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Philos Trans A Math Phys Eng Sci. 2016 Oct 28; 374(2079): 20150380.doi: 10.1098/rsta.2015.0380

PMCID: PMC5031645PMID: 27644966

Quantitative mass spectrometry of unconventional human biologicalmatricesEwelina P. Dutkiewicz and Pawel L. Urban

Department of Applied Chemistry, National Chiao Tung University, 1001 University Rd, Hsinchu 300, TaiwanInstitute of Molecular Science, National Chiao Tung University, 1001 University Rd, Hsinchu 300, Taiwan

e-mail: plurban@nctu.edu.twOne contribution of 19 to a theme issue ‘Quantitative mass spectrometry’.

Accepted 2016 Jun 2.

Copyright © 2016 The Author(s)

Published by the Royal Society. All rights reserved.

Abstract

The development of sensitive and versatile mass spectrometric methodology has fuelled interest in theanalysis of metabolites and drugs in unconventional biological specimens. Here, we discuss theanalysis of eight human matrices—hair, nail, breath, saliva, tears, meibum, nasal mucus and skinexcretions (including sweat)—by mass spectrometry (MS). The use of such specimens brings a numberof advantages, the most important being non-invasive sampling, the limited risk of adulteration and theability to obtain information that complements blood and urine tests. The most often studied matricesare hair, breath and saliva. This review primarily focuses on endogenous (e.g. potential biomarkers,hormones) and exogenous (e.g. drugs, environmental contaminants) small molecules. The majority ofanalytical methods used chromatographic separation prior to MS; however, such a hyphenatedmethodology greatly limits analytical throughput. On the other hand, the mass spectrometric methodsthat exclude chromatographic separation are fast but suffer from matrix interferences. To enabledevelopment of quantitative assays for unconventional matrices, it is desirable to standardize theprotocols for the analysis of each specimen and create appropriate certified reference materials.Overcoming these challenges will make analysis of unconventional human biological matrices morecommon in a clinical setting.

This article is part of the themed issue ‘Quantitative mass spectrometry’.

Keywords: clinical analysis, toxicology, hyphenated techniques, mass spectrometry, non-invasivesampling

1. Introduction

Unconventional biological matrices have recently attracted much attention. They are perceived as

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carriers of chemical information on the human organism. Such matrices (collected in a non-invasivemanner) include hair, nail, breath, saliva, tears, meibum, nasal mucus and skin excretions. The greatestinterest comes from the fields of clinical chemistry [1,2], toxicology [3] and forensics [4]. This interestcan be—in part—attributed to the success of mass spectrometry (MS). In fact, MS is a versatiletechnique of chemical analysis—often used in molecular characterization of biological matrices [5].Development of very sensitive analytical methods, based on MS, enables detection of varioussubstances at very low concentrations [6]. Thus, it is often possible to detect trace quantities of a drugafter the administration of a single dose. Most clinical studies are based on the analyses of standardmatrices such as blood, urine, faeces or tissue biopsy material. However, quantitative analysis ofmolecules present in the unconventional matrices can provide relevant complementary information.Thus, in recent years, the analysis of unconventional specimens gained importance.

In the areas of clinical analysis, toxicology and forensics, both the identities and the quantities ofchemical substances matter. For example, certain trace essential metals (e.g. manganese) should bepresent at low concentrations in the human body. However, when their concentrations exceed certainthresholds, they become harmful or even poisonous [7]. Then again, low-molecular-weight compounds—present in certain types of cells, tissues or organs—help to maintain physiological homeostasis.However, their elevated concentrations may indicate a state of disease [8]. Thus, measurements ofrelative or absolute quantities of substances in human biological specimens can help to understand theinner workings of biological systems, and to identify abnormal physiological states. The assaysinvolving unconventional specimens target various drugs of abuse (cocaine, opiates, amphetamines andcannabinoids) [1,4,9,10], pharmaceuticals [2], metabolites of ethyl alcohol (fatty acid ethyl esters andethyl glucuronide) [11], doping agents [12], environmental contaminants (e.g. pesticides, heavy metals,organochlorides) [13,14] as well as essential and toxic elements [15].

The decision whether to choose conventional or unconventional biological specimens for analysis isinfluenced by several factors. If a patient (or a doctor) had a choice which biological matrix to choosefor examination, they would consider invasiveness, stability, possible contamination or adulteration,extent of sample preparation and the ability to provide retrospective results (figure 1). Undoubtedly, abig advantage of testing unconventional specimens is that they can easily be collected in a non-invasivemanner. The non-invasive specimen collection is especially important during routine tests, whenmultiple specimens need to be obtained from the same person or when under-age patients need to beexamined. Another important advantage is that the risk of adulteration or substitution of theunconventional specimen is very low because its collection can be directly observed (which is unlikelyin the case of urine collection). This feature is very important, especially in the case of anti-dopingscreening conducted during sporting events. In the case of hair and nail specimens, benefits include alarge temporal window of chemical monitoring and the possibility to perform retrospective analysis[16]. Even after a single dose of a drug, a small amount of an analyte can be detected long (evenseveral months) after the drug administration [17]. Retrospective analysis is especially useful in casesof suspected drug abuse or intoxication with sedatives in drug-facilitated crimes [18]. On the otherhand, analysis of hair or nails of mothers and newborns can bring insights into fetal stress related to amother's stressful life [19], administration of drugs [20] or consumption of alcohol [21] duringpregnancy.

