Exposure of Male Farmers and Nonfarmers to Neonicotinoid Pesticides in the South-West and Littoral Regions of Cameroon: A Comparative Study.

Pesticides, especially the newly developed neonicotinoids, are increasingly used in many countries around the world, including Cameroon, to control pests involved in crop destruction or disease transmission. Unfortunately, the pesticides also pose tremendous environmental problems because a predominant amount of their residues enter environmental matrices to affect other nontargeted species including humans. This therefore calls for continuous biomonitoring of these insecticides in human populations. The present study sought to assess the neonicotinoid insecticide exposures in two agrarian regions of Cameroon, the South-West region and Littoral region. The study involved 188 men, including 125 farmers and 63 nonfarmers. Spot urine samples were obtained from these subjects and subjected to liquid chromatographic-tandem mass spectrometric analysis for concentrations of neonicotinoid compounds, including acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, nitenpyram, thiamethoxam, and N-dm-acetamiprid. Neonicotinoid compounds were detected in all study participants, and residues of all the screened pesticides were detected among participants. N-dm-Acetamiprid and imidacloprid were the most prevalent among the subjects (100.0% and 93.1%, respectively), whereas nitenpyram was less common (3.2%). The median values of imidacloprid and total urinary neonicotinoid concentrations were elevated among farmers (0.258 vs. 0.126 µg/L and 0.829 vs. 0.312 µg/L, respectively). Altogether the findings showed that both the farmer and nonfarmer study populations of Cameroon were exposed to multiple residues of neonicotinoids, with relatively higher levels of pesticides generally recorded among farmers. Although exposure levels of the neonicotinoids were generally lower than their respective reference doses, these results warrant further research on the health risk evaluation of multiple residues of the pesticides and reinforcement of control measures to minimize the exposure risks, especially among farmers. Environ Toxicol Chem 2024;43:952-964. © 2024 SETAC.

[Study on determination of 2-thioxothiazolidine-4-carboxylic Acid in urine by high performance liquid chromatography].

Objective: To establish a high performance liquid chromatography method for the determination of 2-thioxothiazolidine-4-carboxylic acid (TTCA) in urine. Methods: After acidification with hydrochloric acid, TTCA in urine was first extracted by ethyl acetate with excessive sodium chloride, then gradient separated by a symmetry C18 column and then detected by a diode array detector. The quantification was based on a working curve of external standard method. Results: The linear relationship of TTCA in urine was good in the range of 0.03-10.00 mg/L, and the correlation coefficient was 0.9999. The detection limit and minimum quantitative concentration of TTCA in urine were 0.008 mg/L and 0.027 mg/L. The intra-assay precision of the method was 0.9%-1.4%, the inter-assay precision was 1.3%-3.5%, and the average recovery was 85.0%-92.7% while the concentrations of TTCA in urine was 0.8, 2.0 and 8.0 mg/L, respectively (n=6) . Conclusion: The gradient elution high performance liquid chromatography method has simple operation and high sensitivity, and it is suitable for the determination of TTCA on a low level in urine for occupational workers exposure to carbon disulfide.

Study on determination of 2-thioxothiazolidine-4-carboxylic Acid in urine by high performance liquid chromatography / 中华劳动卫生职业病杂志

Objective: To establish a high performance liquid chromatography method for the determination of 2-thioxothiazolidine-4-carboxylic acid (TTCA) in urine. Methods: After acidification with hydrochloric acid, TTCA in urine was first extracted by ethyl acetate with excessive sodium chloride, then gradient separated by a symmetry C18 column and then detected by a diode array detector. The quantification was based on a working curve of external standard method. Results: The linear relationship of TTCA in urine was good in the range of 0.03-10.00 mg/L, and the correlation coefficient was 0.9999. The detection limit and minimum quantitative concentration of TTCA in urine were 0.008 mg/L and 0.027 mg/L. The intra-assay precision of the method was 0.9%-1.4%, the inter-assay precision was 1.3%-3.5%, and the average recovery was 85.0%-92.7% while the concentrations of TTCA in urine was 0.8, 2.0 and 8.0 mg/L, respectively (n=6) . Conclusion: The gradient elution high performance liquid chromatography method has simple operation and high sensitivity, and it is suitable for the determination of TTCA on a low level in urine for occupational workers exposure to carbon disulfide.

