Development of a Universal Second-Tier Newborn Screening LC-MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids.

First-tier MS-based newborn screening by flow injection analysis can have high presumptive positive rates, often due to isomeric/isobaric compounds or poor biomarker specificity. These presumptive positive samples can be analyzed by second-tier screening assays employing separations such as liquid chromatography-mass spectrometry (LC-MS/MS), which increases test specificity and drastically reduces false positive referrals. The ability to screen for multiple disorders in a single multiplexed test simplifies workflows and maximizes public health laboratories' resources. In this study, we developed and validated a highly multiplexed second-tier method for dried blood spots using a hydrophilic interaction liquid chromatography (HILIC) column coupled to an MS/MS system. The LC-MS/MS method was capable of simultaneously detecting second-tier biomarkers for maple syrup urine disease, homocystinuria, methylmalonic acidemia, propionic acidemia, glutaric acidemia type 1, glutaric acidemia type 2, guanidinoacetate methyltransferase deficiency, short-chain acyl-CoA dehydrogenase deficiency, adrenoleukodystrophy, and Pompe disease.

Multiplexing Homocysteine into First-Tier Newborn Screening Mass Spectrometry Assays Using Selective Thiol Derivatization.

BACKGROUND: Classical homocystinuria (HCU) results from deficient cystathionine ß-synthase activity, causing elevated levels of Met and homocysteine (Hcy). Newborn screening (NBS) aims to identify HCU in pre-symptomatic newborns by assessing Met concentrations in first-tier screening. However, unlike Hcy, Met testing leads to a high number of false-positive and -negative results. Therefore, screening for Hcy directly in first-tier screening would be a better biomarker for use in NBS. METHODS: Dried blood spot (DBS) quality control and residual clinical specimens were used in analyses. Several reducing and maleimide reagents were investigated to aid in quantification of total Hcy (tHcy). The assay which was developed and validated was performed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS). RESULTS: Interferents of tHcy measurement were identified, so selective derivatization of Hcy was employed. Using N-ethylmaleimide (NEM) to selectively derivatize Hcy allowed interferent-free quantification of tHcy by FIA-MS/MS in first-tier NBS. The combination of tris(2-carboxyethyl)phosphine (TCEP) and NEM yielded significantly less matrix effects compared to dithiothreitol (DTT) and NEM. Analysis of clinical specimens demonstrated that the method could distinguish between HCU-positive, presumptive normal newborns, and newborns receiving total parenteral nutrition. CONCLUSIONS: Here we present the first known validated method capable of screening tHcy in DBS during FIA-MS/S first-tier NBS.

Combining First and Second-Tier Newborn Screening in a Single Assay Using High-Throughput Chip-Based Capillary Electrophoresis Coupled to High-Resolution Mass Spectrometry.

BACKGROUND: Most first-tier newborn screening (NBS) biomarkers are evaluated by a 2-min flow injection analysis coupled to tandem mass spectrometry (FIA-MS/MS) assay. The absence of separation prior to MS/MS analysis can lead to false positives and inconclusive results due to interferences by nominal isobars and isomers. Therefore, many presumptive positive specimens require confirmation by a higher specificity second-tier assay employing separations, which require additional time and resources prior to patient follow-up. METHODS: A 3.2-mm punch was taken from dried blood spot (DBS) specimens and extracted using a solution containing isotopically labeled internal standards for quantification. Analyses were carried out in positive mode using a commercially available microfluidic capillary electrophoresis (CE) system coupled to a high-resolution mass spectrometer (HRMS). RESULTS: The CE-HRMS platform quantified 35 first- and second-tier biomarkers from a single injection in <2-min acquisition time, thus, successfully multiplexing first- and second-tier NBS for over 20 disorders in a single DBS punch. The CE-HRMS platform resolved problematic isobars and isomers that affect first-tier FIA-MS/MS assay specificity, while achieving similar quantitative results and assay linearity. CONCLUSIONS: Our CE-HRMS assay is capable of multiplexing first- and second-tier NBS biomarkers into a single assay with an acquisition time of <2 min. Such an assay would reduce the volume of false positives and inconclusive specimens flagged for second-tier screening.

High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase.

In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents.

A high-throughput UHPLC-MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents.

