Su Youn Ahn

Determination and validation of tetrodotoxin in human whole blood using hydrophilic interaction liquid chromatography–tandem mass spectroscopy and its application

A sensitive analytical method was developed for the quantitative determination of tetrodotoxin (TTX), a powerful sodium channel blocker, in human postmortem whole blood. The sample mixture was cleaned up using cation exchange SPE catridge after protein precipitation by methanol and then separated on a PC-HILIC (phosphorylcholine hydrophilic interaction liquid chromatography) column (150 mm × 2.0mm i.d., 5 μm) using a isocratic elution of 1% acetic acid and acetonitrile. The identification of TTX was performed on tandem mass spectrometry with electrospray ionization interface in positive ion mode. The retention time of voglibose (internal standard) and TTX was 5.1 and 6.0 min, respectively. TTX and internal standard (voglibose) were monitored and quantitated using the ion transitions: the respective precursor to product ion combinations, m/z 320/302 for TTX and m/z 268/92 for voglibose in the multiple reaction monitoring (MRM) mode. The recovery of TTX and voglibose was 61.4% and 62.8%, respectively and the good accuracy (97.7-103.9%), linearity (2-1200 ng/mL) and reproducibility were shown in this method. The limit of detection and limit of quantification were 0.32 ng/mL and 1.08 ng/mL, respectively. This method was applied in the case of three fishermen who were poisoned (including one death) by unknown fish on their boat in October 2010. In this case, the levels of TTX were 27.2, 30.0 and 29.7 ng/mL in heart blood, peripheral blood and serum of a victim, were 3.1 and 12.1 ng/mL in peripheral blood and 3.9 and 12.8 ng/mL in serum of two survivors, respectively.

Determination of Flavonoid Glycosides, Polymethoxyflavones, and Coumarins in Herbal Drugs of Citrus and Poncirus Fruits by High Performance Liquid Chromatography–Electrospray Ionization/Tandem Mass Spectrometry

A high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry method was developed and validated for the determination of flavonoid glycosides, polymethoxyflavones, and coumarins in herbal medicines of Citrus fruits and peels and Poncirus fruits. The analytes were rutin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, tangeretin, imperatorin, phellopterin, and isoimperatorin, and, together with puerarin (the internal standard), were separated in short run time using a gradient flow of acetonitrile and 0.1% formic acid. Mass spectrometry detection was performed in the multiple reaction monitoring mode. Ion detection was operated alternately between negative and positive ion modes in a single injection. The main fragmentation characteristics of the analytes were investigated to discriminate between isomeric pairs. Validation of the analytical method showed linearity, r > 0.999; accuracy, 96.2 to 103.4%; and precision, relative standard deviation <4.2%. The method was employed to determine these compounds in the following herbal drugs: Citri Unshius Pericarpium, Citri Unshius Pericarpium Immaturus, Ponciri Fructus Immaturus, Aurantii Fructus Immaturus, and Aurantii Fructus. The analytical results showed that there were significant differences between different varieties, different herbal origins, and production areas. This study demonstrated that this method may be used to monitor quality control of the herbal drugs and perform chemotaxonomic investigations.

Development of a liquid chromatography-tandem mass spectrometry method for the determination of Grayanotoxins in rat blood and its application to toxicokinetic study.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid-phase extraction using PEP solid-phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C18 (100 × 2.1 mm, 2.6 µm) reversed-phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1-100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra-day and inter-day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively.

Analysis of rocuronium in human whole blood and tissues using liquid chromatography-tandem mass spectrometry.

A rapid, accurate and sensitive liquid chromatography-tandem mass spectrometry method has been developed for the determination of a quaternary nitrogen muscle relaxant, rocuronium, in human blood. The procedure involves protein precipitation with chloroform and trichloroacetic acid, and purification using methanol. The chromatography was performed using a phenyl-hexyl column (150 × 2.0 mm i.d., 3 µm; Phenomenex) with a mobile phase consisting of 5 mM ammonium formate (pH 3.0) and acetonitrile. Multiple reaction monitoring was used for quantification. The assay was linear over a concentration range of 4-500 ng/mL for rocuronium with R(2) ≥ 0.998. The recoveries for this compound ranged from 96.0 to 109.1%. The intra-day and inter-day precision was less than 10.5% and the accuracy ranged from 106.6 to 114.9%. The validated method was applied to quantify the content of rocuronium in blood and a variety of tissues of a victim suspected of overdose. In conclusion, the method was successfully applied for the analysis of rocuronium in biological samples for forensic toxicology.