Dear Ramona,

Please try this USP method

Diluted phosphoric acid:10ml in 100 ml solution.

Solvent :1ml triethylamine to 1000ml water adjust its pH to 5.0 with dilute phosphoric acid.

Mobile phase:prepare a mixture of above mentioned solvent and methanol(5:1).

SAMPLE:25mg Meropenem/50ml in above mentioned solvent.(use this solution immediately)

DETECTOR UV/VISIBLE:300nm

column:4.6mmx25cm ,C18 5 micron

Did you try to derivatize the acid with a benzoyl group and/or use TFA as modifier to maintain pH ? (0.1%).

Dear Ramona,

Please check the follwing reference for guidance. It contains a discussion of the problems you seem to be encountering.

Here is the publication

Hiiii

there may be more than one lambda max for single compound, i have seen structure of your compound & it have definitely another lambda that may 298(that you selected). Your drug nature is slightly basic to neutral , & soluble in water.first you try with water & alcohol mixure, put the ratio of water more than alcohol.

Tell me which column you have used then i will further suggest you accordingly

i would suggest you try the following which worked for metrizamide, and therefore the column would be a partisil 10 ODS column 254x4.6 which is normally good UV detector at 244nm and a flow rate of 2ml/min and a mobile phase of methanol 4.5, water 95 and acetic acid 0.5 i think this will definately work, if you have the access of a guard column it would be good too hope this will help

I would suggest using a mild gradient from 95:5 to 70:30 of dilute phosphate (~20-50mM) to actonitrile or methanol according to Luisa Rizza, but perhaps looking at a 200nm wavelemgth absorption or 298nm if that is a maxima for metrizamide. The intial gradient should be 15mins, and if you achieve baseline separation, vary it to sharpen your peak up, even running a final larger organic content. Running 50% organic as you report is way too high for an isocratic run.

If you are trying to develop the method of determination of impurities of meropenem, then you should follow Pharmacopoeic methods (USP, Ph.Eur. etc.(if its exist)). If you try to find an assay method of meropenem, then I'm agree with all this methods above of the colleagues.

Dear Amir; Thanks for the detailed suggestion. I shall try it, but can I use diluted orthophosphoric acid?

Dear Luisa: Thnx. I have actually tried that method yesterday. Today I tried changing the ratio the other way around (while maintaining the other parameters), ACN:KH2PO4 (90:10) and got my peak, but the lowest detection (without splitting of peak) was 2mcg... A bit too high I think...

Here are some references which will be of great help. Check them out.

Chen, X., Liang, H., Qin, W.

Determination of the concentration of meropenem in human plasma by HPLC method

(2012) Pharmaceutical Care and Research, 12 , 316-318.

Verdier, M.-C., Tribut, O., Tattevin, P., Tulzo, Y.L., Michelet, C., Bentué-Ferrer, D.

Simultaneous determination of 12 β-lactam antibiotics in human plasma by high-performance liquid chromatography with UV detection: Application to therapeutic drug monitoring

(2011) Antimicrobial Agents and Chemotherapy, 55 (, 4873-4879.

Dailly, E., Bouquié, R., Deslandes, G., Jolliet, P., Le Floch, R.

A liquid chromatography assay for a quantification of doripenem, ertapenem, imipenem, meropenem concentrations in human plasma: Application to a clinical pharmacokinetic study

(2011) Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 879 , 1137-1142.

Narala, S.R., Saraswathi, K.

RP-HPLC and visible spectrophotometric methods for the estimation of meropenem in pure and pharmaceutical formulations

(2011) International Journal of ChemTech Research, 3 (2), pp. 605-609.

Kameda, K., Ikawa, K., Ikeda, K., Morikawa, N., Nakashima, A., Ohge, H., Sueda, T.

HPLC method for measuring meropenem and biapenem concentrations in human peritoneal fluid and bile: Application to comparative pharmacokinetic investigations

(2010) Journal of Chromatographic Science, 48 , 406-411.

Berthoin, K., Le Duff, C.S., Marchand-Brynaert, J., Carryn, S., Tulkens, P.M.

Stability of meropenem and doripenem solutions for administration by continuous infusion

(2010) Journal of Antimicrobial Chemotherapy, 65 (5), art. no. dkq044, pp. 1073-1075.

Denooz, R., Frippiat, F., Charlier, C.

Simultaneous quantification of five β-lactam antibiotics in human plasma by HPLC-DAD: Clinical application for ceftazidime treatment in intensive care units

(2010) Acta Clinica Belgica, 65 (SUPPL. 1), pp. 24-30.

