Determination of Phenylbutazone in muscle by LC-MS-MS

[Alita]

Good day! I´m new in the area of developing methods by LC-MS-MS. We have an Agilent 6410 (upgrated) and I´m trying to develop Phenylbutazone in bovine muscle. I looked for information on many papers before starting with the assays, and I´ve seen that some of them use positive mode and others negative mode for PBZ in ESI, why is this? wich mode should I used? I optimized the compounds and I see PBZ also in both of them (Precursor ion 307 and 309 respectively)
I´m using HSS-T3 column Waters, 2,1 x 100 mm, 3,5 uM, and the mobile phases are a) 0,1% formic acid in water b) Acetronitrile, in isocratic mode (60%B - 40%A). I´m using Phenilbutazone-13C12 as IS.
Another problem that I found is that in the negative mode two peaks appears, one in 0,8 minutes and the other one at 4,1 minutes, and when I run different concentrations metanolic solutions of PBZ to figure it out wich peak is the PBZ..both of them change in the same proportions.
Could you give me some tips in order to get better results and understand better the behavior of PBZ?

Thank you very much!
Best regads
Alicia

[JMB]

The choice of +ve or -ve ionization mode is usually governed by two factors,

1) Required sensitivity, which will be indicated by S/N ratio for equal sample loadings,

2) Presence of possible interferences derived from the sample matrix.

If there is already a robust (low RSD;meets sensitivity needs) literature method for phenylbutazone in muscle, I would strongly suggest that you start with that.

The RT 0.8 min peak sounds like a very hydrophilic impurity, possibly formed by hydrolysis of the pyrazolidinedione ring. You should be able to confirm or discount this by a full-scan MS expt. Do you already have MS data for both peaks ? Are the UV spectra of the two peaks similar ?

Regards,

JMB

[Alita]

Thank you very much JMB for your answer.

Effectively as you said, I optimized both Phenylbutazone in positive and negative mode, and when running a methanolic solution of the standard I saw the compound in both modes

But when I inject muscle samples at 50 ng/g only the negative mode was sensitive enough to see the compound. Also I could check that the retention time was 4 minutes and not the peak at 0,8 minutes. Now I´m struggling with diferent extraction methods to see Phenylbutazone at 5 ng/g in muscle .

Thank you very much for your reply.

Best regards
Alicia

[JMB]

Alicia,

I would suggest that you first optimize the HPLC conditions and MS parameters to detect a pure 5 ng/g standard (for LOD you need S/N > or equal to 3:1; for LOQ > or equal to 10:1) ---- until the LC-MS system can do that, do not even think about a muscle extract.

1) What MS scan range are you using ---SIM for [M-H]- at m/z 307, narrow scan e.g. m/z 305-310 or wide scan e.g m/z 250-325 ? Or are you using MS/MS of m/z 307 ?

2) Because ESI depends on the formation of very small, charged droplets it helps to reduce the surface tension of the aqueous/organic mobile phase. Substituting MeOH for MeCN will require the organic % to be increased to give the same RT, and should give a stronger signal.

JMB

[dr_Pyrex]

I would suggest our publication describing determination of NSAIDs (with phenylbutazone) in animal muscles:
http://www.ncbi.nlm.nih.gov/pubmed/20579495

We use negative ionisation. It gives lower abundance but it is more specific in matrix samples.
Please be careful about degradation of phenybutazone in the solution and during sample preparation. It is quite common problem for "bute" analysts.