Talanta Open 4 (2021) 100056

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Talanta Open
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Development of in-vitro release testing method for permethrin cream

formulation using Franz Vertical Diffusion Cell apparatus by HPLC
Lakshmi Narasimha Rao Katakam a, *, Naresh Kumar Katari b
a
Department of Chemistry, J. N. T. University, Kukatpally, Hyderabad - 500072, Telangana, India
b
Department of Chemistry, GITAM University, Hyderabad, Telangana, India-500085

A R T I C L E I N F O A B S T R A C T

Keywords: A new analytical method is developed for a generic drug product equivalent to the Reference Listed drug of
Vertical Diffusion Cell Permethrin Cream formulations for an in-vitro release study using Franz Vertical Diffusion Cell apparatus in
In vitro Release Testing association with the analytical quantitation by High-Performance Liquid Chromatography. The importance of the
Franz Vertical Diffusion Cell
in-vitro release study is to evaluate the formulation Q1/Q2 Sameness, where the test and reference products are
Permethrin
HPLC
proved to be qualitatively and quantitatively the same. The methodology has been evaluated with respect to
Cream Formulation Specificity, Linearity, the limit of quantitation (LOQ), limit of detection (LOD), inter-day Precision, intermediate
Precision, Accuracy, and solution stability. The separation is achieved by using column Inertsil ODS-3V, 4.6 mm
x 150 mm, a 5.0 µm column used, and column and sample temperature maintained at 35◦ C and 10◦ C, respec­
tively. The in-vitro drug product is developed as per Product-Specific Guidance on Acyclovir Cream, 2016 and
USP general chapter <1724> semisolid drug products-performance tests and analytical method validations
evaluated as per International Conference on Harmonization (Q2) methodology.

1. Introduction it acts as a preservative [1]. The importance of the In-Vitro Release

Testing (IVRT) study is to evaluate the formulation Q1/Q2 Sameness,
Permethrin (PRM) is a yellow to light orange-brown, low melting where the test and RLD products are proved to be qualitatively and
solid. It is a 3:1 mixture of the trans and cis isomers of the pyrethroid 3- quantitatively the same. Q3 Similarity is the physicochemical properties
(2,2-dichloroethenyl)-2,2 dimethylcyclopropanecarboxylic acid, (3- of test and RLD products proved to be similar scientifically [2]. IVRT is a
phenoxyphenyl) methyl ester, or viscous liquid. 5% topical cream (50 well-established method for characterizing and evaluating the perfor­
mg/gram) is an off-white, vanishing cream base that acts as a carbicidal mance of semi-solid dosage forms. It is a sensitive and discriminating
agent to treat infestation with scabies [1]. method that is responsive to physicochemical changes in semi-solid drug
product formulation. Therefore, IVRT serves as a valuable tool to
demonstrate the comparative drug release rates between the reference
and test product formulations. Topical development is the shortest route
between the treatment area and application point and mimics topicals
diffusing into the skin and helps determine release rate. The pivotal
IVRT study comparing the drug release rates between the test and
Reference listed drug (RLD) products is to be performed in a compatible
manner with the statistical analysis method specified in the United
The topical cream is developed with the use of Inactive Ingredients States Pharmacopeia (USP) General Guidance Chapter [3]. The IVRT
viz. Medium-chain triglycerides, Isopropyl myristate, Carbomer homo­ methodologies were three types and the typical advantages/disadvan­
polymer type B, glycerin, mono-and diglycerides, lanolin alcohols, tages with comparison was summarized in the Table 1. Based on the
mineral oil, polyoxyethylene (20) cetyl ethers, sodium hydroxide, and overview of the techniques, the advanced Franz cell diffusion cell was
purified water with the addition of Formaldehyde 1 mg (0.1%) in which chosen to develop this methodology.

