone furoate.
10 of 13 PATEL and PATEL

T A B L E 4 Result Data of linearity, precision, the limit of reveal that this approach is sensitive for determining MOM
detection (LOD), the limit of quantification (LOQ), and robustness and OLO simultaneously.
for Mometasone furoate (MOM) and Olopatadine HCl (OLO).
PARAMETERS OLOa MOMb
Linearity range (µg/mL) 33.25–199.5 2.5–15 3.2.5 Accuracy
slope 25374 37021
Intercept 78554 23917 A recovery study was used to determine the method’s accu-
Determination coefficient 0.9997 0.9994 racy. In the pre-quantified sample solution, a standard
Precision Repeatability
preparation of MOM and OLO at three levels with 50, 100 &
150% working solution was mixed. This solution was exam-
% RSD (n = 6) 0.9644 0.4008
ined according to the procedures outlined in section 2.4.4.
Intraday
An accuracy study was repeated three times, with the %
% RSD (n = 3) 0.51–0.88 0.34–0.71
recovery and % RSD of recovered amounts were calculated,
Interday
and the results presented in Table 5. At each additional
% RSD (n = 3) 0.99–1.03 0.85–1.06 concentration, the spiked drug recovered well, demonstrat-
LODc (n = 3) 0.32 0.16 ing that the procedure was accurate. For MOM and OLO,
LOQd (n = 3) 0.97 0.48 the mean recoveries were 99.49 ± 0.14 and 99.98 ± 0.22,
Robustness % RSD (n = 3) respectively.
Flow rate: 0.8 mL/min 0.76 0.41
1.2 mL/min 0.39 0.51
Mobile phase composition 0.53 0.29 3.2.6 Specificity
(W:M:ACN) 0.46 0.38
23:19:58 The peak due to OLO and MOM was well resolved from
24:23:53
any other peak due to the blank solution. The blank solu-
Wavelength: 236 nm 0.58 0.39 tion does not show any interference at the retention time
240 nm 0.43 0.45
of OLO and MOM.
Column temperature: 35◦ C 0.63 0.43
45◦ C 0.60 0.49
OLOa : Olopatadine HCl, MOM.
b
3.2.7 Robustness
: Mometasone furoate, LOD.
c
: limit of detection, LOQ.
d
: limit of quantification. The effect of deliberate changes in mobile phase com-
position, column temperature, absorbance maxima, and
recurrent study of standard MOM (5 µg/mL) and OLO flow rate were studied. These slight variations in chro-
(66.5 µg/mL). matographic conditions did not result in any significant
variation in peak area, tailing factor, and theoretical
Intermediate precision plate (Table 4). This indicates the developed analytical
The designed approach was proven to be precise, with low method is fairly robust enough to analyze both drugs
% RSD values. A study of intra-day variations and inter- simultaneously.
day variations demonstrated intermediate precision. The
% RSD was computed using replicate observations of three
working solutions (2.5, 5, and 7.5 µg/mL) for MOM and 3.3 Stability of solution
(33.25, 66.5, and 99.75 µg/mL) for OLO. Table 4 displays the
findings of intraday and interday precision has a high level Sample solutions of MOM and OLO were tested over a 24-h
of repeatability. The designed approach was determined to period at room temperature to demonstrate their stability.
be reliable, with % RSD values for intermediate precision It was found that % content of OLO and MOM calculated
studies that were acceptable. against the freshly prepared standard solution of OLO at 0,
8, 16, and 24 h were 99.84, 98.35, 97.76, and 97.25, respec-
tively, and MOM at 0, 8, 16, and 24 h were 99.53, 98.26,
3.2.4 LOD and LOQ 97.48, and 97.21, respectively. According to the findings, no
notable variations in the content of the standard drug were
In Table 4, the LOD and LOQ for MOM and OLO are found, and the sample spot of the peak area stayed nearly
shown. The equations were used to compute the LOD and unchanged, indicating that at room temperature solutions
LOQ for MOM and OLO. The LOD and LOQ statistics show stability for at least 24 h.
PATEL and PATEL 11 of 13

TA B L E 5 Result Data of recovery for Mometasone furoate (MOM) and Olopatadine HCl (OLO).
Amount of Amount of Total amount Mean%
Sample taken standard recovered Recovery ± SD
Drug Level (µg/mL) added (µg/mL) (µg/mL) (n = 3)
Olopatadine I (50%) 66.5 33.25 99.74 99.99 ± 0.17
hydrochloride II (100%) 66.5 66.5 133.12 100.16 ± 0.29
III (150%) 66.5 99.75 166.25 99.92 ± 0.22
Mometasone I (50%) 5 2.5 7.48 98.65 ± 0.11
furoate II (100%) 5 5 10.6 100.2 ± 0.14
III (150%) 5 7.5 12.52 99.82 ± 0.19

3.4 Combined mixture analysis deviation. The accuracy of the presented method is demon-
strated by the low % RSD value of intermediate precision.
In order to determine MOM and OLO in a combined The limits for detection and quantification describe how
mixture, the proposed method was used. The assay find- sensitive the approach is. The analytical findings demon-
ings were closer to the label claim, demonstrating that strate that the proposed approach could be used to quantify
the approach can be used to estimate MOM and OLO MOM and OLO simultaneously in a combined mixture.
simultaneously (Table 6). The described HPLC approach is simple, accurate,
precise, and sensitive. The development of a less time-
consuming, simple technique with improved sensitivity
4 CONCLUSION was prioritized. As a result, the suggested technique can
be adopted for routine MOM and OLO in their combined
An HPLC approach for estimating MOM and OLO in a mixture.
combined mixture has been designed and verified accord-
ing to ICH standards. The HPLC method is very simple and AC K N OW L E D G M E N T S
specific as both peaks are well separated with a total run- The authors wish to thank USV Pharmaceutical Ltd.,
time of 10 min, which makes it especially suitable for rou- Mumbai, and Vadish Pharma Pvt. Ltd., Mehsana for pro-
tine quality control analysis work. Based on the findings viding pure active pharmaceutical ingredients for research
of the combined mixture analysis utilizing the provided work. We are heartily thankful to the Department of Qual-
approach, it can be stated that the approach has a linear ity Assurance, Shree S. K. Patel College of Pharmaceutical
response for 2.5–15 and 33.25–199.5 µg/mL for MOM and Education & Research, Ganpat University for providing
OLO, respectively. The correlation coefficient was found permission and all the facilities to carry out the research
to be greater than 0.9994 and 0.9997 which was within the work.
limits specified (NLT 0.99). This gives confidence that the
response and concentration are directly proportional. The C O N F L I C T O F I N T E R E S T S TAT E M E N T
recovery study’s findings indicate that the designed proce- The authors declare no conflict of interest.
dure is accurate (99.49%–99.98%) and has a low standard
F U N D I N G I N F O R M AT I O N
No specific grant for this research was provided by fund-
TA B L E 6 Combined mixture analysis. ing organizations in the public, private, or not-for-profit
Label claim Qty. found % Assay (%) sectors.
Sample MOM OLO MOM OLO MOM OLO
no. (µg) (µg) (µg) (µg) (%) (%) D A T A AVA I L A B I L I T Y S T A T E M E N T
1 50 665 48.60 649.62 97.20 97.68 Research data are not shared.
2 50 665 49.49 657.10 98.98 98.15
3 50 665 48.89 660.98 97.78 99.85 ORCID
4 50 665 48.93 664.86 97.86 100.0 Bhoomi Patel https://orcid.org/0000-0001-7033-9921
5 50 665 49.74 654.90 99.48 98.46
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