Examination of the traction effect in an artificial muscle-type dynamic traction orthosis using computed tomography.

BACKGROUND: Dart-throwing motion from radial dorsiflexion to palmar flexion has recently attracted attention as a functional movement direction of the wrist joint. We developed a novel artificial muscle-type dynamic traction orthosis (DTSaM) that includes these movements. This prospective crossover controlled study aimed to compare the traction effects in the presence and absence of DTSaM using computed tomography. METHODS: Healthy participants with no history of finger disease (6 men: 6 fingers, 4 women: 4 fingers; average age [range]: 29.4 [34-24] years) were examined. The distance and area of the joint space between the radiolunate (RL) and capitolunate (CL) joints were evaluated using 2 types of computed tomography: automatic movement and DTSaM. RESULTS: Participants with DTSaM showed more dilated joint space distance on the dorsal and central sides ( p < 0.05) and larger joint space areas on the dorsal side ( p < 0.05) of the RL and CL joints than those without orthosis. Significant differences in the magnitude of change in each joint were observed between the RL and CL joints regarding the joint space distance on the dorsal ( p = 0.021) and central ( p = 0.038) sides and the joint space area on the dorsal side ( p = 0.044). CONCLUSIONS: The movement of the CL joint is important in the dorsiflexion direction. Our results suggest that the dilated CL joint allows dorsiflexion and that combined traction and dart-throwing motion exercises may be possible for wrist joint contracture.

Analysis of a New Artificial Muscle Type Dynamic Orthosis for Wrist Joint Disease Using a Three-dimensional Motion Analyzer.

OBJECTIVES: Recently, the dart-throwing motion (DTM) has attracted attention as a functional direction of wrist joint motion. Consequently, we devised a new artificial muscle-type dynamic orthosis (Dynamic Traction Splint by Artificial Muscle, DTSaM) to reproduce DTM. This study analyzed the automatic motion of the wrist joint using a three-dimensional motion analysis system to assess how closely the DTSaM replicates DTM. METHODS: The DTSaM orthosis incorporates two McKibben-type rubber artificial muscles, and measurements were performed using image analysis software and a three-dimensional motion analysis system. The wrist radial angle (WRA) was defined as the angle between the line connecting the head of the index finger metacarpal to the radial styloid process and the line connecting the radial styloid process to the lateral epicondyle of the humerus. WRAs were investigated from 60° of palmar flexion to 60° of dorsiflexion. RESULTS: For dorsiflexion, comparisons of radial deviation and wrist ulnar angle (WUA) between the motion obtained using the DTSaM orthosis and active DTM showed a significant difference (P <0.05) at 30° and 60°, respectively. For palmar flexion, the same comparison showed a significant difference for both the ulnar and radial deviations (30°, P <0.05; and 60°, P <0.01, respectively). Furthermore, WUA showed a significant difference at 50° (P <0.05). Intraclass correlation coefficient analyses yielded good reliability with an average value of ≥0.8. CONCLUSION: The DTSaM orthosis produces a motion similar to the DTM. It is hoped that the use of the DTSaM orthosis will help to shorten the treatment period for patients with wrist disease.

Indices reflecting muscle contraction performance during exercise based on a combined electromyography and mechanomyography approach.

Electromyography (EMG) and mechanomyography (MMG) have been used to directly evaluate muscle function through the electromechanical aspect of muscle contraction. The purpose of this study was to establish new absolute indices to describe muscle contraction performance during dynamic exercise by combining EMG and displacement MMG (dMMG) measured simultaneously using our previously developed MMG/EMG hybrid transducer system. Study participants were eight healthy male non-athletes (controls) and eight male athletes. EMG and dMMG of the vastus medialis were measured for 30 s during four cycles of recumbent bicycle pedaling (30, 60, 90, and 120 W) and on passive joint movement. Total powers were calculated based on the time domain waveforms of both signals. Muscle contraction performance was verified with the slope of regression line (SRL) and the residual sum of squares (RSS) obtained from EMG and dMMG correlation. EMG and dMMG has increased with the work rate. Force and EMG were similar between groups, but dMMG showed a significant difference with load increase. Athletes had significantly higher SRL and significantly lower RSS than controls. The average value divided by SRL and RSS was higher in athletes than in controls. The indices presented by the combined approach of EMG and dMMG showed a clear contrast between the investigated groups and may be parameters that reflect muscle contraction performance during dynamic exercise.

Molecular Structure of Gardenia Blue Pigments by Reaction of Genipin with Benzylamine and Amino Acids.

