This thesis deals with the development and validation of Bioanalytical assay method used for the estimation of Ofloxacin in biological fluids. Before discussing the experimental results, a brief introduction for method development, biopharmaceutical analysis and preliminary treatment of biological samples, extraction procedures for drugs and metabolites from biological samples and estimation of drugs in biological sample by LC-MS/MS for Ofloxacin.
Bio-availability and bio-equivalence studies require very precise and accurate assay methods that are well validated to quantify drugs in biological samples. The assay methods have to be sensitive enough to determine the biological sample concentration of the drug and/or its metabolite(s) for a period of about five elimination half-life after dosage of the drug. The assay methods also have to be very selective to ensure reliable data, free from interference of endogenous compounds and possible metabolites in the biological samples. In addition, methods have to be as robust and cost effective as possible, making of particular importance to bioequivalence studies. Above all, the assay methods must be able to withstand the scrutiny of national drug registration authorities who judge them on the basis of criteria established by international consensus.
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Bioanalytical chemistry is the qualitative and quantitative analysis of drug substances in biological fluids (mainly plasma and urine) or tissue. It plays a significant role in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data1. The main phases that comprise bioanalytical services are,
Sample analysis (method application).
Owing to increased interdependence among countries in recent times it has become necessary for results of many methods to be accepted internationally. Consequently, to assure common level of quality, the need for and use of validated methods has increased 2.
Whatever way the analysis is done it must be checked to see whether it does what it was intended to do; i.e. it must be validated. Each step in the method must be investigated to determine the extent to which environment, matrix, or procedural variables can affect the estimation of analyte in the matrix from the time of collection up to the time of analysis 3.
A full validation requires a high workload and should therefore only start when promising results are obtained from explorative validation performed during the method development phase. The process of validating a method cannot be separated from the actual development of method conditions, because the developer will not know whether the method conditions are acceptable until validation studies are performed 2. Method development clears the way for the further processes on the validation stage. It must be recognized that proper validation requires a lot of work. However, this effort is repaid by the time saved when running the method routinely during sample analysis.
1.0 BIOPHARMACEUTICAL ANALYSIS
1.1. NEED FOR BIOPHARMACEUTICAL ANALYSIS
Methods of measuring drugs in biological media are increasingly important related to following;
Bioavailability and Bioequivalence Studies,
New Drug Development,
Research in Basic Biomedical and Pharmaceutical Sciences.
1.2. ASSAY OF DRUGS AND THEIR METABOLITES3
A number of allusions have been made to methods that distinguish drugs from their metabolites. Drug metabolism reactions can be divided into phase I and phase II categories. Phase I typically involves oxidation, reduction, and hydrolysis reactions. In contrast, phase II transformations entail coupling or condensation of drugs.This involves glucoronidation, sulfation, aminoacid conjugation, acetylation, and methylation. Except for reduction processes, most phase I and phase II reactions yield metabolites that are more polar and hence more water soluble than the parent drug. Assays must distinguish between drug and its metabolites. If this fact is ignored, erroneous data may be generated.
1.3. ANALYSIS OF DRUGS IN VARIOUS BIOLOGICAL MEDIA
The most common samples obtained for biopharmaceutical analysis are blood, plasma and urine. Faeces are also utilized, especially if the drug or metabolite is poorly absorbed or extensively excreted in the bile. Other media that can be utilized includes saliva, and tissue.
The choice of sampling media is determined largely by the nature of the drug study. All most the drug levels in a clinical pharmacokinetic study demand the use of blood, urine, and possibly saliva. A bioavailability study may require drug level data in blood and/or urine whereas a drug identification or drug abuse problem may be solved with any one type of biological sample.
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Detection of a drug or its metabolite in biological media is usually complicated by the matrix. Because of this, various types of cleanup procedures involving techniques such as solvent extraction and chromatography are employed to effectively separate drug components from endogenous biologic material. The ultimate sensitivity and selectivity of the assay method may be limited by the efficiency of the cleanup methodology.
If the blood is allowed to clot and is then centrifuged, about 30 to 50% of the original volume is collected as serum (upper level). Thus, plasma generally is preferred because of its greater yield from blood. Blood, serum or plasma samples can be utilized for drug studies and may require protein denaturation steps before further manipulation.
