Prediction of clozapine metabolism by on-line electrochemistry/liquid chromatography/mass spectrometry

Combining electrochemical conversion, liquid chromatography and electrospray ionization mass spectrometry (EC/LC/ESI-MS) on-line allows the rapid identification of possible oxidation products of clozapine (CLZ) in the absence and in the presence of glutathione. CLZ is, depending on the applied potential, oxidized to various products in an electrochemical flow-through cell using a porous glassy carbon working electrode. Several hydroxylated and demethylated species are detected on-line using LC/MS. While hydroxy-CLZ is most abundant at a potential of 400 mV, demethylation occurs more readily at higher potentials (at around 700 mV versus Pd/H₂ reference). In the presence of glutathione (GSH), various isomeric glutathione adducts and respective products of further oxidation can be identified. The thioadducts are characterized by tandem MS. Mono-GSH and bis-GSH derivatives can be seen in the chromatograms. The results correlate well with the cyclic voltammetric profile of CLZ. The data are relevant from a pharmacological point of view, since similar metabolites (phases I and II) have been reported in the literature. The EC/LC/MS and EC/MS methods should be valuable tools that can be used to anticipate and understand the metabolization patterns of molecules of pharmacological interest and to point out reactive intermediates.     

New designer drug 1-(3-trifluoromethylphenyl) piperazine (TFMPP): gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry studies on its phase I and II metabolism and on its toxicological detection in rat urine

Studies are described on the phase I and II metabolism and the toxicological analysis of the piperazine-derived designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP) in rat urine using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). The identified metabolites indicated that TFMPP was extensively metabolized, mainly by hydroxylation of the aromatic ring and by degradation of the piperazine moiety to N-(3-trifluoromethylphenyl)ethylenediamine, N-(hydroxy-3-trifluoromethylphenyl)ethylenediamine, 3-trifluoromethylaniline, and hydroxy-3-trifluoromethylaniline. Phase II reactions included glucuronidation, sulfatation and acetylation of phase I metabolites. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of TFMPP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of TFMPP in human urine.     

Three-step drug extraction from a single sub-millimeter segment of hair and nail to determine the exact day of drug intake

Hair and nails are often used to prove drug intake over several months. However, it is impossible to determine the day of drug intake by conventional segmental analysis of bulk samples. To improve this segmental analysis, we prepared accurate 0.4-mm hair and 0.2-mm nail segments, which correspond to their respective growth rates of 1–2 days, using a tissue slicer. The aim of this study was to develop an efficient method to extract drugs from a single sub-millimeter segment of hair and nail. Hair and nails were collected from a subject who was administered a single dose of chlorpheniramine. Four drug extraction methods based on different principles such as sonication, microwaves, micropulverization, and alkaline dissolution were compared. Short-duration sonication followed by long-duration soaking served the aim. Drug extraction from a sub-millimeter segment was performed in three steps as follows: a segment was first washed, followed by sonication for 10 min soaking in the extraction solution for 24 h. The drug concentrations in the three extracts from each segment were quantified using high performance liquid chromatography-tandem mass spectrometry. Each concentration was displayed on a single hair strand and a single nail block so that the first, second, and third extracts corresponded to components on the surface, in the outer layer, and within the sample, respectively. The distribution of chlorpheniramine in a hair successfully reflected the intake history. This method can be used in the future to measure the detailed distribution of drugs in a single hair and nail.     

Analysis of the pharmacokinetics and metabolism of aloe‐emodin following intravenous and oral administrations in rats

Aloe‐emodin, a natural polyphenolic anthraquinone, has shown various beneficial bioactivities in vitro. The aim of this study was to investigate the pharmacokinetics and metabolism of aloe‐emodin. Aloe‐emodin was intravenously and orally administered to rats. The concentrations of aloe‐emodin and rhein, a metabolite of aloe‐emodin, were determined by HPLC method prior to and after hydrolysis with β‐glucuronidase and sulfatase/β‐glucuronidase. The results showed that the systemic exposures of aloe‐emodin and its metabolites were ranked as aloe‐emodin glucuronides (G) > rhein sulfates (S) > aloe‐emodin > rhein and rhein G when aloe‐emodin was given intravenously. In contrast, when aloe‐emodin was administered orally, the parent form of aloe‐emodin was not absorbed per se, and the systemic exposures of its metabolites were ranked as aloe‐emodin G > rhein G > rhein. In conclusion, the metabolites of aloe‐emodin are more important than the parent form for the bioactivities in vivo. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Applications of LC/MS in structure identifications of small molecules and proteins in drug discovery

