Trace determination of beta-lactam antibiotics in surface water and urban wastewater using liquid chromatography combined with electrospray tandem mass spectrometry.

A sensitive and reliable method using liquid chromatography-electrospray tandem mass spectrometry has been developed and validated for the trace determination of beta-lactam antibiotics in natural and wastewater matrices. Water samples were enriched by solid-phase extraction. The analytes included amoxicillin (AMOX), ampicillin (AMP), oxacillin (OXA), cloxacillin (CLOX) and cephapirin (CEP). Average recoveries of beta-lactams (BLs) in fortified samples were generally above 75% (except amoxicillin) with the standard deviations lower than 10% in water matrices. Amoxicillin was not quantified due to poor recovery (less than 40%) in the investigated water matrices. Matrix effects were found to be minimal when measuring these compounds in water matrices. The accuracy, within- and between-run precision of the assay fell within acceptable ranges of 15% absolute. The method detection limit (MDL) was estimated to range between 8 and 10 ng/L in surface water, 13 and 18 ng/L in the influent and 8 and 15 ng/L in the effluent from a wastewater treatment plant. A large number of actual water samples were analyzed using this method in order to evaluate the occurrence of the beta-lactams in a river and a wastewater treatment plant in northern Colorado. Most of the samples were negative for all analytes. These compounds were found at 15-17 ng/L in the three influent samples and at 9-11 ng/L in three surface water samples out of a total of 200 samples. This indicates that contamination by beta-lactam antibiotics is of minor importance to the small mixed-watershed.

Rapid analysis of trace levels of antibiotic polyether ionophores in surface water by solid-phase extraction and liquid chromatography with ion trap tandem mass spectrometric detection.

The occurrence of antibiotics in surface and ground water is an emerging area of interest due to the potential impacts of these compounds on the environment. This paper details a rapid, sensitive and reliable analytical method for the determination of monensin A and B, salinomycin and narasin A in surface water using solid-phase extraction (SPE) and liquid chromatography-ion trap tandem mass spectrometry (LC-MS-MS) with selected reaction monitoring (SRM). Several product ions as sodiated sodium salts for MS-MS detection have been identified and documented with their proposed fragmentation pathways. Statistical analysis for determination of the method detection limit (MDL), accuracy and precision of the method is described. The average recovery of ionophore antibiotics in pristine and wastewater-influenced water was 96.0+/-8.3% and 93.8+/-9.1%, respectively. No matrix effect was seen with the surface water. MDL was between 0.03 and 0.05 microg/L for these antibiotic compounds in the surface water. The accuracy and day-to-day variation of method fell within acceptable ranges. The method is applied to evaluate to the occurrence of these compounds in a small watershed in Northern Colorado. The method verified the presence of trace levels of these antibiotics in urban and agricultural land use dominated sections of the river.

Solid-phase extraction-high-performance liquid chromatography-ion trap mass spectrometry for analysis of trace concentrations of macrolide antibiotics in natural and waste water matrices.

A method using solid-phase extraction (SPE) combined with high-performance liquid chromatography-ion trap tandem mass spectrometry (LC-MS-MS) has been developed for determination of trace concentrations of erythromycin-H2O (ETM-H2O), roxithromycin (RTM) and tylosin (TLS) in natural and waste water matrices. These macrolides (MLs) were extracted from water samples using Oasis HLB cartridges, and the average recovery was 93.6 +/- 8.6, 92.1 +/- 10.0, and 94.3 +/- 8.9% for ETM-H2O, RTM and TLS in surface water, respectively. For water from the influent of a wastewater treatment plant (WWTP), the average recovery was 84.8 +/- 14.0, 83.2 +/- 13.1, and 86.1 +/- 13.4% for ETM-H2O, RTM and TLS, respectively. Method detection limits in a natural water matrices were 0.07, 0.03, and 0.05 microg/l for ETM-H2O, RTM, and TLS, respectively. Fragment or product ions from MS spectra using in-source collision-induced dissociation and MS-MS spectra have been identified. The accuracy and day-to-day variation of the method fell within acceptable ranges. The method was evaluated by studying the occurrence of the three macrolides on a river and a WWTP in northern Colorado. None of the antibiotics were detected in the stream except immediately downstream of a WWTP, a result consistent with their presence in the influent and effluent of the treatment facility.

