Prevalence and determinants of post-COVID-19 syndrome among patients 6 months post-discharge from a teaching hospital in South India.

Background: The coronavirus disease 2019 (COVID-19) infection has affected millions of people worldwide in the last 4 years. Among those infected, the long-term COVID-19 syndrome, in which symptoms of COVID-19 persist for a variable period, is posing new challenges to the health system, but few studies are available in India that examine the post-COVID-19 syndrome, that is, 6 months and beyond COVID-19 infection. This study aimed to find the prevalence and determinants of post-COVID-19 syndrome among patients 6 months and beyond their infection. Methodology: This cross-sectional study was conducted among 300 patients who were admitted and discharged from Government Medical College, Thrissur, at least 6 months before with a diagnosis of COVID-19 infection. The data collection was performed using a semi-structured interview schedule through a telephonic interview. The analysis was performed using the Statistical Package for the Social Sciences (SPSS) software. Results: 21% of the patients studied had at least one persistent symptom at the end of 6 months, and 4.1% had more than one symptom. Among the symptoms persisting most commonly were fatigue (11%) and dyspnea (7.7%). Other than persisting symptoms, 21% of patients developed exertional dyspnea and 19% developed sleep disturbances during the 6 months after discharge. The factors that were associated with persistent symptoms in univariate analysis were increasing age, presence of chronic obstructive pulmonary disease (COPD), presence of chronic kidney disease (CKD), and admission to the intensive care unit (ICU). Conclusion: The study concludes that one-fifth of the patients still suffer from post-COVID-19 syndrome even 6 months after the COVID-19 infection. Our health systems should be prepared for the long-term management of COVID-19-infected people and prepare policies for the same.

Amphi-Luminescent MoS2 nanostructure for photocatalytic splitting of water and removal of Methylene Blue.

Chemical dyes used in the textile industries are one of the major pollutants in water. Methylene blue (MB) is a commonly seen dye that creates hazardous health problems. In this article the photocatalytic degradation of MB by the nanocatalyst MoS2 (Nano-MoS2) and carbon dot (C Dots) incorporated MoS2 (Nano-CD-MoS2) is reported. The photocatalytic degradation of MB is analyzed based on the electron-hole recombination rate of the catalyst. Photoluminescence emission exhibited by the catalyst is used as a key indicator to probe the electron-hole recombination rate. Nano-MoS2 was synthesized hydrothermally at 180 0C for 8 h from ammonium tetra thiomolybdate (ATTM). C Dot was prepared following a green root from ash guard extract which later mixed with Nano-MoS2 and kept in an autoclave at a temperature 140 °C for 4 h to get Nano-CD-MoS2. The photoluminescence (PL) and photocatalytic behavior of Nano-MoS2 and Nano-CD-MoS2 and their application for water splitting and water purification are reported. The incorporation of graphene and artificial C Dot into MoS2 nanostructures are reported to increase the conductivity and active edge sites of MoS2 that enhances the photocatalytic action. Since green C Dots are eco-friendly and easily synthesizable than artificial C Dots, as a novel study, this article investigated the influence of green C Dots on the PL and photocatalytic performance of nanosized MoS2. Nano-MoS2 and Nano-CD-MoS2 exhibited both upconversion and downconversion PL; accordingly the nanostructures were termed as amphi-luminescent. The amphi-luminescence property widens the photon absorption range and hence enhances the catalytic degradation of dyes. Nano-MoS2 which exhibited lesser intensity of amphi-luminescence emission compared to Nano-CD-MoS2 showed better results in degradation of MB. C Dots may bind with the valence band electrons of MoS2, resulting in the reduction of dangling bonds. Dangling bonds can trap photo-induced excitons to hinder the rate of electron-hole recombination. So, fast electron-hole recombination occurs in Nano-CD-MoS2 than Nano-MoS2. Fast electron-hole recombination supports radiative electron-hole recombination while suppresses the non-radiative energy transfer of electrons and causes high PL intensity. However, according to the energy level diagram, Nano-MoS2 with minimal electron-hole recombination rate is more favorable for O2/O2-,.OH/ OH- and.OH/H2O reactions that facilitate MB degradation. Photocatalytic activity of catalysts were confirmed by measuring the photocurrent from a simple custom-made two-electrode water photolysis cell where the nanocatalysts were dispersed in electrolyte. Lead and steel rods were used as electrodes. Multimeter was used to measure current. Nano-MoS2 exhibited better performance with a maximum photocurrent of 141 µA. Influence of green C Dots in energy levels, PL and photocatalysis of MoS2 and mechanisms of PL and degradation of MB are thoroughly investigated in this article.

