Systematic Reversed-Phase Liquid Chromatography Method Development for Pharmaceutical Degradation: A study on Mometasone Furoate
(2025) KASM01 20251Centre for Analysis and Synthesis
- Abstract
- HPLC is one of the most widely used analytical techniques in the pharmaceutical industry used for separation and identification of compounds in complex formulations.
Despite its advantages, HPLC method development remains challenging. This is due to the need to optimize multiple interdependent parameters simultaneously, while gaining high precision, reproducibility, and efficiency. Even minor changes to a single parameter can significantly impact the entire outcome, making the process complex. More traditional HPLC method development approaches, such as trial-and-error, are inefficient and often lacks reproducibility. While pharmacopeial methods provide a good foundation for analysis, they are not always suitable for samples with... (More) - HPLC is one of the most widely used analytical techniques in the pharmaceutical industry used for separation and identification of compounds in complex formulations.
Despite its advantages, HPLC method development remains challenging. This is due to the need to optimize multiple interdependent parameters simultaneously, while gaining high precision, reproducibility, and efficiency. Even minor changes to a single parameter can significantly impact the entire outcome, making the process complex. More traditional HPLC method development approaches, such as trial-and-error, are inefficient and often lacks reproducibility. While pharmacopeial methods provide a good foundation for analysis, they are not always suitable for samples with increased complexity arising from sources of impurities and degradation products, showcasing the need for systematic optimization strategies.
The aim of this work is to develop and optimize a systematic HPLC method development using experimental design principles to improve resolution of present chemical substances, robustness of the method, and separation efficiency in pharmaceutical analysis.
The study follows a four-phase approach: screening of different columns including different stationary phase chemistries and column dimensions, screening of eluent conditions, optimization using iterative testing, and lastly, robustness testing to evaluate sensitivity to minor variations. A skin-cream formulation with the active drug mometasone furoate, which is a neutral and hydrophobic compound, including common impurities and degradation products, was used as a model sample for the study.
Screening of four different stationary phases using RP-HPLC identified Super C18 and Super Phenyl Hexyl columns to be better fitted for further testing due to improved impurity resolution. Four different particle sizes with similar dimensions were also tested, where 3 µm particles yielded the best efficiency and peak shapes. Out of six different injection volumes, 8 µL gave optimal peak symmetry. Mobile phase optimization with linear gradients of twelve different compositions found that combinations involving water, acetonitrile, and either THF or methanol gave the highest theoretical plate counts and resolution. Method validation showed better precision and reproducibility with the water-acetonitrile-methanol system, although complete resolution of all impurities was not achieved.
Systematic method development using experimental design improved chromatographic efficiency and robustness, enhancing impurity resolution for mometasone furoate. While fully improved impurity resolution was not completed during this time span, the developed method and optimization plan offer a structured approach to improve reproducibility and efficiency in pharmaceutical quality control. (Less) - Popular Abstract (Swedish)
- Inom läkemedelsindustrin är det väldigt viktigt att göra en noggrann kontroll av läkemedel så att de är tillräckligt rena för personen som ska använda dem. En vanlig analysteknik i industrin är HPLC (High Performance Liquid Chromatography), som används för att separera och identifiera olika ämnen i exempelvis krämer eller tabletter. Analysmetoden används för att upptäcka orenheter i produkterna.
Att utveckla effektiva HPLC-baserade metoder för renhetsbestämning av läkemedel är ofta svårt och tidskrävande. Traditionellt har man testat sig fram för att ta fram färdiga HPLC metoder, vilket kan fungera, men är rätt ofta ineffektivt och räcker inte alltid till när man ska upptäcka små mängder av föroreningar i mer komplexa prover. Därför... (More) - Inom läkemedelsindustrin är det väldigt viktigt att göra en noggrann kontroll av läkemedel så att de är tillräckligt rena för personen som ska använda dem. En vanlig analysteknik i industrin är HPLC (High Performance Liquid Chromatography), som används för att separera och identifiera olika ämnen i exempelvis krämer eller tabletter. Analysmetoden används för att upptäcka orenheter i produkterna.
Att utveckla effektiva HPLC-baserade metoder för renhetsbestämning av läkemedel är ofta svårt och tidskrävande. Traditionellt har man testat sig fram för att ta fram färdiga HPLC metoder, vilket kan fungera, men är rätt ofta ineffektivt och räcker inte alltid till när man ska upptäcka små mängder av föroreningar i mer komplexa prover. Därför behövs det ett mer strukturerat sätt att utveckla metoderna.
