International Journal of
Molecular Sciences
Article
HPLC Method Validation for the Estimation of Lignocaine HCl,
Ketoprofen and Hydrocortisone: Greenness Analysis Using
AGREE Score
Tariq Mehmood 1 , Sana Hanif 1, * , Faiza Azhar 2 , Ijaz Ali 3 , Ahmed Alafnan 4 , Talib Hussain 4 , Afrasim Moin 5 ,
Mubarak A. Alamri 6 and Muhammad Ali Syed 1, *
Citation: Mehmood, T.; Hanif, S.;
Azhar, F.; Ali, I.; Alafnan, A.; Hussain,
T.; Moin, A.; Alamri, M.A.; Syed,
M.A. HPLC Method Validation for
the Estimation of Lignocaine HCl,
Ketoprofen and Hydrocortisone:
Greenness Analysis Using AGREE
Score. Int. J. Mol. Sci. 2023, 24, 440.
https://doi.org/10.3390/
ijms24010440
Academic Editor: William C.
(Trey) Putnam
Received: 24 November 2022
Revised: 19 December 2022
Accepted: 21 December 2022
Published: 27 December 2022
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2023, 24, 440. https://doi.org/10.3390/ijms24010440
1 Department of Pharmaceutics, Faculty of Pharmacy, The University of Lahore, Lahore 54590, Pakistan
2 Punjab University College of Pharmacy, University of The Punjab, Faisalabad 38000, Pakistan
3 Faculty of Pharmaceutical Sciences, GC University Faisalabad, Faisalabad 38000, Pakistan
4 Department of Pharmacology and Toxicology, College of Pharmacy, University of Hail,
Ha’il 81442, Saudi Arabia
5 Department of Pharmaceutics, College of Pharmacy, University of Ha’il, Ha’il 81442, Saudi Arabia
6 Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University,
Alkarj 11323, Saudi Arabia
* Correspondence: pharmacist.sas22@gmail.com (S.H.); ma.pharmacist@hotmail.com (M.A.S.)
Abstract: In the current study, the reversed-phased high-pressure liquid chromatography (RP-HPLC)
method was proposed for the estimation of lignocaine hydrochloride (LIG), hydrocortisone (HYD)
and Ketoprofen (KET) according to International Conference for Harmonization (ICH) Q2 R1 guide-
lines, in a gel formulation. The chromatographic evaluation was executed using Shimadzu RP-HPLC,
equipped with a C8 column and detected using UV at 254 nm wavelength, using acetonitrile and
buffer (50:50) as a mobile phase and diluent, at flow rate 1 mL/min and n injection volume of 20 µL.
The retention time for LIG, HYD, and KET were 1.54, 2.57, and 5.78 min, correspondingly. The resul-
tant values of analytical recovery demonstrate accuracy and precision of the method and was found
specific in identification of the drugs from dosage form and marketed products. The limit of detection
(LOD) for LIG, HYD, and KET were calculated to be 0.563, 0.611, and 0.669 ppm, while the limit of
quantification (LOQ) was estimated almost at 1.690, 1.833, and 0.223 ppm, respectively. The AGREE
software was utilized to evaluate the greenness score of the proposed method, and it was found
greener in score (0.76). This study concluded that the proposed method was simple, accurate, precise,
robust, economical, reproducible, and suitable for the estimation of drugs in transdermal gels.
Keywords: HPLC Method validation; ICH guidelines; triple drug; lignocaine HCl; hydrocortisone;
ketoprofen; greenness determination
1. Introduction:
Dermatological disorders such as eczema and dermatitis, caused by pathogens, can
be acute by scratching or picking the infected areas of the skin that might be painful for
the patient [1]. Various topical dosage forms containing steroids, anesthetics, analgesics
in combination with antibiotics are used for the treatment of such diseases [2,3]. It not
only reduces the pain but also minimizes the disorder-related symptoms such as edema
and itching.
Lignocaine (LIG), which is an amide type anesthetic, is a local anesthetic that numbs
the tissues in a target site [4]. It functions by inhibiting the transport of sodium ions via the
voltage-gated channels, thereby, it relieves the symptoms of pain as it is locally released
from the delivery [5]. Methods have been reported in the literature for the detection of
LIG. Numerous instrumentation techniques are reported in literature for the detection
of the drug through liquid chromatography equipped with a UV detector and MS/MS
spectrometer [6,7], UV spectrophotometry [8], and capillary electrophoresis [9].
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023, 24, 440 2 of 11