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Figure 1.

Factors influencing the decision whether to choose conventional or unconventional biological specimens forclinical analysis, toxicology and forensics. (Online version in colour.)

Analysis of every biological matrix has its own advantages and shortcomings. The biggest challenge inchemical analysis of unconventional specimens is that the analytes are often present at very lowconcentrations (down to the picogram per millilitre level) [22]. Moreover, in some cases, only smallamounts of the biological material can be collected. For example, the typical total volume of tearspresent in one eye ranges from approximately 5 to 10 µl [23]. In the case of hairs and nails, owing tothe complexity of the keratinized matrix, specimens require extensive and time-consuming preparation.Although non-invasive hair or nail collection is a great advantage, such matrices are exposed tocontamination from the environment, food or cosmetics. Another major problem is the lack of properstandardization of protocols for analysis of unconventional matrices and the lack of certified referencematerials (CRMs)—required for conducting high-quality analyses (nowadays, only CRMs for hair areavailable [24,25]). However, the Society of Hair Testing has published several guidance documentsdescribing the examination of drugs and doping agents in hair [26]. Thus, hair analysis has alreadyfound multiple applications in clinical and forensic toxicology as a complementary methodology tostandard blood and urine analyses. More information about distinct advantages and challenges in theanalysis of unconventional matrices, discussed in this review, is provided in table 1.

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Table 1.

Application of MS in chemical analysis of unconventional human biological specimens. VOCs,volatile organic compounds.

Open in a separate window

— largetemporalwindow ofdetection— detectionof a singledose of a druglong afteradministration

— extensiveand time-consumingsamplepreparation— bias dueto differencesin melanincontent andhair dying

— chronicdrug abuse— alcoholabuse— dopingcontrol

— drugs ofabuse— alcoholmetabolites— dopingagents— metallicspecies

breath 16 160— real-timeanalysis— breath-to-breathresolution

— dilutionandcontamination— normalizationof thesampledvolume

— diagnosisof diseases— drug abuse

— VOCs— drugs ofabuse— therapeuticdrugs

saliva 14 182— lowproteincontent— evaluation ofprotein-unboundconcentrationof a drug— screening fora parent drug

— shortretention timeof drugs— contaminationwith food andother debrisfrom mouth— for somedrugs, lowcorrelationbetweensaliva andbloodconcentrations

— pharmacokineticstudies— smokers/non-smokersdiscrimination— oral cancerdiagnosis— periodontitisdiagnosis

— therapeuticdrugs— drugs ofabuse— hormones— proteins— VOCs— metallicspecies

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Number of papers listed in the PubMed database. Searched phrases: ‘name of the specimen’ and ‘mass’ and‘spectrometry’ and (‘quantitative’ or ‘quantification’ or ‘quantity’ or ‘quantify’ or ‘quantified’ or ‘quantitate’or ‘quantitated’ or ‘quantifying’) and (‘human’ or ‘humans’) in the title of the paper (T) or title/abstract (T/A).The results of the search also include analyses of metallic species and large molecules. In the case of ‘mucus’,only the number of papers related to nasal mucus is given. The database was accessed on 30 May 2016.

The past two decades have brought various developments related to mass spectrometric analysis ofunconventional biological matrices. The analyses presented below were mostly based on thehyphenation of liquid or gas chromatography (LC, GC) with MS [4,12]. The disclosed methodsprovide good analytical sensitivity and quantitative capabilities and enable analysis of multiplesubstances in single chromatographic runs. Quantification by MS can be achieved because (within acertain concentration range) the intensity of the signal related to an analyte is proportional to theamount of that analyte. Nevertheless, to obtain quantitative results, additional analyses with knownamounts of the standard compounds must be performed (calibration, standard addition). Beside LC-and GC-MS, other MS-related techniques and pre-treatment stages have also been used, for example:inductively coupled plasma (ICP), laser desorption/ionization (LDI), desorption electrospray ionization(DESI), proton transfer reaction (PTR), selected-ion flow-tube, ion mobility and isotope ratio. Forinstance, ICP-MS enabled simultaneous multi-element analyses with very low detection limits, evendown to the ppt level. In one study, an ICP-MS analysis of two hairs obtained from NapoleonBonaparte revealed very high concentrations of arsenic (42.1 and 37.4 ng mg ), which wereapproximately 40 times higher than the normal values [27]. This result supported the hypothesis ofNapoleon's possible intoxication with arsenic, although it did not clarify the cause of his death. Matrix-assisted laser desorption/ionization (MALDI-MS) has been employed, for example, to imageincorporation of a drug into the hair matrix [28], and to identify fungal species in infected nails [29].However, quantitative capabilities of this technique are generally considered to be limited (forexamples of quantitative applications of LDI-MS techniques, see other articles in this theme issue). Inone impressive demonstration, DESI-MS was applied for in vivo analysis of metabolites and drugssampled directly from the surface of fingertip [30]. PTR-MS was used to monitor the breath of apatient undergoing surgery [31]. On the other hand, isotope-ratio MS was used to determine theisotopic composition of C, N, H and O in stable matrices such as hair and nail [32].

