A simple and sensitive assay for the quantitative analysis of paclitaxel in human and mouse plasma and brain tumor tissue using coupled liquid chromatography and tandem mass spectrometry.

The development and validation of an assay for the determination of paclitaxel in human plasma, human brain tumor tissue, mouse plasma and mouse brain tumor tissue is described. Paclitaxel was extracted from the matrices using liquid-liquid extraction with tert-butyl methyl ether, followed by chromatographic analysis using an alkaline eluent. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicate that calibration standards in human plasma can be used to quantify paclitaxel in all tested matrices. In human samples, the validated range for paclitaxel was from 0.25-1000 ng ml(-1) using 200 microl plasma aliquots and from 5 to 5000 ng g(-1) using 50 microl tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma). In mice, the ranges were 1-1000 ng ml(-1) and 5-5000 ng g(-1) using 50 microl of mouse plasma and 50 microl of tumor homogenate aliquots (0.2 g tissue ml(-1) control human plasma), respectively. The method can be applied to studies generating only small sample volumes (e.g. mouse plasma and tumor tissue), but also to studies in human plasma requiring a lower limit of quantitation. The assay was applied successfully to several studies with both human and mouse samples.

Switching from an analogous to a stable isotopically labeled internal standard for the LC-MS/MS quantitation of the novel anticancer drug Kahalalide F significantly improves assay performance.

The importance of a stable isotopically labeled (SIL) internal standard for the quantitative LC-MS/MS assay for Kahalalide F in human plasma is highlighted. Similar results can be expected for other LC-MS/MS assays. Therefore, we emphasize the need for an SIL internal standard for accurate and precise LC-MS/MS assays of drugs in biological matrices.

Simple and sensitive liquid chromatographic quantitative analysis of the novel marine anticancer drug Yondelis (ET-743, trabectedin) in human plasma using column switching and tandem mass spectrometric detection.

The development of a simple and sensitive assay for the quantitative analysis of the marine anticancer agent Yondelis (ET-743, trabectedin) in human plasma using liquid chromatography (LC) with column switching and tandem mass spectrometric (MS/MS) detection is described. After protein precipitation with methanol, diluted extracts were injected on to a small LC column (10 x 3.0 mm i.d.) for on-line concentration and further clean-up of the sample. Next, the analyte and deuterated internal standard were back-flushed on to an analytical column for separation and subsequent detection in an API 2000 triple-quadrupole mass spectrometer. The lower limit of quantitation was 0.05 ng mL(-1) using 100 micro l of plasma with a linear dynamic range up to 2.5 ng ml(-1). Validation of the method was performed according to the most recent FDA guidelines for bioanalytical method validation. The time needed for off-line sample preparation has been reduced 10-fold compared with an existing LC/MS/MS method for ET-743 in human plasma, employing a labor-intensive solid-phase extraction procedure for sample pretreatment. The proposed column switching method was successfully applied in phase II clinical trials with Yondelis and pharmacokinetic monitoring.

Quantitative analysis of the novel anticancer drug ABT-518, a matrix metalloproteinase inhibitor, plus the screening of six metabolites in human plasma using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry.

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the quantitative analysis of the novel anticancer drug ABT-518 and the screening of six potential metabolites in human plasma has been developed and validated to support a phase I study with the drug. ABT-518 is an inhibitor of matrix metalloproteinases, which are associated with tumor growth and development of metastasis. Plasma samples were prepared for analysis using a simple solid-phase extraction method on phenyl cartridges. LC separation was performed on a Zorbax extend C18 column (150 x 2.1 mm i.d., 5 microm particle size) using a mobile phase of methanol-aqueous 10 mM ammonium hydroxide (80:20, v/v) pumped at a flow-rate of 0.2 ml min(-1). An API2000 triple-quadrupole mass spectrometer was used for specific and sensitive detection. The best chromatographic speed (total run time 8 min) and peak shapes were obtained by employing an alkaline mobile phase (pH in aqueous phase approximately 10). Furthermore, an alkaline eluent was favored in order to obtain a better overall sensitivity for the protonated analytes. The dynamic range was from 10 to 1000 ng ml(-1) from 500 microl of plasma for ABT-518 and the metabolites were detected at levels of the same order of magnitude. Inter-assay accuracies for ABT-518 at five concentration levels were between -9.24 and 6.93% and inter-assay precisions were always <10.7%. Analyte stability was not critical during either storage or processing. This method was successfully applied in a phase I clinical study of ABT-518. The active drug, ABT-518, and all of the metabolites included in the assay could be identified in plasma from dosed patients. We believe that the method described in this paper using an alkaline mobile phase in combination with a basic stable analytical column may also be generally useful for the bioanalysis of other basic drugs.