Similarly, the Ketoprofen (KET) drug (2-(3-benzoylphenyl)-propionic acid) is one of

the effective NSAIDs that has significant importance in joint pain therapy [10,11]. It is ad-
vantageous over other NSAIDs because it is non-sedative and lacks addictive potential [12].
It is a white crystalline powder that is freely soluble in organic solvents such as alcohol,
dichloromethane, and acetone while insoluble in water [13]. A thorough existing litera-
ture survey documented that some techniques that include UV-spectrophotometry [14,15],
HPLC [16,17], micellar electrokinetic chromatography [18], capillary zone electrophore-
sis [19], polarography [20], quantitative Fourier transformation infrared spectrophotome-
try [21], and electrochemical methods [22] are available for the estimation of KET.
The pharmaceutical products containing corticosteroids in combination with local
anesthetics and analgesics have been used in the form of tablets, creams and injections re-
spectively [23]. Hydrocortisone (HYD), a synthetic glucocorticosteroid and a corticosteroid
ester play a significant role in the reduction of inflammation by immune suppression. It is
also used for the treatment of various skin disorders such as allergies, eczema, dermatitis,
and rashes. It is absorbed and reduces inflammation, itching or redness [24]. The chemical
name of the hydrocortisone is 11-β, 17-α, 21-trihydroxypregn-4-ene-3, 20-dione with the
chemical formula C23 H32 O6 [25]. It is insoluble in water but soluble in organic solvents [26].
Various methods have been reported on the determination of HYD, alone or in combination
with other drugs. For instance, with LIG (200 nm), the analyte was developed using acetone,
ammonia, and chloroform using thin layer chromatography [23,27,28]. A method was also
found for the estimation of KET and LIG [29], however, no method was found to the best of
knowledge of the authors for the simultaneous estimation of forth-mentioned drugs with
HYD. However, no method has been developed earlier to separate these drugs in a short
time without affecting peak shape and resolution. Henceforth, the current study was the
continuation of existing literature regarding method development and validation of the
Lignocaine hydrochloride, Ketoprofen and hydrocortisone. Initially, the pre-set optimized
instrumental settings wet tests for suitability of the method. Afterwards, it was evaluated
for validation parameters according to the ICH guidelines [30]. Then the dissolution test of
the dosage form was applied to estimate the concentration of drugs. Eventually, greenness
estimation of the developed conditions was performed to evaluate the environmental
friendliness score of the devised method.

2. Results and Discussion

The current study was designed to devise the analysis time of Lignocaine hydrochlo-
ride, Ketoprofen and Hydrocortisone since no method was found in the literature for
the simultaneous estimation of drugs. Therefore, the optimization of chromatographic
conditions was done to study the impact of diluent concentration in the mobile phase and
changing pH. The output of system suitability, validation, application, and greenness is
as follows.

2.1. Development and Optimization of Method

Based on the nature of the analyte, the selection of the HPLC gradient system is the
prime and substantial step before the quantitative determination of drug in its commercial
dosage. In the current study, reverse phase HPLC method was proposed and optimized
using a HPLC system attached with an Agilent® C8 column (250 mm × 4.6 mm × 4.6 mm,
5 µm). The chromatogram obtained from the fourth-mentioned instrumental conditions
with LIG initially, followed by HYD and KET in the last (Figure 1).
Int. J. Mol. Sci. 2023, 24, 440 3 of 11

Figure 1. Typical chromatogram depicting the peaks of lignocaine (LIG) HCl, hydrocortisone (HYD)
and ketoprofen (KET) for the devised HPLC method at respective concentrations of 80%.

