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Two Fatal Cases of Caffeine Poisoning Shimane J. Med. Sci., Vol.37 pp.103-107, 2020 Two Fatal Cases of Caffeine Poisoning Sultana Jannatul Ferdoush JERIN1）, Kaori KIMURA-KATAOKA1）, Junko FUJIHARA1）, Rahman MA- MUNUR1）, Gong AO1）, Sadayuki HASHIOKA2）, Mutsumi HORI3）, Masanori HASEGAWA3）, Haruo TAKESHITA1） 1)Department of Legal Medicine, Shimane University School of Medicine, Izumo 693-8501, Japan 2)Department of Psychiatry, Shimane University School of Medicine, Izumo 693-8501, Japan 3)Criminal Investigation Laboratory, Shimane Prefectural Police H.Q.,690-0038,Japan （Received August 24, 2020; Accepted October 6, 2020） A caffeine overdose is most likely to result from the INTRODUCTION consumption of nutritional supplements or caffeine pills. Herein, we describe two fatal cases of caffeine intoxi- Caffeine （1,3,7-trimethylxanthine） is classified as cation. Case 1: A Japanese man in his 30s was found a weakly basic natural alkaloid with a molecular dead, with two bottles of water and unopened tablets for weight of 196.21 g / mol and is contained in cof- motion sickness near his body. Caffeine concentrations fee, chocolate, cocoa, black tea, green tea, and many were 99.6 μg / mL in the cardiac blood and 169.1 μg / other products. It is one of several methylxanthines mL in stomach contents. Case 2: A Japanese man in his known to have pharmacological effects including 40s was found dead, with an empty bottle of caffeine central nerve stimulation, cardiac stimulation, the and six empty boxes of Travelmin（ a motion sickness relaxation of smooth muscles such as bronchi, and medication） in a trash can. Caffeine concentrations diuresis. Caffeine moderately stimulates the cen- were 79.6 µg / mL in the cardiac blood and 876 µg / tral nervous system to awaken, reduce fatigue, and mL in the stomach contents. Fatal caffeine poisoning stimulate cognitive activity. The range of the safe is comparatively uncommon, but it has recently been administration of caffeine is relatively wide, and it increasing due to the easy availability. For the preven- is unlikely that poisoning will be caused by coffee tion of caffeine overdoses, there should be heightened ［1］. After water, coffee is the most popular bever- public awareness of the potential dangers of a caffeine age and is consumed worldwide in daily amounts of overdose. roughly 1.6 billion cups［ 2］. Caffeine is one of the drugs most readily avail- Keywords: caffeine, poisoning, fatal, LC/MS, GC/ able to the general public. It is often used to relieve MS drowsiness and reduce anxiety and fatigue. Caffeine stimulates the nervous system by blocking the ef- fects of the neurotransmitter adenosine, but high doses of caffeine may have unpleasant and even dangerous side effects ［3］. Intentional poisoning by caffeine consumption has recently become com- paratively common. When consumed in relatively insignificant doses, the side effects of caffeine are milder, but when a high dose is ingested, this meth- ylxanthine alkaloid becomes profoundly toxic and can result in death ［4］. Several reports of caffeine Corresponding author: Haruo Takeshita, MD, PhD overdoses have been published despite the rarity of Department of Legal Medicine, Shimane University Fuculty of caffeine intoxication. Here, we report two cases of Medicine, fatal caffeine intoxication. 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan Tel: +81-853-20-2156 Fax: +81-853-20-2155 Email: [email protected] 103 104 TAKESHITA et al. CASES MATERIALS AND METHODS Case 1 Sample preparation A Japanese man in his 30s was found dead out- Whole blood and stomach contents of each case side a church by some tourists. On a nearby bench, were collected at autopsy and kept frozen at −80℃ there were two bottles of water, unopened tablets until analysis. The extraction for narcoleptics, ata- of Semper QT （a medication for motion sickness）, ractics, and agrichemicals performed using Extrelut and a bag with his wallet and a pessimistic note. NT-3 polyethylene columns（ Merck, Darmstadt, Ger- Semper QT contains d-chlorpheniramine maleate and many）. Briefly, ammonium chloride/ammonia buffer scopolamine hydrobromide. There was some vomit （pH 9.5） was added to the blood sample, and the under a bench a short distance away from where mixture was loaded onto Extrelut NT-20 columns. the man was discovered（ Fig. 1a）. After 15 min, the mixtures were eluted from the columns using methylene chloride/isopropanol（ 85:15 Case 2 a v/v）. The solvent was distilled and then dissolved A Japanese man in his 40s did not arrive at work in methanol. The stomach contents were mixed with on Monday morning as expected, and he was found distilled water and acetonitrile. Anhydrous sodium dead on the floor of his apartment house that after- sulfate/anhydrous sodium acetate （4:1 v/v） was noon. There was an empty bottle