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of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
Electronic Supplementary Material
Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices
Ewelina P. Dutkiewicz1 and Pawel L. Urban1,2*
1
Department of Applied Chemistry, National Chiao Tung University
1001 University Rd., Hsinchu, 300, Taiwan
2
Institute of Molecular Science, National Chiao Tung University
1001 University Rd., Hsinchu, 300, Taiwan
Procedures and applications
Below we provide examples of MS techniques, which are commonly used in clinical analysis, toxicology, and
forensics of unconventional human biological matrices. Interestingly, in some reports, the results of analyses of
unconventional matrices are compared with the results of analyses carried out on conventional matrices such as
blood and urine.
a) Liquid chromatography – mass spectrometry
By the end of the 20th century, liquid chromatography coupled with mass spectrometry became the first choice for
quantitative analysis of complex liquid-phase samples containing a wide range of analytes, especially non-volatile
compounds [1]. Unconventional human biological specimens can readily be analysed by LC-MS following
appropriate preparation to isolate the analytes and to bring them to the liquid phase. Various interfaces and ion
sources can be used. They normally provide high robustness and good analytical sensitivity. For example, trace
amounts of a drug could be detected in a nail specimen after administration of only one oral dose. In one study,
Madry et al. [2] investigated incorporation of zolpidem into fingernails, following administration of a 10-mg dose,
by means of HPLC-QQQ-MS/MS. Interestingly, three maxima of drug concentration (along time axis) were
observed for most of the subjects involved in this study. The first maximum was detected already 24 h after the
drug intake; the second one, 2-3 weeks after the intake; and the third one, 3 months after the intake (see Figure 2
in the main text). Based on the results of additional experiments, the first (highest) peak was attributed to the drug
excreted with sweat and sebum, which adsorbed on the nail. The second peak was attributed to the drug released
from the nail bed. This mechanism explains also why the drug could be detected between peak 1 and 3. The third
peak was linked to the incorporation of the drug into the newly formed nail matrix. In this report, the detection
window of zolpidem in fingernail was  3.5 months, while limit of quantification (LOQ) was 0.1 pg mg-1 nail.
The amount of specimen taken for analysis was  5 mg.
Stable metabolites of ethyl alcohol in nails can be used as indicators of chronic alcohol abuse. For instance,
Morini et al. described a method for detection of ethyl glucuronide (EtG) using HPLC-QQQ-MS/MS [3]. The
LOQ of EtG was 10 pg mg-1 nail, while the mass of specimen used for analysis was 20-30 mg. Signalconcentration dependence was linear within the range of 10-500 pg mg-1. The amount of EtG was found to be
higher in nails than in hair. The two above methods were validated. Typically, high selectivity and good
chromatographic separation of isomers could be achieved when using derivatising agents. Some of the derivatising
agents also enhance ionisation of the analytes; thus, improving analytical sensitivity. For example, trace amounts
of ibuprofen isomers (a common anti-inflammatory drug) were quantified in saliva by HPLC-QQQ-MS/MS after
derivatisation with (S)-1-(4-dimethylaminophenylcarbonyl)-3-aminopyrrolidine (DAPAP) [4]. The LOQ of this
method was 0.15 fmol, what corresponds to 30 fg of drug. In other work, Gao et al. developed HPLC-APCIQQQ-MS/MS methods for quantitative analysis of endogenous steroid hormones in hair [5] and saliva [6]. These
hormones can be regarded as biomarkers of psychosocial stress. Hair specimens were washed and extracted with
an organic solvent prior to analysis, while proteins were removed from saliva by precipitation. In both protocols,
on-line solid-phase extraction (SPE) was performed by column switching. Nonetheless, the column switching
© The Authors under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which
permits unrestricted use, provided the original author and source are credited.
Supplementary material for E. P. Dutkiewicz and P. L. Urban, 2016, Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
significantly reduced sample throughput. LOQs for seven steroid hormones in hair matrix were reported to be 
0.1 pg mg-1. In the case of saliva, LOQs were  5 pg mL-1.
Human tear metabolome was investigated by Chen et al. by means of HPLC-ESI-Q-TOF [7]. In their
report, tears were collected using the Schirmer’s strip. Metabolites were extracted with the mixture of an organic
solvent and water. Wide range of compounds (e.g. amino acids, nucleotides, nucleosides) were identified by MS
coupled with RP and HILIC columns. Although this work does not present quantitative data, it brings new insights
into the tear metabolome. It was hypothesised that some of these metabolites can be used to investigate ocular
diseases. Interestingly, the glucose level in tear fluid can possibly be an indicator of blood glucose concentration.
