A need has existed for the accurate identification of monofluoroacetate (MFA) poisoning in southern Africa. The development of a new method for the determination of MFA has made the analysis of a variety of biological samples (n=50) feasible. The method has been used in the laboratory over 24 months. Monofluoroacetate was present in 66% of samples from cases of suspected poisoning, reflecting the extent of the problem. Stability of MFA in samples was also determined so as to have a time-bound baseline for the acceptance of samples submitted. It was found that there was a decrease in the level of MFA and, after 14 days at room temperature, only 50% of the spiked dose could be identified. It is suggested that samples be analyzed within 7 days of mortality if they not kept frozen.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Botha, C.J. (Christoffel Jacobus)

McCrindle, Robert Ian

Minnaar, P.P.

Naude, T.W.

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.A need has existed for the accurate identification of monofluoroacetate (MFA) poisoning in southern Africa. The development of a new method for the determination of MFA has made the analysis of a variety of biological samples (n=50) feasible. The method has been used in the laboratory over 24 months. Monofluoroacetate was present in 66% of samples from cases of suspected poisoning, reflecting the extent of the problem. Stability of MFA in samples was also determined so as to have a time-bound baseline for the acceptance of samples submitted. It was found that there was a decrease in the level of MFA and, after 14 days at room temperature, only 50% of the spiked dose could be identified. It is suggested that samples be analyzed within 7 days of mortality if they not kept frozen.mn201

