Analytical Techniques for Isoniazid: A Systematic Review of UV Spectrophotometry and HPLC Methods

A first-line antibiotic for both active and latent Mycobacterium tuberculosis (TB) infections is isoniazid (INH). It functions as a prodrug when the enzyme is activated, producing radicals that prevent the formation of mycolic acid, an essential part of the mycobacterial cell wall. Strong bactericidal effects are provided by this mechanism, especially when combined with other TB drugs. With the chemical formula C?H?N?O, isoniazid is structurally a hydrazide derivative of isonicotinic acid. Its simple structure allows it to function as a prodrug by substituting a hydrazide group for the pyridine ring. Under typical circumstances, it is stable, soluble in water, and mostly acetylated in the liver. The pKa value of the compound is 2.48. UV-VIS spectroscopy is a trustworthy technique for figuring out solute concentrations in analytical applications by measuring light absorption based on the Beer-Lambert law in the visible, ultraviolet, and near-infrared spectrums. This method is prized for its affordability, speed, precision, and ease of use. A sophisticated chromatography method called High-Performance Liquid Chromatography (HPLC) is used to separate, identify, and measure substances in mixtures. The analyte's interactions with the stationary phase, mobile phase, and solvent determine the retention periods, which are measured by a detector, a stationary phase column, and a high-pressure pump for the mobile phase. Water and methanol are frequently used solvents in HPLC, and gradient elution improves separation effectiveness. ^[1,2,3]

Isoniazid Drug Profile

Structure of Isoniazid

Table 1: Isoniazid Drug Profile

The way that chemical compounds absorb UV or visible light and cause electrons to travel between energy levels is measured by UV-visible spectroscopy. Excitation to higher energy states produces absorption spectra, whereas relaxation back to lower states produces emission spectra. This idea is the foundation of molecular spectroscopy. By injecting the sample components into a column with a porous stationary phase and forcing a liquid mobile phase through at high pressure, High-Performance Liquid Chromatography (HPLC) separates the components of the sample. Solute affinity is the basis for separation; longer retention periods are associated with stronger interactions. When compared to traditional chromatography, HPLC uses smaller particles for faster and more effective analysis. ^[4,5,6]

Figure 1:  Schematic diagram of UV spectrophotometerr

Table 2: The wavelength of measurement with the solvent

Absorption intensity transmission and UV spectrum

1) Bathochromic shift: also called a redshift, The migration of absorption towards a longer wavelength.
2) Hypochromic shift:  also called a blueshift, happens when the absorption shifts to a shorter wavelength.
3) Hyperchromic effect: The absorption peak's strength increases.
4) Hypochromic change: A decrease in the absorption peak's intensity. ^[7]

Figure No.2: Shifts in Absorption and

1. Chromophores
2. Auxochrome

Table 3: UV Segments and Their Wavelength Regions

Instrumentation

Light source

1. Hydrogen Lamp
2. Deuterium Lamp
3. Tungsten Lamp

(4) Xenon Discharge Lamp ^[8]

Monochromator

Sample Cell                                                   

Detector ^[9]

Theories of Spectroscopy

Beer's Law

A solution's absorbance (A) is directly proportional to its concentration (c) and route length (b). The equation is A = a.b.c. Use c = A / (ab) to get concentration (c), where A stands for absorbance, a for molar absorptivity, and b for path length.

Lambert's Law

The absorbing medium's thickness directly correlates with the light's drop in intensity.^[10]

Law of Beer-Lambert

According to the Beer-Lambert equation, the sample's absorbance (A) is proportional to its concentration (c) and route length (b). A = a.b.c is the formula, where an is the absorptivity. Absorbance quantifies the amount of light that the sample absorbs. ^[11]

Types of Spectroscopies

1. According to the Study Level:

Molecular Spectroscopy: UV Spectroscopy, Colorimetry, Infrared Spectroscopy, and Fluorimetry

1. Atomic Spectroscopy
2. Atomic Absorption Spectroscopy
3. Flame Photometry

2. Based on the Interaction with Electromagnetic Radiation (EMR)

1. Absorption Spectroscopy
2. Emission Spectroscopy

3. Based on the Type of Energy Changes Studied

1) Electronic Spectroscopy

2) Magnetic Spectral Analysis ^[12]

Method development in UV spectroscopy

The evolution of UV spectroscopy techniques
1. Choosing the Wavelength (λmax)

2. Creating Standard Solutions

3.Choosing the Solvent

4.Development of the Calibration Curve

5.Verifying the Linearity Range

6.Analysis of the Sample

7.Optimizing Instruments

8.Correction to Baseline

9. Verification of Methods

Validation Parameter

Linearity

Purpose-Identifying if the UV absorbance is proportionate to the analyte concentration is the goal.

