Applications of Mass Spectrometry for Clinical Diagnostics: The Influence of Turnaround Time.

Despite recent technological advances in diagnostics, clinical microbiology and pathology remain labor intensive, as they still rely heavily on traditional methods such as cell culturing, phenotyping, and biochemical testing. This is due, in part, to the fact that these traditional techniques are cheaper, detect only viable bacteria, and yield isolates that can further be characterized and studied. The desire to improve turnaround times resulted in the use of molecular techniques that are based mainly on PCR. However, the application of PCR to clinical samples has many potential pitfalls due to the susceptibility of PCR to inhibitors, contamination, and experimental conditions. Hybrid techniques based on cooperative integration with MS allow both cultured-based and PCR-based MS analysis for diagnosis of infections caused by bacteria, viruses, and fungi. The Vitek MS platform is a MALDI-TOF MS system that allows for the chemical information in cultured organisms, such as bacteria [ 202 ] , yeast [ 203 ] , filamentous bacteria, and fungi [ 204 ] samples, to be analyzed. The mass spectra output typically involves protein profiles, which are then compared to reference library [ 205 , 206 ] ; the library can be developed in-house for specific organisms or obtained commercially. Integrating MALDI-TOF MS with microbiology in this manner has the potential to significantly reduce analysis times since the only pre-analytical step required is the transfer of the organisms to a MALDI plate followed by the application of a MALDI matrix. The platform requires a minimum of 105 colony forming units, a number can only be obtained in >24 h laboratory culture. Recent efforts to automate liquid medium systems are expected to increase the throughput of this platform [ 207 ] . PLEX-ID, on the other hand, is an ESI-MS based platform that detects nucleic acid amplicons from bacteria, fungi, and virus organisms present in clinical samples [ 208 , 209 ] . This platform allows for direct analysis of these samples without the initial culture step, but it requires multiplexed PCR amplification steps. Identification is made by comparing nucleic acid composition with a reference library. Compared to the Vitek MS platform, the PLEX-ID system requires longer pre-analytical steps (4 – 6 h) to extract and get the DNA sample ready for amplification. MS has not only proliferated in microbiological laboratories in recent years, but it is now being used in histopathology for tissue characterization. The motivation has been to make accurate diagnosis in a shortest amount of time possible. For example, a major challenge for surgeons is differentiating between cancerous and normal tissue. Two MS-based molecular detection strategies are currently being developed to address this challenge: one that provides real-time data during operation and one that assists pathologists in making rapid decisions after the tissue has been removed. We provide a brief history here focusing on contributions made by direct analysis with ambient ionization MS. Shortly after the development DESI and DART, Takáts et al [ 101 ] , introduced a novel mass spectrometry ionization technique, rapid evaporative ionization mass spectrometry (REIMS, Figure 10A ) in 2009. The current design utilizes electrosurgery, which causes evaporation of the tissue upon contact, producing surgical smoke that is collected and transported to a mass spectrometer for characterization of the chemical composition in the smoke. What is interesting is that the analysis of the smoke extract by the mass spectrometer occurs instantaneously, during the period where the tissue is being cut, giving a response and guiding the surgeon on tumor margins. This ambient ionization platform was the first hand-held system developed for ex vivo and in vivo analysis and intraoperative application, it is now widely known as the intelligent knife (iKnife). The diagnostic accuracy of REIMS was successfully implemented in real-time analysis of colorectal cancer [ 30 ] . In another recent study [ 28 ] , the iKnife technology was used to analyze tissue samples supplied by a total of 198 ovarian cancer patients. Cross-validation with separate normal tissues samples yielded 97.4% sensitivity and 100% specificity. Histological agreement between iKnife and histopathologist was also very good ( P < 0.001). High quality identification relies on special-evaluation software that compares collected spectra with library. The iKnife technology is currently being developed by the Waters Corporation, which include database development and device optimization. Another direct analysis platform for tissue profiling involves the MasSpec Pen ( Figure 10B ), which was recently developed Eberlin’s group [ 210 ] . The MasSpec Pen is a handheld ambient ionization device based on nondestructive analysis of tissues using liquid-liquid extractions. Briefly, the pen electrode is placed on a tissue sample where a small droplet of water comes in contact with its surface and after a few seconds it is transferred to the MS inlet, containing diagnostic proteins, lipids, and metabolites, for analysis. Using this device, the MasSpec Pen nondestructively evaluated 253 human tissue samples (normal and cancerous) showing its ex vivo probing diagnostic capabilities. Recently, Sans et al [ 211 ] performed rapid tissue diagnosis of 192 ovarian, fallopian tube, and peritoneum tissue samples using the MasSpec Pen. It was coupled to both a linear ion trap mass spectrometer (LIT) and an Orbitrap and achieved similar metabolic profiles of ovarian cancer samples further validating its compatibility with various mass spectrometer platforms. The high performance and versatility, coupled with nondestructive and direct analysis, show the potential of the MasSpec Pen for further studies. There are still various challenges that will have to be addressed in order to utilize the MasSpec Pen for ovarian cancer diagnosis in the operating room, such as in vivo analysis and extensive analysis of benign ovarian samples. The PRISM lab in Lille, France, also introduced SpiderMass [ 212 ] , which was designed for in vivo and real time analysis. Like the iKnife and MasSpec Pen, SpiderMass utilizes a microsampling probe with a transfer line attached directly a MS inlet ( Figure 10C ). The novelty of SpiderMass, however, stems from how ions are formed. It is considered a water-assisted laser ablation/desorption process, where an IR-tuned laser excites water molecules found within human tissues to generate and desorb ions, instead of using electrosurgery or liquid-liquid extractions. Mass spectrometry imaging (MSI) is growing into a vital analytical tool that can assess the spatial distribution of lipids, proteins, biomarkers, and other large biomolecules in biological samples. These unique abilities have not gone unnoticed and have enhanced MSI’s role in the clinical field, due in part to three common MSI ion sources that include MALDI-MSI, DESI-MSI, and SIMS-MSI. While all three ionization sources are useful for various applications, there are some considerations that need to be made. For example, MALDI-MSI requires proper application of the matrix for proper ionization so it is critical that the matrix is optimized for individual molecules. DESI-MSI and SIMS-MSI require little to no sample preparation and do not need a matrix, but they are limited in the molecules they can ionize. Similarly, SIMS and MALDI both require high vacuum for efficient ionization [ 213 ] . Even with these considerations, many groups have found them useful in being able to image tissue samples for clinical research. The use of MALDI-MSI has emerged as a powerful technique in disease biomarker discovery. In a recent study, Black et al [ 214 ] , designed a new antibody panel based (APB) mass spectrometry platform that coupled traditional MALDI-MSI with protein capture for an array of antibodies in patient biofluid samples ( Figure 11A ). This newly developed platform expanded the analysis of potentially hundreds of different N-glycoproteins in one imaging run. However, this method is limited by the number of antibodies that can be added to a panel, limiting the number of glycoproteins that can be probed per analysis. MALDI-MSI has also been applied for detecting prognostic markers that can be used to predict outcomes of potential cancer patients. Hinsch et al [ 215 ] applied MALDI-MSI to study biologically relevant colorectal cancer masses from formalin fixed tissues to aid in disease prognosis in a more efficient way than univariate analyses. Several other studies have also shown the validity of MALDI-MSI for various cancers such as triple-negative breast cancer (TNBC) [ 216 ] , head and neck cancer tissue microarrays (TMAs) for biomarker prediction [ 217 ] , and pediatric brain samples [ 218 ] . DESI-MSI has also been on the forefront of early-stage diagnostic because of its nondestructive and rapid detection capabilities. For example, Margulis et al [ 219 ] were able to utilize this imaging to distinguish between skin basal cell carcinoma (BCC), common cancer, and normal skin from 86 human specimens. Sans et al [ 220 ] , also used DESI-MSI to investigate ovarian tumors and characterize the metabolic profiles of diseased state and normal tissues to identify predictive markers of cancer aggressiveness. These methods can enable rapid multiplex mapping of metabolites and lipids in fresh tissue specimens and can detect the distributions of various species between cancerous and normal prostate tissues [ 221 ] . Zhang et al [ 222 ] , demonstrated DESI-MSI capabilities by mapping lipid alteration of lymph node tissue to aid diagnosis of human thyroid and breast cancer. It was noted that section size and its influence on MS imaging time could be a limitation for rapid lymph node evaluations. In addition to providing the metabolic profile of biomolecule, DESI-MSI can also offer the spatial information for the same biomolecule samples. Recently, Dória et al [ 223 ] utilized this method to investigate the lipid distribution of different gynecological samples. The results of this study concluded that it has the capability to differentiate between tumor tissues. Recent work aims to enhance the performance reliability in order to identify benign and cancer tissue at 150 μm spatial resolution. For example, the Berman group analyzed prostate cancer samples and utilized over 900 spatially resolved spectra for accurate and high-resolution metabolomic profiling of prostate cancer [ 224 ] . As imaging techniques have become increasingly relevant in the biological field, SIMS-MSI has laid stock in the field as well. This technique has shown increasing potential due to its scale lateral spatial resolution, which is the highest reported of the MSI methods [ 225 ] . SIMS imaging has been used to characterize anionic lipid species such as cardiolipins (CLs), which are of low abundance in brain tissue. The recent introduction of gas-cluster ion beams (GCIB) has shifted the SIMS field from fragment detection to molecular profiling and subcellular molecular imaging. For example, Tian et. al [ 226 ] ( Figure 11B ) employed a GCIB source to enable SIMS imaging in both native and injured brain tissue. This study demonstrates successful imaging of larger lipids with less damage to higher molecular mass lipid molecules and furthers the work being done for high resolution SIMS studies. Very recently, the authors were able to further implement this source to decipher the distribution of CLs, phosphatidylethanolamines, and other lipids in subcellular compartments of mouse hippocampal neuronal cells and rat brain tissues [ 227 ] .

