Hair Analysis for Drug Facilitated Sexual Assault Cases
Angelina Mauriello
George Mason University
Drugs in Sexual Assault: An Introduction
Sexual assault is the most commonly acknowledged offence associated with drug facilitated crime. Typical drugs used in drug facilitated crime are likely to have one or more of the following properties: cause sedation, cause amnesia, odorless and tasteless, dissolve readily in a beverage, and rapidly absorbed after oral administration (Drug-Facilitated Sexual Assault, 2017, p. 2). The types of substances actually found in casework range from alcohol, Rohypnol, GHB (Gamma Hydroxybutyric Acid), ketamine and other sedating drugs like ecstasy (Drug-Facilitated Sexual Assault, 2017, p. 2). Alcohol remains the most commonly used drug in crimes of sexual assault. It is a legal over the counter depressant drug controlled by the ATF that primarily affects the Central Nervous System, specifically the brain. Rohypnol is a Schedule IV central nervous system depressant that belongs to a class of drugs known as benzodiazepines. It is odorless, colorless, and tasteless. It is used as a sedative hypnotic and a pre-anesthetic commonly misused as a “date rape” drug to incapacitate a person targeted for sexual assault. When mixed with alcohol or other drugs may lead to respiratory depression, aspiration, and even death (Drug-Facilitated Sexual Assault, 2017, p. 2). GHB is a Schedule I that can induce a sense of euphoria and intoxication and is abused for their central nervous system depressant effect. An overdose from GHB may result in respiratory depression, coma, and even death (Eckenrode Powerpoint). Ketamine is a Schedule III hallucinogen that causes the person to feel as if their mind is “separated” from the body. The drug causes a combination of amnesia and hallucinations. Also, it stops the feeling of pain and lowers the heart rate leading to oxygen starvation to the brain and muscles (Eckenrode Powerpoint). Ecstasy is a Schedule I that has hallucinogenic and amphetamine-like effects. It causes increased heart rate and blood pressure, muscle tension, teeth grinding, nausea, increased body temperature, confusion, anxiety, paranoia, decreased inhibitions, and increased self-awareness. Abuse of this drug may result in seizures, muscle breakdown, stroke, kidney failure, CV problems, and brain damage (Eckenrode Powerpoint). An ideal drug or alcohol will cause diminished capacity, which will present the victim as easy causing no memory of the offence, or will decrease their consciousness of the person resulting in them participating in activities which they normally would not. In some situations, the victim cannot recall the event that occurred under the influence of drugs due to the effects they bring (Drug-Facilitated Sexual Assault, 2017, p. 5). In these types of cases, timing is critical. It is important to report and get checked out at the earliest opportunity when you think something is wrong. Different types of samples taken for evidence have different time spans on being able to detect the drug. One sample of evidence that can be included is hair.

Collection and Analysis of Hair
Hair can be used as a tool for identifying past drug exposure. Drugs in the bloodstream travel into the blood capillaries at the hair follicle and are incorporated into the newly forming hair shaft where they are stable (Scott, 2009). Head hair grows at an average rate of 1 cm each month. As the hair grows, a history and record of drug use is by using this average hair growth rate. A sample cut from the posterior vertex region of the head, close to the scalp, is preferred as this region of the scalp is associated with least variation in growth rates (Scott, 2009). The amount of hair required for an analysis is a “lock of hair,” or a pencil thickness of hair. It is important to collect a sufficient hair sample in order to carry out routine tests and to allow for a confirmation test by a second laboratory (Scott, 2009). A hair collection kit with clear instructions for collection should be provided while following chain of custody procedures, and wear gloves when handling hair. The collection kit should include: chain of custody form, foil and collection envelope, security seal, evidence bag, transportation envelope, instructions for the collection of a hair sample (Scott, 2009). The color, length, site of collection and any obvious cosmetic treatments should be recorded. The samples must be stored in a dry environment at room temperature. The timing of the hair collection is very important. In cases where early reporting has occurred, an initial sample should be collected immediately and a second sample taken one month later (Scott, 2009). Drugs recently incorporated into hair from the bloodstream will take approximately one week to emerge from the scalp and can then be used for collection. Therefore, the sample taken up to 5 days after the offence will not contain any drug ingested at the time of the offence. The second sample having grown for an additional month will contain the portion of hair which was forming at the time of the offence (Scott, 2009). Because of this, it is recommended that a hair sample should be taken one month after the alleged offence. In cases where reporting occurs more than one month after the alleged offence, the hair sample should be taken as soon as possible. Drugs are relatively stable once incorporated into the hair shaft, however, hair treatments and normal hygienic washing regimes remove some of the incorporated drug (Scott, 2009). Typically, drugs can be detected in hair for up to two months. Head hair is the preferred sample, however, if head hair is not available alternative collection sites should be considered including pubic, underarm and beard hair. Growth rates and other characteristics of hair from these alternate sites differs from head hair. If hair analysis has to be performed, preliminary appropriate washing of the sample is mandatory in order to minimize the risk of surface contamination (Scott, 2009). Hair segmentation is also highly recommended. Segments provide a stronger level of evidence and can assist in establishing a tighter timeframe during which ingestion occurred.

