A Concise Review on Bioanalytical Method Development and Validation with Special Emphasis on Sample Preparation

The main reason for evaluating the existence of drug in bodily fluids in the initial stages of toxicology and forensic medicine was to assess the risk of overdose. The requirement to gauge drug levels in living fluids was exacerbated in the 1930s with the scientific discovery of pharmacokinetic. The development of chromatographic methods, such as paper chromatography, in the 1940s allowed for the separation of drugs from their metabolites. Pharmaceuticals in biological fluids were measured using thin layer chromatography, which was developed later in the 1950s and primarily used for the separation of radiolabeled metabolites. The development of a trustworthy bio analytical method or procedure is essential during preclinical studies and clinical stages of the creation of medications. Consequently, it is well acknowledged that in order to prove the method's effectiveness and dependability, preparing samples and method validation are necessary. Numerous excellent review articles addressing different scientific and technological facets of bio analysis have been added to the literature in recent years. From the time of discovery to be point of market authorization, it is now widely accepted that bio analysis is essential to the pharmacokinetic and pharmacodynamics characterization of novel chemical entities [1]. By using bio analytical procedures, drug concentration and their byproducts in a living matrix (serum, urine, plasma, saliva etc.) can be determined. It is a vital phase in the formulation of a medication. In spite of the use of chromatographic instruments, such as LC-MS (Liquid chromatography-Mass spectroscopy) and HPLC (High performance liquid chromatography), the concentration of a living matrix can be estimated. Bio analysis operates an enormous part in the research of toxicological inspection, pharmacokinetics, and pharmacodynamics during drug development [2]. A biological matrix for a medication is collected, processed, stored, and examined as part of the process. Bio analytical technique validation is the process of recording a particular laboratory study that has been development and approved in order to demonstrate drug substance in a specified living matrix [3]. To confirm that a method for quantitatively evaluating analytes in the biological matrix is reliable and repeatable for its intended use, several examples of “bio analytical method validation” procedures are presented. Finding the concentration of drugs in biological fluid using such techniques is necessary for a number of studies, including those involving drug concentration and its metabolic products, bioavailability, bioequivalence, pharmacokinetics, innovative drug development, critical biomedical and pharmaceutical sciences research, and surveillance of therapeutic drugs [4, 5]. Developments in analytical methodology and validation carry out essential tasks in drug discovery, development, and production. An analytical measure's primary goal is to obtain enduring, accurate, and logical knowledge. In order to accomplish this goal, established analytical techniques are crucial. Analytical results' uniformity, legitimacy, and standard can be chosen using the validation technique's results [6].  The evolution required to support particular research for the various degrees of validation to prove the method's validity Bio analytical procedures were validated during:

•While creating and applying a new bio analytical technique.
•To analyze a novel drug entity.

•To strengthen an existing technique by including metabolite measurement.

•The dissemination of bio analytical techniques among analysts or laboratories [7].

Plasma, urine, cerebral spinal fluid (CSF), organs like the liver and brain, and cell-based in vitro samples are examples of common biological matrices. There are now three types of methods that are employed to support these analyses. Chromatography based methods include ligand-based techniques such as radio immune assay (RIA) and enzyme linked immune sorbent assay (ELISA), gas chromatography with electrochemical detector (GC-ECD), high performance liquid chromatography with ultraviolet detector (HPLC-UV) and mass spectrometry-based techniques comparable to GC-MS and LC-MS [8]. One indirect way to determine a medicine's bioavailability is to look for the drug in urine. Drugs that have not been absorbed following an oral dosage or that have been ejected by biliary secretion following systemic absorption may be represented by estimates of drug in feces. The salivary levels of drug reflect the concentration of free medication instead of whole blood drug level since only the free drug since only the free drug diffuses into the saliva. Consequently, the exact proportion of drug concentration in plasma to saliva is frequently less than 1. The easiest way of evaluating a drug’s pharmacokinetics in the human body is by assessing the amount present in the bloodstream, serum or plasma. Drug concentration changes in plasma will correlate with changes in tissue drug concentration if the drug in plasma and the tissues are in dynamic equilibrium [9]. Among the methods frequently employed in bio analytical research are:

