Determination of chlorothalonil in vegetables/fruits using liquid chromatography-tandem mass spectrometry with a discharge adapter interface.

RATIONALE: Chlorothalonil (CHT), a broad-spectrum fungicide, has been employed widely to control foliar diseases, whereas with a major metabolite of polar 4-hydroxychlorothalonil (CHT-4-OH), only an acceptable nonpolar CHT residue is allowed by most countries. This study involves the method development for CHT residue in vegetables/fruits using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a novel modified discharge-adaptor (DA) interface. METHODS: CHT residue was analyzed using LC-MS/MS with DA interface (LC-DA-MS/MS), developed in our previous works. A DA was placed on the electrospray tip to switch the ionization modes. A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was applied to extract CHT residue of vegetables/fruits efficiently with less sample preparation time and analysis cost. RESULTS: CHT and CHT-4-OH spiked in four different vegetables/fruits were extracted using the modified QuEChERS method. After LC with isocratic elution, CHT and CHT-4-OH were separated within 3 min. Using LC-DA-MS/MS, the ion signals of CHT were improved two to three times, and the limit of quantification of 5 ng/g and linearity (r2 > 0.99) in the range of 5-200 ng/g were achieved using 10 g of vegetables/fruits. The precision and accuracy were within 15% each. The modified QuEChERS and LC-DA-MS/MS were applied to examine eight field-grown vegetables/fruits; 9.5 and 2588.9 ng/g of CHT were detected in two vegetables/fruits. CONCLUSION: LC-DA-MS/MS combined with modified QuEChERS was successfully applied to determine CHT residue <10 ng/g in vegetables/fruits and with satisfied validation results. The developed method could reduce both analysis cost and time, attributing to simplifications in modified QuEChERS, isocratic elution, and DA interface in LC-DA-MS/MS.

Determination of glyphosate, aminomethylphosphonic acid, and glufosinate in river water and sediments using microwave-assisted rapid derivatization and LC-MS/MS.

Glyphosate (N-phosphonomethyl glycine) and glufosinate (ammonium dl-homoalanin- 4-methyl phosphinate) are nonselective, broad-spectrum, and highly polar herbicides that are wildly used for weed control in aquatic systems and vegetation control in non-crop areas. Aminomethylphosphonic acid (AMPA) is the major degradation product of glyphosate. To address the concerns to its environmental residue and the possible adverse effects, the analytical methods by using microwave-assisted derivatization were developed for determining glyphosate, AMPA, and glufosinate in river water and sediments. The methods applied the 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) derivatization for the analytes. The microwave heating is first-time applied to reduce the FMOC-reaction time of glyphosate, AMPA, and glyphosate in the environmental samples to less than 2.5 min. The microwave-assisted methods were successfully validated for river water and sediment. The linear ranges of 7.8-2000.0 ng/L and 0.78-100.0 ng/g were achieved by using 10 mL of water and 2 g of sediments. Glyphosate was found in 30/32% and 25/32% of 32 water and 32 sediments at 27.1-1353.9 ng/L and 2.4-189.6 ng/g levels. AMPA was found in 30/32% and 30/32% of 32 water and 32 sediments at 60.2-1509.0 ng/L and 1.8-233.6 ng/g levels. Glyphosate was found in 10/32% of 32 water at 14.8-503.1 ng/L levels. No glufosinate residue was observed for 32 sediments. The residues of glyphosate and AMPA were wildly detected in the river waters and sediments near the agricultural regions, and glufosinate was less detected. This is the first study that reported herbicide levels in water and sediment from Taiwan rural areas using microwave-assisted rapid derivatization, useful information for environmental management.

Multichannel nanoplasmonic platform for imidacloprid and fipronil residues rapid screen detection.

In recent years, imidacloprid and fipronil have been reported to harm beneficial insects, such as honey bees, and to potentially pose risks to mammals, including humans. Considering their widespread use and potential minimum toxic range from 10 ppb to 1 ppm (species dependent), a simple, rapid, sensitive, and reliable method for screening and detection is urgently needed. Here, we present a surface plasmon resonance (SPR)-based nanoplasmonic chip integrated with a multichannel spectral imaging system to detect ecosystem-harming pesticides. The pre-modification of the designed mercapto-haptens reduced detection time to 2.5 h. Moreover, owing to the multichannel configuration, it was possible to introduce an internal standard analytical method to effectively reduce matrix interference in real samples; thus, the concentration of the target pesticide could be determined more precisely. The strong linearity of the spiked sample test results indicated high accuracy in quantifying target pesticides. Considering the limit of detection (~10 ppb), the cutoffs for detection and quantification were set at 15 and 45 ppb, respectively, and were used as the detection criteria. The detection results of the blind tests of real samples were also compared with those of liquid chromatography electrospray ionization tandem mass spectrometry (standard method) and were highly consistent. The custom-made integrated SPR system allows much simpler, label-free, high-throughput, and reliable on-site identification and quantification of imidacloprid and fipronil. All test results validated the platform's capability in the on-site rapid screening and detection of pesticide residues at the parts per billion and parts per million levels.

