MSc Chemistry

Analytical Chemistry

Literature Thesis

Investigating potential modes of detection for

chromatography-on-a-chip devices.

by

Christian Binder

June 2014

Supervisor:

Dr. Michelle Camenzuli

List of tables.

Table 5.1: Part of a table that indicates (with a +) when a colour reaction occurs between an analyte

(right) and a prepared reagent (A.1, A.2, etc.). Diazepam and Acetaminophen, for example, give

different reaction patterns. Reproduced from ref. [54].

Table 5.2: A number of LOD values of interest for different analytes and tests. Reproduced from ref.

[9g].

Table 7.1: Detection limits for UV and C4D detection for a variety of compounds. Reproduced from

ref. [13].

Attachment 1: Overview of the Rf for different analytes depending on the solved used in the method.

Reproduced from ref. [57][58]. * have an additional spot at Rf 0.68 caused by the reagent.

Attachment 2: Overview of found LOD values per detection mode.

List of figures.

Figure 1.1: The structures of GHB and GABA. Reproduced from ref. [6a].

Figure 1.2: Heroin and morphine. Reproduced from ref. [6b].

Figure 1.3: Methamphetamine and amphetamine. Reproduced from ref. [6c].

Figure 3.1: Example of a portable mass spectrometer, the Mini 11 mass spectrometer weighing 4kg.

Reproduced from ref. [38].

Figure3.2: Schematics for ESI (above) and APCI (below) ionization sources. Reproduced from ref. [44].

Figure 4.1: Structure of the conductivity cell. Reproduced from ref. [17].

Figure 4.2: The working principle of the developed competitive immunoassay method for impedance

detection of methamphetamine (MET) concentration. The gold nanoparticles (AuNPs) are introduced

into the electro-microchip, forming a bridge that allows for the transport of electrons. Reproduced

from ref. [16].

Figure 4.3: Overview of the creation of the immunosandwich for the optical detection of silver films.

Reproduced from ref. [50].

Figure 6.1: A detection method making use of a paper interface and Raman spectroscopy. A) shows

the structure of the printed paper. B) illustrates the use as a surface swab for sampling while c)

shows the ability for concentration of the taken sample by lateral flow. D) is the detection of the

dipstick with the Raman probe. Reproduced from ref. [65].

Figure 6.2: A smartphone with accessory that allows fluorometry. Reproduced from ref.[68].

Figure 7.1: A CE and potentiometry detector setup that indicated the potentiometry detector as an

ion selective electrode (ISE) . Reproduced from ref.[12].

Figure 7.2: A C4D detector (right) with the sensor on the left. Dimensions (H/L/W) are 105/250/250

mm (3 kg) and 10/28/23 mm respectively. Reproduced from ref. [80].

Figure 7.3: Voltammogram of Morphine [M], Noscapine [N] and Heroin [H], with an overlap of M and

H. A graphene nanosheets-glassy carbon modified electrode is used for the acquisition of signal d.

Reproduced from ref.[81].

Figure 7.4: Voltammograms of Morphine (a) and Codeine (b). Reproduced from ref. [75] .

List of abbreviations

6-MAM – Monoacetylmorphine. SERS – Surface enhanced raman spectroscopy.

APCI – atmospheric pressure chemical ionisation. SPR – Surface plasmon resonance.

AuNPs – Gold nanoparticles. TMB – Tetramethylbenzidine.

BSA – Bovine serum albumin. TLC – Thin layer chromatography.

CE – Capillary electrophoresis. UV – Ultra violet.

C4D – Capacitive contactless conductivity detection.

ELISA – Enzyme-linked immunosorbent assay.

ESI – Electrospray ionisation.

FTIR ATR - Fourier-transform infrared attenuated total reflection.

GABA - Gamma-aminobutyric acid.

GBL - Gamma-butyrolactone.

GC – Gas chromatography.

GHB – Gamma-hydroxybutyric acid.

HPLC – High-performance liquid chromatography.

HRP – Horseradish peroxidase.

IS – Internal standard

ISE – Ion selective electrode.

LC – Liquid chromatography.

LIF – Laser induced fluorescence.

LOD – Limit of detection.

LOQ – Limit of quantification.

M – Molar concentration: mole/l

MA/MET – Methamphetamine.

MCE – Microchip capillary electrophoresis.

MDA - Methylenedioxyamphetamine

MS – Mass Spectrometry.

MTBE - methyl tert-butyl ether.

MTBSTFA - N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide

Summary.

Lab-on-a-chip devices are interesting. They do not just help the world, and scientists, by required less

material and chemicals, but also take over tasks such as mixing and diluting. Analysis that previously

have been confined to labs can be taken into the field. This can save time, money and lives, especially

in countries with poor infrastructure or a general lack of equipment.

In this literature study a variety of detection techniques will be studied with the intention of coupling

one with liquid chromatography, ending with a recommendation for the design of a lab-on-a-chip

drug testing device. This device has to fulfil certain expectations:

- The size of a mobile phone.

- Portable and able to function without external equipment.

- Able to detect different types of drugs and their metabolites.

However for now this device only has to be able to give a positive or negative results. Further testing

should still be done in dedicated labs. The detection methods that are reviewed can be grouped into

five categories: mass spectrometry (MS), immunoassays, colour tests, spectroscopy and

electroanalytical detection. Things such as separation methods, chemical requirements and chip

design, while important, are not covered in the thesis.

Three different drugs are used as keywords, both to narrow down the literature search and for

comparison purposes. The three drugs used in the search are gamma-hydroxybutyric acid (GHB),

heroin and methamphetamine. On occasion related drugs(such as morphine) are used.

A set of criteria is used to evaluate the found information. The criteria being:

1. Miniaturization potential: Can the method of detection be scaled down to produce a product

of the wanted physical dimensions and is it robust enough for field work?

2. Sample versatility: What kind of samples can be used for detection and is the sample pre-

treatment something that can be done in the field, either by a person or by the chip.

3. Selectivity: How many different compounds can be detected during one run?

4. Sensitivity: How sensitive is the detection method? What are the parameters such as

detection limit, bias and precision?

Criterion two is proven to be of less worth, none of the found techniques are expressly stated to be

unusable for a type of sample (blood, urine, hair), as long as it can pass through a liquid

chromatography column.

The first criterion rules out MS and spectroscopy techniques. Neither appears to be available in the

desired size or less. Criterion three removes immunoassay and colour tests as options, for both can

only detect anticipated compounds. While this removes potentiometric options of electroanalytical

detection, amperometry, conductivity and voltammetry detection is still possible. Voltammetry may

even be able to identify compounds based on their voltammograms.

Table of contents

Chapter 1: Introduction. .......................................................................................................................... 1

Chapter 2: Samples and sample preparation. ......................................................................................... 4

Chapter 3: Mass Spectrometry ................................................................................................................ 9

Chapter 4: Immunoassay. ...................................................................................................................... 12

Chapter 5: Colour tests. ......................................................................................................................... 16

Chapter 6: Spectroscopy ....................................................................................................................... 19

Chapter 7: Electroanalytical .................................................................................................................. 23

Chapter 8: Conclusion. .......................................................................................................................... 28

References and Literature ..................................................................................................................... 29

Attachment 1: Overview of Rf values for TLC. ...................................................................................... 35

Attachment 2: Overview of LODs. ......................................................................................................... 36

1

Chapter 1: Introduction.

