Hypoglycin A (HGA) is the toxic principle in ackee (Blighia sapida Koenig), a member of the Sapindaceae family, originating from West Africa and imported into the tropical regions of the Caribbean and Central America. Ackee is a nutritious and readily available fruit that is a staple of the Jamaican working-class and rural population. The aril of the unripe fruit has high concentrations of HGA, the cause of Jamaican vomiting sickness, which is very often fatal. HGA is also present in the samara of the sycamore maple (Acer pseudoplatanus L.) which causes equine atypical myopathy, often fatal for horses. The aim of this study was to develop a method for quantifying HGA in blood that would be sensitive enough to provide toxicological evidence of ackee or maple poisoning.

Given the size and the polarity of HGA (zwitterionic compound), the preparation of the biological sample (0.25mL) consisted of a solid phase extraction using CHROMABOND^® HILIC cartridges (Macherey-Nagel^®). The sample was vortex mixed with isopropyl alcohol (4.75mL) and centrifuged, the supernatant being applied to the cartridges. Elution was then performed with methanol. In a first stage of the method development, analysis was carried out using hydrophilic interaction liquid chromatography (bare silica) coupled with high-resolution mass spectrometry detection (HILIC-HRMS) (HGA was not retained in reversed-phase conditions). The chromatographic peak was perfectly resolved but there was considerable interference from some isobaric compounds extracted from the matrix. Therefore, we resorted to a derivatization of the compounds which discriminate the impurities depending on their chemical groups: the eluate was derivatized with dansyl chloride. Analysis could be carried out using a technique of ultra-high performance liquid chromatography coupled with HRMS detection (UHPLC-HRMS) in very classic conditions. Separation was performed using a C₁₈ column for a gradient mobile phase composed of acetonitrile and a 2mM ammonium formate buffer at pH=3.0. The chromatographic run time was 10min. Spectrometric detection was performed using a quadrupole-Orbitrap high-resolution detector (Q Exactive™; Thermo Scientific) after ionization by heated electrospray in positive-ion mode. The mass spectrometer operated in full-scan mode and targeted-MS² mode alternately. MS scans (373 – 456 amu) were acquired with a mass resolution of 70000. The pseudomolecular ion [M+H]⁺ of dns-HGA (375.1369 amu ± 10ppm) was used for quantification. The full-scan product ion spectrum of the compound (resolution of 17500) was used to confirm the identity of the toxin.

The method was validated in the whole blood with between- and within-day RSD (relative standard deviation) less than 12.3% and 11.9% (accuracy between 89.4% and 100.8%). The calibration curve was linear for concentrations ranging from 0.8 to 500μg/L. The detection limit was 0,35μg/L. HGA was quantified in dried arils of unripe ackee fruit harvested in Suriname by the standard method addition. The concentration was 11,7mg/g.

A previous method of quantification of HGA in blood was published in 1977 [Fincham, West Indian Med J, 1977, 26: 62- 65]. This method was limited by insufficient sensitivity (1,4mg/L) given the rapid metabolization of the compound. We present the first validated method, applicable in forensic toxicology, for quantifying HGA in whole blood.