Multilaboratory Collaborative Study of a Nontarget Data Acquisition for Target Analysis (nDATA) Workflow Using Liquid Chromatography-High-Resolution Accurate Mass Spectrometry for Pesticide Screening in Fruits and Vegetables.

Nontarget data acquisition for target analysis (nDATA) workflows using liquid chromatography-high-resolution accurate mass (LC-HRAM) spectrometry, spectral screening software, and a compound database have generated interest because of their potential for screening of pesticides in foods. However, these procedures and particularly the instrument processing software need to be thoroughly evaluated before implementation in routine analysis. In this work, 25 laboratories participated in a collaborative study to evaluate an nDATA workflow on high moisture produce (apple, banana, broccoli, carrot, grape, lettuce, orange, potato, strawberry, and tomato). Samples were extracted in each laboratory by quick, easy, cheap, effective, rugged, and safe (QuEChERS), and data were acquired by ultrahigh-performance liquid chromatography (UHPLC) coupled to a high-resolution quadrupole Orbitrap (QOrbitrap) or quadrupole time-of-flight (QTOF) mass spectrometer operating in full-scan mass spectrometry (MS) data-independent tandem mass spectrometry (LC-FS MS/DIA MS/MS) acquisition mode. The nDATA workflow was evaluated using a restricted compound database with 51 pesticides and vendor processing software. Pesticide identifications were determined by retention time (tR, ±0.5 min relative to the reference retention times used in the compound database) and mass errors (δM) of the precursor (RTP, δM ≤ ±5 ppm) and product ions (RTPI, δM ≤ ±10 ppm). The elution profiles of all 51 pesticides were within ±0.5 min among 24 of the participating laboratories. Successful screening was determined by false positive and false negative rates of <5% in unfortified (pesticide-free) and fortified (10 and 100 µg/kg) produce matrices. Pesticide responses were dependent on the pesticide, matrix, and instrument. The false negative rates were 0.7 and 0.1% at 10 and 100 µg/kg, respectively, and the false positive rate was 1.1% from results of the participating LC-HRAM platforms. Further evaluation was achieved by providing produce samples spiked with pesticides at concentrations blinded to the laboratories. Twenty-two of the 25 laboratories were successful in identifying all fortified pesticides (0-7 pesticides ranging from 5 to 50 µg/kg) for each produce sample (99.7% detection rate). These studies provide convincing evidence that the nDATA comprehensive approach broadens the screening capabilities of pesticide analyses and provide a platform with the potential to be easily extended to a larger number of other chemical residues and contaminants in foods.

Outbreak of Severe Hypoglycemia After Ingestion of a Male Enhancement Supplement - Virginia, August-November 2019.

In August 2019, the Virginia Poison Center (VPC) and the Blue Ridge Poison Center (BRPC) were contacted concerning patients experiencing repeated episodes of marked hypoglycemia following ingestion of a male enhancement supplement tablet marketed as "V8" in convenience stores in central Virginia. Over the following 3 months, the Virginia Department of Agriculture and Consumer Services (VDACS) and the Virginia Department of Health (VDH) conducted an investigation and identified 17 patients meeting the case definition (severe hypoglycemia within 48 hours of consuming an over-the-counter male enhancement supplement in a man with no history of use of insulin or other medication used to control blood glucose). Analysis of the V8 tablets revealed that most contained glyburide, a sulfonylurea oral hypoglycemic used in the treatment of diabetes and associated with prolonged hypoglycemia following overdose (1). To stem this outbreak, V8 was removed from stores when found, and public service announcements were released. The public health implications of V8 use include the potential for substantial morbidity from hypoglycemic episodes and the potential for mortality if health care services are not accessed in a timely manner when hypoglycemia occurs. The presence of V8 in the market poses a serious threat to public health because of its potentially life-threatening adverse effects.

Collaborative Public Health Investigation of Clenbuterol-Adulterated Heroin Outbreak-Richmond, Virginia, March-April 2015.

CONTEXT: In March 2015, the Virginia Department of Health (VDH) was alerted by the Virginia Poison Center of a 6-patient cluster treated for severe clinical presentations after using heroin. Patients' symptoms were atypical for heroin use, and concern existed that patients were exposed to heroin that had been adulterated with or replaced by another substance. OBJECTIVE: To understand the extent and characterization of the outbreak and implement response measures to prevent further cases. The purpose of this report is to highlight the collaborative nature of a public health investigation among a diverse group of stakeholders. DESIGN: Active surveillance and retrospective case finding. SETTING: Richmond metro area community and hospitals. PARTICIPANTS: Regional poison centers, the Division of Consolidated Laboratory Services, the Department of Behavioral Health and Developmental Services, community partners, local law enforcement, and multiple VDH divisions. INTERVENTION: Outbreak investigation, communication to public health professionals, clinicians, and the community, and liaising with the local law enforcement. MAIN OUTCOME MEASURES: Outbreak control. RESULTS: Laboratory confirmation of clenbuterol in clinical specimens implicated it as the heroin adulterant. Thirteen patients met clinical and epidemiologic criteria for exposure to clenbuterol-adulterated heroin. All patients were associated with a localized area within Richmond, and patient interviews elucidated heroin supplier information. VDH collaborated with local law enforcement agents who investigated and arrested the supplier, leading to cessation of the outbreak. CONCLUSION: This outbreak highlights the value of policies and practices that support an integrated outbreak response among public health practitioners, poison center staff, laboratorians, clinicians, law enforcement agents, community groups, and other agencies. Collaboration enabled implementation of effective control measures-including those outside the purview of the health department-and should be standard practice in future outbreaks involving illicit substances.

