Using portable CE instruments for determining banned

compounds in situ.

Mihkel Kaljurand, Prof. emeritus

TalTech, Faculty of Science, Department of Chemistry and Biotechnology

Tallinn Estonia

Reproducibility crisis and Open-source hardware

• The reproducibility crisis is an ongoing methodological crisis in science in which scholars have found that the results of many scientific studies are difficult to replicate.

• One of the ways to deal with the reproducibility crisis is to use open-source hardware.

• The open-source hardware usually means that information about the hardware is easily discerned so that others can make it.

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Proof of concept and open-source hardware• Proof of concept is a realization of a certain method or idea in order

to demonstrate its feasibility

• Proof of the concept is more significant than open-source hardware

• James Jorgenson gave proof of the concept of capillary electrophoresis: anybody who has HV power supply, capillary and detector can make CE instrument.

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Picture of the very first CE instrument in Estonia:HWPS –from colour TVCapillary - gift from a fellow postdoc (Cifuentes)Detector –a HPLC florescencedetector

Capillary electrophoresis

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The development of commercial CE instrumentation has stagnated.

• There are only two main suppliers of CE instrumentation (Agilent and Beckman Coultier),

• Much of CE research has shifted to solving various problems in applied science.

• It might be argued that there is no longer much to invent in CE, but this is not true.

• One of the advantages of this technique (that is often overlooked) is the opportunity to develop portable CE field instruments.

• There is urgent need for field analytical instrumentation

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Portable CE can be easily constructed

• Although modern GC and HPLC combine separation and detection in one instrument, the development of a portable chromatograph for field use is technically challenging and examples of this approach are rare.

• A strong push towards portable CE instruments has led to a variety of successful devices.

• Portable CE can be easily constructed, and several groups have reported designs for portable instruments that use either a CCD detector or advanced optics based on light emitting diodes (LED).

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Portable CE instruments developed at TalTech (Tallinn University of Technology)

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Banned and Regulated Compounds• Banned compounds

• Chemical/Biological Warfare agencies (CWA and BWA)

• Toxic industrial chemicals (TIC)

• Explosives

• Illicit drugs

• There are two main categories of banned compound testing • Screening tests are, qualitative tests conducted to identify classes of chemicals

present in the sample and (typically) are done using immunoassay.

• Confirmatory tests are used for further analysis of a sample – to confirm a positiive/negative, result and (typically) are done using GC-MS or HPLC-MS.

• On site confirmatory test is highly desired • rapid answer

• cheap

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Preparations for expedition: Heidi Eeva and Petr

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Operating on a site

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Simulation of CWA attack (spraying of phosphonic acids)

Sample collection

Extraction of CWA from soil

Sample filtration and injection

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Robustness of operation on a site is the main issue.

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CE analysisExtraction of CWA Filtration

Operating on a site has to be done in a protective suit

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CE is highly sensitive to parameter changes and it is not a reproducible technique

• Sources of low reproducibility in portable CE:

• No temperature control

• Manual sample introduction

• Operating in the field (rain, wind, sunlight)

• Sample matrix effects

• Degradation of capillary performance

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0 1 2 3 4 5 6 7 8 9 10-6

time (min)

raw data

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Ways of improving CE performance:

• Internal standards

• Software means

• Improving of sampling (e.g using Upchurch flow metering valve)

• Operator training

• Disposable capillaries

• ????

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Ways of improving CE performance: Internal standards and warping of time axis

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Raw data std=20%

R² = 0,9285

Corrected std=5 %

R² = 0,9966

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0.5 mM CWA degradation products applied to sand (left) and moss (right)

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time (min)

Standards

Sample

Blank

BGE 15 mM MES/His; 20kV.

1- sodium decanoate; 2 – pinacolyl methylphosphonic acid; 3 – 1-butylphosphonic acid; 4 –propylphosphonic acid; 5 – ethylmethylphosphonic acid; 6 – methylphosphonic acid; 7- lacticacid; 8- salicylic acid

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Standards

Sample

Blank

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Separation of hydrolysis products of nerve agents sarin, soman and VX in a concrete matrix (left) and hydrolysis products of VX in various matrices(right)

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Separation voltage: −16 kV, BGE: 7.5mMMES/HIS, pH 6, C4D detection. Tested in the NATO

CWA testing site near Vyskov (Czech Republic) under the license of the Ministry of Defence and

Armed Forces of the Czech Republic ( Kubán et. al., J. Chromatogr. A, 1218 (2011) 2618.

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BWA detection is a real challenge.CZE technology can still be used

• Detecting of simulants of specific biological toxins is simple

• Using of bio-toxin simulants (same mass and pI than biological toxins) for method development Ovalbumin (as ricin) Trypsinogen (as Staphylococcal

enterotoxin B) IgG (as botulinum toxin)

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“Towards the Monitoring of Dumped Munitions Threat” (MODUM)

• A project financed by the NATO Science for Peace and Security (SPS) program (2013-2015).

• Establishing and testing analysis protocols for nerve agents, Mustard Gases and Adamsite.

