Detection of 30 Fentanyl Analogs by Commercial Immunoassay Kits.

Health-care workers, laboratorians and overdose prevention centers rely on commercial immunoassays to detect the presence of fentanyl; however, the cross-reactivity of fentanyl analogs with these kits is largely unknown. To address this, we conducted a pilot study evaluating the detection of 30 fentanyl analogs and metabolites by 19 commercially available kits (9 lateral flow assays, 7 heterogeneous immunoassays and 3 homogenous immunoassays). The analogs selected for analysis were compiled from the Drug Enforcement Administration and National Forensic Laboratory Information System reports from 2015 to 2018. In general, the immunoassays tested were able to detect their intended fentanyl analog and some closely related analogs, but more structurally diverse analogs, including 4-methoxy-butyryl fentanyl and 3-methylfentanyl, were not well detected. Carfentanil was only detected by kits specifically designed for its recognition. In general, analogs with group additions to the piperidine, or bulky rings or long alkyl chain modifications in the N-aryl or alkyl amide regions, were poorly detected compared to other types of modifications. This preliminary information is useful for screening diagnostic, forensic and unknown powder samples for the presence of fentanyl analogs and guiding future testing improvements.

Synthesis and Storage Stability of Diisopropylfluorophosphate.

Diisopropylfluorophosphate (DFP) is a potent acetylcholinesterase inhibitor commonly used in toxicological studies as an organophosphorus nerve agent surrogate. However, LD50 values for DFP in the same species can differ widely even within the same laboratory, possibly due to the use of degraded DFP. The objectives here were to identify an efficient synthesis route for high purity DFP and assess the storage stability of both the in-house synthesized and commercial source of DFP at the manufacturer-recommended storage temperature of 4°C, as well as -10°C and -80°C. After 393 days, the commercial DFP stored at 4°C experienced significant degradation, while only minor degradation was observed at -10°C and none was observed at -80°C. DFP prepared using the newly identified synthesis route was significantly more stable, exhibiting only minor degradation at 4°C and none at -10°C or -80°C. The major degradation product was the monoacid derivative diisopropylphosphate, formed via hydrolysis of DFP. It was also found that storing DFP in glass containers may accelerate the degradation process by generating water in situ as hydrolytically generated hydrofluoric acid attacks the silica in the glass. Based on the results here, it is recommended that DFP be stored at or below -10°C, preferably in air-tight, nonglass containers.

Simultaneous measurement of tabun, sarin, soman, cyclosarin, VR, VX, and VM adducts to tyrosine in blood products by isotope dilution UHPLC-MS/MS.

This work describes a new specific, sensitive, and rapid stable isotope dilution method for the simultaneous detection of the organophosphorus nerve agents (OPNAs) tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VR, VX, and VM adducts to tyrosine (Tyr). Serum, plasma, and lysed whole blood samples (50 µL) were prepared by protein precipitation followed by digestion with Pronase. Specific Tyr adducts were isolated from the digest by a single solid phase extraction (SPE) step, and the analytes were separated by reversed-phase ultra high performance liquid chromatography (UHPLC) gradient elution in less than 2 min. Detection was performed on a triple quadrupole tandem mass spectrometer using time-triggered selected reaction monitoring (SRM) in positive electrospray ionization (ESI) mode. The calibration range was characterized from 0.100-50.0 ng/mL for GB- and VR-Tyr and 0.250-50.0 ng/mL for GA-, GD-, GF-, and VX/VM-Tyr (R(2) ≥ 0.995). Inter- and intra-assay precision had coefficients of variation of ≤17 and ≤10%, respectively, and the measured concentration accuracies of spiked samples were within 15% of the targeted value for multiple spiking levels. The limit of detection was calculated to be 0.097, 0.027, 0.018, 0.074, 0.023, and 0.083 ng/mL for GA-, GB-, GD-, GF-, VR-, and VX/VM-Tyr, respectively. A convenience set of 96 serum samples with no known nerve agent exposure was screened and revealed no baseline values or potential interferences. This method provides a simple and highly specific diagnostic tool that may extend the time postevent that a confirmation of nerve agent exposure can be made with confidence.

Determination of enantiomeric composition by negative-ion electrospray ionization-mass spectrometry using deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors.

The ability to use mixtures of deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors for the determination of enantiomeric composition by electrospray ionization-mass spectrometry is demonstrated. For each experiment, two N-(3,5-dinitrobenzoyl)amino acids were chosen such that each would have opposite selectivity for the enantiomers of the analyte. Electrospray ionization-mass spectrometry, monitored in the negative ion mode, of solutions containing the two N-(3,5-dinitrobenzoyl)amino acids, sodium hydroxide, and the analyte, in a one-to-one mixture of methanol and water, afford peaks in the mass spectrum that correspond to the deprotonated 1:1 analyte-selector complexes. The ratio of the intensities of the complexes in the mass spectrum can be related to the enantiomeric composition of the analyte. Additionally, the sense and extent of chiral recognition is consistent with chromatographic observations, using chiral stationary phases derived from N-(3,5-dinitrobenzoyl)amino acids. Each analysis of enantiomeric composition requires less than 10 s to complete, indicating that this method has great potential for the development of fast-/high-throughput chiral analyses.

Tertiary amine appended derivatives of N-(3,5-dinitrobenzoyl)leucine as chiral selectors for enantiomer assays by electrospray ionization mass spectrometry.

Derivatives of the chiral selector N-(3,5-dinitrobenzoyl)leucine were prepared and used as chiral selectors for enantiomer discrimination in single-stage electrospray ionization mass spectrometric experiments. The chiral selectors were designed to remove the ionization site from the sites required for effective chiral recognition. Addition of a chiral analyte to a solution of the two pseudoenantiomeric chiral selectors, which differ in absolute stereochemistry and the length of the tether connecting the tertiary amine site used for ionization via protonation and the rest of the chiral selector, affords selector-analyte complexes in the electrospray ionization mass spectrum where the ratio of these complexes is dependent on the enantiomeric composition of the analyte. The relationship between the ratio of the selector-analyte complexes in the electrospray ionization mass spectrum and the enantiomeric composition of the analyte can be used to relate the extent of enantioselectivity that is being observed and for quantitative enantiomeric composition determinations. Investigations into the scope and limitations of this method, plus a comparison to the enantioselectivities observed by chiral HPLC using a N-(3,5-dinitrobenzoyl)leucine-derived chiral stationary phase, is presented.

Determination of abamectin in soil samples using high-performance liquid chromatography with tandem mass spectrometry.

Abamectin, which is comprised of a mixture of avermectins B1a and B1b, is a natural pesticide used as an anti-parasitic agent in livestock, ornamental, and agricultural crops, which can potentially be transported to aquatic systems. These compounds are highly toxic to both aquatic vertebrates and invertebrates at low concentrations in water. This investigation developed high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) techniques to support automated extraction by an accelerated solvent extraction (ASE) system and chromatographic techniques to measure residues of avermectins in complex soil samples. HPLC along with atmospheric pressure chemical ionization (APCI) MS/MS was used for separation and determination of avermectin isomers in soil samples. Average method recovery for abamectin by UV was 91%, while detection by MS/MS resulted in a 68% recovery for abamectin. Individual method recoveries by MS/MS were 53.6% for avermectin B1a and 36.8% for avermectin B1b. The use of tandem technology eliminated matrix interferences and resulted in an approximately eight-fold increase in sensitivity.