In this review, we summarize the major trends in quantitative chemical analysis of unconventionalhuman biological matrices by means of MS. First, we discuss specific aspects of quantitative MSapplied to unconventional biological matrices. Second, we provide a brief description of each of thesematrices, emphasizing distinct advantages and disadvantages of using every type of matrix. We alsodiscuss the main directions in the research on MS analysis of those matrices (see also table 1). Owingto space constraints, examples of procedures and applications of MS methods are described in moredetail in the electronic supplementary material. Finally, we draw conclusions from the past experiencein the area, and outline future trends in quantitative analysis of unconventional human biologicalmatrices. Note that the procedures involving human subjects described below should be performed byqualified and licensed medical professionals in agreement with the relevant regulations.

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Go to:2. Important aspects of quantitative mass spectrometry of unconventionalbiological matrices

(a) Sample versus specimen

Before moving on to further discussion, it is necessary to note that the terms ‘specimen’ and ‘sample’refer to two different concepts [33]. ‘Specimen’ is defined as ‘a specifically selected portion of amaterial taken from a dynamic system’, while ‘sample’ is defined as ‘a portion of material selectedfrom a larger quantity of material’ [34]. Owing to the intrinsic inhomogeneity of dynamic biologicalsystems (tissues, biofluids), it is virtually impossible to collect two biological specimens with exactlythe same chemical composition. To perform reliable analysis, it is important to ensure collection of‘representative specimens’, which reflect momentary compositions of the target tissues or biofluids,relevant to the physiological state of the subject.

(b) Specimen collection

One of the advantages of mass spectrometric analysis is that only a small amount of secondary sample(pretreated specimen) is required (e.g. approx. 100 µl in typical LC-MS analysis). However, in the caseof some unconventional human biological matrices, even collection of comparable volumes may becumbersome. The ‘low-volume problem’ is particularly related to collection of sweat, tears, saliva andnasal mucus. Although there exist methods to stimulate tissues for excretion of larger volumes ofsweat, tears and saliva, it must be ensured that such methods do not significantly affect the analysisresult. For example, a simple method to induce sweating is to do physical exercise. However, physicalactivity may affect the composition of the skin excretions. One standardized method usespharmacological stimulation of sweating. It involves application of an alkaloid (pilocarpine) to the skin[35]. In this approach, pilocarpine is first administered to the skin by means of iontophoresis carriedout over 30 min. Subsequently, within the next 30 min, approximately 50–60 µl of sweat fluid iscollected. A commercial system called Macroduct is used to collect such volumes of sweat. The assayfacilitates diagnosis of cystic fibrosis in infants [36]. Nonetheless, this approach is considered to beinvasive to the human body because an electric potential (a few tens of volts at a current of approx. 1.5 mA) is applied to the skin to facilitate iontophoresis of the alkaloid drug. Moreover, the collected sweatspecimens become contaminated with large amounts of pilocarpine. Alternatively, special collectors—in the form of an absorbing wipe, pad, patch, sponge or strip—can be used to obtain small amounts ofsweat, tears, saliva and nasal mucus. In these cases, high recovery of analytes from the samplingmaterial has to be ensured. Moreover, the absolute concentration of analytes may be difficult todetermine, because the volume of the collected specimen is hard to estimate.

(c) Specimen preparation and matrix effects

When a sufficient amount of a representative specimen has already been collected, one can performdirect mass spectrometric analysis without extensive sample preparation [37]. In the cases of nail andhair, extensive sample preparation is normally inevitable. Most importantly, the target analytes need tobe extracted from a rigid keratinized matrix. Some analytical platforms are more compatible with suchsolid specimens. In the case of analysis by MALDI-MS, hair and nail specimens only need to be rinsed,and an organic matrix needs to be deposited to promote absorption of photonic energy and ionization.

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Small volumes of liquid-phase specimens of sweat, tears or saliva can be directly injected to the LC orGC columns. In many cases, only dilution and centrifugation are necessary. Removal of solid particlesis critical to prevent clogging of the flow lines. Single-step protein precipitation can be performed withan organic solvent [38,39]. Certainly, such simplified sample treatment can still shorten the lifetimes ofchromatographic columns.

