Development of a Quantification Method for New Drugs of Abuse in Serum Using UPLC – MS/MSAuthor: Luiza Tworek

Dept. of Laboratory MedicineProgramme in Biomedical Laboratory ScienceDegree project, C-level, 15 points Biomedical Laboratory Science

Spring term 2012

Tutor:Olof Beck, professor, Department of Clinical Pharmacology Pharmacology laboratory, Karolinska University Hospital, Huddinge

Co – Tutor: Yasir Ahmad Al – Saffar, BMADepartment of Clinical Pharmacology Pharmacology laboratory, Karolinska University Hospital, Huddinge

Dept. of Laboratory Medicine

Programme in Biomedical Laboratory Science

Degree project, C-level

Biomedical Laboratory Science

Spring term 2012

Development of a Quantification Method for New Drugs of Abuse in Serum Using UPLC – MS/MS

Abstract

Abuse of new unregulated synthetic psychoactive substances, also called designer drugs is a growing problem that brings a need for new sensitive and efficient method of analysis. The aim of this study was to develop and validate an ultra – performance liquid chromatography tandem mass spectrometry method (UPLC – MS/MS) for new drugs of abuse. Twelve new designer drugs were determined with this method methylenedioxypyrovalerone, bk-N-methylbenzodioxolylpropylamine, methylone, para-fluoroamphetamine, 4-methylmethcathinone, 5,6-methylenedioxy-2-aminoindane, para-methoxymethcathinone, naphylprovalerone, 4-hydroxyl-N-methyl-Nethyltryptamine, 2-diphenylmethylpiperidine, O-desmethyl- cis -tramadol and metoxetamine. Samples were prepared by protein precipitation using acetonitrile. Chromatographic separation was performed on Acquity UPLC BEH C18 (2.1 mm x 100 mm) particles size 1.7 µm using 0.1% formic acid in water and 100% acetonitrile in a gradient mode at a flow rate of 0.500 mL/min, injection volume 2 µL.The mass spectrometric analysis used electrospray ionization and compounds were detected with MS/MS with multiple reaction monitoring for two transitions.Calibration curves were linear (r2 > 0.99) in the concentration range 0 – 500 ng/ml in serum. Limit of detection and quantification was documented for each analyte. QC samples intra – and inter – assay precision and repeatability were satisfactory (CV < 10%). The developed method was successfully applied on, serum samples from urine - positive patient (n = 57). This study showed that this method is fully reproducible and specific for all analytes and can be introduced to routine analysis.

Abbreviations:..........................................................................................................31. Summary:.............................................................................................................52. Introduction:.........................................................................................................63. Materials and methods.......................................................................................10

3.1 Chemicals and reagents................................................................................103.2 Stock and standard solutions...................................................................103.3 Patient samples.............................................................................................113.4 Instrumentation............................................................................................11

3.4.1 LC conditions........................................................................................123.4.2 MS/MS conditions................................................................................12

3.5 Sample preparation - protein precipitation:.................................................133.6 Method validation........................................................................................13

3.6.1 Recovery and matrix effect...................................................................143.6.2 Accuracy, precision...............................................................................143.6.3 Calibration curve; linearity...................................................................143.6.4 Limit of detection and limit of quantification.......................................153.6.5 Selectivity and specificity.....................................................................15

3.6.5.1 Interferences...................................................................................153.6.5.2 Matrix effect...................................................................................15

3.6.6 Carryover..............................................................................................163.6.7 Sample stability.....................................................................................16

3.7 Application of method.................................................................................163.8 Statistical methods...................................................................................17

4 Results and discussion...................................................................................174.1 Sample preparation......................................................................................184.2 LC optimization...........................................................................................194.3 Internal Standards........................................................................................214.4 MS/MS conditions.......................................................................................23

4.4.1 Product ions determination...................................................................244.4.2 MRM establishment..............................................................................26

4.5 Method validation........................................................................................294.5.1 Recovery (see also section 4.1 Sample preparation).............................29

4.5.2 Matrix effect..............................................................................................29

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4.5.2 Carryover..............................................................................................304.5.3 Accuracy and precision.........................................................................314.5.4 Limit of detection and quantification....................................................324.5.5 Calibration curve; linearity...................................................................334.5.6 Selectivity and specificity.....................................................................354.5.7 Sample stability.....................................................................................37

4.5.8 Application of method..............................................................................37Conclusion.............................................................................................................385. References..........................................................................................................39Appendix I.............................................................................................................41Appendix II............................................................................................................44Appendix III...........................................................................................................45

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Abbreviations:

2- DPMP Desoxypipradol (2 – diphenylmethylpiperidine)

4 – FA 4 – fluoroamphetamine

4 – OH – MET 4 – hydroxyl – N – methyl – N –ethyltryptamine

AcCN Acetonitrile

BEH Ethylene – bridged hybrids

CV Coefficient of variation

ESI Electron spray ionization

FA Formic acid

IS Internal Standard

LC Liquid chromatography

LGC Laboratory of The Government Chemist

LOD Limit of detection

LOQ Limit of quantification

O – DM – TRA O – desmethyl – cis – tramadol

MDAI 5,6 – methylenedioxy – 2 – aminoindane

MDPV Methylenedioxypyrovalerone

ME Matrix effect

3

SRM Selected reaction monitoring (in this text replaced by MRM (multiple reaction monitoring))

MS Mass spectrometry

M/Z ratio Mass to charge ratio

QC Quality control

R2 Correlation coefficient

RE Recovery

RT Retention time

SKL Statens krimineltekniska laboratorium

S/N ratio Signal to noise ratio

STRIDA Samverkansprojekt kring toxicitetutredning och riskbedömning av Internetdroger baserat på Laboratorieanalyser

UPLC Ultra Performance Liquid Chromatography

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1. Summary:

