Ambient Ionization Mass Spectrometry for Point-of-Care Diagnostics and Other Clinical Measurements

Christina R. Ferreira,1 Karen E. Yannell,1 Alan K. Jarmusch,1 Valentina Pirro,1 Zheng Ouyang,1,2

and R. Graham Cooks1*

BACKGROUND: One driving motivation in the develop-ment of point-of-care (POC) diagnostics is to convenientlyand immediately provide information upon which health-care decisions can be based, while the patient is on site.Ambient ionization mass spectrometry (MS) allows directchemical analysis of unmodified and complex biologicalsamples. This suite of ionization techniques was introduceda decade ago and now includes a number of techniques, allseeking to minimize or eliminate sample preparation. Suchapproaches provide new opportunities for POC diagnosticsand rapid measurements of exogenous and endogenousmolecules (e.g., drugs, proteins, hormones) in small vol-umes of biological samples, especially when coupled withminiature mass spectrometers.

CONTENT: Ambient MS-based techniques are applied in di-verse fields such as forensics, pharmaceutical development,reaction monitoring, and food analysis. Clinical applica-tions of ambient MS are at an early stage but show promisefor POC diagnostics. This review provides a brief overviewof various ambient ionization techniques providing back-ground, examples of applications, and the current state oftranslation to clinical practice. The primary focus is on paperspray (PS) ionization, which allows quantification of ana-lytes in complex biofluids. Current developments in theminiaturization of mass spectrometers are discussed.

SUMMARY: Ambient ionization MS is an emerging tech-nology in analytical and clinical chemistry. With appropri-ate MS instrumentation and user-friendly interfaces forautomated analysis, ambient ionization techniques can pro-vide quantitative POC measurements. Most significantly,the implementation of PS could improve the quality andlower the cost of POC testing in a variety of clinical settings.© 2015 American Association for Clinical Chemistry

According to the American Clinical Laboratory. Associ-ation, over 7 billion laboratory tests are performed annu-ally in the US. Nevertheless, the current emphasis isshifting toward point-of-care (POC)3 testing in nonlabo-ratory settings (e.g., clinician’s office, ambulance, insitu), to empower clinicians in making fast decisions,simplify healthcare delivery, and address challenges onhealth disparities (1 ).

Laboratory tests are often performed on blood andurine samples and employ immunoassays or colorimetricscreening (2 ) that can also be used for on-site testing.Profiling and quantification of biomolecules and syn-thetic drugs are best done by mass spectrometry (MS),usually hyphenated with chromatographic separationtechniques [e.g., liquid chromatography (LC)]. In spiteof the expense and complexity of the instrumentationand the extensive sample pretreatment required beforeanalysis, the enormous diversity of molecules detectablefrom complex biological samples—ranging from smallsynthetic drugs to intact proteins and viruses—justifiesthe key role that MS-based techniques play in clinicallaboratory testing (3 ). Hyphenated MS methods providehigh throughput, great versatility, selectivity, accuracy,and precision in analytical measurements as well as mul-tiplexing capabilities. These features often greatly exceedthose of immunoassays. However, the expense and com-plexity of the instrumentation and analytical protocolsmake the translation of current hyphenated MS tech-niques into POC testing unlikely. This is because therequirements are very different from those of laboratorytesting, especially in (a) the limited time for sample prep-aration, which precludes extraction, preconcentration,and reconstitution processes, (b) the individualized na-ture of the measurements, for which the analytical mea-surements are specific to a particular patient and dis-persed instruments are operated at low efficiency even ifthey are capable of high throughput when used in a batchmode (e.g., LC-MS/MS), (c) the physical size limitations,

1 Department of Chemistry and Center for Analytical Instrumentation Development(CAID), Purdue University, West Lafayette, IN; 2 Weldon School of Biomedical Engineer-ing and Department of Electrical and Computer Engineering, Purdue University, WestLafayette, IN.

* Address correspondence to this author at: Department of Chemistry and Center for Ana-lytical Instrumentation Development (CAID), Purdue University, 560 Oval Dr., WestLafayette, IN 47907. Fax 1 765 494-9421; e-mail cooks@purdue.edu.

