1.1 PHARMACEUTICAL ANALYSIS
Pharmaceutical chemistry, which deals with the science of chemical substance extraction, preparation, purification, and estimate utilised in the creation of medications or pharmacological products, includes pharmaceutical analysis as a crucial component. It has played a significant role in the pharmaceutical industry's transformation and has emerged as its mainstay.
1.2 Method Development [1-4]
The discovery, development, and production of novel medications and medicines depend heavily on the creation and validation of analytical methods. These procedures are employed to guarantee the efficacy, identification, purity, and performance of pharmaceutical items. When creating new analytical techniques, there are numerous things to take into account. The physiochemical characteristics of the analyte (pKa, logP , and solubility) are first gathered and are helpful in identifying which modality of detection could be appropriate for the study. Validating an analytical method for stability indications takes up most of the analytical development effort. The primary active medications, any reaction impurities, and any accessible synthesis intermediates and degradants are separated and quantified using a recognised analysis method. There are two main areas in which chemical analysis is typically used.
Analysis, both
(i) Quantitative;
(ii)Qualitative
An indication of the chemical species identity in the sample is provided by qualitative analysis. HPLC, Gas chromatography, TLC, Ion chromatography, and Column chromatography are a few examples of various quantitative chromatography-based analytical procedures.
1.3 Chromatography [5]
The foundation of the chromatographic process is the interaction of a mobile phase with a stationary phase. The factors that determine these differences in contact include polarity, pH, size, functional groups, and electric charge (in the case of ionic compounds).
One type of chromatography called liquid chromatography (LC) is an analytical method for breaking down a mixture in solution into its constituent parts.
The phrase "high performance liquid chromatography" (HPLC) refers to a type of liquid chromatography in which a stationary phase-containing column is mechanically pumped full of liquid mobile phase. Thus, an injector, a pump, a column, and a detector make up an HPLC instrument.
1.3.1 High Performance Liquid Chromatography (HPLC) [5]
One of the most important and practical instruments for quantitative analysis is HPLC. In contrast to normal phase, which is used with a non-polar mobile phase, reverse phase chromatography uses a polar mobile phase with a non-polar stationary phase. For both quantitative and qualitative analysis, HPLC is always employed in conjunction with another analytical method
1. High-performance liquid chromatography (HPLC) is the primary and essential analytical technique used in all phases of drug discovery, development, and manufacture in the contemporary pharmaceutical business.
2. HPLC is the preferred technique for determining a new chemical entity's peak purity, observing variations in reactions during synthetic processes or scale-ups, assessing novel formulations, and performing quality control and assurance on the finished pharmaceutical goods.
3. Attempting to isolate and measure the primary medication, any reaction contaminants, all accessible synthesis intermediates, and any degradants is the aim of the HPLC approach.
4. High Performance Liquid Chromatography is currently one of analytical chemistry's most potent instruments. Any material that can dissolve in a liquid can be used to separate, identify, and measure the chemicals present in it.
5. The most precise analytical technique for both quantitative and qualitative drug product analysis, as well as for assessing the stability of drug products, is high performance liquid chromatography(HPLC).
1.3.2 Importance of polarity in HPLC [6]
Polarity affects the stationary and mobile phases' eluting power or solvent strength. Polarity affects the stationary and mobile phases' eluting power or solvent strength.
1.3.4 Separation mechanism in HPLC
Compounds can be separated as illustrated in Figure 1 because molecules move through a column at different rates depending on their affinities with the stationary and mobile phases. As a result of these interactions, the molecules move at different rates and can be separated as illustrated in Figure 1. 3.
