Determination of a novel xanthine derivative, MKS 213–492, in plasma by high-performance liquid chromatography with electrochemical detection

A reversed-phase liquid chromatographic method with coulometric detection has been developed for the determination of free MKS 213–492 (a novel xanthine derivative) in plasma. This method is based on a simple and rapid plasma extraction procedure using precolumn-switching. The oxidation of this pharmacologically active xanthine derivative was optimized with respect to applied potential, pH of mobile and rinsing phase and rinsing time. The detection limit for MKS 213–492 was found to be 54 pg/injection.     

Use of Hantzsch reaction for quantitation of milnacipran as a magic treatment for fibromyalgia syndrome in human plasma and urine

A new fluorimetric procedure is described for analysis of milnacipran in its bulk, tablet dosage forms, as well as in biological human samples such as plasma and urine. The suggested method relies on the construction of a derivative with strong fluorescence called dihydropyridine derivative. This derivative resulted from the interaction of the primary amino group in the studied drug and acetylacetone/formaldehyde in McIlvaine buffer (pH 5). The fluorescent dihydropyridine derivative was measured at 470 nm. Influences of experimental variables namely pH, reagent concentration and temperature were examined and optimized. The calibration curve showed linearity over the range of 0.15–1.25 μg ml^?1 of milnacipran with an R² value of 0.9998. The detection limit was 0.02 μg ml^?1 and the determination limit was 0.07 μg ml^?1. The developed procedure was successfully used in the assay of the studied drug in Avermilan® tablets with excellent selectivity. In addition, the reaction was applied to estimate the drug in spiked human plasma and urine with mean percentage recoveries of 100.04 ± 1.61 and 99.78 ± 0.81% for urine and plasma, respectively.     

The measurement of nanogram amounts of piperidine in tissues by gas chromatography.

A gas chromatographic method is presented which allows the determination of piperidine in tissue in nanogram quantities. Reaction of the perchloric acid tissue extracts with 3,5-dinitro-4-chloro-benzotrifluoride followed by extraction with hexane, results in a derivative of piperidine suitable for electron capture detection. The specificity of the procedure is confirmed by combined gas chromatography - mass spectrometry. Using this procedure the mean concentration of piperidine in whole mouse brain was found to be 219 pmoles per gram of tissue. No significant difference between the concentration of piperidine in the brains of active and behavioral sleeping mice could be found.     

ADP-ribosylation of isolated rat islets of Langerhans

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.     

Phenylalanine in whole blood and plasma — a candidate reference method

A method for the quantification of phenylalanine in whole blood and plasma by isotope dilution gas chromatography—mass spectrometry is presented. The use of an uncommon derivative allows a simple extraction procedure and the most basic mass spectrometer. The relative standard deviation was found to be 1.3% within-batch and 2.1% between-batch under optimum conditions, and the detection limit was found to be 7 pmol injected. The ruggedness of the procedure and sample handling conditions were also examined.     

Fluorimetric detection of octopamine in high-performance liquid chromatography and its application to the assay of dopamine β-monooxygenase in human serum

A high-performance liquid chromatographic procedure is described for the determination of octopamine. The method, which is based on the separation on a microparticulate bonded strong cation-exchange resin and measurement of the native fluorescence, has been applied to give a sensitive assay of dopamine β-monooxygenase (EC 1.14.17.1) activity in human serum with tyramine as the substrate. The procedure, which has been designed for use with an-automatic sampler, has a detection limit of about 50 pmoles of octopamine, and the analysis time is approximately 10 min per sample.     

Gas chromatographic determination of guanadrel in plasma and urine

To evaluate the pharmacokinetics and drug availability from various dosage formulations, a method for the determination of guanadrel, (1,4-dioxaspiro[4,5]dec-2-ylmethyl)guanidine, in plasma and urine was required. a gas chromatographic procedure, based on formation of a hexafluoroacetylacetone derivative in a two-phase system of water and toluene, was developed. The limit of determination of the method is 5 ng/ml guanadrel in plasma and 15 ng/ml guandrel in urine. Statistical analyses indicate average recoveries of 98.1 ± 18.0 and 104.4 ± 15.6% from plasma and urine, respectively. Mass spectrometric analyses, in conjunction with gas chromatography, confirmed the specificity of the method for intact drug. The procedure was applied successfully to drug absorption studies in humans.     

Determination of low levels of poly(ethylene glycol) 400 in plasma and urine by capillary gas chromatography-selected ion-monitoring mass spectrometry after solid-phase extraction

A convenient and sensitive method for the quantitative determination of poly(ethylene glycol) 400 in plasma and urine with capillary gas chromatography-mass spectrometry has been developed. The sample preparation involves solid-phase extraction with subsequent derivatization with heptafluorobutyric anhydride, which proved to give the most stable derivative. The derivatization procedure was optimized using experimental design, and different solid-phase extraction columns were evaluated. The limit of quantitation was 1 μmol/l (0.4 μg/ml) for both plasma and urine.     