Growth and productivity of Haematococcus pluvialis and Coelastrella saipanensis by photosystem modulation for understanding the heterotrophic nutritional strategy for bioremediation application.

In this study, Haematococcus pluvialis and Coelastrella saipanensis were evaluated for heterotrophic nutrition potential in dairy waste medium by blocking the PSII using DCMU. The study was done by four sets of experiments. In the first set, in the different concentrations DCMU-treatments, 20µL showed pronounced effect in H. pluvialis and C. saipanensis as 89 % and 83% decrease in cells (>30 and > 250 cells/mL) compared to control (536 ± 12.35 × 104 and 1167 ± 15.35 × 104 cells/mL, respectively). Damage to the PS II by DCMU interrupted the growth, which in turn produced a significant drop in the number of cells. In the second round of experiment, growth of algae in various dairy waste concentrations suggest that dairy wastewater (DWW) provides enough nutrients to produce 35.71 % and 64.74 % more cells in H. pluvialis and C. saipanensis, respectively compared to the control. In the third set, high DCMU concentration was added to microalgae cultures in DWW to assess the heterotrophic nutrition potential. Growth in cell number 34.4 ± 19 and 617.46 ± 60.44 cells/mL was recorded in H. pluvialis and C. saipanensis when grown control medium whereas addition of DCMU reduced the cell number to 1.53 ± 0.75 and 55.13 ± 0.75 cells/mL on 15th day, respectively. This shows cells in cultures treated with DCMU reveal that algae can sustain their metabolic activity by utilizing the nutrients of dairy waste inhibiting photosystem. Fourth round of experiments found that microalgae could resume their growth and productivity by adapting to heterotrophic nutritional behaviour when DCMU given in mild dose at different time interval. This study conclude as C. saipanensis grows more readily by absorbing dairy waste nutrients than H. pluvialis. Therefore, C. saipanensis is an excellent choice for wastewater treatment through sustainable environmentally benign process after scale-up investigation. These results provide useful information to advance to molecular study for measuring microalgae's capability for bioremediation application.

Potential Implications of Vouacapan Compounds for Insecticidal Activity: An In Silico Study.

BACKGROUND: From the fruits and seeds of the species of Pterodon, it is possible to obtain two main products: essential oil and oleoresin. In oleoresin, numerous vouacapan compounds have been demonstrated to have biological potential, including insecticidal activity. OBJECTIVE: In silico studies were performed to identify potential candidates for natural insecticides among the vouacapans present in the genus Pterodon. MATERIALS AND METHODS: Molecular docking and molecular dynamics studies were performed to analyze the interaction of vouacapan compounds with acetylcholinesterase of Drosophila melanogaster. Pharmacokinetic parameters regarding physicochemical properties, plasma protein binding, and activity in the central nervous system were evaluated. The toxicological properties of the selected molecules were predicted using malathion as the reference compound. RESULTS: 6α,7ß-dimethoxivouacapan-17-ene (15) showed a high number of interactions and scores in molecular docking studies. These results suggested that this compound exhibits an inhibitory activity of the enzyme acetylcholinesterase. This compound showed the best results regarding physicochemical properties, besides presenting low cutaneous permeability values, suggesting null absorption. Molecular dynamics studies demonstrated few conformational changes in the structure of the complex formed by compound 4 and acetylcholinesterase enzyme throughout the simulation time. CONCLUSION: It was determined that compound 4 (vouacapan 6α,7ß,17ß,19-tetraol) could be an excellent candidate for usage as a natural insecticide.