In a world that is trying to become greener, lab-on-chip applications are becoming increasingly

interesting. Their advantages do not solely lie in a decrease in the costs of materials and chemicals,

because they can also be designed to mix and dilute samples and acting as a reactor[1]. Techniques

previously confined to labs can, with sufficient research, be brought into the field, saving valuable

time. Especially in countries with poor infrastructure or a general lack of equipment, a portable

system could give answers that cut costs and save lives.

In this literature study a variety of detection techniques will be studied with the intention of coupling

one with liquid chromatography, ending with a recommendation for the design of a lab-on-a-chip

drug testing device. This device should be of the same dimensions as a modern mobile phone, able to

function without external equipment and ready to detect different types of drugs and their

metabolites. The final product has to be able to, at the very least, give a yes or no when analysing for

drugs, further testing should still be done in dedicated labs. The detection methods that will be

reviewed can be grouped into five categories: mass spectrometry (MS), immunoassays, colour tests,

spectroscopy and electroanalytical detection.

Things such as separation methods, chemical requirements and chip design, while important, are not

covered in this thesis.

In the beginning of the study, three different drugs were used as keywords, not only to narrow down

the search in the vast sea of information of drug detection, but also to allow for a greater ease in the

comparing of those techniques, as long as the found material allows for it. The three drugs used in

the search were gamma-hydroxybutyric acid (GHB), heroin and methamphetamine. Sometimes

related drugs (such as morphine and amphetamine) will be used if they are relevant and the three

initially chosen drugs to not give an equivalent of information.

GHB:

Odourless, colourless and with just a slightly salty taste, it is used as a date-rape drug through

addition to beverages, and will lower the victims inhibitions and relax their muscles[2]. Other effects

may include short-term amnesia, confusion and respiratory depression.[3]. However that does not

mean that GHB is a compound foreign to the human body[4]. GHB itself is a naturally occurring

metabolite of gamma-aminobutyric acid (GABA) and can be found in the whole body but primarily

the brain, where it affects the rate of serotonin metabolism[4]. Its exact metabolic pathways are not

yet understood[4]. Some detection methods may convert the GHB in a sample into gamma-

butyrolactone (GBL)[2][5]. GHB derivatives and precursors can be found performing other functions

such as solvents and in the treatment of narcolepsy and alcoholism[4]. See figure 1.1 for the

structures of GHB and GABA.

Figure 1.1: The structures of GHB and GABA. Reproduced from ref. [6a].

2

A variety of commercial tests are available for personal safety in addition to laboratory test that

more specialised equipment such as UV spectrophotometers or fluorescence detectors[2][4].

However the personal safety tests are intended for drinks while the specialized instruments are not

yet in a portable format.

Heroin.

A highly addictive drug that can be metabolized into compounds such as morphine and 6-

acetylmorphine, the latter being an interesting marker since it has no natural source[7]. Heroin

produces its effect when it arrives in the brain and is converted. Use may create feeling of euphoria,

cloud the mind and cause a dry mouth[8]. Regular use will change how the brain functions, creating a

tolerance towards heroin[8]. Other than the dependence, chronic users may suffer from issues such

as collapsed veins, constipation, liver and kidney diseases in addition to the risks of Hepatitis and HIV

infections if needles are shared or toxic contaminants in the heroin[8].

A common screening test is the mixing of a sample with a colour changing reagent, such as Mandelin

or Marquis, and observing the colour change[10].However there is the disadvantage that colour tests

can react with other compounds, so additional steps are needed for complex samples, such as blood

and urine[10]. Other examples of detection methods are chemiluminescence and CE

potentiometry[10][11][12]. As with GHB, there no examples of portable devices for field use. See

figure 1.2 for the structures of heroin and morphine.

Figure 1.2: Heroin and morphine. Reproduced from ref. [6b].

Methamphetamine.

Methamphetamine belongs to the amphetamines, a group of synthetic drugs with relatively similar

chemical properties[13]. Once taken they will create feelings of pleasure and well being, with the

possible side effects of delusions, depression and hallucinations[13]. Other side effects, some after

extended use, are: damage the heart, restricted blood flow, involuntary muscle contractions and

overheating[14]. Methamphetamine has a hunger suppressing effect and through that may lead to

malnourishment. [14]

Potential ways to detect methamphetamine are with GC-MS analysis of nails, CE with conductivity

detection, Immunoassays and Immunosensors[13][15][16][17][18]. As with the previous drugs, none

of these are stated to exist as a portable device. But at least conductivity measurements and

immunoassays appear to be small enough, MS devices are in general relatively large. See figure 1.3

for the structures of methamphetamine and amphetamine.

3

Figure 1.3: Methamphetamine and amphetamine. Reproduced from ref. [6c].

To make a sensible recommendation in regards to a detection method for chip-LC, four criteria will

be kept in mind. The criteria are:

1. Miniaturization potential: Can the method of detection be scaled down to produce a

product of the wanted physical dimensions and is it robust enough for field work?

2. Sample versatility: What kind of samples can be used for detection and is the sample pre-

treatment something that can be done in the field, either by a person or by the chip.

3. Selectivity: How many different compounds can be detected during one run?

4. Sensitivity: How sensitive is the detection method? What are the parameters such as

detection limit, bias and precision?

After this introduction a separate chapter will be dedicated towards possible samples (blood, urine,

hair, etc.) and the sample preparation required prior to detection. In other words, the second

criterion has its own chapter. The other criteria are contained in the detection method chapters, but

are not always clear cut paragraphs. Lastly in regards to criterion four, how values such as limit of

detection are reported vary depending on the writer and sample. A hair analysis may give drug per

weight of hair while a concentration in blood can be weight per volume. For ease of comparison all

the values will be translated (based on sample preparation and injection volume) into a molar

concentration (M).

4

Chapter 2: Samples and sample preparation.

There are a wide variety of different sample types that can be encountered, ranging from street

samples from drug seizures to the biological, such as hair and urine .Even if narrowed down to those

that can be collected with little to no training on the street, at an interview or during a drug bust, we

still have a lot of choice. This chapter contains information about various sample types that can be

taken from a human body, with varying degrees of required training. Detection methods that start

with drugs in powder or tablet form are rarely mentioned in the literature.

Blood.

Blood is a preferred sample for analysis it has a wide variety of analytical methods and a vast amount

of reference data, greater than any other type of sample[9a][19]. However the time window for

analysis of drugs is fairly narrow, depending on the drugs the window can range from 6-8 hours, for

GHB, up to 24-36 hours[9b].

The collection of blood samples requires personnel with at least some medical training[9c]. Both for

proper handling and acquisition of the samples, but proper handling if the possibility exists that there

are infectious agents present[9c]. This goes for practically all liquid samples taken from a human

body[9c]. Chemicals used for disinfection prior to the extraction of blood may have an effect on the

samples[9a].

Blood is a complex sample, only a component of it is used for detection with LC-MS methods[20]. The

component is serum, blood plasma that that has been rid of clotting proteins. But even then the

serum has to be treated for actual injection and detection. The next step is an extraction.

This can be done either with column, such as a Bond Elute Certify that extract (cationic) drugs from

the sample that can then be eluted[21].

A different way to extract drugs it to add methyl tert-butyl ether (MTBE) to a volume of samples that

is then centrifuged[22]. From the upper phase a fraction is collected and evaporated to dryness using

a speed vac[22]. The resulting pellet is dissolved in 3M HCl n-butanol and incubated at 50 °C[22]. The

solvent is removed using vacuum concentration after which 1:1 (v/v) acetonitrile:water is added. This

mixture is centrifuged and injected into a HPLC-MS[22].

