Development of a Screening Method for Drugs of Abuse by ...

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Student Theses John Jay College of Criminal Justice

Summer 8-2-2019

Development of a Screening Method for Drugs of Abuse by Direct Development of a Screening Method for Drugs of Abuse by Direct

Analysis of Dried Urine Spots Coupled to Mass Spectrometry Analysis of Dried Urine Spots Coupled to Mass Spectrometry

Melanie Goldstein John Jay College of Criminal Justice, [email protected]

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Development of a screening method for drugs of abuse by direct analysis of dried urine

spots coupled to mass spectrometry

A Thesis Presented in Partial Fulfillment of the Requirements for the Degree of

Master of Science in Forensic Science

John Jay College of Criminal Justice

The City University of New York

Melanie Goldstein

August, 2019

Development of a screening method for drugs of abuse by direct analysis of dried urine

spots coupled to mass spectrometry

Melanie Goldstein

This thesis has been presented to and accepted by the office of Graduate Studies, John Jay College of Criminal Justice in partial fulfillment of the requirements for the degree of Master of Science in Forensic Science.

Thesis Committee:

Thesis Advisor: Dr. Marta Concheiro-Guisan

Second Reader: Dr. Shu-Yuan Cheng

Third Reader: Dr. Daniel Torres-Rangel

Table of Contents Pages

Acknowledgments i

Abstract ii

Introduction 1 – 5

Methods and Materials 5 – 9

Results and Discussion 10 – 30

Conclusion 31 – 32

References 32 – 36

Appendix 37 – 48

i

Acknowledgements

First, I would like to thank my thesis advisor Dr. Marta Concheiro-Guisan for

giving me the opportunity to work on this project and guiding me through the process. I

would also like to thank Dr. Shu-Yuan Cheng and Dr. Daniel Torres-Rangel for taking

the time to be my readers, provide their feedback, and making the completion of my

thesis project possible. Finally, I am so grateful to my parents and husband for supporting

me throughout all of my studies. Without their encouragement and support, this

accomplishment would not have been possible. Thank you.

ii

Abstract

Fast and easy screening procedures are essential in any forensic toxicology

laboratory to differentiate negative samples from presumptive positive cases.

Immunoassay techniques, such as enzyme multiplied immunoassay technique (EMIT),

are routinely employed as screening procedures. However, these techniques lack

specificity (only differentiate group of drugs and not individual compounds) and it is

difficult to add new compounds to the panel. Direct analysis of dried urine spots (DUS)

by mass spectrometry (MS) offers a novel strategy to overcome these issues. DUS offer

an improved storage alternative for biological samples, reducing costs and space

requirements. In this work, an original method to screen for 15 common drugs of abuse in

dried urine spots is described. The drug groups included opioids, prescription opioids,

amphetamines and cocaine. Using a thin-layer chromatography-mass spectrometry (TLC-

MS) interface, DUS samples were directly sampled and mass spectral data were

analyzed. The method allows for analysis of DUS samples in less than five minutes and

only 20 µL of sample is required for analysis. The method was validated according to

SWGTOX guidelines, including limit of detection (LOD) and interference studies. The

LOD ranged from 100 to 1,000 ng/mL, depending on the compound. The performance of

the DUS screening method was compared to EMIT. The DUS method was more specific

than the immunoassay screening but less sensitive than EMIT for certain analytes

including morphine, hydrocodone, codeine, and 6-monoacetylmorphine. While this DUS

screening method is rapid and easy to perform, urinary matrix components can possibly

interfere with analytes and complicate results.

1

Introduction

Dried blood spot (DBS) sampling is a well-established technique which has been

used for screening of neonatal metabolic disorders for decades. Recently, there has been

increased interest in the use of DBS as an alternative to conventional venous blood

sampling, and also as a storage procedure of other types of biological matrices, dried

matrix spot (DMS), to support pharmacokinetic and toxicokinetic studies in therapeutic

drug monitoring (TDM) and for the detection of drugs of abuse in forensic settings

(Antunes, Charao, & Linden, 2016; Oliveira, Henion, & Wickremsinhe, 2014; Stove,

Ingels, De Kesel, & Lambert, 2012). DBS sampling has many advantages over traditional

blood collection, which include a less invasive sampling technique and reduced blood

volume requirements. In addition, DBS and DMS techniques offer a low biohazard risk,

reductions in sample processing, storage and transportation costs (Sadones, Capiau, De

Kesel, Lambert, & Stove, 2014), and improved stability for some drugs and metabolites

(Abu-Rabie & Spooner, 2009; Oliveira et al., 2014).

DMS are gaining popularity in toxicology laboratories and have been employed

as alternative sample collection and storage procedure for confirmatory analysis and for

screening procedures. DMS have been employed in blood (Berm et al., 2014; H. Lee et

al., 2015), urine (Y. Lee, Lai, & Sadrzadeh, 2013; Otero-Fernandez et al., 2013), plasma

and other matrices (Ayre, Chaudhari, Jagdale, & Agrawal, 2018). The typical DMS work

flow in a confirmatory analysis involves punching a disk from the center of the spot,

transferring the disk to a tube, and extracting the sample with a solvent containing an

internal standard. Furthermore, additional sample preparation and cleanup such as liquid-

liquid or solid-phase extraction is often required prior to analysis by liquid

2

chromatography tandem mass spectrometry (LC-MSMS). Although DMS offers several

advantages as highlighted above, the DMS confirmatory analysis may be time consuming

and tedious, especially when a large number of samples need to be prepared for analysis

(Oliveira et al., 2014).

A suitable direct analysis technique for DMS samples without sample pre-

treatment and extraction could simplify sample preparation and provide significant cost

and time savings. Also, the elimination of the need for the sample separation by liquid

chromatography (LC) would simplify the procedure (Abu-Rabie & Spooner, 2009).

Although the lack of sample pre-treatment, extraction and chromatographic separation

may decrease the sensitivity and specificity of the technique as confirmatory tool, it may

be used as screening procedure. Toxicology laboratories routinely employ immunoassays

(EMIT, ELISA), gas-chromatography mass spectrometry (GC-MS), or HPLC with diode-

array detection (DAD) to perform drug screening tests. Each of these techniques have

limitations including lack of specificity and difficulty of incorporating new targets

(immunoassays), or labor-intensive sample preparation and instrument maintenance (GC-

MS and HPLC-DAD), thus there is a need to develop novel techniques that provide a

simpler, faster, sensitive and more cost-effective screening method that would enable

toxicology labs to operate more efficiently (Gaugler, Rykl, Grill, & Cebolla, 2018; Jett,

2017).

Several surface-sampling probes exist that could be used for the direct analysis

with MS detection. These probes could enable the direct analysis of DMS without sample

preparation, extraction, and chromatographic separation. Two examples of these probes

include the liquid microjunction-surface sampling probe (LMJ-SSP), which can be

3

applied to extraction of analytes from the surfaces of hard and nonporous materials like

glass or metal, and the sealing surface sampling probe (SSSP), used on porous surfaces,

including most filter papers, such as DBS filter paper (Deglon, Thomas, Mangin, &

Staub, 2012). Luftmann (2004) described the development of a device which allowed for

the direct sampling of thin layer chromatography (TLC) plates to an electrospray

ionization (ESI) mass spectrometer and an automated TLC-MS interface (Luftmann,

2004; Luftmann, Aranda, & Morlock, 2007). This type of probe was a sealing surface

sampling probe (SSSP). Van Berkel et al (2002, 2009) also described the development of

a combined surface sampling probe/electrospray emitter to directly sample TLC plates by

ESI mass spectrometry. His group developed the liquid microjunction surface sampling

probe (LMJ-SSP), which was shown to be capable of direct mass spectrometric analysis

of drugs and metabolites in DBS samples and whole mouse thin tissue sections (Van

Berkel & Kertesz, 2009; Van Berkel, Sanchez, & Quirke, 2002)

Other examples of direct sampling and ionization techniques include Direct

Analysis in Real Time (DART) and paper spray ionization (PSI). Beck, et al (2016)

developed a method to screen for and confirm methadone in untreated urine specimens

using DART coupled with both time-of-flight and triple quadrupole linear ion trap (Q-

TRAP) mass spectrometers. DART-MS allows for ambient ionization of samples and

does not rely on chromatography for separation. It relies on the mass spectrometer for

separation, which allows for data acquisition in real time (Beck, Carter, Shonsey, &

Graves, 2016). Jeong, et al. (2016) developed a rapid and direct paper spray ionization-

mass spectrometry (PSI-MS) method for quantitative analysis of ephedrine,

pseudoephedrine, norpseudoephedrine, and methylephedrine in human urine. Paper spray

4

ionization generates ions by applying high voltage to a triangular piece of filter paper

wetted with solvent. PSI-MS allows for direct analysis of a sample without complex

preparation and chromatographic separation, which allows for reduced sample

preparation and analytical times. Their method employed high-resolution mass

spectrometry (Orbitrap) with a triple-quadrupole mass spectrometer which allowed for

confirmation of the ephedrines tested (Jeong et al., 2016).

Mass spectrometry has become essential in toxicological analysis. Conventional

laboratory scale mass spectrometers are combined with gas or liquid chromatography,

making them very bulky. Thus, there is a desire to miniaturize MS systems so that the

systems can fit on a bench-top or be portable so that they can be used in the field for

seized substance identification, food and cosmetics analysis, and point-of-care analysis

(Bu, Regalado, Hamilton, & Welch, 2016; Eikel, Prosser, & Henion, 2015; Lawton et al.,

2017; Ma et al., 2015; Ma et al., 2016; Ma & Ouyang, 2016). Analysis by miniature and

compact mass spectrometers may play a unique role in bioanalysis in that it allows users

to obtain MS results at their own benches without a need to send samples to analytical

laboratories. One such compact mass spectrometer (CMS) system is the Advion

Expression CMS. The Advion system is a compact version of a single quadrupole MS

instrument which can be coupled to the PlateExpress TLC plate reader, which allows for

rapid and direct ESI-MS analysis of spots on a TLC plate (Bu, Yang, Gong, & Welch,

2014). If the TLC-CMS interface could be applied to DMS cards, it could be possible to

develop a direct analysis of DMS samples to screen for drugs of abuse.

The goal of our project was to develop and validate a new screening method

which directly samples DUS by employing the PlateExpress TLC plate reader coupled to

5

a portable single quadrupole mass spectrometer. The method allowed the screening of 15

drugs and metabolites, including opioids, amphetamines, and cocaine. DUS were directly

extracted and analyzed, eliminating the punching and subsequent extraction steps that are

involved in the manual work flow for DMS analysis. This novel methodology in direct

sampling and ionization has the potential to provide significant time and cost savings

while greatly simplifying analysis in the toxicology laboratory.

Methods and Materials

1. Reagents and Materials

TLC plates, Analtech TLC UniplatesTM high performance silica gel thin layer

chromatography plates (150 micron, 10x20 cm), and WhatmanTM 903 protein saver cards

were purchased from Fisher Scientific (Hampton, NH). Morphine, 6-

monoacetylmorphine (6-AM), codeine, hydrocodone, oxycodone, fentanyl, norfentanyl,

methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine,

benzoylecgonine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine

(MDA), and 3,4-methylenedioxymethamphetamine (MDMA) 1 mL ampoule standards in

methanol at 1 mg/mL were purchased from Cerilliant (Round Rock, TX). Synthetic urine

was purchased from Ricca Chemical Company (Pocomoke City, MD). Authentic urine

samples were provided by volunteers in the laboratory. The amphetamine, cocaine,

methadone, and opiate EMIT II Assay kits were purchased from Siemens (Munich,

Germany).