Maternal urinary concentrations of organophosphate ester metabolites: associations with gestational weight gain, early life anthropometry, and infant eating behaviors among mothers-infant pairs in Rhode Island.

BACKGROUND: Organophosphate esters (OPEs)-used as flame retardants and plasticizers-are associated with adverse pregnancy outcomes such as reduced fecundity and live births and increased preterm delivery. OPEs may interfere with growth and metabolism via endocrine-disruption, but few studies have investigated endocrine-related outcomes. The objective of this pilot study (n = 56 mother-infant pairs) was to evaluate associations of OPEs with gestational weight gain (GWG), gestational age at delivery, infant anthropometry, and infant feeding behaviors. METHODS: We quantified OPE metabolites (bis-2-chloroethyl phosphate [BCEP], bis (1,3-dichloro-2-propyl) phosphate [BDCPP], diphenyl phosphate [DPHP]) in pooled maternal spot urine collected throughout pregnancy (~ 12, 28, and 35 weeks' gestation). We obtained maternal sociodemographic characteristics from questionnaires administered at enrollment and perinatal characteristics from medical record abstraction. Trained research assistants measured infant weight, length, head and abdominal circumferences, and skinfold thicknesses at birth and 6 weeks postpartum. Mothers reported infant feeding behavior via the Baby Eating Behavior Questionnaire (BEBQ). Using multiple linear regression, we assessed associations of log2-transformed maternal urinary OPE metabolites with GWG, gestational age at delivery, infant anthropometry at birth, weekly growth rate, and BEBQ scores at 6 weeks postpartum. We used linear mixed effects (LME) models to analyze overall infant anthropometry during the first 6 weeks of life. Additionally, we considered effect modification by infant sex. RESULTS: We observed weak positive associations between all OPE metabolites and GWG. In LME models, BDCPP was associated with increased infant length (ß = 0.44 cm, 95%CI = 0.01, 0.87) and weight in males (ß = 0.14 kg, 95%CI = 0.03, 0.24). BDCPP was also associated with increased food responsiveness (ß = 0.23, 95%CI = 0.06, 0.40). DPHP was inversely associated with infant abdominal circumference (ß = - 0.50 cm, 95%CI = - 0.86, - 0.14) and female weight (ß = - 0.19 kg, 95%CI = - 0.36, - 0.02), but positively associated with weekly growth in iliac skinfold thickness (ß = 0.10 mm/wk., 95%CI = 0.02, 0.19). Further, DPHP was weakly associated with increased feeding speed. BCEP was associated with greater infant thigh skinfold thickness (ß = 0.34 mm, 95%CI = 0.16, 0.52) and subscapular skinfold thickness in males (ß = 0.14 mm, 95%CI = 0.002, 0.28). CONCLUSIONS: Collectively, these findings suggest that select OPEs may affect infant anthropometry and feeding behavior, with the most compelling evidence for BDCPP and DPHP.

Quantification of 16 urinary biomarkers of exposure to flame retardants, plasticizers, and organophosphate insecticides for biomonitoring studies.

Chlorinated alkyl and non-chlorinated aryl organophosphate flame retardants (OPFRs) and some brominated flame retardants (FR) were introduced as replacements for polybrominated diphenyl ethers (PBDEs) after PBDEs phase-out in 2004 and 2013. Organophosphorous (OP) insecticides are mainly used in agricultural settings since the Food Quality Protection Act of 1996 phased-out most residential uses of OP insecticides in the United States. Urinary metabolites of FRs and OPs are known exposure biomarkers to FRs and OP insecticides, respectively. For large population-based studies, concurrent quantification of these metabolites using a small urine volume is desirable, but until now was not possible. We developed an analytical approach to quantify in 0.2 mL urine 10 FRs and six OP insecticide metabolites: diphenyl phosphate, bis(1,3-dichloro-2-propyl) phosphate, bis(1-chloro-2-propyl) phosphate, bis(2-chloroethyl) phosphate, dicresyl phosphates, dibutyl phosphate, dibenzyl phosphate, 2,3,4,5-tetrabromobenzoic acid, 2-((isopropyl)phenyl)phenyl phosphate, 4-((tert-butyl)phenyl)phenyl phosphate, dimethyl phosphate, diethyl phosphate, dimethyl thiophosphate, dimethyl dithiophosphate, diethyl thiophosphate, and diethyl dithiophosphate. The method relies on enzymatic deconjugation, automated off-line solid phase extraction, high-performance liquid chromatography, and isotope dilution tandem mass spectrometry. Detection limits ranged from 0.05 to 0.5 ng mL-1, accuracy from 89 to 118%, and imprecision was <10%. . This method is the first to quantify simultaneously trace levels of 16 biomarkers of FRs and OP insecticides in only four drops of urine. We confirmed the method suitability for use in large epidemiological studies to assess background and occupational exposures to these classes of environmental pollutants by analyzing 303 samples collected from the general population and a group of firefighters. FR metabolite and DAPs concentrations in the general population group were lower than in the firefighters group, and within the ranges reported in the U.S. general population and other non-occupationally exposed populations.