Quantitative analysis of ES-285, an investigational marine anticancer drug, in human, mouse, rat, and dog plasma using coupled liquid chromatography and tandem mass spectrometry.

A method was developed for the quantitative analysis of the novel anticancer agent ES-285 (spisulosine; free base) in human, mouse, rat, and dog plasma using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in order to support pre-clinical and clinical studies with the drug. Sample preparation was carried out by protein precipitation with acetonitrile, containing isotopically labeled (d(3)) ES-285 as internal standard. Aliquots of 10 micro l of the supernatant were injected directly on to an Inertsil ODS-3 column (50 x 2.0 mm i.d., 5 micro m). Elution was carried out using methanol-10 mM ammonium formate (pH 4) in water (80 : 20, v/v) pumped at a flow-rate of 0.2 ml min(-1) with a run time of 8 min. Multiple reaction monitoring chromatograms obtained on an API365 triple-quadrupole mass spectrometer were used for quantification. The lower limit of quantitation (LLOQ) was 10 ng ml(-1) in human, mouse, rat, and dog plasma and the linear dynamic range extended to 500 ng ml(-1). A full validation of the method was performed in human plasma, and partial validations were performed in mouse, rat and dog plasma. Accuracies and precisions were <20% at the LLOQ concentration and <15% for all other concentrations in all matrices. ES-285 was stable during all steps of the assay. Thus far this method has been used successfully to analyze over 500 samples in pre-clinical trials, and will be implemented in the planned clinical phase I studies.

Quantitative analysis of the novel depsipeptide anticancer drug Kahalalide F in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray ionization tandem mass spectrometry.

Kahalalide F (KF) is a novel cyclic depsipeptide anticancer drug, which has shown anticancer activity both in vitro and in vivo especially against human prostate cancer cell lines. To characterize the pharmacokinetics of KF during a phase I clinical trial in patients with androgen refractory prostate cancer, a method was developed and validated for the quantitative analysis of KF in human plasma using high-performance liquid chromatography (HPLC) coupled to positive electrospray ionization tandem mass spectrometry (ESI-MS/MS). Microbore reversed-phase liquid chromatography (LC) performed with mobile phases containing trifluoroacetic acid, an additive commonly used for separating peptides, resulted in substantial suppression of the signal for KF on ESI-MS/MS. An alternative approach employing a basic mobile phase provided an excellent response for KF when detected in the positive ion mode. Plasma samples were prepared for LC MS/MS by solid-phase extraction on C(18) cartridges. The LC separation was performed on a Zorbax Extend C(18) column (150 x 2.1 mm i.d., particle size 5 micro m) with acetonitrile -10 mM aqueous ammonia (85 : 15, v/v) as the mobile phase, at a flow-rate of 0.20 ml min(-1). A butyric acid analogue of KF was used as the internal standard. The lower limit of quantitation (LLQ) using a 500 micro l sample volume was 1 ng ml(-1) and the linear dynamic range extended to 1000 ng ml(-1). The inter-assay accuracy of the assay was -15.1% at the LLQ and between -2.68 and -9.05% for quality control solutions ranging in concentration from 2.24 to 715 ng ml(-1). The inter-assay precision was 9.91% or better at these concentrations. The analyte was stable in plasma under all relevant conditions evaluated and for a period of 16 h after reconstituting plasma extracts for LC analysis at ambient temperature.