It is also possible to, once a plasma sample has been spiked with internal standard (IS), diluted the

sample with a phosphate buffer to load it on a conditioned solid-phase cartridge[23]. Analytes are

eluted with a dichloromethane/isopropanol/25% ammonia (80:20:2 v/v/v) mixture[23]. The eluate is

then evaporated under a nitrogen stream at 30 °C while HCl/2-propanol (3:1) is added to prevent

volatiles (such as amphetamines) from vanishing[23]. Dried residue is dissolved in mobile phase of

which a fraction is injected[23].

5

Urine.

Plentiful and easy to analyse with potentially greater concentrations than can be found in blood and

a larger time window from 8-12 hours for GHB, and 2-3 days for other basic drugs[9b].

Urine requires an extraction just as blood does, but not the removal of proteins[21]. A Bond Elute

Certify column can be used here as well[21]. The other possible method of extraction is similar to one

described for blood[22]. HCl (0.1 M) and ethylacetate are added to a sample that is then shaken

vigorously for a few minutes at room temperature and centrifuged[22]. Any IS should be added prior

to the extraction[22]. From the upper layer a volume is collected and evaporated until dryness[22].

The residue is dissolved in 3M HCl n-butanol and incubated at 50 °C[22]. Jjust as with the blood, the

solvent is removed and the resulting pellet is dissolved in acetonitrile/water of which a fraction is

injected[22].

Dilution is the preferred sample pre-treatment for immunoassay methods, and colour tests may just

require an adjustment in pH, as far as colour allows it[24][25].

Beverages.

Spiking beverages is a common method of administration for drugs such as GHB and

Flunitrazepam (Rohypnol), this has led to a variety of commercial tests, these however are not as

sensitive as laboratory methods[9b]. Drinks that have been tampered with may show changes in

colour, cloudiness or opacity, debris either floating or on the bottom of the container, or oily

globules suspended in the beverage[9b].

Either liquid samples of the beverages, or residue scraped from the container can be used for

detection, depending on the method. [9b]

Saliva.

Compared with blood or urine, saliva is much easier to collect because it only needs an absorptive

material, but may still pass on infectious agents and should be handled with care[9c][26]. Ideally the

donor shouldn’t smoke, drink or eat for 10 to 15 minutes prior to sampling[9c]. The use of saliva is

becoming more interesting because new developments allow the sensitivity that detection in saliva

requires[9c]. Unless anxiety, medical conditions or drug use inhibit the production of saliva, an adult

may produce between 500 and 1500 ml per day[9c][26]. If needed the production can be stimulated

further by chewing gum or rubber, or through acidic sweats or a citric acid solution[26].The latter

being much more effective, resulting in a production of 5-10 ml/min while chewing may at best

produce 1-3ml/min[26]. Stimulation will give more sample, less variability in the pH of taken samples

and lower the intersubject variability in the saliva/plasma ratio[26].

The saliva/plasma (S/P) concentration ratio is a value that is used to extrapolate the concentration of

a drug in plasma from the concentration in saliva[9c][26]. The plasma concentration indicates the

free drug concentration that can cross the blood-brain barrier and affect the nervous system

there[9c]. There are many factors that influence the S/P ratio, not just the chemical properties of the

6

drugs[9c][26]. Smoking a drug vs. intravenous injection will give different results, and will also affect

how long the drug can be detection in the saliva (up to 24 hours and 30 minutes respectively)[9c].

This also means that rinsing the mouth or brushing teeth may not be able to clear the saliva collected

at a later date of drugs.

Samples are taken by using cotton pads to absorb oral fluid that is then squeezed into a glass

container as opposed to directing donors to drool into a container[27]. An amount of samples is

taken and saturated ammonium carbonate solution (pH 9.3) is added and vortex mixed[27]. Then

ethyl acetate:hexane (4:3) is added and again vortexed, followed by stirring. From the organic phase

a fraction is taken and is concentrated to dryness under nitrogen at 50 °C[27]. The residue is

resuspended in 250 µl of mobile phase containing IS[27].

Sweat.

Used to a lesser degree than the above mentioned samples. It can collected by wiping a cotton fleece

moistened with isopropanol over skin[21].

Hair.

Hair can be used to test for drugs, but not for the same reason that blood, urine or oral fluids would

be used. Hair production is far slower, 1 cm per month for head hair, other types of hair can be even

slower[9d]. This means that the upper quarter of a 4 cm long hair would give an indication about the

drug use 3-4 months ago. A hair taken when a person is suspected of recent drug use may therefor at

best function as a baseline for future detection, or show the difference between chronic use and

single exposure[9d]. There is however a lot of hair on a head and it means that a practically identical

sample can be taken at a later date, depending on how much hair is needed for detection[9d]. It

should be noted that different types of hair have different growth patterns and will show different

concentrations[9d]. Different individuals can have different drug and metabolite concentration ratios

when comparing hair and blood, unfortunately the exact mechanics of how drugs are incorporated in

hair are not sufficiently clarified[9d][28].

What makes hair a harder sample to analyse than blood or urine is that while the drugs of interest

are stable in the hair, holding the drugs for years under favourable conditions, they have to be

released[9d]. The stability of drugs in hair depend on factors such as hair structure, colour and

cosmetically treatments[9d]. Releasing the drugs from the hair can be done through extraction with

solvents, hydrolysis or by digesting the keratin. The method used to free the drugs for analysis will

have to be carefully chosen so it doesn’t influence the concentrations [9d].

The first step in the preparation of a hair sample, is the decontamination. This can be done in several

ways:

- With a volume of methylene chloride for 2-5 minutes[29][30].

- With water and acetone (1 minute and two times 1 minute respectively)[31].

- Water, acetone and then hexane, each for 2 minutes[32].

7

- Washed with 0.1% sodium dodecyl sulphate, deionized water and acetone [33].

- The hair is added to 0.1M phosphate buffer and mixed for 1 minute before the buffer is

removed. The hair is then washed with isopropanol and dichloromethane.[23]

- Hair is added to a fritted reservoir that can be fitted with a luer plug, 2-propanol and a

disposable stir-bar are added prior to plugging. Samples are incubated at 37 °C while stirring.

The plug is removed and the 2-propanol is collected under vacuum in a glass tube. The reservoir

is now filled with 0.01M phosphate buffer, plugged and incubated at 37 °C. The buffer is

collected as before with the 2-propanol, the buffer wash is repeated two more times and

combined.[34]

After the decontamination, the hairs are dried and cut into fragments, varying in size from <1mm to

2 mm[29][31][32][33].Depending on what is to be analysed, a wash step can be kept for further

analysis[32]. The now washed, dried and fragmented hair is prepared further for injection in one of

many ways:

- Digestion with NaOH at 75 °C for 40 minutes[29]. After allowing it to H2SO4 and ethyl acetate is

added after which the mixture is agitated and centrifuged[29]. The supernatant (organic layer) is

dried and reconstituted with an amount of mobile phase, of which a fraction is injected[29].

- Incubated for 18 hours in methanol/acetonitrile/2mM ammonium formate with gentle shaking

at 37 °C, followed by[31]. The liquid phase is collected and the incubation is repeated. Both

extracts are united and evaporated under nitrogen to a 0.5 ml volume, after which a part

injected[31].

- Enzymatic digestion solution and IS are added and incubated overnight at 37 °C while stirring[34]. After incubation the digest is collected under vacuum in a glass tube and washed with acetate buffer[34]. Additional acetate buffer is added to the tube and the whole is gently stirred for a few seconds[34]. The digest is poured into a SPE cartridge and then washed with water, HCl and methanol before vacuum drying[34]. The analytes are eluted with several times with methylene chloride/2-propanol/ammonium hydroxide[34]. Then MTBSTFA is added and then evaporated to dryness with nitrogen at 55 °C. It is reconstituted with mobile phase, vortexed and centrifuged before being transferred into autosampler microvials for analysis[34].