In general, a lack of extensive sample preparation reduces the overall time of analysis and minimizesthe biases related to multi-step sample-handling procedures. Nonetheless, MS is vulnerable to matrixinterferences [40]. Matrix components often suppress or enhance ionization of the analytes. Highamounts of non-volatile salts (e.g. NaCl or KCl) in the specimens such as sweat, tears or mucus causesignificant ion suppression. In addition to the negative effect of ion suppression on analysis, thevariable chemical composition of biological specimens can considerably affect the quality of the MSresults, and lower their usefulness while drawing clinically relevant conclusions. The commonsolutions to the matrix-interference problem in MS include: (i) specimen/sample dilution, (ii) removingmatrix salts or macromolecules prior to analysis (e.g. by extraction), and (iii) application ofchromatographic separation. It is also possible to compensate for possible matrix effects related tovariability and instability of the ionization process by using isotopically labelled chemical standards.Such standards are added to the prepared specimen. The signal of the isotopically labelled standard isthen used to normalize the signal of the analyte. Isotopically labelled standards are chemically identicalto the analytes. Thus, they generally give better results than chemical standards that are structurallydifferent from the analytes. Unfortunately, the cost of isotopically labelled chemicals is high while theiravailability is limited. One imaginable solution for an analytical laboratory is to hire an organic chemistwho could synthesize such isotopically labelled standards for specific analyses.

(d) Recovery

Another challenge in the analysis of unconventional specimens is the efficient extraction of theanalytes of interest from the matrix or from the material used for specimen collection (e.g. absorbingwipe, pad, patch or strip). However, the efficiency of extraction may be limited due to the physical andchemical properties of the analyte and the internal structure of the specimen matrix. Certain amounts ofthe analytes can be lost through adsorption on surfaces, and degraded during extensivesample/specimen manipulation and prolonged storage. Only high recovery of the analyte ensuresaccurate and precise quantification. Thus, there is a need to develop efficient methods of extraction andconcentration of trace molecules of interest present in unconventional matrices. Isotopically labelledstandards are very useful to evaluate the recoveries of the sample treatment steps.

(e) Verification of the results

Validation of the analytical methodology is normally required to ensure high reliability of thequantitative analysis. Bioanalytical method validation normally includes the assessment of: selectivity,limit of quantification, limit of detection, sensitivity, calibration range, accuracy, precision(repeatability and reproducibility), matrix effects and stability of the analyte in the biological matrix[41,42]. During the validation process, a high-quality standard of the analyte of interest is spiked into abiological matrix. A matrix comparable to the matrix of the studied specimens should be used exceptthat it should not contain the analyte species. In the case of unconventional matrices, it might be

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problematic to find a blank matrix (especially to study endogenous substances). Owing to the limitedavailability of blank matrices, isotopically labelled standards are recommended for validation of MSmethods. They can be added to the specimen matrix that contains certain amounts of the targetanalytes. In some cases, alternative solutions to the above problem exist. For example, artificial tearsolution, which is easily accessible in many pharmacies (as an over-the-counter medication), can beused as a blank matrix during method development for tear analysis [43]. However, artificial tearsolution contains mostly water and salts (e.g. NaCl and KCl), while real tear fluid contains variousorganic molecules. In one study, an artificial sweat solution containing ammonium chloride, lactic acid,urea, acetic acid and sodium chloride dissolved in deionized water (pH adjusted to 4.7) was prepared[44]. In another report, pigs' hoofs were used as a substitute for human nail matrix [45]. In addition, inthe final step of the validation process, CRMs are normally required. However, as we mentioned above,nowadays, only CRMs for hair are available. Alternatively, reference materials can be prepared in-house. For example, matrix-matched standards in a tablet form were prepared to enable quantitativeanalysis by means of direct solid sampling with laser ablation ICP-MS [46].

An important point in the evaluation of MS results for quantitative analysis is to verify the presence ofadduct ions and the occurrence of fragmentation during and after ionization (in-source decay and post-source decay). In fact, in some cases, quantitative analyses are based on such adduct or fragment ionsignals (single/multiple reaction monitoring mode). In these cases, one needs to select theadduct/fragment signals that represent high sensitivity (i.e. correlate with concentrations of the originalanalytes) and high repeatability.

3. Unconventional matrices in clinical analysis, toxicology and forensics

Because most of the unconventional matrices, highlighted here, are quite different in terms of theirmain chemical composition and physical properties, the early stages of analysis procedures have to becustomized to specific matrices. However, the secondary samples derived from the unconventionalmatrices are more similar to one another. Therefore, they can be subjected to detection using very fewstandard techniques (especially, LC-MS and GC-MS; see the electronic supplementary material).Below, we provide a brief summary of the characteristics of unconventional human biological matricesobtained in a non-invasive manner. Their composition, origin and functions are summarized. We alsodiscuss the main directions in the research on MS analysis of those matrices, which are related to thefields of clinical analysis, toxicology and forensics. In addition, we summarize the advantages andshortcomings of the analyses of each matrix (see also table 1).