Genipin was reacted with benzylamine and several amino acids to prepare gardenia blue (GB). The time-course of GB formation with benzylamine was monitored by high-performance liquid chromatography (HPLC), liquid chromatography time-of-flight mass spectrometry (LC-TOFMS), and 1H and 13C NMR measurements. In this experiment, we determined the molecular structures of some intermediates using accurate masses and additional NMR techniques such as heteronuclear multiple bond correlation (HMBC). GBs with amino acids (GB-AAs) were characterized by both liquid and solid-state NMR measurements. Interestingly, many significant peaks appeared in the solid-state NMR spectra, although the 13C NMR spectra from solution samples did not show any distinct peaks. Therefore, we determined that GB-AAs had an alternating copolymer structure composed of methyne and 5H-2-pyrindine, which was substituted by amino acids at N atom and linked with methyne at 5 and 7 positions. To confirm this molecular structure, the pyrolysis gas chromatography-mass spectrometry (GC-MS) measurement of GB-AAs was carried out, and 5H-2-pyrindine and its methyl derivatives were formed as main pyrolysis products from the polymer chains.

A High-Fat and High-Cholesterol Diet Induces Cardiac Fibrosis, Vascular Endothelial, and Left Ventricular Diastolic Dysfunction in SHRSP5/Dmcr Rats.

AIM: Non-alcoholic steatohepatitis (NASH) increases cardiovascular risk regardless of risk factors in metabolic syndrome. However, the intermediary factors between NASH and vascular disease are still unknown because a suitable animal model has never been established. The stroke-prone (SP) spontaneously hypertensive rat, SHRSP5/Dmcr, simultaneously develops hypertension, acute arterial lipid deposits in mesenteric arteries, and NASH when feed with a high-fat and high-cholesterol (HFC) diet. We investigated whether SHRSP5/Dmcr affected with NASH aggravates the cardiac or vascular dysfunction. METHOD: Wister Kyoto and SHRSP5/Dmcr rats were divided into 4 groups of 5 rats each, and fed with a SP or HFC diet. After 8 weeks of HFC or SP diet feeding, glucose and insulin resistance, echocardiography, blood biochemistry, histopathological staining, and endothelial function in aorta were evaluated. RESULTS: We demonstrate that SHRSP5/Dmcr rats fed with a HFC diet presented with cardiac and vascular dysfunction caused by cardiac fibrosis, endothelial dysfunction, and left ventricular diastolic dysfunction, in association with NASH and hypertension. These cardiac and vascular dysfunctions were aggravated and not associated with the presence of hypertension, glucose metabolism disorder, and/or obesity. CONCLUSIONS: SHRSP5/Dmcr rats may be a suitable animal model for elucidating the organ interaction between NASH and cardiac or vascular dysfunction.

Estimating the minimum stimulation frequency necessary to evoke tetanic progression based on muscle twitch parameters.

The summation of the muscle force caused by an increase in the firing rate is named a tetanic contraction (tetanus), and the minimum stimulation frequency necessary to evoke an unfused/fused tetanus is related to the contraction time (CT) and relaxation time (RT) of the twitch. In particular, the fusion index (FI) is a very useful indicator, and it is used to evaluate the change in the muscle fiber component ratio. However, the measurement of the FI is invasive, because most patients experience pain during the electrical stimulation for tetanus. We expect that the twitch parameters CT and RT can substitute for the FI in the future. We found that the minimum stimulation frequency necessary to evoke the unfused/fused tetanus can be estimated from the twitch parameters as a first step. The results showed that (1) the minimum stimulation frequencies calculated from twitch parameters during unfused/fused tetanus were very similar to those calculated from FI parameters, and (2) they were also strongly correlated with FI parameters regardless of fiber components. The basic characteristics of tetanic progression in different fiber types could be estimated from twitch parameters.

Effect of different surgical procedures on the accuracy of prediction of the plasma concentration of fentanyl: comparison between mastectomy and laparoscopic prostatectomy.

