Imipenem and Cilastatin for Injection USP

[syx]

Dear members,
We need any suggestion about column suitable for the assay of Imipenem and Cilastatin for Injection USP (and the catalog number).
Mobile phase – Dissolve 2.0 g of sodium 1-hexanesulfonate in 800 mL of pH 6.8 buffer, adjust with 0.5N sodium hydroxide or 0.5M phosphoric acid to a pH of 6.8 +/- 0.1, and dilute with pH 6.8 buffer to make 1000 mL of solution. (Note: without any portion of organic solvent)
Chromatographic system – The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm x 30-cm column that contains packing L1, and is maintained at a temperature of 50 +/- 1.0 degree. The flow rate is about 2 mL per minute.

I think I found to much extreme parameter values here: high temperature, high flow rate of 100% aqueous mobile phase in a 30-cm column.

Best regards,
Siswanto Tanuatmojo

[Mark Tracy]

Actually, you don't have that big of a problem. First, at 50 °C, the viscosity of water is 0.55 so pressure won't be a big deal; in fact it is about the same as methanol at 20 °C. Second, hexanesulfonate is a good enough wetting agent that dewetting should not be a serious concern.

Hydrolysis of the bonded phase will occur, but at a rate you probably can live with. With the 100% aqueous mobile phase, any hydrolyzed C18 will stay on the column until you wash it with organic solvent. Of more concern is dissolution of the silica substrate; there are several columns on the market with improved resistance and I'm sure that the vendors won't be shy about mentioning their products. (Mine is Acclaim PA2, but since it isn't L1, you don't want it.)

A few miscellaneous comments: if your column oven does not have a mobile phase pre-heater, get one. Degas your mobile phase to prevent outgassing in the detector, and it also helps prevent column dewetting.

[A.L.N. MURTHY]

Dear Sir,
We recommend to use a column with hydrophillic endcapping , in which matting effect of C18 ligands will not happen with 100 % aquoeus mobile phase. We can offer you CHEMSIL AQUA column for your application ,which can be used up to 60deg.C without any trouble. For more details you can contact me.
A.L.N. MURTHY
CHIEF EXECUTIVE
CHEMINDIA
302, UBAS, BABUKHAN ESTATE,
BASHEERBAGH, HYDERABAD 500 001. INDIA
TEL: + 91-40-55904780/ 55904781
FAX: +91-40-27244169
EMAIL: kchemindia@hotmail.com

[Uwe Neue]

The column that is recommended for this assay by the USP is microBondapak C18

[syx]

I do not have any idea while trying to make mobile phase adjustment for this method.

Mobile phase:
Dissolve 2.0 g of sodium 1-hexanesulfonate in 800 mL of pH 6.8 buffer, adjust with 0.5N sodium hydroxide or 0.5M phosphoric acid to a pH 6.8 +/- 0.1, and dilute with pH 6.8 buffer to make 1000 mL of solution. Filter this solution through a filter of 0.5 um or finer porosity, and degas. Make adjustments if necessary.

System suitability requirements:
For each peak:
Column efficiency NLT 600 theoretical plates
Tailing factor (10%) NLT 1.5
Relative standard deviation for replicate injections is not more than 2.0%

Chromatogram (using ‘absolutely new’ column):



Result:
Peak 1 – meet the criteria of SST, retention time: 5.999 minutes
Peak 2 – failed in tailing factor (10%):1.57. Retention time: 17.741 minutes

How could I make adjustment to mobile phase? I do not see any point that could be adjusted. The most common parameter to be adjusted is solvent strength, but in this case the mobile phase is 100% buffer solution. pH? The range is only between 6.7 and 6.9, no significant different would be found.

[Uwe Neue]

Just make sure that you are calculating the tailing factor as the USP prescribes it. Different ways of calculating it give different values.

[syx]

The formula is W 0 .1 / 2f, where W 0 .1 is the width of the peak at 10% height.

[syx]

I have tried to calculate manually and compare the result using asymmetry and tailing factor (10%) formula to cilastatin’s peak. The results are:
Asymmetry: 1.97
Tailing factor (10%): 1.49 (closer to the result of the software)
I am not sure about the accuracy of my calculation.

Here is the structure of dehydropeptidase inhibitor, cilastatin. It may be a chelating agent?

[Rafael Chust]

First of all, L1 maybe described as ANY C18 column... according to USP itself!

I have found a lot of incongruences with USP methods - sometimes it looks like a very quickly developed method just to get peaks and, if you follow the conditions as they are, your column will last a couple of injections!

