reserpine solution for sensitivity

[tedinelkgrove]

I have a waters quattro micro. We have traditionally used 1pg/uL of resperpine to verify instrument sensitivity, lab performance SOP. However, results have been hit and miss. The waters solution comes in an opaque plastic bottle in the PM kits, prepared in a solution of ACN/H2O with 1% ammonium acetate. I don't believe there is an acid component listed on the spec sheet. The ammonium acetate is a buffer, I believe suitable for stablizing solutions in the pH range 6-8 (?). So not sure where the proton donor comes in for ESI? I am assuming this solution is bad from the start, being it is exposed to light and heat in a PM kit on a shelf somewhere for who knows how long.

So I have been made resposnible for developing a home brew of reserpine solution for our testing.

I found some older recipes for reserpine in my personal library, and each noted the importance of storing reserpine in light resistant vials, cold storage, and making up dilutions fresh from the stock solution. I am thinking of buying powdered (99+% purity) and making stock solution with acetic acid and methanol, then making fresh dilutions when needed with the mobile phase. We use an isocratic method (no column) to deliver sample, consistenting of 70% MeOH, 30% H2O, with I believe .1-0.05% formic (proprietary clinical solution, so not absolutely sure).

Any suggestions/guidance would be greatly appreciated.

[Alp]

If your SOP specifies you use a reserpine solution from a specific source then you should do so, otherwise you will have to modify your SOP.

If you are planning to switch from long time use of a commercial reserpine solution and go with solution diluted from stock, you are likely going to have to implement an SOP for the preparation of the stock, storage of the stock, dilution of the stock and long term testing of the stock.

Perhaps you just want to make a solution to compare with the commercial solution, in order to confirm your suspicion that it may be "expired".

If it were me making the solution, I would prepare it as close to the commercial solution as possible.
If the commercial solution is in a certain makeup of ACN and Aq. AmAc (1%) then I would likely prepare the stock in 100% ACN and dilute down in the proper makeup of ACN/Aq. AmAc. If there was a solubility issue, or stability issue in a certain solvent then I would use another solvent (MeOH and/or water) but I would try to avoid addition of acid unless I was sure it would not catalyze a degradation of the analyte.

Introducing an acid or changing the solution makeup can cause a shift in sensitivity. I would assume you would want the response to be as close to what your were getting in the past.

[tedinelkgrove]

Yes, this will be a comparison study, but from experience I think the results will confirm my hypothesis. From there we can make appropriate submissions for the SOP to be modified.

The results with the Waters solution are fairly poor in my opinion. Waters Spec is 20:1 for 1 pg/uL. The SOP says we double the signal to noise, perhaps to compensate for lack of back pressure associated with not using a column(?). Last week I obtained signal to noise of ~12, so according the SOP it is 24. Not great. And it varies substantially.

I did a test a while back with 2 different unexpired lots of the "Waters MS Setup Solution" containing reserpine. Both diluted/sonicated side-by-side and sampled immediately via autosampler. One produced s/n of 30, the other 75. I am sure the instrument precision is good, as our application internal standards and QCs are within acceptable limits. One would assume variations in signal intensity would result in outliers.

But the real question is for the seasoned LCMS analysts: what is the optimal solution (MeOH or ACN) for making a stock solution for use with our eluent? Add acid to final dilution prior to testing, or stock solution?

Here is info on reserpine. Seems like some acetic acid would help with solubility. The pure alkaloid obtained from Rauwolfia serpentina. is insoluble in water, freely soluble in chloroform and acetic acid, and very slightly soluble in alcohol and ether. The pKa is 6.6. In solution it unstable in the presence of alkali.

[lmh]

(1) pumping without a column, and no back pressure. You are right this is a problem, if you are using a low-pressure mixing quaternary pump. Binary high-pressure mixing pumps don't suffer. If you are using a quaternary pump with a gradient proportioning valve before the pump, you should buy a back-pressure regulator and fit it in place of the column. Alternatively you can use a long piece of thin tubing. If you do neither, it is likely your pump is supplying a fairly random mixture of any of the solvents... (you can test this by doing a gradient test with no back-pressure if you like; it will probably fail dismally).

(2) Alp's right; if you are looking to see if your instrument is performing in spec, you have to follow Waters' SOP as closely as possible.

(3) In theory, the solvent you are ionising the reserpine in is the solvent it was dissolved in, as there is no column, but in practise it will mix with the running solvent, which is why (1) and (2) are important.

(4) You don't need a strong proton donor (acid) for electrospray to work. Sugars will give a nice positive signal in water, even though they are insignificantly ionised in water, and water is only a weak source of protons. The electrospray process is electrochemical, and will form ions if ions are possible in solution.

(5) doubling the signal to noise is probably a piece of history related to pharmacopoia definitions: some people look at noise as the distance from bottom of baseline to top of baseline, while peak height is measured from the middle, so the logical view is that the bottom-to-top noise is twice what it ought to be if you are comparing with a peak. It really doesn't matter how you define things, but to know if your instrument is OK, you need to stick to the definition Waters used.

(6) sometimes reserpine dissoves a bit better if you sonicate it.

[tedinelkgrove]

Thank you for the reply. We use a binary pump, and the manufacturer spec calls for running the method at .2mL/min.

It looks like the majority rules. I thought about it and Waters bases their s/n spec not only on the [reserpine], but also on of those specific chemicals and amounts.

Thanks for the input. Your guidance will help in convincing my managers to stay with the Waters SOP and not deviate. I guess as long as we are getting 20:1 and it meets spec, that is all that truly matters.