Abuse of new unregulated synthetic psychoactive substances, also called designer drugs, is a growing problem that brings a need for new sensitive and efficient methods of analysis. The aim of this study was to develop and validate an ultra – performance liquid chromatography tandem mass spectrometry method (UPLC – MS/MS) for new drugs of abuse. Twelve new designer drugs were determined with this method methylenedioxypyrovalerone, bk-N-methylbenzodioxolylpropylamine, methylone, para-fluoroamphetamine, 4-methylmethcathinone, 5,6-methylenedioxy-2-aminoindane, para-methoxymethcathinone, naphylprovalerone, 4-hydroxyl-N-methyl-Nethyltryptamine, 2-diphenylmethylpiperidine, O-desmethyl- cis -tramadol and metoxetamine. Samples were prepared by protein precipitation using acetonitrile. Chromatographic separation was performed on Acquity UPLC BEH C18 (2.1 mm x 100 mm) particles size 1.7 µm using 0.1% formic acid in water and 100% acetonitrile in a gradient mode at a flow rate of 0.500 mL/min, injection volume 2 µL. The mass spectrometric analysis used electrospray ionization and compounds were detected with MS/MS with multiple reaction monitoring for two transitions.Calibration curves were linear (r2 > 0.99) in the concentration range 0 – 500 ng/ml in serum. Limit of detection and quantification was documented for each analyte. QC samples intra – and inter – assay precision and repeatability were satisfactory (CV < 10%). The developed method was successfully applied on serum samples from urine - positive patient (n = 57). This study showed that this method is fully reproducible and specific for all analytes and can be introduced to routine analysis.

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Key words: designer drugs, fragmentation, chromatography, mass spectrometry, validation

2. Introduction:

Abuse of new unregulated synthetic psychoactive substances, also called “designer

drugs” or “legal highs” is a dramatically increasing problem, which brings a need

for a new sensitive and efficient method of analysis.

Designer drugs are analogues or derivatives of illegal substances classified as

narcotics. Their chemical structures are changed with modifications such as

addition, substitution, deletion of one or more chemical groups. Designer drugs

have similar psychoactive effects to the illegal variant. Although these drugs are

considered a safe alternative to real narcotics, there are many cases of lethal

intoxications. These recreational substances can be easily obtained through the

internet and they are much cheaper than classical drugs. The problem however is

their high toxicity and lack of sufficient pharmacological information (1–4).

This need led to the beginning of a new project called STRIDA

(Samverkansprojekt kring toxicitetutredning och riskbedömning av Internetdroger

baserat på Laboratorieanalyser) that involves cooperation between Karolinska

University Laboratory and the Swedish Poison Information Centre. STRIDA’s

main goals are to gather information about new incoming designer drugs, their

toxicity and to develop new methods allowing for easy and efficient detection (3).

Designer drugs can be classified according to chemical structure or based on

clinical effects (2). In this paper classification according to chemical structure is

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used, based on the review article “Clinical toxicology of newer recreational

drugs” S.L. Hill, S.H.L. Thomas (2).

Hill and Thomas categorize designer drugs in four distinct chemical families,

piperazines, phenetylamines, tryptamines, piperidines and non-classified

compounds (Figure 1).

Figure 1Classification of designer drugs according to chemical structure, A. piperazines, B. phenetylamines, C. tryptamines, D. piperidines.

Substances used in this study can be classified as follows: phenetylamines

(MDPV, methylone, butylone, 4-fluoroamphetamin, MDAI, methedrone,

mephedrone, naphyrone); tryptamines (4-OH-Met); piperidines (2-DPMP) and

non-classified (O-DM-Tra, MEX). Chemical structures are presented in figure 2.

Phenetylamines are a large group that includes illegal drugs of abuse such as

amphetamine, metamphetamine and a great number of their derivatives. Most of

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the phenetylamines in this study belong to β-cathinones that are plasma membrane

catecholamine uptake transporters inhibitors. Psychoactive effects are similar to

those produced by amphetamine and MDMA (ecstasy). The most common

symptoms are hallucinations, motorical problems, tachycardia and temperature

rise (3).

4-OH-Met (tryptamine) is a potent hallucinogenic drug that is not classified as a

narcotic in Sweden. STRIDA had 10 registered cases of intoxication with this

substance during 2010. Mydriasis, tachycardia, agitation are those symptoms

which were usually observed in patients (3).

Piperidines are a relatively new group of designer drugs. Psychoactive activity is

probably an effect of inhibition of the dopamine reuptake transporters. There is no

efficient data about piperidines toxicity. The few cases that were registered

showed symptoms such as agitation, tachycardia and mydriasis (2).

O-DM-Tramadol is a metabolite of a common analgesic tramadol. Tramadol is a

µ-opioid receptor agonist and inhibits the reuptake of serotonin and

norepinephrine. O-DM-Tramadol has approximately 300 times higher receptor

affinity than the mother substance (5). This substance is found in a mixture, called

Krypton, with the herbal drug Kratom. Krypton is sold on the internet in large

quantities and because of O-DM-Tramadol it can leads to unintentional overdose

(6).

Methoxetamine, MEX, is an analog of ketamine. Ketamine is a highly addictive

psychedelic drug. There is no scientific information about MEX pharmacology or

toxicity. All described MEX effects come from web forums. Because of a great

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similarity between MEX and ketamine, psychoactive effects of MEX can be

assumed to be related to those of ketamine (7).

Figure 2 Chemical structures of designer drugs used in this study.

The aim with this study was to develop, optimize and validate a quantification

method for new designer drugs in serum based on a presently validated method in

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urine (8). The measured drug concentration should correlate between drug

toxicology and patient’s symptoms.

3. Materials and methods

3.1 Chemicals and reagents

2 – DPMP (2 – diphenylmethylpiperidine) and O – DM – cis – Tramadol (O –

desmethyl – cis - tramadol) were purchased by LGC Standards (Laboratory of The

Government Chemist). 4 – OH – MET (4 – hydroxyl – N – methyl – N –

ethyltryptamine) was provided by THC – Pharm. Mephedrone, Methedrone,

MDPV (Methylenedioxypyrovalerone), Butylone, Methylone, 4 – FA (4 –

fluoroamphetamine) were provided by SKL (Statens krimineltekniska

laboratorium). Naphyrone was purchased from Internet characterised by SKL.