Received June 17, 2015; accepted August 14, 2015.Previously published online at DOI: 10.1373/clinchem.2014.237164

3 Nonstandard abbreviations: POC, point-of-care; MS, mass spectrometry; LC, liquid chro-matography; PS, paper spray; DESI, desorption electrospray ionization; DBS, dried bloodspots; MRM, multiple reaction monitoring; DOPA, dihydroxyphenylalanine; DART, directanalysis in real time; APTDCI, atmospheric pressure thermal desorption chemical ioniza-tion; LTP, low temperature plasma; SPE, solid phase extraction; Chol, cholesterol; BA,betaine aldehyde; FDA, Food and Drug Administration.

Clinical Chemistry 62:1000–000 (2016) Reviews

1

http://hwmaint.clinchem.org/cgi/doi/10.1373/clinchem.2014.237164The latest version is at Papers in Press. Published October 14, 2015 as doi:10.1373/clinchem.2014.237164

Copyright (C) 2015 by The American Association for Clinical Chemistry

and (d) the need for analytical simplicity and automation.New developments in ambient ionization techniques andMS miniaturization (4 ) present an opportunity for trans-lation of MS technology to POC testing (5 ). The termambient ionization refers to a group of ionization tech-niques that produce gas-phase ions in the open air, re-moving chromatographic separation and minimizingprior sample preparation. Such ionization techniquespromote straightforward sample introduction and analy-sis that emphasize simplicity, low cost, and speed (6 ).

This review begins with a brief overview of ambientionization techniques. The clinical implementation andpotential of ambient ionization is illustrated by focusingon paper spray (PS) ionization, highlighting the ability toperform quantitative analysis of small molecules in min-ute volumes of biofluids. We discuss the capability ofperforming reactive ambient ionization, i.e., chemicalderivatization during ionization, to improve chemicalspecificity and sensitivity. Furthermore, the current stateof miniature MS development is described, because it is afundamental element of an ambient MS-based POC sys-tem. Finally, some challenges and future directions infurther developing ambient MS for POC applications arediscussed.

Ambient Ionization MS

Ambient ionization was introduced over a decade agowith desorption electrospray ionization (DESI) (7 ).Since then, a number of ambient techniques have beendeveloped that differ in ionization method (e.g., spray-based, plasmabased, laserbased) and in the degree towhich desorption and ionization are coupled (8 ), butthey all share the capability of generating gas-phase ionsdirectly from untreated samples, greatly reducing oreliminating analyte extraction and prior separation (9 ).Simplicity and rapid analysis are emphasized as charac-teristics of the ambient techniques (scores of which havebeen reported, as shown in Table 1 in the Data Supple-ment that accompanies the online version of this article athttp://www.clinchem.org/content/vol62/issue1), whichmake them well suited in POC applications. They all usethe sensitivity and specificity of MS, and rely on mass-to-charge ratios and/or fragmentation to acquire informa-tion on individual components of mixtures. Extensiveliterature exists on qualitative and quantitative analysis ofendogenous biomolecules and on therapeutic and illicitdrugs, performed in both a targeted and untargeted fash-ion. A selection of techniques with potential for use inPOC applications is listed in Table 1, with some detailsregarding target analytes, biological matrix, and analyti-cal methodology.

Briefly, DESI generates ions via direct desorptionand ionization using charged solvent droplets that impacta sample surface. Tissue analysis by DESI-MS, particu-