1.3.5 Types of HPLC [5]
Types are broadly divided in two main types:
A. Based on modes of chromatography
a) Normal-Phase Chromatography (Np HPLC)
The mobile phase in Np HPLC is a non-polar solvent (such as hexane, heptane, etc.), while the stationary phase is polar in nature. Non-polar chemicals elute first and move more quickly in this method. Less affinity between the solute and stationary phase is the reason of this. Due to their greater affinity for the stationary phase, polar molecules are maintained on the column for longer periods of time. For extremely hydrophobic substances, Np is the preferred technique, mostly utilising non-polar solvents. For instance, compounds such as naproxcinod , drotaverine HCl, and omeprazole are examined by Np HPLC.
b) Reversed-Phase HPLC (Rp HPLC)
As opposed to normal-phase HPLC, reversed-phase chromatography is based mostly on dispersive forces (hydrophobic or Van der Waals interactions). In RP-HPLC, where water-based solutions are generally used, the polarity of the mobile and stationary phases are switched, resulting in a hydrophobic surface for the stationary phase and a polar surface for the mobile phase. The most admired and widely used chromatographic mode is unquestionably reversed-phase HPLC. Roughly 90% of low-molecular-weight sample studies are carried out with RP HPLC. Analytes that are less polar, or more hydrophobic, are finally eluted because they are more attracted to and spend more time associated with the hydrophobic bonded phase (fig. 1. 4).
1.4. HPLC method development [10]
According to the HPLC concept, a liquid phase (mobile phase) is pumped through a porous material column at a higher pressure than the stationary phase, which is filled with the sample solution. The solute is adsorbed on the stationary phase according to its affinity for the stationary phase, which is the separation principle that is used. (Fig. 1.2)
The HPLC technology has the following characteristics.
- Small diameter, Stainless steel, Glass column
- Rapid analysis
- High Resolution
- Relatively higher mobile phase pressure
- Controlled flow rate of mobile phase
1.4.1. Physicochemical properties of drug molecules [10]
When developing a method, a medicinal molecule's physicochemical characteristics are crucial. Physical characteristics of the drug molecule, such as its solubility, polarity, pH, and pKa. The solvent and mobile phase composition are determined by the polarity. The solubility of the analyte determines the choice of diluents. Generally speaking, a substance's pH value determines how basic or acidic it is. Sharp, symmetrical peaks are frequently observed in HPLC when ionizable analytes are selected at the appropriate pH.
1.4.2. Selection of chromatographic conditions [10]
To get the first "scouting" chromatograms of the sample, a set of beginning settings (detector, column, mobile phase) is chosen during the first technique development process. These are typically based on reversed-phase separations using a UV-detected C18 column. This is the time to decide whether to design a gradient approach or an isocratic method.
1.4.3. Selection of Column [10,12]
The column is the main component of an HPLC system. Of course, the first and most important component of a chromatograph is a column. A well-chosen column can result in a strong chromatographic separation, which offers a trustworthy and accurate analysis. Particle size, retention capacity, stationary phase chemistry, and column dimensions must all be taken into account when selecting the ideal column for a given application. An HPLC column consists of three basic parts: the stationary phase, the matrix, and the hardware. For the support of the stationary phase, there are various kinds of matrices available, such as silica, polymers, alumina, and zirconium. The most used matrix for HPLC columns is silica. Strong, simple to derivatize, produced with a constant sphere size, and resistant to compression under pressure are the qualities of silica matrices. Chemical stability exists in silica. To low pH systems and the majority of organic solvents. Propyl (C3), Butyl (C4), and Pentyl (C5) phases are helpful for ion-pairing chromatography (C4), peptides with hydrophobic residues, and other big compounds. These phases are also commonly used reverse phase columns. Non-polar solutes are often retained less well in C3-5 columns than in C8 or C18 phases. The applications of octyl (C8, MOS) phases are broad. Although not as retentive as the C18 stages, this phase is nevertheless very helpful for steroids, nucleosides, and medications. Stable and repeatable columns are crucial to prevent issues with irreproducible sample retention during method development.
1.4.4 Selection of Chromatographic mode [13]
Chromatographic modes determined by the polarity and molecular weight of the analyte. The most used method for tiny organic compounds, reversed-phase chromatography (RPC), will be the main topic of all case studies. Ion-pairing reagents or buffered mobile phases are frequently used in RPC separation procedures for ionizable substances (acids and bases) in order to maintain the analytes' non-ionized state.