* Corresponding author.
E-mail address: [email protected] (L.N.R. Katakam).

https://doi.org/10.1016/j.talo.2021.100056
Received 13 June 2021; Received in revised form 6 July 2021; Accepted 6 July 2021
Available online 11 July 2021
2666-8319/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
L.N.R. Katakam and N.K. Katari Talanta Open 4 (2021) 100056

Table 1
Comparison of IVRT Methodologies.
Significant Vertical Diffusion Cell Immersion Cell USP Apparatus 4
Parameters

Technical Difference Cylindric structured Franz Cell Diffusion with Dissolution of mini/regular Cylindric structured vessel Flow through Cell with cone structured cell
cell orifice as 38 mm through USP Apparatus 1 or 2 diameter has 22.6 mm
Cell volume Capacity 3 mL, 7 mL and 12 mL 150 mL - 200 mL Cell flow volume is nominally at 8, 16 and 24
ml/min
Operating Either at 32◦ C or at 37◦ C Either at 32◦ C or at 37◦ C Either at 32◦ C or at 37◦ C
temperatures
RPM Nominal at 600 RPM Nominal at 25 RPM and/or 50 RPM Flow rate exists instead RPM
Sample size NLT 200 mg 300 mg to 2 g 400 mg to 1200 mg
Advantages/ • Sensitive technique • Insensitive technique • Sensitive technique
Disadvantages • Advanced IVRT • Conventional IVRT • Advanced IVRT
• Compatible for very low/high dosage • Compatible for high dosage strengths • Compatible for very low/high dosage
strengths • Not feasible for micro sample collection forms
• Feasible for micro sample collection • Not compatible for multiple cell volumes • Feasible for micro sample collection
• Compatible for multiple cell volumes • Less clog formation as the sample cell lines are optimally • Compatible for multiple flow rates
• Possible clogs formation in the diffusion high diameter and easy to clean • Possible clogs formation in the flow
cells • Rugged discriminating method through cells
• Robust and rugged descriminating method • Robust and rugged discriminating method