larly DESI imaging, can provide exogenous drug and/ordrug metabolite distributions (10 ). It also provides diag-nostic information on human brain cancers (11 ) anddelineates tumor margins in brain, kidney, and liver viadetection of altered lipid profiles that reflect the struc-tural composition of cellular membranes. Moving to-ward POC diagnostics, DESI has been shown to have apotential application in screening of inborn errors of me-tabolism (12 ) and therapeutic drug monitoring (10, 13 )by means of direct detection of free amino acids anddrugs in dried blood spots (DBS), respectively. In theDBS study, the DESI sprayer was moved laterally to rap-idly scan the DBS spots and detect target analytes in themultiple reaction monitoring (MRM) mode. In theseearly applications, ion suppression due to matrix effectswas highlighted as the biggest analytical challenge fordirect surface analysis of DBS by ambient MS (10, 13 ).The selection of the solvent system for the DESI sprayallows targeting of the method toward specific analytes,while other parameters like pneumatic pressure (e.g., ni-trogen) and geometry of the spray affect the efficiency ofthe desorption/ionization process and the spatial resolu-tion in the case of DESI imaging experiments (14 ). Morerecently, DESI has been used for therapeutic monitoringof salicylic acid using a 3-layer DBS paper card. In thisexperiment, blood (6 �L) was applied on a card yieldingDBS with an average diameter of 9 mm that was analyzedby DESI-MS (15 ). A linear response was achieved overthe concentration range 10–2000 mg/L, with relativeSDs �14% and a limit of quantification of 10 mg/L(15 ). In another experiment, nanospray desorption elec-trospray ionization was reported for chiral analysis (bythe kinetic method) of ibuprofen, dihydroxyphenylala-nine (DOPA), and ephedrine in DBS (13 ).

Direct analysis in real time (DART) has been ap-plied to DBS for newborn screening of phenylketonuria(16 ) and to conduct pharmacokinetic/toxicokineticstudies without additional manipulation of the samples(17 ). In DART, a discharge occurs far from the samplesurface, and a stream of heated gas is used to carry theactive species toward the sample. During transit, meta-stable helium atoms originating in the plasma react withambient water, oxygen, or other atmospheric compo-nents to produce the reactive ions (6 ).

Laser diode thermal desorption–atmospheric pres-sure chemical ionization has been used to quantify met-formin and sitagliptin in mouse and human DBS (18 ).An atmospheric pressure thermal desorption chemicalionization (APTDCI) interface has been described forprofiling of free carnitine, acylcarnitines, and sterols indried blood and plasma spots (19 ). The mechanism ofAPTDCI involves analyte desorption due to the nitrogenflow, and then analyte gas-phase ionization by APCIfrom a corona discharge. A liquid microjunction surfacesampling probe has also been applied to DBS samples

Reviews

2 Clinical Chemistry 62:1 (2016)

Tabl

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Point-of-Care Testing by Ambient Mass Spectrometry Reviews

Clinical Chemistry 62:1 (2016) 3

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Reviews

4 Clinical Chemistry 62:1 (2016)

with a chip-based nanoelectrospray infusion system (20 ).This experiment, also known as nanoDESI (nanosprayDESI), provides desorption by means of 2 small glasstubes or capillaries in series, which allows a continuousstream of solvent to be brought in contact with the sur-face of the sample.

Low temperature plasma (LTP) uses an electric fieldto generate a plasma that interacts directly with the sam-ple being analyzed to thermally desorb and ionize surfacemolecules. Biological samples or skin can be analyzedwithout electrical shock or perceptible heating, includinga proof-of-concept of detection of cocaine directly fromhuman skin reported by Harper et al. (21 ). A handheldLTP probe has also been successfully developed for min-iature MS applications and used for in situ testing (e.g.,detection of pesticide residues) but not yet applied toclinical diagnostics.

Other ambient techniques, such as atmosphericpressure solids analysis probe (22 ), wooden-tip electros-pray (23 ), and touch spray (24 ) introduced the conceptof adopting the substrate itself as the means for specimensampling and ionization, allowing for straightforwardhandling and analysis of intact biofluids, which is idealfor POC testing. Recently, touch spray analysis directlyfrom medical swabs was reported for noninvasive oralfluid analysis for qualitative detection of illicit drugs(25 ), as well as the detection of bacteria causing strepthroat via lipid profiling of pathogenic microorganisms(Fig. 1) (26 ).

