Development and Validation of an HPLC Method for the Simultaneous Estimation of Dolutegravir and Rilpivirine in Bulk and Pharmaceutical Dosage Forms

Pharmaceutical analysis is an essential discipline within pharmaceutical sciences that ensures the identity, purity, safety, and efficacy of drug substances and pharmaceutical formulations. It encompasses a wide range of analytical techniques used throughout drug development, manufacturing, and quality control processes. The primary objective of pharmaceutical analysis is to guarantee that drugs meet predefined specifications and regulatory requirements. (1,2,3,5,6)

Importance of High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC) is one of the most widely used analytical techniques for the separation, identification, and quantification of components in complex mixtures. It is especially valuable in pharmaceutical analysis due to its ability to handle thermally unstable, non-volatile, and high molecular weight compounds. (4,11,13,14)

HPLC offers several advantages, including: (4,13,14)

• High resolution and selectivity
• Rapid analysis with reproducible results
• Capability to analyze multiple components simultaneously
• High sensitivity for trace-level detection
• Compatibility with a wide range of detectors

INTRODUCTION TO HPLC

HPLC is an advanced form of column chromatography that operates under high pressure to increase the efficiency and speed of the separation process. It has become a standard method for analyzing complex mixtures in various industries, particularly pharmaceuticals. (4,13,14)

Principles of HPLC: The principle of HPLC is based on the partitioning of components between a stationary phase (usually packed inside a column) and a mobile phase (solvent that moves through the column). The different affinities of each component for the stationary and mobile phases cause them to elute at different times, known as retention times. (4,14)

Types of HPLC:

1. Normal Phase HPLC: Utilizes a polar stationary phase and a less polar mobile phase.
2. Reverse Phase HPLC (RP-HPLC): The most common type, uses a non-polar stationary phase and a polar mobile phase.
3. Ion Exchange HPLC: Separates ions and polar molecules based on their charge.
4. Size Exclusion HPLC: Also known as gel permeation or gel filtration, separates molecules based on size.
5. Affinity HPLC: Uses the specific interactions between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. (4,11,13,14)

Applications of HPLC:

1. Pharmaceuticals: Used for the analysis of active pharmaceutical ingredients (APIs), excipients, and formulations.
2. Environmental Monitoring: Detects pollutants and contaminants in water, air, and soil.
3. Food Industry: Analyzes food components for quality control and to ensure compliance with regulations.
4. Clinical Research: Assists in the study of biological samples, such as plasma, for drug development and disease markers.
5. Industrial Applications: Used in the production of chemicals and other materials to monitor process streams and ensure product purity.

HPLC is valued for its accuracy, precision, and ability to analyze a wide range of compounds. Its versatility makes it an indispensable tool in scientific research and quality control laboratories worldwide. (4,5,11,13)

System Suitability:  

According to the USP, system suitability tests are an integral part of chromatographic methods. These tests are used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed. System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples constitute an integral system that can be evaluated as a whole.  The purpose of the system suitability test is to ensure that the complete testing system (including instrument, reagents, columns, analysts) is suitable for the intended application. (9)

Similar to the analytical method development, the system suitability test strategy should be revised as the analysts develop more experience with the assay. (3,9)

System suitability is the checking of a system to ensure system performance before or during the analysis of unknowns. Parameters such as plate count, tailing factors, resolution and reproducibility (%RSD retention time and area for six repetitions) are determined and compared against the specifications set for the method. These parameters are measured during the analysis of system suitability "sample" that is a mixture of main components and expected by-products. Below Table lists the terms to be measured and their recommended limits obtained from the analysis of the system suitability sample as per current FDA guidelines on "Validation of Chromatographic Methods". (3,9)

 Accuracy: Defined as the closeness of agreement between the actual (true) value and mean analytical value obtained by applying the test method a number of times. Accuracy is acceptable if the difference between the true value and mean measured value does not exceed the RSD values obtained for repeatability of the method. (1,12)

Precision: It is a measure of degree of repeatability of an analytical method under normal operation and it is normally expressed as % of relative standard deviation (% RSD). This involves (1,12)

1. Repeatability
2. Reproducibility
3.  Intermediate precision

                   % RSD = 100 S/X

Where,            S = Standard deviation

            X = Mean

 It is determined at three levels.