Urinary concentrations of neonicotinoid insecticides were related to renal tubular dysfunction and neuropsychological complaints in Dry-zone of Sri Lanka.

Neonicotinoids are systemic insecticides used since the 1990's , that possess renal tubular toxicity. We conducted a field-based descriptive study in the North Central Dry-zone of Sri Lanka, where chronic kidney disease (CKD) of unknown etiology has been increasing since the 1990's. To elucidate the relationship between renal tubular dysfunctions and urinary neonicotinoids concentrations, we collected spot urine samples from15 CKD patients, 15 family members, and 62 neighbors in 2015, analyzed two renal tubular biomarkers, Cystatin-C and L-FABP, quantified seven neonicotinoids and a metabolite N-desmethyl-acetamiprid by LC-MS/MS; and we investigated their symptoms using a questionnaire. Cystatin-C and L-FABP had a positive correlation (p < 0.001). N-Desmethyl-acetamiprid was detected in 92.4% of the urine samples, followed by dinotefuran (17.4%), thiamethoxam (17.4%), clothianidin (9.8%), thiacloprid and imidacloprid. Dinotefuran and thiacloprid have never been registered in Sri Lanka. In High Cystatin-C group (> 70 µg/gCre, n = 7), higher urinary concentration of dinotefuran (p = 0.009), and in Zero Cystatin-C group (< LOQ, n = 7), higher N-desmethyl-acetamiprid (p = 0.013), dinotefuran (p = 0.049), and thiacloprid (p = 0.035), and more complaints of chest pains, stomachache, skin eruption and diarrhea (p < 0.05) were found than in Normal Cystatin-C group (n = 78). Urinary neonicotinoids may be one of the potential risk factors for renal tubular dysfunction in this area.

Comprehensive characterization of the peroxisome proliferator activated receptor-δ agonist GW501516 for horse doping control analysis.

According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organism's metabolism. Even if urinary metabolism of G501516 has been extensively studied in human subjects, knowledge on GW501516 metabolism in horses remains limited. To fight against doping practices in horses' races, GW501516 metabolism has to be studied in horse urine to identify and characterize the most relevant target metabolites to ensure an efficient doping control. In this article, in vitro and in vivo experiments have been conducted using horse S9 liver microsome fractions and horse oral administration route, respectively. These investigations determined that the detection of GW501516 must be performed in urine on its metabolites because the parent molecule was extremely metabolized. To maximize analytical method sensitivity, the extraction conditions have been optimized. In accordance with these results, a qualitative analytical method was validated to detect the abuse of GW501516 based on its most relevant metabolites in urine. This work enabled the Laboratoire des Courses Hippiques (LCH) to highlight two cases of illicit administration of this forbidden molecule in post-race samples.

Metabolic study of GW1516 in equine urine using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry for doping control.

RATIONALE: The use of GW1516, a peroxisome proliferator-activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine. METHODS: Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein-precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) with a Q-Exactive mass spectrometer. Monoisotopic ions of GW1516 and its metabolites were monitored from the full-scan mass spectral data of pre- and post-administration samples. A quantification method was developed and validated to establish the excretion profiles of GW1516, its sulfoxide, and its sulfone in equine urine. RESULTS: GW1516 and its nine metabolites [including GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), methyl 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylate (MMTC), 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), and M1 to M4] were detected in post-administration urine samples. GW1516 sulfoxide and GW1516 sulfone showed the longest detection times in post-administration urine samples and were therefore recommended as potential screening targets for doping control purposes. Quantitative analysis was also conducted to establish the excretion profiles of GW1516 sulfoxide and GW1516 sulfone in urine. CONCLUSIONS: For the purposes of doping control of GW1516, the GW1516 sulfoxide and GW1516 sulfone metabolites are recommended as the target analytes to be monitored in equine urine due to their high specificities, long detection times (1 and 4 weeks, respectively), and the ready availability of their reference materials.

High-throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction.

In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high-throughput assay based on liquid chromatography-triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96-well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5-ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96-well collection plate. With a 10-min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti-Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02-0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

N-methylmalonamic acid (NMMA) as metabolite of methylisothiazolinone and methylchloroisothiazolinone in 24-h urine samples of the German Environmental Specimen Bank from 2000 to 2017 - exposure and time trends.

