Analytical Method Development and Validation by UPLC

Abstract

Ultra-Performance Liquid Chromatography (UPLC) represents a major advancement of high-performance liquid chromatography (HPLC) by employing sub-2 ?m stationary-phase particles and ultra-high-pressure pumping. The reduced diffusion path and enhanced mass transfer yield markedly higher column efficiency, increased peak capacity, and substantially shorter run times, enabling rapid, high-resolution separations with improved sensitivity. Typical configurations use sub-2 ?m C18 or hybrid-phase materials, small-diameter columns, and gradient elution with volatile mobile-phase modifiers, with detection primarily by UV–Vis, mass spectrometry, or tandem MS for enhanced selectivity in complex matrices. Advantages over conventional HPLC include faster analyses, reduced solvent consumption, and improved quantitative performance for trace-level analytes across diverse fields such as pharmaceutical analysis, clinical chemistry, environmental monitoring, and food safety. However, the elevated operating pressures demand robust instrumentation, durable columns, and careful method transfer and maintenance. This overview discusses the principles, instrumentation, column chemistries, method development considerations (gradient optimization, pH, temperature), and detection strategies, illustrated by representative applications, and addresses current limitations and future directions for UPLC in routine analytical workflows.

Keywords

UPLC, Method Development, Validation

Introduction

This sophisticated system takes lower time of separation. High-performance Liquid Chromatography, on the basis, is more fine-tuned, perfect, and sensitive. Adsorption, partition, ion exchange, and gel achromatism  Principle. Separation of in this kind of chromatography is composites are processed according to their respective polarities. These composites' interaction with the stationary phase of the column is also considered. Equipment for Conducting high-performance liquid chromatography comprises A pump( for pushing the analyte and mobile phase through the column), a sensor, and a stationary phase. HPLC is the most commonly used separation technique for  detection , separating, and quantifying drugs.  to get the most out of the procedure, and a number of chromatographic parameters were investigated, including sample pretreatment, mobile phase, column selection, detector selection (1). Ultra-performance liquid chromatography( UPLC) is a new order of separation fashion grounded upon well- established principles of liquid chromatography, which utilizessub-2 μm patches for stationary phase. These patches operate at elevated mobile phase direct rapidity to affect dramatic increase in resolution, perceptivity and speed of analysis Because of its speed and perceptivity, this fashion is gaining considerable attention in recent times for medicinal and biomedical analysis(2). It improves in three areas Chromatographic resolution, speed and perceptivity analysis. It uses fine patches and saves time and reduces solvent consumption UPLC is comes from HPLC (3)) It is a method of separating a mixture of components into individual components through a porous medium under the influence of the solvent (4). Lower injection volume is needed for UPLC which results in advanced effectiveness and increase in resolution. The advanced column temperature reduces the mobile phase density performing in the high prolixity measure and inflow rate without significant loss in  and increase in column hack pressure(5).The immaculate separation method UPLC has many advantages like robustness, case of use, changeable sensitivity and selectivity but the main limitation is lack of efficiency in comparison to gas chromatography Or capillary electrophoresis (6).

PRINCIPLE:

UPLC uses sub.2um particle technology in its stationary phase. The system follows the principle postulated by Van Demeter equation that basically describe a relationship among the flow rate and height of the plate. The equation for the same is as under:

H =A+B/V +CV

Were, A, B and C are constant.

A= Eddy diffusion, H HETP, B= Longitudinal diffusion, C=Equilibrium mass transfer V=flow rate.

The relationship between direct inflow rate and plate height( HETP or column effectiveness). It was set up that HETP decreases to a minimal value and also increases with adding inflow rate(6). In UPLC chromatogram it's set up that better resolution and separation are set up as compared to HPLC along with perform more sensitive analysis, reduce consumption of detergent and has high speed of analysis(7). The use of UPLC has helped in the detection of drug metabolites and enhancement of the quality of separation spectra(8). The technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles and/or higher flow rates for increased speed, with superior resolution and sensitivity.(9)

For enhancement of the effectiveness of UPLC following measures can be taken

1. Reduction of the mobile phase’s density by applying high temperature, which increases the inflow rate significantly, eventually dwindling the backpressure.
2. Use of monolithic columns that provides better resolution and increased perceptivity, leading to dropped solvent consumption.(10)

INSTRUMENTATION:

Pumping Device:

UPLC pumps ideally deliver solvent at around 15000 psi having its highest efficiency across a 15 cm long column that is packed with 1.7um particles.