Along these lines, Taormina et al. developed an HPLC-ESI-MS method for quantification of glucose with the
LOQ of 10 μmol L-1 [8]. They collected a tiny volume (1 μL) of tears using a microcapillary, what did not cause
any apparent irritation of the eye. Tear glucose concentration in one fasting subject, sampled multiple times, was
within the range 13-51 μmol L-1. On the other hand, Lam et al. used HPLC-ESI-QQQ-MS to investigate tear
lipidome [9]. In this comprehensive report, tears were collected by means of two different techniques: by a
microcapillary (with pre-flushing the ocular surface with saline solution, and without pre-flushing), and using the
Schirmer’s strip. Lipids were further extracted by means of the modified Folch method. Separation of polar lipids
(e.g. phosphatidylcholines) was achieved using normal-phase chromatography on a silica column, while
separation of less polar lipids (e.g. wax esters) was achieved using reversed-phase chromatography on a C18
column. Moreover, HPLC-APCI-QQQ-MS was also used to analyse free cholesterols and cholesteryl esters (for
which high ion suppression was observed in ESI). Identification of lipid species was achieved by conducting highmass-resolution HPLC-ESI-Orbitrap-MS measurements. The authors reported the absolute amounts of different
classes of lipids in tear specimens collected with different techniques. The highest absolute amounts of lipids were
captured by the Schirmer’s strip method (~ 0.18 μmol mL-1). It is believed that, in this method, the volume of the
collected tear fluid is greater than in the other methods. Nevertheless, the presence of lipids belonging to the same
classes was confirmed with various methods. Overall, in that work, more than 600 lipids species belonging to 17
major lipid classes were identified.
Tear specimens can also be used to study drug pharmacokinetics. For instance, Hirosawa et al. determined
concentrations of the two common anti-inflammatory drugs (ibuprofen and loxoprofen), administered orally, in
tear and plasma specimens [10]. Low-volume specimens of the tear matrix (10 μL) were obtained using a
micropipette. Subsequently, the original specimens were diluted, centrifuged, and the supernatant aliquots were
injected into a HPLC-ESI-QQQ-MS system. The LOQs computed for the two drugs were ~ 0.02 μg mL-1. The
maximum concentration of ibuprofen in both, tears and plasma, were achieved in 3 h, while the maximum
concentration of loxoprofen was achieved in 0.5 h after administration. The concentration of loxoprofen in tears
was found to be higher than the concentration of ibuprofen. The concentrations in plasma showed the opposite
trend. This finding may be explained with different tear secretion profiles of the two investigated drugs.
Concheiro et al. proposed an HPLC-ESI-QQQ-MS method to simultaneously quantify various stimulants
(e.g. buprenorphine, methadone, cocaine, opiates, and caffeine) and their metabolites in skin excretions [11].
During the method development, samples containing 14 standards of drugs and their metabolites were deposited
on a commercial sweat collection patch moistened with artificial sweat (prepared in-house), and air-dried. The
analytes were subsequently extracted from the patch with a buffer (sodium acetate), cleaned and pre-concentrated
on an SPE cartridge. The eluate was dried, and the residue was reconstituted with aqueous solution of formic acid
(0.1%). The analytes were separated on a RP column. LOD for most of the tested analytes was 1 ng per patch
(LOQ for ecgonine methyl ester was 5 ng per patch). This method was applied in the analysis of 16 sweat patches
(each worn for ~ 6 days, on average) exposed to one subject receiving controlled buprenorphine (BUP) treatment
(15.6 mg day-1). BUP was detected in 12 out of 16 patches at the level of 1-2 ng per patch (close to the LOD
value), while a metabolite of BUP was detected only in one patch. Cotinine (metabolite of nicotine) was detected
in all the analysed 16 patches, suggesting that the subject consumed tobacco.
Application of ultra-high pressure liquid chromatography (UHPLC) provides a high chromatographic
resolution and shortens the analyses. Multiple classes of compounds (including endogenous metabolites) in nails
have been detected using UHPLC-MS. For instance, analysis of derivatised DL-amino acids from nails was
carried out by Min et al. by means of UHPLC-TOF-MS [12]. The same type of apparatus was used to determine
dicarbonyl intermediates of advanced glycation end-products (AGEs) in nails [13]. In other report, Tegethoff et
al. focused on foetal steroids – dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS).
These steroids are regarded as potential biomarkers of response to maternal stress [14]. The study involved nail
specimens obtained from 80 infants. It took advantage of an UHPLC-QQQ-MS method. The median
concentration of DHEA in the specimens obtained from the infants exposed to stress (880 pg mg-1) was much
higher than the median concentration of DHEA in the specimens obtained from the infants not exposed to maternal
stress (400 pg mg-1). However, median DHEAS concentrations were found to be similar for both groups. An
analytical method, combining UHPLC-QQQ-MS/MS with alkaline digestion and liquid-liquid extraction, was
proposed by Shi et al. [15]. The authors detected and quantified triclosan (TCS) and triclocarban (TCC) – common
antimicrobial agents used in cosmetics and personal care products. In that study, isotopic dilution (using 13C-
© The Authors under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which
permits unrestricted use, provided the original author and source are credited.