2.2. System Suitability

The system suitability was checked by performing the experiment and identifying
the changes in separation, asymmetry of the peaks and retention times. The standard
solution was injected five times while the sample solution was injected two times. The peak
areas, resolution, number of theoretical plates, retention times and peak asymmetry were
measured for the standard as well as sample solution. The retention time of LIG, HYD and
KET under the optimum conditions were 1.54, 2.57, and 5.78 min respectively. The num-
ber of theoretical plates were always over 6000 in all chromatographic runs (Table 1).
The peak may be broadened due to multiple factors such as large injection volume, column
deterioration, molecular weight of the drug and build-up of contamination in column
inlet [31].

Table 1. System suitability and limit parameters of the proposed method.

Parameters LIG HYD KET

System suitability
Retention time (min) 1.54 2.57 5.78
Tailing factor 1.39 1.19 1.10
Plate count (USP) 6167 6094 6003
Resolution (USP) - 9.4 15.7
Linearity
Linear function y = 3175.2x + 116.67 y = 1 × 106 x + 526,211 y = 6 × 106 x + 3 × 106
Coefficient of linear regression (r2 ) 0.9994 0.9990 0.9997
Linearity range (ppm) 0.6–56 0.6–56 0.2–100
LOD (ppm) 0.563 0.611 0.223
LOQ (ppm) 1.690 1.833 0.669

The system suitability parameters were tested according to the United States Phar-
macopoeia (USP). The working standard solution of Lignocaine HCl, Ketoprofen and
hydrocortisone was introduced into HPLC to assess their retention time, and also chro-
matographic purity and UV spectrum. Once symmetrical peaks were obtained, the five
standard injections were injected and their number of theoretical plates, tailing factor, reso-
Int. J. Mol. Sci. 2023, 24, 440 4 of 11

lution and RSD were calculated. The parameters of system suitability concluded that the
chromatographic conditions were satisfactory for the method development and validation.
The tailing factor was also found in between 1–2, which is considered acceptable. Similarly,
the resolution of the peaks was greater than six. There should be a reasonable value not
less than four which could be a possibility that the peaks may be too close to each other.
However, if the retention time is too high, for instance greater than 20 min, it may result in
a laborious as well as costly analytical procedure. Hence, it is considered good if the peaks
are separated from each other that the peaks may be distinctly separated.

2.3. Method Validation

The validation factors are as follows.

2.3.1. Linearity
The linearity measured each for LIG and HYD, in the current study, ranged from 0.6
to 56 ppm, and for KET, it was 0.2–100 ppm. A good linear relationship was observed in
the analytical calibration curve constructed for Lignocaine hydrochloride, Hydrocortisone
and Ketoprofen even though the two latter drugs were hydrophobic and less soluble in
pure water. The correlation coefficient was in between 0.9990 to 0.9997, which is considered
satisfactory (Table 1).

2.3.2. Accuracy
Accuracy was performed by measuring the analytical concentrations by deliberate
addition to the 80% concentration of each of the analyte to form 100% and 120% (Table 2).
Outcomes (Table 3) as relative standard deviation have shown a value of less than 2 in
all the experimental runs for either analyte. This percentage recovery was within the
acceptance criteria of ≤2 [32]. Therefore, the proposed method was found accurate so
that it could be further utilized for the estimation of Lignocaine HCl, Hydrocortisone, and
Ketoprofen simultaneously.

Table 2. Theoretical contents and dose levels of analyte prepared in the study against the respective
mean area of retentive peaks at 100% analytical concentration.

Parameters LIG HYD KET

Theoretical Theoretical
Dose Level Mean Area Theoretical Mean Area Mean Area
Contents Contents
(%) (uA) Contents (ppm) (uA) (uA)
(ppm) (ppm)
80 16 105,831.08 16 3,411,719 40 5,949,786
100 20 132,123.7 20 4,259,328 60 7,446,541
120 24 158,540.0 24 5,111,193 80 8,861,380

Table 3. Accuracy of the devised method at 80, 100, and 120% analytical concentrations.

