I'm trying to develop an HPLC method to determine MEROPENEM concentration in blood samples. A strange phenomenon has happened on preliminary testing to determine retention times of meropenem. I´m using phosphate buffer (KH2PO4) 10mM pH 6.5, flow rate 1mL/min.UV at 299 nm. C18-4.5-150 mm particle size 5 m, column. A sample of analyte (50 ppm) in water was injected on LC system with a nice Peak in a retention time of 4.8-5.0. Up to here everything is OK. Then, I add 1mL of ACN to a 0.7 mL of water sample containing 50 ppm of meropenem and the chromatogram shows two peaks. First on solvent front (1.9 mins) and pretty big and a second peak on 4.8-5.0 where i hope. This second peak have less area than the single peak. I think that my compound is not well buffered and ionized form predominates. Any idea about this?
Hi Carlos!
Is your method isocratic or gradient? What is the initial mobile phase composition? The 1.9 min is approx. the dead time for your column. Isn't it the just the "solvent" peak, the baseline disturbance caused by the injection? It shouldn't be a large peak though.
Is the phenomenon repeatable?
In case of a gradient separation, is the re-equilibration time sufficient?
Inject the acetonitrile alone, check for impurities.
What is the injection volume? Dissolve the analyte in the (initial) mobile phase. If you want to use ACN for protein precipitation, then you might want to do additional dilution with the mobile phase. If you are concerned about diluting your samples, then just blow the solvent off.
Good luck!
Szabi
Your problem is the addition of the ACN to your sample. What is happening is that the composition of your sample is no longer the same as your initial mobile phase. The meropenem is then not retained on the column as passes straight through the column. Best to avoid the addition of the ACN but if it is needed keep as small as possible.
Hi Teodoro. The sample is stable in water and the hidroorganic solution. I watch it by cheking the abs spectrum of both solutions and measuring analytical signal (Abs) at the maximum of 299 nm. Thanks.
Hi szabolcs. It is an isocratic method. Composition of mobile phase is 8% organic and 92% aqueous. The phenomenom is repeatable when I treat the sample with ACN but it dissapears when I only Inject the aqueous sample. I check for imputiries and this is not the problem. I inject 20 microlitres. Thanks.
I am looking for use other Clean-up procedures like precipitation of proteins with Trichloroacetic acid at various concentrations...
Please check the stability your analyte, I think you need to stabilize the analyte which could be done by stabilizing agent like 1 mol/L 3-morpholinopropanesulfonic acid and solubilize the sample completely before enrichment by Rp Cartridge or ultrafiltration. How about using sodium/K phosphate 10 mM, pH around 7.5 and also Methanol as eluting buffers. Find the link below which may help you
http://chromsci.oxfordjournals.org/content/48/5/406.full.pdf
What is the initial mobile phase for LC elution. One solvent is 10mM KH2PO4, another solvent should be ACN or MeOH. If you are using almost 100% 10mM KH2PO4, then you will have problem by injecting sample containing over 50% ACN.
You will need to dry the sample and reconstitute in water or the mobile phase buffer.
IF you use TCA (trichloroacetic acid), meropenem will degraded significantly in 1-2 hrs. You can try to neutralize your sample to check stability, or alternatively, USE ACN to precipitate but dried and reconstituted to your initial mobile phase or just water.
Ok. Thank you for everyone, I´think I have a solution. After protein precipitation step with ACN (1:1) I dilute supernatant by a supernatant:buffer ratio of 1:3 to decrease organic concentration on injected sample. I dilute sample significantly but it is compensated by an increase of injection volume. I´m making now my first injections of blood samples spiked with meropenem with this clean-up procedure and I have a nice peak at time 6.5 and only one peak appears.
Thank you very much to all. I´ll proceed with method validation now.
WHen USing ACN to precipitate protein, the ratio is typically ACN:sample >=2.5:1. If you use 1:1 ratio, it may not be sufficient.
Hi Luisheng. When I use ACN for protein precipitation I always use this ratio and I have not any problem with this step. Today I have injected all day with this procedure and column pressure its constant. Did you have any problem?. However Thanks.
Dear Carlos,
Pl. check the possibility of precipitation of plasma sample on account of acetonitrile.
Sample clean up is important and possibility of hump peaks if the sample is not clean.
Dear Carlos Ezquer
I have suggestions:
1- Mobile phase configuration may be KH2PO4/ACN (80:20 or 90:10) or you can try gradient system with these solvents
2- You can work your serum samples:
a) To a 0.5 mL aliquot of serum sample 0.5 mL acetonitrile are added to precipitate protein and the mixture is vortexed for one minute, then it is centrifuged at 3500 rpm for 10 min. and then supernatant is injected onto HPLC system
b) or extraction solvents are evaporated and dissolved with above mobile phase and injected onto HPLC system
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