Plasma membrane vesicles prepared from unadhered monocytes: Characterization of calcium transport and the calcium ATPase

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.     

Determination of the new podophyllotoxin derivative NK 611 in plasma by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method has been developed for the determination of the new podophyllotoxin derivative NK 611 in plasma samples. A solid—liquid extraction procedure with C₁₈ extraction columns was used for extraction of plasma samples containing NK 611. The adsorbed NK 611 was eluted from the extraction columns with methanol—acetonitrile (50:50, v/v). The elution liquid was injected into a reversed-phase system consisting of a Chrompack C₁₈ column. The mobile phase was acetonitrile—20 mM phosphate buffer, pH 7 (30:70, v/v). The UV detection mode allows sensitive determination of NK 611 in plasma within phase I trials. The limit of detection was 10 ng/ml, the limit of quantitation 35 ng/ml (for 1 ml of extracted plasma and 20-μl injection volume). The calibration curve is linear within the concentration range 100–1000 ng/ml. The recovery of NK 611 from spiked plasma samples was approximately 80%.     

Effects of oral and intravenous administrations of dopamine and L-dopa on plasma levels of two isomers of dopamine sulfate in man

The levels of two isomers of dopamine sulfate, dopamine-3-O-sulfate (DA3S) and dopamine-4-O-sulfate (DA4S), in human plasma were measured by HPLC-fluorometry. The basal plasma levels of DA3S and DA4S in the early morning were 13.8 +/- 1.9 and 3.2 +/- 0.5 pmoles/ml, respectively (means +/- S.E.M.). Oral administrations of dopamine (50 mg/body) and 1-dihydroxyphenylalanine (L-DOPA, 250 mg/body) increased the plasma levels of these dopamine sulfates almost 100-fold to 1807 +/- 266 and 1674 +/- 195 pmoles/ml of DA3S, and 466 +/- 83 and 321 +/- 76 pmoles/ml of DA4S. Intravenous dopamine infusion (5 micrograms/kg/min for 30 min) markedly increased the plasma free dopamine concentration, as expected, but increased the levels of DA3S and DA4S only slightly to 110 +/- 32 and 25 +/- 9 pmoles/ml, respectively. In contrast, intravenous L-DOPA (25 mg/body) resulted in a slight increase of free dopamine followed by marked increases of DA3S and DA4S to 691 +/- 219 and 139 +/- 40 pmoles/ml, respectively. These data indicate that O-sulfation of dopamine, especially 3-O-sulfation, is the main pathway for metabolism of intravenously and orally administered L-DOPA and orally ingested dopamine. This sulfation is suggested to occur in the gut wall.     

Enantioselective LC/MS method for the determination of an antimalarial agent Fenozan B07 in dog plasma

A chiral liquid chromatography/mass spectrometry (LC/MS) bioanalytical procedure has been developed for the analysis of the antimalaric agent Fenozan B07 in dog plasma. Normal-phase chromatography involving a phenylcarbamate derivative of cellulose coated on silica gel as the chiral stationary phase was used to resolve (-)-(S,S)-B07 from (+)-(R,R)-B07. The enantiomers were detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operated in the negative ion mode. A mass spectrum, characterized by a base peak of m/z 285, was obtained for each enantiomer. The m/z 285 ion was very specific for the analysis of both enantiomers in the plasma. The selected ion monitoring analysis of the plasma samples was therefore performed at m/z 285 for quantitative purposes. The enantiomers were extracted from the plasma in a basic medium and purified by solid-phase extraction using a hydrophilic-lipophilic balanced sorbent. A lower limit of quantification of 2 ng/mL in plasma was achieved for both enantiomers. The quantitative procedure reported in this study was highly specific and sensitive, and was validated according to the FDA guidance on bioanalytical method validation.     

Quantitative determination of imidazenil,a novel imidazobenzodiazepine car☐amide derivative,by normal-phase high-performance liquid chromatography

Imidazenil, an imidazobenzodiazepine car☐amide derivative, is a novel anxiolytic and anti-convulsant agent recently characterized as a partial allosteric modulator of GABA_A receptors. Owing to the pharmacological and pharmacokinetic importance of plasma-level determination, a HPLC method has been developed. Imidazenil was extracted from a plasma sample after a partition with diethyl ether, using alprazolam as internal standard. The analysis was performed by a normal-phase HPLC method with UV detection at 255 nm. The limit of quantitation was 6 ng corresponding to 30 ng/ml of plasma concentration. This procedure has been successfully applied to the quantitation of imidazenil plasma levels in primarily pharmacokinetic studies after a single i.v. and an oral administration of the compound to the rat.     

