Pharmacokinetic study of isatin in dog plasma by liquid chromatography tandem mass spectrometry.

AIM: A sensitive and selective method was developed and validated to study the pharmacokinetics of isatin. METHODS: The blood samples were pretreated by protein precipitation method using methanol. Quetiapine was used as an internal standard. After pretreatment, the samples were assayed by LC/MS/MS method and the pharmacokinetic parameters were calculated by WinNonlin 5.2 using non-compartment model. The separation was performed on a Venusil XBP PH column (5 µm, 2.0×100 mm) with an isocratic mobile phase consisted of methanol-water (containing 50 mM ammonium formate) (65:35, v/v) at a flow rate of 0.3 mL/min. The Agilent G6410B triple quadrupole LC/MS system was operated under the multiple reactions monitoring mode (MRM) using the electrospray ionization technique in positive mode. RESULTS: The lower limits of quantification (LLOQ) of the analyte of the method was 10 ng/mL. The method was linear with correlation coefficient >0.995. The intraday and interday accuracy and precision of the assay were acceptable. CONCLUSION: This method has been applied successfully to a pharmacokinetic study involving the oral and intravenous administration of isatin to beagle dogs.

Enzymatic detection and quantification assay of isatin, a putative stress biomarker in blood.

Isatin is an endogenous inhibitor of monoamine oxidase B and is found in human blood and tissue. Increased levels of isatin have been linked to stress and anxiety in rodents and humans; however, the metabolism of isatin in humans is largely unknown. We have developed a fluorescence-based enzymatic assay that can quantify isatin in blood samples. A phase extraction of isatin followed by a second phase extraction combined with an enzymatic reaction performed by an isatin hydrolase is used to extract and quantify isatin in whole blood samples. This results in a purity of more than 95% estimated from RP-HPLC. The hydrophobic molecule isatin is in equilibrium between an organic and aqueous phase; however, conversion by isatin hydrolase to the hydrophilic product isatinate traps it in the aqueous phase, making this step highly specific for isatin. The described protocol also offers a novel method for fast and efficient removal of isatin from any type of sample. The isolated isatinate is converted chemically to anthranilate that allows fluorescent detection and quantification. Pig plasma isatin levels are quantified to a mean of 458 nM ± 91 nM. Biophysical characterization of the isatin hydrolase shows enzymatic functionality between pH 6 and 9 and at temperatures up to 50 °C. Isatin hydrolase is highly selective for manganese ions with a dissociation constant determined to be 9.5 µM. We deliver proof-of-concept for the enzymatic quantification of isatin in blood and provide a straightforward method for further investigation of isatin as a biomarker in human health.

Biomimetic approach to perophoramidine and communesin via an intramolecular cyclopropanation reaction.

[reaction: see text] Starting from tryptamine 4 and isatin 5, a biomimetic approach to the pentacyclic substructure 1 of perophoramidine and communesin was developed. The key steps were to create a stable three/six bicyclic system 2 on the 2,3-double bond of an indole derivative 3 by an intramolecular cyclopropanation, followed by ring opening of the resulting cyclopropane ring with the in situ generated amine group of an aniline.

A microtitre format assay for proline in human serum or plasma.

BACKGROUND: Recent studies have suggested that low serum proline concentration may be associated with low bone mineral density. However, further investigation of this association has been hampered by the lack of a relatively high throughput assay for proline in biological fluids. Here we report a sensitive and specific microtitre plate format assay for proline which exploits the chemical interaction between proline and isatin. METHODS: Human serum or plasma is deproteinised by incubation with sodium citrate buffer pH 4.1 at 95 degrees C, and the supernatant is reacted with isatin at 95 degrees C for 3 h. The resultant blue coloured product is quantitated sprectrophometrically. RESULTS: This assay yields a linear standard curve in the range 15 micromol/l to 1 mmol/l (r=0.998+/-0.002; n=8 determinations) with a sensitivity of 31+/-11 micromol/l. None of the other proteogenic amino acids are detected (<0.3% detection at 10 mmol/l) and the closely related metabolite hydroxyproline is only very weakly detected (3% detection at 10 mmol/l). Using human serum, the assay has linear dilution characteristics and a mean spike recovery of 107+/-5%. Repeated re-measurement of the same serum sample yields an intra-assay coefficient of variation (CV) of 4.8% and an inter-assay CV of 6.1%. CONCLUSIONS: This method provides the first reliable micro-titre format assay for proline in human serum.

Effect of acute stress and gender on isatin in rat tissues and serum.

Isatin is an endogenous indole present in mammalian tissues and fluids. This study was designed to test the effect of both acute stress (AS) and gender on isatin's levels in rat serum, heart and brain, using a HPLC-UV detection method. Basal brain isatin levels were higher in females than in males. AS resulted in increased isatin levels in male serum, heart and brain, and in female serum and heart, but not in the brain. The percentage increases were greater in males than in females in all three sources. After stress, there were significant correlations between isatin levels in the serum and the heart. However, there were no correlations between brain levels and those in the other sources. These results consolidate previous evidence that isatin levels are altered by stress and suggest that brain isatin is under separate control from that in the periphery.

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation.

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.

Determination of isatin in urine and plasma by high-performance liquid chromatography.

A method for the detection and determination of isatin (indole-2,3-dione) in urine and plasma by high-performance liquid chromatography has been developed. It consists of a two-step purification using two different columns with UV detection. With this method, we have reconfirmed that isatin is present in human urine. We have also demonstrated that isatin is present in human plasma and that the isatin levels in spot urine samples reflect the plasma isatin levels. In the present report we describe a rapid and sensitive means of determining urine and plasma isatin for laboratories equipped with a high-performance liquid chromatography system.

Binding (in vitro) of some antimitotic isatin derivatives to human serum albumin.

The binding of isatin and its mustard N-Mannich base, considerably biologically active compounds, to human serum albumin has been studied by equilibrium dialysis and ultrafiltration. The influences of ligand and macromolecule concentration, temperature and pH of the incubation medium have been demonstrated. The Scatchard plot of isatin binding to albumin shows a biphasic curve which indicates the presence of at least two different binding sites on albumin molecule. One site with a higher affinity, K1 = 2.25 X 10(3) M and n1= 25, and the other site with a lower affinity, i.e. higher capacity. In the cases of mustard Mannich base we could demonstrate the same type of curve, K1 = 2.20 X 10(5) M and n1 = 1.0, whereas another site has a lower affinity and greater number of binding sites.