Epithelial folds following slippage of LASIK flap.

We present a case of a slipped corneal flap after laser-assisted in-situ keratomileusis, LASIK. A 22-year-old, female with myopia and a refractive error of -5.50 D sph OU had an uneventful LASIK performed on her left eye with a Chiron Automated Corneal Shaper (ACS) and a VISX-Star Excimer laser using a nasal hinged flap. On the first postoperative day, slit lamp examination revealed 4.0 mm slippage of the corneal flap. The flap was rolled and folded on itself within the inferior cul-de-sac. With the aid of the operating microscope, the flap was repositioned on the stromal bed. Immediately following the repositioning, many folds were still visible despite centering the flap on the stromal bed and performing stretching maneuvers. Detailed examination showed the folds to be of edematous epithelial origin. The underlying stromal bed was flat. Within days following repositioning, the folds had disappeared and the flap assumed the typical postoperative LASIK appearance. The final uncorrected visual acuity returned to 20/20.

Fine lattice lines on the corneal surface after laser in situ keratomileusis (LASIK).

PURPOSE: To present an example of a pattern of lines resembling fine lattice on the corneal surface subsequent to laser in situ keratomileusis (LASIK). This subtle phenomenon may be relatively common and may affect visual outcome. METHOD: Case report. RESULTS: A 41-year-old year old man with high myopia and best-corrected visual acuity of 20/20 +2 in each eye underwent laser in situ keratomileusis (LASIK). No operative or postoperative complications occurred. No striae were evident on slit-lamp examination with direct illumination and retroillumination at the time of surgery or in the postoperative period. Postoperative uncorrected visual acuity was 20/25 with a best-corrected spectacle correction of 20/25 in both eyes. Fine lines in a lattice pattern were seen only with fluorescein dye in the precorneal tear film as areas of "negative stain" within the LASIK flap. With tear film supplementation, the lines were less evident and visual acuity improved. One year postoperatively, his uncorrected visual acuity was 20/25 in both eyes. The best-corrected spectacle visual acuity was RE: 20/20 -2, LE: 20/25. The fine lines were still present within the flap. A soft contact lens improved visual acuity to 20/20 in both eyes. Although all four puncta were occluded, he had no epiphora. CONCLUSION: Fine lines in a lattice pattern that may represent folds in the epithelium or Bowman layer may be present within the flap after LASIK and may adversely affect visual acuity. They may be visible as areas of negative stain with fluorescein dye in the precorneal tear film in the absence of any striae visible in the flap. These superficial lines have been seen more in patients with high degrees of correction and in patients with dry eye. If visual acuity is affected, it may be improved with punctal occlusion, tear supplements, or a contact lens.

Folds and striae in laser in situ keratomileusis flaps.

PURPOSE: To classify striae and folds seen after laser in situ keratomileusis. METHODS: The authors' experience and review of the literature form the basis for the proposed classification. RESULTS: Five types of folds and striae are described: loose epithelium, hinge ridge, hinge fold, deep stromal striae, deep epithelial/Bowman's striae. CONCLUSION: Linear striae in laser in situ keratomileusis flaps have a variety of appearances and different postoperative courses that require accurate diagnosis and management.

Preclinical toxicology studies with the new dopamine agonist pergolide. Acute, subchronic, and chronic evaluations.

