Simultaneous analysis of haloperidol, its three metabolites and two other butyrophenone-type neuroleptics by high performance liquid chromatography with dual ultraviolet detection.

We investigated simultaneous determination of haloperidol (HAL), its three metabolites [reduced HAL (R-HAL), 3-(4-fluorobenzoyl)propionic acid (FBPA) and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP)] and two related compounds [spiperone (SPI) and droperidol (DRO)] in phosphate-buffered saline using high-performance liquid chromatography (HPLC) coupled with dual ultraviolet detection (220 and 250 nm). Retention times of HAL, R-HAL, FBPA, CPHP, SPI and DRO were 16.8, 11.8, 10.2, 4.1, 12.6 and 8.3 min, respectively. Their lower limits of detection were 7.5, 14, 4.5, 12, 10 and 20 ng/mL in the same order. The coefficients of variation for their intra- and inter-day assays were less than 7.8 and 9.4%, respectively. Of the other centrally acting drugs, only amoxapine interfered with the peak of DRO. Using our procedure, the binding study of tested compounds to synthetic melanin, human serum albumin and alpha1-acid glycoprotein was performed by determining the unbound concentration to total concentration ratio. These results indicated that simultaneous assay of HAL, R-HAL, FBPA, CPHP, SPI and DRO in phosphate-buffered saline by HPLC equipped with dual ultraviolet detection is simple, sensitive and reproducible. Also, our assay system can be applied to the binding study of these compounds to synthetic melanin, human serum albumin and alpha1-acid glycoprotein.

Atmospheric pressure laser desorption/chemical ionization mass spectrometry: a new ionization method based on existing themes.

We have designed and constructed an atmospheric pressure laser desorption/chemical ionization (AP-LD/CI) source that utilizes a laser pulse to desorb intact neutral molecules, followed by chemical ionization via reagent ions produced by a corona discharge. This source employs a heated capillary atmospheric pressure inlet coupled to a quadrupole ion trap mass spectrometer and allows sampling under normal ambient air conditions. Preliminary results demonstrate that this technique provides approximately 150-fold increase in analyte ions compared to the ion population generated by atmospheric pressure infrared matrix-assisted laser desorption/ionization (AP-IR-MALDI).

Identification of a D1 dopamine receptor, not linked to adenylate cyclase, on lactotroph cells.

1. We studied the lactotroph cells of the rat by both in vivo and in vitro pharmacological techniques for the presence of D1-receptors. Both approaches revealed the presence of D2-receptor, stimulated by quinpirole (resulting in an inhibition of prolactin secretion) and blocked by domperidone. 2. Administration of fenoldopam, the most selective D1-receptor agonist currently available, resulted in a dose-dependent decrease of prolactin secretion in vivo (after pretreatment with alpha-methyl-p-tyrosine) and in vitro (cultured pituitary cells). This increase was dose-dependently blocked by the selective D1-receptor antagonist, SCH 23390, and although the effect of fenoldopam was less than that obtained by D2-receptor stimulation, these data suggest that a D1-receptor also controls prolactin secretion. 3. In order to detect the location of these dopamine receptors, autoradiographic studies were performed by use of [3H]-SCH 23390 and [3H]-spiperone as markers for D1- and D2-receptors, respectively. Specific binding sites for [3H]-SCH 23390 were demonstrated. Fenoldopam dose-dependently reduced [3H]-SCH 23390 binding, but had no effect on [3H]-spiperone binding. Immunocytochemical labelling of prolactin cells after incubation with [3H]-SCH 23390 revealed that the granulae and hence, D1 binding sites were present on the lactotroph cells. 4. Radioligand binding studies performed on membranes from anterior pituitary cells revealed the presence of the D2-receptor (54 fmol mg-1 protein) with a Kd of 0.58 nM for [3H]-spiperone, but failed to detect D1-receptors. 5. Finally, we studied the effect of dopamine and of fenoldopam on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of anterior pituitary cells. Although cyclic AMP increased upon prostacyclin administration, indicating an intact adenylate cyclase system, fenoldopam failed to increase the cyclic AMP production. 6. It is tempting to speculate that fenoldopam reduces prolactin secretion through interaction with a non-cyclase-linked D1-receptor on the lactotroph cells.