Fig No. 1 Techniques used in Bio analysis

1 Biomatrices that is pertinent to bio analysis

Bio analytical investigation needs the examination of several types of biological matrices. Furthermore, the challenges presented by each matrix vary. For example, plasma has a higher content of phospholipid than urine, which has a higher percentage of salt [3]. Numerous bio fluids including blood, blood products, plasma, serum, spit, urine etc. are used in traditional bio analysis [4]. In recent times, biological specimens have also been generated from human excrement, hair, and breast milk [10]. Here are a few examples of biological matrices utilized in bio analysis:

Fig No. 2 Biological matrices utilized in bio analysis

2 Bio analytical sample preparation-

Sample preparation is the process of purifying a sample before analysis or concentrating a sample to improve detection. The term bio analytical sample preparation refers to this type of procedure when the sample is physiological fluids such as urine, serum or plasma. A crucial step in the drug discovery and development process, the measurement of drug concentrations in living fluids provides the information necessitate comprehending the time duration of drug action, or PK, in humans as well as livestock [11, 12]. In terms of the amount of the required and the difficulty of removing the desired analytes from the matrix, the sample preparation phase of the analysis if frequently the most crucial and challenging. Furthermore, every matrix presents different difficulties. For instance, the entire bloodstream contains red blood cells that often need to be lysed, urine has an elevated level of salt, plasma has a lot of phospholipids, and elsewhere [13].

There are two or more reasons why sample preparation is required [14]

• To minimize the analytes endogenous interferences as much as possible
• To increase the threshold for detection of the system by enriching the sample with regards to the analytes.
• It also ensures that the preferred column and mobile phase can be used with the injecting material.

OBJECTIVE

One of the most important steps in chromatographic analysis is sample preparation and treatment. 

• Clear up by eliminating unnecessary sample matrix elements.
• Boost the Detection Limits
• Enhance Specificity
• Increase the Reproducibility
• Improve Recovery
• extend the life of the instrument
• Lower backpressure and fouling in the LC system.

Too dirty- Includes additional sample matrix elements that impede the analysis.

Too dilute- The concentration of the analytes or analytes is insufficient for quantitative detection.

The current sample matrix is detrimental to the chromatographic column or system or incompatible with it.

2.3 Safety Measures for Sample Preparation-

• The lab should be furnished with all the equipment required for emergencies.
• All applicable GLP (Good Laboratory Practices) and SOP (Standard Operating Procedures) that need to be followed when preparing samples should be thoroughly explained to staff members.
• Employees should receive training and be equipped with the necessary safety precautions.
• Take extra caution when handling biological matrices to prevent any health risks.
• Sample preparation should not be hurried because they are valuable.

2.4 Methods for preparing samples:

The preparation of the sample is a vital stage in the analytical procedure. Procedures for separating and concentrating analytes from intricate biological matrices like blood, urine, and tissues are included. It can be challenging to detect and measure target analytes in biological samples due to the presence of common interfering substances such proteins, lipids, and salts. Therefore, appropriate sample preparation methods are essential for removing contaminants, obtaining analytes, and improving detectability, ultimately guaranteeing precise and repeatable results [15, 16]. These days, there is no doubting the increasing demand for useful and ecologically friendly sample preparation techniques. Solvent extraction methods including Liquid-Liquid extraction (LLE), Solid Liquid extraction (SLE), and Liquid Phase Micro extraction (LPME) are very beneficial for bio analytic applications. These methods generate interest in pharmaceutical production and reduce the cost of drug development [17]. When automation is implemented, high sample throughput can be achieved, guaranteeing improved accuracy and little production of hazardous waste [10].