- Extraction with methanol followed by extraction with 1.4% HCL/methanolic solution, both

under sonication[32]. Each extraction solution is evaporated under nitrogen at 40 °C[32]. If

decontamination wash solutions are to be analysed, a volume is evaporated under nitrogen at

40 °C overnight[32]. Residues (both hair and wash) are dissolved in of methanol and diluted with

ammonium formate buffer adjusted to pH 3 with formic acid. 10 µl is injected for analysis.[32].

- Hair is extracted with a solution of internal standard in methanol:acetonitrile:20mM ammonium

formate ratio and sonicated at room temperature[33]. The mixture is centrifuged at after which

the supernatant is removed and air-dried, the resulting residue is reconstituted in a

H20:Acetonitrile resuspension solvent[33]. The sample is then diluted 100 fold with blank matrix

that has the same concentration of IS before injection[33].

8

Nails.

Comparable to hair, nails are composed of keratin and show the same tendency to incorporate drugs

in their structure, however at a slower rate[35].. Drug ratios may be different compared to hair and

concentrations lower[35]. And resembling how the type and location of hair has an effect on

concentrations in hair, it appears that nail clipping from fingers have higher concentrations than

those from toes[9d][36]. Considering the composition, nails should undergo treatments similar to

hairs prior to detection.

9

Chapter 3: Mass Spectrometry

Mass Spectrometry (MS) is a widely used technique that finds us in many area, among them:

environmental monitoring, food regulation, forensics and homeland security[37][38]. It is a label free

technique that can be used for the detection of any molecule as long as it can be ionized[39]. It is

capable of giving information on the mass, concentration and identity of analytes[39][37]. Optical

techniques in comparison require that the analytes expected in the sample, are given detectable

properties[39]. For our end product we will focus on Liquid Chromatography coupled to MS (LC-MS).

In this section the potential of MS for lab-on-chip applications will be discussed.

Miniaturization of a MS system comes with the usual advantages of portability, less sample use and

lowered power consumption. The decrease in the required power also comes from the fact that less

power is needed to keep up the vacuum required by the MS. The current disadvantages of

miniaturized MS systems that are available in a portable format, is that they are limited to non-polar

and thermally stable organic compounds[40][41].

In a direct comparison of a LC-MS and a LC-chip/MS (for the analysis of steroids), the LOD of the chip

system was higher than the conventional system, 10-150*10-12 M and 50-300*10-12 M

respectively[41]. This could indicate that all miniaturized systems suffer from reduced sensitivity.

The problem is that there are portable MS systems, but they have not yet reached the dimensions

that we prefer (figure 3.1)[38]. There are no references that mention the bottleneck lying either with

the ion source or the mass detector, two of the components that together with the mass analyser

form the MS system[38][42].

Figure 3.1: Example of a portable mass spectrometer, the Mini 11 mass spectrometer weighing 4kg.

Reproduced from ref. [38].

The ion sources commonly used for LC-MS are electrospray ionisation (ESI) and atmospheric-pressure

chemical ionisation (APCI), ESI being more sensitive while APCI works better for molecules that do

not ionise as readily[9e]. The designs of either ionization source do not seem to require a significant

size, if they do then the references remain quiet about them (figure 3.2 for schematics of the

designs). The mass detector merely has to be able to record charges that are induced, or produced,

when an ionized molecule (or a fragment), passes the detector or strikes it. As before, no mention is

made about the detector being an important bottle neck. Neither a faraday cup or an electron

10

multiplier looks as if a certain degree of bulk is needed[43]. However the third (or middle) part of the

MS, the mass analyser has a link between performance and size[42]. In particular quadrupoles are of

interest because of their simplicity and innate compactness[42]. Further miniaturization lowers the

internal volume and surface area, the lower volume adds robustness to the device, while the

reduction in surface area results in less memory effect and through that faster response in

concentration changes[42]. It is however stated by S. Boumsellek et al. that “the reduced surface

area of the sensor inlet leads to a total signal loss by a factor of n2”, with n being the scale down

factor of complete charged particle device[42].

All this considered miniaturizing an MS system is possible, and arguably preferable for the MS itself,

but not to the degree that the first criteria requires.

Figure3.2: Schematics for ESI (above) and APCI (below) ionization sources. Reproduced from ref. [44].

The selectivity of a Mass Spectrometer is higher than optical-based techniques and is one of the

reasons MS has replaced other analytical techniques, although it should be noted that an internal

standard is needed to ensure the best accuracy[39][9e]. In many references there is little to no

attention spent on the selectivity of MS methods. However additional MS dimensions (such as

tandem MS) have an increased selectivity[45].

The possible detection limits depends on the kind of drugs type of sample.

The LOD for GHB found in Serum can range from 2.8*10-7 M to 9*10-6 M[22][46]. Urine has a slightly

higher LOD at 1.24*10-5 M[22]. The values found in a method for detection in hair are given in a per

hair concentration, having an LOD of 0.5 ng/mg and a linear range from 0.6 to 50ng/mg[29].

Considering the method uses 25 mg of hair and reconstituted the residue in 50 µl mobile phase, the

LOD should be about 2.4*10-6 M[29].

11

Methamphetamines, detected in hair, have an even lower LOD than GHB, around 0.003 ng/mg[31].

With 20 mg of hair in a final volume of 0.5 ml, the inject concentration would be around 8.0*10-8 M.

In one instance LOD as low as 0.1-0.75 pg/mg are noted for cocaine, cocaine metabolites and

amphetamines[33].This method uses only 2 mg of hair in 50 µl, and a 100 fold dilution step,

translating roughly to an injection LOD concentration of 1.32*10-11 M for cocaine and 2.96*10-11 M

for amphetamines at the lowest[33]. Plasma and urine have been reported to have a LOQ (10 times

standard deviation of the blank vs. LOD that has 3 times standard deviation) of 20 ng/ml for 3,4-

Methylenedioxyamphetamine (MDA) and 10 ng/ml for other amphetamines, 1.12*10-4 M and

7.41*10-5 M respectively[21]. The LOQ for oral fluids and sweat lies around 1-5 ng/ml for several

other amphetamines, with no specific being mentioned[21]. Assuming amphetamine, the LOQ in

molar concentration would be 7.41*10-6 M to 3.70*10-5 M.

Heroin has been found to have a LOD of 0.26 ng/ml (7,04*10-7 M)in oral fluid, comparable to four

amphetamines mentioned in the same article.[27] The metabolic product morphine has a LOD of

0.65 ng/ml (2.28*10-6 M)[27].

12

Chapter 4: Immunoassay.

In comparison to the mass spectrometers of the previous chapter, Immunoassay based detectors

offer a much smaller and cheaper alternative, due to less expensive instrumentation and required

time[16][17][18]. There are commercially available portable immunoassay tests, such as pregnancy

tests, indicating that a mobile phone sized end product is possible[9f]. Experimental immunoassay

methods that are designed with a conductivity measurement cell end up with cell volumes around

0.09 cm3.[17]. Size is less of a problem compared to MS.

The strength and weakness of an Immunoassay lies in the antibodies that are needed for detection.