BACKGROUND: The accuracy of simulation-predicted fentanyl concentration in different types of surgical procedure is not fully understood. We wished to estimate the effect of different types of surgical procedure on the accuracy of such simulations. FINDINGS: Fifty patients who had undergone elective mastectomy or laparoscopic prostatectomy (American Society of Anesthesiologists physical status = I-II) were enrolled. Anesthesia was maintained throughout surgery with sevoflurane and a bolus infusion of fentanyl. A maintenance infusion was administered with 8 mL/kg/h Ringer's acetate solution from the start of anesthesia to completion of blood sampling. An infusion to compensate for blood loss was administered (one to two volumes of hydroxyethyl starch). A blood sample was drawn every 30 min during anesthesia.We measured the plasma concentration of fentanyl in 358 samples from 50 patients. The plasma concentration of fentanyl was correlated significantly with the simulated predicted fentanyl concentration (r = 0.734, P < 0.01) but 36.0% of all samples had a difference greater than ±0.5 ng/mL. Approximately 0.3 ng/mL of a fixed bias was shown throughout mastectomy. During laparoscopic prostatectomy, the fixed bias gradually became negative from ≈0.3 to -0.3 ng/mL as the sampling stage proceeded. CONCLUSIONS: The predicted concentration of fentanyl was significantly correlated with the plasma concentration of fentanyl (r = 0.734). However, there were different patterns of a fixed bias between mastectomy and laparoscopic prostatectomy groups. We should pay attention to this tendency among different surgical procedures. TRIAL REGISTRATION: UMIN000005110.

Electrically induced mechanomyograms reflect inspiratory muscle strength in young or elderly subjects.

BACKGROUND: Respiratory muscle strength has been used as a tool for evaluating respiratory rehabilitation in chronic obstructive pulmonary disease. However, mouth pressure measurement evaluated by maximum expiratory mouth pressure (PEmax) or inspiratory mouth pressure (PImax) offers an indirect method for measuring respiratory muscle strength. We demonstrated the evaluation of diaphragm contractility using a mechanomyogram (MMG), which is the mechanical signal generated by the motion of the diaphragm induced by the electric stimulation of the phrenic nerve. METHODS: Study participants were 21 young and 20 elderly subjects with no symptoms of respiratory disease. The elderly subjects were divided into non-smoker or smoker groups. The smoker group was defined as subjects having a Brinkman Index of greater than 300. We measured basic spirometric parameters, mouth pressure (PEmax, PImax), and diaphragmatic MMG. RESULTS: Diaphragmatic MMG showed more clear contrast between young subjects and elderly non-smoker or smoker subjects than the conventional method for respiratory muscle contraction (PEmax, PImax). In addition, the diaphragmatic MMG strongly correlated with inspiratory muscle strength. CONCLUSIONS: Diaphragmatic MMG may reflect diaphragmatic contractility more directly and sensitively than the conventional method.

Pneumatic-type dynamic traction and flexion splint for treating patients with extension contracture of the metacarpophalangeal joint.

BACKGROUND: Collateral ligament shortening causes extension contractures of the metacarpophalangeal joint, and dynamic flexion splinting has been widely used to treat these contractures; however, there are various problems with these approaches. We developed a novel, pneumatic-type dynamic traction and flexion splint to solve these problems. CASE DESCRIPTION AND METHODS: A total of 25 fingers were treated with the dynamic traction and flexion splint for 8 weeks. Every 2 weeks, the average metacarpophalangeal joint flexion angle, total active motion, grasp strength, and pain scores were assessed. FINDINGS AND OUTCOMES: The finger flexion angle was significantly greater at the final evaluation, starting after 6 weeks of treatment (p < 0.05), than prior to treatment. Similarly, the total active motion results improved significantly over 8 weeks. CONCLUSIONS: Our results show that use of the dynamic traction and flexion splint improves patient finger functioning and flexural angle. CLINICAL RELEVANCE: The dynamic traction and flexion (DTF) splint appears to be effective for treating patients.

Serum concentration of fentanyl during conversion from intravenous to transdermal administration to patients with chronic cancer pain.

BACKGROUND: To the best of our knowledge, there have been no reports on the pharmacokinetics and pharmacodynamics during the conversion from continuous intravenous infusion (CII) to transdermal fentanyl administration. The primary objective of the present study was to clarify the pharmacokinetic characteristics during this conversion. A secondary objective was to identify an association between serum albumin and the absorption of fentanyl from the transdermal patch. METHODS: A prospective study was conducted from February 2010 to August 2011 that enrolled 19 patients with chronic cancer pain. Patients were classified into 2 study groups according to body mass index and albumin level. All patients received the conversion from CII to transdermal fentanyl using a 2-step taper of CII over 6 hours. Comparisons of efficacy, toxicity, and serum fentanyl concentrations between study groups were analyzed at baseline, 3, 6, 9, 12, 15, 18, and 24 hours after initiation of the conversion. RESULTS: The dose-adjusted serum fentanyl concentrations for all patients were significantly decreased at 15 to 24 hours after conversion compared with baseline, although pain intensity and the number of rescue events remained stable during the conversion. The dose-adjusted serum fentanyl concentrations at 9 to 24 hours were significantly reduced in the low albumin group compared with the normal albumin group (P<0.05). CONCLUSIONS: Our study demonstrated that the dose-adjusted serum fentanyl concentrations remained relatively stable, and pain intensity and the number of rescue events remained stable during conversion. Hypoalbuminemia was strongly associated with poor absorption of transdermally administered fentanyl.