Pergolide (LY127809, CAS 66104-23-2), a dopamine agonist for the treatment of Parkinson's disease, was evaluated for toxicity in acute, subchronic, and chronic studies. Acute toxicity tests using oral, intravenous and intraperitoneal routes were conducted in rats, mice, rabbits, and dogs. The acute oral median lethal doses (MLD) ranged from 8.4 to 33.6 mg/kg in Wistar and Fischer 344 rats, and from 54.0 to 87.2 mg/kg in ICR mice. Oral doses of 20 and 25 mg/kg produced no mortality in rabbits or dogs, respectively. The MLD by the iv route ranged from 0.59 to 0.87 mg/kg for Fischer 344 rats and from 11.6 to 37.1 mg/kg for ICR mice. The predominant signs of toxicity in the acute studies included hyperactivity, poor grooming, ptosis, aggressive behavior, increased gnawing activity, tremors, convulsions, and emesis. In the subchronic and chronic studies, Fischer 344 rats, B6C3F1 mice, and beagle dogs were administered pergolide either by gavage or in the diet for up to 1 year. Daily doses in these studies ranged up to 20 mg/kg for rats, 45 mg/kg for mice, and 5 mg/kg for dogs. The predominant treatment-related effects seen in these studies were attributable to the pharmacologic activity of pergolide. These consisted primarily of CNS-mediated clinical signs in rats and dogs, weight loss or decreased weight gain, emesis in dogs, and inhibition of lysis of corpora lutea with a corresponding increase in the weight of the uterus and ovaries. Pergolide treatment was not associated with any specific target organ toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of high exposure concentrations of inhaled low-toxicity dust on pulmonary function in guinea pigs.

The acute pulmonary function response to graded levels of a low toxicity dust was studied in guinea pigs. Four groups of five male guinea pigs each were exposed to mean concentrations of 0, 0.25, 1.01 and 5.39 mg Foundry Hill Clay l-1 air with mass median aerodynamic diameters of 2.6, 4.6 and 6.7 microns, respectively. There was a 15-min pre-exposure period to clean air, a 1-h exposure to the test atmosphere and then a 1-h recovery period with exposure to clean air. Concentration-related changes, compared to the pre-exposure period, occurred with a rapid onset in a number of parameters. Generally, the severity of observed effects increased with exposure time and, therefore, with inhaled dose. Statistically significant changes (P < 0.05) were observed in tidal volume, dynamic compliance, dynamic resistance, flow, pressure and minute volume during the last 15 min of exposure. The observed changes were consistent with acute bronchoconstriction. These effects reversed rapidly and there were no significant changes 1 h post-exposure. These results suggest that adverse physiological responses of short duration can occur when animals are exposed via inhalation to low-toxicity materials in the concentration range 0.25-5 mg l-1.

Assessment of the blood-aqueous barrier by fluorophotometry following poly(methyl methacrylate), silicone, and hydrogel lens implantation in rabbit eyes.

Intraocular lenses of silicone, hydrogel, or acrylic materials that can be implanted through small incisions are being proposed as an alternative to conventional poly(methyl methacrylate) lenses. The potential of each of these materials to stimulate intraocular inflammation is important in their selection as an implantable material. To investigate the potential of each material, we assessed by clinical slitlamp examination, fluorophotometry, and histopathology the inflammatory response induced in the rabbit eye following phacoemulsification and implantation of hydrogel, silicone, or poly(methyl methacrylate) intraocular lenses. All lenses seemed to be equally well tolerated. In general, the degree of inflammation seen clinically decreased over the four-month study; however, anterior segment fluorophotometry showed continued mild interruption of the blood-aqueous barrier in all lens groups. Fluorophotometry is a sensitive method to assess persistent subclinical anterior segment inflammation.

Sensitive, specific radioimmunoassay for quantifying pergolide in plasma.

Pergolide, a synthetic ergoline with potent dopaminergic activity, is used to treat Parkinson disease. The low plasma concentrations of pergolide achieved during therapy complicate the development of a method for its analysis. Because radioimmunoassay successfully measures other structurally related ergolines in physiological fluids, we undertook the development of a radioimmunoassay of pergolide. The detection limit of the radioimmunoassay is 21 ng/L with an optimal working range from 100 to 1000 ng/L. We maximized assay specificity by using a monoclonal antibody that displayed low cross-reactivity with pergolide sulfoxide, a major metabolite found in animals. The radioimmunoassay has performed acceptably for > 2 years during toxicology studies with rats and rhesus monkeys and in clinical studies involving patients with Parkinson disease. We consider the radioimmunoassay a valid method for quantifying therapeutic concentrations of pergolide in plasma.

Effect of silicone elastomer contact lens wear on endothelial cell morphology in aphakic eyes.

