Standard Addition HPLC Calibration Method (Spiking)

The standard additions calibration method is used to determine the concentration of an analyte which is in a sample matrix such as is commonly found when working with clinical, biological or food samples. To rule out interference from other components in the sample matrix, the sample is “spiked” and the detector response is monitored for any change which is not due to the change in sample concentration. The fortified standards created by spiking the original samples is useful when no version of the sample matrix is available without the analyte for use as a true blank. 

Common “Spiking” Standard Addition Procedure Example:

Divide up your sample evenly into FIVE volumetric flasks, representing five different final concentrations of spiked samples. Into four of the flasks add the same volume of increasing linear levels of the analyte to create four different calibration levels (e.g.  spiked amounts such as 25, 50, 75 and 100 units). Into the fifth flask add the same volume of diluent, without any spiked component, to serve as the ‘0’ or blank equivalent std. Next, extract all samples, inject and analyze the data per the usual method. A calibration curve of this new data set should show a linear relationship. If so, and your method is validated, you may be able to use just one spiked sample along with your regular samples to show that the reported concentration of your sample is not effected by the matrix and save yourself a lot of work (because you would need to prepare these standards and run a calibration curve each time).