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Bio Analytical MethodsPosted on 2018-07-14 12:19:58

Bio analytical Methods:

Analytical methods used for characterization, release, and stability testing of biotechnological/biological products are often automatically referred to as “bio analytical” methods. Bio analytical studies are typically conducted under GLPs, whereas biotech product release and stability tests follow GMP quality requirements.

A bio analytical method is used for “quantitative determination of drugs and/or metabolites in biological matrices such as blood, serum, plasma, urine, tissue and skin samples.” The applications of such methods are “pharmacology, bioavailability, bioequivalence, pharmacokinetic, and toxicology studies” conducted in humans and animals. Bio analytical methods are not intended for elucidating quality parameters (e.g., identity, purity) of a biotech product; they’re intended to determine the quantity of a drug (or the presence of induced antibodies) in biological samples.

For that reason, technologies used to perform bio analytical methods vary according to the molecular entity’s nature. With chemical products, the biological components of test samples can be removed by precipitation or extraction, allowing remaining small molecule(s) to be analysed with technologies such as LC–MS or GC–MS. Biotech products and antiproduct antibodies, bio analytical methods require technologies that can specifically measure one biological moiety (the protein(s) of interest) in the presence of a biological milieu (which contains many proteins).

The most accurate term for the analytical methods used to assess the physiochemical parameters of these products is thought by many to be bio molecular methods.

An appropriate internal standard will give a measure of control for extraction. It is an essential component of a robust high throughput bio analytical method. 

The stable labelled isotopes available to incorporate in a given molecule (drug or drug metabolite) are deuterium (2H or D), 13C and 15N. Generally, because of the abundance of hydrogen in organic molecules, the use of deuterium is preferred compared to 13C and 15N, which are generally more expensive solutions for stable labelled internal standards. For this reason, the term deuterated internal standards is often used.

Deuterated internal standards in Bio analysis:

Internal standards play critical roles in ensuring the accuracy of reported concentrations in bio analysis. Generally, because of the abundance of hydrogen in organic molecules, the use of deuterium is preferred.

D2O relatively more viscous than H2O has higher melting and boiling points while differences in other physical properties are not marked. D2O exhibits about 2600 times neutron-moderating ability compared to H2O. This has contributed to the revolution in nuclear power production as D2O provided an optimized source as a moderator in pressurized heavy water reactor (PHWR). D2O also attracted immediate attention of biologists. The earlier studies were carried out in a variety of organisms ranging from microbes to mammals. During the last two decades major attention has been focused on applications of D2O in medicine and industry besides its use in understanding reaction mechanisms in biological and physical sciences. 

 Ideally in bio analysis, a deuterated internal standard will have the same extraction recovery, ionization response in ESI mass spectrometry and the same chromatographic retention time. An important characteristic of a deuterated internal standard is that it should co-elute with the compound to be quantified. Also it should also contain enough mass increase to show a signal outside the natural mass distribution of the analyte. With this fact in mind, the design of a suitable deuterated internal standard can become a real challenge simply because the analyte of interest contains two chlorine atoms, for instance, and will need a +6 or +7 mass increase to show a signal not interfering with the analyte. Bio analysis will be greatly improved by the use of deuterated internal standards. The chromatography time will be reduced and your assay will be more robust, as it will increase the throughput and lower your rejection rates.

Internal standards play critical roles in ensuring the accuracy in LC-MS bio analysis.

With separation methods coupled to non-specific detection methods, the internal standard is usually an analogue of the analyte that is sufficiently separated from it. 

This substance can then be used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the analyte concentration of the standards. This is done to correct for the loss of analyte during sample preparation or sample inlet.

The method of internal standards is used to improve the precision of quantitative analysis. An internal standard is a known concentration of a substance that is present in every sample that is analysed.

The concept of an internal standard (IS) is quite simple — just add a known amount of the IS to every sample, both calibrators and unknowns, and instead of basing the calibration on the absolute response of the analyte, the calibration uses the ratio of response between the analyte and the IS.