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Figure 1: Collection of hair
Techniques for Analysis
Chromatography is a separation process which depends upon the differential distribution of sample components between a moving phase and a stationary phase (Eckenrode Powerpoint). There exist many different kinds of chromatography techniques, but in particular, hair analysis for drugs uses the following techniques: GC-MS, GC-MS-MS and LC-MS-MS. GC-MS begins with the gas chromatograph, where the sample is volatized. The sample is vaporized and separates its various components using a capillary column packed with a stationary (solid) phase (ThermoFisher Scientific). The compounds are propelled by an inert carrier gas such as argon, helium or nitrogen. As the components become separated, they elute from the column at different times, which is generally referred to as their retention times (ThermoFisher Scientific). Once the components leave the GC column, they are ionized by the mass spectrometer using electron or chemical ionization sources. The ionized molecules are then accelerated through the instrument’s mass analyzer, and are separated based on their different mass-to-charge (m/z) ratios (ThermoFisher Scientific). The final steps of the process involve ion detection and analysis of the mass to charge ratio represented by a peak. Using computer libraries of mass spectra we can identify and quantitate unknown compounds and analytes. GC-MS-MS is the same as a GC-MS but with an extra step. When the components leave the column, they enter the tandem mass spectrometer (MS-MS) which consists of two scanning mass analyzers separated by a collision cell instead of just one mass analyzer (ThermoFisher Scientific). Fragments selected in the first mass analyzer are reacted with an inert gas in the collision cell, resulting in further fragmentation. These daughter product ions are then resolved in the third quadrupole for analysis (ThermoFisher Scientific). “Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) is a powerful analytical technique that combines the separating power of liquid chromatography with the highly sensitive and selective mass analysis capability of triple quadrupole mass spectrometry” (EAG LABORATORIES). A sample solution containing analytes of interest are pumped through a stationary phase (LC column) by a mobile phase flowing through at high pressure. Chemical interaction between the components of the sample, the stationary phase and the mobile phase affects different migration rates through the LC column affecting a separation (EAG LABORATORIES). After elution from the LC column, it is directed to the mass spectrometer. The mass spectrometer for this system has an ionization source where the LC column waster is nebulized, desolvated and ionized creating charged particles (EAG LABORATORIES). These charged particles then migrate under high vacuum through a series of mass analyzers (quadrupole) by applying electromagnetic fields. A specific mass/charge precursor ion is targeted to pass through the first quadrupole, excluding all others. In the collision cell, the selected mass/charge ions are then fragmented into product ions by collision with an inert gas (EAG LABORATORIES). The third quadrupole is used to target specific product ion fragments. The resulting isolated product ions are then quantified with an electron multiplier. This transition of ions from the precursor to product ion is highly specific to the structure of the compound of interest and therefore provides a high degree of selectivity (EAG LABORATORIES).

