Chromatography Forum

I am interested in the analytical method to detect salicylic acid in animal tissues by HPLC.
It’s very difficult to work with these analites because there is not almost publications about salicylic acid and acetylsalicylic acid determination in animal tissues.
I would appreciate so much if you could notice me if there is some method or some publication about how to detect both analites in animal tissues.

Thank you in advanced.

Jordi.

This is a fairly easy analyte to deal with on the HPLC side.
We routinely use a prodigy ods3 (phenomonex) 5u 150mm X 4.6mm for this analysis. 233nm UV detection. Phosphate buffer pH 2.3 40% / Methanol 60% isocratic, with a 10ul injection.

The hard part of this analysis is going to be your sample preparation.

Our assays usually end up with concentrations about 0.4mg/ml. This gives us around 3/4 of an AU at 233nm. I am not sure what your detection ranges will be but hopefully your sample prep will be able to extract them reliably from the tissue samples.

Good Luck
Rick

We routinely assay for salicylic acid both in products and at trace levels. We use a C18 column with acetonitrile and water/acetic acid eluent, isocratic. We use UV wavelength of 304 nm which may not provide as much raw sensitivity, but would provide much more specificity than 233 nm so would likely provide better results. The USP also details a similar procedure, look there under "salicylic acid", related compounds assay, or for salicylic acid gels, lotions, etc. We usually inject 3 ul or 5 ul.

Thank you for your answers,

We need to obtain a range of 100 ug/l from pig tissue samples (liver, kidney, muscle).
Our method basically works with Acetonitrile 20 %/ acidified water to pH 2.5, wavelenght 235 nm. We detect the analyte but we have some problems with recoveries using solid phase extraction (OASIS HLB cartridge 60 mg): no reproducibility at all.

Do you know any reference article about salicylic and acetylsalicylic acid and extraction from tissues or you have any idea about how to extract them with good results?

We are looking forward your answers!

See you.

We often analyze for salicylic acid, however our matrix's are usually in pharmaceuticals. As for your second analyte acetyl salicylic acid I wouldn't see much of a problem. It is very similar to SA and because of this you just need to adjust your chromatographic conditions so that you get a separation that is acceptable.

I personally have no experience with extraction of analytes from tissues... I have done some work with solid phase extraction. As long as your conditioning and elution procedures are well developed, (you can test for repeatablity and recovery from the cartridge with known concentration solutions of your analytes). Then the problem seems to point back at your sample prep.

So it seems like these pigs must have had headaches huh? eating a lot of asprin

Rick

HPLC method of Merck could be adapted into tissues

IS=ethyl 4-hydroxylbenzoate 208ug/ml

Extraction from blood plasma: denaturation with 1.5 ml of cold 5%HClO4
SFE kolumn: Lichrolut RP-18, 100mg, 1 ml, Cat. No 19855

Conditioning: 1ml acetonitril and 1 ml H2O
Loading supernatant
Cleanig: 0.5 ml H2O
Drying:

Elution: 0.5 ml A/B/C (50/45/5) A=Acetonitrile B=H2O C=HCL 32%
kat Nr 322

Analytical column: CichroCART 250-4 LiChrospher RP-18 5um +precolumn
Cat Nr 50833 and 50962

Mobile phase: A/B/C (70/25/5) isocratic gradient

A=KH2PO4/H3PO4 buffer 0.01 M pH 2.3
B=Acetonitrile
C=Methanol

Detection 237 Flow=1 ml/min room temperature
Elution order: acetyl salicylic acid, salicylic acid, 4-hydroxybenzoate

For compounds with such a high polarity, I dould not recoomend to use the Oasis HLB. The Oasis MAX (anion-exchanger) is much more suitable. You can find suitable protocols on the website. They would be roughly as follows: dilute the plasma sample 1:1 with ammonia, and load this sample onto the ion exchanger. Wash with additional ammonia. Then wash with methanol, and finally elute in methanol with formic acid. You can find more details on the website.

In our department we determine veterinary drugs residues in animal tissues and in food of animal origin. NSAIDs is one of these drugs. We have procedures for plasma, milk and tissues. Chromatography is relatively easy step of analysis but (as wrote Jordi) sample preparation is critical step. For plasma and milk only acetonitile extraction can be use, but for tissues very robust procedure have to be applied. Unfortunatelly sample preparation for plasma is not suitable for tissues.
For few months we tried to develop multiresidue method (10 drugs) for determiantion NSAIDs in liver for HPLC - UV and DAD. We have some good results but only with LC-MS/MS method, despite liquid-liquid extraction with acetonitrile, deffatting with hexane and SPE with C18 cartridges are used.
Muscles are quite nice matrix, but liver is horrible

Dr. Pyrex is correct. I misread your post. However, we have done sample cleanup from animal tissue samples as well. I still recommend to look at the Waters website for the Oasis applications booklet.

Here is more detailed information: the relevant details for many different applications can be found in the Oasis Applications Notebook. It is a rather giant pdf, but it is worth it.

the website for the notebook is www.waters.com/WatersDivision/pdfs/720000609EN.pdf

You may need to sign in first before you will be able to get to the notebook.

Thank you all for your interest to answer my questions.
Uwe, we will try to do the method described in Waters Notebook (page 118, I suppose...) We tried this method but only with Oasis MCX cartridge and results didn't better that obtained with HLB cartridges, now we will prove both cartridges together at the same time.
Uwe you used MCX or MAX cartridges? You recommend me to use MAX but Waters method is described using MCX.
We need to obtain 100 ppb concentrations using HPLC technique and I don't know if it's possible to achieve this. Also we want to get recoveries about 70-80 %.
Dr. Pyrex there is any published method of your NSAIDs determination in HPLC-UV?
Analysis from plasma sample was very easy to validate it but in tissue samples (muscle, liver, kidney) is really being very very strong and difficult not for chromatographic conditions but for extraction sample conditions.

Salicylic acid forms an anion, therefore I recommend to use the Oasis Mixed-Mode Anion Exchanger (MAX). The methodologies of the anion-exchanger and the cation exchanger are symmetrical with respect to pH.

Furthermore, finetuning of the generic method is possible for your salicylic acid. Use the same wash protocol, followed by a more specific elution protocol with less organic solvent.

Jordi,

we also had some experience with SSA in porcine tissue. Be aware of the fact that salicylates are of frequent occurence in nature, so biological matrices can contain low concentrations of these compound (plasma around <50 ng/ml, pending the feeding of the experimental animals). Moreover, it revealed that salicylic acid could behave as a sticky component, resulting in incidental poor precision. There was an irreproducible build up in the LC system we used . Hope this helps.

Regards Bert

Dear Bert, do you have any experience in Aspirin quantification in porcine kidney anf liver samples by HPLC?
We also have problems with reproducibility and I think there is no acetylsalicylic acid in nature.

Thank you for your answer.

Jordi Lopez