Methylisothiazolinone (MI) and the mixture of methylchloroisothiazo¬linone/methylisothiazolinone (MCI/MI, 3:1) are widespread biocides used in cosmetics, household products, paints or as disinfectant in air-conditioning systems. Exposure to these compounds has raised concerns due to their sensitizing potential, as rates of skin sensitization were reported to increase in the last decade. We have analyzed N-methylmalonamic acid (NMMA), a common metabolite of MI and MCI in 24-h urine samples of the German Environmental Specimen Bank collected from 480 participants (240 male/240 female) between the years 2000 and 2017. Using these data, we were able to calculate the overall daily intake of MI and/or MCI/MI (3:1) of the study participants and point out time trends. NMMA was determined in all urine samples investigated above the LOQ of 0.5 µg/L urine. Median and 95th percentile level of NMMA in all 24-h urine samples was 4.1 µg/g creatinine and 8.5 µg/g creatinine, respectively. This would correspond to a median and 95th percentile daily intake of 0.35 µg/kg bw and 0.71 µg/kg bw for exclusive uptake of MI and 0.64 µg/kg bw and 1.28 µg/kg bw for exclusive uptake of MCI/MI (3:1). We noted only slight variations over time for median exposures, but an increasing time trend in the 95th percentile exposure between 2006 and 2011 with a decrease in recent years, probably reflecting regulatory measures on MI and MCI/MI (3:1) in cosmetic products. Increasing knowledge on determinants of exposure to MI and/or MCI/MI (3:1) would be necessary to further lower exposure to these sensitizing compounds.

Biomonitoring method for neonicotinoid insecticides in urine of non-toilet-trained children using LC-MS/MS.

There is a growing appreciation of the importance of determining chemical exposure levels in early childhood, as well as in embryonic and foetal life, which are now widely believed to be essential for gaining insight into potential health risks associated with these chemicals. To facilitate the assessment of exposure to neonicotinoid insecticides (NEOs) in non-toilet-trained children, a new method using disposable diapers (nappies) was developed for the simultaneous determination of the NEOs acetamiprid and its metabolite N-desmethylacetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam (NEO biomarkers). The urine absorbed in disposable diapers was extracted with acetone (diaper urine) and was cleaned using a solid-phase extraction column, before analysis with LC-MS/MS. The absolute recoveries of NEO biomarkers were 19-50%. Good results were observed for the linearity of the matrix-matched calibration curves (r2 = 0.983-0.996; concentration range LOQ-20 µg L-1) and the precision of intra-day (% relative standard deviation (%RSD): 3.3-12.7%) and inter-day (%RSD: 4.3-19.5%) analyses. The lowest and highest limits of detection of the developed method were 0.07 µg L-1 for acetamiprid and 0.75 µg L-1 for clothianidin. The developed method was applied for the evaluation of fifty diapered three-year-old children in Japan. Importantly, the study revealed relatively high detection rates for dinotefuran and N-desmethylacetamiprid; 84% and 78% respectively. The highest geometric mean of dinotefuran urinary concentration was 2.01 µg L-1. Thus, a method for determining NEO biomarkers in urine extracted from disposable diapers was established. This is the first report on the simultaneous quantitative analysis of NEO biomarkers of diaper-absorbed urine samples.

Accuracy of a Rapid Diagnostic Test for the Presence of Direct Oral Factor Xa or Thrombin Inhibitors in Urine-A Multicenter Trial.

The rapid determination of the presence of direct oral anticoagulants (DOACs) in a patient remains a major challenge in emergency medicine and for rapid medical treatment decisions. All DOACs are excreted into urine. A sensitive and specific point-of-care test has been developed to determine whether they are present in patient urine samples. This prospective multicenter study aimed to demonstrate at least 95% correct positive and negative predictive results for factor Xa and thrombin inhibitors in urine samples using DOAC Dipstick pads compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (NCT03182829). Nine hundred and fourteen subjects were included and 880 were evaluated per protocol (factor Xa inhibitors apixaban, edoxaban, and rivaroxaban: n = 451, thrombin inhibitor dabigatran: n = 429) at 18 centers. The sensitivity, specificity, accuracy, and predictive values and agreement between methods for determination of factor Xa inhibitors were at least noninferior to 95% with a 0.5% margin and of thrombin inhibitor superior to 97.5%. These results were compared with LC-MS/MS results in the intention-to-analyze cohort (all p < 0.05). The receiver operating curve showed c-values of 0.989 (factor Xa inhibitors) and 0.995 (thrombin inhibitor). Visual evaluation of the factor Xa and thrombin inhibitor pads was not different between centers. Qualitative determination of both types of DOACs was accurate using the DOAC Dipstick compared with using LC-MS/MS. The high predictive values may impact laboratory and clinical decision-making processes.