These antibodies must be produced for each analyte and may have poor specificity, sensitivity to the

test matrix or a short shelf life[9f]. Immunoassays may also have problems with analytes of low

molecular weight[18]. This issue is not just in detection, but in production as well since the target

molecule has to be an immunogen and this is not always the case for smaller molecules[18]. A

solution to this is the linking of the molecule of interest to a carrier protein[18].

In addition to the right type of antibody, there are applications in need of a reporter. This reporter is

the component that results in a detectable signal[9f]. This could be a radioactive label, something

that produces chemiluminescence or colloidal particles for an electrochemical detection[9f][16].

It is possible to use an enzyme in the detection. The so called enzyme-linked immunosorbent assay

(ELISA) links an enzyme to an antibody (or antigen) that reacts with a colourless substance to

produce a detectable colour[16]. ELISA makes use of a microtitre plate, which may not be the easiest

to method for the portable detector[47]. A construction using microchannels however would be

more practical. In these kinds of optical methods, the detection can be done with a

spectrophotometer[48].

Electrochemical detection methods may not have the same problems, as can be seen in figure

4.1[17]. The antibodies are immobilized on the electrode and the conductivity only decreases in the

presence of the target analyte (in this case methamphetamine) [17]. On the other hand, using the

previously mentioned colloidal particles, it is possible to measure the impedance (conductivity-1),

where an increase in conductivity is correlated to the amount of colloidal particles that are bound to

the electrode[16]. In addition, it is possible to do this in a “competitive assay”, were analyte in the

sample and analyte immobilized to the electrode compete for particles (ex. Gold) bound to

antibodies (Figure 4.2) [16]. It is possible to further enhance the sensitivity by increasing the

diameter of the colloidal particles. [16]

13

Figure 4.1: Structure of the conductivity cell. Reproduced from ref. [17].

Figure 4.2: The working principle of the developed competitive immunoassay method for impedance

detection of methamphetamine (MET) concentration. The gold nanoparticles (AuNPs) are introduced

into the electro-microchip, forming a bridge that allows for the transport of electrons. Reproduced

from ref. [16].

The existence of conductivity (impedance) detection suggest that electrochemical detection methods

are possible as well[49]. In contrast to the impedance detector, the antibody used for

electrochemical detection is immobilized on the electrode and there is no colloidal metal, but

Horseradish peroxidase (HRP) that is coupled to the competitive analyte[49]. When a

14

tetramethylbenzidine substrate (TMB) is added, the HRP catalyzes the reduction and amplifies the

current signal[49].

Slightly resembling the impedance detection, is an optical technique that uses immobilized

antibodies and antibodies with gold labelling to form a sandwich around analyte (Figure 4.3).

[50]After the formation silver-ions are reduced onto the gold particles[50].The resulting film of silver

can be measured with light-emitting diodes and photodetectors, or even be examined by eye[50].

Figure 4.3: Overview of the creation of the immunosandwich for the optical detection of silver films.

Reproduced from ref. [50].

A different optical technique that does not require a reporter component bound to analyte and

antibody, is surface plasmon resonance (SPR)[51]. In this method an antibody is immobilized on a

transducer surface (gold) and the act of an analyte binding to the antibody changes the biomolecular

interactions that take place in the close vicinity of the transducer surface[51].

The detection is done by aiming a polarized beam of light with an appropriate wavelength and at a

specific angle, through a prism, at a thin metal film (the transducer)[51][52]. This causes an

oscillation in the charge density inside the metal, decreasing the intensity of the reflected

light.[51][52]. The angle of minimum light reflection is called the SPR intensity[51][52]. Once analyte

is bound to the antibody connected to the transducer, there will be a change in the properties of the

metal, changing the SPR angle[51][52]. In other words, binding analyte to the transducer changes the

refractive index[53].

In short, the possible applications of immunoassays are quite varied. But with their selectivity (based

on antibody quality) comes the problem that every possible analyte needs its own antibody. And

while it is possible to have a method that can detect multiple types, any additional antibody would

require time and effort[50]. That the detector is left blind to unexpected drugs could also be

considered a disadvantage.

A more environmental concern is that there is little mention in the literature about the regeneration

of the immunoassay for multiple use. When it is, it states that analyte immobilization is the better

choice (vs. antibody immobilization) and that the antigen-antibody binding determines what kind of

15

eluent should be used for regeneration[51]. Considering this, it may mean that the regeneration of a

system that hold several antigen-antibody bindings becomes more difficult as the number of bindings

increases[51]. It is also possible that some regeneration eluents, glycine-HCl and ethanolamine are

mentioned, might prove impractical for a field situation[51]. All this will result in either additional

maintenance (dedicated to regeneration) in the field or at a different location, or in disposable

immunoassays[51].

A last mark against coupling an immunoassay to a LC-chip, is the lack of literature were an

immunoassay is preceded by a liquid chromatography step. When immunoassays and LC are

mentioned, it is for a comparison of the methods.

As for the detection limits, they are around 1 ng/ml (6.7*10-6 M) for methamphetamine in urine,

about 10 pg/ml (3.5*10-11 M) for morphine in urine, 1.3 µg/ml (1.24*10-5 M) for GHB using a

method meant for both urine and blood analysis[18][16][48][24]. In the case of methamphetamine

also dependant on the concentration of gold-antibody particles that are used in the method[16].

16

Chapter 5: Colour tests.

Colour tests are analysis methods that make use of a reagent that is added to a sample of interest to

produce a colour. Furthermore colour tests (or chemical spot tests) are meant to be observed with

the naked eye[54]. This mean that this type of test is inexpensive in instrumentation, use minimal

reagents and are simple to perform, meaning that end users need minimal training[9g][54]. The tests

can also be done quite quickly, some only taking a few minutes[25]. Although the addition of colour

reagents mean that the sample is diluted. In some cases such as GHB, colour tests are available

commercially for personal safety[2]. On the other hand this kind of testing lacks accuracy, either

because it relies on subjective interpretation of colour that is further influence by other compounds

in the sample, drug concentrations, whether the drug is a salt or free base, or an instability in the

colour producing complexes[54]. Although there is a system that can be used to describe the results

of colour tests by using ten different colours that are combined, if needed, to indicate variations in

hue or colour changes over time[9g]. Therefor evaluation using a reference list (with colours and

hues) may require checking for more than one colour[9g]. Colour tests may not be precise enough

for definitive measurements, but should suffice for screening and preliminary testing[54]. Samples

should be handled carefully since parameters such as pH can influence resulting colours[9g].

The tests can be carried out either in clear containers, on white glazed porcelain tiles that allow a

better assessment by providing a uniform background or with paper strips[9g][25]. Colour tests

designed for drugs work on about 1mg of analyte, either dried or in a solution (usually water)[9g]. A

sample, preferably with an identical matrix, that does not contain the compound of interest should

be tested as well to confirm that colour changes are due to the compound of interest[9g]. Because

reagents can produce colour when in contact with many different analytes, a battery of colour tests

should be used (Table 5.1)[54].

17

Table 5.1: Part of a table that indicates (with a +) when a colour reaction occurs between an analyte

(right) and a prepared reagent (A.1, A.2, etc.). Diazepam and Acetaminophen, for example, give

different reaction patterns. Reproduced from ref. [54].

