Applications of ambient ionization mass spectrometry in 2021: An annual review

Abstract

Ambient ionization mass spectrometry (AIMS) has revolutionized the field of analytical chemistry, enabling the rapid, direct analysis of samples in their native state. Since the inception of AIMS almost 20 years ago, the analytical community has driven the further development of this suite of techniques, motivated by the plentiful advantages offered in addition to traditional mass spectrometry. Workflows can be simplified through the elimination of sample preparation, analysis times can be significantly reduced and analysis remote from the traditional laboratory space has become a real possibility. As such, the interest in AIMS has rapidly spread through analytical communities worldwide, and AIMS techniques are increasingly being integrated with standard laboratory operations. This annual review covers applications of AIMS techniques throughout 2021, with a specific focus on AIMS applications in a number of key fields of research including disease diagnostics, forensics and security, food safety testing and environmental sciences. While some new techniques are introduced, the focus in AIMS research is increasingly shifting from the development of novel techniques toward efforts to improve existing AIMS techniques, particularly in terms of reproducibility, quantification and ease-of-use.

Abbreviations
• AFADESI
• air flow-assisted DESI
• AIMS
• Ambient ionization mass spectrometry
• APCI
• atmospheric pressure chemical ionization
• ASAP
• atmospheric pressure solids analysis probe
• 4-ATP
• 4-aminothiophenol
• BFRs
• brominated flame retardants
• CBD
• cannabidiol
• DART
• direct analysis in real time
• DBD
• Dielectric barrier discharge
• DESI
• desorption electrospray ionization
• DPA
• diphenylamine
• ESI
• electrospray ionization
• EVs
• extracellular vesicles
• GC-MS
• gas chromatography-mass spectrometry
• HCD
• hollow cathode discharge
• HDX
• hydrogen-deuterium exchange
• LAESI
• Laser ablation ESI
• LESA
• liquid extraction surface analysis
• MS
• mass spectrometry
• MSI
• mass spectrometry imaging
• nanoDESI
• Nanospray DESI
• NMR
• nuclear magnetic resonance
• OSA
• obstructive sleep apnea
• PESI
• probe ESI
• PFAS
• polyfluoroalkyl substances
• PLS-DA
• partial least squares-discriminant analysis
• PSI
• paper spray ionization
• REIMS
• rapid evaporative ionization mass spectrometry
• SAWN
• surface acoustic wave nebulization
• SESI
• Secondary ESI
• SPME
• solid phase microextraction
• THC
• Δ-9- tetrahydrocannabinol
• t-SPESI
• tapping-mode scanning probe ESI
• TWIMS
• travelling wave ion mobility spectrometry

INTRODUCTION

For decades, mass spectrometry (MS) has been an invaluable component of the analytical chemist's toolkit, providing a means of compound detection and identification across numerous areas of research. In the early 2000s, the field of MS experienced a major revolution with the introduction of the first ambient ionization techniques. Through the development of desorption electrospray ionization (DESI)¹ and direct analysis in real time (DART),² analytical chemistry entered a new age. Both of these novel techniques allowed for the desorption and ionization of analytes directly from the surface of an untreated sample. DESI uses an electrospray-based ionization mechanism and DART applies a plasma for analyte ionization. The potential of this new class of techniques was quickly realized across the analytical community.

Ambient ionization MS (AIMS) techniques enable the atmospheric desorption and ionization of sample analytes in their native environment,³ and the advantages of AIMS over traditional benchtop techniques are plentiful. Time-consuming extraction and concentration steps can be reduced or even eliminated, the removal of chromatography enables sample analysis in a matter of seconds and the nature of ambient ion sources makes them ideal for on-site analysis when coupled with portable or transportable instruments. As such, an ever-increasing number of scientific fields have realized the potential applications of AIMS, particularly in disease diagnostics, forensics and homeland security and environmental sciences. All of these fields, and many more, have been hindered by the limitations of traditional benchtop MS techniques and have the potential to be greatly enhanced by rapid, on-site analysis. AIMS-based disease diagnostics has the potential to significantly reduce wait times for test results with point-of-care testing, criminal investigations could be resolved faster by the analysis of forensic evidence at the crime scene and the on-site analysis of environmental samples could be achieved without the cumbersome shipment of samples from remote locations.

Following the inception of AIMS almost 20 years ago, the field of ambient ionization has rapidly expanded. Each year brings novel techniques, improvements in existing methodologies and exciting new applications as increasingly more fields of research discover the potential advantages of AIMS. This review will provide an overview of AIMS applications throughout 2021 and acts as a continuation to the previous review on literature published in 2020.⁴ Search terms included ‘ambient ionization’ and individual AIMS technique names using the PubMed database during October and November 2021. A brief overview of the techniques covered in this review is included in the subsequent sections, followed by the detailed coverage of recent applications in a number of key fields of research.

AMBIENT IONIZATION MS TECHNIQUES

Since the development of the first ambient ionization techniques, dozens of variants have been developed and shared across the analytical community.⁵ Despite the broad range of techniques available, all AIMS technologies can typically be divided into three broad classes. Many popular AIMS techniques fall into the class of solid-liquid extraction techniques, which involves the extraction or desorption of analytes from a sample surface followed by ionization, typically using a charged spray such as in electrospray ionization (ESI). Plasma-based ionization techniques use an electrical discharge to produce reactive ions to facilitate ionization, with ionization mechanisms akin to those occurring in atmospheric pressure chemical ionization (APCI). Laser-based techniques utilize an UV or infrared laser to facilitate sample analysis, using highly focused lasers to ablate and desorb analytes from a sample.

As one of the first ambient ionization techniques to be developed, DESI is undoubtedly amongst the most renowned and well-developed AIMS methods. DESI utilizes a steam of charged microdroplets that collides with the surface of the sample, causing the production of secondary droplets containing desorbed and ionized analytes.¹ With the DESI system positioned directly in front of the mass spectrometer inlet, ions are drawn into the MS for detection. Over time, various modifications have been made to DESI. Several recent studies have also utilized air flow-assisted DESI (AFADESI), a method incorporating a high-flow air stream intended to supplement extraction of analytes and facilitate the transportation of droplets containing desorbed analytes over longer distances.^6–9 Nanospray DESI (nanoDESI) employs two small capillaries to form a liquid micro-junction on the sample surface and a nebulizing gas to produce charged droplets, enabling localized liquid extraction of materials.¹⁰ Since their conception, DESI and nanoDESI are now amongst the most commonly utilized techniques for MS imaging (MSI).¹¹

Paper spray ionization (PSI) has become a particularly popular tool due to its low cost and the ease of use. PSI utilizes a triangular paper substrate positioned in front of the MS inlet onto which a small volume of liquid sample is added, followed by a spray solvent.¹² Upon application of a high voltage to the paper, ESI occurs at the pointed tip of the substrate, producing ions via a similar mechanism to traditional ESI. The primary benefit of PSI over other electrospray-based AIMS techniques is the use of the paper substrate, which acts as a filter to retain certain components of complex biological matrices, such as whole cells or other large biological molecules, which could cause matrix effects. In 2015, Kim and Cha developed a modification of PSI referred to as cone spray ionization.¹³ This technique is essentially a three-dimensional version of PSI, consisting of a paper cone as opposed to the flat triangular paper substrates typically employed. Whereas paper spray generally requires the addition of a liquid sample to the paper substrate prior to analysis, cone spray enables the analysis of untreated solid material, which can be simply added into the interior of the cone. A spraying solvent is added to the tip of the cone and a high voltage is applied to induce electrospray.

An alternative ESI-based AIMS technique gaining increasing popularity is probe ESI (PESI), first developed by Hiraoka et al. in the mid-2000s.¹⁴ The technique uses a grounded solid needle which is touched to the surface of a solid or liquid sample, causing the transfer of analytes to the tip of the needle. The needle is then positioned in front of the MS inlet, either manually or via an automated system, and the application of a high voltage results in the formation of electrospray from the needle tip. The technique offers a simple means of sample analysis without the need for spray solvents and can be purchased as a commercially available product or easily constructed. Another probe-based ESI technique is pressure probe ESI, or PPESI. This technique was originally designed to assess cell characteristics, such as hydration and pressure, but its use as an ambient ionization tool was soon realized. The probe consists of a microcapillary controlled by a piezo manipulator. This enables the tip location and penetration depth to be finely controlled, to the extent that individual cells may be sampled.¹⁵ The tip of the capillary penetrates the sample, trapping a small volume in the microcapillary tip. As the tip is moved from the sample to the MS inlet, a high voltage is applied to an internal electrode, inducing analyte ionization.

Liquid extraction surface analysis (LESA), developed by van Berkel et al., enables analyte extraction via the formation of a liquid junction between the sample surface and a conductive pipette tip.¹⁶ The solvent is repeatedly re-aspirated against the sample surface for several seconds before the pipette tip is robotically repositioned in front of the MS inlet where electrospray occurs. The technique enables rapid and automated surface sampling and has been shown to be particularly promising in the imaging of biological tissues. The technique has recently been further developed into microLESA, which was developed to reduce the sampling area targeted by traditional LESA.¹⁷ Somewhat similar to LESA is the MasSpec Pen, which also uses a liquid-solid extraction process for ambient sampling.¹⁸ During the operation of the MasSpec Pen, the solvent is drawn through an easy-to-use handheld device which is manually placed in contact with the sample surface for analyte extraction. The solvent containing extracted analytes is then drawn up toward the mass spectrometer for ionization and analysis. Finally, some AIMS techniques have been developed with gas-phase analytes specifically in mind. Secondary ESI (SESI) is an AIMS technique specific to the analysis of gaseous samples and vapours. It utilizes two sprayers positioned in front of the mass spectrometer inlet, one of which introduces gas-phase analytes and the other a spray solvent to produce charging agents. As the two sprays collide, charge is transferred and ionization occurs.¹⁹

Although solid-liquid extraction techniques account for a large proportion of AIMS applications (see Table 1), plasma-based techniques are an alternative popular choice. This class of ambient ion source uses electrical discharges to produce plasma containing electrons, metastable species and radicals to induce ionization. Perhaps the most widely utilized is DART, one of the pioneering ambient ionization techniques develop by Cody et al. in the early 2000s.² The DART ion source applies an electrical discharge to a stream of inert gas, typically nitrogen or helium. This results in the formation of electronic or vibronic excited metastable species which undergo various ion-molecule reactions, either with atmospheric reagent ions or directly with analyte molecules. DART is a particularly versatile technique that can be readily applied to solid, liquid and gaseous samples alike, and is one of the few AIMS techniques to be commercialized.