Various applications of heavy water in biology, medicine and industry -

 Heavy Water exhibits different chemical and physical properties compared to normal water due to difference in the zero point energy arising out of the isotope effects. The isotope effect gives rise to difference in bond energies and this leads to the different chemical behaviour of heavy water. Due to the difference in bond energy D2O exhibit different deuterium bonding effects also (hydrogen bonding in case of normal water) giving rise to changes in composite molecular structure and behaviour. In broader sense, the effects of D2O on living systems can be categorized into two types:-

1. "Solvent Isotope Effect," based on the properties of D2O molecule as a whole in particular its effects on the structure of water and the biological macro-molecules.

2. "Deuterium Isotope Effect" (DIE), resulting from the ability of D2O to replace H with D in biological molecules. The C-D bond is several times stronger than the C-H bond and thus more resistant to enzymatic and even to chemical cleavage.

 In humans, studies on the use of D2O as a tracer in clinical nutrition and for determining normal parameters of human physiology, among infants, pregnant and lactating mothers as well as healthy adults . 

The major advantages of D2O as a tracer are that it is non-radioactive and more importantly with almost instant access to all body compartments, tissues and cell types. A number of methods based on nuclear magnetic resonance and spectrophotometry have increased the sensitivity of detection and deuterium is now a well-accepted tracer in humans. These investigations establish the safety of D2O in humans at low-level exposures.

Deuteration of drugs to slow down the pathway(s) of their metabolism. The altered metabolism of some of the drugs may lead to increased duration of their pharmacological action and/or reduction of toxic manifestations. 

Unique property of D2O relates to the enhancement of thermal stability of macromolecules, cells and tissues. This property has been utilized to enhance the thermo stability of certain vaccines e.g. oral polio vaccine or other macromolecules which need freezing temperature.

Deuteration of anticancer drugs reduces side effects while retaining their therapeutic efficacy while, D2O enhances therapeutical potential of photosensitizers. Thus, there is wider interest in exploring the application of D2O in cancer therapy employing different approaches.

1. Future Perspective: D2O exhibits several biological effects, independent of H2O and some of these have enormous possibilities of application in medicine and technology. 

2. Shelf-life Extension: Thermo stabilization of vaccines being used in the country is an important field with enormous possibilities and deserves to be considered on priority. Since regulatory obligations may be relatively less stringent the vaccines currently in use in poultry or for cattle, farm and domestic animals form the areas of choice. Likewise, preservation of cells, organs and industrial enzymes is another area of profound interest worth exploring.

3. Drug Development and Therapy: The recent approval of clinical trials by the Canadian and US Agencies for deuterated drugs opens up enormous avenues for deuteration of drugs already in use in the country. This includes all classes of drugs, whose biological efficacy can be enhanced. A comprehensive programme for synthesis of deuteration of drugs, their pharmacokinetic profile and detailed pharmaco therapeutic studies can be very rewarding. Likewise, in the area of cancer therapy, several strategies such as deuteration of anti-cancer drugs to enhance efficacy and reduce side effects use of D2O to slow down proliferation of malignant cells and enhance BNCT or photo dynamic therapy are promising. The anti-hypertensive effects of D2O are dramatic and make a case for a detailed pharmacological evaluation of D2O.

4. Biotechnology: Lower organisms including algae and bacteria can adapt to grow almost in 100% D2O. Since, distinct morphological features are associated with microbial adaptation, the phenomenon is an excellent model to explore the basic biology of stress and adaptive responses. Some of these organisms are being explored as sources of a number of deuterated molecules for industrial, biotechnological and medical applications. To illustrate the point, deuterated glycerol has interesting potential uses as a precursor of transparent plastics and other compounds. 

5. Industry: The different behaviour arising out of the isotope & deuterium bonding effects have found potential use in various industrial activities. Using some of the fundamental differences between hydrogen and deuterium a number of applications have been developed in the high technology area. Some of these are in the field of optical fibres [improved intensity and better transmission characteristics of deuterated PMMA (Poly methyl meta acrylate) compared to conventional one], optical recording systems (improved storage capacity as a result of sharper and uniform optical recording density with deuterated polymeric substance) and better quality semi-conductors with reduced stress induced leakage currents across the metal oxide gates. Deuterated compounds like lubricant additives have also been reported to provide excellent properties vis-a-vis the normal lubricants. The deuterated polymers exhibit interesting physico–chemical properties providing enormously better features for future technology.