Jennifer M. Colby

Comparison of Information-Dependent Acquisition on a Tandem Quadrupole TOF vs a Triple Quadrupole Linear Ion Trap Mass Spectrometer for Broad-Spectrum Drug Screening

BACKGROUND Liquid chromatography high-resolution mass spectrometry (LC-HRMS) with untargeted data collection is especially attractive for general unknown drug screening owing to its ability to identify unexpected compounds. LC-HRMS offers several advantages over traditional selected reaction monitoring (SRM) techniques and could be an ideal screening platform as long as its analytical performance is comparable to that of SRM-based methods. METHODS We developed a broad-spectrum drug screen on a high-resolution mass spectrometer [tandem quadrupole time-of-flight (QqTOF)] that collected data in an untargeted manner and compared its performance to a nominal mass instrument [triple quadrupole linear ion trap (QqLIT)] that collected data in a targeted manner. Both methods used information-dependent acquisition of product ion spectra. We evaluated the lower limits of detection and matrix effects for each method and compared their ability to identify drugs in 100 routine clinical urine samples. Additional information (patient prescription history, drug screening results, etc.) was used to confirm discordant results. RESULTS QqLIT was slightly more analytically sensitive than QqTOF; however, this difference did not significantly affect compound identification in patient samples. QqLIT identified 596 drugs in the urine samples, of which 531 (89%) were confirmed. QqTOF identified 515 drugs, of which 500 (97%) were confirmed. There were 562 instances of a confirmed drug (68 unique drugs) in the 100 urine samples; the methods were concordant in 469 of these instances. CONCLUSIONS Overall, QqTOF performed similarly to QqLIT and could serve as an alternative method for general unknown screening.

Cardiogenic shock after use of fluoroamphetamine confirmed with serum and urine levels

Abstract Context. 4-Fluoroamphetamine (4-FA) is a para-substituted phenethylamine-type synthetic stimulant that has in recent years gained popularity through internet blogs and market share according to confiscated drug data. No serious toxicity has previously been reported. We report a case of a young man who developed severe toxicity and cardiogenic shock after using 4-FA, with laboratory confirmation. Case details. An 18-year-old man presented to the emergency department with vomiting, shortness of breath, chest tightness, and altered mental status about 5 h after using a new and unfamiliar street drug. Two days prior, he had received naltrexone intramuscular injection as part of an opioid addiction treatment program and was taking fluoxetine and trazodone. Five hours after presentation, he developed cardiogenic shock requiring intraaortic balloon pump, inotropic and ventilatory support. An echocardiogram showed left ventricular (LV) hypokinesia, sparing the apex and ejection fraction (EF) = 10%. Comprehensive toxicology serum testing revealed FA, naproxen, trazodone, and cotinine. The 4-FA urine level was 64,000 ng/ml and serum level was 118 ng/ml. With slow recovery, the patient was discharged after 2 weeks of hospitalization. Discussion. Although no previously reported 4-FA clinical poisoning cases have been published for comparison, by examining 4-FA pharmacology compared with other stimulant drugs, and given this patients presentation and echocardiogram suggestive of reverse takotsubo cardiomyopathy we suspect the toxic mechanism was an acute cardiomyopathy caused by 4-FA catecholamine-induced myocarditis and/or small vessel myocardial ischemia. Conclusion. Recreational use of 4-FA may present with life threatening toxicity including cardiomyopathy, cardiogenic shock, and pulmonary edema.

Cross-Reactivity of Acetylfentanyl and Risperidone With a Fentanyl Immunoassay

Fentanyl and its analogs, such as acetylfentanyl, have become a concern for potential abuse. Fentanyl compliance monitoring and urine drug testing are becoming increasingly necessary; however, a limited number of fentanyl immunoassays have been validated for clinical use. The purpose of this study was to validate the use of the DRI® fentanyl immunoassay, determine the potential cross-reactivity of acetylfentanyl and other pharmaceuticals, and investigate acetylfentanyl use in San Francisco. All urine toxicology samples from patients presenting to the emergency department were analyzed using the fentanyl immunoassay for 4 months. Positive samples were analyzed qualitatively using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for fentanyl, fentanyl metabolites, fentanyl analogs and greater than 200 common drugs and metabolites. Subsequently, quantitative analysis was performed using LC-tandem mass spectrometry (LC-MS-MS). Acetylfentanyl, risperidone and 9-hydroxyrisperidone were found to cross-react with the fentanyl immunoassay. No acetylfentanyl was detected in our emergency department patient population. The fentanyl immunoassay demonstrated 100% diagnostic sensitivity in a subset of urines tested; however, the specificity was only 86% due to seven false-positive samples observed. Five of the seven samples were positive for risperidone and 9-hydroxyrisperidone. The DRI® fentanyl immunoassay can be used to screen for fentanyl or acetylfentanyl; however, confirmatory testing should be performed for all samples that screen positive.