If plasma or serum is used for the procedure, the fresh whole blood should be centrifuged immediately at 4000rpm for approximately 5 to 10 min, and the superÂnatant should be transferred by means of a suitable device, such as a Pasteur pipette, to a clean container of appropriate size for storage.
Urine is easiest to obtain from the patient and also permits collection of large and frequently more concentrated samples. The lack of protein in a healthy individual's urine obviates the need for denaturation steps. Because urine samples are readily obtained and often provide the greatest source of metabolites, they are frequently analyzed in drug metabolism studies.
With humans, faeces are collected in an aluminium foil pan placed under a toilet seat. Once collected, the foil is folded around the material and the sample lyophilized. Faecal specimens contain high protein content, and difficulties arise in their handling and analysis (even after Lyophilization) because of the large ratio of solid mass to drug. Denaturation of protein is usually required before further manipulations are begun.
Saliva and biological media obtained from humans when constant ratio between plasma and salivary levels of certain drugs exists via non invasive sampling techniques. Saliva is advantageous in drug studies done with children. Although the concentrations of drugs in saliva are rarely equal to those in plasma, a constant ratio (over an effective therapeutic range) permits calculation of plasma levels based on salivary analysis.
Separation or isolation of drugs and metabolites from biologic samples is performed in order to partially purify a sample. In this manner, an analyst can obtain the selectivity and sensitivity needed to detect a particular compound and can do so with minimum interference from components of the more complex biological matrix. The number of steps in a separation procedure should be kept to a minimum to prevent loss of drug or metabolite. Sometimes, the separation steps are preceded by a sample pretreatment.
1.4. STORAGE REQUIREMENTS FOR BIOLOGICAL SAMPLES
In order to avoid decomposition or other potential chemical changes in the drugs to be analyzed, biological samples should be frozen immediately upon collection and thawed before analysis. When drugs are susceptible to plasma esterases, the addition of esterase inhibitors, such as sodium fluoride, to blood samples immediately after collection helps to prevent drug decomposition .
When collecting and storing biological samples, the analyst should be wary of artifacts from tubing or storage vessels that can contaminate the sample. For example, plastic-ware frequently contains the high boiling liquid bis (2-ethylhexyl) phthalate; similarly, the plunger-plugs of vacutainers are known to contain tri-butoxyethyl phosphate, which can interfere in certain drug analysis.
1.5. PRELIMINARY TREATMENT OF BIOLOGICAL SAMPLES
In most cases, preliminary treatment of a sample is needed before the analyst can proceed to the measurement step. Analysis is required for drug in samples as diverse as plasma, urine, faeces, saliva, bile, sweat, and seminal fluid. Each of these samples has its own set of factors that must be considered before an appropriate pretreatment method can be selected. Such factors as texture and chemical composition of the sample, degree of drug-protein binding, chemical stability of the drug, and types of interferences can affect the final measurement step.
1.5.1. PROTEIN PRECIPITATION OR DENATURATION3
Biological materials such as plasma, faeces, and saliva contain significant quantities of protein, which can bind a drug. The drug may have to be freed from protein before further manipulation. Protein denaturation is important, because the presence of proteins, lipids, salts, and other endogenous materials in the sample can cause rapid deterioration of HPLC columns and also interfere the assay.
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Protein denaturation procedures include the use of tungstic acid, ammonium sulfate, heat, alcohol, trichloroacetic acid, and perchloric acid.
Methanol and acetonitrile frequently have been used as protein denaturants of biological samples. Methanol sometimes is preferred because it produces a flocculent precipitate and not the gummy mass obtained with acetonitrile. Methanol also gives a clearer supernatant and may prevent the drug entrapment that can be observed after acetonitrile precipitation.
Ultrafiltration and dialysis procedures also have been used to remove proteins from biological fluids. These procedures are not widely used because they are slow.
1.5.2. HYDROLYSIS OR CONJUGATES
The presence-of drug metabolites as conjugates, such as glucuronides and sulfates, in biological samples cannot be ignored. The effect of a drug depends to a considerable extent on the biotransformation that occurs in the human body. Therefore, it may be important to isolate the actual conjugates. Samples containing either glucuronide acetals or sulfate esters are usually pretreated using enzymatic or acid hydrolysis. The unconjugated metabolites that result from the hydrolysis procedure are less hydrophilic than their conjugates and usually can be extracted from the biological matrix.