Numerous bio analytical sample preparation methods are available to extract and concentrate analytes from biological matrices. Here are a few common techniques:

Fig No. 3 Sample preparation methods

1. Liquid-liquid extraction (LLE)-

Its foundation lies in the understanding of analytes molecule partitioning equilibrium and difference solubility between aqueous and organic states (the sample). Liquid-Liquid extraction is a technique used to extract a substance from a single liquid state to another. LLE (Liquid-Liquid extraction) is the process of separating substances based on how soluble they are in two distinct immiscible fluids, usually aqueous and organic solvent-based [8]. Modern, improved methods including single drop liquid phase micro extraction, assisted membrane extraction, and (LPME) (liquid phase micro extraction) have replaced liquid extraction [17]. However, there are several disadvantages to LLE, including as low/variable recovery, matrix effects in LC-MS (Liquid Chromatography- Mass Spectroscopy) methods, poor selectivity, and the need for a large sample volume. Another drawback of LLE is its inability to remove a large range of molecules with different lipophilicity, such as hydrophilic or water-soluble compounds from the matrix. There have been recent attempts to address the constraints related to LLE [18].

Fig No. 4 Liquid- liquid Extraction Process

2. Solid-phase Extraction (SPE)-

SPE has gained popularity as a popular and successful method for removing analytes from complicated materials since the 1970s. The most widely used method for quick and precise sample preparation at the moment is solid phase extraction. A benefit of using solid-phase extractions (SPE) in many sample preparation techniques nowadays is that they may be automated and processed in parallel. For the extraction and quantification of analyze trace elements in a range of sample matrices, SPE has developed into a potent instrument. Because of its adaptability, SPE can be used for numerous kinds of processes, including trace prosperity and the purification procedure [14]. One of the earliest innovations in getting samples ready was the SPE technique, which helped to address the drawbacks of the LLE and PP. SPE, operates on the same affinity-based isolation mechanism as liquid chromatography. Analytes retention and elution from biological fluid, elimination of pollutants and interfering chemicals, and sample concentration are the fundamental objectives of SPE [18].

Steps involved in SPE-

Fig No. 5 Steps involved in SPE

1. Conditioning

The initial step of SPE involves wetting the sorbent with a solvent to condition or equilibrate the cartridge or column. This demonstrate that the cartridge or column is activated using an organic solvent, a packing material wetting agent. Water or an aqueous buffer is added to the column to start the right adsorption process.

2. Loading

Percolation through the solid phase of the analytes- containing loading solution is the next stage. Thus, some contaminants and the analytes are retained by the sorbent. After the pit has been fixed, the samples are fed into the column by gravity, vacuum, or pump.

3. Washing

To get rid of contaminants, the sorbent is cleansed. The analytes is preserved while the matrix interference is eliminated.

4. Elution

The analytes is collection during the elution step, which is the last phase.

Fig No. 6 Solid Phase Extraction Process

5. Protein precipitation technique-

Protein precipitation, which encompasses eliminating proteins from sophisticated biological fluids like blood, blood products, or urine, is a frequently implemented sample preparation technique in bio analytical the field of chemistry particularly for filtering and insisting analytes. The purpose of this method is to eliminate proteins that might interfere with further analytical procedures, such as mass spectrometry or chromatography [19]. Particularly popular in bio analytical arrives PP is one of the promptly and little time-consuming sample preparation techniques. Proteins prevalent in biometrics incur denaturation (loss of tertiary and secondary structures) as a result of external stressors like heat, strong acids, or bases, or, more often, the use of organic solvents like acetonitrile or methanol. The majority of bio analytical techniques involve adding at least three parts organic solvent to one part bio matrix, then centrifuging and cyclo mixing. Protein pellets are formed by centrifugation, and the supernatant is removed for bio analytical quantification [18]. The analytes must readily dissolve in the reconstituting solvent in order for this approach to work. Early in the downward purifying procedure, precipitation is commonly used to reduce the volume and increase the protein's purity before any chromatography stages are done. Precipitation typically yields both concentration and purification. Moreover, precipitating agents that produce a more stable product than the soluble version can be chosen [14].

Extraction of human plasma by PPT

Fig No. 7 Extraction of human plasma by PPT

Fig No. 8 Protein Precipitation Technique

6. Chromatography Method-

Numerous reported methods are still being produced as a result of the quantitative examination of chemicals of biomedical relevance using chromatographic technique. Some guarantees regarding the caliber of the work, especially the accuracy of the data produced in living matrix materials, must be given for these techniques to be beneficial [17].