Supplementary material for E. P. Dutkiewicz and P. L. Urban, 2016, Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
labeled analytes as internal standards) was implemented to compensate for matrix effects, and to ensure accurate
quantification of the analytes in 20 nail specimens. The limits of quantification for TCS and TCC were reported
as 2.0 and 0.2 ng mg-1, respectively. Importantly, in all the above four reports [12–15], the proposed analytical
methods were validated, and used to quantify analytes.
In other report, Takayama et al. described an UHPLC-QQQ-MS/MS method for quantification of 12
polyamines in saliva specimens [16]. Polyamines were derivatised with 4-(N,N-dimethylaminosulfonyl)-7-fluoro2,1,3-benzoxadiazole (DBD-F), and successfully separated on a reversed-phase column. The LODs calculated for
all of the studied polyamines were below 0.1 fmol. On the basis of the results of a screening involving cancer
patients before and after the operation, as well as healthy subjects, the authors developed a discrimination equation.
The methodology could become a tool for diagnosis of breast cancer using saliva specimens. An UHPLC-QQQMS/MS method was also developed to determine the ratios of endogenous D,L-lactic acid in saliva as a possible
biomarker of diabetes [17]. L-lactic acid (a more abundant isomer) is produced from pyruvic acid under anaerobic
conditions, while D-lactic acid is produced in the glyoxalase pathway and via hepatic ketone metabolism. It was
reported earlier, that, due to an increased flux of glucose, concentration of D-lactic acid is increased in the serum
of diabetic animals and humans. Small amounts of saliva were collected onto filter paper ( = 5 mm) [17]. Dand L-lactic acid present in the saliva spots were extracted with acetonitrile, and derivatised with (S)-1-(4,6dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy), synthesised in house. Separation of the
derivatised D,L-lactic acid isomers was achieved using a reversed-phase column. Very low LOQs were reported
for both isomers: ~ 100 amol. High D/L ratio of lactic acid was observed in the saliva specimens obtained from
diabetic patients, while low D/L ratios were observed in the specimens obtained from healthy subjects [17]. The
dried saliva spot (DSS) sampling can be regarded as a very convenient technique because the resulting spots do
not occupy a lot of space during storage, and – in general – they do not need to be refrigerated (although some
labile compounds may still be decomposed during long storage).
To improve the sensitivity and selectivity of simultaneous determination of 9 endogenous polyamines in
nail, Min et al. combined nano-flow chip-LC with Q-TOF-MS/MS [18]. Highly polar polyamines were labelled
with a fluorogenic reagent, and separated on a reversed-phase chip column. Implementation of this method led to
a finding that 3 polyamines (putrescine, N1-acetylputrescine, and spermine) tend to accumulate in nails of cancer
patients. This finding suggests that nail specimens may be a useful target for detection of cancer biomarkers.
b) Gas chromatography – mass spectrometry
While LC-MS is particularly suitable for analysis of non-volatile analytes in the liquid phase, gas chromatography
coupled with mass spectrometry (GC-MS) is a robust platform for analysis of volatile compounds in the gas phase.
Many analysts implement the popular GC-Q-MS instruments with electron impact ionization (EI) to detect
commonly abused drugs in various biological matrices. In numerous reports, quantitative data are provided along
with partial or full validation of the proposed methodologies. For example, nail specimens – obtained from the
adults participating in the Edinburgh Drug Addiction Study – were used to detect cannabinoids [19]. In fact, that
study was one of the early efforts toward detection of cannabinoids in nails. The analysis procedure consisted of
several steps. First, fingernail clippings were decontaminated. Subsequently, alkaline digestion was performed
(for 30 min). Then, cannabinoids were extracted with ethyl acetate. Later on, the organic phase was separated
from the aqueous phase, the solvent was evaporated, and the solid residue was subjected to derivatisation with
N,O-bis(trimethyl-silyl)trifluoroacetamide (BSTFA) containing trimethylchlorosilane (TMCS). The results
obtained with GC-Q-MS were compared with the results of a radioimmunoassay (RIA). Δ9-Tetrahydrocannabinol
(THC) was quantified by GC-MS method, while THC and its metabolites (all the structurally related analytes)
were quantified with the RIA method. The cannabinoids were detected by both methods in nails of the same six
subjects. LOD for THC in the GC-MS method was reported to be ~ 0.1 ng mg-1 nail (10 mg of nail specimens
were used). In other work, Milman et al. developed a method for quantification of THC, cannabidiol (CBD),
cannabinol (CBN), and their metabolites (11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC
(THCCOOH)) in saliva [20]. Simultaneous analysis of those compounds was difficult due to the different
physicochemical properties and concentration ranges. THC, CBD, CBN, and 11-OH-THC are typically present
in saliva at much higher concentrations (ng mL-1) than THCCOOH (pg mL-1). THCCOOH is not present in
cannabis smoke and it can be detected only in the specimens obtained from active smokers. Thus, detection of
THCCOOH can be used to distinguish active and passive smokers. In that study, SPE was used to fractionate
saliva matrix. THC, CBD, CBN, and 11-OH-THC were eluted from the SPE sorbent with the mixture of hexane,
acetone, and ethyl acetate, while THCOOH was eluted with the mixture of hexane, ethyl acetate, and acetic acid.