A nonspecific acid hydrolysis can be accomplished by heating a biological sample for 30 min at 90 to 100Â°C in 2 to 5N hydrochloric acid. Upon cooling, the pH of the sample can be adjusted to the desired level and the metabolite removed by solvent extraction. Particularly stable conjugates sometimes require hydrolysis in an autoclave.
For samples containing insoluble protein, such as muscle or other related tissues, a homogenization or solubilizing step using 1N hydrochloric acid may be required before treating the sample further. For gelatinous samples such as seminal fluid or sputum, liquefaction is achieved via sonication. A solid sample such as faeces can be homogenized with a minimum amount of methanol. Homogenization is usually performed with a blade homogenizer (e.g., Waring Blender).
2.0. EXTRACTION PROCEDURES FOR DRUGS AND METABOLITES FROM BIOLOGICAL SAMPLES
After pre treatment of biological material, the next step is usually the extraction of the drugs from the biological matrix. All separation procedures use one or more treatments of matrix-containing solute with some fluid. As extracting solvents are liquid and the biological sample solid (e.g., lyophilized faeces), it is an example of liquid-solid extraction. If the extraction involves two liquid phases, it is an example of liquid-liquid extraction.
2.1. LIQUID-SOLID EXTRACTION
Liquid - solid extractions occur between a solid phase and a liquid phase, either phase may initially contain the drug substance. Among the solids that have been used successfully in the extraction (usually via adsorption) of drugs from liquid samples are XAD-2 resin, charcoal, alumina, silica gel, and aluminum silicate. Sometimes the drugs are contained in a solid phase, such as in lyophilized specimens. Liquid-solid extraction is often particularly suitable for polar compounds that would otherwise tend to remain in the aqueous phase. The method could also be useful for amphoteric compounds that cannot be extracted easily from water.
Factors governing the adsorption and elution of drugs from the resin column include solvent polarity; flow rate of the solvent through the column, and the degree of contact between the solvent and with the resin beds.
In the adsorption process, the hydrophobic portion of the solute that has little affinity for the water phase is preferentially adsorbed on the resin surface while the hydrophilic portion of the solute remains in the aqueous phase. Alteration in the lipophilic / hydrophilic balance within the solute or solvent mix, and not within the resin, affects adsorption of the solute.
Biological samples can be prepared for cleanup by passing the sample through the resin bed where drug (metabolite) components are adsorbed and finally eluted with an appropriate solvent. The liquid-solid extraction method provides a convenient isolation procedure for blood samples, thus avoiding solvent extraction, protein precipitation, drug losses, and emulsion formulation. It is possible; however, that strong drug-protein binding could prevent sufficient adsorption of the drug to resin.
2.2. DEHYDRATION METHODS.
An aqueous biological sample is treated with a sufficient quantity of anhydrous salt (sodium or magnesium sulfate) to create a "dried" mix. This mix is then extracted with a suitable organic solvent to remove the desired drug or metabolite.
2.3. LIQUID-LIQUID EXTRACTION
Liquid-liquid extraction is probably the most widely used technique because the analyst can remove a drug or metabolite from larger concentrations of endogenous materials that might interfere with the final analytical determination.
The technique is simple, rapid, and has a relatively small cost factor per sample.
The extract containing the drug can be evaporated to dryness, and the residue can be reconstituted in a smaller volume of a more appropriate solvent. In this manner, the sample becomes more compatible with a particular analytical methodology in the measurement step, such as a mobile phase in LCMS/MS determinations.
The extracted material can be reconstituted in small volumes (e.g., 100 to 500 Âµl of solvent), thereby extending the sensitivity limits of an assay. It is possible to extract more than one sample concurrently. Quantitative recoveries (90% or better) of most drugs can be obtained through multiple or continuous extractions.
Partitioning or distribution of a drug between two possible liquid phases can be expressed in terms of a partition or distribution coefficient, usually called partition coefficient is constant only for a particular solute, temperature, and pair of solvents used. By knowing the P value for the extracted drug and the absolute volumes of the two phases to be utilized, the quantity of drug extracted after a single extraction can be obtained. In multiple extractions methodology, the original biological sample is extracted several times with fresh volumes of organic solvent until as much drug as possible is obtained. Because the combined extracts now contain the total extracted drug, it is desirable to calculate the number of extractions necessary to achieve maximum extraction.