A robust high-throughput sample preparation and liquid chromatography/tandem mass spectrometry method for the quantitation of ß-lyase metabolites of sulfur mustard as 1,1'-sulfonylbis-[2-(methylthio)ethane] in human urine.

RATIONALE: Sulfur mustard (HD) is a major chemical warfare agent threat to humans. Since World War I, several incidents of human exposure to sulfur mustard have been reported. In order to assist health professionals during an exposure event and support biological monitoring, a rapid analytical method is required to measure the exposure of humans to HD. METHOD: The ß-lyase metabolites of HD, 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) and 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) were reduced to the single biomarker, 1,1'-sulfonylbis-[2-(methylthio)ethane] (SBMTE), using titanium(III) chloride. High-throughput sample preparation was performed on a Tecan Freedom EVO liquid handler and analysis was performed by electrospray ionization liquid chromatography and tandem mass spectrometry (LC/MS/MS) in the multiple-reaction monitoring mode. RESULTS: Each analytical run consisted of a matrix blank, calibration standards (0.1-100 ng/mL), low quality controls (QCs), 2.5 ng/mL, and high QCs, 25.0 ng/mL, of SBMTE in human urine. The method was validated with 20 analytical runs performed by four analysts. The mean calculated concentrations of the low and high QCs were 2.52 and 25.5 ng/mL with relative standard deviations of 3.6% and 2.3%, respectively. CONCLUSION: This semi-automated method has few manual transfer steps, thus minimizing common manual errors and saving time. Therefore, this method would be very helpful to responding laboratories in a large-scale exposure event related to HD.

High-throughput sample preparation and simultaneous column regeneration liquid chromatography-tandem mass spectrometry method for determination of nitrogen mustard metabolites in human urine.

Nitrogen mustards (NMs) are known to have DNA alkylation and strong vesicant properties. Their availability to terrorist organizations makes them a potential choice for chemical attacks on civilian populations. After an exposure, it is difficult to measure NMs directly because of their rapid metabolism in the human body. Therefore to determine an individual's level of exposure to NMs, it is necessary to analyze for NM metabolites being excreted by the body. The metabolites of NMs are generated by a hydrolysis reaction, and are easily detectable by liquid chromatography tandem mass spectrometry (LC-MS/MS). This work is focused on the development of a high-throughput assay for the quantitation of N-ethyldiethanolamine (EDEA) and N-methyldiethanolamine (MDEA) metabolites of bis (2-chloroethyl) ethylethanamine (HN1) and bis (2-chloroethyl) methylethanamine (HN2), respectively. The method uses automated 96-well plate sample preparation of human urine samples and a 2-position 10-port switching valve to allow for simultaneous regeneration of the liquid chromatography (LC) columns. Using this method, over 18 h was saved through the reduction of sample preparation and analysis time when compared to a conventional method for 96 samples. The validated method provided excellent accuracy for both EDEA (100.9%) and MDEA (100.6%) with precision better than 5.27% for each analyte.

Liquid chromatography/mass spectrometry characterization of Escherichia coli and Shigella species.

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories.

Biological monitoring of uranium exposure in south central Virginia.

In May 2004, two groundwater wells in Dinwiddie County, Virginia were found to have natural uranium levels either at or above the EPA recommended limit of 30 microg/l. As a result, a stop drinking water advisory was issued until a water treatment system could be installed to remove the uranium. In response to residents' concerns, and uncertainty of exposures, affected individuals were asked to participate in a voluntary epidemiological investigation of uranium uptake and 1-year uranium retention study. This study had two primary objectives: quantification of the uranium load on the participants, as expressed by their urine uranium concentration, and retention after 1 year of no exposure. A first-morning void urine specimen, along with survey information, was collected from 156 participants in May 2004, with a second collection occurring 12 months later of 91 participants. The samples were analyzed for uranium by ICP/MS, pH, creatinine by the Jaffe method, and RBP by LIA after both collections. A reduction of one order of magnitude for the geometric mean urine uranium concentration was observed, from 0.100 microg/g creatinine to 0.011 microg/g creatinine in 1 year. Comparatively, NHANES has reported that the geometric mean for all participants, ages 6 years and older, is 0.008 microg/g creatinine, with the 95th percentile being 0.040 microg/g creatinine. None of the second round specimens showed a urine uranium concentration higher than baseline for an individual.