THC, CBD, CBN, and 11-OH-THC were derivatised with BSTFA (with TMCS), while THCCOOH was
derivatised with hexafluoroisopropanol. GC-Q-MS with EI ionisation was used for analysis of most of the
analytes, while chemical ionisation (CI) was used for analysis of fluorinated derivatives of THCOOH. LOQs for
THC, CBD, CBN, and 11-OH-THC were reported to be in the range of 0.5-1.0 ng mL-1, while LOQ for
THCCOOH was 7.5 pg mL-1. In other study, nails collected from adults enrolled in a methadone-maintenance
© The Authors under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which
permits unrestricted use, provided the original author and source are credited.
Supplementary material for E. P. Dutkiewicz and P. L. Urban, 2016, Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
program were analysed for the presence of methadone [21]. That study was one of the first efforts toward detection
of methadone in nails. The results obtained with GC-Q-MS were compared with the results of an enzyme
immunoassay (EIA). The mean concentration of methadone in fingernail clippings determined by GC-Q-MS was
26.9 ng mg-1, while the concentration of methadone determined by EIA was 32.8 ng mg-1.
The usefulness of nail specimens from newborns to monitor drug exposure during pregnancy was explored
by Mari et al. in a study involving over 50 infants [22]. Drugs including cocaine, benzoylecgonine, morphine,
methadone, caffeine, nicotine, and cotinine, were determined in this study. Derivatisation with BSTFA (with
TMCS) was performed prior to GC-Q-MS analysis. LOQs for all the analytes, except for methadone, were
estimated to be 0.025 ng mg-1. LOQ for methadone was 0.05 ng mg-1. Interestingly, cocaine and morphine were
only found in the nails of abandoned newborns, while methadone, caffeine, and nicotine were found in the nails
of both abandoned infants as well as those born in a local hospital. The above result suggests that many of the
mothers who had abandoned their infants were most probably drug users. In other work, Engelhart et al. detected
drugs of abuse (cocaine and opiates) in post-mortem nails removed from cadavers of suspected drug users [23].
Solid-phase and liquid-liquid extractions were performed to isolate analytes, prior to derivatisation with N-methylN-(trimethyl-silyl)trifluoroacetamide (MSTFA), and GC-Q-MS analysis. Interestingly, in their later work,
concentrations of drugs in post-mortem fingernails were found to be higher than in toenails [24].
A few examples of detection of endogenous metabolites in nails can also be found in literature. Two
secondary metabolites, testosterone and pregnenolone – which are also determined to verify steroid abuse in
athletes – were detected in nail specimens obtained from both men and women [25]. GC-Q-MS analysis was
performed after derivatisation of the analytes with the MSTFA reagent. In other work, Δ6-monounsaturated fatty
acids were detected in hair and nail specimens using GC-Q-MS after derivatisation with 4,4dimethyloxazoline [26]. In this work, an ionic liquid-coated capillary was used to achieve baseline separation
between cis-6 and cis-8 monounsaturated fatty acid derivatives. However, absolute amounts of fatty acids were
not determined.
A GC-Q-MS method was used by Ammazzini et al. for quantification of trace amounts of thiocyanate in
the saliva specimens obtained from smoking and non-smoking subjects [27]. Thiocyanate is a metabolite of
cyanide – a toxic compound present in tobacco smoke. Thiocyanate from saliva was converted into volatile ethyl
thiocyanate with a derivatising agent – triethyloxonium tetrafluoroborate. Thus, headspace sampling could be
implemented. Isotope dilution, with the aid of 13C-labelled thiocyanate, was implemented during quantification.
The LOQ of this method was reported to be 25 ng g-1. The actual concentrations of thiocyanate in the “real” saliva
specimens were found to be several orders of magnitude higher (10-184 μg g-1) than the LOQ. The saliva
specimens were diluted before analysis.
In-vivo thin film solid-phase microextraction (TF-SPME) of saliva, combined off-line with GC-EI-Q-MS
and HPLC-Orbitrap-MS, was implemented by Bessonneau et al. [28]. Hydrophilic/lipophilic-balanced (HLB)
particles preloaded with internal standards were selected as the extraction phase. During in-vivo sampling, the TFSPME probe was placed under the tongue of the subject, and kept in mouth (immersed in saliva) for 5 minutes.