TABLE 1 Summary of techniques covered throughout this review

Technique	Abbreviations	Classifications
Air flow-assisted desorption electrospray ionization	AFADESI	Liquid extraction
Atmospheric pressure chemical ionization	APCI	Plasma
Atmospheric pressure solids analysis probe	ASAP	Plasma
Coated blade spray	CBS	Liquid extraction
Cone spray ionization	CSI	Liquid extraction
Direct analysis in real time	DART	Plasma
Dielectric barrier discharge	DBD	Plasma
Desorption electrospray ionization	DESI	Liquid extraction
Hollow cathode discharge	HCD	Plasma
Laser ablation electrospray ionization	LAESI	Ablation
Liquid extraction surface analysis	LESA	Liquid extraction
MasSpec Pen	N/A	Liquid extraction
Nanospray desorption electrospray ionization	nanoDESI	Liquid extraction
Open port sampling interface	OPSI	Liquid extraction
Paper spray ionization	PSI	Liquid extraction
Probe electrospray ionization	PESI	Liquid extraction
Picolitre pressure probe electrospray ionization mass spectrometry	picoPPESI	Liquid extraction
Rapid evaporative ionization mass spectrometry	REIMS	Other
Surface acoustic wave nebulization	SAWN	Other
Secondary electrospray ionization	SESI	Other
Thread spray ionization	TSI	Liquid extraction
Tapping-mode scanning probe electrospray ionization	t-SPESI	Liquid extraction

Aside from DART, there are also multiple options for homemade plasma-based ambient ion sources that can be designed and modified to suit the needs of a particular experiment. Dielectric barrier discharge (DBD) ion sources utilize two electrodes, one a stainless-steel needle or wire and the other a copper strip separated by an insulating barrier such as glass.²⁰ Upon application of a high voltage between the two electrodes, a low-temperature plasma forms that can be applied directly to the surface of liquid or solid samples, or into which gas-state samples can be introduced. DBD ion sources consist of very few components and can be constructed very cheaply compared to commercialized sources. Numerous similar plasma-based ion sources have also been developed with slightly different geometries and mechanisms, such as low-temperature plasma and hollow cathode discharge (HCD).²¹

Finally, the atmospheric pressure solids analysis probe (ASAP) consists of a sampling tool to collect material for direct exposure to an APCI source.²² The technique is easy-to-use, requiring the operator to simply dip the tip of the probe into the sample before inserting the assembly into the mass spectrometer. A heated stream of gas desorbs volatile and semi-volatile analytes from the surface of the probe and subsequent ionization via APCI occurs. The device can be readily applied to solid or liquid samples, however, unlike other plasma-based ambient ionization sources, is not suited to direct surface analysis.

Electrospray and plasma-based techniques are by far the most commonly utilized in the field of ambient ionization, though alternative techniques incorporate laser ablation for analyte desorption and ionization. Laser ablation ESI (LAESI) ablates the surface of a sample using a mid-infrared laser.²³ This produces a plume of desorbed molecules which are subsequently ionized by charged solvent droplets produced by electrospray. Diode lasers have also been evaluated as a means of sample desorption for AIMS applications. Diode lasers are compact, cost-effective and do not require a high power input to operate, thus, are well suited to use in ion sources intended for use outside of the laboratory.²⁴ Techniques employing diode lasers operate in a similar fashion to LAESI, directing the laser at the sample surface to induce ablation prior to ionization by an electrospray plume. Although less commonly utilized, the primary advantage of laser-based techniques is the ability to perform highly focused sampling, particularly ideal for MS imaging purposes, and the ability to decouple the desorption and ionization processes.

Finally, rapid evaporative ionization MS (REIMS) is an ambient ionization technique somewhat different to other techniques, and as such, does not fit within the primary aforementioned classes. REIMS utilizes a type of electrocautery knife akin to those used in surgery for the removal of biological tissues. The knife causes vaporization of the sample, producing a plume containing gas-phase analyte ions which are drawn into the mass spectrometer. REIMS was specifically developed for the real-time analysis of healthy and cancerous tissues during surgery, but has since found its place in the analysis of a wider range of samples.²⁵

The diversity of ambient ionization techniques extends far beyond those covered in this section, which solely represents techniques used in the studies explored in this review, as summarized in Table 1 and Figure 1. The subsequent sections will discuss applications of AIMS in a number of key fields of research throughout 2021, alongside the highlighting of key technological developments within the field.

APPLICATIONS OF AMBIENT IONIZATION MASS SPECTROMETRY Biomedical applications: Disease diagnostics

Disease diagnostics is undoubtedly one of the most common applications of AIMS. A major bottleneck in medical treatment is the timely diagnosis of disease and infection, which often relies on time-consuming laboratory analyses such as blood tests and tissue biopsies. Ever since the introduction of ambient ionization, there has been a concerted effort to develop rapid diagnostics based on disease-induced changes in the human metabolome.

It comes as no surprise that a primary clinical application of AIMS research is in the field of cancer. The ability to identify biomarkers associated with cancer and furthermore develop strategies for the rapid detection of such biomarkers would potentially be a game changer in the field of cancer diagnostics. For example, the development of diagnostic and prognostic strategies for prostate cancer has been a matter of great interest due to the high level of risk it poses within the adult male population. Ambient ionization is an attractive tool for this purpose, offering the potential to develop rapid techniques to detect and categorize prostate cancer without the need for invasive medical procedures. In a recent study by Mahmud et al., PSI was employed in the analysis of urine to differentiate between healthy controls and patients with prostate cancer.²⁶ Following the direct analysis of raw urine, multivariate analysis demonstrated metabolic profiles specific to the urine of prostate cancer patients. Partial least squares-discriminant analysis (PLS-DA) enabled not only the differentiation between the urine of healthy controls and prostate cancer patients, but also patients of different disease states as classified by the Gleason score, the standard grading system for prostate cancer classification. The technique used and the cancer-specific biomarkers identified could serve as the basis for new diagnostic strategies for prostate cancer categorization. Numerous other AIMS techniques have been applied to detect cancer-related biomarkers in biological samples, including PESI for the detection of colorectal liver metastasis²⁷ and DESI for prostate cancer and lymphoma detection.^28,29

Not only can AIMS be used for identifying cancerous tissue based on unique metabolic profiles, it can also be used to gain insight into the biological environment of malignant growths. Song et al. developed a DESI-MSI hydrogen-deuterium exchange (HDX) method to study the acidic tumour microenvironment to measure the pH of the tumor region, a factor which crucially affects the progression and malignancy of a cancerous growth.³⁰ With this technique, the HDX of metabolites could be observed by incorporating D₂O into the DESI spraying solvent. Species carrying hydroxyl groups respond particularly well to the HDX process and can be used as pH indicators based on the extent of the HDX. In the case of this study, choline was selected as the indicator. Cancer cells have been found to grow and survive in more acidic environments, with varying pH influencing the metabolic phenotype of different cells, thus, the ability to explore this space could provide valuable insight into cancer progression and invasion.

Though clinical applications of AIMS are often applied to whole sections of biological tissues, some studies have focused on the analysis of single cells. An active capillary DBD ionization system was recently developed for the high-throughput analysis of single cells, with a particular focus on pancreatic cancer cells.³¹ The setup consisted of a capillary through which cell suspension was introduced before reaching the DBD ion source, with ionization occurring within the capillary. The output of the capillary was directly connected to the MS inlet. This technique enabled the high-throughput metabolic profiling of single cells at a rate of 38 cells per minute, producing unique mass spectra that enabled the differentiation of cell types. Furthermore, through studying the lipid profile of pancreatic cancer cells, signs of abnormal lipid metabolism in pancreatic cancer cells could be observed.