2. Urine sample preparation

6

To determine the limit of detection (LOD) or cut-off for each drug in methanol,

synthetic urine, and authentic urine, samples were prepared at 100, 250, 500, 1,000, and

10,000 ng/mL. The synthetic and authentic urine samples were prepared by spiking 200

µL of the samples to with the appropriate amount of 100,000, 10,000 or 1,000 ng/mL

standard needed to reach the desired concentration.

3. Blind urine sample preparation

To test the performance of the screening test, 5 urine samples were fortified with

different drugs (methamphetamine, amphetamine, morphine, hydrocodone,

benzoylecgonine, MDMA, methadone, EDDP, or oxycodone) at 500 ng/mL. These

samples were prepared by another member of the laboratory and were analyzed by the

screening method and by EMIT.

4. TLC spot preparation

The methanolic drug standards were diluted to 100,000 ng/mL, and these

solutions were spotted onto the TLC plates using micro capillary tubes. The spot was

circled with pencil and allowed to dry for approximately two minutes prior to analysis.

5. DMS preparation

For the methanolic drug samples, 20 µL of sample was dispensed into the center

of the spot on the card and allowed to dry for 10 min at room temperature before analysis.

For the synthetic and authentic urine samples, 20 µL of sample was dispensed into the

center of the spot on the card and were allowed to dry overnight at room temperature

prior to analysis.

7

To determine cut-off intensities and conduct interference studies, the spot was cut

from the card after drying, and it was placed on a clean microscope slide for analysis to

avoid cross-contamination between spots.

6. Instrumentation

The instrument employed was a PlateExpress TLC plate reader coupled to the

Expression compact mass spectrometer (CMS), both from Advion (Ithaca, NY) (Figure

1). The Plate Express is a device that provides a simple, semi-automated means of

obtaining mass spectra directly from TLC plates by direct solvent extraction of

compounds. The plate reader has an elution head which lowers itself onto the spot of

interest, presses firmly into the plate to form a seal, then flows solvent over the spot to

extract the sample and sends this solution to the mass spectrometer (Advion, 2015)

(Figure 2). The solvent that we employed was 0.1% formic acid in acetonitrile at a flow

rate of 2.5 mL/min.

The Advion Expression CMS is a single quadrupole MS. We employed the

electrospray ionization (ESI) source in positive mode. Data was acquired in scan mode,

with a mass range from 88 to 500 m/z. The ESI parameters were as follows: capillary

temperature 200 °C, capillary voltage 150.0 V, source voltage offset 20.0 V, source

voltage span 30.0 V, ESI source gas temperature 200 °C, and ESI voltage 3,500 V. The

MS parameters used were as follows: extraction electrode 9 V, hexapole bias 8 V,

hexapole RF offset 0, hexapole RF span 140, ion energy offset -1.2, ion energy span 0,

resolution offset -0.01, resolution span 1.1, and detector gain 1,100 V.

8

Figure 1. (A). TLC-MS system used in this work. On the left is the Advion CMS and on the right is the PlateExpress TLC plate reader. (B). Close-up view of PlateExpress extraction device. (Advion, 2015).

Figure 2. Schematic view of PlateExpress elution head. The blue line shows extraction solvent flowing down to the sample spot of interest. The green line shows the solution formed by the extraction solvent flowing back to the mass spectrometer (Advion, 2015).

9

EMIT screening was performed using a VivaTM Drug Testing System from

Siemens (Munich, Germany). Five hundred µL of each authentic urine sample was used

to screen for amphetamines, cocaine, methadone, and opiates. For amphetamine

screening, a 1,000 ng/mL cut-off concentration was used. For cocaine, methadone, and

opiate screening a 300 ng/mL cut-off concentration was used.

7. Validation of DUS screening method

The screening assay was validated according to the SWGTOX guidelines

(Scientific Working Group for Forensic Toxicology, 2013). To validate the screening

method, we evaluated the limit of detection LOD or cut-off of each analyte of interest in

methanol, synthetic urine, and authentic urine, and conducted interference studies. In

order to determine the LODs for each drug in a particular matrix, DMS spots were

prepared at 10,000, 1,000, 750, 500, 250, and 100 ng/mL. Additional samples were

prepared at 200 ng/mL for cocaine and benzoylecgonine. The lowest concentration at

which the compound could be repeatedly and reliably detected was designated as the

LOD. In order to assess the possibility of endogenous interferences, blank urine samples,

which had not been fortified with any drug, were tested. Any peaks detected in the blank

with a mass-to-charge ratio identical to one of the analytes of interest was designated as

endogenous interference. Additional compounds that could possibly account for any

observed interferences (endogenous and exogenous interferences), due to their similar

molecular weight to the target analytes, were identified by searching the Human

Metabolome Database using the LC-MS search function (Wishart et al., 2018).

10

Results and Discussion

1. TLC Analysis

Prior to analysis of drugs extracted from DMS cards, each drug standard (100,000

ng/mL) was spotted on and extracted from a TLC plate. The mass spectrum of each pure

drug was obtained in order to determine the target ion m/z of interest (Table 1).

Table 1. Mass spectral data of the analytes of interest extracted from TLC plates. No fragmentation was observed for analytes with no information in the product ion column

Drug Molecular Weight (g/mol)

Precursor Ion (m/z)

Product Ion(s) (m/z)

Morphine 285.3 286.1 - 6-AM 327.3 328.1 -

Oxycodone 315.3 316.2 - Hydrocodone 299.3 300.2 -

Codeine 299.3 300.2 - Fentanyl 336.4 337.3 -

Norfentanyl 232.3 233.1 - Methadone 309.4 310.2 265.0

EDDP 277.4 278.1 - Amphetamine 135.2 136.1 119.1, 91.1

Methamphetamine 149.2 150.0 119.1, 91.1 MDMA 193.2 194.1 163.0 MDA 179.2 180.0 163.0

Cocaine 303.3 304.2 182.1 Benzoylecgonine 289.3 290.1 168.0

By extracting the drug standards from TLC plates, we were able to analyze the

mass spectral data for each analyte, including the parent ion and determine if any

fragmentation occurred. This information was helpful in determining which ions to

monitor in the DUS samples and for establishing what criteria should be met in order to

determine if a sample was positive. Morphine, 6-AM, oxycodone, hydrocodone, codeine,

fentanyl, norfentanyl, and EDDP did not undergo any fragmentation. Therefore, we could

only rely on the detection of the parent ion when screening unknown samples.

11

Methadone, MDMA, MDA, cocaine, and benzoylecgonine each displayed one precursor

and one fragment ion. The presence of a characteristic fragment ion improves the

specificity of the analysis, because we can rely on the presence and intensity of both ions

when making conclusions. Amphetamine and methamphetamine displayed a precursor

and two fragment ions (Table 1).

2. Determination of Limit of Detection on DMS cards

To determine if the Whatman 903 card had any effect on the mass spectral data,

we applied 20 µL of each drug standard at 100,000 ng/mL to the center of the spot and

directly analyzed the spot using the TLC-CMS interface. All parent and fragment ions

observed when using the TLC plate were observed when extracting the methanolic

standards from the card. We determined the LOD for each methanolic drug standard on

the DMS card (Table 2). This gave us an idea of the instrumental LOD and informed us

of what concentrations to proceed with when determining the LOD in synthetic and

authentic urine. For example, the LOD of the oxycodone, hydrocodone, and codeine

standards on the DBS cards were all 500 ng/mL. Therefore, the lowest concentration

synthetic and authentic urine samples prepared for these drugs were 500 ng/mL. These

results were mostly in agreement with LOD data published by the manufacturer

(Orlowicz et al., 2019).

Table 2. Mass spectral and LOD data for methanolic drug standards extracted from the Whatman 903 cards.

Drug Precursor Ion (m/z)


LOD (ng/mL)

Peak Intensity at LOD

Morphine 286.1 - 100 1.4E6 6-AM 328.1 - 100 6.7E5

Oxycodone 316.2 - 500 1.3E5 Hydrocodone 300.2 - 500 1.2E5

12

Codeine 300.2 - 500 2.4E5 Fentanyl 337.3 - 100 2.9E5

Norfentanyl 233.1 - 100 2.4E5 Methadone 310.2 265.0 100 1.4E6

EDDP 278.1 - 100 1.2E6 Amphetamine 136.1 119.1, 91.1 100 6.4E5

Methamphetamine 150.0 119.1, 91.1 100 3.3E5 MDMA 194.1 163.0 100 5.8E5 MDA 180.0 163.0 100 1.6E5

Cocaine 304.2 182.1 100 6.1E5 Benzoylecgonine 290.1 168.0 100 3.9E5

After determining the LOD of each drug in methanol, samples were prepared in

synthetic urine. The mass spectral data and LOD of each drug in synthetic urine is

summarized in Table 3. In synthetic urine, the LOD for morphine, 6-AM, amphetamine,

methamphetamine, MDMA, and MDA increased from 100 ng/mL to 250 ng/mL. In

addition, the 182.1 m/z product ion of cocaine was no longer detectable in synthetic urine.

Table 3. Mass spectral and LOD data for dried synthetic urine samples.



LOD (ng/mL)

Peak Intensity at LOD

Morphine 286.0 - 250 6.6E5 6-AM 328.1 - 250 1.2E5




EDDP 278.1 - 100 2.4E5 Amphetamine 136.1 119.1, 91.1 250 1.8E5

Methamphetamine 150.1 119.1, 91.1 250 3.2E5 MDMA 194.1 163.0 250 1.4E5 MDA 180.0 163.0 250 1.5E5

Cocaine 304.2 100 4.8E5 Benzoylecgonine 290.1 168.0 100 3.8E5

13

Finally, drug samples were prepared in negative authentic urine samples. To determine

the LOD , each drug was prepared in three different authentic urine samples and analyzed

by TLC-CMS. Mass spectral and LOD data is summarized in Table 4. The LOD for

morphine, 6-AM, amphetamine, methamphetamine, MDMA, and MDA increased from

250 ng/mL in synthetic urine to 500 ng/mL in authentic urine. The LOD for oxycodone,

hydrocodone, and codeine increased from 500 ng/mL in synthetic urine to 1,000 ng/mL

in authentic urine. The LOD for cocaine and benzoylecgonine increased from 100 ng/mL

in synthetic urine to 200 ng/mL in authentic urine. In addition, the product ions for

amphetamine and methamphetamine were no longer detectable in authentic urine. Matrix

components and urinary metabolites may impact the ability to detect the target analyte.

The significant increase in LOD for these drugs and inability to detect certain ions is

likely caused by matrix effects due to the complex chemical composition of urine. The

LOD and fragmentation pattern for each analyte in authentic urine was used to determine

the cut-off intensity and ions to be monitored when screening unknown samples (Table

5).

Table 4. Mass spectral and LOD data for dried authentic urine samples.



LOD (ng/mL)

Peak Intensity at LOD (cut-off intensity)

Morphine 286.1 - 500 1.75E6 6-AM 328.1 - 500 2.33E5




EDDP 278.1 - 100 1.32E6 Amphetamine 136.1 - 500 3.68E5

Methamphetamine 150.0 - 500 3.63E5 MDMA 194.1 163.0 500 1.18E5

14

MDA 180.0 163.0 500 2.38E5 Cocaine 304.2 - 200 2.10E5

Benzoylecgonine 290.1 168.0 200 2.41E5

Table 5. Cut-off intensities for each individual drug in authentic urine.

