LC-MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum

Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.

Orofacial dyskinesia induced by nasal Ritalin(R) (methylphenidate) sniffing: A rare case report from Switzerland

Ritalin® (methylphenidate) is an amphetamine-like prescription stimulant commonly used in the treatment of attention deficit hyperactivity disorder in children and adults. Recently, the recreational use of Ritalin has increased, particularly among young adults. Well-known symptoms of intoxication include signs of sympathetic nervous stimulation, such as agitation, anxiety, tachycardia, hypertension, headache, tremor, and dizziness. This case report describes oral dyskinesia as a rare presentation of Ritalin intoxication, with the review of pathophysiology and some epidemiological data.

Functional effect of FGF2- and FGF8-expanded ventral mesencephalic precursor cells in a rat model of Parkinson's disease

Ventral mesencephalic (VM) precursor cells are of interest in the search for transplantable dopaminergic neurons for cell therapy in Parkinson's disease (PD). In the present study we investigated the survival and functional capacity of in vitro expanded, primary VM precursor cells after intrastriatal grafting to a rat model of PD. Embryonic day 12 rat VM tissue was mechanically dissociated and cultured for 4 or 8 days in vitro (DIV) in the presence of FGF2 (20 ng/ml), FGF8 (20 ng/ml) or without mitogens (control). Cells were thereafter differentiated for 6 DIV by mitogen withdrawal and addition of serum. After differentiation, significantly more tyrosine hydroxylase-immunoreactive (TH-ir), dopamine-producing neurons were found in FGF2- and FGF8-expanded cultures compared to controls. Moreover, expansion for 4 DIV resulted in significantly more TH-ir cells than expansion for 8 DIV both for FGF2 (2.4 fold; P<0.001) and FGF8 (3.8 fold; P<0.001) treated cultures. The functional potential of the expanded cells (4 DIV) was examined after grafting into striatum of aged 6-hydroxydopamine-lesioned rats. Amphetamine-induced rotations performed 3, 6 and 9 weeks postgrafting revealed that grafts of FGF2-expanded cells induced a significantly faster and better functional recovery than grafts of FGF8-expanded cells or control cells (P<0.05 for both). Grafts of FGF2-expanded cells also contained significantly more TH-ir cells than grafts of FGF8-expanded cells (P<0.05) or control cells (P<0.01). In conclusion, FGF2-mediated pregrafting expansion of primary VM precursor cells considerably improves dopaminergic cell survival and functional restoration in a rat model of PD.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

Effects of GDNF pretreatment on function and survival of transplanted fetal ventral mesencephalic cells in the 6-OHDA rat model of Parkinson's disease

Transplantation of fetal dopaminergic (DA) neurons offers an experimental therapy for Parkinson's disease (PD). The low availability and the poor survival and integration of transplanted cells in the host brain are major obstacles in this approach. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with growth- and survival-promoting capabilities for developing DA neurons. In the present study, we examined whether pretreatment of ventral mesencephalic (VM) free-floating roller tube (FFRT) cultures with GDNF would improve graft survival and function. For that purpose organotypic cultures of E14 rat VM were grown for 2, 4 or 8 days in the absence (control) or presence of GDNF [10 ng/ml] and transplanted into the striatum of 6-hydroxydopamine-lesioned rats. While all groups of rats showed a significant reduction in d-amphetamine-induced rotations at 6 weeks posttransplantation a significantly improved graft function was observed only in the days in vitro (DIV) 4 GDNF pretreated group compared to the control group. In addition, no statistical significant differences between groups were found in the number of surviving tyrosine hydroxylase-immunoreactive (TH-ir) neurons assessed at 9 weeks posttransplantation. However, a tendency for higher TH-ir fiber outgrowth from the transplants in the GDNF pretreated groups as compared to corresponding controls was observed. Furthermore, GDNF pretreatment showed a tendency for a higher number of GIRK2 positive neurons in the grafts. In sum, our findings demonstrate that GDNF pretreatment was not disadvantageous for transplants of embryonic rat VM with the FFRT culture technique but only marginally improved graft survival and function.

Detection and quantification of 56 new psychoactive substances in whole blood and urine by LC-MS/MS

BACKGROUND New psychoactive substances (NPS) have become increasingly prevalent and are sold in internet shops as 'bath salts' or 'research chemicals' and comprehensive bioanalytical methods are needed for their detection. METHODOLOGY We developed and validated a method using LC and MS/MS to quantify 56 NPS in blood and urine, including amphetamine derivatives, 2C compounds, aminoindanes, cathinones, piperazines, tryptamines, dissociatives and others. Instrumentation included a Synergi Polar-RP column (Phenomenex) and a 3200 QTrap mass spectrometer (AB Sciex). Run time was 20 min. CONCLUSION A novel method is presented for the unambiguous identification and quantification of 56 NPS in blood and urine samples in clinical and forensic cases, e.g., intoxications or driving under the influence of drugs.