Internal Standards

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 standards is often used.

Deuterated internal standards

Ideally in bioanalysis, 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.

Your bioanalysis 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.