Organophosphorus nerve agents (OPNAs) are toxic compounds that are classified as prohibited Schedule 1 chemical weapons. In the body, OPNAs bind to butyrylcholinesterase (BChE) to form nerve agent adducts (OPNA-BChE). OPNA-BChE adducts can provide a reliable, long-term protein biomarker for assessing human exposure. A major challenge facing OPNA-BChE detection is hydrolysis (aging), which can continue to occur after a clinical specimen has been collected. During aging, the o-alkyl phosphoester bond hydrolyzes, and the specific identity of the nerve agent is lost. To better identify OPNA exposure events, a high-throughput method for the detection of five aged OPNA-BChE adducts was developed. This is the first diagnostic panel to allow for the simultaneous quantification of any Chemical Weapons Convention Schedule 1 OPNA by measuring the aged adducts methyl phosphonate, ethyl phosphonate, propyl phosphonate, ethyl phosphoryl, phosphoryl and unadducted BChE. The calibration range for all analytes is 2.00-250. ng/mL, which is consistent with similar methodologies used to detect unaged OPNA-BChE adducts. Each analytical run is 3 min, making the time to first unknown results, including calibration curve and quality controls, less than 1 h. Analysis of commercially purchased individual serum samples demonstrated no potential interferences with detection of aged OPNA-BChE adducts, and quantitative measurements of endogenous levels of BChE were similar to those previously reported in other OPNA-BChE adduct assays.

Supplemental Learning in the Laboratory: An Innovative Approach for Evaluating Knowledge and Method Transfer.

The Multi-Rule Quality Control System (MRQCS) is a tool currently employed by the Centers for Disease Control and Prevention (CDC) to evaluate and compare laboratory performance. We have applied the MRQCS to a comparison of instructor and computer-led pre-laboratory lectures for a supplemental learning experiment. Students in general chemistry and analytical chemistry from both two- and four-year institutions performed two laboratory experiments as part of their normal laboratory curriculum. The first laboratory experiment was a foundational learning experiment in which all the students were introduced to Beer-Lambert's Law and spectrophotometric light absorbance measurements. The foundational learning experiment was instructor-led only, and participant performance was evaluated against a mean characterized value. The second laboratory experiment was a supplemental learning experiment in which students were asked to build upon the methodology they learned in the foundational learning experiment and apply it to a different analyte. The instruction type was varied randomly into two delivery modes, participants receiving either instructor-led or computer-led pre-laboratory instruction. The MRQCS was applied and determined that no statistical difference was found to exist in the QC (quality control) passing rates between the participants in the instructor-led instruction and the participants in the computer-led instruction. These findings demonstrate the successful application of the MRQCS to evaluate knowledge and technology transfer.

Quantification of metabolites for assessing human exposure to soapberry toxins hypoglycin A and methylenecyclopropylglycine.

Ingestion of soapberry fruit toxins hypoglycin A and methylenecyclopropylglycine has been linked to public health challenges worldwide. In 1976, over 100 years after Jamaican vomiting sickness (JVS) was first reported, the cause of JVS was linked to the ingestion of the toxin hypoglycin A produced by ackee fruit. A structural analogue of hypoglycin A, methylenecyclopropylglycine (MCPG), was implicated as the cause of an acute encephalitis syndrome (AES). Much of the evidence linking hypoglycin A and MCPG to these diseases has been largely circumstantial due to the lack of an analytical method for specific metabolites. This study presents an analytical approach to identify and quantify specific urine metabolites for exposure to hypoglycin A and MCPG. The metabolites are excreted in urine as glycine adducts methylenecyclopropylacetyl-glycine (MCPA-Gly) and methylenecyclopropylformyl-glycine (MCPF-Gly). These metabolites were processed by isotope dilution, separated by reverse-phase liquid chromatography, and monitored by electrospray ionization tandem mass spectrometry. The analytical response ratio was linearly proportional to the concentration of MCPF-Gly and MCPA-Gly in urine from 0.10 to 20 µg/mL with a correlation coefficient of r > 0.99. The assay demonstrated accuracy ≥80% and precision ≤20% RSD across the calibration range. This method has been applied to assess exposure to hypoglycin A and MCPG as part of a larger public health initiative and was used to provide the first reported identification of MCPF-Gly and MCPA-Gly in human urine.

Resolving powers of >7900 using linked scans: how well does resolving power describe the separation capability of differential ion mobility spectrometry.

