Abstract
Forensic veterinary toxicology is a growing field of interest. In The Netherlands only a veterinarian is
allowed to take blood samples. This is a problem, since veterinarians are not always available when an
acute intoxication takes place. Therefore, there is a need for toxicological analysis of alternative
matrices. Saliva can be an interesting and animal friendly alternative. It can be sampled relatively easy
without a veterinarian and it is a “mirror” of blood since it has comparable metabolism characteristics.
Acute intoxication of animals can take place by intake of all kinds of toxic compounds. Three main
compound groups were included in this study; pesticides, natural toxins and veterinary drugs. These
compounds can be toxic for animals due to accidentally intake during feeding or negligence of the owner
of the animal. They can also be administered on purpose like poisoning or overdosing.
The aim of this research project was to develop a multi-residue method for the analysis of
animal saliva using Liquid Chromatography – Mass Spectrometry (LC-MS). The development had to cover
the entire procedure; saliva sampling, samples pre-treatment, LC-MS analysis and data interpretation.
The method was validated according an in-house validation protocol based on official international
legislation. As model animal saliva, calf saliva has been used, because the availability of calf saliva for
this research was significantly higher compared to that of other animals.
A sampling method was developed with two types of sampling devices, one for small volume and
one for larger volume saliva sampling. The main reason for sample pre-treatment is to get rid of proteins
which are present in the saliva. To achieve this a protein precipitation method using acetonitrile was
developed and tested. A microLC-microfluidics-QTOF-MS method was developed for the identification of
the compounds in saliva. With the microLC-microfluidics technique a relatively high sensitivity can be
obtained using small sample injection volume and low flow rates. The microfluidics system consists of a
Waters IonKey system, which includes an analytical column in a chip device (iKey), using ESI as an
ionization technique. The IonKey source is placed in front of a QTOF MS. The acquisition method used is
a so called MSe method where a full scan and a fragmentation scan are acquired alternately. In this way
next to full scan data there is also fragmentation scan data available which can be used for more reliable
identification of the compounds.
A targeted library was developed using UNIFI, new MS software from Waters Corporation. The
library includes retention times, monoisotopic masses and if present fragment ions of the toxic
compounds which were included in the research project.
The developed method was validated according international legislation (2002/657/EC) to test
the characteristics of the method. For a screening method the limit of detection (CCß), selectivity and
ruggedness of the method and stability of the compounds and sample extracts needs to be validated.
The validation was performed on three days with 20 different blank saliva samples. The samples were
spikes at two levels, 0.1 and 1 mg/liter. These correspond with the CCß levels of the validated
compounds. The validation results showed that with the method 203 compounds can be detected in
saliva with a CCß of 0.1 or 1 mg/l. The method was not selective for twelve compounds, interfering
peaks were found in the chromatograms. The method was rugged for two tested variations on the
sample pre-treatment and sample extracts were proven to be stable for one week in the freezer.
Within this research project a screening method for more than 200 toxic compounds was
successfully developed. The method consists of a sampling procedure, sample pre-treatment and
analyzing method using microLC-microfluidics-QTOF-MS. Additionally, the method was validated
according European legislation.