A new electrospray ionization source (emitter) has been developed which allows the effective utilization of very small sample volumes at much lower flow rates than previously demonstrated. A small diameter etched-tip capillary has been incorporated into a pressure-infusion electrospray ionization source. The ability to electrospray aqueous solutions without the use of an ancillary sheath flow is demonstrated with several biopolymers. High signal intensities and stable signals are observed for this source in a comparison with a standard methanolsheath source. Electrospray ionization mass spectrometry (ESI-MS)has become an important analytical technique for chemical, biochemical and biological materials.'.' The further application of ESI-MS to small-volume and difficult-to-analyze samples will require a source that utilizes sample efficiently, affords high sample sensitivity and provides a stable electrospray for extended periods of time. Several approaches have been reported for either the continuous delivery of sample or of a sample plug to an ESI source, primarily based upon pressure-driven flow. One of the most widely used approaches, initially developed at this lab~ratory,~ involves the use of a coaxial sheath liquid. The coaxialsheath-liquid ESI source design was originally developed to facilitate interfacing of capillary electrophoresis (CE) and mass spectrometry and provides considerable flexibility in solvent choice and flow rate.3.4 The use of a sheath liquid, however, results in several disadvantages. These include the unavoidable addition of ionic and neutral species through the sheath liquid, which compete in the ESI process for available charge. The maximum sensitivity obtainable is lowered, to an extent which depends on the details of mixing of the analyte and sheath-liquid streams. Past experience in our laboratory has shown that stable signals from sheath-liquid type sources can, in certain applications, be difficult to obtain for extended periods of time. Obtaining a stable electrospray with a water sheath, when non-denaturing solvent conditions are desired and/or required, is often particularly difficult to achieve. Chowdhury and Chait have described a nonsheath ESI source constructed from a highperformance liquid chromatogaphy (HPLC) syringe that functions at conventional electrospray flow rates.' The syringe needle tip was electropolished to a tapered point which enabled the stable electrospraying of proteins from aqueous solutions. Wahl and coworkers have recently reported the use of a non-sheath source incorporating an etched tip at the terminus of small-i.d. fused silica capillaries for CE-MS applications, and have achieved subfemtomole detection of protein^.^,^ The combination of low flow rates with small i.d. fusedsilica capillaries resulted in high ESI-MS sensitivity due to an increase in sample ionization efficiency.A non-sheath source for pressure-infusion ESI-MS, * Author to whom correspondence should be addressed.incorporating a small-diameter silica capillary with an etched terminus ...

We report a method based on mass spectrometry for the characterization of noncovalent complexes of proteins with mixtures of ligands; this method is relevant to the study of drug leads and may be useful in screening libraries for tight-binding compounds. It is based on the ability of electrospray ionization (ESI)* 1•2 3to generate ions of intact noncovalent complexes in the gas phase3-5 and of Fourier transform ion cyclotron resonance

ABSTRACT:Studies were designed to quantitatively assess the mRNA expression of 1) 10 cytochrome P450 (P450) enzymes in human cornea, iris-ciliary body (ICB), and retina/choroid relative to their levels in the liver, and of 2) 21 drug transporters in these tissues relative to their levels in human small intestine, liver, or kidney. Potential species differences in mRNA expression of PEPT1, PEPT2, and MDR1 were also assessed in these ocular tissues from rabbit, dog, monkey, and human. P450 expression was either absent or marginal in human cornea, ICB, and retina/choroid, suggesting a limited role for P450-mediated metabolism in ocular drug disposition. In contrast, among 21 key drug efflux and uptake transporters, many exhibited relative expression levels in ocular tissues comparable with those observed in small intestine, liver, or kidney. This robust ocular transporter presence strongly suggests a significant role that transporters may play in ocular barrier function and ocular pharmacokinetics. The highly expressed efflux transporter MRP1 and uptake transporters PEPT2, OCT1, OCTN1, and OCTN2 may be particularly important in absorption, distribution, and clearance of their drug substrates in the eye. Evidence of cross-species ocular transporter expression differences noted in these studies supports the conclusion that transporter expression variability, along with anatomic and physiological differences, should be taken into consideration to better understand animal ocular pharmacokinetic and pharmacodynamic data and the scalability to human for ocular drugs.

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Purpose. p38 mitogen-activated protein kinase (MAPK) is known to play a regulatory role in inflammatory processes in disease. Inflammation has been linked also to the development of diabetic retinopathy in rodents. This study was conducted to evaluate the effect of a p38 MAPK inhibitor on the development of early stages of diabetic retinopathy in rats. Methods. Streptozotocin-diabetic rats were assigned to two groups-treated with the p38 MAPK inhibitor PHA666859 (Pfizer, New York, NY) and untreated-and compared with age-matched nondiabetic control animals. Results. At 2 months of diabetes, insulin-deficient diabetic control rats exhibited significant increases in retinal superoxide, nitric oxide (NO), cyclooxygenase (COX)-2, and leukostasis within retinal microvessels. All these abnormalities were significantly inhibited by the p38 MAPK inhibitor (25 mg/kgBW/d). At 10 months of diabetes, significant increases in the number of degenerate (acellular) capillaries and pericyte ghosts were measured in control diabetic rats versus those in nondiabetic control animals, and pharmacologic inhibition of p38 MAPK significantly inhibited all these abnormalities (all P < 0.05). This therapy also had beneficial effects outside the eye in diabetes, as evidenced by the inhibition of a diabetes-induced hypersensitivity of peripheral nerves to light touch (tactile allodynia). Conclusions. p38 MAPK plays an important role in diabetes-induced inflammation in the retina, and inhibition of p38 MAPK offers a novel therapeutic approach to inhibiting the development of early stages of diabetic retinopathy and other complications of diabetes.