Eunyoung Han

Evaluation of postmortem redistribution phenomena for commonly encountered drugs

We described the findings of a study into the post-mortem redistribution (PMR) of 76 drugs found in 129 drug-related cases between 2006 and 2009. Seventy six drugs (psychotropic drugs (n=14), antidepressants (n=9), sedatives (n=6) and so on) were simultaneously quantified in cardiac and peripheral blood by gas chromatography-mass spectrometry (GC/MS) or liquid chromatography-tandem mass spectrometry (LC/MS/MS). The absence, possibility or presence of PMR of drugs was determined according to the ratios of cardiac to femoral blood concentrations (C/P ratios). Proxyphylline (C/P ratio: 0.85) showed no PMR; carbamazepine was not subject to PMR; a potential for PMR of lorazepam and mirtrazapine cannot be excluded; chlordiazepoxide is subject to PMR; acetaminophen and alprazolam exhibit minimal PMR; amitriptyline and benztropine exhibit PMR. Codeine (C/P ratio: 4.9), zolpidem (C/P ratio: 3.74), chlorpromazine (C/P ratio: 2.97), fluoxetine (C/P ratio: 2.83) and propranolol (C/P ratio: 2.72) had the largest C/P ratios. Postmortem drug concentrations showed variations depending on sampling sites and characteristics of the drugs. It is continuously necessary to analyze commonly used or abused drugs in simultaneously collected cardiac and peripheral blood to establish significant reference values for PMR. These findings can be used to reach a conclusion about the cause and manner of death.

Hair analysis and self-report of methamphetamine use by methamphetamine dependent individuals

The questions of whether the dose of drug that is consumed corresponds to drug concentration levels in hair and how results of hair analyses can be interpreted are still debated. The aim of this study was to investigate (1) whether there is a correlation between doses of Methamphetamine (MA) use and MA concentration levels in hair and (2) whether results of hair analyses can be used to estimate dose, frequency, and patterns of MA use. In this study, segmental hair analysis was performed through consecutive 1cm as well as 1-4 cm (=3 cm) segmental hair lengths. MA dependent individuals (n=9) provided information on doses (0.25-4 g/day) of MA use as well as the frequency of MA use. The concentrations of MA and its metabolite amphetamine (AP) in hair were determined using gas chromatography/mass spectrometry (GC/MS). One-way analysis of variance (ANOVA) test was performed to evaluate whether MA and AP concentrations in consecutive 1cm length segmental hair were consistent with the history of MA use. The cumulative doses of MA use calculated from the daily dose and the frequency during 1-4 months were well correlated to the concentrations of MA and AP in 1-4 cm segmental hair length (correlation coefficient, r=0.87 for MA and r=0.77 for AP). The results from this study show the patterns and histories of MA use from MA dependent individuals and could assist in the interpretation of hair results in forensic toxicology as well as in rehabilitation and treatment programs.

Real-time concurrent monitoring of apoptosis, cytosolic calcium, and mitochondria permeability transition for hypermulticolor high-content screening of drug-induced mitochondrial dysfunction-mediated hepatotoxicity.

A quantitative high-content screening (HCS) was suggested for the real-time monitoring of drug-induced mitochondrial dysfunction-mediated hepatotoxicity. This HCS is very advantageous in that it allows simultaneous observation of drug-induced activations of hepatotoxic pathways using hypermulticolor cellular imaging. The mitochondrial permeability transition (MPT), cytosolic calcium, and caspase-3 were selected as functional markers to verify drug-induced hepatotoxicity and were concurrently monitored in HepG2 cells in a real-time manner. Nefazodone, tolcapone, and troglitazone caused mitochondrial dysfunction and subsequent apoptotic HepG2 cell death in addition to marked cytosolic calcium increase. On the other hand, extrinsic pathway-mediated apoptotic cell death was monitored when HepG2 cells were treated with piroxicam. It was found that piroxicam-treated HepG2 cells showed apoptotic cell death without the MPT formation, while a cytosolic calcium increase was clearly observed. This finding was confirmed by the caspase-8 inhibition assay. These results demonstrated the unique potential of real-time hypermulticolor HCS to screen hepatotoxic drugs at the in vitro stage rather than the later in vivo stage based on an animal model and to ultimately reduce the probability of drug failure.