Exposure to organophosphate flame retardant chemicals in the U.S. general population: Data from the 2013-2014 National Health and Nutrition Examination Survey.

BACKGROUND: Use of organophosphate flame retardants (OPFRs) including tris(1,3-dichloro-2-propyl) phosphate, triphenyl phosphate, tris(1-chloro-2-propyl) phosphate, and tris-2-chloroethyl phosphate, in consumer products is on the rise because of the recent phase out of polybrominated diphenyl ether (PBDE) flame retardants. Some of these chemicals are also used as plasticizers or lubricants in many consumer products. OBJECTIVES: To assess human exposure to these chlorinated and non-chlorinated organophosphates, and non-PBDE brominated chemicals in a representative sample of the U.S. general population 6years and older from the 2013-2014 National Health and Nutrition Examination Survey (NHANES). METHODS: We used solid-phase extraction coupled to isotope dilution high-performance liquid chromatography-tandem mass spectrometry after enzymatic hydrolysis of conjugates to analyze 2666 NHANES urine samples for nine biomarkers: diphenyl phosphate (DPHP), bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), bis-(1-chloro-2-propyl) phosphate (BCIPP), bis-2-chloroethyl phosphate (BCEP), di-n-butyl phosphate (DNBP), di-p-cresylphosphate (DpCP), di-o-cresylphosphate (DoCP), dibenzyl phosphate (DBzP), and 2,3,4,5-tetrabromobenzoic acid (TBBA). We calculated the geometric mean (GM) and distribution percentiles for the urinary concentrations (both in micrograms per liter [µg/L] and in micrograms per gram of creatinine). We only calculated GMs for analytes with an overall weighted frequency of detection >60%. For those analytes, we also a) determined weighted Pearson correlations among the log10-transformed concentrations, and b) used regression models to evaluate associations of various demographic parameters with urinary concentrations of these biomarkers. RESULTS: We detected BDCIPP and DPHP in approximately 92% of study participants, BCEP in 89%, DNBP in 81%, and BCIPP in 61%. By contrast, we detected the other biomarkers much less frequently: DpCP (13%), DoCP (0.1%), TBBA (5%), and did not detect DBzP in any of the participants. Concentration ranges were highest for DPHP (<0.16-193µg/L), BDCIPP (<0.11-169µg/L), and BCEP (<0.08-110µg/L). Regardless of race/ethnicity, 6-11year old children had significantly higher BCEP adjusted GMs than other age groups. Females had significantly higher DPHP and BDCIPP adjusted GM than males, and were more likely than males to have DPHP concentrations above the 95th percentile (odds ratio=3.61; 95% confidence interval, 2.01-6.48). CONCLUSIONS: Our results confirm findings from previous studies suggesting human exposure to OPFRs, and demonstrate, for the first time, widespread exposure to several OPFRs among a representative sample of the U.S. general population 6years of age and older. The observed differences in concentrations of certain OPFRs biomarkers by race/ethnicity, in children compared to other age groups, and in females compared to males may reflect differences in lifestyle and exposure patterns. These NHANES data can be used to stablish a nationally representative baseline of exposures to OPFRs and when combined with future 2-year survey data, to evaluate exposure trends.

