Oral Fluid Method Validation for Bowling Green State University

ORAL FLUID METHOD VALIDATION FOR BOWLING GREEN STATE UNIVERSITY

Nathan Bunch

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

May 2020

Committee:

Jon Sprague, Advisor

Phillip Gibbs

Nathan Bunch

Jon Sprague, Advisor

Oral fluid (OF) is rapidly becoming a new media for assisting law enforcement in determining if a subject is driving under the influence of drugs (DUID). Preliminary research shows that drugs can be identified in OF in conjunction with blood, and drug concentrations in

OF and blood correlate. Despite availability of several roadside devices to test for drugs in OF, the roadside devices are considered a presumptive test. The results from these roadside tests must be confirmed with a validated liquid chromatography- mass spectrometer (LC-MS) instrumentation. A validated instrument for confirmation of OF results is important, and this study validates BGSU’s Shimadzu 8050 LC-MS. For validation, the instrument must pass various guidelines set by Scientific Working Group for Forensic Toxicology (SWGTOX)

Standard Practices for Method Validation in Forensic Toxicology and others for accuracy, precision, linearity, Limit of Detection (LOD) and Limit of Quantitation (LOQ), carryover, interference, stability, and matrix effects. Due to the Covid-19 global pandemic, only accuracy, precision, linearity, and LOD and LOQ were accessed. The validation studies were conducted over five days (not consecutive) with two runs being conducted during each 24-hour period for a total of 10 runs. A total of 81 different analytes were accessed. The 81 analytes covered a broad range of drugs with abuse potential. The results of the validation study showed that the instrument is highly precise for the vast majority of analytes, but the cannabinoids, particularly delta-9-tetrahydrocannabinol (THC), were troublesome. Linearity for all analytes were accessed using the R^2 of the calibration curve, and all analytes were above the 0.95 limit. The LOD and

LOQ study proved that the cutoff for each analyte is higher than the factor of 2 limit for cutoff/LOQ. The method proved to be an overwhelming success for all analytes. iv

Dedicated to my family, Trevor Bunch, Patricia Bunch, James Bunch, and Emily Jones for providing me with structure and support throughout my life, and to all my friends, mentors, and teachers who have helped and guided me along the way. v ACKNOWLEDGMENTS

When I started as a young 18-year-old college student in the fall of 2013, I had no idea what I wanted to do with my life. It’s an unusually vulnerable position that many students encounter. I bounced around several majors and two universities before finally landing on

Forensic Science at BGSU. Having a great support structure of family, friends, and mentors kept me on the right track towards success.

First and foremost, I would like to thank Dr. Jon Sprague. Dr. Sprague is a family friend and to have him double as a mentor is extremely fortunate. It was his insight into the world of forensics and pharmacology that really pushed me towards majoring in forensic science at

BGSU, and eventually a Master of Science in forensic science at BGSU. Dr. Sprague exemplifies excellence, is confidently humble, and has truly taught me that actions speak louder than words.

Next, I would like to thank the entire Sprague family for a lifetime of support and friendship. I am eternally grateful to have known their family throughout my life. During the last semester of my undergraduate career, I was taking a Monday night class and a class early

Tuesday morning. I was living at home during this time, which was a 40-minute drive from

BGSU. The Sprague family graciously invited me to stay at their home in Bowling Green on

Monday nights after my night class. I will never forget this kind gesture and I cannot thank them enough.

I would like to thank Dr. Travis Worst for being a great mentor and role model for my forensic science career at BGSU. Dr. Worst is someone who can keep a conversation with anyone. He is always thinking about how to improve his students’ life. I truly appreciate his commitment to me and my well-being as a student and more so, as a person. I am grateful to have gotten to know Dr. Worst. vi I would like to thank Dr. Phillip Gibbs for being another great mentor in my life. Almost all of the knowledge I have gained about chromatography and mass spectrometry is due to Dr.

Gibbs’ teaching and mentorship. He is willing to explain and teach about the instrumentation whenever possible, and best of all, he really cares if you learn it. There is no way I would have been able to complete this project without him.

I would like to thank Mrs. M. Michele Nagel. She is a blessing for anyone working in the forensic science department. This project would not have been possible without her.

I would like to thank Dr. Crystal Oechsle for teaching me how to read and understand scientific articles. Her help made this research much easier.

Lastly, I would like to thank my research partner, Ms. Latisha Pipes. We became de facto research partners due to the nature of the studies we were working on. While we tend to be polar opposites, I would not have chosen anyone else. I believe all good relationships require some conflict and I am very appreciative to have met Latisha. I cannot thank her enough for putting up with me for the past two years. Her hard work and endless effort have greatly influenced me to become a better student and researcher.

I am notorious for not expressing the proper gratitude, so if I have not acknowledged anyone else that has helped me on this journey, I truly am sorry. Just know, that I am grateful for the sacrifices people have made to get me to where I am today. This gratitude extends to my family. Where they have made great sacrifices to raise me and help put me through college. I am a better person because of my family. Thank you. vii

LIST OF ABBREVIATIONS

OF: oral fluid

DUID: driving under the influence of drugs

DRE: drug recognition expert

LC-MS: liquid chromatography mass spectrometry

GC-MS: gas chromatography mass spectrometry

SWGTOX: scientific working group for forensic toxicology

LOD: limit of detection

LOQ: limit of quantitation

OMT: oral mucosal transudate

OSHP: Ohio State Highway Patrol

CO: cutoff

HiQC: high quality control

LoQC: low quality control

NegQC: negative quality control

SST (CO): system suitability test (cutoff)

30% (CO): 30% (cutoff)

BGSU: Bowling Green State University

EI: electron impact ionization

TOF MS: time-of-flight mass spectrometry

SIMS: secondary ion mass spectrometry

NASA: National Aeronautics and Space Administration

QMS: quadrupole mass spectrometer viii QIT: quadrupole ion trap

CID: collision-induced dissociation

CI: chemical ionization

ESI: electrospray ionization

IRMS: isotope ratio mass spectrometry

MALDI: matrix-assisted laser desorption ionization

ME: matrix effects

CC: calibration curve

QC: quality control

%CV: coefficient variable

RSS: residual sum of squares

RS: residual sum

6-MAC: 6-monoacetylcodeiene

6-MAM: 6-monoacetylmorphine

EDDP: 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine

MDA: methylenedioxyamphetamine

MDMA: methylenedioxymethamphetamine

MDPV: methylenedioxypyrovalerone

PCP: Phencyclidine

THC: delta-9-tetrahydrocannabinol

THC-COOH: 11-nor-9-carboxy-delta-9-tetrahydrocannabinol

THC-OH: 11-hydroxy-delta-9-tetrahydrocannabinol ix

TABLE OF CONTENTS

Page

CHAPTER I: INTRODUCTION AND BACKGROUND ...... 1

1.1 Current Methods for Drug Identification and Determination of

Impaired Drivers ...... 1

1.2 Oral Fluid ...... 2

1.3 OF Collection Methods ...... 3

1.4 Does OF Accurately Depict Blood Drug Concentrations? ...... 4

1.5 Advantages and Disadvantages of Oral Fluid for Drug Analysis ...... 5

1.6 Preliminary Results ...... 6

1.7 Purpose ...... 9

1.8 Approach ...... 9

CHAPTER II: INSTRUMENTATION AND THEORY ...... 11

2.1 Immunoassay ...... 11

2.2 Mass Spectrometer ...... 12

2.2.1 History ...... 12

2.3 Liquid Chromatography and Gas Chromatography ...... 18

2.3.1 Liquid Chromatography- Tandem Mass Spectrometry ...... 19

2.3.2 Gas Chromatography- Mass Spectrometry ...... 22

2.3.3 Liquid Chromatography- Tandem Mass Spectrometry vs Gas

Chromatography-Mass Spectrometry ...... 23

CHAPTER III: MATERIALS AND METHODS ...... 25

3.1 Drug Standards ...... 25 x

3.2 Internal Standard Mix ...... 25

3.3 Oral Fluid Matrix ...... 25

3.4 Calibrators ...... 26

3.5 Instrumentation ...... 26

3.5.1 Method Setup ...... 26

3.5.2 LC-MS/MS ...... 27

3.6 Methodology ...... 28

3.7 Data Analysis ...... 28

3.8 Limit of Detection and Limit of Quantitation ...... 30

CHAPTER IV: RESULTS ...... 31

4.1 R^2 ...... 31

4.2 HiQC ...... 33

4.2.1 HiQC Batch Average ...... 36

4.2.2 HiQC Intra-Day ...... 39

4.2.3 HiQC Inter-Day ...... 42

4.3 LoQC ...... 45

4.3.1 LoQC Batch Average ...... 48

4.3.2 LoQC Intra-Day ...... 51

4.3.3 LoQC Inter-Day ...... 54

4.4 SST (CO) ...... 57

4.4.1 SST (CO) Batch Average ...... 60

4.4.2 SST (CO) Intra-Day ...... 63

4.4.3 SST (CO) Inter-Day ...... 66 xi

4.5 30% (CO) ...... 69

4.5.1 30% (CO) Batch Average ...... 72

4.5.2 30% (CO) Intra-Day ...... 75

4.5.3 30% (CO) Inter-Day ...... 78

4.6 LOD and LOQ ...... 81

CHAPTER V: DISCUSSION ...... 86

5.1 Cannabinoids’ Challenges ...... 86

5.2 Barbiturates ...... 87

5.3 Low HiQC Concentration for Several Analytes ...... 87

5.4 0.75x Calibrator Issues ...... 88

5.5 Patterns Observed ...... 88

5.6 LOQ ...... 89

5.7 Success of the OF Method ...... 90

5.8 Recommendations ...... 91

5.9 Future Work ...... 92

REFERENCES ...... 93

APPENDIX A: AVERAGE OF:BLOOD CONCENTRATION RATIO ...... 100

APPENDIX B: LIST OF STANDARDS ...... 101

APPENDIX C: LIST OF INTERNAL STANDARDS ...... 104

APPENDIX D: MASS SPECTROMETER SETTINGS ...... 106 xii

LIST OF TABLES

Table Page

1 OSHP Study ...... 7

4.1 R^2 ...... 31

4.2 HiQC ...... 34

4.2.1 HiQC Batch Average ...... 37

4.2.2 HiQC Intra-Day ...... 40

4.2.3 HiQC Inter-Day ...... 43

4.3 LoQC ...... 46

4.3.1 LoQC Batch Average ...... 49

4.3.2 LoQC Intra-Day ...... 52

4.3.3 LoQC Inter-Day ...... 55

4.4 SST (CO) ...... 58

4.4.1 SST (CO) Batch Average ...... 61

4.4.2 SST (CO) Intra-Day ...... 64

4.4.3 SST (CO) Inter-Day ...... 67

4.5 30% (CO) ...... 70

4.5.1 30% (CO) Batch Average ...... 73

4.5.2 30% (CO) Intra-Day ...... 76

4.5.3 30% (CO) Inter-Day ...... 79

4.6 LOD and LOQ ...... 82

CHAPTER I: INTRODUCTION AND BACKGROUND

Oral fluid (OF) is rapidly becoming a new media for assisting law enforcement in determining if a subject is driving under the influence of drugs (DUID) [SWGTOX, Ellefsen K.

N. et al. (2016), Tang M. H. et al. (2018)]. With cannabis and other drugs like psychedelic mushrooms becoming decriminalized in many cities and states, the increase in drivers under the influence of these psychoactive substances is a growing problem [Ellefsen K. N. et al. (2016),

Hartman R. L. et al. (2016), Swortwood M. J. et al. (2017)]. The European Union (EU) conducted a study on Driving Under the Influence of Drugs, Alcohol, and Medicine (DRUID), which found that the overall detection rate of illegal substances in the general driving population was 1.9% [Tang M. H. et al. (2018)]. In seriously injured drivers, the detection rate for illegal substances was 2.3%-12.6% [Tang M. H. et al. (2018)]. In the United States, the National

Roadside Survey from 2013-2014 found that drug prevalence in weekend nighttime drivers increased to 20.0% (with 48% of detected drugs being cannabis related), up from 16.3% in 2007

[Hartman R. L. et al. (2016)]. The breathalyzer is a well-established device to determine the current blood-alcohol content of the subject, but this method does not work well with other psychoactive substances. To complement the breathalyzer and attempt to discover other psychoactive substances causing impairment, other methods of detection are needed. Currently, law enforcement uses Drug Recognition Experts (DRE) and bodily fluids (blood, urine, and more recently OF) to detect psychoactive substances and determine if the driver is impaired

[Ellefsen K. N. et al. (2016), Hartman R. L. et al. (2016), Blencowe T. et al. (2010)].

1.1 Current Methods for Drug Identification and Determination of Impaired Drivers

Law enforcement currently utilizes a DRE to determine if someone is too impaired to drive [Hartman R. L. et al. (2016)]. After detainment from the arresting officer, the suspect is 2 evaluated by a DRE [Hartman R. L. et al. (2016)]. A DRE is a law enforcement officer who is specifically trained to determine the if and which psychoactive substance is causing the impairment [Hartman R. L. et al. (2016)]. The DRE’s determination is based on various sobriety tests which capitalize on the physical and psychological symptoms associated with specific drug families such as miosis, mydriasis, rebound mydriasis, latency of pupil response, recent track marks, tongue discoloration/residue, sense of time, balance, and overall cognition and consciousness [Hartman R. L. et al. (2016)]. Based on their observations, the DRE can determine the family of the psychoactive substance causing the impairment. The DRE categorizes the psychoactive substance into separate categories: Depressant, Stimulant, Hallucinogen,

Dissociative Anesthetic, Narcotic Analgesics, Inhalants, and Cannabis [Hartman R. L. et al.

(2016)]. Their observations and conclusions are verified by a urine and/or blood toxicology screen. Urine drug analysis is used in law enforcement to rapidly determine the active drug causing impairment [Blencowe T. et al. (2010)]. Coupled with a DRE observation, blood analysis is used as confirmation [Gjerde H. et al. (2015)]. Currently, blood is the gold standard for identifying drugs in bodily fluids [Gjerde H. et al. (2015), Elmongy H. et al. (2016)]. Blood offers the unique ability to determine and quantify the metabolites and the parent drug that are influencing the user [Elmongy H. et al. (2016)]. Blood provides a window to the active compounds present in the brain and throughout the body. The blood samples are analyzed and quantified using a liquid chromatography- mass spectrometer (LC-MS) [Elmongy H. et al.

(2016)].

1.2 Oral Fluid

OF is the composite liquid that is present throughout the oral cavity. OF is the mixture of saliva, oral mucosal transudate (OMT), mucin, bacteria, leukocytes, and epithelial cells. Saliva is 3 the primary component of OF and it is a complex mixture of parotid, sublingual, submandibular, and minor salivary glands secretions. OMT is the fluid that is made from the passive transport of serum components through the oral mucosa [Oral Fluid Sampling System, Prickett J. et al.,

Stoykova S. et al. (2016)]. Drugs appear in OF through passive diffusion or active secretion from serum to oral mucosa [Stoykova S. et al. (2016)]. Only unbound and unionized drugs will cross epithelial cell membranes, making the drug’s pKa an important factor for determining concentration [Cone E. J. (2007)].

1.3 OF Collection Methods

There are a few ways to stimulate the production of saliva: pharmacologically, mechanically, chemically. To stimulate the production of saliva pharmacologically, muscarinic receptor agonists are typically prescribed [Łysik et al. (2019)]. These medications increase parasympathetic release of acetylcholine and subsequent binding to the M1 and M3 muscarinic receptors. Parasympathomimetic agents can induce a plethora of adverse reactions; therefore limiting their overall utility as a sialogogue. Mechanical stimulation of saliva involves the act of chewing, swishing, talking, and other general mouth movements [Proctor et al. (2006)].

Chemical stimulation of saliva is typically done by acidic stimulation. Acidic models change the environment of the oral cavity in terms of pH. Several oral fluid collection devices fall under the latter two categories (mechanical or chemical stimulation). The method used for the patient samples in the validation study incorporates both mechanical and chemical stimulation. The swishing mechanism and the introduction of citric acid with the OF buffer synergistically promote saliva production. 4

1.4 Does OF Accurately Depict Blood Drug Concentrations?

Determining the concentration of a psychoactive substance is highly advantageous, because of the ability to determine if the drug is pharmacologically active [Elmongy H. et al.

(2016)]. The concentration of a drug in blood and OF is greatly dependent on the pH of the blood and OF, drug molecule characteristics (pKa, lipophilicity, and charge), protein binding affinities, route of administration, and OF flowrate. [Stoykova S. et al. (2016), Drummer O. H. et al.

(2006)]. Time from consumption to testing and route of administration are major factors for comparing OF concentrations and blood concentrations [Gjerde H. et al. (2015), Elmongy H. et al. (2016), 9]. Cone and Huestis, 2007, explain that the pKa of a drug is very important because it determines how much of the drug will be unionized, allowing it to cross the epithelial cell membranes into the oral cavity [Cone E. J. (2007)]. The species of drug (acidic, basic, or neutral) will determine the ionization of the drug in plasma [Cone E. J. (2007)]. Basic drugs with a higher pKa will have greater ionization in plasma than basic drugs with a lower pKa. Acidic drugs with a lower pKa will have greater ionization of the drug in plasma than acidic drugs with a higher pKa. Because of the acidic nature of saliva, basic drugs are found in higher concentrations than acidic drugs [Cone E. J. (2007)]. Drummer, 2006, created a list of selected drugs with the average OF-to-blood concentration ratios, shown in Appendix A [Drummer O. H. et al. (2006)].

Overall, research has found that blood and OF concentrations of drugs correlate but are highly dependent on time of consumption, route of administration, pKa of the drug, pharmacokinetics of the drug, and physiological differences between individuals [Gjerde H. et al. (2015), Elmongy H. et al. (2016), Swortwood M. J. et al. (2017), Stoykova S. et al. (2016), Drummer O. H. et al.

(2006), Cone E. J. (2007)]. 5

1.5 Advantages and Disadvantages of Oral Fluid for Drug Analysis

OF is currently being researched and implemented in law enforcement procedures when determining and identifying psychoactive substances causing impairment [Elmongy H et al.

(2016)]. The relationship between OF and blood has several advantages. Drawing blood from a suspected driver under the influence can take a few hours. During that time, the levels of the drug that are relevant to intoxication can drop significantly [Swortwood et al. (2017)]. OF can be collected rapidly on the roadside, which can provide a better interpretation of intoxication. The connection between OF and blood components allows for the inference of the current drug of influence from OF. Because of the relationship of blood and OF, detection of drugs in the OF can provide insight towards the drugs currently affecting the brain. While there is a connection between urine and blood, urine analysis shows previous history of a drug of influence, but not necessarily the current drug of influence. Urine analysis is comprised analyzing metabolized analytes or analytes that have undergone glucuronidation from passage through the liver and kidneys. When coupled with a confirmatory blood analysis, OF offers an advantage over urine due its ability to be collected and analyzed for drugs on the roadside similar to a breathalyzer for alcohol impairment. [Blencowe T. et al. (2010), Elmongy H et al. (2016)]. The disadvantage of urine is its inability to be implemented on the roadside by a responding officer, because urine requires a private area and an officer of the same sex to administer the urine toxicology screen

[Ellefsen K. N. et al. (2016), Tonnes S. W. et al. (2005)]. OF’s unique ability to be implemented as a viable roadside bodily fluid, allows the officer to quickly determine impairment and identify if an impairing substance was consumed [Ellefsen K. N. et al. (2016), Elmongy H et al. (2016),

Tonnes S. W. et al. (2005)]. For example, the Michigan State Police is adopting the Alere DDS2 as their OF roadside drug test [Oral fluid roadside analysis pilot program. Michigan State Police 6

(2019)]. The Alere DDS2 uses an immunoassay similar to urine toxicology screens [Oral fluid roadside analysis pilot program. Michigan State Police (2019)]. A disadvantage of OF comes with the collection. Xerostomia (dry mouth) is a common adverse reaction to many drugs of abuse which can limit the amount of OF that can be obtained from a subject. Collection devices for OF are highly variable between devices [Wu (2012)]. While more research and development are required for OF to increase its sensitivity and selectivity with impairing drugs, its ease of access and ability to be implemented on the roadside is why it is overtaking urine as a drug testing standard.

1.6 Preliminary Results

I have completed prior research at BGSU using the Shimadzu 8050 LC-MS with OF and blood as the testing media. For this prior research, two roadside OF devices were tested for the

Ohio State Highway Patrol (OSHP) and the results of the devices, OF matrix on LC-MS, urine toxicology screen, and DREs were compared to blood as the “gold standard.” The blood was taken at the same time as the device utilization, which was analyzed on the LC-MS. In light of this research, it would be beneficial to validate the methods that were used to conduct the OHSP study and further studies. Table 1 shows the preliminary results from the OSHP study:

Table 1: OSHP Study

Percent Agreement with Blood LCMS

DRE

Score Value THC Opioid Stimulant Depressant

1 69.4 53.3 42.1 11.5

2 12.2 33.3 5.3 0

3 18.4 13.3 52.6 88.5

1 = DRE and Blood Agree

2 = DRE (yes) and Blood (no)

3 = DRE (no) and Blood (yes)

Urine MEDTox

Score Value THC Opioid Stimulant Depressant

1 79.4 71.4 85.7 29.6

2 15.9 14.3 2.4 0

3 4.8 14.3 11.9 70.4

1 = MEDTox and Blood Agree

2 = MEDTox (yes) and Blood (no)

3 = MEDTox (no) and Blood (yes)

DDS2

Score Value THC Opioid Stimulant Depressant

1 69.6 0 63.4 11.1

2 5.4 0 0 0

3 25.0 100 36.6 88.9

1 = DDS2 and Blood Agree

2 = DDS2 (yes) and Blood (no)

3 = DDS2 (no) and Blood (yes)

EZ Saliva

Score Value THC Opioid Stimulant Depressant

1 0 16.7 58.1 0

2 0 0 4.7 0

3 100 83.3 37.2 100

1 = EZ and Blood Agree

2 = EZ (yes) and Blood (no)

3 = EZ (no) and Blood (yes)

Oral Fluid LC/MS

Score Value THC Opioid Stimulant Depressant

1 88.1 66.7 94.1 84

2 7.1 25 5.9 12

3 4.8 8.3 0 4 9

1 = OF and Blood Agree

2 = OF (yes) and Blood (no)

3 = OF (no) and Blood (yes)

*Table 1: Percent agreement of results from OSHP study. “Oral Fluid LC/MS” pertains to validation study proposal.

The results in section “Oral Fluid LC/MS” in Table 1, show the capability of the OF method. The section shows a percent agreement between OF and blood in four separate drug categories when analyzed on the LC-MS. If a drug under one of the four categories was identified in OF and blood (Score Value 1), it is said that OF and blood agree. When OF and blood agreed, a percent agreement between the two was established and the percent agreement was the overall determining factor for OF’s ability to detect drugs also found in blood, with blood being the standard for comparison.

1.7 Purpose

Despite this preliminary research, the OF method validation for BGSU’s Shimadzu 8050

LCMS has not been done. The purpose of this research is to validate the OF method for BGSU and validate the results of this preliminary research.

1.8 Approach

In order to validate the OF method, a specific approach was needed to meet the validation goal. The approach of this research was to create a calibration model and use quality controls as indicators and arbiters of the quality of data and apply theory of Mass Spectrometry and Liquid

Chromatography when interpreting data received by the calibration model. The decision was made to set criteria to interpret the data, in-part, by using the Scientific Working Group for

Forensic Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic

Toxicology. With a great significance of data interpretation using a ±20% Coefficient of 10

Variation (%CV) to determine if the data is acceptable and can be used to validate the OF method. As previously stated, quality controls will be used to determine if data is acceptable and if the data can be used for validation. The quality controls used in the calibration model are explained in the “Materials and Methods” section. The next step in validation is to determine the

Limit of Detection (LOD) and Limit of Quantitation (LOQ). After LOD and LOQ determination, carryover is assessed determining the Negative Quality Controls (NegQC), which are injected after the highest concentrated quality control (HiQC). Next, real patient samples in a double- blind study are analyzed to determine the ability of a researcher to decide and match patient samples on BGSU’s LC-MS with the same patient samples from another LC-MS. By fulfilling this set criteria, the OF method can be validated for BGSU, and future research using the

Shimadzu 8050 LC-MS at BGSU can have their research backed by a validated method.

CHAPTER II: INSTRUMENTATION AND THEORY

2.1 Immunoassay

Due to the popularity of immunoassay devices in toxicology screening, it is worth noting the mechanism and issues regarding immunoassay drug testing. The immunoassay drug test is a technique used commonly for urine and OF drug screens. The immunoassay operates by interaction of antibodies and drugs of interest in a biological fluid. Depending on whether or not the drug in the biological fluid binds to the antibodies on the immunoassay test strip, the test can show if the drug is present or not [Blencowe T. et al. (2010), Cone E. J. (2007), Dickson et al.