Drug Ion m/z Concentration (ng/mL)

Average signal intensity

Morphine 286.1 500 1.75E6 6-AM 328.2 500 2.33E5

Oxycodone 316.1 1000 2.20E5 Hydrocodone 300.2 1000 2.39E5

Codeine 300.2 1000 2.19E5 Fentanyl 337.3 100 6.92E5

Norfentanyl 233.1 100 2.84E5

Methadone 310.2 100 2.03E6 265.0 100 1.32E6

EDDP 278.1 100 1.52E6 Amphetamine 136.1 500 3.68E5

Methamphetamine 150.1 500 3.63E5

MDMA 194.1 500 1.18E5 163.1 500 2.63E5

MDA 180.0 500 2.38E5 163.1 500 7.70E5

Cocaine 304.2 200 2.10E5

Benzoylecgonine 290.0 200 2.41E5 168.1 200 1.65E5

3. Interference Studies

Because this method does not involve sample pre-treatment or chromatographic

separation, identification relies solely on the mass spectrometer. Urine is a biological

waste material which may contain metabolic breakdown products from food, drinks,

drugs, environmental contaminants, endogenous waste products, and bacterial by-

products (Bouatra et al., 2013). Thus, it was important to perform interference studies.

Interferences are non-targeted analytes, such as matrix components, impurities, other

drugs or metabolites, which may impact the ability to detect or identify a targeted analyte

15

(Standard Working Group for Forensic Toxicology, 2013). Interferences to target

analytes can arise from several different situations: intra-target interferences can arise

from analytical targets of this method with the same parent ion and/or interferences can

arise from endogenous or exogenous substances potentially present in the urine samples.

Ten negative authentic urine samples were analyzed prior to fortification in order

to assess possible endogenous interferences. These data are summarized in Table 6.

Table 6. Peak intensity of the ions of interest detected in each negative authentic urine samples (n=10).

Ion m/z Negative urine samples

1 2 3 4 5 6 7 8 9 10 136 1.40E5 1.4E5 ND 1.5E5 2.0E5 3.0E5 1.3E5 1.4E5 1.6E5 2.4E5 150 ND 2.4E5 ND ND 1.3E5 2.2E5 2.5E5 2.7E5 2.0E5 2.8E5 163 ND 1.9E5 1.6E5 ND 1.8E5 1.8E5 ND 1.7E5 2.4E5 1.7E5 168 1.8E5 1.7E5 2.3E5 2E5 ND ND ND ND ND 1.2E5 180 ND ND ND ND ND ND ND ND ND 1.9E5 194 ND ND ND ND ND ND ND ND ND ND 233 ND 1.5E5 ND ND 1.5E5 1.9E5 ND ND ND 1.5E5 265 9.3E5 8.7E5 6.1E5 8.3E5 9.1E5 6.9E5 3.7E5 8E5 5.5E5 7.8E5 278 3.3E5 ND ND ND ND ND ND ND ND ND 286 1.7E6 1.7E6 1.2E6 1.9E6 1.3E6 8.9E5 9E5 2.3E6 1.6E6 2.1E6 290 3.2E5 3E5 2.2E5 2.3E5 1.4E5 ND 1.8E5 3.9E5 2.7E5 3.4E5 300 1.3E5 ND ND 1.8E5 1.4E5 1.6E5 ND ND ND ND 304 ND ND ND ND ND ND ND ND ND ND

310.2 1.5E5 1.2E5 2.8E5 3.9E5 1.6E5 1.8E5 ND ND ND ND 316.2 1.4E5 ND 1.7E5 1.7E5 1.4E5 1.8E5 ND ND 1.8E5 ND 328 2.6E5 1.2E5 2.7E5 3E5 1.7E5 2.4E5 ND 3.3E5 3.1E5 ND 337 ND ND ND ND ND ND ND ND ND ND

ND-Not detected

The 136.1 m/z peak for amphetamine was detected in 9 of the 10 blank urine

samples. The 150.0 m/z peak for methamphetamine was detected in 7 of the 10 blank

urine samples. The 310.2 m/z peak for methadone was detected in 6 of the 10 blank urine

samples. The 316.2 m/z peak for oxycodone was detected in 6 of the 10 blank urine

samples. The 328.2 m/z peak for 6-AM was detected in 8 of the 10 blank urine samples.

16

The 290 and 168 m/z peaks for benzoylecgonine presented a complication. The 290 m/z

peak was detected in 9 of the 10 blank urine samples and for 5 of these the 168 m/z peak

was also detected. Therefore, we cannot rely on the fragmentation pattern to distinguish

between benzoylecgonine and possible interferences, so we had to rely solely on the

intensity of the peaks to make a determination of positive or negative. In addition, the

286.0 m/z peak for morphine was detected in all 10 blank urine samples and at very high

intensities (average signal intensity of 1.56E6). This apparent endogenous interference

makes accurate detection of morphine by this method very difficult and unreliable.

Without a more thorough analysis of the chemical composition of each urine

sample we are unable to determine exactly what these interference compounds are. The

chemical complexity of urine poses a challenge because other compounds may

complicate results and lead to false positives. To investigate other exogenous or

endogenous compounds found in urine that could cause interferences with the developed

drug screening method, the Human Metabolome Database was searched using the LC-

MS search function (Wishart et al., 2018). The results of the search are summarized in

Table 7. This list is limited to compounds with a parent ion that shares a m/z with an

analyte of interest and does not include all possible adducts that could appear.

Table 7. Possible interferences identified for each target ion using the Human Metabolome Database.

Target analyte Possible MS Interference compound Amphetamine

(136 m/z) Homocysteine

Adenine

Methamphetamine (150 m/z)

NAPQI Penicillamine L-Methionine

Sevelamer Cathinone

17

Phentermine

MDMA/MDA (163 m/z product ion)

D-4-hydroxy-2-oxoglutarate 3-hydroxymethylglutaric acid

2-hydroxyadipic acid Levoglucosan

Benzoylecgonine

(168 m/z product ion)

Quinolinic acid N-acetyltaurine

Thioguinine 8-Hydroxyguanine

Isopyridoxal

MDA (180 m/z)

Hippuric acid Acetylisoniazid Glucosamine Mexiletine

Methylephedrine Methoxyphenamine

Rimantadine Memantine

MDMA (194 m/z)

3-Anilino-4-oxobutanoic acid Methyl hippurate

2-Methylhippuric acid 3-Carbamoyl-2-phenylpropionaldeyde

4-Hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one Phenylacetylglycine

m-Methylhippuric acid

Norfentanyl

(233 m/z)

Vanillin 4-sulfate (2E, 11Z)-5-[5-(methylthio)-4-penten-2-ynyl]-2-

furanacrolein (2S,3S,4S)-5,7,9,11-Tridecatetrayne-1,2,3,4-tetrol

Phenobarbital Nalidixic Acid

Methadone (265 m/z product ion)

Methylgallic acid-O-sulfate Thioxanthine monophosphate

3-Methoxy-4-hydroxyphenylglycol sulfate 2’,2’-Difluorodeoxyuridine

Dapsone hydroxylamine Sulfamerazine

N-acetylcystathionine

EDDP (278 m/z)

Amitriptyline Venflaxine

DOPA sulfate Azathioprine

Ethylmethylthiambutene Entecavir

Maprotiline

18

N-Desmethylterbinafine Perhexiline

Morphine (286 m/z)

7-aminoclonazepam Hydromorphone

Cladribine Faropenem Letrozole

Probenecid Isothipendyl Norcodeine

Norhydrocodone N-monodesmethyl-rizatripan

Mepyramine

Benzoylecgonine (290 m/z)

Quinethazone Chlphedianol

Cofedanol Norcocaine

Chloropyramine Hydroxylated N-acetyl desmethyl frovatriptan

Hyoscyamine Donepezil metabolite M4

Atropine Dyclonine

Hydrocodone/Codeine (300 m/z)

Chlordiazepoxide Metoclopramide

Imipenem N-desalkylpropafenone N-depropylpropafenone

Cocaine (304 m/z)

Clofarabine Chlorambucil Flumazenil

Pipemidic acid Ezogabine

Phenoxybenzamine Fenoterol

7-hydroxytodolac Alpha-noroxycodol

Scopolamine Hydromorphinol

Vildagliptin

Methadone (310 m/z)

Fluoxetine Lamivudine-monophosphate

2,8-bis-trifluoromethyl-4-quinoline carboxylic acid

3-oxobrimonidine

19

Hydroxylumiracoxib Glycodiazine

Ketoifen Metixene Nadolol

Metipranolol Diphenodol Diclomine

Oxycodone (316 m/z)

Clonazepam Bromazepam

Efavirenz Chlorprothixene Codeine N-oxide

Rotigotine Alizapride Mitiglinide Saxagliptin

6-monoacetylmorphine (328 m/z)

Diloxanide Acetominophen glucuronide

Desethylamodiaquine Naloxone

Dimenoxadol Butorphanol

Norelgestromin Butorphanol

Fentanyl (337 m/z)

Captopril-cysteine disulfide Berberine

Acebutolol Acetyl-alpha-methylfentanyl

To determine the if the presence of other drugs in each class have an effect on the

signal, four mixtures were prepared and analyzed in ten different authentic urine samples.

Mixtures 1-10A contained 500 ng/mL of amphetamine, methamphetamine, MDMA, and

MDA (Table 8). Mixtures 1-10B contained 200 ng/mL of cocaine and benzoylecgonine

(Table 9). Mixtures 1-10C contained 1,000 ng/mL of morphine, 6-AM, oxycodone,

hydrocodone, codeine, fentanyl, norfentanyl, methadone, and EDDP (Table 10). Mixtures

1-10D contained 100 ng/mL of fentanyl, norfentanyl, methadone, and EDDP (Table 11).

20

When compared to the individual drugs, the average signal intensity for amphetamine

and methamphetamine decreased in the mixture. In addition, MDMA and MDA were

only detected in 5 of the 10 urine samples tested. This indicates that the drugs are likely

interfering with each other resulting in ion suppression.

Table 8. Signal intensities for each of the ions monitored for amphetamine, methamphetamine, MDMA, and MDA in mixtures 1-10A.

Drug Ion 1A 2A 3A 4A 5A 6A 7A 8A 9A 10A Average signal

Amphetamine 136.1 2E5 ND 2E5 1.5E5 2.7E5 1.6E5 1.5E5 2.1E5 1.9E5 2E5 1.92E5

Methamphetamine 150.1 1.8E5 3.2E5 1.5E5 ND 2.2E5 2.4E5 1.4E5 2.6E5 1.4E5 3.1E5 2.18E5

MDMA 194.1 1.2E5 ND 8E4 ND ND 1.1E5 1.3E5 ND ND 1.5E5 1.18E5

MDA 180.0 ND ND 1.4E5 ND ND 1.9E5 2.6E5 2E5 ND 3E5 2.38E5

MDMA/MDA 163.1 2.1E5 3.2E5 1.4E5 1.9E5 1.8E5 3.3E5 1.6E5 1.9E5 3E5 3.6E5 2.38E5

ND-Not detected.

For mixture B, no significant difference in signal intensity was observed when

compared to the average signal intensity for cocaine and benzoylecgonine individually.

Therefore, it does not appear that these two compounds interfere with each other.

Table 9. Signal intensities for each of the ions monitored for cocaine and benzoylecgonine in mixtures 1-10B.

Drug Ion 1B 2B 3B 4B 5B 6B 7B 8B 9B 10B Average signal

Cocaine 304.2 ND ND 5E5 2.6E5 3.5E5 1.4E5 ND 3.4E5 3.5E5 2.3E5 3.1E5

Benzoylecgonine 290.0 ND 2.5E5 7.1E5 4.3E5 ND 2E5 1.7E5 ND ND ND 3.52E5 168.1 1.7E5 1.5E5 2E5 1.9E5 3.5E5 ND 1.7E5 1.5E5 3E5 2E5 1.98E5

ND-Not detected.

Regarding mixture C, the data shows that some of the drugs are affected by the

presence of other compounds in the mixture. Hydrocodone and codeine both have the

same parent ion, so they are intra-target interferences and cannot be distinguished by this

method. Oxycodone was only detected in four out of 10 of the samples, which indicates

that the other compounds are interfering with the ionization and detection of this analyte.