Methoxetamine was approved by Läkemedelsverket/SKL. MDAI was supplied by

Chiron. Acetonitrile (AcCN) and formic acid (FA) were provided by VWR

International and IT Baker,Tamro. Water purified with the Milli – Q Water

purification system was used throughout the experiment.

Serum used throughout the experiment was supplied by Department of

Transfusion Medicine, Huddinge.

Stock solution of methamphetamine-d5, pethidine-d4 and psilocin-d10 were

purchased by SKL.

3.2 Stock and standard solutions

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Reference substances, methanolic solutions, methoxetamine (conc. 2.044 mg/mL),

methedrone (conc. 1.2467 mg/mL), mephedrone (conc. 0.536 mg/mL), naphyrone

(conc. 0.81 mg/mL), butylone (conc. 1.057 mg/mL), O – DM – cis – Tramadol

(conc. 1000 µg/mL), MDAI (conc. 1000 µL/mL), 4 – OH – MET (conc. 893.14

µg/mL), 4 – FA (conc. 1.1528 mg/mL), 2 – DPMP (conc. 964.2 µl/mL) were

kindly supplied by Pharmacological Laboratory (Karolinska University Hospital,

Huddinge).

Stock solution at concentration 40 µg/mL was prepared by diluting reference

substances in 0.1% FA.

Internal standard (IS) working solution at concentration 1000 ng/mL was prepared

by diluting from methamphetamine-d5 in methanol (conc. 100 µg/mL),

phetidine-d4 in methanol (conc. 1.0 mg/mL) and psilocin-d10 (conc. 100 µg/mL)

stock solutions in 0.1% FA.

All solutions were stored at – 20 °C.

3.3 Patient samples

57 patient samples used during the experiment were left-over aliquots from the

routine flow. All samples showed positive result in urine for at least one analyte.

Serum samples were stored at - 80ºC until use.

3.4 Instrumentation

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All analyses were performed using UPLC – MS/MS Waters system. System

contained Waters Acqutiy UPLC system, Waters Acquity Autosampler, Xevo –

TQ MS # VBA 492 and MassLynx 4.1 SCN810 software.

Optimal instrumental parameters used under this study were based on already

known, designer drugs in urine by Department of Pharmacology Laboratory

(Karolinska University Hospital, Huddinge) (8). New MS/MS condition for

methamphetamine – D5 were determined using screening mode, MassLynx

software (Table I).

3.4.1 LC conditions

Reversed phase column, Acquity UPLC BEH C18 (2.1 mm x 100 mm) with

particles size 1.7 µm, was used to separation of analytes. The flow rate 0.600

mL/min and 0.500 mL/min were tested with an injection volume of 2 µL. Final

flow rate was set at 0.500. Two gradient elution were tested using two mobile

phases A (0.1% formic acid in water) and B (100% acetonitrile). Final gradient

was carried out at a 4 % B, increasing to 45 % B by 2.7 min. At 3.7 min was set to

95 % B for 0.4 min and then returned to starting conditions. Target column

temperature was set at 60.0 °C. Run time was 4.5 min.

Strong needle wash and weak wash was used to avoid carry-over between

injections.

3.4.2 MS/MS conditions

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Optimal MS/MS parameters used under this study were based on already known,

designer drugs in urine by Department of Pharmacology Laboratory (Karolinska

University Hospital, Huddinge) (8).

MS/MS was operated in positive ESI (electron spray ionization) mode with

multiple reaction monitoring (MRM) using three time segment. Ionization source

capillary voltage was set at 500 V. Cone voltage were set at 14.00 V. Extractor

was set at 3.00 V. Source temperature was set at 150.0 °C and desolvation

temperature at 600 °C. Argon was used as a collision gas with flow set at 0.15

mL/Min.

MRM (multiple reaction monitoring) time segments for analytes detection (two

transitions/analyte, one transition/IS) were maintained at (1) 0 – 2.48 min, (2) 2.48

– 3.14 min and (3) 3.14 – 4.50 min.

Methamphetamine – d5 trace and quantification ion was determined in an

injection experiment using Waters Xevo TQ MS Detector software function. The

sample spiked with 100 µL IS was screened to establish presence of a

methamphetamine-d5 molecule (protonated molecule mass: 154.39 g/mole).

Thereafter, the methamphetamine-d5 product ions were isolated and their

molecular masses were determined.

3.5 Sample preparation - protein precipitation:

IS, sample and AcCN proportion used under this study were based on already

known, designer drugs in urine by Department of Pharmacology Laboratory

(Karolinska University Hospital, Huddinge) (8).

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50 µL of IS and 400 µL of AcCN was added to 100 µL of a serum sample

followed by centrifugation at 3050 x g for 10 minutes. 300 µL of the supernatant

was transferred to a vial and analysed.

3.6 Method validation

A partial validation was performed according to the FDA Guidance for Industry,

Bioanalytical Method Validation (9)

3.6.1 Recovery and matrix effect

For determination of recovery (RE) and matrix effect (ME), three sets of extracts

were prepared, each in triplicates. (1) included analytes at concentration of 350

ng/mL, spiked with serum, prepared according to protein precipitation method,

there after centrifugation 500 µL of aliquot was diluting with 100 µL 0.1%FA.

(2) 0 - samples prepared as before, there 500 µL of supernatant was added to 100

µL of solution at concentration 350 ng/mL in 0.1%FA.

(3), 1 mL working solutions at concentration of 350 ng/mL in 0.1% FA was used.

Samples were mixed with 50 µL IS, 400 µL ACCN and vortexed.

3.6.2 Accuracy, precision

Accuracy and precision experiment was performed by preparing quality control

samples (QC) in number of 5 at 3 different concentrations: 25; 50; 500 ng/mL

which were analysed for the period of 5 days. All samples were prepared

according to protein precipitation method.

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3.6.3 Calibration curve; linearity

Calibration standards with concentration of 5; 10; 25; 50; 100; 150; 250; 350; 500

ng/mL analytes in 0 – serum were used. All standards included 50 µL IS (1000

ng/mL). Standards were prepared and analysed as an unknown samples using

protein precipitation method. This experiment was performed by preparing six

calibration curves.

