A strategy for high-throughput analysis of levosimendan and its metabolites in human plasma samples using sequential negative and positive ionization liquid chromatography/tandem mass spectrometric detection.

Levosimendan (Simdax) is an approved drug in approximately 40 countries and currently in phase III clinical studies in the USA and Europe. An accurate, high-throughput and rugged assay is critical to support these clinical trials. Due to the mechanism of drug metabolism, the drug and its active metabolites often have significant differences in their chemical properties. In order to achieve high assay throughput and low sample volumes, a single bioanalytical assay for the drug and its metabolites is preferred. However, this need may prevent the optimization of both high-performance liquid chromatography (HPLC) and mass spectrometric ionization conditions. The chemical properties of levosimendan are significantly different from those of its two active metabolites, OR-1855 and OR-1896. Here, we present a novel strategy for high-throughput analysis of levosimendan and its metabolites. A 96-well liquid/liquid extraction procedure was developed for sample preparation. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) system with two separate mobile phases, shared backwash solvent and conditioning solvent, was developed to perform sequential LC separation for levosimendan and the metabolites. Levosimendan was eluted by 5 mM ammonium acetate in 33.3% acetonitrile and detected using negative ionization mode MS/MS monitoring. The metabolites were eluted by 5 mM ammonium acetate and 0.2% acetic acid in 20% acetonitrile and detected with positive ionization mode MS/MS monitoring. The method has been demonstrated to have excellent precision and accuracy, with high assay ruggedness during method validation and clinical sample analysis. The linear dynamic ranges were approximately 200-50,000 pg/mL for levosimendan and approximately 500-130,000 pg/mL for both metabolites. The coefficient of determination (r2) for all analytes was greater than 0.9985. The intra-assay %CVs for QC samples were from 0.9% to 2.0% for levosimendan, 0.9% to 3.2% for OR-1855, and 0.4% to 4.9% for OR-1896. The inter-assay %CVs for QC samples were from 1.2% to 1.8% for levosimendan, 1.3% to 2.7% for OR-1855, and 1.4% to 3.4% for OR-1896. The mean % biases for QC samples were from 1.5% to 5.5% for levosimendan, -1.4% to 2.6% for OR-1855, and -0.3% to 4.5% for OR-1896. By using a single extraction approach coupled with sequential LC/MS/MS analysis for levosimendan and its metabolites, the assay maintained high throughput and low sample volume usage.

A high-throughput liquid chromatography/tandem mass spectrometry method for simultaneous quantification of a hydrophobic drug candidate and its hydrophilic metabolite in human urine with a fully automated liquid/liquid extraction.

ABT-869 (A-741439) is an investigational new drug candidate under development by Abbott Laboratories. ABT-869 is hydrophobic, but is oxidized in the body to A-849529, a hydrophilic metabolite that includes both carboxyl and amino groups. Poor solubility of ABT-869 in aqueous matrix causes simultaneous analysis of both ABT-869 and its metabolite within the same extraction and injection to be extremely difficult in human urine. In this paper, a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method has been developed and validated for high-speed simultaneous quantitation of the hydrophobic ABT-869 and its hydrophilic metabolite, A-849529, in human urine. The deuterated internal standards, A-741439D(4) and A-849529D(4), were used in this method. The disparate properties of the two analytes were mediated by treating samples with acetonitrile, adjusting pH with an extraction buffer, and optimizing the extraction solvent and mobile phase composition. For a 100 microL urine sample volume, the lower limit of quantitation was approximately 1 ng/mL for both ABT-869 and A-849529. The calibration curve was linear from 1.09 to 595.13 ng/mL for ABT-869, and 1.10 to 600.48 ng/mL for A-849529 (r2 > 0.9975 for both ABT-869 and A-849529). Because the method employs simultaneous quantification, high throughput is achieved despite the presence of both a hydrophobic analyte and its hydrophilic metabolite in human urine.

Simultaneous quantitation of dexmedetomidine and glucuronide metabolites (G-Dex-1 and G-Dex-2) in human plasma utilizing liquid chromatography with tandem mass spectrometric detection.

