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HEXELS 2 PRESSURE



(10) This technique does not require a matrix coating, but initial reports from
the analysis of tissue from drug dosed animals indicate that overall sensitivity
may need improvement and detection of glucuronides known to be present in the
tissue have so far been unsuccessful. Newer atmospheric pressure surface
sampling/ionization techniques, (6-8) like desorption electrospray ionization
(DESI)-MS, (9) have also been demonstrated for determining drug related
materials in thin tissue sections. (4) This may be because phase II metabolites,
like glucuronides and glutathione conjugates, are relatively fragile and do not
survive the laser desorption/ionization process intact.

Also second, most MALDI-MS analyses report detection of parent drug and some
phase I metabolites, but apparently only rarely are phase II metabolites
reported. First, MALDI-MS requires the careful coating of the tissue with a
matrix compound prior to the MS analysis. (3-5) While this technique continues
to mature, some lingering limitations are apparent. While this conventional
procedure might be fully automated, a direct surface sampling, ionization, and
analysis method by mass spectrometry of such thin tissue sections, including the
same tissues used for WBA study, would save time and other resources by
shortening the sampling and extraction steps of a quick first pass look for drug
and metabolite distributions.Ĭurrently, the most widely used technique for
molecular identification of drug related materials directly from animal thin
tissue sections is matrix-assisted laser desorption/ionization mass spectrometry
(MALDI-MS). Currently, the particular molecular forms and quantity of the
drug-related material present must be determined from punched samples for areas
of interest in the same tissue sections (e.g., radioactive “hot spots”) or from
whole organ tissue homogenates from separate animals, which are analyzed with
conventional sample extraction, cleanup and high-performance liquid
chromatography−mass spectrometry (HPLC−MS) or tandem mass spectrometry (MS/MS).
(1, 2) Inherently, WBA cannot distinguish between the parent drug and the
metabolites of that drug. In the area of drug discovery, the distribution of
total drug related compounds in thin tissue sections is often determined using
whole-body autoradiography (WBA) employing radiolabeled drugs. The ability to
directly and efficiently sample from thin tissue sections via a liquid
extraction and then perform a subsequent liquid phase separation increases the
utility of this liquid extraction surface sampling approach. These drug and
metabolite data obtained using the LMJ surface sampling/HPLC−MS method and the
results achieved by analyzing similar samples by conventional extraction of the
tissues and subsequent HPLC−MS analysis were consistent. Confirming the presence
of one or the other or both of these glucuronides in the extract from the
various organs was not possible without the separation. In addition, two
isomeric phase II metabolites of propranolol (an aliphatic and an aromatic
hydroxypropranolol glucuronide) were observed in the chromatograms of the
extracts from lung, kidney, and liver. The parent drug was observed in the
chromatograms of the surface sampling extracts from all the organs of the dosed
mouse examined. To illustrate the utility of coupling a separation with this
direct liquid extraction based surface sampling approach, four different organs
(brain, lung, kidney, and liver) from whole-body thin tissue sections of
propranolol dosed and control mice were examined. In this work, a commercially
available autosampler was adapted to perform direct liquid microjunction (LMJ)
surface sampling followed by a high-pressure liquid chromatography (HPLC)
separation of the extract components and detection with electrospray ionization
mass spectrometry (ESI-MS).





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