Abstract Recent US legislation permitting recreational use of marijuana in certain states brings the use of marijuana odor as probable cause for search and seizure to the forefront of forensic science, once again. This study showed the use of solid-phase microextraction with multidimensional gas chromatography—mass spectrometry and simultaneous human olfaction to characterize the total aroma of marijuana. The application of odor activity analysis offers an explanation as to why high volatile chemical concentration does not equate to most potent odor impact of a certain compound. This suggests that more attention should be focused on highly odorous compounds typically present in low concentrations, such as nonanal, decanol, o-cymene, benzaldehyde, which have more potent odor impact than previously reported marijuana headspace volatiles.

Citation: Rice S, Koziel JA (2015) Characterizing the Smell of Marijuana by Odor Impact of Volatile Compounds: An Application of Simultaneous Chemical and Sensory Analysis. PLoS ONE 10(12): e0144160. https://doi.org/10.1371/journal.pone.0144160 Editor: John I. Glendinning, Barnard College, Columbia University, UNITED STATES Received: September 4, 2015; Accepted: November 14, 2015; Published: December 10, 2015 Copyright: © 2015 Rice, Koziel. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: The authors have no support or funding to report. Competing interests: The authors have declared that no competing interests exist.

Introduction Americans know the Fourth Amendment of the U.S. Constitution protects citizens from unreasonable search and seizure, without a warrant, by government bodies. Landmark legal cases have set a precedent of what is deemed probable cause (S1 Table). Courts are challenged to be consistent with using odor of marijuana as probable cause when recreational use is now legal in some states and illegal at the federal level. Previous research has been conducted, identifying the volatile organic compounds (VOC) present in the headspace of marijuana. The major components of total VOC in headspace of the plant material has been reported to consist of limonene [1–5], α-pinene [1, 3, 4, 6], β-pinene [1, 3, 4, 6], β-myrcene [1, 3–5], β-ocimene [2, 4], β-caryophyllene [2, 4–6], α-caryophyllene [4, 6], α-phellandrene [4], 3-carene [4], α–terpinene [4], terpinolene [4], terpineol [5], linalool [4, 5], α-cadinene [4]. With improved analytical techniques, the list of identified compounds is increasing, starting from 20 compounds in 1973 [1] with an addition of 10 new compounds since [1–6]. Even though more compounds have been identified, it has not increased understanding of forensic odor. To date, a total of approximately 31 compounds are known to be emitted from marijuana [1–6]. Solid phase microextraction (SPME) was used as a non-destructive, non-invasive, sampling device to collect volatiles permeated through packaging and responsible for ‘characteristic’ aroma of marijuana. The use of micro-sampling techniques in forensic science has been reviewed in Kabir (2013) [7]. SPME is favored due to a smaller requirement on sample size, eliminated use of organic solvents, portability, and lends itself to automation [7]. SPME is also best at reducing matrix effects inherent in forensic work with blood, plasma, and urine [8]. Headspace (HS) sampling using SPME for characterization of volatile organic compounds (VOC) has been used to characterize explosives [9], confiscated 3, 4-methylenedioxy-N-methylamphetamine (MDMA a.k.a. Ecstasy), amphetamine [10], and cocaine [11]. The upsurge in the use of SPME as an all-in-one sample preparation, cleanup, and pre-concentration of volatiles in forensics highlights its importance to the field. There are some clear favorites in instrumentation being used for analysis of headspace VOC emitted from marijuana. Gas-chromatography (GC) was used to try and distinguish marijuana of different geographic origins, with unsuccessful results for classification [12]. GC tandem mass-spectrometry (MS) was used to characterize volatile oil composition of dried and fresh marijuana buds [13], and to discern differences between volatile compounds found in male and female marijuana plants of Northern Lights and Hawaiian Indica [14]. Volatile composition of entire inflorescences of hemp have been analyzed by GC-MS [15], even with ultrasound-assisted extraction [4]. Dogs trained for specific odor detection (e.g. narcotics, explosives, cadavers) are the current benchmark used in the law enforcement community [16–18]. A study by Macias, et al. in 2008 [17] showed that a mixture of α-pinene, β-pinene, myrcene, limonene, and β-caryophyllene associated with marijuana showed low alert responses when field tested on narcotic detection dogs. None of the dogs alerted to Sigma Pseudo Marijuana scent [17] (Sigma Aldrich, St. Louis, MO, USA). In a separate study by Jezierski (2014) [18] comparing dogs trained and tested with illicit drugs, (i.e., 68 Labrador retrievers, 61 German shepherds, 25 terriers and 10 English cocker spaniels), it was found that German shepherds were superior scent dogs and terriers were inferior at detecting drugs. The researchers tested 5 types of illicit drugs and found that marijuana was the easiest for all dogs to detect, followed by hashish, amphetamine, cocaine, and lastly heroin. In over 1000 trials, the dogs found the hidden drugs within 64 sec and an 87.7% accuracy rate (5.3% false positive) [17]. It has also been shown that the dog handler may also affect alert responses, with a failure rate of 85% false positives during search of a clean room [19]. With such a large range of variability, research is warranted on discovering what triggers an alert from the dogs. Rice (2015) [20] and Rice and Koziel (2015) [21] have reported on the usefulness of using OAV to characterize forensic odor from drugs, and offer an explanation on why current surrogate scent training tools may not be effective for canines [21]. Is human sense of smell any better? In a situational based study by Dotty in 2004, subjects were asked to smell a garbage bag containing 5 pounds of marijuana, and a garbage bag of crushed newspapers [22]. All human subjects could identify the bag containing marijuana. Could these same people detect marijuana smell sitting in the driver’s compartment, with the marijuana in a garbage bag inside the car trunk? False positives (9.36%) was the same as true positives (12.97%), with p > 0.20, meaning there were no significant difference in detecting the marijuana bag versus the newspaper bag. Next, the researchers wanted to know if budding and non-budding marijuana plants produce similar odors (i.e. mature versus non-mature plants, respectively)? A tomato plant was used as the negative control. All participants found mature (budding) plant volatiles more intense (p< 0.025) suggesting the buds hold the odorous compounds. Intensities of immature cannabis did not differ significantly from the tomato plant. Lastly, the researchers wanted to test if the smell of marijuana can be distinguished when it is mixed with diesel exhaust. The rates of detection when combined with diesel exhaust were not significant [22]. Limited work has been published on canine and human detection of marijuana odor, yielding mixed results and high variability. A thorough, analytical approach to the investigation of marijuana odor detected by humans is warranted, if and when more states seek to legalize recreational use. The objectives of this study were to (1) identify odorous compounds emitted from marijuana using multidimensional gas chromatography (MDGC) tandem mass spectrometry coupled with simultaneous human olfaction and (2) show an application and novelty of odor activity values (OAV) to better understand the ‘characteristic’ aromas of marijuana (3) explore aromatic compounds that are emitted through packaging typical in illicit distribution of marijuana. The working hypothesis is that simultaneous chemical and sensory analysis can indicate the identity of aromatic compounds that are responsible for the characteristic smell of marijuana. This information is needed to (a) better understand which compounds are really responsible for the “characteristic” aroma of marijuana, (b) provide additional insight into aroma perception by applying a method (i.e., OAV) established in food and beverage field in a new setting (i.e., forensic sciences), and (c) investigate how marijuana packaged for illicit distribution can smell differently according to these OAV. Odor activity value Odor perception is multi-faceted and this laboratory has highlighted this complexity, showing the role of highly odorous compounds present at extremely low concentrations [23]. There are two big hurdles when using GC for characterization of odorous compounds, sufficient resolution between aromatic compounds, and co-elution of two or more of these compounds. A GC using a non-polar column connected in series to a polar analytical column can account for such occurrences [24, 25]. The use of state-of-the-art simultaneous MDGC-MS-O allows researchers to separate, at high resolution, odors that may not be separated on a single column, and to detect compounds [24] based on their OAV. This report is the first instance of using MDGC-MS-O to characterize the odor of marijuana. Since the introduction of GC—olfactometry (GC-O), intensity and odor character of an individual compound has been better described [26]. Patton and Josephson originally presented the concept of the OAV [27]. A caveat is offered for equating high chemical concentration to high odor impact. The quantitative measurements of chemical concentration have been the primary data collected to date, while qualitative measurements of odor character has been largely ignored in analysis of marijuana odor. (1) where ODT is odor detection threshold and defined as the concentration a compound is detected by 50% of the population [28] OAV has been used extensively in the food and beverage industry to characterize aroma of bread, beef, coffee, beer [29] and wines [30, 31] and more recently odor emissions from animal buildings [32]. This report is the first application of OAV to characterize marijuana. This paradigm shift from concentration based (i.e., high concentration equates to potent odor) to OAV based aroma detection of marijuana and associated odor perception can help extend the knowledge of marijuana odor and its role in forensic science.