Pancreatic polypeptides affect luteinizing and growth hormone secretion in rats

We have examined the effects of third cerebroventricular (3V) injections of avian and bovine pancreatic polypeptide (APP and BPP) and the C-terminal hexapeptide amide of human PP (CHPP) on the secretion of anterior pituitary hormones in conscious ovariectomized rats. Injection of APP (2.0 micrograms; 472 pmoles) or BPP (5.0 micrograms; 1191 pmoles) decreased plasma levels of luteinizing hormone (LH) when compared to pre-injection levels in these animals or to saline-injected controls. The lower dose of BPP (0.5 micrograms; 119 pmoles) decreased plasma LH versus pre-injection levels and control animals, however, these effects diminished at later times. Plasma growth hormone (GH) also decreased following 3V injections of APP (2.0 micrograms) or BPP (5.0 micrograms). The lower dose of BPP (0.5 microgram) initially inhibited GH release, however, this effect was rapidly reversed and GH levels were significantly greater than those in controls at 60 and 120 min. Injections of BPP or APP did not alter prolactin (PRL) or thyroid stimulating hormone (TSH) secretion. Administration of 2.0 micrograms and 0.2 microgram of CHPP (2488 and 249 pmoles) produced no significant effects on plasma LH, GH, PRL or TSH. APP and BPP had no consistent effects on hormone secretion from dispersed anterior pituitary cells. The results indicate that APP and BPP exert potent central effects which inhibit LH and GH release from the pituitary gland.     

Measurement of gamma-aminobutyric acid (GABA) in blood.

Blood GABA levels can be readily determined using a radioreceptor assay or gas chromatography-mass spectrometry. After withdrawal of blood, GABA levels remain stable with 25–50% of the GABA in whole blood found in the plasma fraction. Whole blood GABA concentrations range from 500 pmoles/ml to 1200 pmoles/ml in 8 mammalian species with human values being about 900 pmoles/ml. $\text{in vivo}$ administration of aminooxyacetic acid increases both blood and brain GABA levels to a similar extent.     

Bradykinin-induced changes in inositol trisphosphate mass in MDCK cells

Bradykinin produces increases in cytosolic calcium in MDCK cells. We have extracted and separated Inositol 1,4,5 trisphosphate by HPLC and after-acid hydrolysis and conversion to the hexatrifluoro-acetyl derivative quantitated by negative ion chemical ionization mass spectrometry the mass of inositol trisphosphate in MDCK cells. Bradykinin causes an increase in the mass of Inositol trisphosphate from basal levels of 152 pmoles/mg cell protein to 537 pmoles/mg cell protein by 10 secs of stimulation. We conclude that bradykinin stimulates PLC hydrolysis of PIP2 with rapid release of IP3 in sufficient amount to account for the increase in cytosolic Ca++.     

Gas chromatography chemical ionization mass spectrometric analysis of diminazene in plasma

A quantitative and sensitive method was developed for the determination of diminazene in plasma. The assay involves the reduction of diminazene to 4-aminobenzamidine and 4-hydrazinobenzamidine. The latter is further reduced to give an additional mole of 4-aminobenzamidine which is extracted, acetylated and condensed with hexafluoroacetylacetone to form a volatile derivative that is subsequently analyzed by gas chromatography chemical ionization mass spectrometry. 4-Aminobenzylamidine was synthesized and used as an internal standard. The method is reproducible and its sensitivity limit using 1 ml of plasma is 0.1 microgram diminazene ml-1. This sensitivity limit is sufficient to detect plasma levels in cattle following therapeutic doses of the drug.     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Determination of adenosine kinase activity by high-pressure liquid chromatography

High-pressure liquid chromatography (reverse-phase mode) was used to assay adenosine kinase in cell and tissue extracts. The method is optimized for a rapid and selective analysis using 6-methylthiopurine riboside as substrate, isocratic elution and detection at 300 nm. A complete separation of substrate and product is achieved in about 3 min with no interference by other UV-absorbing compounds; the limit of detection is 20 pmoles.     

Pro-Leu-Gly-NH2 and pareptide inhibit development of tolerance to haloperidol catalepsy in the mouse

Single doses of MIF-1 (0.03-2.0 mg/kg, SC) and chronic pretreatments with MIF-1 (0.03-2.0 mg/kg, SC, BID, 3 1/2 days) or pareptide (0.25 mg/kg, SC, BID, 3 1/2 days) did not affect the acute cataleptic response to haloperidol in the mouse. Chronic pretreatment with haloperidol (8.0 mg/kg, IP, BID, 3 days) decreased the duration of catalepsy in mice given smaller challenge dose of haloperidol (2.0 or 3.0 mg/kg, IP) 15 hours after the last pretreatment injections. Administration of either MIF-1 or pareptide to mice also chronically pretreated with haloperidol antagonized the development of tolerance.