With advancements in ionization methods and instrumentation, liquid chromatography/mass spectrometry (LC/MS) has become a powerful technology for the characterization of small molecules and proteins. This article will illustrate the role of LC/MS analysis in drug discovery process. Examples will be given on high-throughput analysis, structural analysis of trace level impurities in drug substances, identification of metabolites, and characterization of therapeutic protein products for process improvement. Some unique MS techniques will also be discussed to demonstrate their effectiveness in facilitating structural identifications.     

Quantification of lurasidone, an atypical antipsychotic drug, in rat plasma with high-performance liquid chromatography with tandem mass spectrometry

A liquid chromatography–tandem mass spectrometric (LC/MS/MS) method was developed for the determination of an atypical antipsychotic drug, lurasidone, in rat plasma. The method involves the addition of acetonitrile and ziprasidone (internal standard) solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on a column packed with octadecylsilica (5 μm, 2.0 × 50 mm) with 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phase and the detection was performed using tandem mass spectrometry by multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear (r = 0.9982) over the concentration range 0.002–1 μg/mL. The intra‐ and inter‐assay precisions were 1.7 and 8.6%, respectively. The accuracy range was from 90.3 to 101.8%. The lower limit of quantification was 2.0 ng/mL using 50 μL of rat plasma sample. The developed analytical method was successfully applied to the pharmacokinetic study of lurasidone in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Electrochemistry meets enzymes: instrumental on-line simulation of oxidative and conjugative metabolism reactions of toremifene

The metabolism of the selective estrogen receptor modulator toremifene was simulated in an on-line electrochemistry/enzyme reactor/liquid chromatography/mass spectrometry system. To simulate the oxidative phase I metabolism, toremifene was oxidized in an electrochemical (EC) flow-through cell at 1,500 mV vs. Pd/H₂ to its phase I metabolites, some of which are reactive quinoid species. In the presence of glutathione-S-transferase (GST), these quinoid compounds react with glutathione, which is also the common detoxification mechanism in the body. While reacting with glutathione, the chlorine atom is eliminated from the toremifene moiety. Due to higher conversion rates, GST supplied in continuous flow proved to be more efficient than using immobilized GST on magnetic microparticles. In the absence of GST, not all GSH adducts are formed, proving the necessity of a phase II enzyme to simulate the complete metabolic pathway of xenobiotics in an on-line EC/LC/MS system. Figure Mass voltammogram of toremifene     

Fenton’s reagent-mediated degradation of residual Kraft black liquor

In this work, the effect of Fento’s reagent on the degradation of residual Kraft black liquor was investigated. The effect of Fenton’s reagent on the black liquor degradation was dependent on the concentration of H₂O₂. At low concentrations (5 and 15 mM) of H₂O₂, Fenton’s reagent caused the degradation of phenolic groups (6.8 and 44.8%, respectively), the reduction of reaction medium pH (18.2%), and the polymerization of black liquor lignin. At a high concentration (60 mM) of H₂O₂, Fenton’s reagent induced an extensive degradation of lignin (95–100%) and discoloration of the black liquor. In the presence of traces of iron, the addition of H₂O₂ alone induced mainly lignin fragmentation. In conclusion, Fenton’s reagent and H₂O₂ alone can degrade residual Kraft black liquor under acidic conditions at room temperature.     

