The highest and lowest drug concentration standards are used to check whether the analytical HPLC method can accurately and reliably quantify the drug at both extremes of concentration. For the preparation of HD and LD, dilution of secondary standard is carried out. 

Importance of Highest drug concentration standard (HD)

The HD is used to determine if any impurity peak is interfering with the main standard peak as higher concentration make such interferences easier to detect as can be seen in figure below. Additionally, peak tailing and drug-column affinity can also be assessed by using the HD standard. 

Importance of Lowest drug concentration standard (LD)

The LD is used to determine how accurately and precisely the HPLC detector can detect and measure the lowest drug concentration. It also helps initially to assess the signal-to-noise ratio which is important for the subsequent estimation of limit of detection and limit of quantification. 

HD and LD preparation

• Select appropriate dilution ratio
• Select the size of volumetric flask 
• Transfer required volume of SS for highest and lowest drug concentration into the respective selected volumetric flasks
• Make up each volumetric flask to volume with diluent
• Inject both standards into HPLC

Use Highest (HD) and Lowest drug (LD) concentration standards calculator to find out how much volume of secondary standard is required to prepare the HD and LD also what will be the concentration of HD and LD standards.

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