2.3.1. FACTORS AFFECTING THE PARTITION COEFFICIENT4
Factors that influence partition coefficient and hence recovery of drugs in liquid-liquid extraction are choice of solvent, pH, and ionic strength of the aqueous phase. In almost all cases, one of the liquid phases is aqueous because of the nature of a biological sample. The second liquid is selected by the analyst. It is highly desirable to select an organic solvent that shows greater affinity for the drug analyzed, yet leaves contaminants or impurities in the aqueous or biological phase. The solvent should be immiscible with an aqueous phase, should have less polarity than water, and should solubilize the desired extractable compound to a large extent. It should also have a relatively low boiling point so that it can be easily evaporated if necessary. Other considerations are cost, toxicity, flammability, and the nature of the solvent. If larger numbers of samples are to be extracted, the volume of solvent needed per sample can affect the overall cost of the assay procedure.
It is generally accepted that diethyl ether and chloroform are the solvents of choice for acidic and basic drugs, respectively, especially when the identity of the drugs in the samples are unknown. In these cases, any chemically neutral drugs are extracted into either solvent depending on their relative partition tendencies.
Proper pH adjustment of a biological sample permits quantitative conversion of an ionized drug to an un-ionized species, which is more soluble in a nonpolar solvent and therefore, extractable from an aqueous environment. In analysis, do determine a known drug or metabolite, the proper pH for extraction can be calculated from the Henderson-Hassel Balch equation using the pKa of the compound. If the species to be analyzed is unknown, the pH must be approximated based on the chemical nature of the suspected agent.
Third Factor influencing extractability of drugs from biological samples is ionic strength. Addition of highly water-soluble ionized salts, such as sodium chloride, to an aqueous phase creates a high degree of interaction between the water molecules and the inorganic ions in solution. Fewer water molecules are free to interact with an unionized drug. Therefore, the solubility of the drug in the aqueous phase decreases, thereby increasing the partitioning or distributing in favor of the non-polar or organic phase. The technique is commonly called "salting out."
Either mechanical or manual tumbling, rocking, or vigorous shaking of the samples can accomplish mixing of the aqueous organic phases . The percent recovery of a drug vs. time and/or type of mixing should be investigated for each biological sample. In many cases, vigorous shaking of a sample should be avoided because it leads to emulsification, which can be intractable for centrifugation. Emulsification is often observed when organic solvents are used at basic pH whereas certain organic solvents such as n-hexane and diethyl ether are less emulsion-prone.
Certain types of amphoteric drugs or drugs that possess extreme water solubility are not amenable to classic solvent extraction. In these cases, other types of analytical methodology such as ion-pairing must be adopted.
The technique of back-extraction can be applied with success to the analysis of drugs in biological samples. The purpose of the methodology is to further purify an extract by removing either drug or impurities by additional extractions.
3.0. CHROMATOGRAPHIC METHODS5
The presence of metabolites or more than one drug in a biological sample usually demands a more sophisticated separation for their measurement especially, when two or more drugs are of similar physical and chemical nature. Chromatography is a separation technique that is based on differing affinities of a mixture of solutes between at least two phases. The result is a physical separation of the mixture into its various components. The affinities or interactions can be classified in terms of a solute adhering to the surface of a polar solid (adsorption), a solute dissolving in a liquid (partition), and a solute passing through or impeded by a porous substance based on its molecular size (exclusion).
3.1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY5
HPLC is directly derived from classic column chromatography in that a liquid mobile phase is pumped under pressure rather than by gravity flow through a column filled with a stationary phase. This has resulted in a sharp reduction in separation time, narrower peak zones, and improved resolution. The mobile phase is placed in a solvent reservoir for pumping into the system. In the case of liquid-solid HPLC, solvents are chosen from the elutropic series. A solvent system is usually degassed by vacuum treatment or sonication before use.
3.2 LIQUID CHROMATOGRAPHY MASS SPECTROMETRY4, 5
Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for nonvolatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins.
Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors. Some of these generate two dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode array UV-Vis detectors, generate three-dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time.