Differential ion mobility spectrometry (DIMS) separations are described using similar terminology to liquid chromatography, capillary electrophoresis, and drift tube ion mobility spectrometry. The characterization and comparison of all these separations are typically explained in terms of resolving power, resolution, and/or peak capacity. A major difference between these separations is that DIMS separations are in space whereas the others are separations in time. However, whereas separations in time can, in theory, be extended infinitely, separations in space, such as DIMS separations, are constrained by the physical dimensions of the device. One method to increase resolving power of DIMS separations is to use helium in the DIMS carrier gas. However, ions have a greater mobility in helium which causes more ions to be neutralized due to collisions with the DIMS electrodes or electrode housing, i.e. the space constraints. This neutralization of ions can lead to the loss of an entire peak, or peaks, from a DIMS scan. To take advantage of the benefits of helium use while reducing ion losses, linked scans were developed. During a linked scan the amount of helium present in the DIMS carrier gas is decreased as the compensation field is increased. A comparison of linked scans to compensation field scans with constant helium is presented herein. Resolving powers >7900 are obtained with linked scans. However, this result highlights the limitation of using resolving power as a metric to describe DIMS separations.

Probing Mobility-Selected Saccharide Isomers: Selective Ion-Molecule Reactions and Wavelength-Specific IR Activation.

Differential Ion Mobility Spectrometry (DIMS) provides orthogonal separation to mass spectrometry, and DIMS combined with the high sensitivity of a quadrupole ion-trap is shown to be useful for the separation and identification of saccharides. A comprehensive analysis of the separation of anomers (α- and ß-methylated glucose) and epimers (α-methylated glucose and mannose) ionized with Li(+), Na(+), and K(+) is performed. DIMS separation is found to be better for saccharides cationized with the two latter species. The corresponding resolving power for the two glucose anomers with Na(+) is found to be very close to the corresponding drift-tube IMS value. The lithiated complexes are investigated further using a combination of infrared spectroscopy integrated to ion-trap mass spectrometry and quantum chemical calculations. Together with DIMS, consistent results are obtained. It is found that two competing structural motifs might be at play, depending on the subtle balance between the maximization of the coordination of the metal cation and the intrinsic conformational energetics of the saccharide, which is for a large part driven by hydrogen bonding. The comparison of simulated and observed spectra clearly shows that a band at ∼3400 cm(-1) is specific to a structural motif found in the lithiated glucose complexes, which could explain the trends observed in the DIMS spectra of the saccharide complexes. It is shown that DIMS-MS/MS using wavelength specific IR activation would provide a new orthogonal dimension to mass spectrometry.

Differential ion mobility spectrometry coupled to tandem mass spectrometry enables targeted leukemia antigen detection.

Differential ion mobility spectrometry (DIMS) can be used as a filter to remove undesired background ions from reaching the mass spectrometer. The ability to use DIMS as a filter for known analytes makes DIMS coupled to tandem mass spectrometry (DIMS-MS/MS) a promising technique for the detection of cancer antigens that can be predicted by computational algorithms. In experiments using DIMS-MS/MS that were performed without the use of high-performance liquid chromatography (HPLC), a predicted model antigen, GLR (FLSSANEHL), was detected at a concentration of 10 pM (20 amol) in a mixture containing 94 competing model peptide antigens, each at a concentration of 1 µM. Without DIMS filtering, the GLR peptide was undetectable in the mixture even at 100 nM. Again, without using HPLC, DIMS-MS/MS was used to detect 2 of 3 previously characterized antigens produced by the leukemia cell line U937.A2. Because of its sensitivity, a targeted DIMS-MS/MS methodology can likely be used to probe for predicted cancer antigens from cancer cell lines as well as human tumor samples.

Hydrodynamic versus size exclusion chromatography characterization of alternan and comparison to off-line MALS.

Alternan is an ultrahigh molar mass polysaccharide composed of alternating α-(1→3) and α-(1→6) repeat units and that also possesses long-chain branching. Its molar mass distribution (MMD) can extend into the hundreds of millions of grams per mole. Characterizing alternan by means of size exclusion chromatography (SEC) is a lengthy process and an incomplete one because even under the best possible experimental conditions, the polysaccharide appears to degrade during its passage through the SEC columns. As an alternative to SEC, we have investigated the use of hydrodynamic chromatography (HDC) as a possible characterization technique for alternan. Results from packed-column HDC with multiangle static light scattering (MALS) detection compare favorably to results from off-line MALS analysis, and HDC results are obtained in a fraction of the time needed for SEC. The largest molar mass alternan did appear to undergo some degradation during HDC as a result of interstitial stresses in the column bed. This could be remedied by the use of columns with larger packing particles. Evidence of long-chain branching in alternan is provided via a comparison of the intrinsic viscosities and viscometric radii of the polysaccharide to those of a pullulan standard and of a theoretical pullulan of molar mass equal to those of the alternans examined.