Dear Jordi: Thanks for the reply. No (followed the method of the Mendez paper, as mentioned by Luisa), but I could try with TFA. May I know how is this different and how does it influence peak production?

Dear Remi: Thanks for the paper. I currently use C18 column and tried the similar buffer before, with pH 6 and 6.5 on isocratic. I shall opt for gradient with this method and ones suggested by others.

Dear Ram Singh: thanks for the reply. The drug is also soluble in methanol but not in acetonitrile, I tried solubilizing before. I tried higher amount of water to organic, but was not successful either. I use the C18. 250 x4.6x5 column...

Dear Paul. Thanks for responding. If its possible, I shall give this a try.

Dear Leo: Thanks. I shall try it. I see...

Dear Bocancea: Im rather determining an assay method for the drug, for later detection from release studies. Thanks.

If this will deepen my understanding on HPLC... Does it matter in which solution the drug is dissolved in and how does molarity play a role in the buffer phase solvent for peak production? I am rather new to the system. Thanks.

Thanks Hassan. Will check them out. :)

I

i will suggest to change your column & use C-8 column , because you have tried gradient. it may separate your drug from solvent peak

use mobile phase in different ratios of methanol & water(neutral pH)

i will suggest to change your column & use C-8 column , because you have tried gradient. it may separate your drug from solvent peak

use mobile phase in different ratios of methanol & water(neutral pH)

I suggest you should study the pH-effect of mobile phase (k' vs pH). The Meropenem shows a Bronsted acid-base behavior. In a large range of pH, the main species of this compound are ionic (in low pH, cation and in high pH, anion), and so, the peak will be near solvent peak. You should find the pH-range where the zwitterion specie is the major in solution. I don't have pK1, pK2 and pK3 of compound, but I believe the ideal pH should be over 6.5. You must have care: don't use pH of mobile phase larger than the maximum pH of column.

Dear Ram: Ill see what I can do.

Dear Andre: Thanks. I shall study the pH characteristic further.

Thanks.

Change your column. You got solution definitely & use c-8 column

check your HPLC column, if you use the RP column, you should choose mobile phase a aqueous phase and a organic phase, eg, Methanol and water. Both of mobile phase are organic phase, which can not help your HPLC method.

Hi Ming Liu. Sorry I guess I put it the wrong way. I actually used the organic (of the ones mentioned and their ratio) and water as the mobile phase (mentioned above as well).

refer USP monograph as they have used different MP(pH 5.0) ,FR(1.6mL/min) & WL (220nm), try with USP conditions, modify accrodingly to your requirement therafter if necessary.

you can check pH column filtration kit and dosage also play a important role during you analysis.

Ramona Khanum I have given you the method did you try,Dear Ramona,

Please try this USP method

Diluted phosphoric acid:10ml in 100 ml solution.

Solvent :1ml triethylamine to 1000ml water adjust its pH to 5.0 with dilute phosphoric acid.

Mobile phase:prepare a mixture of above mentioned solvent and methanol(5:1).

SAMPLE:25mg Meropenem/50ml in above mentioned solvent.(use this solution immediately)

DETECTOR UV/VISIBLE:300nm

column:4.6mmx25cm ,C18 5 micron

Dear all,

I have gotten my peak for Meropenem and I'm in the 3rd day of validation, with the following method:-

Phase:

Aqueous - KH2PO4 buffer, pH 3 adjusted with othophosphoric acid, 0.03M, added with 0.1% triethylamine.

Solvent - Acetonitrile

Ratio - 20 (Buffer): 80 (Actn)

FR:1ml/min

WL: 298nm

Sample: Drug dissolved in UPW, stock conc was 1mg/ml

Tempt:25C

Column: C18, 250x4.6x5

Sample volume: 20microliter

Thanks everyone. I learned alot and had more references to modify my method for the drug detection.

Ramona

Dear Ramona,

I have given you a method & you may try on this method :

Column – 4.6mm X 250mm, 5 µm (e.g. ODS ,Hypersil)

Wave length – 300 nm

Flow rate – 1.3ml/ min (Adjust the flow rate if necessary)

Injected Volume – 20 µL

Retention Time – 5 to 8 minute.

Run Time – 1.5 times of main peak

Mobile phase:-

Dilute 15ml of tetrabutyl ammonium hydroxide solution (25% in water) with water to 750ml. Adjust pH with dilute phosphoric acid (1 in 10) to 7.5±0.1. Add 150ml of Acetonitrile &100ml of Methanol mix & degas.

Sample Preparation - 0.11mg/ml in Water

Hi Anuj. Thanks a lot for the suggestion but I have solved the problem (gotten the peaks) as I mentioned just above your reply. Thanks again and have a nice day.