ISSN: 2155-9872
Journal of Analytical & Bioanalytical Techniques
Open Access
 
 
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
 
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Development and Validation of Analytical Methods for Pharmaceuticals

Nishant Toomula1*, Arun Kumar2, Sathish Kumar D3 and Vijaya Shanti Bheemidi4
1Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam, India
2Department of Biochemistry, GITAM University, Visakhapatnam, India
3Department of Biotechnology, University of Hyderabad, Hyderabad, India
4Department of Biotechnology, Nottingham Trent University, Nottingham, United Kingdom
Corresponding Author : Nishant Toomula
Department of Biotechnology
GITAM Institute of Technology, GITAM University
Visakhapatnam, India
E-mail : nishanththumula@gmail.com
Received October 15, 2011; Accepted December 02, 2011; Published December 06, 2011
Citation: Nishant T, Arun Kumar, Sathish Kumar D, Vijaya Shanti B (2011) Development and Validation of Analytical Methods for Pharmaceuticals. J Anal Bioanal Tech 2:127. doi: 10.4172/2155-9872.1000127
Copyright: © 2011 Nishant T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Related article at
DownloadPubmed DownloadScholar Google

Visit for more related articles at Journal of Analytical & Bioanalytical Techniques

Abstract

Analytical methods development and validation play important roles in the discovery, development, and manufacture of pharmaceuticals. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products. This review gives information regarding various stages involved in development and validation of analytical methods like LC, HPLC, MS.

Keywords
High performance liquid chromatography (HPLC); Liquid- liquid extraction (LLE); UV detector; Mass Spectrometry; NMR; limit of detection (LOD); Limit of quantitation (LOQ)
Introduction
Analytical method development
Analytical chemistry deals with methods for identification, separation, and quantification of the chemical components of natural and artificial materials [1]. The choice of analytical methodology is based on many considerations, such as: chemical properties of the analyte and its concentration [2], sample matrix, the speed and cost of the analysis, type of measurements i.e., quantitative or qualitative and the number of samples. A qualitative method yields information of the chemical identity of the species in the sample. A quantitative method provides numerical information regarding the relative amounts of one or more of the analytes in the sample.
The steps of method development and method validation depend upon the type of method being developed. However, the following steps are common to most types of projects:
• Method development plan definition
• Background information gathering
• Laboratory method development, it includes various stages namely sample preparation, specific analytical method, detection and data processing
• Generation of test procedure
A well-developed method should be easy to validate. A method should be developed with the goal to rapidly test preclinical samples, formulation prototypes, and commercial samples. There are five common types of analytical methods, each with its own set of validation requirements:
• Identification tests
• Potency assays
• Quantitative tests for impurities
• Limit test for the control of impurities
• Specific tests
The first four tests are universal tests, but the specific tests such as particle-size analysis and X ray diffraction are used to control specific properties of the active pharmaceutical ingredient (API) or the drug product [3,4].
The most widely used methods for quantitative determination of drugs and metabolites in biological matrices such as blood, serum, plasma, or urine includes Gas chromatography (GC), High-performance liquid chromatography (HPLC) [5,6], Thin layer chromatography (TLC), combined GC and LC mass spectrometric (MS) procedures such as LC-MS [7,8], LC-MS-MS [9,10], GC-MS [11,12], and GC-MSMS, techniques like NMR is used for structure identification.
Chromatography in different forms is the leading analytical method for separation of components in a mixture. The chromatographic procedure for the separation of substances is based on differences in rates of migration through the column arising from different partition of the compounds between a stationary phase (column packing) and a mobile phase transported through the system [13]. Chromatographic methods can be classified according to the physical state of the mobile phase into the following basic categories: gas chromatography (GC), supercritical fluid chromatography (SFC) and liquid chromatography (LC). The technique was originally developed by the Russian botanist M.S. Tswett in 1903 [14,15].
 
