Development and Validation of Stability Indicating UPLC Method for the Simultaneous Estimation of Drugs in Combined Dosage Forms using Quality by Design Approach

P. Geetha Bhavani¹*, D. Akila Devi²

¹Research Scholar, Department of Pharmaceutics, School of Pharmaceutical Sciences,

Vels Institute of Science Technology and Advanced Studies, Pallavaram, Chennai - 600117.

²Associate Professor, Department of Pharmaceutics, School of Pharmaceutical Sciences,

Vels Institute of Science Technology and Advanced Studies, Pallavaram, Chennai - 600117.

*Corresponding Author E-mail: akilaajcp@gmail.com

ABSTRACT:

UPLC is a modern technique which gives a new direction for liquid chromatography. UPLC refers to ultra performance liquid chromatography, which enhance mainly in three areas: speed, resolution and sensitivity. Ultra performance liquid chromatography (UPLC) applicable for particle less than 2μm in diameter to acquire better resolution, speed, and sensitivity compared with high-performance liquid chromatography (HPLC). The concept of “Quality by Design” (QbD) is an approach which covers a better scientific understanding of critical process and product qualities, designing controls and tests based on the scientific limits of understanding during the development phase and using the knowledge obtained during the life-cycle of the product to work on a constant improvement environment.

KEYWORDS: Ultra performance liquid chromatography, resolution.

INTRODUCTION:

A precise measurement of drug levels both in a pharmaceutical industry’s perspective and health care setup is the need of the hour. Pharmaceutical companies spend extravagantly and also untiringly, day in and day out, to delineate a single successful drug moiety from thousands of lead compounds. In this pursuit, they rely on bio-analytical techniques which could help them in separating the most active elements from the crude mixtures.

And also, later on in pre-clinical and clinical testing accurate measurement of drug levels in biological tissues using suitable Liquid chromatography are indispensable. Moreover, a treating physician depends on drug levels especially for those drugs with narrow therapeutic margin. Though the qualitative and quantitative analytical methods existing today are more sophisticated and complex, they actually originated and evolved from the roots of chromatography. Hence, chromatography still prevails as the most significant analytical method in molecular chemistry despite being primitive. This current indisputable status of chromatography is reflected by the fact that majority of the present techniques is based on the principle of chromatography.

At the early stage of drug discovery when a large number of closely related compounds is synthesized, these are required to be separated. Its identification and purity testing is very essential. Revealing the purity and/or impurity of the synthetic compounds is inadequate without the chromatography. During the various preclinical and clinical closely related components of complex mixtures; many of this separation are impossible by other means. Chromatography is not restricted to analytical separations. It may be used in the preparation of pure substances, the study of kinetics of reactions, structural investigations on the molecular scale, and the determination of physicochemical constants, including stability constants of complexes, enthalpy, entropy, and free energy. Chromatography is the separation and identification technique. Structurally or chemically similar components of homogeneous mixture can be separated using this technique. Separation is based upon component’s relative ability to adsorb and/or partition between mobile phase and the stationary phase. Depending upon separation principle, geometry of method, mode of chromatography, the technique is classified in various types. Consequently, the separation of the analyte from potential interference is quite often a rate limiting step in the research.⁽¹⁾

UPLC and Quality by Design:

UPLC is a modern technique which gives a new direction for liquid chromatography. UPLC refers to ultra performance liquid chromatography, which enhance mainly in three areas: “speed, resolution and sensitivity. Ultra performance liquid chromatography (UPLC) applicable for particle less than 2μm in diameter to acquire better resolution, speed, and sensitivity compared with high-performance liquid chromatography (HPLC).⁽²⁾

UPLC is a new separation technique with increased speed, sensitivity and resolution. The performance of a column can be measured in terms of the height equivalent to the theoretical plates (HETP or H), which is calculated from the column length (L) and the column efficiency, or number of theoretical plates (N). N is calculated from an analyst’s retention time (tR) and the standard deviation of the peak (σ).