A combined analytical method for biological monitoring of arsenic, benzene and polycyclic aromatic hydrocarbons in human urine by liquid chromatography tandem mass spectrometry.

An analytical method for the biomonitoring of arsenic, benzene and polycyclic aromatic hydrocarbons (PAHs) in human urine was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The urinary metabolites of monomethylarsonic acid (MMAA), dimethylarsonic acid (DMAA), s-phenylmercapturic acid (S-PMA), and 1-hydroxypyrene (1-OHP) were selected as the corresponding marker compounds. After enzymatic deconjugation, 4 mL urine sample was extracted by 2 steps of solvent extractions. The 1-OHP was first extracted with n-hexane with 10% ethyl acetate, and MMAA, DMMA, and S-PMA were then extracted in a 2nd extraction/back-extraction using chloroform/ammonium bicarbonate aqueous solution. The two extracts were mixed and analyzed by LC-MS/MS. The method was validated with spiked urine samples. The obtained linear ranges (r2 > 0.99) of the urinary markers were 2-64 ng/mL MMAA, 1-64 ng/mL DMAA, 0.78-100 ng/mL S-PMA and 0.05-6.4 ng/mL 1-OHP. The measured accuracy (% Error) and precision (CV%) were -16.67 to 29.17% and 2.03-30.99% (3 spiked levels, 6 replicates), respectively. The method was applied to 10 real urine samples, and the presence of MMAA, DMAA, S-PMA and 1-OHP were clearly detected. The detected concentrations were BQL-8.38 ng/mL of MMAA, BQL-10.71 ng/mL of DMAA, BQL-2.55 ng/mL of SPMA, and BQL-0.17 ng/mL of 1-OHP, which were all consistent with the reported background levels.

Induced pluripotent stem cell-derived endothelial progenitor cells attenuate ischemic acute kidney injury and cardiac dysfunction.

BACKGROUND: Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI), which is associated with high morbidity and mortality. AKI is a serious and costly medical condition. Effective therapy for AKI is an unmet clinical need, and molecular mechanisms underlying the interactions between an injured kidney and distant organs remain unclear. Therefore, novel therapeutic strategies should be developed. METHODS: We directed the differentiation of human induced pluripotent stem (iPS) cells into endothelial progenitor cells (iEPCs), which were then applied for treating mouse AKI. The mouse model of AKI was induced by I/R injury. RESULTS: We discovered that intravenously infused iEPCs were recruited to the injured kidney, expressed the mature endothelial cell marker CD31, and replaced injured endothelial cells. Moreover, infused iEPCs produced abundant proangiogenic proteins, which entered into circulation. In AKI mice, blood urea nitrogen and plasma creatinine levels increased 2 days after I/R injury and reduced after the infusion of iEPCs. Tubular injury, cell apoptosis, and peritubular capillary rarefaction in injured kidneys were attenuated accordingly. In the AKI mice, iEPC therapy also ameliorated apoptosis of cardiomyocytes and cardiac dysfunction, as indicated by echocardiography. The therapy also ameliorated an increase in serum brain natriuretic peptide. Regarding the relevant mechanisms, indoxyl sulfate and interleukin-1ß synergistically induced apoptosis of cardiomyocytes. Systemic iEPC therapy downregulated the proapoptotic protein caspase-3 and upregulated the anti-apoptotic protein Bcl-2 in the hearts of the AKI mice, possibly through the reduction of indoxyl sulfate and interleukin-1ß. CONCLUSIONS: Therapy using human iPS cell-derived iEPCs provided a protective effect against ischemic AKI and remote cardiac dysfunction through the repair of endothelial cells and the attenuation of cardiomyocyte apoptosis.

Epinecidin-1 has immunomodulatory effects, facilitating its therapeutic use in a mouse model of Pseudomonas aeruginosa sepsis.

Antimicrobial peptides (AMPs) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of epinecidin-1 against a multidrug-resistant clinical isolate of P. aeruginosa (P. aeruginosa R) and a P. aeruginosa strain from ATCC (P. aeruginosa ATCC 19660) in vivo. The MICs of epinecidin-1 against P. aeruginosa R and P. aeruginosa ATCC 19660 were determined and compared with those of imipenem. Epinecidin-1 was found to be highly effective at combating peritonitis infection caused by P. aeruginosa R or P. aeruginosa ATCC 19660 in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that epinecidin-1 enhances the rate of survival of mice infected with the bacterial pathogen P. aeruginosa through both antimicrobial and immunomodulatory effects.

Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry.

Sinensetin (SIN), one of the major polymethoxyflavones (PMFs) contained mainly in the citrus peels, has been reported to possess various bioactivities, including antifungal, antimutagenic, anticancer, and anti-inflammatory activities. Although the biotransformation of SIN in fungi and insects has been reported, the information about the metabolism of SIN in mammals is still unclear. In this study, formation of SIN metabolites in rats was investigated. Four isotope-labeled SINs ([4'-D3]SIN, [3'-D3]SIN, [5-D3]SIN, and [6-D3]SIN) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. The administered compound and four SIN metabolites were detected in rat urine. These metabolites were identified as 4'-hydroxy-5,6,7,3'-tetramethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 6-hydroxy-5,7,3',4'-tetramethoxyflavone, and 7-hydroxy-5,6,3',4'-tetramethoxyflavone sulfate.