The application of AIMS in clinical diagnostics has largely focused on the development of cancer detection strategies, however, other diseases and conditions of interest have also been examined. In a recent study, ambient ionization was utilized to map metabolic networks in the brain with the aim of understanding the role of metabolic network dysfunctions in diseases such as Alzheimer's.³² AFADESI was used to image metabolites throughout the rat brain, resulting in the identification of a range of metabolite classes and metabolites related to specific pathways such as the purine metabolic pathway and NT-regulated metabolic pathways. The methodology was furthermore applied to the analysis of brain sections from a scopolamine-treated Alzheimer rat model to demonstrate the ability of the technique to detect metabolic dysfunctions. Zhang et al. explored the potential of ambient ionization to image metabolic signatures in the brain, specifically to better understand the lipid metabolism dysfunction associated with Alzheimer's disease.³³ DESI-MS was applied to the brain tissue of an Alzheimer's mouse model, detecting significant changes in a range of compounds including fatty acids, cholines, glycerides and phosphatidylethanolamines. Although based on a murine model, the study demonstrates the use of DESI in exploring lipid metabolic pathways that could be associated with Alzheimer's disease. Similarly, AIMS has also been applied to study the metabolic composition of brains from a murine model of Parkinson's to study amino acid neurotransmitters in animals suffering from neurodegenerative diseases.³⁴ The study combined a Katritzky reaction-based derivatization protocol with PSI. This incorporated a derivatization solution sprayed onto the biological tissue prior to analysis by PSI, facilitating the ionization of amino acids. Derivatization using the Katritzky reaction resulted in a 10-fold increase in amino acid concentration, enabling the detection of gamma-aminobutyric acid and glycine, two compounds of particular importance in the study of Parkinson's disease. Finally, Pruski et al. used DESI-MS for the analysis of vaginal swabs from pregnant patients to study the contribution of the vaginal microbiome to pre-term birth risks.³⁵ The study discovered that specific metabolite signatures could predict both the composition of the vaginal microbiome in addition to host inflammatory status, linked with the risk of pre-term birth. This demonstrates a unique approach to classifying pre-term birth risks, crucial in the selection of appropriate preventative treatments.

In the development of rapid diagnostic tests, there has been a particular push toward non-invasive techniques, with exhaled breath becoming a popular choice for research efforts. Obstructive sleep apnoea (OSA) is a respiratory disease associated with various metabolic and cardiovascular problems. It is a particularly underdiagnosed disease, and the procedures that are available for diagnosis are expensive and time-consuming. SESI-MS was recently applied to the analysis of exhaled breath in a cohort of suspected OSA patients.³⁶ A classification model was built using data from a previous study as a training set, and was subsequently applied to data from this independent validation study. Based on the analysis of breath from patients and healthy controls, metabolic differences were detected associated with the disease, in addition to biomarkers related to disease severity. The study concluded that SESI coupled with high resolution MS could offer an OSA screening method substantially faster than current diagnostic strategies. SESI permits real-time breath monitoring and substantially faster analysis when compared to traditional breath analysis methods which involve collection of exhaled breath onto sorbent materials, or into Tedlar bags, prior to analysis by gas chromatography-MS (GC-MS).

Although techniques, such as SESI, are more widely used in the ambient ionization of breath-borne metabolites, a recent study by Li et al. developed a paper spray-based method for the analysis of exhaled breath.³⁷ Participants were asked to provide exhaled breath samples into Tedlar bags and a gas-tight syringe containing treated test paper was used to extract a small volume of exhaled breath from the bags, enabling gas-phase analytes to be captured by the test paper. The paper substrate was then retrieved and analysed directly via the application of a high voltage to the paper in front of the MS inlet. This technique was developed to specifically enable the analysis of aldehydes in breath, which have been highlighted as potential biomarkers for lung cancer. The extraction is based on the capture of gas-phase aldehydes via the Schiff base reaction, induced by the presence of 4-aminothiophenol applied to the paper substrate prior to sample collection. The novel technique was applied to the exhaled breath of lung cancer patients and healthy volunteers, showing a significant increase in several aldehydes in the breath of cancer patients, in line with previous research in this area. This study demonstrates an interesting new method for exhaled breath analysis, though the need for the reactive 4-aminothiophenol treatment may limit the analysis to specific compounds.

Finally, given the recent COVID-19 pandemic, it is not surprising that the creation of rapid diagnostics for detection of SARS-CoV-2 infection has been at the forefront of clinical research. In a recent study, a modified version of the MasSpec Pen was used for the direct analysis of nasopharyngeal swabs collected from COVID-19 positive patients and healthy controls, sampling a total population of 244 individuals.³⁸ Using the mass spectral data generated, particularly lipid profiles, two models were developed. The first aimed to differentiate between symptomatic PCR-positive patients and asymptomatic healthy individuals, and the second to distinguish symptomatic PCR-positive patients from symptomatic PCR-negative patients. The two models exhibited accuracies of 83.5 and 78.4%, respectively, demonstrating the potential for rapid MS-based strategies for the identification of patients suffering from COVID-19 and similar diseases.

The development of AIMS techniques for disease diagnostics or the detection of diseased tissues has naturally extended to tools intended for real-time in vivo use by surgeons in the operating room. REIMS, also known as the Intelligent Knife or iKnife, was specifically designed with the operating room in mind, and has seen increased use for various surgical applications. A recent study applied REIMS to the analysis of human aortic tissue to differentiate between healthy tissue and tissue from patients suffering from thoracic aortic aneurysms.³⁹ During thoracic surgery, surgeons will often make intraoperative decisions based on the appearance of the aorta, a potentially subjective practice. A standardized method to chemically differentiate healthy and unhealthy tissue would reduce, if not remove, this subjectivity. In this study, mass spectra were collected from samples of aneurysmal human thoracic aorta tissue and healthy aorta tissue (samples from 44 and 13 patients) and analysed by REIMS, following which PLS-DA models were developed to differentiate between the two sample types. The model was able to discriminate aneurysmal from normal tissue with an accuracy and precision of 88.7 and 85.1%, respectively, and furthermore differentiate tissue from patients with different aortic valve diseases (bicuspid or tricuspid aortic valve). This demonstrates a further potential application of REIMS in the operating room, in addition to, the most common application of cancerous tissue detection.

Manoli et al. integrated REIMS with the Harmonic scalpel, a next-generation laparoscopic surgical tool that uses ultrasound to dissect tissues.⁴⁰ This was applied to the direct analysis of various porcine tissues, with multiple points sampled across the surface of the tissues. Tissue-specific lipid profiles were produced, enabling the differentiation between tissues from the muscle, liver, colon and small intestine with an accuracy of 100%. The study also evaluated the use of REIMS with other electrosurgical dissection tools, showing distinct mass spectral differences from samples depending on the type of tool used, believed to be due to different mechanisms of aerosolization and droplet formation. Whereas the use of the Harmonic scalpel resulted in high intensities of diglycerides and triglycerides, other surgical tools produced high abundances of glycerophospholipids. Chen et al. developed a string sampling probe designed to be incorporated into endoscopes to perform in vivo chemical analysis during endoscopic procedures.⁴¹ The device consists of medical-grade silk moved by a stepping motor which places the probe in contact with a surface of interest during the procedure (Figure 2). This contact allows analytes from the surface to adhere to the string and be transported to the ion inlet tube, where analytes are subsequently ionized by an electrospray ion source. In this study, the new technique was demonstrated with the in vivo analysis of gastric mucosa of a mouse using a gastrointestinal endoscope two meters in length. Finally, Fu et al. developed a new method referred to as coupling ultrasonic sputter desorption for the in vitro and in vivo analysis of biological tissues, demonstrating the use of the technique with liver, brain, kidney and lung tissues.⁴² The technique promises to be another potential tool for in situ analysis of biological tissues. Whereas the majority of AIMS techniques established for the operating room are intended for manual use by the surgeon, technologies have also been developed for use with surgical robots such as the Da Vinci robot. In a recent study, SpiderMass technology was coupled with a robotic arm for the topographical imaging of biological tissue.⁴³ This enabled the production of 3D topographical images of various biological matrices including the whole body of a mouse during a post-mortem examination.

FIGURE 1. Percentage of papers covered in this annual review using each AIMS technique (above) and percentage of papers in specified fields of research (below)

FIGURE 2. Photographs of the string sampling probe, a device developed for in situ sampling during endoscopy procedures. Reprinted with permission from Chen et al., 2021.41 Copyright 2021 American Chemical Society

While many AIMS applications intended for use in the operating room have been developed, research has now reached a crucial point where the new technologies can be ‘field-tested’ during real operations. In a recent study by Brown et al., a mobile DESI-MS system was developed for the classification of diffuse gliomas in the human brain.⁴⁴ Diffuse gliomas are the most common type of malignant brain tumor and often result in a poor prognosis. Accurate diagnosis of suspected diffuse glioma is essential for ensuring the development of appropriate treatment strategies to improve patient outcomes. In this study, the MS system was specifically developed for use in the operating room and used to analyse small tissue biopsies from a cohort of 49 patients with suspected diffuse glioma undergoing tumor resection surgery. Using statistical modelling to classify tissue samples, the study demonstrated assessment of isocitrate dehydrogenase mutation (an important enzyme linked to glioma development) with a diagnostic accuracy of 94%. The method was also used to estimate tumor cell infiltration, which it achieved with an accuracy of 83 and 81%, depending on the chemical target of the analysis. Various other laboratory-based AIMS techniques have also been applied to the classification of brain tumor tissue including DESI,⁴⁵ REIMS,⁴⁶ PESI⁴⁷ and LESA alongside 3DOrbi SIMS.⁴⁸

In another study in which MS was extended to the operating room, King et al. demonstrated the use of the MasSpec Pen for the rapid assessment of tumor tissue during pancreatic surgeries.⁴⁹ The hand-held device was utilized both in the laboratory with a collection of banked human tissues and in the operating room during 18 pancreatic surgeries to characterize the metabolic and lipid profiles of dozens of tissue biopsies. Following the development of a classification model, in vivo analysis achieved a 93.8% overall agreement with post-operative pathology reports, demonstrating the potential suitability of the MasSpec Pen for use in evaluating suspect tissues mid-surgery.

Biomedical applications: Drug monitoring

The biomedical applications of ambient ionization techniques have largely cantered around disease diagnostics, though their use in real-time drug monitoring has also emerged. Therapeutic drug monitoring involves the measurement of specific medications in the body, and is typically conducted to evaluate whether or not appropriate levels of active ingredients are being reached to confirm that a patient has been taking prescribed medications, and to assess potential cases of drug efficacy as well as potential patient overdose. For this reason, ambient ionization techniques would be well-suited to the development of techniques for the rapid real-time monitoring of drugs in the body.