H=L/N ------------------------------------------------------- (1)

N=(tR/σ)2 --------------------------------------------------- (2)

The van Deemter equation is the empirical formula that describes the relationship between linear flow velocity (μ) and column efficiency, where A, B, and C are constants related to the mechanistic components of dispersion.

H=L/N=A+B/μ+Cμ -(3) (Vandeemter Equation)

According to the van Deemter plot, column efficiency is inversely proportional to the particle size (dp) (Equation 4), so by decreasing the particle size there is an increase in efficiency. Since resolution is proportional to the square root of N (Equation 5), decreasing particle size increases resolution. Also, by using smaller particles, analysis time can be decreased without sacrificing resolution, because as particle size decreases, column length can also be reduced proportionally to keep column efficiency constant. By using the same HPLC mobile phase and flow rate, UPLC™ reduces peak width and produced taller peaks which increased the S/N 1.8 to 8 fold, improving both sensitivity and resolution.

Nα1/dp ----------------------------------------------------- (4)

R=√N/4(α-1/α)(k/k+1)------------------------------------ (5)

Also according to the van Deemter plot, use of particles smaller than 2μm produces no loss in column efficiency with increasing flow rates. However, by increasing flow rates to decrease analysis time, there is a corresponding increase in system pressure. As a result, a system capable of withstanding the proper pressures while still maintaining efficiency is required. As well, a mechanically stable column is needed.⁽³⁾

Application:

In pharmaceutical industry the demand of UPLC analysis is very high, because of the unique features of UPLC like high resolution in chromatogram, short time analysis which make more analytical work in less time with valuable, reliable and authentic data.

Scientist can generate more accurate data by UPLC in faster way. UPLC technique is used for the analysis of herbal product.

In analytical laboratory the demand of UPLC is very high because the method developed are accurate and précised and also this expand the research information of the analyte in nano level. By this method the standard of analysis in every respect like qualitative, quantitative and complexity of sample can be differentiate in very high standard.

The UPLC/MS system is used to generate a data which solved the complexity of the compound. By using MS as a detector with UPLC the interpretation of analysis is go to depth. Such analysis is very use full in bio-analytical field.

The unique features of UPLC that high resolution and speedy analysis also very helpful in pharmacokinetic studies like – adsorption, distribution, metabolism and excretion (ADME). ADME studies measure physical and chemical properties of compound. UPLC/MS method saves time. For the drug development and formulation process, profiling, detecting and quantifying drug substances and their impurities can be performed very accurately

Ultra-high-performance liquid chromatography (UPLC) has marked a radical change by opening new doors for analyst to fetch rapid analytical separation techniques without sacrificing high-quality results obtained earlier by high performance liquid chromatography (HPLC). The immaculate separation method UPLC has many advantages like robustness, ease of use, changeable sensitivity and selectivity but the main limitation is lack of efficiency in comparison to gas chromatography or capillary electrophoresis. UPLC is a derivative of HPLC whose underlying principle is that as column packing particle size decreases, efficiency and resolution increases. If we decrease particle size less than 2µm, the efficiency shows a significant gain1 by making use of the smaller particles, the speed of analysis and peak capacity i.e., number of peaks resolved per unit time, can be prolonged to the maximum values and these values are much better than the values achieved earlier by HPLC. Waters changed the landscape and future of chromatography with the ACQUITY Ultra Performance LC (UPLC) system. Chromatographers need no longer choose between the speed of short columns and the resolution of long columns. Separations scientists can now enjoy the best of both worlds; speed and resolution with the bonus of increased sensitivity. UPLC delivers more information faster without compromising data integrity. Waters ACQUITY UPLC systems are holistically designed to dramatically improve resolution, sample throughout and sensitivity.

Key innovations include:

· Small, pressure tolerant particles

· High pressure fluidic modules

· Minimized system volume

· Negligible carry over

· Reduced cycle times

· Fast response detectors

· Integrated system software and diagnostics.

UPLC applies the same principle as HPLC, the difference is the use of substance 2-µm particle columns in a system holistically designed to maximize the advantages of these columns, creating a powerful, robust and reliable solution. The familiar design of UPLC H-class’s Quaternary Solvent Manager (QSM) and Sample Manager (SM-FTN), with flow-through needle design, gives all the flexibility and usability of your current HPLC while still achieving the highly efficient separations that only UPLC can provide.