Accuracy LIG HYD KET

Concentration Recovery % ± Recovery % ± Recovery % ±
Amount (ppm) Amount (ppm) Amount (ppm)
Level (%) RSD RSD RSD
80 15.91 99.38 ± 0.62 16.0 100.00 ± 1.16 40.25 100.50 ± 1.44
100 20.0 100.0 ± 0.46 20.14 100.50 ± 0.91 60.30 100.20 ± 1.58
120 23.93 99.58 ± 0.51 24.01 100.00 ± 1.29 79.86 99.83 ± 0.38

2.3.3. Precision
Repeatability and intermediate precision were evaluated by five injections of sample
solutions at prepared concentrations (Table 2). Response indicated that the RSD values
were less than 2% and the proposed method was found to be precise for the quantification
of Lignocaine HCl, Hydrocortisone and Ketoprofen (Table 4).
Int. J. Mol. Sci. 2023, 24, 440 5 of 11

Table 4. Precision of the current HPLC method indicated at different days and analysts.

Machine/Day 1 & 2 LIG HYD KET

Concentration Level Recovery % ± Recovery % ± Amount Recovery % ±
Amount (ppm) Amount (ppm)
(%) RSD RSD (ppm) RSD
80 Analyst 1 & 2 15.84 99.01 ± 1.28 16.05 100.32 ± 1.76 39.63 99.08 ± 1.08
100 19.87 99.39 ± 1.47 19.16 99.58 ± 0.73 59.68 99.47± 0.97
120 24.18 100.76 ± 1.10 23.80 99.19 ± 0.90 80.08 100.10 ± 0.58
Inter day precision
Machine/Day 3 & 4 LIG HYD KET
Content Content Content
Concentration level Recovery % ± Recovery % ± Recovery % ±
recovered recovered recovered
(%) RSD RSD RSD
Analyst (ppm) (ppm) (ppm)
3&4
80 15.99 99.95 ± 0.92 16.09 100.11± 0.37 39.89 99.74 ± 1.18
100 19.94 99.71 ± 1.46 19.85 99.27 ± 1.77 60.18 100.30 ± 0.75
120 23.87 99.47 ± 0.60 23.96 99.85 ± 0.91 79.34 99.18 ± 0.31

Accuracy and precision were tested at three different concentration levels over a
range of 80, 100 and 120%. The obtained percentage recovery was in between 99 to 101%,
which was under the acceptance range. Usually, there are three stages of precision i.e.,
repeatability, reproducibility, and intermediate precision [33]. Repeatability of the proposed
method was performed by injecting six sample solutions three times in HPLC. Intermediate
precision was carried out on the sample solution on different days by two different analysts.
The RSD values were not more than 2% for Lignocaine HCl, Ketoprofen and hydrocortisone
which were in accordance with USP i.e., RSD ≤ 2%. It can be observed that the recovery
of the analyte was independent of day, analyst, and machine, which concludes that the
method was precise.

2.3.4. Robustness
The deliberate changes in chromatographic conditions such as change in pH of the
mobile phase (2.5 and 3.5), temperature (20 and 30 ◦ C), and flow rate (0.85 and 1.15 mL/min)
were done to detect robustness of the developed method (Table 5). The 100% dose level
of the analyte under investigation were selected for calculating robustness. Results has
shown that that the recovered analytical concentration of drugs was unaffected by small
changes in the conditions and no significant change was noticed in the chromatogram.
Henceforth, the developed method was found to be robust with the exception that when
the wavelength was changed to above and below the integral whole numbers (nm), issues
of detection were formed. Therefore, we can report that the working wavelength (λ) was
found to be effective in accurate recovery of the analyte.

Table 5. Outcome of robustness of the proposed method.

LIG HYD KET

Parameters
% ± RSD % ± RSD % ± RSD
Optimized conditions at 100% analytical dose = pH 3.0, 30 ◦ C and 1.00 mL/min
Flow rate
0.85 mL/min 100.33 ± 0.95 100.21 ± 1.67 99.15 ± 1.24
1.15 mL/min 98.89 ± 1.36 99.57 ± 1.18 99.90 ± 0.66
Temperature range
20 ◦ C 100.26 ± 1.74 100.49 ± 1.14 100.14 ± 0.86
30 ◦ C 100.58 ± 1.09 99.41 ± 0.68 98.69 ± 0.70
Mobile phase
pH 2.5 99.51 ± 1.38 98.47 ± 1.62 99.35 ± 1.41
pH 3.5 98.86 ± 0.91 100.01 ± 0.75 99.74 ± 0.58
Int. J. Mol. Sci. 2023, 24, 440 6 of 11