Propofol is a commonly used drug for general anaesthesia and, therefore, maintaining suitable concentrations in the body through the duration of medical procedures is critical. Recently, a PSI strategy was developed to enable the rapid detection of propofol in whole blood.⁵⁰ Although the method necessitated some sample preparation in the form of vortexing the whole blood with methanol prior to application to the paper substrate, an overall analysis time of less than 2 minutes per sample was achieved, providing a suitable method for rapid near-real-time drug monitoring. Following laboratory validation with spiked samples, the technique was furthermore applied to blood samples retrieved from three patients undergoing anaesthesia. When MS quantification was compared with the pharmacokinetic models typically used for establishing target blood drug concentrations, it was found that detected concentrations were consistently higher than predicted concentrations. This was attributed to individual patient differences that are not taken into consideration in traditional models, a weakness that could be resolved by mass spectrometric analysis.

DESI-MS imaging has also been utilized for therapeutic drug monitoring. Most recently, it was used to monitor the brain distribution of fluoropezil, an acetylcholinesterase inhibitor development for Alzheimer's disease.⁵¹ Following drug dosing, brain slices were obtained 1 and 3 h after administration and subjected to analysis by DESI-MS coupled with ion mobility spectrometry to reduce background and matrix effects. Imaging of the brain showed the rapid diffusion of fluoropezil across the entirety of the brain, with a particular concentration in the striatum. This was confirmed by LC-MS/MS, demonstrating DESI-MS to be a suitable alternative technique to traditional methods for the determination of drug concentrations and distribution within the brain. Although not suitable for monitoring drugs in living patients, the technique could still provide valuable insight into the biological distribution of drugs following administration and could be considered for the potential of measuring other drugs or compounds in a post-mortem setting. Yamamoto et al. have also evaluated the use of DESI for drug monitoring in rat models.⁵² The authors used the technique to image the spatial distribution of the corticosteroid ciclesonide in the lungs of rats following dosing via inhalation. Imaging revealed the presence of differential distribution of ciclesonide and its metabolites throughout the airway and lungs. The study revealed that the primary drug was largely localized to the airway epithelium, whereas metabolites were distributed throughout the tissues of the lung. The use of DESI to image inhaled drug distributions in this manner offers a valuable tool for studying and improving the efficiency of particulate drug delivery.

Whereas therapeutic drug monitoring applications of AIMS typically use blood, or in some cases, biological tissues, a recent study by Chen et al. used exhaled breath to monitor the pharmacokinetic profile of the drug venlafaxine, a widely utilized anti-depressant.⁵³ This drug has been found to exhibit a large variability in efficacy across patients, thus, necessitating individual-specific dosing. Using SESI-HRMS, real-time monitoring of exhaled breath of mice was performed to measure the presence and abundance of venlafaxine in the breath, which was detected immediately after administration of the dose. Although venlafaxine is not volatile, it could still be detected via exhaled breath particles rather than as a gaseous molecule. For validation, blood was also analysed and concentrations across biospecimens were found to be in good agreement with one another. Although a mouse model was used in this instance, the research does demonstrate the potential for SESI to be used for non-invasive real-time drug monitoring in the breath of humans to assess inter-individual drug dosage needs. In a similar fashion, another study utilized SESI-MS to monitor exhaled breath metabolites in human participants following the ingestion of a peppermint oil capsule, demonstrating the ability to study washout profiles of metabolites over time.⁵⁴

AIMS has also been utilized to monitor the spatial distribution of drugs across the skin. Using DESI-MSI, the cutaneous spatial distribution of two topical drug creams applied to porcine skin was imaged, demonstrating heterogeneity in the distribution of clinically equivalent formulations.⁵⁵ This indicates AIMS could provide valuable insight in the development of skin medicinal products to ensure different batches and brands of the same drug are achieving the same distribution of active ingredient.

Forensics and security

The time-sensitive nature of forensic analysis has resulted in a broad effort to develop rapid analytical tools that could be used for on-site analysis of potential forensic evidence. The development of such techniques has the potential to reduce laboratory backlogs, rapidly provide police forces with crucial information and ultimately speed up the completion of investigations. Furthermore, traditional analytical techniques, such as GC- and LC-MS, often necessitate sample destruction, which can be particularly problematic in criminal investigations where evidence preservation is of the utmost importance.

The two primary areas of application of AIMS in forensic science are drugs and explosives. In the analysis of illicit drugs using AIMS, PSI has been a particularly popular choice of method, in addition to the implementation of various modifications to improve the applicability of the technique to forensic evidence. Huang et al. used silver-impregnated paper as the substrate to semi-quantitatively detect Δ-9- tetrahydrocannabinol (THC) and cannabidiol (CBD).⁵⁶ THC and CBD can be challenging to differentiate between using standard PSI, due to their identical molecular weights and similar fragmentation patterns. The addition of silver into the paper results in the production of silver ion adducts with different fragmentation patterns, caused by the two compounds exhibiting a differential affinity for the silver ions. The resulting method enabled the differentiation of THC from CBD with an LOD of 6 ng/mL and comparable accuracy, precision and working range to an UHPLC-UV method used as a benchmark. In another study utilizing paper spray for illicit drug analysis, the authors used a paper substrate with a pressure-sensitive adhesive coating for the collection of trace residues of drugs.⁵⁷ The paper can be used to swab suspect surfaces to collect residues invisible to the eye and then be positioned directly in front of the MS inlet for rapid analysis. The method was evaluated with 10 illicit drugs of interest which were collected from a variety of forensically relevant surfaces including fabric, glass and concrete. Analytes were detected in the picogram range using a benchtop mass spectrometer and the low nanogram range using a portable instrument. In other studies, De silva et al. used a microporous polyolefin silica-based synthetic PSI source to reduce matrix effects and increase ion formation in the screening of 212 synthetic opioids,⁵⁸ whereas Skaggs et al. conducted a systematic study of paper substrate-solvent combinations in the analysis of illicit drugs and chemical warfare agents to determine optimal conditions for different compound types.⁵⁹ Aside from paper spray, DART-MS has also been readily utilized in the rapid analysis of drugs, including for the detection of synthetic cathinones in blood and urine,⁶⁰ the analysis of benzodiazepines in plasma,⁶¹ the detection of cannabinoids in various complex matrices⁶² and for the analysis of illicit drugs in electronic cigarette aerosols.⁶³

Recently, the applications of ambient ionization to drugs analysis have extended beyond the laboratory, with research focusing on the development of hand-held tools and methods that could be used in the field for the analysis of real-world samples to truly test the feasibility of such techniques. One recent study developed a hand-held laser diode thermal DESI MS method for the rapid analysis of solid illegal materials.⁶⁴ The ion source consists of a hand-held diode laser operating at 940 nm for analyte desorption that is positioned directly in front of the MS inlet. In this work, the beam was directed at untreated pill and powder samples containing illegal growth promoters that could be abused in competitive sports. The device is inexpensive and battery-powered, thus, could be readily used away from the laboratory, and easy-to-use for non-specialists. It was coupled with a benchtop high-resolution QTOF in addition to a transportable quadrupole mass spectrometer to demonstrate applicability in the field. In a similar effort, Feider et al. recently applied the MasSpec pen to the analysis of forensically relevant compounds such as drugs.⁶⁵ Although originally developed for the analysis of biological materials for disease diagnostics, this study integrated the pen with a sub-APCI source, which was applied to the direct analysis of drugs, such as cocaine and oxycodone, in addition to other materials of interest. With a sampling time of less than 20 s and LODs in the high picogram to low nanogram range, this easy-to-use hand-held device could be readily employed in the field, if coupled with appropriate portable instrumentation.

Outside the laboratory, a recent pilot study employed paper spray MS for quantitative on-site drug screening at a supervised drug consumption site in Vancouver, Canada.⁶⁶ During the 2-day study, 113 samples were submitted for analysis and the method targeted 49 specific drugs in addition to an untargeted screen to capture unknown components. Of the samples submitted, only 78% contained the drug expected by the user, demonstrating the uncertainty in drug authenticity experienced by users. Fentanyl, a particularly potent synthetic opioid that has become a drug of major concern in recent years, was detected in 44% of all samples. In another study, discarded drug packaging was seized from multiple large public events, such as music festivals, in addition to public locations in which illicit drug use was prevalent.⁶⁷ Material was collected from packaging using cotton swabs, which were directly exposed to the ion source of a DART. In total, 1362 individual pieces of packaging were analysed, some on-site using transportable MS and some off-site in the laboratory. In total, 92.2% of samples yielded a positive result for one of the 15 drugs or adulterants screened for, with commonly detected drugs including cocaine, MDMA, ketamine and various novel psychoactive substances. Studies, such as this highlight the potential benefits of implementing AIMS strategies in real-world drug screening, both to ensure the safety of drug users and support police intelligence regarding the chemical makeup of drugs in circulation.