(2006), Wu et al. (2013)]. The immunoassay test is not a perfect method though. Many issues arise with the test because of the inherent nature of the test. For example, cross-reactivity is a major issue with the immunoassay test. Various analytes with similar chemical structures can interact with the antibodies on the test strip, giving a false positive test result [Dickson et al.

(2006), Wu et al. (2013), Krasowski et al. (2020), Anizan S. et al. (2014), Cao et al. (2015),

Yuan C. et al. (2012)]. THC is a major issue for quantitation in general, because of THC’s affinity with binding to other surfaces during the collection process. Due to the nature of THC, immunoassay tests have difficulties in identifying the analyte because of major analyte losses during the collection process. In the “Preliminary Results” section, three immunoassay devices were tested. The MEDTox tested urine, and the EZ-Saliva II and Alere DDS2 tested OF. THC clearly had the highest losses because of the low detection from the immunoassay devices when compared to the detection in blood. The lowered sensitivity of the immunoassay drug test categorizes the test as a presumptive test and cannot be used alone as a confirmatory test [Cao et al. (2015)]. 12

2.2 Mass Spectrometer

In order to fully realize the importance, significance, and theory of the mass spectrometer, it is necessary to understand the history of the instrument. By providing the history of the mass spectrometer, the application of a variety of scientific principles by many different scientists throughout the instrument’s history can be appreciated.

2.2.1 History

The Mass Spectrometer (MS) is widely considered the most sensitive and specific analytical instrument and is used in a wide range of professions and applications [Maher S. et al.

(2015)]. Despite seeming technologically advanced, the MS is a relatively old instrument, first pioneered by Sir J.J. Thomson in 1913 [Maher S. et al. (2015)]. The principles discovered by

Faraday, Maxwell, and Dalton, and the scientific advancements by Geissler, Crookes, and

Goldstein in the late 19th century laid the foundation for the success of the MS. The cathode-ray tube was used by Goldstein to discover that “canal rays” traveled in the opposite direction to the negatively charged particles of the cathode rays and determined that the “canal rays” must be positively charged. Perrin, in 1895, confirmed that the “canal rays” were positively charged, and discovered that the charge magnitude was approximately equal to that of the negatively charged cathode rays. Perrin’s confirmation and discovery led to Thomson discovering the electron and receiving the Nobel Prize in Physics in 1906. By confirming that cathode rays consisted of negatively charged particles, Thomson was able to measure the ratio of the electric charge of a particle to its mass (e/m). Thomson further experimented with the cathode rays by deflecting the cathode rays with a magnet to see if the charge and rays could be separated. He discovered that were not able to separate and determined that they must be the same thing. To confirm this conclusion, Thomson deflected the cathode rays with a magnet away from the detector and 13 observed no signal. When he deflected the cathode rays towards the detector, the signal increased. Thomson’s next experiment utilized a similar approach carried out by Hertz that attempted to deflect cathode rays by applying an electric field between a pair of metal plates.

Thomson observed the cathode ray become deflected by the electrically charged metal plates.

Thomson’s third experiment utilized a combination of electric and magnetic fields. By using the two fields in conjunction, he was able to infer the e/m of the electrons. Thomson adjusted the magnetic field strength between the metal plates, in order to cancel out the magnetic field and electric field, which in turn canceled out the deflection of the cathode ray. Using the force law from Lorentz, Thomson was able to calculate the mean velocity of the particles. Thomson proceeded to calculate the angle of ray deflection from the electric field, by using the length, separation, voltage, horizontal speed of the ray, and the e/m. From all of his work, Thomson had found the nature of cathode rays, but it was not until Wien, when the nature of the “canal rays” was found. Wien utilized a magnetic and electric field to measure the deflection of “canal rays”.

Wien found that the “canal rays” had a lower velocity and smaller e/m than the cathode rays.

Wien identified the “canal rays” to be composed of positive particles (known as the proton) and that the particles have the same mass as the hydrogen atom. Following the work of Wien,

Thomson began to work with positive rays using a photographic plate for a method of detection.

The photographic plate allowed Thomson to distinguish different “electric atomic weights”, where he compared the mass-to-charge (m/z) ratio of different compounds to the m/z of hydrogen. He oriented the magnetic and electric fields to produce right-angled deflections, which produced a parabolic curve on the photographic plate for identical species of different speeds.

The parabolic lines represented the different “electric atomic weights” of the residual gases that 14 became ionized inside the chamber deflected onto the photographic plate [Maher S. et al.

(2015)].

In 1912, Thomson invented what he called a “parabola spectrograph” which was the world’s first scanning mass spectrometer. By doing so, he replaced the photographic plate with a parabolic slit in a metal plate and placed a Faraday cup connected to an electroscope behind the plate, which allowed him to measure relative abundance of the ion. When Thomson adjusted the magnetic field, the positive ion beam would be deflected through the slit and he was able to measure the intensity. Once he plotted the mass spectrum of ion abundance versus the relative mass, Mass Spectrometry was born [Maher S. et al. (2015)].

After Thomson’s work, Dempster, in 1918, constructed the magnetic sector analyzer. His magnetic sector analyzer laid the groundwork for electron impact ionization (EI), as well as his basic design is still used in magnetic sector instruments today. Dempster’s instrument accelerates ions from an ion source, through a narrow slit. Then the ions are deflected by a magnetic field into an analyzer region, and only ions of a certain m/z pass through a second slit. Then the charge of the ions is measured by an electrometer. Magnetic sector instruments are able to separate ions in a magnetic field based on their charge and momentum. The principles behind the magnetic sector instruments are based on the Lorentz force and angular momentum. The Lorentz force is defined by the equation [Syed et al (2011)]: F=q(E+v×B) where v is the instantaneous velocity of the particle, q is electric charge of the particle, E is the strength of electric field, and B is the strength of magnetic field. Dempster’s work was followed by Bleakney, who developed the electron impact ion source (EI). EI works by having energized electrons interact with gas phase neutral atoms to produce ions. Bleakney improved Dempster’s instrument by separating the fields controlling the electron and ion beams, which improved molecular ionization 15 measurements. The mass spectrum that resulted included fragmentation pattern that could characterize a certain compound, essentially giving a compound’s “fingerprint” [Maher S. et al.

(2015)].

Moving ahead, the 1940s, 1950s, and 1960s featured many improvements and new techniques in mass spectrometry. The first being the usage of “dynamic” instruments. The

“dynamic” instrument uses varying magnetic and electric fields to focus ions of certain m/z to a detector, which allows for quick identification of a wider range of compounds. Prior to the

1950s, “static” instruments, as opposed to “dynamic” instruments represented the majority of mass spectrometers. The term “static” referred to mass spectrometers that had magnetic and electric fields that remained constant as the ion passed through the instrument. Using the dynamic technique, Stephens discovered a way to identify compounds with time dispersion. This later became known as time-of-flight (TOF) MS. A TOF MS, uses the differences in ion flight time through a region free of an electric field. The theory behind the TOF MS is that ions of the same kinetic energy, but have different masses take a different amount of time to travel across the region [Maher S. et al. (2015)].

Another important technique was developed in the late 1940s for surface science analysis. This technique was called secondary ion mass spectrometry (SIMS). SIMS works by shooting a solid sample surface with a focused primary ion beam, which causes the emission of secondary ions that are distinctive of that sample. Thomson made mention of this technique in

1910, but it did not become reality until 1949 when Herzog and Vichböck performed the first experiments. The technique became so useful for many applications, that NASA used SIMS to analyze moon rocks [Maher S. et al. (2015)]. 16

In 1953, Paul and Steinwedel, became enshrined in MS history when they described a new concept in mass analysis called the quadrupole mass spectrometer (QMS). The QMS was a new type of dynamic instrument that could separate ions based on their stability in a quadrupolar field. The quadrupolar field is created by a combination of sinusoidal and static voltage potentials. The QMS consists of four parallel electrodes, with a hyperbolic cross section, that are positioned radially and equally spaced around a central axis. Paul and Steinwedel’s QMS concept did not use magnetic fields, but only electric fields to separate the ions. They filed for patents for the QMS and the quadrupole ion trap (QIT). The QIT uses a three-dimensional field to trap ions and differs from the QMS by using three electrodes. In 1963, Von Zahn, a co-worker of Paul, invented the monopole which was an adaptation of the QMS. The monopole had one circular electrode and an angled v-shaped electrode that produced one-fourth of the QMS field.

The instrument garnered much interest initially, presumably due to less parts involved, but eventually fell out of favor for the QMS because of low sensitivity and poor peak shape. QMS is a popular instrument with gas chromatography (GC). The combination of GC and QMS are a very effective means for identification of compounds. The QMS allows for fast scanning speeds, which couples well with the GC. The GC separates the compound mixture into its components, then the MS acts as a detector to identify the components. The combination is often referred to as the “gold standard” for forensic drug analysis [Maher S. et al. (2015)].

The 1960s provided significant advancements for the MS. One notable advancement was the invention of the tandem mass spectrometer (MS/MS). The MS/MS allows for multiple stages of the MS to be carried out in a single run. When scanning for product ions, the typical MS/MS begins by selecting a precursor ion with a mass analyzer. Then, the precursor ion is fragmented by Collision-induced dissociation (CID). CID is a method that dissociates a projectile ion (in this 17 case, a precursor ion) into smaller fragments by colliding it with a neutral species (noble gases helium or argon are popular). After fragmentation, mass analysis of the product ions is carried out. The resultant mass spectrum of the fragments of a specific precursor ion provides important information for the structure and identification of the primary ion [Maher S. et al. (2015)].

The 1960s saw great advancements for ion sourcing as well. Many different sourcing techniques were utilized, which widened the range of samples that could be analyzed and in turn widened the applicability. In addition to EI as a source, Chemical ionization (CI) was developed.

CI works by injecting a reagent gas into the ionization source and raising the gas pressure. The increased pressure increases the probability of forming protonated or deprotonated molecular ions. CI was termed as a “soft” ionization method, because it does not break chemical bonds and leaves more of the molecular ion, compared to EI. EI is termed as a “hard” ionization method, because it breaks chemical bonds and creates ionized fragments. Another significant soft ionization method was the development of electrospray ionization (ESI) by Dole et al in 1968.

While highly complex, the ESI essentially has an analyte solution sprayed from a small diameter electrode tip via an applied high voltage. The charged droplets are produced at the electrode’s tip, and through solvent evaporation and multiple charge-induced droplet disintegration, small and highly charged droplets are formed. From these charged droplets, gas phase ions are produced and are taken into the MS through a vacuum [Maher S. et al. (2015), Vachet (2003),

Chong et al. (2018), Loos et al. (2016)].

Moving along to the 1970s, a significant development was made by coupling liquid chromatography (LC) and MS. This combination provides high selectivity and sensitivity for complex mixtures, including biological samples. Another significant advancement for MS was made in the 1970s, when the triple-quadrupole was invented by Morrison and incorporated into 18

MS/MS by Enke and Yost. The triple-quadrupole MS is made up of three quadrupoles placed in series, where the middle quadrupole is the collision cell and CID takes place [Maher S. et al.

(2015), Vachet (2003)].

Through the 1980s to present day, many developments were made with the MS using many of the foundational techniques developed by earlier research. These developments lead to new techniques such as isotope ratio mass spectrometry (IRMS) and matrix-assisted laser desorption ionization (MALDI). New applications for the MS utilizing the various techniques included: genomics, archeology, imaging mass spectrometry that can provide crucial details for disease diagnosis, coupled with electrosurgery to provide rapid biological tissue analysis, and

(my personal favorite) the miniature MS that can perform analysis in situ [Maher S. et al. (2015),

Vachet (2003)].

By going through the long history of the MS from its initial inception through cathode ray tubes to analyzing biological tissue for electrosurgery, it helps form a certain respect to the scientists that contributed to the development of the instrument. Understanding the history of the

MS allows for the understanding of the theory behind the MS, why it was developed, and its applications in various fields and research.

Looking at the many applications for the MS and how it can be coupled with different separation techniques, the application of the MS is crucial for my research. Coupled with the LC, the MS allowed for me to detect various analytes within a biological sample (OF) with a high degree of sensitivity and specificity.

2.3 Liquid Chromatography and Gas Chromatography

There are currently a variety of chromatography coupled mass spectrometry techniques for clinical and forensic toxicology [Stout et al. (2010), Holčapek et al. (2012)]. LC-MS/MS and 19

GC-MS are very popular techniques used for separation of compounds in complex mixtures, identifying, and analyzing psychoactive substances. While the MS portion is very similar (except for the addition of another quadrupole in the LC-MS/MS), the ion source and chromatography techniques are very different [Wu (2013), Stout et al. (2010), Ojanperä et al (2012), Wu et al.

(2012)]. The two techniques that will be reviewed and accessed are LC-MS/MS and GC-MS, both using quadrupole MS, in terms of OF analysis.

2.3.1 Liquid Chromatography- Tandem Mass Spectrometry

An overview of LC and GC is necessary for accessing LC-MS/MS and GC-MS, beginning with LC. LC-MS/MS is a relatively new technique compared to GC-MS/MS. LC operates with compounds in the liquid phase. The invention of the ESI by Dole et al., was the starting point of LC’s ability to be coupled with MS. The ESI allowed for a continuous liquid flow onto the mass spectrometer [Maher S. et al. (2015)]. Modern techniques for separation, identification, and analysis of psychoactive substances formed once the triple-quadrupole

MS/MS was invented and coupled with LC [Wu et al. (2012), Maher S. et al. (2015)].

Starting at the beginning, preparation of samples for the LC-MS/MS are fairly simple depending on the matrix of the sample. To begin sample preparation, the matrix of the sample must be filtered, and the sample is extracted as to prevent unwanted constituents interfering with the analysis of the sample. OF extraction method is relatively simple method and boils down to adding the sample to a filter vial and loading the sample onto the instrument. Essentially,

OF offers an easy “dilute-and-shoot” method. Other matrices, such as blood, require more comprehensive extraction processes [Wu et al. (2012), Holčapek et al. (2012), Cao et al. (2015)].

Once filtration has taken place, a standard curve containing various concentrations of known 20 standards is prepared. The standard curve allows for the quantification of an analyte within the sample.

The LC can use a variety of mobile phases. When running in a gradient elution method, mobile phases typically consist of miscible solvents such as water with an organic solvent. [Wu et al. (2012), Yabre et al. (2018)]. Acetonitrile and methanol are common organic solvents. The aqueous mobile phase can be combined with an acid to increase separation by adjusting the pH

[Yabre et al. (2018)]. Once preparation is complete, the sample is injected into the instrument and mixed with the mobile phases to carry the sample into the column.

The column is considered the stationary phase of the instrument, and the stationary phase typically contains a solid phase non-polar substance. The samples within the mobile phase interact with the stationary phase of the instrument and can “stick” to the stationary phase for a certain amount of time [Peters (2012), Stout et al. (2010), Wu et al. (2012)]. The time that the sample is injected to the time the sample hits the detector is called the retention time, and the retention time can vary between different compounds based on how strongly the compound interacts with the stationary phase. The retention time is largely governed by the analyte’s polarity [Peters (2012), Stout et al. (2010), Wu et al. (2012)].

While C18 columns are considered a standard for LC, there are alternative stationary phases that can be used to invoke alternative interactions with certain analytes in order to increase selectivity [Bell et al. (2017), Ferro et al. (2019)]. Alternative columns have been introduced to improve retention times and selectivity for specific analytes, and this is generally completed by the addition of functional groups (e.g. biphenyl, polar-embedded alkyl, fluorophenyl) to the stationary phase. The addition of functional groups to the stationary phases promotes stronger interactions with specific analytes through different intermolecular forces 21

[Bell et al. (2017), Ferro et al. (2019)]. Such that, silica-based additions to the C18 columns, attributed to better selectivity and improved peak shape with more aqueous based mobile phases

[Ferro et al. (2019)]. The C18 column interacts through intermolecular forces. The C18 column is consistent and efficient at separating hydrophobic molecules, but the column is less efficient at separating hydrophilic molecules [Bell et al. (2017), Ferro et al. (2019)]. An alternative to the

C18 is a silica-based biphenyl column. The biphenyl column replaces the eighteen-carbon alkyl chain from the C18 column, and instead has a biphenyl molecule attached to the silica-base [Bell et al. (2017)]. The substitution of biphenyl introduces pi-pi interactions for aromatic compounds

[Bell et al. (2017)]. The pi-pi interactions from the biphenyl, greatly increase the retention and selectivity of aromatic compounds [Bell et al. (2017)]. Including the pi-pi interactions, the biphenyl column has a high degree of hydrogen bonding capability [Bell et al. (2017)].

After the sample finishes interacting with the stationary phase, the sample enters the ion source. With the invention of ESI, the MS had the ability to analyze non-volatile compounds continuously in the liquid phase. Once ionized by the ESI, the ions enter the first quadrupole of the MS via vacuum. The first quadrupole separates the ions by size according to their m/z, acting as a filter. The quadrupoles are charged with an alternating and direct electric current and ions passing through quadrupole form trajectories. Unstable ions are lost in the vacuum, but ions with a stable trajectory reach the electron multiplier within the quadrupole. The electron multiplier is a device that converts captured ions into an electrical current. Within the electron multiplier, the user can vary voltages between the quadrupoles, which stabilizes the trajectories different sized ions in succession. GC-MS has a single quadrupole mass detector, where the LC-MS/MS has two quadrupoles in tandem- hence the second “MS” in the name. In the LC-MS/MS, the first quadrupole is identical to the quadrupole in the GC-MS [Wu et al. (2012)]. After the first 22 quadrupole filters out unstable ions, the next detector allows only the stable precursor ions, typically the molecular ions, to enter. The second quadrupole is where the fragmentation takes place by CID. The precursor ions in this area are fragmented into the product ions. Next, the third quadrupole filters out the product ions using similar methods as the first quadrupole. After the product ions pass through the third quadrupole, they reach the detector. The detector uses the selected product ions to measure and link the ions back to the precursor ion. Due to the addition of the second and third quadrupole, the specificity of the LC-MS/MS is higher than the GC-MS

[Wu et al. (2012)].

2.3.2 Gas Chromatography- Mass Spectrometry

Continuing the with the overview, the GC-MS operates differently than LC, because it operates under gas phase and the analyte must be volatile [Wu et al. (2012)]. Retention times

(similar to LC) are governed by the interaction of the sample with the stationary phase but differ as volatility also affects the analyte’s retention time. Sample preparation for the GC differs from

LC, because the analyte must be volatile [Stout et al (2010), Wu et al. (2012)]. If the analyte is not volatile, a derivatization reaction is required. Derivatization involves chemically modifying the analyte into a new compound that has a much lower boiling point. Next, the sample is injected into the GC, where the sample is volatized. The gas-phase sample is mixed with a unreactive carrier gas, such as helium. The carrier gas carries the sample through a column where the interactions between the sample and the stationary phase occur. Once through the stationary phase, the sample reaches the ion source. The sample is ionized and enters the MS. As described with the LC, the sample on the GC-MS travels through the quadrupole to the detector

[Wu et al. (2012)]. 23

2.3.3 Liquid Chromatography- Tandem Mass Spectrometry vs Gas Chromatography- Mass

Spectrometry

While similarities exist in the MS portion, this is only true for the first MS, as the LC-

MS/MS has another MS in tandem with the first. The addition of a second MS allows for better specificity, because of the ability of the second detector to link specific fragments of product ions back to a precursor ion. The theory that the product ions, because of specific fragmentation patterns, must come from a specific precursor ion allows for the higher specificity [Wu et al.

(2012)].

GC-MS has high separating power and high selectivity through the GC and EI respectively. GC-MS was the gold standard technique for compound identification in clinical and forensic toxicology settings [Peters (2012)]. The GC-MS can improve its limitations with hydrophilic, thermolabile, and non-volatile compounds with extensive sample preparation, e.g. derivatization. LC-MS/MS eliminates the need for derivatization, because it is not operating under gaseous conditions, rather liquid conditions. There is no need for concern about the volatility of a compound [Peters (2012)]. The lack of concern about volatility of compounds for

LC-MS/MS is greatly realized in terms of biological fluid analysis. LC-MS/MS offers a quick and easy “dilute-and-shoot” method. Owing to the high popularity of the LC-MS/MS in toxicological laboratories, the “dilute-and-shoot” method allows for high through-put of biological fluids [Deventer et al. (2014)].

Another advantage in terms of OF analysis, is the LC-MS/MS ability to quantify the analytes present in OF. With THC becoming legal in many states, local and state governments are wanting to set a limit of THC concentration in biological fluids to declare impairment

[Ellefsen K. N. et al. (2016), Hartman R. L. et al. (2016), Swortwood M. J. et al. (2017)]. When 24 concentration limits are set, there becomes a pressing necessity for a reliable quantitation method.

LC-MS/MS is not impervious to limitations, however. A major issue concerning LC-

MS/MS is the instrument’s susceptibility to matrix effects (MEs). MEs encompass areas of the ionization process to the co-eluting compounds to the sample matrix itself. The MEs associated with LC-MS/MS can lead to ion enhancement (increased intensity) or to ion suppression

(decreased intensity) [Peters (2012)]. Mechanisms regarding MEs were categorized by Trufelli et al. The first being a competition of analyte and co-eluting compound for available charges during the ESI process. The second being reduced efficiency of droplet formation from higher viscosity and surface tension of the droplet. The third being the formation of solid analyte particles with non-volatile additives. The fourth being ion pair formation of an analyte and co-eluting compound [Trufelli et al. (2011)].

CHAPTER III: MATERIALS AND METHODS

3.1 Drug Standards

Drug standards in methanol were purchased from Cerilliant (Round Rock, Texas) and can be found in Appendix B. Purchasing compounds allows for verification of the standards and concentration of standards from an independent third-party source. The standards were mixed into various stock solutions, and the corresponding stock mixture were diluted to create the calibrators and controls. While caffeine is not considered a drug of abuse, it is present in the panel. Caffeine’s presence in the panel enables the calculation of the collection volume from the

OF solution wash in patient samples and helps control for different collection volumes.

Furthermore, caffeine acts a “pseudo” internal standard because of the commonality associated with the analyte’s use.

3.2 Internal Standard Mix

An internal standard mix containing deuterated compounds of the standard drug mixture, also purchased from Cerilliant (Round Rock, Texas). The list of internal standards can be found in Appendix C. The internal standards are used to quantify the standards using the relative abundance ratio between the two. Several standards shared the same internal standard e.g.

Alprazolam, Diazepam, and Triazolam, share Diazepam-D5 as the internal standard. The internal standards were structurally similar to the standards, and their elution times were the same or very close together. The internal standards were added at a consistent amount to correct for absorptive loss and matrix effects for the standards.

3.3 Oral Fluid Matrix

The OF matrix was prepared by a mixture of artificial saliva and OF buffer at a ratio of

1:2 respectively. The artificial saliva was obtained by Pickering Laboratories (Mountain View, 26

California) and the OF buffer was obtained from Clinical Lab Consulting, LLC (Dublin, Ohio).

The OF buffer contains citric acid, water, sucralose, and red food dye, and is formulated to a pH of 6.5.

3.4 Calibrators

Calibrator stocks were prepared in amber-glass vials at increasing concentrations of 40 ng/mL, 75 ng/mL, 100 ng/mL [Cut-off (CO)], 200 ng/mL, 300 ng/mL, 1000 ng/mL, 5000 ng/mL, and 10000 ng/mL, using the standard stock mixture. Apart from the calibrators, controls were prepared using control stocks. The concentrations of the controls are as follows: High

Quality Control (HiQC): 2500 ng/mL, Low Quality Control (LoQC): 60 ng/mL, Negative

Quality Control (NegQC): 0.0 ng/mL (Methanol only), System Suitability Test (SST CO): 100 ng/mL, 30% Cut-off (30% CO): 30 ng/mL. The calibrator stocks were diluted with the OF solution to create a calibrators at 0.40 ng/mL, 0.75 ng/mL, 1.0 ng/mL, 2.0 ng/mL, 3.0 ng/mL,

10.0 ng/mL, 50.0 ng/mL, and 100 ng/mL. The control stocks were also diluted with the OF solution to create controls at: 25 ng/mL (HiQC), 0.6 ng/mL (LoQC), 0.0 ng/mL (NegQC), 1.0 ng/mL (SST CO), and 0.3 ng/mL (30% CO). The HiQC is set at 25 ng/mL because it falls right in the middle of the calibration curve. The LoQC is set just below cut-off at 0.6 ng/mL, and the

30% CO is set well-below cut-off at 0.3 ng/mL near the LOQ of each analyte to demonstrate the accuracy of the calculated LOQ. Both of those QCs can represent the quality of data below the cut-off point by in terms of signal-to-noise ratio, ensuring that the cut-off point is set at the point at which signal-to-noise ratio does not become a major issue in reading the signal from each compound.