2.3.5. Specificity
Specificity of the proposed method was assessed by evaluating the interference of
excipients used in the pharmaceutical dosage form as placebo formulation. It was found
from the sample run of placebo that when hydroxylpropylmethyl cellulose and Carbopol®
containing formulations were analyzed, no interfering peak was observed at the peak
retention times of either drug. It added to the findings that the proposed method could
be utilized for the estimation of Lignocaine HCl, Hydrocortisone, and Ketoprofen in
transdermal delivery (Figure 1).

2.3.6. LOD and LOQ

The LOD and LOQ was measured statistically on the basis of the calibration curve
data [34,35]. The measured LOD values for LIG, HYD, and KET were found to be 0.563,
0.611, and 0.669 ppm, whereas the corresponding LOQ values were for 1.690, 1.833, and
0.223 ppm for LIG, HYD, and KET (Table 2).

2.4. Greenness Evaluation Using AGREE® Score

The greenness is an assessing procedure to demonstrate the extent of greenness present
in the sample. Although it was not a mandatory parameter to be tested for the validation,
it was performed additionally on the instrumental conditions to ensure that the developed
method was not too toxic or dangerous when evaluated on ecological scale. It is a safer
prerequisite to estimate the method both for validation as well as for safety extent before
using it practically, though the theoretical guidelines have been detailed in the literature [36].
An AGREE pictogram is more meaningful since it uses contour to score an element or
parameter and it defines the aggregated safety score after penalties reduction to deviate
from safety. Generally, a score above 0.7 is considered green as depicted in the scale
(Figure 2). Out of the responses of 12 parameters pertaining to the developed instrumental
method, it was found that the AGREE score was in close agreement with the green zone as
pointed with the black line in the scale which depicts that the developed method was safer
to the environment.

Figure 2. Greenness of the proposed HPLC method using AGREE score.

2.5. Application to Transdermal Gels

The formulated gels, as prepared by the authors, were evaluated in terms of drug
concentrations and it was found that the method was simple and made it easy determine
the drugs from the aliquots of dissolution sampling at defined intervals. The drug release
data however, will be presented as a separate study by the authors. Moreover, the peak
times remained undisturbed when compared with standard values. Moreover, when the
drugs in the marketed product were analysed, the analytical concentration of drugs were in
Int. J. Mol. Sci. 2023, 24, 440 7 of 11

accordance with the compendial limits in USP. The amount found for each drug separately
in commercial dosage forms contained Lignocaine HCl, Ketoprofen and Hydrocortisone in
the respective amounts of 99.80, 100.09 and 97.38% (Table 6).

Table 6. Assay of commercial tablets according to the developed method.

Active Brand Labelled Claim Amount Found (%) USP Interpretation

Lignocaine HCl Lignocaine Gel® 2.0% w/v 99.80 ± 0.83 in limits
Ketoprofen Profenid Gel® 2.5% w/v 100.09 ± 1.07 in limits
Hydrocortisone Hydrocortisone 1% Gel 1.0% w/v 97.38 ± 2.15 in limits

3. Materials and Methods

3.1. Materials
Lignocaine (LIG) as hydrochloride (HCl) pure was kindly donated by Hebei Guanlang
Biotechnology® Co., Ltd. (Hebei, China), while hydrocortisone (HYD) and ketoprofen
(KET) were generously delivered by Selmore® Pharma Pvt. Ltd. (Lahore, Pakistan).
The respective drugs were procured from XiaoGan ShenYuan ChemPharm® Co., Ltd.
(XiaoGancity, Hubei, China) and Reyoung Pharmaceutical® Co., Ltd. (Shandong, China).
Likewise, potassium dihydrogen phosphate and acetonitrile were purchased from Fluka®
(Buchs, Switzerland) and ISOLAB® (Eschau, Germany), respectively. All other chemicals,
reagents and/or solvents were of analytical HPLC grade and used as received. Similarly,
deionized distilled water was used throughout the experiment unless otherwise described.