Given the time-sensitive nature of explosives detection, the potential benefits of rapid analysis techniques that can be used in situ are plentiful. As such, several developments have been made in the use of AIMS for the detection of explosives. A recent study by Burns et al. compared two ambient ionization techniques, ASAP and SESI, for the detection of trace levels of RDX, TNT, HMTD, PETN and Tetryl.⁶⁸ Samples were introduced into the ion source with either a glass capillary rod or a glass fibre swab, depending on the technique. Both techniques were able to detect all tested explosives, though SESI exhibited less variability, whereas ASAP generally achieved a lower limit of detection. In this study, the ion sources were coupled with a transportable mass spectrometer to demonstrate the feasibility of on-site explosives analysis with multiple ion source options. Forbes et al. applied open port sampling interface MS to explosives analysis.⁶⁹ OPSI enables the direct analysis of solid or liquid samples via an open port into which the material is directly exposed. A constant flow of solvent through the port forms a meniscus at the opening, which extracts analytes directly from the surface of the sample and draws them into the ion source of the mass spectrometer. In this study, explosives (RDX, PETN and TNT) were applied to the OPSI inlet for rapid analysis, demonstrating a limit of detection of 0.11, 0.21 and 0.19 ng, respectively. The system was also tested with narcotics and inorganic oxidizers, highlighting its versatility for a range of forensically important applications.

Numerous other AIMS techniques have also been utilized in explosives detection. Ninomiya et al. have employed various methods for the analysis of explosives such as TNT and RDX, including DBD and both AC and DC corona discharge ionization.^70,71 Habib et al. analysed TNT, RDX, PETN and NG (nitroglycerin) using HCD, a plasma-based technique suitable for the simultaneous detection and quantification of multiple explosives,⁷² in addition to ultrasonic cutter blade coupled with DBD for the analysis of explosives and drugs.⁷³ DART-MS has been used by multiple groups for explosives detection, including in the evaluation of TATP released from training aids for explosive detection canines,⁷⁴ and coupled with infrared thermal desorption for the analysis of black powders and black powder substitutes.⁷⁵ PSI has been applied to the analysis of peroxide explosives in biological matrices, highlighting the potential for AIMS to be used to demonstrate exposure to explosive materials in the body.⁷⁶ Similarly, modified versions of PSI have also been used in the detection of chemical warfare agents. Cone spray ionization was recently applied to the detection of trace amounts of chemical warfare agents in various solid matrices, such as soil, sand and gravel, achieving detection limits at parts-per-trillion concentrations.⁷⁷ This research has been conducted with a particular focus on developing portable techniques for use by the military for real-time testing of potentially hazardous materials or environments.

Forensic applications of ambient ionization are by no means confined to drugs and explosives. The benefits of AIMS have recently been demonstrated in the field of forensic document examination, particularly with regard to writing sequence identification. In other words, differentiating between handwriting produced at different times. Luo et al. imaged printed seals and handwritten signatures using AFADESI, a variation of DESI which utilizes a high-speed air flow for improved ionization efficiency and sensitivity.⁷ By applying chemometrics to the dataset, it was possible to develop models capable of differentiating between stamps and handwriting produced at different points in time. Another recent study also applied nanoDESI to the depth-dependent analysis of ink in handwriting samples, demonstrating the ability to differentiate between individual pen strokes based on variations in the chemical profiles.⁷⁸ This application could be beneficial in differentiating between handwriting produced at different times and using different inks, thus, identifying illicit alterations of written documents.

Finally, DART-MS in particular has been applied to a range of forensically relevant sample types. This technique has been demonstrated in the analysis of sexual lubricants to evaluate storage conditions for sexual assault evidence,⁷⁹ the analysis of animal products to battle illegal trading,^80,81 polymeric carpet fibres for material identification⁸² and seed-based toxins encountered in cases of poisoning.⁸³ Gao et al. combined REIMS with multivariate analysis to differentiate between leather products sourced from different animals, a method that would be beneficial in leather product testing and authenticity confirmation.⁸⁴

Food and agriculture

A primary area of application of AIMS has been in the analysis of food and agricultural products. The food and agriculture industry necessitates the use of high-throughput analytical testing to promptly detect food contamination and adulteration to avoid the rollout of hazardous products to consumers, but is currently heavily reliant on slower techniques such as liquid chromatography. As such, rapid AIMS techniques have swiftly found their place in this field of work, though further development is needed to achieve the robust and reproducible standards of traditional techniques currently utilized in food testing.

A particular concern in the provision of fresh produce is the presence of residual pesticides in fruits and vegetables, and the rapid detection of potentially harmful compounds before consumption is of the utmost importance. Jeng et al. developed a thermal desorption-based probe sampling technique to achieve molecular imaging of pesticide residues in fruits.⁸⁵ This technique consists of a stainless steel sampling probe which is manually applied to the sample surface and positioned in front of the MS inlet. The probe is heated to thermally desorb collected analytes which are ionized in an ESI plume. In this study, the probe was applied to the surface analysis of pesticide-treated strawberries, imaging the spatial distribution of numerous pesticide compounds. GC-MS and LC-MS analyses were performed concurrently to validate the results. With this method, analysis can be performed in the lab or remotely by sampling with the probe off-site and subsequently transporting collected samples to the laboratory for subsequent analysis. PSI has also been applied for the detection of pesticides in fruits. A recent study developed a corona-discharge assisted paper spray ion source integrated with a thermal desorption probe.⁸⁶ Contrary to standard paper spray techniques in which the analyte solution is added to the paper substrate, here samples were collected and introduced via the thermal desorption probe, which was scratched across the surface of a lemon and positioned in front of the MS inlet. As the probe heats, desorbed analytes are ionized by ions generated by the paper spray ion source. In this version of paper spray, the ionization method consists of both ESI and corona discharge, extending the range of analyte compounds ionized and detected. As such, various pesticides could be simultaneously detected, including polar compounds such as acetamiprid and pyridaben and less polar analytes such as chlorfenapyr and pyriproxyfen.

In the field of food safety testing, concerns have been raised over the migration of contaminants from food packaging into food products. As such, an increasing number of countries are implementing limits on the permitted concentration of certain compounds in food materials. In recent years, the contamination of food with brominated flame retardants has occurred, which is attributed to improperly recycled plastics containing waste from electronic equipment. In a recent study, DART-HRMS was used to analyse potential contaminants in food packaging, known as food contact articles.⁸⁷ Items, such as bottles, travel mugs and plastic utensils, were purchased from various retail stores in the United States, targeting a variety of colours and polymer materials with different countries of origin. Approximately 10% of articles tested contains brominated flame retardants, the majority of which were known potential contaminants in food packaging. The samples were furthermore subjected to analysis by GC-MS to both validate the identification of compounds and quantify BFR levels. Another study by Osorio et al. evaluated DART, ASAP and LC-MS in the determination of oligomer migration from food contact materials into food.⁸⁸ The study particularly focused on poly-lactic acid-based biopolymers, which are advantageous in that they can be produced from renewable sources. However, during the manufacture of these polymers, oligomers can also be formed, which are considered to be ‘non-intentionally added substances’ which have the potential to migrate from food contact materials into food. In this study, biopolymer cups and dishes were acquired from a polymer manufacturing company and simulant migration substances were formed to mimic the migration of compounds from the polymers into food. Analysis was initially performed using LC-MS to confirm the identity of oligomers, after which DART and ASAP were applied. Both AIMS techniques were able to rapidly detect a range of oligomers, though DART detected compounds in the m/z 50–1000 range, whereas ASAP was more suited to the detection of smaller molecules. These studies demonstrate the potential for AIMS techniques to be utilized at manufacturing sites or in regulatory screening processes to rapidly confirm the safety of food packaging based on the presence of harmful or prohibited chemicals.

AIMS has furthermore proven to be a beneficial technique in the analysis of food products for the purpose of authentication and quality control. The MasSpec Pen was recently demonstrated in the authentication of meat and fish products.⁸⁹ Meat fraud is an increasing concern in some parts of the world, typically seen in the form of the replacement of one type of meat with a cheaper alternative. In this study, direct analysis was performed on several animal products, including grain- and grass-fed beef, venison, salmon and trout. Using classification models, the discrimination of meat was achieved with an accuracy of 100% for the distinction of beef versus venison, 84% for classifying different fish types and 95% for the beef model. With an analysis time of 15 s per sample, an easy-to-use sampling mechanism and high accuracy models, the MasSpec Pen has been shown to be a promising tool for food authentication. REIMS has also been demonstrated in meat analysis.⁹⁰ As the global transportation of food worldwide has become commonplace, it is more important than ever before to ensure the appropriate storage conditions of food products throughout their journey. This is particularly true of raw meat products, which must be maintained under specific storage conditions free from significant temperature fluctuations, which can cause problems ranging from poor quality due to structural damage of the meat to safety concerns resulting from degradation. In this study, REIMS was utilized for the lipidomic profiling of fresh and frozen-thawed beef muscle to evaluate the chemical variations caused by consecutive freeze-thaw cycles. Using multivariate analysis, fresh and frozen-thawed samples could be rapidly differentiated with an accuracy of 92–100%, with discrimination primarily based on changes in the content of fatty acids and phospholipids (Figure 3). Various AIMS techniques have also been utilized to detect the presence of toxins in food products, such as LAESI to detect mycotoxins in mold-infected fruit,⁹¹ DART-MS coupled with a smartphone-based lateral flow to detect mycotoxin contamination⁹² and immuno-magnetic blade spray (a coated blade spray-based technique) to detect the marine toxin domoic acid.⁹³

FIGURE 3. Representative MasSpec Pen mass spectra from meat products, PCA plot differentiating samples, and image showing direct analysis of beef sample using the MasSpec Pen. Reprinted with permission from Gatmaitan et al., 2021.89 Copyright 2021 American Chemical Society

Whereas an important focus of AIMS in food and agriculture is from a safety and authentication perspective, ambient MS techniques also have their use in detecting potentially healthy and harmful natural products in food and plants, evaluating flavour compounds in food products, and studying the metabolomics of agricultural products and plants. PSI has been used to quantify flavonoids in citrus beverages⁹⁴ and analyse plants containing pharmacologically active ingredients,⁹⁵ whereas DART-MS has been widely applied for the analysis of active ingredients in herbal supplements and plants.^96–101 These two techniques have also been utilized in the evaluation of sour compounds in tea and wine,¹⁰² to measure artificial sweeteners in alcoholic beverages¹⁰³ and for the study of post-harvest quality of vegetables.¹⁰⁴ REIMS has also been applied to the study of omega-7 phospholipids in marine consumer products.¹⁰⁵ Finally, exploring the metabolome of plants and agricultural products can provide insight into factors affecting growth and quality. Picolitre pressure-probe electrospray-ionization MS (picoPPESI) MS has been used to study the effects of temperature on rice yield and quality,¹⁰⁶ in addition to, profiling apple metabolites¹⁰⁷ and DESI have been used to image plant hormones during development.¹⁰⁸

Environmental

A primary feature of ambient ionization is the ability to couple the ionization sources with portable mass analysers for on-site analysis. As such, the ability to perform in situ analysis at remote locations has made AIMS techniques particularly attractive in environmental sciences. The use of AIMS has the potential to not only eliminate the need for transportation of bulk samples, such as water and soil, back to the laboratory for analysis, but also enable the rapid analysis of complex matrices without the extensive preparation steps often required for environmental materials.