PS for POC Testing

Ambient ionization MS can provide qualitative andquantitative results for clinically relevant analytes in bio-logical matrices. Endogenous and exogenous analytesmay be investigated quickly to create a quality POC assaythat answers the necessary questions for diagnosis. Qual-itative analysis by ambient ionization MS seeks to estab-lish whether or not one or more substances are present.These assays may have legal or clinically relevant thresh-olds that must be met analytically, representing the cut-offs for discrimination between undetected and positiveresults (i.e., concentrations below or above particular val-ues). Simple, straightforward, and rapid techniques, suchas spraying directly from medical swabs (25, 26 ), areideal for on-site emergency toxicological screens or road-side drug testing. Providing the patient with the best carejustifies the ranking of speed over the most precise ana-lytical result, which is often unnecessary, costly, and slow.Additionally, the ease of sample collection can translateinto better patient compliance.

Certainly, there are important questions that requirequantitative answers, and PS (27 ) is currently one of theambient techniques most developed and investigated forsuch tasks. Quantification of small analytes in complexmatrices is performed by ambient MS using workflowssimilar to those for LC-MS/MS (Fig. 2). In ambient ion-ization MS, quantification involves minimal sample han-dling, usually just internal standard addition, followed by

Fig. 1. Strep throat diagnosis.(A), Schematic of the standard on-site procedure for rapid immunoassay testing. (B), Procedure based on touch spray MS with medical swabsdescribed in Jarmusch et al. (26 ). Fig. was adapted from (26 ) and reproduced with permission from The Royal Society of Chemistry.

Point-of-Care Testing by Ambient Mass Spectrometry Reviews

Clinical Chemistry 62:1 (2016) 5

mass spectral analysis in which chemical specificity is usu-ally achieved by MRM, i.e., PS-MS/MS of particulartransitions using triple quadrupole mass analyzers. Thelack of multistep offline sample preparation and chroma-tography confers simplicity and speed. Specific examplesof analytes quantified in biological matrices by PS-MS/MS are listed in Table 1.

Certainly, the absence of chromatographic separa-tion requires alternative solutions to achieve chemicalspecificity, as the recognition of isobaric, isomeric, andchiral species is critical in many clinical applications (e.g.,identification of dextromethorphan vs levomethorphan).Ambient ionization relies only on the acquisition of MSn

data for specificity, which is based on characteristic frag-mentation patterns. However, scans that are multidi-mensional in mass (MSn) are available to efficientlyscreen drugs in an untargeted fashion or search for desig-nated classes of compounds in mixtures. Such multidi-mensional scans (e.g., precursor ion scan, product ionscan, neutral loss scan) are useful in screening knownillicit compounds, but they are also capable of screeningfor classes of compounds, as in detection of minor mod-ifications to molecules due to metabolic processes or in-tentional chemical modification (e.g., designer syntheticdrugs). These are otherwise difficult tasks in clinical–toxicological investigations (see online SupplementalFig. 1). Reactive ionization experiments described laterrepresent an alternative way of increasing chemicalspecificity.

PS ionization generates ions directly from paper sur-faces cut into a triangular shape, usually cellulose filterpaper (e.g., Whatman paper) of various thicknesses. Theproperties of the paper influence the performance of PS.Recently, Zheng et al. reported detailed methodology toprepare different types of paper substrates without com-plex surface coating procedures with the aim of enhanc-ing PS performance and facilitating its use (28 ). A fewmicroliters of biofluid (usually �10�L) is spotted ontothe paper substrate and then solvent (typically methanol,acetonitrile, or a combination of these with deionized

water and/or doped with formic acid or other modifiers)and high voltage (about 3.5 kV) is applied to the paper(13, 27 ). High voltage creates a strong electric field at thetip of the paper triangle causing field emission of solutiondroplets from which gas phase ions are generated (Fig.3A). The biological matrix (e.g., proteins and salts) inter-acts with and is partially retained in the cellulose-basedpaper, thereby minimizing the need for multistep samplepretreatment, especially when dried sample spots are an-alyzed or when coagulant agents that are not soluble inthe solvent used are added to quickly clot blood and allowfor analysis of nondried samples (29 ).

PS is simple and robust and a suitable ionizationmethod for a variety of clinical analytes measured in bi-ological fluids in less than 1 minute (27–32). In contrastto traditional chromatographic MS techniques, whichare well suited for cases for which time is less of a concernand clinicians do not need immediate test results, PS canprovide rapid optimal results in the opposite situationseven without reaching the extreme analytical perfor-mance of more exhaustive extraction and purificationtechniques.