Today TLC is rapidly becoming a routine analytical technique due to its advantages of low operating costs, high sample throughput and the need for minimum sample preparation. The major advantage of TLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC thus reducing the analysis time and cost per analysis [16,17]. An enhanced form of thin layer chromatography (TLC) is called as High performance thin layer chromatography (HPTLC) [18,19]. A number of enhancements can be made to the basic method of thin layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements.
Liquid chromatography can be categorized on the basis of the mechanism of interaction of the solute with the stationary phase as: adsorption chromatography (liquid-solid chromatography), partition chromatography (liquid-liquid chromatography), ion-exchange chromatography (IEC), size exclusion chromatography (SEC) and affinity chromatography.
Early work in liquid chromatography was based on highly polar stationary phases, and nonpolar solvents served as mobile phases, this type of chromatography is now referred to normal-phase liquid chromatography (NPLC) [20]. Chromatography on bare silica is an example of normal-phase chromatography. In reversed-phase high performance liquid chromatography (RP-HPLC), the stationary phase is nonpolar [21,22], often a hydrocarbon, and the mobile phase is relatively polar [23]. In RP-HPLC, the most polar component is eluted first, because it is relatively most soluble in the mobile phase.
The definite break-through for liquid chromatography of low molecular weight compounds was the introduction of chemically modified small diameter particles (3 to 10μm) e.g., octadecyl groups bound to silica in the late 1960s. The new technique became rapidly a powerful separation technique and is today called high performance liquid chromatography (HPLC).
HPLC-UV diode-array detection (DAD) [24,25] and HPLC-MS techniques take advantage of chromatography as a separation method and DAD or MS as identification and quantification methods. The HPLC equipment consists of a high-pressure solvent delivery system, a sample auto injector, a separation column, a detector (UV or DAD) a computer to control the system and display results.
Ultra performance liquid chromatography (UPLC) is a recent technique in liquid chromatography, which enables significant reductions in separation time, solvent consumption and analysis time as compared to the conventional HPLC [26,27].
Sample preparation
The purpose of sample preparation is to create a processed sample that leads to better analytical results compared with the initial sample. The prepared sample should be an aliquot relatively free of interferences that is compatible with the HPLC method and that will not damage the column [28]. The main sample preparation techniques are liquid-liquid extraction (LLE) [29,30] and solid-phase extraction (SPE) [31]. In these methods the analyte of interest was separated from sample matrix, so that as few potentially interfering species as possible are carried through to the analytical separation stage.
Detection
After the chromatographic separation, the analyte of interest is detected by using suitable detectors. Some commercial detectors used in LC are: ultraviolet (UV) detectors [32], fluorescence detectors, electrochemical detectors, refractive index (RI) detectors and mass spectrometry (MS) detectors. The choice of detector depends on the sample and the purpose of the analysis.
The UV detectors are the most common HPLC detectors since they are robust, cheap, easy to handle, and since many solutes absorb light in this frequency range [33,34]. The ordinary UV detector measures the absorbance at one single wavelength at the time. A diode-array detector (DAD) can measure several wavelengths at the same time, and since no parts are moved to change wavelength or to scan, there are no mechanical errors or drift with time.
DAD detectors have been proposed for various applications, such as preliminary identification of a steroidal glycoside in seed [35], peptide mapping [36], assay of sulfamethazine in animal tissues [37], or identification of pesticides in human biological fluids [38].
HPLC with a mass spectrometer detector (LC-MS) [39,40] showed superior sensitivity and selectivity compared to HPLC-UV methods [41].
Mass Spectrometry: Mass spectrometry (MS) is a widely used detection technique that provides quantitative and qualitative information about the components in a mixture [42]. In qualitative analysis it is very important to determine the molecular weight of unknown compound and MS is capable of that. MS is also more sensitive than an UV detector for quantification. An MS detector consists of three main parts: the ionization source where the ions are generated, the mass analyzer, which separates the ions according to their massto- charge ration (m/z), and the electron multiplier (detector). There are several types of ion sources, which utilize different ionization techniques for creating charged species. Three popular ionization techniques are: electrospray ionization (ESI) [43], atmospheric pressure chemical ionization (APCI) and matrix-assisted laser desorption (MALDI). Electrospray is the most widely used ionization technique when performing LC-MS [44-47].
NMR: Nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool to determine the structure of compounds [48,49]. This nondestructive spectroscopic analysis can reveal the number of atoms and their connectivity’s, and thus the conformations of the molecules.
Near infrared (NIR) spectroscopy is a quick, non-destructive method that is amenable for spot analysis application. In the last two decades, it has been increasingly used in pharmaceutical analysis [50].
Method validation
“Validation of an analytical method is the process by which it is established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application - “[51].
The methods were validated according to International Conference on Harmonization (ICH) guidelines for validation of analytical procedures [52,53]. Validation is required for any new or amended method to ensure that it is capable of giving reproducible and reliable results, when used by different operators employing the same equipment in the same or different laboratories [54]. The type of validation program required depends entirely on the particular method and its proposed applications.
Typical analytical parameters used in assay validation include:
• Accuracy
• Precision
• Specificity
• Detection Limit
• Quantitation Limit [55]
• Linearity
• Range
• Robustness
Accuracy: Accuracy is a measure of closeness between the measured and real value [56].
Precision: Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions of repeatability [57], intermediate precision reproducibility.
Specificity: Specificity is the ability to measure the desired analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc [58].
Detection limit: The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.
Can be determined
– Visually
– Signal to Noise ratio [59]
– Standard Deviation of the Response and the Slope [60]
Quantitation limit: The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.
Linearity: The linearity of an analytical procedure is its ability to obtain test results which are directly proportional to the concentration of analyte in the sample. Test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares.
Range: The range of an analytical procedure is the interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Robustness: The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters [61] and provides an indication of its reliability during normal usage.
Only specificity is needed for an identification test. However, the full range of specificity, accuracy, linearity, range, limit of detection (LOD) [62], limit of quantitation (LOQ) [63], precision, and robustness testing is needed for more-complex methods such as quantitative impurity methods.
Conclusion
Recent development in pharmaceutical and biotechnological field generates a cumulative demand for analytical methods. Rapid and accurate quantification of the substrate and drug product is important in the process development. Improvements in analytical instrumentation leads to development of new techniques like isocratic and gradient RP-HPLC, which evolved as the primary techniques for the analysis of nonvolatile APIs and impurities. These analytical methods are critical elements of pharmaceutical development so it is very important to develop efficient and accurately validated analytical methods to develop safe and effective drugs.
 
