Some Important Things To Observe In Internal Standard

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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

more expensive solutions for stable labelled internal standards. For this reason, the term Deuterated
Internal Standard is often used.
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