Figure 2: Gas Chromatography Diagram Figure 3: Example GC-MS

Figure 4: LC-MS-MS Diagram
Techniques and Analysis for GHB in Hair
Hair strands were obtained from a female victim who had claimed to have been sexually assaulted after drinking a soft drink spiked with a drug. She had no memory of the crime and went to the police the day after the event occurred. After contact with the police, this laboratory recommended to wait for about one month in order to have the corresponding growing hair between the root and the tip (Kintz, Cirimele, Jamey, ; Ludes, 2003). Full-length hair samples (8 cm long) were taken at the surface of the skin from the vertex and stored in plastic tubes at room temperature (Kintz et al. 2003). Controlled hair specimens of twelve laboratory personnel were collected to establish the physiological GHB concentrations, as well as a sample from a subject receiving a 25 mg/kg dose. To extract the GHB, the hair was decontaminated twice using 5 mL of methylene chloride for two minutes at room temperature and then cut into 3-mm segments over a length of 3 cm (ten segments). A longer decontamination time will reduce the concentration of GHB in the hair. About 5 to 10 mg of decontaminated hair were incubated in 0.5 mL 0.01 N NaOH, 16 h at 56°C, in the presence of 10 ng of GHBd6 used as an internal standard (Kintz et al 2003). After cooling, the homogenate was neutralized with 0.5 mL 0.01 N HCl, and 3 mL of ethyl acetate were added together with 0.1 mL of 0.01 M H2SO4 (Kintz et al. 2003). After agitation and centrifugation, the organic phase was evaporated to dryness under nitrogen flow. The residue was derivatized by adding 20 micro liters BSTFA + 1% TMCS and 20 micro liters ethyl acetate, then incubated for 20 min at 60°C (Kintz et al. 2003) 1 micro liter aliquot of this derivatized extract was injected into the column of a gas chromatograph (6890 Series). The flow of carrier gas used was helium at a flow rate of 1 mL/min. The injector temperature was 270°C, the column oven temperature was programmed to rise from an initial temperature of 100°C, maintained for 1 min, to 295°C at 30°C/min, and maintained at 295°C for the final 5 min (Kintz et al. 2003). The detector was a Finnigan TSQ 700 operated in the electron ionization mode and in selected reaction monitoring. The precursor ions, m/z 233 and 239 for GHB and the internal standard, were selected in the first quadrupole. The common product ions, m/z 147 and 148, were selected in the third quadrupole after collision with argon at a cell pressure at 0.62 mTorr (Kintz et al. 2003). Quantitation of GHB and its internal standard were performed in selective ion monitoring mode and retention times and ions monitored were recorded. In normal population, the GHB concentration in the hair segment is always lower than 20 ng/mg. In the case of GHB exposure, the concentration is clearly much higher, allowing a biological discrimination between exposed and non-exposed (Kintz, 2016). Sweat and probably sebum contaminations are likely contributors to the elevated GHB concentration. This new approach allows toxicologists to obtain a much better discriminating ratio between the two populations and should be used in routine (Kintz, 2016). As GHB is endogenous it is expected to be positive throughout the hair. Given the average hair growth rate in adults is 1 cm per month, if a sample of hair was not collected right away, the hair segment would have grown out further. Every segment of the hair will be positive for GHB, however, an elevated level can be discovered in just one segment. Depending on the location of the segment in the hair, a time frame of alleged ingestion can be determined. This allows an increase in the detection window compared to detecting GHB in blood and urine with only a 6-12 hour window (Kintz, 2016).

Figure 5: Data
Techniques and Analysis for Ketamine in Hair
Hair strands were obtained from self-confessed ketamine abusers. Drug free hair samples were obtained from laboratory personnel and were used for calibration, quality controls, and validation (Leong, Tan, Lui, ; Lee, 2005). Quantitation of ketamine and norketamine was performed in selective ion monitoring mode and retention times and ions monitored were recorded. Some of the drug free samples were spiked with ketamine and norketamine to be used as well. Two or more hair segments of one to three centimeters were obtained from each hair sample. All hair samples were first washed with 10 mL each of methylene chloride, distilled water, and methanol (Leong et al. 2005). After, they were dried in the oven at 60°C, and then were pulverized using a ball mill mixer into fine powder. About 25 mg of the pulverized hair sample was weighed into a glass tube. Fifty microliters of internal standard solution of ketamine and norketamine was added to the hair sample followed by 1 mL of 0.5M of hydrochloric acid (Leong et al. 2005). The mixture was vortexed and incubated overnight at 45°C and then cooled to room temperature. The suspension was neutralized using 1 mL of 0.5M of sodium hydroxide and buffered with 1 mL of ammonium buffer (Leong et al 2005). The suspension was centrifuged at 2500 rpm for 10 minutes, and the supernatant liquid was transferred to another tube for solid-phase extraction. The C-18 disposable extraction cartridge was conditioned by sequential rinsing with 3 mL of methanol and 3 mL of distilled water (Leong et al. 2005). The sample solution was then loaded into the conditioned cartridges and washed with 3 mL of distilled water and twice with 3 mL of a methanol water mixture. The drugs of interest were eluted with 3 mL of methanol. The eluate was collected in a clean tube and evaporated to dryness at 40°C under a stream of nitrogen (Leong et al. 2005). The residue was reconstituted in 50 microliters of methanol and transferred to a vial for GC-MS analysis (Leong et al 2005). One microliter of the sample extract was injected into the GC-MS. The GC oven was maintained at 75°C for 0.5 minutes, increased to 180°C, held at this temperature for 7.0 minutes, increased to 260°C, and maintained at this final temperature for 1.42 min (Leong et al 2005). From the results, a correlation between the amount of ketamine detected and the frequency of abuse was observed.