3.2. HPLC Instrumental Conditions

Briefly, Shimadzu® LC20 pump (Machine 1 and 2) as well as Waters® 2695 (Machine 3
and 4) were utilized for optimizing instrumental conditions and HPLC method conditions
for the drugs. For analysis, the gradient mobile phase was employed with a rheodyne
type sample injector and equipped with an SPD-20 A variable wavelength detector. To
accomplish this, the temperature of the Agilent® C8 A2010150 X046 (150 mm × 4.6 mm,
5 µm) column was maintained at 30 ◦ C with a flow of the mobile phase at 1 mL/min from
the column. The injection volume of sample was preset at 20 µL. The elution was detected
at 254 nm with a runtime for 10 min. The data integration and acquisition was performed
via Lab solutions® software version 3.2.

3.3. Preparation of Mobile Phase and Standard Solution

The solvent mixture as mobile phase comprised of an equal volumetric ratio of ace-
tonitrile and phosphate buffer (pH 3.0) under ambient conditions, where the phosphate
buffer was prepared by dissolving 7.2 g of potassium dihydrogen phosphate in 500 mL of
deionized water. The final pH was adjusted to 3.0 with 10% o-phosphoric acid.
The standard solution was prepared by dissolving 20 mg each for LIG as well as HYD
and 50 mg for KET in 100 mL of a volumetric flask containing deionized water (labelled
as ‘A’). Then 5 mL of ‘A’ was transferred in a 25 mL of volumetric flask and the remaining
volume was filled with the mobile phase. The solution was then filtered through 0.45 µ
Millipore® syringe filters before analysis.

3.4. System Suitability Parameters

The developed analytical method was then validated in accordance with International
Conference of Harmonization (ICH) Q2 R1 guidelines. To be brief, the system suitability
was evaluated by identifying the retention time, tailing factor, and resolution of the peaks
as well as the number of theoretical plates (USP) [37]. According to United States Pharma-
copoeia, the tailing factor (Tf) as well as coefficient of the peak symmetry were calculated
(Equation (1)):
( a + b)
Tf = (1)
2a
Int. J. Mol. Sci. 2023, 24, 440 8 of 11

where ‘a’ is the distance from the front half of the peak to the peak midpoint (upright from
the peak highest point) that is evaluated at 5% of peak altitude and b is the distance from
the peak center (perpendicular from the peak maximum point) to the trailing edge of the
peak estimated at 5% of peak height [38]. Similarly, resolution between the peaks were
estimated using Equation (2):

(tR2 − tR1)
Rs = (2)
0.5(tW1 + tW2)

where tR2 and tR1 are the retention times of drug 1 and drug 2 for respective peaks 1 and 2.
Whereas, tW1 and tW2 are the corresponding peak width.

3.5. ICH Validation Parameters

The ICH guidelines provide a detailed procedure for estimating the validation of the
devised method. It includes linearity, accuracy, precision (reproducibility and repeatability),
limit of detection (LOD), limit of quantification (LOQ), robustness, and specificity [39].

3.5.1. Linearity
Linearity was measured by injecting series of solutions diluted from the standard
solution ‘A’ to prepare a desirable analytical concentration and peak area were then deter-
mined respectively. The concentration ranges prepared in the study for the linearity range
determination has been tabulated in Table 2.

3.5.2. Accuracy
The accuracy of the devised method was measured by estimating the percentage
recoveries of the solutions presented in Table 1, which shows the percentage of dose
prepared at different levels. Briefly, the amount of drug concentration was deliberately
added to the 80% dose solution to form 100% and 120% concentrations. It was then
estimated using the devised methods and outcome was analyzed.

3.5.3. Precision
The precision was measured by analyzing the samples at define concentrations of
each drug simultaneously i.e., 80%, 100%, and 120% (Table 1). For repeatability and
reproducibility, the analysis was performed on different instruments, at different days and
by two or more independent analysts [40].

3.5.4. Robustness
To assess the robustness, deliberate variations in the processing conditions were
formed, which were minute changes in pH, temperature, flow rate and mobile phase
composition.

