Relative to liquid chromatography coupled to electrospray ionization (ESI), paper spray ionization (PSI) can more rapidly ionize analytes from complex matrices with less sample preparation and less expensive instrumentation. However, matrix effects can lead to ion suppression and poorer sensitivity. To overcome these problems, we combined on-paper faradaic ion concentration polarization (f-ICP), a form of electrokinetic stacking, with PSI to preconcentrate analytes directly on the spray substrate prior to ionization. Electrokinetic stacking also enables desalting because of the separation of analyte molecules from small ions like sodium. 3D-printed cartridges containing chemically modified PTFE papers were utilized with a high voltage isolated power supply to perform on-paper stacking from the same device as paper spray. In this study, ion suppression was compared for paper spray with and without electrokinetic stacking in the presence of five salts and artificial urine at varying concentrations. The signal enhancement achieved for f-ICP/PSI relative to normal PSI was also quantified. Finally, the effect of salt type and concentration on stacking time was assessed. Increasing salt concentrations generally results in longer stacking times and increased currents, indicative of increasing quantities of charge being desalted. In the presence of single salt-containing matrices, f-ICP/PSI frequently decreased ion suppression and gave a signal enhancement of 2 to >200× for small molecule drugs compared to unstacked paper spray (uPSI). In artificial urine, a more complex matrix with various salts and metabolites, f-ICP/PSI did not reliably decrease matrix effects due to creatinine and other matrix components costacking with the analytes. Nevertheless, f-ICP/PSI still resulted in signal enhancements of 8 to >50× relative to uPSI due to analyte preconcentration from electrokinetic stacking. Overall, this research demonstrated that f-ICP/PSI results in significant sensitivity improvements relative to uPSI.

Less expensive, faster, and laborious protocols are needed for protein and peptide mass spectrometry (MS) for therapeutic protein characterization. This work describes a simple paper-based device that couples electrokinetic (EK) stacking via faradaic ion concentration polarization (f-ICP) with paper spray (PS) MS to enrich proteins and peptides on paper in minutes. The EK-PS device consists of a three-dimensional (3D)-printed holder with two polytetrafluoroethylene (PTFE) filter papers that act both as the media for electrokinetic stacking by f-ICP and as the supports for paper spray ionization. Modification of the PTFE filters via silanization improves MS ionization and imparts a positive surface charge to enable electro-osmotic flow. A potential of ∼280 V along the length of the papers induces electrokinetic stacking, whereas simultaneously floating the device at 4000 V generates online ionization via paper spray. Stacking and elution of proteins and peptides, which occurs by a combination of electro-osmotic flow and electrophoresis, takes ∼5-10 min and improves detection sensitivity by over 10-fold. In the analysis of tryptic protein digests and glycosylation profiling of monoclonal antibodies, electrokinetic stacking increases peptide identifications ∼9-fold and enables the detection of glycoforms above 3% relative abundance. The method is relatively simple and rapid, which may be useful for monitoring protein manufacturing.

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.

We developed a simple, paper-based device that enables sensitive detection by mass spectrometry (MS) without solid phase extraction or other sample preparation. Using glass fiber filter papers within a 3D printed holder, the device employs electrokinetic manipulations to stack, separate, and desalt charged molecules on paper prior to spray into the MS. Due to counter-balanced electroosmotic flow and electrophoresis, charged analytes stack on the paper and desalting occurs in minutes. One end of the paper strip was cut into a sharp point and positioned near the inlet of a MS. The stacked analyte bands move toward the paper tip with the EOF where they are ionized by paper spray. The device was applied to analysis of PFAS in tap water with sub part-per-trillion detection limits in less than ten minutes with no sample pretreatment. Analysis of opioids in urine also occurs in minutes. The crucial parameters to enable stacking, separation, and MS ionization of both positively and negatively charged analytes were determined and optimized. Experimental and computational modeling studies confirm the electrokinetic stacking and analyte transport mechanisms. On-paper separations were carried out by stacking analyte bands at different locations depending on their electrophoretic mobility, achieving baseline separation in some cases.

Mass spectrometry (MS) is a powerful analytical technique that typically involves sample preparation and online analytical separation before MS detection. Traditional methods often face bottlenecks in sample preparation and analytical separation, despite the rapid detection capabilities of MS. This review explores the integration of electrokinetic manipulations directly with the ionization step to enhance MS performance, focusing on methods that eliminate or simplify sample preparation and separation processes. Techniques such as paper spray, electrophoresis in nanoelectrospray ionization (nESI) emitters, induced nESI, counterflow gradient electrofocusing, and in-syringe electrokinetics are highlighted for their ability to combine extraction and ionization in a single step, significantly improving throughput. The review delves into the use of electric fields during sample preparation and separations for these methods, demonstrating the efficiency of electrophoretic methods in driving extractions, crude separations, desalting, and enhanced sensitivity. The integration of these methods directly with MS ionization aims to enhance the analytical capabilities of mass spectrometry, while reducing costs and increasing throughput relative to traditional approaches.