(a) Hair and nail

Hair consists of two parts: hair shaft and hair follicle. The hair shaft is a thin flexible cylindricalstructure composed of dead keratinized epithelial cells, while the hair follicle contains living epithelialcells. A blood capillary system surrounds each hair follicle providing necessary nutrients. Hair folliclesare directly connected to sebaceous (oil) gland ducts. Each hair shaft consists of three axial layers:medulla (inner layer), cortex (middle layer) and cuticle (outer layer). Hair matrix is composed ofproteins (65–95%, mainly keratins), water, lipids and minerals. The average rate of hair growth isapproximately 0.4–0.5 mm d [47]. Similar to hair, nails (at the tips of fingers and toes) are composedof a keratinized matrix. Each nail consists of three sections: nail plate, nail matrix and nail bed. The

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average rate of fingernail growth is estimated to be around 3.0 mm per month, whereas the rate oftoenail growth is around 1.1 mm per month [48]. In both hair and nail matrices, keratins form longfibres, which are bound together via disulfide bridges. Those bridges make hair and nail matrices verydurable. Various xenobiotic compounds (including drugs) are incorporated into the keratinized matrix.Thus, it is very difficult to alter the chemical compositions of hair and nail specimens.

Owing to the most consistent hair growth being on the vertex of the head, hair specimens are usuallycollected from this location. Several hair shafts can simply be cut out (as close to the scalp as possible),or they can be plucked out from the scalp (using tweezers) [28,47]. Nail specimens are normallycollected by clipping. Both finger nail and toe nail specimens can be collected for chemical analysis.Preparation of hair and nail specimens for analysis includes decontamination, homogenization,extraction/digestion, preconcentration and clean-up [47,49]. The rigid structure of the keratinizedmatrix can be broken down by overnight alkaline (with NaOH) or acidic (with HCl, H SO or HNO )digestion. However, following such digestion, some components of interest may be decomposed. Thus,alternative methods of extraction are occasionally implemented; for example, ultrasound-assistedextraction with methanol (after micropulverization of the specimen) [50,51]. To remove matrixinterferences (especially after digestion), subsequent clean-up by solid-phase extraction (SPE) isnecessary [48]. In fact, such extensive preparation of hair and nail specimens is laborious, time-consuming and may introduce errors to the final result [52]. Less extensive specimen preparation isrequired for imaging MS—in which case, longitudinally sectioned hair specimens are often used [28].Hair and nail specimens are very stable. In general, they can be stored and transported at roomtemperature, while the risk of matrix and analyte degradation is low. Because of the high stability, post-mortem specimens can be collected and analysed [53]. One of the problems related to hair analysis isthe fact that dying, bleaching or exposure to heat or sunlight may affect the results [54,55]. Owing tothese factors, analytes can decompose, while other chemicals (including drugs of abuse) may adsorb onthe hair surface leading to erroneous results [56]. There are also concerns about the rates of analyteincorporation into hair matrix. These rates may depend on the type of hair. For example, some drugsbind to melanin, which contributes to higher concentrations of those drugs in dark hair than in lighter-coloured hair [3]. The advantage of analysis of nails (when compared with hair) is the lack of thepossible bias due to different melanin content in the specimens obtained from different individuals[48].

MS analyses of hair specimens include screening for drugs of abuse (cocaine, opiates, amphetaminesand cannabinoids) [4,57], pharmaceuticals, stable metabolites of alcohol (fatty acid ethyl esters andethyl glucuronide) [11] as well as doping agents [26,47]. Analysis of nail specimens can providecomplementary information to that obtained from analysis of hair specimens [58,59]. Hair and nailanalyses extend the temporal profiling of exposure to xenobiotic substances by up to several months.As a result, chronic abuse of drugs and alcohol or bioaccumulation of environmental contaminants canbe verified. Moreover, short-term abstinence can be confirmed [11]. Interestingly, several maxima of adrug concentration in time were observed for the non-benzodiazepine hypnotic drug zolpidem, detectedin a nail specimen (following administration of a single dose; figure 2) [17]. The detection window ofthis drug in the fingernail was appproximately 3.5 months (for further description of this work, see theelectronic supplementary material). On the other hand, hair and nail specimens collected within severalmonths after birth can be used as indicators of the exposure to drugs in utero [20]. Moreover, toxic

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metallic species [60–62], pesticides and persistent organic pollutants [13,51,63] in hair and nailspecimens can be investigated. Levels of essential elements in hair and nail are also of interest [15]. Inaddition, nail specimens are studied to investigate nail disorders, such as fungal infections [29].

Open in a separate windowFigure 2.