In the literature, few analytical methods were published on different performed by total area counts of Cis and Trans form of PRM, as the
drug formulations in-vitro release methods by Franz Vertical Diffusion peaks elute at different retention times.
Cell (VDC) Apparatus [4-6] based on the recently published FDA guid­ Vision Microette Franz Diffusion Cell Apparatus with six VDCs
ance on semi-solid formulations. Several other methodologies are pub­ operated in parallel, 15 mm Orifice, 7 mL Volume. Each cell was
lished for the quantitative determinations of PRM in human/rat plasma equipped with a magnetic stir bar on a six-cell drive system. Vision®
[7-9] and analytical applications using various formulation matrices Microette™ for programming and dispensing diffusion medium into
[10-20]. The in-vitro release testing method is essential for the phar­ vessel programmed with circulating water bath to maintain the constant
maceutical industries per allowable Scale Up and Post Approval Changes cell temperature. Vision AutoFill to fill the vial through auto-sampler.
(SUPAC) guidance of the process changes through the product lifecycle The diffusion cell apparatus was set at 600 RPM, and the cell tempera­
without compromising the quality to determine product sameness with tures were maintained at 32 ± 0.5◦ C. The VDC system was allowed to
the referenced subject test product approved by FDA earlier stage. equilibrate for approximately 30 min. Four hundred milligrams of the
However, none of the methods is suitable for the subject analytical cream sample was applied onto the 0.45 µm PVDF membrane (pre-
method to determine the in-vitro release testing due to insensitivity, soaked filter in the receptor medium for 30 min). Samples of the relevant
poor selectivity, and poor column life (as the release media is developed receptor medium were collected from each of the 6 VDCs simultaneously
using a saline buffer with organic media). Hence, a novel, sensitive and at 0.5, 1, 2, 4, and 6 h after applying the PRM cream. The sampling port
selective IVRT method is developed for determination of PRM in Cream was flushed with 2.0 mL of rinse volume before sample collection of 0.6
formulation using Franz Vertical Diffusion Cell Apparatus by HPLC. The mL.
analytical method validations evaluated as per International Conference The release rates were calculated by applying the Higuchi equation
on Harmonization Q2(R1) Validation of Analytical Procedures - text and as mentioned below:
methodology.
Q = SQRT(t.(2ADCs)) (1)
2. Materials and methods In Eq. (1), Q [μg/cm2] is the amount of drug released per unit area of
exposure, t [minutes] is the time, D [m2/s] denotes the diffusion coef­
2.1. Chemicals and reagents ficient, CS [μg/cm3] is the solubility of a drug in the formulation and A
[μg/cm3] is the initial drug concentration in the product.
PRM (purity-99.8%) primary standard was procured from SRIKEM
Laboratories Pvt. Ltd, Mumbai, India. Analytical reagents of sodium 2.3. Preparation of Franz Cell Diffusion media
phosphate dibasic, methanol, ethanol, sodium chloride, potassium
chloride, mono-basic potassium phosphate, and Ortho phosphoric acid Accurately weigh and transfer about 8.0 g of sodium chloride, 0.2 g
was procured from Sigma Aldrich, USA. Alcohol 200 proof, hydrochloric of potassium chloride, 1.44 g of disodium phosphate, and 0.24 g of
acid is procured from VWR international, USA. A 0.45 µm nylon filter mono-basic potassium phosphate in a 1000 mL volumetric flask. Add
was procured from Pall India PVT for receptor media and mobile phase 800 mL of purified water (Milli-Q) and mixed well. The pH of phosphate
filtrations. Ltd, Mumbai, India. For diffusion study, 0.45 µm PVDF buffer saline is adjusted to 7.4 with diluted hydrochloric acid and dilute
membrane is procured from Durapore EMD Millipore, Millipore, Mum­ to volume with water. Combine 20 volumes of pH 7.4 buffer with 80
bai, India. Propylene glycol procured from EMD Millipore USA and volumes of ethanol. Filter the receptor media through a 0.45 µm nylon
Reference listed drug (RLD) Elimite cream is obtained from Prestium membrane filter before use.
Pharma, Inc, USA. Micropipettes are procured from Eppendorf, Con­
necticut, USA. 2.4. Chromatographic conditions

2.2. HPLC and VDC instrumentation The separation of PRM (Cis and Trans form) was achieved by using
10 mM Dibasic potassium phosphate buffer, pH adjusted to 7.0 using
The chromatographic experiments were performed using Waters dilute Ortho phosphoric acid solution thereafter the addition of meth­
HPLC Separation (model 2695) System equipped with UV or PDA de­ anol in the ratio of 10: 90 (v/v) as mobile phase at a flow rate of 1.0 mL
tector capable of detection wavelength 235 nm. The chromatographic min − 1 with an isocratic elution method. Inertsil ODS-3V, 4.6 mm x 150
system used Empower 3 Software for the acquisition, processing, and mm, 5.0 µm column used and the temperature of column and sample
reporting of chromatographic data. The quantitation of PRM is maintained at 35◦ C and 10◦ C, respectively. Detection and quantitation

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L.N.R. Katakam and N.K. Katari Talanta Open 4 (2021) 100056

