Determination of Grayanotoxins in Biological Samples by LC-MS/MS

Abstract

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

A case of human poisoning by grayanotoxins following honey ingestion: elucidation of the toxin profile by mass spectrometry

“…For all peaks, which were identified as potential grayanane-type toxins by HRMS, product ion spectra were recorded. Those compounds displaying fragmentation patterns similar to the GrTx III standard (Figure 2) or as described in the literature (Holstege et al 2001) were confirmed as grayanane-type toxins. Results are listed in Table 2.…”

“…A study of 10 honey samples from the Black Sea region revealed GrTx I concentrations between 1.5 and 39.3 mg kg −1 and GrTx III concentrations between 0.3 and 35.1 mg kg −1 , while GrTx II was only detected at very low concentrations (Kaplan et al 2014). Furthermore, a detailed report on the quantitative mass spectrometric determination of GrTx in biological samples and the description of product ion spectra is given by Holstege et al (2001). This paper describes the case of a 56-year-old man who caused a traffic accident after eating two tablespoons of wild Turkish honey.…”

“…The common core structure of these toxins ( Figure 1 and Table 1) results in a similar and thus predictable mass spectrometric fragmentation. All toxins exclusively exhibit successive losses of water arising from collision-induced dissociation during electrospray ionisation (Holstege et al 2001). Therefore, toxins were easily identified by exact mass determination of several typical in-source fragments resulting from repeated losses of water.…”

Mad honey: uses, intoxicating/poisoning effects, diagnosis, and treatment

“…Other possible techniques for the detection of grayanotoxin are HPLC, LCMS/MS, nuclear magnetic resonance, and infrared-based technologies. 64,90,[107][108][109][110]130 For corroborative evidence, pollen grains from Rhododendron (its owers are rich in grayanotoxin) can be investigated occasionally in honey samples. Cardiac ischemia and rhythm abnormalities should be checked by ECG.…”

Mountain laurel toxicosis in a dog

“…If clinical signs for grayanotoxin poisoning are also present, a diagnosis of this toxicosis can be made . The most common test used currently by most diagnostic labs is the rapid liquid chromatography/tandem mass spectrometry method, which detects 0.2 μg/g of feces or rumen contents and 0.05 μg/mL in urine and other fluids …”