A Comprehensive Review of RP-HPLC In Bioanalytical Method Validation and Sample Preparation

1. High Performance Liquid Chromatography

HPLC is powerful and widely used approach for isolating, determining, and measuring particular components in a liquid mixture. [1] When assessing new formulations, monitoring reaction changes throughout synthesis processes or scale up, verifying the peak purity of novel chemical entitis, and performing quality control and assurance on the finished drug products, HPLC is the preferred technique. [2] It is a more advanced form of a liquid chromatography that uses high pressure to move a solvent (the mobile phase) through a stationary phase packed column. HPLC has isolated each individual chemical component from the sample mixture based on its unique affinities for the mobile phase or adsorbent substance in the column, causing various constituents to separate in the column, causing various constituents to separate as they travel at different velocities. [3]

1. 1. Principle

HPLC works on the basis of separating components interactions with a stationary phase as they get carried by a mobile phase. Small particle size of stationary phase which gives high surface area to make separation more specific and precise. The use of micro syringes allows samples to be injected into pumps that provide high pressure flow of the mobile phase. [4] When small volume of analyte is injected into column the components will move with different affinity in column and separate out with different retention time and on recorder will give distinct and resolved peaks which is used in analysis of analyte. [5]

1. 2. Classification of HPLC can be done as [6]

HPLC is often divided into two subclasses according on the mode of operation:

1. NP-HPLC

 The term “Normal Phase High Performance Liquid Chromatography” (NP-HPLC) refers to methods where the mobile phase is less polar than the stationary phase. In NP- HPLC SiO2, NH2, -CN, NO2, ALO3 and diol are used as stationary phase and cyclohexane [7-8]

2. RP-HPLC

In RP-HPLC mobile phase used is polar or slightly polar, but the SP is non poIar. Separation is primarily based on hydrophobic interactions. [9] Non-polar analytes in the polar mobile phase are attracted to and interact effectively with non-polar SP, leading to longer retention. Polar analytes have weak interactions with the SP and elute quickly as they are more soluble in the polar MP. [10]

1. 3. Instrumentation

The common parts of HPLC instrumentation are

1. Solvent reservoir
2. Pumps
3. Sample injector
4. Column
5. Detector

Figure 1: Schematic diagram of HPLC

1. Solvent reservoir

A reservoir made of glass holds the contents of the mobile phase. The polar and non- polar liquid components that make up the mobile phase, or solvent, in HPLC are often mixed together, and the amounts of these components vary based on the sample’s makeup. Usually polar and non-polar solvents are stored separately in different container and used in proper ratio in isocratic and gradient elution. [11-12]

2. Pumps

Pumps are designed to prevent pulsation during the change in composition of mobile phase. HPLC pump continuously pump the mobile phase towards the column with constant pressure and constant flow rate of pump may up to 200 bar depending upon flow rate. Depending on needs of the analysis, the operational pressure limits can vary greatly, ranging from 2000 to 5000 psi in normal analytical operation. [13-14] Three commonly used pump types are

• Constant pressure pump: This uses pressure from a gas cylinder to generate a steady, continuous flow rate across the column. The solvent chamber can be quickly refilled due to the valve configuration. To produce high liquid pressures, a low-pressure gas source is required.
• Syringe type pump: The column receives the steady flow rate through a controlled anchor mechanism. Adjusting the motor’s voltage determines the solvent supply rate. Significant drawbacks include restricted solvent storage and restricted gradient operation.
• Resiprocating piston pump: These uses the rotating action of a piston in a hydraulic chamber to deliver the solvents. Because one pump is in the delivery cycle and the other is in the filling cycle, reciprocating pump systems provide smooth solvent supply. Both gradient operation and high pressure output at a consistent flow rate are achievable.

3. Sample Injector

Sample injector used to add the sample to the active mobile phase. Liquid samples with a volume of 0.1-100 mL can be loaded into an HPLC injector with excessive pressure (in the range of to 4,000 psi) and satisfactory reproducibility. When a user has to detect a more number of samples, an auto sampler is an automatic version. Samples are injected into the mobile phase stream at a fixed volume using injectors. To maintain a high degree of accuracy, injection must be inert and repeatable. [15-16] The sample can be introduced into the injection port in three crucial ways.