Table 2 2.6. Standard preparation

System suitability results.
S. Component Retention % % USP Tailing 2.6.1. Stock standard preparation
No. Name Time (min) RSDa Recoveryb (NMT 2.0) Accurately weigh and transferred 42 mg of PRM (Trans and Cis
1 PRM Cis isomer 7.45 0.3 1.1 isomer) reference standard (previously warmed using water bath at 70-
2 PRM Trans 8.88 0.5 1.2 85◦ C for homogenization of the semisolid form then after cooled to room
Isomer temperature prior to weigh) into a 25 mL volumetric flask. Add about 20
3 PRM Cis+Trans N/A 0.4 0.4 mL of ethanol and sonicated to form a miscible liquid. Dilute to volume
Isomer
with ethanol and mix well. Stock standard concentration is about 1.68
mg mL− 1.
a
PRM for five replicate standard injections should be NMT 3.0.
b
% recovery determined with placebo spiked with standard preparation
should be between 85%-115%. 2.6.2. Working standard preparation
Transferred 10.0 mL of stock standard solution into 50 mL of volu­
of the main active pharmaceutical ingredient was achieved with an in­ metric flask and dilute to volume with diffusion medium and mix well.
jection volume of 10 µL using a UV detector at 235 nm. The total runtime The working standard concentration is about 336 μg mL− 1. Transfer 2.0
of the chromatography is 15 minutes. mL of the stock standard into a 10 mL volumetric flask. Diluted to vol­
ume with placebo receptor fluid solution and mix well. The system
suitability results and evaluation is shown in Table 2. The chromato­
2.5. Blank and Placebo preparation gram of Blank, Placebo, and Standard preparations is presented in
Figure 1.
Collected the receptor fluid in all the cells after 360 minutes time
point of placebo analysis and mixed well. Transfer 0.1 mL of solution
into a 1.5 mL vial containing 1.4 mL of diffusion medium and mix well.
Diffusion media is used as a blank solution.

Fig. 1. Representative Chromatogram of Standard Solution.

Fig. 2. Representative Chromatogram-Diffusion sample @ 360 min.

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L.N.R. Katakam and N.K. Katari Talanta Open 4 (2021) 100056

Table 3 2.7. Sample preparation

IVRT Method development and optimization.
Diffusion medium Study drug Inference Used the spatula supplied with the diffusion cell system to carefully
time release on smooth the sample (about 300-500 mg) over the membrane evenly.
over 6 cells Apply the glass disk to the top of the dosage chamber. Ensure that the
Water and ethanol up to 3-6% • The vertex formation (with stirrers are not rotating and a positive meniscus covering the top of each
(1:1) 360 foam heap) was observed in diffusion cell. Place the thumb on edge of dosage wafer and diffusion
min VDC after 1-2 hr at 600 rpm cell, and roll the sample assembly onto top of the cell. Slide the clamps
Water and ethanol up to 15-18% and continued until the end of
(3:7) 360 the test run due to poor solu­
slowly into place, inspect the membrane for any air bubbles, and ensure
min bility of the drug. that the membrane has not moved and should be verified for the other
• Drug release observed < 20%. five diffusion cells. Start the diffusion system for sample preparation and
• No difference observed collect the samples by the programmable auto-sampler.
between the two mediums.
Prepare 30 HPLC auto-sampler vials with caps and place these into
Water and up to 2-4% • The vertex formation (with
methanol (1:1) 360 foam heap) was observed in appropriate tray slots for the diffusion cell sampling unit. Collect 0.6 mL
min VDC after 1-2 hr at 600 rpm of sample from each of the six diffusion cell receptor compartments at
Water and up to 12-16% and continued until the end of 30, 60, 120, 240, and 360 minutes. Diluted 0.1 mL of stock sample to
methanol (3:7) 360 the test run due to poor solu­ respective stock sample into 1.5 mL vial containing 1.4 mL of diffusion
min bility of the drug.
• Drug release observed < 20%.
medium and mixed well. Analyzed sample using HPLC analysis to
Water, propylene up to 15-20% • The vertex formation (with determine PRM released at each time interval. The chromatogram of
glycol and 360 foam heap) was observed in sample preparation at 360 minutes is presented in Figure 2.
ethanol (2:2:6) min VDC after 2-3 hrs at 600 rpm
Water, propylene up to 17-21% and continued until the end of
glycol and 360 the test run due to poor solu­
3. Results and discussion
ethanol (1:3:6) min bility of the drug.
• Drug release observed < 25%. The development and optimization for the drug diffusion method
• Drug release is the not was split into two categories. One is the Franz cell diffusion method, and
proportional to the rate
the other is HPLC method. The diffusion method development and
limiting step corresponding
time intervals. optimization is achieved by comparing the results of reference drug
pH 7.4 saline buffer up to 14-18% • The vertex formation (with product formulation versus test sample drug product on the finalized
and ethanol (4:6) 360 foam heap) was observed in diffusion method conditions. The HPLC method was optimized using the
min VDC after 2-3 hr at 600 rpm test sample collected from the various diffusion media under the
pH 7.4 saline buffer up to 20-26% and continued until the end of
and ethanol (3:7) 360 the test run due to poor solu­
development process.
min bility of the drug.
• Drug release observed < 30%.
• Drug release is the rate
3.1. Franz cell diffusion system method optimization
limiting step corresponding
time intervals. 3.1.1. Receptor medium
pH 7.4 saline buffer up to 24-33% • No vertex formation (with Solubility of a drug in receptor medium was preliminarily optimized
and ethanol (2:8) 360 foam heap) was observed in
using the solvents viz. ethanol, methanol, and propylene glycol in
(Finalized min VDC throughout the time
diffusion media) intervals at 600 rpm. different volume proportions and the evaluation summary presented in
• Homogeneous and transparent Table 3. Ethanol was found to be optimal solubility rate and solvent
solution visually observed in compatibility with aqueous buffers; therefore, ethanol was used for
the vessel. further media optimizations. The optimized volume proportions with
• Drug release observed < 35%.
individual solvent compositions with water did not obtain the expected
• Drug release is the rate
limiting step corresponding to drug solubility rate with time. Due to these observations, the diffusion
the time intervals and also medium was further optimized using a slightly basic pH 7.4 saline buffer
correlated to the reference (prepared using sodium chloride, monobasic potassium phosphate, and
drug product.
dibasic sodium phosphate) with a variable volume proportion of
ethanol. It evaluated the test versus reference drug product formulation.