3.5 Instrumentation

3.5.1 Method Setup 27

Prior to conducting the validation runs, the Shimadzu 8050 LC-MS had to be optimized for the OF method. Proper optimizations allow for the maximum sensitivity to be achieved for the instrument. Preparation for an optimization simply involved diluting each analyte in 100% methanol, and further diluting in a 50:50 mixture of both mobile phases (0.1% acetic acid:100% methanol). Optimization runs consisted of the following: The monoisotopic mass was calculated for each analyte. The ion masses (precursor and product) were calculated from the most abundant primary mass. A determination was made as to run in negative mode of positive mode for each analyte. Next, the first quadrupole was optimized by voltage to select the precursor ion.

The collision cell was optimized by voltages to select the proper product ion. The third quadrupole was optimized by voltages to select the product ion. Once the ions were optimized, each compound was optimized by retention times on from the LC to time of detection. Lastly, the dwell times (number of scans) were adjusted at specific time intervals according to retention times of each analyte.

3.5.2 LC-MS/MS

The Shimadzu 8050 LC-MS was used to conduct this research. The LC portion used a

Raptor Biphenyl column with a Raptor guard column cartridge. Guard column cartridges were replaced every 3-4 runs to maintain high sensitivity. Column conditions were consisted of a temperature of 70 C. Two mobile phases were used which consisted of 0.1% acetic acid and

100% methanol at HPLC grade or higher. 0.1% acetic acid was used instead of the more popular formic acid, due to acetic acid’s increase sensitivity for negative ion mode analytes as well as its higher gas phase proton affinity [Hua Jenke (2012)]. Chromatography was conducted using a gradient method ending with 100% methanol. Method run-time was 10 minutes per injection.

Batch run-time was 7.5 hours. A batch-run consisted of two null injections, six methanol 28 injections, followed by two calibration curve (CC) injections (using the same CC, denoted at

CC1 and CC2), with four methanol injections in between the curves, and ending with 2 methanol injections.

The MS/MS portion consisted of a triple quadrupole tandem MS. ESI was used as the ionization source. The source was cleaned with LC-MS grade water and 100% LC-MS grade

Methanol between every batch. The MS/MS operated under the set parameters shown in the

Appendix D.

3.6 Methodology

The entire data set was produced over the course of 5 days (although not consecutive days due to various circumstances). A total of 10 runs were conducted throughout the 5 days. The procedure went as follows:

1. Day 1 (Consists of a 24-hour period): Prepare a (CC).

2. Conduct the first batch run

3. After the first batch run concludes, check data quality using the R^2 values (>0.95) as

determining factor of acceptability.

4. If data is deemed acceptable, re-run the batch for Day 1 to gather data for intra-day

precision studies.

5. Day 2: Prepare a new CC.

6. Repeat steps 2 through 5 until Day 5.

3.7 Data Analysis

To conduct data analysis for validation, Shimadzu’s LabSolutions Insight program was used. On Insight, the CC and QCs were analyzed using the R^2 values to determine the quality of linearity. The linearity was weighted towards the lower end of concentrations as the majority 29 of the data resides at and below 10.0 ng/mL, near cut-off. The linearity was weighted using: y =

1/(x^2). Next, accuracy was analyzed by comparing the found concentration and the theoretical concentration. Any calibrator that was above the ±20% threshold, [determined by the SWGTOX guidelines (1)] was excluded and the CC was recalculated. The limit of calibrators that could be excluded was two. A limit of two was set to keep variability for linearity.

Data analysis was further conducted on Insight and Microsoft Excel. Accuracies of each QC of each analyte were analyzed on both programs. For each QC of each analyte, multiple precision studies were conducted. The Coefficient Variable (%CV) was used to determine precision, and the quality of precision was decided by a limit of ±20% [determined by the

SWGTOX guidelines]. The %CV was calculated by the average concentration and standard deviation of the population of interest, where the standard deviation was divided by the average concentration. The quotient was converted to a percentage by multiplying by 100. The precision studies were conducted as follows:

1. Overall precision of the QC batch: Average of Day 1 through Day 5 / Standard Deviation

of Day 1 through Day 5 = CV * 100 = %CV

2. Precision between each QC from the CC1 and CC2 of the intra-day runs: Average:

[Average of Day 1 (Run 1: CC1, Run 2: CC1)] and [Average of Day 1 (Run 1: CC2, Run

2: CC2)] … [Average of Day 5 (Run 1: CC2, Run 2: CC2)], Standard Deviation/ Average

= CV * 100 = %CV

3. Intra-day precision: Average of Intra-day: [Day 1 (Run 1: Average of CC1 and CC2)] and

[Day 1 (Run 2: Average of CC1 and CC2)] … [Day 5 (Run 2: Average of CC1 and

CC2)], Standard Deviation of Intra-day/Average of Intra-day = CV * 100 = %CV 30

4. Inter-day precision: Average of Inter-day: [Average of Day 1 (Run 1 and Run 2)] …

[Average of Day 5 (Run 1 and Run 2)], Standard Deviation of Inter-day/ Average of

Inter-day = CV * 100 = %CV

3.8 Limit of Detection and Limit of Quantitation

LOD and LOQ were determined using the residual sum of squares (RSS) of each run and the slope of the line using Clinical Lab Consulting Validation Methods and ICH Harmonised

Tripartite Guideline as a guide. Additionally, Heym explains the reasoning behind using RSS as a statistical method for determining LOD and LOQ. Instead of using the Residual Sums (RS)

(the deviation from the line), the RSS is used. The RS ends up at zero, because of some residuals being positive deviations and some residuals being negative deviations, which end up cancelling out. The RSS squares the RS, so all residuals are positive ending with a positive sum. Also, the

RSS can be seen as “rewarding” residuals that are small (less than 1) and “penalizing” residuals that are large (greater than 1) [Heym (2018)].

LOD was calculated using:

LOD = 3.3 * [Standard deviation of the response/slope of the calibration curve for each analyte]

LOQ was calculated using:

LOQ = 10 * [Standard deviation of the response/slope of the calibration curve for each analyte]

The LOQ is expected to be at a higher concentration than the LOD considering the factors used in the determination calculations of each. The cutoff value should exceed the LOQ by at least 2 times [Gibbs (2020), ICH (1996)]. The 2.0 cutoff/LOQ ratio is set to account for decreased sensitivity throughout the validation study as the source becomes dirty from various injections of different matrices.

CHAPTER IV: RESULTS

4.1 R^2

The R^2 values are shown in Table 4.1. There are a few notable observations. The first notable observation is all analytes had an average R^2 value above 0.97, which is above the 0.95 threshold. The second notable observation is THC has a relatively high %CV compared to the other analytes. The third observation is THC has a minimum R^2 below the 0.95 cut-off at

0.912096. The fourth observation is MDPV has a high %CV relative to the other analytes. The last notable observation is MDPV has a minimum R^2 just below cut-off at 0.944298.

Table 4.1: R^2

Analyte Average R^2 %CV Min R^2 Max R^2 10,11-Dihydro-10-Hydroxycarbamazepine 0.989345 0.28% 0.984579 0.994239 2-Hydroxyethylflurazepam 0.992947 0.37% 0.986267 0.998548 4-Methylephedrine 0.987112 0.64% 0.975246 0.997354 6-MAC 0.993025 0.32% 0.986932 0.997790 6-MAM 0.991158 0.32% 0.983619 0.994990 7-Aminoclonazepam 0.992591 0.29% 0.988910 0.998366 Alfentanil 0.990050 0.41% 0.982370 0.995011 Alprazolam 0.992186 0.27% 0.986975 0.995494 Amo-Pentobarbital 0.992070 0.42% 0.983216 0.997404 Amphetamine 0.993733 0.24% 0.988399 0.997657 Benzoylecgonine 0.992296 0.36% 0.984654 0.997018 Buprenorphine 0.991084 0.55% 0.980379 0.996531 Bupropion 0.991122 0.35% 0.986791 0.998789 Butabarbital 0.991540 0.32% 0.986539 0.995632 Butalbital 0.992075 0.38% 0.983168 0.996483 Caffeine 0.991332 0.55% 0.977262 0.997434 Cannabidiol 0.983205 0.58% 0.972482 0.990927 Carisoprodol 0.989967 0.54% 0.982996 0.997255 Clonazepam 0.993473 0.28% 0.988605 0.997509 Cocaethylene 0.991147 0.38% 0.987072 0.996890 Cocaine 0.992562 0.36% 0.986935 0.998714 Codeine 0.993517 0.28% 0.989642 0.998313 Cotinine 0.992080 0.35% 0.986970 0.996320 32

Desalkylflurazepam 0.991008 0.36% 0.984295 0.994784 Desmethyltapentadol 0.992428 0.43% 0.983285 0.999126 Dextromethorphan 0.992400 0.46% 0.984580 0.998661 Dextrorphan 0.988999 0.42% 0.978751 0.995012 Diazepam 0.992699 0.24% 0.988800 0.997801 Dihydrocodeine 0.991439 0.40% 0.983002 0.998774 Ecgonine methyl ester 0.986037 0.37% 0.977002 0.992274 EDDP 0.987669 0.57% 0.980423 0.997512 Ephedrine 0.992923 0.36% 0.987278 0.997904 Fentanyl 0.990628 0.34% 0.983645 0.995030 Flurazepam 0.987767 0.43% 0.981135 0.993833 Gabapentin 0.992596 0.35% 0.986652 0.996126 Hydrocodone 0.993285 0.31% 0.986503 0.997151 Hydromorphone 0.994108 0.32% 0.986501 0.998817 Hydroxyalprazolam 0.990835 0.39% 0.981796 0.995485 Hydroxymidazolam 0.989308 0.46% 0.982531 0.995879 Hydroxytriazolam 0.991319 0.37% 0.985416 0.997849 Ketamine 0.991891 0.34% 0.984460 0.996882 Lorazepam 0.989727 0.46% 0.983392 0.997717 MDA 0.991838 0.26% 0.988011 0.997149 MDMA 0.991223 0.35% 0.982810 0.995319 MDPV 0.981545 1.30% 0.944298 0.989364 Meperidine 0.992003 0.40% 0.984457 0.998813 Meprobamate 0.992860 0.47% 0.984459 0.998499 Methadone 0.989010 0.39% 0.983414 0.995104 Methamphetamine 0.991535 0.38% 0.987145 0.999145 Methylone 0.992005 0.27% 0.986116 0.996169 Midazolam 0.994081 0.21% 0.989620 0.996815 Morphine 0.994125 0.30% 0.990396 0.999693 Nalbuphine 0.992049 0.26% 0.988624 0.996258 Norbuprenorphine 0.992806 0.56% 0.976762 0.997069 Norcodeine 0.992361 0.37% 0.985233 0.997357 Nordiazepam 0.993747 0.30% 0.989642 0.999542 Norfentanyl 0.991761 0.29% 0.987146 0.996558 Norhydrocodone 0.994232 0.43% 0.984599 0.999589 Norhydromorphone 0.983863 0.42% 0.975642 0.990502 Norketamine 0.993045 0.40% 0.985520 0.999755 Normeperidine 0.990832 0.30% 0.984253 0.994764 Noroxycodone 0.992265 0.47% 0.984204 0.999312 O-Desmethyl-cis-Tramadol 0.983686 0.49% 0.976847 0.994113 Oxazepam 0.990779 0.38% 0.984977 0.997719 33

Oxycodone 0.991962 0.28% 0.987180 0.995692 Oxymorphone 0.990320 0.29% 0.985400 0.994717 PCP 0.991965 0.30% 0.985930 0.996264 Pentazocine 0.981834 0.35% 0.975644 0.987223 Phenobarbital 0.993216 0.51% 0.983140 0.999312 Phentermine 0.994233 0.33% 0.988597 0.999145 Pregabalin 0.991649 0.31% 0.984670 0.994709 Secobarbital 0.991631 0.67% 0.976363 0.999073 Sufentanil 0.981514 0.71% 0.963867 0.989214 Tapentadol 0.991154 0.34% 0.984412 0.995342 Temazepam 0.991746 0.34% 0.986479 0.996203 THC 0.972387 2.20% 0.912096 0.992322 THC-COOH 0.987658 0.46% 0.976918 0.993663 THC-OH 0.981243 0.90% 0.957570 0.989629 Tramadol 0.989885 0.31% 0.984686 0.996728 Triazolam 0.992873 0.28% 0.988601 0.998429 Zaleplon 0.992013 0.29% 0.985744 0.995882 *Table 4.1: Shows the linearity of each analyte tested over the validation period. The table shows the average R^2 of each analyte, variation (%CV), minimum R^2 observed, and the maximum R^2 observed. Values below the 0.95 cut-off are highlighted in red.

4.2 HiQC

The HiQC data is summarized and shown in Table 4.2. A few notable observations were made. Overall, only 3 analytes had a %CV higher than 10%. The analytes above 10% CV were

Cannabidiol, THC, and THC-OH, where THC was the only analyte above the 20% CV cut-off proposed by SWGTOX. Another notable observation is that many of the analytes for each HiQC calculation (Batch, Intra-day, Inter-day) had a lower concentration than the theoretical concentration of 25.0 ng/mL. However, the analytes had a low %CV i.e. the precision was high, but accuracy was low for the related analytes.

Table 4.2: HiQC

HiQC Week Standard Analyte Average Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 27.4399 2.0682 7.54% 2-Hydroxyethylflurazepam 24.4053 1.6442 6.74% 4-Methylephedrine 23.5797 1.7661 7.49% 6-MAC 25.8251 1.9678 7.62% 6-MAM 24.6564 1.9365 7.85% 7-Aminoclonazepam 21.1310 1.6265 7.70% Alfentanil 23.3290 1.8833 8.07% Alprazolam 17.9466 1.4569 8.12% Amo-Pentobarbital 24.6139 2.2136 8.99% Amphetamine 19.7322 1.4643 7.42% Benzoylecgonine 29.5492 2.0825 7.05% Buprenorphine 24.5002 2.3527 9.60% Bupropion 28.5904 2.3449 8.20% Butabarbital 24.4825 2.2858 9.34% Butalbital 17.6390 1.5325 8.69% Caffeine 20.3117 1.7280 8.51% Cannabidiol 21.9074 4.0084 18.30% Carisoprodol 25.4491 2.5257 9.92% Clonazepam 24.2353 1.7281 7.13% Cocaethylene 20.3220 1.5380 7.57% Cocaine 24.5105 1.8737 7.64% Codeine 23.2472 1.7249 7.42% Cotinine 22.6220 1.7687 7.82% Desalkylflurazepam 22.4992 1.6615 7.38% Desmethyltapentadol 21.0134 1.5353 7.31% Dextromethorphan 23.8875 1.9001 7.95% Dextrorphan 21.7313 1.5710 7.23% Diazepam 22.5074 1.6096 7.15% Dihydrocodeine 17.7550 1.1908 6.71% Ecgonine methyl ester 16.4694 1.3026 7.91% EDDP 22.0251 1.4590 6.62% Ephedrine 20.7228 1.5394 7.43% Fentanyl 23.8947 1.8852 7.89% Flurazepam 19.4921 1.4229 7.30% Gabapentin 22.6096 1.6836 7.45% Hydrocodone 21.0275 1.4934 7.10% 35

Hydromorphone 23.5575 1.7196 7.30% Hydroxyalprazolam 19.2572 1.8712 9.72% Hydroxymidazolam 19.0869 1.6736 8.77% Hydroxytriazolam 26.2716 2.1092 8.03% Ketamine 22.0861 1.7246 7.81% Lorazepam 25.3304 1.8622 7.35% MDA 23.1978 1.6043 6.92% MDMA 17.2164 1.2930 7.51% MDPV 20.1000 1.6739 8.33% Meperidine 21.0276 1.6735 7.96% Meprobamate 25.1749 2.2237 8.83% Methadone 22.5776 1.6758 7.42% Methamphetamine 23.6994 1.8589 7.84% Methylone 28.7061 2.2566 7.86% Midazolam 20.8279 1.5374 7.38% Morphine 23.4501 1.7682 7.54% Nalbuphine 20.2683 1.6771 8.27% Norbuprenorphine 25.2439 2.1163 8.38% Norcodeine 22.9177 1.6432 7.17% Nordiazepam 21.6495 1.6173 7.47% Norfentanyl 19.0672 1.4056 7.37% Norhydrocodone 19.7509 1.6327 8.27% Norhydromorphone 21.9866 1.8775 8.54% Norketamine 15.2939 1.1407 7.46% Normeperidine 23.2692 1.6384 7.04% Noroxycodone 21.3741 1.5687 7.34% O-Desmethyl-cis-Tramadol 22.8497 2.0237 8.86% Oxazepam 25.2743 1.9301 7.64% Oxycodone 20.2937 1.4919 7.35% Oxymorphone 20.5991 1.5411 7.48% PCP 22.6349 1.7287 7.64% Pentazocine 25.4936 2.1677 8.50% Phenobarbital 25.9151 2.2627 8.73% Phentermine 22.7900 1.7324 7.60% Pregabalin 23.7604 1.7622 7.42% Secobarbital 21.8405 1.7730 8.12% Sufentanil 24.4926 2.0916 8.54% Tapentadol 25.7321 1.9955 7.75% Temazepam 21.2082 1.4270 6.73% THC 27.1231 7.2590 26.76% THC-COOH 21.2637 2.0759 9.76% 36

THC-OH 22.3021 2.3046 10.33% Tramadol 23.8124 1.9403 8.15% Triazolam 20.0315 1.5669 7.82% Zaleplon 24.4137 1.7556 7.19% *Table 4.2: The HiQC theoretical concentration was 25.0 ng/mL. The average HiQC concentration over the validation period is show under the “Week Average” column. The average was calculated by taking the average of each duplicate HiQC from each run. Standard deviation was calculated by taking the standard deviation of the calculated average of each duplicate. %CV was calculated by dividing the Standard Deviation by the Average.

4.2.1 HiQC Batch Average

The HiQC Batch Averages data is summarized in Table 4.2.1. Cannabidiol and THC were the only analytes over 10% CV and THC was over the 20% limit. As highlighted in the previous section “HiQC”, the concentration for many analytes was lower than expected, but they were still precise.

Table 4.2.1: HiQC Batch Average

HiQC Batch Averages Standard Average Analyte Deviation %CV (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 27.4399 1.8829 6.86% 2-Hydroxyethylflurazepam 24.4053 1.3647 5.59% 4-Methylephedrine 23.5797 1.6542 7.02% 6-MAC 25.8251 1.8318 7.09% 6-MAM 24.6564 1.7666 7.16% 7-Aminoclonazepam 21.1310 1.5401 7.29% Alfentanil 23.3290 1.7467 7.49% Alprazolam 17.9466 1.2933 7.21% Amo-Pentobarbital 24.6139 1.9537 7.94% Amphetamine 19.7322 1.3805 7.00% Benzoylecgonine 29.5492 1.9145 6.48% Buprenorphine 24.5002 2.1334 8.71% Bupropion 28.5904 2.2129 7.74% Butabarbital 24.4825 1.8524 7.57% Butalbital 17.6390 1.2828 7.27% Caffeine 20.3117 1.5873 7.81% Cannabidiol 21.9074 3.9833 18.18% Carisoprodol 25.4491 2.0483 8.05% Clonazepam 24.2353 1.6220 6.69% Cocaethylene 20.3220 1.4725 7.25% Cocaine 24.5105 1.7946 7.32% Codeine 23.2472 1.5493 6.66% Cotinine 22.6220 1.6341 7.22% Desalkylflurazepam 22.4992 1.5606 6.94% Desmethyltapentadol 21.0134 1.4222 6.77% Dextromethorphan 23.8875 1.7256 7.22% Dextrorphan 21.7313 1.4225 6.55% Diazepam 22.5074 1.5173 6.74% Dihydrocodeine 17.7550 1.1125 6.27% Ecgonine methyl ester 16.4694 1.2075 7.33% EDDP 22.0251 1.4033 6.37% Ephedrine 20.7228 1.3830 6.67% Fentanyl 23.8947 1.7512 7.33% Flurazepam 19.4921 1.3080 6.71% Gabapentin 22.6096 1.5177 6.71% 38

Hydrocodone 21.0275 1.4172 6.74% Hydromorphone 23.5575 1.5600 6.62% Hydroxyalprazolam 19.2572 1.5672 8.14% Hydroxymidazolam 19.0869 1.3760 7.21% Hydroxytriazolam 26.2716 1.6019 6.10% Ketamine 22.0861 1.6137 7.31% Lorazepam 25.3304 1.6903 6.67% MDA 23.1978 1.4846 6.40% MDMA 17.2164 1.1989 6.96% MDPV 20.1000 1.3615 6.77% Meperidine 21.0276 1.5446 7.35% Meprobamate 25.1749 2.0230 8.04% Methadone 22.5776 1.6118 7.14% Methamphetamine 23.6994 1.7734 7.48% Methylone 28.7061 2.1606 7.53% Midazolam 20.8279 1.3376 6.42% Morphine 23.4501 1.6623 7.09% Nalbuphine 20.2683 1.4623 7.21% Norbuprenorphine 25.2439 1.9374 7.67% Norcodeine 22.9177 1.4058 6.13% Nordiazepam 21.6495 1.5090 6.97% Norfentanyl 19.0672 1.2991 6.81% Norhydrocodone 19.7509 1.5210 7.70% Norhydromorphone 21.9866 1.6472 7.49% Norketamine 15.2939 1.0877 7.11% Normeperidine 23.2692 1.5314 6.58% Noroxycodone 21.3741 1.2889 6.03% O-Desmethyl-cis-Tramadol 22.8497 1.8907 8.27% Oxazepam 25.2743 1.8059 7.15% Oxycodone 20.2937 1.3794 6.80% Oxymorphone 20.5991 1.3850 6.72% PCP 22.6349 1.6313 7.21% Pentazocine 25.4936 2.0448 8.02% Phenobarbital 25.9151 2.0968 8.09% Phentermine 22.7900 1.5150 6.65% Pregabalin 23.7604 1.6489 6.94% Secobarbital 21.8405 1.6012 7.33% Sufentanil 24.4926 1.8556 7.58% Tapentadol 25.7321 1.8550 7.21% Temazepam 21.2082 1.3236 6.24% THC 27.1231 6.9963 25.79% 39

THC-COOH 21.2637 1.8381 8.64% THC-OH 22.3021 2.1551 9.66% Tramadol 23.8124 1.8098 7.60% Triazolam 20.0315 1.3969 6.97% Zaleplon 24.4137 1.6521 6.77% *Table 4.2.1: Batch averages were calculated by averaging the average of the first set of each duplicate and the average of the second set of each duplicate for the 24-hour period. Standard deviation was calculated by taking the standard deviation of the averages calculated from the batch averages. The %CV was calculated by dividing the standard deviation by the batch averages.

4.2.2 HiQC Intra-Day

The HiQC Intra-Day data is summarized in Table 4.2.2. Cannabidiol and THC were, again, the only analytes over 10% CV, with THC being over the 20% limit at 26.43% CV.

