Development and validation of RP-HPLC method for simultaneous estimation of minoxidil and Aminexil in topical formulation.

In RP- HPLC method based on separation of chemical impurities.

1. Development & validation of RP-HPLC method for simultaneous
estimation of minoxidil and aminexil in topical formulation
Presented by
Narwade Aarti Namdev
B pharm 4
th
year 8 th semester
Shri Sambhaji college of pharmacy khadkut , Nanded
Under the guidance of
Ms.Deshmukha s.s.
Asst.professor
Quality Assurance

2. •Content
•Introduction
•Aim & Objective
•Literature & Review
•Plan of work
•Drug profile
•Material & methods
•Result and conclusion
•Reference

3. •Introduction
•Pharmaceutical analysis defined as application of analytical
procedure used to determine the purity, safety and quality of
drug and chemicals. Analytical chemistry is sub discipline of
chemistry that has broad mission of understanding the chemical
composition of all matter and developing tool and experiments
to make either qualitative or quantitative measurements.
•Analysis can be divided in to two classes :
1) Quality analysis. 2)Quantitative analysis.
•Qualitative analysis gives an indication of the identity of the
chemical species in the sample.
•Quantitative analysis estimates, how much quantity is present
in a mixture.

4. •RP-HPLC stands for Reverse Phase High-Performance Liquid Chromatography,
which is a widely used chromatographic technique for the separation of polar and
non-polar compounds based on their hydrophobicity.
•The development of an effective RP-HPLC method involves several key steps,
including the selection of an appropriate column, mobile phase, and detection method.
The column is the heart of the RP-HPLC system and is selected based on the
properties of the analytes and the separation mechanism.
•The mobile phase is typically composed of a polar solvent, such as water, and an
organic solvent, such as acetonitrile or methanol. The ratio of the two solvents can be
adjusted to optimize the separation of analytes based on their hydrophobicity.
•To optimize the RP-HPLC method, a systematic approach is typically employed, such
as Design of Experiments (DoE) or Quality by Design (QbD). These approaches
involve the variation of multiple parameters simultaneously to identify the most
significant factors that affect the separation and quantification of analytes.
•This allows for the determination of the optimal conditions for the RP-HPLC method,
such as the flow rate, column temperature, and gradient elution profile.

5. •Recent advances in RP-HPLC method development have also
focused on the use of artificial intelligence, such as machine learning
and deep learning, to optimize the method development process.
These approaches can expedite the development of an effective RP-
HPLC method by reducing the time and resources required for
method optimization.

6. • Aim & objective
•Aim:
Development and validation of analytical method for
simultaneous estimation of minoxidil and Aminexil in
topical solution by HPLC.
•Objective:
1.The objective was to studied a simple and rapid
HPLC method for minoxidil and aminexil.
2. The developed method will be validated in
accordance to ICH guidelines.
3.To studied and validated method will be applied for
assay of topical solution.

7. 1.kishore et al.., a RP-HPLC method was developed for the simultaneous estimation
of adapalene and benzoyl peroxide in a topical gel. The method utilized a C18
column, methanol-water mobile phase, and UV detection at 215 nm. The method was
validated for linearity, precision, accuracy, and specificity.
2.Baheti et al.., This study developed an RP-HPLC method for the determination of
tazarotene in a topical formulation. The method utilized a C18 column and a mobile
phase consisting of acetonitrile and phosphate buffer, and was validated for
specificity, linearity, precision, and accuracy.
3.Vishwanath et al.., This study developed and validated an RP-HPLC method for the
simultaneous determination of metronidazole and methylparaben in a topical gel
formulation.
4.Chen et al.., RP-HPLC method was developed and validated for the determination of
minocycline in a topical gel. The method used a C18 column, acetonitrile-water
mobile phase, and UV detection at 254 nm
5.Dongre et al.., RP-HPLC method was developed and validated for the simultaneous
estimation of tazarotene and mometasone furoate in a topical cream. The method
used a C18 column, acetonitrile-water mobile phase, and UV detection at 243 nm..
•Literature review

8. •Plan of work
1 Literature survey.
.2. Procurement of chemicals and drugs.
.3. Quantitative evaluation: The development of HPLC method.
4. Validation of the developed method.
• Accuracy &Precision
• Linearity
• Ruggedness
• Robustness
5. Application of proposed method to standard laboratory mixture.
.6. Application of proposed method to marketed formulation.
7. Results and discussion.