Reference standards

Quality control samples spiked with reference standards and norms for calibration are employed to analyze medications and their metabolites in bodily fluids. The findings of the research may vary depending on how pure the reference standard used to create the spiked samples was. Therefore, solutions of known concentrations must be prepared using certified quantitative referenced standards of established integrity and quality. The reference standard and the substance being investigated should, if at all possible, match. If this isn’t feasible, a known-purity, predictable [20]. Typically, three different kinds of Reference standards are employed.

• Make use of standards of reference that come from reputable economic sources and are commercially available.
• Approved reference criteria 
• Additional, verified-purity material that has been specially manufactured by a nonprofit organization or testing lab [21].

7. Ligand Binding Assay-

Microbiological and LBA are likewise subject to the previously established guideline and additional bio analytical validation measures. When validation the process, it is important to consider the unique features of the various design combinations of these assays. Important reagents like matrices, antibodies, traces, and reference standards need to be properly stored under specified circumstances. When essential reagents change, assay validations become crucial [22].

8. Supercritical fluid extraction-

Bio analytical samples can be produced by using a sophisticated and robust technique known as supercritical fluid extraction (SFE). Relying on supercritical fluids, predominantly carbon dioxide (CO?), it extracts desired compounds from elaborate matrices. The fluid may be extracted selectively and efficiently without the necessity of a solvent owing to its supercritical state, which encompasses the properties of a liquid and a gas. This reflects the accuracy of this approach [23].

1. Dried saliva spot (DSS)

DSS is a method that is barely invasive to look into the oral cavity's distribution of salivary proteins. Several benefits of DSS might involve their affordability and relatively straightforward, effortless sample collection. Comparing sample preparation and extraction processes to other methods, DSS is beneficial and feasible method [24]. DSS may be crucial for assessing lactate and a migraine medication component in diabetic patients, as well as for analyzing local anesthetics (such as lidocaine) [25]. Paper microfluidic devices have recently been shown to detect glucose and lactose in saliva samples [26] cortisol and caffeine in biological matrices [27]

10. Single drop micro extraction [SDME]-

Several desirable analytes can be isolated from complicated matrices using SDME, a practical and affordable miniaturized tool. In order to overcome the issue of solvent evaporation, Liu and Dasgupta initially proposed SDME as an additional extraction technique. The analyte in SDME is split between an organic solvent micro drop called acceptor phase and an aqueous sample called donor phase. A single drop of SDME used as the extraction media. In most instances, the micro drop is composed of an organic solvent [28].

11. Stir bar Sorptive micro extraction (SBSME)-

Sorptive extraction is the basis of SBSME. The only distinction is that the deposition position is different from that in the SPME. Two essential SBSME processes are extraction and desorption. Throughout the analysis, a stir bar coated with polydimethylsiloxane was submerged deep within the sample solution. Approximately 125 milliliters of sorbent are used in commercial SBSME, which exceeds than SPME. Highly hydrophilic substances are unable to be analyzed with SBSME due to the hydrophobic property of the covering material [29].

50. Electro membrane micro extraction (EME)-

EME is a mass transfer-based micro extraction technique that works over an SLM. To shorten the extraction time, EME was added. The extraction process primarily relies on electrokinetic movement inside an electrical field. Due to its ability to reduce the equilibrium time during SLM, EME has the benefit over the HF-LPME approach. The key application of EME is for the extraction of intensely polar basic analytical substances. Both cations and anions can be removed from electrified metabolic matrices that are complex using EME, which has been demonstrated to be a practical partitioning method. EME is primarily being utilized on ionized substances from different matrices that are moderately lipophilic, like organic ions [30].

1000. Micro Extraction by Packed Sorbent (MEPS)-

Today, MEPS is a popular technique for preparing tiny samples for biological analysis that offers advantages like automation, simplicity, and cost-effectiveness. MEPS generally consist of four steps: sample installation with cleaning, elution and sorbent post- cleaning. MEPS use a small volume of solvent (10–50 mL) to elute the analytes. Sorbent ingredients are put into the MEPS cartridge to ensure proper operation. Among the sorbents used in MEPS are cation exchangers, reduced graphene oxide, graphene oxide and organic monoliths [31, 32].