To improve the UPLC efficiency following measures need to be performed:

1. Elevated temperature range should be employed, which will allow high flow rate of mobile phase by reducing its viscosity and thus it will significantly reduce back pressure.

2. Monolithic columns should be used, which consist of one piece of solid that possesses interconnected skeletons and interconnected flow paths (through-pores). UPLC is a technique which comprises the above-mentioned features and stands better than HPLC in many ways as it shows better chromatographic resolution, performs more sensitive analysis, consumes less time, reduces solvent consumption and has high analysis speed.⁽³⁾

In all the chromatographic techniques, the sample is dissolved in a mobile phase which may be a liquid, a gas, or a supercritical fluid. This mobile phase is allowed to run over the stationary phase which is fixed in place on a solid surface. The two phases are selected so that the components of the sample distribute themselves between the mobile and stationary phase to varying degrees. Those components strongly retained by the stationary phase move slowly with the flow of mobile phase and elutes later on. In contrast, components that are weakly held by the stationary phase travel rapidly down the column and elutes first. This difference in migration rates make the sample components to separated into discrete zones which can be analyzed qualitatively or quantitatively. There are various parameters, based upon it the chromatographic methods.

High performance liquid chromatography (HPLC) is a well-known technique that has been used in laboratories worldwide from more than last 30 years. The factor responsible for the development of the technique was evolution of packing materials used to effect the separation.⁽³⁾

The efficiency of HPLC increased as particle sizes of the column packing decreased from 10 [micro] m in the 1970s to 3.5 Nun in the 1990s. This is shown by lower values of HETP (height equivalent to a theoretical plate) for van Demeter plots of HETP (column efficiency) versus mobile phase flow rate in units of linear velocity ([mu], mm/s; Figure 1). In this particle size range, and even down to 2.5 Nun particles used in shorter columns in the early 2000s, it was found that HETP decreases to a minimum value and then increases with increasing flow rate. However, with the 1.7 [micro] m particles used in UPLC, HETP is lowered compared to the larger particles and does not increase at higher flow rates. This allow faster separations to be carried out on shorter columns and/or with higher flow rates, leading to column increased resolution between specific peak pairs and increased peak capacity, defined as the number of peaks that can be separated with specified resolution in a given time interval.

Comparison between HPLC over UPLC

Parameters	HPLC Assay	UPLC Assay
Column	XTerra, C18, 50x 4.6mm	Aquity UPLC BEH C18, 50x2.1mm
Particle size	4µm particles	1.7µm particles
Flow rate	3.0 ml per min	0.6 ml per min
Injection Volume	20 µl	3 µl partial loop fill or 5 µl full loop fill
Total run time	10 min	1.5 min
Theoretical plate count	2000	7500
Column Temperature	30° C	65° C
Maximum back pressure	35-40 Mpa	103.5 Mpa
Resolution	Less	High
Method Development Cost	High	Low

Advantages of UPLC:

Various advantages of UPLC are as follows:

· Require less run time and enhance sensitivity.

· Provides the selectivity, sensitivity, and dynamic range of LC analysis.

· In chromatogram resolved peaks are obtained.

· Multi residue methods are applied.

· Speedy analysis, quantify accurately analytes and related products.

· Uses of fine particle (2μm) for packing of stationary phase make analysis fast.

· Time and cost both are reduced.

· Consumption of solvents is less.

· More products are analyzed with existing resources.

· Increases sample throughput and enables manufacturers to produce more material that consistently meet or exceeds the product specifications, potentially eliminating variability, failed batches, or the need to re-work material.

· Delivers real-time analysis in step with manufacturing processes.

· Assures end-product quality, including final release testing.⁽⁴⁾

Validation:

The developed method can be validated according to the ICH guidelines.