Microdosed Cocktail of Three Oral Factor Xa Inhibitors to Evaluate Drug-Drug Interactions with Potential Perpetrator Drugs.

OBJECTIVES: The aim of this study was to prove the suitability of simultaneously administered microdoses of the factor Xa inhibitors (FXaIs) rivaroxaban, apixaban and edoxaban (100 µg in total). To evaluate drug-drug interactions, the impact of ketoconazole, a known strong inhibitor of cytochrome P450 3A4 and P-glycoprotein, was studied. METHODS: In a crossover clinical trial, 18 healthy volunteers were randomized to the two treatments using microdoses of rivaroxaban, apixaban and edoxaban alone and when coadministered with ketoconazole. Plasma and urine concentrations of microdosed apixaban, edoxaban and rivaroxaban were quantified using a validated ultra-performance liquid chromatography-tandem mass spectrometry assay with a lower limit of quantification of 2.5 pg/ml. RESULTS: The microdosed FXaI cocktail showed similar pharmacokinetic parameters compared with published data, using normal therapeutic doses of each FXaI. Ketoconazole significantly increased exposure, with geometric mean AUC ratios of 1.90 (apixaban), 2.35 (edoxaban) and 2.27 (rivaroxaban). CONCLUSION: The microdosed FXaI cocktail approach was able to precisely predict the drug interaction with ketoconazole. This is the first study that has been conducted to evaluate drug-drug interactions with a drug class, and the low administered doses also allow evaluation in vulnerable target populations. STUDY PROTOCOL: EudraCT 2016-003024-23.

Development of magnetic carbon nanotubes for dispersive micro solid phase extraction of the cyanide metabolite, 2-aminothiazoline-4-carboxylic acid, in biological samples.

2-aminothiazoline-4-carboxylic acid (ATCA) is a minor metabolite of cyanide and is suggested to be a promising biomarker for cyanide exposure due to its specificity to cyanide metabolism and its excellent short- and long-term stability during storage. In this study, magnetic carbon nanotubes, including magnetic multi-walled carbon nanotubes (Mag-MWCNT) and magnetic single-walled carbon nanotubes (Mag-SWCNT) were synthesized as a novel sorbent for dispersive micro solid phase extraction (d-µSPE) to extract ATCA from biological matrices. ATCA spiked deionized water samples with the addition of the isotopic internal standard (ATCA - 13C, 15N) were subjected to Mag-CNT/d-µSPE to confirm extraction efficiency of this new technique. The extracted ATCA was derivatized and quantitated using gas chromatography/mass spectrometry (GC/MS) analysis. The extraction parameters were optimized and a detection limits of 15 and 25 ng/mL were obtained for synthetic urine and bovine blood respectively with a linear dynamic range of 30-1000 ng/mL. The optimized Mag-CNT/d-µSPE method facilitated efficient extraction of ATCA using 2 mg of Mag-MWCNT with a 10-minute extraction time. The current assay was also found to be effective for the extraction of ATCA with average recoveries of 97.7 ±â€¯4.0% (n = 9) and 96.5 ±â€¯12.1% (n = 9) from synthetic urine and bovine blood respectively. The approach of using Mag-CNT to facilitate d-µSPE offered a novel alternative to extract ATCA from complex biological matrices.

Simultaneous determination of nine neonicotinoids in human urine using isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry.

