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The eyes are in continuous motion. A robust system required to make spatial separation judgments should be resistant to the positional noise produced by such movements. Two parallel lines have been jittered horizontally in order to assess the impact of stimulus movement on the retina. Jitter that maintains the separation between the two lines has minimal effect on separation discrimination thresholds, regardless of whether the targets in the two eyes jitter in a positively correlated, negatively correlated or uncorrelated manner. Presenting both eyes with line pairs but only altering the separation in one eye in the second interval yields poorer performance. However, if one eye receives a change in separation while the other views a dark screen then that monocular threshold is very similar to the binocular threshold. These results are most simply explained by procedures which average the monocular separation estimates.

This work describes a system to interface either microbore or packed capillary gradient liquid chromatography (LC) to fast atom bombardment mass spectrometry (FAB-MS). The interface incorporates on-line ultraviolet detection and post-column matrix addition to enable independent optimization of both LC and FAB-MS. The glycopeptide antibiotic teicoplanin was chosen as a model system because this group of compounds places severe demands on the chromatographic separation and is difficult to analyze by FAB-MS. For both microbore and capillary LC, high-quality mass spectra of the major components in teicoplanin were obtained; however, the increased sensitivity of the capillary system allowed spectra to be obtained at low picomole concentrations. The sensitivity and ease of use make capillary LC the preferred system for use in LC-FAB-MS.

Three polypeptide neurotoxins (curtatoxins) were isolated from the venom of the spider Hololena curta by reverse-phase high performance liquid chromatography, gel permeation, and ion-exchange chromatography. The purified toxins induced an immediate paralysis in the cricket Acheta domestica that resulted in desiccation and death of the insect within 24-48 h (LD50 congruent to 4-20 micrograms/g); this toxic effect is consistent with irreversible presynaptic neuromuscular blockade. Curtatoxins are a class of sequence-related, cysteine-rich, carboxyl-terminal amidated polypeptides of 36 to 38 amino acid residues. The cysteine residues are conserved at identical sequence positions among these polypeptides and form 4 intramolecular disulfide bonds. Hydropathy calculations show that the toxins have an internal hydrophobic region flanked by hydrophilic and oppositely charged amino- and carboxyl-terminal ends. By analogy to other cysteine-rich arthropod venom proteins, the folded structure of the curtatoxins is likely important for their target specificity and mode of action at the neuromuscular junction.

The structure elucidation of drug metabolites directly from urine by tandem mass spectrometry (MS/MS) for a new bronchodilator is described. When urine samples from human subjects dosed with 400 mg of MDL 257 were examined by MS/MS, three major urinary metabolites previously characterized in animal studies were confirmed and two previously unsuspected metabolites were identified. Using the operational modes of a triple stage quadrupole mass spectrometer, it is possible both to detect and to identify possible metabolites. Since the pure drug and its metabolites often contain common structural daughter ions, the parent spectra of these common daughter ions should contain some or all of the molecular ions of possible metabolites. Daughter spectra of these suspected molecular ions were obtained and the resulting daughter spectra were interpreted for structural information of suspected metabolites. This study confirms the utility of MS/MS to do rapid metabolic profiling and identification directly from complex samples such as urine, with minimal time for sample preparation and analysis. This technique can provide unique and complimentary data when combined with the more classical approaches such as HPLC profiling, isolation, and off-line spectroscopy.

An automated analytical procedure for the determination of enoximone, a new cardiotonic agent, and its major oxidative metabolite in plasma using robotic technology is described. A Zymark robot is used to perform all the operations of solid phase extraction including column pretreatment, internal standard addition, sample mixing, sample pipetting, column rinsing, drying and sample elution. The processed sample is injected directly into the high performance liquid chromatography system which is equipped with an ultraviolet absorption detector. The assay has good precision and accuracy, equivalent to the manual method it replaces, and yet provides higher throughput of sample.

A fully-automated system incorporating a Nier—Johnson geometry mass spectrometer and an on-line computer designed originally to collect high-resolution data has been modified to acquire and reduce metastable ion data produced by the Barber—Elliott—Major defocusing technique. Factors affecting system performance have been identified, and a variety of operating modes are proposed.