Components of validation:

· System suitability

· Accuracy

· Precision

· Linearity and Range

· Specificity

· Robustness

· Ruggedness

· Limit of detection

· Limit of quantitation

· Stability of samples, reagents, instruments.

i) System suitability:

Prior to the analysis of samples each day, the operator must establish that the HPLC system and procedure are capable of providing data of acceptable quality. This is accomplished with system suitability experiments, which can be defined as tests to ensure that the method can generate results of acceptable accuracy and precision. The USP defines parameters that can be used to determine system suitability prior to analysis. These parameters include,

· Plate number (N)

· Tailing factor (k and/or α)

· Resolution (R_S)

· Relative standard deviation (RSD) of peak height or peak area for repetitive injection

ii) Accuracy:

The ICH defines the accuracy of an analytical procedure as the closeness of agreement between the values that are accepted either as conventional true values or an accepted reference value and the value found. Accuracy is usually reported as percent recovery by assay, using the proposed analytical procedure, of known amount of analyte added to the sample. The ICH also recommended assessing a minimum of nine determinations over a minimum of three concentration levels covering the specified range (e.g., three concentrations/three replicates).

iii) Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samples of the same homogeneous sample under prescribed conditions. Precision is usually investigated at three levels: repeatability, intermediate precision, and reproducibility.

a) Repeatability (Precision):

Repeatability is a measure of the precision under the same operating conditions over a short interval of time. It is sometimes referred to as intraassay precision. Two assaying options are allowed by the ICH for investigating repeatability:

1. A minimum of nine determinations covering the specified range for the procedure (e.g., three concentrations/three replicates as in the accuracy experiment), or

2. A minimum of six determinations at 100% of the test concentration.

The standard deviation, relative standard deviation (coefficient of variation), and confidence interval should be reported as required by the ICH.

b) Intermediate Precision:

Intermediate precision is defined as the variation within the same laboratory. The extent to which intermediate precision needs to be established depends on the circumstances under which the procedure is intended to be used. Typical parameters that are investigated include day-to-day variation, analyst variation, and equipment variation. Depending on the extent of the study, the use of experimental design is encouraged. Experimental design will minimize the number of experiments that need to be performed.

c) Reproducibility:

Reproducibility measures the precision between laboratories as in collaborative studies. This parameter should be considered in the standardization of an analytical procedure (e.g., inclusion of procedures in pharmacopoeias and method transfer between different laboratories). To validate this characteristic, similar studies need to be performed at other laboratories using the same homogeneous sample lot and the same experimental design.

iv) Linearity:

The ICH defines the linearity of an analytical procedure as the ability (within a given range) to obtain test results of variable data (e.g., absorbance and area under the curve) which are directly proportional to the concentration (amount of analyte) in the sample. Quantitation of the analyte depends on it obeying Beer’s law and is linear over a concentration range. Linearity is usually demonstrated directly by dilution of a standard stock solution. It is recommended that linearity be performed by serial dilution of a common stock solution.

v) Range:

The range of a method can be defined as the lower and upper concentrations for which the analytical method has adequate accuracy, precision, and linearity. While a desired concentration range is often known before starting the validation of a method, the actual working range results from data generated during validation studies.

vi) Specificity:

Specificity is the ability to assess unequivocally an analyte in the presence of components that may be expected to be present. In many publications, selectivity and specificity are often used interchangeably. The specificity of a test method is determined by comparing test results from an analysis of samples containing impurities, degradation products, or placebo ingredients with those obtained from an analysis of samples without impurities, degradation products, or placebo ingredients. For the purpose of a stability indicating assay method, degradation peaks need to be resolved from the drug substance.

vii) Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in the analytical procedure parameters. The robustness of the analytical procedure provides an indication of its reliability during normal use. The evaluation of robustness should be considered during development of the analytical procedure. Common variations that are investigated for robustness include filter effect, stability of analytical solutions, extraction time during sample preparation, pH variations in the mobile-phase composition, variations in mobile-phase composition, columns, temperature effect, and flow rate.

viii) Ruggedness:

It is defined as the reproducibility of results when the method is performed under actual use conditions. This includes different analysts, laboratories, columns, instruments, sources of reagents, chemicals, solvents, and so on. Method ruggedness may not be known when a method is first developed, but insight is obtained during subsequent use of that method.

ix) Limit of detection:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. Detection limit (DL) corresponds to the concentration that will give a signal-to-noise ratio of 3: 1. It can be determined by the following equation.