Fig. 3. Representative Graph of Diffusion cell (Temperature Variation).

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L.N.R. Katakam and N.K. Katari Talanta Open 4 (2021) 100056

Fig. 4. Representative Graph of Diffusion cell (Formulation Composition).

Fig. 5. Representative Graph of Diffusion cell (Stirring Rate Variation).

Fig. 6. Representative Graph of Diffusion cell (Release Rate).

The temperatures of the receptor medium was set at steady temperature determination. The binding of PRM to membranes was evaluated by
of 32±1◦ C as the drug product is for cutaneous applications. immersing each of the three membranes in 10 mL of identical test so­
lutions (35 μg mL-1 PRM in 0.9% sodium chloride solution) at 32 ± 1◦ C
3.1.2. Membrane inertness and solubility of the API in the receptor medium for 6 h. As a control, the same test solution without an immersed
The membrane inertness was optimized and evaluated using filters membrane, prepared in triplicate, was allowed to equilibrate for 6 h at
such as 0.45 µm PVDF and 0.45 µm Nylon of low binding capacity, 32 ± 1◦ C. Subsequently, the concentrations of all six test solutions were
chemical compatibility with receptor medium, and free resistance to the determined to calculate the recovery relative to the control by dividing
diffusion rate of the drug. A durapore EMD membrane with a receptor the mean concentration of the solutions with immersed membranes by
medium as pH 7.4 buffer and ethanol (2:8) was used for IVRT method the mean concentration of the control solutions. The solubility of PRM in