Electron impact mass spectrometry of substituted 1,3,8-triazaspiro[4,5]decan-4-ones.

The neuroleptic butyrophenone drug spiroperidol (8-[4-fluorophenyl)-4-oxobutyl]-1-phenyl-1,3,8-triazaspiro [4,5]decan-4-one) and related compounds are valuable tools for mapping the dopamine receptors in the brain. During the development of the radiochemical synthesis of these compounds with very short-lived isotopes suitable for positron emission tomography studies, positive electron impact mass spectrometric information was obtained. Ion series are present in the spectra of all 12 compounds studied, which unambiguously allow recognition of type and site of substitution on the spiro system.

Interactions of nicotinamide with dopamine receptors in vivo.

[3H]-Spiperone (20 microCi/kg, 0.0003 mg/kg, SC) was administered to mice. Relative decreases in the 2-hr ratio of accumulation of this dopamine receptor radioligand in the dopaminergic corpus striatum ("specific" plus "nonspecific binding") and the nondopaminergic cerebellum ("nonspecific binding" only) were used to evaluate nicotinamide for possible effects on the dopamine receptor. The nicotinamide-treated animals were also observed for signs of catalepsy. Pretreatment for 30 min with IP doses of 200 and 500 mg/kg reduced accumulation in both areas approximately the same as judged from striatum:cerebellum ratios, which did not differ significantly from controls. However, at 1000 mg/kg, although nicotinamide decreased [3H]-spiperone accumulation in both striatum and cerebellum, "specific binding" was affected more than "nonspecific binding," as judged from a statistically significant decrease in the striatum:cerebellum ratio. This dose also produced a cataleptic state. Nicotinamide at high doses might have some antagonistic effect on dopamine receptors in mice as judged from the greater effect on accumulation of [3H]-spiperone in striatum ("specific binding") than in cerebellum ("nonspecific binding") which appeared to correlate somewhat with the production of a cataleptic state.

Effect of lipophilicity on the in vivo localization of radiolabelled spiperone analogues.

A series of N-alkylated and para-brominated analogues of spiperone were synthesized to ascertain the effect of lipophilicity on the in vivo localization of neuroleptics. While the IC50 for D2 receptor binding was within the same order of magnitude for these compounds, the calculated octanol-water partition coefficients varied 300-fold. When the in vivo distribution data for 77Br-labelled compounds were compared with previous data for 18F- and 11C-labelled analogues it was seen that the highest cerebral uptake was for bromospiperone, but the optimum striatum-to-cerebellum and brain-to-blood concentrations were achieved by N-methyl spiperone. The implications of these results for radiopharmaceutical design or medicinal chemistry are discussed.

An easily constructed and inexpensive three-layer cassette for tritium-sensitive film autoradiography.

This report describes the design, construction, and use of a three-layer photographic cassette that is suitable for tritium-sensitive film autoradiography. The cassette can be produced easily and at a fraction of the cost of commercially available cassettes. The three-tiered design allows for the simultaneous exposure of 108 standard-sized glass microscope slides to three 20 X 34 cm pieces of 3H-Ultrofilm (LKB). The small between- or within-film variation of gray values recorded for either background, tritium-containing standards, or neighboring brain sections labeled with 3H-spiroperidol demonstrates the reliability of the cassette for autoradiographic quantification of tritium label.

Increased 3H-spiperone binding sites in mesolimbic area related to methamphetamine-induced behavioral hypersensitivity.

The specific 3H-spiperone binding to membrane homogenates of the striatum, mesolimbic area, and frontal cortex was examined in two groups of rats pretreated once daily with saline or 4 mg/kg of methamphetamine (MAP) for 14 days. At 7 days following cessation of chronic pretreatment, all rats received an injection of 4 mg/kg of MAP and were decapitated 1 hr after the injection. In the chronic saline-pretreatment group, the single administration of MAP induced significant changes in the number (Bmax) of specific 3H-spiperone binding sites (a decrease in the striatum and an increase in the mesolimbic area and frontal cortex), but no significant changes in the affinity (KD) in any brain area. The chronic MAP pretreatment markedly augmented the changes in Bmax in the striatum and mesolimbic area. The increase in specific 3H-spiperone binding sites in the mesolimbic area is discussed in relation to MAP-induced behavioral hypersensitivity.