As illustrated in Figure 1 , the field of clinical diagnostics with MS has seen a shift in recent years towards developing various POC devices as a means of streamlining the processes of sample collection and analysis in non-traditional lab settings [ 96 – 98 ] . The overall goal of POC testing is twofold: i) to reduce the time and cost required for sample collection and analysis and ii) to provide the patient with results faster so appropriate medical decisions can be made in a timely fashion. It is also important that the results themselves are easy to understand for non-experts to interpret. Traditional methods (e.g. LC-MS, PCR, and immunological assays), while sensitive and relatively selective, still require expert knowledge, large sample sizes, extensive sample preparation, and offline separation/extraction steps. This not only affects the total analysis time but makes it difficult for high-throughput testing. In order to address these challenges for MS analysis, ambient ionization techniques have been implemented. These easy-to-use techniques offer the advantage of being able to directly analyze raw samples, while maintaining sensitivity and selectively. It is also possible to store and analyze samples on the same substrate [ 99 , 100 ] . Ambient ionization was first introduced with desorption electrospray ionization (DESI) in 2004 [ 101 ] and direct analysis in real time (DART) in 2005 [ 102 ] and over the past 15 years, numerous techniques have been developed. Under the umbrella of ambient ionization, techniques are classified according to their desorption agent, such as spray-based (e.g., DESI [ 101 ] and nano-DESI [ 103 ] ), plasma-based (e.g., DART [ 102 ] and low-temperature plasma [ 104 ] ), laser-based (e.g., laser ablation electrospray ionization [ 105 ] and electrospray-assisted laser desorption ionization [ 106 ] ), and substrate-based (e.g., paper spray [ 107 ] and thread spray [ 108 , 109 ] ). Depicted in the outer circle of Figure 6A are a larger scheme of agents for sampling/ionization currently being used in direct analysis. Common to all of these methods is the fact that they operate under the general principle of producing gas-phase ions from raw, untreated samples, examples of which are provided in the middle circle of Figure 6A , including biofluids (blood, urine, and oral fluid) and various human tissues (e.g. liver and brain). By avoiding sample preparation steps ( Figure 6B , lower sequence), ambient ionization MS can produce results in less than two minutes. Due to the reduced analysis time, minimal solvent and instrumentation requirements, these methods also provide outstanding field capabilities, especially when combined with portable mass spectrometers. Therefore, the range of patients that can be reached for clinical studies/analysis can be large as illustrated in the inner circle of Figure 6A . There are many ambient ionization methods that rely on the desorption mechanism to provide direct MS analysis. Each method, either spray-based, plasma-based, laser-based, or a combination thereof has its own characteristics and properties such as imaging capabilities, mass range (i.e., analysis of small or large molecules), reactive modes, and analyte types (i.e. polar or non-polar). Recent reviews have covered this field extensively including specific clinical applications for various ambient ionization techniques [ 99 , 100 , 110 – 113 ] . Therefore, a general outline of the field will be provided here, focusing on the most studied methods and recent developments. DESI employs charged liquid droplets derived from an electrospray emitter to desorb analytes present on an ambient surface. The initial liquid droplets arriving at the sample surface form a thin film in which the analyte is dissolved. Subsequent droplets impact the thin film producing secondary droplets containing analyte ions, transferring them to the mass spectrometer. Hence, the solubility of analytes in the spray solvent is an important parameter in the DESI process [ 114 – 116 ] as well as a higher-primary droplet kinetic energy (~500 MeV [ 117 ] ) to facilitate the release and transfer of the secondary droplets. Droplet kinetic energy is derived from the use of high nebulizer gas pressure (N 2 , 150 psi) providing a primary droplet velocity of ~100 m/s. Though high energies are involved, it dissipates very quickly during droplet impact with surface, and as a result, shorter spray distances must be used to capture the low velocity secondary droplets (<5 m/s) that contain the analyte of interest [ 117 ] . Nano-desorption electrospray ionization (nano-DESI) is a variant of the DESI experiment that uses liquid microjucntions to facilitate analyte desorption/extraction, transfer, and ionization for direct analysis by MS. That is, instead of using high velocity electrosprayed droplets, nano-DESI employs a large solvent droplet that bridges two capillaries positioned on top of the sample. One of the capillaries delivers the solvent droplet onto the sample and after analyte dissolution, the extract is then transferred to the mass spectrometer via the second capillary, which is also in contact with the solvent. Since splashes are eliminated in nano-DESI, it affords a higher sampling spatial resolution of ~50-100 μm [ 118 ] compared with the typical 200 μm resolution in DESI [ 119 ] and 1000 μm in liquid microjunction surface sampling probe (LMJ-SSP) [ 120 ] . Related methods like single-probe mass spectrometry have shown even better resolution of ~10 μm [ 121 ] . DESI and other related spray-based ambient ionization techniques are highly compatible with clinical studies due to their applicability to raw biofluid and tissue analysis, which give insights for disease diagnosis and prognosis. Importantly, they can be operated either in imaging or profiling modes. For example, DESI [ 122 ] and LMJ-SSP [ 123 ] are routinely applied for direct analysis of DBS samples in profiling mode, where the sampling liquid is simply scanned across the sample while the mass spectrometer collects the analyte extract in real-time. Direct tissues analysis by ambient ionization is perhaps the most impactful and active field of application in recent years. Consequently, several novel ambient ionization techniques have been developed, which will be discussed later under the tissue imaging and profiling section. In the current section, however, we focus on general metabolite/small molecule screening from biofluids and tissues using spray-based ambient ionization methods of which DESI has played significant role. For example, Pirro et al [ 124 ] used intraoperative DESI to detect gliomas and tumors in the brain and spinal cord to assess surgical margins. This not only brings DESI into the operating room, but also establishes a platform that can help identify the type of tissue, how advanced the tumor is, and monitor any mutations via the use of molecular measurements. Lee et al [ 125 ] applied a thermal DESI methodology to detect psychoactive drugs in blood and gastric fluids. In this study, the authors were able to detect sub ppm concentrations of the target analytes, concentrations that warrant immediate medical attention. Chagovets et al [ 126 ] , similarly, developed a tissue spray methodology for lipid profiling for endometriosis. Here, eutopic and ectopic tissues were successfully differentiated by the abundance of lipids belonging to three specific classes, phosphatidylcholines, sphingomyelins, and phosphoethanolamines. Ambient ionization also enables the screening of skin metabolites. In a study by Dutkiewicz et al [ 127 ] , hydrogel micropatch probes were used to sample lesioned and healthy skin and the collected sweat samples present in the hydrogel were directly analyzed using nano-DESI MS, which provided information on the alterations in skin metabolome caused by psoriasis. The hydrogel patches offered a non-invasive means of sample collection and a surface where direct MS analysis could occur. Overall, DESI and related methods are sensitive and take a fraction of the time and resources, compared to traditional methods, making it a viable and efficient alternative to be used in clinical diagnostics. DART is the most widely used plasma-based ambient ionization method. In DART, heated metastable helium gas (He*), generated from a glow discharge, is used to desorb analytes present on an ambient surface. Like the spray-based methods, sample preparation is not required for DART analysis. Instead, complex mixtures including biofluids (blood, plasma, urine, etc.) [ 128 – 130 ] , medicine tablets, and hair samples can all be analyzed directly without sample pre-treatment. DART is particularly versatile because it embodies three distinct ionization mechanisms that enable the analysis of various kinds of small molecules, including polar and non-polar species [ 14 , 129 , 131 ] . The first and predominant mechanism involves proton transfer reactions that produce (M+H) + ions after analytes interact with protonated water clusters, H + (H 2 O) n , formed from a cascade of events in the plasma. The second mechanism entails Penning ionization in which the excited state metastable helium gas accepts an electron into its ground-state atomic orbital from the analyte (M), resulting in concomitant formation of its molecular ion (M •+ ), and the release of an electron that was initially present in excited-state orbital of helium. Usually, the released electron is thermalized due to high collisions at atmospheric pressure where the DART experiment is performed, but depending on the analyte of interest, electron capture ionization is also feasible, serving as the third ionization mechanism. Other plasma-based ambient ionization methods include atmospheric pressure solid probe (ASAP) [ 132 ] , low-temperature plasma probe (LTP) [ 104 ] , and flowing