Chromatography Forum

Hello everyone,

I am trying to quantify castor oil extracted from plasma and I have been having problems with the extraction method.

Initial conditions:
-column: NovaPak C18 3.9x300
-mobile phase: 80:20 ACN:THF, 0.500 mL/min isocratic flow
-run time: 20 min
-elution time: 12.5 min
-neats: 250-5000mcg/mL in MeOH (found linearity among neats but not when extracted)

Failed extraction methods:
SIMPLE METHODS-10:1 cold MeOH/or ACN:plasma sample (500:50), incubated for 5 min, vortex 1 min, centri. 10 min, remove 520 mcL, dry under nitrogen stream, reconst. in 80:20 ACN:THF

DETAILED METHOD A-Citation:
Sparreboom, A., van Tellingen, O., Huizing, M.T., Nooijen, W.J., Beijnen, J.H. Determination of polyoxyethyleneglycerol triricinoleate 35 (Cremophor EL) in plasma by pre-column derivatization and reversed-phase high-performance liquid chromatography. Journal of Chromatography B, 681 (1996) 355-362.

Sample Extraction for Castor Oil Assay
**Steps taken from literature and slightly modified due to equipment**
1. 60ul of sample is added to 200ul of alcoholic KOH in a 2.0 ml microtube
2. The mixture is deproteinized by vortexing for 30 s followed by centrifuge @ 2500 rcf for 10 min.
3. 200ul of supernatant is transferred to a 10 ml glass test tube with PTFE covered screw cap and saponification is performed by heating @ 100 C for 30 min on a heating block.
4. After cooling 200ul of 1.0M aqueous HCL and 2ml of chloroform are added and the reaction solutions are mixed vigorously for 2 min, sonicated for 5 min, followed by centrifugation @ 460 rcf for 5 min.
5. 1600ul of the organic the organic layer is pipetted from the bottom and transfered into a clean 4.6 ml glass tube.
6. The solvent is evaporated by nitrogen stream @ 43 C and the residue is reconstituted in 500ul of benzene by sonication for 5 min.
7. To each sample 500ul of 2.0% (v/v) of oxalyl chloride in benzene is added , vortexed for 1 min and the mixture is incubated @ 70 C in a temperature controlled waterbath.
8. The organic solvent is evaporated by nitrogen stream and the residue is reconstituted in 100ul of benzene, followed by adding of 100ul of 40mM 1-naphthylamine in benzene and 10ul of 5.6% (v/v) triethylamine in benzene. The mixture is, sonicated for 5 min, vortexed for 30s and incubated for 30 min @ 37C.
9. The sample is again evaporated to dryness under nitrogen stream, redissolved in 300ul of methanol-acetonitrile- water (72:13:15), sonicated for 5 min and vortexed for 30s.
10. 300ul is transferred to a 300ul HPLC insert vial and 20ul is injected into the HPLC system

-DETAILED METHOD B:
1. 500 uL of castor oil/plasma sample is transfered to a clean 15mL cetrifuge tube.
2. 2,000 uL of ACN is added, vial is screw capped and vortexed for 30 sec, incubated for 10 min on ice, and vortexed for 1 min.
3. 5 mL of n-butyl chloride is added, vortexed for 30 sec, incubated for 10 min on ice, and vortexed for 1 min.
4. tube is spun down @ 4,000 rpm for 20 min
5. 6,000 uL of supernatant is transfered to a clean glass test tube
6. test tube is dried under nitrogen stream
7. 20uL of HCL is added, 500uL of 80:20 ACN:MeOH, vortex for 1 min, sonicate for 30, vortex for 1 min, sonicate for 20 min, and vortex for 30 sec.
8. 300 uL is injected.

Sorry for the long post. I wanted to give as much detail as possible. Thanks in advance for you help.

Larry

you got to understand what's going on in every step of the operation such the 3rd is detecting the 1-naphthylamine derivative and the 4th the castor oil ester(the assumed status in the plasma). You have to use different standard for qualification.
I also believe you can cut (maybe) half of the unnecessary operations such as many times sonication and vortex.
Good Luck

I mean for quantification

I don't see anything obviously wrong with method B, but I wonder what is the HCl for in step 7.

The simple method failed because 10% water is enough to make lipids insoluble in methanol or acetonitrile.

Method A has a couple things to watch out for. Be sure that at step 4 the pH is strongly acidic otherwise the fatty acids won't dissolve in CHCl3. Also, the acyl chlorides formed in step 7 are rather sensitive to water or alcohols.

Is there some reason why you are not using the classic Fatty Acid Methyl Ester method with a GC?

Thank you for your responses.

ym3142-I forgot to add that I was using different conditions for Method A. As for B I am still toying with the conditions.

Mark-I added HCL because the precipitate remained adhered to the test tube. Was this a mistake? Also, we don't have a GC in our lab, just three LC's and a ZQ. Will the method work LC?

Thanks in advance

Larry

The FAME method won't work for LC/UV because it has no chromophores.

I looked up ricinoleic acid. It is 12-hydroxy,9-octadecenoic acid. It probably reacts with itself in Method A step 7.

The HCl in B7 may or may not be a mistake. Try without it.

Hi Larry

When you say that you have problems with the extraction, does this mean that you are not seeing any peaks, not the peaks you expect, or that you have poor repeatability or poor recovery.

What happens if you start off with pure castor oil ?

Peter

The method was originally setup without HCL and I was unable to reconstitute the precipitate. It was added later. Should I sonicate the samples for a longer period? Each sample takes an hour to completely dry.

For method A, yes, in step 7 & 8 the purpose is produce a naphthyl group to enable detection at 280nm. However, I have been unsuccesful at recovering linear amounts (neat and plasma samples)

Initial conditions for A:
UV detection: 280nm
Mobile Phase: methanol – acetonitrile – 10 mM potassium phosphate buffer (pH 7.0) (72:13:15)
Column: Spherisorb: S5 ODS1 4.0 mm x 125 mm C18

I feel A is the better of the two, but I think unless conditions are exact, there is a large margin for error.