Table 4.2.2: HiQC Intra-Day

HiQC Intra-day Standard Average Analyte Deviation %CV (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 27.4399 1.8293 6.67% 2-Hydroxyethylflurazepam 24.4053 1.5689 6.43% 4-Methylephedrine 23.5797 1.6651 7.06% 6-MAC 25.8251 1.8278 7.08% 6-MAM 24.6564 1.8013 7.31% 7-Aminoclonazepam 21.1310 1.4382 6.81% Alfentanil 23.3290 1.7029 7.30% Alprazolam 17.9466 1.3439 7.49% Amo-Pentobarbital 24.6139 2.1272 8.64% Amphetamine 19.7322 1.3845 7.02% Benzoylecgonine 29.5492 1.9319 6.54% Buprenorphine 24.5002 2.0960 8.55% Bupropion 28.5904 2.2444 7.85% Butabarbital 24.4825 2.1494 8.78% Butalbital 17.6390 1.3071 7.41% Caffeine 20.3117 1.6143 7.95% Cannabidiol 21.9074 3.9880 18.20% Carisoprodol 25.4491 2.2362 8.79% Clonazepam 24.2353 1.6256 6.71% Cocaethylene 20.3220 1.4861 7.31% Cocaine 24.5105 1.7986 7.34% Codeine 23.2472 1.6103 6.93% Cotinine 22.6220 1.6551 7.32% Desalkylflurazepam 22.4992 1.5637 6.95% Desmethyltapentadol 21.0134 1.3856 6.59% Dextromethorphan 23.8875 1.7830 7.46% Dextrorphan 21.7313 1.4670 6.75% Diazepam 22.5074 1.5146 6.73% Dihydrocodeine 17.7550 1.0810 6.09% Ecgonine methyl ester 16.4694 1.2170 7.39% EDDP 22.0251 1.4061 6.38% Ephedrine 20.7228 1.4622 7.06% Fentanyl 23.8947 1.7888 7.49% Flurazepam 19.4921 1.3310 6.83% Gabapentin 22.6096 1.5854 7.01% 41

Hydrocodone 21.0275 1.4225 6.76% Hydromorphone 23.5575 1.6341 6.94% Hydroxyalprazolam 19.2572 1.7209 8.94% Hydroxymidazolam 19.0869 1.5157 7.94% Hydroxytriazolam 26.2716 1.7941 6.83% Ketamine 22.0861 1.6190 7.33% Lorazepam 25.3304 1.7259 6.81% MDA 23.1978 1.4839 6.40% MDMA 17.2164 1.2362 7.18% MDPV 20.1000 1.5484 7.70% Meperidine 21.0276 1.6051 7.63% Meprobamate 25.1749 1.9745 7.84% Methadone 22.5776 1.6576 7.34% Methamphetamine 23.6994 1.7748 7.49% Methylone 28.7061 2.1320 7.43% Midazolam 20.8279 1.4247 6.84% Morphine 23.4501 1.6320 6.96% Nalbuphine 20.2683 1.5613 7.70% Norbuprenorphine 25.2439 1.8673 7.40% Norcodeine 22.9177 1.5138 6.61% Nordiazepam 21.6495 1.5036 6.95% Norfentanyl 19.0672 1.2888 6.76% Norhydrocodone 19.7509 1.5034 7.61% Norhydromorphone 21.9866 1.7195 7.82% Norketamine 15.2939 1.0630 6.95% Normeperidine 23.2692 1.5396 6.62% Noroxycodone 21.3741 1.4633 6.85% O-Desmethyl-cis-Tramadol 22.8497 1.9440 8.51% Oxazepam 25.2743 1.7936 7.10% Oxycodone 20.2937 1.3914 6.86% Oxymorphone 20.5991 1.4433 7.01% PCP 22.6349 1.6414 7.25% Pentazocine 25.4936 2.0207 7.93% Phenobarbital 25.9151 2.0702 7.99% Phentermine 22.7900 1.5305 6.72% Pregabalin 23.7604 1.6240 6.84% Secobarbital 21.8405 1.5748 7.21% Sufentanil 24.4926 1.9662 8.03% Tapentadol 25.7321 1.8760 7.29% Temazepam 21.2082 1.3368 6.30% THC 27.1231 7.1692 26.43% 42

THC-COOH 21.2637 1.9540 9.19% THC-OH 22.3021 2.2285 9.99% Tramadol 23.8124 1.8384 7.72% Triazolam 20.0315 1.4271 7.12% Zaleplon 24.4137 1.6443 6.74% *Table 4.2.2: HiQC Intra-day are looking at the variations within each duplicate. The averages were calculated by averaging the measured concentration of each set in the duplicate for a run, then taking the overall average. The standard deviation was calculated by taking the standard deviation of the calculated averages with each duplicate. The %CV was calculated by dividing the standard deviation by the average.

4.2.3 HiQC Inter-Day

The HiQC Inter-Day data is summarized in Table 4.2.3. Cannabidiol and THC were above 10% CV for the inter-day calculations, with THC being above the 20% limit at 25.58%

CV.

Table 4.2.3: HiQC Inter-Day

HiQC Inter-Day Standard Average Analyte Deviation %CV (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 27.4399 1.7456 6.36% 2-Hydroxyethylflurazepam 24.4053 1.3298 5.45% 4-Methylephedrine 23.5797 1.5931 6.76% 6-MAC 25.8251 1.7116 6.63% 6-MAM 24.6564 1.7012 6.90% 7-Aminoclonazepam 21.1310 1.4012 6.63% Alfentanil 23.3290 1.6286 6.98% Alprazolam 17.9466 1.2518 6.98% Amo-Pentobarbital 24.6139 1.8811 7.64% Amphetamine 19.7322 1.3344 6.76% Benzoylecgonine 29.5492 1.8021 6.10% Buprenorphine 24.5002 1.9884 8.12% Bupropion 28.5904 2.1388 7.48% Butabarbital 24.4825 1.7130 7.00% Butalbital 17.6390 1.2286 6.97% Caffeine 20.3117 1.5145 7.46% Cannabidiol 21.9074 3.9685 18.11% Carisoprodol 25.4491 2.0022 7.87% Clonazepam 24.2353 1.5425 6.36% Cocaethylene 20.3220 1.4428 7.10% Cocaine 24.5105 1.7505 7.14% Codeine 23.2472 1.4888 6.40% Cotinine 22.6220 1.5705 6.94% Desalkylflurazepam 22.4992 1.4836 6.59% Desmethyltapentadol 21.0134 1.2888 6.13% Dextromethorphan 23.8875 1.6466 6.89% Dextrorphan 21.7313 1.3559 6.24% Diazepam 22.5074 1.4639 6.50% Dihydrocodeine 17.7550 1.0318 5.81% Ecgonine methyl ester 16.4694 1.1506 6.99% EDDP 22.0251 1.3790 6.26% Ephedrine 20.7228 1.3278 6.41% Fentanyl 23.8947 1.6774 7.02% Flurazepam 19.4921 1.2407 6.36% Gabapentin 22.6096 1.4609 6.46% 44

Hydrocodone 21.0275 1.3752 6.54% Hydromorphone 23.5575 1.4988 6.36% Hydroxyalprazolam 19.2572 1.5326 7.96% Hydroxymidazolam 19.0869 1.3296 6.97% Hydroxytriazolam 26.2716 1.5098 5.75% Ketamine 22.0861 1.5494 7.02% Lorazepam 25.3304 1.5981 6.31% MDA 23.1978 1.4098 6.08% MDMA 17.2164 1.1709 6.80% MDPV 20.1000 1.2479 6.21% Meperidine 21.0276 1.4894 7.08% Meprobamate 25.1749 1.8692 7.42% Methadone 22.5776 1.5989 7.08% Methamphetamine 23.6994 1.7166 7.24% Methylone 28.7061 2.0778 7.24% Midazolam 20.8279 1.3203 6.34% Morphine 23.4501 1.5680 6.69% Nalbuphine 20.2683 1.3868 6.84% Norbuprenorphine 25.2439 1.7730 7.02% Norcodeine 22.9177 1.3203 5.76% Nordiazepam 21.6495 1.4344 6.63% Norfentanyl 19.0672 1.2085 6.34% Norhydrocodone 19.7509 1.4222 7.20% Norhydromorphone 21.9866 1.5850 7.21% Norketamine 15.2939 1.0258 6.71% Normeperidine 23.2692 1.4650 6.30% Noroxycodone 21.3741 1.2268 5.74% O-Desmethyl-cis-Tramadol 22.8497 1.8391 8.05% Oxazepam 25.2743 1.7015 6.73% Oxycodone 20.2937 1.2991 6.40% Oxymorphone 20.5991 1.3133 6.38% PCP 22.6349 1.5700 6.94% Pentazocine 25.4936 1.9441 7.63% Phenobarbital 25.9151 1.9715 7.61% Phentermine 22.7900 1.4053 6.17% Pregabalin 23.7604 1.5618 6.57% Secobarbital 21.8405 1.4851 6.80% Sufentanil 24.4926 1.8364 7.50% Tapentadol 25.7321 1.7871 6.94% Temazepam 21.2082 1.2663 5.97% THC 27.1231 6.9388 25.58% 45

THC-COOH 21.2637 1.7810 8.38% THC-OH 22.3021 2.1162 9.49% Tramadol 23.8124 1.7528 7.36% Triazolam 20.0315 1.3567 6.77% Zaleplon 24.4137 1.5726 6.44% *Table 4.2.3: HiQC Inter-day studies the variation between each day of runs. The average was calculated by taking the average of each 24-hour period (2 runs in each 24 -hour period). The standard deviation was calculated by taking the standard deviation of each 24-hour period. The %CV was calculated by dividing the standard deviation by the average.

4.3 LoQC

The LoQC data is summarized and shown in Table 4.3. The LoQC has a theoretical concentration of 0.6 ng/mL and cut-off is at 1.0 ng/mL. Higher %CV is expected for all analytes.

There were 9 analytes that had a calculated %CV above 20%. The analytes: Butabarital

(23.20%), Butalbital (20.92%%), Caffeine (21.14%), Flurazepam (28.60%), Secobarbital

(24.45%), THC (99.52%), THC-OH (28.19%), and Tramadol (21.91%).

Table 4.3: LoQC

LoQC Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.6234 0.0294 4.71% 2-Hydroxyethylflurazepam 0.5954 0.1045 17.56% 4-Methylephedrine 0.6114 0.0659 10.79% 6-MAC 1.2546 0.0587 4.68% 6-MAM 0.6691 0.0441 6.59% 7-Aminoclonazepam 0.6023 0.0268 4.45% Alfentanil 0.5682 0.0240 4.23% Alprazolam 0.6027 0.0170 2.82% Amo-Pentobarbital 1.1022 0.1240 11.25% Amphetamine 0.5739 0.0374 6.52% Benzoylecgonine 0.5611 0.0250 4.45% Buprenorphine 0.5689 0.0689 12.11% Bupropion 0.5316 0.0280 5.27% Butabarbital 0.5135 0.1191 23.20% Butalbital 0.5104 0.1068 20.92% Caffeine 0.4890 0.1034 21.14% Cannabidiol 0.6945 0.1158 16.67% Carisoprodol 1.1645 0.1001 8.60% Clonazepam 0.5698 0.0401 7.04% Cocaethylene 0.6523 0.0346 5.30% Cocaine 0.6413 0.0231 3.61% Codeine 0.4943 0.0351 7.10% Cotinine 0.5676 0.0364 6.42% Desalkylflurazepam 0.5931 0.0375 6.33% Desmethyltapentadol 0.5785 0.0267 4.61% Dextromethorphan 0.5778 0.0267 4.62% Dextrorphan 0.6283 0.0274 4.37% Diazepam 0.6256 0.0225 3.60% Dihydrocodeine 0.6032 0.0501 8.30% Ecgonine methyl ester 0.5891 0.0252 4.27% EDDP 1.2422 0.1338 10.77% Ephedrine 0.5362 0.0355 6.62% Fentanyl 0.5792 0.0239 4.12% Flurazepam 0.5066 0.1449 28.60% Gabapentin 0.5485 0.0545 9.94% 47

Hydrocodone 0.5414 0.0195 3.59% Hydromorphone 0.6053 0.0316 5.23% Hydroxyalprazolam 0.5648 0.0456 8.07% Hydroxymidazolam 0.5948 0.0370 6.22% Hydroxytriazolam 0.5761 0.0546 9.48% Ketamine 0.5437 0.0263 4.83% Lorazepam 0.5392 0.0650 12.06% MDA 0.5452 0.0321 5.89% MDMA 0.5730 0.0333 5.82% MDPV 0.5738 0.0471 8.20% Meperidine 0.5767 0.0313 5.43% Meprobamate 1.0432 0.1030 9.87% Methadone 0.6584 0.0486 7.38% Methamphetamine 0.5762 0.0363 6.29% Methylone 0.6635 0.0409 6.17% Midazolam 0.6326 0.0279 4.42% Morphine 0.5831 0.0334 5.73% Nalbuphine 0.5870 0.0626 10.66% Norbuprenorphine 1.2865 0.1658 12.89% Norcodeine 0.6137 0.0650 10.60% Nordiazepam 0.5665 0.0253 4.47% Norfentanyl 1.0412 0.0445 4.28% Norhydrocodone 0.6522 0.0633 9.71% Norhydromorphone 0.6513 0.0389 5.97% Norketamine 0.5778 0.0306 5.29% Normeperidine 0.6163 0.0234 3.80% Noroxycodone 0.5871 0.0764 13.01% O-Desmethyl-cis-Tramadol 0.9721 0.0293 3.01% Oxazepam 0.5843 0.0361 6.18% Oxycodone 0.5853 0.0411 7.02% Oxymorphone 0.5742 0.0334 5.81% PCP 0.5847 0.0290 4.95% Pentazocine 0.5736 0.0324 5.66% Phenobarbital 0.6042 0.0883 14.62% Phentermine 0.5425 0.0978 18.03% Pregabalin 0.5707 0.0567 9.93% Secobarbital 0.5516 0.1349 24.45% Sufentanil 0.6345 0.0201 3.17% Tapentadol 0.5543 0.0243 4.38% Temazepam 0.5448 0.0415 7.62% THC 0.3871 0.3852 99.52% 48

THC-COOH 0.5992 0.0911 15.20% THC-OH 0.6130 0.1728 28.19% Tramadol 0.5264 0.1153 21.91% Triazolam 0.5785 0.0441 7.63% Zaleplon 0.5678 0.0485 8.54% *Table 4.3: The average LoQC concentration over the validation period is show under the “Average” column. The average was calculated by taking the average of each duplicate LoQC from each run. Standard deviation was calculated by taking the standard deviation of the calculated average of each duplicate. %CV was calculated by dividing the Standard Deviation by the Average.

4.3.1 LoQC Batch Average

The LoQC Batch Average data is summarized and shown in Table 4.3.1. THC and THC-

OH were the only analytes measured to have a %CV above 20%. That being said, THC was well above 20% at 52.57%.

Table 4.3.1: LoQC Batch Average

LoQC Batch Averages Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.6234 0.0226 3.62% 2-Hydroxyethylflurazepam 0.5954 0.0846 14.20% 4-Methylephedrine 0.6114 0.0557 9.12% 6-MAC 1.2546 0.0464 3.70% 6-MAM 0.6691 0.0388 5.80% 7-Aminoclonazepam 0.6023 0.0224 3.71% Alfentanil 0.5682 0.0203 3.57% Alprazolam 0.6027 0.0145 2.41% Amo-Pentobarbital 1.1022 0.0717 6.50% Amphetamine 0.5739 0.0323 5.63% Benzoylecgonine 0.5611 0.0216 3.85% Buprenorphine 0.5689 0.0557 9.79% Bupropion 0.5316 0.0224 4.21% Butabarbital 0.5135 0.0790 15.38% Butalbital 0.5104 0.0454 8.89% Caffeine 0.4890 0.0612 12.52% Cannabidiol 0.6945 0.0777 11.19% Carisoprodol 1.1645 0.0681 5.85% Clonazepam 0.5698 0.0355 6.23% Cocaethylene 0.6523 0.0293 4.49% Cocaine 0.6413 0.0187 2.91% Codeine 0.4943 0.0166 3.37% Cotinine 0.5676 0.0282 4.98% Desalkylflurazepam 0.5931 0.0187 3.15% Desmethyltapentadol 0.5785 0.0203 3.51% Dextromethorphan 0.5778 0.0192 3.32% Dextrorphan 0.6283 0.0202 3.22% Diazepam 0.6256 0.0148 2.36% Dihydrocodeine 0.6032 0.0350 5.80% Ecgonine methyl ester 0.5891 0.0219 3.71% EDDP 1.2422 0.1016 8.18% Ephedrine 0.5362 0.0220 4.11% Fentanyl 0.5792 0.0163 2.81% Flurazepam 0.5066 0.0906 17.89% Gabapentin 0.5485 0.0470 8.57% 50

Hydrocodone 0.5414 0.0153 2.82% Hydromorphone 0.6053 0.0175 2.89% Hydroxyalprazolam 0.5648 0.0375 6.64% Hydroxymidazolam 0.5948 0.0266 4.48% Hydroxytriazolam 0.5761 0.0404 7.02% Ketamine 0.5437 0.0210 3.87% Lorazepam 0.5392 0.0351 6.51% MDA 0.5452 0.0139 2.55% MDMA 0.5730 0.0272 4.75% MDPV 0.5738 0.0294 5.13% Meperidine 0.5767 0.0214 3.71% Meprobamate 1.0432 0.0707 6.78% Methadone 0.6584 0.0389 5.90% Methamphetamine 0.5762 0.0210 3.64% Methylone 0.6635 0.0341 5.14% Midazolam 0.6326 0.0150 2.37% Morphine 0.5831 0.0258 4.42% Nalbuphine 0.5870 0.0347 5.90% Norbuprenorphine 1.2865 0.1424 11.07% Norcodeine 0.6137 0.0411 6.70% Nordiazepam 0.5665 0.0200 3.53% Norfentanyl 1.0412 0.0357 3.43% Norhydrocodone 0.6522 0.0505 7.74% Norhydromorphone 0.6513 0.0236 3.62% Norketamine 0.5778 0.0234 4.05% Normeperidine 0.6163 0.0187 3.03% Noroxycodone 0.5871 0.0549 9.35% O-Desmethyl-cis-Tramadol 0.9721 0.0128 1.31% Oxazepam 0.5843 0.0291 4.98% Oxycodone 0.5853 0.0318 5.43% Oxymorphone 0.5742 0.0245 4.26% PCP 0.5847 0.0130 2.23% Pentazocine 0.5736 0.0140 2.45% Phenobarbital 0.6042 0.0671 11.11% Phentermine 0.5425 0.0556 10.26% Pregabalin 0.5707 0.0456 7.99% Secobarbital 0.5516 0.0992 17.98% Sufentanil 0.6345 0.0153 2.41% Tapentadol 0.5543 0.0209 3.77% Temazepam 0.5448 0.0334 6.13% THC 0.3871 0.2035 52.57% 51

THC-COOH 0.5992 0.0690 11.51% THC-OH 0.6130 0.1467 23.92% Tramadol 0.5264 0.0792 15.04% Triazolam 0.5785 0.0188 3.24% Zaleplon 0.5678 0.0398 7.01% *Table 4.3.1: Batch averages were calculated by averaging the average of the first set of each duplicate and the average of the second set of each duplicate for the 24-hour period. Standard deviation was calculated by taking the standard deviation of the averages calculated from the batch averages. The %CV was calculated by dividing the standard deviation by the batch averages.

4.3.2 LoQC Intra-Day

The LoQC Intra-Day data is summarized and shown in Table 4.3.2. Caffeine, THC, and

THC-OH were all above 20%, with THC calculated at 78.35%.

Table 4.3.2: LoQC Intra-Day

LoQC Intra-Day Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.6234 0.0203 3.25% 2-Hydroxyethylflurazepam 0.5954 0.0834 14.00% 4-Methylephedrine 0.6114 0.0598 9.79% 6-MAC 1.2546 0.0527 4.20% 6-MAM 0.6691 0.0353 5.28% 7-Aminoclonazepam 0.6023 0.0238 3.94% Alfentanil 0.5682 0.0207 3.64% Alprazolam 0.6027 0.0155 2.58% Amo-Pentobarbital 1.1022 0.0914 8.29% Amphetamine 0.5739 0.0315 5.49% Benzoylecgonine 0.5611 0.0239 4.25% Buprenorphine 0.5689 0.0615 10.80% Bupropion 0.5316 0.0250 4.71% Butabarbital 0.5135 0.0834 16.25% Butalbital 0.5104 0.0829 16.25% Caffeine 0.4890 0.0989 20.23% Cannabidiol 0.6945 0.0913 13.15% Carisoprodol 1.1645 0.0883 7.58% Clonazepam 0.5698 0.0363 6.37% Cocaethylene 0.6523 0.0303 4.65% Cocaine 0.6413 0.0188 2.93% Codeine 0.4943 0.0297 6.01% Cotinine 0.5676 0.0302 5.32% Desalkylflurazepam 0.5931 0.0352 5.94% Desmethyltapentadol 0.5785 0.0237 4.10% Dextromethorphan 0.5778 0.0245 4.24% Dextrorphan 0.6283 0.0211 3.36% Diazepam 0.6256 0.0185 2.96% Dihydrocodeine 0.6032 0.0449 7.45% Ecgonine methyl ester 0.5891 0.0239 4.07% EDDP 1.2422 0.1285 10.34% Ephedrine 0.5362 0.0279 5.21% Fentanyl 0.5792 0.0208 3.59% Flurazepam 0.5066 0.0937 18.50% Gabapentin 0.5485 0.0497 9.05% 53

Hydrocodone 0.5414 0.0146 2.70% Hydromorphone 0.6053 0.0259 4.28% Hydroxyalprazolam 0.5648 0.0367 6.49% Hydroxymidazolam 0.5948 0.0291 4.89% Hydroxytriazolam 0.5761 0.0443 7.69% Ketamine 0.5437 0.0221 4.06% Lorazepam 0.5392 0.0609 11.30% MDA 0.5452 0.0171 3.13% MDMA 0.5730 0.0285 4.97% MDPV 0.5738 0.0373 6.50% Meperidine 0.5767 0.0277 4.80% Meprobamate 1.0432 0.0818 7.85% Methadone 0.6584 0.0459 6.97% Methamphetamine 0.5762 0.0304 5.27% Methylone 0.6635 0.0319 4.80% Midazolam 0.6326 0.0245 3.87% Morphine 0.5831 0.0181 3.11% Nalbuphine 0.5870 0.0591 10.07% Norbuprenorphine 1.2865 0.1262 9.81% Norcodeine 0.6137 0.0587 9.57% Nordiazepam 0.5665 0.0187 3.30% Norfentanyl 1.0412 0.0415 3.98% Norhydrocodone 0.6522 0.0517 7.93% Norhydromorphone 0.6513 0.0351 5.40% Norketamine 0.5778 0.0271 4.69% Normeperidine 0.6163 0.0187 3.03% Noroxycodone 0.5871 0.0554 9.44% O-Desmethyl-cis-Tramadol 0.9721 0.0264 2.72% Oxazepam 0.5843 0.0253 4.33% Oxycodone 0.5853 0.0348 5.94% Oxymorphone 0.5742 0.0254 4.42% PCP 0.5847 0.0251 4.29% Pentazocine 0.5736 0.0309 5.38% Phenobarbital 0.6042 0.0704 11.66% Phentermine 0.5425 0.0680 12.54% Pregabalin 0.5707 0.0438 7.68% Secobarbital 0.5516 0.0874 15.84% Sufentanil 0.6345 0.0170 2.68% Tapentadol 0.5543 0.0215 3.89% Temazepam 0.5448 0.0330 6.05% THC 0.3871 0.3033 78.35% 54

THC-COOH 0.5992 0.0768 12.81% THC-OH 0.6130 0.1250 20.40% Tramadol 0.5264 0.0812 15.44% Triazolam 0.5785 0.0403 6.97% Zaleplon 0.5678 0.0429 7.56% *Table 4.3.2: LoQC Intra-day are looking at the variations within each duplicate. The averages were calculated by averaging the measured concentration of each set in the duplicate for a run, then taking the overall average. The standard deviation was calculated by taking the standard deviation of the calculated averages with each duplicate. The %CV was calculated by dividing the standard deviation by the average.

4.3.3 LoQC Inter-Day

The LoQC Inter-Day data is summarized and shown in Table 4.3.3. THC was the only analyte above 20% CV at 20.53%.