An antioxidant Trolox restores decreased oral absorption of cyclosporine A after liver ischemia-reperfusion through distinct mechanisms between CYP3A and P-glycoprotein in the small intestine.

Oxidative stress is a critical mediator of various injuries following ischemia-reperfusion (I/R) associated with organ transplantation. Although oral bioavailability of cyclosporine A (CsA) was decreased by increased first-pass metabolism through CYP3A and P-glycoprotein (P-gp) specifically in the upper small intestine after liver I/R, the mechanism responsible for them remained to be clarified. In the present study, the effect of Trolox (an α-tocopherol analogue) on the decreased oral absorption of CsA through elevated intestinal CYP3A and P-gp after liver I/R and their regulations were investigated. Rats were subjected to 60 min of liver ischemia followed by 12h of reperfusion. Trolox was administered intravenously 5 min before reperfusion. Trolox diminished the increased malondialdehyde and total glutathione levels in plasma by liver I/R and concomitantly prevented the decreased area under the blood concentration-time curve of orally administered CsA as well as initial absorption rate of CsA from upper small intestine. The elevated CYP3A mRNA and activity in the upper small intestine as well as expression levels of P-gp in upper, middle, and lower small intestines after liver I/R were attenuated by Trolox administration. The elevations of CYP3A levels specifically in the upper small intestine of I/R rats were correlated with the lithocholic acid levels in the bile. These results demonstrate that Trolox ameliorates the decreased oral absorption of CsA through elevated intestinal CYP3A and P-gp by preventing oxidative stress, where the biliary lithocholic acid may be responsible for the elevated transcription of CYP3A specifically in the upper small intestine after liver I/R.

Simultaneous determination of five polyether ionophores using liquid chromatography with one-step fluorescent derivatization.

We present a selective method for simultaneous determination of five polyether ionophores such as salinomycin (SAL), monensin (MON), narasin (NAR), semduramicin (SEM) and lasalocid (LAS) in aquatic samples using a liquid chromatography with one-step fluorescent derivatization of 2-(4-hydrazinocarbonyl-phenyl) 4,5-diphenylimidazole (HCPI) and 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride hydrochloride (DIB-Cl). Fluorescent one-step derivatization for SAL, MON, NAR and SEM using HCPI and for LAS using DIB-Cl was monitored by an LC/fluorescence detector (E(x), 340 nm; E(m), 465 nm). Chromatographic separation was performed on a TSK-GEL ODS-120T (4.6 × 150 mm, 3 µm) column using a mobile phase of 0.1% formic acid in acetonitrile and 0.5 mM ammonium formate in water (70/30, v/v). The limits of detections were 0.01 µg/mL (50 pg) for LAS, 0.05 µg/mL (250 pg) for SAL, NAR and SEM, and 0.1 µg/mL (500 pg) for MON, respectively. The recoveries for water samples were indicated to be the range of 79.6 ± 6.4 - 99.0 ± 4.4% with associated precision values (between-day for 3 days) for repeatability. Based on solid-phase extraction, the limit of quantitation values indicated 0.1 ng/mL for SAL, MON, NAR and SEM, and 0.01 ng/mL for LAS in water samples.

On-line solid-phase extraction LC-MS/MS for the determination of Ac-SDKP peptide in human plasma from hemodialysis patients.

We developed a high-throughput method based on on-line solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) to determine N-terminal thymosin-ß fragment peptide (N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) in human plasma samples. Quantification of Ac-SDKP was performed using direct injection for on-line SPE based on C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac-SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3-100.4% with inter-day (relative standard deviation, RSD, 0.4-14.1%) and intra-day (RSD, 0.8-19.7%) assays. This method was applied to the measurement of Ac-SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre-hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post-hemodialyzed subjects, n = 9). All plasma Ac-SDKP levels were decreased by dialysis. Thus, plasma Ac-SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis.