Segmental Hair Analysis for 11-Nor-Δ9-Tetrahydrocannabinol-9-Carboxylic Acid and the Patterns of Cannabis Use

Cannabis is the most widely abused drug in the world. The purpose of this study is to detect 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THCCOOH) in segmental hair and to evaluate the patterns of cannabis use. We investigated the relationship between the concentrations of THCCOOH in hair and the self-reported use data and the route of administration. For this purpose, the hair samples were washed, digested with 1 mL of 1 M NaOH at 85°C for 30 min along with the internal standard, THCCOOH-d₃ (2.5 pg/mg) and extracted in 2 mL of n-hexane-ethyl acetate (9:1) twice after adding 1 mL of 0.1N sodium acetate buffer (pH = 4.5) and 200 µL of acetic acid. The organic extract was transferred and evaporated and the mixture was derivatized with 50 µL of pentafluoropropionic anhydride and 25 µL of pentafluoropropanol for 30 min at 70°C. Reconstituted final extract was injected into the gas chromatography-tandem mass spectrometer operating in the negative chemical ionization mode. In segmental hair analysis, the concentrations of THCCOOH decreased from the proximal to distal segments. The concentrations of THCCOOH in hair and the self-reported dose and frequency of administration from cannabis users were not well correlated because of the low accuracy and reliability of the self-reported data. However, this study provides preliminary information on the dose and frequency of administration among cannabis users in our country.

Simultaneous analysis of Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol in hair without different sample preparation and derivatization by gas chromatography–tandem mass spectrometry

The present study describes a gas chromatography/tandem mass spectrometry-negative ion chemical ionization assay (GC/MS/MS-NCI) for simultaneous analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair. Each hair sample, of approximately 20mg, was weighed and the sample was dissolved in 1ml of 1M sodium hydroxide (30min at 85°C) in the presence of THC-d(3) and THCCOOH-d(3). For the analysis of THC, hair samples were extracted with n-hexane:ethyl acetate (9:1) two times; acetic acid and sodium acetate buffer were added for the analysis of THCCOOH, and hair samples were re-extracted with n-hexane:ethyl acetate (9:1) two times. The extracts were then derivatized with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH). This method allowed the analysis of THC and THCCOOH using the GC/MS/MS-NCI assay. This method was also fully validated and applied to hair specimens (n=54) collected from known cannabis users whose urine test results were positive. The concentrations of THC and THCCOOH in hair ranged from 7.52 to 60.41ng/mg and from 0.10 to 11.68pg/mg, respectively. In this paper, we simultaneously measured THC and THCCOOH in human hair using GC/MS/MS-NCI without requiring different sample preparation and derivatization procedures. The analytical sensitivity for THCCOOH in hair was good, while that for THC in hair needs to be improved in further study.

Effects of repeated hair washing and a single hair dyeing on concentrations of methamphetamine and amphetamine in human hairs

The effects of repeated hair washing and a single hair dyeing on concentrations of methamphetamine (MA) and amphetamine (AM) in hair samples of MA addicts were studied. Thirty-one MA positive hair samples collected from male (n = 24, 24-51 yrs) and female abusers (n = 7, 17-46 yrs) were evaluated for MA and AM concentrations changes after repeated hair washing and a single hair dyeing. Thirty-one MA positive hair samples, no additional treatment hair sample group (NAT), were treated in vitro with liquid soap or three kinds of hair dyes which were black, brown and yellow color hair dye, respectively. Quantitation of AM and MA in hair samples was utilized GC-MS using selected ion monitoring. MA and AM concentrations in NAT were 10.41 ± 8.91 ng/mg (range 1.50-30.0 ng/mg) and 2.24 ± 2.75 ng/mg (range 0.41-12.90 ng/mg). And, their concentrations were decreased about 23.3 ± 4.5% (range 16.7-32.8%) in hair repeated washing group (WAS) and 32.6 ± 4.82 (22.2-41.9) in three kinds of a single hair dyeing groups in comparison to original concentrations of MA and AM in NAT. A statistically significant difference was found between NAT and WAS or three hair dyeing groups (p < 0.01), but not between WAS and three hair dyeing groups, and not between each hair dyeing groups with each three kinds of hair dyes (p > 0.05).

Deaths from recreational use of propofol in Korea

Propofol, a short-acting and sedative-hypnotic agent, induces and maintains anesthesia. Since it is known to produce mild euphoria and hallucinations, the recreational use of propofol has been a big issue in Korea. Furthermore, many deaths have occurred due to its abuse and misuse. In order to study the prevalence of abuse and deaths due to propofol, all autopsy cases conducted between 2005 and 2010 at the NFS (National Forensic Service, Korea) were monitored by checking its concentrations in the blood. Propofol was detected in 131 cases (0.88%) out of 14,673 autopsied cases within 6 years. Propofol alone was detected in 49 of 131 fatal cases, while the combination of drugs was detected with propofol in the remaining 82 cases. The concentrations of propofol from autopsied cases ranged from 0.05 to 8.83 mg/L (mean 1.66; median 0.9) and from 0.08 to 8.65 mg/L (mean 1.71; median 1.05) in the heart (n=31) and the femoral blood (n=32), respectively. The investigation of the ratio of heart to femoral blood and the difference between the concentrations in heart and femoral blood (n=15) from the same body revealed the ratio from 0.45 to 3.66 (mean 1.53; median 1.40). The autopsy resulted in accidental death after self-administration in 16 autopsied cases among 131 autopsied cases. In 16 cases, their ages ranged from 17 to 56 and 75% of them were in their 20s and 30s and 75% were female. Half of them were medical personnel including 19% of doctors and 38% of nurses. The combination of drugs was detected in 6 cases. Fluoxetine was detected in three and vecuronium was detected in two along with propofol. The cause of death in 14 cases was drug intoxication, while that in 2 cases was hanging. Due to its prevalence, Korea has become the first country that regulates propofol as a psychotropic substance.