Acceptance Criteria-2 (correlation coefficient) must be greater than 0.999.a linear correlation, within a certain range, between concentration and absorbance.

Table 4 : Different Linearity range

Purpose- To measure how close the experimental values are to the true value.

Acceptance Criteria- Accuracy should be within 98%-102% of the expected value for pharmaceutical compounds, although criteria may vary depending on the application and regulatory guidelines.

Precision

Purpose- To evaluate the precision under the same operating conditions over a short period.

Acceptance Criteria- Relative standard deviation (RSD) should be ≤ 2%.

Relative standard deviation % = standard deviation Mean × 100

Detection Limit (LOD)

Purpose- To determine the lowest amount of analyte that can be detected but not necessarily quantified.

Acceptance Criteria- LOD can be determined by signal-to-noise ratio, typically S/N ≥ 3.

The LOD was calculated according to following equation:

LOD = 3.3 × SD  σ

Where, SD = standard deviation of response, σ = slope of regression line

Quantitation Limit (LOQ)

Purpose- Finding the lowest concentration of analyte that can be detected but not necessarily quantified is the goal of the detection limit (LOD).
Acceptance Criteria: Signal-to-noise ratio, usually S/N ≥ 3, can be used to calculate LOD.
The following formula was used to determine the LOD:
LOD = where σ is the slope of the regression line and SD is the response standard deviation.
Limit of Quantitation (LOQ)
The goal is to ascertain the minimum quantity of analyte that can be quantitatively quantified with a level of accuracy and precision that is acceptable.
Acceptance Criteria: A signal-to-noise ratio of S/N > 10 is normally required for LOQ.
The following formula was used to determine the LOQ: LOQ = 10 × SD  σ
LOQ = where σ is the regression line's slope and SD is the response's standard deviation
Robustness
The goal is to determine whether the method can withstand minor, intentional adjustments to its parameters, such as wavelength and solvent composition.

Range

Purpose: To identify the concentration range over which the technique exhibits satisfactory linearity, accuracy, and precision.
Acceptance Criteria: Must meet regulatory requirements or encompass 80%–120% of the intended concentration.

Stability of Analytical Solution

Purpose: To ascertain whether the analytical solution is stable throughout time. Acceptance Criteria: Over a predetermined amount of time (e.g., 24 hours), the answer must be within ±2% of the original reading.

ICH Guidelines (ICH Q2R1) for Analytical Procedure and Validation:

The method used to carry out the analysis is referred to as the analytical technique. The procedures required to carry out each analytical test should be thoroughly explained. The preparation of the reagents, the reference standard, the sample, the usage of the equipment, the creation of the calibration curve,use of the formulae for the calculation.

Advantages of UV-Visible spectroscopy

1. High accuracy
2. Robust operation
3. Simplicity
4. Cost-effectiveness

Disadvantages of UV-Visible spectroscopy

1. Chromophore limitation
2. Sample state
3. Preparation time

Application Of UV-Vis Spectroscopy:

Detection of Impurities: Identifying contaminants in samples.

Structural Elucidation: Determining the structure of organic compounds through absorption characteristics.

Quantitative Analysis: Measuring concentrations of substances in solutions.

Qualitative Analysis: Identifying compounds based on their UV-Vis spectra.

Chemical Analysis: Analysing various chemical substances and reactions.

Pharmaceutical Analysis: Quantifying active pharmaceutical ingredients and other components.

Dissociation Constants: Measuring the dissociation constants of acids and bases.

Molecular Weight Determination: Estimating the molecular weights of compounds.

HPLC Detection: Serving as a detector in high-performance liquid chromatography (HPLC). ^[13]

High Performance Liquid Chromatography (HPLC)

Figure 3: Flow ddiagram of HPLC

Instrumentation

Solvent Reservoir-

Pump

Sample Injector                                                                                                                             Column

 Detector

Normal Phase Chromatography

Principle: the non-polar analytes elute first while polar analytes are retained longer by a polar stationary phase.
Application: Best suited for preparative-scale HPLC, ideal for isomeric combination separation, and a backup choice for lipophilic chemicals.
mobile phase: Non-polar solvents, such as hexane and dichloromethane.

stationary phase: Polar materials, such as alumina and silica gel. ^[17]

Reverse phase chromatography

principle: is the separation of non-polar analytes from the non-polar stationary phase by hydrophobic interactions.
Application: It works well with a variety of substances, such as hydrophobic, lipophilic, and nonvolatile molecules.