Mass spectrometers also generate three dimensional data. In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample.
Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analysis.
For most compounds, a mass spectrometer is more sensitive and far more specific than all other LC detectors. It can analyze compounds that lack a suitable chromophore. It can also identify components in unresolved chromatographic peaks, reducing the need for perfect chromatography.
Some mass spectrometers have the ability to perform multiple steps of mass spectrometry on a single sample. They can generate a mass spectrum, select a specific ion from that spectrum, fragment the ion, and generate another mass spectrum; repeating the entire cycle many times. Such mass spectrometers can literally deconstruct a complex molecule piece by piece until its structure is determined.
Mass spectral data complements data from other LC detectors. While two compounds may have similar UV spectra or similar mass spectra, it is uncommon for them to have both.
4.0. ESTIMATIONÂ OFÂ DRUGSÂ INÂ BIOLOGICAL SAMPLES BY LC-MS/MS
MS has emerged as an ideal technique for the identification of such structurally diverse metabolites. When coupled with online HPLC the technique is extremely robust, rapid, sensitive, and easily automated. Not surprisingly, LC/MS/MS have become the methods of choice for pharmacokinetic studies, yielding concentration versus time data for drug compounds from in vivo samples such as plasma.
LC-MS instrument consist of three major components
LC (to resolve a complex mixture of components)
An interface (to transport the analyte in to the ion source) of a mass spectrometer
Mass spectrometer (to ionize and mass analyze the individually resolved components)
Reverse phase (RP) HPLC is a widely pretended mode of chromatography and is a major contributing factor to advances made in several areas of pharmaceutical science. Mobile phase composition is a very critical in achieving selectivity in RP-HPLC separation. Although a large number of buffer system have been used in conventional RP-HPLC, only the volatile ion paring reagent can be used in LC-MS analysis.
4.1. IONIZATION TECHNIQUES8,9
Interface is used for transporting the analyte into the ion source of a mass spectrometry. The different types of ionization techniques are ESI, APCI, APPI most commonly used ionization techniques.
4.1.1. ELECTROSPRAY IONIZATION (Turbo spray) 8, 9
Electrospray relies in part on chemistry to generate analyte ions in solution before the analyte reaches the mass spectrometer. The LC eluent is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field causes further dissociation of the analyte molecules.
The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentration in the droplets increases. Eventually, the repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected (desorbed) into the gas phase. These ions are attracted to and pass through a capillary sampling orifice into the mass analyzer.
Some gas-phase reactions, mostly proton transfer and charge exchange, can also occur. Between the times, ions are ejected from the droplets and they reach the mass analyzer.
4.1.2. ATMOSPHERIC PRESSURE CHEMICAL IONIZATION8, 9
In APCI, the LC eluent is sprayed through a heated (typically 250Â°C - 400Â°C) vaporizer at atmospheric pressure. The heat vaporizes the liquid. The resulting gas-phase solvent molecules are ionized by electrons discharged from a corona needle. The solvent ions then transfer charge to the analyte molecules through chemical reactions (chemical ionization).
The analyte ions pass through a capillary sampling orifice into the mass analyzer. APCI is applicable to a wide range of polar and nonpolar molecules. It rarely results in multiple charging so it is typically used for molecules less than 1,500Âµ. Due to this, and because it involves high temperatures, APCI is less well-suited than electrospray for analysis of large biomolecules that may be thermally unstable. APCI is used with normal-phase chromatography more often than electrospray is because the analytes are usually nonpolar.
4.1.3. ATMOSPHERIC PRESSURE PHOTO IONIZATION8, 9
Atmospheric pressure photo ionization (APPI) for LC-MS/MS is a relatively new technique. As in APCI, a vaporizer converts the LC eluent to the gas phase. A discharge lamp generates photons in a narrow range of ionization energies. The range of energies is carefully chosen to ionize as many analyte molecules as possible while minimizing the ionization of solvent molecules. The resulting ions pass through a capillary sampling orifice into the mass analyzer.
APPI is applicable to many of the same compounds that are typically analyzed by APCI. It shows particular promise in two applications, highly nonpolar compounds and low flow rates (<100 Âµl/min), where APCI sensitivity is sometimes reduced.
