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Hello,
I'm looking for ideas of how to approach extraction of a highly hydrophobic compound from plasma and
other tissue matrices. This compound has a clogP of 7.8 (Chemdraw) and is 99.9% plasma protein
bound. Our absolute extraction efficiencies using standard acetonitrile precipitation range from
5-20% but this efficiency seems to be declining over time. We have tried liquid-liquid extraction
with dichloromethane and a number of other organic solvents at various ratios for precipitation. We
are looking into acid extraction and high salt extraction at present. I cannot locate the data at
present but I think in the past we have tried spiking compound into already extracted blank plasma
and found what we think is ion suppression because recovery is poor relative to compound spiked into
solvent only. However, clean up of organic precipitated plasma containing compound by SPE does not
improve recovery. I think we are seeing mostly poor extraction of the compound from protein as well
as possibly some ion suppression from matrix. Any ideas for improving recovery would be greatly
appreciated.
Thank you,
Noelle
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Do you have a chromatographic method (LCMS?) for this compound? If so, what are the conditions?
Lee
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An Agilent (Santa Clara, CA) XDB C18 column (50 X 4.6 mm, 5 micron packing) was used for
chromatography with the following conditions: Buffer A: dH20 + 0.1% formic acid, Buffer B:
methanol + 0.1% formic acid, 0 - 1. 5 min 5% B, 1.5 - 2.5 min gradient to 100% B, 2.5 - 3.5 min
100% B, 3.5 to 3.6 min gradient to 5% B, 3.6 to 4.5 min 5% B.
(Shimadzu Prominence LC). Get nice sharp peak around 3.26 min. I think we have played around with
increasing initial organic (B) concentrations but may be room for additional work.
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Ok, yes it is very non-polar if the compound doesn't even elute during your gradient, but during the
100% hold. Are there any acidic or basic functional groups on the molecule? If so, how many of each?
Have you tried a Hexane liquid/liquid? Or a 30:70 water-saturated ethyl acetate:hexane
liquid/liquid? Have you tried both acidic and basic pH aqueous adjustments in your liquid/liquid
experiments?
Lee
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Hi Noelle,
I guess as you mentioned compound is very hydrophobic, you can try hexane with combination to ETHYL
ACETATE OR DICHLOROMETHANE for LLE method to improve the extraction efficiency. Addition of 1 % OPA
in water of Formic acid (5%) or 0.1 N HCL can also be tried based on the stability in acidic
condition. It may improve the extraction by making the drug free from plasma proteins.
Regards,
Manoj
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For increasing the extraction recovery, it may help to compare Protein Precipitation with:
a) Acetonitrile : plasma, 4:1
b) 0.5% Acetic Acid in Acetonitrile : plasma 4:1
c) Methanol : plasma 4:1
d) 0.5% Acetic Acid in Methanol : plasma 4:1 (this may possibly show the highest recovery)
e) SPE with Oasis HLB: Load plasma previously mixed with 0.5 % Acetic Acid, 1:1 for about 5 minutes
at medium speed. If the final volume of the mixture is too large for a thorough mixing in a 96-well
plate (and not contaminate from well to well), it helps to prepare the mix in Matrix or Micronic
cluster tubes, cap them, and mix vigorously either on a multi tube vortex-mixer or an overhead
mixer.
For each of the tests, extracting double blank and using it to spike post extraction will help
evaluate the extraction efficiency, not including the matrix effect.
For matrix effect, I would suggest using acetonitrile instead of methanol in the mobile phase.
Monitor the phospholipids transitions (several publications show what the major MRM transitions are,
or even monitoring 184 - 184 may help by using some strong conditions in the source to generate
in-source fragmentation). Adapt the gradient based on the elution of the phospholipids, trying to
keep them away from the analytes. Inject neat solvent (same composition as the extracted samples) a
few times after each sample, to monitor the elution of phospholipids on subsequent injections. If
the level of phospholipids after injecting neat solution is high, a 100% methanol step added after
the high organic step in the gradient and held for about 1.5 min (needs to be optimized based on
flow rate, column length etc) will help remove phospholipids. I've has success separating
hydrophobic compounds from phospholipids with ACE columns.
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How about trying the dilution with either acidic or basic water solution depending on your compound
is a base or an acid, then using LLE.
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Hi Noelle,
From bioanalysis point of view, your compound is very hydrophobic with clogP of 7.8 and it will have
strong interaction with C18 stationary phase although the peak shape was nice. You may want to try C
8 column and elevate your column temperature to improve the recovery from LC column.
Best of Luck!
Xiaohui Chen
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Hi Noelle,
As you doubt the there might be a matrix ion suppression, it is my advice to check infusion matrix
effect. A good exercise to determine ME. As a second thought, you can try acid precipitation with
perchloric acid or n-Hexane for LLE, as your analyte is highly hydrophobic.
Pavan Kumar Prathipati
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