References
































































Select your language of interest to view the total content in your interested language
 
Share This Article
   
 
   
 
Relevant Topics
Disc Affinity Purification
Disc Analytical Biochemistry Techniques
Disc Analytical Chemistry
Disc Analytical Chromatography
Disc Analytical Techniques
Disc Anthropology Biomedicine
Disc Applied Biomedicine
Disc Bioanalysis
Disc Bioanalysis Methods
Disc Bioanalytical Chemistry
Disc Bioanalytical Method Validation
Disc Bioanalytical Techniques
Disc Biomedical Chromatography
Disc Biomedicine and Pharmacotherapy
Disc Biomolecules
Disc Blood Biochemistry
Disc Capillary Electrochromatography
Disc Capillary Electrophoresis
Disc Carbohydrates Biochemistry
Disc Cellular and molecular Biochemistry
Disc Chemometric Analysis
Disc Chromatographic Techniques
Disc Chromatography
Disc Computational Biomedicine
Disc Deuterium Exchange Mass Spectrometry
Disc Differential Scanning Calorimetry
Disc Drug Resistance
Disc Electron Capture Dissociation Mass Spectroscopy
Disc Electrophoresis
Disc Electrospray Tandem Mass Spectrometry Newborn Screening
Disc Extraction Chromatography
Disc Filtration
Disc Fishery biochemistry
Disc Flow Injection Analysis
Disc Food Biochemistry
Disc Fourier Transform Mass Spectrometry
Disc GC-MS
Disc Gas Chromatography
Disc Gas Chromatography Mass Spectrometry
Disc Gravimetric Analysis
Disc HPLC
Disc HPTLC
Disc Heterocyclic Compounds
Disc Imaging Mass Spectrometry
Disc Immuno Affinity Chromatography
Disc Infrared Spectroscopy
Disc Inorganic biochemistry
Disc Ion-exchange chromatography
Disc LC-MS
Disc LC-MS principles
Disc Liquid Chromatography
Disc Liquid Chromatography Mass Spectrometry
Disc Liquid Liquid Extraction
Disc MALDI
Disc Marine Biomedicine
Disc Mass Spectrometry
Disc Mass Spectrometry in Medicine
Disc Mass Spectroscopy
Disc Mass Spectroscopy in Forensic Studies
Disc Medical Biology
Disc Membrane Biochemistry
Disc Method Validation
Disc Molecular Biomedicine
Disc Molecular Epidemiology
Disc NMR Applications in Biomedicine
Disc Negative Results in Biomedicine
Disc Pesticides Biochemistry
Disc Pharmaceutical Analytical Techniques
Disc Pharmaceutical Sciences
Disc Preparative Biochemistry
Disc Protein Biochemistry
Disc Protein Folding by Mass Spectrometry
Disc Protein Mass Spectrometry
Disc Protein Purification
Disc Qualitative Analysis
Disc Separation Techniques
Disc Soil Biochemistry
Disc Spectrophotometry
Disc Spectroscopy
Disc Sports Biomedicine
Disc Super Critical Fluid Chromatography
Disc Synthetic Organic Chemistry
Disc Systems Biomedicine
Disc Tandem Mass Spectrometry
 