atmospheric pressure afterglow (FAPA) [ 133 ] , just to name a few. What sets DART apart from all the other plasma-based methods is that ionic species in the initial glow discharge are filtered and only the uncharged He* is allowed to interact with the analyte, limiting unwanted reactions and enabling the generation of ions of interest with high efficiency. In terms of application, DART and its related methods are widely used in the field of forensics [ 14 , 134 – 137 ] . Recently, however, Augusto Gómez-Ríos et al [ 138 ] used DART for semi-quantitative analysis of drugs of abuse in oral fluids from a SPME fiber. In this case, a SPME fiber was introduced into an oral fluid sample and placed in front of the DART source for subsequent desorption and ionization of the target analytes, yielding detection limits as low as 10 ng/mL for heroin, cocaine, and methamphetamine. Fast untargeted metabolic screening was also achieved from untreated serum using transmission-mode DART [ 139 ] . Analysis of DBS by DART was recently demonstrated by Wang et al [ 140 ] , who used the ion source to detect phenylketonuria (PKU) for newborn screening. The authors used DBS samples taken from a newborn screening program and cut out each disc to perform a liquid-liquid extraction before using a DIP- it tips autosampler for MS analysis. L-phenylalanine, a marker for PKU was detected; assay was performed in as little as 18 seconds per sample, showing that DART can be used in screening studies while maintaining sensitivity and reproducibility. Similarly, direct analysis of samples present on paper substrates has also been demonstrated with LTP probe [ 141 ] . In an attempt to analyze tissue samples, a robotic arm was developed, which can pick up the tissue residues and subsequently expose the sample to the DART source [ 142 ] . With proper computer programing that correlates sample pick-up position with time of MS analysis, the approach is expected to open unique opportunities to use diffused gaseous probe ions for chemical imaging. A recent study by our group introduced functionality in ionizing analytes present in raw biofluid samples by plasma [ 143 ] . Here, in-capillary liquid-liquid extraction was used to purify the analyte during sample loading into a nano-electrospray ionization (nESI) glass capillary. Through a non-contact spray mode, a corona discharge was created in close proximity to the tip of the nESI emitter. Analytes delivered by the electrospray droplets were ionized by the plasma and subsequently transferred to the mass spectrometer for characterization and quantification. Like DART and related methods, the plasma generated in the nESI experiment enabled both polar (cocaine and caffeine) and non-polar (vitamin D 2 and β-estradiol) analytes to be efficiently detected from the same sample. The integration of the in-capillary liquid-liquid extraction reduced matrix effects and enabled ultra-sensitive detection down to pg/mL levels. A recent development in ambient ionization methodology allows for samples present on a given substrate to be extracted, ionized, and transferred to the mass spectrometer in a single step. In other words, while the desorption-based methods rely on separate desorption and ionization steps, requiring special efforts to generate a suitable desorption agent (e.g., high energy liquid droplets, plasma, or lasers), the substrate-based ambient ionization methods involve some form of an online extraction and chromatographic effect to liberate the analyte from the complex mixture. Nebulizing gases are not required, which further simplifies the experimental setup, improving field applicability. Typically, porous cellulose-based substrates (e.g., paper, thread, toothpick) are employed but the use of conductive metal blades have also been reported. Each substrate offers some unique benefits in terms of sample type, sampling mode, and integrated sample pre-treatment capabilities and while the sensitivity may differ between substrate types, they each offer the advantage of rapid turnaround times. Paper spray (PS) ionization was the first substrate-based ambient ionization method to be introduced in 2010 [ 144 ] . The ionization mechanism for this technique mimics the traditional electrospray process where ions are formed at the tip of a conductive surface. For PS-MS experiments, paper (unmodified and modified) triangles are cut and used as the ESI emitter. The sample is dried onto the triangles and upon application of an appropriate solvent and high DC voltage, analyte ions are produced and transferred to the MS. Not only does using paper substrates allow for sample storage and direct analysis, but they have the added benefit of being able to separate the collection and analysis steps. Due to its wide availability and low-cost, paper substrates can be utilized in low resource settings for sample collection and the dried biofluid present can be shipped to a laboratory for analysis. In a proof-of-concept study, Yannell et al [ 145 ] used DBS devices to collect blood samples from patients to detect imatinib, a chemotherapy drug, and its main metabolite, N -desmethyl-imatinib. After a patient pricked their finger, 10 μL of blood was collected onto a paper triangle and the dried blood samples were transported to a laboratory for direct PS-MS analysis. Imatinib was quantified on different devices between 2.5 – 60 ng/mL and N-desmethyl-imatinib between 6-100 ng/mL in whole blood. This showed that the sample collection step could in fact be separate from the analysis step and that using a PS-MS platform would be able to detect low concentrations of this drug. Aside from blood and plasma testing [ 146 ] , urine is another suitable matrix used in ambient ionization experiments. These samples can be collected non-invasively and, in comparison to whole blood, offer a simpler matrix, making analyte extractions more efficient without having to pre-treat the raw sample. Michely et al [ 147 ] coupled PS to high resolution MS/MS for toxicological screening of drugs of abuse and compared their results to LC-MS/MS data collected after common urine pre-treatment workflows. The PS high resolution methodology was able to detect 73% of the drugs, comparable to LC-MS/MS studies. This validates the use of PS-MS as an alternative strategy for drug screening while being simple, less labor intensive, and rapid. Another clinical application of PS-MS is studying enzymatic activity – changes of which can be used as biomarkers for diagnosis and prognosis. Yan et al [ 148 ] developed a protocol for studying aspartate aminotransferase (AST), an enzyme used to detect liver damage. Typically, measuring enzymatic activity requires multiple derivatization steps, but in this paper-based methodology, enzymatic reactions were initiated in Eppendorf tubes before adding an internal standard and depositing the product onto a paper triangle for PS-MS analysis. To determine enzymatic activity, the formation of L-glutamate and L-2-aminoadipic was monitored and the kinetics of the reaction at concentrations of AST representative of different physiological stages of liver damage (healthy to life-threatening) were measured. This method is not only effective in monitoring enzymatic activity but could also easily be translated for use with a miniature MS for in situ measurements. Another recent study describes a complete paper-based protocol based on a simple 3D-printed PS-MS reactor for the detecting the activity of butyrylcholinesterase (BuChE) enzyme in human serum, an enzyme that can be used to indicate degenerative diseases, liver damage, and organophosphate poisoning. Here, Yang et al [ 149 ] circumvented the common obstacles of BuChE detection, such as tedious sample pretreatment and denaturation, by performing an online reaction on the surface of paper strips and detecting the low molecular weight hydrolysis products via MS. To amplify the detection signal, the reaction strips were coated with gold nanoparticles before immobilizing the substrate, 4-mercaptobutyrylcholine, on their surfaces reaching detection limits as low as 0.0303 U/mL (units per milliliter) in serum samples. The development of this low cost, reusable, and sensitive platform opens new opportunities for future 3D printed PS-MS experiments for enzymatic activity studies and disease diagnostics. PS-MS has also been applied to differentiating between pathogenic and harmless bacteria, providing another layer to clinical diagnostics. Common methods of identifying pathogens use clinical cultures and antigen tests. Cultures can take anywhere between 2 and 3 days and can be easily contaminated, while antigen tests lack sensitivity and specificity. Chamberlain et al [ 150 ] circumvented these disadvantages by using paper to analyze both whole cells and cell lysate for two strains of mammalian intestinal bacterium Oxalobacter formigenes – a microorganism with the potential for use as a probiotic therapy for kidney stones, Figure 7A . With this ambient ionization platform, the authors were able to identify, using multivariate statistical analysis, 23 mass spectrometric features that could be used as biomarkers for these strains which could help provide insights on pathogenesis – a method that could be further modified for other clinically relevant pathogens. Newer substrate-based ambient techniques have explored the advantages of surface modifications for selective microextractions in complex matrices. One such technique, coated blade spray (CBS) [ 151 ] , uses a sword-like shaped piece of stainless steel, coated at the tip with an appropriate extractive material, as its source for generating a spray, ( Figure 8A,i ). Typically, the tip of the coated blade is dipped into a sample and vortexed to facilitate the extraction the analyte from the complex matrix, adsorbing it to the blade’s