Reagent

A.1 A.2 A.3 A.4 A.5 A.6

Acetaminophen - - - + - +

Alprazolam - - - - - -

Aspirin - - - + + -

Baking soda - - - - - -

Brompheniramine maleate + - - + - -

Chlordiazepoxide HCl + - - - - -

Chlorpromazine HC. + - - + + +

Contac - - - + - -

Diazepam - - - - - -

Doxepin HCl + - - + + +

Dristan - - - + + +

Ephedrine HCl + - - - - -

Exedrine - - - + + +

In the cases that there is overlap between analytes resulting from drug use and analytes that can be

found naturally in a human body, a test can be designed to compensate for this. For example a test

with a detection limit for a GHB that lies at 0.1 mg/ml in urine (9.61*10-4 M), while the physiological

concentration that naturally occurs in a human body is around 0.5-2.0 µg/ml (4.8-19.2*10^-6), and

therefor will not give a positive result[55]. The limits of detection for compounds of interest depend

on the analyte and the colour tests that are used[54]. LODs typically lie between in the µg, with d-

methamphetamine HCl (the hydrochloride salt) at 100 µg and Heroin at 20-200 µg with no sample

volume stated, see table 5.2 for a small overview[54][9g].

Table 5.2: A number of LOD values of interest for different analytes and tests. Reproduced from ref.

[9g].

Colour test Analyte LOD in µg

Mandelin's test

Amphetamine-HCL 10

Heroin 20

Metamphetamine 150

Morphine 5

Marquis

Metamphetamine-HCl 5

Morphine 5

Amphetamine-HCl 10

Heroin 10

Sodium nitroprusside-acetone Metamphetamine 5-30

But there still is a problem since the compositions of real life samples are unknown, it is possible that

other components in the sample influence the test to produce a false result or making it unreadable.

18

For example a colour test with Froehde reagent would result in a red colouration for amphetamine

and a purple-red colour for heroin, this may be difficult to observed[9g].

It is in that case that a chromatography step is of use, and while there are no explicit mentions of a

LC-chip with colour testing, thin layer chromatography (TLC) however may prove useful. In some

applications TLC separations can occur by essentially the same physical process as LC[9h].

TLC is a low-cost, easy maintenance, analysis technique suitable for the analysis of a large number of

samples[56]. The retardation factor (Rf) is a value used in TLC that describes the distance travelled by

a compound versus the distance travelled by eluent[57][58]. It can be used to identify unknown

analytes in a sample, although standards should be used alongside the sample, in the same system,

to improve accuracy, see attachment 1 for an overview of Rf values[9h][57][58]. It should be noted

that some reagents used for colour tests and TLC can suffer from poor selectivity or sensitivity[58].

One more reason to perform a battery of tests when analysing samples.

But it is not just the reagents that depend on the analytes, different compounds of interest and the

colour reagents require different mobile phases and solvents, adding additional analysis steps that

either need to be performed by the technician or by the device used for analysis[9h][57] .

The testing of a sample using TLC usually requires the application of a sample “spot” on the TLC

plate, and depending on the method may require evaporation of the sample solvent, but not

always[9h][57].The colour reagent can be added before separation or after. A method with after

colouration may require a brief stay an oven-like environment, or at least a drying period, before

addition of colour[9h][57] [59]. This adds inconvenience for a portable system that is meant for field

use. The TLC separations, coloured or not, can be archived for re-evaluation at a different time and

place, or even be used to collect fractions of a sample.

There is the possibility to use optical detectors for colour tests and TLC, however that will be

discussed in Chapter “Spectroscopy”.

19

Chapter 6: Spectroscopy

The immediate advantage that spectroscopy has over colour tests (with and without TLC), is that the

detector is an objective instrument and not a subjective human. Spectroscopy itself covers many

different analytical methods such as UV-Vis absorbance, fluorescence, chemiluminescence and

infrared. The use of visible and ultraviolet light for absorbance detection is widespread when coupled

to chromatography methods, despite that other methods (such as fluorescence) have better

sensitivity and specificity[9i][60]. That fact is the reason why the other optical techniques will be

considered. Commercially available spectrophotometers are also generally bigger than the desired

mobile phone size and there is no information on why the dimensions of the device are what they

are[61][62]. It should also be noted that miniaturization of a system that makes us of spectroscopy

also leads to a reduction in optical path length, which may weaken the method altogether[12][63].

The techniques will be reviewed after the following examples of spectroscopy that are coupled to

microchip (viable) components.

X. Weng et al. developed a polydimethylsiloxane (PDMS) microfluidic chip with fluorescence

detection using 410 nm light[64]. It can be designed to use a gentle application of force by just

pressing a location with a finger to pump necessary substrate towards the analyte[64]. Heat may be

needed to speed up the chemical reaction between analyte and substrate[64].

Surface enhanced raman spectroscropy (SERS) on a paper-based microfluidic device, a dipstick, that

is manufactured with an inkjet-printer, which presents a useful and rapid application for on-site

analysis[65]. It is a low-cost, less sensitive alternative to other SERS methods that provide a highly

sensitive analysis at a higher cost[65]. Material used for the other SERS methods may also suffer from

a short shelf life[65]. The dipsticks are also able to process much larger volumes of sample in

comparison to conventional SERS substrates, and can be used as surface swabs for the collection of

trace analytes because of the flexible nature (Figure 6.1)[65]. A last advantage is that the paper

device can improve signal intensity by concentrating the analyte through a lateral-flow

operation[65].

20

Figure 6.1: A detection method making use of a paper interface and Raman spectroscopy. A) shows

the structure of the printed paper. B) illustrates the use as a surface swab for sampling while c)

shows the ability for concentration of the taken sample by lateral flow. D) is the detection of the

dipstick with the Raman probe. Reproduced from ref. [65].

Fluorescence

Fluorescence detectors are a tool with high sensitivity, fast response time and technical simplicity[2].

The sensitivity is around 10 fold that of UV-absorption, which usually has an LOD in the range of 10-6 -

10-5 M[60][67].However the difficulty for fluorescence lies in that some methods require the

choosing of an appropriate dye, the available choices numbering around 5500 that then need to be

screened for fluorescence intensity and optimal working conditions such as solvent[2]. GHB would be

a compound that needs a dye, while methamphetamine related compounds do not[2][58]. Certain

dyes may even require additional steps in the analysis procedure due to solvent incompatibilities,

such as in one case for the detection of gamma-butyrolactone (GBL) in real drinks because the

presence of alcohol[2].

Detecting several different compounds with one device may therefor require that a selection of dyes

is added to the sample. This adds an additional level of complexity since the dyes, or the dye addition

system, must insure that the selected reagents do not negatively affect the analysis procedure.

A further leap in the sensitivity of fluorescence can be done through Laser Induced Fluorescence (LIF)

spectroscopy[60].This can result in LOD’s that lie in the range of 10-12 - 10-10 M[66][67]. It can still

suffer from problems such as scattered excitation light that can interfere with the measurements and

deteriorate the S/N (signal/noise) ratio, despite using a laser.[66]. However LIF with a PDMS/Glass

chip that uses small-angle optical deflection to minimize interference from all light resulting from

scattering, reflection or refraction[66]. Diode-LIF detection systems focus on near-infrared and red

21

regions of the spectrum where background noise is low, but lacks labelling dyes for wider

application[66]. The size of the detector remains a problem, as previously mentioned for

spectrophotometers, but while not strictly mobile phone sized or for the detection of drugs, an

accessory has been presented by J. Canning et al. that allows a smart phone to act as a fluorometer

(figure 6.2)[68].

Figure 6.2: A smartphone with accessory that allows fluorometry. Reproduced from ref.[68].

Chemiluminescence.

An alternative to Fluorescence is Chemiluminescence which has high sensitivity, a simple optical

structure, a wide linear working range and low background noise[67][69]. The LOD values for heroin,

morphine and codeine lie in the range of 10-8 to 10-9 M, methamphetamine has values of 0.002-0.041

ng/mg in hair (shaft, injection concentration around 8.37*10-8 to 1.7*10^-6 M) and 0.001-0.008 ng/ml

(6.7*10-9 M -5.36*10-8 M) in blood[10][70].

