Jesse C. Seegmiller

A SECM Study of Heterogeneous Redox Activity at AA2024 Surfaces

Scanning electrochemical microscopy (SECM) was used to spatially resolve the heterogeneous cathodic activity at AA2U24 surfaces. Experiments used a 10 μm diameter Pt microelectrode in a solution containing the protonated form of (dimethylamino) methylferrocene [DMAFc + , i.e., the Fe(II) state]. The tip was brought near the alloy surface while held at a potential of 0.65 V vs. SCE, where DMAFc + is oxidized to DMAFc 2+ [i.e., the Fe(III) state]. The AA2024 substrate was held at -0.75 V, where the DMAFc 2+ produced near the probe tip is reduced back to DMAFc + at regions on the surface that were cathodically active. Lateral variation in the DMAFc + oxidation current at the tip arises either from a decrease of the diffusive delivery of DMAFc + to the tip due to proximity to the surface or from a positive feedback mechanism in which the DMAFc 2+ that had been oxidized at the probe tip was reduced at the substrate. The images show locally high redox reactivity which is attributed to second phase, intermetallic inclusions. Comparison of the SECM images with scanning electron microscopy-energy dispersive spectroscopy images shows that the regions of high redox reactivity correlate with the locations of the intermetallic particles.

Quantification of Urinary Albumin by Using Protein Cleavage and LC-MS/MS

BACKGROUND Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). METHODS We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, (15)N-labeled recombinant HSA ((15)NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides (42)LVNEVTEFAK(51) and (526)QTALVELVK(534) from albumin and (15)NrHSA were referenced to the calibration curve. RESULTS The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13-200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%-11.0% for the peptide (42)LVNEVTEFAK(51), and 1.9%-12.3% for the (526)QTALVELVK(534) peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%-14.8% for the (42)LVNEVTEFAK(51) peptide, and 6.4%-14.1% for the (526)QTALVELVK(534) peptide. For the (42)LVNEVTEFAK(51) peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R(2) value of 0.97 and a regression line of y = 0.99x + 23.16. CONCLUSIONS Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a (15)NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Mechanism of Action of Corrosion Protection Coating for AA2024-T3 Based on Poly(aniline)-Poly(methylmethacrylate) Blend

A corrosion protection coating for AA2024-T3 composed of a blend of camphorsulfonate-doped poly(aniline) (PANI) and poly(methylmethacrylate) (PMMA) is described. The open-circuit potential (OCP) for the bare and coated alloy in H 2 SO 4 solutions is presented vs. time. Raman spectroscopy revealed the oxidation state of the PANI component of the blend, both on undamaged films and as a function of distance away from a scratch on purposefully damaged films. The open-circuit and Raman data show galvanic coupling between the coating and the alloy, causing reduction of the PANI component. Scanning electrochemical microscopy was used to monitor H 2 evolution over a scratch on the surface of either PANI blend or PMMA films coated on AA2024. These experiments show that the scratched surface is highly active toward H 2 evolution when coated with only a PMMA film. In contrast, the blend coating drastically suppresses the amount of H 2 evolution in a scratch. This suppression of H 2 evolution was mimicked by poising the OCP of the PMMA-coated alloy at the same value at which it is poised by the PANI-containing film. It is shown that camphorsulfonate anion has no inhibitory effect on H 2 evolution in scratches on PMMA-coated AA2024.

Comparison between immunoturbidimetry, size-exclusion chromatography, and LC-MS to quantify urinary albumin.

BACKGROUND The accurate and precise measurement of urinary albumin is critical, since even minor increases are diagnostically sensitive indicators of renal disease, cardiovascular events, and risk for death. To gain insights into potential measurement biases, we systematically compared urine albumin measurements performed by LC-MS, a clinically available immunoturbidimetric assay, and size-exclusion HPLC. METHODS We obtained unused clinical urine samples from 150 patients who were stratified by degrees of albuminuria (<20 mg/L, 20-250 mg/L, >250 mg/L) as determined by the immunoturbidimetric assay used in our clinical laboratory (Roche Hitachi 912). Urine albumin was then remeasured via LC-MS and HPLC (Accumin) assays. RESULTS The immunoturbidimetric assay, calibrated using manufacturer-supplied serum-derived calibrators (Diasorin), underestimated albumin compared with LC-MS. After calibration with purified HSA, this immunoturbidimetric assay correlated well with LC-MS. HPLC overestimated albumin compared with both LC-MS and immunoturbidimetry. The current LC-MS and HPLC assays both performed poorly at concentrations <20 mg/L. CONCLUSIONS Efforts are needed to establish gold-standard traceable calibrators for clinical assays. LC-MS is a specific method to quantify albumin in native urine when concentrations exceed 20 mg/L, and therefore could be employed for standardization among assays.