surface. After a brief washing period, a spray solvent and voltage are applied for MS analysis. Aside from the geometrical specifications for the method, stainless steel gives another cost-effective alternative to the use of paper and the total analysis time per sample can be done in under 5 minutes. For example, Tascon et al [ 152 ] used CBS to quantify voriconazole, an antifungal medication, in plasma samples. Traditional extraction and sampling methods were compared with coated blade spray – traditional methods typically require large sample volumes (300 μL) and need to be vortexed for proper extraction, while CBS only uses 10 μL samples and vortexing is not necessary before MS/MS analysis. Quantification was successful down to 0.1 μg/ mL with a total time per analysis being 120 s. Tascon et al [ 202 ] furthered their coated blade spray technology for high throughput (96-well plate) analysis for drug monitoring in urine and plasma samples. Increasing the sample size to 96 at a time allowed for the same level of sensitivity for the quantification of 18 compounds (under 10 ng/mL LOQs) occurring in a fraction of the time (55 sec for high throughout analysis vs. 120 sec for conventional CBS analysis per sample). So et al [ 153 ] demonstrated the use of surface-modified aluminum foil substrates for the analysis of polyaromatic hydrocarbons (PAHs), phosphopeptides, and proteins in biological and clinical samples. The enrichment of analytes of low polarity, a limitation of the traditional ESI process, shows that ambient ionization techniques are versatile, yet still simple in execution. Other surface modifications that have been used in recent years incorporate molecular imprinted polymers (MIPs) cross-linked to membranes. The advantage of using this type of substrate is that it creates a specific site for analyte extraction and the issue of non-uniform analyte diffusion on hydrophilic paper is eliminated, allowing for sample deposition to be done reproducibly. MIP substrates can also be operated at lower voltages than traditional PS methods, which is applicable for field analyses. Li et al [ 154 ] used molecularly imprinted membranes (MIMs) for the quantification of drugs in blood, improving sensitivity 10- to 50-fold. Similarly, Tavares et al [ 155 ] employed MIPs for cocaine analysis in oral fluid and found LOQ values as low as 1 ng/mL. The use of cartridges with MS has also been gaining popularity for automation of substrate-based techniques. Zhang et al [ 156 ] used a 3D printed cartridge with a carbon nanotube (CNT)-coated porous polyethylene spray tip to detect intact proteins – apolipoprotein c1, hemoglobin A1C, and variants of transthyretin – relevant to obesity, diabetes, and hereditary amyloidosis in plasma samples. The novelty of this method stems from the use of an antibody column to achieve selective and sensitive detection of these proteins, while incorporating protein enrichment and ionization in the same device. Needles can also be used to facilitate sampling and ionization of analytes in clinical samples for in situ experiments for solid and liquid samples [ 157 , 158 ] . One such method, touch spray (TS), uses a needle probe to sample material and upon the addition of solvent and voltage, desorbs and transfers ions for MS analysis via field-induced droplet emission ( Figure 8A,ii ). Kerian et al [ 159 ] described various applications that TS has been used for, such as tissue sampling, in vitro detection of bacteria, and trace detection of illicit drugs from various surfaces, and has shown that the rapid and reproducible sampling/ionization method has promise for further clinical diagnostic testing. Similarly, probe electrospray ionization [ 160 ] (PESI) has been employed for metabolic profiling [ 161 ] and screening studies [ 162 ] ( Figure 8A,iii ). This method utilizes a needle probe that collects small volumes of a sample from its surface before applying a high voltage to generate charged analyte species. For example, Johno et al [ 163 ] applied a PESI method to detect new biomarkers for atherosclerosis in plasma samples. The authors were able to distinguish between samples with and without atherosclerosis by combining this ionization source to machine learning, finding that cholesterol sulfate and PE 18:0/20:4 (a phospholipid) could serve as indicators for the condition independent of diet. Wei et al [ 164 ] have also incorporated needles for analyzing samples. They directly sprayed tissue samples from the tips of acupuncture needles to detect small molecules and lipids specific to human lung squamous cell carcinomas. This not only requires no sample preparation but also allows for the direct analysis of cells, enhancing the level of chemical information obtained in each spectrum. As an extension of the PS-MS platform, research groups have started to look towards fiber-like substrates for direct analysis. These newer substrate-based ambient ionization methods operate under the premise that cutting a substrate to a sharp tip, or other pointed geometries, is not necessary since there are always protruding sub-fibers on porous materials that can serve as “natural sharp tips” and from which ionization can occur. An example involves the thread spray platform ( Figure 8B ) for direct analysis of clinically relevant samples developed by our group. Here, Swiner et al [ 108 ] used cotton thread, both with and without surface modifications, for the detection of illicit drugs in blood microsamples. While the hydrophilic, unmodified surface, allowed for proper detection and identification of the targeted drug analytes to the parts-per-billion range, the hydrophobic surface modification improved the detection limit to parts-per-quadrillion concentrations in 10 μL blood samples. We showed that this ultra-sensitivity is due to a delayed extraction mechanism that is only possible with substrates like thread. That is, with the thread substrate inserted into a glass capillary, solvent evaporation is limited creating the opportunity to effectively control extraction time; it was demonstrated that an optimal extraction of 60 s is able to enrich the analyte before the spray voltage is applied. This enrichment, or delayed extraction, step is not possible with planar porous substrates like paper because under this geometry the spray solvent evaporates very quickly, necessitating that spray voltage and solvent be applied simultaneously. Performing ionization during the time of solvent application limits extraction efficiency and hence limits the ion yield. The 60 s delayed extraction step used in thread spray enable up to 80% extraction efficiency for hydrophilic compounds like benzoylecgonine (Log P : −0.59) and 60% for hydrophobic analytes such as diazepam (Log P : 2.82) when using treated hydrophobic thread substrates. Aside from the online liquid-liquid extraction capabilities, we also demonstrated that thread substrates can serve not only as a sample collection and analysis medium, but as an effective storage medium where labile compounds can be stabilized for up to 6 weeks. The ultra-sensitive abilities of the thread spray platform have the potential to enable tissue residues, mimicked by agarose beads in our study, to be collected (after pushing the thread through the sample once) and analyzed directly by MS [ 108 , 109 ] . Other materials such as cotton swabs and polymeric fibers have been found to be suitable for direct MS experiments. For example, Morato et al [ 165 ] used a swab touch spray (TS) method to detect 30 common drugs of abuse from oral fluid previously collected on the VAM device ( Figure 7B ). The touch spray platform can analyze solid and liquid samples without sample pretreatment steps. This method allows for noninvasive, in vivo sampling and a single cotton swab for both sample collection and direct analysis. Another use of cotton swabs with MS was performed for mucosal diagnostics. Here, Pruski et. al [ 166 ] used medical swabs for sample collection coupled to DESI to differentiate between bacterial species induced by pregnancy and those induced by bacterial vaginosis. This method allows for sampling from the urogenital tract, oral, and nose cavities, without having to use a matrix, like in MALDI, or be limited to cultivatable bacteria. The direct MS approach enabled the profiling of mucosal metabolites as a function of patient physiological changes. VAM was also utilized by Hect et al [ 167 ] , in a technique called “sponge spray”, to detect penicillin G, an antibiotic used to treat critically ill patients, from serum and urine samples. Other fiber-like substrates that are being used are SPME fibers. SPME originally was used as an extraction method in conjunction with analytical techniques like GC- and LC-MS, but in more recent years, the technique has been used in extraction and direct analysis experiments. The versatility of the coatings for the fibers, either commercially or homemade, enhances the extraction capabilities for analyses of analytes belonging to various chemical classes. Gómez-Rios et al [ 168 ] coupled an open port probe to a bio-SPME fiber in order to detect doping agents in urine samples. This method incorporates short extraction (≤ 5 min) and desorption (5s) times to yield detection limits under the minimum required performance levels without the need for a chromatographic separation. It is also high throughput with a moderate cost per analysis. For nonpolar analyte detection, Yang et al [ 169 ] used SPME fibers, coupled to nano-ESI, to detect OH-PAHs in the urine samples of smokers and non-smokers. Similar to other SPME methodologies, the overall analysis time is low and with detection limits as low as 0.05 ng/mL shows that the incorporation of substrate-based ionization techniques can be used as an efficient screening platform for clinical studies.