PS has been used for direct analysis of dried samplespots, including whole blood, plasma, urine, and oralfluid, making it particularly amenable to POC testing(33 ), which would benefit synergistically from the ad-vantages of dried spot analysis (20 ) and ambient MS.Similarly to dried spot analysis, the position of the spoton the paper surface, type of paper substrate and its thick-ness, sample volume, solvent used for analysis, solventapplication, and internal standard addition are all fea-tures that need to be optimized.

Quantitative performance using PS-MS/MS hasbeen demonstrated for a range of analytes (Table 1). Theimmunosuppressant drug tacrolimus has been quantifiedby PS-MS/MS in the therapeutic range of 1.5–30 ng/mL,an assay typically performed by either immunoassay orLC-MS/MS (Fig. 3B). Accuracy and precision were com-parable to LC-MS/MS techniques (34 ). Currently, ef-forts are being made to enhance the multiplexing capa-

Fig. 2. Comparison of sample treatment, MS analysis, and data output between LC-MS/MS and ambient MS/MS for quantificationof small molecules.These are characteristic features but exceptions occur. IS, internal standard.

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bilities of PS-MS/MS because clinical testing often relieson the identification of a pattern of molecules (e.g., tox-icological screening needs to cover a wide range of com-pounds). Recently a PS-MS/MS method was developedto detect 8 traditional illicit drugs in whole blood (35 ).As PS-MS/MS develops, novel means of improving ana-lytical performance (e.g., improved accuracy and impre-cision, reduced matrix effects, lower limits of detection)have been reported, while retaining the rapid andstraightforward characteristics of ambient techniques. Ina recent study, a solid phase extraction (SPE) column wasintegrated with the paper spray cartridge to perform ex-traction and preconcentration before PS and quantifyalprazolam, atenolol, carbamazepine, diazepam, and sul-famethazine from bovine plasma. Using isotope-labeledinternal standards, limits of detection �3.0 ng/mL wereobtained (36 ). Another sample preparation method,coated blade spray ionization, which uses a solid-phasemicro extraction coated metal substrate shaped to apoint, allows sample extraction followed by direct ioniza-tion. Using this technique, Gomez-Rıos et al. were ableto detect 1.5 ng/mL of cocaine and diazepam in plasmaand urine samples (37 ). In these techniques a largeramount of sample (�100�L) is used than in traditionalPS (typically 2–10 �L). An alternative to preconcentra-tion techniques is reactive ambient ionization, in whichchemical derivatization to generate a more favorable formof the analyte ions is performed simultaneously with ion-ization, as described below.

Reactive Ambient Ionization MS

Ambient ionization allows on-line derivatization to beperformed concurrently with ionization. Reaction prod-ucts can be generated on the millisecond timescale ofionization and transferred directly to the mass spectrom-eter. The speed of the reactions is the result of reactionrate acceleration in microdroplets(38, 39 ). On-line der-ivatization has several advantages that are beneficial forambient ionization and avoid the need for any extraction,clean-up, and desalting process before analysis: (a) in-creased ionization efficiency and minimized ion suppres-sion in complex biological matrices (40 ), (b) enhancedchemical specificity to distinguish structural isomers orrecover detailed structural information, e.g., doublebond positions in lipids (41, 42 ) or peptide and proteincharacterization (43 ), and (c) enhanced structural infor-mation via MS/MS fragmentation. Reactive ambient MShas been reported for compounds with a range of func-tional groups including aldehydes, ketones, alcohols,amines, thiols, and alkenes (38 ). Table 2 lists examples ofonline derivatization reactions used in PS, DESI, andLTP and their target analytes. Other types of reactionscan also be used in different ambient techniques, likephotoinitiated reactions in plasmas and discharges.

