Analysis of pesticides by LC-electrospray-MS with postcolumn removal of nonvolatile buffers.

Liquid chromatographic (LC) separations for pesticides and many other compounds make use of nonvolatile buffers in the mobile phase. The coupling of LC with mass spectrometry (MS) does not allow the use of nonvolatile buffers. Substitution with volatile buffers is possible, but changes in chromatographic retention and resolution can result even if pH is held constant. The postcolumn removal of nonvolatile buffers using a commercially available ion suppressor is evaluated for the analysis of carbamate pesticides. The suppressor efficiently removes phosphate anions from an LC mobile phase. Most compounds show an increased signal by factors of 2-7 after postcolumn phosphate removal. The suppressor has little effect on the chromatographic parameters of some compounds, while serious negative effects are noted for others. Some compounds will give poor results due to adsorption or retention by the suppressor. The results indicate that such a device may be useful for the LC-MS analysis of some pesticides using nonvolatile buffers.

Evaluation of electrospray transport CID for the generation of searchable libraries.

This paper reports the preliminary investigation of performance-based standard conditions that have been developed for electrospray ionization mass spectrometry. Using performance-based standard criteria, reproducible spectra can be obtained by CID in the electrospray-transport region and searched using a database created using the same performance criteria. To generate library-searchable mass spectra, the instrument was tuned to standard conditions that correspond to low, mid, and high fragmentation energies. The instrument was tuned using ion ratios relative to a given peak in a tune compound for each energy level. The library was evaluated using a set of 22 benzodiazepines. The CID libraries were found to be reproducible, both on the same instrument and on different instruments of the same type. Also, the libraries were found to be independent of flow rate and solvent system. The library was expanded to include 16 sulfonylurea herbicides and tested using spiked water samples. Performance of the library was tested over a concentration range of 2 orders of magnitude using sulfonylurea standards.

Heterophilic binding of L1 on unmyelinated sensory axons mediates Schwann cell adhesion and is required for axonal survival.

This study investigated the function of the adhesion molecule L1 in unmyelinated fibers of the peripheral nervous system (PNS) by analysis of L1- deficient mice. We demonstrate that L1 is present on axons and Schwann cells of sensory unmyelinated fibers, but only on Schwann cells of sympathetic unmyelinated fibers. In L1-deficient sensory nerves, Schwann cells formed but failed to retain normal axonal ensheathment. L1-deficient mice had reduced sensory function and loss of unmyelinated axons, while sympathetic unmyelinated axons appeared normal. In nerve transplant studies, loss of axonal-L1, but not Schwann cell-L1, reproduced the L1-deficient phenotype. These data establish that heterophilic axonal-L1 interactions mediate adhesion between unmyelinated sensory axons and Schwann cells, stabilize the polarization of Schwann cell surface membranes, and mediate a trophic effect that assures axonal survival.

Combinatorial peptide on-resin analysis: optimization of static nanoelectrospray ionization technique for sequence determination.

The optimizations of static nanoelectrospray parameters to determine peptide or mimetic sequences released from resin were explored. Several different manufacturers of probe tips were utilized and a method was developed for the direct analysis of bead-bound peptides by nanoelectrospray. The method involved minimum sample handling to assure maximum recovery from individual beads. Parameters that were explored included an inside and outside wash of the probe tip, the distance from the probe housing to the probe tip, source temperature, drying gas flow, individual tips and presence of beads. The same soluble synthetic peptide was used in all comparisons, which had a molecular weight of 717 amu. The discovery of the sequence of a bead-bound peptide was achieved. The parameters that were found to effect signal were outside wash, presence of bead and distance. There was the need for pneumatic assist to initiate electrospray on some occasions, although this generally resulted in unsatisfactory performance.

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase.

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[14C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[14C]ricinoleoyl-sn-glycero-3-phosphocholine, 14C-labeled 2-linoleoyl-phosphatidylcholine (PC), and 14C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A2 strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[14C]ricinoleoyl-PC and [14C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC-->2-ricinoleoyl-PC-->ricinoleate-->triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.

Polarization of myelinating Schwann cell surface membranes: role of microtubules and the trans-Golgi network.

Schwann cells polarize their surface membranes into several biochemically and ultrastructurally discrete regions of the myelin internode. To form these membrane domains, Schwann cells must sort, transport, and target membrane proteins appropriately. In this study, microtubule disassembly, confocal microscopy, and electron microscopic immunocytochemistry were used to investigate mechanisms involved in targeting P0 protein (P0), the myelin-associated glycoprotein (MAG), and laminin to different plasma membrane domains in myelinating Schwann cells from 35-d-old rat sciatic nerve. After microtubule disassembly by colchicine, all three proteins accumulated in Schwann cell perinuclear cytoplasm, indicating that microtubules are necessary for their transport. The distributions of Golgi membranes, endoplasmic reticulum, and intermediate filaments were also altered by colchicine treatment. Electron microscopic immunocytochemical studies indicated that P0 and MAG are sorted into separate carrier vesicles as they exit the trans-Golgi network. Following microtubule disassembly, P0-rich carrier vesicles fused and formed myelin-like membrane whorls, whereas MAG-rich carrier vesicles fused and formed mesaxon-like membrane whorls. Microtubule disassembly did not result in mistargeting of either P0 or MAG to surface membranes. These results indicate that following sorting in the trans-Golgi network, certain carrier vesicles are transported along the myelin internode on microtubules; however, microtubules do not appear to target these vesicles selectively to specific sites. The targeting of P0-, MAG-, and laminin-rich carrier vesicles to specific sites most likely occurs by ligand receptor binding mechanisms that permit fusion of carrier vesicles only with the appropriate target membrane.

Pharmacokinetics and metabolism of a 4'-methylthio derivative of propranolol in the dog.

The purpose of this study was to examine the effect of a methylthio substituent in the metabolically most active position of propranolol, i.e. the 4'-position, on the pharmacokinetics and metabolism of this drug in the dog. The kinetics of 4'-methylthiopropranolol (MTP) were compared to those of propranolol following simultaneous iv doses of labeled drug and oral doses of unlabeled drug. MTP had a significantly larger volume of distribution and a longer half-life, and demonstrated a greater accumulation by red blood cells and cardiac conductile tissue than propranolol, effects which presumably are due to a higher lipophilicity of MTP. The greatest effect was on the oral clearance, which was substantially lower for MTP (1.6 vs. 5.5 liters/min) with an associated higher bioavailability (23.1 vs. 10.9%). Studies of MTP metabolism using radiolabeled drug showed that MTP, like propranolol, was eliminated entirely by metabolism. About 70% of the urinary radioactivity was extractable into ethyl acetate at pH 9.8 and pH 2.0. The extractable metabolites were separated by HPLC and identified by GC/MS, direct probe MS, and comparison with authentic compounds. Eleven metabolites were identified as sulfoxides and, in particular, sulfones of MTP and its N-dealkylated and subsequently deaminated glycollic and lactic acid metabolites. The nonextractable urinary radioactivity (30%) was isolated by DEAE-Sephadex chromatography and identified by HPLC/MS as four glucuronic acid conjugates. In contrast to propranolol, there was no evidence of aromatic carbon oxidation for MTP. These observations suggest that the markedly decreased oral clearance of MTP compared to propranolol is due to qualitatively altered metabolism from a highly efficient aromatic carbon oxidation for propranolol to a less efficient sulfur oxidation for MTP.