Application of in-sample calibration curve methodology for regulated bioanalysis


Bioanalytics is an essential tool in drug discovery and development for determining the concentration of drugs and their metabolites as well as various pharmacodynamic biomarkers in biological fluids. In these analyses, scientists use developed and validated methods to quantitatively detect analytes and metabolites within biological matrices such as serum, plasma, urine, cerebrospinal fluid (CSF), etc. A critical component of any bioanalytical program includes bioanalytical method validation, ensuring quantitative results demonstrate accuracy, precision, selectivity, and stability so the accuracy of sample analysis results can be justified.

Efficient Bioanalytical Techniques to remove unwanted components and to selectively extract the compounds of interest are an important part of the bioanalysis. The liquid-liquid extraction, protein precipitation and solid-phase extraction techniques are being utilized less frequently. The development of novel sample preparation techniques such as use of selective sorbents, molecularly imprinted polymers has been rapid in bioanalysis. Bioanalytical Techniques Journals deal with these various advanced techniques.


Bioanalytical liquid chromatography-mass spectrometry is a technique that uses liquid chromatography with the mass spectrometry. LC-MS is commonly used in laboratories for the quantitative and qualitative analysis of drug substances, drug products and biological samples. LC-MS has played a significant role in evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. Through LC-MS biological samples are determined throughout all phases of method development of a drug in research and quality control.

Method Development:

Method of analysis are being routinely developed, improved, validated, collaboratively studied and applied. Chromatographic separations are mainly required which depend on the samples to be analyzed. The chromatographic procedure is important for the systemic approach to LC-MS/MS method development. In most cases as desired separation can be achieved easily with only a few experiments. In other cases a considerable amount of experimentation may be needed.

Procedure for Method Development

  • Collect the physicochemical properties of drug molecules from the literature.
  • Determine solubility profile
  • MS scanning and optimization
  • Mobile phase selection
  • Selection of extraction method and optimization
  • Selection of chromatographic method (based on solubility study, retention of compound)

Reversed Phase Chromatography: Reversed phase packing’s such as C18, C8 are the most popular and most widely used for reversed phase. In addition to these C4, C2 and phenyl bonded are also available. Reversed phase sorbents generally involves conditioning with an organic solvent (e.g. methanol) followed by an aqueous solvent (e.g. water).

Normal Phase Chromatography: Normal phase packing’s include silica, amino and alumina. Normal phase packing generally requires conditioning with a non polar solvent and elution is carried with polar solvents. Compounds which are with basic pH functional groups are retained by silica. However, polar compounds are irreversibly retained on a silica surface and in this case amino may be used.

High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography is a form of column chromatography used frequently in bio chemistry and analytical chromatographic packing material (stationary phase),a pump that moves the mobile phase(s) through the column, and a detector that shows the retention time of the molecules, retention time varies depending on the interactions between the stationary phase, the molecules being analyzed and the solvent(s) used. Bionalytical method development is the process of creating a procedure to enable a compound of interest to be identified and quantified in a matrix. By using biological products can be measured by several methods and the choice of bioanalytical method involves several considerations of quantitative or qualitative measurement, and precision are required with necessary equipment. The bioanalytical chain describes the process of method development by biological samples includes sampling, sample preparation, separation, detection and evaluation of the results.


The need for sound bioanalytical methods is well understood and appreciated in the discovery phase and during the preclinical and clinical stages of drug development. Therefore, it is generally accepted that sample preparation and method validation are required to demonstrate the performance of the method and the reliability of the analytical results. The acceptance criteria should be clearly established in a validation plan, prior to the initiation of the validation study. The developed assay should be sufficiently rugged that it provides opportunities for minor modifications and/or ease of adoptability to suit other bioanalytical needs such as applicability to a drug–drug interaction study, toxicokinetic study as well as for characterization of the plasma levels of the metabolites. For bioanalytical liquid chromatographic methods, sample preparation techniques, the essential validation parameters with their guidelines and suggested validation work in drug discovery and development phase have been discussed here.


Hanna Marin

Journal Manager

Journal of Analytical and Bioanalytical Techniques

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