Instrumental Tools for Express Analysis of Lacrimal Fluids

Valentina Vassilenko

1,3

, Mónica Silva

, Ricardo Alves

and João O’Neill

1,2

Centre of Physics and Technological Research (CeFITec), Faculty of Sciences and Technology,

NOVA University of Lisbon, Campus FCT UNL, 2829-516 Caparica, Portugal

Faculty of Medical Sciences, NOVA University of Lisbon, Campo dos Mártires da Pátria, 130, 1169-056, Lisboa, Portugal

NMT, S.A, Edifício Madan Parque, Rua dos Inventores, 2825-182, Caparica, Portugal

Keywords: Diagnostic, Lacrimal Fluid, Ion Mobility Spectrometry, MCC-IMS, Volatile Organic Compounds, VOCs.

Abstract: Analysis of lacrimal fluid is under attention of scientists and physicians as an open “window” for non-

invasive assessment to relevant information about the health status. In the present work we propose an

innovative method for the analysis of volatile metabolites present in the lacrimal fluid by non-invasive, fast

and inexpensive technique: Ion Mobility Spectrometry coupled to a Multi-Capillary Column (MCC-IMS).

Experimental protocol for lacrimal fluid collection and its further analysis by MCC-IMS was developed. For

the first time this technology was used for the analysis of tears from healthy and diabetic person for a “proof

of concept” purpose. Obtained experimental result showed that proposed method is suitable for the

sensitive in-situ express analysis of Volatile Organic Compounds (VOCs) from lacrimal fluid and have a

promising diagnostic potential.

1 INTRODUCTION

The financial costs related to the diagnostic and

treatment of several diseases such as diabetes,

cancer, pulmonary diseases and others are extremely

important and need to be carefully controlled. Even

the conventional analysis of biological matrices, like

blood and urine, require long time and complex

reagents that increase the cost of this analysis.

Beyond that, the collection of blood samples almost

every is an invasive procedure and need a

specialized medical staff and conditions. Therefore,

nowadays the medical community is interested in

new non-invasive, accurate, time and cost saving

methods of analysis for diagnostic or screening

purpose.

Among the existent biological matrices, the

lacrimal fluid has been shown to have the essential

characteristics to perform the non-invasive analysis.

The tear is an extracellular fluid that covers the

surface epithelial cells and forms the anterior

component of the ocular surface. The lacrimal fluid

lubricates and prevents the dehydration of the eye.

Several compounds are present in this matrix

such as: amino acids, glucose, proteins and

electrolytes (Beuerman and Zhou, 2012). These

matrix elements vary from person to person in the

range of concentrations from parts-per-billion (ppb)

or microgram/litre (μg/l) to parts-per-trillion (ppt) or

nanogram/litre (ng/l) and are related to the health or

metabolic condition of each individual.

Thus the sensitive in-situ express analysis of

Volatile Organic Compounds (VOCs) present in the

matrix of lacrimal fluid is a very interesting issue.

Currently, this analysis is not very common in the

clinical practice. Mainly due to the difficulties on

analysis of analites in such low concentration, lack

of information about the detected metabolites and

due to the inexistence of a uniform method of

analysis.

In the present feasibility study, a lacrimal fluid

from patients with diabetes and from the healthy

persons was analyzed in order to find common

and/or discriminating volatile organic compounds.

The main objective was the characterization of tears

matrix by Ion Mobility Spectrometry, but not the

identification of the analytes and determination of

their concentration. As far as we know these are the

first results of the investigations of lacrimal fluids by

IMS technology.

220

Vassilenko V., Silva M., Alves R. and O’Neill J..

Instrumental Tools for Express Analysis of Lacrimal Fluids.

DOI: 10.5220/0004329602200224

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2013), pages 220-224

ISBN: 978-989-8565-34-1

 2013 SCITEPRESS (Science and Technology Publications, Lda.)

2 EXPERIMENTAL

2.1 Ion Mobility Spectrometry

Among several techniques that are used for direct

analysis of VOCs, there is one that stands out due to

its high sensitivity, low cost, portability and

simplicity: the ion mobility spectrometry. The ion

mobility spectrometry (IMS) is based on the drift of

ions according to their mobility in the gas phase at

ambient pressure, under the influence of an electric

field (Stach and Baumbach, 2002).

Figure 1: Physical principle of the IMS. The ionized ions

enter the drift region and are separated according to their

mass and structure.

The major component of the IMS is the

spectrometer: a measuring tube that consists of a

reaction chamber (also called ionization chamber),

in which the ions are generated by a β-radiation

source (3H - tritium), and a drift region, at the end of

which a detector is positioned (Fig.1). The reaction

chamber and the drift region are separated by a

shutter grid, called Bradbury-Nielsen grid. This

element is responsible for controlling the passage of

the ion cloud formed in the ionization region.

In the drift region, there’s an external electric

field that is responsible for the movement of the

formed ions in this chamber. Also, a drift gas

(usually nitrogen, but also ambient air can be

utilized) is pumped into the spectrometer from the

detector’s side.