A strategy for high-speed countercurrent chromatography purification of specific antioxidants from natural products based on on-line HPLC method with radical scavenging assay.

We have proposed a novel and first strategy of high-speed countercurrent chromatography (HSCCC) purification for the efficient and effective discovery of antioxidant from natural product based on on-line HPLC method with radical scavenging assay. To achieve a strategy for HSCCC purification, the antioxidants in materials are identified by on-line HPLC with DPPH radical scavenging assay. Then, the optimal condition of target peaks would be investigated for the two-phase solvent system, and purified by HSCCC. In this study, the specific antioxidants in red cabbage, perilla and elderberry pigments were evaluated by on-line HPLC with DPPH radical scavenging assay, and purified by HSCCC technique. Specific antioxidants could be rapidly pinpointed in complex mixtures by on-line HPLC with DPPH radical scavenging assay. Then, the optimal two-phase solvent systems were investigated using these HPLC peaks. Finally, the purification of these nine antioxidants form three mixtures were performed by HSCCC. Using mass spectrometric analysis, these antioxidants were confirmed to cyanidin-based anthocyanin from red cabbage and elderberry pigments, and luteolin-based flavones from perrilla pigment. Due to the advantages derived from on-line HPLC with DPPH radical scavenging assay and HSCCC technique, a rapid, efficient and effective strategy has been developed for the discovery of antioxidants from natural products.

Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry.

We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.

Screening assay of angiotensin-converting enzyme inhibitory activity from complex natural colourants and foods using high-throughput LC-MS/MS.

Inhibition of angiotensin-converting enzyme (ACE) by various foods decreases the blood pressure. ACE inhibitors derived from natural components may be of therapeutic value in preventive medicine. In this study, we report a novel screening assay of ACE inhibitors from complex natural colourants and foods that employ solid phase extraction (SPE), high-throughput liquid chromatography (LC) separation, and stable isotope dilution electrospray tandem mass spectrometry (SID-ESI-MS/MS). When a target sample was subjected to N-Hippuryl-His-Leu (HHL) and ACE in phosphate buffer (pH 7.4), generated hippuric acid (HA) was extracted by SPE. LC/SID-ESI-MS/MS detection of HA allowed us to accurately identify the effects of complex substances such natural colourants and foods that inhibit the ACE of HHL. The major HA and HA-d5 fragment ions at m/z 180→105 and 185→110 in the multiple reaction monitoring (MRM) mode can quantify levels that are lower than other methods. The LC/SID-ESI-MS/MS method described here is a rapid, selective, sensitive, and highly reproducible method for the determination of HA in various samples. Based on the assay developed, all samples such as natural colourants, infant formula, soy paste, ketchup, mayonnaise, wheat flour, orange juice, supplement drink, tea, and coffee could be accurately measured for ACE inhibition in various matrices. High-throughput LC/SID-ESI-MS/MS assay has no limitations in the evaluation of inhibition activity in various natural samples such as colour, high-matrix, and processed foods.

Quantification of N-acetyl-seryl-aspartyl-lysyl-proline in hemodialysis patients administered angiotensin-converting enzyme inhibitors by stable isotope dilution liquid chromatography-tandem mass spectrometry.

We developed a sensitive, selective and accurate method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to determine N-terminal thymosin-ß peptides of Ac-SDKP and Ac-ADKP in human plasma samples. Quantification of Ac-SDKP and Ac-ADKP was performed using solid phase extraction (SPE) based on C(18), reversed phase LC separation, and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) and Ac-ADKP-d(7) were synthesized for the internal standards. These MRM monitoring ions were m/z 488→129 (quantitative ion)/226 for Ac-SDKP, m/z 496→137 for Ac-SDKP-(13)C(6), (15)N(2), m/z 472→129 (quantitative ion)/226 for Ac-ADKP, and m/z 479→129 for Ac-ADKP-d(7), respectively. Lower limit of quantitation (LLOQ) of Ac-SDKP and Ac-ADKP was 0.1ng/mL in human plasma. Recovery values were ranged from 94.7% to 106.3% for inter- (RSD: 0.6-3.5%) and intra- (RSD: 0.4-4.9%) day assays. Plasma Ac-SDKP levels were significantly higher in hemodialyzed subjects treated with angiotensin-converting enzyme inhibitors of enalapril (27.3±24.6ng/mL, n=10) and trandolapril (12.3±16.9ng/mL, n=18) than healthy (0.4±0.2ng/mL, n=7) and hemodialyzed subjects (0.6±0.2ng/mL, n=34). This analytical method would be useful to measure N-terminal thymosin-ß peptides in human plasma for the clinical study.