Effect of anti-inflammatory activity of ranolazine in rat model of inflammation.

BACKGROUND & OBJECTIVES: Inflammatory processes are a recognized feature of atherosclerotic lesions. Ranolazine inhibits the inflammatory markers such as C-reactive protein, interleukins-1 and -6 and tumour necrosis factor-alpha. The present study was planned to evaluate the effect of anti-inflammatory activity of ranolazine in acute and sub-acute models of inflammation in rats and compare the same with that of control (gum acacia 1%) and aspirin (standard anti-inflammatory drug). METHODS: Adult male Wistar rats (150-180 g) were used for the study. They were divided into three groups (n=6). One per cent gum acacia (control), aspirin (200 mg/kg body weight) and ranolazine (180 mg/kg body weight) were given orally. Acute inflammation was induced by injecting carrageenan in the left hind paw. Paw oedema volume and percentage inhibition were measured. Subacute inflammation was induced by implanting foreign bodies subcutaneously. Percentage inhibition of granuloma dry weight and haematoxylin and eosin stained sections of granulation tissue were studied. RESULTS: In acute and subacute model study, ranolazine significantly (P <0.01) decreased the paw oedema volume and granuloma dry weight as compared to control and it was comparable to that of aspirin and histopathological sections showed a decrease in granulation tissue formation as compared to control. INTERPRETATION & CONCLUSIONS: Ranolazine demonstrated significant anti-inflammatory activity in acute and subacute models of inflammation and needs further evaluation for its use in reducing atherosclerosis.

Variability and predictors of urinary concentrations of organophosphate flame retardant metabolites among pregnant women in Rhode Island.

BACKGROUND: Organophospate flame retardants (PFRs) are chemicals of emerging concern due to restrictions on polybrominated diphenyl ether flame retardant formulations. We describe the occurrence, variability, and predictors of urinary metabolites of PFRs among pregnant women. METHODS: In 2014-2015, 59 women from Providence, RI provided up to 3 spot urine samples during pregnancy (~12, 28, and 35 weeks' gestation). We created a pooled urine sample per woman and measured nine relevant metabolites in individual and pooled samples. We used linear mixed models to calculate intraclass correlation coefficients (ICCs) across the 3 measurements and to assess sociodemographic and dietary predictors of PFRs. RESULTS: The median (IQR) of bis-2-chloroethyl phosphate (BCEP), bis(1,3-dichloro-2-propyl) phosphate (BDCPP), and diphenyl phosphate (DPhP), the metabolites most frequently detected, from pooled samples were: 0.31 µg/L (0.17-0.54), 1.18 µg/L (0.64-2.19), 0.93 µg/L (0.72-1.97), respectively. We observed fair to good reproducibility for BCEP (ICC = 0.50), BDCPP (ICC = 0.60), and DPhP (ICC = 0.43), and excellent agreement between the urinary flame retardant metabolite concentrations averaged across pregnancy versus pooled urine sample concentrations for BCEP (ICC = 0.95), BDCPP (ICC = 0.89), and DPhP (ICC = 0.93). Adjusting for pertinent sociodemographic factors and gestational week of urine collection, each 1 kg increase in pre-pregnancy weight was associated with greater BCEP (1.1%; 95% CI: 0.1, 2.1), BDCPP (1.5%; 95% CI: 0.3, 2.7), and DPhP (0.5%; 95% CI: 0.0, 1.1). Dietary factors were generally not associated with urinary flame retardant metabolites. CONCLUSIONS: Urinary concentrations of BCEP, BDCPP, and DPhP were frequently detected among women in this pilot study and had fair reproducibility across pregnancy. Body size may be an important predictor of urinary flame retardant metabolite concentrations.

Quantification of three chlorinated dialkyl phosphates, diphenyl phosphate, 2,3,4,5-tetrabromobenzoic acid, and four other organophosphates in human urine by solid phase extraction-high performance liquid chromatography-tandem mass spectrometry.

