ABSTRACT
A new derivatization and extraction technique termed as dispersive derivatization liquid-liquid extraction (DDLLE) speeds up the analysis process by removing the requirement for drying of the sample. The derivatization process takes place at the interface between the analyte containing aqueous phase and derivatization agent laden organic phase. The organic phase is highly dispersed using disperser solvent so that the total surface area is large. The derivatizing agent used is 1-(heptafluorobutyryl)imidazole and the resulting heptafluorobutyryl (HFB) derivatized analytes are partitioned into the organic phase. In addition to reduced sample preparation time, for some of the analytes, the HFB derivatives provide better spectral differentiation between isomers than conventional trimethylsilyl (TMS) derivatives. Method parameters for the DDLLE, such as extraction, and disperser solvent and their volume, type and amount of base, amount of heptafluorobutyrylimidazole and extraction time were optimized on diisopropylaminoethanol (DiPAE), ethyldiethanolamine (EDEA), triethanolamine (TEA) and thiodiglycol (TDG). The DDLLE was also used on various real world samples, which also includes few OPCW organized proficiency test and a spiked urine sample. The observed limit of detection (LOD) with 1mL of sample for DDLLE in full scan with AMDIS was 10ng/mL and with methane chemical ionization, multiple reaction monitoring (MRM) was 100pg/mL, i.e., 100fg on-column.
Subject(s)
Chemical Fractionation/methods , Chemical Warfare Agents/isolation & purification , Ethanolamines/isolation & purification , Phosphoramide Mustards/chemistry , Water Pollutants, Chemical/isolation & purification , Chemical Fractionation/instrumentation , Chemical Warfare Agents/analysis , Ethanolamines/analysis , Limit of Detection , Mustard Plant , Phosphoramide Mustards/isolation & purification , Water Pollutants, Chemical/analysisABSTRACT
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 [alpha, beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