In all cases, the nature of the analyte(s) and the separation conditions has a strong influence on which ionization technique: electrospray, APCI, or APPI will generate the best results. The most effective technique is not always easy to predict.
4.2. MASS ANALYZER (Quadrupole) 8, 9
A quadrupole mass analyzer consists of four parallel rods arranged in a square. The analyte ions are directed down the center of the square. Voltages applied to the rods generate electromagnetic fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a given time. Quadrupoles tend to be the simplest and least expensive mass analyzers.
Quadrupole mass analyzers can operate in two modes:
Scanning (scan) mode
Selected ion monitoring (SIM) mode
In scan mode, the mass analyzer monitors a range of mass-to-charge ratios. In SIM mode, the mass analyzer monitors only a few mass to- charge ratios.
SIM mode is significantly more sensitive than scan mode but provides information about fewer ions. Scan mode is typically used for qualitative analyses or for quantitation when all analyte masses are not known in advance.
SIM mode is used for quantitation and monitoring of target compounds.
4.3. APPLICATIONS OF LC-MS/MS
Selective detection of compounds in a complex mixture
Efficient analysis of biological samples
To identify degradation products in stability studies
Identification of metabolites
Quantification of compounds in biological matrix.
5.0. QUANTITATIVE ANALYSIS
Three methods are generally used for quantitative analysis. They are the external standard method, the internal standard method and the standard addition method.
5.1. EXTERNAL STANDARD METHOD
The external standard method involves the use of a single standard or up to three standard solutions. The peak area or the height of the sample and the standard used are compared directly or the slope of the calibration curve based on standards that contain known concentrations of the compounds of interest.
5.2. INTERNAL STANDARD METHOD
A widely used technique of quantitation involves the addition of an internal standard to compensate for various errors. In this approach, a known compound of a fixed concentration is added to the known amount of samples to give separate peaks in the chromatograms, to compensate for the losses of the compounds of interest during sample pretreatment steps. Any loss of the component of interest will be accompanied by the loss of an equivalent fraction of internal standard. The accuracy of this approach obviously dependents on the structural equivalence of the compounds of interest and the internal standard.
The requirements for an internal standard must
Give a completely resolved peak with no interferences,
Elute close to the compound of interest,
Behave equivalent to the compounds of interest for analysis like pretreatments, derivative formations, etc.,
Be added at a concentration that will produce a peak area or peak height ratio of about unity with the compounds of interest,
Not be present in the original sample,
Be stable, unreactive with sample components, column packing and the mobile phase and
Be commercially available in high purity.
Free from Drug-drug intraction
The internal standard should be added to the sample prior to sample preparation procedure and homogenized with it. Response factor is used to determine the concentration of a sample component in the original sample. The response factor (RF) is the ratio of peak areas of sample component (Ax) and the internal standard (ISTD) obtained by injecting the same quantity.
6. METHOD DEVELOPMENT4, 8,9, 10,11
The method development and establishment phase defines the chemical assay.
A bioanalytical method is a set of all procedures involved in the collection, processing, storing, and analysis of a biological matrix for an analyte methods employed for quantitative determination of drugs and their metabolites in biological fluids are the key determinants in generating reproducible and reliable data that in turn are used in the evaluation and interpretation of bioavailability, bioequivalency and pharmacokinetics.
Method development involves evaluation and optimization of the various stages of sample preparation, chromatographic separation, detection and quantification. To start these works an extensive literature survey, reading work done on the same or similar analyte and summarizing main starting points for future work is of primary importance. Based on the information from the survey, the following can be done.
Choice of instrument that is suitable for the analysis of analyte of interest.
Choice of the column associated with instrument of choice, the detector and the mobile phase.
Choice of internal standard, (It must have similar chromatographic properties of analyte.)
Choice of extraction procedure, (which is time economical, gives the highest possible recovery without interference and has acceptable accuracy and precision.)
Another important issue in method development stage is the choice of internal versus external standardization. Internal standardization is common in bioanalytical methods especially with chromatographic procedures. For internal standardization, a structural or isotopic analogue of the analyte is added to the sample prior to sample pre-treatment and the ratio of the response of the analyte to that of the internal standard is plotted against the concentration. Another important point is that the tests performed at the stage of method development should be done with the same equipment that will actually be used for subsequent routine analysis. The differences found between individual instruments representing similar models from the same manufacturer is not surprising and should be accounted.