We prospectively evaluated the effect of extended silicone elastomer contact lens wear on the endothelial mosaic of aphakic subjects. Endothelial cell photography and central corneal thickness measurements were performed on 23 eyes of 15 aphakic subjects (mean age 60 +/- 10 years). These measurements were made before lens wear (n = 23) and were repeated after 1 year (n = 19) and again after 5 years (n = 10) of lens wear. No statistically significant change in mean endothelial cell size or coefficient of variation of cell size was found. Central corneal thickness was significantly decreased at 1 year and hexagonality was significantly increased at 5 years. Thus, the changes in endothelial morphology usually associated with contact lens wear did not occur with silicone lenses, which are highly permeable to oxygen.

Endothelial morphologic features and function after long-term extended wear of contact lenses.

We performed anterior segment fluorophotometry and endothelial cell photography on 11 subjects who had used extended-wear contact lenses for at least two years. The lenses had been removed for cleaning at intervals of one to four weeks. Forty subjects of similar age who did not wear contact lenses served as a control group. The coefficient of variation of cell size was increased in the contact lens group compared with the control group; no significant difference in mean endothelial cell size was found. The percentage of seven-sided cells making up the endothelial cell mosaic was increased in the contact lens group compared with the control group (15% vs 12%, respectively). No difference in corneal clarity, central corneal thickness, endothelial permeability to fluorescein, or rate of flow of aqueous humor was found between the groups. There was a significant correlation between duration of lens wear and mean endothelial cell size. Extended-wear contact lens usage induces morphological changes in the corneal endothelium; these changes may progress as the duration of lens wear increases. We detected no functional impairment, however.

Variations in human corneal endothelial cell morphology and permeability to fluorescein with age.

Fluorophotometry with topically applied fluorescein and endothelial cell photography were performed on 80 normal subjects (age 5-79 yr). Variations in endothelial cell morphology and function, flow of aqueous humor, and intraocular pressure were recorded. The mean endothelial cell size was 332.3 +/- 46.3 micron 2. A 28% increase in endothelial cell size was measured over the eight decades (r = 0.53, P less than 0.001). The coefficient of variation of cell size also increased with age (r = 0.41, P less than 0.001). The percentage of hexagonal endothelial cells decreased by 14% (r = -0.48, P less than 0.001), while the percentage of pentagonal and heptagonal cells increased by 50% (r = 0.44, P less than 0.001) and 40% (r = 0.33, P less than 0.002), respectively, with age. The mean endothelial permeability to fluorescein was 4.03 +/- 0.63 x 10(-4) cm min-1. A 23% increase in endothelial permeability with age was observed (r = 0.44, P less than 0.001). No change in central corneal thickness or endothelial pump rate was found. Flow of aqueous humor remained stable with age, despite a 25% increase in intraocular pressure (r = 0.50, P less than 0.001). Polarization of fluorescence of fluorescein in the corneal stroma decreased with age (r = -0.46, P less than 0.001). We conclude that with age the human corneal endothelium becomes morphologically less regular and may become more permeable to fluorescein.

Effect of long-term contact lens wear on corneal endothelial cell morphology and function.

Anterior segment fluorophotometry (topical) and central endothelial cell photography were performed on 40 long-term (2-23 years) contact lens wearers (four groups of ten each: hard, soft, gas permeable, and gas permeable plus prior lens usage) and 40 non-contact lens wearers of similar ages. Morphologically, the endothelial cells of contact lens wearers showed greater variability in size and shape compared to controls. The mean endothelial cell size in contact lens wearers (307 +/- 35 micron2) was smaller than that of controls (329 +/- 38 micron2, P less than 0.01). There was an increase in the coefficient of variation of cell size of the contact lens group (0.35 +/- 0.06 versus 0.25 +/- 0.04 for controls, P less than 0.0001). The endothelial cell mosaic contained a smaller percentage of hexagonal cells in contact lens wearers (66 +/- 8) compared to controls (71 +/- 7, P less than 0.01). There was a compensatory increase in five-sided cells. Functionally, there was no difference in corneal clarity, central corneal thickness or endothelial permeability to fluorescein (3.78 +/- 0.57 X 10(-4) cm/min versus 3.85 +/- 0.55 X 10(-4) cm/min for controls) between the two groups. Aqueous humor flow was increased 7% in contact lens wearers. We found no correlation between oxygen transmissibility, estimated underlying oxygen tension, or duration of wear of the contact lenses and any morphologic or functional variable. We also found no differences between the four groups of contact lens wearers except that the gas permeable lens wearers had more hexagonal and less pentagonal cells. Long-term contact lens wear induces morphologic changes in the corneal endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

The clinical measurement of endothelial permeability.

