Paper Spray Mass Spectrometry with On-Paper Electrokinetic Manipulations: Part-Per-Trillion Detection of Per/Polyfluoroalkyl Substances in Water and Opioids in Urine.

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.

Combining presumptive color tests, pressure-sensitive adhesive-based collection, and paper spray-mass spectrometry for illicit drug detection.

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.

Insects as Chemical Sensors: Detection of Chemical Warfare Agent Simulants and Hydrolysis Products in the Blow Fly Using LC-MS/MS.

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.

Enhancing Nonfouling and Sensitivity of Surface-Enhanced Raman Scattering Substrates for Potent Drug Analysis in Blood Plasma via Fabrication of a Flexible Plasmonic Patch.

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.

Pressure-Sensitive Adhesive Combined with Paper Spray Mass Spectrometry for Low-Cost Collection and Analysis of Drug Residues.

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.

A statistical approach to optimizing paper spray mass spectrometry parameters.

RATIONALE: Paper spray mass spectrometry (PS-MS) was used to analyze and quantify ampicillin, a hydrophilic compound and frequently utilized antibiotic. Hydrophilic molecules are difficult to analyze via PS-MS due to their strong binding affinity to paper substrates and low ionization efficiency, among other reasons. METHODS: Solvent and paper parameters were optimized to increase the extraction of ampicillin from the paper substrate. After optimizing these key parameters, a Resolution IV 1/16 fractional factorial design with two center points was employed to screen eight different design parameters simultaneously. RESULTS: Pore size, sample volume, and solvent volume were the most significant factors affecting average peak area under the curve (AUC) and the signal-to-blank (S/B) ratio for the 1 µg/mL ampicillin calibrant. After optimizing the key parameters, a linear calibration curve with a range of 0.2 µg/mL to 100 µg/mL was generated (R2  = 0.98) and the limit of detection (LOD) and lower limit of quantification (LLOQ) were calculated to be 0.07 µg/mL and 0.25 µg/mL, respectively. CONCLUSIONS: The statistical optimization procedure undertaken here increased the mass spectral signal intensity by more than a factor of 40. This statistical method of screening followed by optimization experiments proved faster and more efficient, and produced more drastic improvements than typical one-factor-at-a-time experiments.

Optimization of electromagnetic hot spots in surface-enhanced Raman scattering substrates for an ultrasensitive drug assay of emergency department patients' plasma.

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.

Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry.

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 anti-fungal 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 (R2 = 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 µg/mL, 0.1-10 µg/mL, 0.1-10 µg/mL, 0.1-10 µg/mL, and 0.1-10 µg/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.

Targeted Protein Detection Using an All-in-One Mass Spectrometry Cartridge.

We developed a simple 3D printed cartridge for mass spectrometry (MS) targeted detection of plasma proteins, including post-translational modifications (PTMs). The cartridge uses an integrated antibody enrichment column to preconcentrate the protein target as well as a novel built-in substrate to ionize the protein targets for MS detection. We show several examples of using this cartridge to perform rapid detection of clinically significant proteoforms from plasma samples.

Forensic Sampling and Analysis from a Single Substrate: Surface-Enhanced Raman Spectroscopy Followed by Paper Spray Mass Spectrometry.

Sample preparation is the most common bottleneck in the analysis and processing of forensic evidence. Time-consuming steps in many forensic tests involve complex separations, such as liquid and gas chromatography or various types of extraction techniques, typically coupled with mass spectrometry (e.g., LC-MS). Ambient ionization ameliorates these slow steps by reducing or even eliminating sample preparation. While some ambient ionization techniques have been adopted by the forensic community, there is significant resistance to discarding chromatography as most forensic analyses require both an identification and a confirmation technique. Here, we describe the use of a paper substrate, the surface of which has been inkjet printed with silver nanoparticles, for surface enhanced Raman spectroscopy (SERS). The same substrate can also act as the paper substrate for paper spray mass spectrometry. The coupling of SERS and paper spray ionization creates a quick, forensically feasible combination.

