A sensitive and rapid HPLC-MS/MS method for the quantitative determination of trace amount of bromocriptine in small clinical prolactinoma tissue.

Usually, insufficient intratumoral concentration of therapeutic drugs is one of the reasons for tumor treatment failure. However, little is known about intratumoral distribution of bromocriptine in non-responding prolactinomas because of extremely low drug concentration and small prolactinoma tissue samples. In this study, a sensitive, rapid and high-throughput quantitative bioanalytical method has been established by using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination of bromocriptine at trace level in human prolactinoma tissue. As little as 20 mg (wet weight) tissue sample was required and total analysis time was 6 min in this method. The assay quantifies over a linear range of 50 fg/mg to 5 pg/mg, and has a 25 fg/mg limit of detection at a signal/noise ratio of 3. This validated method was successfully used to quantitatively determine bromocriptine in clinical post-operative bromocriptine-sensitive and -resistant prolactinomas. The results revealed bromocriptine concentration in resistant prolactinomas (0.49-1.25 pg/mg) was significantly higher than that in sensitive prolactinomas (0.057-0.47 pg/mg). These results provided direct evidence to demonstrate the reseaon for failure of bromocriptine treatment in some patients with prolactinoma was "intrinsic" tumor (cell) resistence, rather than insufficient drug concentration in tumor tissue. Additionaly, this HPLC-MS/MS method has been shown to be suitable for bromocriptine analysis in small amount tissue sample and could be adapted for therapeutic drug monitoring of other clinical medicine.

Differential pulse voltammetric determination of the dopaminergic agonist bromocriptine at glassy carbon electrode.

The electrochemical oxidation of bromocriptine at glassy carbon electrode has been carried out in Britton-Robinson (B-R) buffer solutions in the pH range 2.0-11.0 employing cyclic, linear sweep and differential pulse voltammetry (DPV). Bromocriptine showed one well-defined oxidation peak accompanied by a smaller one. The oxidation process was found irreversible. For analytical purposes, the well-resolved diffusion controlled voltammetric peak at pH 5 was critically investigated. The linear relationship between peak current height and bromocriptine concentration allowed the differential pulse voltammetric determination of the drug over a wide concentration range, from 0.04 to 5.00 microg ml(-1) with a detection limit of 0.01 microg ml(-1). A relative standard deviation of 1.44% at 0.1 microg ml(-1) level was obtained. The proposed DPV method was successfully applied for the individual tablet assay to verify the uniformity content of bromocriptine in commercial tablets.

High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit.

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid-liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-microm Nova-Pak C18 column (150x3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2-10 microg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1-50 microg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 microg/l for plasma and vitreous humour using a 1-ml sample and was 1 microg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100-102% for plasma, 91-106% for aqueous humour and 96-111% for vitreous humour. The between-day recovery (n=9) was 90-114% for plasma, 83-115% for aqueous humour and 90-105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7-7.0% and 8.1-13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.

Enzyme immunoassays for bromocriptine and its metabolites.

We have developed two bromocriptine enzyme immunoassays with different specificities for applications in human and animal pharmacokinetic studies. The first assay uses antibodies directed against the cyclopeptide structure of bromocriptine, and is specific for untransformed bromocriptine. The second assay uses antibodies directed against the bromolysergic part of the molecule and allows the measurement of both bromocriptine and its metabolites. Enzymatic tracers were obtained by covalent coupling of bromocriptine analogs to acetylcholinesterase from the electric eel Electrophorus electricus. Both assays have a limit of detection of 10 pg/ml and a limit of quantification of 50 pg/ml. The specificity of the assays was determined following fractionation by high-performance liquid chromatography of rat samples obtained after administration of bromocriptine.

Structural elucidation of two novel ergot alkaloid impurities in alpha-ergokryptine and bromokryptine.

Two novel natural derivatives (2 and 3) of the ergot alkaloid alpha-ergokryptine (1), as well as their synthetically brominated analogues (5 and 6) were isolated and identified by NMR and MS methods. Compounds 2 and 5 contain what appears to be a so far unknown natural amino acid building block. Complete 1H and 13C NMR assignments are given for compounds 1-6.

Rapid identification of ergot derivatives by 1H-NMR spectroscopy.

The 400 MHz 1H-NMR spectra of some therapeutically important ergot derivatives (three bases, four protonated bases and four dihydroergoline salts) are analysed in terms of the low field chemical shift region (above 5 ppm), common resonances of rings C and D (below 5 ppm) and C-8 substituent features. Attention is drawn to data of specific analytical value, and a scheme for the rapid identification of members of this group of ergots proposed. Features which provide evidence of the solute conformation of ring D, and isomerization to less active C-8 epimers are also emphasized.

Mass spectrometry as an aid to the identification of ergots and dihydroergots: comparison of hard and soft ionization techniques.

An analysis of the 70 eV electron impact (EI) and fast atom bombardment (FAB) mass spectral features of a variety of ergoline and dihydroergoline derivatives of therapeutic importance is presented with emphasis upon analytical utility. Derivatives which carry non-peptide based C-8 substituents are fully characterized by EI-MS through provision of molecular wieght evidence and fragment ions diagnostic of both the ergoline skeleton and the C-8 substituent. Peptidic ergolines and dihydroergolines are poorly characterized by EI-MS, but their FAB-MS clearly reveal [M + 1]+ (high intensity) and [M - 1]- (high to low intensity) ions in positive and negative ion spectra, respectively. Negative FAB spectra of salts also display diagnostic anion-base conjugate ions.

Mapping of intracellular halogenous molecules by low and high resolution SIMS microscopy.