5
L.N.R. Katakam and N.K. Katari Talanta Open 4 (2021) 100056

Table 4 the receptor medium was evaluated in triplicate by dissolving PRM in

HPLC Method development and optimization. the receptor medium to yield a saturated solution. A homogenous so­
Mobile phase Column Inference lution was achieved by stirring for 5.5 h at 600 rpm and 32 ± 1◦ C and
allowing the solution to remain at 32 ± 1◦ C overnight. Aliquots of the
Dibasic potassium Inertsil • The PRM isomeric peaks were
phosphate (pH 3.0) /Hypersil/ eluted about 60-75 minutes on supernatant were withdrawn, filtered (Nalgene syringe filter, 0.2 μm, 25
and methanol (50:50) Symmetry subject columns. mm, SFCA membrane, Nalgene, Thermo Scientific), diluted, and
Dimension: • Enantiomeric (cis and trans) peaks analyzed to determine the concentration of dissolved PRM.
4.6 mm x 150 were co-eluted with distorted
mm, 5.0 µm peaks on subject columns.
Dibasic potassium Inertsil • The PRM isomeric peaks were
3.1.3. Sampling intervals
phosphate (pH 3.0) /Hypersil/ eluted about 35-45 minutes on The % drug release is the rate limiting step corresponding to the time
and acetonitrile Symmetry subject columns. intervals for the test sample and reference drug product, which produces
(50:50) Dimension: • Enantiomeric (cis and trans) peaks slow and consistent release of the drug concentration during the study.
4.6 mm x 150 were co-eluted with distorted
First sample point was selected after the diffusion cell has reached a
mm, 5.0 µm peaks on subject columns.
• Acetonitrile found poor baseline steady state of diffusion at about 30 min. The last sample should be
in the obtained chromatography. during the steady state and before excessive drug depletion occurs
Dibasic potassium Inertsil • The PRM isomeric peaks were through the membrane (up to 360 min). The comparative membranes
phosphate (pH 7.0) /Hypersil/ eluted about 30-40 minutes on all between 0.45 µm PVDF and 0.45 µm Nylon are evaluated, and the ob­
and methanol (50:50) Symmetry subject columns.
Dimension: • Enantiomeric peaks (cis and trans)
tained results found optimally faster drug depletion in the PVDF mem­
4.6 mm x 150 were optimally separated in the brane. The optimization of drug diffusion rate is evaluated and achieved
mm, 5.0 µm inertsil column. at about 10 to 35% with the specified 0.45 µm PVDF membrane at a time
• Poor peak shape and low interval up to 6 h.
theoretical plates were observed
on all subject columns.
• Found more suitability in the 3.1.4. Stirring rate
obtained chromatography High stirring rate may result in a change at the membrane and re­
Dibasic potassium Inertsil • The PRM isomeric peaks were ceptor media interface, which may affect diffusion. The lower stirring
phosphate (pH 7.0) /Hypersil/ eluted about 30-45 minutes on all rate would impact the non-homogeneity in the receptor medium. The
and acetonitrile Symmetry subject columns.
optimal stirring rate at rpm of 500, 600, and 700 were evaluated to
(50:50) Dimension: • Enantiomeric (cis and trans) peaks
4.6 mm x 150 are co-eluted with distorted peaks obtain the diffusion rate of the drug (about 35%) with respect to the time
mm, 5.0 µm on Hypersil and Symmetry and prevent the media disruption throughout the run. The receptor
columns. medium and donor chamber performance found optimal and robust at a