Recommended Journals
Disc Chromatography Journal
Disc Analytical Biochemistry Journal
Disc Pharmaceutical Analysis Journal
Disc Pharmaceutical Analytical Chemistry Journal
Disc Mass Spectrometry Journal
Disc Bioanalysis Journal
  View More»
 
Recommended Conferences
Disc 2nd International Conference on Current Trends in Mass Spectrometry
July 20-22, 2016 Chicago, USA
Disc World Congress on Chromatography
September 21-23, 2016 Amsterdam, Netherlands
Disc  7th International Conference and Exhibition on Analytical & Bioanalytical Techniques
September 28-30, 2016 Orlando, Florida, USA
Disc 3rd Asia Pacific Mass Spectrometry Congress
October 10-12, 2016 Kuala Lumpur, Malaysia
Disc International Pharmaceutical Method Development and Validation Conference
November 24-25, 2016 Dubai, UAE
View More»
 
Article Tools
Disc Export citation
Disc Share/Blog this article
 
Article usage
  Total views: 11272
  [From(publication date):
December-2011 - May 07, 2016]
  Breakdown by view type
  HTML page views : 7524
  PDF downloads :3748
 
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

 
OMICS International Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
 
 
OMICS International Conferences 2016-17
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings
 
 

Contact Us

Agri, Food, Aqua and Veterinary Science Journals

Dr. Krish

agrifoodaquavet@omicsinc.com

1-702-714-7001 Extn: 9040

Clinical and Biochemistry Journals

Datta A

clinical_biochem@omicsinc.com

1-702-714-7001Extn: 9037

Business & Management Journals

Ronald

business@omicsinc.com

1-702-714-7001Extn: 9042

Chemical Engineering and Chemistry Journals

Gabriel Shaw

chemicaleng_chemistry@omicsinc.com

1-702-714-7001 Extn: 9040

Earth & Environmental Sciences

Katie Wilson

environmentalsci@omicsinc.com

1-702-714-7001Extn: 9042

Engineering Journals

James Franklin

engineering@omicsinc.com

1-702-714-7001Extn: 9042

General Science and Health care Journals

Andrea Jason

generalsci_healthcare@omicsinc.com

1-702-714-7001Extn: 9043

Genetics and Molecular Biology Journals

Anna Melissa

genetics_molbio@omicsinc.com

1-702-714-7001 Extn: 9006

Immunology & Microbiology Journals

David Gorantl

immuno_microbio@omicsinc.com

1-702-714-7001Extn: 9014

Informatics Journals

Stephanie Skinner

omics@omicsinc.com

1-702-714-7001Extn: 9039

Material Sciences Journals

Rachle Green

materialsci@omicsinc.com

1-702-714-7001Extn: 9039

Mathematics and Physics Journals

Jim Willison

mathematics_physics@omicsinc.com

1-702-714-7001 Extn: 9042

Medical Journals

Nimmi Anna

medical@omicsinc.com

1-702-714-7001 Extn: 9038

Neuroscience & Psychology Journals

Nathan T

neuro_psychology@omicsinc.com

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

John Behannon

pharma@omicsinc.com

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

social_politicalsci@omicsinc.com

1-702-714-7001 Extn: 9042

 
© 2008-2016 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version