Determination of famprofazone, amphetamine and methamphetamine in liver samples using enzymatic cell dispersion and SPE-LC-ESI-MS.

The use of small amounts of sample presents advantages in chromatographic analyses that have made this a current trend following the development of increasingly sensitive analytical techniques. Biological sample preparation methods, especially for rigid or semi-rigid matrices, are also under constant development, focusing on a more efficient extraction and in obtaining cleaner residues for analysis. In this context, the aim of this study was to present a validated a liquid chromatography-mass spectrometry (LC-MS) method for the quantification of famprofazone and its metabolites, methamphetamine and amphetamine in liver, using enzymatic cell dispersion promoted by collagenase, followed by protein precipitation and solid phase extraction (SPE) for sample extraction, concentration and clean-up. Potentially relevant variables for enzymatic cell dispersion concerning efficiency, such as enzyme concentration, temperature, buffering, agitation, and mechanical effect of stainless-steel spheres were assessed. Recovery evaluations were performed during the optimization of each step to ensure minimal loss of analytes. Linearity, the limit of detection (LOD) and limit of quantification (LOQ), stability, carryover, matrix effect, precision and bias were evaluated using fortified blank samples. An authentic sample was obtained from a controlled daily oral administration of 200 mg famprofazone to pigs for five days. The procedure was optimized for 500 mg of liver tissue, obtaining 99.9 ± 9.3% of digested collagen and 90.2 ± 1.7% of dispersed cells, without the tissue losses that usually ensue during crushing or grinding processes. Precision (CV%) was ≤ 10% and bias was ≤ 13% for all analytes. The LOQ was 5 ng/g for all analytes. The mean famprofazone concentration was 9.3 ± 0.53 ng/g, and mean metabolite concentrations were 16.7 ± 1.67 and 24.3 ± 1.36 ng/g for amphetamine and methamphetamine, respectively.

Simultaneous measurement of perillyl alcohol and its metabolite perillic acid in plasma and lung after inhalational administration in Wistar rats.

The inhalational administration of drugs is a practical and non-invasive approach with the potential to reduce side effects and with a quick onset of therapeutic activity. Perillyl alcohol (POH) is a monoterpene with antitumor activity that currently is undergoing clinical evaluation as an inhalational anticancer agent. A detection method was developed that will be applicable to pharmacokinetic studies of not only POH, but also its longer-lived main metabolite, perillic acid (PA), in lung tissue and plasma after inhalational delivery. The anticancer activity of POH was investigated in vitro with the use of various lung cancer cell lines. Toxicity was established by a standard MTT assay, and apoptosis markers were analyzed by Western blot. For the detection of POH and PA in lungs and plasma, albino Wistar rats were used that were exposed to POH inhalation. Tissues were subjected to chromatographic separation on an Agilent Zorbax Eclipse XDB C18 column, followed by detection of absorption in the ultraviolet (UV) range. In vitro, POH exerted cytotoxic activity against six different lung tumor cell lines, and apoptotic cell death was indicated by induction of active caspase 3 and cleavage of poly (ADP-ribose) polymerase 1 (PARP1). These results demonstrate that inhalational delivery of POH results in effective biodistribution and metabolism of POH in the systemic circulation. In addition, our study introduces a simple, rapid HPLC-UV method with high accuracy for simultaneous detection of POH and its metabolite PA in plasma, and for sensitive detection of PA in lung tissue, which should prove useful for applications in clinical studies.

Postmortem analysis of famprofazone and its metabolites, methamphetamine and amphetamine, in porcine bone marrow.

Forensic toxicologists typically work with body fluids, such as blood and urine, or visceral tissues. The analysis of alternative samples, such as bone marrow, can be requested when the commonly used samples are unavailable due to an extended time lapse between the time of death and collection of the material to be analysed. In this study, a method for the analysis of the lipophilic drug famprofazone (FA) and its metabolites, methamphetamine (MA) and amphetamine (AM), in bone marrow was developed, validated and applied to bone marrow from pigs given controlled doses of famprofazone. This method involves enzymatic bone-cleaning, fragmentation of the bones with the assistance of a micro electric motor, optimization of clean-up and LLE (liquid/liquid extraction) conditions and determination by GC/MS. After evaluation through statistical tests, such as Shapiro Wilk for normality and Cochran for homoscedasticity, a linear model was applied in the range of 100 (LOQ) - 2000 ng g-1. Inter-day precision and bias was always < 4.6%. In real sample analysis, bone marrow FA and MA concentrations ranged from 103 to 232 and from 174 to 267 ng g-1, respectively; AM was not detected. The obtained results are useful for application in forensic toxicological protocols (human autopsy cases) and as a starting point for the development of further analytical tools.

Particle-size distribution (PSD) of pulverized hair: A quantitative approach of milling efficiency and its correlation with drug extraction efficiency.

Different types of hair were submitted to different milling procedures and their resulting powders were analyzed by scanning electron microscopy (SEM) and laser diffraction (LD). SEM results were qualitative whereas LD results were quantitative and accurately characterized the hair powders through their particle size distribution (PSD). Different types of hair were submitted to an optimized milling conditions and their PSD was quite similar. A good correlation was obtained between PSD results and ketamine concentration in a hair sample analyzed by LC-MS/MS. Hair samples were frozen in liquid nitrogen for 5min and pulverized at 25Hz for 10min, resulting in 61% of particles <104µm and 39% from 104 to 1000µm. Doing so, a 359% increment on ketamine concentration was obtained for an authentic sample extracted after pulverization comparing with the same sample cut in 1mm fragments. When milling time was extended to 25min, >90% of particles were <60µm and an additional increment of 52.4% in ketamine content was obtained. PSD is a key feature on analysis of pulverized hair as it can affect the method recovery and reproducibility. In addition, PSD is an important issue on sample retesting and quality control procedures.

Fast and sensitive collagen quantification by alkaline hydrolysis/hydroxyproline assay.

A preparative protein alkaline hydrolysis procedure, as part of a spectrophotometric collagen quantification method, is presented. The procedure is suitable for small amounts of fresh solid or liquid samples. Various aspects of the procedure, such as the NaOH concentration, time needed to hydrolyse different collagen contents, buffer strength of the reagent solution, pH control of the hydrolysate and spectrophotometric conditions, were evaluated. Compared to other procedures that use alkaline hydrolysis, the sensitivity of this procedure was increased by a factor of 5. Compared to the conventionally used Association of Official Analytical Chemists (AOAC) acid hydrolysis method, the reaction time was reduced from 16 h to 40 min and the amount of sample from 4 g to 3-20 mg, producing equivalent results when applied to porcine liver and sausage samples.

Quantitative GC-ECD analysis of halogenated metabolites: determination of surface and within-thallus elatol of Laurencia obtusa.

Information on natural concentrations or variability of secondary metabolites in marine organisms may be important both to ecological/evolutionary and applied approaches. A gas chromatographic procedure with an electron capture detector (GC-ECD) was developed to quantify the sesquiterpenoid elatol at the surface and within-thalli of 70 specimens of the red seaweed Laurencia obtusa. The concentration of elatol was highest within-thalli [9.89 mg g(-1) of L. obtusa, dry weight (d.w.)], compared to lower values found at the surface [0.006 mg g(-1) of L.obtusa (d.w.), or 0.5-10.0 ng cm(-2)]. This method provides a rapid and inexpensive quantification of small quantities of elatol, and probably may also be used to quantify other halogenated compounds usually found in red seaweeds.