21

In addition, the average signal intensity of morphine in mixture C was significantly less

than the average signal intensity of morphine alone. The signal intensities for fentanyl,

norfentanyl, methadone, and EDDP were also lower than expected at a concentration of

1,000 ng/mL, indicating that some ion suppression could be occurring.

Table 10. Signal intensities for each of the ions monitored in mixtures 1-10C.

Drug Ion 1C 2C 3C 4C 5C 6C 7C 8C 9C 10C Average signal

Morphine 286.1 3.8E5 7.1E5 3.9E5 6.5E5 1E6 7.4E5 3.2E5 7.5E5 8.7E5 3.6E5 6.17E5

6-AM 328.2 1.7E5 ND 1.3E5 2.4E5 2.3E5 1.9E5 1.7E5 3.2E5 2.4E5 1.1E5 2E5

Oxycodone 316.1 1.4E5 ND ND 1.4E5 ND 1.6E5 1.7E5 ND ND ND 1.53E5

Hydrocodone/Codeine 300.2 1.3E5 ND ND 2.1E5 1.7E5 2.3E5 2.4E5 ND 4.6E5 4E5 2.63E5

Fentanyl 337.3 3.8E5 2.9E5 6.3E5 8.5E5 3.3E5 1E6 2E5 4E5 1.1E6 8.9E5 6.07E5

Norfentanyl 233.1 1.8E5 3E5 1.7E5 1.3E5 1.5E5 2.1E5 ND ND 1.9E5 1.4E5 1.84E5

Methadone 310.2 1.3E6 7.9E5 2.1E6 3.4E6 1.2E6 2.5E6 6E5 1.5E6 2.2E6 2.1E6 1.77E6

265.0 9.8E6 6.1E5 9.3E5 1.7E6 1.2E6 1.8E6 4.1E5 1.2E6 1.8E6 1.9E6 1.25E6

EDDP 278.1 1E6 1E6 1.6E6 2.9E6 1.2E6 3.4E6 4.9E5 1.9E6 3.2E6 2.6E6 1.93E6

Regarding mixture D, all four compounds displayed a decreased signal intensity

at 100 ng/mL when analyzed as a mixture. Therefore, these compounds may interfere

with each other when present in a mixture.

Table 11. Signal intensities for each of the ions monitored in mixtures 1-10D.

Drug Ion 1D 2D 3D 4D 5D 6D 7D 8D 9D 10D Average signal

Fentanyl 337.3 ND ND 1.7E5 ND ND 2.3E

5 ND 1.5E5

2.7E5

3.1E5 2.26E5

Norfentanyl 233.1 ND 1.5E5

1.9E5

1.5E5

2.2E5

2.1E5

1.8E5 ND ND 1.3E

5 1.76E5

Methadone 310.2 3.3

E5 2E5 6E5 4.5E5

3.5E5

5.8E5

2.9E5

7.9E5

8.4E5

6.5E5 5.08E5

265.0 6.2E5

3.9E5

4.2E5

3.6E5

5.8E5

6.7E5

3.6E5

6.2E5

5.1E5

6.5E5 5.18E5

EDDP 278.1 1.6E5 ND 2.2E

5 ND ND 6E5 1.6E5

4.7E5 6E5 6.6E

5 4.1E5

22

4. Blind testing and EMIT results

Five urine samples were prepared for screening with our DUS method and labeled

cases 1 through 5. The same procedure was followed with the case samples as with the

other authentic urine samples. Three spots were prepared for each case and the average

ion intensity was compared to the established cut-off intensity in order to make a

determination of positive or negative. If the average peak intensity for the unknown case

sample was above the cut-off intensity, the sample was designated as positive for that

particular substance. For the compounds that displayed fragmentation, an additional

requirement for making a determination of positive was that both the parent and product

ions be detected.

For Case 1, the results indicated that the sample was positive for amphetamine,

methamphetamine, and benzoylecgonine (Table 12). For Cases 2 and 3, none of the

analytes displayed a peak intensity above the cut-off value so they were designated as

negative for all analytes (Tables 13 and 14). For Case 4, the results indicated that the

sample was positive for MDMA (Table 15). For case 5, the results indicated that the

sample was positive for methadone and EDDP (Table 16).

Table 12. Results obtained for Case 1.

Drug Ion Case 1 Spot 1

Case 1 Spot 2

Case 1 Spot 3 Average Cut-off

intensity Result

Morphine 286.1 2E6 1.3E6 1.0E6 1.43E6 1.75E6 N 6-monoacetylmorphine 328.2 1.8E5 ND 1.6E5 1.7E5 2.33E5 N

Oxycodone 316.1 ND ND ND - 2.20E5 N Hydrocodone/Codeine 300.2 ND ND ND - 2.19E5 N

Fentanyl 337.3 ND ND ND - 6.92E5 N Norfentanyl 233.1 1.8E5 1.6E5 1.5E5 1.63E5 2.84E5 N

Methadone 310.2 2.4E5 1.8E5 ND 2.1E5 2.03E6 N 265.0 1.1E6 8.0E5 7.7E5 8.9E5 1.32E6 EDDP 278.1 ND ND ND - 1.52E6 N

Amphetamine 136.1 3.7E5 3.8E5 4.4E5 3.97E5 3.68E5 P

23

Methamphetamine 150.1 4.2E5 4.3E5 4.1E5 4.2E5 3.63E5 P MDMA 194.1 ND ND ND - 1.18E5 N MDA 180.0 ND ND ND - 2.38E5 N

MDMA/MDA 163.1 2.7E5 2.4E5 2.6E5 2.57E5 2.63E5 Cocaine 304.2 ND ND ND - 2.10E5 N

Benzoylecgonine 290.0 3.3E5 2.3E5 2.1E5 2.57E5 2.41E5 P 168.1 2.4E5 2.1E5 1.5E5 2.0E5 1.65E5 ND-not detected; N-negative; P-positive



Case 2 Spot 2


intensity Result

Morphine 286.1 1.1E6 9.2E5 7.7E5 9.3E5 1.75E6 N 6-monoacetylmorphine 328.2 ND ND ND - 2.33E5 N


Fentanyl 337.3 ND ND ND - 6.92E5 N Norfentanyl 233.1 1.4E5 ND ND - 2.84E5 N

Methadone 310.2 ND ND ND - 2.03E6 N 265.0 5.2E5 3.9E5 3.5E5 4.2E5 1.32E6 EDDP 278.1 ND ND ND - 1.52E6 N

Amphetamine 136.1 2.7E5 1.9E5 1.7E5 2.1E5 3.68E5 N Methamphetamine 150.1 3.1E5 ND ND - 3.63E5 N

MDMA 194.1 ND ND 1.5E5 - 1.18E5 N MDA 180.0 2.2E5 2.0E5 2.4E5 2.1E5 2.38E5 N

MDMA/MDA 163.1 2.1E5 ND ND - 2.63E5 Cocaine 304.2 ND ND ND - 2.10E5 N

Benzoylecgonine 290.0 2.2E5 1.8E5 1.8E5 1.93E5 2.41E5 N 168.1 1.7E5 ND ND - 1.65E5 ND-not detected; N-negative



Case 3 Spot 2


intensity Result


Oxycodone 316.1 ND ND ND - 2.20E5 N Hydrocodone/Codeine 300.2 ND ND ND ND 2.19E5 N

Fentanyl 337.3 ND ND ND ND 6.92E5 N Norfentanyl 233.1 1.3E5 1.3E5 ND 1.3E5 2.84E5 N

Methadone 310.2 ND ND ND ND 2.03E6 N 265.0 4.7E5 5.8E5 5.2E5 5.23E5 1.32E6 EDDP 278.1 ND ND ND - 1.52E6 N

Amphetamine 136.1 3.0E5 3.2E5 2.1E5 2.77E5 3.68E5 N Methamphetamine 150.1 3.4E5 2.9E5 1.8E5 2.70E5 3.63E5 N

MDMA 194.1 ND ND ND - 1.18E5 N MDA 180.0 1.6E5 1.6E5 1.6E5 1.6E5 2.38E5 N


24

Benzoylecgonine 290.0 2.4E5 2.3E5 2.3E5 2.33E5 2.41E5 N 168.1 1.5E5 1.6E5 1.8E5 1.63E5 1.65E5 ND-not detected; N-negative.



Case 4 Spot 2


intensity Result

Morphine 286.1 9.8E5 7.2E5 8.3E5 8.43E5 1.75E6 N 6-monoacetylmorphine 328.2 ND ND 1.6E5 - 2.33E5 N


Fentanyl 337.3 ND ND ND - 6.92E5 N Norfentanyl 233.1 1.3E ND ND - 2.84E5 N

Methadone 310.2 2.8E5 ND ND - 2.03E6 N 265.0 2.6E5 3.9E5 4.6E5 3.7E5 1.32E6 EDDP 278.1 3.1E5 ND ND - 1.52E6 N

Amphetamine 136.1 3.7E5 1.7E5 2.3E5 2.57E5 3.68E5 N Methamphetamine 150.1 2.6E5 ND 1.9E5 2.25E5 3.63E5 N

MDMA 194.1 ND 1.2E5 1.3E5 1.25E5 1.18E5 P MDA 180.0 ND 1.6E5 2.0E5 1.80E5 2.38E5 N

MDMA/MDA 163.1 1.9E5 2.1E5 2.6E5 2.2E5 2.63E5 Cocaine 304.2 ND ND 2.2E5 - 2.1E5 N

Benzoylecgonine 290.0 2.2E5 1.7E5 1.8E5 1.9E5 2.41E5 N 168.1 2.0E5 1.4E5 1.9E5 1.77E5 1.65E5 ND-not detected; N-negative; P-positive.



Case 5 Spot 2


intensity Result



Fentanyl 337.3 ND ND ND - 6.92E5 N Norfentanyl 233.1 1.3E5 1.7E5 ND 1.63E5 2.84E5 N

Methadone 310.2 2.8E6 2.9E6 2.9E6 2.87E5 2.03E6 P 265.0 2.6E6 2.7E6 3.0E6 2.77E6 1.32E6 EDDP 278.1 3.1E6 3.2E6 3.9E6 3.4E6 1.52E6 P

Amphetamine 136.1 3.7E5 3.5E5 2.4E5 3.3E5 3.68E5 N Methamphetamine 150.1 2.6E5 3.0E5 1.7E5 2.43E5 3.63E5 N

MDMA 194.1 ND ND ND - 1.18E5 N MDA 180.0 ND 1.9E5 ND - 2.38E5 N


Benzoylecgonine 290.0 2.2E5 2.0E5 2.1E5 2.1E5 2.41E5 N 168.1 2.0E5 1.8E5 2.0E5 1.93E5 1.65E5 ND-not detected; N-negative; P-positive.

25

After employing the screening assay to test case samples 1 through 5, EMIT was

performed on each sample (Table 17), and the results of the immunoassay screen were

compared to the results of the DUS screen we developed, and to the actual results (Table

18).

Table 17. Results of EMIT screening of cases 1-5

EMIT ASSAY Results

Case 1 Case 2 Case 3 Case 4 Case 5

Amphetamines N N N N N

Cocaine N N P P N

Methadone N N N N P

Opiates N P N N N

Table 18. Summary of screening results compared to actual drug composition of sample.

Amphetamine (A); methamphetamine (MA); benzoylecgonine (BE); hydrocodone (HC);

oxycodone (OC).