• Loop Injection: In this type, fixed volume loop injector is used to introduce a fixed quantity of volume.
• Valve Injection: This makes an use of an injection valve to introduce an alterable volume.
• On column Injection: In which, a syringe is used to insert a varied volume through a septum.

4. Column

Made mostly of clean stainless steel, columns have internal dimensions of 2 to 5 mm in width and 50 to 300 mm in length. Columns are packed with a SP that contains particles between 3 to 10 µm. Microbore columns have internal diameter of less than 2 mm. During the analysis, maintaining a constant temperature for both the column and the MP is adequate. [17] Various column types are- 1) Guard columns and 2) Analytical columns [18]

Guard column: It prolongs the lifespan of analytical column by eliminating impurities and particulate, matter from solvents. It has same composition compared to analytical column, but it has large particle size.

Analytical column: It is referred to as heart of HPLC, as from it mobile phase continuously passes. The length of column can be vary from 10 to 30 cm with diameter of 4 to 10 mm.

5. Detector

Every molecule that elutes from the chromatographic column can be identified by the HPLC detector. The detectors include electrochemical, ultraviolet spectroscopy, mass spectrometric, fluorescence, and evaporative light scattering detectors are utilized.  Detector supplied an output from the detector to a computer or recorder, which produces the graph, or a liquid chromatogram of the detector output. Both the necessary sensitivity and a particular response are provided by a detector for the components that the column separates. [19]

Table 1: Detector and their applications

1.4 Application of HPLC [20]

1. Pharmaceutical application

HPLC has a high linear dynamic range and reliable quantitative precision and accuray, it can be used to quantify various substances in a single run. A useful method for preparing samples for solid dosage forms in aqueous solutions that have been altered with acetonitrile or methanol. There are several ways to separate chiral substances into their respective enantiomers using HPLC. Precolumn derivatization is one method of creating diastereomers.

2. Manufacturing

There are several uses for HPLC in experimental and therapeutic science. This technique is commonly used in pharmaceutical manufacturing as it is an accurate way to determine and confirm the purity of the product. HPLC is not often the primary method used in the production of bulk pharmaceutical compounds, although its ability to yield very pure and superior products. Unfortunately, HPLC tends to increase specificity, precision and accuracy at the expense of increased cost. 

3. Research [21]

Research may determine the concentration of potential medicinal candidates, such as asthma drugs and antifungal treatments, using similar assays techniques. When attempting to determine the identify of a species, this method necessitates the use of standard solutions, because purity is crucial in research, it is employed as a technique to verify the outcomes of synthesis procedures. It is definitely useful in observing a variety of species in sample collection also.

4. Medical

Drug analysis is one application of HPLC in medicine, but it is more closely related to nutrient analysis. The most common medium for detecting medication concentrations is urine, but usually medical analyses utilizing HPLC use blood serum as the sample. Other techniques, such as immunoassays, for detecting chemicals that are relevant for clinical research has been evaluated against HPLC. In one instance, the sensitivity of HPLC and competitive protein binding assays (CPBA) for vitamin D detection was evaluated.

6. Foods

High-performance liquid chromatography has improved food analysis in ways that are desired. In general, food matrices are complicated, and extracting analytes is a challenging process. Trace elements often contain both unwanted and useful components, and conventional separation and evaluation techniques are not accurate or precise enough to make matters more problematic.

1.5 System Suitability Parameters

A crucial component of the liquid chromatographic approach is a system suitability test. They are used in analysis to make sure that the chromatographic system has adequate resolution and reproducibility. The test’s foundation is the idea that the apparatus, electronics, investigative process, and tester under analysis form a single, integrated system that should be assessed as such. [22] System performance before or during analysis is confirm by determining the parameters such as resoIution, pIate count, reproducibility and taiIing factor. [23]

1. Retention time (RT)

 Retention time is the interval of time between the injection site and peak maximum appearance. Additionally, it can refer to the time it takes for half of a component to come out from a column. The measuring units are minutes and seconds.