3.6.4 Limit of detection and limit of quantification

Limit of detection (LOD) and limit of quantification (LOQ) were determined

theoretically using data for calibration solution 5 from “Calibration curve;

linearity”. Thereafter the empirical value of LOD and LOQ were established by

analysis of serum samples spiked to obtain following concentrations (ng/mL)

between 0.02 – 10.0. Signal to noise ratio (s/n ratio) was calculated for each

analyte using MassLynx software s/n ratio function.

3.6.5 Selectivity and specificity

3.6.5.1 Interferences

The potential for interference was investigated by testing ten serum samples

collected from patients there other xenobiotic than analytes of interest were

present (sertraline, clozapine, ganciclovir, lamictal, valproate, vancomycin,

zuclopenthixol, haloperidol, rifabutin/rifampicin, meropenem), ten blanks

collected from 10 different drug -free sources and ten calibration solutions each

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spiked with one of the following reference substances: sertraline, clonazepine,

ganciclovir, buprenorphine, aripirazole, azole – antimycotica, all common

benzodiazepines, haloperidol/ zuclopenthixol, rifabutin/rifampicin,

meropenem/ceftazedim.

3.6.5.2 Matrix effect

Ion suppression was determined for MDAI, methylone and methedrone by

simultaneously injection and infusion of 10 µg/min of two samples, one blank and

one spiked with analytes. Two sets were needed to perform the experiment. Set 1

contained 0-serum sample and set 2 consisted of 0.1 % FA as a blank, in both sets

were used the same sample containing 1000 µL 0.1%FA spiked with MDAI,

methylone, methedrone reference substances to obtained concentration of 100

ng/mL.

3.6.6 Carryover

Carryover experiment was performed by analysing two blank samples after the

analysis of each solution at concentration of 5000; 1000; 500 ng/mL.

3.6.7 Sample stability

Short-term stability was tested for one batch consisted of 57 patient samples

extracted as described before, calibration curve solutions 5 – 500 ng/mL and 0 –

serum. After the first run, samples were stored in 72 hours in autosampler at 8 ºC

and run again with a new prepared calibration curve.

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3.7 Application of method

Serum samples from 57 patients, which showed positive results in urine for at

least one analyte, were tested. Tested batch included a new prepared calibration

curve solutions 5 – 500 ng/mL, one blank and following numbers of positive

samples: 2 of methylone, 10 of O – DM –tramadol, 3 of 4 – fluoroamphetamine, 1

of methedrone, 4 of butylone, 2 of mephedrone, 13 of metoxetamine, 14 of

MDPV, 5 of 2 – DPMP, 2 of naphyrone and 12 of 4- OH – Met. All samples were

prepared and analysed as described before.

3.8 Statistical methods

The regression analysis was used to calculate correlation coefficient for

calibration curve for each analyte. To determine method repeatability (coefficient

of variation, CV %), inter - assay precision (CV %) and intra – assay precision

(CV %) one - way ANOVA with 5% significance level was used.

4 Results and discussion

This report describes an optimization and validation of a screening method for

new designer drug in serum using highly selective and sensitive method UPLC-

MS/MS. This new method is based on an existing one for those drugs in urine.

The experiment included adjustment of chromatographic parameters and

optimization of mass spectrometry.

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In the last few years, a number of LC – MS methods have been developed and

validated for different designer drugs both for urine, plasma and serum. This

newly developed method for UPLC MS/MS is simple, rapid and uses simple

sample preparation. It allows detection of 12 analytes belonging to different

chemical classes. The method is developed for serum which decreases problems

with samples collection in acute intoxication cases. Use of tandem MS allows for

determination of a great number of compounds and samples, which is required for

routine analysis. Sample preparation is simple that minimizes errors. Analysis is

performed automatically, and once optimized it does not need further adjustments.

Validation procedure confirmed that this method is specific for the analytes and

suitable for routine use. Recovery results were not as expected but on acceptable

levels. Inter -, intra – assay precision and method repeatability values showed

results between 0.2 – 9.0 % variance. Method accuracy values were between 96 –

108%, which is highly satisfactory. Measuring range was established at 0 – 500

ng/mL. With this method a concentration as low as <5 ng/mL can be detected for

most analytes. This study showed that analytes can be identified in samples as old

as 2 years, when stored properly. Short–term stability results did not shown any

significant differences in peak areas. Long–term stability tests should be

performed to established samples durability when stored at -20ºC and at rum

temperature.

4.1 Sample preparation

Samples were prepared using simple protein precipitation with AcCN. All twelve

analytes prepared according to protein precipitation could be separated and

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identified. Drying with nitrogen gas of supernatant and resuspension with

10%AcCN in 0.1% FA was tested and results showed no differences in recovery

results. Moreover, a difficulty was observed that 4 – OH – MET could not be

detected. Choice of the sample preparation method was mainly based on recovery

results which refer to extraction efficiency (1). Precipitation with AcCN showed

recovery higher than 85%, which is satisfactory. However, the expected value of

recovery is higher than 92% with this method of purification (10). Sample

purification from potential interference sources is a crucial step preceding liquid

chromatography and mass spectrometry. Preparation accomplished high sample

purity by precipitation of proteins with AcCN. Polson et al. showed in theirs

study that efficiency of protein precipitation with AcCN is approximately 92% at

volume ratio > 2:1 (AcCN; sample). AcCN is the most common organic

precipitant which does not interfere with analytes (10). Moreover, pure mobile

phase used as a precipitant causes greater ionization effects according to Polson et

al. This procedure also allowed less time for sample preparation by excluding the

drying and resuspension steps. However, the high AcCN volume ratio which was

used in this experiment did influence chromatography and will be discussed in the

next section.

4.2 LC optimization

Reversed phase column, Acquity UPLC BEH C18 (2.1 mm x 100 mm) particle

size 1.7 µm, was used throughout the experiment to separate analytes.