Concentration of the drug zolpidem in fingernail clippings collected from four subjects (a–d) after ingestionof a single dose (10 mg) of zolpidem. Three maxima of the drug concentration in time were observed for mostsubjects. Peak 1 corresponds to the drug excreted in sweat and sebum, and adsorbed on the nail; peak 2corresponds to the drug released from the bed of the nail; while peak 3 corresponds to the drug incorporatedinto the newly formed nail matrix. (Adapted with permission from [17]. Copyright © 2013 Wiley.) (Onlineversion in colour.)

(b) Saliva and nasal mucus

Saliva is viscous oral fluid composed mostly of water (99%). It also contains salts, proteins (enzymes),peptides, hormones, lipids, sugars as well as epithelial cells, food debris and microorganisms. Saliva issecreted primarily by three major salivary glands: the parotid, submaxillary and sublingual glands. The

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main functions of saliva are: maintenance of the mucosa, involvement in chewing, mineralization ofteeth, control of microorganisms, taste perception and digestion [64]. The flow rate of saliva variesfrom zero to several millilitres per minute, depending on the emotional state and stimulation [65].Nasal mucus is a gelatinous fluid produced by mucous membranes in the nasal passages. It iscomposed mostly of water (95%) but it also contains salts, proteins (enzymes) and epithelial cells.Mucus protects the respiratory system by blocking pathogenic antigens and additionally humidifies,cools or heats, and cleans the inhaled air [66].

Relatively large volumes of saliva (more than 1 ml) can be collected directly by expectoration(spitting). Special saliva collectors in the form of an absorbing wipe, pad or sponge can also beimplemented [64]. Recently, dried saliva spot [67] and solid-phase microextraction [68] samplingtechniques were proposed. Similar to the dried blood spot sampling method, the dried saliva spotsampling method facilitates storage and transport of the specimens from the point of collection to thelaboratory. On the other hand, a solid-phase microextraction probe enables in vivo sampling andextraction (due to intrinsic selectivity). Usually, subjects are restricted from eating, drinking orbrushing their teeth prior to saliva collection. The advantage of saliva analysis in pharmacokineticstudies is that there is a lower concentration of proteins in saliva than in plasma. Thus, the probabilityof binding a drug to the proteins is reduced. In consequence, biologically active forms of protein-unbound drugs (or their metabolites) can be quantified. Nonetheless, in the case of some drugs, thereare substantial differences between concentrations in saliva and blood [69]. As expected, correlationbetween saliva and blood concentrations is drug-dependent [64]. Another limitation of saliva analysisis that drugs can be monitored over shorter periods than in the case of urine analysis. Furthermore, thistype of specimen can be easily contaminated with food debris. Saliva specimens do not normallyrequire extensive treatment prior to MS analysis. Typically, only solid particles are removed bycentrifugation. In several studies, proteins were precipitated with small amounts of an organic solventprior to analysis of low-molecular-weight drugs [38,70]. In addition, SPE may be performed topreconcentrate and purify the analytes [69]. Nasal mucus can be collected by blowing out the nose overa beaker or by using a swab. Nostrils can be pre-washed with saline solution prior to specimencollection.

Saliva is normally used in the assays targeting drugs of abuse (amphetamines, opioids and cannabis)[71,72], therapeutic drugs [67,70,73], alcohol (ethanol), hormones (cortisol) and steroids [64]. Salivarymetabolomics can provide an outlook for diagnosis of oral cancer and periodontal disease [74,75]. Infact, many researchers are interested in the composition of saliva proteome [76]. Saliva can also beanalysed to monitor human exposure to toxic elements such as arsenic [77]. MS analysis of nasalmucus has not yet become popular. In one study, volatile organic compounds were analysed in nasalmucus specimens [78]. Some of these compounds were produced by the bacteria causing sinusinfections. Nasal mucus can also be used to investigate the exposure of human subjects to aircontaminants; for example, traces of cocaine dust in forensic laboratories [79].

(c) Breath

Exhaled breath is composed of water vapour, volatile organic compounds (e.g. metabolites of ingestedfood and drugs, metabolites produced by bacteria in the gut and airways, or environmental

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contaminants), inorganic gases (e.g. carbon dioxide and nitric oxide) and non-volatile particles [80].Breath sampling and analysis can be carried out by several methods. In the first method, the examinedsubject is normally asked to breathe through a special device comprising a mouthpiece and sorbent forseveral minutes. The compounds of interest are adsorbed on a sorbent (e.g. solid-phase microextractionsorbent), and later desorbed at an elevated temperature. The second method involves collection of theexhaled breath into a container or a bag with a known volume. The collected breath specimen is thenreleased to the ion source of a mass spectrometer [81]. In the third method, the exhaled breath iscondensed in a precooled device, and collected as a liquid sample (the so-called ‘breath condensate’)[82]. Breath sampling can be performed without any restrictions regarding the time and frequency. Thatpoint is a big advantage of sampling and analysis of breath specimens.