9. •Drug profile
•Minoxidil is a medication used primarily for the treatment of hair
loss and high blood pressure. It was originally developed as an oral
medication for the treatment of hypertension, but its effect on hair
growth was discovered as a side effect. Minoxidil is a vasodilator,
meaning it relaxes and widens blood vessels, which allows for
increased blood flow. When applied topically to the scalp, it can
stimulate hair growth and slow down hair loss.
•minoxidil is thought to work by increasing blood flow and oxygen
supply to hair follicles, as well as by prolonging the anagen (growth)
phase of the hair cycle. It also may have a direct effect on the hair
follicle, promoting the proliferation of dermal papilla cells and
stimulating the production of extracellular matrix proteins.

10. • Name - minoxidil
• Chemical name - 2, 4-diamino-6-piperidinopyrimidine-3-oxide
• Structural formula
• Melting point -248°c
• Solubility - soluble in water
• Appearance - white crystalline powder
• Molar mass -209.253g\mol

11. •Aminexil, 2,4-diaminopyrimidine-3-N-oxide,is a newer drug that is most
commonly found in combination With minoxidil in marketed liquid
formulations.
• Minoxidil and Aminexil together exerts synergistic action by increasing the
overall therapeutic outcome of the combined formulation, which therefore
remains to be clinically more efficacious in individuals with any stage of
hair loss. Aminexil occurs as an odourless white crystalline powder, slightly
soluble in water.
•Aminexil is a vasodilator, meaning it widens the blood vessels and provides
better blood flow to the hair follicles. This prevents hair loss and stimulates
re-growth resulting in longer, thicker and increased numbers of hair.
•Minoxidil is available as a topical solution or foam in various strengths,
typically ranging from 2% to 5%. It is applied directly to the scalp once or
twice a day, and results can typically be seen within three to six months of
use. It is generally considered safe and well tolerated, although some users
may experience side effects such as scalp irritation, itching, and drynes.

12. • Name-- aminexil
• Chemical name – 2, 4-diaminopyrimidine-3-N-oxide
• Structural formula -
• Melting point – 110-218°c
• Solubility -- slightly soluble in water
• Appearance –white crystalline powder
• Molar mass—129.245g/mol

13. •Material & Methods
• Material of HPLC-
1.Millipore water - 99.9% pure HPLC
2.Methanol - 99.9% pure HPLC
3.Acetonitrile - 99.9%pure HPLC
•Methods
• Selection of wavelength
•Preparation of mobile phase
•Preparation of standard solution
•Preparation of working sample
•Validation system suitability
•Precision repeatability
•Selection of detector

14. •Result & discussion
•A RP-HPLC method was developed and validated for simultaneous
determination of minoxidil and aminexil in a combined topical liquid
formulation.
• Isocratic separation was achieved on a C18 column (250 mm × 4.6 mm i.d.,
particle size 5 μm, ThermoHypersil BDS) using a mobile phase consisting of
methanol-phosphate buffer (pH 3.0, 60:40, v/v) at a flow rate of 1 mL/min and
UV detection at 250 nm.
• Linearity was observed over the concentration range of 1.0-2.0 mg/mL for both
minoxidil (r2 = 0.99) and aminexil (r² = 0.99). The average percentage recovery
of the method was 101.0 % with a relative standard deviation of 0.14 %.
•The limits of detection (LODs) were 0.7346 μg/mL and 0.2423 μg/mL for
minoxidil and aminexil and limits of quantification (LOQs) were 0.7346 μg/mL
and 0.2423 μg/mL, respectively. The method was validated as per ICH
guidelines.

15. :

16. 1.Sheri, M. M., & El-Kousy, S. M. (2017). “Stability-indicating RP-HPLC method for
the determination of pyrantel pamoate in bulk and pharmaceutical formulation”.
Journal of Analytical Science and Technology, 8(1), 2.75. .
2.Aggarwal, G., Bhatia, M. S., Singh, G., & Kumar, V. (2017). “Development and
validation of an RP-HPLC method for the simultaneous determination of lopinavir and
ritonavir in a pharmaceutical formulation”. Journal of Liquid Chromatography &
Related Technologies, 40(4), 222-229.6. 57.
3.Reddy, P. S., Ramya, K. V. M., & Rao, J. V. (2019). " Development and validation of a
new stability-indicating RP-HPLC method for the simultaneous determination of
tazarotene, clindamycin phosphate, and betamethasone dipropionate in topical gel
formulation". Journal of Chromatographic Science, 57(5), 427-436.58.
4. Bhadra, S., Thakur, A., Jain, P., & Garg, S. (2018). "Development and validation of a
stability-indicating RP-HPLC method for simultaneous estimation of clindamycin
phosphate and adapalene in a topical gel formulation". Journal of Liquid
Chromatography & Related Technologies, 41(10), 580-589.
•Reference