Stationary phase: Non-polar materials, such as C18 and C8-bonded silica. Polar solvent is the mobile phase. ^[18]

Size-Exclusion Chromatography

Principle: Using gel matrices, separation is carried out according to molecular size.

Application: Determining polysaccharide molecular weights and amino acid structures.

mobile phase: Depending on the kind of gel and analyte, the mobile phase may consist of either aqueous or non-aqueous solvents.

Stationary phase: semi-rigid gels such as polystyrene and alkyle-dextran, or gel materials such as agarose, polyacrylamide, and dextran (soft gel). ^[19]

Ion-exchange chromatography:

Principle: The interaction between charged solutes and oppositely charged groups on the stationary phase is the foundation of the retention

Application: In biology and industry, common applications include protein purification, water deionisation, carbohydrate analysis, and ionic compound separation.
Mobile phase: Is an aqueous solution that contains salts to regulate ionic strength and buffers to alter pH.
Stationary Phase: A resin or matrix with covalently bonded charged groups, such as cationic (like quaternary ammonium) or anionic (like sulfonic acid) functional groups, is known as the stationary phase. ^[20]

Affinity Chromatography

Principle: Target molecules and immobilized ligands must form a specific bond in order for separation to occur.

Applications: The separation of biomolecules, enzyme isolation, and protein purification.

Mobil phase: A buffer system that is tailored to the conditions of binding and elution.

stationary phase: A solid matrix having covalently bonded ligands, such as agarose or sepharose, is known as the stationary phase. ^[21]

Method Development of HPLC

1. Assessing the drug's physicochemical characteristics to inform the choice of solvent and technique.
2. Choosing the detector, column, and mobile phase, among other basic chromatographic parameters.
3. Modifying the parameters of the analysis approach in light of initial findings.
4. Getting the sample ready to make sure it works with the selected technique.
enhancing the process to increase resolution, accuracy, and precision.

Understanding the physicochemical properties of a drug molecule

Selection of chromatographic conditions

The first step in developing a chromatographic separation method is choosing the beginning conditions, which in reversed-phase separations usually involve a C18 column with UV detection.

Selection of column

The steady retention of the sample is guaranteed by a repeatable stationary phase. Purified silica columns, such as C8 and C18, are frequently employed for basic chemical separation because of their strength and stability in organic solvents. But compounds based on silica can dissolve at pH values higher than 7. For the separation of ionizable substances, such bases and acids, reversed-phase chromatography (RPC) is frequently utilized. Utilizing buffered mobile phases or ion-pairing agents to inhibit ionization and improve separation, it takes use of the polarity and molecular weight of the analyte.

Selection of Chromatographic Mode

The polarity and molecular weight of the analyte determine the chromatographic modes. Using buffered mobile phases or ion-pairing reagents, reversed-phase chromatography (RPC) is a popular method for tiny organic molecules because it efficiently separates ionizable chemicals like bases and acids.

Buffer Selection

The ideal pH range for reversed-phase chromatography on silica-based packing materials is typically between 2 and 8, therefore choosing a buffer is mostly based on that value. When a buffer's pKa is around the target pH—ideally, within two pH units of the intended mobile phase pH—it controls pH the best. The following general rules apply when choosing a buffer:

Table 7: Buffer used in HPLC

General Considerations for Buffer Selection

Phosphate buffers dissolve more readily in water and methanol. Chromatographic problems such as tailing and changes in selectivity can be brought on by hygroscopic salt buffers. Ammonium salts dissolve better in organic/water-soluble phases. Microbial development is encouraged by low organic buffered phases, which impacts column performance. Phosphate buffers reduce the life of HPLC columns by dissolving silica at pH >7; organic buffers are recommended instead. The release of CO? causes ammonium bicarbonate buffers to change pH during a 24- to 48-hour period. ^{[22, 23,24]}

Concentration of Buffers

Generally, 10–50 mm of buffer concentration is adequate for small molecule analysis. Generally speaking, the mobile phase should not have more than 50% organic content. The most used buffer systems for reversed-phase HPLC are phosphoric acid and its potassium or sodium salts. ^[25]

Selection of Mobile Phase

Table 9:  Mobile Phases Used During the HPLC Method Development Process.

Selection of detectors

Table 10: Detectors Used in HPLC

HPLC columns with a larger internal diameter are utilized in industrial settings, whilst those with a smaller ID have a lower loading capacity but consume less solvent and are more sensitive. ^[26]

Pore Size:

Larger pores improve kinetics for larger analytes, while smaller pores provide a greater surface area but are better suited for smaller analytes. Because the inner surface area of the particle is significantly bigger than the outer surface, there is a considerable interaction between the analytes and the inner surface.