The detection of oxidized or reduced polycyclic ar-omatic quinones, compounds implicated in carcinogen-esis and in the pathogenesis of respiratory diseases, wasperformed by reactive PS using minute volumes (2 �L) ofurine, serum, and cultured cells. A cysteamine reagent

Fig. 3. (A) Schematic of PS analysis.(B) Performance of PS-MS/MS method for quantification of tracrolimus compared with 2 immunoassay tests, and an LC-MS/MS methoddeveloped in-house and in a reference laboratory. Fig. adapted and reproduced with permission from Shi et al. (34 ).

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was used to derivatize the quinones. The products werequantified by MS/MS using just a single internal stan-dard. In urine, the limits of quantification for 1,4-naphthoquinone and 1,4-anthraquinone deposited onpaper were 1.35 and 2.64 ng (absolute quantity) (44 ).

Compounds that have permanent charges give thebest responses in MS, so charge labeling is an impor-tant class of derivatization reactions. Online chargelabeling of cholesterol (Chol) can be achieved usingbetaine aldehyde (BA) as the chloride salt to generatethe precharged ester. This reaction was first demonstratedin a DESI tissue imaging experiment (45 ), and it allowedcholesterol, which is poorly ionized in both positive andnegative ion modes, to be readily detected via its productin the positive ion mode. Derivatization of cholesterol inhuman serum was achieved using a solution of BA di-rectly by PS. Without the derivatization reagent,cholesterol-related peaks, such as the protonated mole-cule [Chol � H]� (m/z 387), its dehydration product[Chol � H [mnus] H2O]� of m/z 369, and the sodiumadduct [Chol � Na]� of m/z 409, were not detected. Butafter online derivatization, the reaction product [Chol �BA]� was observed at m/z 488 and its identity confirmedby collision-induced dissociation tandem mass spectrom-etry (45 ).

The analysis of cortisol in human oral fluid by quan-titative reactive PS-MS/MS is yet another example of theapplicability of ambient ionization in improving patientcare. Cortisol is important in the diagnosis of many dis-eases and may have prognostic value in critically ill pa-tients. The timescales for testing are wideranging, frommedical emergency to long-term hormone concentrationmonitoring (46 ). Cortisol concentrations, which plum-met as a result of an adrenal crisis, represent a medicalemergency and clearly need to be determined urgently.

By contrast, the long-term monitoring of a patient withAddison disease merely requires periodic confirmationthat the concentration falls within an expected range(47 ). In both of these cases, a semiquantitative POCdetermination that allows rapid and confident assess-ment is all that is required. As shown schematically inonline Supplemental Fig. 2, reactive PS-MS/MS can beperformed by adding Girard’s Reagent T onto Whatmanpaper to which 5 �L of oral fluid was previously spot-ted, to derivatize the ketone functional group of cor-tisol. The prechargedhydrazone product of the reactionneeds only to be desorbed for mass spectral analysis. De-tection of cortisol is achieved via MRM (m/z 476.2 �417.2) of the cortisol-Girard’s Reagent T product using atriple-quadrupole mass spectrometer. Quantification inthe nanogram per milliliter range is achieved by means ofthe standard addition method because cortisol is natu-rally present in oral fluid. The reactive PS-MS/MSmethod results in improved performance compared toPS-MS/MS with no derivatization, and is thus relevant toPOC diagnostic applications.

Miniature Mass Spectrometers

Implementation of miniature MS instruments and theirinterfacing with ambient ion sources (48, 49 ) representsan ideal combination of technologies for POC testing innonlaboratory settings (Fig. 4). Development of minia-ture MS instrumentation began in the early 1990s(50, 51 ). In the 2000s, the reduction of MS system sizecontinued until a handheld mass spectrometer was cre-ated (52, 53 ). Substantial obstacles to miniaturized MSperformance were overcome with the introduction ofimproved pumping technology, minimization of elec-tronics, and, especially, improved atmospheric sampling.

