Use of Diagnostic Ions for the Detection of Fentanyl Analogs in Human Matrices by LC-QTOF.

To combat the ongoing opioid epidemic, our laboratory has developed and evaluated an approach to detect fentanyl analogs in urine and plasma by screening LC-QTOF MS/MS spectra for ions that are diagnostic of the core fentanyl structure. MS/MS data from a training set of 142 fentanyl analogs were used to select the four product ions and six neutral losses that together provided the most complete coverage (97.2%) of the training set compounds. Furthermore, using the diagnostic ion screen against a set of 49 fentanyl analogs not in the training set resulted in 95.9% coverage of those compounds. With this approach, lower reportable limits for fentanyl and a subset of fentanyl-related compounds range from 0.25 to 2.5 ng/mL in urine and 0.5 to 5.0 ng/mL in plasma. This innovative processing method was applied to evaluate simulated exposure samples of remifentanil and carfentanil in water and their metabolites remifentanil acid and norcarfentanil in urine. This flexible approach enables a way to detect emerging fentanyl analogs in clinical samples.

Application of the fentanyl analog screening kit toward the identification of emerging synthetic opioids in human plasma and urine by LC-QTOF.

Human exposures to fentanyl analogs, which significantly contribute to the ongoing U.S. opioid overdose epidemic, can be confirmed through the analysis of clinical samples. Our laboratory has developed and evaluated a qualitative approach coupling liquid chromatography and quadrupole time-of-flight mass spectrometry (LC-QTOF) to address novel fentanyl analogs and related compounds using untargeted, data-dependent acquisition. Compound identification was accomplished by searching against a locally-established mass spectral library of 174 fentanyl analogs and metabolites. Currently, our library can identify 150 fentanyl-related compounds from the Fentanyl Analog Screening (FAS) Kit), plus an additional 25 fentanyl-related compounds from individual purchases. Plasma and urine samples fortified with fentanyl-related compounds were assessed to confirm the capabilities and intended use of this LC-QTOF method. For fentanyl, 8 fentanyl-related compounds and naloxone, lower reportable limits (LRL100), defined as the lowest concentration with 100 % true positive rate (n = 12) within clinical samples, were evaluated and range from 0.5 ng/mL to 5.0 ng/mL for urine and 0.25 ng/mL to 2.5 ng/mL in plasma. The application of this high resolution mass spectrometry (HRMS) method enables the real-time detection of known and emerging synthetic opioids present in clinical samples.

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.

Determination of Fentanyl Analog Exposure Using Dried Blood Spots with LC-MS-MS.

Fentanyl, and the numerous drugs derived from it, are contributing to the opioid overdose epidemic currently underway in the USA. To identify human exposure to these growing public health threats, an LC-MS-MS method for 5 µL dried blood spots (DBS) was developed. This method was developed to detect exposure to 3-methylfentanyl, alfentanil, α-methylfentanyl, carfentanil, fentanyl, lofentanil, sufentanil, norcarfentanil, norfentanyl, norlofentanil, norsufentanil, and using a separate LC-MS-MS injection, cyclopropylfentanyl, acrylfentanyl, 2-furanylfentanyl, isobutyrylfentanyl, ocfentanil and methoxyacetylfentanyl. Preparation of materials into groups of compounds was used to accommodate an ever increasing need to incorporate newly identified fentanyls. This protocol was validated within a linear range of 1.00-100 ng/mL, with precision ≤12% CV and accuracy ≥93%, as reported for the pooled blood QC samples, and limits of detection as low as 0.10 ng/mL. The use of DBS to assess fentanyl analog exposures can facilitate rapid sample collection, transport, and preparation for analysis that could enhance surveillance and response efforts in the ongoing opioid overdose epidemic.

Investigation of dried blood sampling with liquid chromatography tandem mass spectrometry to confirm human exposure to nerve agents.

A method was developed to detect and quantify organophosphate nerve agent (OPNA) metabolites in dried blood samples. Dried blood spots (DBS) and microsampling devices are alternatives to traditional blood draws, allowing for safe handling, extended stability, reduced shipping costs, and potential self-sampling. DBS and microsamplers were evaluated for precision, accuracy, sensitivity, matrix effects, and extraction recovery following collection of whole blood containing five OPNA metabolites. The metabolites of VX, Sarin (GB), Soman (GD), Cyclosarin (GF), and Russian VX (VR) were quantitated from 5.0 to 500 ng mL-1 with precision of ≤16% and accuracy between 93 and 108% for QC samples with controlled volumes. For unknown spot volumes, OPNA metabolite concentrations were normalized to total blood protein to improve interpretation of nerve agent exposures. This study provides data to support the use of DBS and microsamplers to collect critical exposure samples quickly, safely, and efficiently following large-scale chemical exposure events.