3.3 × SD

DL= ––––––––––––.

where SD is the standard deviation of the response near DL and S is the slope of the linearity curve near DL.

x) Limit of quantification:

The quantification limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be determined quantitatively with suitable precision and accuracy. Quantification limit is defined as the concentration of related substance in the sample that will give a signal-to-noise (S/N) ratio of 10 : 1. The LOD and LOQ values determined during method validation are affected by the separation conditions: columns, reagents and especially instrumentation and data systems, Instrumental changes, particularly pumping systems and detectors, or the use of contaminated reagents. It can be determined by the following equation.⁽⁵⁾

QL=10 × SD

QUALITY BY DESIGN (QbD) APPROACH:

Definition [ICH Q 8(R1)]:

A Systematic approach for development that begins with predefined objectives and emphasizes of the product and process understanding as well as process control, based on sound science and quality risk management.

Definition [FDA PAT Guidelines, Sept. 2004]:

A system for designing, analyzing and controlling manufacturing through timely measurements (i.e. during processing) of critical quality and performance attributes of new and in-process materials and processes, with the goal of ensuring final product safety.

The concept of “Quality by Design” (QbD):

It is defined as an approach which covers a better scientific understanding of critical process and product qualities, designing controls and tests based on the scientific limits of understanding during the development phase and using the knowledge obtained during the life-cycle of the product to work on a constant improvement environment.

International Conference of Harmonization (ICH):

Relevant documents from the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. (ICH)

· Pharmaceutical Development Q8 (R2)

· Quality Risk Management Q9

· Pharmaceutical Quality System Q10

The difference between QbD for NDA and ANDA products is most apparent at the first step of the process. For an NDA, the target product profile is under development while for the ANDA product the target product profile is well established by the labeling and clinical studies conducted to support the approval of the reference product.

Seven steps of quality by design start up plan

1. Hire an independent Quality by design expert.

2. Audit your organization and process with the expert conducting a gape analysis.

3. Hold a basic quality by design workshop with all your personal.

4. Review the expert’s report and recommendation.

5. Draft an implementation plan, timelines and estimated costs.

6. Assign the resources (or contract out).

7. Retain the independent expert as your “Project Assurance” advisor.⁽⁶⁾

Advantages of QbD:

Benefits for Industry:

· Better understanding of the process.

· Less batch failure.

· More efficient and effective control of change.

· Return on investment/cost savings.

Additional opportunities:

· An enhance QbD approach to pharmaceutical development provides opportunities for more flexible regulatory approaches.

Ex: Manufacturing changes within the approved design space without further regulatory review.

· Reduction of post-approval submissions.

· Better innovation due to the ability to improve processes without resubmission to the FDA when remaining in the Design Space.

· More efficient technology transfer to manufacturing.

· Greater regulator confidence of robust products.

· Risk-based approach and identification.

· Innovative process validation approaches.

· Less intense regulatory oversight and less post-approval submissions.

· For the consumer, greater drug consistency.

· More drug availability and less recall.

· Improved yields, lower cost, less investigations, reduced testing, etc.

· Time to market reductions: from 12 to 6 years realized by amongst others.

· First time right: lean assets management.

· Continuous improvement over the total product life cycle (i.e. controlled, patient guided variability).

· Absence of design freeze (no variation issues).

· Less validation burden.

· Real time controls (less batch controls).