Neonicotinoids (neonics), a class of systemic insecticides, have been frequently detected in pollen, vegetables, and fruits. Recently, an increasing concern has been aroused for human exposure to neonics. However, biological monitoring for quantifying body burden of neonics has rarely been reported. In this study, we developed an isotope-dilution ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify nine neonics, including acetamiprid (ACE), thiamethoxam (THIAM), imidacloprid (IMIP), clothianidin (CLO), flonicamid (FLO), thiacloprid (THIAC), dinotefuran (DIN), nitenpyram (NIT), and imidaclothiz (IMIT) in urine. The limits of quantification were 0.1 µg/L for ACE, FLO, DIN, NIT and IMIT, and 0.2 µg/L for THIAM, IMIP, CLO, and THIAC. The overall recoveries were 80.8-103%, 81.5-91.7% and 83.0-92.3% for QA/QC samples fortifying at 1, 25, and 100 µg/L levels, respectively. UPLC/MS/MS method was used to analyze urine samples obtained from 10 children in Hangzhou, China. The detection frequencies were 80% for ACE and IMIP, 70% for THIAM and CLO, 20% for DIN and IMIT and 10% for THIAC. FLO and NIT were not detected in those urine samples. The data provided here will be helpful for conducting biological monitoring of neonics exposure in the future.

Metabolism and disposition of the anticancer quinolone derivative vosaroxin, a novel inhibitor of topoisomerase II.

Background Vosaroxin is a first-in-class anticancer quinolone derivative that is being investigated for patients with relapsed or refractory acute myeloid leukemia (AML). The primary objective of this study was to quantitatively determine the pharmacokinetics of vosaroxin and its metabolites in patients with advanced solid tumors. Methods This mass balance study investigated the pharmacokinetics (distribution, metabolism, and excretion) of vosaroxin in cancer patients after a single dose of 60 mg/m2 14C-vosaroxin, administered as short intravenous injection. Blood, urine and feces were collected over 168 h after injection or until recovered radioactivity over 24 h was less than 1% of the administered dose (whichever was earlier). Total radioactivity (TRA), vosaroxin and metabolites were studied in all matrices. Results Unchanged vosaroxin was the major species identified in plasma, urine, and feces. N-desmethylvosaroxin was the only circulating metabolite detected in plasma, accounting for <3% of the administered dose. However, in plasma, the combined vosaroxin + N-desmethylvosaroxin AUC0-∞ was 21% lower than the TRA AUC0-∞ , suggesting the possible formation of protein bound metabolites after 48 h when the concentration-time profiles diverged. The mean recovery of TRA in excreta was 81.3% of the total administered dose; 53.1% was excreted through feces and 28.2% through urine. Conclusions Unchanged vosaroxin was the major compound found in the excreta, although 10 minor metabolites were detected. The biotransformation reactions were demethylation, hydrogenation, decarboxylation and phase II conjugation including glucuronidation.

Quantification of N-methylmalonamic acid in urine as metabolite of the biocides methylisothiazolinone and chloromethylisothiazolinone using gas chromatography-tandem mass spectrometry.

Methylisothiazolinone and the mixture of chloromethylisothiazolinone/methylisothiazolinone (MCI/MI, 3:1) are widespread biocides used in cosmetic and household products. Due to their skin permeability, they might be taken up by the general population via use of products containing these biocides. As both compounds are known skin sensitizers, the use of these products is under discussion by regulatory agencies. In order to evaluate the possible uptake of MI and/or MCI/MI by human biomonitoring, we have developed and validated a highly sensitive and specific GC/MS/MS-method for the quantification of N-methylmalonamic acid (NMMA), a known metabolite of MI and MCI in urine of rats. After freeze-drying of urine, the analyte is derivatised with pentafluorobenzyl bromide in anhydrous solution and the PFB-derivative is extracted into n-hexane. After concentration, the derivative is finally quantified by GC/MS/MS in EI-mode using 13C3-NMMA as internal standard. The limit of quantification for NMMA was 0.5ngmL-1 urine. Precision within and between-series was determined to range between 3.7-10.9% using native and spiked quality control samples. Accuracy ranged between 89 and 114%. In a pilot study we applied this method to spot urine samples of 63 persons not knowingly exposed to MI and/or MCI/MI. NMMA was quantifiable in every urine sample analysed, with no significant difference in urinary levels between male and female participants. The median (95th percentile) levels for urinary NMMA were 3.6 (7.4) ngmg-1 creatinine and 2.9 (9.1) ngmg-1 creatinine for males (n=32) and females (n=31), respectively. In a volunteer experiment, a relation of exposure to MI and/or MCI/MI and subsequent NMMA-excretion was shown. Our method is the first to report human urinary background levels of NMMA. However, the possibility of formation and urinary excretion of NMMA within physiological processes cannot be ruled out.