Studies were conducted in New Zealand White rabbits to assess the seminal transfer, vaginal absorption, and placental transfer of a therapeutic monoclonal antibody (T-IgG4). T-IgG4 was administered by intravenous injection (IV) in males and by IV and intravaginal routes in females. Low levels of T-IgG4 were excreted into seminal plasma (100- to 370-fold lower than serum concentrations) and absorbed following vaginal dosing (three orders of magnitude lower than IV administration). On gestation day 29 (GD29), fetal serum T-IgG4 levels were 1.5-fold greater than maternal levels following IV dosing. The fetal T-IgG4 exposure ratio for seminal transfer vs. direct maternal IV dosing was estimated to be 1.3×10−8. Applying human serum T-IgG4 exposure data to the model, the estimated human T-IgG4 serum concentration from seminal transfer was 3.07×10−7μg/mL, an exposure level at least 1000-fold lower than the T-IgG4-ligand dissociation constant (Kd) and at least seven orders of magnitude lower than the in vivo concentration producing 20% inhibition of the target (EC20). These data indicate that excretion of a T-IgG4 into semen would not result in a biologically meaningful exposure risk to the conceptus of an untreated partner.

The thermospray mass spectra of the phytoestrogens have intense protonated molecular ions but contain few or no ions indicative of structure. Tandem mass spectrometry (MS/MS) was used to obtain daughter ion spectra containing ions unique to the different structural characteristics of each phytoestrogen subclass and was used both to confirm identification and propose structures for unknowns. In addition to unique daughter ion spectra, MS/MS was used as a class identifier to detect phytoestrogens through the neutral loss of 56 (due to consecutive losses of CO) that is common to all members of this family. Several sources of soy protein were investigated to confirm the presence or absence of phytoestrogens. In one preparation investigated, daidzein and genistein were detected as well as an unknown phytoestrogen of the Biochanin A subclass. This unknown has been tentatively identified as 6,7-dihydroxy-4'-methoxyisoflavone using its daughter ion spectrum.

Intraperitoneal injection of zymosan (1 mg in 0.5 ml saline) in mice induces a transient writhing response accompanied by the synthesis of small amounts of prostaglandin E2(PGE2, less than 2 ng) and larger amounts of PGI2 (200 ng per mouse), measured as its non-enzymatic breakdown product, 6-keto-PGF1 alpha. Although both centrally-acting analgesics (morphine, clonidine) and prostaglandin biosynthesis inhibitors (aspirin, indomethacin, ibuprofen) blocked the writhing response to intraperitoneal injection of zymosan, only the latter reduced prostaglandin levels in the peritoneal cavity. The writhing response correlated equally well with PGE2 levels and 6-keto-PGF1 alpha levels when data from mice treated with centrally-acting analgesics were excluded. However, intraperitoneal injection of PGI2, but not PGE2, reversed the analgesia induced by indomethacin in zymosan-injected mice. Centrally-acting agents, but not ibuprofen, blocked the ability of PGI2 to reverse the analgesic activity of indomethacin. PGI2 (2 micrograms per mouse), injected intraperitoneally in otherwise untreated mice, induced writhing. These data indicate that PGI2 is the prostaglandin involved in mediation of the writhing response to zymosan and that prostaglandin biosynthesis inhibitors, but not centrally-acting analgesics, exert their analgesic activity by reducing the peritoneal level of PGI2. It is possible that PGI2 may have the ability to stimulate pain receptors directly in the mouse peritoneal cavity, in addition to its previously recognized ability to sensitize pain receptors to other pain-producing stimuli.

A method for the routine analysis of phenylthiocarbamyl (PTC) amino acids by thermospray high-performance liquid chromatography-mass spectrometry (TSP LC-MS) is described. Data were acquired on a small dedicated TSP LC-MS system in which the temperature of the vaporizer and ion source block were optimized. PTC-amino acids exhibited unique TSP mass spectra containing sufficient fragment ions to determine structural data. Therefore, using this method the amino acids contained in the acid hydrolysates of unique and modified peptides were able to be positively identified. Additionally, the amino acid composition of peptides as determined by TSP LC-MS in the selected ion monitoring mode corresponded well with the theoretical value. The detection limits for the PTC-amino acids were at the low picomole level.

The use of high-performance liquid chromatography and fast atom bombardment mass spectrometry are shown to be an efficient combination for investigating protease-mediated digestion of synthetic analogs of the peptide hormone ANF (atrial natriuretic factor). As examples of the reported methodology, rANF5-23-NH2 and rANF7-23-NH2 were digested with the endopeptidase thermolysin. These truncated analogs were selected to investigate metabolism within the disulfide-linked core of ANF, particularly at the Cys7-Phe8 bond. While this position was the site of initial hydrolysis for rANF5-23-NH2 (t1/2 = 0.5 min), the Cys7-Phe8 bond remained intact for all observed degradation products of rANF7-23-NH2 (t1/2 = 16 min). These findings suggest that improved stability towards endopeptidase-mediated core hydrolysis may be conferred to analogs of ANF by removal of the first six residues from the N-terminus.