Pump Pressure:

Pumps come in a variety of pressure capabilities, and the system's effectiveness is correlated with their capacity to provide a steady flow rate. For instance, two micrometers. ^[27]

System Resolution:

The formula =2(t2-t1)/(W2-W1) is used to determine the resolution between two components in an HPLC system. where W1 and W2 are the peak widths and t1 and t2 are the retention times. In general, a resolution factor larger than 2.0 is regarded as appropriate. ^[28]

System Precision:

The relative standard deviation (RSD) of the peak responses (area or height) following several injections is used to calculate the system precision. If the RSD is 2% or below, five replicates are usually employed; if it is greater than 2%, six repetitions are needed.

Asymmetry factor:

The formula T=W 0.05 /2f is used to determine the peak asymmetry or tailing factor, where W 0.05 is the peak width at 5% of the peak height. Reduced resolution and precision are the results of increased asymmetry. ^[29]

Method of validation parameter

Specificity: The capacity to distinguish the analyte with clarity when anticipated components such as contaminants or degradation products are present is known as specificity. ^[30]
Range and Linearity: The ability of a method to produce findings proportionate to the analyte concentration within a certain range is measured by its linearity. Typically, to evaluate this, standard solutions at different concentrations are analyzed, and a calibration curve is produced. ^[31]
Accuracy: Trueness, which is frequently evaluated as accuracy, is the degree to which measured values resemble the true value. By contrasting the measured value with a reference standard, accuracy is ascertained. The usual accuracy limitations for pharmacological compounds and products are 98.0–102.0% and 97.0–103.0%, respectively. An average recovery range of 50–150% can be suitable for determining impurities. ^[32]

Calculate The Relative Standard Deviation (RSD) By Following The Equation Below:

relative standard deviation % =  standard deviationMean × 100

Precision: A bio-analytical method's precision is the consistency of results from several measurements of the same homogeneous material under predetermined conditions. It shows the random error. Usually stated as the relative standard deviation (RSD) or coefficient of variation (%CV), it evaluates the variability (scatter) in concentrations from replicate samples.

% C V = standard deviation males X 100 is the formula used to determine the %CV. [33]

Detection Limit (LOD):

The smallest amount of an analyte in a sample that can be detected but not precisely measured is known as the detection limit of an analytical procedure. It is a crucial indicator of the sensitivity of the procedure since it denotes the lowest concentration at which the analyte may be accurately identified.

The LOD was calculated according to following equation:  LOD = 3.3 × SD  σ

Limit of Quantification (LOQ):

The limit of quantification, or LOQ, is a measurement of an analytical technique's ability to accurately and precisely quantify an analyte. It can be computed as follows:  LOQ = 10 × SD  σ

Where, SD = standard deviation of response σ = slope of regression line

Robustness: An analytical procedure is said to be robust if it can withstand minor, deliberate adjustments to its parameters, demonstrating its dependability under typical operating circumstances. ^[34,35]
System Suitability: Tests for system suitability make sure that the sensitivity, resolution, and repeatability of a chromatographic system are sufficient for the planned analysis. These tests evaluate the system's entire performance, taking into account the electronics, equipment, processes, and samples. Peak resolution, theoretical plate count, peak tailing, and capacity are frequently assessed metrics. ^[36]

Advantages of HPLC

1. High repeatability.

2. Extremely sensitive

3. Ongoing column effluent monitoring.

4. Useful for sorting and evaluating extremely complicated combinations.

5. Excellent accuracy and efficiency.

6. Rapid resolution.

Disadvantages of HPLC

1. Because HPLC requires expensive equipment and organic solvents, it can be costly.
2. Needs frequent maintenance and a steady power source.
3. It could be difficult to debug or create new techniques.
4. Expensive and complicated operations.

Pharmaceutical Applications

1. Dissolution tests of pharmacological dose forms in tablets.
2.  Regulation of medication stability and assessment of shelf life.
3. Determining the Active Component
4. Determining the active pharmacological component
5. Quality Control for Pharmaceuticals

b) Food Applications

1) HPLC is frequently used to ensure quality control and analyze food goods.

2) Good for testing chemicals that are labile in complicated matrices.

3) Examines pollutants, food additives, and natural substances including carbohydrates, fats, proteins, and amino acids.

4) Performs multiresidue testing for pesticides and pollutants.

c) Forensics Applications

1) medicines found in biological specimens.
2) Performs forensic analysis on textile dyes.
3) Identifies cocaine in blood samples and its metabolites.

d) Clinical Applications

1) Applied to routine analysis and clinical research.
2) Assesses glycated hemoglobin to track diabetes patients' long-term glucose management.
3) Aids in determining the origins of poisoning. ^[37,38]

CONCLUSION