19

Ethylene – bridged hybrids (BEH), provided by Waters, is a technology used to

create particles that allow performance at difficult conditions such as high

pressure, temperature and wide pH range (11). 0.1% FA (A) and 100% AcCN (B)

was used as mobile phases according to Water recommendation. 0.1% FA is the

first choice of mobile phase A because of low molecular weight, which decrease

interferences and background noise. AcCN, used as mobile phase B, has a high

efficiency of ESI as mentioned before, good elution strength and applies less

pressure to the column. However, AcCN has a tendency to cause peak tailing or

fronting. In this study, a significant fronting was observed for IS substance

psilocin – d10 (RT 1.35) (figure 9). Psilocin – d10 is the first eluting compound

which indicates that high AcCN volume ratio causes rapid elution and a

noticeable peak fronting (12). This problem can be solved by adding 0.1 % FA to

the supernatant at the last stage of sample preparation or gradient adjustment.

Figure 3 Chromatogram of psilocin - d10, RT 1.37. A. Calibration solution 25; B. Calibration solution 250. Upper chromatogram shows samples diluting with 100 µL 0.1%FA, lower shows standard sample.

One attempt, as described before, was performed to analyse 0.1% FA influence on

analyte response and peak shape (figure 9). Results from the experiment showed

more symmetric-shaped peak but it was still not satisfactory. This experiment

should be tested with different 0.1% FA: supernatant ratio.

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The mobile phase gradient and flow rate was adjusted to optimize analytes

separation. The goal was to avoid elution of analytes or IS with the void volume

and to achieve a good separation in a short run time. The flow rate was set at

0.500 mL/min to achieve rapid analysis, duration time 4.5 min. Final gradient was

optimized according to analysis duration time as following 0 – 0.5 min: 4 % B;

0.5 – 2.7 min: 45 % B; 2.7 – 4.0 min: 95 % B; 4.1 – 4.5 min: 4 % B (figure 3).

Analytes retention times (RT) were measured for each analyte and summarized in

Figure 4.

In this experiment, no analyte was eluted with the void volume. All analytes were

fully separated and no double peaks were observed.

Figure 4 Mobile phases gradient (%B) used in chromatography throughout the experiment.

4.3 Internal Standards

Internal standard is essential to quantify an analyte. In ideal situation, IS should be

a compound that has as similar as possible chemical structure and properties to the

analyte. IS should also give a similar response but different enough to be

21

recognized by instrument (13). In this case, it was not possible to choose a more

suitable IS for each substance. Mostly because, reference substance for new

designer drugs do not exist and a procedure to acquire a certificate for a new

reference product is complicated and expensive (14).

Psilocin – d10 and Pethidine – d4 and Methamphetamine – d5 were chosen as IS

due to their retention times, stability, chemical similarities to the analytes as well

as availability. A retention time for each IS was measured under the same

conditions as the analytes and determined to 1.35 min Psilocin d-10, 1.98 min

Methamphetamine d-5, 2.78 min Pethidine d – 4 (figure 4).

22

Figure 5 Retention times were measured for each analyte (A) and IS (B). To elution 0.1 % FA and 100 % AcCN were used as mobile phases.

23

4.4 MS/MS conditions

Mass spectrometry function is based on three fundamental units: ion source,

analyzer and detector. The knowledge of their function is important to achieve

good optimization of the instrument leading to good results (15).

The ion source used was electrospray ionization (ESI) operating in positive mode

which was the most suitable for the analyte chemical structures. All ESI

parameters were optimized to accomplish the most effective ionization of the

analytes without any fragmentation. The schematic of a ESI with all used

parameters is presented in figure 11 (picture modifide from 16). To maximize the

operating parameters it is important to be sure that analytes are ionized. The main

problem that can occur is when there is too much interfering molecules in the

matrix that compete with the analytes. This mechanism is known as ion

suppression and must be taken into consideration when experiments are designed.

Ion suppression mechanism will be described in later section.

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Figure 6 (A) Schematic representation of ESI with accurate parameters which were used throughout the experiment. (B) Schematic of the mechanism of ion formation using ESI.

4.4.1 Product ions determination

Two transitions were used as analyte determination for each substance. All used

transitions were based on earlier method for urine (8). Experiment was performed

only for methamphetamine – d5. Trace and quantification ion were established by

screening the sample with MS/MS software. Three fragments were detected:

91.85; 121.02; 98.95. The fragment: 121.02 was chosen as a trace and

quantification ion.

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To determine a unique trace and quantification ion (daughter ions) is essential to

identify compounds such as designer drugs. The main reason is that the analyzed

substances can have similar structure to the endogenous compounds or medicine.

Cleavage pattern is important to determine right ions and depends on the chemical

structure of the analyte, atoms and groups and theirs position. Fragmentation

pattern is presented in an example in Figure 13 (17).

Figure 7 Fragmentation pattern for MDPV (Mol. Mass 275.343 g/mol) and methylone (Mol. Mass 207.23 g/mol).

In some cases, when compounds have isomers, a specific isomer cannot be easily

differentiated. Fluoroamphetamine is one of those substances.

Fluoroamphetamine has three isomers para (4) -, meta (3) - and orto (2) –

fluoroamphetamine. All three isomers give fragments which have exactly the

same molecular masses (figure 14, modified from (18–20)) (21,22)

26

Figure 8 Isomers of fluoroamphetamine. Two transitions resulting from fragmentation are presented.

In this study 4 – fluoroamphetamine was included as an analyte. However, when

patient sample is tested the results should indicate that the sample can contain any

one of those three isomers. In cases where patients claim to have consumed 4 –

fluoroamphetamine, the same precaution must be taken because no isomer

determination can be made.

4.4.2 MRM establishment

The principles of tandem mass spectrometry are fragmentation of an ionized

analyte and detection of those fragments with reference to mass-to-charge ratio

(m/z ratio). In tandem mass spectrometry the first quadruple (Q1) acts as a filter

with fixed voltage, there the protonated mother molecule is selected.