Pumps used in UPLC can be classified as :

• Constant Flow Pump, which is used for all typical UPLC applications.
• Constant Pressure Pump used mainly for column packing.(11)

Sample Injection:

In UPLC, sample preface is critical. Conventional injection faucets, either automated or homemade, are n't designed and hardened to work at extreme pressure. To cover the column from extreme pressure oscillations, the injection process must be fairly pulsefree and the swept volume of the device also needs to be minimum to reduce implicit band spreading. A fast injection cycle time is demanded to completely subsidize on the speed swung by UPLC, which in turn requires a high sample capacity. Low volume injections with minimum carryover are also needed to increase perceptivity. There are also direct injection approaches for natural samples.(7)

History of Small Particle :

Using the sub 2um particle technology in the stationary phase of UPLC, the efficiency is increased and retained. There is no observation of diminishing efficiency after that. Also, the speed and the peak capacities are taken Into new limits with the Introduction of smaller particles. As the particle size in the stationary phase decreases, the linear velocity or the flow rate increases. Once the optimized flow rate is obtained, it remains constant. As the particle size in the column increases more than 1.9 um decrease in efficiency can be seen after a certain period. For proper retention instead of commercially available non-porous small particles, novel porous particles can withstand high pressure. Sol-gel synthesis containing columns with carbon in methyl groups form makes it mechanically strong and efficient and can also work in a wide range of pH.(12)

Detector:

After separation, the analyte needs to be detected both qualitatively and quantitatively. For this purpose, the UPLC system can be equipped with PDA, ELS, TUV, and FLR detectors individually or in combination

1. PDA (Photodidode Array)Detector

This is basically an optical detector that has the capacity of absorbing UV-Visible light ranging from 190-500um.(13)

2. ELS (Evaporative Light Scattering) Detector:

This technique basically involves the mixing of the solvents with a carrier gas which is inert in its nature. This is followed by forced nebulization after elution is completed which aids in separation of the liquids to minute aerosolized droplets. Moreover, the droplets are also allowed to pass through a heated tube as because the mobile phase evaporates and the droplets becomes smaller that is further pushed by the carrier gas towards the detection zone.(14)

3. TUV (Tunable Ultraviolet) Detector:

The ACQUITY UPLC TUV Detector is dual wavelength ultraviolet/visible (UV/Vis) detector and tunable that offers optimal resolution, linearity, and sensitivity separations via UPLC. (15)

Column:

The packing materials in UPLC columns are approximately 1.7um which gives the benefit of faster separation. As the bonded stationary phase is required to provide selectivity and retention, the particles are connected in the matrix. Different column types are made by ACQUITY, available in the market, and can be used for the UPLC technique.

Commonly used UPLC column are equipped with the following technologies:

• BEH technology: B?? ?18, ??? ??, ?EH phenyl, BEH shielded RP18 columns
• Protein separation technology: BEH200, BEH123, BEH450, SEC columns
• Peptide separation technology: BEH 300, BEH
• 130 Oligoneuclotides separation technology: OST C18 Columns
• Glycin separation technology: BEH glycan column

a. ??? ?18 Columns:

Acquity UPLC BEH C18 columns having a particle size of 1.7um high degree of peak symmetry, chemical stability, and efficiency. They work in the pH range of 1-12 and temperature up to 80° C. (16).

b. ??? ?8 Columns :

When compared to C18 columns, Cs columns provide low hydrophobicity, causing lower retention and rapid elution due to the presence of shorter aralkyl chain length. But this column also provides good peak symmetry, stability, and efficiency.(17)

c. BEH shield RP 18 columns:

This variant of columns basically have a polar group that is embedded which in turn combines the hydrophilicity a polar group embedded with the hydrophobicity of a straight- chain alkyl ligand C18. (18)

d. BEH phenyl columns:

Due to pi-pi interactions, complementary selectivity to straight chain alkyl phases is given by this column.(19)

e. BEH200, BEH123, BEH450 Columns:

The determinations are provided accurately by this column which is faster than traditional HPLC SEC assays. (20)

Advantages :

• Drop in consumption of mobile phase volume by at least 80 compared to HPLC.
• Dropped run time and cost of operation.
• Lower injection volume is needed.”
• Greater Signal to Noise rate( S/ N) due to the reduction in band broadening thereby adding the
• Perceptivity.
• Because of better chromatographic peak resolution, the problem of ion repression fromco-eluting
• Peaks is greatly reduced. Faster resolving power.”
• The advanced column temperature minimizes the mobile phase density performing in the high prolixity measure without significant loss in effectiveness and increase in column back pressure.(21)

Disadvantages:

• Expensive Equipment -Costs more than HPLC.
• More Maintenance checks Requires frequent system.
• Shorter Column Life High pressure wears out columns faster.
• Not Always Compatible methods need adjustments. Some HPLC.(22)

Application:

Bottom of Form

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• Natural product and herbal medicine :

Ultra Performance Liquid Chromatography has the ability to provide high quality of separation and detection capability of active compound which is present in mixture.

Examples:

1. Ultra Performance Liquid Chromatography is used for multiple components for quantitative analysis in example analysis of Hyangsapyeongwisan which is traditional medicine and used in gastric disease.
2. Ginseng species.
3. analysis of ascorbic acid and dehydroascorbic
4. acid in liquid and solid vegetable samples for detection of pyrriozidine alkaloids in herbal medicines

• Identification of Metabolites:

UPLC/ MS/ MS32 offers unmatched perceptivity and delicacy in biomarker discovery.

Examples:

1. Fungal secondary metabolites,
2. UPLC- MSE was used for rapid-fire discovery and characterization of verapamil metabolites in rats
3. C) UPLC- pater- MS/ MS was used in the metabolic of the medicinal lawn Eleusine indica

• Drug Discovery:

Useful in medicine discovery process. UPLC system by using acquity BEH C18 column that system is briskly and sensitive as compare to HPLC system. Affect with lower reagents bring and employed in internal quality control in different lozenge type.

Examples:

1. Mango splint tea metabolites”,
2. Analysis of fenofibrate in mortal Tube.
3. Determination of Mesa amine related contaminations from medicine sproduct by reversed phase validated UPLC system”

• Method Development:

Validation to reduce cost and improving opportunities for business  success

Examples:

1. Butoconazole in active ingredient
2. Cefditoren pivoxil in API (active pharma Ingredient).
3. Glibenclamide in rat plasma.
4. UPLC method determination of sofosbuvir and daclatasvir in human plasma for therapeutic drug monitoring

• Impurity profile:

Reversed phase UPLC methods are highly useful for quantitative determination of active pharmaceutical compound.

Examples:

1. Impurities in Maraviroc,
2. Determination of products and process impurities of asenapine maleate in asenapine sublingual tablets by UPLC”,
3. Impurities of halobetasol propionate in cream.

• Amino acid determination:

 The UPLC Also suitable for analysis of different amino acids by dependable, fast with high perceptivity and reputability” coupling with MS technologies. The styles are

Exemplifications

1. System confirmation for amino acids
2. Quantification of sulphur amino acids in submarine pets For quantify amines and amino acids in mortal complaint phenotyping. UHPLC- UV was applied for the analysis of total amino acid in child formulas and adult nutritional.