The subcellular distribution of halogenous molecules has been studied by SIMS microscopy in cultured cells of a human breast carcinoma (MCF-7 cell line). Two instruments of microanalysis were used. A low lateral resolution ion microscope (SMI 300 CAMECA) and a prototype scanning ion microscope equipped with a cesium gun that gives high lateral resolution images. This apparatus has been developed by G Slodzian, in Onera Laboratories (Office National d'Etudes et de Recherches Aérospatiales). Molecules studied by low lateral resolution ion microscope were halogenous steroids: fluorometholone, triamcinolone, bromocriptine and bromoandrosterone. Analytical images show that the first two compounds are mainly localized in the nuclear structure of MCF-7 cells whereas the last two molecules are localized in cytoplasm of these cells. Images were obtained with a resolution of 1 micron. With the scanning ion microscope, it is now possible to obtain images at the ultrastructural level. Four analytical images can be simultaneously obtained by a single scan of the imaged area, corresponding to a depth of erosion of the section of ten nm. The intranuclear distributions of three pyrimidine analogs, 5-bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine and 5-fluorouracil have been studied in phase S and M of MCF-7 cells and these images have been compared to the distribution of sulfur, nitrogen and phosphorus. All these images have been obtained with a lateral resolution better than 100 nm.

Experience with routine applications of liquid chromatography-mass spectrometry in the pharmaceutical industry.

The combination of liquid chromatography and mass spectrometry (LC-MS) has been established to complement gas chromatography (GC)-MS in the analysis of non-volatile and labile drugs in complex materials. The possibilities of LC-MS in the pharmaceutical industry for the analysis of drug substances and dosage forms, metabolism studies and the elucidation of the structures of materials of biological origin are discussed. Instrumental requirements, limitations and applications of LC-MS are considered and experiences with LC-MS in routine applications are reported. Preliminary results obtained with thermospray LC-MS are compared with those using a direct liquid inlet interface.

Selective regional effect of various neuroactive drugs on bromocriptine concentration in the brain of rats.

Bromocriptine (2-Br-alpha-ergocryptine), a partial ergoline derivative, is a dopamine agonist which has been used successfully in the treatment of hyperprolactinemia, acromegaly and Parkinson's disease. The main targets for the action of the drug are the hypothalamic, hypophyseal pathway and the striatum. These regions contain different populations of neurons which interact with each other in a complex way. In order to check the mechanism of these interactions in rats, we administered different neuroactive drugs together with bromocriptine. After a single intraperitoneal injection, bromocriptine concentration in the striatum was 13.1 +/- 2.9 ng/mg protein, and in the hypothalamus 13.9 +/- 0.8 ng/mg protein. The largest increase in the bromocriptine content in the striatum was found after the concomitant administration of naloxone, an opiate receptor blocker (21.2 +/- 2.5 ng/mg protein). The largest increase of the bromocriptine content in the hypothalamus was found after the concomitant injection of methysergide, a serotonin receptor blocker (27.8 +/- 2.6 ng/mg protein). Amantadine, diazepam and haloperidol caused the largest decrease in the two regions. The mechanism of interaction and therapeutic implication of these findings are discussed.

Effect of various neuroactive drugs on bromocriptine concentration in the striatum of rats.

Parkinsonian patients are usually given several drugs concomitantly with bromocriptine, with variable results. The possibility of interference of these drugs with the availability of bromocriptine in the brain was investigated by measuring bromocriptine concentrations in the striatum in rats. After a single injection, bromocriptine concentration in the striatum was 13.1 +/- 2.9 ng/mg protein. Naloxone, an opiate receptor blocker, was found to produce the largest increase in bromocriptine content (21.7 +/- 2.5 ng/mg protein). Amantadine, diazepam and haloperidol produced the largest decreases (3.2 +/- 0.9, 3.3 +/- 0.8 and 4.4 +/- 1.2 ng/mg protein, respectively). Rats given L-dopa or benzodiazepine also showed slightly lower levels of bromocriptine.

Liquid chromatography-mass spectrometry in the pharmaceutical industry: objectives and needs.

The role of liquid chromatography-mass spectrometry (LC-MS) in the analysis of drugs is discussed. The main fields of application are thermally labile compounds, compounds with low volatility and compounds with rather high molecular weights, all of which are not generally suitable for analysis by combined gas chromatography-mass spectrometry. The objectives, needs, limitations and abilities of LC-MS for the analysis of by-products, degradation products, traces of drug substances for pharmacokinetic studies and metabolites in complex matrices are presented. The LC-MS coupling is discussed as a sophisticated LC detector for sensitive and selective quantitative determinations or as an on-line sample-introduction system for the mass spectrometer to obtain structural information for identification or structural elucidation. LC-MS combined with a flow-switching technique can be used for the analysis of mixtures containing large amounts of components which otherwise would be detrimental to the LC-MS technique. With flow-injection techniques the LC-MS interface is used as a sample-introduction system with possibilities for sample preparation, sample clean-up and chemical derivatization.

Bromocriptine concentration in saliva plasma after long-term treatment of patients with Parkinson's disease.

Salivary and plasma concentrations of bromocriptine (BCT), a dopamine agonist, were measured by gas chromatography in four patients with Parkinson's disease. All the patients had been on mono-therapy with BCT for years, and during the 3 weeks prior to the investigation they received constant but individually different dosage regimens. Paired samples of pure, parotid, serous saliva and of blood were collected hourly during one eight hour dose interval. The concentrations of BCT in saliva were very low and there was a ten-fold range in the areas under the salivary and plasma concentration/time curves. It is concluded that in clinical practice measurement of BCT in saliva is not suitable for exact estimation of the plasma concentration of BCT. Using the measured salivary pH and the plasma BCT concentration, calculations based on the Henderson-Hasselbalch equation showed that the assumption of about 99% plasma protein binding of BCT best fited the observed concentrations of BCT in saliva.