Fragmentation of the chosen molecule occurs in the collision cell with an inert gas

(the second quadruple, Q2). The third quadruple like Q1 is a fixed filter where

trace and quantification ions are selected (figure 12, modified from 15 ) (15).

When Q1 and Q3 are simultaneously set to obtain different m/z, it allows

27

scanning specific ions of interest. This highly selective system is called selected

(multiple) reaction monitoring (SRM/MRM).

In this study three MRM time segments were used, created according to retention

time of analytes (see figure 2 and table 1 ) (23).

Figure 9 Schematic of tandem mass spectrometer.

Fragments which were detected by each function and theirs conditions were

summarized in Table I.

Table I Summary of MRM function parameters and transition for each analyte and IS used

throughout the experiment.

No Parent (Da) Daughter (Da)QuantifierQualifier

Cone (V) Collision (eV)

Compound (equivalent IS)

Time segment 1

1 153.98 108.70 25.00 21 4- fluoroamphetamine

(pethidin –d4)137.00 9

2

155.08 121.02 20.00 10.00 Metamphetamine-D5

3 177.98 102.80 22.00 25.00 MDAI

28

(psilocin – d10)

160.90 11.00

4 178.01 118.98 20.00 20.00 Mephedrone

(pethidin –d4)144.91 20.00

5 194.07 161.00 22.00 19.00 Methedrone

(metamphetamine – d5)176.00 13.00

6 207.98 131.94 20.00 26.00 Methylone

(metamphetamine – d5) 190.04 12.00

7 215.20 164.00 22.00 19.00 Psilocin–D10

8 219.20 132.00 23.00 27.00 4 – OH – MET

(psilocin – d10)160.00 19.00

9 222.10 174.10 28.00 17.00 Butylone

(pethidin –d4)204.10 13.00

10 250.12 57.80 22.00 15.00 O – DM – Tramadol

(metamphetamine – d5)232.10 11.00

Time segment 2

1 248.19 175.00 30.00 19.00 Methoxetamine

(pethidin –d4)203.00 13.00

2 252.25 224.10 44.00 23.00 Phetidine-D4

3 276.29 126.00 38.00 25.00 MDPV

(pethidin –d4)135.00 25.00

Time segment 3

1 252.14 128.30 36.00 25.00 2-DPMP

29

(pethidin –d4)166.90 25.00

2 282.04 126.11 32.00 28.00 Naphyrone

(pethidin –d4)154.93 28.00

4.5 Method validation

4.5.1 Recovery (see also section 4.1 Sample preparation)

The efficiency of the preparation method was evaluated by performing recovery

test for each analyte and IS. The results obtained for samples spiked in serum

were compared with the results obtained for post – spiked samples. To calculate

the recovery the ratio of the peak areas were used:

RE = [Area (1) /area (2)]*100%, there area was calculated as mean of 3 samples.

Recovery for all analytes and IS were higher than the minimal required ratio

which is 75%. Recovery data is summarized and shown in Table II. Data used to

recovery calculation is displayed in Appendix I.

4.5.2 Matrix effect

Matrix effect (ME) is a common problem when biological samples are analyzed.

Presences of endogenous molecules like plasma proteins, phospholipids or other

xenobiotic are a great interference source which must be eliminated or decreased.

The sample cleanup and the chromatography are crucial to minimize this problem.

Matrix effect was calculated by comparison of peaks are for post - spiked samples

and samples spiked with 0.1% FA:

ME = [Area (2)/Area (3)]*100%, there area was calculated as mean of 3 samples.

30

Results were higher than 100 % for MDAI and 4 – OH –Met, two analytes that

elutes first. For remaining ten analytes, calculated matrix effect was between 88

and 94 % (Table II).

Table II Recoveries (%) were calculated for each analyte and IS. Matrix effect (%) was calculated for analytes.

Analyte Recovery (%) Matrix

effect (%)

Analyte Recovery

(%)

Matrix

effect (%)

MDPV 86 93 4 –FA 87 89

O – DM –cis –

Tramadol

86 94 Mephedrone 86 93

4 – OH – MET 94 147 MDAI 87 119

2 – DPMP 90 89 Methedrone 86 93

Butylone 87 91 Metoxetamine 87 93

Methylone 85 93 Naphyrone 92 88

IS Recovery (%)

Phetidine – d4 97

Psilocin – d10 94

Methamphetamin

e – d5

99

4.5.2 Carryover

31

Carryover is a problem that can have a great effect on the accuracy and precision.

The carryover detected in this experiment was considered to be a result of needle

contamination by a previous sample at a high concentration (24).

Carryover was tested for samples at concentration 5000, 1000 and 500 ng/mL.

Results were calculated by comparison peak area of analyte in blank with peak

area of analyte in high concentrated solution that was analysed before blank

sample. Calculation showed that carry over values were below 0.30 % (between

0.02 – 0.27) and only three analytes, O – DM –tramadol, 4 – OH – met, butylone

showed no or non-significant carryover (ng/mL) when compared with empirical

LOD and LOQ. Other analytes had carryover (ng/mL) higher than empirical LOD

or LOQ. However, after establishing cutoff and method precision (determined

from QC samples), carryover results were considered to be non-significant.

Calculated data (percentage, ng/mL) is presented in Appendix II.

4.5.3 Accuracy and precision

Five replicates of quality control (QC) samples at three different concentrations

were analysed to determine intra – assay, inter – assay precision and method

repeatability. All results were expresses as a coefficient of variance (CV %)

calculated for QC concentrations. Accuracy for each concentration was calculated

as deviation of the mean from the nominal value. All accuracy and precision

results were within the reference intervals of 20 % for lower calibration solutions

and 15% for other concentrations (Table III).

Table III Accuracy (deviation %) and precision calculation (CV %).