Polybrominated diphenyl ethers (PBDEs), produced as flame retardants worldwide, have been phased-out in many countries, and chlorinated and non-chlorinated organophosphates and non-PBDE brominated formulations (e.g., Firemaster 550 (FM550)) have entered the consumers' market. Recent studies show that components of organophosphate esters and FM550 are frequently detected in many products common to human environments. Therefore, urinary metabolites of these compounds can be used as human exposure biomarkers. We developed a method to quantify nine compounds in 0.4 mL urine: diphenyl phosphate (DPhP), bis(1,3-dichloro-2-propyl) phosphate (BDCPP), bis-(1-chloro-2-propyl) phosphate, bis-2-chloroethyl phosphate, di-p-cresylphosphate, di-o-cresylphosphate (DoCP), di-n-butyl phosphate, dibenzyl phosphate (DBzP), and 2,3,4,5-tetrabromobenzoic acid. The method relies on an enzymatic hydrolysis of urinary conjugates of the target analytes, automated off-line solid phase extraction, reversed phase high performance liquid chromatography separation, and isotope dilution-electrospray ionization tandem mass spectrometry detection. The method is high-throughput (96 samples/day) with detection limits ranging from 0.05 to 0.16 ng mL-1. Spiked recoveries were 90-113 %, and interday imprecision was 2-8 %. We assessed the suitability of the method by analyzing urine samples collected from a convenience sample of adults (n = 76) and from a group of firefighters (n = 146). DPhP (median, 0.89; range, 0.26-5.6 ng mL-1) and BDCPP (median, 0.69; range, 0.31-6.8 ng mL-1) were detected in all of the non-occupationally exposed adult samples and all of the firefighter samples (DPhP [median, 2.9; range, 0.24-28 ng mL-1], BDCPP [median, 3.4; range, 0.30-44 ng mL-1]); DBzP and DoCP were not detected in any samples.

Homology modeling and docking studies on oxidosqualene cyclases associated with primary and secondary metabolism of Centella asiatica.

Centella asiatica is a well-known medicinal plant, produces large amount of triterpenoid saponins, collectively known as centelloids, with a wide-spectrum of pharmacological applications. Various strategies have been developed for the production of plant secondary metabolites in cell and tissue cultures; one of these is modular metabolic engineering, in which one of the competitive metabolic pathways is selectively suppressed to channelize precursor molecules for the production of desired molecules by another route. In plants the precursor 2,3-oxidosqualene is shared in between two competitive pathways involved with two isoforms of oxidosqualene cyclases. One is primary metabolic route for the synthesis of phytosterol like cycloartenol by cycloartenol synthase; another is secondary metabolic route for the synthesis of triterpenoid like ß-amyrin by ß-amyrin synthase. The present work is envisaged to evaluate specific negative modulators for cycloartenol synthase, to channelize the precursor molecule for the production of triterpenoids. As there are no experimentally determined structures for these enzymes reported in the literature, we have modeled the protein structures and were docked with a panel of ligands. Of the various modulators tested, ketoconazole has been evaluated as the negative modulator of primary metabolism that inhibits cycloartenol synthase specifically, while showing no interaction with ß-amyrin synthase. Amino acid substitution studies confirmed that, ketoconazole is specific modulator for cycloartenol synthase, LYS728 is the key amino acid for the interaction. Our present study is a novel approach for identifying a suitable specific positive modulator for the over production of desired triterpenoid secondary metabolites in the cell cultures of plants.

Two-dimensional high performance liquid chromatography separation and tandem mass spectrometry detection of atrazine and its metabolic and hydrolysis products in urine.