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Onderstepoort Journal of Veterinary Research, 67:27-30 (2000) Investigation of biological samples for monofluoroacetate and Dichapetalum cymosum poisoning in southern Africa P.P MINNAAR1, R.I. McCRINDLE2, T.W. NAUDE3 and C.J. BOTHA1 ABSTRACT MINNAAR, P.P., McCRINDLE, R.I. , NAUDE, T.w. & BOTHA, C.J. 2000. Investigation of biological samples for monofluoroacetate and Dichapetalum cymosum poisoning in southern Africa. Onderste-poort Journal of Veterinary Research, 67:27- 30 A need has existed for the accurate identification of monofluoroacetate (MFA) poisoning in southern Africa. The development of a new method for the determination of MFA has made the analysis of a variety of biological samples (n = 50) feasible. The method has been used in the laboratory over 24 months. Monofluoroacetate was present in 66 % of samples from cases of suspected poisoning , re-flecting the extent of the problem. Stability of MFA in samples was also determined so as to have a time-bound baseline for the acceptance of samples submitted . It was found that there was a decrease in the level of MFA and, after 14 days at room temperature, only 50 % of the spiked dose could be identified. It is suggested that samples be analyzed within 7 days of mortality if they not kept frozen . Keywords: Dichapetalum cymosum, gifblaar, liquid chromatography, monofluoroacetate poisoning INTRODUCTION Synthetically-produced monofluoroacetate (MFA or Compound 1080; CFH2COO-) is a banned substance in South Africa in terms of the Hazardous Substance Act (Act 15 of 1973). This is mainly due to its physi-cal, chemical and toxicological properties, i.e. ex-treme toxicity, water solubility, colourlessness, taste-lessness, difficulty of detection, and a latent period between ingestion and development of clinical signs. The potential of secondary pOisoning also exists. In countries such as New Zealand, compound 1080 is still used extensively as a predacide and rodenticide, and for the control of problem animals (Rammel & Flemming 1978). 1 Department of Pharmacology and Toxicology, Faculty of Vet-erinary Science, University of Pretoria , Private Bag X04, Onderstepoort, 0110 South Africa 2 Department of Chemistry and Physics, Technikon Pretoria, Pri-vate Bag X680, Pretoria, 0001 South Africa 3 Toxicology Section, OnderstepoortVeterinary Institute, Onder-stepoort, 0110 South Africa Accepted for publication 17 December 1999-Editor Despite being banned in South Africa, compound 1080 is used for the malicious poisoning of dogs, cats and other animals. Typical clinical signs of poi-soning include severe convulsions and muscular fi-brillations (Osweiler, Buck & Van Gelder 1985). In South Africa, the substance is possibly obtained from neighbouring countries where MFA is not banned, from expired stock purchased prior to it having been banned, or from possible illegal production of the compound. Dichapetalum cymosum, known colloquially as "gif-blaar" (which means poison leaf), is an indigenous shrub which contains monofluoroacetate. Gifblaar poisoning often results in acute death of ruminants, particularly cattle, in southern Africa. Mortalities in ru-minants have been reported from Gauteng, Mpuma-langa, North West, and Northern Provinces in South Africa. There are also reports of mortalities from Zim-babwe, Botswana and Namibia (Kellerman, Naude & Fourie 1996). Gifblaarpoisoning is considered the fourth most important plant poisoning syndrome in South Africa (Kellerman et al. 1996). Mortality as a result of ingestion of the leaves of this plant occurs mainly during the months of August to November, as 27 Biological samples for monofluoroacetate and Dichapetalum cymosum poisoning in South Africa well as in March (Steyn 1949, Kellerman, Coetzer & Naude 1988). The duration of the clinical signs of gifblaar poison-ing in cattle is extremely short (Steyn 1949; Egyed & Shlosberg 1973; Kellerman et al. 1988). Veterinary practitioners usually do not observe the animal alive. In dogs, the clinical signs of MFA are of somewhat longer duration and resemble those manifested in strychnine or convulsive pesticide poisoning. Unless fragments of the gifblaar plant are found in the ru-men , the diagnosis can only be based on clinical signs and circumstantial evidence. The aim of this investigation was to apply a modified high-performance liquid chromatographic (HPLC) method for the determination of MFA (Minnaar, McCrindle, Naude, Botha, Swan & De Beer 2000) to specimens received for diagnostic purposes by a veterinary laboratory, and to estimate the lability of MFA in them over a period of time. The specimens originated from a variety of animal species, chiefly cattle, as well as two from humans, animal baits and water. MATERIALS AND METHODS The method used for determining MFA (Minnaar et al. 2000) was modified and used over a period of 24 months (1997/98) in 50 cases of suspected gifblaar or synthetic MFA poisoning . The reported method was modified by changing the eluent composition from 0,02 M H3P04 to a solution comprised of 0,04 M H3P04 and 5 % acetonitrile. The flow-rate was also decreased from 0,8-0,4 mQ/min. The limit of detec-tion for MFA was established at 12 IJg/Q. Analysis was done on submitted samples, which consisted mainly of rumen or stomach contents, kidney or liver, origi -nating from cheetah (n= 1), bird (n= 1), horse (n= 1), human (n=2) , sheep (n=2) , cat (n=7) , dog (n=13) and cattle (n= 17). In addition , five specimens were of bait and one comprised water. The samples were extracted in an aqueous solution and analyzed di-rectly without any chemical clean-up. Rumen or stomach contents from the animals or humans were initially analyzed, but if found to be negative, the liver (if available) was also analyzed for the presence of MFA. To determine the stability of MFA in biological sam-ples, fresh rumen content and liver samples from a bovine were collected from an abattoir and sufficient MFA was added to make the concentration of MFA in the rumen sample 7 IJg/mQ and in the liver sample 5 IJg/mQ. These two spiked samples were then ho-mogenized and each was divided into ten equal aliquots. One aliquot each of rumen content and liver were immediately analyzed. Another aliquot of each was placed in a freezer at - 23 °C. All the remaining aliquots were left at room temperature (approxi-mately 10-27 °C day-time temperature) and ana-lyzed periodically during the following 20 weeks (Ta-ble 2) . The MFA concentrations of the frozen aliquots were determined after 14 d. Absorbance 0,00 20,00 29,99 0,0000 0,0500 0,1000 -"C=============================~- 10,15 10,53 0,0000 19,31 25,68 27,27 15,24; 15,49 