One area of particular interest in environmental sciences is the rapid screening of water supplied for potentially harmful contaminants. Min et al. applied PSI to the rapid analysis of surfactants in pond water samples.¹⁰⁹ Surfactants are extensively used in the production of a wide range of products, including textiles, pharmaceuticals, and cosmetics, and are one of the emerging water contaminants of concern. Using PSI-MS in place of LC-MS, the traditional technique for such analyses, a range of cationic and anionic surfactants could be simultaneously rapidly detected, with LODs as low as 0.05 mg/L. Ambient ionization has also been used to develop a technique for the rapid detection of fluorine in water. Nebulization-DBD ionization was applied to the analysis of mineral, rain and river water, the latter of which was specifically collected from near an industrial site.¹¹⁰ The study demonstrated the ability to detected fluorine in water matrices at sub-nanogram per litre levels.

In a recent study by Jackson et al., enhanced thread spray ionization was applied to the detection of pesticides in complex matrices.¹¹¹ Thread spray is a relatively new substrate-based ionization method, developed further in this work via the modification of the glass capillary surrounding the thread to enhance signal intensity. The technique was applied to the analysis of the pesticide glyphosate, a widely used herbicide, and its metabolite in environmental surface water samples obtained from a river. Using standard addition calibration to compensate for matrix effects from the water samples, the concentration of glyphosate in the river samples was estimated to be 12.2 µg/mL. This study also demonstrated the potential use of the technique in the surface analysis of fresh produce, monitoring the presence of diphenylamine, a pesticide commonly applied to apples to prevent browning. Thread spray was used to first detect diphenylamine on the surface of fruit to differentiate between organic and non-organic fruit, then to study the penetration of the chemical deeper into the fruit.

DART-MS has been a popular choice for the analysis of water samples, and was recently applied to the detection of anatoxins, a type of cyanobacterial neurotoxins sometimes found in drinking water reservoirs.¹¹² Anatoxins are a challenging class of compounds to be analysed by traditional LC-MS methods, thus, alternative screening approaches are desired. The DART-MS technique was used to screen 30 cyanobacterial culture samples for the presence of different anatoxins, demonstrating excellent agreement between DART-MS and the traditional LC-MS technique. Cobo-Golpe et al. used DART to quantify levels of triclosan, a synthetic anti-microbial agent used in various consumer products, in wastewater and other complex matrices.¹¹³ The technique was able to detect triclosan in raw and treated wastewater in good agreement with LC-MS. DART-MS has also been applied to the analysis of pesticides and pharmaceuticals in surface water, though using a solid phase microextraction (SPME) extraction step in an attempt to improve reproducibility by reducing background interferences.¹¹⁴

Cone spray ionization has been used in the analysis of per- and polyfluoroalkyl substances (PFAS). PFAS are widely used chemicals in a range of different consumer and industrial products and, in recent years, have been shown to accumulate in humans and animals following ingestion or other means of exposure. Since the 1960s, PFAS-containing substances, such as aqueous film forming foams, have been readily used as flame retardants and fire extinguishers, particularly by the military. As such, significant concerns have been raised regarding the contamination of soil and water with PFAS and related chemicals. Brown et al. used 3D-printed conductive plastic cones to perform cone spray ionization MS on solid environmental materials, such as soil, sand and clay, containing 11 different PFAS compounds.¹¹⁵ The method was first validated using materials spiked with analytical standards before being tested with genuine soil samples collected from an aqueous film forming foams site. The technique allowed solid material to be added to the cones and analysed in less than 2 min, achieving detection limits as low as 100 ppt, depending on the compound and type of solid material.

Many applications of AIMS in environmental sciences have focused on the detection of contaminants that may be harmful to humans. However, ambient ionization may also be useful in the study of ecosystems to monitor biodiversity and changes in biological systems. There have been significant declines in coral reef populations over recent decades as a result of various anthropogenic effects such as pollution and climate change. As such, the fast and efficient identification and cataloguing of existing coral reef populations is important to understand their decline and implement strategies to prevent further harm. Traditional techniques, such as morphological assessment and DNA profiling, would be greatly supplemented by a rapid and less subjective technique. In a recent study, DART-HRMS was used to chemically profile 61 coral samples across 22 different species by exposing coral tissue segments directly to the DART ion source.¹¹⁶ Chemical profiles were subjected to processing by kernel discriminant analysis, enabling genus-level differentiation with accuracies of 87 to 97%. Through the creation of a database of coral species chemical profiles, the rapid identification of species could be achieved.

Biological materials

Biological material remains to be one of the most difficult samples matrices to work with. Analyses by traditional techniques often require extensive sample preparation and extraction prior to analysis, and the complex nature of such materials can necessitate multiple methods and techniques to characterize the diverse range of compound classes present. As such, direct analysis MS has the potential to eliminate the need for these destructive and time-consuming steps, making biological MS more efficient.

In particular, the ability to rapidly collect chemical information from minute surface areas has enabled researchers to perform mass spectral imaging, which has been particularly useful in the analysis of biological materials such as human tissues. DESI-based techniques have been widely used in the imaging of proteins. Hale et al. used nanoDESI for the imaging of proteins and protein complexes in rat kidney tissue with the aim of improving upon the relatively low-resolution LESA-MS method typically used by the group for tissue imaging.¹¹⁷ nanoDESI was found to offer considerable improvements in imaging resolution, enabling finer details of the tissue to be revealed. The same group then coupled nanoDESI with travelling wave ion mobility spectrometry (TWIMS) for protein imaging.¹¹⁸ The addition of TWIMS to ambient MS workflows can offer both improved specificity via the reduction of chemical noise in addition to the improved determination of protein structure due to the ability of collisional cross-section calculation. The technique was applied to rat kidney tissue sections, successfully detecting proteins across a wide range of molecular weights from 5 to 43 kDa. The study also compared results with those from native LESA-MS experiments, demonstrating no additional error resulting from the use of TWIMS. In other studies, DESI-MS has been applied to image cranial mesenchyme of mouse embryos,¹¹⁹ to the analysis of liver and lung tissues in combination with the elemental mapping technique particle-induced X-ray emission,¹²⁰ to study lipid profiles in human hippocampus tissue from patients with temporal lobe epilepsy,¹²¹ and mouse tissues following different diet treatments.¹²²

Over time, modifications have been made to DESI to improve weaknesses of the technique, one of these alternative techniques is AFADESI. In the diagnostic study of biological tissues, lipids can play an important role in the study of diseases such as cancer. However, lipids are often present as different isomers, which can be difficult, if not impossible to differentiate between using traditional MSI techniques. In a recent study, Zhang et al. developed an AFADESI method to study oxidative reactions to detect C = C double-bond isomers of unsaturated lipids.⁸ The technique involved a short period of accelerated oxidation of mouse lung cancer tissues, following by rapid imaging. The ratio of two isomers was found to be reduced in tissue regions containing a concentration of cancer cells, demonstrating the ability to image the distributional difference in lipid isomers across healthy and cancerous tissues. AFADESI has recently been applied in various other MSI applications, including the study of metabolic changes in the brain tissue of rats with diabetic encephalopathy,⁹ and multiple organ tissues from rats using a unique hydrogel-assisted chemical derivatization method to improve visualization of poorly ionizable molecules.¹²³ Although DESI has been the ambient technique of choice in tissue imaging, other methods have also recently been applied, including tapping-mode scanning probe ESI (t-SPESI) for the analysis of mouse brain tissue.¹²⁴

More recently, imaging technology has developed to enable topological imaging of 3D objects. A laser-assisted REIMS technique was recently developed to profile uneven surfaces and produce 3D maps.¹²⁵ With this method, the sample is automatically moved in relation to a surgical laser and the MS inlet, allowing rapid chemical profiling of multiple points across the surface of the sample. In this study, the technique was applied to the analysis of biological materials, such as a human femoral head, marrowbone, and apple, to demonstrate its applicability to large, uneven surfaces with complex matrices (Figure 4). With the automated system, a 3D image composed of hundreds of datapoints could be produced in as little as 1 h. 3D MSI techniques, such as laser-assisted REIMS technique, could allow for the automated chemical profiling of large and uneven surfaces, providing new insight into molecular distributions across biological materials. This kind of information could be particularly beneficial in understanding the chemical basis of structural changes in biological materials such as osteoarthritis in bones.