Materials and Methods The marijuana samples were obtained from Iowa Division of Criminal Investigation (Iowa DCI), Drug Identification Section. Marijuana was available in various states of seizure and included: 1) a US military-style duffel bag filled with marijuana weighing ~ 50 kg; 2) 1 gram air-dried marijuana (loose); 3) 1 gram of the same air-dried marijuana placed in a plastic zip-top sandwich bag (bagged). Carboxen/Polydimethylsiloxane (PDMS), 85 μm Stableflex, 24 gauge solid-phase microextraction (SPME) fibers were used (Sigma-Aldrich, St. Louis, MO, USA). Briefly, experimental conditions were as follows: the drugs were placed in separate, pre-cleaned and baked 16 ounce (473 mL) mason jars with modified lids. The Carboxen/PDMS fibers were exposed to the headspace and volatiles were collected. Marijuana samples were placed in the sample jars and sealed, with the exception of the marijuana in a duffel bag (S1 and S2 Figs). Immediately, SPME fibers were inserted into the sample port and exposed for 5 min, 1 h, and 68 h at ambient temperature. When the extraction step was completed, the SPME fiber was retracted, wrapped in pre-baked aluminum foil, placed in a pre-cleaned mason jar, and transported back to the laboratory in a cooler on ice. In the laboratory, fibers loaded with VOC were stored in a 4°C refrigerator until analysis, wrapped in the foil and sealed in a clean mason jar. SPME fibers were exposed to the heated injection port of the MDGC-MS-O for thermal desorption and analysis. MDGC-MS-O analysis was performed on an Agilent 6890 GC, with a restrictor guard column, non-polar capillary column (BP-5, 56 m x 530 μm inner diameter x 1.00 μm thickness, SGE, Austin, TX, USA) and polar capillary column (BP-20, 25 m x 530 μm inner diameter x 1.00 μm thickness, SGE, Austin, TX, USA) connected in series. Outflow from analytical column was held at 7.0 mL/min. Sample flow was split 3:1 via open split interface to the sniff port and mass spectrometer, respectively, as determined by restrictor column inner diameter. Desorption time was 2 min in splitless mode at 270°C under flow of helium carrier gas (99.995% purity). Subsequent analysis of the same fiber immediately afterward, revealed no carry over. The oven temperature was programmed as follows: 40°C for 3.00 min, then increased to 220°C at a rate of 7.00°C per min, and held for 11.29 min (40 min total run time). The carrier gas was set at constant pressure at the midpoint (junction point of the non-polar and polar column) at 5.8 psi (0.395 atm). Restricted transfer line to the MS was set at 240°C; restricted transfer line to the sniff port was set at 240°C with humidified air flow. MS heated zones were 150°C for the quadrupole and 230°C for the source. The MS source was electron ionization mode with ionization energy set at 70 eV. Mass acquisition range m/z 33.0–280.0 u. Tentative identification of VOC was performed using the Automatic Mass Spectral Deconvolution and Identification System (AMDIS) (National Institute of Standards and Technology, Gaithersburg, MD) and six specialty mass spectral libraries provided derived from the NIST05/EPA/NIH mass spectral database. It was not appropriate to use retention indexes (Kovats RI) for identification due to the configuration of the capillary columns, but known retention times of standards previously analyzed on this system were also used for identification. One panelist was trained using odorous chemical standards on MDGCMS-O. Sample availability and time allotted at the crime lab allowed for limited fibers (i.e., limited replicates). A single panelist analyzed all sample fibers in the open experiments. A maximum of 6 sample fibers was analyzed per day. There were three parameters recorded for perception of odorants during olfactometry work outlined in this study. The first parameter was detectability, defined here as the minimum concentration of the odorant needed to be recognized. Secondly, intensity for each aroma note was recorded, and defined here as the perceived strength of the aroma event. Guidelines for a unitless, relative intensity scale were as follows: not present = 0, faint = 25, distinct = 50, strong = 75, intense = 100. The last parameter measured was character, or aroma descriptor, as described by the trained panelist. A descriptor of “characteristic” was used when an odor was distinguished to represent the overall aroma of the sample. Area under the peak of each aroma event in the aromagram is calculated as Aroma Area = Width x Intensity x 100, where width is the length of time in min that an aroma persisted.