In the case of ex-vivo extraction, saliva was collected by spitting. In that case, the probe was used to perform
“static” extraction, also during 5 minutes. In order to enable LC-MS analysis, metabolites were extracted from
the probe with the mixture of acetonitrile and water (60 min). For GC-MS analysis, the probe was introduced
directly to the instrument, and metabolites were desorbed thermally. Numerous compounds were detected with
the two analytical platforms. 107 metabolites were detected using the LC-MS method, while 26 metabolites were
detected using the GC-MS method (without derivatisation). The results from the in-vivo and ex-vivo sampling
showed some differences in relation to the amounts of the sampled compounds. Furthermore, the authors
developed and validated an LC-MS method for simultaneous quantification of 49 prohibited substances (such as
cannabinoids, steroids, and other stimulants) with the LOQs ranging from 0.004 to 0.98 ng mL1 [28].
Nakajima et al. investigated concentration of an anti-epileptic drug – valproic acid – in tears and plasma
with the use of GC-CI-Q-MS [29]. In this study, eleven epileptic patients received a single dose of valproic acid
(400-800 mg). Tears were collected with the Schirmer’s strip, and then – with no need for extraction of the
specimens – a solution of derivatising agent (pentafluorobenzyl bromide) was added. The mean concentration of
the free drug in tears was 4.2 μg mL-1, while in plasma it was 7.5 μg mL-1. The concentration in tears correlated
very well with the concentration in plasma. In fact, concentration of free valproic acid (not bound to proteins) is
regarded as a good indicator of therapeutic efficacy [29].
Kintz et al. published numerous reports covering analysis of various drugs of abuse in skin excretions. For
example, in one report, they developed a GC-EI-Q-MS method to quantify heroin and its metabolites (6acetylmorphine and morphine) in the specimens obtained from 14 subjects participating in a heroin maintenance
program [30]. At first, sweat patches were affixed onto the skin. Afterwards, subjects received 2-3 intravenous
doses of heroin hydrochloride during one day (80-1000 mg person-1). The patches were removed after 24 h, and
skin excretions were extracted from the adsorbent pads with acetonitrile (30 min). Subsequently, the organic
solvent was evaporated. Heroin was analysed directly after reconstitution of the residue with fresh acetonitrile,
while 6-acetylmorphine and morphine were derivatised with BSTFA (mixed with TMCS). The LODs were 0.5,
© The Authors under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which
permits unrestricted use, provided the original author and source are credited.
Supplementary material for E. P. Dutkiewicz and P. L. Urban, 2016, Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
1.0, and 1.0 ng per patch for heroin, 6-acetylmorphine, and morphine, respectively. The recorded amounts (ng per
patch) fell in the ranges: 2-96 for heroin, 0-25 for 6-acetylmorphine, and 0-11 for morphine. The parent drug
appeared to be the major analyte in the skin excretions. In this study, no apparent correlation was observed
between the dose of the administered drug and its amount recovered from the sweat patches [30].
Exhaled breath condensate specimens were analysed by means of GC-EI-Q-TOF-MS by Peralbo-Molina
et al. [31]. In their report, the specimen preparation strategy and the data processing method were fine-tuned to
match the requirements of non-targeted metabolomic studies. Two extraction techniques (liquid-liquid extraction
and solid-phase extraction) were compared in terms of analyte coverage. Liquid-liquid extraction with hexane
was found to be the most efficient. Over 50 volatile and non-volatile metabolites were tentatively identified in the
breath condensate specimens.
c)
Direct mass spectrometry
Secondary electrospray ionisation mass spectrometry (SESI-MS) can be used for rapid detection of volatile
compounds [32]. In this approach, charged droplets created by ESI interact with neutral gaseous sample molecules
introduced into the ESI chamber. The advantage of this system is that gaseous sample/specimen does not undergo
any pre-treatment. Thus, fast and sensitive analysis of vapours from saliva or breath specimens can be carried out.
In one report, bacteria-specific volatile metabolites, present in saliva, were analysed to investigate an
inflammatory disease – periodontitis [33]. 10 mL of headspace vapour was transferred using a gas-tight syringe
into a reaction chamber, where (with the assistance of ESI) secondary ions were formed. Spectra were recorded
within few seconds. Metabolic profiles were constructed for real saliva samples and four individual strains of
cultured bacteria. By implementing high-resolution mass spectrometric detection (SESI with Orbitrap analyser),
the authors identified 120 bacteria-specific compounds. 18 of these compounds were present at a higher level in
the saliva specimen from a patient suffering from periodontitis than in the saliva specimens obtained from healthy
subjects.