The endothelium maintains corneal deturgescence in part by serving as a barrier to fluid movement into the cornea. This barrier function is estimated by measuring the permeability of the endothelium to small, nontoxic, fluorescent molecules such as fluorescein and carboxy-fluorescein. After topical or systemic application of the dye, its concentration in the cornea and the anterior chamber is measured noninvasively at intervals over several hours with a fluorophotometer. Endothelial permeability to the fluorescent dye is calculated from the changes in its concentration in the cornea and anterior chamber over time. The permeability of the endothelium to the small fluorescent molecules is assumed to be proportional to its permeability to water, and thus provides an estimate of the barrier function of the endothelium. This paper reviews the theory and technique of clinical fluorophotometry and discusses published data concerning the effects on endothelial permeability of aging, contact lens wear, medication, and surgery.

Effect of body position on intraocular pressure and aqueous flow.

The relationship between intraocular pressure and the rate of aqueous humor formation was studied in human subjects. Intraocular pressure was altered by changing the subject's gravity-dependent body position. Aqueous humor flow was measured fluorophotometrically. An average change of intraocular pressure of 2.4 +/- 1.2 mmHg and 11.2 +/- 2.7 mmHg was recorded in subjects whose body position was alternated +/- 15 degrees from horizontal and +/- 50 degrees from horizontal. No change of the rate of fluorescein clearance was observed for the milder change of position. A small change of fluorescein clearance was noted in response to the steeper change of position. Changing intraocular pressure seems not to induce compensatory changes in aqueous flow. Therefore we conclude that aqueous formation is relatively pressure-insensitive, a conclusion one would draw from clinical instances of angle-closure glaucoma or as in other systems of secretion, such as the toad bladder or the cerebrospinal fluid, where alterations in pressure do not necessarily influence flow. If there is a regulator of aqueous flow it is not responsive to the changes in intraocular pressure brought about in this study. If aqueous flow regulates intraocular pressure or compensates for changes in intraocular pressure, the signal is not small changes in pressure.

Topical forskolin (colforsin) and aqueous flow in humans.

A 1% forskolin (nonproprietary name, colforsin) suspension was instilled in one eye each of 15 normal human subjects in a single-dose experiment under the following three conditions: during the day, during the night while asleep, and following pretreatment with timolol maleate. The rate of flow was 2.6 +/- 0.13 microL/min (mean +/- SE) in the afternoon in both the treated and the untreated eyes. During sleep at night the flow was lower than in the afternoon in both the placebo-treated eye, 1.5 +/- 0.09 microL/min, and the forskolin-treated eye, 1.3 +/- 0.09 microL/min. Timolol pretreatment reduced the flow to 1.6 +/- 0.08 microL/min in the placebo-treated eye and 1.6 +/- 0.10 microL/min in the forskolin-treated eye. No statistically significant effects of forskolin on flow were observed under any of the conditions. Forskolin caused transient hyperemia in all subjects. The experiment confirmed previous reports of differences in the rate of aqueous flow at different times of day and the effect of the beta-adrenergic blocker timolol on the rate of aqueous humor flow.

Pulmonary mechanics in conscious Fischer 344 rats: multiple evaluations using nonsurgical techniques.

An adaptation of the method reported by Amdur and Mead (1958, Amer. J. Physiol. 192, 364-368) was used to evaluate pulmonary mechanics in conscious rats, utilizing a flow plethysmograph and intraesophageal catheter to determine compliance, resistance, respiratory flow, thoracic pressure, tidal volume, frequency, and minute volume. The procedure did not require surgery, and repeat evaluations were made in conscious rats. Real time calculations of pulmonary parameters were accomplished with an analog computer interfaced with a digital computer for display and data storage. Multiple evaluations (1, 3, 6, 13, 18, 24, 30, and 36 weeks) in 40 rats are reported. The utility of the system in establishing a base line against which pulmonary impairment, related to the effects of known pulmonary toxicants, e.g., ANTU and NO2, could be measured, was evaluated.