2. Theoretical plates (N)

An alternative name for it is column efficiency. Wherever the distribution of the tester between liquid-liquid or solid- solid segment takes place, a column can be seen of as consisting of a large no. of theoretical plates. To determine TP formula is given below:      N=16RTW2

Where RT= Retention time and w= Width at the peak’s base.

As the no. of TP in a column rises, it increases its separation efficiency. Efficient separation results in clear, well-defined peaks, and improved resolution between distinct analytes. There should be more than 2000 theoretical plates.

3. Resolution

Resolution, as used in HPLC analysis, is the chromatographic system’s capacity to isolate and differentiate between two neighboring peaks in a chromatogram. The precision and dependability of the analytical results are directly impacted by this parameter, making it an essential one in HPLC. Using the following formula, the resolution (R) between two neighboring peaks is determined:

R=2 (t2- _t1)w1+w2

Where w2 and w1 = Widths at the bases of the 2 & 1 peak, respectively.

while t1 and t2 = Retention times of the 1 and 2 compounds, respectively.

4. Tailing Factor

The tailing factor (TF), a numerical quantity that measures the amount of tailing in an HPLC peak, is used to evaluate the peak shape of a chromatographic peak. It is computed using the following formula:

T=w0.052F

Where F is the length of the peak height from the baseline to the peak maximum, and W_0.05 is the peak’s width at 5% height. A totally symmetrical peak is represented by a TF of 1, whereas tailing is indicated by values greater than1 and fronting is shown by values less than 1. [24]

5. Selectivity (α)

This measure of peak spacing is sometimes referred to as the separation factor and is written as

∝=K2'K'1

Table No.1: HPLC parameters and standard acceptance range

2. Analytical method development using RP-HPLC

Analytical techniques are always being created, refined, verified, jointly researched, and used. These created techniques are subsequently compiled in sizable compendia like USP, BP and IP, among others. Usually, it just takes a few attempts to get the necessary separation. Typically, method development entails choosing the method requirements and determining the kind of instrumentation to use and why. During the HPLC method development phase, choices for detectors, mobile phase, column, and method quantitation must be made. The optimal stationary phase, column, detector, internal diameter for columns, and mobile phase all be selected when creating n new HPLC techniques. This is because development involves consideration of all characteristics related to any method. There are several steps in the analytical strategy for developing an HPLC method, as seen figure 3. [25-26]

Figure 2: Flow Chart for Method Development by RP-HPLC

3. Bioanalytical method development

Analytes in certain biological fluids, including blood plasma, serum, or urine, are measured quantitatively. Pharmacokinetic and toxicokinetics studies are conducted both clinically and non-clinically to assess the safety and effectiveness of drugs. Using bioanalysis to assess characteristics in vivo interactions between drugs, bioavailability, and bioequivaIence. The examination of analytes and their metabolites, endogenous compounds, and biomarkers from biological fluid is done using a bioanalytical approach. [27]

Figure 3: Procedure for bioanalytical method development

1. 1.  Sample Preparation

Aim of sample preparation is to extract analyte from the biological matrix, which can be injected into the chromatographic apparatus. Techniques for preparing drug and metabolite samples from biological sources traditionally, the following extraction procedures are used to isolate the analyte from the biological tissue. [28]

Figure 4: Sample preparation techniques

1. 1. 1. Liquid extraction (LLE)

The foundation of LLE is analyte molecules partition equilibria and varying solubility between the aqueous and organic phases. A substance’s transition from one liquid phase to another is commonly referred to as liquid liquid extraction. LLE is regarded as being inexpensive and capable of producing clean extracts and good analyte recoveries. LLE technique is a widely used sample preparation technique in regulated biological analysis. It may be necessary to acidify, basify, or use small amounts of more polar solvents in the extraction process in order to simultaneously obtain high recoveries for the primary analyte, metabolites, and related chemicals. [29]