Table 4.3.3: LoQC Inter-Day

LoQC Inter-Day Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.6234 0.0170 2.72% 2-Hydroxyethylflurazepam 0.5954 0.0755 12.68% 4-Methylephedrine 0.6114 0.0526 8.61% 6-MAC 1.2546 0.0416 3.31% 6-MAM 0.6691 0.0314 4.70% 7-Aminoclonazepam 0.6023 0.0202 3.35% Alfentanil 0.5682 0.0182 3.21% Alprazolam 0.6027 0.0137 2.28% Amo-Pentobarbital 1.1022 0.0543 4.93% Amphetamine 0.5739 0.0281 4.89% Benzoylecgonine 0.5611 0.0213 3.79% Buprenorphine 0.5689 0.0517 9.09% Bupropion 0.5316 0.0193 3.63% Butabarbital 0.5135 0.0277 5.40% Butalbital 0.5104 0.0283 5.55% Caffeine 0.4890 0.0603 12.32% Cannabidiol 0.6945 0.0695 10.00% Carisoprodol 1.1645 0.0552 4.74% Clonazepam 0.5698 0.0333 5.84% Cocaethylene 0.6523 0.0254 3.90% Cocaine 0.6413 0.0154 2.40% Codeine 0.4943 0.0076 1.55% Cotinine 0.5676 0.0228 4.01% Desalkylflurazepam 0.5931 0.0169 2.85% Desmethyltapentadol 0.5785 0.0184 3.18% Dextromethorphan 0.5778 0.0165 2.85% Dextrorphan 0.6283 0.0128 2.04% Diazepam 0.6256 0.0125 1.99% Dihydrocodeine 0.6032 0.0333 5.52% Ecgonine methyl ester 0.5891 0.0207 3.51% EDDP 1.2422 0.0966 7.77% Ephedrine 0.5362 0.0155 2.89% Fentanyl 0.5792 0.0140 2.41% Flurazepam 0.5066 0.0469 9.26% Gabapentin 0.5485 0.0442 8.06% 56

Hydrocodone 0.5414 0.0114 2.11% Hydromorphone 0.6053 0.0085 1.40% Hydroxyalprazolam 0.5648 0.0294 5.20% Hydroxymidazolam 0.5948 0.0221 3.72% Hydroxytriazolam 0.5761 0.0318 5.52% Ketamine 0.5437 0.0172 3.17% Lorazepam 0.5392 0.0289 5.36% MDA 0.5452 0.0119 2.18% MDMA 0.5730 0.0250 4.36% MDPV 0.5738 0.0198 3.45% Meperidine 0.5767 0.0183 3.18% Meprobamate 1.0432 0.0566 5.42% Methadone 0.6584 0.0378 5.74% Methamphetamine 0.5762 0.0180 3.13% Methylone 0.6635 0.0241 3.63% Midazolam 0.6326 0.0124 1.96% Morphine 0.5831 0.0095 1.63% Nalbuphine 0.5870 0.0311 5.29% Norbuprenorphine 1.2865 0.1039 8.08% Norcodeine 0.6137 0.0373 6.08% Nordiazepam 0.5665 0.0163 2.88% Norfentanyl 1.0412 0.0328 3.15% Norhydrocodone 0.6522 0.0423 6.49% Norhydromorphone 0.6513 0.0191 2.93% Norketamine 0.5778 0.0208 3.61% Normeperidine 0.6163 0.0156 2.53% Noroxycodone 0.5871 0.0452 7.70% O-Desmethyl-cis-Tramadol 0.9721 0.0095 0.97% Oxazepam 0.5843 0.0163 2.79% Oxycodone 0.5853 0.0295 5.05% Oxymorphone 0.5742 0.0207 3.61% PCP 0.5847 0.0122 2.08% Pentazocine 0.5736 0.0116 2.03% Phenobarbital 0.6042 0.0471 7.80% Phentermine 0.5425 0.0371 6.83% Pregabalin 0.5707 0.0378 6.63% Secobarbital 0.5516 0.0625 11.34% Sufentanil 0.6345 0.0129 2.03% Tapentadol 0.5543 0.0200 3.61% Temazepam 0.5448 0.0291 5.34% THC 0.3871 0.0795 20.53% 57

THC-COOH 0.5992 0.0636 10.61% THC-OH 0.6130 0.1146 18.70% Tramadol 0.5264 0.0554 10.53% Triazolam 0.5785 0.0131 2.27% Zaleplon 0.5678 0.0369 6.49% *Table 4.3.3: LoQC Inter-day studies the variation between each day of runs. The average was calculated by taking the average of each 24-hour period (2 runs in each 24 -hour period). The standard deviation was calculated by taking the standard deviation of each 24-hour period. The %CV was calculated by dividing the standard deviation by the average.

4.4 SST (CO)

The SST (CO) data is summarized and shown in Table 4.4. The SST (CO) has a theoretical concentration of 1.0 ng/mL, which is set at the cut-off. Ideally, %CV should be below

20%. %CV for SST (CO) is expected to be higher than the HiQC data sets, but lower than LoQC and 30% (CO). There were 7 analytes that were calculated to have a %CV above 20%. The analytes included: Butalbital (21.53%), Cannabidiol (29.31%), Dextromethorphan (20.07%),

Normeperidine (21.54%), Secobarbital (27.80%), THC (73.86%), and THC-OH (32.71%).

Table 4.4: SST (CO)

SST (CO) Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 1.0459 0.0495 4.73% 2-Hydroxyethylflurazepam 1.0798 0.0961 8.90% 4-Methylephedrine 1.0332 0.0681 6.59% 6-MAC 1.0875 0.0731 6.73% 6-MAM 1.0269 0.0642 6.25% 7-Aminoclonazepam 1.0473 0.0537 5.13% Alfentanil 1.0448 0.0457 4.37% Alprazolam 1.0550 0.0633 6.00% Amo-Pentobarbital 1.0776 0.1896 17.59% Amphetamine 1.0481 0.0526 5.02% Benzoylecgonine 1.0664 0.0643 6.03% Buprenorphine 1.0366 0.1332 12.85% Bupropion 1.0230 0.0592 5.79% Butabarbital 1.0398 0.1798 17.29% Butalbital 0.9500 0.2046 21.53% Caffeine 1.0051 0.0798 7.94% Cannabidiol 1.0223 0.2997 29.31% Carisoprodol 1.0763 0.0822 7.64% Clonazepam 1.0354 0.0548 5.29% Cocaethylene 1.0124 0.0512 5.05% Cocaine 1.0333 0.0418 4.04% Codeine 1.0442 0.0866 8.29% Cotinine 1.0535 0.0602 5.71% Desalkylflurazepam 1.0562 0.0507 4.80% Desmethyltapentadol 1.0594 0.0493 4.65% Dextromethorphan 1.0189 0.2046 20.07% Dextrorphan 1.0459 0.0446 4.26% Diazepam 1.0872 0.0568 5.23% Dihydrocodeine 1.0742 0.0949 8.84% Ecgonine methyl ester 1.0305 0.0515 4.99% EDDP 0.9564 0.1419 14.84% Ephedrine 1.0815 0.0548 5.07% Fentanyl 1.0397 0.0474 4.56% Flurazepam 1.0265 0.0526 5.12% Gabapentin 1.0520 0.1023 9.73% 59

Hydrocodone 1.0438 0.0401 3.85% Hydromorphone 1.0532 0.0651 6.18% Hydroxyalprazolam 1.0560 0.0657 6.22% Hydroxymidazolam 1.0653 0.0711 6.68% Hydroxytriazolam 1.0763 0.0756 7.02% Ketamine 1.0704 0.0626 5.85% Lorazepam 1.0260 0.0918 8.95% MDA 1.0493 0.0591 5.63% MDMA 1.0339 0.0538 5.20% MDPV 0.9873 0.0629 6.37% Meperidine 1.0277 0.0460 4.48% Meprobamate 1.0361 0.0962 9.28% Methadone 0.9777 0.0716 7.32% Methamphetamine 1.0474 0.0733 7.00% Methylone 1.0317 0.0627 6.08% Midazolam 1.0646 0.0500 4.70% Morphine 1.0832 0.0575 5.31% Nalbuphine 1.0762 0.0830 7.72% Norbuprenorphine 1.1122 0.1305 11.73% Norcodeine 1.1032 0.0817 7.41% Nordiazepam 1.0673 0.0557 5.22% Norfentanyl 1.0683 0.0499 4.67% Norhydrocodone 1.0686 0.0740 6.93% Norhydromorphone 1.0488 0.0490 4.68% Norketamine 1.0541 0.0538 5.11% Normeperidine 1.0589 0.2280 21.54% Noroxycodone 1.0671 0.0997 9.34% O-Desmethyl-cis-Tramadol 1.0334 0.0530 5.13% Oxazepam 1.0502 0.0606 5.77% Oxycodone 1.0680 0.0504 4.72% Oxymorphone 1.0428 0.0643 6.17% PCP 1.0452 0.0499 4.78% Pentazocine 1.0282 0.0425 4.13% Phenobarbital 1.1140 0.1502 13.48% Phentermine 1.0183 0.0811 7.96% Pregabalin 1.0717 0.0769 7.18% Secobarbital 0.9094 0.2528 27.80% Sufentanil 0.9987 0.0564 5.65% Tapentadol 1.0448 0.0415 3.98% Temazepam 1.0499 0.0776 7.39% THC 0.6895 0.5093 73.86% 60

THC-COOH 1.0632 0.1049 9.87% THC-OH 0.8456 0.2766 32.71% Tramadol 1.0465 0.0482 4.60% Triazolam 1.0720 0.0664 6.20% Zaleplon 1.0443 0.0861 8.25% *Table 4.4: The average SST (CO) concentration over the validation period is show under the “Average” column. The average was calculated by taking the average of each duplicate SST (CO) from each run. Standard deviation was calculated by taking the standard deviation of the calculated average of each duplicate. %CV was calculated by dividing the Standard Deviation by the Average

4.4.1 SST (CO) Batch Average

The SST (CO) Batch Average data is summarized and shown in Table 4.4.1. Cannabidiol

(29.62%), Secobarbital (21.85%), THC (61.75%), and THC-OH (27.22%) all had a %CV above

20%, with THC well above 20%.

Table 4.4.1: SST (CO) Batch Average

SST (CO) Batch Averages Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 1.0459 0.0434 4.15% 2-Hydroxyethylflurazepam 1.0711 0.0702 6.56% 4-Methylephedrine 1.0183 0.0539 5.30% 6-MAC 1.0534 0.0614 5.83% 6-MAM 1.0280 0.0410 3.99% 7-Aminoclonazepam 1.0394 0.0406 3.91% Alfentanil 1.0381 0.0391 3.77% Alprazolam 1.0543 0.0546 5.18% Amo-Pentobarbital 1.0165 0.1487 14.63% Amphetamine 1.0395 0.0499 4.80% Benzoylecgonine 1.0431 0.0583 5.59% Buprenorphine 1.0086 0.0980 9.72% Bupropion 1.0001 0.0463 4.63% Butabarbital 1.0418 0.1283 12.32% Butalbital 0.9863 0.1325 13.43% Caffeine 1.0086 0.0693 6.87% Cannabidiol 0.9849 0.2917 29.62% Carisoprodol 1.0842 0.0482 4.44% Clonazepam 1.0373 0.0526 5.07% Cocaethylene 1.0054 0.0429 4.26% Cocaine 1.0184 0.0386 3.79% Codeine 1.0620 0.0644 6.07% Cotinine 1.0488 0.0531 5.06% Desalkylflurazepam 1.0442 0.0423 4.05% Desmethyltapentadol 1.0449 0.0388 3.71% Dextromethorphan 0.9694 0.1412 14.56% Dextrorphan 1.0351 0.0418 4.03% Diazepam 1.0665 0.0461 4.32% Dihydrocodeine 1.0609 0.0834 7.86% Ecgonine methyl ester 1.0184 0.0464 4.56% EDDP 0.9969 0.1132 11.36% Ephedrine 1.0803 0.0378 3.50% Fentanyl 1.0255 0.0455 4.44% Flurazepam 1.0138 0.0474 4.67% Gabapentin 1.0582 0.0958 9.06% 62

Hydrocodone 1.0334 0.0338 3.27% Hydromorphone 1.0500 0.0506 4.82% Hydroxyalprazolam 1.0718 0.0413 3.85% Hydroxymidazolam 1.0735 0.0566 5.27% Hydroxytriazolam 1.0774 0.0476 4.42% Ketamine 1.0570 0.0561 5.30% Lorazepam 1.0251 0.0559 5.45% MDA 1.0311 0.0500 4.85% MDMA 1.0156 0.0453 4.46% MDPV 0.9587 0.0495 5.16% Meperidine 1.0254 0.0311 3.03% Meprobamate 1.0415 0.0782 7.50% Methadone 0.9656 0.0617 6.38% Methamphetamine 1.0231 0.0427 4.18% Methylone 1.0150 0.0349 3.44% Midazolam 1.0566 0.0469 4.43% Morphine 1.0679 0.0525 4.91% Nalbuphine 1.0613 0.0586 5.52% Norbuprenorphine 1.0607 0.1042 9.83% Norcodeine 1.0702 0.0725 6.78% Nordiazepam 1.0617 0.0489 4.61% Norfentanyl 1.0557 0.0436 4.13% Norhydrocodone 1.0470 0.0607 5.79% Norhydromorphone 1.0324 0.0380 3.68% Norketamine 1.0373 0.0427 4.11% Normeperidine 0.9948 0.1632 16.40% Noroxycodone 1.0500 0.0817 7.78% O-Desmethyl-cis-Tramadol 1.0348 0.0404 3.90% Oxazepam 1.0480 0.0484 4.62% Oxycodone 1.0475 0.0427 4.07% Oxymorphone 1.0337 0.0552 5.34% PCP 1.0276 0.0447 4.35% Pentazocine 1.0165 0.0300 2.95% Phenobarbital 1.1050 0.0724 6.55% Phentermine 1.0026 0.0522 5.21% Pregabalin 1.0506 0.0561 5.34% Secobarbital 0.8834 0.1930 21.85% Sufentanil 0.9953 0.0515 5.18% Tapentadol 1.0391 0.0379 3.65% Temazepam 1.0516 0.0698 6.63% THC 0.7127 0.4401 61.75% 63

THC-COOH 1.0413 0.0794 7.62% THC-OH 0.8986 0.2446 27.22% Tramadol 1.0323 0.0370 3.58% Triazolam 1.0680 0.0465 4.36% Zaleplon 1.0228 0.0660 6.45% *Table 4.4.1: Batch averages were calculated by averaging the average of the first set of each duplicate and the average of the second set of each duplicate for the 24-hour period. Standard deviation was calculated by taking the standard deviation of the averages calculated from the batch averages. The %CV was calculated by dividing the standard deviation by the batch averages.

4.4.2 SST (CO) Intra-Day

The SST (CO) Intra-Day data is summarized and shown in Table 4.4.2. Cannabidiol

(29.88%), Secobarbital (24.75%), THC (63.92%), and THC-OH (27.37%) all had a %CV above

20%, with THC well above 20%.

Table 4.4.2: SST (CO) Intra-Day

SST (CO) Intra-Day Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 1.0459 0.0430 4.11% 2-Hydroxyethylflurazepam 1.0711 0.0697 6.51% 4-Methylephedrine 1.0183 0.0556 5.46% 6-MAC 1.0534 0.0548 5.21% 6-MAM 1.0280 0.0529 5.14% 7-Aminoclonazepam 1.0394 0.0478 4.60% Alfentanil 1.0381 0.0434 4.18% Alprazolam 1.0543 0.0610 5.78% Amo-Pentobarbital 1.0165 0.1438 14.14% Amphetamine 1.0395 0.0417 4.01% Benzoylecgonine 1.0431 0.0578 5.54% Buprenorphine 1.0086 0.1129 11.19% Bupropion 1.0001 0.0499 4.99% Butabarbital 1.0418 0.0945 9.07% Butalbital 0.9863 0.1280 12.98% Caffeine 1.0086 0.0730 7.24% Cannabidiol 0.9849 0.2943 29.88% Carisoprodol 1.0842 0.0626 5.77% Clonazepam 1.0373 0.0528 5.09% Cocaethylene 1.0054 0.0460 4.58% Cocaine 1.0184 0.0378 3.71% Codeine 1.0620 0.0649 6.11% Cotinine 1.0488 0.0583 5.56% Desalkylflurazepam 1.0442 0.0422 4.04% Desmethyltapentadol 1.0449 0.0451 4.31% Dextromethorphan 0.9694 0.1395 14.39% Dextrorphan 1.0351 0.0418 4.04% Diazepam 1.0665 0.0499 4.68% Dihydrocodeine 1.0609 0.0859 8.09% Ecgonine methyl ester 1.0184 0.0472 4.63% EDDP 0.9969 0.1201 12.05% Ephedrine 1.0803 0.0483 4.47% Fentanyl 1.0255 0.0446 4.34% Flurazepam 1.0138 0.0496 4.89% Gabapentin 1.0582 0.0865 8.18% 65

Hydrocodone 1.0334 0.0339 3.28% Hydromorphone 1.0500 0.0533 5.08% Hydroxyalprazolam 1.0718 0.0532 4.96% Hydroxymidazolam 1.0735 0.0540 5.03% Hydroxytriazolam 1.0774 0.0632 5.87% Ketamine 1.0570 0.0584 5.53% Lorazepam 1.0251 0.0839 8.19% MDA 1.0311 0.0509 4.94% MDMA 1.0156 0.0452 4.45% MDPV 0.9587 0.0519 5.42% Meperidine 1.0254 0.0455 4.44% Meprobamate 1.0415 0.0831 7.98% Methadone 0.9656 0.0690 7.15% Methamphetamine 1.0231 0.0495 4.84% Methylone 1.0150 0.0536 5.28% Midazolam 1.0566 0.0388 3.67% Morphine 1.0679 0.0481 4.50% Nalbuphine 1.0613 0.0717 6.76% Norbuprenorphine 1.0607 0.0807 7.61% Norcodeine 1.0702 0.0670 6.26% Nordiazepam 1.0617 0.0523 4.92% Norfentanyl 1.0557 0.0473 4.48% Norhydrocodone 1.0470 0.0625 5.97% Norhydromorphone 1.0324 0.0388 3.76% Norketamine 1.0373 0.0460 4.43% Normeperidine 0.9948 0.1629 16.37% Noroxycodone 1.0500 0.0804 7.66% O-Desmethyl-cis-Tramadol 1.0348 0.0514 4.97% Oxazepam 1.0480 0.0549 5.24% Oxycodone 1.0475 0.0357 3.41% Oxymorphone 1.0337 0.0581 5.62% PCP 1.0276 0.0433 4.22% Pentazocine 1.0165 0.0376 3.70% Phenobarbital 1.1050 0.0528 4.78% Phentermine 1.0026 0.0683 6.81% Pregabalin 1.0506 0.0559 5.32% Secobarbital 0.8834 0.2186 24.75% Sufentanil 0.9953 0.0515 5.18% Tapentadol 1.0391 0.0364 3.50% Temazepam 1.0516 0.0690 6.56% THC 0.7127 0.4556 63.92% 66

THC-COOH 1.0413 0.0757 7.27% THC-OH 0.8986 0.2460 27.37% Tramadol 1.0323 0.0420 4.07% Triazolam 1.0680 0.0617 5.78% Zaleplon 1.0228 0.0754 7.37% *Table 4.4.2: SST (CO) Intra-day are looking at the variations within each duplicate. The averages were calculated by averaging the measured concentration of each set in the duplicate for a run, then taking the overall average. The standard deviation was calculated by taking the standard deviation of the calculated averages with each duplicate. The %CV was calculated by dividing the standard deviation by the average.

4.4.3 SST (CO) Inter-Day

The SST (CO) Inter-Day data is summarized and shown in Table 4.4.3. Cannabidiol

(29.28%), THC (55.47%), and THC-OH (25.50%) were above 20% CV, with THC well above

20%.

Table 4.4.3: SST (CO) Inter-Day

SST (CO) Inter-Day Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 1.0459 0.0409 3.91% 2-Hydroxyethylflurazepam 1.0711 0.0572 5.34% 4-Methylephedrine 1.0183 0.0476 4.67% 6-MAC 1.0534 0.0488 4.64% 6-MAM 1.0280 0.0325 3.16% 7-Aminoclonazepam 1.0394 0.0377 3.63% Alfentanil 1.0381 0.0372 3.59% Alprazolam 1.0543 0.0526 4.99% Amo-Pentobarbital 1.0165 0.1153 11.34% Amphetamine 1.0395 0.0403 3.88% Benzoylecgonine 1.0431 0.0522 5.00% Buprenorphine 1.0086 0.0825 8.18% Bupropion 1.0001 0.0383 3.83% Butabarbital 1.0418 0.0657 6.31% Butalbital 0.9863 0.0438 4.44% Caffeine 1.0086 0.0673 6.67% Cannabidiol 0.9849 0.2884 29.28% Carisoprodol 1.0842 0.0388 3.58% Clonazepam 1.0373 0.0514 4.95% Cocaethylene 1.0054 0.0397 3.95% Cocaine 1.0184 0.0349 3.43% Codeine 1.0620 0.0565 5.32% Cotinine 1.0488 0.0521 4.97% Desalkylflurazepam 1.0442 0.0356 3.41% Desmethyltapentadol 1.0449 0.0342 3.27% Dextromethorphan 0.9694 0.0930 9.59% Dextrorphan 1.0351 0.0400 3.87% Diazepam 1.0665 0.0398 3.73% Dihydrocodeine 1.0609 0.0759 7.15% Ecgonine methyl ester 1.0184 0.0446 4.38% EDDP 0.9969 0.0985 9.88% Ephedrine 1.0803 0.0349 3.23% Fentanyl 1.0255 0.0429 4.18% Flurazepam 1.0138 0.0449 4.43% Gabapentin 1.0582 0.0816 7.71% 68

Hydrocodone 1.0334 0.0303 2.93% Hydromorphone 1.0500 0.0429 4.09% Hydroxyalprazolam 1.0718 0.0227 2.12% Hydroxymidazolam 1.0735 0.0417 3.89% Hydroxytriazolam 1.0774 0.0388 3.60% Ketamine 1.0570 0.0538 5.09% Lorazepam 1.0251 0.0433 4.23% MDA 1.0311 0.0417 4.04% MDMA 1.0156 0.0399 3.93% MDPV 0.9587 0.0379 3.96% Meperidine 1.0254 0.0309 3.01% Meprobamate 1.0415 0.0645 6.19% Methadone 0.9656 0.0597 6.18% Methamphetamine 1.0231 0.0269 2.63% Methylone 1.0150 0.0298 2.93% Midazolam 1.0566 0.0365 3.46% Morphine 1.0679 0.0444 4.16% Nalbuphine 1.0613 0.0537 5.06% Norbuprenorphine 1.0607 0.0547 5.16% Norcodeine 1.0702 0.0626 5.85% Nordiazepam 1.0617 0.0476 4.48% Norfentanyl 1.0557 0.0412 3.90% Norhydrocodone 1.0470 0.0499 4.77% Norhydromorphone 1.0324 0.0247 2.40% Norketamine 1.0373 0.0371 3.58% Normeperidine 0.9948 0.1169 11.75% Noroxycodone 1.0500 0.0729 6.94% O-Desmethyl-cis-Tramadol 1.0348 0.0398 3.85% Oxazepam 1.0480 0.0432 4.13% Oxycodone 1.0475 0.0297 2.83% Oxymorphone 1.0337 0.0530 5.12% PCP 1.0276 0.0404 3.93% Pentazocine 1.0165 0.0253 2.49% Phenobarbital 1.1050 0.0185 1.67% Phentermine 1.0026 0.0386 3.85% Pregabalin 1.0506 0.0422 4.02% Secobarbital 0.8834 0.1511 17.10% Sufentanil 0.9953 0.0477 4.79% Tapentadol 1.0391 0.0354 3.41% Temazepam 1.0516 0.0666 6.34% THC 0.7127 0.3954 55.47% 69

THC-COOH 1.0413 0.0724 6.95% THC-OH 0.8986 0.2292 25.50% Tramadol 1.0323 0.0298 2.88% Triazolam 1.0680 0.0446 4.18% Zaleplon 1.0228 0.0569 5.56% *Table 4.4.3: SST (CO) Inter-day studies the variation between each day of runs. The average was calculated by taking the average of each 24-hour period (2 runs in each 24 -hour period). The standard deviation was calculated by taking the standard deviation of each 24-hour period. The %CV was calculated by dividing the standard deviation by the average.

4.5 30% (CO)

The 30% (CO) data is summarized and shown in Table 4.5. 30% (CO) has a theoretical concentration of 0.3 ng/mL, where cut-off is at 1.0 ng/mL. The expectation is that the %CV for all analytes is much higher than HiQC, SST (CO), and LoQC due to the 30% (CO) QC being the lowest concentrated QC. As expected, the %CV was higher for all analytes and the 30% (CO)

QC had the most analytes above 20% CV. The analytes were: Amo-Pentobarbital (27.26%),

Butabarbital (37.91%), Butalbital (38.65%), Caffeine (23.75%), Cocaine (20.49%), EDDP

(25.27%), Lorazepam (21.88%), Norbuprenorphine (20.68%), Norcodeine (20.78%),

Normeperidine (21.02%), Noroxycodone (20.31%), Phenobarbial (26.13%), Phentermine

(23.69%), Secobarbital (35.02%), THC (150.78%), THC-COOH (34.36%), and THC-OH

(42.70%).