Direct Analysis of Aerosolized Chemical Warfare Simulants Captured on a Modified Glass-Based Substrate by "Paper-Spray" Ionization.

Paper spray ionization mass spectrometry offers a rapid alternative platform requiring no sample preparation. Aerosolized chemical warfare agent (CWA) simulants trimethyl phosphate, dimethyl methylphosphonate, and diisopropyl methylphosphonate were captured by passing air through a glass fiber filter disk within a disposable paper spray cartridge. CWA simulants were aerosolized at varying concentrations using an in-house built aerosol chamber. A custom 3D-printed holder was designed and built to facilitate the aerosol capture onto the paper spray cartridges. The air flow through each of the collection devices was maintained equally to ensure the same volume of air sampled across methods. Each approach yielded linear calibration curves with R2 values between 0.98-0.99 for each compound and similar limits of detection in terms of disbursed aerosol concentration. While the glass fiber filter disk has a higher capture efficiency (≈40%), the paper spray method produces analogous results even with a lower capture efficiency (≈1%). Improvements were made to include glass fiber filters as the substrate within the paper spray cartridge consumable. Glass fiber filters were then treated with ammonium sulfate to decrease chemical interaction with the simulants. This allowed for improved direct aerosol capture efficiency (>40%). Ultimately, the limits of detection were reduced to levels comparable to current worker population limits of 1 × 10-6 mg/m3.

Detection of chemical warfare agent simulants and hydrolysis products in biological samples by paper spray mass spectrometry.

Paper spray ionization coupled to a high resolution tandem mass spectrometer (a quadrupole orbitrap) was used to identify and quantitate chemical warfare agent (CWA) simulants and their hydrolysis products in blood and urine. Three CWA simulants, dimethyl methylphosphonate (DMMP), trimethyl phosphate (TMP), and diisopropyl methylphosphonate (DIMP), and their isotopically labeled standards were analyzed in human whole blood and urine. Calibration curves were generated and tested with continuing calibration verification standards. Limits of detection for these three compounds were in the low ng mL-1 range for the direct analysis of both blood and urine samples. Five CWA hydrolysis products, ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), isobutyl methylphosphonic acid (iBuMPA), cyclohexyl methylphosphonic acid (CHMPA), and pinacolyl methylphosphonic acid (PinMPA), were also analyzed. Calibration curves were generated in both positive and negative ion modes. Limits of detection in the negative ion mode ranged from 0.36 ng mL-1 to 1.25 ng mL-1 in both blood and urine for the hydrolysis products. These levels were well below those found in victims of the Tokyo subway attack of 2 to 135 ng mL-1. Improved stability and robustness of the paper spray technique in the negative ion mode was achieved by the addition of chlorinated solvents. These applications demonstrate that paper spray mass spectrometry (PS-MS) can be used for rapid, sample preparation-free detection of chemical warfare agents and their hydrolysis products at physiologically relevant concentrations in biological samples.

Development of a Paper Spray Mass Spectrometry Cartridge with Integrated Solid Phase Extraction for Bioanalysis.

A novel paper spray cartridge with an integrated solid phase extraction (SPE) column is described. The cartridge performs extraction and pre-concentration, as well as sample ionization by paper spray, from complex samples such as plasma. The cartridge allows for selective enrichment of target molecules from larger sample volumes and removal of the matrix, which significantly improved the signal intensity of target compounds in plasma samples by paper spray ionization. Detection limits, quantitative performance, recovery, ionization suppression, and the effects of sample volume were evaluated for five drugs: carbamazepine, atenolol, sulfamethazine, diazepam, and alprazolam. Compared with direct paper spray analysis of dried plasma spots, paper spray analysis using the integrated solid phase extraction improved the detection limits significantly by a factor of 14-70, depending on the drug. The improvement in detection limits was, in large part, due to the capability of analyzing larger sample volumes. In addition, ionization suppression was found to be lower and recovery was higher for paper spray with integrated SPE, as compared to direct paper spray analysis. By spiking an isotopically labeled internal standard into the plasma sample, a linear calibration curve for the drugs was obtained from the limit of detection (LOD) to 1 µg/mL, indicating that this method can be used for quantitative analysis. The paper spray cartridge with integrated SPE could prove valuable for analytes that ionize poorly, in applications where lower detection limits are required, or on portable mass spectrometers. The improved performance comes at the cost of requiring a more complex paper spray cartridge and requiring larger sample volumes than those used in typical direct paper spray ionization.

