Studies on the metabolism of mitragynine,the main alkaloid of the herbal drug Kratom,in rat and human urine using liquid chromatography‐linear ion trap mass spectrometry

Mitragynine (MG) is an indole alkaloid of the Thai medicinal plant Mitragyna speciosa (Kratom in Thai) and reported to have opioid agonistic properties. Because of its stimulant and euphoric effects, Kratom is used as a herbal drug of abuse. The aim of the presented study is to identify the phase I and II metabolites of MG in rat and human urine after solid‐phase extraction (SPE) using liquid chromatography‐linear ion trap mass spectrometry providing detailed structure information in the MSⁿ mode particularly with high resolution. The seven identified phase I metabolites indicated that MG was metabolized by hydrolysis of the methylester in position 16, O‐demethylation of the 9‐methoxy group and of the 17‐methoxy group, followed, via the intermediate aldehydes, by oxidation to carboxylic acids or reduction to alcohols and combinations of some steps. In rats, four metabolites were additionally conjugated to glucuronides and one to sulfate, but in humans, three metabolites to glucuronides and three to sulfates. Copyright © 2009 John Wiley & Sons, Ltd.     

Fast and sensitive liquid chromatography/electrospray mass spectrometry method to study ocular penetration of EDL‐155, a novel antitumor agent for retinoblastoma in rats

Our group has used the tetrahydroisoquinoline derivative EDL‐155 to treat glioblastoma in animal models and it is currently being evaluated in the treatment of ocular cancers. The purpose of this study was to develop a rapid and sensitive liquid chromatography and tandem mass spectrometry (LC‐MS/MS) method to study the plasma and vitreous humor disposition of EDL‐155 in rats. Animals received a single periocular injection of EDL‐155 (20 mg/kg). Animals were sacrificed at specified times (5, 60, 120, 240 and 360 min) and plasma and vitreous humor samples were obtained. EDL‐155 was isolated by protein precipitation and the extracts were analyzed by reversed‐phase high‐pressure liquid chromatography (HPLC) with MS/MS detection. A structurally similar analog was used as internal standard (IS). The chromatographic run time was 3.5 min per injection. The mass spectrometer was operated in positive‐ion, multiple reaction monitoring (MRM) mode. The mass transitions monitored were m/z332.2 → 167.2 (EDL‐155) and m/z391.2 → 200.2 (IS). The lower limit of quantification (LLOQ) was 0.1 ng/ml in both vitreous humor and plasma. The method was validated for selectivity, linearity, accuracy and precision in rat vitreous humor and partially validated for accuracy and precision in rat plasma. The ion suppression, recovery and stability of the analyte in the biological matrix were also tested. The assay was rapid, sensitive and robust enough to support EDL‐155 ocular penetration studies in a rodent model of intraocular cancer. Application of this method revealed that EDL‐155 was rapidly passed into the vitreous humor following periocular administration. Further, vitreous humor exposure exceeded systemic exposure by approximately sevenfold. High local concentrations coupled with minimal systemic exposure supports further testing of EDL‐155 as localized therapy for intraocular cancers. Copyright © 2009 John Wiley & Sons, Ltd.     

Self‐Propelled Polymer Nanomotors

Polymer conformations: The picture shows instantaneous conformations of product molecules produced in a catalytic reaction at the head of a polymer chain. The extended (left) and collapsed (right) polymers correspond to chains in good and poor solvent conditions, respectively. The product molecule concentration gradient leads to a directed force on the polymer, so that it functions as a self‐propelled nanomotor.

     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

Simultaneous extraction of tocotrienols and tocopherols from cereals using pressurized liquid extraction prior to LC determination

Tocopherols and tocotrienols have been simultaneously determined in food samples using a rapid and simple analytical method including pressurized liquid extraction (PLE) and LC with electrochemical detection. Separation was carried out on a Phenomenex Synergi 4 μm Hydro‐RP 80A column, using a solution of 2.5 mM acetic acid/sodium acetate in methanol/water (99:1, v/v) as mobile phase at a flow rate of 1.0 mL/min. Column temperature was maintained at 30°C. Detection was performed by coulometric detection at 500 mV except for (β+γ)‐tocotrienol, in wheat and rye samples, which was at +350 mV. A palm oil containing a relatively large amount of γ‐tocotrienol and lower concentrations of α‐ and δ‐tocotrienols and α‐ and γ‐tocopherols was used to provide reference retention times for the tocotrienols. Analyte quantification was performed using the external standard method. The calibration equations of tocopherols were used to quantify both tocopherols and their corresponding tocotrienols. The extraction recoveries obtained using the optimized PLE conditions were in the 80–114% range, with RSDs lower than 15%. The method was successfully applied to the determination of tocotrienols and tocopherols in cereal (wheat, rye, barley, maize and oat) and palm oil samples.     

LC‐MS of streptomycin following desalting of a nonvolatile mobile phase and pH gradient

Streptomycin (SM) is composed of streptidine, streptose and N‐methyl glucosamine sugar moieties. For the determination of SM and its related substances, an ion‐pair LC‐UV detection method using a Supelcosil LC‐ABZ column was developed previously. While analyzing commercial samples, several unknown compounds were detected. Most of these compounds are not yet characterized. In this study, the above LC method was coupled to MS for impurity profiling in a selected commercial sample. However, it could not be directly coupled to MS due to the presence of the nonvolatile salt, buffer and ion‐pair reagent in the mobile phase. So, for structural characterization, each peak eluted from the nonvolatile eluent system was collected and transferred to MS after the desalting process. In total, 16 compounds were studied, 15 compounds including 12 unknowns could be identified.     

