A rapid method for the separation and analysis of leaked and liposomal entrapped phosphoramide mustard in plasma

doi:10.1016/0731-7085(94)00044-1

Journal of Pharmaceutical and Biomedical Analysis

Volume 12, Issue 8, August 1994, Pages 961-968

https://doi.org/10.1016/0731-7085(94)00044-1 Get rights and content

Abstract

Pharmacokinetic studies of liposomal drugs should include simultaneous determination of leaked and entrapped drug in biological specimens. Due to the limited stability of many liposomal preparations in biological samples, a rapid analytical procedure is often necessary. Phosphoramide mustard (PM), a key cytotoxic metabolite of a widely used alkylating drug cyclophosphamide, has recently been entrapped into a liposomal formulation and the preparation has been found to be rather unstable in plasma. We have, therefore, developed a rapid method for the separation of liposome-associated PM from the unassociated drug and a method for their quantitation in plasma. This method involves the use of size exclusion mini-gel column and requires minutes to process. Due to the use of internal standards, this method tolerates low recovery and requires the collection of a single fraction of each of liposome-associated PM and the unassociated drug. The recovery of liposomal PM from the first fraction of the gel column was found to be 82.4 ± 7.9% (SD, n = 8), whereas that of liposome-unassociated PM from the major fraction was 16.8 ± 2.8% (SD, n = 8). However, the low recovery problem of liposome-unassociated PM was circumvented by adding the internal standard [α,β-²H₈] PM prior to separation, thus compensating for the loss of liposome-unassociated PM due to incomplete collection. Two types of standard curve were constructed for quantitation of liposome-associated PM and unassociated PM and the linearity for both was excellent. Assay validation indicated that within-run RSD values at 213 ng, 426 ng and 1065 ng for liposomal PM were 4.2, 4.3 and 3.0%, respectively. For liposome-unassociated PM at 100 ng, 200 ng and 500 ng levels, within-run RSD values of 9.7, 3.6 and 2.1% respectively, were found. Between-run RSD values was 2.9% for liposome-associated PM and 6.3% for unassociated PM. For total PM in plasma, the within- and between-run RSD values were 10.3 and 11.3%, respectively, at 100 ng ml⁻¹ level. This method was applied to separate liposome-associated PM from unassociated PM in the plasma obtained from rats following intravenous administration of liposomal PM at 5 mg kg⁻¹. Surprisingly, liposomal PM was found to be quite stable in vivo since about 70–90% of total plasma PM levels was accounted for by PM associated with the liposomal form at least up to a 1-h period of observation.

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^☆: Presented in part at the Analysis and Pharmaceutical Quality Section of the Eighth Annual American Association of Pharmaceutical Scientists Meeting, November 1993, Orlando, Florida, USA.

¹: College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

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A rapid method for the separation and analysis of leaked and liposomal entrapped phosphoramide mustard in plasma☆

Abstract

J. Mol. Biol.

Cancer Treat. Rep.

Pharmacol. Ther.

Anal. Biochem.

Biochim. Biophys. Acta

J. Clin. Pharmacol.