Amoxicillin trouble with LC/MS/MS - Antibiotics development

[Alita]

Hello dear friends:

This same issue I posted also in the Sample Preparation subject, (I don´t know if I´m able to do that), but is a concern with the both topics (LC/MS/MS and sample preparation).

I´m trying to develop a method for the determination of antibiotics in kidney samples. The antibiotics and their respective LMR are:
* Penicillin G - 50 ppb
* Amoxicillin - 50 ppb
* Tetracicline, Oxitetracicline and Clortetracicline - 600 ppb
* Erythromicin - 200 ppb
* Tylosin - 100 ppb

Equipment: Agilent LC/MS/MS 6460 (UHPLC 1290)
Column: Phenomenex Gemini 3 u - 110 A, 100 mm x 2 mm
Mobile phase: a) 0,1% HCOOH in water B) Methanol
Ramp:
0,5 min 90A-10B
1 min 80A - 20 B
4 min 75% A - 25%B
8 min 40A - 60 B
9 min 5% A - 95B
9,5 min 5% A - 95% B
10 min - 90%A - 10 %B

Injecting the antibiotics in mobile phase at 100 ppb I don´t have any trouble seeing all the compounds!!

The trouble is when I inject the kidney extract, I loose the Amoxicillin, who has a retention time of 1,6 minutes.

The extraction procedure is the following:
- 5 grams of sample
- Add 1% Oxalic acid in Acetonitrile. Mix
- Add 5 grams of NaSO4. Vortex and centrifugate
- Pass the supernatant to another tube that contains 500 mg of Bondensil-C18. Mix and centrifugate
- Dilute 750 uL of the extract with 250 uL of water, filter with PTFE and inject.

All the other antibiotics looks great! but as I mentioned, in matrix I don´t see the amoxicillin, I´m injecting at 250 ppb in kidney and see a small peak. The transitions are:
366 - 349 Fragmentor: 60 and CE: 5
366 - 208 Fragmetor: 60 and CE: 5
366 - 133.9 Fragmentor: 60 and CE: 20
366 - 114 Fragmentor: 60 and CE: 15

I´ve tryed also another kind of extraction, using SPE column with Strata-X-AW, but the same trouble appears, the other antibiotics looks good but The amoxicillin doesn´t appear

I´ve tryed changing the proportion of the dilution of the extract, add mobile phase instead of water, add 1%HCOOH in water instead of water, etc...but nothing seems to work.

I don´t believe that the cromatography is the trouble, because I see a beatiful peak of amoxicillin injecting without matrix, but I would like you to help me to figure out in which step of the extraction i could be loosing it

Thank you very much!
Best regards
Alita

[James_Ball]

Have you tried spiking the extract just before injection to see if it is simply a recovery problem or an extraction from matrix problem?

Can you alter your starting mobile phase composition to try to hold the Amoxicillin on the column longer in case there are other compounds eluting at the same time which are causing suppression of signal?

Also try a spike before and after the final cleanup procedure to be sure you are not losing it there.

[Alita]

Dear James, thank you for your answer.

I did now what you told me. I added 100 uL of a solution of 1 ppm of Amoxicillin to a vial and added 900 uL of the kidney extract that already contained 50 ppb of Amoxicillin and the Amoxicillin peaks appeared!

According with the procedure extraction that I mentioned before, wich step could I change in order to see the Amoxicillin? or wich other changes could I do?

Changing the mobile phase composition will help in this case? Now that we have seen that we´re loosing it in some step of the extraction procedure

Thank you!
Regards

[James_Ball]

Next try this step:

The extraction procedure is the following:
- 5 grams of sample
- Add 1% Oxalic acid in Acetonitrile. Mix
- Add 5 grams of NaSO4. Vortex and centrifugate

Spike sample with 1ppm standard


- Pass the supernatant to another tube that contains 500 mg of Bondensil-C18. Mix and centrifugate
- Dilute 750 uL of the extract with 250 uL of water, filter with PTFE and inject.

Add the spike here to see if you still get the response for Amoxicillin. If you do not, then the C-18 is retaining it, if not then the problem is before this step.

Also you may want to try other filter materials instead of PTFE. Some analytes can adsorb onto PTFE, though it is the least likely material to cause problems.

[Alita]

James, I´ll do exactly as you said and will let you know the results.

I did have try with PVDF filters and had the same results, even did a centrifugation step at 4400 rpm and injecting the supernatant, but still without response for the Amoxicillin. Despite this, the area of the other antibiotic did get bigger without the filtration step.

Thank you!!

[Indumini]

I am also facing same problem with amoxicillin. Standard is giving very good peak. But meat spike sample extraction is not giving a peak. Anyone knows how to extract amoxicillin from meat using spe clean up kits?

[suman_k81]

Dear James, i Think this is a matrix problem so please check below method and apply ,i think your problem may be resolve:-
2 gm of minced and mixed Meat sample were weighed into a 20-mL glass centrifuge tube, and the internal standard solution was added (200μ L of ampicillin working solution). The sample was extracted by shaking using a Reax shaker for 10 min with 10 mL of a freshly prepared 0.2 M phosphate buffer solution (pH=8.0±0.5)containing 2% of sodium chloride. Following centrifugation for 15 min at 4,000× g , the supernatant was transferred into a new tube and submitted to a clean-up step using SPE with Oasis® HLB cartridges. The cartridges were preconditioned with methanol (3 mL), water (3 mL) and 2% sodium chloride (1 mL). After passing the aqueous meat extract through the columns using gravity, the cartridges were washed with water (2 mL) and then dried under reduced pressure for approximately 10 min. The elution was performed with acetonitrile (3 mL). At the same time, in order to compare and choose which sorbent gives better results, the same SPE procedure was performed with Sep-Pack® C18 columns. The eluate was evaporated to dryness under a gentle stream of nitrogen. The residue was re-dissolved with water (400 μL) and transferred to HPLC vials

Thanx
Suman Kr Jha