Determination of selected β-receptor antagonists in biological samples by solid-phase extraction with cholesterolic phase and LC/MS

A new method is presented for the determination of five selected β-receptor antagonists by HPLC, which emphasizes sample preparation via retention on a new type of silica gel sorbent used for solid-phase extraction (SPE). Sorbents of this type were obtained by the chemical modification of silica gels of various porosities by cholesterol ligands. The cholesterol-based packing material was investigated by spectroscopic methods and elemental analysis. The recoveries obtained with the extraction procedure were optimum over a relatively broad sample pH range (3.08–7.50). Analytical factors such as the sample loading, the washing step and elution conditions, the concentration of β-receptor antagonists to be extracted, and the type of sorbent were found to play significant roles in the sample preparation procedure and would therefore need to be controlled to achieve optimum recoveries of the analytes. Under optimum conditions, the recoveries of nadolol, acebutolol, esmolol, oxprenolol and propranolol from spiked buffers, blood and urine were reproducible and dependent on the polarity or hydrophilicity of the compounds. The above analytes were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV and ESI-ion trap mass spectrometry (MS) detection. The described method was found to be suitable for the routine measurement of compounds that are both polar and basic, and can be applied for the analysis of biological samples such as urine and blood in clinical, toxicological or forensic laboratories. The recovery measurements were performed on spiked human urine and serum, and on real samples of mouse blood serum.     

The design of a multi-dimensional LC-SPE-NMR system (LC(2)-SPE-NMR) for complex mixture analysis

In this communication, we describe the design of an online multi-chromatographic approach to the routine NMR analyses of low-level components ( approximately 0.1%) in complex mixtures. The technique, termed LC(2)-SPE-NMR, optimally combines multi-dimensional liquid chromatography with SPE technology for isolating, enriching and delivering trace analytes to the NMR probe. The fully automated LC(2)-SPE-NMR system allows for maximal loading capacity (in the first, preparative LC dimension), close to optimal peak resolution (in the second, analytical LC dimension) and enhanced sample concentration (through SPE). Using this system, it is feasible to conveniently conduct a wide range of NMR experiments on, for example, drug impurities at the low microgram per milliliter level, even for components poorly resolved in the first dimension. Such a sensitivity gain significantly elevates the analytical power of online NMR technology in terms of the level at which substances of pharmaceutical significance can be structurally characterized.     

Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry

A method for the determination of ethyl glucuronide (EtG) in hair samples, using liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS), was developed and validated. The treatment of hair samples was as follows: to 100 mg of washed (dichloromethane followed by methanol, 1 ml each) and cut (1-2 mm) material, 700 microl of water, 20 microl of internal standard solution (pentadeuterated EtG, D(5)-EtG, 500 microg/l) and 20 microl of methanol were added. Samples were incubated at 25 degrees C overnight and then ultrasonicated for 2 h. Finally, 8 microl of the centrifuged solution (13,000 rpm) were analyzed by LC/ESI-MS/MS in negative ion mode. The surviving ions of EtG and D(5)-EtG were monitored together with the following MRM transitions: m/z 221 --> 75, m/z 221 --> 85 (EtG) and m/z 226 --> 75, m/z 226 --> 85 (D(5)-EtG). The method exhibited a mean correlation coefficient better than 0.9998 over the dynamic range (3-2000 pg/mg). The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 3 and 2 pg/mg respectively. The intra- and interday precision and accuracy were studied at four different concentration levels (3, 5, 56 and 160 pg/mg) and were always better than 7% (n = 5). Matrix effects did not exceed 20%. The method was applied to several hair samples taken from autopsies of known alcoholics, from patients in withdrawal treatment, from social drinkers, from adult teetotalers and from children not exposed to ethanol, with EtG concentrations globally ranging from < or =2 to 4180 pg/mg.     

Incorporation of a nanosplitter interface into an LC-MS-RD system to facilitate drug metabolism studies

In the work reported here, a novel interface, the nanosplitter, is incorporated into the drug metabolism laboratory in order to enhance the analytical capabilities of detecting and identifying drug-related metabolites to support drug metabolism studies during the drug development process. When an existing LC-MS-radiometric detector (RD) system is coupled with this nanosplitter, the system becomes capable of performing dynamic microspray under a typical analytical LC method. With the superior MS sensitivity offered by this system, most of the analytical LC methods developed for metabolite profiling can then be easily adopted for metabolite identification work. The improvement of these analytical capabilities can streamline the entire process of the drug metabolism study. In the experiments presented here, the nanosplitter interface coupled with analytical HPLC systems (e.g. 4.6 x 250 mm column @ 1 ml/min) demonstrated significant increases in MS signal (2x to 40x peak area) when compared to the standard LC-MS interface for both in vitro and in vivo metabolism studies. Furthermore, this signal gain facilitated the MS detection of additional metabolites (observed in the radiometric trace) that were below the MS level of detection when using the standard interface.