Development and application of an HILIC-MS/MS method for the quantitation of nucleotides in infant formula.

A method for the quantitation of nucleotides (adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and inosine 5'-monophosphate (IMP)) in infant formula was developed by hydrophilic interaction chromatography with tandem mass spectrometry (HILIC-MS/MS). The internal standards used (AMP-13C10, 15N5; GMP-13C10, 15N5; UMP-13C9, 15N2; CMP-13C9, 15N3) were prepared with centrifugal ultrafiltration (CUF). Data acquisition was achieved by using multiple reaction monitoring (MRM) of product ions of protonated molecules of the five nucleotides generated by the positive-ion ESI. HILIC conditions were performed with 30 mmol/L ammonium formate in water (pH 2.5, adjusted with formic acid) and methanol. The LOD and LOQ were 5-10 µg/mL and 10-30 µg/mL for standard solution, respectively. Recovery for intra- and interday assays ranged from 98.1 to 108.9% (RSD: 0.7-5.4%) spiked with three concentration levels (5, 25, and 250 µg/g powder infant formula). This method could be applied for the determination of nucleotides in infant formula samples. The detected concentrations of five nucleotides ranged from not detected (n.d.) to 278 µg/g powder infant formula. The total nucleotide level ranged from n.d. to 600.2 µg/g powder infant formula.

Simple and novel screening assay of natural antioxidants for Cu(II) ion/adrenaline-mediated oxidation of N-terminal amyloid beta by liquid chromatography/mass spectrometry.

In the present study, a novel assay for screening inhibitors of Cu(II) ion/adrenaline-mediated oxidative modification of N-terminal amyloid beta (Abeta) peptides was developed using liquid chromatography with mass spectrometry (LC/MS). The physiological condition of Cu(II) ion/adrenaline in buffer (pH 7.4) at 37 degrees C for 90 min revealed a specific modification of N-terminal Abeta peptides, such as Abeta1-6, Abeta1-40, and Abeta1-42, using trypsin digestion and LC/MS detection of the modified Abeta peptide. When this oxidative modification of the shorter N-terminal Abeta1-6 was subjected to LC/MS, single charged ions from native peptide ([M+H]+, m/z 774) were observed at m/z 729 and 685, corresponding to a decrease in mass of 45 and 89 Da, respectively, as compared with the original peptide. To determine the effect of specific antioxidants, a screening assay to find inhibitors of Cu(II) ion/adrenaline-mediated oxidation was developed based on the response ratio of m/z 685 to 774. LC/MS detection of the modified peptides allowed us to identify antioxidants that inhibit oxidative modification of Abeta1-6 model peptide. The oxidative modification of Abeta1-6 was inhibited by curcumin but not an isoflavone or catechin mixture or saponin or capsaicin, revealing a clear difference between antioxidants that inhibit oxidative modification and other antioxidants. This novel assay may allow for the identification of antioxidants that protect against oxidative modification of Abeta and other proteins related to oxidative stress by adrenaline and Cu(II) ions under normal physiologic conditions.

[LC/ESI-MS/MS method for the simultaneous determination of macrocyclic lactone parasiticides in livestock products and fish].

An LC/ESI (positive-mode)-MS/MS method was developed for simultaneous quantification of 8 macrocyclic lactones (abamectin B1a, abamectin 8,9-Z isomer B1a, emamectin benzoate B1a, emamectin benzoate 8,9-Z isomer B1a, ivermectin, eprinomectin B1a, doramectin and moxidectin) in animal tissues, egg, milk and honey. The separation was achieved on a TSK-GEL ODS 100 V column (2.0 x 50 mm, 3 microm) with a mobile phase consisting of 0.1% formic acid in acetonitrile, and 0.1 mM ammonium formate-0.1% formic acid in water, at a flow rate of 0.2 mL/min with gradient elution. Linear calibration plots were obtained with high correlation coefficients (r=0.998-0.999). The LOQ and LOD ranged from 0.02-1.5 ng/mL and 0.1-5 ng/mL, respectively. Average recoveries were in the range of 70.8-117.1% with associated RSD values<15% (n=10) for repeatability and reproducibility. The spiking levels for recoveries and RSDs met the validation criteria for Japanese maximum residue limits (MRLs). Based on these results, the proposed analytical method has been proven to be highly efficient and suitable for routine determinations of macrocyclic lactones in animal food matrices.