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.

Illicit drug trafficking and abuse is a significant public safety and health concern. Color tests are commonly used for drug screening, but their poor specificity results in false positives. This study demonstrates the combination of drug residue collection using pressure-sensitive adhesive paper, on-paper color testing, and post-reaction analysis by paper spray mass spectrometry (PS-MS) on both portable and benchtop ion trap MS. All steps, including residue collection, color testing, and paper spray analysis, were performed on the same piece of paper. Three common color tests were investigated: the cobalt thiocyanate test for cocaine, the Simon test for methamphetamine, and the Marquis test for phenethylamine stimulants and opiates. The detection threshold for color tests ranged from 1.25 to 10 μg on paper. Drug residues were successfully confirmed by paper spray MS at the color test threshold in all cases, except for heroin after reaction with the Marquis reagent, when using the portable MS. In this case, the MS detection threshold was 4-fold higher than the color test threshold. The stability of the color test products was assessed through a time study. Drug residues could be detected by MS at least 24 hours after reaction. A series of realistic samples, including false positives, were analyzed to demonstrate the technique's utility in real-world scenarios. Overall, combining color tests with PS-MS offers a rapid, low-cost method for the collection and analysis of illicit drugs.

The rise of fentanyl and fentanyl analogs in the drug supply pose serious threats to public health. Much of these compounds enter the United States through shipping routes. Here we provide a method for fentanyl screening and analysis that utilizes pressure-sensitive adhesive (PSA) lined paper to recover drug residues from parcel-related surfaces. The paper used is commercially available repositionable notes (also called post-it or sticky notes). From this paper, mass spectra were obtained by paper spray-mass spectrometry (PS-MS), where PSA paper served as both a sampling and analysis substrate. Seven fentanyl-related compounds were analyzed: fentanyl, 4-anilino-N-phenethylpiperidine (4-ANPP), N,1-diphenethyl-N-phenylpiperidin-4-amine (phenethyl-4-ANPP), valerylfentanyl, 4-fluoroisobutyrylfentanyl (4-FIBF), carfentanil, and p-fluorofentanyl. These compounds were recovered by PSA paper and identified by PS-MS from packaging tape and plastic at 50 ng and from cardboard and shipping labels at 100 ng. The impact of cutting agents on PS-MS analysis of fentanyl analogs was explored. No trends of analyte suppression were found at high concentrations of the cutting agents caffeine, diphenhydramine, and lidocaine when recovered from surfaces. A cartridge that required no precise cutting of PSA paper prior to sampling or analysis was evaluated for use in PS-MS for fentanyl screening. Recovery and detection of fentanyl from plastic sheeting was demonstrated with this cut-free cartridge. The cut-free cartridge showed somewhat less consistency and lower analyte signal than the standard cartridge, but performance was suitable for potential screening applications. In combining PSA surface sampling with PS-MS for drug screening, both sampling and detection of fentanyl-related compounds is simple, rapid, and low-cost.

Microneedles are widely used substrates for various chemical and biological sensing applications utilizing surface-enhanced Raman spectroscopy (SERS), which is indeed a highly sensitive and specific analytical approach. This article reports the fabrication of a nanoparticle (NP)-decorated microneedle substrate that is both a SERS substrate and a substrate-supported electrospray ionization (ssESI) mass spectrometry (MS) sample ionization platform. Polymeric ligand-functionalized gold nanorods (Au NRs) are adsorbed onto superhydrophobic surface-modified polydimethylsiloxane (PDMS) microneedles through the control of various interfacial interactions. We show that the chain length of the polymer ligands dictates the NR adsorption process. Importantly, assembling Au NRs onto the micrometer-diameter needle tips allows the formation of highly concentrated electromagnetic hot spots, which provide the SERS enhancement factor as high as 1.0 × 106. The micrometer-sized area of the microneedle top and high electromagnetic field enhancement of our system can be loosely compared with tip-enhanced Raman spectroscopy, where the apex of a plasmonic NP-functionalized sharp probe produces high-intensity plasmonic hot spots. Utilizing our NR-decorated microneedle substrates, the synthetic drugs fentanyl and alprazolam are analyzed with a subpicomolar limit of detection. Further analysis of drug-molecule interactions on the NR surface utilizing the Langmuir adsorption model suggests that the higher polarizability of fentanyl allows for a stronger interaction with hydrophilic polymer layers on the NR surface. We further demonstrate the translational aspect of the microneedle substrate for both SERS- and ssESI-MS-based detection of these two potent drugs in 10 drug-of-abuse (DOA) patient plasma samples with minimal preanalysis sample preparation steps. Chemometric analysis for the SERS-based detection shows a very good classification between fentanyl, alprazolam, or a mixture thereof in our selected 10 samples. Most importantly, ssESI-MS analysis also successfully identifies fentanyl or alprazolam in these same 10 DOA plasma samples. We believe that our multimodal detection approach presented herein is a highly versatile detection technology that can be applicable to the detection of any analyte type without performing any complicated sample preparation.