Table 4.5: 30% (CO)

30% CO Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.3448 0.0162 4.70% 2-Hydroxyethylflurazepam 0.3277 0.0596 18.19% 4-Methylephedrine 0.3431 0.0529 15.42% 6-MAC 0.6596 0.0603 9.15% 6-MAM 0.3630 0.0323 8.88% 7-Aminoclonazepam 0.3094 0.0283 9.13% Alfentanil 0.3110 0.0103 3.32% Alprazolam 0.3191 0.0365 11.43% Amo-Pentobarbital 0.4980 0.1358 27.26% Amphetamine 0.3073 0.0540 17.57% Benzoylecgonine 0.3103 0.0248 7.99% Buprenorphine 0.3117 0.0592 19.01% Bupropion 0.2939 0.0169 5.75% Butabarbital 0.2815 0.1067 37.91% Butalbital 0.2824 0.1091 38.65% Caffeine 0.3401 0.0808 23.75% Cannabidiol 0.3207 0.0563 17.57% Carisoprodol 0.5701 0.0774 13.58% Clonazepam 0.3048 0.0403 13.21% Cocaethylene 0.3342 0.0256 7.65% Cocaine 0.3262 0.0668 20.49% Codeine 0.2551 0.0357 14.01% Cotinine 0.2970 0.0332 11.17% Desalkylflurazepam 0.3115 0.0287 9.21% Desmethyltapentadol 0.3080 0.0266 8.65% Dextromethorphan 0.3143 0.0202 6.44% Dextrorphan 0.3429 0.0190 5.54% Diazepam 0.3332 0.0247 7.41% Dihydrocodeine 0.3458 0.0500 14.45% Ecgonine methyl ester 0.3415 0.0164 4.80% EDDP 0.5871 0.1484 25.27% Ephedrine 0.2786 0.0248 8.90% Fentanyl 0.3081 0.0121 3.91% Flurazepam 0.3001 0.0098 3.26% Gabapentin 0.3169 0.0497 15.67% 71

Hydrocodone 0.2880 0.0169 5.88% Hydromorphone 0.3292 0.0302 9.16% Hydroxyalprazolam 0.2998 0.0353 11.77% Hydroxymidazolam 0.2923 0.0312 10.66% Hydroxytriazolam 0.3076 0.0563 18.31% Ketamine 0.2902 0.0183 6.30% Lorazepam 0.3010 0.0659 21.88% MDA 0.3082 0.0308 9.98% MDMA 0.3141 0.0227 7.23% MDPV 0.3481 0.0635 18.25% Meperidine 0.3122 0.0179 5.73% Meprobamate 0.5336 0.0885 16.59% Methadone 0.3369 0.0326 9.67% Methamphetamine 0.2975 0.0399 13.43% Methylone 0.3614 0.0288 7.97% Midazolam 0.3229 0.0115 3.57% Morphine 0.3060 0.0480 15.68% Nalbuphine 0.3209 0.0532 16.58% Norbuprenorphine 0.6288 0.1301 20.68% Norcodeine 0.3384 0.0703 20.78% Nordiazepam 0.3033 0.0194 6.40% Norfentanyl 0.5525 0.0310 5.60% Norhydrocodone 0.3432 0.0378 11.00% Norhydromorphone 0.3877 0.0467 12.05% Norketamine 0.3025 0.0228 7.52% Normeperidine 0.3361 0.0706 21.02% Noroxycodone 0.2917 0.0592 20.31% O-Desmethyl-cis-Tramadol 0.5468 0.0508 9.29% Oxazepam 0.3197 0.0346 10.83% Oxycodone 0.3159 0.0246 7.80% Oxymorphone 0.3062 0.0270 8.81% PCP 0.3120 0.0190 6.09% Pentazocine 0.3390 0.0429 12.64% Phenobarbital 0.3483 0.0910 26.13% Phentermine 0.3215 0.0762 23.69% Pregabalin 0.3015 0.0414 13.72% Secobarbital 0.3167 0.1109 35.02% Sufentanil 0.3516 0.0116 3.29% Tapentadol 0.3062 0.0173 5.64% Temazepam 0.3004 0.0228 7.57% THC 0.1348 0.2033 150.78% 72

THC-COOH 0.2943 0.1011 34.36% THC-OH 0.3120 0.1332 42.70% Tramadol 0.3222 0.0224 6.96% Triazolam 0.3007 0.0332 11.02% Zaleplon 0.3040 0.0451 14.84% *Table 4.5: The average 30% (CO) concentration over the validation period is show under the “Average” column. The average was calculated by taking the average of each duplicate 30% (CO) from each run. Standard deviation was calculated by taking the standard deviation of the calculated average of each duplicate. %CV was calculated by dividing the Standard Deviation by the Average

4.5.1 30% (CO) Batch Average

The 30% (CO) Batch Average data is summarized and shown in Table 4.5.1. Butabarbital

(22.95%), Butalbital (22.65%), Caffeine (20.26%), EDDP (20.12%), Secobarbital (30.44%),

THC (163.35%), THC-COOH (29.20%), and THC-OH (29.41%) were all above a 20% CV.

Table 4.5.1: 30% (CO) Batch Average

30% CO Batch Averages Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.3448 0.0116 3.36% 2-Hydroxyethylflurazepam 0.3334 0.0434 13.01% 4-Methylephedrine 0.3317 0.0369 11.11% 6-MAC 0.6424 0.0514 8.01% 6-MAM 0.3619 0.0159 4.39% 7-Aminoclonazepam 0.3084 0.0144 4.68% Alfentanil 0.3060 0.0084 2.73% Alprazolam 0.3170 0.0319 10.06% Amo-Pentobarbital 0.5430 0.0935 17.23% Amphetamine 0.3065 0.0453 14.79% Benzoylecgonine 0.3056 0.0194 6.35% Buprenorphine 0.3087 0.0520 16.86% Bupropion 0.2878 0.0106 3.67% Butabarbital 0.2990 0.0686 22.95% Butalbital 0.2997 0.0679 22.65% Caffeine 0.3187 0.0646 20.26% Cannabidiol 0.3254 0.0343 10.55% Carisoprodol 0.5772 0.0467 8.09% Clonazepam 0.3024 0.0311 10.29% Cocaethylene 0.3342 0.0152 4.55% Cocaine 0.3118 0.0443 14.20% Codeine 0.2459 0.0257 10.46% Cotinine 0.2905 0.0296 10.18% Desalkylflurazepam 0.3065 0.0241 7.87% Desmethyltapentadol 0.3023 0.0244 8.08% Dextromethorphan 0.3114 0.0150 4.82% Dextrorphan 0.3414 0.0170 4.98% Diazepam 0.3262 0.0172 5.27% Dihydrocodeine 0.3358 0.0342 10.20% Ecgonine methyl ester 0.3358 0.0146 4.35% EDDP 0.5587 0.1124 20.12% Ephedrine 0.2795 0.0169 6.03% Fentanyl 0.3041 0.0095 3.12% Flurazepam 0.2985 0.0052 1.75% Gabapentin 0.3054 0.0338 11.07% 74

Hydrocodone 0.2882 0.0106 3.68% Hydromorphone 0.3215 0.0162 5.04% Hydroxyalprazolam 0.2990 0.0202 6.77% Hydroxymidazolam 0.2894 0.0276 9.55% Hydroxytriazolam 0.3116 0.0445 14.28% Ketamine 0.2845 0.0170 5.99% Lorazepam 0.2871 0.0378 13.18% MDA 0.3015 0.0174 5.76% MDMA 0.3146 0.0191 6.06% MDPV 0.3355 0.0259 7.71% Meperidine 0.3043 0.0148 4.85% Meprobamate 0.5624 0.0624 11.10% Methadone 0.3353 0.0290 8.66% Methamphetamine 0.3046 0.0276 9.07% Methylone 0.3504 0.0206 5.89% Midazolam 0.3198 0.0080 2.51% Morphine 0.3048 0.0391 12.82% Nalbuphine 0.3229 0.0323 9.99% Norbuprenorphine 0.6566 0.1007 15.34% Norcodeine 0.3235 0.0301 9.32% Nordiazepam 0.2997 0.0139 4.64% Norfentanyl 0.5475 0.0273 4.99% Norhydrocodone 0.3348 0.0229 6.83% Norhydromorphone 0.3739 0.0293 7.84% Norketamine 0.3034 0.0170 5.60% Normeperidine 0.3147 0.0496 15.76% Noroxycodone 0.2922 0.0401 13.73% O-Desmethyl-cis-Tramadol 0.5440 0.0329 6.05% Oxazepam 0.3144 0.0250 7.96% Oxycodone 0.3110 0.0154 4.95% Oxymorphone 0.3088 0.0231 7.48% PCP 0.3087 0.0166 5.38% Pentazocine 0.3324 0.0289 8.69% Phenobarbital 0.3342 0.0570 17.07% Phentermine 0.3128 0.0295 9.42% Pregabalin 0.3176 0.0350 11.01% Secobarbital 0.3084 0.0939 30.44% Sufentanil 0.3489 0.0095 2.71% Tapentadol 0.3024 0.0147 4.87% Temazepam 0.2999 0.0189 6.32% THC 0.0955 0.1560 163.35% 75

THC-COOH 0.2821 0.0824 29.20% THC-OH 0.2873 0.0845 29.41% Tramadol 0.3119 0.0140 4.50% Triazolam 0.3010 0.0198 6.59% Zaleplon 0.3085 0.0401 13.00% *Table 4.5.1: Batch averages were calculated by averaging the average of the first set of each duplicate and the average of the second set of each duplicate for the 24-hour period. Standard deviation was calculated by taking the standard deviation of the averages calculated from the batch averages. The %CV was calculated by dividing the standard deviation by the batch averages.

4.5.2 30% (CO) Intra-Day

The 30% (CO) Intra-Day data is summarized and shown in Table 4.5.2. Butabarbital

(29.35%), Butalbital (29.35%), Caffeine (21.51%), EDDP (23.10%), Lorazepam (20.96%),

Phenobarbital (20.37%), Phentermine (22.85%), Secobarbital (32.35%), THC (172.00%), THC-

COOH (33.77%), and THC-OH (39.28%) were all above 20% CV.

Table 4.5.2: 30% (CO) Intra-Day

30% (CO) Intra-Day Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.3448 0.0128 3.70% 2-Hydroxyethylflurazepam 0.3334 0.0371 11.13% 4-Methylephedrine 0.3317 0.0458 13.80% 6-MAC 0.6424 0.0506 7.88% 6-MAM 0.3619 0.0283 7.83% 7-Aminoclonazepam 0.3084 0.0254 8.24% Alfentanil 0.3060 0.0070 2.30% Alprazolam 0.3170 0.0344 10.86% Amo-Pentobarbital 0.5430 0.1040 19.15% Amphetamine 0.3065 0.0501 16.36% Benzoylecgonine 0.3056 0.0230 7.52% Buprenorphine 0.3087 0.0466 15.09% Bupropion 0.2878 0.0127 4.40% Butabarbital 0.2990 0.0878 29.35% Butalbital 0.2997 0.0880 29.35% Caffeine 0.3187 0.0686 21.51% Cannabidiol 0.3254 0.0383 11.76% Carisoprodol 0.5772 0.0511 8.85% Clonazepam 0.3024 0.0388 12.83% Cocaethylene 0.3342 0.0239 7.16% Cocaine 0.3118 0.0487 15.61% Codeine 0.2459 0.0262 10.67% Cotinine 0.2905 0.0264 9.08% Desalkylflurazepam 0.3065 0.0247 8.06% Desmethyltapentadol 0.3023 0.0231 7.66% Dextromethorphan 0.3114 0.0187 6.02% Dextrorphan 0.3414 0.0166 4.86% Diazepam 0.3262 0.0193 5.92% Dihydrocodeine 0.3358 0.0318 9.47% Ecgonine methyl ester 0.3358 0.0152 4.54% EDDP 0.5587 0.1290 23.10% Ephedrine 0.2795 0.0187 6.71% Fentanyl 0.3041 0.0099 3.24% Flurazepam 0.2985 0.0079 2.64% Gabapentin 0.3054 0.0442 14.48% 77

Hydrocodone 0.2882 0.0162 5.61% Hydromorphone 0.3215 0.0269 8.36% Hydroxyalprazolam 0.2990 0.0327 10.92% Hydroxymidazolam 0.2894 0.0300 10.36% Hydroxytriazolam 0.3116 0.0511 16.39% Ketamine 0.2845 0.0161 5.65% Lorazepam 0.2871 0.0602 20.96% MDA 0.3015 0.0249 8.26% MDMA 0.3146 0.0215 6.84% MDPV 0.3355 0.0604 17.99% Meperidine 0.3043 0.0082 2.70% Meprobamate 0.5624 0.0654 11.63% Methadone 0.3353 0.0319 9.50% Methamphetamine 0.3046 0.0357 11.71% Methylone 0.3504 0.0249 7.12% Midazolam 0.3198 0.0102 3.19% Morphine 0.3048 0.0438 14.39% Nalbuphine 0.3229 0.0497 15.38% Norbuprenorphine 0.6566 0.1074 16.36% Norcodeine 0.3235 0.0642 19.86% Nordiazepam 0.2997 0.0161 5.36% Norfentanyl 0.5475 0.0269 4.92% Norhydrocodone 0.3348 0.0301 9.00% Norhydromorphone 0.3739 0.0439 11.75% Norketamine 0.3034 0.0207 6.82% Normeperidine 0.3147 0.0491 15.61% Noroxycodone 0.2922 0.0414 14.17% O-Desmethyl-cis-Tramadol 0.5440 0.0503 9.25% Oxazepam 0.3144 0.0268 8.52% Oxycodone 0.3110 0.0198 6.36% Oxymorphone 0.3088 0.0210 6.81% PCP 0.3087 0.0140 4.53% Pentazocine 0.3324 0.0419 12.62% Phenobarbital 0.3342 0.0681 20.37% Phentermine 0.3128 0.0715 22.85% Pregabalin 0.3176 0.0318 10.02% Secobarbital 0.3084 0.0998 32.35% Sufentanil 0.3489 0.0093 2.67% Tapentadol 0.3024 0.0149 4.94% Temazepam 0.2999 0.0201 6.71% THC 0.0955 0.1642 172.00% 78

THC-COOH 0.2821 0.0953 33.77% THC-OH 0.2873 0.1129 39.28% Tramadol 0.3119 0.0193 6.19% Triazolam 0.3010 0.0301 10.00% Zaleplon 0.3085 0.0409 13.25% *Table 4.5.2: 30% (CO) Intra-day are looking at the variations within each duplicate. The averages were calculated by averaging the measured concentration of each set in the duplicate for a run, then taking the overall average. The standard deviation was calculated by taking the standard deviation of the calculated averages with each duplicate. The %CV was calculated by dividing the standard deviation by the average.

4.5.3 30% (CO) Inter-Day

The 30% (CO) Inter-Day data is summarized and shown in Table 4.5.3. Secobarbital

(27.02%), THC (141.36%), THC-COOH (28.51%), and THC-OH (26.38%) were above 20%

CV.

Table 4.5.3: 30% (CO) Inter-Day

30% (CO) Inter-Day Standard Average Analyte Deviation CV% (ng/mL) (ng/mL) 10,11-Dihydro-10-Hydroxycarbamazepine 0.3448 0.0075 2.19% 2-Hydroxyethylflurazepam 0.3334 0.0295 8.84% 4-Methylephedrine 0.3317 0.0302 9.12% 6-MAC 0.6424 0.0436 6.79% 6-MAM 0.3619 0.0076 2.11% 7-Aminoclonazepam 0.3084 0.0116 3.75% Alfentanil 0.3060 0.0065 2.13% Alprazolam 0.3170 0.0308 9.72% Amo-Pentobarbital 0.5430 0.0705 12.98% Amphetamine 0.3065 0.0425 13.86% Benzoylecgonine 0.3056 0.0179 5.85% Buprenorphine 0.3087 0.0432 13.98% Bupropion 0.2878 0.0039 1.34% Butabarbital 0.2990 0.0514 17.19% Butalbital 0.2997 0.0410 13.67% Caffeine 0.3187 0.0598 18.77% Cannabidiol 0.3254 0.0316 9.70% Carisoprodol 0.5772 0.0259 4.49% Clonazepam 0.3024 0.0309 10.22% Cocaethylene 0.3342 0.0137 4.11% Cocaine 0.3118 0.0316 10.13% Codeine 0.2459 0.0168 6.83% Cotinine 0.2905 0.0249 8.58% Desalkylflurazepam 0.3065 0.0209 6.82% Desmethyltapentadol 0.3023 0.0213 7.06% Dextromethorphan 0.3114 0.0136 4.38% Dextrorphan 0.3414 0.0155 4.53% Diazepam 0.3262 0.0117 3.57% Dihydrocodeine 0.3358 0.0217 6.46% Ecgonine methyl ester 0.3358 0.0134 3.99% EDDP 0.5587 0.1026 18.37% Ephedrine 0.2795 0.0105 3.76% Fentanyl 0.3041 0.0083 2.72% Flurazepam 0.2985 0.0020 0.67% Gabapentin 0.3054 0.0290 9.49% 80

Hydrocodone 0.2882 0.0098 3.40% Hydromorphone 0.3215 0.0129 4.01% Hydroxyalprazolam 0.2990 0.0194 6.48% Hydroxymidazolam 0.2894 0.0268 9.25% Hydroxytriazolam 0.3116 0.0414 13.30% Ketamine 0.2845 0.0157 5.51% Lorazepam 0.2871 0.0338 11.78% MDA 0.3015 0.0120 3.99% MDMA 0.3146 0.0182 5.79% MDPV 0.3355 0.0213 6.34% Meperidine 0.3043 0.0065 2.13% Meprobamate 0.5624 0.0443 7.87% Methadone 0.3353 0.0285 8.50% Methamphetamine 0.3046 0.0233 7.64% Methylone 0.3504 0.0156 4.46% Midazolam 0.3198 0.0067 2.10% Morphine 0.3048 0.0354 11.63% Nalbuphine 0.3229 0.0317 9.82% Norbuprenorphine 0.6566 0.0909 13.84% Norcodeine 0.3235 0.0250 7.72% Nordiazepam 0.2997 0.0115 3.84% Norfentanyl 0.5475 0.0255 4.65% Norhydrocodone 0.3348 0.0191 5.69% Norhydromorphone 0.3739 0.0257 6.87% Norketamine 0.3034 0.0168 5.52% Normeperidine 0.3147 0.0344 10.92% Noroxycodone 0.2922 0.0348 11.91% O-Desmethyl-cis-Tramadol 0.5440 0.0327 6.02% Oxazepam 0.3144 0.0227 7.21% Oxycodone 0.3110 0.0122 3.93% Oxymorphone 0.3088 0.0198 6.43% PCP 0.3087 0.0122 3.95% Pentazocine 0.3324 0.0280 8.43% Phenobarbital 0.3342 0.0415 12.42% Phentermine 0.3128 0.0233 7.46% Pregabalin 0.3176 0.0279 8.80% Secobarbital 0.3084 0.0833 27.02% Sufentanil 0.3489 0.0078 2.24% Tapentadol 0.3024 0.0129 4.25% Temazepam 0.2999 0.0172 5.72% THC 0.0955 0.1350 141.36% 81

THC-COOH 0.2821 0.0804 28.51% THC-OH 0.2873 0.0758 26.38% Tramadol 0.3119 0.0093 2.99% Triazolam 0.3010 0.0191 6.34% Zaleplon 0.3085 0.0359 11.63% *Table 4.5.3: 30% (CO) Inter-day studies the variation between each day of runs. The average was calculated by taking the average of each 24-hour period (2 runs in each 24 -hour period). The standard deviation was calculated by taking the standard deviation of each 24-hour period. The %CV was calculated by dividing the standard deviation by the average.

4.6 LOD and LOQ

The LOD and LOQ data is summarized and shown in Table 4.6. As expected, the LOD of each analyte is lower than the LOQ of each analyte. The minimum ratio of Cutoff/LOQ is above the set lowest limit of 2.0 (Cutoff/LOQ). 34 analytes were below a 20% CV for the LOQ.

Phenobarbital (45.7%) and Secobarbital (44.5%) are the only analytes above a 40% LOQ %CV.

At such low concentrations (less than half of the cutoff), a higher %CV is expected.

Table 4.6: LOD and LOQ

LOD and LOQ Average Cutoff LOQ Max Min LOD Analyte LOQ Cutoff/LOQ (ng/mL) CV% Ratio Ratio (ng/mL) (ng/mL) 10,11-Dihydro-10- 0.162 1.0 6.2 13.1% 8.7 4.9 0.053 Hydroxycarbamazepine 2-Hydroxyethylflurazepam 0.136 1.0 7.4 29.1% 15.0 4.8 0.045 4-Methylephedrine 0.180 1.0 5.6 26.7% 11.1 3.9 0.059 6-MAC 0.127 1.0 7.9 23.1% 14.2 5.8 0.042 6-MAM 0.146 1.0 6.8 18.4% 8.9 4.8 0.048 7-Aminoclonazepam 0.132 1.0 7.6 19.3% 14.2 6.3 0.044 Alfentanil 0.155 1.0 6.5 19.7% 9.4 4.7 0.051 Alprazolam 0.136 1.0 7.3 15.9% 9.9 5.8 0.045 Amo-Pentobarbital 0.157 1.0 6.4 26.3% 11.2 4.4 0.052 Amphetamine 0.122 1.0 8.2 20.4% 12.8 5.7 0.040 Benzoylecgonine 0.133 1.0 7.5 20.8% 12.2 5.3 0.044 Buprenorphine 0.156 1.0 6.4 30.2% 9.7 4.0 0.051 Bupropion 0.144 1.0 6.9 22.2% 16.5 5.3 0.048 Butabarbital 0.168 1.0 6.0 21.0% 8.6 4.2 0.055 Butalbital 0.152 1.0 6.6 24.7% 10.4 4.0 0.050 Caffeine 0.150 1.0 6.7 33.9% 12.2 3.7 0.049 Cannabidiol 0.236 1.0 4.2 18.7% 6.5 3.3 0.078 Carisoprodol 0.159 1.0 6.3 32.1% 12.6 4.3 0.052 Clonazepam 0.123 1.0 8.1 19.7% 11.5 6.2 0.041 Cocaethylene 0.146 1.0 6.8 26.1% 10.3 5.0 0.048 Cocaine 0.134 1.0 7.5 29.1% 16.0 5.4 0.044 Codeine 0.128 1.0 7.8 19.8% 13.9 6.0 0.042 Cotinine 0.135 1.0 7.4 19.9% 10.5 5.8 0.045 83

Desalkylflurazepam 0.144 1.0 7.0 18.9% 9.2 5.3 0.047 Desmethyltapentadol 0.130 1.0 7.7 30.5% 19.4 4.7 0.043 Dextromethorphan 0.142 1.0 7.0 32.4% 15.6 4.6 0.047 Dextrorphan 0.165 1.0 6.1 19.7% 8.7 4.2 0.054 Diazepam 0.131 1.0 7.7 19.2% 14.2 6.3 0.043 Dihydrocodeine 0.140 1.0 7.1 27.4% 17.6 4.7 0.046 Ecgonine methyl ester 0.184 1.0 5.4 12.6% 7.5 4.3 0.061 EDDP 0.176 1.0 5.7 25.5% 11.5 4.1 0.058 Ephedrine 0.128 1.0 7.8 24.8% 12.5 5.9 0.042 Fentanyl 0.150 1.0 6.7 18.3% 9.4 5.2 0.049 Flurazepam 0.174 1.0 5.7 20.7% 8.3 4.4 0.057 Gabapentin 0.137 1.0 7.3 22.5% 9.9 5.3 0.045 Hydrocodone 0.127 1.0 7.9 21.6% 12.5 5.7 0.042 Hydromorphone 0.116 1.0 8.6 27.4% 16.6 5.7 0.038 Hydroxyalprazolam 0.150 1.0 6.7 18.9% 9.2 4.9 0.050 Hydroxymidazolam 0.161 1.0 6.2 23.5% 9.6 4.3 0.053 Hydroxytriazolam 0.147 1.0 6.8 26.8% 14.3 4.8 0.048 Ketamine 0.139 1.0 7.2 18.1% 10.2 5.3 0.046 Lorazepam 0.154 1.0 6.5 26.3% 13.9 4.7 0.051 MDA 0.140 1.0 7.1 16.0% 12.4 6.0 0.046 MDMA 0.144 1.0 7.0 16.7% 8.8 5.0 0.047 MDPV 0.220 1.0 4.5 34.5% 6.2 2.3 0.073 Meperidine 0.135 1.0 7.4 25.7% 16.6 5.3 0.044 Meprobamate 0.126 1.0 8.0 37.5% 17.0 5.3 0.041 Methadone 0.162 1.0 6.2 20.3% 8.5 4.6 0.053 Methamphetamine 0.139 1.0 7.2 26.6% 19.6 5.4 0.046 Methylone 0.138 1.0 7.2 12.6% 9.2 5.6 0.046 Midazolam 0.120 1.0 8.3 17.0% 10.2 6.5 0.040 Morphine 0.114 1.0 8.8 32.0% 35.2 6.2 0.038 Nalbuphine 0.144 1.0 6.9 17.6% 9.3 5.3 0.048 84

Norbuprenorphine 0.139 1.0 7.2 33.9% 11.4 3.7 0.046 Norcodeine 0.136 1.0 7.3 23.1% 11.1 5.0 0.045 Nordiazepam 0.119 1.0 8.4 28.0% 26.8 6.5 0.039 Norfentanyl 0.139 1.0 7.2 15.2% 9.7 5.8 0.046 Norhydrocodone 0.112 1.0 8.9 39.3% 28.2 5.3 0.037 Norhydromorphone 0.214 1.0 4.7 14.2% 6.0 3.6 0.071 Norketamine 0.123 1.0 8.2 33.0% 36.6 5.5 0.040 Normeperidine 0.148 1.0 6.7 17.0% 9.2 4.9 0.049 Noroxycodone 0.132 1.0 7.6 35.2% 23.5 5.3 0.044 O-Desmethyl-cis-Tramadol 0.204 1.0 4.9 18.3% 8.6 3.8 0.067 Oxazepam 0.147 1.0 6.8 21.7% 12.9 5.4 0.049 Oxycodone 0.137 1.0 7.3 16.3% 10.1 5.8 0.045 Oxymorphone 0.151 1.0 6.6 13.7% 8.5 5.5 0.050 PCP 0.140 1.0 7.2 20.6% 10.9 5.2 0.046 Pentazocine 0.216 1.0 4.6 10.9% 5.8 3.7 0.071 Phenobarbital 0.122 1.0 8.2 45.7% 23.6 4.9 0.040 Phentermine 0.118 1.0 8.5 34.8% 18.0 5.4 0.039 Pregabalin 0.152 1.0 6.6 14.8% 8.5 5.3 0.050 Secobarbital 0.144 1.0 7.0 44.5% 17.2 3.7 0.047 Sufentanil 0.225 1.0 4.5 19.4% 6.0 3.2 0.074 Tapentadol 0.146 1.0 6.9 18.8% 9.7 4.9 0.048 Temazepam 0.138 1.0 7.2 21.4% 10.8 5.7 0.046 THC 0.349 2.0 5.7 29.8% 10.7 3.6 0.115 THC-COOH 0.186 1.0 5.4 21.7% 7.7 3.7 0.061 THC-OH 0.246 1.0 4.1 23.7% 6.1 2.6 0.081 Tramadol 0.157 1.0 6.4 16.3% 10.0 5.0 0.052 Triazolam 0.129 1.0 7.8 19.0% 14.4 6.2 0.042 Zaleplon 0.140 1.0 7.1 18.5% 10.3 5.5 0.046 *Table 4.6: LOD and LOQ. “Average LOQ” is the average LOQ determined over the validation period. “Cutoff” is the determined cutoff for each analyte. Note: THC (2.0 ng/mL) is the only analyte above the 1.0 ng/mL cutoff. “Cutoff/LOQ” describes the ratio of 85 the LOQ to the set cutoff. The goal is to have a ratio above 2.0. “LOQ %CV” is the variation of each analyte’s LOQ over the validation period. “Max Ratio” and “Min Ratio” describe the maximum ratio of cutoff/LOQ and the minimum ratio of cutoff/LOQ found for each analyte over the entire validation period. “LOD” describes the average limit of detection for each analyte over the validation period. Note: LOD is rughly 1/3 of the L

CHAPTER V: DISCUSSION

5.1 Cannabinoids’ Challenges

Quantitation of THC was especially difficult. The R^2 values for THC were consistently under the 0.95 correlation value set using the SWGTOX standards, without removing calibrators

[SWGTOX]. Table 2 shows that THC had only a single run of a R^2 value below 0.95.