7.0 METHOD VALIDATION 2, 9,10
The search for the reliable range of a method and continuous application of this knowledge is called validation. It can also be defined as the process of documenting that the method under consideration is suitable for its intended purpose.
Method validation involves all the procedures required to demonstrate that a particular method for quantitative determination of the concentration of an analyte (or a series of analytes) in a particular biological matrix is reliable for the intended application. Validation is also a proof of the repeatability, specificity and suitability of the method.
Bioanalytical methods must be validated if the results are used to support the registration of a new drug or a new formulation of an existing one. Validation is required to demonstrate the performance of the method and reliability of results. If a bioanalytical method is claimed to be for quantitative biomedical application, then it is important to ensure that a minimum package of validation experiments has been conducted and yields satisfactory results.
The guideline for industry by FDA states that the fundamental parameters of validation parameters for a bioanalytical method validation are accuracy, precision, selectivity, sensitivity, reproducibility and stability. Typical method development and establishment for bioanalytical method includes determination of (1) selectivity, (2) accuracy, (3) precision, (4) recovery, (5) calibration curve, and (6) stability.
For a bioanalytical method to be considered valid, specific acceptance criteria should be set in advance and achieved for accuracy and precision for the validation of the QC samples.
Validations are subdivided into the following three categories:
This is the validation performed when developing and implementing a bioanalytical method for the first time. Full validation should be performed to support pharmacokinetic, bioavailability, and bioequivalence and drug interaction studies in a new drug application (NDA).
Partial validations are performed when modifications of already validated bioanalytical methods are made. Partial validation can range from as little as one intra-assay and precision determination to a nearly full validation. Some of the typical bioanalytical method changes that fall into this category include bioanalytical method transfer between laboratories or analyst, change in methodology, change of matrix within species, change of species within matrix. The decision of which parameters to be revalidated depend on the logical consideration of the specific validation parameters likely to be affected by the change made to the bioanalytical method.
Cross validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies. An example of cross validation would be a situation when the original validated bioanalytical method serves as the reference and the revised bioanalytical method is the comparator.
A method is said to be specific if it produces a response for only a single analyte. Method selectivity is the ability of a method to produce a response for the target analyte distinguishing it from all other interferences. Interferences in biological samples arise from a number of endogenous (analyte metabolite, degradation products, co-administered drugs and chemicals normally accruing in biological fluids) and exogenous sources (impurities in reagents and dirty lab-ware). Zero level interference of the analyte is desired, but it is hardly ever the case. The main thing one must take care of is that, the response of the LLOQ (Lower Limit of Quantification) standards should be greater than the response from the blank biological matrix by a defined factor. If all the efforts to get rid of interferences in the chromatographic process fail, changing to a more selective detector such as Mass Spectrometry (MS) or MS-MS may give a better result.
The following practical approach may be used during method development to investigate the selectivity of methods.
Processing blank samples from different sources will help to demonstrate lack of interference from substances native to the biological sample but not from the analyte metabolite. Processing of reagent blank in the absence of biological matrix is normally adequate to demonstrate selectivity with regard to exogenous interferences mentioned above.
Although it would be preferable that all tested blanks, if obtained under controlled conditions, be free from interferences, factors like food and beverage intake and cigarette smoking can affect selectivity. Evaluation of a minimum of six matrix sources to approve the selectivity of the method.
The precision of a bioanalytical method is a measure of random error and is defined as the agreement between replicate measurements of the same sample. It is expressed as the percentage coefficient of variance (% CV) or relative standard deviation (R.S.D.) of the replicate measurements.
% CV = ---------------------
7.2.1 INTRA-ASSAY PRECISION
This is also known as repeatability i.e. the ability to repeat the same procedure with the same analyst, using the same reagent and equipment in a short interval of time, e.g. within a day and obtaining similar results.
7.2.2 INTER-ASSAY PRECISION
The ability to repeat the same method under different conditions, e.g. change of analyst, reagent, or equipment or on subsequent occasions, e.g. over several weeks or months, is covered by the between batch precision or reproducibility, also known as inter -assay precision. The reproducibility of a method is of prime interest to the analyst since this will give a better representation of the precision during routine use as it includes the variability from a greater number of sources.