Analyzer performance (e.g., sensitivity) and stability are typically governed by sample handling, which in turn dominates the reliability of the system and validity of results. Since the primary information obtained from mass spectrometers is the mass-to-charge ( m/z ) ratio, the first step in this experiment involves the ionization of the analyte and subsequent transfer of the derived ions into the gas-phase where m/z is measured under high vacuum. The traditional methods for the ionization of liquid samples include electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) [ 43 – 46 ] . These are spray-based ion sources and are therefore subject to matrix effects. The analyzer performance is affected because of the high noise that can emanate from improperly treated samples. While high performance is desirable, the associated sample treatment, handling, and preparation steps are the main bottleneck in analytical laboratories, representing the slowest steps of the chemical analysis process and limiting turnaround times. For clinical biofluid samples (e.g., oral fluid, urine, whole blood, plasma, and cerebrospinal fluid), the sample preparation steps involve dilution with specific solutions/reagent to precipitate proteins, which can then be removed via solid-phase extraction (SPE). After protein removal, the sample is typically subjected to liquid/liquid extraction (LLE) to separate polar and non-polar components. These fractions must be dried and reconstituted into a solvent that is suitable to the analyzer of interest. SPE and LLE methods are powerful in their own regard, due to their low cost, and ability to be used in conjunction with various detection techniques. Despite promising advances in these methodologies, there are still some drawbacks, such as the lengthy protocol (the collective process can take up to 4 h) [ 47 ] , lack of adherence to green chemistry due to the use of harsh organic solvents, and large solvent consumption [ 48 ] . For this reason, the microscale counterparts are also used extensively, including solid-phase microextractions (SPME) and liquid-liquid microextractions (LLME) [ 49 ] . Recently, a much greener sample preparation procedure known as QuEChERS (quick, easy, cheap, effective, rugged, and safe) has been introduced that can afford clean extracts from complex samples (e.g., tissues and whole blood) in less than 30-45 min [ 50 ] . Originally developed for the extraction of agricultural contaminants, QuEChERS combines the two conventional extraction processes (i.e., SPE and LLE) into a robust procedure, where optimized quantities of acetonitrile solvent, inorganic salts (e.g., MgSO 4 or NaCl), and C-18 sorbent are used in a dispersive solid-phase extraction (d-SPE) process ( Figure 2 ). Different d-SPE kits are commercially available and can be optimized for improving analytes recovery [ 51 – 53 ] . Although QuEChERS is fast and extracts are typically analyzed by liquid chromatography (LC)-MS, the incorporation of centrifugal separations steps makes it hard to automate, limiting high throughput analysis. The RapidFire system offers an alternative where a robotic liquid-handling system is combined with on-line SPE for rapid mobile phase exchange and is interfaced with the mass spectrometer via conventional ESI. The unique feature of the RapidFire technology is that an automated SPE system is used in place of LC, yielding analytical results in less than 30 s from complex biological matrices. The importance of RapidFire MS in clinical settings has wide-reaching impact, from preventative measures [ 54 ] to decreasing false-positive rates in screenings [ 55 ] . For example, Leveridge et al [ 56 ] showed the RapidFire platform’s capability of acting as a screening tool for potential enzyme inhibitors to help with the control or mediation of particular neurological disorders. In the study, the RapidFire platform was used to screen potential substrates for leucine-rich repeat kinase 2 (LRRK2), which is associated with Parkinson’s disease. The RapidFire platform allowed screening of 100,000s of molecules at a time. Haslam et al [ 57 ] coupled the RapidFire platform with matrix assisted laser desorption (MALDI) MS and successfully screened for small molecules and peptides in patient samples. It was demonstrated that the use of MS as the tool of analysis is an important improvement over other analytical tools (e.g. fluorescence-based technologies) because these methods are susceptible to interferences, many analytes need to be modified for analysis, and confirmation assays are often required to validate library hits. The RapidFire based MS high-throughput screening method was compared to more traditional techniques and found that the analysis speed was approximately 1.2 seconds per sample (comparable to fluorescence-based techniques); analyte modification was not necessary, sample volume was greatly reduced, and the cost was three cents per well, indicating great improvement to what is typically done. Veach et al [ 58 ] showed that using the RapidFire platform was more than 40-times faster than conventional methods (i.e., coupling extractions to LC/MS), 21 seconds per sample vs. 990 seconds per sample, for their system of interest. While all systems entail different preparations and analyses, simplification of the methods and a reduction of steps will help to improve the accuracy and reduce analysis time. It is common practice to subject SPE/LLE and QuEChERS extracts to further separation before MS analysis. Although this adds extra time to the analytical process, it is necessary because the extracts typically contain several chemical species that can still present matrix effects if subjected to direct infusion MS analysis. One of the most common separation techniques used is LC. This analytical technique has been applied to a wide range of applications and when combined with MS, it offers excellent analyzer performance such as improved sensitivity, accuracy, and efficiency for both small organic compounds and biomolecular species. For example, in a recent study Li et al [ 59 ] demonstrated the coupling of SPE with LC-MS for enhanced detection of neurotransmitters in biofluids. The optimized SPE parameters included pH and elution solvent conditions, which ensured sufficient extraction of both catecholamines and monoamine neurotransmitters in urine samples. Through this simple and low cost SPE method, the quantification of serotonin and catecholamines was demonstrated, while reducing sample consumption, sample preparation, and overall analysis time. Aside from the analysis of small molecules, the application of LC to separate large biomolecules for subsequent detection by MS is becoming increasing popular. In this case the mechanisms of molecular separation involve affinity techniques, capillary electrophoresis (CE), and size exclusive chromatography (SEC). Each separation technique can be executed using native or denaturing conditions, depending on information needed, which may include protein 3D conformation or primary sequencing data, respectively [ 60 , 61 ] . In a specific example, Lanshoeft et al [ 62 ] used a hybrid LC-MS method to separate antibodies, circumventing laborious bottom-up MS approaches. This hybrid ligand binding assay LC-high resolution MS workflow (LBA-LC-HRMS) used an affinity capture process followed by elution of the immunoglobulins via acid dissociation with de-glycosylation for quantifying intact antibodies ( Figure 3A ). While LC-MS is a powerful tool for separating a wide array of species, its inability to offer baseline separation for large molecules, within a reasonable analysis time, using conventional bonded-phase LC columns is still a limitation. For those types of analytes, SEC is very useful. SEC separates species based on the differences in hydrodynamic volumes and filters proteins through pores of well-defined sizes. It is widely used in the biopharmaceutical industry, due to its applicability in separating high molecular weight species. In clinical oncology, being able to differentiate between classes of antibodies is vital to understanding the biology of a