Comparison of Urinary Albumin Quantification by Immunoturbidimetry, Competitive Immunoassay, and Protein-Cleavage Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND Increased urinary albumin excretion is a well-documented diagnostic and prognostic biomarker for renal disease. Urinary albumin is typically measured in clinical settings by immunoassay methods. However, neither a reference method nor a urine albumin calibration reference material is currently available. METHODS We quantified urinary albumin in patient samples by using 3 commercially available reagent systems: DiaSorin SPQ and Beckman Coulter LX 20 (immunoturbidimetric), and Siemens Immulite (competitive immunoassay). Results were compared to values obtained by protein-cleavage liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS In general, results from the 3 immunoassays agreed with results from LC-MS/MS. However, the SPQ results showed a negative bias across all ranges of albuminuria [(0-200 mg/L, y = 0.91x - 3.74 (CI 0.86-0.96); > 200 mg/L, y = 0.88x - 40.30 (CI 0.76-1.00)], whereas the LX 20 showed minimal bias in the 0-200 mg/L range [y = 0.97x - 88 (CI 0.92-1.02)] and the Immulite assay showed positive bias in the 0-200 mg/L range [y = 1.15x - 4.38 (CI 1.09-1.20)]. CONCLUSIONS These results showed a reasonable quantification of urinary albumin by representative polyclonal and monoclonal immunoassays compared to an LC-MS/MS assay. In addition, the results do not suggest the presence of nonimmunoreactive albumin in urine. However, differences in analytic performance between assays support the need for a reference calibration material and reference method to standardize clinical laboratory measurements of urinary albumin.

Discordance Between Iothalamate and Iohexol Urinary Clearances

BACKGROUND Iothalamate and iohexol are contrast agents that have supplanted inulin for the measurement of glomerular filtration rate (GFR) in clinical practice. Previous studies have noted possible differences in renal handling of these 2 agents, but clarity about the differences has been lacking. STUDY DESIGN Study of diagnostic test accuracy. SETTING & PARTICIPANTS 150 participants with a wide range of GFRs were studied in an outpatient clinical laboratory facility. INDEX TESTS Simultaneous urinary clearances of iothalamate, iohexol, and creatinine. REFERENCE TEST None. OUTCOME Relative differences between the urinary clearances. Iohexol and iothalamate in plasma and urine were assayed concurrently by a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. RESULTS Mean iohexol, iothalamate, and creatinine clearances were 52±28 (SD), 60±34, and 74±40 mL/min/1.73 m(2), respectively. The proportional bias of iohexol to iothalamate urinary clearance was 0.85 (95% CI, 0.83-0.88) and was proportional across the GFR range. The mean proportional bias of iohexol clearance compared with creatinine clearance is 1.27 (95% CI, 1.20-1.34), whereas that of iothalamate clearance compared with creatinine clearance is 1.09 (95% CI, 1.03-1.15). LIMITATIONS Lack of reference standard. CONCLUSIONS This study reveals a significant and consistent difference between urinary clearances of iothalamate and iohexol. Comparison of studies reporting renal clearance measurements using iohexol versus iothalamate must account for this observed bias.

Iothalamate Quantification by Tandem Mass Spectrometry to Measure Glomerular Filtration Rate

BACKGROUND Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radiocontrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. METHODS Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8-487.0, 614.8-456.0, 614.8-361.1, and 614.8-177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. RESULTS Mean recovery was 90%-110% in both urine and plasma matrices. Imprecision was <or=15% for the m/z 614.8-487.0 and 614.8-456.0 transitions over a 10-day period at 1 mg/L. Method comparison for 159 patient samples (53 clearances) provided the following Passing-Bablok regressions: plasma iothalamate LC-MS/MS (y) vs CE-UV (x), y = 0.99x + 0.36; urine iothalamate LC-MS/MS vs CE-UV, y = 1.01x + 0.31; corrected GFR LC-MS/MS vs CE-UV, y = 1.00x + 0.00. Interfering substances prevented accurate iothalamate quantification by CE-UV in 2 patients, whereas these samples could be analyzed by LC-MS/MS. CONCLUSIONS Iothalamate can be quantified by LC-MS/MS for GFR measurement. This method circumvents potential problems with interfering substances that occasionally confound accurate GFR determinations.