Introduction: Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several singlestranded RNA viruses, including the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2). It is currently the only FDA-approved antiviral agent for the treatment of individuals with COVID-19 caused by SARSCoV-2. However, remdesivir pharmacokinetics/pharmacodynamics (PK/PD) and toxicity data in humans are extremely limited. It is imperative that precise analytical methods for the quantification of remdesivir and its active metabolite, GS-441524, are developed for use in further studies. We report, herein, the first validated antiviral paper spray-mass spectrometry (PS-MS/MS) assay for the quantification of remdesivir and GS-441524 in human plasma. We seek to highlight the utility of PS-MS/MS technology and automation advancements for its potential future use in clinical research and the clinical laboratory setting.Methods: Calibration curves for remdesivir and GS-441524 were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Briefly, plasma samples were spotted on a paper substrate contained within pre-manufactured plastic cassette plates, and the spots were dried for 1 h. The samples were then analyzed directly for 1.2 min utilizing PS-MS/MS. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer utilizing automated technology. Results: The calibration ranges were 20 - 5000 and 100 - 25000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within +/- 15%. Plasma calibrant stability was assessed and degradation for the 4 degrees C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 degrees C. No interference, matrix effects, or carryover was discovered during the validation process.Conclusions: PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir and GS-441524, which may be useful for clinical PK/PD, therapeutic drug monitoring (TDM), and toxicity assessment, particularly during the current COVID-19 pandemic and future viral outbreaks.

Forensic and environmental sciences often rely on chromatographic separations coupled to mass spectrometry to detect contaminants in complex matrices. However, these methods require lengthy analysis times and sample preparation that is not suitable for analysis in the field. In this work, two analytical methods were combined that are known for their potential for portable analysis. The ambient ionization technique, paper spray mass spectrometry (paper spray-MS) was coupled to paper-based surface enhanced Raman spectroscopy (pSERS) to detect toxic organophosphorus molecules from the same substrate, with a total analysis time of less than five minutes. The coupling of these techniques presents a potential for portable Raman screening followed by MS confirmation in a field-forward laboratory. A cartridge insert was designed and 3D printed to facilitate the sample collection and analysis for PS-MS and pSERS. Three chemical warfare agent simulants: dimethyl methylphosphonate (DMMP), diethyl phosphoramidate (DEPA), and diisopropyl methylphosphonate (DIMP) were included in the method due to having similar chemistries to G- and V-series chemical warfare agents (CWAs). Organophosphorus pesticides, malathion and dichlorvos, with similar mechanisms of action to the CWAs, were also included in the method. Because CWAs quickly degrade in the environment, the CWA hydrolysis products, ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), pinacolyl methylphosphonic acid (PinMPA), methylphosphonic acid (MPA), 2-Diethylaminoethanethiol (EDA), and 2-Diisopropylaminoethanethiol (IDA) were also studied. A mixture of the analytes was used to create calibration curves using the dual-polarity, PS-MS method with sub-ng to low ng limits of detection. A dilution series, spanning 3 orders of magnitude, was made using pSERS, also with low ng limits of detection. These experiments show the potential and feasibility for PS-MS coupled to pSERS to be used to rapidly, screen and confirm the presence of organophosphorus molecules, in complex matrices, with portable instrumentation.

Robust sample collection and identification of trace amounts of compounds outside of a laboratory environment is a challenge facing military users, first responders, and law enforcement An increasing number of portable instruments are being developed to focus on improving on-site sampling and analysis. A weighty obstacle for these field-focused systems is the ability to detect trace amounts of analytes from complex matrices. Previous work has shown the benefits of utilizing pressure-sensitive adhesive (PSA) coated paper for collection combined with paper spray ionization mass spectrometry (PS-MS) for the identification of trace amounts of small molecule. In this work, analysis of explosives captured on a PSA substrate via a portable instrument was examined. Positive identification was achieved for TNT, HMX, and RDX when sampling from surfaces containing less than 1 mg of each explosive. It was also determined that mixtures of the explosives and illicit drugs could be identified even with the presence of interferents. Additional experiments were performed to extract and reanalyze substrates, as well as longevity stability studies. The results demonstrate the potential value that PSA substrates combined with PS-MS can provide in a field forward or first responder setting.