patient. Due to the sheer quantity of antibody formats, however, it is increasingly difficult to quantitatively measure and separate these species. SEC has been employed to deal with this issue and methods are constantly being improved to enhance the sensitivity of the chromatographic system. Goyon et al [ 63 ] found that ultra-high pressure SEC (UHP-SEC) can reduce separation times and can greatly improve peak separation and subsequent species identification for 30 FDA and EMA approved products. Additionally, the study of extracellular vesicles (EV) for disease diagnosis has grown rapidly in recent years due to the valuable information that can obtained from their contents. Given their complex composition, SEC methods have been used to separate the many species present in these samples. Lane et al [ 64 ] proposed a workflow that reduces the background from plasma, proving the importance of SEC as a viable method to enrich EVs from small volumes of complex biofluids ( Figure 3B ). Baranyai et al [ 65 ] showed that it does offer advances in the reduction of albumin impurities and an increase in the number of isolated exosomes from blood plasma, showing it’s protein purification capabilities. Recently, there has also been a push to enhance CE capabilities in the field of biological sample analysis. The usual limitations of CE, being compatible with only polar, charged species, are not an issue for biofluid samples, as these species often are highly polar or can be modified to be charged. In clinical settings, CE-MS has become a powerful tool for analyzing metabolites in complex biological samples for disease diagnosis. For example, urine is an ideal biofluid because it can be collected non-invasively, but it suffers from having a high salt concentration. Nevertheless, CE is able to separate these species from the analyte of interest. It offers high separation efficiency, compared to traditional LC, rapid analysis times, low sample consumption, and can be used for the profiling of polar and nonvolatile metabolites in complex aqueous sample matrices without pretreatment or derivatization [ 66 ] . The Maráková group looked at using CE-MS as a tool to better characterize thiopurine drugs in human urine samples [ 67 ] . Another biofluid that can be used for analysis of disease biomarkers is oral fluid, which can also be collected non-invasively. Asai et al [ 68 ] investigated working with oral fluid samples from patients with and without pancreatic cancer to see if N 1 -acetylspermidine can be used as a reliable metabolite biomarker. They found that with CE-MS, 292 metabolites could be separated and quantified from oral fluid samples and that differences between groups with and without pancreatic cancer existed. Specifically, there seemed to be a great difference between polyamines and modified polyamines (e.g. acetylation of spermine, N 1 -acetylspermidine, and N 1 -acetylspermine) between the groups. These results seem promising in establishing a potential screening biomarker for pancreatic cancer that can be obtained noninvasively. For clinical applications, high throughput diagnostic testing is directly influenced by turnaround times. LC, a technique widely used in the bioanalytical and clinical fields, has separation times anywhere from 10 minutes up to an hour. However, particularly in clinical settings, there have been many sample preparation steps that have greatly extended this analysis time, some adding hours to the total time [ 62 ] . SEC is known to be an improvement over the previously laborious and slow ultra-centrifugation methodologies, which takes hours to complete [ 64 ] , but it also suffers from the lack of ability to be used with high-throughput screening because high pressures are needed to warrant the appropriate resolution for complex matrix analyses. Like with LC, depending on column length, mobile phase composition, and analyte of interest, this type of separation can take anywhere from minutes [ 69 ] to up to an hour [ 65 ] . CE-MS is not limited by resolution, having the best resolving power of the separation techniques, but it does suffer from the fact that elution times can be up to half an hour and there is a two minute washing step required between samples [ 67 ] . To improve these separation techniques to be compatible with high throughput testing, coupling them to other sample collection and extraction techniques would be beneficial. Clinical turnaround time can be categorized into three distinct phases as illustrated in Figure 4 : pre-analytical, analytical, and post-analytical. Like the analytical phase (described in the previous section), the pre-analytical phase not only contributes to the waiting time, but it can also affect analyzer performance. In this regard, volume of sample and sample integrity during transport are the most important factors. To avoid scenarios such as low sample volumes, which can increase the time it takes to obtain results (e.g., by asking patient to come back for another round of sample collection), vacutainers that collect up to 10 mL of blood per tube are typically used. While this sample collection procedure is applicable for adults (average weight of 70 kg) having an average body blood volume of 5 L, the use of vacutainers may not be appropriate for infants (weighing ~3 kg) whose total blood volume is ~250 mL. Instead, the dried blood spots (DBS) sample collection methodology is used for newborn screening programs [ 70 – 74 ] . It is important to note that without temperature control, biological specimens degrade very quickly, accounting for >60% of all experimental variations [ 75 – 77 ] , making cold storage of vacutainers and DBS cards a requirement [ 78 – 80 ] . Recently, our group has shown that sample degradation in DBS is caused by the diffusion/absorption of the blood in the hydrophilic paper card, which predisposes the analyte to oxidative stress after a brief exposure to ambient air. To overcome these issues, the use of modified hydrophobic paper substrates was proposed [ 81 , 82 ] . That is, instead of drying as a 2D spot on the hydrophilic paper substrate, blood samples placed on hydrophobic paper bead up, due to a mismatch in surface tension, which dries to give 3D spheroid (or mold). The 3D spheroid, having a reduced surface area-to-volume ratio (compared with DBS), provides a critical radius of insulation [ 83 , 84 ] that can increase the spheroid’s resistance to thermal conduction. We have simulated a finite element analysis of thermal energy flux from surrounding ambient air for spheroid and DBS using approximate geometries and confirmed the spheroid’s enhanced thermal protection over a given time period ( Figure 5 ) [ 82 ] . Experimentally, we have observed enhanced stability for labile organic compounds like cocaine and diazepam when storing blood in the dry-state without cold storage. Other means of improving analyte stability in the dry state include lyophilization, vetrification, silk- or silk-based blood stabilization strategies [ 85 – 87 ] , all of which are labor and resource intensive. The paper-based DBS sampling method is attractive not only because it can be applied in resource-limited settings but also for the fact only micro-liter blood samples are collected, which can be expected to improve patient compliance for disease monitoring. However, inherent limitation such as hematocrit effect limit analytical performance in terms of quantification. This limitation has encouraged the development of newer microsampling techniques that overcome hematocrit effects by sampling known volumes of blood. These include capillary microsampling (CMS) [ 88 – 91 ] and volumetric absorptive microsampling (VAM) [ 92 – 95 ] . Though CMS and VAM offer efficient pre-analytical strategies, turnaround times can still be limited because the collected small blood volumes must be converted (including dilution) into a form that can be handled by traditional analyzers that are designed for large sample volumes. Therefore, there is a need to develop methods that can offer direct analysis of small volumes of complex mixture without time-consuming sample preparation.

