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A Highly Sensitive LC-MS/MS method for the quantification of Free Amphotericin B in human plasma

28 Feb 2020

A Highly Sensitive LC-MS/MS method for the quantification of Free Amphotericin B in human plasma

Structure: 
            

Introduction: 
Amphotericin B is used for serious fungal infections and leishmaniasis. Amphotericin B has severe side effects, while administrated as intravenously it has also associated with multiple organ failure. Kidney damage is frequently reported side effect. With Liposomal formulation of Amphotericin B less toxicity has been reported and can be tolerated by the patient and has fewer adverse side effect.

Liposomes in the study samples are very fragile and break when exposed to freezing, vigorous shaking and when all activities are not performed under controlled conditions.

The breaking of liposome results in improper concentration data for both free and encapsulated amphotericin B and more impeccable to the concentration of free amphotericin B as its concentration in the study samples is significantly less as compared to the concentration of encapsulated Amphotericin B.

As per recommendation for liposomal Amphotericin B formulation from USFDA, bio-equivalence criteria is based on two analytes i.e. free and encapsulated Amphotericin B. Thus it becomes important that free Amphotericin B is accurately estimated from the collected samples.

To overcome this issue and to have more realistic and accurate method of estimation of free (un-encapsulated amphotericin B), Lambda has developed a method which takes care of the freezing related and other molecule related issues.

Liposomes in the study samples are very fragile and break when exposed to freezing, vigorous shaking and when all activities are not performed under controlled conditions.

Method: 
The method developed at Lambda involves the usage cryo protectant agent as a stabilizer. This cryo protectant agent is added to the separated plasma samples at the time of sample collection itself. This prevents the leakage of encapsulated amphotericin B to free Amphotericin B (un-encapsulated doxorubicin) post freezing thereby making the free doxorubicin levels more accurate and reliable. 

Briefly, the method for the quantification of Free Amphotericin B involves solid phase extraction using Natamycin as internal standard (ISTD). Chromatography was achieved on Biphenyl column with a mobile phase consisting of ammonium formate and Methanol, with total run-time of 3.5 minute.

This new method was validated in terms of linearity, stabilities, carry-over effect, reinjection reproducibility, precision, accuracy and selectivity. The linearity ranged from 10 to 5000 ng/mL. The method exhibited very good precision and accuracy at all concentration levels.

This validated method has been successfully applied to the analysis of study samples in which incurred sample reproducibility experiment was performed and found well within the acceptable criteria which proves method reproducibility for incurred samples.

Conclusion: 
A selective, precise, accurate and highly sensitive LC-MS/MS method is now available for the quantification of Free Amphotericin B in human plasma. 

Separate validated method also available for Quantification of encapsulated and Total Amphotericin B suitable for liposomal formulation studies.





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