Catching Fakes: New Markers of Urine Sample Validity and Invalidity.

Urine drug testing is common for workplace drug testing, prescription management, emergency medicine and the criminal justice system. Unsurprisingly, with the significant consequences based upon the results of urine drug testing, a donor in need of concealing the contents of their sample is highly motivated to cheat the process. Procedures and safeguards ensuring sample validity are well known, and include measuring sample temperature at the time of collection, and laboratory measurements of creatinine, specific gravity and pH. Synthetic urine samples are available and are designed to deceive all aspects of urine drug testing, including validity testing. These samples are sophisticated enough to contain biological levels of creatinine, and are at a physiological pH and specific gravity. The goal of our research was to develop new procedures designed to distinguish authentic samples from masquerading synthetic samples. We aimed to identify substances in commercial synthetic urines not expected to be present in a biological sample distinguishing fake specimens. Additionally, we aimed to identify and employ endogenous compounds in addition to creatinine for identifying biological samples. We successfully identified two compounds present in synthetic urines that are not present in biological samples and use them as markers of invalidity. Four new endogenous markers for validity were successfully evaluated. Validity assessment was further aided by monitoring metabolites of nicotine and caffeine. When the method was applied to patient samples, 2% of samples were identified as inconsistent with natural urine samples, even though they met the current acceptance criteria for creatinine, pH and specific gravity.

Quantification of vosaroxin and its metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine using high-performance liquid chromatography-tandem mass spectrometry.

Vosaroxin is a first-in-class anticancer quinolone derivative topoisomerase II inhibitor that is currently in development in combination with cytarabine for the treatment of acute myeloid leukemia (AML). To investigate vosaroxin pharmacokinetics (PK) in patients, liquid chromatography tandem mass spectrometry (LC-MS/MS) assays to quantify vosaroxin and the two metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine were developed and validated. Immediately after collection the samples were stored at -80°C. Prior to analysis, the plasma samples were subjected to protein precipitation and the urine samples were diluted. For both assays the reconstituted extracts were injected on a Symmetry Shield RP8 column and gradient elution was applied using 0.1% formic acid in water and acetonitrile-methanol (50:50, v/v). Analyses were performed with a triple quadruple mass spectrometer in positive-ion mode. A deuterated isotope of vosaroxin was used as internal standard for the quantification. The validated assays quantify vosaroxin and N-desmethylvosaroxin in the concentration range of 2-500ng/mL in plasma and urine. For O-desmethylvosaroxin the concentration range of 4-500ng/mL in plasma and urine was validated. Dilution integrity experiments show that samples can be diluted 25 fold in control matrix prior to analysis. The expanded concentration range for plasma and urine for vosaroxin and N-desmethylvosaroxin is therefore from 2 to 15,000ng/mL and in plasma for O-desmethylvosaroxin from 4 to 15,000ng/mL.

Are MUPs a Toxic Waste Disposal System?

Male house mice produce large quantities of major urinary proteins (MUPs), which function to bind and transport volatile pheromones, though they may also function as scavengers that bind and excrete toxic compounds ('toxic waste hypothesis'). In this study, we demonstrate the presence of an industrial chemical, 2,4-di-tert-butylphenol (DTBP), in the urine of wild-derived house mice (Mus musculus musculus). Addition of guanidine hydrochloride to male and female urine resulted in an increased release of DTBP. This increase was only observed in the high molecular weight fractions (HMWF; > 3 kDa) separated from male or female urine, suggesting that the increased release of DTBP was likely due to the denaturation of MUPs and the subsequent release of MUP-bound DTBP. Furthermore, when DTBP was added to a HMWF isolated from male urine, an increase in 2-sec-butyl-4,5-dihydrothiazole (SBT), the major ligand of MUPs and a male-specific pheromone, was observed, indicating that DTBP was bound to MUPs and displaced SBT. These results suggest that DTBP is a MUP ligand. Moreover, we found evidence for competitive ligand binding between DTBP and SBT, suggesting that males potentially face a tradeoff between eliminating toxic wastes versus transporting pheromones. Our findings support the hypothesis that MUPs bind and eliminate toxic wastes, which may provide the most important fitness benefits of excreting large quantities of these proteins.