Due to the applied electrical field and the

opposite gas flow, the ions are vulnerable to

collisions with the drift gas molecules and are

separated according to their structure, charge and

mass, reaching the detector (Faraday plate) at

different times. Ideally, all the analyte molecules in

the sample considered for analysis are totally

separated.

The detected ions generate an ion mobility

spectrum, which shows signals registered at different

times (ms) with the corresponding intensities (V).

Those intensities are proportionally related to the

concentration of each compound, meaning that

higher intensities correspond to higher

concentrations.

The combination of IMS with a multi-capillary

column (MCC-IMS) allows a pre-separation of the

sample, through a gas chromatography technique

(Baumbach, 2009). This provides an increasing of

selectivity and the advantage of an immediate

twofold separation of VOCs with visualisation in a

three-dimensional chromatogram, as represented in

the Figure 2.

IMS has a wide range of applications. Initially, it

was used to detect explosives and chemical warfare

agents, but the same principle has been applied for

medical applications, and to give information about

nutrition, oral hygiene and environmental conditions

(Eiceman, 2005).

Figure 2: Typical MCC-IMS chromatogram showing

characteristic signals of biological matrix.

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221

2.2 Other Analysis Techniques

There are other techniques that are also suitable for

the volatile organic compounds analysis, like GC-

MS, SIFT-MS and PTR-MS.

GC-MS (mass spectrometry with gas

chromatography) is a technique that enables the

separation and identification of volatile organic

compounds and some volatile inorganic compounds

of a gas mixture (Dolan, Newman and Stauffer,

2007). This technique needs a long time to perform

the analysis and pre-concentration (Blake, Monks

and Ellis, 2009).

SIFT-MS (mass spectrometry associated with a

selected ion flow tube) is a technique that uses

precursor ions to ionize gases in a gas sample. It is a

technique that cannot be miniaturized and it is less

sensitive than the MCC-IMS (Spanel and Smith,

1996).

PTR-MS (mass spectrometry by proton transfer

reaction) is a technique that enables the

identification of volatile organic compounds mostly

from natural sources. It uses a vacuum system and

for that reason it cannot be miniaturized (Blake et.

al., 2009).

Compared with other methods of VOC analysis,

ion mobility spectrometry (IMS) stands out due to

its high sensitivity, low cost, portability and

simplicity. The fact that is does not require vacuum

or further sample preparation and the analysis is

performed in a few minutes makes this technique

suitable to be used in hospitals and healthcare

centers.

2.3 Materials and Methods

The samples were collected from 9 diabetic patients

and 9 health individuals according to the standard

protocol procedures with sterile tear flow test strips

(by Sno*Strips) based on the technique of the

Schirmer’s Test (Zhou and Beuerman, 2012). In this

test, the sterile strip is placed in the outer lower

eyelid of each eye during 3 minutes. After this time,

the strip is collected and stored into vials which are

closed with screw caps with a silicone septum.

The reason why the strip is placed in a specific

part of the eyelid is related to some facts:

 The lacrimal gland which is responsible for the

production of the aqueous layer of the lacrimal fluid

is located at the superior temporal portion of each

eye,

 The lacrimal channels are located at the nasal

portion of each eye and communicate with the

lacrimal sac, which could cause a contamination of

the lacrimal sample with nasal compounds.

 The cornea is an extremely sensitive eye region

that could be negatively affected by the strip contact.

A strip with collected lacrimal fluid was placed

in a 20 mL vial, sealed and heated in an Analogic

Heating Plate from VWR®, at 60ºC during 10

minutes. The vial was connected to the MCC-IMS

through a needle that was inserted across the silicone

septum. Figure 3 shows the experimental scheme

considered in this study

After the 10 min of equilibrium headspace the

carrier gas (N

, 25 mL min−1) transferred the

injected sample to the MCC for separation. Then,

the separated analyte, was driven into the ionization

chamber of the ion mobility spectrometer.

Figure 3: Experimental scheme considered in this study.

Analyses were performed on a MCC-IMS

apparatus fabricated by Gesellschaft für Analytische

Sensorsysteme (G.A.S. mbH, Dortmund, Germany).

The multicapillary column (Multichrom, Ltd.,

Novosibirsk, Russia) has a length of 20 cm, a

volume of 0.45 mL, providing a high sample

capacity for preseparation.. The detector was

equipped with a Tritium ioniztion asource (St.

Petersburg, Russia) with an activity of 300 MBq. A

sample inlet lets a continuous stream of nitrogen 5.0

(Air Liquide, Portugal) at 25 mL min−1 to pass

through the ionization chamber where ions are

formed and focused to a shutter grid. A drift gas

flow rate of 500 mL min−1 was used to provide a

good separation of ions and to reduce the flush time

between consecutive measurements.

All experimental parameters of MCC-IMS , such

as drift gas and carrier gas flow rates, injection

volume, grid pulse and system temperature, were

optimized in order to obtain the better spectra.

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3 RESULTS AND DISCUSSION

All spectra were recorded in the positive ion mode.