Table 1. List of compounds extracted from different biological matrices and sample preparation methods.

3 Bio analytical method development-

The process employed to develop a method to identify and measure a mysterious substance or novel element in a matrix has been designated as bio analytical method development. A compound can be evaluated in a variety of ways, and choosing the type of analytical technique depends on several factors, including the chemical properties of the analyte, concentration of substances, the specimen matrix, technique and instrument costs, the speed and duration of the analysis, the degree of qualitative and quantitative evaluation, accuracy and necessary instruments [7]. A collection of all the steps required to gather, process, store, and analyze a biological matrix for an analyte may be referred to as a bio analytical method. The techniques employed for the numerical evaluation of medications and their related metabolites in biological samples are essential for producing accurate and repeatable information which is subsequently utilized for the assessment and derivation of pharmacokinetics, biological equivalents, and bioavailability. [66]. When developing new drugs, a methodical approach is crucial. Three crucial, interconnected elements make up method development:

• Analyte detection,
• analyte separation, and
• sample preparation [67]

3.1 Steps involved in method validation

The method development process involves several steps, which are as follows: [68, 69]

Fig No. 9 Steps involved in method development

Step 1: Selecting the technique and sample information

A review of the literature will be done in order to obtain firsthand knowledge of the drug's composition and pharmacokinetic parameters. The literature review of the particular drug analyte serves as the foundation for gathering data on the physicochemical characteristics of the analyte and related compounds for the development of the analytical method. For instance, solubility, polarity, size, form, structure, partition coefficient, dissociation constant and functional groups of molecules, among others. Choose the internal standard for the analytes similar chemical structure and physicochemical characteristics.

Step 2: Choosing the initial method condition

A prerequisite for the analytical procedure is the selection of a dilution agent depending on the solubility of the drug ingredient, pharmaceutical consequence, and referencing norms. Run time and peak-to-peak resolution are measured during this phase. The liquid solution should be used to determine the lowest amount of analyte.

 Step 3: Verifying the analytical method in an aqueous standard

The analytical method should be tested in aqueous standards before being examined in a biological matrix. The highest and lowest concentrations are among the minimum four concentrations of aqueous calibration curve standards that are prepared. The highest permitted concentration standard can be found using C max, while the lowest dosage standard can be found through preliminary study. By taking into account each calibration curve standard, find the correlation coefficient. It is imperative that the correlation coefficient (r) be at least 0.99. Adjust the mass spectral characteristics, the moving phase and chromatography environments, such as Buffer strength, column, rate of flow, Mobile phase, pH, wavelength, column oven temp. etc., if required to achieve the clear resolution with the required sensitivity.

Step 4: Develop and optimize the sample processing procedure

As soon as the aqueous standards instrumental method is finished, create a matrix sample. Data from the literature survey on the physical and chemical characteristics of the analyzer and internal standard, such as structure, functional groups, pH, partition coefficient, dissociation constant, polarity, and solubility, are used to set and optimize sample preparation methods, such as PP (Protein Precipitation), SPE (Solid phase extraction) and LLE (Liquid-liquid extraction).

Step 5: Verifying the analytical technique in the biological matrix

It is essential to confirm the accuracy, precision and recovery of the suggested bio analytical approach utilizing matrix samples before completing the pre-validation procedure. Prefer protein precipitation when the drug's sensitivity is higher, and monitor recovery, accuracy, and interferences. Choose liquid-liquid extraction when the drug's sensitivity is lower, and be sure to check for interferences, recovery, and precision. Solid phase extraction is preferred for improved sensitivity, recovery, precision, and less interferences when liquid-liquid extraction yields lower recovery and reproducibility. Next, during the pre-validation phase, this bio-analytical approach checks for the validation parameter.

Step 6: Pre-validation

If the technique has been demonstrated shown to be reliable, proceed with pre-validation by developing a concise protocol that covers the details of collecting the sample, experimental circumstances, and procedure parameters. It is important to assess selectivity, accuracy, precision, and recovery characteristics during the pre-validation phase.