Breath analysis can be used to monitor compounds of endogenous and exogenous origin, which hasalready been summarized in numerous review papers [80,83–86]. Potential biomarkers present inexhaled breath can be used for early diagnosis of diseases (especially lung-related) [87,88], and intoxicological investigations (e.g. exposure to tobacco smoke) [83]. Analysis of breath is often used tostudy the metabolism of administered drugs [81]. The great advantage of breath analysis is thepossibility to detect volatile compounds in real time. In one spectacular demonstration, real-time breathsampling, followed by on-line MS analysis, was performed on a patient undergoing surgery [89].However, when collecting breath specimens, the impact of various contaminants from the environmentmust be considered because these contaminants may influence the results of breath analysis.

(d) Tears and meibum

Tears are formed from the aqueous fluid that overlays the eye's surface. Tear fluid is composed mostlyof water, but also contains lipids, proteins, amino acids, sugars and electrolytes. According to the so-called three-layer model, the tear film consists of an inner mucin layer, a middle aqueous layer and anouter lipid layer [23]. Each of these layers has a different origin—the mucin layer comes from gobletcells, the aqueous layer is secreted by the lacrimal glands, while the lipid layer is mostly produced bymeibomian glands. Tears lubricate and hydrate the membranes forming the ocular surface, protectagainst various pathogens and provide nutrients to the corneal epithelium. Meibum is an oily substanceproduced by the meibomian glands (type of sebaceous gland) present on the upper and lower eyelids[23,90]. Once secreted, meibum mixes with tears and forms the outer layer of the tear film. The typicaltotal volume of tears present in one eye ranges from approximately 5 to 10 µl, while the secretion rateis of the order of approximately 1.2 µl min . Basal tears are composed of the fluid that normallycovers the eye's surface, while reflex tears are produced in response to irritation of the eyes by foreignobjects [23].

Tears are normally collected using one of the three available methods. In the first method, a 10 µl glassmicrocapillary is placed very close to the eye without touching the eyeball [91,92]. The second methodinvolves flushing the eye with saline solution, and collecting the after-wash liquid [92]. Notably, in thismethod, the tear fluid is significantly diluted. The third method implements the so-called Schirmer'sstrip [92,93]. This can be used to collect tear fluid from subjects with dry-eye syndrome. In thismethod, a strip of filter paper is placed inside the lower eyelid, and is left there for 5 min to absorb thetear fluid. If basal tears need to be collected, a local anaesthetic may be applied to reduce the release of

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reflex tears. Afterwards, tear components are extracted from the paper strip using a solvent. Puremeibum specimens can be obtained by applying a small physical force (e.g. by tweezers) to the eyelid,and then collecting the excreted fluid with a spatula. Tear fluid specimens do not normally requireextensive sample preparation prior to MS analysis. In many cases, specimens are diluted with organicsolvents, and solid particles are removed by centrifugation. According to the Folch method [94], lipidscan be extracted from tears and meibum using biphasic extraction (with a mixture of chloroform andmethanol, followed by addition of a buffer) [95].

Tear fluid is used to investigate various ocular diseases, including dry-eye syndrome [96]. Numerousreports focus on lipidomic analyses of human tear and meibum [90]. Some of the recent studies havedemonstrated major differences in the lipid composition of tear fluid and meibum fluid [92,97].Moreover, other endogenous metabolites, such as amino acids or glucose, can be detected in tear fluidby means of MS [91,98]. Protein content as well as the presence of therapeutic drugs in eye-derivedspecimens are also of great interest [43,99].

(e) Skin excretions

Human skin is coated with a film containing numerous compounds, which can altogether be regardedas ‘skin excretions’. Some of these compounds are secreted by specialized glands (predominantly,eccrine (sweat) and sebaceous (oil) glands), while others are produced during the breakdown ofproteins in the outermost layer of the skin [100]. Sweat fluid is mostly composed of water. However, italso contains mineral elements (e.g. sodium and potassium) and organic compounds such as proteins,amino acids, lactic acid and urea [101,102]. The typical sweating rate (without stimulation) rangesfrom approximately 300 to 700 ml d [3]. Sebum is an oily substance mostly composed of lipids. Infact, different compounds present on the skin surface have diverse distributions (influenced by skincells, microbes and daily routines) [103]. The main function of sweating is to protect the human bodyfrom overheating. Skin excretions (including sweat) also moisturize the skin, and protect it fromevaporation of water.