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Explosives

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DynamitePETNTNTRDXPENODetonatorANFOV40C4

Illicit drug abuse is a problem

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Roadside testing of illicit drugs

• Ethanol is an easy compound to analyse.

• No analyzer of exhaled breath for roadside testing of drug abuse exists

• Analysis of exhaled breath condensate is done by HPLC/MS

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Oral fluid is preferable to urine or blood.its collection can be observed by the officer, results are comparable to blood and urine testing.

DrugWipe can detect

cannabis,

cocaine,

crack cocaine,

heroin,

morphine,

amphetamines,

MDMA, and

benzodiazepines.

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„DrugWipe“ lateral flow device for saliva

There are more than a dozen drug testing devices on the international market. ▪ Weight 1-5 kg,

▪ Analysis time 7-12 min,

▪ Price10-40 EURO

Firma Sensitivity, the probability of a positive result if the driver has drugs in his/her body

Specificity, the probability of a negative result if

the driver does not have drugs in his/her body

Dräger Drug Test 5000®; Alere DDS2®; DrugWipe 5S®; RapidSTAT®;

40-90% 50-100%

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Disadvantages

• Cross-reactivity,

• Limited specificity, and false positive responses.

• The interpretation of the results is somewhat subjective and may be even more challenging when it is dark.

• Confirmatory tests are performed by HPLC and GC-MS,

HPLC-MS 100K EURO

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Roadside drug tests would encourage the police to lay more charges.

• It will have a significant preventive impact by increasing the likelihood of being caught.

• The police will be confident to charge suspected drivers.

• Roadside drug tests would predictably save both time and money by simplifying drug enforcement procedures and by limiting the need for drug recognition experts.

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CE-FD instrument and excitation spectra of illicit drugs

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(A) Excitation spectra of illegal drugs and excitation filters sets (228FS25, FF01-276/SP and ET240-40bp), (B) emission spectra of illegal drugs and emission filters (FEM1, FEM2, FEM3, FEM4, and *FEM5 is up to 600 nm): 1, PMA and PMMA in water; 2, AMP, METH, 3, cocaine in water; 4, THC in ethanol; 5, CBD in ethanol; 6, MDMA, MDEA, MDA in water.

(A) Optical layout of the detector: (1) flash xenon lamp, (2, 4, 7, 11) lenses, (3) set of two bandpass filters and a short-pass filter, (5) capillary, (6) spherical mirror, (8) long-pass filter, (9) changeable emission filters, (10) filter wheel, (12) diaphragm, (13) PMT window. (B) CE-FD instrument:

Analysis workflow

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Electropherograms of illegal drugs in acetonitrile obtained with emission filters FEM1, FEM2, and FEM3:

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(A). Electropherograms of illegal drugs in acetonitrile obtained with emission filters FEM1, FEM2, and FEM3: (B) Drug abuse suspected user’s oral fluid samples eletropherograms at emission filter FEM3 #S5, #S1, #S14, #S21- suspects, corrected by IS1 and IS2 peaks migration time and IS1 peak height.

1, AMP (66 mg/L); 2, METH (66 mg/L); 3, MDA (3.3 μg/L); 4, MDMA (3.3 μg/L); 5, MDEA (3.3 μg/L); 6, cocaine (83 μg/L); 7, cocaethylene (100 μg/L); IS1, allocryptomine (33 μg/L); IS2, benzylamine (300 mg/L).

cocaine, cocaethylene,

3,4- methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxeamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxy-N-methylamphetamine (PMMA), amphetamine (AMP), methamphetamine (METH),

tetrahydrocannabinol (THC) cannabidiol (CBD).

CE analysis of oral fluid specimens.

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Time, min

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Peaks: 1- neutral compound from Salivette® swab, 2 – cannabidiol (CBD), 3 – tetrahydrocannabinol (THC), 4 – IS, 5 – saliva endogenous compound.

a – blank oral fluid

b - after smoking of a “LM blue” cigarette

c - after smoking of a cannabis cigarette

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Saliva speciments from street: tetrahydrocannabinol (THC)

THC, 13 ppm

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BGE - MeOH/ACN 1:1, 2.5mM NaOH (pH=11), L(tot)/ L(ef)= 62/50cm, tinj=20s. λ(ex)/λ(em) = 280/307 nm

THC, 3 ppm

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Weekend Festival Baltic: August, 2016-2018; Pärnu, Estonia

• 75 000 participants

• About one hundred suspects

• Detected illicit drugs• Cannabis (THC)

• Amphetamine

• Cocaine

• GHB (traces from red wine?)

• https://geenius.ee/uudis/geeniuse-video-vaata-kuidas-weekendil-uut-narkomeetrit-testiti/,

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Problems: interferents I

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Inte

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1 - IS1 (benzylamine)2 - Amphetamine3 – tyramine4 – METH, 5 – MDA,

6 – MDMA, 7 – MDEA, 8 - Cocaine9 - Cocethylene10 - IS2 (allocryptopine)

Current protocol requires experienced analytical chemist

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Oral fluid collection Centrifugation of cotton pad

Application of ACN

Second centrifugation

Manual manipulations with CE instrument

Interpretation of electropherogram

Coming Soon?: An Analyser for Pot

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