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Validation is a process to show that your assay is measuring what you think it's measuring (http://ac.els-cdn.com/S1570023215000720/1-s2.0-S1570023215000720-main.pdf?_tid=a5103056-37fa-11e5-96cb-00000aab0f26&acdnat=1438398715_80d6e6a8d5fb17ca02125ce52c091edf). You need to have an analyte standard of the analyte of interest which is distiguishable from the real chemical. This can be done by using a deuterated isotope of your analyte of interest or a chemiical with very similar physical and chemical characteristics. Several concentrations of the analyte standard should be tested which span the concentrations you expect to find in your specimen. So you are making a calibration curve. The analyte standard should be added at the begining of your detection process and in the same matrix as you will be testing (blood, urine, etc...). You go through extractions and derivitization just as you would your specimen. The internal control is added at a known ccentration to the speciment too, which will be used to calculate loss during extraction and other sample processing. You then have to repeat this calibration curve several times, so you can run some statistics and determine you limit of detection and limit of quantitation. Also as part of your calibration curve, you need a blank with no analyte control, but it will need to still have an internal control.