• Determination of Pesticides:

Combination of UPLC-MS/MS is effective for determination of pesticides. The instrument technique provides highly accurate with less matrix result

Examples:

1. For pesticide analysis in different fruit and vegetable
2. Analysis of residual pesticides and mycotoxins in cannabis
3. Pesticides analysis of vegetables by UPLC in combination with mass spectrometry. (23)

ANALYTICAL METHOD DEVELOPMENT ON UPLC:

When there no definitive techniques are present, new methodologies are being progressed for evaluation of the novel product. To investigate the presence of either pharmacopeial or non-pharmacopoetal product novel techniones are developed to reduce the value besides time for higher precision and strength These methodologies optimised and valid through preliminary runs. Alternate ways are planned and place into practice to exchange the present procedure within the comparative laboratory information with all accessible merits and demerits.(24)

Necessity of system development

Medicine evaluation exhibits the identity characterization and resolution of the medicines in combination like lozenge forons and organic fluids. At some point of producing fashion and development of medicine the top purpose of logical strategies is to Induce data regarding effectiveness , contamination, bioavailability and stability and effect of manufacturing parameter to varify that production of drug product is steady. (25)

Critic befare the development of new technologies, do n't forget below citation criteria

1. Is this fashion ponemes the needful perceptivity?
2. Is this system sufficiently picky fur direct use without hindrance by means of the contrary olement within the sample
3. Is the delicacy and perfection realizable with this fashion?
4. Are the reagents and outfit needed on this system available or attained at a reasonable price?
5. Is the time requires to perform this fashion applicable. (26)

Way for developing a system

Colorful way is involved in the development of a logical

System are as follows:

• Characterization of analyte and standard

All the known necessary data concerning the analyte and its structure that’s to mention the physical and chemical parcels similar as solubility, optic isomerismi, etc., are collected.

The standard analyte Is equal to 100 chastity is acquired Necessary arrangement is be created for the proper storehouse( refrigerator, desiccators, and freeser)

In the sample matris, when multiple corridor are to to be be measured the quantum of rudiments is observed properly presenting the information and the accesibility of standard are calculated

Ways like spectroscopy( UV-Visible, FTIR, infinitesimal immersion spectroscopy, etc.), high- performance liquid chromate- graphy and gas chromatography so on and, are still about formerly coordinated with the stability of samples.(27)

• Requirement of the techniques:

Requirement of analytical methodology is essential to build up the analytical fig of advantage like linearity, selectivity, specificity,  range, accuracy, precision LOD, LOQ etc. shall be outlined. (28)

• Literatare survey and prior methods:

All the data of literature related to the drug are reviewed for its physical and chemical properties, manufacturing, solubility and applicable analytical ways with reference to relevant hooks, journals, united states pharmacopeia/national formulary(USP/NF), association of official agricultural chemists (ADA) and american society for testing and materials (ASTM) pahlications and it is ustremely convenient to look Chemical Abstracts Service automatic computerized literature.(29)

• Opting the system:

Vilting the data attained from the literature, the methodology evolving since the system in being modified wherever demanded. Occasionally, it’s important to acquire fresh Instrumentation to produce, alter or replicate and validate being procedures for analytes and tests.(30)

• Proper instrumentation and initial studies:

Installation qualification (IQ), operation qualification (OQ), and performance qualification (PQ) of instrument pertinent to research standard methodology is examined by appropriate set up of instruments. (31)

• Optimization:

 While performing optimization, once a parameter is modified at a time, and a group of conditions are discerned, before exercising trial and error approach. This work is demanded for accomplished grounding on a scientific systematized system plan properly each necessary points and proved with relation to dead ends.(32)

• Proper documentation of analytical of merits:

 The true determined analytical of benefit consisting of LOD, LOQ, cost linearity and evaluation time and planning of samples, etc. are alao recorded.(33)

• Evaluation of produced technique with actual specimen:

The specimen solution needs to prompt specific, complete recognition of the peak interest of the medication other than all different matrix parts. (34)

VALIDATION

Confirmation is a conception developed in the United States in 1978. The conception of confirmation has been broaden over the times to achieve numerous conditioning like from logical styles used to control quality of medicine substances and medicine products up to computerized systems for clinical trials, process control or labelling, confirmation is stylish seen as a necessary and high part of cGMP. The word confirmation means evaluation of validity or the act of proving effectiveness. Confirmation is a platoon work involving people from different branches of shops System confirmation is a” process of establishing proved substantiation” Method thing document that provides a high position of guarantee that the product( outfit) will meet the conditions of the asked logical operations.(35)

Importance of validation:

• Assurance of quality.
• Minimal batch failure
• Reduction in rejections
• Improved efficiency and productivity
• Increased output
• Reduced testing in process and in finished goods. (36)

Types of validation :

There are four types of validation:

1. Equipment validation.

1. Design Qualification
2. Installation Qualification
3. Operational Qualification
4. Performance Qualification

2. Process validation

1. Prospective validation
2. Retrospective validation
3. Concurrent validation
4. Revalidation

3. Analytical method validation

4. Cleaning validation. (37)

Types of analytical procedures to be validated:

1. Identification tests
2. Quantitative tests for impurities content
3. Limit tests for the control of impurities

Steps in method validation:

1. Define the scope, purpose and applications of the method.
2. Define the performance parameters and its acceptance criteria.
3. Define validation experiments.
4. Verify rulated performance characteristics of equipment.
5. Qualify materials, ex. Standards and reagent
6. Perform pre-validation experiments.
7. Adjust method parameters or/and acceptance criteria if required.
8. Perform full internal (and external) validation experiments.
1. Develop SOPs for implementing the method in the routine
10. Define criteria for revalidation
11. Define type and frequency of system suitability texts arud/or Ana lytical Quality Control (AQC) checka for the routine.
50. Document valulation experiments and results in the validation.(38)

Parameters (components) of method validation

• Accuracy
• Precision
• Linearity
• Limit of detection
• Limit of quantitation
• Specificity
• Range
• Robustness

1) Accuracy:

Accuracy is defined as the closeness of the test results to the true value.(39) Accuracy is expressed as the nearness of agreement between the values found and values that are already available. It can also be defined as the closeness between the true value and the observed value. It is sometimes called as trueness, and it could be determined by using at least 9 determinations over a minimum of 3 concentration over the specified range.(40)

2) Precision :

Precision is defined as the measuresment of closeness of agreement for multiple measurements on the same sample. The precision is expressed as the relative standard deviation. (41) The exactness of an analytical procedure expresses the nearness of agreement (degree of scatter) between a group of measurements obtained from different sampling of a uniform sample underneath the prescribed conditions.(42)

3) Linearity :

Linearity is the ability of analytical procedure in obtain a response that is directly proportional to concentration (amount) of analyte in the sample. Linearity is expressed as the confidence limit around the slope of the regression line.(43) Linearity may be characterized as the capacity of a analytical technique to  produce outcomes which  are directly related to the concentration of an analyte .(44)

4) Limit of detection (LOD):

LOD is defined as lowest amount (concentration) of analyte in a sample that can be detected or identified not quantified. LOD Dis expressed as a concentration at a specified signal noise ratio, usually 3:1.

 LOD= 3.3×S/SD.(45)

Lowest quantity of an analyte which may be detected by the chromatographical separation however it is not necessary  that this quantity will quantify as a precise value. Blank resolution is Injected and peak to peak quantitative noise relation we have calculate from blank chromatograms.(46)

5) Limit of Quantitation :

LOQ is defined as lowest amount (concentration) of analyte is a sample that can be quantified. For LOQ ICH has recommended a signal: noise ratio 10:1.

LOQ =10×S/SD.(47)

Some usual ways, styles for the assessment of LOD and LOQ are as follows

• Visaal examination,
• gesture to noise rate,
• Standard divagation of the blank, and
• Retrogression line at low attention.(48)

6) Specificity :

Particularity is defined as the capability of an logical system to measure the analyte easily in the presence of other factors.

This description has following recrimination:

• Identification
• Chastity tasta
• Assay (49)

CONCLUSION:

Ultra-Performance Liquid Chromatography (UPLC) is a highly efficient analytical technique that provides faster, more sensitive, and higher-resolution separations compared to conventional HPLC. By utilizing smaller particle sizes and operating at higher pressures, UPLC significantly reduces analysis time and solvent consumption while improving peak capacity and detection limits. Its enhanced performance makes it a powerful tool for quantitative and qualitative analysis in pharmaceuticals, biotechnology, environmental testing, and food chemistry. Overall, UPLC offers superior analytical accuracy, reliability, and throughput, establishing it as a preferred method for modern chromatographic applications.

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