The continued rise in overdoses, driven by fentanyl and novel psychoactive substances (NPS), underscores the need for improved methods for drug screening. Traditional analytical techniques involving chromatography can be time-consuming and require sample preparation. Paper spray mass spectrometry (PS-MS) can rapidly detect analytes from complex matrices. However, challenges such as matrix effects can lead to higher detection limits, prompting improvements aimed at preconcentrating or cleaning up samples. While most methods focus on plasma analysis, there is a need to further simplify sample preparation, particularly for whole blood. Here, we report on three-dimensional (3D)-printed devices that preconcentrate drugs from whole blood for PS-MS analysis. An uncontrolled amount of blood was added to the blood reservoir part of the cartridge. The capillary fills and then rotates to sit on top of the SPE column. The blood wicks through the SPE to the waste pad, and the sample is left to dry until analysis. The dry SPE holder is "snapped" in the paper spray cartridge. A variety of parameters were optimized to improve manufacturing and the performance of the device, including waste pad substrates, SPE sorbents, and binders; the size of the SPE compartment; the amount of SPE; a water wash step; and blood volume. Performance was tested with 21 different drugs including opioids like fentanyl and isotonitazene, cathinones, designer and prescription benzodiazepines, cocaine, methamphetamine, and synthetic cannabinoids. Dried blood samples were found to be stable for at least 14 days. The detection limits were at single digit or subng/mL levels or lower for all analytes for 70 μL blood sample, with a median decrease of 9-fold compared to paper spray without SPE.

Drug overdoses have risen dramatically in recent years. We developed a simple nontargeted method using a disposable paper spray cartridge with an integrated solid phase extraction column. This method was used to screen for ~160 fentanyl analogs, synthetic cannabinoids, other synthetic drugs, and traditional drugs of abuse in over 300 authentic overdose samples collected at emergency departments in Indianapolis. A solid phase extraction step was implemented on the paper spray cartridge to enable subnanograms per milliliter synthetic drugs screening in plasma. Analysis was performed on a quadrupole orbitrap mass spectrometer using the sequential window acquisition of all theoretical fragment ion spectra approach in which tandem mass spectrometry was performed using 7 m/z isolation windows in the quadrupole. Calibration curves with isotopically labeled internal standards were constructed for 35 of the most frequently encountered synthetic and traditional illicit drugs by US toxicology labs. Additional qualitative-only drugs in a suspect screening list were also included. Limits of detection in plasma for synthetic cannabinoids ranged from 0.1 to 0.5 and 0.1 to 0.3 ng/mL for fentanyl and its analogs and between 1 and 5 ng/mL for most other drugs. Relative matrix effects were evaluated by determining the variation of the calibration slope in 10 different lots of biofluid and found to be between 3% and 20%. The method was validated on authentic overdose samples collected from two emergency departments in Indianapolis, Indiana, from suspected or known overdoses. Commonly detected synthetic drugs included fentanyl related substances, designer benzodiazepines such as flubromazolam, and the synthetic cannabinoid 5F-PB-22.

Paper spray mass spectrometry is an ambient ionization technique capable of the direct ionization of analyte from a bio-fluid spot on a paper substrate. Different solvents and types of paper can have different interactions with the analytes of interest; therefore, they can significantly impact the analyte signal and the assay as a whole. In this study, we examined the effects of substrate-solvent composition on signal intensity, blank signal intensity, and signal-to-blank ratio for a variety of pharmaceutical drugs, illicit drugs, chemical warfare agent (CWA) simulants, and CWA hydrolysis products. The analytes were prepared either neat or spiked into human plasma and deposited on a variety of modified paper substrates. Extraction occurred with a range of different solvents. Optimizing the substrate-solvent combination improved the signal-to-blank ratio for all compounds ranging from 1 to 7,964 factor improvement, with the substrate providing a more impactful improvement. Aprotic solvents, such as tetrahydrofuran and ethyl acetate, tended to produce optimal signal-to-blank ratios, while carbon sputtered and glass fiber substrates were the top paper substrates. The research presented herein illustrates the need for systematic optimization of substrate and spray solvent combinations to achieve the best detection limits for paper spray analysis.

Surface-enhanced Raman scattering (SERS) is an ultrasensitive analytical technique, which is capable of providing high specificity; thus, it can be used for toxicological drug assay (detection and quantification). However, SERS-based drug analysis directly in human biofluids requires mitigation of fouling and nonspecificity effects that commonly appeared from unwanted adsorption of endogenous biomolecules present in biofluids (e.g., blood plasma and serum) onto the SERS substrate. Here, we report a bottom-up fabrication strategy to prepare ultrasensitive SERS substrates, first, by functionalizing chemically synthesized gold triangular nanoprisms (Au TNPs) with poly(ethylene glycol)-thiolate in the solid state to avoid protein fouling and second, by generating flexible plasmonic patches to enhance SERS sensitivity via the formation of high-intensity electromagnetic hot spots. Poly(ethylene glycol)-thiolate-functionalized Au TNPs in the form of flexible plasmonic patches show a twofold-improved signal-to-noise ratio in comparison to triethylamine (TEA)-passivated Au TNPs. Furthermore, the plasmonic patch displays a SERS enhancement factor of 4.5 ×107. Utilizing the Langmuir adsorption model, we determine the adsorption constant of drugs for two different surface ligands and observe that the drug molecules display stronger affinity for poly(ethylene glycol) ligands than TEA. Our density functional theory calculations unequivocally support the interaction between drug molecules and poly(ethylene glycol) moieties. Furthermore, the universality of the plasmonic patch for SERS-based drug detection is demonstrated for cocaine, JWH-018, and opioids (fentanyl, despropionyl fentanyl, and heroin) and binary mixture (trace amount of fentanyl in heroin) analyses. We demonstrate the applicability of flexible plasmonic patches for the selective assay of fentanyl at picogram/milliliter concentration levels from drug-of-abuse patients' blood plasma. The fentanyl concentration calculated in the patients' blood plasma from SERS analysis is in excellent agreement with the values determined using the paper spray ionization mass spectrometry technique. We believe that the flexible plasmonic patch fabrication strategy would be widely applicable to any plasmonic nanostructure for SERS-based chemical sensing for clinical toxicology and therapeutic drug monitoring.

