Some Important Things To Observe In Internal Standard

1. Some Important Things To Observe In Internal Standard +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Used as internal standard for method validation in Bioanalytical Studies as comparison for formulation while filings in USFDA, UKMHRA, WHO. An internal standard in analytical chemistry is a chemical substance that is added in a constant amount to samples, the blank and calibration standards in a chemical analysis. 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 Internal Standard is a compound that is very similar, but not identical to the chemical species of interest in the samples, as the effects of sample preparation should, relative to the amount of each species, be the same for the signal from the internal standard as for the signal(s) from the species of interest in the ideal case. Adding known quantities of analyte(s) of interest is a distinct technique called standard addition, which is performed to correct for matrix effects. This ratio for the samples is then used to obtain their analyte concentrations from a clibrationcurve. The internal standard used needs to provide a signal that is similar to the analyte signal in most ways but sufficiently different so that the two signals are readily distinguishable by the instrument. For example deuterated chlorobenzene (C6D5Cl) is an internal standard used in the analysis of volatiles on GC-MS because it is similar to Chlorobenzene but does not occur naturally. Norleucine is also a popular internal standard for the analysis of amino acids via GC-MS. In NMR spectroscopy, e.g. of the nuclei 1H, 13C and 29Si, frequencies depend on the magnetic field, which is not the same across all experiments. Therefore, frequencies are reported as relative differences to the internal standard tetra methyl silane (TMS). This relative difference to TMS is called chemical shift, and measured in parts per million.In practice, the difference between the signals of common solvents and TMS are known, and since modern instruments are capable of detecting the small quantities of protonated solvent present in commercial deuterated solvents no TMS need be added. By specifying the lock solvent to be used, modern spectrometers are able to correctly reference the sample; in effect, the solvent itself serves as the internal standard.In chromatography, internal standards are used to determine the concentration of other analytes by calculating response factor. The internal standard selected should be again similar to the analyte and have a similar retention time and similar derivitization. It must be stable and must not interfere with the sample components. An internal standard is used when performing bioanalysis with mass spectrometry detection. An appropriate internal standard will give a measure of control for extraction, HPLC injection and ionization variability. It is an essential component of a robust high throughput bioanalytical method. The best internal standard for bioanalysis is an isotopically labelled version of the molecule you want to quantify. 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

2. more expensive solutions for stable labelled internal standards. For this reason, the term Deuterated Internal Standard is often used. +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ VIVAN Life Sciences Pvt. Limited. Ph. : +91(22) 2587 0080/81/82 Fax : +91(22) 2587 0084 Website : http://www.vivanls.com e Mail : info@vivanls.com