LC-MS/MS analysis of pholedrine in a fatal intoxication case.

Pholedrine (4'-hydroxymethamphetamine) is a cardiovascular agent exerting hypertensive and adrenergic effects. High doses may cause a drop in the peripheral circulation blood flow and increase blood pressure, heart rate and body temperature up to a state of central respiratory paralysis. A 15-year-old girl who suffered from heavy agitation and hallucinations was admitted to the intensive care unit in a comatose state. The clinical findings included a maximum heart rate of 170 bpm and a body temperature of 43.8 degrees C. Resuscitation measures were in vain and abandoned after approximately 2h. A toxicological emergency analysis using GC/MS revealed a considerable amount of pholedrine in blood and urine. A method for determining pholedrine in human body fluids utilizing high-performance liquid chromatography (HPLC)/tandem mass spectrometry (LC-MS/MS) with a turbo ion-spray source was developed, using D11-methamphetamine and D5-methylenedioxymethamphetamine as internal standards. Samples were prepared by SPE extraction using SPEC-C18AR/MP3((R)) columns, which yielded the best extraction recovery (67%). Chromatographic separation was achieved at pH 5 on an RP-18 stationary phase applying gradient elution from 50 to 70% of B (methanol/acetonitrile 3/1 (v/v), 0.02% acetic acid) in A (5mM ammonium acetate/acetonitrile 95/5 (v/v), 0.02% acetic acid). Supra-pure acetic acid was added to the post-column effluent with a flow rate of 0.2 microl/min to optimize ionization. Detection was carried out in the positive ionization, multiple reaction monitoring (MRM) mode. The chromatograms showed no interference from other substances. The limit of detection (LOD, S/N=3) of pholedrine was 0.8 ng/ml and its lower limit of quantification (LLOQ, S/N=10) 3ng/ml. The calibration curve was linear (r=0.999) in the range 1-100 ng/ml. Samples with higher concentrations were diluted to suit the working range. The intra-day R.S.D. between 5 and 80 ng/ml were 3.8-8.7% and the inter-day R.S.D. between 5 and 100 ng/ml were 6.7-10.7%. The pholedrine concentrations in blood and urine collected when the girl was still alive were 16.1 microg/ml (R.S.D. 10.5%) and 1120 microg/ml (R.S.D. 8%), respectively. In post-mortem samples, they were 23.0 microg/ml (R.S.D. 5.1%) in heart blood and 27.3 microg/g (R.S.D. 6.6%) in the liver.

[Case report. Phenprocoumon (Marcumar, Falithrom) as an unusual reason for coumarin poisoning in a dog]. / Phenprocoumon (Marcumar, Falithrom) als ungewöhnliche Ursache einer Kumarinvergiftung beim Hund.

Coumarin poisoning in dogs is not unusual and is in most cases caused by warfarin, a coumarin derivative which is used as a rodenticide. Competitive inhibition of vitamin K with an incomplete synthesis of the coagulation factors II, VII, IX and X can lead to a significant bleeding tendency. We observed a 3-year old male West Highland White Terrier with a reduced general condition and dyspnoea together with a massive haemothorax. Administration of vitamin K1 (3 mg/kg) led to a rapid improvement of the condition. Coagulation analysis revealed a prolonged activated recalcification time (ARCT), prothrombin time (PT) and aPTT with uncharacteristic thrombin time (TT); factor II, VII and X activities were reduced while factor V activity was normal, all of which are characteristic for coumarin poisoning. HPLC did not reveal the presence of warfarin but of phenoprocoumon, a drug used for thromboembolic prophylaxis in humans. This observation has not been described for dogs to date.

Quantitative determination of piritramide in human serum applying liquid chromatography-two-stage mass spectrometry.

A method for the determination of the synthetic narcotic analgesic piritramide in human serum utilizing high-performance liquid chromatography with atmospheric pressure chemical ionization two-stage mass spectrometry (HPLC-APCI-MS-MS) is presented. Pipamperone is used as the internal standard. Serum samples are prepared by liquid-liquid extraction under basic conditions with 1-chlorobutane. The chromatographic separation is achieved on an RP-18 stationary phase applying gradient elution with methanol-0.02% trifluoroacetic acid in water. Detection is carried out in the MS-MS single reaction monitoring mode of the ion-trap mass spectrometer. The limit of detection is 0.3 ng/ml and the calibration covers the range of 1-80 ng/ml. The intra-day RSDs are 6.1 to 7.3% over the calibration range, whereas the inter-day RSDs are 9.6 to 12.8%.

Uptake and subcellular distribution of 65zinc in brain structures during the postnatal development of the rat.

The uptake ratio of 65zinc into the postnatally developing hippocampal formation, cerebellum and remainder of the brain showed only minor differences, whilst following subcellular fractionation a marked decrease in 65zinc uptake from postnatal day 15 to adulthood was found in nuclear sediments (hippocampal formation 44.3%, cerebellum 27.5%), along with an increase in synaptosomal fractions on the hippocampal formation by 55.4% and of the cerebellum by 37.1%. The shift of zinc out of the perikarya into terminal fields during the postnatal development without substantial alterations of the net zinc content is supposed to be a consequence of a functional relation of zinc to synaptic transmission processes.