Quantitation of Fentanyl Analogs in Dried Blood Spots by Flow-Through Desorption Coupled to Online Solid Phase Extraction Tandem Mass Spectrometry.

An automated dried blood spot (DBS) elution coupled with solid phase extraction and tandem mass spectrometric analysis for multiple fentanyl analogs was developed and assessed. This method confirms human exposures to fentanyl, sufentanil, carfentanil, alfentanil, lofentanil, α-methyl fentanyl, and 3-methyl fentanyl in blood with minimal sample volume and reduced shipping and storage costs. Seven fentanyl analogs were detected and quantitated from DBS made from venous blood. The calibration curve in matrix was linear in the concentration range of 1.0 ng/mL to 100 ng/mL with a correlation coefficient greater than 0.98 for all compounds. The limit of detection varied from 0.15 ng/mL to 0.66 ng/mL depending on target analyte. Analysis of the entire DBS minimized the effects of hematocrit on quantitation. All quality control materials evaluated resulted in <15% error; analytes with isotopically labeled internal standards had <15% RSD, while analytes without matching standards had 15-24% RSD. This method provides an automated means to detect seven fentanyl analogs, and quantitate four fentanyl analogs with the benefits of DBS at levels anticipated from an overdose of these potent opioids.

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.

Bridging the Gap between Sample Collection and Laboratory Analysis: Using Dried Blood Spots to Identify Human Exposure to Chemical Agents.

Public health response to large scale chemical emergencies presents logistical challenges for sample collection, transport, and analysis. Diagnostic methods used to identify and determine exposure to chemical warfare agents, toxins, and poisons traditionally involve blood collection by phlebotomists, cold transport of biomedical samples, and costly sample preparation techniques. Use of dried blood spots, which consist of dried blood on an FDA-approved substrate, can increase analyte stability, decrease infection hazard for those handling samples, greatly reduce the cost of shipping/storing samples by removing the need for refrigeration and cold chain transportation, and be self-prepared by potentially exposed individuals using a simple finger prick and blood spot compatible paper. Our laboratory has developed clinical assays to detect human exposures to nerve agents through the analysis of specific protein adducts and metabolites, for which a simple extraction from a dried blood spot is sufficient for removing matrix interferents and attaining sensitivities on par with traditional sampling methods. The use of dried blood spots can bridge the gap between the laboratory and the field allowing for large scale sample collection with minimal impact on hospital resources while maintaining sensitivity, specificity, traceability, and quality requirements for both clinical and forensic applications.

Evaluation of Multiple Blood Matrices for Assessment of Human Exposure to Nerve Agents.

Biomedical samples may be used to determine human exposure to nerve agents through the analysis of specific biomarkers. Samples received may include serum, plasma, whole blood, lysed blood and, due to the toxicity of these compounds, postmortem blood. To quantitate metabolites resulting from exposure to sarin (GB), soman (GD), cyclosarin (GF), VX and VR, these blood matrices were evaluated individually for precision, accuracy, sensitivity and specificity. Accuracies for these metabolites ranged from 100 to 113% with coefficients of variation ranging from 2.31 to 13.5% across a reportable range of 1-100 ng/mL meeting FDA recommended guidelines for bioanalytical methods in all five matrices. Limits of detection were calculated to be 0.09-0.043 ng/mL, and no interferences were detected in unexposed matrix samples. The use of serum calibrators was also determined to be a suitable alternative to matrix-matched calibrators. Finally, to provide a comparative value between whole blood and plasma, the ratio of the five nerve agent metabolites measured in whole blood versus plasma was determined. Analysis of individual whole blood samples (n = 40), fortified with nerve agent metabolites across the reportable range, resulted in average nerve agent metabolite blood to plasma ratios ranging from 0.53 to 0.56. This study demonstrates the accurate and precise quantitation of nerve agent metabolites in serum, plasma, whole blood, lysed blood and postmortem blood. It also provides a comparative value between whole blood and plasma samples, which can assist epidemiologists and physicians with interpretation of test results from blood specimens obtained under variable conditions.

Comparison of two automated solid phase extractions for the detection of ten fentanyl analogs and metabolites in human urine using liquid chromatography tandem mass spectrometry.