Analyte Repeatability Inter – assay Intra – assay Accuracy (deviation %)

32

(CV %) precision (CV %) precision (CV %)

25 50 500 25 50 500 25 50 500 25 50 500

MDAI 4.7 6.3 5.3 5.7 4.4 3.7 7.4 6.3 5.3 108 106 98.8

O – DM –Tramadol 5.7 3.3 2.6 0.2 4.6 2.7 5.7 5.67 3.7 99.2 96.9 100.2

4 – OH – MET 5.7 5.7 4.3 4.0 6.2 3.8 5.7 8.44 5.7 100.3 96.3 103.9

2 – DPMP 6.1 3.5 3.0 3.7 4.5 1.1 6.1 5.66 3.0 103 100.7 99.4

Butylone 7.4 4.9 4.9 3.6 3.1 3.1 7.4 4.89 4.9 103.2 100.5 100.1

Methylone 5.5 3.7 3.3 1.7 4.9 2.5 5.5 6.14 3.3 98.5 98.8 102

4 –FA 5.6 3.8 2.8 6.2 3.7 4.5 8.3 5.31 5.3 107.4 102.3 99.3

Mephedrone 6.3 3.7 2.6 1.1 4.0 1.4 6.3 5.47 2.6 103.4 101.8 98.4

MDPV 5.4 2.7 2.7 5.1 5.9 3.1 7.4 6.51 4.1 102.8 99.7 100.5

Methedrone 6.0 3.1 2.4 5.9 4.2 2.1 8.4 5.25 2.4 102.3 98.6 101.3

Metoxetamine 5.1 3.4 2.3 7.3 4.1 2.4 8.9 5.29 3.3 103.8 99.7 99.9

Naphyrone 5.8 4.0 3.1 5.6 4.0 2.1 8.0 5.64 3.1 104.1 100.6 100.7

4.5.4 Limit of detection and quantification

LOD and LOQ values were established theoretically from calibration solution 100

using following equations 3.3*standard derivation (SD)/slope and 10*SD/slope.

SD were calculated for six samples of calibration solution 100 and slope were

calculated as a medium slope for six calibrations curves (25). Theoretical LOD

and LOQ were corrected empirically and adjust to instrument capacity. The

theoretically calculated values were below 0.1 ng/mL for LOD and below 0.2

ng/mL for LOQ. However, empirical values established using MassLynx software

at s/n ratio ≥ 3 for LOD were 0.02 – 0.75 ng/mL and s/n ratio ≥ 10 for LOQ were

33

0.1 – 5.0 ng/mL (figure 5) for all analytes except O – DM –Tramadol. LOD and

LOQ for O – DM – Tramadol were 10 and 25 respectively (Table IV). The main

reason that O-DM-Tramadol shows high LOD and LOQ values is problem with

fragmentation.

Figure 10 Determination of s/n ratio for establishment of LOQ for naphyrone (both transitions are shown). Sample concentration 0.02 ng/mL.

4.5.5 Calibration curve; linearity

Six calibration curve were analysed and a correlation coefficient (r2) were

calculated to be higher than 0.99 for each analyte. Calibration curve for Butylone

is shown in figure 6. The correlation coefficient for each analyte is shown in

Table IV.

34

0 100 200 300 400 500 6000

20000400006000080000

100000120000140000160000180000200000

f(x) = 380.664540710285 x + 453.501637722722R² = 0.99995433348773

BUTYLON

BUTYLON Linear (BUTYLON )

Figure 11 Calibration curve (f (concentration) = area) for Butylone, correlation coefficient R2 = 1.

Table IV Calibration curves correlation coefficient, empirical limit of detection and limit of quantification (ng/mL).

Analyte Correlation coefficient (r2) LOD (ng/mL) LOQ (ng/mL)

MDAI 0.997 0.02 0.75

O – DM –cis –

Tramadol

0.999 10 25

4 – OH – MET 0.998 0.75 5.0

2 – DPMP 1.0 0.1 0.75

Butylone 1.0 2.0 5.0

Methylone 0.999 0.5 2.0

4 –FA 0.998 0.5 5.0

Mephedrone 1.0 0.75 2.0

MDPV 1.0 0.02 0.1

35

Methedrone 0.999 0.5 5.0

Metoxetamine 0.999 0.02 0.1

Naphyrone 1.0 0.02 0.1

4.5.6 Selectivity and specificity

Selectivity and specificity was tested for the method by analysis of samples

containing different xenobiotic and ten calibration solutions spiked with known

drugs other than analytes of interests. Moreover, blanks collected from ten

different sources were analysed, for the potential interferences. Chromatograms

example for butylone are presented in figure 7. Analysis of above-mentioned

solutions showed no interference with tested analytes. The method is found highly

selective.

36

Figure 12 Chromatograms for interferences experiment presented for butylone (2 transitioner):patinet sample (sertraline), calibration solution (sertraline), blank.

Ion suppression was tested for MDAI, Methedrone and Methylone to establish a

presence of any source of interference from substances that can exist in a new

matrix, serum. Ion suppression or enhancement (loss/gain in signal) is one

mechanism which is a form of matrix effect. There are many possibilities that can

cause ME. The most well-known cause is the competition between the analyte and

matrix molecule in the ion source. Matrix molecules can co-elute with the analyte

and be easier ionized that causes decreased ionization of molecule of interest

(26,27).

Results of the experiment showed that a significant the ion suppression exist

between 0.4 – 0.9 min. No significant ion suppression was detected what is shown

37

for MDAI (transitions 102.8) in Figure 8.Chromatograms can be found in

appendix III.

Figure 13 Ion suppression for MDAI (102.8 transition is shown). Multiple reaction chromatograms for postcolumn infusion for MDAI, methylone and methedrone. 0.1 % FA is a reference sample (red), matrix 0 - serum (green) and peak for MDAI obtained by standard analysis of calibration solution 100 (violet).

4.5.7 Sample stability

Short – term stability test was performed by analyzing 57 patient samples

containing 68 analytes (se section 3.6 “Sample stability”), thereafter stored in

autosampler at 8ºC for 72 hours and analyzed again. Only two samples showed

significant differences, one consisting mephedrone and one O – DM –tramadol.

Mephedrone was detected on day 1 but not after 72 hours. O – DM –tramadol was

not detected on day one but could be detected after 72 hours. Both samples

showed concentrations below the cutoff, this could explain the results. 53 samples

38

showed positive results there analyte could be detected on both occasions and 14

that gave negative results on day 1 and after 72 hours.