Case DUS screen results EMIT Results Actual

1 Positive for A Positive for MA Positive for BE

Negative for all tests Positive for A Positive for MA

2 Negative for all analytes Positive for opiates Positive for morphine Positive for HC

3 Negative for all analytes Positive for cocaine Positive for BE

4 Positive for MDMA Positive for cocaine Positive for BE Positive for MDMA

5 Positive for methadone Positive for EDDP Positive for methadone

Positive for methadone Positive for EDDP Positive for OC

Regarding our DUS screening method, Case 1 was actually positive for

amphetamine and methamphetamine. Therefore, the detection of amphetamine and

26

methamphetamine were true positives but the detection of benzoylecgonine was a false

positive. The Case 2 sample contained morphine and hydrocodone at 500 ng/mL. Five-

hundred ng/mL is below the LOD for hydrocodone in our method, so it was not detected

and, as described earlier, the endogenous interference(s) at 286 m/z complicate the

interpretation of results for morphine. The Case 3 sample contained benzoylecgonine at

500 ng/mL. However, the peak intensities for both of the ions of interest, while close to

the cut-off, were below the cut-off intensity and a determination of negative was made.

The Case 4 sample contained benzoylecgonine and MDMA at 500 ng/mL. The results

indicated a positive result for MDMA but the peak intensities for the benzoylecgonine

peaks were below the cut-off, so it was designated as negative for that substance. The

Case 5 sample contained 500 ng/mL of methadone, EDDP, and oxycodone. The results of

the screening method indicated a positive result for methadone and EDDP, but were

negative for oxycodone. Five-hundred ng/mL is below the LOD for oxycodone in

authentic urine for this method, and therefore, it was not detected. Overall, the DUS

screening method developed resulted in 5 true positives, 59 true negatives, 1 false

positive, and 5 false negatives.

Regarding the EMIT screening results, for the case 1 sample, the EMIT results

indicated that the sample was negative for amphetamines, cocaine, methadone and

opiates, even though actually contained both amphetamine and methamphetamine. This

false negative is due to the concentration of the analytes. The amphetamines EMIT screen

was performed at cut-off concentration of 1,000 ng/mL, which is based on the signal

produced by a 1,000 ng/mL d,l-amphetamine calibrator. Amphetamine and

methamphetamine were present in the case 1 sample at a concentration below the cut-off

27

concentration and therefore were not detected. The EMIT results for Case 2 showed that

the sample was positive for opiates and the sample did contain morphine and

hydrocodone. Using a cut-off concentration of 300 ng/mL, morphine will be detected by

the opiate EMIT screen at a concentration of 300 ng/mL (morphine is used as the

calibrator) and hydrocodone will be detected at a concentration of 247 ng/mL (Siemens,

2017). This result is a true positive. However, the EMIT opiate assay is not specific

enough to indicate which opiates are actually present in the sample. For Case 3, the

EMIT results indicated that the sample was positive for cocaine. The assay actually

detects the presence of the cocaine metabolite, benzoylecgonine, which was present.

Therefore, this result was a true positive. For Case 4, the EMIT results indicated that the

sample was positive for cocaine metabolite. However, it was unable to detect MDMA.

According to Siemens (2017), MDMA will produce a positive result in the amphetamine

assay at a concentration of 34,300 ng/mL when using a cut-off concentration of 1,000

ng/mL. The extremely high concentration of MDMA required to produce a positive

signal resulted in a false negative. For Case 5, the EMIT results indicated that the sample

was positive for methadone, which was accurate. However, the EMIT methadone assay is

specific for methadone only, thus, EDDP was not detected. The results showed that the

sample was negative for opiates as well, despite the presence of oxycodone. At a cut-off

concentration of 300 ng/mL, oxycodone will be detected in the opiate screen at a

concentration of 1,500 ng/mL. Since the concentration of oxycodone in the Case 5

sample was 500 ng/mL, it was not detected, and this resulted in a false negative. Overall,

the EMIT screening method resulted in 4 true positives, 13 true negatives, 0 false

positives, and 3 false negatives.

28

When comparing the results of our DUS screening method to the results of the

EMIT assays, the advantages and drawbacks of each method are highlighted. In general,

our DUS screening method is more specific than the EMIT assays. Among the four

EMIT assays we used, only the methadone and cocaine metabolite assays are specific for

a particular analyte. The opiate assay can produce a positive result for numerous natural

and semi-synthetic opioids. However, as seen with oxycodone, they do not all produce a

positive result at the cut-off concentration. According to Siemens’ cross-reactivity data

(2017), when using the 300 ng/mL cut-off level, 6-AM, codeine, hydrocodone, and

oxycodone will give an assay response equal to the morphine cut-off calibrator at a

concentration of 435, 102-306, 247, and 1,500 ng/mL, respectively. Thus, the opiate

assay is not equally sensitive for all opiates. Furthermore, the opiate assay does display

cross reactivity with some non-opiate drugs such as the antibiotics ofloxacin and

levofloxacin and the emergency opiate antagonist naloxone. In contrast, the DUS method

that we developed can distinguish between the different opiates, with the exception of

isobaric compounds such as hydrocodone and codeine. Also, EMIT is more sensitive for

morphine, 6-AM, codeine and hydrocodone but our method is more sensitive for

oxycodone. Another disadvantage of the EMIT opiate assay is that it does not detect

synthetic opiates and their metabolites such as fentanyl, norfentanyl, methadone, and

EDDP, which are compounds that our DUS method is highly sensitive for.

When comparing the DUS method with EMIT screening for amphetamines, our

method appears to be superior at the 1,000 ng/mL EMIT cut-off level. First, our DUS

method is more sensitive for amphetamine, methamphetamine, MDA, and MDMA than

the EMIT assay. All four amphetamine compounds assayed were detectable at 500

29

ng/mL using the DUS method. For the EMIT amphetamine assay, methamphetamine,

MDA, and MDMA give an assay response equal to the 1,000 ng/mL amphetamine cut-

off calibrator at 2100, 6500, and 34,300 ng/mL, respectively (Siemens, 2017). Just as

with the opiate assay, the EMIT amphetamine assay is not equally sensitive for all of the

amphetamines and very high urine concentrations are required to produce a signal above

the cut-off level for MDA and MDMA. In addition, the amphetamine assay also displays

cross-reactivity with substances that are not amphetamines such as the antidepressant

bupropion, the anti-malarial drugs chloroquine and quinacrine, the Alzheimer’s drug

donepezil, the heart medication mexiletine, the beta-blocker propranolol, and the MAO-B

inhibitor selegiline (Siemens, 2017). The greater sensitivity and specificity for the

amphetamine drugs tested in the developed DUS screening method appear to indicate that

our method is superior for the detection of amphetamines compared to the EMIT assay.

Based on the failure to accurately detect and identify benzoylecgonine in the DUS

samples using the developed screening method, the EMIT cocaine metabolite assay

appears to be the superior method to screen for cocaine use. When analyzing the

unfortified authentic urine samples, possible interferences were detected for the

benzoylecgonine ions of interest more often than not. Despite being able to accurately

detect cocaine with our method, it is too unreliable to make determinations about the

presence of benzoylecgonine in urine. In addition, the EMIT cocaine assay also provides

an assay response equal to the 300 ng/mL benzoylecgonine calibrator for cocaine at 40-

119 ng/mL and ecgonine at 7-20 ng/mL. No other compounds were reported to display

cross-reactivity for this assay (Siemens, 2017). Therefore, the EMIT cocaine metabolite

30

assay is more reliable for the detection of benzoylecgonine and more sensitive for

cocaine as compared to our DUS method.

Finally, our DUS screening method appears to be superior to the EMIT

methadone assay. The EMIT methadone assay is specific to methadone only and does not

detect the metabolite EDDP. Our screening method was consistently able to detect

methadone and EDDP in the fortified authentic urine and case 5 samples. The ability to

detect both methadone and EDDP in urine may be useful for certain applications such as

monitoring methadone compliance (George & Braithwaite, 1999). In addition, our DUS

screening method is more sensitive for methadone than the EMIT assay. The LOD for

methadone and EDDP for the DUS screening method was determined to be 100 ng/mL

while the cut-off level for EMIT methadone assay is 300 ng/mL. The higher sensitivity

and ability to detect EDDP in addition to methadone are advantageous features of our

DUS screening method as compared to traditional EMIT screening.

While both our DUS method and EMIT screening have their advantages and

disadvantages, our method has the major advantage of easily incorporating new analytes

into the assay. EMIT and other immunoassays rely on antibody-antigen interactions

(Hoofnagle & Wener, 2009). Therefore, development a new EMIT assay requires the

development of new antibodies specific for a particular drug or drug class. This is

difficult, costly, and extremely time consuming (Hoofnagle & Wener, 2009). Therefore,

mass spectrometry based screening methods, such as the screening method presented

here, may offer a solution to the problems associated with immunoassay screening

including a lack of specificity, cross-reactivity, and difficulty adding new targets.

31

Conclusion

Drug screening is an essential part of toxicological analysis, but it can be time

consuming and costly. Direct sampling of dried matrix spots and analysis by mass

spectrometry provides an alternative method for drug screening that is simple, fast, and

cost effective. We developed a method for the direct analysis of DUS samples using a

TLC-CMS interface for opioids, amphetamines and cocaine screening. While the DUS

method developed and described in this work does have potential, it also has

shortcomings. When using the TLC-CMS interface no chromatography is used so all

compounds are subjected to mass spectrometric analysis simultaneously. This problem is

exacerbated by direct analysis because urinary matrix components are also present in the

sample. The inability to distinguish analytes of interest from urinary matrix components

can lead to false positives, especially at lower concentrations. However, it does have

certain advantages over traditional immunoassay screening methods such as EMIT

because it has the potential to give more specific results rather than giving a result for a

broad group of drugs, such as opioids. In addition, new drugs and metabolites can easily

be added to our method and validated. It is difficult and time consuming to develop a new

immunoassay and validate it because these tests rely on the development of specific

antibodies to bind each drug. This makes our DUS superior for screening for drugs like

fentanyl and its metabolite norfentanyl, for which there is no commercially available

EMIT assay kit.

The sensitivity and specificity of the DUS method presented could be improved

by introducing a sample pre-treatment step, chromatographic separation, or employing a

high resolution mass spectrometer. However, all of these would increase sample

32

preparation and analysis time and high-resolution mass spectrometers are much larger

than the CMS system used in this work. Since the method described in this work is a

screening method, all results need to be followed by a confirmatory analysis by GC-MS

or LC-MS/MS so further investigation and optimization of instrumental parameters

should be conducted in order to decrease the rate of false positives and negatives. Despite

these shortcomings, direct sampling of DMS coupled to mass spectrometry has great

promise for the future of toxicological screening.

33

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37

Appendix

Figure A1. Mass spectrum of 6-monoacetylmorphine in methanol at 100 ng/mL.

Figure A2. Mass spectrum of 6-monoacetylmorphine in synthetic urine at 250 ng/mL.

Figure A3. Mass spectrum of 6-monoacetylmorphine in authentic urine at 500 ng/mL.

Figure A4. Mass spectrum of amphetamine in methanol at 100 ng/mL.