Table 1.1- Physical properties organic solvent commonly used for HPLC
|
Solvent
|
MW
|
BP
|
RI
(25 0C)
|
UV
cut-off (nm)
|
Density
g/ml
(25 0C)
|
Viscosity
(25 0C)
|
Dielectric constant
|
|
Acetonitrile
|
41.0
|
82
|
1.342
|
190
|
0.787
|
0.358
|
38.8
|
|
Dioxane
|
88.1
|
101
|
1.420
|
215
|
1.034
|
1.26
|
2.21
|
|
Ethyl acetate
|
88.1
|
77
|
1.372
|
256
|
0.901
|
0.450
|
6.02
|
|
Methanol
|
32.0
|
65
|
1.326
|
205
|
0.792
|
0.584
|
32.7
|
|
THF
|
72.1
|
66
|
1.404
|
210
|
0.889
|
0.51
|
7.58
|
|
Water
|
18.0
|
100
|
1.333
|
170
|
.0998
|
1.00
|
78.5
|
1.4.6 Selection of detector and wavelength [14]
Detectors for mass spectrometry (MS), electrochemistry, ultraviolet (UV), fluorescence, and refractive index (RI) are a few types of detectors frequently used in LC. Choosing a detector is contingent upon the type of sample and the analysis's goal. It is necessary to select a wavelength that will allow for the majority of analytes to respond adequately.
1.4.7 Developing the approach for analysis [15]
Several chromatographic parameters, such as mobile phase pH and flow rate, column choice, and mobile phase flow rate, were selected during the development of the RP-HPLC analytical method. Trials are used to choose each of these characteristics, and then the system suitability parameters are taken into account. The retention time (which should be more than 5 minutes), the theoretical plates (which should be more than 2000), the tailing factor (which should be less than 2), the resolution (which should be more than 5) and the percentage R.S.D. of the analyte peak area (which in standard chromatograms should not be more than 2.0%) are typical parameters of system suitability. When two components are being estimated simultaneously, the detection wavelength is often the isobestic point.
1.4.8 Sample preparation [10]
Several chromatographic parameters, such as mobile phase pH and flow rate, column choice, and mobile phase flow rate, were selected during the development of the RP-HPLC analytical method. Trials are used to choose each of these characteristics, and then the system suitability parameters are taken into account. The retention time (which should be more than 5 minutes), the theoretical plates (which should be more than 2000), the tailing factor (which should be less than 2), the resolution (which should be more than 5) and the percentage R.S.D. of the analyte peak area (which in standard chromatograms should not be more than 2.0%) are typical parameters of system suitability. When two components are being estimated simultaneously, the detection wavelength is often the isobestic point.
1.4.9 Method optimization [10]
It is necessary to consider the compositions of the stationary phase and mobile phase. Since mobile phase parameter optimisation is more straightforward and convenient than stationary phase optimisation, it is always given priority. When optimising liquid chromatography (LC) techniques, the many elements of the mobile phase that determine acidity, solvent, gradient, flow rate, diluents, and solvent type are the main control variables. Column dimensions, flow rate, and column-packing particle size are among the characteristics that are involved. Changes to these parameters won't impact selectivity or capacity factor.
1.4.10 Method Validation [5-10]
The process of demonstrating that an analytical method is appropriate for its intended use is known as method validation. Any new or modified procedure must be validated to make sure it can produce repeatable and trustworthy results when utilised by various operators using the same equipment in various laboratories.
Analytical results' consistency, dependability, and quality can all be assessed using technique validation results. It is necessary to validate or revalidate analytical methodologies.
Guidelines for pharmaceutical methods of analysis from the Food and Drug Administration (FDA), International Conference on Harmonisation (ICH), and United States Pharmacopoeia (USP) offer a framework for carrying out these kinds of validations.
1.5 Parameters used for Assay Validation
The validation of the assay procedure can be carried out as per ICH guidelines using the following parameters.
