Milk samples preparation

[gruby]

Has anyone, any materials concerning preparation of breast-milk samples for HPLC analysis of hyperforins?

[Uwe Neue]

I do not have an example specifically for hyperforins in breast milk, but we have worked out generally applicable sample preparation techniques for many different matrices using the Oasis family of sorbents. Typical matrices are blood samples, meat, vegetables, or water. Since the methods are generic, I am sure that you can find a method that is suitable for this problem. Look at the Waters website!

[gruby]

I was looking but i found nothing applicable...

[SIELC_Tech]

Hyperforins are very hydrophobic compounds, If you are looking to separate them from proteins in the breast milk you can try to use Primesep D column wich is designed for direct injection of biological samples (blood, urine, etc.). Proteins are positevely charged as well as the surface of silica (when you use acidic modifier like formic acid, TFA or sulfuric acid). Proteins will come in a void and hyperforin will retain by hydrophobic mechanism. You can use 50 mm column for this analysis. In case of UV detection you can use sulfuric, phosphoric acid and TFA. In case of ELSD or LC/MS you can use TFA or formic acid. Here is the link (although it is for plasma analysis but same principles applied to your separation too):

http://allsep.com/Technology_DirectPlasmaAnalysis.php

[HW Mueller]

But milk is stuffed with lipids, that should create a mess.
Also, Sielc_Tech, do you have some data on this positive silica surface, I have seen only confusing material on this.

[SIELC_Tech]

My assumption was that in the case of breast milk you can “slice and diceâ€

[Uwe Neue]

Nothing??? I don't know about that...
I looked for "milk", found 250 records. One of the top records, a "Comparison and Critical Evaluation of Six Published Extraction and Cleanup Procedures for Aflatoxin in Liquid Milk" in turn had a link to "The Book on SPE Method Development" by Dr. Patrick McDonald. This took about two minutes on my slow home computer.
I then searched for Oasis applications and method information. Among the 238 results offered was an Oasis applications notebook with how-to-do things for horse urine, beef kidney, whole blood, river water, fruit, meat, milk,...

It does not have hyperforins in breast milk, but the information is sufficiently generic such that a chemist can build his own application from the information provided.

[HW Mueller]

Sielc_Tech, you also have gobs of triglycerides (butter) and even cholesterol esters in milk.
What about the SiOH2+, or whatever, surface?

[SIELC_Tech]

Dear HW,

The stationary phase has aminogroup shielded by ligand, it is positively charged when you use something like TFA, formic or sulfuric acid (as well as other amino compounds in breast milk-if any). Just check our website. I am not saying that you will see only target compound using this appoach but you can "slice" the sample to remove compounds with different polarity. I am sure that with this approach you will see a few peaks on chromatogram but the point is that with this approach you can avoid sample preparation and analyze hyperforin.

[HW Mueller]

Ok, I thought that you have evidence for SiOH2+, etc.
Just checked the Geigy tables (Wissenschaftliche Tabellen Geigy):
Mother´s milk:
13.4-82.9 g/L lipids. Cholesterols 88-202 mg/L, Lipid phosphorus 7-14 mg/L
Cow´s milk:
34-61 g/L lipids, Chol. 70-170 mg/L, Lipid phos. 53-70 mg/L

[SIELC_Tech]

Based on this content of the breast milk I would try to trap lipids with the guard column as described in the newsletter

http://allsep.com/brochures/SielcNewsletter_0904.pdf

[gruby]

to HW Mueller: can You tell me what do You think about this method:
1 freeze drying of milk sample,
2 extraction of dry residue with non-polar solvent (hexane:ethyl acetate 9:1.. or hexane alone ) (solution of lipids and hyperforins in solvent)
3 centrifuge this solution in low temperature (-10 C ) (to make lipids solid )
4 evaporate supernatant (solvent) and dissolve the residue to analysis in mobile phase

[HW Mueller]

I don´t know what hyperforin is (structure, etc.), but if it is nearly as hydrophobic as tryglycerides, and in very low concentration, you may need some chemistry as one separation step. I would think freeze drying is useless, and precipitation of lipids from nonpolar solvents?? Maybe some would oil out? But probably together with hyperforin?
SPE could have similar problems, but if you are lucky.......
We have separated lipids of plasma/serum using TLC (for FA analysis with GC), but there was not such a huge lopsidedness of triglycerides.

[SIELC_Tech]

Hans,

Hyperforin is hydrophobic too (I send you a structure via email) with formula C35H52O4 and sample preparation is going to be a very tricky, unfortunately I don't have breast milk in my possession to try Primesep approach, may be I'll try cow milk instead to show applicability of the approach.

[Uwe Neue]

I had some time tonight, so I looked up what there was in the Oasis Applications Notebook. There was an application for Tetracyclines in milk. While tetracyclines are not hyperforins, they are full of phenol functions and are probably very similar in hydrophobicity to hyperforins.

The method goes as follows: 15 mL milk are diluted with 15 mL McIlvain buffer (mixture of citrate and phosphate) at pH 4.1. Centrifuge at 800 rpm and remove the lipid layer. The aqueous phase is processed with SPE. After the conditioning of the SPE cartrige (3cc/60mg) with methanol and water, 15 mL of the centrifuged sample are added. Polar stuff is washed out with 1.5 mL of 5% methanol in water, then the analyte is reextracted from the SPE cartridge with methanol.

I would do exactly the same thing with the hyperforins, and work from there if the method needs improvements.