Chemical and enzymatic stability of a cyclic depsipeptide, the novel, marine-derived, anti-cancer agent kahalalide F.

Kahalalide F is a cyclic depsipeptide isolated from the Hawaiian mollusk Elysia rufescens. This compound is under present phase I clinical investigations as an anti-tumor drug. The role of possible metabolic reactions of this drug in (pre-)clinical investigations has not yet been explored. The first results for kahalalide F in this field of research are given in this paper. The chemical degradation of kahalalide F was investigated under acid, neutral and alkaline conditions using high-performance liquid chromatography with ultraviolet detection. The half-lives at 80 degrees C were 1.1, 20 and 8.6 h at pH 0, 1 and 7, respectively. At 26 degrees C and pH 11, the half-life was 1.65 h. At pH 7 and 11, only one reaction product of kahalalide F was observed, kahalalide G, the hydrolyzed lactone product of kahalalide F. At pH 0 and 1, additional reaction products emerged. Metabolic conversion of kahalalide F was tested in vitro using three different enzyme systems based on pooled human microsomes, pooled human plasma and uridine 5'-diphosphoglucuronyl transferase, respectively. The incubated samples were analyzed using the same chromatographic technique as for the degradation samples. Biotransformations were not observed under these conditions and, therefore, it is concluded that kahalalide F is a metabolically stable drug.

Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry.

The dissection of specific and nonspecific protein complexes in the gas phase is studied by collisionally activated decomposition. In particular, the gas phase dissection of multiple protonated homodimeric Human Galectin I, E. Coli Glyoxalase I, horse heart cytochrome c, and Hen egg Lysozyme have been investigated. Both the Human Galectin I and E. Coli Glyoxalase I enzymes are biologically active as a dimer, exhibiting molecular weights of approximately 30 kDa. Cytochrome c and Lysozyme are monomers, but may aggregate to some extent at high protein concentrations. The gas phase dissociation of these multiple protonated dimer assemblies does lead to the formation of monomers. The charge distribution over the two concomitant monomers following the dissociation of these multiple protonated dimers is found to be highly dissimilar. There is no evident correlation between the solution phase stability of the dimeric proteins and their gas-phase dissociation pattern. Additionally, in the collisionally activated decomposition spectra diffuse ion signals are observed, which are attributed to monomer ions formed via slow decay of the collisionally activated dimer ions inside the reflectron time-of-flight. Although, the formation of these diffuse metastable ions may complicate the interpretation of collisionally activated decomposition mass spectra, especially when studying noncovalent protein complexes, a simple mathematical equation may be used to reveal their origin and pathway of formation.

Growth temperature regulation of host-specific modifications of rhizobial lipo-chitin oligosaccharides: the function of nodX is temperature regulated.

Lipo-chitin oligosaccharides (LCOs) are usually produced and isolated for structural analysis from bacteria cultured under laboratory rather than field conditions. We have studied the influence of bacterial growth temperature on the LCO structures produced by different Rhizobium leguminosarum strains, using thin-layer chromatographic, high-performance liquid chromatographic, and mass spectrometric analyses. Wild-type R. leguminosarum bv. viciae A1 was shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C, indicating that the activity of nodX (a gene encoding an LCO O-acetyl transferase) is temperature dependent. Interestingly, symbiotic resistance genes sym1 and sym2 found in primitive pea cultivars are also temperature sensitive, only being active at low temperatures, at which they block nodulation by R. leguminosarum bv. viciae strains lacking nodX. We therefore propose that the gene-for-gene relationship between plant and bacterium has a temperature-sensitive mechanism as an adaptation to environmental conditions. An R. leguminosarum bv. trifolii strain was also shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C. The major components synthesized by the two strains are produced at both temperatures but in different relative amounts, while some minor components are only produced at one of the two temperatures.

Characterization of glyoxalase I (E. coli)-inhibitor interactions by electrospray time-of-flight mass spectrometry and enzyme kinetic analysis.

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-(2-[3-(hexyloxy)benzoyl]-vinyl)glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.