Examining the QCs with THC showed high variability from all tested methods. The %CV for

THC was above the 20% limit for every QC tested. This trend continued even with raising the cutoff level of THC to 2.0 ng/mL. Two other Cannabinoids (THC-OH and Cannabidiol) in the analyte panel posed similar challenges as THC. THC-OH only showed issues with variability from all the QCs expect the HiQC. The R^2 values for THC-OH were above the 0.95 limit for each run. Cannabidiol had a relatively high %CV compared to the other analytes for the HiQC, but it was below the 20% limit. Interestingly, Cannabidiol had a lower %CV for the LoQC and

30% CO (below 20%) than the SST CO, which was above 20%. The R^2 values for Cannabidiol were all above the 0.95 limit. THC-COOH did not have the quantitation issues seen in the other cannabinoids. The lower correlation values and high %CV of THC and the other cannabinoids can be explained by the analyte’s documented absorptive losses [Drummer O. H. et al. (2006),

Hayley et al. (2018), Fabritius et al. (2013), Garrett (1974)]. THC’s inclination to “stick” to various surfaces, is the major contributor to the absorptive losses [Garrett (1974)]. THC has relatively high lipophilicity compared to analytes including other cannabinoids [Garrett (1974),

Fabritius et al. (2013)]. The high lipophilic nature of THC can lead to interactions between THC and non-polar surfaces such as plastic. Due to the processing procedures an OF sample, as shown in the “Materials and Methods” section, there are many surfaces that the analyte is exposed to that could possibly contribute to absorption. Among the many surfaces, plastic surfaces were 87 present in the processing procedures, which contribute the greatest absorptive losses for cannabinoids [Garrett (1974)]. The pipetting procedure utilized plastic pipette tips and the

Thompson filter vial used for injection was plastic, with a plastic filter. The multiple exposures to plastic surfaces and other surfaces likely contributed to the absorptive loses found with THC.

The absorptive losses of other cannabinoids in the panel can be attributed to their high lipophilicity [Garrett (1974)].

5.2 Barbiturates

While staying well below the 20% %CV limit for the HiQC, many of the barbiturates in the analyte panel had high %CV (above 20%) for the other QCs. Amo-Pentobarbital was below the 20% limit for the LoQC and SST CO but was above the limit for 30% CO. Butabarbital was above the 20% limit for the LoQC and SST CO but was below the limit for SST CO. Butalbital and Secobarbital were above the 20% limit for all of the QCs (except HiQC). Phenobarbital was below the 20% limit for all QCs. The barbiturates were analyzed in negative ion mode and because of the decreased sensitivity involved with negative ion mode analysis, the variability issues seen near cutoff levels can be attributed to decreased sensitivity in negative ion mode.

5.3 Low HiQC Concentration for Several Analytes

Several analytes in the panel were found to have low HiQC concentrations. While they were within the acceptable variance of 20%, the concentrations were consistently on the low end as shown in Tables 3A-3D.

It is worth noting how to interpret the data. When interpreting the data, it is important to consider how the data presents itself. Precision and accuracy play a major role with interpretation of data. For example, if multiple runs show a concentration outside the range of ±20% from the theoretical concentration and the measured concentrations are highly scattered, the data would be 88 interpreted as imprecise and inaccurate. The lack of tight grouping between concentrations means the measurements are imprecise. The large difference between the measured concentration and the theoretical concentration means the measurements are inaccurate.

In terms of the low concentrations observed with the HiQCs, the concentrations were observed to be consistently low throughout the entire group of runs. The concentrations were consistently low, but they were in a tight grouping. The tight grouping and consistency of the results indicate that the concentrations were precise, but because they were low, they were not accurate. Because the concentrations of the HiQCs were precise, but not accurate, the interpretation of the data would indicate an issue with preparation of the HiQC stock solutions and not the instrument.

5.4 0.75x Calibrator Issues

The 0.75x calibrator was systematically below 80.0% accuracy. The low accuracy of the

0.75x calibrator lowered the R^2 values of several analytes because of the high weight given to the lower concentrated calibrators. Since 0.75 ng/mL is below the 1.0 ng/mL cut-off, removing the calibrator can be expected for reliable quantitation. By removing the calibrator for affected analytes during data analysis, the R^2 values improved significantly. The low concentrations of the 0.75x calibrator were likely caused by a preparation of the stock solution for that specific calibrator as explained in the “Low HiQC Concentration for Several Analytes” section.

5.5 Patterns Observed

Data analysis showed clear patterns for certain analytes such as barbiturates and cannabinoids explained in the “Barbiturates” and “Cannabinoids’ Challenges” sections respectively. The %CV for analytes under the barbiturates and cannabinoids drug categories was notoriously high as the concentrations neared and fell below the cut-off levels. The pattern of a high %CV (above the 20% limit) as the concentrations of QCs fell below the 1 ng/mL cut-off 89 continued for other analytes as well. In order of highest to lowest, starting with the highest concentrated QC, HiQC (25 ng/mL), the only analyte above the 20% limit was THC. The next highest concentrated QC was the SST CO, which is at a 1 ng/mL (same as cut-off) concentration.

The analytes above the 20% limit included: Butalbital Cannabidiol, Dextromethorphan,

Normeperidine, Secobarbital, THC, and THC-OH. 5 of the 7 analytes belonged to the barbiturate and cannabinoid drug categories. Moving to the next highest concentrated QC which is concentrated below the cut-off of 1 ng/mL, LoQC (0.6 ng/mL), the analytes above the 20% limit included: Butabarbital, Butalbital, Caffeine, Flurazepam, Secobarbital, THC, THC-OH, and

Tramadol. 5 of the 8 analytes belonged to the barbiturate and cannabinoid drug categories.

Finishing with the lowest concentrated QC, 30% CO (0.3 ng/mL), the analytes above the 20% limit included: Amo-Pentobarbital, Butabarbital, Butalbital, Caffeine, Cocaine, 2-Ethylidiene-

1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), Lorazepam, Norbuprenorphine, Norcodeine,

Normeperidine, Noroxycodone, Phentermine, Secobarbital, THC, THC-COOH, and THC-OH.

Of the 16 analytes above the 20% limit, 7 analytes were either a barbiturate or cannabinoid where issues with precision were expected. Of the 81 analytes in the panel, only 16 analytes were above the 20% limit at 30% of the cut-off. The pattern of precision lowing as the concentration of analytes fell below the cut-off of 1 ng/mL can be seen. As the concentration is lowered, the number of analytes having a %CV above the 20% increased. In other words, a correlation exists between precision and concentration. As concentration increases, precision increases (%CV decreases).

5.6 LOQ

The LOQ results showed that all analyte’s cutoff exceeded the LOQ by at least a factor of

2, and it is worth noting that all analytes exceeded cutoff by a factor of 4. The lowest cutoff/LOQ 90 was THC-OH (4.1) and cannabidiol (4.2). If THC had the original cut-off of 1.0 ng/mL, the cutoff/LOQ ratio would be 2.9, which would make THC the analyte with the lowest ratio. THC also had the highest average LOQ of all analytes with an average LOQ of 0.349 ng/mL and a

%CV of 29.8%. THC’s LOQ exceeds the 30% (CO) QC (0.3 ng/mL). Because of these results, increasing the cutoff for THC was critical. The next 2 highest average LOQs were THC-OH

(0.246 ng/mL) and cannabidiol (0.236 ng/mL). The lowest cutoff/LOQ ratios and average LOQs were cannabinoids. The results are expected, as explained in the “Cannabinoids’ Challenges” section, and are further indications that cannabinoids pose issues for quantitation at low concentrations.

5.7 Success of the OF Method

The drug panel consists of many common drugs of abuse and seeing the overwhelming success of these analytes shows the reliability of the method. Linearity was measured using the

R^2 value for each analyte. The method utilizes a maximum of 16 data points from each calibration curve (each calibrator ran in duplicate). All analytes (81 out of 81) had a R^2 value above 0.97, and it is worth noting the cutoff of acceptability for R^2 from SWGTOX is 0.95.

Observing the lowest QC, the 30% (CO) QC (0.3 ng/mL), 65 of the 81 analytes in the panel had a %CV below 20%. At the SST (CO) QC (1.0 ng/ml), 74 of the 81 analytes in the panel had a

%CV below 20%. Even at cutoff, the vast majority of the analytes in the panel were below the

%CV limit of 20%. Furthermore, to show perspective of the success of the OF method, a 20%

CV at 1.0 ng/mL means an average variation of 0.2 ng/mL per run. In terms of biological samples, a variation of 0.2 ng/mL is a very miniscule amount. Despite there being 7 analytes that had a %CV above 20% at the cutoff, it is safe to say that even at cutoff levels the method was an overwhelming success. 91

Throughout the validation, THC was shown to be problematic in terms of variability, but it is worth noting that the cutoff for THC was not changed to 2.0 ng/mL until after the validation runs. The original cutoff of THC was 1.0 ng/mL, like all of the other analytes in the panel. THC was prepared and analyzed (only LOD and LOQ analysis had THC at a cutoff of 2.0 ng/mL) at a

1.0 ng/mL cutoff. The Substance Abuse and Mental Health Services Administration sets a cutoff for confirmatory THC detection at 2.0 ng/mL [Swortwood et al. (2017)]. While the EU DRUID project used a 1.0 ng/mL cutoff. Swortwood et al. (2017) showed that a 5.0 ng/mL cutoff for

THC analysis increased sensitivity and specificity significantly (relative to 1.0 ng/mL and 2.0 ng/mL) when confirming different roadside OF devices. Relative to other methods, a 2.0 ng/mL cutoff for THC shows that the validated OF method can be considered a successful method.

5.8 Recommendations

THC repeatedly had quantitation issues throughout the validation study. I think raising the cutoff for THC to 2.0 ng/mL was a wise move because it increased the signal to noise for the

LOQ. The goal is to increase sensitivity and the ability to quantitate at much lower concentrations. THC has well-documented stability issues, as described in “Cannabinoids’

Challenges”. The issue with THC is that it experiences significant absorptive losses in its over time, especially in plastic. There are two logical directions to take to help mitigate losses. The first would be to use low-binding containers. Low-binding containers may prevent major losses of THC over time. The caveat to using low-binding containers is that many are internally coated with a low-binding substance. Since the MS is a very sensitive instrument, a low-binding substance may cause unintended matrix effects for THC or increase the background noise, which would hamper quantitation. The second would be to make the matrix more lipophilic. This would 92 increase THC’s affinity for the matrix and help mitigate the loss of THC into the container. The caveat to this direction is, again, matrix effects on THC.

5.9 Future Work

Due to the unforeseen developments of the 2020 Covid-19 global pandemic, further studies for BGSU’s OF method could not be completed. The incomplete studies include:

Carryover, Patient Samples, Matrix Effects, and Interference. These studies will be completed as soon as the “Stay-at-Home” order by Governor DeWine is lifted.

In conclusion, the validation studies performed for BGSU’s OF method, show that the

Shimadzu LC-MS 8050 can perform routine drug identification and quantitation over a vast number of analytes. The results obtained throughout the validation achieved expectations and concur with other validations [Gibbs, (2020)]. A complete validation for BGSU’s instrument allows for roadside drug testing to be coupled with confirmatory instrument. Despite the validation limitations, OF is a viable method for drug identification and quantitation and can be a reliable matrix for future research. 93

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APPENDIX A: AVERAGE OF:BLOOD CONCENTRATION RATIO

Average OF: Blood Drug (type) Concentration Ratio Alcohol 1.07 Barbituates 0.3 Buprenorphine 1 Codeine (basic) 4 Methamphetamine 2 (basic) MDMA (basic) 7 Cocaine (basic) 3 Diazepam (acidic) 0.01 - 0.02 Methadone (basic) 1.6 Morphine (basic) 0.8 THC (neutral) 1.2 101

APPENDIX B: LIST OF STANDARDS

Stock Cerrilliant Analyte Concentration ID (µg/mL) 10,11-Dihydro-10- D-091 1,000 Hydroxycarbamazepine M-158 4-Methylephedrine 1,000 F-902 2-Hydroxyethylflurazepam 1,000 M-158 4-Methylephedrine 1,000 A-053 6-MAC 1,000 A-009 6-MAM 1,000 A-916 7-Aminoclonazepam 1,000 A-071 Alfentanil 1,000 A-903 Alprazolam 1,000 A-020 Amo-Pentobarbital 1,000 A-007 Amphetamine 1,000 B-004 Benzoylecgonine 1,000 B-044 Buprenorphine 1,000 B-034 Bupropion 1,000 B-024 Butabarbital 1,000 B-006 Butalbital 1,000 C-051 Caffeine 1,000 C-045 Cannabidiol 1,000 C-077 Carisoprodol 1,000 C-907 Clonazepam 1,000 C-010 Cocaethylene 1,000 C-008 Cocaine 1,000 C-006 Codeine 1,000 C-016 Cotinine 1,000 D-915 Desalkylflurazepam 1,000 D-052 Desmethyltapentadol 1,000 D-013 Dextromethorphan 1,000 D-034 Dextrorphan 1,000 D-907 Diazepam 1,000 D-019 Dihydrocodeine 1,000 E-001 Ecgonine methyl ester 1,000 E-022 EDDP 1,000 E-011 Ephedrine 1,000 102

F-013 Fentanyl 1,000 F-003 Flurazepam 1,000 G-007 Gabapentin 1,000 H-003 Hydrocodone 1,000 H-004 Hydromorphone 1,000 A-907 Hydroxyalprazolam 1,000 H-922 Hydroxymidazolam 1,000 T-911 Hydroxytriazolam 1,000 K-002 Ketamine 1,000 L-901 Lorazepam 1,000 C-089 mCPP 1,000 M-012 MDA 1,000 M-013 MDMA 1,000 M-146 MDVP 1,000 M-035 Meperidine 1,000 M-039 Meprobamate 1,000 M-007 Methadone 1,000 M-009 Methamphetamine 1,000 M-140 Methylone 1,000 M-140 Methylone 1,000 M-908 Midazolam 1,000 M-005 Morphine 1,000 N-051 Nalbuphine 1,000 N-059 Norbuprenorphine 1,000 N-005 Norcodeine 1,000 N-905 Nordiazepam 1,000 N-031 Norfentanyl 1,000 N-053 Norhydrocodone 1,000 N-107 Norhydromorphone 1,000 N-036 Norketamine 1,000 N-089 Normeperidine 1,000 N-011 Noroxycodone 1,000 N-913 Norpropoxyphene 1,000 T-035 O-Desmethyl-cis-tramadol 1,000 O-902 Oxazepam 1,000 O-002 Oxycodone 1,000 O-004 Oxymorphone 1,000 P-007 PCP 1,000 P-073 Pentazocine 1,000 103

P-010 Pentobarbital 1,000 P-008 Phenobarbital 1,000 P-023 Phentermine 1,000 P-066 Pregabalin 1,000 P-011 Propoxyphene 1,000 S-002 Secobarbital 1,000 S-008 Sufentanil 100 T-058 Tapentadol 1,000 T-907 Temazepam 1,000 T-005 THC 1,000 T-019 THC-COOH 1,000 H-027 THC-OH 1,000 T-027 Tramadol 1,000 T-910 Triazolam 1,000 Z-004 Zaleplon 1,000

104

APPENDIX C: LIST OF INTERNAL STANDARDS

Analyte

Caffeine-13C3 6-MAM-D3 Amphetamine-D5 Benzoylecognine-D3 Buprenorphine-D4 Butabarbital-D5 Caffeine-13C-D3 Cannabidiol-D3 Cocaethylene-D3 Cocaine-D3 Codeine-D3 Cotinine-D3 Diazepam-D5 EDDP-D3 Fentanyl-D5 Hydrocodone-D6 Hydromorphone-D6 Ketamine-D4 Lorazepam-D4 Meperidine-D4 Methadone-D3 Methamphetamine- D5 Midazolam-D4 Morphine-D3 Norbuprenorphine- D3 Norcodeine-D3 Nordiazepam-D5 Norhydrocodone-D3 Norketamine-D4 Normeperidine-D4 105

Noroxycodone-D3 Oxazepam-D5 Oxycodone-D6 PCP-D5 Pentobarbital-D5 Phenobarbital-D5 Propoxyphene-D5 Tapentadol-D3 THC-COOH-D9 THC-D3 THC-OH-D3 Zaleplon-D4

106

APPENDIX D: MASS SPECTROMETER SETTINGS

Mass Spectrometer Settings

ISTD Ret. Analyte m/z(1) m/z(2) Group Time

10,11-Dihydro-10- Hydroxycarbamazepine 20 255.2 194.25 3.633 2-Hydroxyethylflurazepam 22 333.2 211.3 5.367 4-Methylephedrine 17 180.2 162.3 1.711 6-MAC 3 342.1 225.3 3.077 6-MAM 1 328.2 165.1 2.002 7-Aminoclonazepam 20 286 222.1 3.611 Alfentanil 4 417.2 268.2 4.324 Alprazolam 9 309 281.1 5.711 Amo-Pentobarbital 30 225.1 182.35 3.999 Amphetamine 2 136.2 65.25 1.18 Benzoylecgonine 3 290.1 168.2 3.018 Buprenorphine 4 468.2 396.15 4.389 Bupropion 3 240.2 184.1 2.945 Butabarbital 5 211.1 168.3 3.309 Butalbital 20 223.1 180.3 3.506 Caffeine 33 195 138.1 2.58 Cannabidiol 6 313.3 245.4 6.383 Carisoprodol 18 261.1 176.25 4.616 Clonazepam 27 316 270 5.051 Cocaethylene 37 318.1 196.25 3.504 Cocaine 38 304 182.05 3.086 Codeine 7 300.1 152.1 1.956 Cotinine 8 177.2 80.2 1.695 Desalkylflurazepam 14 289 140.15 5.313 Desmethyltapentadol 24 208.1 107.25 2.394 Dextromethorphan 29 272 215.15 3.943 Dextrorphan 13 258.3 157.05 2.667 Diazepam 9 285 193.15 5.97 Dihydrocodeine 7 302.1 199.1 1.959 Ecgonine methyl ester 19 200.1 82 0.39 107

EDDP 39 278.1 234.2 4.375 Ephedrine 12 166.2 117.1 1.024 Fentanyl 10 337.1 188.15 4.048 Flurazepam 10 388.1 315.1 4.14 Gabapentin 17 172.2 119.3 1.228 Hydrocodone 11 300.1 199.1 2.153 Hydromorphone 12 286.2 185.1 1.29 Hydroxyalprazolam 18 325 297.15 5.287 Hydroxymidazolam 18 342 202.95 5.172 Hydroxytriazolam 18 358.9 331 5.284 Ketamine 13 238 125.25 2.766 Lorazepam 13 321 229.2 5.091 MDA 17 180.2 135.05 1.504 MDMA 17 194.2 163.15 1.835 MDPV 3 276.1 126.2 3.144 Meperidine 15 248.2 220.1 2.91 Meprobamate 3 219.25 158.1 3.161 Methadone 16 310.3 265.3 4.765 Methamphetamine 17 150.1 119.2 1.57 Methylone 17 208 132.05 1.64 Midazolam 18 326 291 4.844 Morphine 19 286.2 151.9 0.988 Nalbuphine 33 358.1 185.1 2.637 Norbuprenorphine 20 413.9 165.2 3.439 Norcodeine 21 286.2 152.1 1.542 Nordiazepam 22 271 140.1 5.497 Norfentanyl 24 233.2 84.35 2.49 Norhydrocodone 23 286.2 199 1.834 Norhydromorphone 19 272.2 185.1 0.728 Norketamine 24 224 125.15 2.489 Normeperidine 25 234.2 160.15 2.828 Noroxycodone 26 302.2 187.1 1.73 O-Desmethyl-cis-Tramadol 7 250.3 58.3 1.946 Oxazepam 27 287.1 104.25 5.07 Oxycodone 28 316.2 241.2 2.064 Oxymorphone 19 302 227.2 1.101 PCP 29 244.3 86.2 4.002 Pentazocine 20 286.1 218.2 3.427 Phenobarbital 31 231 42.2 3.185 108

Phentermine 17 150.2 133.1 1.675 Pregabalin 2 160.2 83.3 0.853 Secobarbital 31 237.1 194.4 4.321 Sufentanil 18 387.1 238.25 4.53 Tapentadol 33 222.2 107.15 2.59 Temazepam 22 301.1 177.2 5.645 THC 40 315.1 193.35 6.58 THC-COOH 34 343.1 245.3 6.372 THC-OH 41 329.3 268.35 6.275 Tramadol 13 264.3 58.3 2.779 Triazolam 9 342.9 308.1 5.715 Zaleplon 35 306.2 236.3 5.285 Caffeine-13C3 33 198 140.15 2.579 6-MAM-D3 1 331 165.1 1.993 Amphetamine-D5 2 141.1 124.2 1.16 Benzoylecognine-D3 3 293.2 171.05 3.007 Buprenorphine-D4 4 472.2 59.2 4.348 Butabarbital-D5 5 216 173.15 3.31 Caffeine-13C-D3 36 198.9 142.15 2.561 Cannabidiol-D3 6 316 248.15 6.38 Cocaethylene-D3 37 321.1 199.15 3.499 Cocaine-D3 38 307.1 185.15 3.081 Codeine-D3 7 302.95 165.1 1.943 Cotinine-D3 8 180.1 80 1.682 Diazepam-D5 9 290 198.1 5.956 EDDP-D3 39 281.1 234.1 4.375 Fentanyl-D5 10 342.3 188.2 4.032 Hydrocodone-D6 11 306 201.95 2.129 Hydromorphone-D6 12 292.2 185.1 1.272 Ketamine-D4 13 242.2 129.1 2.748 Lorazepam-D4 14 327 281.2 5.072 Meperidine-D4 15 252.1 224.2 2.901 Methadone-D3 16 313.1 268.15 4.755 Methamphetamine-D5 17 155.1 121.2 1.553 Midazolam-D4 18 330.1 295.45 4.822 Morphine-D3 19 289.1 152.1 0.971 Norbuprenorphine-D3 20 417.2 101.05 3.428 Norcodeine-D3 21 289.1 152.1 1.527 Nordiazepam-D5 22 276 140.1 5.475 109