1.5.1. Specificity
The capacity to evaluate the analyte without a doubt in the presence of potentially present components is known as specificity. These could typically contain matrix, degradants, and contaminants. Plate count, tailing factor, and resolution are used to gauge specificity.
i) Identification test
ii) Assay and Impurity Test
1.5.2. Linearity
The capacity of an analytical process to produce test findings that are exactly proportionate to the concentration (amount) of the analyte in the sample, within a specified range, is known as linearity.
1.5.3. Accuracy
The degree to which the value found and the value acknowledged as a conventional true value or an established reference value agree is expressed as the accuracy of an analytical technique. The degree to which the measured value closely resembles the true value for the sample is the method's accuracy. One of four methods is typically used to determine accuracy.
1.5.4. Range
The data from the accuracy and linearity investigations are used to calculate the range. An assay method's range criteria are reached at the acceptable range, which is the concentration interval that produces a precision of 3% RSD and across which linearity and accuracy are acquired according to previously described criteria.
1.5.5 Precision
The degree of agreement between several measurements made from repeated samplings of the same homogeneous material under specified circumstances is expressed as the precision of an analytical method. Typically, the variance, standard deviation, or coefficient of variation of a set of measurements are used to represent the precision of an analytical process.
The degree of reproducibility or repeatability of the analytical procedure under typical operating conditions is measured by precision.
1.5.6. Detection limit
Detection limit is based on the standard derivation of the response and the slope.
Detection limit (or) limit of detection may be expressed as,
DL= [3.3σ/S]
Where,
σ =standard deviation of the response
S =slope of the calibration
1.5.7. Quantitation limit
The quantitation of an analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. Quantitation limit is based on the standard deviation of the response and the slope.
It can be expressed as,
QL = [10σ/S]
where,
σ =standard deviation of the response
S =slope of the calibration curve (of the analyte)
1.5.8. Ruggedness
The consistency of test results under varying conditions, including multiple laboratories, analysts, instruments, and several regulators, as well as elapsed hours, assay temperatures, and days, is measured by ruggedness.
1.5.9. Robustness
A method is considered robust if it can withstand minor variations in parameters such the injection volume, temperature, buffer concentration, mobile phase pH, and percent organic content. The RSD should not exceed 2% as the robustness criterion.
1.5.10. System Suitability Testing
Many analytical techniques include system suitability assessment, which is based on the idea that the tools, electronics, analytical processes, and samples to be examined all make up an integral system that can be assessed as such. The procedure usually entails injecting a standard solution five times and assessing a range of chromatographic parameters, including resolution, area% repeatability, theoretical plate count, tailing factor, etc.
1.6 Stability Studies [20, 21]
The ability of a drug substance or drug product to continue operating within predetermined parameters across retest or expiration dates in order to maintain its identity, strength, quality, and purity is known as stability.
Establishing recommended storage conditions, intervals for repeat testing, and ultimately product shelf-life and expiration dates are made possible by stability testing. Stability studies have applications in the following drug development programme areas in the pharmaceutical industry.
1.6.1 Conducting forced degradation studies [22- 23]
Before formal stability studies begin, forced degradation experiments must to be carried out on each distinct formulation. When a drug substance has decomposed by approximately 10% from its initial amount or after exposure exceeding the energy given by accelerated storage (such as 40 0C for six months), sufficient exposure of the drug substance or drug product has been accomplished. In certain situations, using this general guideline might lead to no degradation at all. The objective is to produce a degradation profile under ICH settings that is similar to what would be seen in formal stability investigations.
There should be more light storage than what the ICH requires. According to the guidelines, research of a medicinal substance's solution phase degradation might be allowed in solution or in suspension.
Although any organic co-solvent has the ability to react with a drug material under specific stress circumstances, the use of inert organic co-solvents may be advised in cases where the drug substance is extremely insoluble. Non-drug substance related peaks for drug products should be distinguished from chemicals related to drug substances.