3.5.5. LOD and LOQ

The limit of quantification (LOQ) and limit of detection (LOD) parameters was mea-
sured from the lowest concentration prepared in the study. The LOD (Equation (3)) and
LOQ (Equation (4)) were evaluated as follows.
σ
LOD = × 3.3 (3)
s
σ
LOQ = × 10 (4)
s
The ‘s’ is slope of the calibration curve and ‘σ’ is the standard deviation of the lowest
concentration of the analyte prepared.
Int. J. Mol. Sci. 2023, 24, 440 9 of 11

3.5.6. Specificity
The specificity of the proposed method was evaluated using a blank mixture of the
analytical solution containing no drug. Any peak was analyzed under devised conditions
that could possibly appear in place of the retention time of the peaks [41].

3.5.7. Method Application to Transdermal Gels

After the analytical method was validated, the instrumental conditions were applied
to determine the concentration from commercial dosage form. To accomplish this, 2 g of
transdermal gel containing the drug equivalent to 20 mg each of LIG and HYD and 50 mg
of the KET was placed in the dissolution vessel according to the method reported. Briefly,
the gels (containing hydroxypropylmethyl cellulose) were placed in basket and immersed
900 mL of phosphate buffer solution, adjusted to pH 7.4 at 50 rpm. The temperature of
whole system was maintained at 37.5 ± 0.5 ◦ C. At defined intervals (0.5, 1, 2, 4 and 8 h),
5 mL of aliquot was diluted with 25 mL of mobile phase to mark up 25 mL. It was then
filtered through 0.45 µm filter papers and analyzed quantitatively.

3.5.8. Statistical Analysis

The statistical analysis was carried out using IBM SPSS v.20 software for various
validation parameters. Values of mean as well as related standard deviation with percentage
of the concentration of analyte were determined.

3.5.9. Greenness Estimation

By using AGREE® software v0.5 beta (Universida de Vigo), the agreement of the
recognized analytical procedure for greenness was estimated [41]. Different factors linked
with the extent to waste substances, toxic materials and different resources were measured
with an already existing standard and penalty on various twelve parameters [36]. After
subtracting penalty points, the acquired scores were displayed at the center of a spherical
pictogram. Green color referred the safety of method developed to the environment while
red color showed the method is not safer to the environment (Figure 2).

4. Conclusions
Herein we proposed a RP-HPLC method for the simultaneous quantification of Ligno-
caine hydrochloride, Ketoprofen, and Hydrocortisone which was not previously found in
the literature. The instrumental conditions were evaluated for validation parameters and
were found to be accurate, precise, subtle, economical, time saving, and robust. The method
provided reasonable resolution and acceptable peak parameters according to USP spec-
ifications for lignocaine hydrochloride, ketoprofen, and hydrocortisone. Moreover, the
validated method was found environment friendly when compared on a greenness scale as
part of ecological safety. The developed method was also tested on self-prepared formula-
tion as it demonstrated no interfering peak of the polymers. Furthermore, the method also
detected the analyte in marketed brands which depicts that the validated method can play
a significant role in the quantification of triple drugs simultaneously named as Lignocaine
hydrochloride, Ketoprofen, and Hydrocortisone. It can be utilized for analysis of dosage
forms (bulk or finish) in the pharmaceutical industry and during stability studies with a
single run time of <10 min.

Author Contributions: Conceptualization, M.A.S.; Methodology, S.H. and A.A.; Software, S.H.;
Validation, T.H.; Formal analysis, S.H.; Investigation, T.M.; Resources, A.A. and A.M; Data curation,
T.M.; Writing—original draft, F.A.; Writing—review & editing, I.A., T.H., A.M., M.A.A. and M.A.S.;
Supervision, M.A.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Int. J. Mol. Sci. 2023, 24, 440 10 of 11

Data Availability Statement: All data contained in the manuscript.

Acknowledgments: This research was funded by Scientific Research Deanship at University of
Ha’il—Saudi Arabia through project number RG-21141.
Conflicts of Interest: The authors declare no conflict of interest.

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