A minimum of four concentrations in the range of expected concentrations is recommended. The %CV determined at each concentration level, should be Â±15 % except for the LLOQ, where it should be Â±20%.
The accuracy of a bioanalytical method is a measure of the systematic error or bias and is defined as the agreement between the measured value and the true value. Accuracy is best reported as percentage bias that is calculated from the formula:
Measured value - True value
% Bias = -------------------------------------- x 100
Some of the possible error sources causing biased measurement are: error in sampling, sample preparation, preparation of calibration line and sample analysis. The method accuracy can be studied by comparing the results of a method with results obtained, by analysis of certified reference material (CRM) or standard reference material (SRM).
Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended.
The mean value should be Â± 15 % of the actual value except at LLOQ, where it should be Â± 20 %.
Absolute recovery of a bioanalytical method is the measured response of a processed spiked matrix standard expressed as a percentage of the response of a pure standard, which has not been subjected to sample pre -treatment and indicates whether the method provides a response for the entire amount of the analyte that is present in the sample.
Extracted sample Response
% Recovery = --------------------------------------- x 100
Un-extracted sample response
Good precision and accuracy can be obtained from methods with moderate recoveries, provided they have adequate sensitivity. Indeed it may be desirable to intentionally sacrifice high recovery in order to achieve better selectivity with some sample extraction procedure.
Solvents such as ethyl acetate normally give rise to high recovery of analyte; however these solvents simultaneously extract many interfering compounds. Therefore, provided that an adequate sensitive detection limit is attained with good precision and accuracy, the extent of recovery should not be considered an issue in bioanalytical method development and validation.
The stability of the analyte is often critical in biological samples even over a short period of time. Degradation is not unusual even when all precautions are taken to avoid specifically known stability problems of the analyte (e.g. light sensitivity). It is therefore important to verify that there is not sample degradation between the time of collection of the sample and their analysis that would compromise the result of the study. Stability evaluation is done to show that the concentration of analyte at the time of analysis corresponds to the concentration of the analyte at the time of sampling.
An essential aspect of method validation is to demonstrate that analyte(s) is (are) stable in the biological matrix and in all solvents encountered during the sample work-up process, under the conditions to which study samples will be subjected.
According to the recommendations on the Washington conference report by Shah et al.(1992), the stability of the analyte in matrix at ambient temperature should be evaluated over a time that encompasses the duration of typical sample preparation, sample handling and run time. Similarly Dagar & Brunett (1995) gave the following details to be investigated.
7.5.1 LONG TERM STABILITY
This is done to assess whether the analyte is stable in the plasma matrix under the sample storage conditions for the time period required for the samples generated in a clinical study to be analysed.
7.5.2 BENCH TOP STABILITY
Six replicates of low (LQC) and high (HQC) quality control samples were left at room temperature for 12 hours (stability samples). A calibration curve and 6 replicates of low and high quality control samples (comparison samples) were freshly processed along with the stability samples and analyzed in a single run. Ofloxacin were found to be stable in human plasma for 12 hours at room temperature.
7.5.3 AUTO SAMPLER STABILITY
Samples prepared at low (LQC) and high (HQC) quality control levels were extracted as per the procedure and kept in the auto sampler (stability samples). A calibration curve were freshly processed and analyzed with 6 replicates of stability samples in a run. Concentrations were calculated to determine % nominal over time Ofloxacin were found to be stable for 12 hours in auto sampler.
7.5.4 FREEZE -THAW STABILITY
This stability test is done to ensure that the sample remains stable after it is subjected to multiple freeze-thaw cycles in the process of the study. This can be done by thawing samples at high and low concentrations unassisted and allowing them to freeze again for at least 12-24 hrs. The cycle is repeated twice and the sample is processed at the end of the four cycles and its result is compared with freshly prepared sample. If the analyte is not stable after four cycles, measures must be taken to store adequate amounts of aliquots to permit repeats, without having to freeze and thaw the sample more than once.
7.6 MATRIX EFFECT
The direct or indirect alteration or interference in response due to the presence of unintended analytes or other interference substance in the sample. The quantitation measure of matrix effect is the matrix factor and was calculated by using following equation.
Peak response in post extracted spiked sample
Matrix Effect = ---------------------------------------------------------- x 100
Peak response in standard solution