Liquid chromatography–tandem mass spectrometry analysis of urinary fluticasone propionate-17beta-carboxylic acid for monitoring compliance with inhaled-fluticasone propionate therapy

BACKGROUND Inhaled corticosteroids including fluticasone propionate (FP) are the most effective treatment for persistent-asthma. Noncompliance ranging from 20% to 80% of treated patients is associated with substantial health care costs, morbidity and fatalities. A noninvasive test to assess FP treatment compliance is needed. The major metabolite of FP is FP-17beta-carboxylic acid (FP17betaCA) and is excreted in urine. This study demonstrates the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure FP17betaCA in urine and evaluation of FP17betaCA urinary elimination. EXPERIMENTAL Fluorometholone was used as the internal standard. After acetonitrile precipitation, samples were extracted with dichloromethane, washed and dried. Reconstituted extract (60 microL) was subjected to reversed-phase chromatography and positive-ion mode LC-MS/MS analysis. Assay precision, linearity, recovery and sample stability were determined. Elimination evaluation included measurement of FP17betaCA in urine collected daily from human subjects before (day 1), during treatment (days 2-5; dose FP-110 microg 2 puffs/day), and following cessation of FP therapy (days 6-14; n=4). RESULTS Linear range of the FP17betaCA assay was 10.3-9510pg/mL. Limit of quantitation (LOQ) was 10.3 pg/mL and recovery ranged from 85.8% to 111.9%. Inter-assay CVs were 7.4-12.0% for FP17betaCA concentrations of 11.1-5117 pg/mL. Urine FP17betaCA was absent in subjects prior to FP therapy, detectable (180-1991 ng FP17betaCA/g creatinine) throughout the dosing period and reached below the LOQ at 6 days after therapy cessation. CONCLUSIONS Measurement of FP17betaCA by LC-MS/MS has acceptable analytical performance for clinical use. These data support the clinical utility of measuring FP17betaCA in urine to monitor patient compliance with FP therapy.

Visualization of Cathode Activity for Fe-Rich and Cu-Rich Intermetallic Particles via Cathodic Corrosion from Dioxygen Reduction at Aluminum Alloy 2024-T3

A wall-jet flow cell was used to study O 2 reduction at AA2024-T3. In buffered O 2 -saturated solution the current density was 2% of the diffusion-limited value and relatively stable with time. In unbuffered solution the current density was 25-50% of the diffusion-limited value. The higher currents under unbuffered conditions are attributed to production of OH - via O 2 reduction at the intermetallic particles (IPs) at AA2024-T3. Trenching was observed around both Cu-rich and Fe-rich IPs. This cathodic corrosion was observed in the absence of chloride and when the alloy was held potentiostatically at open-circuit potential demonstrating that both types of IPs can act as cathodes toward O 2 reduction under these conditions.

Dioxygen Reduction Affects Surface Oxide Growth and Dissolution on AA2024-T3

The influence of dioxygen reduction on oxide growth and dissolution at the surface of AA2024-T3 has been investigated in near-neutral, unbuffered conditions. Dioxygen reduction at Fe and Cu also was examined to compare with the behavior of intermetallic particles in AA2024-T3. Electrodes were exposed alternately to N 2 -saturated or O 2 -saturated 0.1 M Na 2 SO 4 solution in a characterized wall-jet flow cell. Limiting current densities associated with O 2 reduction on Fe and Cu range between 1.5 and 2.0 mA cm -2 , whereas AA2024-T3 displays significantly lower values. AA2024-T3 samples also display potential-dependent current density transients when the solution is alternated between N 2 -saturated and O 2 -saturated supporting electrolyte. The data show that the current density transient behavior is due to competition between interfacial etching of oxide due to high local pH, which is a result of the cathodic oxygen reduction reaction (ORR) at intermetallic particles, and anodic oxide growth on the alloy, which is driven by the electric field produced at a given potential. Scanning electron microscopy shows significant differences in oxide thickness with radial distance from the center of the jet impingement region. Energy-dispersive spectroscopic imaging reveals an increase in near-surface Cu and Fe in areas that experience high current densities.