It has also successfully analysed a variety of compounds when combined with microchip capillary

electrophoresis (MCE)[67]. For signal detection it can use instruments such as a fluorometer or a

photomultiplier tube (PMT)[67]. Unlike Fluorescence, it does not need an excitation source, and

while not as bright it means that the system is more compact[71].

To produce chemiluminescence, a reagent is needed, for example luminol which it emits light in the

wavelength range 425-435 nm[67]. Good contact, and mixing, between analyte and reagent is

important for the strength of the luminescent signal[71]. Note that only a limited amount of

molecules are chemiluminescent[71].

The choice of reagent for chemiluminescence is just as important as colours and dyes in previous

analysis techniques. For example: in the case of heroin detection the chemiluminescence reagents of

choice will be either tris(2,2’-bipyridine)ruthenium(III) (|Ru(bipy)3|3+) or potassium

permanganate[10]. The permanganate requires the heroin to be hydrolized into 6-

monoacetylmorphine (6-MAM) and/or morphine for a reaction[10]. |Ru(bipy)3|3+ may give false

positives when exposed to tertiary amines such as codeine[10].

Drawbacks are rapid reaction time and fast decay of the luminescence. This means that the reactants

need to be mixed quickly and immediately analysed[71].

22

Infrared

Infrared makes use of vibrational (bending and stretching) characteristics of compounds for

identification and in cased such as a Fourier-transform infrared(FTIR) attenuated total reflection

(ATR) spectroscopy (with a one-step extraction) can be done in the field[72]. ATR instruments further

have the ability to dry samples[72]. However the instrumentation used for IR is far too big for a

mobile phone sized device[65]. And none of the literature mentions IR-LC.

LOD values for heroin and cocaine are 9 ng and 15 ng respectively if the paper-based method

mentioned earlier is used, with a sample volume of 5 µl the molar concentrations will be around

5.95*10-6 M and 9.88*10-6 M respectively[65]. The FTIR ATR spectroscopy method can give LOD

values of around 0.74 µg/ml (2.43*10-6 M] cocaine in saliva, 30 minutes after consumption of 40

mg[72].

23

Chapter 7: Electroanalytical

This chapter is about electroanalytical detection methods that are not coupled to an immunoassay.

This means that they do not, in comparison, have the same specificity or complexity. Neither do they

have the same size requirements as an MS system, colour reagents, highly specialized

instrumentation or high-intensity light[73.] A last advantage is that electroanalytical techniques allow

for the analysis of turbid samples, although this is a debatable advantage because the aim is to have

LC prior to the detection[74]. At its most basic, electroanalytical detection requires a sample, two

electrodes, a current and a component that detects changes in the current. This means that

electroanalytical sensors have simple to integrate structures that allow for easy (and cheap)

miniaturization without sensitivity loss[73][75]. Depending on the type of technique, it may even be

many times more sensitive than a UV[76].

The general disadvantage of electroanalytical techniques, which adds inconvenience to a portable

device, is the possibility of electrode surface fouling.[76][77].

Electroanalytical methods are not as widely used as an MS or some kind of spectrometry, but there

are instances where heroin, methamphetamine and in one case GHB have been tested with

electroanalytical techniques[ 88]. This chapter will briefly look at potentiometric, amperometric,

conductometric and voltammetric methods. It is unlikely that any of these techniques will prove

themselves too large for the desired (mobile phone sized) end product[74].

Potentiometry can be considered one of the most sensitive electrochemical (detection making use of

an electrochemical reaction) techniques[12]. The response this method gives is only dependant on

concentration and not on parameters such as detection cell dimensions and an in the case of

conductivity measurements, the flow rates[12][73].

In the case of Sekula et al. where capillary electrophoresis (CE) is used for separation, an electrode is

coated with polymers, resulting in something resembling an ion selective electrode (ISE) Which is not

mentioned in the report itself, but in one of the illustration(see figure 7.1)[12]. The coated electrode

is able to detect heroin (and related compounds) and a variety of amines, there for the detection of

amphetamines would not be farfetched[12]. The lack of full understanding on how the detected

potential is generated, is given as a reason as to why there are not more potentiometry/separation

applications[12].

24

Figure 7.1: A CE and potentiometry detector setup that indicated the potentiometry detector as an

ion selective electrode (ISE) . Reproduced from ref.[12].

Amperometric detection of morphine is promising for a portable and fast sensor, although less

documented[79]. The process of detection is compatible to HPLC and molecularly imprinted

polymers (MIP), the later also happens to be suitable for bio-samples (urine, blood, etc.) because of

its high selectivity for morphine[79] .

Conductometric detection measures a change in resistance (or impedance) that results from a

change in concentration[73]. However the flow rate can have an effect as well, increasing at a low

flow rate and decreasing at a high flow rate[73]. Reasons for this can be streaming potential, heat

convection or deformation in the carbon nanotubes used[73]. A conductivity detector can make use

of electrodes that are in contact with the sample, comparable to potentiometry and amperometry.

However a different take on conductometry is the detection with capacitive contactless conductivity

detection (C4D) [13]. C4D uses two radial metallic electrodes that are fitted around a capillary, voltage

is applied, the current passes through the electrodes, capillary wall and gap between electrodes

before being picked up again[13][80] [13] The differences between the conductivity of the buffer and

the conductivity of the sample are converted into a peak[13] C4D is suitable for a variety of analytes

including amphetamines, inorganic ions, carboxylic acids and explosives[13]. It also happens to be

electrically isolated from the sample and prevents the previously mentioned electrode fouling[80].

See figure 7.2 For the C4D detector.

25

Figure 7.2: A C4D detector (right) with the sensor on the left. Dimensions (H/L/W) are 105/250/250

mm (3 kg) and 10/28/23 mm respectively. Reproduced from ref. [80].

Lastly we have voltammetric detection, with a drawback that may be an advantage. The drawback

for voltammetric techniques, when used for simultaneous detection, is the overlap of

voltammograms (Figure 7.3), the graphene nanosheets also have antifouling properties [81]. With a

separation step in the microchip, these voltammograms could be used as an additional tool for

identification purposes.

Figure 7.3: Voltammogram of Morphine [M], Noscapine [N] and Heroin [H], with an overlap of M and

H. A graphene nanosheets-glassy carbon modified electrode is used for the acquisition of signals c

and d. A and b makes use of differential pulse voltammetry. A and c have an absence of drugs and b

and d have a presence. Reproduced from ref.[81].

In the case of the voltammetry method, modified electrodes are used because unmodified

electrodes used for electroanalysis suffer limitations such as low sensitivity, reproducibility and

stability[81].

26

Specifically mentioned in the case of voltammetry, is the importance of the optimization of the

detection potential because it affects the electrochemical behaviour of analytes[75]. A potential that

is too low will give a signal that is too small, while a high potential gives an increase in the

background current (Figure 7.4)[75].

Figure 7.4: Voltammograms of Morphine (a) and Codeine (b). Reproduced from ref. [75] .

The LOD values for heroin are around 3*10-7 M for amperometric detection (2*10-7 M for morphine),

1.35*10-7 M for potentiometry and about 5*10-7 M for voltammetry.[12][75][81][79]. LODs can be

higher in the case of voltammetry if several compounds are analysed simultaneously[81]. No

detection limits for conductometric detection of heroin were found during the writing of this report.