A technique related to SESI-MS, called extractive electrospray ionisation MS (EESI-MS), has been used
in the in-vivo real-time fingerprinting of exhaled breath [34]. In this approach (EESI-TOF-MS), exhaled air was
directly blown through a transfer line into the ESI source (operated at 80 °C). The analytes present in breath were
ionised through the collisions with charged droplets created by electrospray. Breath fingerprints were recorded
for a subject before and after consuming food, and before and after drinking beer. Interestingly, selective detection
of sulphur compounds present in breath (after eating garlic) could be achieved when solution of silver nitrate was
delivered through the ESI probe. Recently, EESI-IT-MS was applied in the quantitative analysis of nitric oxide
in exhaled breath [35]. Nitric oxide is a breath biomarker of asthma and other pulmonary diseases. In this study,
breath samples were collected into collection bottles (flow of breath was controlled) containing a solution of 2phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reagent. Exhaled nitric oxide was oxidized by
PTIO. As a result, nitrogen dioxide and 1-oxyl-2-phenyl-4,4,5,5-tetramethylimidazoline (PTI) were formed.
Subsequently, the resulting solution was sampled by EESI-MS, and the initial concentration of nitric oxide was
calculated based on the PTI signal. The reported LOD for nitric oxide was ~ 0.02 ppbv.
Direct injection nanoESI-QQQ-MS was used by Saville et al. to investigate phospholipids in tears and
meibum [36]. Prior to analysis, lipids were extracted from the specimens according to the modified Folch protocol
[37]. The researchers identified 35 choline-containing phospholipids in meibum. Notably, 24 of them were also
found in tears. This result suggests that tear phospholipids are derived from meibum. However, in this study, the
authors did not perform quantitative analysis. In other work, ESI-QQQ-MS was used to identify and quantify
major non-polar lipids in tear specimens collected from normal and dry eyes [38]. The specimens were collected
from rabbits and humans using microcapillaries. Lipids were extracted according to the modified Folch protocol
[37]. The lipid profiles of rabbit tears were found to be very similar to those of human tears. To estimate the
concentrations of lipids in tears, the peak intensities were compared with the peak intensities of internal standards
spiked into specimens (four different standards were used for different m/z ranges). The total lipid amount in
normal eyes was on average ~ 7.3 μg μL-1. In the case of dry eyes, it was much higher, ~ 14.0 μg μL-1. This finding
may be related to a lower content of water in the tears obtained from dry eyes than in the tears obtained from
normal eyes.
Some attempts have been made to lift and detect metabolites and drugs directly from the surface of fingertip.
Takáts et al. used a desorption electrospray ionisation MS (DESI-MS) system to perform such in-vivo sampling
[39]. An aqueous solution of ethanol was sprayed onto fingertip of a human subject (after administration of a
single dose (10 mg) of an antihistamine drug). After 40 min, a signal corresponding to this drug could be detected.
Certainly, this approach is spectacular. However, a high risk of electric shock prevents its wide-spread
implementation. More recently, Katona et al. developed a similar DESI-MS system by implementing a high omic
resistor to minimise the risk of electric shock [40]. Numerous reports related to analyses of latent fingerprints can
be found in literature. For example, Muramoto et al. developed a test sample for spatially resolved quantification
of illicit drugs present on fingerprints with the use of two imaging mass spectrometric techniques – secondary ion
© The Authors under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which
permits unrestricted use, provided the original author and source are credited.
Supplementary material for E. P. Dutkiewicz and P. L. Urban, 2016, Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
MS (SIMS) and DESI-MS [41]. At first, an artificial fingerprint was collected onto a solid surface. Then, known
amounts of standard drugs (from 8 pg to 50 ng of cocaine, methamphetamine, and heroin) were printed on the
surface following an array pattern. Amounts of the drugs were increased in each row of the array. Thus, calibration
curves could later be constructed based on the signals recorded at individual spots. In the case of fingerprints
deposited on the surface of silicon and scanned by SIMS-TOF, concentration maps could be generated for all the
three tested drugs with good resolution, and without high ion suppression [41].