Norhydrocodone-D3 23 289.1 202.1 1.82 Norketamine-D4 24 228.2 129.1 2.468 Normeperidine-D4 25 238.1 164.2 2.818 Noroxycodone-D3 26 305.2 230.2 1.711 Oxazepam-D5 27 292 246.15 5.049 Oxycodone-D6 28 322.1 247.2 2.042 PCP-D5 29 249.3 86.2 3.982 Pentobarbital-D5 30 230 187.2 3.992 Phenobarbital-D5 31 236 193.15 3.183 Propoxyphene-D5 32 345 58.2 4.292 Tapentadol-D3 33 225.1 107.15 2.585 THC-COOH-D9 34 352.2 254.2 6.36 THC-D3 40 318.25 196.25 6.58 THC-OH-D3 41 332.2 271.3 6.271 Zaleplon-D4 35 310.2 240.3 5.27

Mass Spectrometer Settings

Ref.(1) Ref.(1) Start Analyte2 Event m/z(1) m/z(2) Time

10,11-Dihydro-10- Hydroxycarbamazepine 255.2 193.1 70 3.466 2-Hydroxyethylflurazepam 333.2 305.25 102 5.2 4-Methylephedrine 180.2 147.25 22 1.544 6-MAC 342.1 165.3 59 2.91 6-MAM 328.2 211.1 33 1.835 7-Aminoclonazepam 286 250 69 3.444 Alfentanil 417.2 197.25 79 4.157 Alprazolam 309 205.1 106 5.544 Amo-Pentobarbital 225.1 42.15 118 3.832 Amphetamine 136.2 119.3 9 1.013 Benzoylecgonine 290.1 82.1 58 2.851 Buprenorphine 468.2 414.35 83 4.222 Bupropion 240.2 130.1 56 2.778 Butabarbital 211.1 42.2 114 3.142 Butalbital 223.1 42.15 116 3.339 Caffeine 195 110 44 2.413 Cannabidiol 313.3 107.3 125 6.216 Carisoprodol 261.1 62.05 86 4.449 110

Clonazepam 316 214.1 92 4.884 Cocaethylene 318.1 82.25 68 3.337 Cocaine 304 82.15 61 2.919 Codeine 300.1 165.1 30 1.789 Cotinine 177.2 98.15 21 1.528 Desalkylflurazepam 289 226.1 101 5.146 Desmethyltapentadol 208.1 51 38 2.227 Dextromethorphan 272 171.1 71 3.776 Dextrorphan 258.3 201.1 48 2.5 Diazepam 285 154.1 109 5.803 Dihydrocodeine 302.1 201.2 31 1.792 Ecgonine methyl ester 200.1 68.1 1 0.223 EDDP 278.1 249.05 81 4.208 Ephedrine 166.2 133.1 6 0.857 Fentanyl 337.1 132.25 75 3.881 Flurazepam 388.1 317 76 3.973 Gabapentin 172.2 93.25 10 1.061 Hydrocodone 300.1 171.2 37 1.986 Hydromorphone 286.2 157.1 12 1.123 Hydroxyalprazolam 325 216.1 100 5.12 Hydroxymidazolam 342 168.1 96 5.005 Hydroxytriazolam 359.1 176.1 98 5.117 Ketamine 238 207.15 50 2.599 Lorazepam 321 194.25 95 4.924 MDA 180.2 132.9 13 1.337 MDMA 194.2 133.15 27 1.668 MDPV 276.1 135.1 62 2.977 Meperidine 248.2 174.2 55 2.743 Meprobamate 219.25 97.25 63 2.994 Methadone 310.3 117.3 88 4.598 Methamphetamine 150.1 65.2 17 1.403 Methylone 208 159.95 18 1.473 Midazolam 326 209.1 90 4.677 Morphine 286.2 165.2 5 0.821 Nalbuphine 358.1 254.1 47 2.47 Norbuprenorphine 413.9 396.4 66 3.272 Norcodeine 286.2 165.1 15 1.375 Nordiazepam 271 165.1 104 5.33 Norfentanyl 233.2 56.05 41 2.323 111

Norhydrocodone 286.2 171.1 26 1.667 Norhydromorphone 272.2 157.1 2 0.561 Norketamine 224 207.15 40 2.322 Normeperidine 234.2 56.2 53 2.661 Noroxycodone 302.2 198.2 24 1.563 O-Desmethyl-cis-Tramadol 250.3 42.3 29 1.779 Oxazepam 287.1 163.25 93 4.903 Oxycodone 316.2 256.2 35 1.897 Oxymorphone 302 198.05 7 0.934 PCP 244.3 159.2 73 3.835 Pentazocine 286.1 69.15 64 3.26 Phenobarbital 231 188.3 113 3.018 Phentermine 150.2 65.1 19 1.508 Pregabalin 160.2 97.3 3 0.686 Secobarbital 237.1 42.15 119 4.154 Sufentanil 387.1 111.15 85 4.363 Tapentadol 222.2 103.1 46 2.423 Temazepam 301 193.3 105 5.478 THC 315.1 123.25 111 6.413 THC-COOH 343.1 191.3 123 6.205 THC-OH 329.3 173.35 121 6.108 Tramadol 264.3 42.3 51 2.612 Triazolam 342.9 315 107 5.548 Zaleplon 306.2 264.3 99 5.118 Caffeine-13C3 198 112.15 43 2.412 6-MAM-D3 32 1.826 Amphetamine-D5 8 0.993 Benzoylecognine-D3 57 2.84 Buprenorphine-D4 80 4.181 Butabarbital-D5 115 3.143 Caffeine-13C-D3 42 2.394 Cannabidiol-D3 124 6.213 Cocaethylene-D3 67 3.332 Cocaine-D3 60 2.914 Codeine-D3 28 1.776 Cotinine-D3 20 1.515 Diazepam-D5 108 5.789 EDDP-D3 82 4.208 Fentanyl-D5 74 3.865 112

Hydrocodone-D6 36 1.962 Hydromorphone-D6 11 1.105 Ketamine-D4 49 2.581 Lorazepam-D4 94 4.905 Meperidine-D4 54 2.734 Methadone-D3 87 4.588 Methamphetamine-D5 16 1.386 Midazolam-D4 89 4.655 Morphine-D3 4 0.804 Norbuprenorphine-D3 65 3.261 Norcodeine-D3 14 1.36 Nordiazepam-D5 103 5.308 Norhydrocodone-D3 25 1.653 Norketamine-D4 39 2.301 Normeperidine-D4 52 2.651 Noroxycodone-D3 23 1.544 Oxazepam-D5 91 4.882 Oxycodone-D6 34 1.875 PCP-D5 72 3.815 Pentobarbital-D5 117 3.825 Phenobarbital-D5 112 3.016 Propoxyphene-D5 77 4.125 Tapentadol-D3 45 2.418 THC-COOH-D9 122 6.193 THC-D3 110 6.413 THC-OH-D3 120 6.104 Zaleplon-D4 97 5.103

Mass Spectrometer Settings

End Event Analyte3 Polarity Time Time

10,11-Dihydro-10- Hydroxycarbamazepine 3.8 0 0.04 2-Hydroxyethylflurazepam 5.534 0 0.034 4-Methylephedrine 1.878 0 0.028 113

6-MAC 3.244 0 0.034 6-MAM 2.169 0 0.028 7-Aminoclonazepam 3.778 0 0.04 Alfentanil 4.491 0 0.036 Alprazolam 5.878 0 0.084 Amo-Pentobarbital 4.166 1 0.054 Amphetamine 1.347 0 0.046 Benzoylecgonine 3.185 0 0.034 Buprenorphine 4.556 0 0.036 Bupropion 3.112 0 0.034 Butabarbital 3.476 1 0.054 Butalbital 3.673 1 0.054 Caffeine 2.747 0 0.032 Cannabidiol 6.55 1 0.074 Carisoprodol 4.783 0 0.036 Clonazepam 5.218 0 0.034 Cocaethylene 3.671 0 0.04 Cocaine 3.253 0 0.034 Codeine 2.123 0 0.028 Cotinine 1.862 0 0.028 Desalkylflurazepam 5.48 0 0.034 Desmethyltapentadol 2.561 0 0.038 Dextromethorphan 4.11 0 0.05 Dextrorphan 2.834 0 0.032 Diazepam 6.137 0 0.084 Dihydrocodeine 2.126 0 0.028 Ecgonine methyl ester 0.557 0 0.302 EDDP 4.542 0 0.036 Ephedrine 1.191 0 0.046 Fentanyl 4.215 0 0.04 Flurazepam 4.307 0 0.04 Gabapentin 1.395 0 0.046 Hydrocodone 2.32 0 0.032 Hydromorphone 1.457 0 0.046 Hydroxyalprazolam 5.454 0 0.034 Hydroxymidazolam 5.339 0 0.034 Hydroxytriazolam 5.451 0 0.034 Ketamine 2.933 0 0.032 Lorazepam 5.258 0 0.034 114

MDA 1.671 0 0.028 MDMA 2.002 0 0.028 MDPV 3.311 0 0.034 Meperidine 3.077 0 0.032 Meprobamate 3.328 0 0.034 Methadone 4.932 0 0.052 Methamphetamine 1.737 0 0.028 Methylone 1.807 0 0.028 Midazolam 5.011 0 0.052 Morphine 1.155 0 0.046 Nalbuphine 2.804 0 0.032 Norbuprenorphine 3.606 0 0.04 Norcodeine 1.709 0 0.028 Nordiazepam 5.664 0 0.046 Norfentanyl 2.657 0 0.032 Norhydrocodone 2.001 0 0.028 Norhydromorphone 0.895 0 0.084 Norketamine 2.656 0 0.032 Normeperidine 2.995 0 0.032 Noroxycodone 1.897 0 0.028 O-Desmethyl-cis-Tramadol 2.113 0 0.028 Oxazepam 5.237 0 0.034 Oxycodone 2.231 0 0.032 Oxymorphone 1.268 0 0.046 PCP 4.169 0 0.04 Pentazocine 3.594 0 0.04 Phenobarbital 3.352 1 0.054 Phentermine 1.842 0 0.028 Pregabalin 1.02 0 0.06 Secobarbital 4.488 1 0.054 Sufentanil 4.697 0 0.036 Tapentadol 2.757 0 0.032 Temazepam 5.812 0 0.084 THC 6.747 0 0.252 THC-COOH 6.539 1 0.074 THC-OH 6.442 1 0.074 Tramadol 2.946 0 0.032 Triazolam 5.882 0 0.084 Zaleplon 5.452 0 0.034 115

Caffeine-13C3 2.746 0 0.032 6-MAM-D3 2.16 0 0.028 Amphetamine-D5 1.327 0 0.046 Benzoylecognine-D3 3.174 0 0.035 Buprenorphine-D4 4.515 0 0.037 Butabarbital-D5 3.477 1 0.022 Caffeine-13C-D3 2.728 0 0.032 Cannabidiol-D3 6.547 1 0.027 Cocaethylene-D3 3.666 0 0.041 Cocaine-D3 3.248 0 0.035 Codeine-D3 2.11 0 0.028 Cotinine-D3 1.849 0 0.028 Diazepam-D5 6.123 0 0.084 EDDP-D3 4.542 0 0.037 Fentanyl-D5 4.199 0 0.041 Hydrocodone-D6 2.296 0 0.032 Hydromorphone-D6 1.439 0 0.046 Ketamine-D4 2.915 0 0.032 Lorazepam-D4 5.239 0 0.035 Meperidine-D4 3.068 0 0.032 Methadone-D3 4.922 0 0.052 Methamphetamine-D5 1.72 0 0.028 Midazolam-D4 4.989 0 0.052 Morphine-D3 1.138 0 0.046 Norbuprenorphine-D3 3.595 0 0.041 Norcodeine-D3 1.694 0 0.028 Nordiazepam-D5 5.642 0 0.046 Norhydrocodone-D3 1.987 0 0.028 Norketamine-D4 2.635 0 0.032 Normeperidine-D4 2.985 0 0.032 Noroxycodone-D3 1.878 0 0.028 Oxazepam-D5 5.216 0 0.035 Oxycodone-D6 2.209 0 0.032 PCP-D5 4.149 0 0.045 Pentobarbital-D5 4.159 1 0.022 Phenobarbital-D5 3.35 1 0.022 Propoxyphene-D5 4.459 0 0.037 Tapentadol-D3 2.752 0 0.032 THC-COOH-D9 6.527 1 0.027 116

THC-D3 6.747 0 0.022 THC-OH-D3 6.438 1 0.027 Zaleplon-D4 5.437 0 0.035

Mass Spectrometer Settings Target Target Q1 Analyte4 Dwell Pre Bias Time 10,11-Dihydro-10- Hydroxycarbamazepine 19 -6 2-Hydroxyethylflurazepam 16 -10 4-Methylephedrine 13 -9 6-MAC 16 -10 6-MAM 13 -8 7-Aminoclonazepam 19 -10 Alfentanil 17 -8 Alprazolam 41 -7 Amo-Pentobarbital 25 8 Amphetamine 22 -7 Benzoylecgonine 16 -15 Buprenorphine 17 -9 Bupropion 16 -7 Butabarbital 25 14 Butalbital 25 8 Caffeine 15 -6 Cannabidiol 35 11 Carisoprodol 17 -8 Clonazepam 16 Cocaethylene 19 -6 Cocaine 16 -9 Codeine 13 -15 Cotinine 13 -9 Desalkylflurazepam 16 -7 Desmethyltapentadol 18 -6 Dextromethorphan 24 -8 Dextrorphan 15 -6 Diazepam 41 -10 Dihydrocodeine 13 -7 Ecgonine methyl ester 150 -6 117

EDDP 17 -8 Ephedrine 22 -9 Fentanyl 19 -10 Flurazepam 19 -9 Gabapentin 22 -5 Hydrocodone 15 -7 Hydromorphone 22 -7 Hydroxyalprazolam 16 -10 Hydroxymidazolam 16 -10 Hydroxytriazolam 16 -11 Ketamine 15 -7 Lorazepam 16 -12 MDA 13 -7 MDMA 13 -10 MDPV 16 -8 Meperidine 15 -9 Meprobamate 16 -11 Methadone 25 -16 Methamphetamine 13 -8 Methylone 13 -11 Midazolam 25 -10 Morphine 22 -7 Nalbuphine 15 -10 Norbuprenorphine 19 -8 Norcodeine 13 -7 Nordiazepam 22 -23 Norfentanyl 15 -7 Norhydrocodone 13 -8 Norhydromorphone 41 -8 Norketamine 15 -12 Normeperidine 15 -7 Noroxycodone 13 -9 O-Desmethyl-cis-Tramadol 13 -5 Oxazepam 16 -7 Oxycodone 15 -12 Oxymorphone 22 -6 PCP 19 -7 Pentazocine 19 -9 Phenobarbital 25 8 118

Phentermine 13 -11 Pregabalin 29 -19 Secobarbital 25 8 Sufentanil 17 -11 Tapentadol 15 -8 Temazepam 41 -7 THC 125 -16 THC-COOH 35 12 THC-OH 35 11 Tramadol 15 -5 Triazolam 41 -10 Zaleplon 16 -15 Caffeine-13C3 15 -14 6-MAM-D3 27 -10 Amphetamine-D5 45 -16 Benzoylecognine-D3 34 -7 Buprenorphine-D4 36 -14 Butabarbital-D5 20 11 Caffeine-13C-D3 31 -15 Cannabidiol-D3 25 11 Cocaethylene-D3 40 -10 Cocaine-D3 34 -10 Codeine-D3 27 -15 Cotinine-D3 27 -9 Diazepam-D5 83 -15 EDDP-D3 36 -11 Fentanyl-D5 40 -8 Hydrocodone-D6 31 -16 Hydromorphone-D6 45 -7 Ketamine-D4 31 -9 Lorazepam-D4 34 -16 Meperidine-D4 31 -13 Methadone-D3 51 -9 Methamphetamine-D5 27 -18 Midazolam-D4 51 -16 Morphine-D3 45 -11 Norbuprenorphine-D3 40 -12 Norcodeine-D3 27 -15 Nordiazepam-D5 45 -8 119

Norhydrocodone-D3 27 -4 Norketamine-D4 31 -7 Normeperidine-D4 31 -14 Noroxycodone-D3 27 -11 Oxazepam-D5 34 -14 Oxycodone-D6 31 -9 PCP-D5 44 -12 Pentobarbital-D5 20 11 Phenobarbital-D5 20 12 Propoxyphene-D5 36 -17 Tapentadol-D3 31 -11 THC-COOH-D9 25 13 THC-D3 20 -24 THC-OH-D3 25 25 Zaleplon-D4 34 -11

Mass Spectrometer Settings Target Target Q3 Analyte5 Collision Pre Bias Energy 10,11-Dihydro-10- Hydroxycarbamazepine -20 -20 2-Hydroxyethylflurazepam -37 -22 4-Methylephedrine -14 -17 6-MAC -27 -16 6-MAM -36 -17 7-Aminoclonazepam -24 -24 Alfentanil -20 -13 Alprazolam -27 -14 Amo-Pentobarbital 11.9 11 Amphetamine -37 -7 Benzoylecgonine -19 -18 Buprenorphine -42 -20 Bupropion -12 -19 Butabarbital 11.4 17 Butalbital 10.5 17 Caffeine -20 -14 Cannabidiol 22.1 11 Carisoprodol -14 -19 120

Clonazepam -24 Cocaethylene -21 -21 Cocaine -20 -19 Codeine -64 -16 Cotinine -24 -17 Desalkylflurazepam -29.4 -14 Desmethyltapentadol -24 -11 Dextromethorphan -24 -15 Dextrorphan -40 -16 Diazepam -31 -21 Dihydrocodeine -33 -21 Ecgonine methyl ester -25 -18 EDDP -31 -17 Ephedrine -21 -12 Fentanyl -23 -20 Flurazepam -24 -16 Gabapentin -18 -13 Hydrocodone -31 -14 Hydromorphone -28 -13 Hydroxyalprazolam -25 -15 Hydroxymidazolam -28 -21 Hydroxytriazolam -27 -24 Ketamine -26.4 -13 Lorazepam -31 -16 MDA -17.8 -14 MDMA -12.5 -17 MDPV -26 -13 Meperidine -22 -11 Meprobamate -5 -16 Methadone -16 -13 Methamphetamine -16 -24 Methylone -26 -14 Midazolam -28 -21 Morphine -59 -28 Nalbuphine -36 -19 Norbuprenorphine -71 -17 Norcodeine -63 -15 Nordiazepam -29 -16 Norfentanyl -18.8 -18 121

Norhydrocodone -29 -10 Norhydromorphone -31 -13 Norketamine -23 -13 Normeperidine -15 -17 Noroxycodone -23 -13 O-Desmethyl-cis-Tramadol -23 -22 Oxazepam -36 -22 Oxycodone -29 -12 Oxymorphone -30.6 -16 PCP -11 -9 Pentazocine -21 -15 Phenobarbital 15.5 14 Phentermine -15 -33 Pregabalin -16 -19 Secobarbital 11.5 8 Sufentanil -19 -17 Tapentadol -26 -11 Temazepam -39 -19 THC -23 -13 THC-COOH 29 29 THC-OH 28 11 Tramadol -23 -22 Triazolam -29 -22 Zaleplon -29 -17 Caffeine-13C3 -19 -25 6-MAM-D3 -40 -17 Amphetamine-D5 -14.6 -13 Benzoylecognine-D3 -18 -18 Buprenorphine-D4 -54 -24 Butabarbital-D5 13 16 Caffeine-13C-D3 -18 -29 Cannabidiol-D3 24 16 Cocaethylene-D3 -20 -30 Cocaine-D3 -20 -12 Codeine-D3 -48 -17 Cotinine-D3 -25 -19 Diazepam-D5 -30 -21 EDDP-D3 -30 -25 Fentanyl-D5 -22 -13 122

Hydrocodone-D6 -30 -21 Hydromorphone-D6 -30 -20 Ketamine-D4 -25 -13 Lorazepam-D4 -23 -14 Meperidine-D4 -20 -16 Methadone-D3 -14 -28 Methamphetamine-D5 -15 -13 Midazolam-D4 -16 -11 Morphine-D3 -60 -16 Norbuprenorphine-D3 -41 -26 Norcodeine-D3 -59 -29 Nordiazepam-D5 -27 -15 Norhydrocodone-D3 -26 -21 Norketamine-D4 -22 -24 Normeperidine-D4 -16 -17 Noroxycodone-D3 -29 -24 Oxazepam-D5 -22 -12 Oxycodone-D6 -28 -17 PCP-D5 -11 -19 Pentobarbital-D5 14 18 Phenobarbital-D5 11 11 Propoxyphene-D5 -23 -22 Tapentadol-D3 -25 -23 THC-COOH-D9 30 19 THC-D3 -26 -13 THC-OH-D3 28 17 Zaleplon-D4 -29 -17

Mass Spectrometer Settings Ref.(1) Ref.(1) Ref.(1) Q1 Ref.(1) Q3 Analyte6 Collision Dwell Time Pre Bias Pre Bias Energy 10,11-Dihydro-10- Hydroxycarbamazepine 19 -6 -35 -21 2-Hydroxyethylflurazepam 16 -10 -23 -22 4-Methylephedrine 13 -9 -22 -15 123

6-MAC 16 -10 -45 -16 6-MAM 13 -12 -27 -22 7-Aminoclonazepam 19 -6 -22 -25 Alfentanil 17 -8 -28 -14 Alprazolam 41 -6 -41 -14 Amo-Pentobarbital 25 8 16.3 14 Amphetamine 22 -7 -13 -13 Benzoylecgonine 16 -11 -30 -9 Buprenorphine 17 -9 -37 -21 Bupropion 16 -7 -36 -13 Butabarbital 25 14 16.1 14 Butalbital 25 14 15.3 14 Caffeine 15 -10 -24 -12 Cannabidiol 35 11 32.3 20 Carisoprodol 17 -8 -17 -24 Clonazepam 16 -41 Cocaethylene 19 -9 -31 -9 Cocaine 16 -9 -30 -17 Codeine 13 -7 -45 -16 Cotinine 13 -7 -22 -11 Desalkylflurazepam 16 -9 -27 -23 Desmethyltapentadol 18 -6 -68 -19 Dextromethorphan 24 -8 -38 -18 Dextrorphan 15 -10 -25 -21 Diazepam 41 -8 -28 -16 Dihydrocodeine 13 -9 -30 -21 Ecgonine methyl ester 150 -10 -44 -15 EDDP 17 -8 -24 -18 Ephedrine 22 -11 -21 -14 Fentanyl 19 -10 -31 -14 Flurazepam 19 -6 -20 -16 Gabapentin 22 -10 -24 -10 Hydrocodone 15 -9 -39 -18 Hydromorphone 22 -8 -38 -16 Hydroxyalprazolam 16 -9 -39 -22 Hydroxymidazolam 16 -7 -36 -18 Hydroxytriazolam 16 -7 -31 -18 Ketamine 15 -7 -14.9 -22 Lorazepam 16 -7 -43 -19 124

MDA 13 -9 -18.8 -14 MDMA 13 -10 -18.8 -14 MDPV 16 -8 -26 -14 Meperidine 15 -9 -20 -18 Meprobamate 16 -18 -16 -19 Methadone 25 -11 -32 -12 Methamphetamine 13 -8 -44 -25 Methylone 13 -11 -18 -17 Midazolam 25 -10 -36 -22 Morphine 22 -8 -42 -33 Nalbuphine 15 -7 -33 -18 Norbuprenorphine 19 -12 -28 -20 Norcodeine 13 -8 -45 -11 Nordiazepam 22 -15 -29 -17 Norfentanyl 15 -12 -24.8 -25 Norhydrocodone 13 -7 -39 -18 Norhydromorphone 41 -8 -38 -16 Norketamine 15 -11 -13 -15 Normeperidine 15 -7 -22 -22 Noroxycodone 13 -7 -44 -21 O-Desmethyl-cis-Tramadol 13 -5 -65 -16 Oxazepam 16 -7 -38 -16 Oxycodone 15 -6 -25 -18 Oxymorphone 22 -9 -43.4 -14 PCP 19 -7 -14 -17 Pentazocine 19 -10 -27 -14 Phenobarbital 25 8 10.5 17 Phentermine 13 -8 -40 -12 Pregabalin 29 -5 -14 -10 Secobarbital 25 8 16.2 14 Sufentanil 17 -6 -38 -12 Tapentadol 15 -11 -36 -11 Temazepam 41 -7 -36 -13 THC 125 -16 -34 -13 THC-COOH 35 16 31 17 THC-OH 35 24 29 30 Tramadol 15 -5 -65 -16 Triazolam 41 -10 -30 -23 Zaleplon 16 -16 -22 -19 125

Caffeine-13C3 15 -11 -23 -20