2E9

4E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00 2.33

Min

TIC Filtered 2019_3_20_6MAM0.25 2019.03.20 14:23:48 6 MAM (0.25 ug/mL) on DBS card;ESI +

100.0%; 328.1

0

50

Intensity

%

100 200 300 400 500

m/z

Spectrum RT 0.00 - 2.61 {317 scans} 2019_3_20_6MAM0.25 2019.03.20 14:23:48 6 MAM (0.25 ug/mL) on DBS card;ESI + Max: 7.8E6

328.2144.9

146.9369.2180.9

329.2196.0154.9 284.3 344.2

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered 6-MAM 0.5_Scan1 2019.04.05 12:15:43 scan MeOH;ESI +

91.8%; 441.4

4.4%; 179.03.8%; 135.9

500,000

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered 6-MAM 0.5_SIM2 2019.04.05 12:15:43 scan MeOH;ESI +

95.2%; 328.1

4.3%; 328.10.5%; 328.1

10

20

Intensity

%

315 320 325 330 335 340

m/z

Spectrum RT 0.00 - 0.94 {65 scans} 6-MAM 0.5_Scan1 2019.04.05 12:15:43 scan MeOH;ESI + Max: 1.4E6

335.2311.2

323.3 337.2

331.2315.0 336.2325.1 328.2 341.2

0

50

Intensity

%

327.4 327.6 327.8 328 328.2 328.4 328.6 328.8

m/z

Spectrum RT 0.01 - 0.96 {66 scans} 6-MAM 0.5_SIM2 2019.04.05 12:15:43 scan MeOH;ESI + Max: 2.6E5

328.1

2E9

4E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00

Min

TIC Filtered 04162019_6-MAM 10-2_Scan1 2019.04.16 10:42:50 6-MAM (10 ug/mL) authentic urine;ESI +

93.1%; 365.1

5.8%; 155.11.1%; 154.9

1E6

2E6

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00

Min

TIC Filtered 04162019_6-MAM 10-2_SIM2 2019.04.16 10:42:50 6-MAM (10 ug/mL) authentic urine;ESI +

99.0%; 328.1

0.5%; 369.10.2%; 369.1 0.2%; 369.1 0.1%; 369.1

0

20

Intensity

%

315 320 325 330 335

m/z

Spectrum RT 0.00 - 2.16 {174 scans} 04162019_6-MAM 10-2_Scan1 2019.04.16 10:42:50 6-MAM (10 ug/mL) authentic urine;ESI + Max: 6.3E5

337.1

314.3

316.3 330.2328.3322.1 327.2315.1

0

50

Intensity

%

310 320 330 340 350 360 370 380

m/z

Spectrum RT 0.01 - 2.18 {175 scans} 04162019_6-MAM 10-2_SIM2 2019.04.16 10:42:50 6-MAM (10 ug/mL) authentic urine;ESI + Max: 1.9E5

328.1

369.1

0E0

5E9

10E9

Intensity

0.00 0.33 0.67 1.00 1.33 1.67 2.00

Min

ΔIC Filtered 2019_3_11_amp_neat 2019.03.11 13:27:31 Neat amp on DBS card;ESI +

98.1%; 91.11.9%; 140.0 0.0%; 154.9

0

50

Intensity

%

50 100 150 200 250 300 350 400 450

m/z

ΔS 1.47 - 1.60 {16 scans} 2019_3_11_amp_neat 2019.03.11 13:27:31 Neat amp on DBS card;ESI + Max: 1.3E2

91.0

132.0154.9 352.292.1 380.3136.0 247.1119.1

38

Figure A5. Mass spectrum of amphetamine in synthetic urine at 250 ng/mL.

Figure A6. Mass spectrum of amphetamine in authentic urine at 500 ng/mL.

Figure A7. Mass spectrum of benzoylecgonine in methanol at 100 ng/mL.

Figure A8. Mass spectrum of benzoylecgonine in synthetic urine at 100 ng/mL.

5E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00

Min

TIC Filtered amp 0.5_Scan1 2019.04.05 11:59:15 scan MeOH;ESI +

87.1%; 365.2

12.9%; 337.1

1E6

2E6

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00

Min

TIC Filtered amp 0.5_SIM2 2019.04.05 11:59:16 scan MeOH;ESI +

35.8%; 136.133.6%; 136.1 30.6%; 91.1

5

10

Intensity

%

100 110 120 130 140 150

m/z

Spectrum RT 0.00 - 1.02 {62 scans} amp 0.5_Scan1 2019.04.05 11:59:15 scan MeOH;ESI + Max: 2.4E6

150.9149.0

146.9136.0

124.0 131.0150.0

98.9 153.0119.0

0

50

Intensity

%

80 90 100 110 120 130 140 150

m/z

Spectrum RT 0.01 - 1.04 {63 scans} amp 0.5_SIM2 2019.04.05 11:59:16 scan MeOH;ESI + Max: 1E6

136.1

91.1

119.1

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

TIC Filtered 0506_AMP 0.1-3 2019.05.07 11:50:15 authentic urine 3;ESI +

87.6%; 102.1

12.4%; 151.1

5

10

Intensity

%

125 130 135 140 145

m/z

Spectrum RT 0.00 - 1.25 {158 scans} 0506_AMP 0.1-3 2019.05.07 11:50:15 authentic urine 3;ESI + Max: 1.2E6

143.1145.0

130.2

147.0124.1121.1

136.0127.1 141.1 144.1

10E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00

Min

TIC Filtered 03052019_BE 2019.03.05 12:40:12 ;ESI +

95.7%; 290.1

3.1%; 144.90.4%; 290.1

0.4%; 144.90.4%; 144.9

0

50

Intensity

%

100 200 300 400 500 600

m/z

Spectrum RT 0.00 - 2.28 {277 scans} 03052019_BE 2019.03.05 12:40:12 ;ESI + Max: 1.8E7

290.1

168.0144.9

353.2146.9181.0 247.2 346.4

231.1 291.2

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

Chromatogram TIC Filtered syn urine BE 2019.07.03 11:47:38 syn urine;ESI +

93.0%; 239.2

7.0%; 144.9

10

20

Intensity

%

280 282 284 286 288 290 292 294

m/z

Spectrum RT 0.00 - 1.47 {183 scans} syn urine BE 2019.07.03 11:47:38 syn urine;ESI + Max: 3.4E6

285.9

287.9

284.2282.1 287.0 290.0289.0283.1 292.0

39

Figure A9. Mass spectrum of benzoylecgonine in authentic urine at 200 ng/mL.

Figure A10. Mass spectrum of cocaine in methanol at 100 ng/mL.

Figure A11. Mass spectrum of cocaine in synthetic urine at 100 ng/mL.

Figure A12. Mass spectrum of cocaine in authentic urine at 200 ng/mL.

Figure A13. Mass spectrum of codeine in methanol at 500 ng/mL.

2E9

4E9

Intensity

c/s

0.00 0.08 0.17 0.25 0.33 0.42 0.50 0.58 0.67 0.75

Min

TIC Filtered 0506_BE 10-3 2019.05.07 12:12:08 authentic urine 3;ESI +

90.5%; 114.1

9.5%; 144.9

10

20

Intensity

%

285 290 295 300

m/z

Spectrum RT 0.00 - 0.78 {102 scans} 0506_BE 10-3 2019.05.07 12:12:08 authentic urine 3;ESI + Max: 6.7E5

286.1

302.3

282.2 297.3 299.3

288.1284.3 287.2 290.1 301.3

20E9

40E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

TIC Filtered 2019_3_11_cocaine_neat 2019.03.11 13:13:25 Neat cocaine on DBS card;ESI +

98.5%; 304.1

0.8%; 154.9 0.6%; 144.9

0

50

Intensity

%

100 150 200 250 300 350 400 450

m/z

Spectrum RT 0.00 - 1.41 {172 scans} 2019_3_11_cocaine_neat 2019.03.11 13:13:25 Neat cocaine on DBS card;ESI + Max: 1.5E8

304.1

182.0

305.1144.9 183.1154.9 180.9146.9 196.0 272.1

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered syn urine cocaine 2019.07.03 11:45:45 syn urine;ESI +

75.8%; 311.1

24.2%; 144.9

5

Intensity

%

296 298 300 302 304 306 308

m/z

Spectrum RT 0.00 - 0.82 {103 scans} syn urine cocaine 2019.07.03 11:45:45 syn urine;ESI + Max: 7.9E5

305.1

304.1

299.1296.2 302.1 309.2

2E9

4E9

6E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33 1.50

Min

TIC Filtered 0430_cocaine 1 2019.04.30 13:04:11 blank syn urine;ESI +

100.0%; 365.1

20

Intensity

%

275 280 285 290 295 300 305 310 315 320

m/z

Spectrum RT 0.00 - 1.59 {207 scans} 0430_cocaine 1 2019.04.30 13:04:11 blank syn urine;ESI + Max: 1.8E6

285.9

287.9

304.1302.1287.0275.1 289.0 312.2284.2 299.1

1E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

TIC Filtered 2019_3_25_codeine_10 2019.03.25 11:40:44 codeine std (10 ug/mL) on DBS card;ESI +

50.0%; 154.9

40.8%; 152.0

9.2%; 154.9

0

50

Intensity

%

100 150 200 250 300 350 400 450

m/z

Spectrum RT 0.00 - 1.25 {152 scans} 2019_3_25_codeine_10 2019.03.25 11:40:44 codeine std (10 ug/mL) on DBS card;ESI + Max: 5.3E6

144.9

146.9154.9 180.9300.1195.9

139.9130.1 284.3157.0

40

Figure A14. Mass spectrum of codeine in synthetic urine at 500 ng/mL.

Figure A15. Mass spectrum of codeine in authentic urine at 1000 ng/mL.

Figure A16. Mass spectrum of EDDP in methanol at 100 ng/mL.

Figure A17. Mass spectrum of EDDP in synthetic urine at 100 ng/mL.

Figure A18. Mass spectrum of EDDP in authentic urine at 100 ng/mL.

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00

Min

Chromatogram TIC Filtered syn urine codeine 2019.07.03 12:03:12 syn urine;ESI +

51.7%; 144.9

48.3%; 311.0

5

10

Intensity

%

290 295 300 305 310

m/z

Spectrum RT 0.00 - 1.11 {139 scans} syn urine codeine 2019.07.03 12:03:12 syn urine;ESI + Max: 1.2E6

287.9311.1

305.1

289.9286.9 299.1288.9 293.1 312.1300.1

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

Chromatogram TIC Filtered codeine 1_Scan1 2019.06.10 12:10:27 authentic urine @ 1ug/mL;ESI +

97.2%; 155.1

1.2%; 144.9 1.1%; 181.0 0.5%; 145.1

2E6

4E6

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

Chromatogram TIC Filtered codeine 1_SIM2 2019.06.10 12:10:27 authentic urine @ 1ug/mL;ESI +

98.8%; 300.2

0.3%; 300.20.3%; 300.20.2%; 300.20.1%; 300.2 0.1%; 300.2 0.1%; 300.2

10

20

Intensity

%

294 296 298 300 302 304 306 308

m/z

Spectrum RT 0.00 - 1.15 {128 scans} codeine 1_Scan1 2019.06.10 12:10:27 authentic urine @ 1ug/mL;ESI + Max: 1E6

302.2

300.2306.1297.1296.2 303.2

0

50

Intensity

%

299.6 299.8 300 300.2 300.4 300.6 300.8

m/z

Spectrum RT 0.01 - 1.19 {131 scans} codeine 1_SIM2 2019.06.10 12:10:27 authentic urine @ 1ug/mL;ESI + Max: 3.8E5

300.2

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00

Min

TIC Filtered eddp std 100 2019.07.03 11:21:06 stds at LOD;ESI +

88.2%; 325.2

5.3%; 152.0 3.7%; 144.91.6%; 144.91.1%; 144.9

50

Intensity

%

150 200 250 300 350 400

m/z

Spectrum RT 0.00 - 1.04 {129 scans} eddp std 100 2019.07.03 11:21:06 stds at LOD;ESI + Max: 1.7E7

144.9

146.9

185.9325.3188.0 297.2151.0 261.0152.0 278.1

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

TIC Filtered 0430_EDDP 0.1 2019.04.30 12:07:11 blank syn urine;ESI +

94.5%; 454.3

5.5%; 130.1

20

40

Intensity

%

260 265 270 275 280 285 290 295 300 305

m/z

Spectrum RT 0.00 - 1.41 {184 scans} 0430_EDDP 0.1 2019.04.30 12:07:11 blank syn urine;ESI + Max: 1.5E6

285.9262.9

288.0264.9278.2

282.2267.0268.0 299.1261.0

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00

Min

TIC Filtered 0506_EDDP 0.1-4 2019.05.07 13:06:21 authentic urine 3;ESI +

94.9%; 114.1

5.1%; 151.8

50

Intensity

%

150 200 250 300 350 400 450

m/z

Spectrum RT 0.00 - 1.09 {142 scans} 0506_EDDP 0.1-4 2019.05.07 13:06:21 authentic urine 3;ESI + Max: 3.4E6

114.1

143.1

155.1 457.4152.0

429.3278.2 325.4 482.4302.3

41

Figure A19. Mass spectrum of fentanyl in methanol at 100 ng/mL.