Linearity: It is the ability of the method to elicit test result that is directly proportional to analytic concentration within a given range. It is generally reported as variance of slope of regression line. It is determined by series of three to six injections of five of more standards. (1,12)

Limit of Detection (LOD): It is defined as the lowest concentration of an analyte in a sample that can be detected but not quantified. LOD is expressed as a concentration at a specified signal to noise ratio. The LOD will not only depend on the procedure of analysis but also on the type of instrument. (1,12)

In chromatography, detection limit is the injected amount that results in a peak with a height at least twice or thrice as high as baseline noise level.

S/N = 2/1 or 3/1

LOQ is expressed as a concentration at a specified signal to noise ratio. In chromatography, limit of quantification is the injected amount that results in a peak with a height, ten times as high as base line noise level.

S/N = 10/1

Robustness: It is the measure of the capacity of the analytical method to remain unaffected by small but deliberate variation in procedure. It provides an indication about variability of the method during normal laboratory conditions. (1,12)

MOLECULAR STRUCTURE OF DOLUTEGRAVIR

 

 

MOLECULAR STRUCTURE OF RILPIVIRINE

 

 

MATERIALS AND METHODS:

Equipment’s and instruments used in the study:

S.No.

S.No	Name	RT(min)	Area    (µV sec)	Height (µV)	Resolution	USP tailing	USP     plate count
1	Dolutegravir	3.602	214546	8021578	4.57	1.06	4596
2	Rilpivirine	5.148	34475	124577		1.2	3153

System Suitability: Tailing factor for the peaks due to Dolutegravir and Rilpivirine in Standard solution should not be more than 2.0.  Theoretical plates for the Dolutegravir and Rilpivirine peaks in Standard solution should not be less than 2000

Calculation: (For Dolutegravir and Rilpivirine)

 

 

Where:

 AT      = average area counts of sample preparation.

 AS      = average area counts of standard preparation.

             WS     = Weight of working standard taken in mg.

  P        = Percentage purity of working standard

                                                                                                                                                                                LC       = Label Claim mg/ml.

Acceptance criteria of System Suitability:

1. Tailing factor should be less than 2
2. Theoretical Plates should be above 2000

RESULTS AND DISCUSSION

TRIAL - 1

Instrument used        : High performance liquid chromatography equipped  with Auto Sampler and PDA

Temperature               :            Ambient

Column                       : Platisil C₁₈, (250×4.6mm, 5mm)

Mobile phase              :  65% Methanol: 35% NaH2PO4 PH-4

 Flow rate                    :           0.9 ml per min

Wavelength                :           250 nm

Injection volume         :           10 ml

Run time                     :           10 min.

TRAIL 2

TRAIL 3

1 VALIDATION PARAMETERS:

Figure 1: Chromatogram for Standard

Figure 2: Chromatogram for Sample

Table 1: Results of Assay for Dolutegravir and Rilpivirine

Figure 3: Calibration graph for Dolutegravir

Figure 4: Calibration graph for Rilpivirine

Table 4: Analytical performance parameters of Dolutegravir and Rilpivirine

CONCLUSION

The developed RP-HPLC method is accurate, precise, linear, robust, and sensitive for the simultaneous determination of Dolutegravir and Rilpivirine. All validation parameters including system suitability, precision, accuracy, linearity, LOD, LOQ, and robustness were found to be within acceptable limits, confirming the suitability of the method for routine quality control analysis. The method is economical, time-efficient, and reproducible, making it highly suitable for regular laboratory use in pharmaceutical industries and research laboratories for the analysis of these drugs in combined dosage forms.

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