Amoxicillin & Clavulanic Acid Plasma method

[sandip.gurav]

Presently Im working on Combination of Amoxicillin & Clavulanic Acid.Following SPE conditions were used to extract both the drugs from plasma
1 mL Methanol (Conditoning)
+
1 ml Water (Eduilibration)
+
200 µl Plasma (Acidified with 0.1 % Formic Acid)
+
Washing with 300 µl ACN
+
Elution with 1 % Ammonia in Methanol

With this method the recovery of Amoxicillin is about 60% but that of Clavulanic acid is 30 %.

Does any one suggest How to Improve the recoery of Clavulanic Acid ?
Clavulanic Acid is highly Water Soluble.
Mobile Phase : 80 (Acetonitrile):20(10 mM Ammonium Acetate)

[SIELC_Tech]

You can do direct plasma analysis without sample prep on Primesep D column. You will retain Amoxicillin based on reverse phase and ion-exchange mechanism and Clavulanic Acid by ion-exchange mechanism. Plasma components will repel from the surface of Primesep D column. Please see following application for analysis of acidic and hydrophobic compounds in plasma:

http://www.sielc.com/Technology_DirectP ... lysis.html

You should be able to do 200 injections without seeing effect of plasma. In our case we used 1:1 dilution, if you dilute it more and inject less you'll be able to make more injections.

Contact me if you have questions

Vlad

[Uwe Neue]

Unfortunately, you did not specify what type of SPE device you are using. Therefore it is difficult to judge what has gone wrong.

I am proposing the following methods:

Method 1:
Device: Oasis HLB (hydrophobic only)
Acidify your plasma sample with phosphoric acid, not with formic acid. Formic acid is less likely to break the interaction of analytes with plasma proteins, and you may loose some compound there. You also need to use a stronger concentration: dilute your plasma sample 1:1 with 4% phosphoric acid.

1. Activate the SPE device with methanol, followed by water or water with phosphoric acid.
2. Add the acidified plasma solution (are you using an internal standard?)
3. Wash with water acidified with phosphoric acid
4. Elute with methanol

Method 2:
Device: Oasis MAX (mixed-mode anion exchanger)

Make you plasma sample basic by adding 5% ammonia.

1. Activate the device with methanol, followed by water with ammonia.
2. Add the basic plasma solution
3. Wash with water with 5% ammonia
4. Wash with methanol with 5% ammonia
5. Elute with 2% formic acid.

I think the second method will give you better results.

When you develop a method, you should collect all fractions to see where you loose analyte, in case that the method does not give you 100% recovery in the target fraction.