Immunoassay testing for barbiturates using alternative matrices in postmortem tissues from cats and dogs

The barbiturate drug pentobarbital is commonly used by veterinarians for the euthanasia of domestic animals. During the veterinary forensic autopsy, it is sometimes necessary to determine whether the animal was chemically euthanized with pentobarbital. The use of a human immunochromatographic test for barbiturate screening utilizing dog or cat urine has been previously validated; however, the use of alternative matrices for this purpose is yet to be explored when urine is not available. Postmortem heart, liver, spleen, skeletal muscle, blood and/or urine samples from 20 dogs and 26 cats with a reported chemical euthanasia status were processed using two different methods, bead homogenization and sonication, and screened for barbiturates using a human immunochromatographic test. There was 100% agreement of the immunochromatographic test results using the sonication method with the reported euthanasia status of both dogs and cats. Using the bead homogenization method, agreement with the reported euthanasia status was 93.3% and 96.7% for dogs and cats, respectively, due to invalid test results from four dog and two cat samples. A subset of liver samples (10 canine and 10 feline) was analyzed via gas chromatography-mass spectrometry, and there was 100% agreement between the immunochromatographic test results and gas chromatography-mass spectrometry results for both cats and dogs. Overall, our results support the use of a variety of alternative matrices for barbiturate screening in cats and dogs.