Comparison of umbilical cord tissue and meconium for the confirmation of in utero drug exposure

OBJECTIVES Drug screening in neonates is traditionally performed using meconium, but cord tissue has been proposed as an alternative specimen. This study compares the detection of drugs in a large number of paired meconium and umbilical cord tissue samples from subjects at risk of in utero drug exposure. DESIGN AND METHODS Physician-ordered toxicology results and clinical information were collected in a retrospective review of subject medical records. All toxicology testing was performed by a national reference laboratory using a combination of immunoassays and chromatography-mass spectrometry. The comparison was limited to drugs present in both cord and meconium panels. RESULTS Overall agreement between cord and meconium ranged from 76% (cannabinoids) to 100% (barbiturates), but Cohens kappa was <65% for 5 of the 6 drug classes we studied. Considering meconium as the gold standard, cord was less sensitive for the detection of 5 of the 6 drug classes, and for the detection of all 5 individual opioids. For 3 of the 5 individual opioids, the concentration of drug measured in meconium did not correlate well with qualitative detection in cord. CONCLUSIONS This study reveals different sensitivities of drug detection in umbilical cord tissue and meconium. For the drugs studied here, meconium provides greater sensitivity, and is likely to remain the specimen of choice when sensitivity is of greatest importance. These results can help clinicians, laboratorians, and epidemiologists to (1) select the most appropriate test to confirm a suspected drug exposure and (2) interpret discordant results when testing is performed in multiple matrices.

Optimization and Validation of High-Resolution Mass Spectrometry Data Analysis Parameters

High-resolution mass spectrometry (HRMS) has gained recognition as a valuable tool for comprehensive drug screening in a variety of biological matrices. HRMS instruments collect untargeted, accurate mass data, which permit identification of known and unknown compounds in a single analytical run. One of the most challenging aspects of implementing an HRMS drug screen is establishing appropriate data analysis parameters for identifying compounds. Unlike other types of mass spectrometry data, guidelines for HRMS data analysis and acceptability criteria have not been established. Although many laboratories have published on the utility of HRMS for drug screening, few have included details on how they determined allowable errors and set positivity criteria. Previously, we developed and validated a comprehensive 169-compound drug screen on a high-resolution quadrupole time of flight mass spectrometer. Here we report the detailed procedure that we used to determine appropriate positivity criteria for our screening procedure. Our approach was empirical; we collected data and analyzed it with commonly available software. We found that a combined scoring approach using a threshold of 70, with 70% weight given to library match and 10% weight given to each of mass error, retention time error and isotope pattern difference provided optimum drug identification efficiency of 99.2%. Our results demonstrate the importance of library matching in accurately identifying compounds, and underscore the utility of robust product ion spectra that contain information on the lineage, mass and relative abundance of fragments. The method we describe is easily adaptable to include alternative parameters that may be available in software associated with a variety of HRMS platforms. With careful selection of error limits and positivity criteria, HRMS instruments are capable of producing high-quality, high-confidence results that may reduce the need for confirmatory testing.

High-Resolution Mass Spectrometry for Untargeted Drug Screening

While gas chromatography-mass spectrometry (GC/MS) continues to be the forensic standard for toxicology, liquid chromatography coupled to tandem MS offers significant operational advantages for targeted confirmatory analysis. LC-high-resolution (HR)-MS has recently been available that offers advantages for untargeted analysis. HR-MS analyzers include the Orbitrap and time-of-flight MS. These instruments are capable of detecting 1 ppm mass resolution. Following soft ionization, this enables the assignment of exact molecular formula, limiting the number of candidate compounds. With this technique, presumptive identification of unknowns can be conducted without the need to match MS library spectra or comparison against known standards. For clinical toxicology, this can greatly expand on the number of drugs and metabolites that can be detected and reported on a presumptive basis. Definitive assignments of the compounds identity can be retrospectively determined with acquisition of the appropriate reference standard.

Rapid Assessment of Drugs of Abuse

Laboratory testing for drugs of abuse has become standard practice in many settings both forensic and clinical. Urine is the predominant specimen, but other specimens are possible including hair, nails, sweat, and oral fluid. Point-of-care test kits provide for rapid analysis at the site where specimens are collected allowing for immediate action on the results. POCT is based on immunochromatography where the drug in the patients sample competes with drug and antibody conjugates in the test to develop or block the development of a colored line. Most POCTs are visually interpreted in a few minutes. The potential for false positives is possible due to drug cross-reactivity with the antibodies in the test. False negatives are also possible due to dilution of the sample and the potential for adulteration or sample substitution by the patient. POCT shows more variability than central laboratory testing because of the variety of operators involved in the testing process, but POCT has good agreement for most tests with mass spectrometry provided comparable cutoffs and cross-reactivity of drugs/metabolites are considered. Validation of the test performance with the intended operators will identify potential interferences and operational issues before implementing the test in routine practice. POCT offers faster turnaround of test results provided the limitations and challenges of the test are considered.

