Optimization of solid phase extraction clean up and validation of quantitative determination of carbazochrome sodium sulfonate in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

A mixed-mode anion exchange solid phase extraction (SPE) method for extraction and clean up of carbazochrome sodium sulfonate (CSS) and (1S)-(+)-10-camphorsulfonic acid (IS) was optimized for quantification by high-performance liquid chromatography/negative electrospray ionization mass spectrometry. The analytes were extracted from 1mL of human plasma via SPE on Oasis(®) WAX cartridge. Chromatographic separation was achieved on a Zorbax SB-Aq (4.6×250mm, 5µm) column under an isocratic condition. Detection was performed using electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. The deprotonated precursor to product ion transitions monitored for CSS and IS was at m/z 299.0→256.0 and m/z 230.9→79.8, respectively. The method was fully validated for its selectivity, sensitivity, precision, accuracy, recovery, matrix effect and stability. Linear range was 0.189-37.8ng/mL with a high square regression coefficient (r=0.9995). The intra-and inter-day precision (RSD, %) ranged from 0.95% to 4.17%, and the intra-and inter-day accuracy was between 95.03% and 105.9%. This method was successfully applied to a bioequivalence study of 90mg CSS formulation in 18 healthy Chinese male subjects under fasting condition.

Determination of carbazochrome by fluorescence quenching method.

A sensitive, simple and selective spectrofluorimetric method for the reaction of carbazochrome (CBZC) and Eosin Y (EY) or Phloxine B (PB) in acidic medium is developed for the determination of carbazochrome in biological fluids, which gives a highly fluorescent derivative measured at 545 and 565 nm at excitation wavelengths of 301 and 305 nm. The fluorescence quenching extent (ΔF) is proportional to the concentration of CBZC for CBZC-EY and CBZC-PB system at the range of 0.03-1.50 µg/mL and 0.08-1.25 µg/mL, respectively. The detection limit is 9.1 ng/mL for EY system and 22.7 ng/mL for PB system. The intra-day and inter-day reproducibility (RSD values) are less than 8.3% under three concentrations. Moreover, the affecting factors of fluorescence intensity of the product are carefully investigated and optimized, as well as the effect of coexisting substances. Judging from temperature, the Stern-Volmer plots and fluorescence emission decay curves, the quenching of fluorescence of EY and PB by CBZC is a static quenching process, caused by electrostatic attraction and aromatic stacking interaction.

Development and validation of a robust LC-MS-MS with atmospheric pressure chemical ionization for quantitation of carbazochrome sodium sulfonate in human plasma: application to a pharmacokinetic study.

A highly selective and sensitive liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS) was developed and validated for the quantitation and pharmacokinetic study of carbazochrome sodium sulfonate in human plasma. Protein precipitation with 14% perchloric acid solution was selected for sample preparation, and amiloride hydrochloride was employed as an internal standard. The analytes were separated on a Hypersil ODS-2 column by a multiple-step linear gradient elution with a mobile phase consisting of 0.2% formic acid solution and methanol pumped at a flow rate of 1.0 mL/min. The determination was optimized and carried out with positive atmospheric pressure chemical ionization by selective reaction monitoring of the ion of m/z 148, the protonated thermodegraded fragment of the free acidic form of carbazochrome sodium sulfonate selected as the parent, and the ion of m/z 107 as the optimum collision induced dissociation (CID) product. The method was fully validated over a concentration range of 0.5-50 ng/mL, with the lower limit of quantitation of 0.5 ng/mL. The application of the LC-MS-MS method was demonstrated for the specific and quantitative analysis of carbazochrome sodium sulfonate in human plasma from a pharmacokinetic study in 24 healthy male Chinese volunteers after a single oral administration of 90 mg carbazochrome sodium sulfonate capsules.

Inactivation of catecholamines by superoxide gives new insights on the pathogenesis of septic shock.

A major feature of septic shock is the development of a vascular crisis characterized by nonresponsiveness to sympathetic vasoconstrictor agents and the subsequent irreversible fall in blood pressure. In addition, sepsis, like other inflammatory conditions, results in a large increase in the production of free radicals, including superoxide anions (O(2)) within the body. Here we show that O(2) reacts with catecholamines deactivating them in vitro. Moreover, this deactivation would appear to account for the hyporeactivity to exogenous catecholamines observed in sepsis, because administration of a superoxide dismutase (SOD) mimetic to a rat model of septic shock to remove excess O(2) restored the vasopressor responses to norepinephrine. This treatment with the SOD mimetic also reversed the hypotension in these animals; suggesting that deactivation of endogenous norepinephrine by O(2) contributes significantly to this aspect of the vascular crisis. Indeed, the plasma concentrations of both norepinephrine and epinephrine in septic rats treated with the SOD mimetic were significantly higher than in untreated rats. Interestingly, the plasma concentrations for norepinephrine and epinephrine were inversely related to the plasma concentrations of adrenochromes, the product of the autoxidation of catecholamines initiated by O(2). We propose, therefore, that the use of a SOD mimetic represents a new paradigm for the treatment of septic shock. By removing O(2), exogenous and endogenous catecholamines are protected from autoxidation. As a result, both hyporeactivity and hypotension are reversed, generation of potentially toxic adrenochromes is reduced, and survival rate is improved.

Measurement of adrenolutin as an oxidation product of catecholamines in plasma.

Using the reverse phase high-performance liquid chromatography (HPLC) with mobile phases composed of simple acids, we have developed an assay technique for the measurement of adrenolutin, one of the oxidation products of catecholamines, in rat plasma. Ion-pairing chromatography permits the separation and quantitation of plasma adrenolutin (microM) in a linear manner. Sample preparation involved the precipitation of plasma proteins with perchloric acid and it is easier to handle a large number of samples at a time. However, we were unable to demonstrate the presence of adrenochrome, another oxidation product of catecholamines, in plasma since adrenochrome was rapidly destroyed in acid as well as in blood and was quickly changed into adrenolutin. Adrenolutin peak in HPLC was confirmed by 1) the retention time; 2) co-injection of adrenolutin and; 3) the appearance of 3H-adrenolutin after injection of 3H-norepinephrine. Administration of different catecholamines as well as adrenochrome and adrenolutin in rats also increased the level of adrenolutin in plasma. Adrenolutin was found to be present in plasma in other species including dog, rabbit and pig. High level of adrenolutin, which may represent total concentration of aminolutin in plasma, suggests the presence of an efficient mechanism for the oxidation of catecholamines under in vivo conditions.

Partial characterization of the 1-anilino-8-naphthalene sulfonate-adrenochrome semicarbazide interaction site in erythrocyte ghost membrane fragments.

The effect of adrenochrome semicarbazide on the conformation of erythrocyte ghost membranes has been studied by ANS fluorescence, lipid and sulfhydryl spin labels and circular dichroism. No large conformational alterations in the membrane were detected by these techniques. Noncompetitive quenching of ANS fluorescence by ADCS suggests ADCS to interact with the membrane at sites close to the ANS binding domain.