Paper spray and extraction spray mass spectrometry for the direct and simultaneous quantification of eight drugs of abuse in whole blood.

Determination of eight drugs of abuse in blood has been performed using paper spray or extraction spray mass spectrometry in under 2 min with minimal sample preparation. A method has been optimized for quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), morphine, cocaine, and Δ9-tetrahydrocannabinol (THC) from a single blood spot. Sample to sample variations of 1-5% relative standard deviation were achieved using stable isotope-labeled internal standards and tandem mass spectrometry. Limits of detection for all drugs were below typical physiological and toxicological levels. Paper spray and extraction spray each used less than 10 µL of whole blood. These methods exhibit the potential for performing rapid and high-throughput assays for selective on-site multicompound quantitative screening of illicit drugs.

Plasma drug screening using paper spray mass spectrometry with integrated solid phase extraction.

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.

Direct quantitative analysis of nicotine alkaloids from biofluid samples using paper spray mass spectrometry.

The determination of tobacco derived nicotine alkaloids in biofluid samples is of great importance to testing for tobacco use, tobacco cessation treatment, and studies on exposure to secondhand smoke. Paper spray mass spectrometry (MS) has been adapted for direct, quantitative analysis of tobacco alkaloids from biofluid samples, such as blood, urine, and saliva in liquid and dried form. Limits of quantitation as low as several nanograms per milliliter were obtained for nicotine, cotinine, trans-3'-hydroxycotinine, and anabasine. Direct analysis of fresh blood samples has also been achieved with improved sensitivity using print paper substrates of high density. Quantitation of the cotinine in the blood of a rat was performed with both direct analysis using paper spray and a traditional analysis protocol using liquid chromatography MS. Comparable results were obtained and the precision of the two methods was similar. The paper spray MS method is rapid and shows potential for significantly improved analytical efficiency in clinical laboratories as well as for point-of-care tobacco use assessment.

Automated and High-Throughput Urine Drug Screening Using Paper Spray Mass Spectrometry.

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.

Pressure sensitive adhesives and paper spray-mass spectrometry for the collection and analysis of fentanyl-related compounds from shipping materials.

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.

Superhydrophobic Surface Modification of Polymer Microneedles Enables Fabrication of Multimodal Surface-Enhanced Raman Spectroscopy and Mass Spectrometry Substrates for Synthetic Drug Detection in Blood Plasma.

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.

Silica coated paper substrate for paper-spray analysis of therapeutic drugs in dried blood spots.

Paper spray is a newly developed ambient ionization method that has been applied for direct qualitative and quantitative analysis of biological samples. The properties of the paper substrate and spray solution have a significant impact on the release of chemical compounds from complex sample matrices, the diffusion of the analytes through the substrate, and the formation of ions for mass spectrometry analysis. In this study, a commercially available silica-coated paper was explored in an attempt to improve the analysis of therapeutic drugs in dried blood spots (DBS). The dichloromethane/isopropanol solvent has been identified as an optimal spray solvent for the analysis. The comparison was made with paper spray using chromatography paper as substrate with methanol/water as solvent for the analysis of verapamil, citalopram, amitriptyline, lidocaine, and sunitinib in dried blood spots. It has been demonstrated that the efficiency of recovery of the analytes was notably improved with the silica coated paper and the limit of quantitation (LOQ) for the drug analysis was 0.1 ng mL(-1) using a commercial triple quadrupole mass spectrometer. The use of silica paper substrate also resulted in a sensitivity improvement of 5-50-fold in comparison with chromatography papers, including the Whatman ET31 paper used for blood cards. Analysis using a hand-held miniature mass spectrometer Mini 11 gave LOQs of 10-20 ng mL(-1) for the tested drugs, which is sufficient to cover the therapeutic ranges of these drugs.