Dexmedetomidine (Dex) (Precedex) is a novel lipophilic imidazole derivative with a high affinity for alpha-2 adrenergic receptors, which exhibits sedative, analgesic-sparing, and sympatholytic properties. The pharmacological effects and therapeutic benefits of this drug have drawn continued interest from the medical community. Here we report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to simultaneously measure the concentrations of dexmedetomidine and its glucoronide metabolites, G-Dex-1 and G-Dex-2, in human plasma samples. A solid-phase extraction method was developed to effectively extract Dex, G-Dex-1, and G-Dex-2 from plasma matrices. An isocratic chromatographic method was developed to achieve baseline separation of G-Dex-1 and G-Dex-2. The linear dynamic range evaluated was 19.08-1908.56 pg/mL for Dex, 65.17-6518.17 pg/mL for G-Dex-1, and 29.42-2943.28 pg/mL for G-Dex-2. The linear correlation coefficient (r) ranged from 0.9944-0.9979 for Dex, from 0.9966-0.9984 for G-Dex-1, and from 0.9939-0.9966 for G-Dex-2. The intra-assay coefficient of variation (CV) was between 2.5-12.5% for Dex, between 5.2-11.0% for G-Dex-1, and between 3.5-12.1% for G-Dex-2. The inter-assay precision of QC samples give % CV ranges from 6.5-9.3% for Dex, from 7.1-10.6% for G-Dex-1, and from 8.2-10.2% for G-Dex-2. The inter-assay accuracies ranged from 102.0-109.3% for Dex, from 95.4-105.6% for G-Dex-1, and from 98.7-115.0% for G-Dex-2.

A strategy of plasma protein quantitation by selective reaction monitoring of an intact protein.

Immunoassays are used extensively in the quantitative analysis of proteins in plasma, urine, and other biological matrixes to support preclinical and clinical studies. Although immunoassays are both sensitive and rapid, difficulties during development of these assays are compounded by the need to have a specific antibody or antigen to the protein of interest. Furthermore, calibration curves of immunoassays are inherently nonlinear, and the technique often detects many structurally related components in addition to the analyte of interest. We have developed a novel strategy of analyzing protein concentrations in plasma by utilizing 96-well solid-phase extraction and LC-MS/MS detection of the intact protein. This strategy has been successfully applied in method development and assay validation of quantitatively analyzing protein rK5 concentrations in monkey plasma samples. Additional techniques such as precolumn regeneration and column heating were also incorporated into the assay. Total run time for each sample was approximately 15 min. An LLOQ of 99.2 ng/mL from a sample volume of 50 microL, corresponding to only 380 fmol (3.97 ng) of the rK5 analyte being injected onto the analytical column (assuming 100% extraction recovery), was obtained. The validated linear dynamic range was between 99.2 and 52 920.0 ng/mL, with a correlation coefficient (r(2)) ranging from 0.9972 and 0.9994. The intraassay CV for this assay was between 0.6 and 3.8%, and the interassay CV was between 1.7 and 3.2%. Interassay mean accuracies were between 101.5 and 104.7%. The assay has proven rugged and specific and has been employed to generate data in support of preclinical studies. This strategy for rK5 assay could be used for the development of bioanalytical assays to provide preclinical and clinical support for other protein drug candidates and, furthermore, for the validation of biomarkers discovered from proteomic research.

Method development for the concentration determination of a protein in human plasma utilizing 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometric detection.

Although liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology has been widely used for quantitative analysis of small organic molecules, it has been a challenging task to quantitatively analyze protein samples utilizing this technology in biological matrices for pre-clinical and clinical studies. Here we present our initial results in method development for the quantitative determination of rK5 protein concentrations in human plasma samples utilizing LC/MS/MS technology. A protein similar in structure to rK5, but with a slightly reduced molecular weight, was used as internal standard. A 96-well solid-phase extraction procedure was developed to effectively extract protein analytes from plasma samples. Quantitative analysis was obtained by a novel approach of protein monitoring that employed selective reaction monitoring (SRM). Even though mass spectrometry of the internal standard protein gave no fragment ions, SRM monitoring greatly reduced background interference. Using samples prepared in human plasma with sodium EDTA as anticoagulant, a correlation coefficient (r(2)) of 0.9940 was obtained by producing a single standard curve with the injection of six rows of standards with a concentration range from 100 ng/mL to 10 microg/mL. The mean analytical recovery for these standards ranged from 91.5 to 103.6%. The CVs for individual standard levels ranged from 3.7 to 20.9%. The experiment was also repeated using samples prepared in human plasma with sodium heparin as anticoagulant, which produced a correlation coefficient (r(2)) of 0.9952 obtained from a single standard curve with the injection of four rows of standards with a concentration range from 50 ng/mL to 10 microg/mL. The mean analytical recovery for the standards ranged from 96.2 to 104.6%. The CVs for individual standard levels ranged from 2.6 to 15.6%.