Atrazine [6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine] is the most widely used herbicide in the United States. In recent years, there has been controversy about atrazine's potential endocrine/reproductive and neurological adverse effects in wildlife and humans. The controversy triggered several environmental and epidemiologic studies, and it generated needs for sensitive and selective analytical methods for the quantification of atrazine, atrazine metabolites, and degradation or hydrolysis products. We developed a two-dimensional high performance liquid chromatography (2D-HPLC) method with isotope dilution tandem mass spectrometry detection to measure atrazine in urine, along with 11 atrazine metabolites and hydrolysis products, including 6-chloro (Cl), 6-mercapto (Mer) and 6-hydroxy (OH) derivatives, and their desethyl, desisopropyl and diamino atrazine analogs (DEA, DIA and DAA, respectively). The 2D-HPLC system incorporated strong cation exchange and reversed phase separation modes. This versatile approach can be used for the quantitative determination of all 12 compounds in experimental animals for toxicological studies. The method requires only 10 µL of urine, and the limits of detection (LODs) range from 10 to 50 µg/L. The method can also be applied to assess atrazine exposure in occupational settings by measurement of 6-Cl and 6-Mer analogs, which requires only 100 µL of urine with LODs of 1-5 µg/L. Finally, in combination with automated off-line solid phase extraction before 2D-HPLC, the method can also be applied in non-occupational environmental exposure studies for the determination of -6-Cl and 6-Mer metabolites, using 500 µL of urine and LODs of 0.1-0.5 µg/L.

High-throughput sample preparation for the quantitation of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea, and propylenethiourea in human urine using 96-well-plate automated extraction and high-performance liquid chromatography-tandem mass spectrometry.

Acephate, methamidophos, o-methoate, and dimethoate are organophosphorus pesticides, and ethylenethiouria and propylenethiourea are two metabolites from the bisdithiocarbamate fungicide family. They are some of the most widely used pesticides and fungicides in agriculture both domestically and abroad. The existing high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the measurement of these compounds in human urine was improved by using a 96-well plate format sample preparation; the use of HPLC-MS/MS was comparable with a concentration range of 0.125 to 50 ng/ml. Deuterium-labeled acephate, ethylenethiouria, and methamidophos were used as internal standards. The sample preparation procedure, in the 96-well format with a 0.8-ml urine sample size, uses lyophilization of samples, followed by extraction with dichloromethane. The analytes were chromatographed on a Zorbax SB-C3 (4.6 × 150 mm, 5.0-µm) column with gradient elution by using 0.1% formic acid in aqueous solution (solvent A) and 0.1% formic acid in methanol (solvent B) mobile phase at a flow rate of 1 ml/min. Quantitative analysis was performed by atmospheric pressure chemical ionization source in positive ion mode using multiple-reaction monitoring of the precursor-to-product ion pairs for the analytes on a TSQ Quantum Ultra HPLC-MS/MS. Repeated analyses of urine samples spiked with high (15 ng/ml), medium (5 ng/ml), and low (1 ng/ml) concentrations of the analytes gave relative SDs of <13%. The limits of detection were in the range of 0.004-0.01 ng/ml. The method also has high accuracy, high precision, and excellent extraction recovery. Furthermore, the improved sample preparation method decreased the cost and labor required while effectively doubling the analytic throughput with minimal matrix effect.

Measurement of ethyl methanesulfonate in human plasma and breast milk samples using high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Ethyl methanesulfonate (EMS) is a mesylate ester, which is known to be a potent mutagen, teratogen, and possibly carcinogen. Mesylate esters have been found in pharmaceuticals as contaminants formed during the manufacturing process and may potentially pose an exposure hazard to humans. We have developed and validated a method for detection of trace amounts (ng/ml levels) of EMS in human plasma and breast milk. The samples were extracted by matrix solid-phase dispersion with ethyl acetate using Hydromatrix and the ASE 200 Accelerated Solvent Extractor. The extracts were separated by high-performance liquid chromatography (HPLC) using a HILIC column. The detection was performed with a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Electron Corporation) using atmospheric pressure chemical ionization in negative-ion mode and multiple reaction monitoring. The use of a surrogate internal standard in combination with HPLC-MS/MS provided a high degree of accuracy and precision. The extraction efficiency was greater than 70%. Repeated analyses of plasma and breast milk samples spiked with high (100 ng/ml), medium (50 ng/ml) and low (5 ng/ml) concentrations of the analytes gave relative standard deviations of less than 12%. The limits of detection were in the range of 0.5-0.9 ng/ml for both matrices.

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography-tandem mass spectrometry.

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.