• Acetonitrile found poor baseline
stirring rate of 600 rpm.
in the obtained chromatography.
Dibasic potassium Inertsil • The PRM isomeric peaks were
phosphate (pH 7.0) Dimension: eluted about 20-30 minutes. 3.1.5. Mass balance of IVRT release sample
and methanol (25:75) 4.6 mm x 150 • Enantiomeric peaks (cis and trans) The mass balance of the test sample was studied on the method
mm, 5.0 µm were optimally separated in precision test run. The residual test sample was collected from the re­
inertsil column.
ceptor chamber of the individual diffusion cell after 360 minutes and
• Peak shape and theoretical plates
were improved. transferred the contents into the respective 50 mL volumetric flask and
Dibasic potassium Inertsil • The PRM isomeric peaks were sonicated to dissolve in ethanol to the volume and filtered through 0.45
phosphate (pH 7.0) Dimension: eluted within 10 min retention µm Nylon filter and injected into the HPLC. The mass balance was
and methanol (10:90) 4.6 mm x 150 time with optimal resolution of >
calculated by the cumulative addition of %drug release and %residual
(Finalized method) mm, 5.0 µm 2.0.
• Peak shape and theoretical plates
mass obtained from the receptor chamber. The mass balance results
were optimally high and found obtained for the 6 cells were found to be 95-97%.
selective chromatography.
• Found standard and sample 3.1.6. Discrimination of diffusion media and similarity factor (f2)
solutions stable for,multiple days.
evaluation
The study was performed on optimized diffusion conditions for the
following categories with 6 VDCs and evaluated the similarity factor
Table 5 against the method precision test sample. The formulation Q1/Q2
Method validation summary. Sameness is evaluated using the test and RLD sample through similarity
Parameters PRM
factor determination. The similarity factor for test versus RLD sample in
final optimized method condition is found to be greater than 50 (against
Linearity
an acceptance criteria of NLT 50).
Range (μg mL-1) 3.35 - 402.7
Correlation coefficient 0.999991
Slope 18523.349021 3.1.7. Diffusion media concentration variability
Limit of Detection (LOD) 1.006781 μg mL-1 The analytical method development was initiated primarily with
S/N ratio at LOD level 45 drug solubility study on different diffusion mediums such as purified
Limit of Quantitation (LOQ) 3.355936 μg mL-1
S/N ratio at LOQ level 180
water and saline solution (pH 7.4), independently with the combination
Accuracy(a) (% of Recovery) of organic solvents such as Ethanol, Methanol, Propylene glycol due to
Accuracy @ 8 μg mL-1 ± SD 102.1 ± 2.1 the enhanced solubility of the drug in the organic medium than aqueous
Accuracy @ 35 μg mL-1 ± SD 99.3 ± 0.9 medium. The summary of the diffusion media evaluation is presented in
Accuracy @ 175 μg mL-1 ± SD 99.6 ± 0.4
Table 3. The final optimized diffusion media considers optimal drug
Accuracy @ 400 μg mL-1 ± SD 101.5 ± 0.6
Precision (b)(%RSD of 12 Slopes) solubility and diffusion rate of the drug formulation with an adequate
Repeatability 5.9 similarity factor comparable to RLD Sample formulation. The optimi­
Intermediate precision 10.1 zation graphs in terms of Temperature Variation ± 2◦ C, Formulation
Composition ± 50%, Stirring Rate Variation ± 100 RPM, Release Rate
(test-1, test-2 vs. RLD) at method precision condition is presented in