In this work, blow flies were investigated as environmental chemical sample collectors following a chemical warfare attack (CWA). Blow flies sample the environment as they search for water and food sources and can be trapped from kilometers away using baited traps. Three species of blow flies were exposed to CWA simulants to determine the persistence and detectability of these compounds under varying environmental conditions. A liquid chromatography mass spectrometry (LC-MS/MS) method was developed to detect CWA simulants and hydrolysis products from fly guts. Flies were exposed to the CWA simulants dimethyl methylphosphonate and diethyl phosphoramidate as well as the pesticide dichlorvos, followed by treatment-dependent temperature and humidity conditions. Flies were sacrificed at intervals within a 14 day postexposure period. Fly guts were extracted and analyzed with the LC-MS/MS method. The amount of CWA simulant in fly guts decreased with time following exposure but were detectable 14 days following exposure, giving a long window of detectability. In addition to the analysis of CWA simulants, isopropyl methylphosphonic acid, the hydrolysis product of sarin, was also detected in blow flies 14 days post exposure. This work demonstrates the potential to obtain valuable samples from remote or access-restricted areas without risking lives.

Paper spray mass spectrometry (PS-MS) has been shown to be a rapid, simple and inexpensive alternative to traditional forensic drug screening methods. It can address the limitations of both immunoassays and chromatography-based techniques due to its non-reliance on sample preparation and its ability to rapidly screen for a wide array of compounds. In this study, an automated PS-MS system was employed to semi-quantitatively screen for 40 commonly abused drugs and metabolites in urine after a 15-min glucuronidase reaction. The target compounds included common prescription opioids, fentanyl and norfentanyl, stimulants including methamphetamine and cocaine, benzodiazepines and antidepressants. The enzyme, buffer and internal standard solution were combined in one spiking solution to minimize sample handling. Analysis was carried out using a commercially available automated paper spray system coupled to a triple quadrupole mass spectrometer. This method may prove useful for clinical and forensic toxicology laboratories as it allows for automated screening of complex samples for drugs without extraction, separation and sample cleanup.

Smith et al. recently reported dye analysis of a carpet fragment thought by some to be from a 15th century silk-knotted Persian carpet [Smith et al., Forensic Science International: Synergy 2021; 3:100130]. Sample threads were found to contain several synthetic dyes from the late 19th century, including Congo red, which was first synthesized in 1883, indicating that the carpet is a modern object. Two mysterious compounds designated as A and B of unknown identity were also detected with Congo red in similar amounts on the object. This paper reports the structural elucidation of A and B using high resolution mass spectrometry and multidimensional NMR spectroscopy. To our knowledge, these compounds have never been reported before. Additionally, experiments are described demonstrating that A and B were products from the reaction of Congo red with formaldehyde. Previously unknown homologs of A and B were also formed when Congo red was reacted with acetaldehyde, pentanal, and hexanal, indicating a common reaction mechanism. Uv–Vis spectra of the homologs suggest that aldehyde exposure can cause an overall color change in objects dyed with Congo red. A historic dye swatch book containing cotton skeins dyed with the colorants benzopurpurine 4B and deltapurpurine 5B, which are of the same chemical class as Congo red, and exhibiting a faded appearance, were found to contain dye derivatives analogous to A and B. Chemical mechanisms proposed for the formation of compounds A and B are discussed, along with the possible origins of the formaldehyde exposure. The concept of ‘chemical fading,’ is adduced to heighten the awareness of the exposure of historic dyed textiles to the harmful effects of formaldehyde, a common museum and household pollutant.

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.

Illicit drug use causes over half a million deaths worldwide every year. Drugs of abuse are commonly smuggled through customs and border checkpoints and, increasingly, through parcel delivery services. Improved methods for detection of trace drug residues from surfaces are needed. Such methods should be robust, fieldable, sensitive, and capable of detecting a wide range of drugs. In this work, commercially produced paper with a pressure-sensitive adhesive coating was utilized for the collection and analysis of trace drug residues by paper spray mass spectrometry (MS). This modified substrate was used to combine sample collection of drug residues from surfaces with rapid detection using a single paper spray ticket. The all-in-one ticket was used to probe different surfaces commonly encountered in forensic work including clothing, cardboard, glass, concrete, asphalt, and aluminum. A total of 10 drugs (acetyl fentanyl, fentanyl, clonazolam, cocaine, heroin, ketamine, methamphetamine, methylone, U-47700, and XLR-11) were evaluated and found to be detectable in the picogram range using a benchtop mass spectrometer and in the low nanogram range using a portable ion trap MS. The novel approach demonstrates a simple yet effective sampling strategy, allowing for rapid identification from difficult surfaces via paper spray mass spectrometry.

Publisher’s Note: This paper, originally published on 12 April 2021, was withdrawn on 5 May 2021 per author request.

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.