Dynamic Phenylalanine Clamp Interactions Define Single-Channel Polypeptide Translocation through the Anthrax Toxin Protective Antigen Channel

Anthrax toxin is an intracellularly acting toxin where sufficient detail is known about the structure of its channel, allowing for molecular investigations of translocation. The toxin is composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxins translocon, PA, translocates the large enzymes, LF and EF, across the endosomal membrane into the host cells cytosol. Polypeptide clamps located throughout the PA channel catalyze the translocation of LF and EF. Here, we show that the central peptide clamp, the ϕ clamp, is a dynamic site that governs the overall peptide translocation pathway. Single-channel translocations of a 10-residue, guest-host peptide revealed that there were four states when peptide interacted with the channel. Two of the states had intermediate conductances of 10% and 50% of full conductance. With aromatic guest-host peptides, the 50% conducting intermediate oscillated with the fully blocked state. A Trp guest-host peptide was studied by manipulating its stereochemistry and prenucleating helix formation with a covalent linkage in the place of a hydrogen bond or hydrogen-bond surrogate (HBS). The Trp peptide synthesized with ʟ-amino acids translocated more efficiently than peptides synthesized with D- or alternating D,ʟ-amino acids. HBS stapled Trp peptide exhibited signs of steric hindrance and difficulty translocating. However, when mutant ϕ clamp (F427A) channels were tested, the HBS peptide translocated normally. Overall, peptide translocation is defined by dynamic interactions between the peptide and ϕ clamp. These dynamics require conformational flexibility, such that the peptide productively forms both extended-chain and helical states during translocation.

Analysis of codeine positivity in urine of pain management patients.

The opioids codeine and morphine have legitimate uses in managing chronic pain conditions, but they are frequently abused. Patients prescribed opioids submit urine samples for medication compliance monitoring, and the interpretation of the results is complex. The purpose of this study was to evaluate the percentage of codeine- and morphine-positive urine drug tests that result from morphine use only, with the positive codeine result arising from low levels of codeine present in pharmaceutical formulations of morphine. This study included 80 urine samples which tested positive for codeine and morphine after pre-analytical hydrolysis and analysis by gas chromatography-mass spectrometry. Quantitative results were correlated with patient prescription information and immunoassay results to classify patients into one of four categories: heroin users (50%), codeine users (34%), codeine and morphine users (5%), and morphine users (11%). The percentage of codeine-positive resulting from morphine use was higher than previous estimates. Urine from patients prescribed morphine only was found to contain codeine at <1% of the morphine concentration, a ratio that was also observed in patients who used heroin. Careful analysis of urine drug testing results, including assessing the ratio of codeine to morphine (C/M), can help providers determine if patients are compliant with their pain management regimens.

Suspect Screening Using LC–QqTOF Is a Useful Tool for Detecting Drugs in Biological Samples

High-resolution mass spectrometers (HRMS), including quadrupole time of flight mass analyzers (QqTOF), are becoming more prevalent as screening tools in clinical and forensic toxicology laboratories. Among other advantages, HRMS instruments can collect untargeted, full-scan mass spectra. These datasets can be analyzed retrospectively using a combination of techniques, which can extend the drug detection capabilities. Most laboratories using HRMS in production settings perform untargeted data collection, but analyze data in a targeted manner. To perform targeted analysis, a laboratory must first analyze a reference standard to determine the expected characteristics of a given compound. In an alternate technique known as suspect screening, compounds can be tentatively identified without the use of reference standards. Instead, predicted and/or intrinsic characteristics of a compound, such as the accurate mass, isotope pattern, and product ion spectrum are used to determine its presence in a sample. The fact that reference standards are not required a priori makes this data analysis approach very attractive, especially for the ever-changing landscape of novel psychoactive substances. In this work, we compared the performance of four data analysis workflows (targeted and three suspect screens) for a panel of 170 drugs and metabolites, detected by LC-QqTOF. We found that retention time was not required for drug identification; the suspect screen using accurate mass, isotope pattern, and product ion library matching was able to identify more than 80% of the drugs that were present in human urine samples. We showed that the inclusion of product ion spectral matching produced the largest decrease in false discovery and false negative rates, as compared to suspect screening using mass alone or using just mass and isotope pattern. Our results demonstrate the promise that suspect screening holds for building large, economical drug screens, which may be a key tool to monitor the use of emerging drugs of abuse, including novel psychoactive substances.