Urinary Concentrations and Antibacterial Activity of BAL30072, a Novel Siderophore Monosulfactam, against Uropathogens after Intravenous Administration in Healthy Subjects.

This annex study to a phase 1 study aimed to correlate urinary concentrations and bactericidal titers (UBTs) of BAL30072, a novel siderophore monosulfactam, in healthy subjects in order to evaluate which dosage of BAL30072 should be investigated in a clinical study on complicated urinary tract infection (UTI). Three cohorts of a total of 19 healthy male subjects were included in the add-on study and received the following BAL30072 dosages. The 1st cohort received 1 g once a day (q.d.) intravenously (i.v.) (1 h) on day 1 and 1 g thrice daily (t.i.d.) on day 2, the 2nd cohort received 2 g q.d. i.v. (1 h) on day 1 and 2 g t.i.d. on day 2, and the 3rd cohort received 1 g q.d. i.v. (4-h infusion) on day 8. Urine was collected up to 24 h after drug administration. UBTs were determined for seven Escherichia coli isolates (three wild type [WT], CTX-M-15, TEM-3, TEM-5, NDM-1), two Klebsiella pneumoniae isolates (WT, KPC), one Proteus mirabilis isolate (WT), and two Pseudomonas aeruginosa isolates (WT, VIM-1 plus AmpC). Urine drug concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The median urinary excretions of BAL30072 ranged between 38% and 46% (3 cohorts). The median UBTs after i.v. administration of 1 or 2 g q.d. and after 1 or 2 g t.i.d. showed positive UBTs for 24 h after the lowest dosage (1 g q.d.) for 5 of 7 of the Enterobacteriaceae strains and after the higher dosage of 2 g administered i.v. t.i.d. for all strains tested. After i.v. infusion of 1 g over 4 h, positive UBTs were demonstrated for three E. coli strains for up to 12 h, for the K. pneumoniae (KPC) strain for up to 8 h, and for the P. aeruginosa (VIM-1 plus AmpC) strain for up to only 4 h. The minimal bactericidal concentrations (MBCs) of the E. coli (NDM-1) strain and the K. pneumoniae (WT) strain correlated well between broth and urine but did not correlate well for the two P. aeruginosa strains. BAL30072 exhibits positive UBTs for 24 h even after a dosage of 1 g administered i.v. q.d. for 5 of 7 Enterobacteriaceae strains and after 2 g administered i.v. t.i.d. for all strains except one P. aeruginosa strain (50% of the time). In general, the UBTs correlated well with the MICs of the Enterobacteriaceae but were lower for P. aeruginosa The clinical efficacy with a dosage regimen of BAL30072 of 2 g administered i.v. t.i.d. should be evaluated in the treatment of complicated UTI.

Biological Monitoring of Human Exposure to Neonicotinoids Using Urine Samples, and Neonicotinoid Excretion Kinetics.

BACKGROUND: Neonicotinoids, which are novel pesticides, have entered into usage around the world because they are selectively toxic to arthropods and relatively non-toxic to vertebrates. It has been suggested that several neonicotinoids cause neurodevelopmental toxicity in mammals. The aim was to establish the relationship between oral intake and urinary excretion of neonicotinoids by humans to facilitate biological monitoring, and to estimate dietary neonicotinoid intakes by Japanese adults. METHODOLOGY/PRINCIPAL FINDINGS: Deuterium-labeled neonicotinoid (acetamiprid, clothianidin, dinotefuran, and imidacloprid) microdoses were orally ingested by nine healthy adults, and 24 h pooled urine samples were collected for 4 consecutive days after dosing. The excretion kinetics were modeled using one- and two-compartment models, then validated in a non-deuterium-labeled neonicotinoid microdose study involving 12 healthy adults. Increased urinary concentrations of labeled neonicotinoids were observed after dosing. Clothianidin was recovered unchanged within 3 days, and most dinotefuran was recovered unchanged within 1 day. Around 10% of the imidacloprid dose was excreted unchanged. Most of the acetamiprid was metabolized to desmethyl-acetamiprid. Spot urine samples from 373 Japanese adults were analyzed for neonicotinoids, and daily intakes were estimated. The estimated average daily intake of these neonicotinoids was 0.53-3.66 µg/day. The highest intake of any of the neonicotinoids in the study population was 64.5 µg/day for dinotefuran, and this was <1% of the acceptable daily intake.