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PK/LC/MS/MS Reverse Phase
Chromatography
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Faster is better! In every aspect of
pharmacokinetic analysis faster is better. Sample sets including
replicates of the standards, QC's and samples often approach 300
individual analyses. If you allow 15 minutes for each sample analysis the total sample
set would take 75 hours of uninterrupted analysis to complete. That particular
study would tie up an instrument in high demand (your mass spectrometer)
for over three days. Some researchers in the field are obtaining analysis cycle times of 1-2
min per sample. The majority of PK/MS labs easily achieve individual
sample turn around
times of 3-4 minutes. That same 300 sample set with a 4 min cycle time
would be finished in 20 hours. One can easily see that every
minute counts. Fast gradients and fast re-equilibrations are achieved
on short 2 mm X 30-50 mm reverse phase columns. A normal
non-PK/LC/MS flow rate for these columns is typically 200 µl/min.
Typical PK/LC/MS flow
rates used with these columns range from 400 to 1000 µl/min. The faster
flow rate helps in speedy elution and re-equilibration. Below is a
typical PK LC/MS experimental description.
PK/LC/MS Method: Column: 2.1 X 50 mm, 5 µm , 100 Å , C18 Column Temp: 40 °C Mobile Phase: A = Water,
0.1% Formic Acid Flow Rate: 400 µl/min Gradient:
Time %B
0
25 (Download this method in MS word
format
Optimizing the Gradient : Of course you will need to optimize this method to your particular compound. We recommend creating a chromatographic database detailing the hydrophobic character of all compounds coming through the lab. This can be accomplished by running the 60/60 gradient recommended in the reverse phase chromatography tutorial on the IonSource web site. Such a chromatographic database will be a valuable resource when you need to pick an internal standard that has a similar hydrophobic character. Once the apparent %B is identified in the long run back off the initial %B in the development of your short run by 10%. For example if the apparent %B is 40% then try and start the PK/LC/MS/MS method at 30%B. This procedure should ensure that your compound sticks to the column.
Optimizing the Column and Mobile Phase : Some compounds may not respond well to the mobile phase described in the example method above. Some compounds may require a different organic modifier or organic. Phosphate is not tolerated by mass spectrometers in general. Some use ammonium acetate, 10-20 mM, at low concentration in the A buffer. Others use combinations of methanol and acetonitrile in the B buffer. If your compound has poor peak shape it may pay to experiment with some or all of these parameters. It often pays to do a column survey to find the column that gives the best recovery and peak shape.
Helpful Tips: 1.) An MS lab can become overwhelmed with chromatographic method development, especially if the lab has 5 to 10 new compound coming in per week. We have found success in combining compounds and their internal standards and performing a combined chromatographic method development. Not all compounds will be amenable to this approach, however, this method has saved us countless days in chromatographic method development. This method evolved from cassette dosing method development when it was found that the method development time for 6 mixed compounds was about the same as for a single compound. 2.) Speed up method development. Many common compounds like ibuprofen already have methods developed, search for articles, search the web and or try to find the method in the publication of the USP. Don't waste time reinventing the wheel! 3.) Compound screening can often be accelerated by performing cassette dosing. Many researchers are reluctant to attempt a cassette experiment for fear of drug-drug interaction. One thing that can be done is to combine compounds post dosing. Combined with the mixed method development described in tip two this approach can save tons of time for an impacted lab. 4.) HPLC autosamplers are notoriously slow, remember to include this time in your re-equilibration, because as the autosampler is doing it's thing the column is being washed at the initial conditions, consider this time as part of the re-equilibration. You may be able to cut a 30-60 seconds from the method re-equilibration time. Every minute counts! One minute cut from an assay with 300 samples is worth 5 hours!!!
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