Table 2. Representative derivatization reactions for biomolecules by ambient MS

FunctionalGroups Reagents Target compounds

Ambient MSmethod

Aldehydes andketones

Girard’s reagent T, hydroxylamine,dinitrophenylhydrazine

Cortisone in oral fluid, steroid hormones [Huanget al. (72)], malondialdehyde in tissue [Girodet al. (77)]

PS, DESI

Alcohols Betaine aldehyde Cholesterol in tissue [Wu et al. (45)] DESI

Phenylboronic acid Saccharides in urine [Gomez-Rıosa et al. (37)] DESI

Amines Bis(sulfosuccinimidyl) suberate,acetone

Cross-linking of peptides containing asparagine,glutamine, arginine, or lysine, amino acids[Gomez-Rıosa et al. (37)]

DESI

Disulfides andthiols

Dithiothreitol Oxidized glutathione and insulin [Peng et al.(43)]

ELDI

Alkenes Ozone, silver nitrate Unsaturated lipids in bacteria, algae and animalpreimplantation embryos [Gonzalez-Serranoet al. (40), Zhang et al. (41), Jackson et al.(42)]

LTP, PS, DESI

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Discontinuous atmospheric pressure introduction was acritical step in this development, allowing externally cre-ated ions to periodically be introduced into the massanalyzer. Today, a range of miniature mass spectrometersis being developed and commercialized, including iontraps of various geometries (toroidal, cylindrical, rectilin-ear) (49, 50, 52, 54 ). Miniature instrumentation basedon other mass analyzers has also been developed, includ-ing TOF (55 ) and single quadrupoles, but these lackMS/MS capabilities and thus are less well suited to com-plex mixture analysis. Recently, a miniature triple quad-rupole with capabilities for selected ion fragmentationand mass analysis was reported (56, 57 ). The detectionlimits of current miniature MS systems are about 1 orderof magnitude less than an analytical lab-scale instrument;however, this difference is expected to decrease and tomatch or exceed the requirements for many clinical ap-plications (57 ). Other desirable aspects are the size,power requirements, random access, easy data analysis,and reliability of the system. Miniaturized MS for POC

measurements should be evaluated in terms of size, cost,and performance, but certainly should be highly auto-mated and robust so that staff with little or no knowledgeof MS can operate the system repeatedly, rapidly, andreliably (49, 58, 59 ). Great effort should be invested indesigning miniature instruments with integrated ambi-ent ion sources for POC systems. Commercialized andpartially automated ambient ionization MS sources existfor DESI (10 ), liquid extraction surface analysis (60 ),and DART but have been coupled only to benchtop MSinstrumentation, although the feasible coupling withminiature instruments (e.g., for DESI, desorption atmo-spheric pressure chemical ionization, and PS) has beenproved with in-house built instruments for research pur-poses (49, 61 ).

The field of ambient miniature MS-based POC sys-tems is at an early stage of development, and only a lim-ited number of techniques have been reported. First, theso-called Mini 12, a miniaturized ion trap with a discon-tinuous atmospheric pressure introduction valve (sche-

Fig. 4. Schematic of PS miniature MS POC system.RF, radiofrequency; RIT, rectilinear ion trap; DAPI, •••; DAQ, data acquisition. Fig. adapted from Li et al. (49 ) and reproduced with permissionfrom the American Chemical Society.

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matics shown in Fig. 4) equipped with PS as ambient ionsource has been used to measure amitriptyline in wholeblood at concentrations as low as 7.5 ng/mL (CV 10%),which is below the therapeutic concentration, provingthe feasibility of the systems currently available (49 ). Thesame apparatus proved suitable for detection of five syn-thetic cannabinoids in blood and urine (59 ). Zhai et al.used a continuous atmospheric pressure ionization iontrap system to identify 4 different chemicals (Gly-Pro-Arg-Pro and Met-Arg-Phe-Ala peptides, rhodamine, andreserpine at 100 �g/mL) in a complex matrix by PS (56 ).The miniature triple quadrupole with a differentialpumping interface allows quantification of small mole-cules, reaching nanogram per milliliter detection for re-serpine. Capabilities for precursor and neutral loss scans,which are well suited for searches for designated classes ofcompounds, were also demonstrated (56, 57 ).