With the development of clinical diagnostic techniques using MS on the rise, there has been a push to incorporate them for field analyses. Typical laboratory instruments are large, heavy, use gas cylinders, and have large power requirements, especially for the vacuum pump systems, so they need to be miniaturized in an effort to cut down on costs and to make them mobile. Systems like the Mini 11 [ 228 – 230 ] and Mini 200 [ 231 ] revolutionized this shift in instrumentation. While practically all types of mass analyzers have been miniaturized, the most prominent ones used are ion traps [ 232 – 235 ] . They are operated at mass ranges lower than TOFs, coupled to ambient ionization methods, and have tandem MS capabilities. There are three main types of atmospheric pressure interfaces (APIs) [ 229 ] used with mini ion traps: membrane inlet (MI), discontinuous atmospheric pressure interface (DAPI), and continuous atmospheric pressure interface (CAPI). Each of these interfaces have different power requirements and ion trap configurations which influences the types of compatible ionization sources. Commercially available mini-MS systems are provided by companies like BaySpec Inc., Torion, Advion, and Microsaic Systems and are used for a wide range of applications. In the realm of diagnostics, coupling mini-MS systems with microfluidic devices is common. These types of devices use small sample volumes, short analysis times, ESI-compatible flow rates, are low cost, and with their small overall size make for easy fitting with smaller instruments. They also offer high separation efficiencies for non-laboratory settings with the potential for automation. Microfluidic chip platforms are a powerful tool that can be used for screening studies and for online droplet processing to aid in the analysis of biological samples, specifically for cellular biology. Earlier work done in this area by Gilliland et al [ 236 ] incorporated a mini cylindrical ion trap (CIT) with a capillary electrophoresis-electrospray ionization (CE-ESI) platform for the analysis of amino acids and peptides, which can be used to gain insight into cellular function ( Figure 12A ). Peptide mixtures were detected at 7 fmol levels at a pressure of 1 Torr, showing that this platform could be an alternative to current LC-MS methods. Lab-on-a-Chip methodologies have also been implemented in the detection of pathogenic microorganisms, as described by Zhang et al [ 237 ] . Additionally, Sun et al [ 238 ] used a plasmonic gold chip platform with LDI-MS for metabolic diagnosis of early stage lung cancer patients ( Figure 12B ). These chip-based technologies offer a level of functionalization abilities and sensitivity that can be applied to multiple diseases. The droplet scale that microfluidic devices are based on also allows for online reactions to occur, similar to the ones that occur in PCR and DNA detection, before MS analysis [ 239 – 244 ] . Generally, droplets containing enzymes of interests can react with substrate-containing droplets before being emitted and ionized via ESI. Shi et al [ 245 ] described multiple ways that microfluidics can be used as microreactors for biomedical applications. For example, ELISA can be implemented with microfluidics for antigen detection. Traditionally, ELISA is performed in a 96-well microtiter plate, but this requires large sample volumes, which gives rise to long analysis times. Using a microfluidic platform not only reduces the sample volume but decreases the reaction time and allows for flexible experimental design. Overall, the use of microfluidics with mini-MS is an emerging field for clinical diagnostics. There still is work that needs to be done in analyzing various biomarkers for clinically relevant diseases before quantification studies for early detection screening can be explored. The customizability of these platforms for separations, online biological reaction screening, and analysis is promising in developing future technologies that can be implemented in the laboratory, operating room, and in the field.

While the use of MS for clinical diagnostics is continually growing there have been strides in developing new platforms that can be used at point-of-care, in the laboratory and in the operating room. The sensitivity and selectivity of the functional MS-based methodologies have allowed for comparable, if not better, detection limits than traditional methods proving that early detection screening is possible. With sample preparation and separation no longer being the limiting steps in analysis, the push for incorporating these technologies for POC testing in the field is eminent, expecting to provided results in real-time.

Immunoassays and polymerase chain reactions (PCR) are among the most common platforms by which diseases are diagnosed in a typical laboratory setting. Immunoassays are based on the binding between a specific antibody and a target antigen, the most common being enzyme-linked immunosorbent assay (ELISA) [ 170 ] . Antigens will selectively bind to an antibody, forming a complex before an enzyme-linked antibody is used for detection, typically via a colorimetric technique. There are many formats of an immunoassay, but overall, they provide great sensitivity and specificity for a wide range of analytes including small organics and large proteins. Conversely, PCR is used to make a large number of copies of short sections of DNA in order to detect particular genes of interest, specifically ones that are associated with cancer, genetic disorders, bacteria, and viral infections [ 171 ] . The most common forms of PCR are real-time PCR and reverse transcriptase PCR (RT-PCR). The main difference between the two is that real-time PCR collects data while the DNA amplification process is taking place and RT-PCR is used to amplify RNA. Though they are highly specific and sensitive, these types of laboratory testing are complicated, labor intensive, and require some degree of expertise to be executed properly [ 170 ] . The method of detection for these tests, commonly colorimetric or fluorescent-based, also has some drawbacks. Spectroscopic techniques have low peak capacity, are typically functional group sensitive, and require special control of experimental conditions. Also, the use of enzymatic reactions for colorimetric signal amplification has limitations. It is difficult to pinpoint the exact endpoint for the reaction and there are specific conditions that must be met to ensure enzymatic activity. For these reasons, coupling immunoassay and PCR to MS is attractive [ 172 , 173 ] . As a detection platform, MS is robust, sensitive, highly mass accurate, and field compatible while giving both molecular and structural information, all important advantages for clinical applications. It also removes the need to rely on a color change, which can be subjective. The most common ionization source and mass analyzer for these studies is matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). MALDI-TOF experiments are typically used for macromolecule analysis, making it a compatible ionization source to use for immunoassay- and PCR-based MS studies. Another MS system involves the use of inductively coupled plasma (ICP)-MS. This is used for elemental analysis since the mechanism of ionization uses a high temperature plasma to atomize the sample to create atomic and small polyatomic ions for detection. ICP-MS methods in this field use elemental mass tags that serve as MS reporters for immunoassay platforms, which will be further discussed in a subsequent section. Immunoprecipitation is one experiment that has been found to benefit from the cooperative integration between immunoassay and MS experiments [ 174 , 175 ] . This is a type of affinity technique that uses an antibody to isolate and precipitate a specific protein antigen from a complex sample that contain thousands of proteins. Bhattacharjee et al [ 176 ] was able to use immunoprecipitation of α-synuclein with LC-MS/MS to compare the different forms of the protein in Parkinson’s disease (PD) patients. Increased levels of α-synuclein was observed in the brain fractions with PD versus the control fractions. Scharf et al [ 177 ] used immunoprecipitation methods with MALDI-TOF for identifying autoantigens that can be used for autoimmune disease detection. Here, 14 novel autoantigens were identified that can be applied to detecting brain disorders, paraneoplastic neurologic syndromes, and autoimmune cerebellar syndromes. MALDI has also been used with a magnetic bead form of immunoprecipitation. Li et al [ 178 ] developed the Bead-Extractor Assisted ready-to-use Reagent System (BEARS) technology for plasma renin activity which is measured in cases of diseases with symptoms of hypotension and hypertension. The use of magnetic beads in this assay allowed for sampling of 96 wells simultaneously ( Figure 9A ), increasing the speed in comparison to manual methods by a factor of 4 while maintaining comparable sensitivity. Gao et al [ 179 ] developed a multiplexed immuno-MALDI-TOF MS method for targeted quantification of inflammatory markers C-reactive protein, serum amyloid A, and calprotectin. The assay allowed for acceptable detection limits (0.01-0.06 μg/mL), low sample consumption (~20 μL), and high throughput analysis (~384 samples per day). Direct detection of proteins is possible by MS however, the use of mass tags allows for signal amplification by which ultra-sensitivity can be achieved as well as reduced instrument requirements. Also, by using paper-based microfluidic platforms, that can handle raw biofluids, assay time can be dramatically reduced. In a recent study [ 180 ] , our group used wax-printed paper-based immunoassay to capture malaria and cancer antigen (CA) 125 directly from serum. Detection of the captured proteins was achieved using novel ionic probe, which was cleaved upon the application of a basic solution. Once cleaved, the ionic probes were detected via a touch paper spray ambient technique. By detecting the low molecular weight ionic probes, instead of the protein antigen, we expect this methodology to enable potable mass spectrometers to indirectly large proteins in the field. Xu et al [ 181 ] also used a sandwich immunoassay platform with rhodamine-based mass tags to aid in signal amplification for an ultrasensitive