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L.N.R. Katakam and N.K. Katari Talanta Open 4 (2021) 100056

Fig. 7. Representative Chromatogram of Linearity_Limit Of Detection.

Figures 3-6. 3.3.1.2. Inter-day accuracy/precision. Inter-day Accuracy was demon­

strated by relative mean error calculated from 12 Slopes of two sample
3.2. HPLC method development and optimization sets obtained by Analyst 1 on Day-1 and Day-2. An Analyst-2 performed
intermediate Precision using the same sample as analyst-1 in the inter-
Based on the poor solubility (≈0.069 μg mL− 1 in aqueous medium) day precision study.
and strong basic characteristics (pka: -3.5) of the drug, the initial HPLC
method development was started with mobile phase buffer prepared at 3.3.1.3. Spiked sample recovery. Method Accuracy was determined by
slight acidic pH (3.0) and neutral pH (7.0) using dibasic potassium spiking known concentrations of PRM at three concentration levels into
phosphate salt with the individual combination of methanol and placebo receptor fluid in triplicate at each concentration. The un-spiked
acetonitrile solvents. Dibasic potassium phosphate was only selected as (control) sample was also analyzed, and no interferences were observed
the preliminary buffer as the drug has a strong basic nature, and the at the retention time of PRM peak. The concentration levels of PRM
chromatography would have better selectivity in terms of rapid elution. correspond to approximately 8 μg mL− 1, 35 μg mL− 1, 175 μg mL− 1 and
The topical cream formulation contains large quantities of non-ionic 400 μg mL− 1. Method precision and recovery results are presented in
and/or long-chain fatty alcohols. A suitable organic solvent medium Table 5.
for the optimal amount of drug solubility for the optimization of both
diffusion media and chromatographic mobile phase well. The C18 3.3.2. Specificity
bonded phase columns of various brands viz. Inertsil, Hypersil, and Specificity study was performed on a placebo (without PRM)
Waters Symmetry at fixed column length (150 mm x 4.6 mm, 5 μm) was formulation using diffusion apparatus up to 360 minutes. No significant
used and evaluated the chromatographic development summarized in interference was observed at the retention time of PRM due to diluent
Table 4. The Cis and Trans isomers have optimal separation in the and placebo.
chromatographic elution and did not result much variation in the
selectivity of this method optimization process. The final optimized 3.3.4. Linearity
chromatography was found selective, rugged, robust, and sensitive Linearity of the method was determined using series of solutions at
quantitation of diffusion test samples with the allowable drug concen­ eight different concentration levels ranging from 3 μg mL− 1 to 400 μg
tration of 8 - 400 μg mL− 1. mL− 1 of PRM USP, reference standard. The limit of quantitation (LOQ)
and limit of quantitation (LOD) of PRM was established based on the
3.3. Method validation ratio of signal/noise 10:1 and 3:1, respectively. The results and repre­
sentative chromatogram are presented in Tables 4 and Figure 7,
The current optimized method was validated as per guidelines of ICH respectively.
[21] and USP Compendia [22], along with referenced literature on
method validation [23-26] studies in current industry practices. 3.3.5. Stability of mobile phase and sample solutions
A working standard solution, QC sample solutions, and sample so­
3.3.1. Method precision and accuracy lution (collected at 360 minutes) were prepared and stored under
Repeatability/Inter-day Precision for the determination of PRM was ambient laboratory conditions, refrigerated conditions, and auto-
performed on the same Lot of Cream sample. sampler chiller conditions. The aged solutions of working standard,
diffusion sample collected at 360 minutes were stored at room tem­
3.3.1.1. Inter-day precision / repeatability. Repeatability/Inter-day Pre­ perature and tested against the initial obtained responses of subject
cision for In-vitro diffusion of PRM from the Cream sample was per­ solutions for day-1, day-2, and day-7, respectively. Both solutions were
formed on the same homogeneous lot by analyst 1 on day-1 (Set 1) and found stable up to 7 days at room temperature condition.
day-2 (Set 2). The in-vitro comparison was done by analyzing samples
(from six vessels) collected each day at five different time points. The 4. Conclusion
amount released at each time point was calculated, the flux curve was
plotted, and slopes for each flux curve were calculated. The % RSD A rapid, simple, selective LC method provides PRM quantitation
should be NMT 25.0 for the Slopes of Flux Curve obtained for samples on without the interference of blank and placebo. The developed method is
Day-1 and Day-2. The results are presented in Table 5. highly selective, reproducible, specific, rapid and robust for

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L.N.R. Katakam and N.K. Katari Talanta Open 4 (2021) 100056

determination of in-vitro release, separation and quantification of PRM [10] Maja Shishovska, Vera Trajkovska, HPLC-method for determination of permethrin
enantiomers using chiral β-cyclodextrin-based stationary phase, Chirality 22 (5)
isomers (Cis and Trans) in the complex cream formulations. The prac­
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Declaration of Competing Interest [14] M.S. Arayne, N. Sultana, F. Hussain, Validated RP-HPLC method for determination
of permethrin in bulk and topical preparations using UV-vis detector,
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49.4.287.
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