A case, where the victim was strangled during sleep after drinking with the suspected culprit on one day of 2015, was submitted for us to discover the truth. The death cause was mechanical asphyxia. Frozen gastric contents and blood of the victim were extracted, ready for toxicological analysis.

1.2.1 Instruments and Setting-up

A Shimadzu 30A-LC (Shimadzu Corporation, Japan) system, equipped with a reversed phase C18 analytical column (100 mm× 2.1 mm and 1.8 μ m) (Zorbax Eclipse XDB-C18), was used for LC analysis. The injection was 2 µ L. The mobile phases were consisted of two eluents: 0.1% formic acid in water as eluent A and the absolute acetonitrile as eluent B. The gradient elution was performed at a flow rate of 0.5 mL/min, starting from 90% A and being kept for 0.6 min, then decreased to 50% A within 0.4 min and maintained for 1.0 min until returning to initial condition in 0.1 min. An equilibration time of 1.9 min was allowed prior to the next injection.

MS analysis was conducted on an API5500 QTRAP (Applied Biosystems Inc., USA) system with an electrospray ionization source (ESI) operated in positive ionization mode. The following parameters were selected for all of the experiments: ion source temperature, 600℃; capillary voltage, 5500 V; desolvation gas, 70 psi; cone gas, 8 psi. The scan mode was multiple reaction monitoring (MRM). The MS/MS parameters targeting at each compound, including retention time (Rt), ion pairs for qualitative and quantitative test, declustering potential and collision energy, were summarized in Table 1.

Table 1

Table 1

Table 1 The MS/MS parameters for determination of melatonin and 6-hydroxy melatonin Compound (Rt, min) Qualitative ion pair Quantitative ion pair Declustering potential (V) Collision energy (V) Melatonin （1.83） 233.1/174 233.1/174 70 18 233.1/159 70 35 6-Hydroxy Melatonin （1.71） 249.1/190.1 249.1/190.1 70 19 249.1/158 70 33

Table 1 The MS/MS parameters for determination of melatonin and 6-hydroxy melatonin

1.2.2 Chemicals and Reagents

Acetonitrile and formic acid, both HPLC grade, were obtained from Merk (Darmstadt, Germany). Melatonin and 6-hydroxy melatonin standards were purchased from TRC Chemical Reagent Limited Corporation (Canada).

1.2.3 Sample Preparation

Stock standard solutions were prepared in acetonitrile at concentration of 1 mg/mL, stored at -20℃. Working solutions were products from the standard stock solutions by appropriate sequential dilutions with distilled water, stored at 4 ℃.

For protein precipitation, 1 mL of blood sample was mixed with 2 mL of acetonitrile, then subjected to vortex for 30 s, sonicated for 10 min and finally centrifuged at 8000 rpm for 20 min. The supernatant of either blood sample or the gastric contents was diluted up to 100 times with distilled water, and followed to undergo LC-MS/MS analysis after filtration through a membrane of pores about 0.22 µ m.

For this case, an LC-MS/MS was decided to detect the drug (poison). Both melatonin and its metabolite 6-hydroxy melatonin were found in blood sample while only the melatonin protomer being left in the stomach contents. The negative and positive control tests confirmed the effectiveness of the method by the blank blood, and further so did it by the spiked blood sample. Extract ion chromatograms were respectively shown for the case blood, blank blood and spiked blood samples in Fig. 1~3.

Fig.1

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	Fig.1 Extract ion chromatograms of both melatonin and 6-hydroxy-melanin obtained from the case-submitted blood sample

Fig.2

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	Fig.2 Extract ion chromatograms from the blank blood sample

Fig.3

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	Fig.3 Extract ion chromatograms of both melatonin and 6-hydroxy-melanin harvested from the spiked blood sample.