4 Bio analytical method validations-

The International Conference on Harmonization (ICH) defines method validation as "the process of establishing documented evidence that gives a high degree of assurance that a specific method or activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics.'' Regulation-enforcement authorities need to be validated. Demonstrating the reliability of a particular technique developed for the quantitative estimation of an analyte in a particular biological matrix is the main objective of method validation [3].

Table 2. The drug’s developmental stage determines he validation selectivity of bio analytical methods

A specific bio analytical technique’s development, validation and application in regular sample evaluation can be categorized as the following

1. Reference Standard Preparation
2. The establishment of assay procedures, the advancement of bio analytical techniques
3. Using an approved bio analytical method to meet the acceptance, requirements for batch or analytical run as well as standard drug analysis [66].

4.1 Types of method validation-

Fig No. 10 Type of method validation

1. Full Validation [8]

When developing and using a biological analytical technique for first time, effective evaluation is required. A novel drug molecule must undergo complete validation. When adding metabolites to an established test for quantification, it is crucial to thoroughly validate the new assay.

2. Partial Validation [70, 71]

The modifications of certified bio analytical techniques that seldom require complete revalidations are often referred to as partial validations. There are various ways to achieve partial validation, ranging from a "nearly" full validation to as little a single test accuracy and precision assessment. Typical bio analytical technique alteration that fall under this heading involve, however aren’t restricted to:

• The sharing of bio analytical methods among facilities or analysts
• Adaptations to analytical methods (such modifying detection systems) Modification of anticoagulant used in biological fluid collecting
• A change in a species’ matrix, such as from human plasma to human urine
• Modifications to the methods for processing samples
• A species shift inside the matrix, such as from rat to mouse plasma
• A shift in the pertinent concentration range
• Small sample size (e.g., research on pediatrics)
• Analyte selectivity is demonstrated when certain metabolites or concurrent medicines are present.

3. Cross validation- [72]                                         

For the following criteria, cross-validation will be carried out.

• In one study or in several investigations, data is generated using two or more analytical techniques.
• In one investigation, data are produced using several analytical methods such as LC-UV versus LC-MS/MS
• A single study's sample analyses are carried out at several different places.
• When many contract laboratories perform sample analysis for a single project

4.2 Steps for method validation: [70]

Fig No. 11 Method validation steps

4.3 Significance and Necessity of validating bio analytical methods [70, 73]

• The production of reliable data that can be effectively understood depends on the use of bio analytical techniques that have been well described and validated.
• It is widely accepted that the methods and approaches employed in biological analysis are at the forefront of technology and are constantly being improved.

• Since the properties of the bio analytical approach differ from analyte to analyte, it could be necessary to create unique validation standards for every analyst.
• The results of the bio analytical techniques are dependable and interpretable.
• Through the use of particular laboratory studies, validation entails proving that the technique's performance features are appropriate and trustworthy for the planned analytical tasks.

5 US FDA bio analytical technique validation recommendations [70, 74, 75]

The USFDA just released the updated 2018 industry advice document on bio analytical validation. Because of this, it would be worthwhile to consider both the foreseeable future and the possible practical effects that the 2018 guideline document may have on lab procedures and the validation of bio analytical methods.

Table 3. US FDA guidelines for bio analytical validation methods

5.1 Validation parameter- [8, 66, 70, 72, 76]

Fig No. 12 Validation parameters

1. Selectivity  

The ability of an analytical technique to detect and measure the analyte of interest regardless of the presence of other components in the sample is known as selectivity. Another definition is the absence of significant interference peaks at the internal standard retention times and the analyte. Selectivity is checked by injecting a blank living matrix like plasma, urine, serum etc. and comparing any contamination at the retention time of the analyte peak to the recommended extraction procedure and chromatographic conditions. Using internal standards, LLOQ samples treated with blank matrix lots are compared.

2. Sensitivity

A method is considered sensitive, according to IUPAC, as cited in Roger Causon (Causon, 1997), if slight variations in concentration result in significant variations in the response function. The gradient of the measurement curve indicates the sensitivity of an analytical procedure. In the biological samples produced for the particular investigation, the concentration to be tested determines the sensitivity needed for a certain response.