Background: Invasive fungal disease is a life-threatening condition that can be challenging to treat due to pathogen resistance, drug toxicity, and therapeutic failure secondary to suboptimal drug concentrations. Frequent therapeutic drug monitoring (TDM) is required for some antifungal agents to overcome these issues. Unfortunately, TDM at the institutional level is difficult, and samples are often sent to a commercial reference laboratory for analysis. To address this gap, the first paper spray-mass spectrometry assay for the simultaneous quantitation of five triazoles was developed. Methods: Calibration curves for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were created utilizing plasma-based calibrants and four stable isotopic internal standards. No sample preparation was needed. Plasma samples were spotted on a paper substrate in pre-manufactured plastic cartridges, and the dried plasma spots were analyzed directly utilizing paper spray-mass spectrometry (paper spray MS/MS). All experiments were performed on a Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer. Results: The calibration curves for the five anti-fungal agents showed good linearity (R-2 = 0.98-1.00). The measured assay ranges (lower limit of quantification [LLOQ]-upper limit of quantitation [ULOQ]) for fluconazole, posaconazole, itraconazole, hydroxyitraconazole, and voriconazole were 0.5-50 mu g/mL, 0.1-10 mu g/mL, 0.1-10 mu g/mL, 0.1-10 mu g/mL, and 0.1-10 mu g/mL, respectively. The inter- and intra-day accuracy and precision were less than 25% over the respective ranges. Conclusions: We developed the first rapid paper spray-MS/MS assay for simultaneous quantitation of five triazole anti-fungal agents in plasma. The method may be a powerful tool for near-point-of-care TDM aimed at improving patient care by reducing the turnaround time and for use in clinical research.

Herein we report the programmable preparation of ultrasensitive surface-enhanced Raman scattering (SERS)-based nanoplasmonic superlattice substrates to assay fentanyl and cocaine (detection and quantification) from 10 μL aliquots of emergency department patient plasma without the need for purification steps. Highly homogeneous three-dimensional (3D) nanoplasmonic superlattices are generated through the droplet evaporation-based self-assembly process of chemically-synthesized, polyethylene glycol thiolate-coated gold triangular nanoprisms (Au TNPs). Close-packed, solid-state 3D superlattice substrates produce electromagnetic hot spots due to near-field plasmonic coupling of Au TNPs, which display unique localized surface plasmonic resonance properties. These uniquely prepared superlattice substrates enable strong SERS enhancement to achieve a parts-per-quadrillion limit of detection using the label-free SERS-based technique. Our reported limit of detection is at least 100-fold better than any known SERS substrates for the drug assay. Importantly, our density functional theory calculations show that a specific electronic interaction between the drug molecule and novel nanoplasmonic superlattice substrates plays a critical role that may trigger achieving this unprecedentedly high sensitivity. Additionally, we show high selectivity of the superlattice substrate in the SERS-based detection of analytes from different patient samples, which do and do not contain target analytes (i.e., fentanyl and/or cocaine). The demonstrated sensitivity and selectivity of 3D superlattice substrates for SERS-based drug analysis in real toxicological samples are expected to advance the field of measurement science, and forensic and clinical toxicology by obviating the need for complicated sample processing steps, long assay times, and the low sensitivity of existing "gold standard" analytical techniques including gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry and enzyme-linked immunosorbent assays. Taken together, we believe that this entirely new and reproducible superlattice substrate for the SERS analysis will aid scientific, forensic, and healthcare communities to battle the drug overdose epidemic in the United States.

Designer drugs, drugs synthesized in a lab that mimic other drugs of abuse, have become a major cause of death in the United States. Limitations in either speed, sensitivity or selectivity of current analytical techniques hinders rapid detection of designer drugs. Paper spray mass spectrometry (PS-MS) is a rapid ambient pressure technique capable of detecting analytes in complex matrices. However, due to a lack of sample cleanup and chromatography, matrix effects can have a significant impact on the detection limits. Previous work has shown that the paper spray substrate has an impact on matrix effects, but the current literature lacks a systematic approach to studying different properties of paper with regards to matrix effects. In this work, the effect of pore size, flow rate, and thickness on ionization efficiency and recovery was assessed. Cellulose thin layer chromatography (TLC) plates were made along with a universal spray cartridge to provide a porous spray substrate similar to paper but with easily controllable properties. It was found that substrates with the highest filtration properties (thicker, slower flow rate, or smaller pore sizes) exhibited lower analyte recovery but improved ionization efficiency. This trend was verified with an offline extraction conducted with a 3D printed centrifuge extractor. Paper/solvent combinations were tested with urine samples to determine if selecting a paper and solvent with better ionization efficiency could improve detection limits. While for some drugs minimizing ionization suppression improved detection limits, other drug targets, like those that were charged at physiological pH, were largely unaffected. For analytes that showed improvement, both the paper and the solvent had an impact, although most of the improvement was due to the solvent.