To avoid contamination of the system with residual

traces from previous runs a system was flushing by

high drift gas flow rate of 500 mL min−1 before

each analysis.

The peak area was used as analytical signal. A

dedicated data acquisition and analysis software

LAV

from G.A.S. was used for analysis of the

results.

A typical spectrum from the performed analysis

is presented in Figure 4. In this map the white colour

represents the highest intensity and saturation and

the black colour represents a negative intensity. The

middle colours vary from blue to green, and then to

pink and red. It was possible to observe several

peaks in the obtained spectra.

Figure 4: Example of a spectrum obtained for one of the

samples analysed. Identified with a P and a number are the

peaks related to the strip analysis, and identified with an L

the peaks of the lacrimal fluid.

To distinguish the peaks related to the lacrimal

fluid and the peaks related to the sterile strip we

have also performed a separate analysis of a single

strip without any tear sample. The peaks in the

Figure 4 identified with P are related to the strip

analysis and the peaks identified with L are related

to the lacrimal fluid.

In the present study were identified 10

characteristic peaks for the strip, and 15 peaks for

the lacrimal fluid. The LAV software were used for

determination of the intensity of the peaks and there

position, determined by the corresponding retention

time (t

) and drift time (t

). The characteristic

parameter of IMS spectra is referred to as ion

mobility, K. It represent as a quotient of ion velocity

and electric field on the tube, and can be calculated

using the measured drift time, td, of an ion through a

drift length, l

, under electric field, E:

However, this ion mobility value can be

normalized to standard gas density, 2.687×1019

molecules/cm, corresponding to T

=273 degree

Kelvin and P

=101325 Pascal, and reported as the

reduced ion mobility, K

(Bensch and Leonhardt ,

2002):

In the Table 1 are represented the calculated

values of reduced ion mobility, as well as the

experimentally determined respective retention

time, drift time and intensities for most relevant

peaks from lacrimal fluids analysis of diabetic and

healthy (control) group.

Table 1: Characteristic parameters for some relevant peaks

obtained from the lacrimal fluid.

Peak t

(s)

(ms)

ࢉ࢓

૛

ࢂ

ି૚

࢙

ି૚

Intensity

(control

group)

Intensity

(diabetes

group)

49,98 8,67 1,26 0,24 0,24

55,20 8,39 1,31

0,29 0,24

106,73 8,48 1,29

0,33 0,54

L11

151,88 13,07 0,84

0,13 0,11

L13

427,39 11,47 0,95

0,10 0,11

L17

428,76 14,88 0,74

0,08 0,08

It was observed that, from the qualitative point of

view, all spectra of lacrimal fluids contain the same

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ௗ

଴

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ௗ

଴

൰൬

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InstrumentalToolsforExpressAnalysisofLacrimalFluids

223

characteristic peaks from the diabetic and healthy

persons. However, some of them represent a

different intensity: the peaks L7 and L11 have a

lower intensity for the diabetes patients, while the

intensity of the L9 is much higher.

These first results show that ion mobility

spectrometry can provides an analysis of lacrimal

fluids by detecting and visualization of volatile

organic compounds.

4 CONCLUSIONS

A first feasibility study with ion mobility

spectrometry was performed to find characteristic

peaks of volatile organic compounds in lacrimal

fluid matrix.

Obtained preliminary results indicate that MCC-

IMS technology is suitable for express analysis of

lacrimal fluid, enabling the detection and recognition

of analytes from this non-invasive matrix which are

relevant in underlying metabolic processes or

diseases.

Further studies with greater numbers of patients

are necessary. Additionally it will be interested to

analyse under the same experimental conditions a

glucose solution (1M) and human. A possibility to

observe coincident peaks of the glucose and insulin

with the analysis of the tear can clarify better if the

glucose level can be monitored through the tear

analysis by IMS.

The different intensities of detected peaks of

insulin and glucose in both control and diabetic

group can indicate a way for probable diagnostic or

disease monitoring

Future experiments shall be performed in order

to allow identification and quantitative analysis of

the compounds present in the lacrimal fluid so that

this method can have clinical application.

The additional testing of exhaled breath by GC-

MS may to relate peaks to corresponding and

chemically identified volatile organic compounds.

ACKNOWLEDGEMENTS

We would like to express our acknowledgments to

all the volunteers because without them this work

would not be possible.

REFERENCES

Baumbach, J. (2009). Ion mobility spectrometry coupled

with multi-capillary columns for metabolic profiling

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Bensch, H. and Leonhardt, M. (2002) Comparison of drift

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(3), 7-10

Blake, R. S., Monks, P. S., & Ellis, A. M. (June de 2009).

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Eiceman, G. (2005). Ion Mobility Spectrometry. Taylor &

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Spanel, P., & Smith, D. (November de 1996). Selected ion

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Medical & biological engineering & computing, pp.

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Stach, J., & Baumbach, J. I. (2002). Ion Mobility

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Stauffer, E., Dolan, J. A., & Newman, R. (2007). Fire

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