Final Remarks

Implementation of ambient methods on miniature in-struments in clinical diagnostics has great potential forPOC testing outside the laboratory environment. Thisapproach not only simplifies the analytical process butalso reduces cost and time of analysis. Such a strategy isapplicable to toxicological screening, therapeutic drugmonitoring, studies of compliance and pharmacokinet-ics, and metabolic screening. In the latter case, POC test-ing can be created not only to identify metabolic aberra-tions indicative of disease (e.g., in newborn screeningapplications) (3 ) but also to recover individual molecularfingerprints that can be used to monitor a patient’s stateof health longitudinally. It is likely that systematic devi-ations in repeated measurements of selected biomoleculesmay reveal the early stages of diseases or physiologicalchanges. Appropriate data handling systems (e.g., multi-variate data analysis) can be developed for decision-making strategies, which do not differ much from thoseused in industrial process monitoring or antidoping con-trols used to create athletes’ biological passports (62 ).

Challenges and limitations still need to be faced.First, detection of protein biomarkers has had limitedsuccess using ambient MS. Proteins are extremely usefulfor early detection of disease with a quite a number ofprotein-based POC diagnostic tests (63 ), because pro-teins play fundamental roles in life processes. Secondly,ultratrace analysis (�ppb) by ambient MS is challenging.Blending online concentration and extraction techniqueswith ambient ionization is a strategy that is already inplace, but certainly improvements in instrumentationwill assist in reaching new limits of detection (now at pgvalues, absolute, for benchtop instruments). Couplingambient MS with ion mobility represents an additionalstrategy to improve analytical performance, providingadditional specificity without sample preparation or a

substantial increase in time of analysis (64 ). The applica-tion of 3D printing technology to MS has allowed thedesign of small and cheap ion focusing devices that can becoupled with miniature instruments and so allow ionseparation at atmospheric pressure, on top of increasingefficiency of ion transmission in air (65, 66 ). Modifica-tions to the PS substrate, like carbon nanotube impreg-nated paper, can advance the development of PS for POCanalysis by reducing the magnitude of the external volt-age needed to create an electrospray to values as low as 3V, and so address any safety concerns (67 ). Lastly, theregulatory environment for MS-based clinical assays iscurrently uncertain, and this extends to new techniquessuch as ambient ionization and POC MS. Nowadays inthe US, almost all clinical assays performed by MS aredeveloped in individual laboratories that are regulated byCenters for Medicare and Medicaid Services withoutFood and Drug Administration (FDA) review. The as-says described in this review have been developed usingresearch-only instruments because only recently have in-strument companies started registering their instrumentsas class I and class II medical devices. Moreover, the re-search is still focused on methodology and instrumenta-tion development, and only rarely has internal validationand proficiency testing been conducted. Method valida-tion and the development of shared protocols to followwill be one step needed as the technology spreads. InOctober 2014, the FDA released a draft framework forregulatory oversight of lab-developed tests, which indi-cates that the agency has plans to establish a regulatoryenvironment for MS-based assays (2, 68 ). The use ofambient MS for a number of applications (especially fo-rensics and tissue analysis) is being stimulated by thepresence of dedicated companies in this area. Companiesinvolved in POC diagnostics may be challenged by theupcoming changes in the regulatory environment, andambient MS may be implemented in future with the aidof commercial kits approved by the FDA.

Author Contributions: All authors confirmed they have contributed to theintellectual content of this paper and have met the following 3 requirements: (a)significant contributions to the conception and design, acquisition of data, oranalysis and interpretation of data; (b) drafting or revising the article for intel-lectual content; and (c) final approval of the published article.

Authors’ Disclosures or Potential Conflicts of Interest: Upon man-uscript submission, all authors completed the author disclosure form. Dis-closures and/or potential conflicts of interest:

Employment or Leadership: Z. Ouyang, Purspec Technologies Inc.Consultant or Advisory Role: None declared.Stock Ownership: Z. Ouyang, Purspec Technologies Inc.Honoraria: None declared.Research Funding: Z. Ouyang, institutional funding from NIH.Expert Testimony: None declared.

Patents: None declared.

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10 Clinical Chemistry 62:1 (2016)

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Reviews

12 Clinical Chemistry 62:1 (2016)