21,38 MFA 0,0500 0,1000 FIG. 1 Chromatogram of bovine rumen contents, after 7,0 IJg/mQ of sodium monofluoroacetate had been added, showing a peak after 21 ,38 min which indicates the presence of MFA 28 IABLE 1 Results of suspected MFA pOisoning In different species Species or sample type Bait Birdc Cata Cattleb Cheetah Doga Horsec Humana Sheepb Water Total a Deliberate poisoning b Natural poisoning Number of samples positive samples Bait (n = 5) Liver (n = 1) Stomach (n = 7) Rumen (n= 17) Stomach (n = 1) Stomach (n = 13 Stomach (n = 1) Liver (n = 1) Stomach (n = 1) Rumen (n= 2) Water (n = 1) 50 C Accidental/secondary poisoning Number of samples (lJg/mQ) 4 1 6 10 -8 1 2 -1 -33 TABLE 2 Stability of MFA in spiked rumen and liver samples Time Recovered MFA conc. Recovered MFA conc . in rumen samples in liver samples (d) (lJg/mQ) (lJg/mQ) 6 6,6 4,4 13 6,1 4,0 20 5,8 3,6 27 5,8 3,4 34 5,8 2,8 56 5,7 2,7 83 5,6 2,6 104 5,6 2,3 RESULTS Results of submitted specimens The modified HPLC method was found to be most effective in separating the important components in the samples analyzed as is exemplified by the chro-matogram of a spiked rumen sample shown in Fig. 1. Sixty-six per cent of the samples submitted were positive for the presence of MFA. Of the 50 cases investigated (Table 1), 20 were submitted for confir-mation of gifblaarpoisoning . All the cattle and sheep were poisoned by ingestion of the actual plant. It was found that if the rumen samples were not 'fresh', MFA detection was unreliable and the lability of specimens was investigated further. Thirty of the specimens submitted were from cases that were suspected of having been poisoned with MFA, and eight dogs and seven cats were found to be positive. This implies that these companion animals, in general , may be considered victims of deliberate poisoning. The MFA concentration in the samples from dogs, consisting mainly of stomach content, liver, and kidney, were found to be high, with a mean MFA concentration of PP MINNAAR et al. MFA concentration in positive 16,2-432,0, x = 253,0 336,0 74,2-210,0, x = 122,8 15,4-966,0, x = 408,9 -38,2-744,0, x = 226,9 34,2 280,0, 500,0, x = 440,0 61 ,2 -x = 235,3 IJg/mQ 227,0 IJg/mQ, whereas the mean from the cat sam-ples were 100,6 IJg/mQ. Most of the incidents of sus-pected deliberate poisoning occurred in the Eastern Cape Province. All 50 samples were submitted 'fresh' frozen or 'fresh' on ice. Results of spiked specimens The concentration of MFA in the spiked rumen and liver samples is given in Table 2. The concentration of MFA in the samples frozen immediately and ana-lyzed after 14 d were within 2 % of those analyzed immediately after the addition of MFA. After 83 d, the concentration of MFA in the rumen samples held at room temperature, stabilized at 85 %, while that of the liver samples decreased to 52 % after 104 d. Fur-ther time lapse revealed no additional changes in the MFA concentration in either the rumen or liver sam-ples. DISCUSSION During the two year period from 1997-1998, MFA has been one of the compounds used to poison dogs and cats maliciously in South Africa. No less than 14 cases have been considered deliberately poisoned with this substance during this period. Non-target ani-mals are usually incidently poisoned by ingestion of poisoned carcasses. It is suspected that the bird, a Blue Crane, died as a result of secondary poisoning after it had fed on a donkey carcase which is thought to have contained MFA although this was never proved. The horse died after eating fodder which is presumed to have been contaminated with gifblaar. The results obtained during the two year period by the modified HPLC method developed by Minnaar et al. (2000) for the determination of MFA in animal tissues and rumen or stomach contents has been 29 Biological samples for monofluoroacetate and Dichapetalum cymosum poisoning in South Africa employed indicate that MFA poisoning, either natu-ral or intended, are of great concern in South Africa . Although compound 1080 is banned in South Africa it would appear, as there is, to our knowledge, no other feasible source of MFA, that there is a supply of it particularly in the southern Eastern Cape Prov-ince and that this substance is being used for the malicious poisoning of animals. The South African Police Services were assisted in their investigations by analyzing two of the samples included in this survey. In order to obtain the best quality material for analy-sis by the modified HPLC method, it is recommended that specimens from cases of suspected MFA poi-soning should comprise, if possible, 100 g each of liver and kidney tissue, and the same amount of stomach or rumen contents. These should be placed separately in scrupulously clean containers, prefer-ably glass or plastic, as soon after the death of the animal as possible, and immediately frozen . These should be submitted in the frozen state to a labora-tory equipped to perform the analYSis preferably within 14 d. 30 REFERENCES EGYED, H.J. & SHLOSBERG, A. 1973. Diagnosis of fie ld cases of sodium fluoroacetate and fluoroacetamide poisoning in ani-mals. Refuah Veterinarith , 30:112-115. KELLERMAN , T.S ., COETZER, J.A.W & NAUDE, T.W 1988. Plant poisoning and mycotoxicoses of livestock in southern Africa. Cape Town: Oxford University Press. KELLERMAN, T.S., NAUDE, T.W & FOURIE, N. 1996. The dis-tribution , diagnoses and estimated economic impact of plant poisonings and mycotoxicoses in South Africa. Onderstepoort Journal of Veterinary Research, 63:65-90. MARAIS, J.S.C. 1944. Monofluoroacetic acid, the toxic principle of Gifblaar Dichapetalum cymosum. Onderstepoort Journal of Veterinary Science and Animal Industry, 20:67-73. MINNAAR , P.P. , McCRINDLE, RI. , NAUDE, T.W , BOTHA, C.J., SWAN, G.E. & DE BEER, WH.J . 2000. A High-Performance Liquid Chromatographic method for the determination of mono-fluoroacetate. Journal of Chromatoghraphic Science, 38: 1. OSWEILER, T.L. , GD. CARSON, BUCK, WB. & VAN GELDER ., G A. 1985. Clinical and diagnostic veterinary toxicology, 2nd ed . Ohio: Kendall/Hunt Publishing . RAMMEL, C.G. & FLEMMING , P.A. 1978. Compound 1080, prop-erties and use of sodium monofluoroacetate in New Zealand . Well ington: New Zealand Min istries of Agriculture and Fisher-ies, Animal Health Division. STEYN , D.G. 1949. Vergiftiging van mens en dier. Pretoria: Van Schaik. 

Investigation of biological samples for monofluoroacetate and Dichapetalum cymosum poisoning in southern Africa

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Investigation of biological samples for monofluoroacetate and Dichapetalum cymosum poisoning in southern Africa

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