Figure A20. Mass spectrum of fentanyl in synthetic urine at 100 ng/mL.

Figure A21. Mass spectrum of fentanyl in authentic urine at 100 ng/mL.

Figure A22. Mass spectrum of hydrocodone in methanol at 500 ng/mL.

Figure A23. Mass spectrum of hydrocodone in synthetic urine at 500 ng/mL.

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered fentanyl std 100 2019.07.03 11:13:22 stds at LOD;ESI +

67.8%; 239.1

19.9%; 144.912.4%; 152.0

1

2

Intensity

%

330 335 340 345

m/z

Spectrum RT 0.00 - 0.89 {111 scans} fentanyl std 100 2019.07.03 11:13:22 stds at LOD;ESI + Max: 3.8E5

337.2

340.2

327.2 341.2

338.3329.1

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered syn urine fentanyl 2019.07.03 12:05:11 syn urine;ESI +

72.7%; 311.1

19.2%; 144.98.0%; 144.9

2

4

Intensity

%

332 334 336 338 340 342 344 346 348

m/z

Spectrum RT 0.00 - 0.82 {103 scans} syn urine fentanyl 2019.07.03 12:05:11 syn urine;ESI + Max: 6.1E5

340.1

337.2

349.1

341.2336.2

338.2 339.1

2E9

4E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33

Min

TIC Filtered 0503_fentanyl 0.1-2 2019.05.03 11:38:59 authentic urine 2;ESI +

94.6%; 144.1

3.8%; 337.41.6%; 151.1

10

20

Intensity

%

330 335 340 345

m/z

Spectrum RT 0.00 - 1.65 {215 scans} 0503_fentanyl 0.1-2 2019.05.03 11:38:59 authentic urine 2;ESI + Max: 6.4E5

337.3

340.2338.3

341.2329.1

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

TIC Filtered 2019_3_25_hydrocodone_0.5 2019.03.25 12:07:17 hydrocodone std (0.5 ug/mL) on DBS card;ESI +

87.6%; 428.3

6.7%; 144.95.7%; 154.9

0

50

Intensity

%

100 150 200 250 300 350 400 450

m/z

Background RT 0.91 - 0.92 {2 scans} 2019_3_25_hydrocodone_0.5 2019.03.25 12:07:17 hydrocodone std (0.5 ug/mL) on DBS card;ESI + Max: 7.4E6

456.3

428.3239.1457.3

429.3144.9

300.1195.9180.9

363.2

1E9

1.5E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered syn urine HC 2019.07.03 12:00:42 syn urine;ESI +

61.1%; 143.1

38.9%; 144.9

5

Intensity

%

295 300 305 310

m/z

Spectrum RT 0.00 - 0.91 {113 scans} syn urine HC 2019.07.03 12:00:42 syn urine;ESI + Max: 7E5

305.1

299.1300.1

293.1

42

Figure A24. Mass spectrum of hydrocodone in authentic urine at 1000 ng/mL.

Figure A25. Mass spectrum of MDA in methanol at 100 ng/mL. Spectrum is zoomed in to show 180.0 m/z peak.

Figure A26. Mass spectrum of MDA in methanol at 100 ng/mL. Spectrum is zoomed in to show 163.0 m/z peak.

Figure A27. Mass spectrum of MDA in synthetic urine at 250 ng/mL. Spectrum is zoomed in to show 180.0 m/z peak.

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered hydrocodone 1_Scan1 2019.06.10 12:00:19 authentic urine @ 1ug/mL;ESI +

87.8%; 155.1

6.2%; 145.1 6.0%; 144.9

2E6

4E6

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

TIC Filtered hydrocodone 1_SIM2 2019.06.10 12:00:19 authentic urine @ 1ug/mL;ESI +

99.5%; 300.2

0.3%; 300.20.2%; 300.2

20

Intensity

%

294 296 298 300 302 304 306 308 310 312

m/z

Spectrum RT 0.00 - 0.94 {105 scans} hydrocodone 1_Scan1 2019.06.10 12:00:19 authentic urine @ 1ug/mL;ESI + Max: 1.2E6

302.3

310.2300.2

304.3303.3299.2296.3

0

50

Intensity

%

299.6 299.8 300 300.2 300.4 300.6 300.8

m/z

Spectrum RT 0.01 - 0.97 {107 scans} hydrocodone 1_SIM2 2019.06.10 12:00:19 authentic urine @ 1ug/mL;ESI + Max: 4.3E5

300.2

1E9

2E9

3E9

Intensity

c/s

0.00 0.08 0.17 0.25 0.33 0.42 0.50 0.58

Min

TIC Filtered MDA std 100 2019.07.03 10:39:25 stds at LOD;ESI +

68.7%; 163.0

19.3%; 144.911.9%; 151.0

10

20

Intensity

%

175 180 185 190

m/z

Spectrum RT 0.00 - 0.60 {66 scans} MDA std 100 2019.07.03 10:39:25 stds at LOD;ESI + Max: 3.5E6

185.9

187.9

171.1

173.0 187.0185.0183.1180.0176.1 182.1

1E9

2E9

3E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

Chromatogram TIC Filtered MDA std 100 2019.07.03 10:39:25 stds at LOD;ESI +

89.0%; 163.0

4.3%; 144.93.5%; 151.0 3.3%; 144.9

10

20

Intensity

%

155 160 165 170 175 180 185 190

m/z

Spectrum RT 0.00 - 1.32 {144 scans} MDA std 100 2019.07.03 10:39:25 stds at LOD;ESI + Max: 2.8E6

185.9151.0

152.0163.0

187.9171.1153.0

155.1

173.0 176.1

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

Chromatogram TIC Filtered syn urine MDA 2019.07.03 11:37:57 syn urine;ESI +

49.2%; 144.9

46.0%; 162.9

4.8%; 144.9

10

20

Intensity

%

180 185 190 195 200

m/z

Spectrum RT 0.00 - 1.33 {165 scans} syn urine MDA 2019.07.03 11:37:57 syn urine;ESI + Max: 2.6E6

185.9

188.0

196.0 198.0180.9 183.0 187.0180.0 189.0 195.0

43

Figure A28. Mass spectrum of MDA in synthetic urine at 250 ng/mL. Spectrum is zoomed in to show 163.0 m/z peak.

Figure A29. Mass spectrum of MDA in authentic urine at 500 ng/mL. Spectrum is zoomed in to show 180.0 m/z peak.

Figure A30. Mass spectrum of MDA in authentic urine at 500 ng/mL. Spectrum is zoomed in to show 163.1 m/z peak.

Figure A31. Mass spectrum of MDMA in methanol at 100 ng/mL.

Figure A32. Mass spectrum of MDMA in synthetic urine at 250 ng/mL. Spectrum is zoomed in to show 194.1 m/z peak.

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

Chromatogram TIC Filtered syn urine MDA 2019.07.03 11:37:57 syn urine;ESI +

49.2%; 144.9

46.0%; 162.9

4.8%; 144.9

5

10

Intensity

%

158 160 162 164 166 168 170 172 174 176

m/z

Spectrum RT 0.00 - 1.33 {165 scans} syn urine MDA 2019.07.03 11:37:57 syn urine;ESI + Max: 1.2E6

163.0

171.1

173.0

165.0160.5 163.9

2E9

4E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67

Min

TIC Filtered 0517_MDA 10-6 2019.05.17 11:56:51 authentic urine 6;ESI +

89.8%; 419.5

5.4%; 152.1

4.8%; 102.1

500,000

Intensity

c/s

176 178 180 182 184 186 188 190 192

m/z

Spectrum RT 0.00 - 1.80 {226 scans} 0517_MDA 10-6 2019.05.17 11:56:51 authentic urine 6;ESI +

177.0

186.0

181.0 183.0 188.0180.1

2E9

4E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67

Min

TIC Filtered 0517_MDA 10-6 2019.05.17 11:56:51 authentic urine 6;ESI +

89.8%; 419.5

5.4%; 152.1

4.8%; 102.1

500,000

Intensity

c/s

158 160 162 164 166 168 170 172

m/z

Spectrum RT 0.00 - 1.80 {226 scans} 0517_MDA 10-6 2019.05.17 11:56:51 authentic urine 6;ESI +

171.1

163.1

160.5157.0 172.15E9

10E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67

Min

TIC Filtered MDMA 2019.04.15 10:52:40 MDMA TLC plate;ESI +

99.0%; 163.1

1.0%; 322.1

0

50

Intensity

%

100 150 200 250 300 350 400 450

m/z

Spectrum RT 0.00 - 1.85 {239 scans} MDMA 2019.04.15 10:52:40 MDMA TLC plate;ESI + Max: 4.6E7

163.0

194.1235.1

164.1322.1195.1145.0 236.2155.1 284.3

5E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00 2.33

Min

TIC Filtered 0430_MDMA_0.1 2019.04.30 11:24:35 blank syn urine;ESI +

95.9%; 318.2

4.1%; 144.8

10

20

Intensity

%

185 190 195 200 205

m/z

Spectrum RT 0.00 - 2.39 {312 scans} 0430_MDMA_0.1 2019.04.30 11:24:35 blank syn urine;ESI + Max: 4.5E5

196.0

185.9 194.1

203.9 206.0198.1

188.0181.0 183.0 205.0

44

Figure A33. Mass spectrum of MDMA in synthetic urine at 250 ng/mL. Spectrum is zoomed in to show 163.0 m/z peak.

Figure A34. Mass spectrum of MDMA in authentic urine at 500 ng/mL. Spectrum is zoomed in to show 194.1 m/z peak.

Figure A35. Mass spectrum of MDMA in authentic urine at 500 ng/mL. Spectrum is zoomed in to show 163.1 m/z peak.

Figure A36. Mass spectrum of methamphetamine in methanol at 100 ng/mL.

Figure A37. Mass spectrum of methamphetamine in synthetic urine at 250 ng/mL. Spectrum is zoomed in to show 150.1 m/z peak.

