Method Development Advice

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

8 posts Page 1 of 1
I am working in a lab that does toxicology testing for drug metabolites in urine.

I have a good understanding of how to perform tests and review the data, but I am really wanting to learn more about the method development side.

We are working with Sciex 5500 LCMS/MS. I was hoping to find someone patient enough to break down the process into simple steps so that I will have enough information to play around with the instrument and learn the rest for myself.

I did search through existing threads and found the Analyst Optimization guide that someone posted, but that seems to be about optimizing compounds for which a method already exists.

What would be the initial steps to develop a method for a compound for which you already know what mobile phases would be best and for which you already have pure standards and a suitable column ready?
Hi alexcowart,

These references may be helpful to you:

http://www.chromatographyonline.com/uni ... analysis-2

https://www.agilent.com/cs/library/esem ... opment.pdf

The "gold standard" text is still:

Practical HPLC Method Development 2nd Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and Joseph L. Glajch, ISBN-13: 978-0471007036.
MattM
Thank you for the response Matt. Those resources seem to be for HPLC method development.

I am more looking for Mass pectrometry method development. For now the two main questions that would help me get started are"

1) How do you decide which masses to try and detect for a given compound? I have been told there is a scan mode but I am not sure how to access that in Analyst software. Would you use the scan mode to see what masses are detected?

2) I found a somewhat helpful document here https://sciex.com/documents/downloads/l ... -guide.pdf It mentions the compound optimization wizard which is a tool to auto detect optimum settings. Would this be an advisable place to start in order to find good settings? I assume I need to tell it what masses to be searching for for this step.

And again, I am just looking for basic information so that I can have enough information to play around and build a basic method and teach myself the rest.
Hi again,

My apology.

(1) Yes, this is the purpose of Scan--to access multiple masses simultaneously whilst searching for ions to monitor. Depending on the ionization mode (as you know the analytes you are assaying for in advance), you can look for [M+H]+ and [M-H]-. Ions from the eluent such as ammonium (and other phenomena within the MS) can complicate this a bit--which is why Scan is valuable.
MattM
(2) I was not able to follow the AB Sciex link, though I did find this, which may not be so helpful to you at the start:

https://sciex.com/videos/method-dev-aWang

Possibly a book by Sparkman could be helpful to you:

Introduction to Mass Spectrometry: Instrumentation, Applications, and Strategies for Data Interpretation, 4th Edition, by J. Throck Watson and O. David Sparkman
ISBN: 978-0-470-51634-8, Nov 2007, 862 pages.
MattM
MattM
alexcowart wrote:
Thank you for the response Matt. Those resources seem to be for HPLC method development.

I am more looking for Mass pectrometry method development. For now the two main questions that would help me get started are"

1) How do you decide which masses to try and detect for a given compound? I have been told there is a scan mode but I am not sure how to access that in Analyst software. Would you use the scan mode to see what masses are detected?

2) I found a somewhat helpful document here https://sciex.com/documents/downloads/l ... -guide.pdf It mentions the compound optimization wizard which is a tool to auto detect optimum settings. Would this be an advisable place to start in order to find good settings? I assume I need to tell it what masses to be searching for for this step.

And again, I am just looking for basic information so that I can have enough information to play around and build a basic method and teach myself the rest.


I use the compound optimization often when developing new methods. You need to put a single compound into a solution that will be similar to the mobile phase composition when it elutes for best results but a mixture of A and B at any ration will help. Make the solution first at 1ppm, then dilute with the mobile phase mixture you use if it gives the error that the signal is too high. You use the syringe pump on the instrument to infuse at a flow rate of 5-10ml/min then use the optimization tool by inputting the molecular weight and a range to scan for it. Just follow the guide and it should take you through the rest. You then can edit the final method that produces to include the HPLC parameters and begin optimizing separations.
The past is there to guide us into the future, not to dwell in.
Thanks a million!
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