For methamphetamine, amperometry gives LOD values of about 1.3 *10-6 M, potentiometry has a

value of 10*10-6 M and voltammetry goes as low as 0.5*10-7 M[77]. The conductometric LOD (with a

C4D) is 0.75 ppm for amphetamine and 0.94 ppm for methamphetamine about 5.56*10-9 M and

6.29*10-9 M if in water[13]. Table 7.1 holds the LOD values, in ppm, for several other drugs for C4D

and UV detection that were done simultaneously.

27

Table 7.1: Detection limits for UV (detector of undetermined size) and C4D detection for a variety of

compounds. Reproduced from ref. [13].

LOD (ppm) for UV and C4D

Sample UV absorbance LOD C4D LOD

2-4-MPEA 0.46 0.38

MBA 0.75 0.83

2-MMA 1.5 2.5

PEA-HCl 1.8 1.3

AM-c-BC 1.7 1.4

MPEA 0.79 0.65

MMBA 0.54 0.58

AMP 0.81 0.75

MA 1.1 0.94

MDMA Peak 1 0.79 2.5

MDMA Peak 2 0.83 2.5

Average 1.0 1.3

No LODs for GHB were mentioned in the literature.

28

Chapter 8: Conclusion.

As was stated in the beginning, four criteria would be used to recommend one type of detection for a

portable, mobile phone sized, lab-on-a-chip device. The second criterion, “Sample versatility”, does

not currently add (or detract) much to (from) any of the techniques value for the desired end

product. There are no methods that are expressly stated to be unsuitable for one or more types of

samples (urine, blood, hair, etc.). Even if there were, all samples require some degree of sample

preparation to pass through an LC-chip. However some possibilities, such as the electroanalytical

detection of GHB, still have gaps in the literature[78].

Criterion one, “Miniaturization potential”, filters out MS, simply because MS instruments have

currently not been miniaturized enough, although portable versions exist. The same concerns exist

for spectrophotometers and the IR detector mentioned earlier (Figure 6.1) also appears to require a

little more space than the desired end product allows. This could mean that fluorescence and

chemiluminescence systems may require comparable dimensions. Also because practically all of the

literature deals with methods performed in the lab, the robustness of portable field applications

remain unknown. Spectroscopy may be an option, but nothing can currently be said about the

quality of a spectroscopic detector that fits inside the dimensions of the lab-on-a-chip device and is

able to detect on the chip. If colour tests are to be used in combination with a LC-chip, then the

volumes will have to be large enough so that the naked eye can observe the results.

This leaves spectroscopy, immunoassays and electroanalytical techniques. The third criterion is

“Selectivity”. Immunoassays and some electroanalytical techniques (such as ISE) have to be

predictive, they should not give a signal unless they are designed to detect a specific compound, as to

spectroscopy methods that require dyes . That is fine if the end device is only meant to detect a

select amount of compounds, however every additional compound that needs to be detected means

one more immunoassay or ISE. Since the end product has separation with a LC-chip the other

electroanalytical methods such as amperometry and conductivity detection can be applied as long as

the identity of compounds can be confirmed, for example by spiking samples or using standards.

Voltammetry can be used to detect several compounds simultaneously without making use of

chromatography, however it means that the voltammograms could be used to identify

compounds[81]. Because it measures amperes versus a variable voltage, it may be difficult to apply it

to a continuous flow LC-chip. Instead a continual flow with breaks that allow for a voltammetric

detection sequence could be applied. If diffusion during the breaks proves a problem, the chip could

be designed to add sections (plugs or droplet) of a liquid that does not mix with the mobile phase or

extracts compounds of interest[82].

The last criterion for review is “Sensitivity”. A number of the LODs found have a concentration of

around 1*10-5 M to 1*10-7. There is no strong reason to favour a different technique than

electroanalytical, especially because there is no certainty that any of the LODs found in the lab

procedures can be achieved in a portable field device. See attachment 2for an overview of LOD

values found in the literature.

29

References and Literature

[1] A. J. deMello, “Control and detection of chemical reactions in microfluidic systems”, Nature.,

volume 442, 27 July 2006, 394-402.

[2] D. Zhai, Y.Q.E. Tan, W. Xu, Y. Chang, “Development of a fluorescent sensor for illicit date rape

drug GHB”, Chem. Commun, 2014, 50, 2904-2906.

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Attachment 1: Overview of Rf values for TLC.

Overview of the Rf for different analytes depending on the solved used in the method. Reproduced

from ref. [57][58]. * have an additional spot at Rf 0.68 caused by the reagent.

Analyte Solvent / Rf values

Isopronanol/ammonia acetone/toluene/ammonia

tert-butanol/ammonia

MDMA 0.32 0.30 0.20

MDA 0.47 0.52 0.32

MBDB 0.49 0.49 0.36

MDEA 0.48 0.49 0.30

HMDMA 0.22 0.27 0.13

MDDA 0.40 0.4 0.29

Analyte Solvent / Rf values

chloroform-methanol n-hexane-chloroform-methanol

d-Amphetamine 0.49 0.43*

l-Amphetamine 0.55 0.47*

dl-Amphetamine 0.49, 0.55 0.43, 0.47*

Analyte Solvent / Rf values

n-hexane-ethyl acetate-acetonitrile-diisopropyl ether

d-Methamphetamine 0.61

l-Methamphetamine 0.57

dl-Methamphetamine 0.57, 0.61

36

Attachment 2: Overview of LODs.

Overview of LODs for MS detection (in M) per related type of sample, *LOQ, **Original LOD given for “Several

types of amphetamines”.

GHB

Blood 2.8*10-7

to 9*10-6

Urine 1.24*10-5

Hair 2.4*10-6

Amphetamine

Blood 7.41*10-5

*,**

Urine 7.41*10-5

*,**

Hair 2.96*10-11

Oral fluid 7.41*10-6

to 3.70*10-5

**

Sweat 7.41*10-6

to 3.70*10-5

**

Methamphetamine

Hair 8.0*10-8

Heroin

Oral fluid 7.04*10-7

Morphine

Oral fluid 2.28*10-6

MDA

Blood 1.12*10-4

Urine 1.12*10-4

Cocaine

Hair 1.32*10-11

Overview of LODs for immunoassay detection (in M) per related type of sample.

GHB Methamphetamine Morphine

Urine 6.7*10-6 3.5*10-11

Blood&Urine 1.24*10-5

37

LOD of colour tests are 9.61*10-4 M for GHB in urine.

Overview of LOD ranges for some types of spectroscopy (in M)

Technique LOD

UV absorbance 10-6 - 10-5

Fluorescence 10-7 - 10-6

LIF 10-12 - 10-10

Overview of LOD range for chemiluminescence (in M) per related type of sample.

Sample Methamphetamine Heroin Morphine

Unspecified 10-9 to 10-8 10-9 to 10-8

Hair 8.37*10-8 to 1.7*10^-6

Blood 6.7*10-9 M -5.36*10-8

Overview of LOD ranges for IR spectroscopy methods (in M).

Technique Heroin Cocaine

Raman paper based 5.95*10-6 9.88*10-6

FTIR ATR 2.43*10-6

Overview of LOD ranges for electroanalytical methods (in M).

Technique Heroin Morphine

Amperometric 3*10-7 2*10-7

Potentiometric 1.35*10-7

Voltammetric. 5*10-7

Technique Methamphetamine Amphetamine

Amperometric 1.3 *10-6

Potentiometric 10*10-6 M

Voltammetric 0.5*10-7 M

Conductometric 6.29*10-9 5.56*10-9