Anyway, I think you should try increase the pH, as your molecule looks basic and the chromatogram shows some typical tailing.

In othe hand, I would recommend a ultrapure silica column, such as Phenomenex Luna or Waters Symmetry, since you can go up to pH 9-10 without major problems - of course Gemini C18 or XBridge would be a must!

[Uwe Neue]

This method is really wacky. You got a compound with mostly acidic functions, which will be negatively charged at pH 6.8, and then there is a negatively charged ion-pairing reagent in the mobile phase. For what purpose???? As far as I am concerned, this is voodoo...

To Rafael: if you believe that any USP L1 method will work on any L1 column, you will end up throwing away a lot of columns. This will be especially true if you use a modern C18 on a method that was developed 30 years ago.

[syx]

This method is really wacky. You got a compound with mostly acidic functions, which will be negatively charged at pH 6.8, and then there is a negatively charged ion-pairing reagent in the mobile phase. For what purpose????

I think the ion pair reagent is used to retain imipenem. It is the peak that comes out earlier in the chromatogram. Here is the structure:

First of all, L1 maybe described as ANY C18 column... according to USP itself!

I do not think so. In this case the only column that could give tolerable back pressure with this mobile phase using flow rate 2 mL/min is Water MicroBondapak (or other with same characteristic with MicroBondapak). The dimension is also appropriate.
If you want to know appropriate columns that are used in USP monographs you may search it in Chromatographic Reagents Used in USP-NF and Pharmacopeial Forum, a publication from USP. They share the information for quick reference.

…as your molecule looks basic and the chromatogram shows some typical tailing.

I tend to call it amphoteric substance. You could find more than one carboxylic structure, one site of this structure is forming salt with sodium ion.

[Uwe Neue]

This does not make it a hell of a lot better. At pH 7, I expect this imipenem thing to be a zwitterion, which does not interact with the ion-pair reagent either.

Nevertheless, even if the method is voodoo:

How long have you equilibrated the column with the ion-pairing reagent? Do you keep the column in the mobile phase (with ion-pair reagent) for storage?

[syx]

I equilibrate using 20 x column volume and then check the stability of retention time by replicate injections. It is ok; there is no significant retention change from both peaks.
After all analysis done, we wash the column with 10% acetonitrile in water for 20 x column volume and then 10 x column volume of acetonitrile. Why should we keep the column in mobile phase that contains 100% buffer solution?

[Rafael Chust]

Just to clarify what I was saying...



USP Forms Working Group on HPLC Column

The United States Pharmacopeia (USP) is pleased to announce that it has formed a USP Working Group on HPLC Columns. The group, which consists of members from the USP Expert Committee on Pharmaceutical Analysis 2, the National Institute of Standards and Technology (NIST), and major column manufacturers, is charged with creating categories in order to subclassify the USP designation L1 for C18 (octadecyl silane) HPLC columns. The subclassification of the L1 columns is meant to help facilitate the selection of alternative columns to be used for tests in accordance with the United States Pharmacopeia and National Formulary (USP-NF) monographs. The working group is starting with the L1 category due to the large number of columns within this classification and plans to extend its work to other column categories in the future.

"The group was created to help users of USP monographs simplify their column selections," said Margareth Marques, USP scientist and working group coordinator. "More than 220 HPLC columns can be classified as L1 for use with USP monographs, and this makes it difficult for an analyst to chose the proper column to use. USP wants to help column users streamline their processes and find alternative column choices."

The working group has decided to use NIST Standard Reference Material 870 to create the subclassifications. They will examine four characteristics (hydrophobicity, chelating, silanol activity, and shape selectivity) for each column, and the results from this study will be used to develop the subclassification criteria.

By this fall, the working group plans to have the necessary data to subclassify the L1 (C18) columns. For further information about the working group, please contact Margareth Marques, scientist at USP, at 301-816-8106 or e-mail her at mrm@usp.org.

[Uwe Neue]

Syx:

20 column volumes is not enough, if the column has not been stored in the mobile phase. It takes longer to equilibrate a column with an ion-pair reagent. This is one of the problems with ion-pair reagents. My suggestion is to equilibrate the column longer and see if the peak shape of the second peak improves. If it indeed does, then do not remove the ion-pair reagent from the column again, unless you want to go through such a long reequilibration again. I do not know if this will fix your problem, since the analyte is not BINDING with the ion-pair reagent, but it may be REPELLED by the ion-pair reagent, and this may create an equilibration problem.

Rafael:

I am very aware what the USP is doing with respect to the subclassification. I have been a member of the working group.