Procedure: The component mixture should be dissolved in an appropriate solvent first, and then an immiscible solvent should be added. To create layers of the two immiscible solvents, properly mix the ingredients and set aside. Based on the partition coefficients of the two immiscible solvents, the mixture's constituent parts will be divided between them. After separating the two layers of immiscible solvents, move and separate the component from each solvent. Hydrophilic substances enter the aqueous phase after extraction, while hydrophobic compounds are found in the organic solvents. By letting the solvent evaporate and then reconstituting the residue with a tiny amount of a suitable solvent, ideally mobile phase, non-polar analytes that have been extracted into the organic phase can be readily recovered. A reverse phase (RP) column can be directly filled with polar analytes that have been extracted into the aqueous phase. [30]

Figure 5: Liquid extraction

1. 1. 2. Solid Phase Extraction (SPE)

In SPE technique in the analyte is eluted selectively after being attached onto a solid substrate and interferences are removed. Conditioning, sample loading, washing, and elution are the four stages that make up the solid phase. It has evolved as a powerful technique for the extraction and measurement of analyse trace components in various sample matrices. The main goal of SPE are sample concentration, removal of contaminants and interfering compounds, and retention and elution of analytes from biological fluid. [31-32]

Steps involved in SPE

1. Conditioning

To condition or equilibrate the cartridge or column, the sorbent is moistened with a solvent in the first stage of SPE. It shows that an organic solvent, a wetting agent for packing materials, is used to activate the cartridge or column. For the proper adsorption process to begin, water or an aqueous buffer is supplied to the column.

2. Loading

The subsequent step involves percolation through the solid phase of the loading solution that that contains the analyte. The sorbent thus retains the analytes and some impurities. The samples are neither pumped, vacuumed, or gravity fed into the column once the pit has been fixed.

3. Washing

The sorbent is cleaned to remove impurities. While the matrix interference is removed, the analytes are kept intact.

4. Elution

The final stage, the elution step, involves collecting the analytes.

Figure 6: Solid phase extraction

1. 1. 3. Protein precipitation

Proteins are routinely removed from analyses using protein precipitation. The process through which proteins in a biomatrix lose their tertiary and secondary structures- known as denaturation- is caused by external stressors such as heat, strong acids or bases, or common organic solvents like methanol or acetonitrile. A quick and simple extraction method for both water loving and water repelling compounds is protein precipitation. Sometimes, to gain higher efficiency protein precipitation technique combine with LLE or SPE for the purpose of extraction of particular medicines and metabolites. [33]

Figure 7: Human plasma extraction procedure

1. 1. 4. Micro-extraction technique

Modern extraction techniques like micro extraction usually result in an extremely small proportion of extracting solvent to sample volume and minimal analyte separation. One effective and relatively recent method for sample preparation is micro extraction. Some advantages of using micro extraction techniques in bioanalysis include high rates of sample preparation, enhanced efficiency of extraction, small sample quantity, accuracy, reduced solvent intake, and a reduced expense. [34]

4. Bioanalytical Method Validation

According to FDA guidelines, validation is the process of producing a registered proof that provides a high degree of assurance that a particular operation will consistently produce a product that satisfies its predetermined parameters, quality specifications, and quality attributes. All of the steps necessary to show that a specific bioanalytical method for the quantitative assessment of an analytes (or group of analytes) concentration in a given biological matrix is dependable for the intended use are included in bioanalytical method validation. A minimum set of validation trials and satisfactory outcomes provide reassurances regarding the method’s reliability and accuracy. [35]

4.1 Need of Bioanalytical Method Validation [36]

• To provide accurate data that can be completely accepted, it is important to implement bioanalytical techniques that have been fully explained and validated.
• Bioanalytical methods and techniques are at the top of technology and are known to be constantly developing and improving.
• It is important to remember that every bioanalytical technique has special characteristics that differ from one analyte to another and that each analyte may require a different set of validation criteria should also be implemented.
• In addition, the study’s final goal may have an impact on the techniques suitability. When analysis of samples for specific research is carried out at different location, inter- laboratory reliability must be established by validating the bioanalytical methods at every site and providing appropriate validation data for different locations.
• Since the properties of the bioanalytical technique differ from analyte to analyte, it could be necessary to define distinct validation criteria for every analyst.
• The bioanalytical techniques produce accurate and satisfactory results.
• Validation is proving that the technique’s performance characteristics are appropriate and reliable for the proposed analytical applications through the use specific laboratory investigations.