· Realistic risk perceptions.⁽⁷⁾

Importance of QbD in Analytical method development:

The application of QbD concept to analytical method is justifiable, because of many variables that significantly affect the method results. These variables are such as instrument settings, sample characteristics, method parameters, and choice of calibration models. Being chromatographic technique is the most common analytical tool in pharmaceutical quality control, and the number of variables involved in analytical method development phase is almost equivalent to the number of variables involved in formulation and development protocols for dosage form. As per FDA, analytical techniques and methods play an essential role in QbD paradigm, and real time release testing and nontraditional testing techniques provide valuable information for in-process control and improvement. Implementation of QbD provides an opportunity to achieve regulatory flexibility but requires high degree of robustness, product quality, and analytical method understanding. To adopt a suitable design of experiments (DOEs) protocol in AQbD approach to identify a validated MODR for high degree of process-product-analytical method understanding is recommended. ⁽⁸⁾

SUMMARY:

Advanced analytical methods were applied successfully for the determination of drugs in the pharmaceutical dosage forms. Stability studies can be applied for the cited drugs gave an indication about their stability under different stress conditions and their shelf life. Moreover we will compare the results obtained from UPLC method.

CONCLUSIONS:

UPLC is an advance technique of liquid chromatography where it takes advantage of innovation in various technologies such as instrumentation and particle size to achieve dramatic increases in resolution, speed and sensitivity of the liquid chromatography. It operates at higher pressure than that used in HPLC and uses fine particles (less than 2.5μm) & mobile phases at high linear velocities. UPLC Technology is now applied throughout the world produce quality data with reproducible and robust methods as compared to the conventional HPLC. UPLC can be hyphenated with other techniques such as Mass spectrometer (MS), Ion chromatograph (IC), Nuclear magnetic resonance spectrometer (NMR) and Infrared spectrometer (IR) etc. This technique provides unique end-to-end solutions for all industries and has found application in various fields such as pharmaceutical, food, environmental, forensic, toxicology and pesticide. An accurate data analysis tool is necessary to evaluate any process or system to assure that it works consistently as intended. Implementing QbD is one of the approaches that devoutly make scientist to understand the process or system closely. Optimizing process by QbD has become mandatory by some of the regulatory guidelines around the globe. Never the QbD approach can also be implemented successfully in analytical method development and optimization. Identifying critical parameters, performing risk assessment studies of critical parameters, using DOE for screening and method optimization are some of the milestones for QbD implementation, whenever magnificently applied will result in a robust method with fewer trials. Analyst also gains confidence in the method performance as the approach provides understanding between the method variables and performance. The overall advantage of the approach is improved method proficiency, reduced variability, less trials hence less method cost and reduced time consumption, knowledge about the extreme limitations of the method which when traversed may lead to method failures and at times method alternatives. Hence this approach can be well practiced for complexed chromatographic method where more number of analytes needs efficient separations.

REFERENCES:

1. Manisha Ch., "Significance of Various Chromatographic Techniques in Drug Discovery and Development.,” IJRPC., 3(2)., 2013.

2. Taleuzzaman M., “Ultra Performance Liquid Chromatography,” AJAPC., 2(6): 1056., 2016.

3. Rekha Priyadarshini., "Chromatography - the essence of bioanalysis.,” EJBPS., 3(2)., 366-377., 2016.

4. Sharath Chandra S., “Switch from HPLC to UPLC: A Novel Achievement in Liquid Chromatography Technique” IJPS., 21(1)., 237-246., 2013.

5. Rajneet K., “Analytical Quality by Design Approach for Development of a Validated Bioanalytical UPLC Method of Docetaxel Trihydrate.,” CPA., vol (11)., 2015.

6. Pavan Kumar V., “Quality by Design Approach (QBD) for Pharmaceuticals”., IJDDR., 2015.

7. Pharmaceutical “Quality By Design” (QbD): An Introduction, Process Development and Applications.

8. Ramalingam P., “Analytical Quality by Design: A Tool for Regulatory Flexibility and Robust Analytics.,” IJAC., 2015.

9. Karmarkar S., “Quality by Design (QbD) Based Development of a Stability Indicating HPLC Method for Drug and Impurities.,” JCS., Vol (49)., 2011.

10. Thakor N S., “Implementing Quality by Design (QbD) in Chromatography., ʺ AJAPC., 4(1): 1078., 2017.

Received on 17.04.2020 Modified on 30.04.2020

Asian J. Pharm. Ana. 2020; 10(3):158-164.

DOI: 10.5958/2231-5675.2020.00029.0