detection. This assay used aptamers immobilized on gold chip, which was in turn placed on indium tin oxide glass to capture the antigens of interest. The rhodamine mass tags were also conjugated on gold nanoparticles, and the addition of which completes the sandwich complex. Immunoassay analysis was achieved via DESI probe ( Figure 9B ), which delivers the appropriate solvent for rhodamine dissociation and ionization. Multiplexed detection was achieved with sensitivities as low as zeptomole for the target antigens, which included CA 125 (a free cancer antigen), carcinoembryonic antigens, and epithelial cell adhesion molecules in cells. Another classic example demonstrating integration of MS with immunoassay is the study by Chu et al [ 182 ] , who used laser-desorption ionization- mass spectrometry (LDI-MS) for the detection of viral infections. LDI-MS operates under similar principles as MALDI-MS, with the exception being that there is no matrix added to the sample prior to analysis. Here, metal nanoparticle mass tags were used that served as MS reporters; the metal nanoparticles were coupled to the detection antibody, which was subsequently used in a sandwich immunoassay. With laser irradiation, the metal nanoparticles undergo photoabsorption and form distinct metal clusters that can be detected with MS. The authors applied this platform to detect Enterovirus 71, Japanese encephalitis virus, and nonstructural protein 1 (NS1) from Zika virus on a cellulose acetate membrane from serum samples. Metal tags for immunoassay and analysis by ICP-MS is also becoming increasing popular [ 183 – 186 ] . While the formation of the immunocomplex is important, the mass reporter used for detection is equally as important. They must be easily ionizable and have compatibility with the antibody, or nanoparticle, used for detection. As mentioned earlier, ICP-MS is used for elemental analysis, so designing tags that have many metal atoms is viable for developing new, sensitive immunoassay-MS methodologies. For example, Hu et al [ 187 ] developed a new elemental mass tag using alkyne-DNA scaffolds and rare earth elements. The authors used PCR to obtain the alkyne-DNA chains from the normal nucleotides and then used click chemistry to metalize the strands before applying them as mass tags in the sandwich immunoassay complex. This tag was applied for the detection of carcinoembryonic antigen, a biomarker for cancers, and proved that its sensitivity and multiplex capabilities warrant further exploration in incorporating it in future ICP-MS-based immunoassay platforms. The integration of MS with PCR workflows has been seen in several research studies as a means of having a more robust detection method without the need for fluorescent or radioactive labeling. Zhu et al [ 188 ] established a platform using spermine-modified nanodiamonds (SP- NDs) to selectively enrich the oligonucleotides related to human papillomavirus (HPV). The authors were able to extract HPV genotype 16 and 18 fragments, obtained via PCR-restriction fragment mass polymorphism, with their SP-NDs and detected them via MALDI-TOF in clinical samples. This method simplifies the analysis workflow and offers a better sensitivity to what is currently used, showing that it could be applied to other virus typing strategies based on DNA enrichment. Beside DNA amplification, PCR can be used to amplify microRNAs (miRNAs) [ 185 – 191 ] , which can be used as biomarkers for cancers due to their role in many biological processes. The challenges in detecting miRNAs using traditional methods alone, like PCR, is data interpretation and the introduction of sequence-specific biases, so any effort to use direct detection methods, like MS, will be beneficial. Shi et al [ 192 ] used a duplex-specific-nuclease (DSN) enzyme-assisted recycling amplification approach to detect multiple miRNAs. In general, since this enzyme favors cleavages of DNA in a DNA-RNA heteroduplex, it can be used to recycle low abundance target miRNAs and produce large amounts of DNA fragments complementary to them – amplifying the signal for MS detection. This workflow was applied to quantify miRNA-141, miRNA-21, and let-7a in cancer cell lines with limits of detection as low as 41 pM (pmol/L). Similar studies conducted by, Kuang et al [ 193 ] applied a DSN-mediated amplification strategy for quantifying miR-200c in breast cancer stem cells ( Figure 9C ). To increase their sensitivity, streptavidin-bound agarose beads were introduced to remove excess DNA-peptide probes before LC-MS/MS analysis. This added step reduced the linear detection range to as low as 1fM (fmol/L) in these cells. A commercial genotyping platform used for cancer research for MALDI-TOF with PCR-MS is Agena Bioscience’s MassARRAY system [ 194 – 198 ] . This system provides a non-fluorescent detection method to accurately analyze PCR samples – endpoint PCR is conducted in a sample vial before transferring a small amount onto a MALDI plate for analysis. Some advantages for MassARRAY include the fast turnaround time (within a day) for analysis, low reagent costs, quantification abilities, multiplexing compatibilities, and the use of the Chip Prep Module makes this platform automated. It has been used by Pesenti et al [ 199 , 200 ] for the simultaneous detection of hotspot mutations related to both papillary thyroid cancer and gliomas, providing mutation detection at frequencies as low as 5% of all alleles. Sutton et al [ 201 ] used a multiplexed system, iPLEX HS, for MassARRAY to detect 10% more mutations than the original platform for low level somatic mutations associated with early detection for lung cancer. The versatility and the ease of execution for the MassARRAY system make it a promising avenue for future PCR-MS studies. Cooperative integration methods are beneficial to the clinical field in that they combine the advantages of traditional techniques with the high sensitivity, selectively, and robustness of mass spectrometry. The shift from using fluorescent and radioactive detection platforms to mass reporters not only enhances sensitivity, but also opens opportunities for multiplexing and field analysis. Future studies for integrating these platforms could include automation and quantification for a wide class of diseases for early detection studies.

The activities of modern society are centered on an obsessive relationship with time, where we put emphasis on speed. Therefore, turnaround times (also response times or waiting times) have become an important concept in the clinical, laboratory, and computational settings. The relationship between turnaround times and efficiency for clinics and laboratories has been well studied and its convergence (or divergence) has important consequences in providing high quality care to patients in the emergency rooms or intensive care units (ICUs). For example, turnaround time becomes a significant issue for a pediatric patient in the ICU because neonates do not have reliable clinical histories and physical symptoms may underrepresent disease severity. Another classic example involves a cancer patient undergoing surgery where accurate assessment of the extent of tumor infiltration can be challenged when using the conventional histopathologic analysis of frozen sections. Traditionally, laboratories define quality of service in terms of the precision and accuracy of the data they provide. However, to the clinician, quality of service encompasses many factors including timeliness, cost, accessibility, and test error [ 1 – 3 ] . The proliferation of point-of-care (POC) devices in clinics is a result of the high demands for short turnaround times since reports delivered on time can lead to improved levels of engagement and increased treatment efficiency [ 4 – 7 ] Despite their popularity, POC tests have limitations and might not always be the best option, especially in terms of accuracy and precision, as compared to centralized-based laboratories. Because of this, current instrumentation for laboratory testing now embodies enhanced functionality, which includes the automation of sample handling/preparation, multiplexing, data analysis, and reporting, providing scientists with a greater range of capabilities to increase the depth and speed of laboratory testing ( Figure 1 ). As an example of this shift in chemical instrumentation, consider the fact that traditional clinical applications of mass spectrometry (MS) (e.g., newborn screening [ 8 – 12 ] , analysis of drugs of abuse [ 13 – 17 ] and steroid analysis [ 18 – 23 ] ) all relied on analyzer capabilities: such as, various tandem MS modes, high resolution, and isotope ratio measurements. With the increased functionality that takes advantage of novel front-end modifications and computational capabilities, MS can now be used for non-traditional clinical analyses, including applications in clinical microbiology for bacteria differentiation and in surgical operation rooms. Specific examples include the (i) RapidFire [ 24 – 26 ] technology, which allows for high throughput analyses by automating solid-phase extraction sample preparation steps and (ii) iKnife technology that provides a clever way to sample tissue residues for direct analysis via a method known as rapid evaporative ionization mass spectrometry (REIMS) [ 27 – 30 ] , allowing for specific cancer diagnosis in real-time during surgery. The current review highlights important recent developments in MS, and how enhanced functionality has not only expanded the speed, but the scope of this analytical instrument in various clinical applications. We focus on developments in sample preparation, direct complex mixture analysis under ambient conditions, profiling and imaging capabilities, miniaturization for POC applications, and other synergistic integrations. It is worth noting that similar advancements have been made in other analytical methods, including microfluidics [ 31 ] , fluorescence [ 32 , 33 ] , multiplexed immunohistochemistry [ 34 , 35 ] and immunoassays [ 36 , 37 ] , Raman [ 38 , 39 ] , and polymerase chain reaction (PCR)-based technologies [ 40 – 42 ] . Among these methods, MS is widely used because of its molecular specificity (i.e., most other methods are functional group specific), easy hyphenation with other analytical techniques, multiplexing capabilities, and miniaturization.