The dependence of the incorporation of methamphetamine into rat hair on dose, frequency of administration and hair pigmentation

In this paper, the incorporation of methamphetamine (MA) into rat hair was studied. The main purpose of this study was to investigate whether MA can be detected or positive hair results can be obtained in hair of rats administered a single dose of MA. The relationship between dose and frequency of administration and the concentrations of MA and its metabolite, amphetamine (AP), in rat hair were evaluated and the MA and AP concentrations in white and pigmented hair were compared. MA was administered to rats as follows: low dose (0.5mg/kg/day), medium dose (2mg/kg/day) and high dose (10mg/kg/day). The frequency of administration was one time per day for 1, 2, 3, 4, 5, 15 and 30 days. Hair and urine samples were collected from rats and analyzed by gas chromatography/mass spectrometry (GC/MS). MA could be identified in pigmented rat hair when MA was administered for 4 or more days at low daily dose and on day 1 following administration of medium and high daily doses. Positive results for MA were obtained from pigmented rat hair when MA was administered for 30 days at low daily dose, for 4 or more days at medium daily dose, or for 2 or more days at high daily dose. The concentrations of MA and AP found in rat hair were proportional to the dose and frequency of administration. The concentrations of MA and AP in pigmented rat hair were 2-10 times higher than those in white rat hair. The results of this study on the incorporation of MA into rat hair can serve as a model to better understand the incorporation of MA into human hair even though there are differences between animal models and human hair.

A multifunctional composite of an antibacterial higher-valent silver metallopharmaceutical and a potent wound healing polypeptide: a combined killing and healing approach to wound care

The present study relates to a combined killing and healing approach for the treatment of infected wounds. Herein we report a multifunctional, including antimicrobial and wound healing, composite containing a conjugate of a bi-valent silver polydiguanide that demonstrated high antibacterial activity in vitro and a potent wound healing polypeptide, histatin-1, for the treatment of infected wounds. The synthesis of silver(II) chlorhexidine [Ag(II)CHX] was accomplished by the oxidation of Ag(I), followed by the complexation of the oxidized metal with chlorhexidine (CHX), whereas the metal complex conjugate of the solid phase-synthesized histatin polypeptide (Hst-1), Hst-1-[Ag(II)CHX], was realized by mixing the starting materials in aqueous solution. The change in the Hst-1 structure upon binding with the silver complex was examined by circular dichroism spectroscopy. The wound healing applicability of the histatin polypeptide and its metal complex conjugate was tested using the synthesized Hst-1 and Hst-1-[Ag(II)CHX] complex on 3T3-L1 preadipocytes in a cell-spreading assay. The antibacterial activity of the silver metal complex and its Hst-1 conjugate was tested against several gram positive and gram negative bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant coagulase negative staphylococcus (MRCNS) by a broth microdilution method. The results of these experiments revealed that the polypeptide and silver(II) polydiguanide complex retained their individual wound healing and antimicrobial activity even in their conjugate. The conjugate of an antibacterial higher-valent silver polydiguanide complex with a potent wound healing polypeptide (Hst-1) showed promise as a new multifunctional therapeutic wherein the killing and healing functions of the constituent materials are preserved together for the development of new-generation wound-care agents.

Thebaine in hair as a marker for chronic use of illegal opium poppy substances

Opium poppy products are often illegally used for both recreational and medicinal purposes. In order to demonstrate the ingestion of opium poppy substances, morphine, codeine and their metabolites have been identified. However, morphine and codeine also originate from the ingestion of therapeutic drugs. Therefore, thebaine, one of the main opium alkaloids, in hair was suggested as a marker for chronic use of illegal opium poppy substances in the present study. First, thebaine was included in the analyte list of our routine analytical method for the simultaneous quantification of codeine, morphine, norcodeine, normorphine and 6-acetylmorphine (6-AM) in hair, which was fully validated previously. Then, the incorporation of thebaine and other opiates into hair and the effect of hair pigmentation were examined using lean Zucker rats with both dark grey and white hair on the same body. Thebaine was also measured in hair samples from actual cases of opium poppy substance use. Consequently, thebaine in hair was demonstrated as a marker of chronic use of illegal opium poppy substances using an animal study and actual cases. Thebaine and other opiates were successfully measured in pigmented hair from rats that ingested raw opium suspension. Moreover, thebaine identified in hair excluded possibility of ingestion of pharmaceutical opiates in actual cases.