I detta arbete togs en mer strukturerad HPLC-strategi fram, i syfte att förbättra förmågan av att separera och upptäcka föroreningar i läkemedel. Undersökningarna gjordes på en hudkräm med det aktiva ämnet Mometasone Furoate och arbetet delades in i fyra olika steg: först gjordes en undersökning av kolonner och mobilfaser, vilka är två delar av analysmetoden som har stor påverkan på separationen av ämnena som undersöks. Därefter optimerades inställningarna så som injektionsvolymer m.m. genom att jämföra replikat, följt av robusthetstester för att se hur känslig metoden är för små förändringar. Slutligen utvärderades resultaten.
Resultaten visade att två kolonner, SuperC18 och Super Phenyl Hexyl, fungerade bäst för att separera föroreningar. Utöver detta, gav generellt mindre partiklar i kolonner och en injektionsvolym på 8 µL av provet bättre resultat och bland de mobilfasblandningarna som testades fungerade en blandning av vatten, acetonitril och metanol bäst.
Trots att alla föroreningar inte kunde separeras helt med bra upplösning, visar studien att en planerad och strukturerad metodutveckling har stor potential att förbättra upplösningen och separationen av orenheter, samtidigt som tiden för utförandet och mängden lösningar minskar. Genom noggrann planering och experimentell undersökning kan optimala HPLC-metoder tas fram mer effektivt, vilket gör analysarbetet mer tillförlitligt och därmed säkrare för patienterna. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9204506
- author
- Nilsson, Nicole LU
- supervisor
- organization
- course
- KASM01 20251
- year
- 2025
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- HPLC, Chromatography, Analytical method, Pharmaceutical analysis, Method development, Analytical chemistry
- language
- English
- id
- 9204506
- date added to LUP
- 2025-06-25 12:02:55
- date last changed
- 2025-06-25 12:02:55
@misc{9204506,
abstract = {{HPLC is one of the most widely used analytical techniques in the pharmaceutical industry used for separation and identification of compounds in complex formulations.
Despite its advantages, HPLC method development remains challenging. This is due to the need to optimize multiple interdependent parameters simultaneously, while gaining high precision, reproducibility, and efficiency. Even minor changes to a single parameter can significantly impact the entire outcome, making the process complex. More traditional HPLC method development approaches, such as trial-and-error, are inefficient and often lacks reproducibility. While pharmacopeial methods provide a good foundation for analysis, they are not always suitable for samples with increased complexity arising from sources of impurities and degradation products, showcasing the need for systematic optimization strategies.
The aim of this work is to develop and optimize a systematic HPLC method development using experimental design principles to improve resolution of present chemical substances, robustness of the method, and separation efficiency in pharmaceutical analysis.
The study follows a four-phase approach: screening of different columns including different stationary phase chemistries and column dimensions, screening of eluent conditions, optimization using iterative testing, and lastly, robustness testing to evaluate sensitivity to minor variations. A skin-cream formulation with the active drug mometasone furoate, which is a neutral and hydrophobic compound, including common impurities and degradation products, was used as a model sample for the study.
Screening of four different stationary phases using RP-HPLC identified Super C18 and Super Phenyl Hexyl columns to be better fitted for further testing due to improved impurity resolution. Four different particle sizes with similar dimensions were also tested, where 3 µm particles yielded the best efficiency and peak shapes. Out of six different injection volumes, 8 µL gave optimal peak symmetry. Mobile phase optimization with linear gradients of twelve different compositions found that combinations involving water, acetonitrile, and either THF or methanol gave the highest theoretical plate counts and resolution. Method validation showed better precision and reproducibility with the water-acetonitrile-methanol system, although complete resolution of all impurities was not achieved.
Systematic method development using experimental design improved chromatographic efficiency and robustness, enhancing impurity resolution for mometasone furoate. While fully improved impurity resolution was not completed during this time span, the developed method and optimization plan offer a structured approach to improve reproducibility and efficiency in pharmaceutical quality control.}},
author = {{Nilsson, Nicole}},
language = {{eng}},
note = {{Student Paper}},
title = {{Systematic Reversed-Phase Liquid Chromatography Method Development for Pharmaceutical Degradation: A study on Mometasone Furoate}},
year = {{2025}},
}