The Effects of Gamma Irradiation on Chemical Biomarker Recovery from Mixed Chemical/Biological Threat Exposure Specimens.

BACKGROUND: Irradiative sterilization of clinical specimens prior to chemical laboratory testing provides a way to not only sterilize pathogens and ensure laboratorian safety but also preserve sample volume and maintain compatibility with quantitative chemical diagnostic protocols. Since the compatibility of clinical biomarkers with gamma irradiation is not well characterized, a subset of diagnostic biomarkers ranging in molecular size, concentration, and clinical matrix was analyzed to determine recovery following gamma irradiation. METHODS: Sample irradiation of previously characterized quality control materials (QCs) at 5 Mrad was carried out at the Gamma Cell Irradiation Facility at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. Following irradiation, the QCs were analyzed alongside non-irradiated QCs to determine analyte recovery between dosed and control samples. RESULTS: Biomarkers for exposure to abrin, ricin, and organophosphorus nerve agents (OPNAs) were analyzed for their stability following gamma irradiation. The diagnostic biomarkers included adducts to butyrylcholinesterase, abrine, and ricinine, respectively, and were recovered at over 90% of their initial concentration. CONCLUSIONS: The results from this pilot study support the implementation of an irradiative sterilization protocol for possible mixed-exposure samples containing both chemical and biological threat agents (mixed CBTs). Furthermore, irradiative sterilization significantly reduces a laboratorian's risk of infection from exposure to an infectious agent without compromising chemical diagnostic testing integrity, particularly for diagnostic assays in which the chemical analyte has been shown to be fully conserved following a 5 Mrad irradiative dose.

An SEC/MALS study of alternan degradation during size-exclusion chromatographic analysis.

Ultrahigh-molar-mass (M) polymers such as DNA, cellulose, and polyolefins are routinely analyzed using size-exclusion chromatography (SEC) to obtain molar mass averages, distributions, and architectural information. It has long been contended that high-M polymers can degrade during SEC analysis; if true, the inaccurate molar mass information obtained can adversely affect decisions regarding processing and end-use properties of the macromolecules. However, most evidence to the effect of degradation has been circumstantial and open to alternative interpretation. For example, the shift in SEC elution volume as a function of increased chromatographic flow rate, observed using only a concentration-sensitive detector, may be the result of degradation or of elution via a nondegradatory slalom chromatography mechanism. Here, using both concentration-sensitive and multiangle static light-scattering detection, we provide unambiguous evidence that the polysaccharide alternan actually degrades during SEC analysis. The decrease in molar mass and size of alternan with increasing flow rate, measured using light scattering, allows ruling out an SC mode of elution and can only be interpreted as due to degradation. These findings demonstrate the extreme fragility of ultrahigh-M polymers and the care that must be taken for accurate characterization.

Designing traceable opioid material§ kits to improve laboratory testing during the U.S. opioid overdose crisis.

In 2017, the U.S. Department of Health and Human Services and the White House declared a public health emergency to address the opioid crisis (Hargan, 2017). On average, 192 Americans died from drug overdoses each day in 2017; 130 (67%) of those died specifically because of opioids (Scholl et al., 2019). Since 2013, there have been significant increases in overdose deaths involving synthetic opioids - particularly those involving illicitly-manufactured fentanyl. The U.S. Drug Enforcement Administration (DEA) estimates that 75% of all opioid identifications are illicit fentanyls (DEA, 2018b). Laboratories are routinely asked to confirm which fentanyl or other opioids are involved in an overdose or encountered by first responders. It is critical to identify and classify the types of drugs involved in an overdose, how often they are involved, and how that involvement may change over time. Health care providers, public health professionals, and law enforcement officers need to know which opioids are in use to treat, monitor, and investigate fatal and non-fatal overdoses. By knowing which drugs are present, appropriate prevention and response activities can be implemented. Laboratory testing is available for clinically used and widely recognized opioids. However, there has been a rapid expansion in new illicit opioids, particularly fentanyl analogs that may not be addressed by current laboratory capabilities. In order to test for these new opioids, laboratories require reference standards for the large number of possible fentanyls. To address this need, the Centers for Disease Control and Prevention (CDC) developed the Traceable Opioid Material§ Kits product line, which provides over 150 opioid reference standards, including over 100 fentanyl analogs. These kits were designed to dramatically increase laboratory capability to confirm which opioids are on the streets and causing deaths. The kits are free to U.S based laboratories in the public, private, clinical, law enforcement, research, and public health domains.