4.2 Parameters of Method Validation [37-38-39]

Analytical method validation is the process to produce results from tests consistently and continuously fulfil predefined criteria. Method validation is the process of verifying that the investigative technique being used for a particular test is appropriate for its intended use. An essential element of any valid study procedure, process validation results can be used to assess the quality, consistency, and dependability of results. All validation parameters are described in this section.

Figure 8: Bioanalytical Validation Parameter.

1. Accuracy

The degree to which a measured value approaches the true or accepted value is known as accuracy. In practical terms, accuracy shows the difference between the actual value and the mean value found. The degree to which test results obtained using an investigative methodology closely resemble the actual value is known as technique’s accuracy. Pure standards of dapagliflozin and vildagliptin at three different concentration levels (80%, 100% and 120%) were added to the tablet sample solution that had been previously analysed in order to evaluate accuracy. At each level, the average recovery should be between 99 and 101% of the drug.

2. Robustness

Robustness is a measure of an analytical technique’s ability to maintain accuracy even after deliberate minor adjustments to its parameters. A techniques robustness is a measure of how sensitive it is to minute changes in temperature, pH levels, mobile phase composition, and other variables that could occur during routine analysis. Systemic adjustments were made to the mobile phase composition, wavelength, and flow rate (±0.1 ml/min) to maximize robustness. RSD was used in the calculation.

3. Linearity

The International Council for Harmonization (ICH) has given a definition, an analytical method’s ability to produce test results that accurately reflect an analyte concentration within a specific range is known as its linearity. To determine the linearity, one can utilize the correlation coefficient. In order to do this evaluation, three to six different standards must be injected at concentrations that ranges from 80% of the lowest expected level to 120% of the highest predicted level, or in ranges like 50-150% or 100-120%.

4. Precision

The degree of agreement between a set of measurements made by repeatedly sampling the same homogenous sample under specified conditions is expressed as the precision of an analytical method, which is a measure of the chance of error. There was precision research investigations conducted both inside and between days. Three distinct drug concentrations were injected into a single intraday precision sample, and a chromatogram was recorded. Similarly, to do inter-day precision analyses, three distinct amounts were injected, and a chromatogram was acquired over the course of two consecutive days. The percentage of coefficient of variance (CV%), or the ratios of standard deviations (SD) to the mean, was used to calculate the intraday and inter-day testing precision.

%CV=Standard DeviationMean×100

5. Limit of Detection (LOD)

It is the lowest detectable but not usually quantifiable concentration of an analyte in a sample. The analysis process and instrument type will determine the detection limit. The detection is commonly expressed as a percentage or parts per billion (ppb) of the test specimen's analyte concentration.

LOD=3.3×Standard deviationSlope

6. Limit of Quantification (LOQ)  

Lower limit of quantification (LOQ) is the smallest amount of a material in a sample that can be measured with acceptable precision and accurately below a technique’s predefined operating parameters. LOQ is an empirical measure or compounds that are found in sample matrices in extremely small amounts, like breakdown products in commonly used therapies and contaminants in bulk medications. The signal-to-noise ratio, standard deviation, and visual assessment techniques are some of the methods used to determine LOQ

LOQ=10×Standard DeviationSlope

7. Reproducibility

“Reproducibility” describes how accurate a procedure is when there are changes in the laboratory, including different days, analysts, and equipment. Each testing site can prepare a total of six sample preparation in accordance with the analytical procedure. Additionally, the results are analysed to ensure statistical consistency across different testing sites. The same approval criteria that apply to intermediate precision also apply to reproducibility.

8. Ruggedness

According to USP, “the ruggedness of an analytical method is denoted by the extent of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions, such as different labs, different analysts, temperature, assay labs, instruments, and different reagents. “Ruggedness is a metric that quantifies a method’s responsiveness to minor variations that may arise during normal analysis, such as slight variations in temperature, mobile phase composition, or pH values.

Table 2: Acceptance criteria for method validation as per ICH guidelines.

5. Application of RP-HPLC in drugs by analysis utilizing different sample preparation techniques.