Nanoelectrospray desorption ionisation MS (nanoDESI-MS) was used to extract on-line, and
subsequently ionise skin metabolites collected with the use of a probe comprising of hydrogel micropatches (see
Figure 3 in the main text) [42]. The probe was affixed onto skin for 10 min and afterwards – without any sample
preparation – it was put in contact with liquid micro-junction of the nanoDESI-MS setup. Using this method, a
chemical fingerprint of skin metabolites could be recorded within several seconds. Relative amounts of six skinrelated metabolites were compared for nine healthy subjects. Quantification of metabolites could be achieved by
addition of known amounts of standard compounds to the subsequent micropatches (each probe contained 3
micropatches), and constructing calibration curves. Hydrogel micropatches were also arranged into micropatcharrayed (5 × 5) pads, and used for spatial and temporal profiling of topical drugs on skin surface [43]. It is
noteworthy that a 3D-printed nanoDESI-MS interface, incorporating a humidity chamber, was implemented in
this work. The humidity chamber enabled longer scans of multiple micropatches without the risk of water
evaporation from the hydrogel matrix.
d) Inductively coupled plasma – mass spectrometry
ICP-MS is the technique of choice when conducting trace elemental analysis. If no speciation analysis is required,
specimens are normally digested in strong acids (e.g. nitric acid, sulphuric acid) prior to analysis. Batista et al.
proposed an alternative specimen preparation. It involves overnight solubilisation in 25% tetramethylammonium
hydroxide [44]. In other report, Samanta et al. analysed arsenic as well as 9 other elements in hair, nails, and skin
scales obtained from subjects intoxicated with arsenic (in an area contaminated by geochemical reactions such as
oxidation of arsenic-rich pyrites) [45]. To validate the ICP-MS method, a human hair CRM was implemented in
this study.
An important advantage of coupling ICP-MS with a separation technique is that one can perform speciation
analysis. Different species of one element leave the separation column at different times, and reach the detector.
However, speciation analysis requires extremely careful specimen/sample preparation to prevent decomposition
of any of the species of interest. For example, an LC-ICP-MS method was developed to investigate arsenic
speciation in saliva specimens obtained from 32 subjects exposed to background levels of arsenic (from a location
in Canada) and 301 subjects exposed to increased concentrations of arsenic (from a location in China) [46]. In
this report, arsenic species were quantified with HPLC-ICP-MS after separation within 5 minutes on a reversedphase (RP) column with addition of tetrabutyloammonium (an ion-pairing reagent) to the mobile phase.
Furthermore, to confirm the identities of arsenic species, a strong anion exchange column was used for separation
(mobile phase for ion-pair chromatography was not compatible with ESI). The eluate from the LC column was
split, and directly infused to ICP-MS and ESI-MS/MS. Additionally, the total amount of arsenic was evaluated
by ICP-MS after digestion of saliva with nitric acid. This protocol was successfully applied for speciation of:
arsenite (AsIII), arsenate (AsV), and their methylation metabolites – monomethylarsonic acid (MMAV) and
dimethylarsinic acid (DMAV). The mean concentrations of arsenic species in the saliva specimens obtained from
the subjects from Canada were in the range 0.1-0.7 μg L-1, while the specimens collected from the subjects from
China had concentrations in the range 0.4-8.1 μg L-1. This report showed that quantification of arsenic in saliva
can assist biomonitoring of human exposure to this harmful element [46].
In other work, toxic methyl mercury was quantified in nails by GC-ICP-MS [47]. Nail specimens were
incubated in tetramethylammonium hydroxide overnight, and subsequently derivatised with sodium
tetraethylborate. The GC instrument was coupled with ICP-Q-MS via a heated interface. The recorded amounts
of methyl mercury were found to be relatively high in the fingernails obtained from the individuals who eat fish
regularly. However, the results were obtained only for 6 persons, so they cannot be generalised. Because CRMs
for methyl mercury in nail were not available, the authors used lyophilised tuna fish in the quality control tests.
In one recent study using ICP-MS, presence of some of the essential elements (Ca, Cr, Mg, Cu, Mn, and Fe) and
toxic metals (Cd, Co, and Pb) in hair and nails was found to be associated with cancer [48]. Quantitative
multielemental analysis by direct solid sampling of hair and nail by laser ablation (LA) with ICP-MS was
described by Rodushkin et al. [49]. Using this type of sampling/analysis, quantification is more difficult than
using conventional methods that involve solubilisation and nebulisation at the ICP torch. LA-ICP-MS requires
solid-state analytical standards and correction for possible variations in the ablation efficiency. For these reasons,
most applications of LA-ICP-MS focus on spatial distribution of elements rather than quantification. However,
absolute quantification could still be done; for example, using an external calibrant in the form of a tablet [49].
© The Authors under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which
permits unrestricted use, provided the original author and source are credited.