4.5.8 Application of method All tested serum samples were collected from urine–positive patients over a 2-

years period (2010 February – 2012 February) and stored at -80ºC. From 68

analytes 53 were detected. Sample concentration cannot be compared because of

different matrices.

In a few cases results from serum samples were different than results in urine.

In two butylone positive urine-samples showed negative result in serum, it was

probably an error in detection in the urine sample. Small peaks at methylone RT

could be seen on chromatograms. However, quantification was not possible.

In one case, the serum sample showed negative for 4-OH-Met and positive for O-

DM-Tramadol when the urine analysis showed the other way about. 4-OH-Met is

an unstable analyte and degrades easily. In low concentration samples detection

can be a problem. O-DM-Tramadol could have been missed in the urine analysis.

The other samples with negative results could not be explained. It was not

possible to attain data about the initially measured concentrations. No further data

analysis could be performed.

Conclusion

By using UPLC with triple quadruple and MRM a highly selective and specific

method could be developed. Two unique transitions could be determined for each

analyte. Analytes that belong to different chemical classes could be detected in

39

matrix with high levels of interference. Quantification of all substances was

possible. The method was validated and successfully applied to patient samples.

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42

Appendix I

id17 name20 desc_sample area 1 area 2 area 3 medel SD

1 MDAI sample 0,1%FA 158725,18

8143556,84

4168809,79

7157030,

612711,4

2 Metylon490882,28

1485187,90

6514767,06

3496945,

815694,1

3 O-DM-tramadol571300,37

5 477815571010,68

8 54004253890,3

4 4-Fluoroamfetamin 91836,102 85299,969 92741,516 89959,24060,32

5 Metedron544774,31

3 532404,5 557968545048,

912783,9

6 Butylon147809,67

2142089,70

3145222,71

9145040,

72864,32

7 Mefedron767760,56

3745136,18

8742869,06

3751921,

913763,4

8 Metoxetamin274498,43

8259491,78

1254188,92

2262726,

410534,0

9 MDPV521053,62

5489798,46

9481832,09

4497561,

420731,1

43

10 2-DPMP146085,31

3135333,62

5133252,78

1138223,

96887,21

11 Nafyron478926,46

9443671,28

1438886,12

5 45382821867,2

12 4-OH-MET102817,22

7 93835,844 104494,18100382,

45731,16

desc_sample_a area 1a area 2a area 3a medel_a SD_a CV_a

sample 350 S 160685,93

8 175203,5158505,78

1164798,40

6 9076,77 5,5388091,06

3422295,84

4385334,62

5398573,84

420590,0

3 5,2428452,53

1464528,65

6433050,34

4 442010,5119636,3

2 4,4

69592,523 75832,438 66968,29770797,752

74553,31

6 6,4

431963,75466428,90

6429198,18

8442530,28

120742,9

6 4,7113827,80

5125429,63

3112735,64

1117331,02

67034,82

6 6,0574303,12

5 641595,5 605377,75607092,12

533678,9

3 5,5207921,67

2227938,95

3207990,70

3214617,10

911537,1

1 5,4381207,78

1429071,34

4390848,62

5400375,91

725314,1

7 6,3108505,30

5119819,53

1109092,52

3112472,45

36369,52

7 5,7358739,09

4398389,15

6359000,40

6372042,88

522816,9

1 6,1136159,18

8145583,62

5136889,59

4139544,13

65243,08

6 3,8

44

desc_sample_b area 1b area 2b area 3b medel_b SD_b CV_bsample 350 B 181926,2 197049 184066,14 187680,443 8183,66

462097,7 483669,69 449306,97 465024,782 17367,4489419 548803,38 492708 510310,125 33376,7

78071,82 85369,852 78168,305 80536,659 4185,95492442,2 531489,13 508358,34 510763,208 19634,3130445,8 140685,33 129003,65 133378,26 6369,06

676786 743009 686150,88 701981,958 35837,7237635,4 261779,11 240481,77 246632,094 13194,7445891,4 493627,19 449171 462896,542 26664122046,4 125892,95 123504,16 123814,492 1941,98395053,7 407281,47 405050 402461,719 6511,84

144993 154642,45 144005,53 147880,333 5876,94

utbyte sample350 S/sample 350 B utbyte sample B/sample 0,1FA87,81 119,5285,71 93,5886,62 94,4987,91 89,5386,64 93,7187,97 91,9686,48 93,3687,02 93,8786,49 93,0390,84 89,5892,44 88,6894,36 147,32

45

ANALYTKONCENTRATIO

N carryover % carryover

ng/mL JON I JON II JON I4-OH-MET 500 - - - 1000 - - - 5000 - - -BUTYLON 500 0,045 - 0,225 1000 0,024 - 0,24 5000 0,028 - 1,4O-DM-TRAMADOL 500 - - - 1000 0,021 - 0,21 5000 0,022 - 1,1MEFEDRON 500 - 0,071 - 1000 0,097 0,068 0,97 5000 0,104 0,047 5,2METEDRON 500 0,044 - 0,22 1000 0,032 0,062 0,32 5000 0,041 0,035 2,05METYLON 500 0,046 0,084 0,23 1000 0,039 0,037 0,39 5000 0,019 0,017 0,954-FLUOROAMFETAMIN 500 0,168 0,245 0,84 1000 0,187 0,172 1,87 5000 0,164 0,168 8,2MDAI 500 0,046 0,134 0,23 1000 0,037 0,087 0,37 5000 0,055 0,053 2,75MDPV 500 0,044 0,059 0,22 1000 0,039 0,053 0,39 5000 0,032 0,027 1,6METOXETAMIN 500 0,053 0,048 0,265 1000 0,04 0,027 0,4 5000 0,012 0,016 0,6NAFYRON 500 0,204 0,233 1,02 1000 0,166 0,171 1,66 5000 0,141 0,11 7,052-DPMP 500 0,264 - 1,32 1000 0,163 - 1,63 5000 0,095 - 4,75

46

Appendix II

Appendix III

47

48