Conclusions Odorous compounds emitted from marijuana were identified using MDGC-MS coupled with simultaneous human olfaction. Over 200 compounds are being added to the list of what is currently known to be emitted from illicitly packaged marijuana. It is suggested that newly packaged marijuana (i.e. packaged or sitting in a room for 5 min) would have a different aroma profile than marijuana that has been stored for a longer period (i.e. packaged or sitting in a room for 68 h) due to the increased number of chemical peaks detected by MDGCMS-O (~20 compounds to ~100 compounds, respectively). Overall odor of marijuana due to compounds emitted is time dependent, but effects of plastic zip-top sandwich bag or cloth duffel bag packaging on compound surrogate concentration were not significant (p <0.05). When simultaneous chemical and sensory analysis was used to analyze headspace volatiles of marijuana emitted through a duffel bag, 9% of the chemicals detected by MS had an associated aroma, 75% of the chemicals detected did not have an aroma detected, and 16% registered low or no chemical signal but an aroma was detected. This phenomenon can be explained by taking into account OAV. The application of OAV to forensic odor is being proposed as a novel approach to investigating forensic odor. More work is needed to establish ~55% of missing ODT and ~41% missing odor description. This reports suggests that highly odorous compounds are not necessarily the most concentrated compounds in headspace. This is the first reported instance of using MDGC-MS tandem simultaneous olfactometry by human nose to characterize the volatiles in the total aroma profile emitted from marijuana in the context of non-destructive, through-packaging analysis of evidence. Shifting the focus to using OAV to evaluate total odor, instead of analyzing the most concentrated compounds, can help forensic investigators understand cadaver odor, drug odor, explosives odor, etc. This draws attention to how training a drug detection dog, handlers, and other law enforcement officers to a handful of compounds does not cover the gamut of VOC found in different conditions of marijuana for illicit distribution.

Acknowledgments The authors would like to acknowledge Iowa Division of Criminal Investigation, Drug Identification Section, for permitting the headspace SPME sampling of marijuana to be conducted in their laboratory.

Author Contributions Conceived and designed the experiments: SR JK. Performed the experiments: SR. Analyzed the data: SR JK. Contributed reagents/materials/analysis tools: JK. Wrote the paper: SR JK.