Rapid quantification of two chemical nerve agent metabolites in serum.

Organophosphorus compounds (OPs) continue to represent a significant chemical threat to humans due to exposures from their use as weapons, their potential storage hazards, and from their continued use agriculturally. Existing methods for detection include ELISA and mass spectrometry. The new approach presented here provides an innovative first step toward a portable OP quantification method that surmounts conventional limitations involving sensitivity, selectivity, complexity, and portability. DNA affinity probes, or aptamers, represent an emerging technology that, when combined with a mix-and-read, free-solution assay (FSA) and a compensated interferometer (CI) can provide a novel alternative to existing OP nerve agent (OPNA) quantification methods. Here it is shown that FSA can be used to rapidly screen prospective aptamers in the biological matrix of interest, allowing the identification of a 'best-in-class' probe. It is also shown that combining aptamers with FSA-CI enables quantification of the OPNA metabolites, Sarin (NATO designation "G-series, B", or GB) and Venomous Agent X (VX) acids, rapidly with high selectivity at detection limits of sub-10 pg/mL in 25% serum (by volume in PBS). These results suggest there is potential to directly impact diagnostic specificity and sensitivity of emergency response testing methods by both simplifying sample preparation procedures and making a benchtop reader available for OPNA metabolite quantification.

Quantification of hypoglycin A and methylenecyclopropylglycine in human plasma by HPLC-MS/MS.

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPG) are naturally-occurring amino acids known to cause hypoglycemia and encephalopathy. Exposure to one or both toxins through the ingestion of common soapberry (Sapindaceae) fruits are documented in illness outbreaks throughout the world. Jamaican Vomiting Sickness (JVS) and seasonal pasture myopathy (SPM, horses) are linked to HGA exposure from unripe ackee fruit and box elder seeds, respectively. Acute toxic encephalopathy is linked to HGA and MCPG exposures from litchi fruit. HGA and MCPG are found in several fruits within the soapberry family and are known to cause severe hypoglycemia, seizures, and death. HGA has been directly quantified in horse blood in SPM cases and in human gastric juice in JVS cases. This work presents a new diagnostic assay capable of simultaneous quantification of HGA and MCPG in human plasma, and it can be used to detect patients with toxicity from soapberry fruits. The assay presented herein is the first quantitative method for MCPG in blood matrices.

Quantification of Ricinine and Abrine in Human Plasma by HPLC-MS-MS: Biomarkers of Exposure to Ricin and Abrin.

Ricin and abrin are toxic ribosome-inactivating proteins found in plants. Exposure to these toxins can be detected using the biomarkers ricinine and abrine, which are present in the same plant sources as the toxins. The concentration of the biomarkers in urine and blood will be dependent upon the purification of abrin or ricin, the route of exposure, and the length of time between exposure and sample collection. Here, we present the first diagnostic assay for the simultaneous quantification of both ricinine and abrine in blood matrices. Furthermore, this is the first-ever method for the detection of abrine in blood products. Samples were processed by isotope-dilution, solid-phase extraction, protein precipitation and quantification by HPLC-MS-MS. This analytical method detects abrine from 5.00 to 500 ng/mL and ricinine from 0.300 to 300 ng/mL with coefficients of determination of 0.996 ± 0.003 and 0.998 ± 0.002 (n = 22), respectively. Quality control material accuracy was determined to have <10% relative error, and precision was within 19% relative standard deviation. The assay's time-to-first result is three hours including sample preparation. Furthermore, the method was applied for the quantification of ricinine in the blood of a patient who had intentionally ingested castor beans to demonstrate the test was fit-for-purpose. This assay was designed to support the diagnosis of ricin and abrin exposures in public health investigations.

Quantitative HPLC-MS/MS analysis of toxins in soapberry seeds: Methylenecyclopropylglycine and hypoglycin A.

Methylenecyclcopropylglycine (MCPG) and hypoglycin A (HGA) are naturally occurring amino acids found in various soapberry (Sapindaceae) fruits. These toxins have been linked to illnesses worldwide and were recently implicated in Asian outbreaks of acute hypoglycemic encephalopathy. In a previous joint agricultural and public health investigation, we developed an analytical method capable of evaluating MCPG and HGA concentrations in soapberry fruit arils as well as a clinical method for the urinary metabolites of the toxins. Since the initial soapberry method only analyzed the aril portion of the fruit, we present here the extension of the method to include the fruit seed matrix. This work is the first method to quantitate both MCPG and HGA concentrations in the seeds of soapberry fruit, including those collected during a public health investigation. Further, this is the first quantitation of HGA in litchi seeds as well as both toxins in mamoncillo and longan seeds.

Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study.

BACKGROUND: Outbreaks of unexplained illness frequently remain under-investigated. In India, outbreaks of an acute neurological illness with high mortality among children occur annually in Muzaffarpur, the country's largest litchi cultivation region. In 2014, we aimed to investigate the cause and risk factors for this illness. METHODS: In this hospital-based surveillance and nested age-matched case-control study, we did laboratory investigations to assess potential infectious and non-infectious causes of this acute neurological illness. Cases were children aged 15 years or younger who were admitted to two hospitals in Muzaffarpur with new-onset seizures or altered sensorium. Age-matched controls were residents of Muzaffarpur who were admitted to the same two hospitals for a non-neurologic illness within seven days of the date of admission of the case. Clinical specimens (blood, cerebrospinal fluid, and urine) and environmental specimens (litchis) were tested for evidence of infectious pathogens, pesticides, toxic metals, and other non-infectious causes, including presence of hypoglycin A or methylenecyclopropylglycine (MCPG), naturally-occurring fruit-based toxins that cause hypoglycaemia and metabolic derangement. Matched and unmatched (controlling for age) bivariate analyses were done and risk factors for illness were expressed as matched odds ratios and odds ratios (unmatched analyses). FINDINGS: Between May 26, and July 17, 2014, 390 patients meeting the case definition were admitted to the two referral hospitals in Muzaffarpur, of whom 122 (31%) died. On admission, 204 (62%) of 327 had blood glucose concentration of 70 mg/dL or less. 104 cases were compared with 104 age-matched hospital controls. Litchi consumption (matched odds ratio [mOR] 9·6 [95% CI 3·6 - 24]) and absence of an evening meal (2·2 [1·2-4·3]) in the 24 h preceding illness onset were associated with illness. The absence of an evening meal significantly modified the effect of eating litchis on illness (odds ratio [OR] 7·8 [95% CI 3·3-18·8], without evening meal; OR 3·6 [1·1-11·1] with an evening meal). Tests for infectious agents and pesticides were negative. Metabolites of hypoglycin A, MCPG, or both were detected in 48 [66%] of 73 urine specimens from case-patients and none from 15 controls; 72 (90%) of 80 case-patient specimens had abnormal plasma acylcarnitine profiles, consistent with severe disruption of fatty acid metabolism. In 36 litchi arils tested from Muzaffarpur, hypoglycin A concentrations ranged from 12·4 µg/g to 152·0 µg/g and MCPG ranged from 44·9 µg/g to 220·0 µg/g. INTERPRETATION: Our investigation suggests an outbreak of acute encephalopathy in Muzaffarpur associated with both hypoglycin A and MCPG toxicity. To prevent illness and reduce mortality in the region, we recommended minimising litchi consumption, ensuring receipt of an evening meal and implementing rapid glucose correction for suspected illness. A comprehensive investigative approach in Muzaffarpur led to timely public health recommendations, underscoring the importance of using systematic methods in other unexplained illness outbreaks. FUNDING: US Centers for Disease Control and Prevention.

Quantification of Hydrazine in Human Urine by HPLC-MS-MS.

Currently used on F-16 fighter jets and some space shuttles, hydrazine could be released at toxic levels to humans as a result of an accidental leakage or spill. Lower-level exposures occur in industrial workers or as a result of the use of some pharmaceuticals. A method was developed for the quantitation of hydrazine in human urine and can be extended by dilution with water to cover at least six orders of magnitude, allowing measurement at all clinically significant levels of potential exposure. Urine samples were processed by isotope dilution, filtered, derivatized and then quantified by HPLC-MS-MS. The analytical response ratio was linearly proportional to the urine concentration of hydrazine from 0.0493 to 12.3 ng/mL, with an average correlation coefficientRof 0.9985. Inter-run accuracy for 21 runs, expressed as percent relative error (% RE), was ≤14%, and the corresponding precision, expressed as percent relative standard deviation (% RSD), was ≤15%. Because this method can provide a quantitative measurement of clinical samples over six orders of magnitude, it can be used to monitor trace amounts of hydrazine exposure as well as industrial and environmental exposure levels.