---
title: How do I develop a sample prep method?
description: This blogs describes how to do systematic sample prep method development for clinical LC-MS/MS assays.
---

[Biotage Blog ](https://www.biotage.com/blog)

# [How do I develop a sample prep method?](https://www.biotage.com/blog/how-do-i-develop-a-sample-prep-method)

 Written by [Stephanie J. Marin](https://www.biotage.com/blog/author/stephanie-j-marin-ph-d) | Feb 2, 2023 2:02:11 PM

This is a question that I am often asked by very smart people that just haven’t had time to learn the process. An optimized sample preparation method is critical for an accurate, specific, robust clinical LC-MS/MS assay. A good sample prep method can improve accuracy and precision, provide longer LC column lifetime, and keep your LC-MS/MS system clean.

Once the LC and MS/MS conditions for the compounds of interest are optimized, and method parameters like desired LOQ/LOD, analytical measurement range and specimen volume used for analysis have been established, sample prep method development can begin.

#### There are many things to consider when developing a sample prep method:

- Do I want minimal clean up or a more specific extraction technique? 
- What interferences are specific to my matrix that I need to be concerned about? 
- What type of sample pretreatment do I need to get the best results from my sample prep?

I use a system called plate mapping to design my experiments. It allows me to systematically test different ranges or variables to optimize an extraction, scout a single variable or many at a time, and save time by generating data on multiple variables at once.

Let’s look at a quick example: a basic drug in plasma. 

The drug has a molecular weight of 300 amu, and has an amine attached to aromatic ring.  The [logP](https://www.biotage.com/blog/what-is-the-role-of-logp-in-sample-prep-methods) is 4.5, and the [pKa](https://www.biotage.com/blog/why-is-ph-adjustment-important-for-sample-prep-methods) of the amine is 8.2. Desired LOQ is 2 ng/mL, and desired specimen volume is 100 – 200 µL of plasma, and I want to do minimal clean up.

Minimal clean-up can be accomplished with a phospholipid depletion/protein removal plate like [Biotage ISOLUTE PLD+](https://www.biotage.com/isolute-pld). According to the package directions, we add acetonitrile to each well of the plate, and then add sample, mix, and elute.  Variables are the addition of 1% formic acid to the acetonitrile, and to vary the acetonitrile: specimen ratio.

I would design a plate map that looks like this:

Samples are run in triplicate at 5-10 times the desired LOQ. In this experiment, we are evaluating 2 different specimen volumes (100 and 200 µL), three different solvent:specimen ratios (3:1, 4:1 and 6:1) and samples analyzed with and without formic acid.

I also analyze an extraction blank, and a negative control (drug free plasma) for each set of conditions. I would also analyze a no matrix control (unextracted control) at the same concentration to assess [process efficiency](https://www.biotage.com/blog/how-to-calculate-percent-recovery-and-matrix-effects-for-your-analytical-assays).  The best option is determined by looking at average area counts and signal:noise for each set of conditions.

Biotage has presented a one-day seminar in April and May 2019 on how to do systematic sample prep method development for clinical LC-MS/MS assays.  If you’d like to learn more, watch the recording of the event clicking on the button below:

 

[Watch The Webinar](https://www.biotage.com/a-systematic-approach-to-sample-prep-method-development-for-clinical-forensic-and-cro-laboratories-on-demand-webinar?hsCtaTracking=3f989ac8-a3e4-4678-a4f1-687eee59f742%7C262ab81e-f13d-470d-b769-86a28eccee1f)

 

[View full post](https://www.biotage.com/blog/how-do-i-develop-a-sample-prep-method)

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