Identification of Unknown Inorganic Compound by Electrospray Mass Spectrometry

Don K. Noot, M.Sc. and Joe D. Kendall DVM, M.Sc., Dipl. ABVT
Toxicology Section, Animal Health Laboratories Branch, Alberta Agriculture

Recently a veterinarian submitted a sample of rumen contents and a sample of unknown white powder to our laboratory for analysis. The history received with the submission indicated that a farmer with a herd of 170 cows had found 12 of his cows dead. The veterinarian stated that the animals had access to old sheds which were littered with debris, including a pail containing an unknown white substance. There was visual evidence that some animals had been licking the contents of the pail. The veterinarian requested nitrate analysis on the rumen contents and the white substance. He was concerned that the white substance may have been fertilizer and that the animals may have died due to nitrate toxicosis.

The submitted material consisted of white crystals which appeared to be hygroscopic (moisture content was determined to be 4%). The crystals were readily soluble in water and the resulting pH of the solution was neutral. Analysis of 14 elements by Inductively Coupled Plasma (ICP) indicated the presence of sodium. Ion chromatography indicated an anion eluting between nitrate and sulfate. Further elemental analysis indicated the compound did not contain carbon, nitrogen or sulfur. At this point, the identity of the anion was still unknown.

The compound was then analyzed by electrospray mass spectrometry (ES/MS) (Micromass Platform II) in both positive and negative ion modes. A water:methanol solution (approximately 50 µg/mL) of the compound was directly infused into the electrospray source at 10 µL/min via syringe pump (Orion Sage M361). ES- spectra indicated a base peak at 83 amu. When the cone voltage was increased to induce fragmentation, an ion at 67 amu appeared, suggesting the loss of oxygen. Clusters of larger ions 106 amu apart were also present (see figure 1). ES+ spectra indicated a strong sodium ion as well as clusters of larger ions 106 amu apart (see figure 2). Ion clusters in both ES- and ES+ indicated the chlorine isotope pattern, with the 83- and 129+ ions containing a single chlorine atom. The identity of the anion was therefore determined to be chlorate (ClO3-). Subsequent analysis of pure sodium chlorate produced mass spectra and anion chromatogram identical to the unknown. Ion compositions are listed in table 1.

Unfortunately, chlorate could not be confirmed to be present in the rumen content, as it was likely reduced. The consumption of sodium chlorate by cattle may result in a chlorate toxicosis which would clinically resemble a nitrate toxicosis. Sodium chlorate was sometimes used as a soil sterilant.

It is interesting to note that while electrospray mass spectrometry is usually utilized in the analysis of organic compounds, it is also amenable to the analysis of inorganic compounds. In this case, the identity of sodium chlorate was elusive by other analytical methods, but very clear by ES/MS. Both the cation and anion were readily determined, and multiple ion clusters produced a unique mass spectrum which was easily interpreted. ES/MS is proving to be a valuable tool for both diagnostics and surveillance in the Veterinary Toxicology lab.

Table 1: sodium chlorate ions by ES/MS

cation (amu) composition anion (amu) composition
23+ Na+ 83- ClO3-
129+ [NaClO3]Na+ 189- [NaClO3]ClO3-
235+ [NaClO3] 2Na+ 295- [NaClO3] 2ClO3-
341+ [NaClO3] 3Na+ 401- [NaClO3] 3ClO3-
(106*n)+23 [NaClO3] nNa+ (106*n)+83 [NaClO3] nClO3-


Figure 1: negative ion electrospray mass spectrum of unknown

Figure 2: positive ion electrospray mass spectrum of unknown


Note: higher mass ranges are magnified to better display chlorine isotope ratios.