FIGURE 4. Sample surface before and after analysis by LA-REIMS with the 3D MS scanner and 3D visualization of molecular distributions across the sample surface. Reprinted with permission from Nauta et al., 2021.125 Copyright 2021 American Chemical Society

As the spatial resolution of ambient ionization MS techniques improves, the ability to apply such techniques to not only the imaging of complex tissues but also the analysis of single cells has become a reality. A dual optical laser-ablation ESI and microscopy ion source was recently used to target single-cell analysis in addition to imaging with high spatial resolution.¹²⁶ The microscopy aspect of the setup permits a smaller LAESI laser beam profile compared to traditional setups, offering improved resolution. The technique was first used to analyse 200 individual Allium cepa (red onion) cells in both positive and negative ion mode, primarily revealing the presence of a series of saccharides. Next, LAESI-MS was used to image compounds in Fittonia argyroneura (nerve plant) leaves, achieving high resolution of 40 µm imaging to reveal the localized concentration of compounds specific to certain physical features in the leaf such as the veins and chloroplast-concentrated inner tissues. In another study, LAESI-MSI was used to measure proteins in tissue-embedded single cells,¹²⁷ in this case using bimodal microscopy imaging was combined by fibre-based LAESI. The technique was applied to the automated analysis of over 1000 single cells, studying both Allium cepa red onion cells and cells from infected soybean root nodule cells. Accurate single-cell sampling as achieved using this method, which could be beneficial for evaluating cell heterogeneity based on different types and concentrations of metabolites.

Aside from tissue imaging, AIMS has also found its use in the direct analysis of biological fluids, which typically require extensive sample pre-treatment prior to analysis. Liu et al. developed capillary PSI, a modification of the traditional paper spray method that exhibits improved tolerance to the complex matrices of biological fluids.¹²⁸ This was applied to the analysis of foetal bovine serum spiked with drugs to evaluate the ability of the technique to detect clinically relevant drugs in complex biological backgrounds. These results were also compared with those obtained by standard PSI, demonstrating the improved spray stability and improve signal intensity. Another PSI variation known as multilayer PSI has also been developed, which employs filter paper with multiple layers to improve reduction of matrix effects.¹²⁹ This was applied to the quantitative analysis of hormones in serum and evaluated the effects of different numbers of paper layers on the sensitivity of the technique when detecting three androgens. Numerous other developments have been made in the use of paper spray for biological fluid sampling, including an alkali metal salt-impregnated paper substrate for the detection of glycans in blood and urine,¹³⁰ and a selenium-based PSI method for the detection of biothiols in biological fluids.¹³¹ Another recent study developed thermal-assisted carbon fibre ionization for the direct analysis of biological fluids.¹³² The carbon fibre ionization ion source consists of a bundle of fine 90% graphite carbon fibres which are positioned in front of the MS inlet. Liquid samples are applied directly to the fibre either using a pipette or by dipping the bundle in the sample directly, whereas solid samples can be applied by wiping the surface of the material with the fibre bundle prior to analysis. A small heater positioned near the fibre tip is used to induce thermal desorption and a high voltage is applied to induce corona discharge for ionization. In this case, raw biological materials, such as blood and saliva, were applied to the fibre and analysed directly. Finally, PESI has been applied to the rapid analysis of human plasma. In a recent study by Bordag et al., a workflow was developed in which samples could be analysed in both positive and negative ionization mode within 2 min, enabling the detection of approximately 1200 unique features across a range of chemical classes.¹³³ The study campaigns for the potential for PESI-MS to become a cost-effective, automated user-friendly technique for routine blood analysis. In addition to the application of AIMS to tissue imaging and biological fluids, recent applications of AIMS also include the study of protein folding and solubility,^134,135 the quantification of extracellular neurotransmitters in brain tissue¹³⁶ and the characterization of metabolites in bacterial extracellular vesicles (EVs).¹³⁷

The ability to perform real-time analysis of bacteria headspace has also been achieved using AIMS. In medical diagnostics, the ability to quickly identify bacterial species is crucial in ensuring the appropriate use of antibiotics, though traditional techniques for species determination can be labour-intensive and subjective. In a recent study by Rosenthal et al., direct analysis MS was used for the rapid analysis of Escherichia coli and Staphylococcus aureus bacterial cultures.¹³⁸ Using a bespoke Petri dish sampling system, VOCs were drawn directly from the headspace of the cultures into the volatile APCI ion source for real-time chemical profiling of VOCs produced by the different strains. The technique was used to collect discrete samples at specific timepoints throughout the bacterial growth in addition to continuous sampling over a 24-h period. By analysing the chemical profiles with multivariate statistics, it was possible to identify characteristic markers specific to species and subsequently differentiate between the sample types.

Staphylococcus aureus is a type of bacteria responsible for hundreds of thousands of hospitalizations annually due to bloodstream infections, which can rapidly spread to different parts of the body. During infection, abscesses can form which offer a refuge for Staphylococcus to evade actions of immune response. In a recent study, microLESA was used to perform spatially targeted protein analysis of these abscesses, characterizing the proteomic composition of different regions of tissue throughout the course of an infection.¹³⁹ In doing so, the authors were able to evaluate bacterial contribution to the development of abscesses and characterize the metabolic processes during the course of an infection and study host immune response.

Havlikova et al. also applied LESA to the analysis of pathogens, specifically the ESKAPE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter cloacae.¹⁴⁰ This group of bacteria are responsible for the majority of drug-resistant infections acquired in hospitals, and thus, their rapid identification is vital for the administration of appropriate treatment. The study applied top-down LESA-MS/MS to the identification of proteins in bacterial cultures, identifying 24 different proteins in the samples. One particular protein, the 50S ribosomal protein L29, was detected in 75% of the ESKAPE species studied, thus, could be used as a potential biomarker for some ESKAPE pathogens.

AIMS has also been used to study pathogens related to cystic fibrosis, a condition in which patients are particularly prone to lung infections. Kaeslin et al. used SESI-HRMS for the direct headspace sampling of six CF-related pathogen cultures, specifically P. aeruginosa, S. pneumoniae, S. aureus, H. influenzae, E. coli and S. maltophilia.¹⁴¹ Analyzing a total of 180 samples, a machine learning algorithm was developed and is capable of differentiating between the pathogens based on distinct chemical signatures. The ability to rapidly differentiate between headspace samples of pathogens in culture may pave the way for the development of breath-based analytical strategies to rapidly identify the cause of patient lung infections.

In a recent study by Tata et al., the technique was used to detect and identify different strains of Legionella pneumophila, a Gram-negative bacteria found in natural bodies of water.¹⁴² If not treated, the bacteria can reach public water supplies and cause Legionnaires' disease, a severe form of pneumonia. Legionella pneumophila consists of multiple sub-types, of which one is responsible for the majority of infections. As such, the rapid differentiation of different Legionella strains could offer an alternative to the biological assays for Legionella detection. In this study, bacterial extracts on melting point tubes were directly introduced into the DART ion source, after which a multi-variate analysis model was built to classify different strains based on their chemical profiles. Using PLS-DA, a model was constructed with an identification accuracy of almost 96%.

Miscellaneous applications

Whereas fields such as disease diagnostics, forensic science and food chemistry, have become some of the primary beneficiaries of ambient ionization, an increasing number of other fields are discovering the power of AIMS.

In recent years, MS has become an advantageous tool in the study of chemical reactions, with AIMS being particularly beneficial in the real-time monitoring of reactants and products. The rapid measurement of reactant products can allow for the faster evaluation of chemical reaction parameters, reducing the optimization bottleneck experienced in organic synthesis. In previous applications of AIMS to reaction monitoring experiments, DESI has been the technique of choice. Fouquet et al. applied reactive-DESI to perform online monitoring of chain solvolysis with the aim of developing a rapid method to study polymer degradation.¹⁴³ The technique was applied to the study of various homopolymers and copolymers of different molecular weights under different chemical conditions, demonstrating reactive-DESI could be used to study the degradation of polymers and assess their susceptibility to erosion. In recent months, alternative ambient ionization techniques have been evaluated for the purpose of reaction monitoring including ambient corona discharge ionization to study the N-alkylation of amines in ionic wind¹⁴⁴ and PSI to study on-surface oxidation reactions.¹⁴⁵

Despite the minimally destructive nature of AIMS, there have been surprisingly few instances of applications to artwork. In a recent study by Astefanei et al., surface acoustic wave nebulization (SAWN)-MS was utilized for the chemical profiling of oil paintings.¹⁴⁶ Modern oil paintings from the 20th and 21st centuries experience various problems of deterioration, such as oxidation and hydrolysis, making cleaning and maintenance of the paintings challenging due to structural damage. Understanding the chemical changes in oil paintings over time and how these changes relate to deterioration may lead to improved cleaning and preservation strategies. In this study, painting swatches were analysed by SAWN-MS in addition to ESI-MS as a control method, after which multi-variate analysis was used to classify samples based on the type of pigment analysed and the water sensitivity of the paint. By measuring chemical markers, such as specific fatty acids and glycerides, it was possible to indicate the extent of oxidation and hydrolysis of the paint, with higher levels of dicarboxylic acid and diacyl- and triacylglycerides and lower levels of short-chain fatty acids correlating with increased oxidation, hydrolysis and water sensitivity. In this work, paint scrapings were removed from the oil paintings prior to analysis, but nevertheless, the study demonstrates the applicability of AIMS to the analysis of artwork.