3. Linearity

The capacity of the procedure to produce test finding that are exactly proportionate to the analyte concentration in the sample is known as linearity. The ICH 7 guidelines state that at least 5 concentrations must be evaluated in order to determine linearity. The simplest model for explaining the concentration-response correlation is the calibration curve. For every analytic agent, the amount needed for the calibration curve should be created in the same live matrix as the sample and calibration curve. If a single calibration curve is unable to adequately depict the entire range, two calibration ranges may be certified. The most popular method for demonstrating the strong correlation between concentration response data is the correlation coefficient. The divergence from the affordable concentration of LLOQ and NMT 15%b of the other standard curve shouldn't be greater than 20%.

Acceptance criteria:

• The nominal concentration of the non-zero calibration standards (CS-1 to CS-6) should be +/- 15%.75% of at least six non-zero standards ought to satisfy the aforementioned requirements.
• For LLOQ- +/- 20% of each validation run's nominal concentration.

4. Precision

Precision is defined as the degree of agreement, or disperse, within a number of observations acquired through repeatedly sampling an identical homogenous sample under predetermined conditions.

The CV must be less than 15% to be accepted. 20% variation is acceptable at LOQ.

5. Accuracy

There should be at least three concentration levels used for accuracy. Result that is adequate in terms of precision, linearity and specificity can be used to infer accuracy for pharmacological compounds. To assess the computed value’s proximity to the real value and its proximity to the nominal values, the spiked placebo samples should be made in triplicate at 80%, 100%, and 120%. The mean value ought to fall within ± 15% of the predicted value, with the exception of LLOQ, where it ought not to differ by more than ± 20%.

Procedure

At least three different concentration levels were administered into six duplicates.

HQC: Near ULOQ

MQC: Middle of calibration curve

LQC: Lower than the three times the LLOQ

Acceptance criteria

Accuracy should be between 85 and 115 %, with the exception of LLOQ between 80 and 120 %. CV should be within 15 %, with the exception of LLOQ 20%.

6. Range

The separation of analytes with high and low concentrations. The concentration range over which an analyte can be measured with a satisfactory level of accuracy and precision is known as the range of a bio analytical examination.

7. Recovery

The efficiency of the extraction is determined by comparing the detector response for the actual concentration of the pure authentic standard with the detector response obtained from a quantity of the analyte injected into and separated from a live matrix. The extraction efficiency of an analytical procedure within its variable bounds is referred to as recovery.

Acceptance criteria

The three distinct concentrations mean recovery percentage should be within 15 %

8. Matrix Effect or Matrix factor or ISTD Normalization

By comparing the response of specimen spiked prior to extraction with the response of the blank matrix sample, to which analyte was introduced at the same minimal concentration just prior to injection, it is possible to determine the effect of matrix ions on the analyte or internal standard matrix effect.

Acceptance criteria

ISTD normalization should be within 15 %.

1. Limit of detection

Limit of detection is the smallest amount of analyte in a sample that can be determined yet cannot be measured under specific experimental conditions. It is also described as the lowest concentration where background noise may be reliably separated from it.

10. Limit of quantification

In a sample, the LLOQ (lower limit of quantification) is the smallest quantity of an element that might be quantitative identified with a reasonable level of precision as well as accuracy. Determining the LLOQ based on accuracy and precision is perhaps the most realistic approach. The smallest amount of a sample that may be assessed with a reasonable level of accuracy and precision is known as the LLOQ (lowest limit of quantification).

11. Ruggedness

The term "ruggedness" refers to a method's resistance to minor variations in temperature, mobile phase composition, pH levels, and other variables that may arise during routine analysis.

Different Analyst

Different Column

Different Instrument

Acceptance criteria

With the exception of LLOQ, where it should be between 80 and 120 percent, accuracy should be between 85 and 115%. With the exception of LLOQ, which should be 20%, CV should be ± 15%.

50. Robustness 

According to ICH guidelines, an analytical technique's robustness is a gauge of how well it can tolerate small but intentional adjustments to method parameters and offers information about how reliable it is under normal operating circumstances.

Table 4. Acceptance criteria for method validation as per ICH guideline [guidelines Q2A] [77]