Hello dear friends:
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