1.6.2 Study Protocol [24]
A general protocol for conducting forced degradation studies, shown in Table1.2
Table 1. 2- General protocol for stress testing of drug substances and drug products
|
Condition
|
Drug Substance
|
Drug Product
|
|
Solid
|
Solution/ Suspension
|
Solid(Tablets, Capsules, Blends)
|
Solution (IV, Oral Suspension)
|
|
Acid/base
|
|
√
|
|
X
|
|
Oxidative
|
X
|
√
|
√
|
√
|
|
Photostability
|
√
|
X
|
√
|
√
|
|
Thermal
|
√
|
|
√
|
√
|
|
Thermal/humidity
|
√
|
|
√
|
|
√=recommended; X=optional, suggested for some compounds.
1.6.3 Conditions for Stress Testing [25]
About 5–20% is the intended goal extent of deterioration. To do this, change the stress conditions, such as the temperature, length of exposure, or amount of the stressing agent (oxidizer, base, acid, etc.). Excessive straining could break down the compound or cause the pertinent principal degradants to break down much more. It's possible that under stressing won't produce significant degradation products. Even if adequate degradation has not been attained, the degradation investigations ought to end after the maximum duration or stress conditions that have been advised.
Table 1.3- Recommended stress conditions for drug substance
|
Stress type
|
Conditions
|
Time
|
|
Acid hydrolysis
|
1 mg/mL in 0.1 N (up to 1.0 N) HCl; RT or higher
|
1–7 days
|
|
4 weeks Base hydrolysis
|
1 mg/mL in 0.1 N (up to 1.0 N) NaOH; RT or higher
|
1–7 days
|
|
Thermal hydrolysis (control)
|
Aqueous Solution; 70 0C
|
1–7 days
|
|
Oxidative/solution
|
O2 + Initiator (AIBN) in acetonitrile/H2O2, (80/20); 40 0C
|
1–7 days
|
|
Oxidative/solution
|
0.3% (up to 3%) H2O2; RT; protected from light
|
Few hours to 7 days
|
|
Thermala
|
70 0C temperature
|
Up to 2 weeks
|
|
Thermal/Humiditya
|
70 0C temperature /75% RH
|
Up to 2 weeks
|
If the solid drug substance is unstable to thermal stress at high temperature due to melting, decomposition, etc. use a lower temperature with longer stress time.
Table 1. 4 Recommended stress conditions for drug product
|
Stress type
|
Conditions
|
Time
|
|
Thermal
|
700C (may vary headspace if oxidation is expected)
|
Up to 3 weeks
|
|
Thermal/humidity
|
700C /75%RH
|
Up to 3 weeks
|
|
Photo-degradation
|
Fluorescent and UV light
|
> 2× ICH
|
Note: To distinguish drug-related degradation products from any non-drug-related degradation products from the excipients or solvents, further conduct stress testing on a placebo as a control.
1.6.4 Reaction Mechanism/Degradation Pathway [26]
Hydrolysis, oxidation, isomerization/epimerization, decarboxylation, rearrangement, dimerization/polymerization, photolysis, and reactions with excipients and salt forms are among the major reaction mechanisms of chemical degradation of pharmaceutical substances. Table 1.5 provides examples.
Table 1.5- Common degradation routes for functional group
|
Functional group
|
Degradation route
|
Degradants
|
|
Acetals
|
Hydrolysis
|
Ketones /aldehydes /alcohols
|
|
Esters/lactones
|
Acids/alcohols
|
|
Amides/lactams
|
Amines/acids
|
|
Alkenes
|
Alcohols
|
|
2◦ and 3◦ Amines
|
Oxidation
(radical, light, metal, peroxide mediated)
|
N-oxide, hydroxylamine
|
|
Thiols
|
Disulfide
|
|
Thioethers
|
Sulfoxide, sulfone
|
|
Alkenes
|
Epoxide
|
|
Allylic Alcohols
|
α, β, unsaturated ketones
|
|
Aldehyde
|
Acids
|
|
Alcohol
|
Ketones, acids
|
|
Oxazoles/imidazoles
|
Various products
|
|
Dienes (able to aromatized)
|
Aromatic rings
|
|
Benzyl/Allylic groups
|
Benzylic/allylic alcohols
|