Identification of 5,7,3',4'-tetramethoxyflavone metabolites in rat urine by the isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry.

5,7,3',4'-Tetramethoxyflavone (TMF), one of the major polymethoxyflavones (PMFs) isolated from Kaempferia parviflor , has been reported possessing various bioactivities, including antifungal, antimalarial, antimycobacterial, and anti-inflammatory activities. Although several studies on the TMF have been reported, the information about the metabolism of TMF and the structures of TMF metabolites is still not yet clear. In this study, an isotope-labeling method was developed for the identification of TMF metabolites. Three isotope-labeled TMFs (5,7,3',4'-tetramethoxy[3'-D(3)]flavone, 5,7,3',4'-tetramethoxy[4'-D(3)]flavone, and 5,7,3',4'-tetramethoxy[5,4'-D(6)]flavone) were synthesized and administered to rats. The urine samples were collected, and the main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry. Five TMF metabolites were unambiguously identified as 3'-hydroxy-5,7,4'-trimethoxyflavone, 7-hydroxy-5,3',4'-trimethoxyflavone sulfate, 7-hydroxy-5,3',4'-trimethoxyflavone, 4'-hydroxy-5,7,3'-trimethoxyflavone, and 5-hydroxy-7,3',4'-trimethoxyflavone.

A discharge adaptor interface for use in liquid chromatography/mass spectrometry.

A discharge adaptor, composed of a metal casing and platinum (Pt) wire needle, was directly attached to an electrospray ionization (ESI) probe tip, to transform the ionization into atmospheric pressure chemical ionization (APCI). Six generic drugs were analyzed with the developed discharge adaptor (DA) and two commercial interfaces. The DA interface produced more intense radical anions, [M]˙⁻, and less sodium adduct ions, [M + Na]⁺, than the ESI interface, whereas almost the same molecular ions were detected as the APCI interface. The effects of solvent and desolvation gas flow in the DA interface were similar to those in the ESI interface, but differed from those in the APCI interface. Better sensitivity of the tested drugs was obtained relative to the commercial APCI interface. For human plasma samples, the DA interface also demonstrated good tolerance to plasma matrices, linearity from 5 or 20 to 500 ng/mL (r² > 0.99) and ruggedness.

Gene expression and localization of the epinecidin-1 antimicrobial peptide in the grouper (Epinephelus coioides), and its role in protecting fish against pathogenic infection.

Epinecidin-1 is an antimicrobial peptide and plays a vital role in protecting fish against pathogenic infection. As a mimic of a grouper epinecidin-1 peptide, it has tertiary structures that closely resemble those of pleurocidin found in the winter flounder (Pleuronectes americanus). The tissue-specific, lipopolysaccharide (LPS)-stimulation-specific, and poly(I):poly(C)-stimulation-specific expressions of the grouper (Epinephelus coioides) epinecidin-1 antimicrobial peptide were determined using a comparative reverse-transcription polymerase chain reaction. Results of the tissue distribution analysis revealed high levels of epinecidin-1 messenger RNA (mRNA) in the head kidneys, intestines, and skin. Expression of epinecidin-1 mRNA was dose-dependently stimulated by both LPS and poly(I):poly(C). Immunohistochemical analysis with the polyclonal antiserum of a grouper epinecidin-1 peptide (rabbit polyclonal antibody) showed that the peptide was localized with the epinecidin-1 antibody in the gills and intestines. Two synthetic peptides of the grouper epinecidin-1 peptide (g-ple 22-51 and g-ple 22-42) and one winter flounder pleurocidin as a control exhibited high antimicrobial activities against gram-negative or gram-positive bacteria. In addition, peptide treatment was effective in promoting a significant increase in fish survival after the injection of Vibrio vulnificus in tilapia (Oreochromis mossambicus) and grouper. These results are relevant to the design of prophylactic and therapeutic strategies to counter bacterial infections, especially for preventing or ameliorating immune defects in fish during bacterial infections.

Measurement of benzylmercapturic acid in human urine by liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry.

A method based on liquid chromatography (reversed-phase)-electrospray (negative) ionization-tandem triple quadrupole mass spectrometry [LC-ESI(-)-MS-MS], in the multiple reaction monitoring mode, was developed for determination of urinary benzylmercapturic acid (BMA). Isotope dilution through introduction of (13)C(6)-labeled BMA was employed as an internal standard, achieving intra- and interrun precision ranges of 2.32-2.95% and 2.24-4.97%, respectively. The calibration curve obtained with BMA-spiked blank human urine was linear from 0.5-120.0 microg/L; the correlation coefficient of the calibration curve was 0.999. The method limit of quantification was 0.5 microg/L; mean BMA recovery was 97.56%. The analytical method was used to analyze urine samples collected from workers involved in the manufacture of adhesive tape who had been exposed to toluene. The method appears to provide sensitive, specific, and reliable testing for urinary BMA to determine occupational exposure to toluene.