of caffeine （100 added to the mixture, and the supernatant after cen- tablets） and six empty boxes of the motion sickness trifugation was filtered using Captiva NDLipids（ Agilent medication Travelmin （36 tablets） in a trash can, Technologies, Santa Clara, CA, USA）. The eluants and 18 empty boxes of Travelmin （108 tablets） in were evaporated under a stream of nitrogen gas, another trash can. Travelmin contains diphenhydr- and the residue was dissolved in acetonitrile for the amine salicylate and diprophylline. A mortar and liquid chromatography/mass spectrometry （LC/MS） pestle, a soup bowl with some white powder, and analysis. an unopened bottle of caffeine （100 tablets） were Caffeine extraction was performed using Extrelut on a table in the room where the man was found NT-20 columns. Briefly, samples were diluted with （Fig. 1b）. distilled water （sample/distilled water, 1:100 v/v）. An internal standard （IS） solution （diazepam-d5） a b Figure 1: a） There were two water bottles, a plastic bag and two medications that are sold for the prevention and palliation of dizziness, nausea, and headache due to motion sickness. b） An empty bottle of caffeine（ 100 tablets）, a mortar, and a bowl on the table. b Caffeine poisoning 105 was added to each sample. Caffeine and the IS so- DB-5 MS column （30m × 0.25 mm inner dia.: lutions were mixed with an ammonium chloride/am- Agilent Technologies）. The column temperature was monia buffer（ pH 9.5）, and the mixture was loaded programmed initially at 60℃ （2 min） to 320℃ at onto Extrelut NT-20 columns. After 15 min, the 10°C/min, which was maintained for 10 min. The caffeine and the IS solution were eluted from the injector and ion-source temperatures were 260℃ columns using methylene chloride/isopropanol（ 85:15 and 200℃, respectively. The MS detection was per- v/v）. The eluants were evaporated under a stream formed with electron ionization（ EI）. of nitrogen gas, and the residue was dissolved in methanol. GC/MS conditions for caffeine Alcohol concentration was measured by the head- Caffeine was determined by gas chromatography- space method and GC-FID detector. Sample 0.2 mL mass spectrometry （HP 7890B/5977A GC/MSD, were placed into sealed vials containing 0.2 mL in- Agilent Technologies）. Compounds were separated ternal standard solution of 0.2% 2-methyl-1propanol using a HP-5 MS column （30m × 0.25 mm inner and incubated at 55°C for 15 minutes, then 0.3 mL dia.: Agilent Technologies）. The column tempera- of the gas phase was injected. The sample was ana- ture was programmed at initial 50℃ （1 min） to lyzed using DB-WAX columns （GC-18A manufac- 100℃ at 20°C/min and finally to 280℃ at 10℃/ tured by Shimadzu Corporation）, inner diameter 0.53 min, which was maintained for 20 min. The injector mm, length 30 m, thickness 1 μm. The Column and ion-source temperatures were 250℃ and 230℃, temperature was maintained at 40°C （1 min hold） respectively. MS detection was performed with EI. →100°C （10°C/min temperature rise）. The Inlet The m/z values 194 and 289 were monitored for temperature was at 200°C. Helium was used as a caffeine and diazepam-d5 （IS）, respectively, in the carrier gas at a flow rate of 1 mL/min. selected ion monitoring mode for quantification. LC/MS conditions for narcoleptics, ataractics, and RESULTS agrichemicals The qualitative analysis was performed using a Case 1 Prominence ultra-fast liquid chromatography（ UFLC） The deceased was 172.0 cm and weighed 55.1 system （Shimadzu, Kyoto, Japan）/linear trap quad- kg. There were slight abrasions and skin discol- rupole （LTQ） （Thermo Fisher Scientific, Waltham, orations on the face, neck, and upper and lower MA）. Compounds were separated using Hypersil limbs, and minor lacerations on the face and intra- Gold™ columns （50 mm × 2.1 mm inner dia. and muscular bleeding on the neck. The major organ 10 mm × 2.1 mm inner dia.; Thermo Fisher Scien- weights were as follows: brain 1450 g, heart 290 g, tific）. The column temperature was programmed at right lung 345 g, left lung 340 g, and liver 1320 g. 40℃. The mobile phase consisted for methanol（ A） Moderate to severe pulmonary edema was confirmed and 10mM ammonium acetate （B）. The gradient pathologically. The cardiac blood contained the sleep profile was as follows: 10% A hold for 2 min, fur- inducer/anticonvulsant nitrazepam, and the stomach ther increased linearly to 94% A from 2 to 23 min. contents contained zopiclone, a sleep disorder-im- After 3 min, the mobile phase returned to 10% A, proving agent. The caffeine concentrations were 99.6 equilibrating for 4 min up to a total run time of 30 μg/mL in the cardiac blood and 169.1 μg/mL in the min. stomach contents. GC/MS conditions for narcoleptics, ataractics, and Case 2 agrichemicals The deceased was 169.0 cm and weighed 73.2 A qualitative analysis of narcoleptics, atarac- kg.
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