It is difficult to collect pure sweat, separately from other skin excretions. Thus, usually a mixture of allskin excretions, lifted from the skin, is analysed. However, in many reports, this mixture is inaccuratelyreferred to as ‘sweat’. One standardized method of pharmacological stimulation of sweating is used tocollect sufficient volumes of sweat-rich fluid (more than 50 µl) [35]. As we mentioned earlier, at first,sweating is induced by pilocarpine iontophoresis. Then, the so-called Macroduct is employed to collectsweat. Smaller volumes of sweat and skin excretions can be collected using various accessories, suchas a semipermeable skin patch (the most widely used), cotton swab, cellulose wipe, textile [104], silicaplate [105] or hydrogel micropatch [106]. A skin patch can be worn for up to a fortnight, while othercollectors are suitable for sampling during short periods of time. Subsequently, skin excretions areextracted from the collector off-line using a solvent. Nonetheless, an on-line extraction method has alsobeen demonstrated [106,107]. In this approach, endogenous metabolites collected with the use ofhydrogel micropatches (figure 3) were extracted on-line, and subsequently ionized at atmosphericpressure (for further description of this work, see the electronic supplementary material). Skinexcretions do not normally require extensive sample preparation prior to MS analysis. Usually,specimens are diluted with an organic solvent, followed by the removal of solid particles by

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centrifugation. However, a high content of salts present in the sweat (which cannot be removed bycentrifugation) may contribute to ion suppression during MS analysis. This is one of the maindisadvantages of using skin excretion specimens in clinical assays.

Figure 3.

Sampling and rapid chemical profiling of skin metabolites collected using a hydrogel micropatch probe. Theprobe was attached to the skin for 10 min. Subsequently, it was analysed by nanospray DESI-MS withoutfurther treatment. Numerous metabolite species were detected. (Adapted with permission from [106].Copyright (2014) American Chemical Society.) (Online version in colour.)

Skin excretions are used to conduct tests for drugs of abuse (amphetamines, cannabis, cocaine andopiates) [104], therapeutic drugs [108] and other xenobiotics [109,110], such as nicotine or caffeine[111,112]. The advantage of skin excretion analysis in drug testing is that non-metabolized drugs arelikely to be excreted with sweat and sebum. This characteristic makes identification of an excreted drugsimpler. Endogenous metabolites excreted by skin may serve as important biomarkers of disease[105,113,114]. Volatile organic compounds emanating from skin, such as alcohols, fatty acids or esters,are also of great interest [115,116]. In the area of forensic science, attention is paid to chemical analysisof latent fingerprints [117].

4. Conclusion and future trends

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The areas of application of mass spectrometric analysis of unconventional biological specimens includeclinical analysis, toxicology and forensics. There has been enormous progress in the development ofanalytical methods targeting unconventional biological specimens. However, many of the publishedprotocols are based on very few instrumental platforms, such as LC-ESI-MS and GC-EI-MS. Certainly,these instruments are adequate for performing quantitative analyses. Nonetheless, they provide limitedanalytical throughput because chromatographic separation takes from a few minutes to several tens ofminutes. Thus, direct mass spectrometric approaches—without the separation step—have beenproposed. Further development of these methods will certainly be directed towards the implementationof portable mass spectrometers in the analysis of unconventional specimens at the points of specimencollection. Nevertheless, adoption of such approaches has to be preceded by comprehensive testsfocused on technical quality.

MS is particularly vulnerable to matrix effects (ion suppression). Thus, variable chemical compositionof biological specimens may considerably affect the analysis results if the separation step is missing.Another obstacle in the implementation of unconventional biological specimens in routine analysis issample pre-treatment. Such specimens are composed of numerous biomolecules with diverse physicaland chemical properties. Their extractability may be limited by the intrinsic structure of the matrix.Hence, there is a need to develop efficient methods of extraction and concentration of trace moleculesof interest. Protocols for handling and detecting multiple analytes at once are desired. Moreover,normalization of the amounts of the collected specimens (e.g. breath, tears and sweat), for absolutequantification, is still problematic. Because of the inter-personal variability (e.g. related to rate ofperspiration, mucus secretion, breath volume), it is necessary to standardize the specimen collectionprotocols. In addition, the metabolites that have equal concentrations across the studied groups ofsubjects may be used for normalization. Further work in this direction is required. Above all, thebiggest shortcomings of the available analytical toolkit include the lack of harmonization of differentsteps (including sample pre-treatment, separation and detection) as well as validation of the developedprocedures. These shortcomings are the main reasons for the limited use of unconventional humanbiological matrices in the clinical setting. One can envision that, in future, several specimens (e.g. hair,nails, breath or skin excretion) will be obtained from a patient to be diagnosed. Subsequently, multipleassays will be performed to produce a comprehensive report on the patient's health.

Supplementary Material

Electronic Supplementary Material:

Click here to view.

Data accessibility

The section ‘Procedures and applications’ has been included in the electronic supplementary material.

Authors' contributions

E.P.D. conducted literature survey and drafted the manuscript. P.L.U. critically discussed the contentand edited the manuscript draft. Both authors gave final approval of the version to be published.

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Competing interests

We have no competing interests.

Funding

E.P.D.: National Chiao Tung University (PhD scholarship). P.L.U.: Ministry of Science andTechnology of Taiwan (grant no. MOST 104-2628-M-009-003-MY4) and National Chiao TungUniversity (intramural grant).

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