Supplementary material for E. P. Dutkiewicz and P. L. Urban, 2016, Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
e)
Other techniques
It is generally known that MALDI-MS is not the prime technique for quantitative chemical analysis. Thus, it has
mostly been used in the studies focused on qualitative analyses of unconventional specimens. However, some
efforts have been made to improve its quantitative capabilities (see the review paper on quantitative MALDI-MS
included in this theme issue). For instance, in one report, Pföhler et al. suggested that MALDI-TOF-MS can be
regarded as a fast, precise, and robust diagnostic tool for investigation of nail disorders such as fungal infections
[50]. The discrimination between non-infectious (e.g. psoriatic) and infectious (affected with onychomycosis) nail
disorders could be achieved on the basis of qualitative nail peptide mass fingerprinting [50]. Furthermore, Alshawa
et al. evaluated MALDI-TOF-MS as a routine tool for identifying fungi in skin, nails, and hair [51]. They
developed a spectral database of 12 different species of fungi, and tested it on a group of 381 clinical isolates of
dermatophytes. Mulvenna et al. utilised MALDI-TOF-MS to investigate peptides in tear specimens [52]. Tears
were collected with a microcapillary, and dialysed to remove salts and other low-molecular-weight compounds
(membrane filter with a molecular weight cut-off mass of 500 Da was used) prior to analysis. Small amounts (1
μL) of pre-treated tear specimens were spotted onto a MALDI plate, air-dried, and covered with a MALDI matrix.
Subsequently, additional amounts (1 μL) of pre-treated tear specimens were dispensed again onto the same sites
(on top of the MALDI matrix). Numerous peptide signals were observed for the specimens collected from 11
subjects. Majority of those signals were detected repeatedly in specimens collected during three days. This result
shows low diurnal variation of tear fluid composition. Nakanishi et al. proposed an approach for quantification of
nicotine from a single longitudinally sectioned hair of smokers [53]. They mixed 13C-labelled standard of nicotine
with MALDI matrix, and sprayed such a cocktail onto the hair specimens. The LOQ of this method was reported
to be 1.6 ng mg-1. Kamata et al. compared the results of MALDI-TOF-MS with the results of a reference LCMS technique to prove quantitative capabilities of the MALDI-TOF-MS approach [54]. Both analyses indicated
that there are two sites of incorporation of a drug (an analogue of methamphetamine) into the hair matrix.
In other work, direct analysis in real time (DART-MS) was used to detect Δ-9-tetrahydrocannabinol in
hair without extensive sample preparation [55]. A different technique – isotope ratio MS (IRMS) – was
implemented by Fraser et al. to profile isotopic compositions of hair and nail specimens [56]. Natural variability
of the isotopic compositions of body tissues (e.g. related to diet) can be used to unravel recent geographical life
history of human subjects. For example, the authors of the cited report suggested that stable isotope profiling may
be an efficient way to detect and identify human remains after mass disasters. In their later work, they found a
correlation between 2H isotopic composition of hair and nail with local tap water [57]. Furthermore, Ehleringer et
al. developed a model to predict the geographic region of origin of the owner of a hair specimen, by correlating
the isotopic ratios of 2H and 18O in hair and local drinking water [58]. They constructed maps of the expected
isotope ratios in hair across 48 states within the USA [58].
Capillary electrophoresis (CE) enables separation of electroactive analytes in nanoliter-volume samples in
microscale capillaries. It can also be coupled with atmospheric pressure ion sources using sheath-flow or
sheathless interfaces. Due to the small volume of sample solution, the CE-MS technique is particularly suitable
for analysing volume- or mass- limited specimens. In one approach, CE-TOF-MS was applied in quantification
of 8 psychoactive agents in hair and serum [59]. Low LOQs were reported for this method: 20-77 pg mg-1 for hair
and 1-4 ng mL-1 for serum. CE-TOF-MS was also used in another report from a metabolomic study of saliva
specimens [60]. The authors screened over 200 subjects to identify biomarkers of oral, breast, and pancreatic
cancers. 57 metabolites were found to be related to the onset of cancer disease in human subjects. Overall, the
results of that study supports the usefulness of saliva matrix in the screening for cancer.
Continuous real-time breath monitoring can be performed by means of proton-transfer-reaction MS (PTRMS). PTR-MS features short response times (required for continuous analysis) and low detection limits (down
to the pptv level). At first, primary hydronium ions (H3O+) are produced in the ion source (by a hollow cathode
discharge) using water vapour as a reagent gas. Afterwards, protons are transferred from H3O+ to the neutral
analyte molecules in a drift tube. Trefz et. al. monitored exhaled breath from mechanically ventilated patients,
who had previously undergone cardiac surgery [61]. A 6-m long heated Silcosteel transfer line was used to connect
a mouthpiece and the PTR-TOF-MS instrument located in another room. Endogenous isoprene was proposed as
a biomarker of increased cardiac output. It was monitored with breath-to-breath resolution. The LOQ of isoprene
was 2.39 ppbv using this method. The authors of that work stress that there might be a significant impact of
contaminants present in the hospital air on the results obtained for the tested patients.
© The Authors under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which
permits unrestricted use, provided the original author and source are credited.
Supplementary material for E. P. Dutkiewicz and P. L. Urban, 2016, Quantitative Mass Spectrometry
of Unconventional Human Biological Matrices, Phil. Trans. R. Soc. A. doi: 10.1098/rsta.2015.0380
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