Two types of automated solid phase extraction (SPE) were assessed for the determination of human exposure to fentanyls in urine. High sensitivity is required to detect these compounds following exposure because of the low dose required for therapeutic effect and the rapid clearance from the body for these compounds. To achieve this sensitivity, two acceptable methods for the detection of human exposure to seven fentanyl analogs and three metabolites were developed using either off-line 96-well plate SPE or on-line SPE. Each system offers different advantages: off-line 96-well plate SPE allows for high throughput analysis of many samples, which is needed for large sample numbers, while on-line SPE removes almost all analyst manipulation of the samples, minimizing the analyst time needed for sample preparation. Both sample preparations were coupled with reversed phase liquid chromatography and isotope dilution tandem mass spectrometry (LC-MS/MS) for analyte detection. For both methods, the resulting precision was within 15%, the accuracy within 25%, and the sensitivity was comparable with the limits of detection ranging from 0.002ng/mL to 0.041ng/mL. Additionally, matrix effects were substantially decreased from previous reports for both extraction protocols. The results of this comparison showed that both methods were acceptable for the detection of exposures to fentanyl analogs and metabolites in urine.

Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry.

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101-105%) and high precision (5-8%) for the detection of these five nerve agent hydrolysis products in serum.

Comparison of high-resolution and tandem mass spectrometry for the analysis of nerve agent metabolites in urine.

RATIONALE: Although use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. High-resolution mass spectrometry (HRMS) was compared to tandem mass spectrometry (MS/MS) analysis for the quantitation of five urinary metabolites specific to VX, Russian VX, soman, sarin and cyclosarin nerve agents. The HRMS method was further evaluated for qualitative screening of metabolites not included in the test panel. METHODS: Nerve agent metabolites were extracted from urine using solid-phase extraction, separated using hydrophilic interaction chromatography and analyzed using both tandem and high-resolution mass spectrometry. MS/MS results were obtained using selected reaction monitoring with unit resolution; HRMS results were obtained using a mass extraction window of 10 ppm at a mass resolution of 50 000. The benchtop Orbitrap HRMS instrument was operated in full scan mode, to measure the presence of unexpected nerve agent metabolites. RESULTS: The assessment of two quality control samples demonstrated high accuracy (99.5-104%) and high precision (2-9%) for both HRMS and MS/MS. Sensitivity, as described by the limit of detection, was overlapping for both detectors (0.2-0.7 ng/mL). Additionally, the HRMS method positively confirmed the presence of a nerve agent metabolite, not included in the test panel, using the accurate mass and relative retention time. CONCLUSIONS: The precision, accuracy, and sensitivity were comparable between the current MS/MS method and this newly developed HRMS analysis for five nerve agent metabolites. HRMS showed additional capabilities beyond the current method by confirming the presence of a metabolite not included in the test panel.

A method for visualization of "omic" datasets for sphingolipid metabolism to predict potentially interesting differences.

Sphingolipids are structurally diverse and their metabolic pathways highly complex, which makes it difficult to follow all of the subspecies in a biological system, even using "lipidomic" approaches. This report describes a method to use transcriptomic data to visualize and predict potential differences in sphingolipid composition, and it illustrates its use with published data for cancer cell lines and tumors. In addition, several novel sphingolipids that were predicted to differ between MDA-MB-231 and MCF7 cells based on published microarray data for these breast cancer cell lines were confirmed by mass spectrometry. For the data that we were able to find for these comparisons, there was a significant match between the gene expression data and sphingolipid composition (P < 0.001 by Fisher's exact test). Upon considering the large number of gene expression datasets produced in recent years, this simple integration of two types of "omic" technologies ("transcriptomics" to direct "sphingolipidomics") might facilitate the discovery of useful relationships between sphingolipid metabolism and disease, such as the identification of new biomarkers.

Lipidomics reveals a remarkable diversity of lipids in human plasma.

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Altered sphingolipid metabolism induced by tumor hypoxia - new vistas in glycolipid tumor markers.

Uncontrolled growth of malignant cells produces hypoxic regions in locally advanced tumors. Recently we showed that tumor hypoxia-induced transcription of multiple genes involved in glycan synthesis, leading to expression of useful glycolipid tumor markers, such as gangliosides having N-glycolyl sialic acid. Our subsequent studies indicated that the ceramide portion of glycolipids, as well as their glycan moiety, was also significantly affected by hypoxia. Tumor hypoxia-induced marked accumulation of sphinganine (dihydrosphingosine) long-chain base, and significant reduction of unsaturated very long-chain fatty acids in the ceramide moiety. Mass-spectrometry, which yields information on both glycan- and ceramide moieties, is expected to be clinically useful in detecting such distinct molecular species of cancer-associated glycolipids having combined alteration in both glycan- and ceramide moieties.

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.

Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS3 protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.