AIMS is increasingly becoming a valuable tool in exploring and understanding the everyday environment humans are exposed to including potentially irritating or hazardous chemicals. Liu et al. utilized DBD-MS to detect the presence of fragrance allergens, volatile compounds commonly used in consumer products that have been known to induce allergic reactions in some people.¹⁴⁷ A custom-built liquid infusion device was developed to evaporate liquid samples and create volatilized compounds that could be encountered in everyday life. Gaseous analytes were drawn into an active capillary plasma ionization source, an ion source based on DBD. Seven common fragrance allergens were evaluated both as analytical standards and in commercially available perfume products, achieving a satisfactory linear range and low limits of detection, sometimes at ppt levels. With high sensitivity, resilience to matrix effects, and a fast analysis time, DBD-MS has been demonstrated to be a strong tool in the detection of airborne allergens.

Exhaled breath has become a sampling medium of particular interest in recent years, primarily in the field of disease diagnostics, where the goal is to develop a rapid, non-invasive method to detect disease-related metabolic changes in the body. In a time when facemasks have become a commonplace part of everyday life, some researchers have explored how the use of facemasks can be utilized to facilitate exhaled breath sampling. Cai et al. developed a paper spray method to monitor human environmental exposure.¹⁴⁸ Paper strips were positioned on the inside and outside surfaces of facemasks worn by participants. The strips were then collected and analysed directly to detect environmental and exhaled analytes absorbed into the paper. The study demonstrates the potential to use AIMS for the development of a wearable sampling device potentially capable of monitoring the health of the wearer through biomarker detection in addition to environmental exposure. Both volatile compounds from the environment and exhaled breath were detected, in addition to non-volatile analytes likely derived from aerosolized saliva. Finally, in a recent study by Nowak et al., SESI-MS was employed to perform real-time measurements of human breath throughout the sleep cycle.¹⁴⁹ Almost 2000 metabolites were studied, demonstrating a complex mediation of major metabolic pathways as numerous metabolites altered their concentration in sleep and wake states. Metabolite analyses were validated by comparison with metabolites in blood and sleep states were verified using polysomnography, a multiparametric test commonly used as a diagnostic tool in the study of sleep disorders. This fascinating use of direct MS demonstrates the potential for AIMS to be used in the real-time monitoring of the human body, providing novel insight into metabolic pathways under different conditions.

Technological advancements

Since the advent of the first two ambient ionization techniques, DESI and DART, dozens of variations and novel techniques have been developed over the past 15 years. Although the design and construction of novel techniques offers exciting developments to the field, a large proportion of recent AIMS developmental research has focused on the improvement of existing techniques. Despite the advantages of AIMS over benchtop instruments, in many ways, ambient ionization cannot yet perform to the same standard as traditional techniques, particularly in terms of reproducibility and robustness. PSI is amongst the most widely used ambient ionization technique and as such the past year has seen a notable effort to improve this technique via modifications to existing methods. PSI has seen numerous developments in the form of modifications to the paper substrate, such as the use of fluorinated boron nitride nanosheet as a paper spray matrix to improve signal intensity and stability in negative ion mode,¹⁵⁰ and the formation of new gold-embedded paper substrates based on covalent organic frameworks for improved detection of non-polar compounds.¹⁵¹ The solvent spray system has been modified through the development of solvent-free reagent deposition methods to make PSI more suitable for point-of-care analysis,¹⁵² in addition to modifications to the structural design of PSI setups by introducing an ambient focusing lens to improve spray stability, signal intensity and detection limits.¹⁵³ Other AIMS techniques have also been the focus of improvement efforts. DART-MS has seen changes in the form of modifications to the inlet system such as the development of a tee-shaped device for the introduction of gaseous samples into the ion source.¹⁵⁴ Finally, the implementation of different internal standard strategies has been explored with DESI, demonstrating optimal and inappropriate methods for the introduction of internal standards to achieve the best quantitative results.¹⁵⁵

Interestingly, there has been a recent increase in the supplementation of ambient ionization techniques with extraction techniques such as SPME. Although this limits one of the primary advantages of AIMS, the ability to perform direct analysis of samples, it has provided a means of performing rapid chromatography-free analysis whilst improving detection limits and reducing background interference. Extraction technologies, such as SPME, have been successfully coupled with a range of AIMS techniques, including PESI for the detection of drugs of abuse,¹⁵⁶ both PESI and CBS to monitor drug biotransformations,¹⁵⁷ DART to detect pesticides and pharmaceuticals in surface water¹¹⁴ and using the SPME device itself as an ESI emitter for direct analysis of biological samples in front of the MS inlet.¹⁵⁸

A primary advantage of AIMS is the potential to take the instrument into the field to perform rapid on-site testing, yet many techniques have not yet been validated in such a way that they are usable outside of the laboratory. As such, recent developments in AIMS have focused on the improvement of existing ambient technologies to improve performance in addition to creating user-friendly devices that could feasibly be used for remote analysis. Currently, many AIMS techniques require high-voltage power supplies, gases and carefully controlled setups to operate. In order for ambient ionization to translate to the field, various aspects of existing technologies need to be modified and refined. Li et al. recently aimed to resolve some of these requirements by developing a hand-powered ion source.¹⁵⁹ The device is based on a piezo-electric element to achieve the transformation of hand mechanical energy into electrical energy and, in this study, was able to generate voltages of over 10 kV. The device was used to power several ion sources, including paper spray and nanoelectrospray, for the analysis of small molecules. Although just a proof-of-concept study, the research does demonstrate potential alternatives to ion sources requiring power supplies, though the mass detector coupled to the ion source would still require mains power. The same group also developed the MasSpec Pointer, a hand-held and wireless ion source based on arc discharge, a device that was produced from a commercially available electronic lighter (Figure 5).¹⁶⁰ Stelmack et al. evaluated the use of PSI coupled with a portable mass spectrometer under field conditions, specifically to assess the ruggedness of the technique under environmental conditions such as different wind speeds and directions in addition to a range of temperature and humidity levels.¹⁶¹ The study concluded that wind speeds greater than approximately 7 mph had substantial effects on signal intensity and duration, and certain wind directions (particularly a flow perpendicular to the ion source) were particularly problematic. The study explores some of the vulnerabilities of ambient ionization sources in the field and highlights points for consideration when moving from the laboratory into the field.

FIGURE 5. Figures depicting the design and appearance of the MasSpec Pointer (A-D), the waveform of the pulsed high-voltage of the device (E), and the use of use of the pointer (F). Reprinted with permission from Li et al., 2021.160 Copyright 2021 American Chemical Society

The development of ambient ionization technologies has reduced sample analysis time down to seconds, meaning that samples can be analysed at a rate faster than ever before. Despite the ability to collect data in such a high-throughput manner, the analysis of that data is not high-throughput and remains to be a major bottleneck. The use of mass spectral reference libraries provides support for the interpretation and identification of unknown compounds, making MS particularly suited to use by non-experts. However, these libraries must be painstakingly generated and, therefore, improved global collaboration for these approaches would greatly benefit the field. Although alternative analytical techniques, such as low field benchtop nuclear magnetic resonance spectroscopy, also utilize spectral library searching, the low resolution and peak overlap obtained with these methods can be problematic, particularly in complex matrices. Peak deconvolution and chemometrics are, therefore, frequently required to identify shifts prior to submission to an nuclear magnetic resonance database. MS, in contrast, offers high mass accuracy and greater resolution, thus, producing higher quality spectra for database matching with less need for data processing and interpretation.¹⁶² Furthermore, with the greater variety of widely available MS databases, identification of unknown analytes can be achieved much more readily.

Despite these benefits, the existence of such libraries is dependent on the time-consuming analysis of reference samples and upload of reference spectra. As such, the contents of spectra libraries are limited, thus, restricting their utility. Recently, Le et al. used a high-throughput DESI system coupled with collision-induced dissociation for the rapid generation of MS/MS data for a large set of compounds.¹⁶³ Thousands of pharmaceutically relevant small molecules were analysed, resulting in the production of over 16 000 high-quality MS/MS spectra. The creation of this library demonstrates the power of AIMS for the rapid creation of mass spectral libraries and lays the groundwork for the development of machine learning algorithms for the interpretation of MS/MS spectra. Over the past decade, the National Institute of Standards and Technology has created and maintained a DART-MS mass spectral database of drugs seized for forensic analysis. Recent work by Sisco et al. further built on that database, developing an automated data evaluation process to assess the quality of mass spectra in the database,¹⁶⁴ in addition to developing a library search algorithm for the identification of mixed samples of illicit drugs.¹⁶⁵ The development of maintenance of AIMS mass spectral databases is an important step in implementing ambient ionization techniques in real-world settings such as pharmaceutical and forensic laboratories.

SUMMARY AND OUTLOOK

The development of AIMS has transformed analytical chemistry, providing scientists with the ability to achieve sample analysis in a simple, rapid and efficient way. Applicable not only in the laboratory but also out in the field, the real-world applications of AIMS are finally being realized. This review has highlighted the use of AIMS across a diverse range of fields including biomedical sciences, environmental monitoring and forensic science. Furthermore, as AIMS becomes more widely used across these fields and many more, the analytical community has made focused efforts to drive forward the improvement of existing techniques, particularly, in terms of quantification, analyte diversity and ease-of-use. As novel techniques are developed and current practices are improved, the impact of AIMS across many fields of study will undoubtedly continue to grow.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

AUTHOR CONTRIBUTION

Stephanie Rankin-Turner: Writing–original draft (lead), Conceptualization (supporting).

James C. Reynolds: Writing—review and editing (equal). Matthew A. Turner: Writing—review and editing (equal). Liam M. Heaney: Conceptualization (lead), Writing—review and editing (equal).

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created.