5E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00 2.33

Min

TIC Filtered 0430_MDMA_0.1 2019.04.30 11:24:35 blank syn urine;ESI +

95.9%; 318.2

4.1%; 144.8

20

40

Intensity

%

165 170 175 180

m/z

Spectrum RT 0.00 - 2.39 {312 scans} 0430_MDMA_0.1 2019.04.30 11:24:35 blank syn urine;ESI + Max: 1E6

171.1

173.0166.0 172.1163.0 165.0181.0 183.0180.0

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

TIC Filtered 0517_MDMA 10-5 2019.05.17 10:56:26 authentic urine 5;ESI +

100.0%; 114.1

10

Intensity

%

185 190 195 200 205 210

m/z

Spectrum RT 0.00 - 1.18 {154 scans} 0517_MDMA 10-5 2019.05.17 10:56:26 authentic urine 5;ESI + Max: 5E5

186.0196.0194.1

188.0 198.1207.1195.2

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

TIC Filtered 0517_MDMA 10-5 2019.05.17 10:56:26 authentic urine 5;ESI +

100.0%; 114.1

20

40

60

Intensity

%

150 155 160 165 170

m/z

Spectrum RT 0.00 - 1.18 {154 scans} 0517_MDMA 10-5 2019.05.17 10:56:26 authentic urine 5;ESI + Max: 1.8E6

152.0 155.1

151.1

171.1

160.4153.0

163.1162.1150.0 165.1

0E0

10E9

20E9

Intensity

0.00 0.33 0.67 1.00 1.33 1.67 2.00 2.33 2.67

Min

ΔIC Filtered 2019_3_6_meth 2019.03.06 14:29:09 ;ESI +

100.0%; 91.1

0

50

Intensity

%

50 100 150 200 250 300 350 400 450

m/z

ΔS 0.55 {1 scans} 2019_3_6_meth 2019.03.06 14:29:09 ;ESI + Max: 3.9E2

91.0

132.0

346.3344.3150.0 247.2119.0

347.392.1 358.3

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

Chromatogram TIC Filtered syn urine Meth 2019.07.03 11:35:39 syn urine;ESI +

100.0%; 150.1

50

Intensity

%

145 150 155 160

m/z

Spectrum RT 0.00 - 0.82 {102 scans} syn urine Meth 2019.07.03 11:35:39 syn urine;ESI + Max: 1.1E7

143.0

144.9

146.9150.1

152.0151.1146.0153.0 155.0 160.2

45



Figure A40. Mass spectrum of methamphetamine in authentic urine at 500 ng/mL.

Figure A41. Mass spectrum of methadone in methanol at 100 ng/mL.

Figure A42. Mass spectrum of methadone in synthetic urine at 100 ng/mL. Spectrum is zoomed in to show 310.2 m/z peak.

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83

Min

Chromatogram TIC Filtered syn urine Meth 2019.07.03 11:35:39 syn urine;ESI +

100.0%; 150.1

5

Intensity

%

110 115 120 125 130

m/z

Spectrum RT 0.00 - 0.82 {102 scans} syn urine Meth 2019.07.03 11:35:39 syn urine;ESI + Max: 8.4E5

119.1

115.1130.2 132.0124.0

122.0114.1

128.2

1E9

2E9

3E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

TIC Filtered METH std 100 2019.07.03 10:32:51 stds at LOD;ESI +

90.7%; 150.0

4.9%; 144.9 3.8%; 144.90.6%; 144.9

10

20

Intensity

%

88 90 92 94 96 98 100 102

m/z

Spectrum RT 0.00 - 1.28 {139 scans} METH std 100 2019.07.03 10:32:51 stds at LOD;ESI + Max: 2.5E6

102.1

101.1

87.2

100.197.189.188.1 91.1

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33 1.50

Min

TIC Filtered meth 7 2019.05.21 12:18:17 authentic urine 7-10;ESI +

94.4%; 114.1

5.6%; 152.0

50

Intensity

%

150 152 154 156 158 160 162 164

m/z

Spectrum RT 0.00 - 1.49 {222 scans} meth 7 2019.05.21 12:18:17 authentic urine 7-10;ESI + Max: 5.9E6

152.0151.0

155.0153.0

157.0153.9150.0 156.0149.0 159.0

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33 1.50

Min

Chromatogram TIC Filtered methadone std 100 2019.07.03 11:18:38 stds at LOD;ESI +

93.5%; 297.2

2.5%; 144.91.3%; 150.91.0%; 144.9 0.9%; 144.90.8%; 144.9

5

10

Intensity

%

260 270 280 290 300 310

m/z

Spectrum RT 0.00 - 1.61 {200 scans} methadone std 100 2019.07.03 11:18:38 stds at LOD;ESI + Max: 1.9E6

297.2261.0

285.9310.2

287.9265.0 305.1284.2 298.3262.9

5E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

TIC Filtered 0430_methadone 0.1 2019.04.30 12:01:52 blank syn urine;ESI +

94.2%; 318.2

5.2%; 130.20.6%; 152.1

10

20

Intensity

%

300 305 310 315

m/z

Spectrum RT 0.00 - 1.45 {190 scans} 0430_methadone 0.1 2019.04.30 12:01:52 blank syn urine;ESI + Max: 7E5

296.2

297.3299.2

310.2 311.2298.3

308.2

46

Figure A43. Mass spectrum of methadone in synthetic urine at 100 ng/mL. Spectrum is zoomed in to show 265.0 m/z peak.

Figure A44. Mass spectrum of methadone in authentic urine at 100 ng/mL.

Figure A45. Mass spectrum of morphine in methanol at 100 ng/mL.

Figure A46. Mass spectrum of morphine in synthetic urine at 250 ng/mL.

Figure A47. Mass spectrum of morphine in authentic urine at 500 ng/mL.

5E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

TIC Filtered 0430_methadone 0.1 2019.04.30 12:01:52 blank syn urine;ESI +

94.2%; 318.2

5.2%; 130.20.6%; 152.1

10

20

Intensity

%

255 260 265 270 275

m/z

Spectrum RT 0.00 - 1.45 {190 scans} 0430_methadone 0.1 2019.04.30 12:01:52 blank syn urine;ESI + Max: 7.5E5

263.0

265.0

261.1268.0

275.1264.0256.3

266.0254.2 273.1

5E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67

Min

TIC Filtered 0503-methadone 0.1-1 2019.05.03 11:10:49 authentic urine 1;ESI +

92.6%; 114.1

6.6%; 143.10.8%; 152.1

10

20

Intensity

%

260 270 280 290 300 310 320 330

m/z

Spectrum RT 0.00 - 1.76 {230 scans} 0503-methadone 0.1-1 2019.05.03 11:10:49 authentic urine 1;ESI + Max: 1.6E6

286.0

265.0256.0

288.0

263.0302.2 334.2

310.2281.0

314.2

2E9

3E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00 2.33 2.67 3.00

Min

TIC Filtered 2019_3_18_morphine_.25 2019.03.18 11:47:05 Neat morphine std (.25 ug/mL) on DBS card;ESI +

68.8%; 155.0

7.2%; 144.96.3%; 154.9

4.2%; 144.9 3.8%; 144.93.5%; 144.9 3.1%; 144.9 3.1%; 145.0

0E0

5E6

Intensity

c/s

100 200 300 400 500

m/z

Spectrum RT 0.00 - 3.03 {367 scans} 2019_3_18_morphine_.25 2019.03.18 11:47:05 Neat morphine std (.25 ug/mL) on DBS card;ESI +

144.9

146.9181.0

196.0205.0155.0151.0 286.0183.0102.1

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

Chromatogram TIC Filtered syn urine morphine 2019.07.03 11:54:59 syn urine;ESI +

86.0%; 239.2

10.4%; 144.93.6%; 144.9

50

Intensity

%

150 200 250 300 350 400

m/z

Spectrum RT 0.00 - 1.29 {161 scans} syn urine morphine 2019.07.03 11:54:59 syn urine;ESI + Max: 9.2E6

144.9

322.0146.9

143.0261.1152.0 239.2 311.1185.9 285.9

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33 1.50

Min

TIC Filtered 0506_morphine 0.25-4 2019.05.07 12:43:51 authentic urine 3;ESI +

96.1%; 239.3

3.9%; 155.1

50

Intensity

%

150 200 250 300 350 400 450

m/z

Spectrum RT 0.00 - 1.62 {210 scans} 0506_morphine 0.25-4 2019.05.07 12:43:51 authentic urine 3;ESI + Max: 1.4E6

239.2311.2 457.4

286.1104.1358.3 482.4

155.0114.1137.1

47

Figure A48. Mass spectrum of norfentanyl in methanol at 100 ng/mL.

Figure A49. Mass spectrum of norfentanyl in synthetic urine at 100 ng/mL.

Figure A50. Mass spectrum of norfentanyl in authentic urine at 100 ng/mL.

Figure A51. Mass spectrum of oxycodone in methanol at 500 ng/mL.

Figure A52. Mass spectrum of oxycodone in synthetic urine at 500 ng/mL.

1E9

2E9

3E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

Chromatogram TIC Filtered norfentanyl std 100 2019.07.03 11:15:59 stds at LOD;ESI +

91.9%; 239.1

8.1%; 144.9

5

10

Intensity

%

215 220 225 230 235 240 245

m/z

Spectrum RT 0.00 - 1.23 {153 scans} norfentanyl std 100 2019.07.03 11:15:59 stds at LOD;ESI + Max: 2E6

239.1

217.0

226.1

240.1229.1212.1 244.9224.0 241.1233.1

2E9

4E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33

Min

Chromatogram TIC Filtered syn urine norfentanyl 2019.07.03 12:07:08 syn urine;ESI +

89.7%; 311.1

10.3%; 144.9

5

Intensity

%

228 230 232 234 236 238 240

m/z

Spectrum RT 0.00 - 1.41 {176 scans} syn urine norfentanyl 2019.07.03 12:07:08 syn urine;ESI + Max: 7.6E5

239.2

241.1229.0

237.1240.0233.0230.1

5E9

Intensity

c/s

0.00 0.33 0.67 1.00 1.33 1.67 2.00

Min

TIC Filtered 0503-norfenatnyl 0.1-1 2019.05.03 11:04:50 authentic urine 1;ESI +

98.6%; 114.0

0.7%; 151.90.7%; 151.9

5

10

Intensity

%

225 230 235 240 245

m/z

Spectrum RT 0.00 - 2.09 {272 scans} 0503-norfenatnyl 0.1-1 2019.05.03 11:04:50 authentic urine 1;ESI + Max: 5.9E5

245.0

229.1

240.1239.2 243.0

224.0 233.1227.0223.1

241.1

1E9

2E9

3E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17 1.33 1.50

Min

Chromatogram TIC Filtered oxy std 500 2019.07.03 11:04:21 stds at LOD;ESI +

86.7%; 239.1

7.4%; 151.15.9%; 144.9

2

4

Intensity

%

310 315 320 325 330 335 340

m/z

Spectrum RT 0.00 - 1.64 {203 scans} oxy std 500 2019.07.03 11:04:21 stds at LOD;ESI + Max: 6.2E5

322.0

340.1

327.1316.1 321.1 323.2 329.0 341.2

1E9

2E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

TIC Filtered syn urine oxy 2019.07.03 11:58:43 syn urine;ESI +

82.4%; 143.0

17.6%; 144.90.0%; 144.9

2

4

6

Intensity

%

310 312 314 316 318 320

m/z

Spectrum RT 0.00 - 1.22 {152 scans} syn urine oxy 2019.07.03 11:58:43 syn urine;ESI + Max: 5.7E5

311.1

316.1

48

Figure A53. Mass spectrum of oxycodone in authentic urine at 1000 ng/mL.

2E9

4E9

6E9

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

Chromatogram TIC Filtered oxycodone 1_Scan1 2019.06.10 11:39:56 authentic urine @ 1ug/mL;ESI +

97.6%; 155.1

1.3%; 144.9 1.1%; 144.9

2E6

Intensity

c/s

0.00 0.17 0.33 0.50 0.67 0.83 1.00 1.17

Min

Chromatogram TIC Filtered oxycodone 1_SIM2 2019.06.10 11:39:56 authentic urine @ 1ug/mL;ESI +

98.9%; 316.2

0.5%; 316.2 0.3%; 316.20.2%; 316.2

10

Intensity

%

312 314 316 318 320 322 324 326 328

m/z

Spectrum RT 0.00 - 1.15 {128 scans} oxycodone 1_Scan1 2019.06.10 11:39:56 authentic urine @ 1ug/mL;ESI + Max: 7.6E5

314.2

318.2320.3

328.2

316.2312.2 321.2 325.2

0

50

Intensity

%

315.6 315.8 316 316.2 316.4 316.6 316.8

m/z

Spectrum RT 0.01 - 1.19 {131 scans} oxycodone 1_SIM2 2019.06.10 11:39:56 authentic urine @ 1ug/mL;ESI + Max: 3.1E5

316.2

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