Imitation of drug metabolism in cell co-culture microcapsule model using a microfluidic chip platform coupled to mass spectrometry

In this work,a multi-functional analysis platform by coupling a microfluidic chip to a mass spectrometry(MS) detector was described.We constructed a three-dimensional tumor-endothelial co-culture model for simulating drug resistance during tumor treatment.On this specially designed integrated platform,the first step was to prepare heterogeneous cell-encapsulated alginate microcapsules for threedimensional co-culture,and the second step was to achieve on-line perfusion culture and continuous drug stimulation on chip.It facilitates cell proliferation analysis and the collection of metabolism medium.After micro solid phase extraction column(SPE) pretreatment,subsequent mass spectrometry could detect drug metabolism.The high activity of two kinds of cells(A549 and HUVEC) shows the biocompatibility of the platform.Paclitaxel was used as a model drug,the distinctions of drug absorption between the mono-culture group and co-culture group were clearly observed by electrospray ionization quadrupole time-of-flight mass spectrometry(ESI-Q-TOF MS).Therefore,the integrated platform has shown promise as a high throughput,low cost for cell metabolism research and drug screening processes.     

HPLC analysis of in vivo intestinal absorption and oxidative metabolism of salicylic acid in the rat

In vivo absorption and oxidative metabolism of salicylic acid in rat small intestine was studied by luminal perfusion experiment. Perfusion through the lumen of proximal jejunum with isotonic medium containing 250 μm sodium salicylate was carried out. Absorption of salicylate was measured by a validated HPLC‐DAD method which was evaluated for a number of validation characteristics (specificity, repeatability and intermediate precision, limit of detection, limit of quantification, linearity and accuracy). The method was linear over the concentration range 0.5–50 μg/mL. After liquid–liquid extraction of the perfusion samples oxidative biotransformation of salicylate was also investigated by HPLC‐MS. The method was linear over the concentration range 0.25–5.0 μg/mL. Two hydroxylated metabolites of salicylic acid (2,5‐dihydroxybenzoic acid and 2,3‐dihydroxybenzoic acid) were detected and identified. The mean recovery of extraction was 72.4% for 2,3‐DHB, 72.5% for 2,5‐DHB and 50.1% for salicylic acid, respectively. The methods were successfully applied to investigate jejunal absorption and oxidative metabolism of sodium salicylate in experimental animals. The methods provide analytical background for further metabolic studies of salycilates under modified physiological conditions.     

Distribution of metoprolol, tramadol, and midazolam in human autopsy material

In this study it was possible to measure the distribution of metoprolol, tramadol, and midazolam in human directly in several compartments. In the legal medicine autopsy material is normally investigated to find out the cause of death. But the results of corresponding toxicology measurements often involve more information. With screening methods drugs were detected without connection to the cause of death. The deceased had either a continual therapeutic treatment, a treatment during an operation, or an unsuccessful urgent therapy. A liquid/liquid extraction and a LC/MS/MS method were developed for the determination of the drug concentrations. Different autopsy materials of about 120 cases were investigated. Most frequently the drugs metoprolol, tramadol, and midazolam could be proved and determined simultaneously. Metoprolol was found in seven cases, tramadol in seven cases and midazolam in thirteen cases. The dosage of the drugs was unknown. Therefore and because of the low number of cases statistic calculations were not meaningful and an individual case study was necessary. In all cases with oral metoprolol application the patients probably took a normal customary continuous dosage. The concentrations of tramadol in blood were in the toxic range in three cases. The distribution of tramadol in the compartments was independent of the dosage. The time between oral intake of metoprolol or tramadol and death was unknown. With the distribution pattern of metoprolol in the compartments it was possible to estimate the duration between drug intake and death. In most cases midazolam was given intravenously during an operation or an unsuccessful urgent therapy. Sometimes the time between dosage and death was documented. The duration between application of the drug and death played the crucial role for the distribution of midazolam in the compartments. Measurements of drug concentrations in human autopsy material deepen the knowledge of the respective drugs' pharmacokinetics.     

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.     

Identification of the major urinary metabolites in man of seven synthetic cannabinoids of the aminoalkylindole type present as adulterants in ‘herbal mixtures’ using LC‐MS/MS techniques

Herbal mixtures, such as ‘Spice’, containing cannabimimetic compounds are easily available on the Internet and have become increasingly popular among people having to undergo urine drug testing, as these compounds are not detected by current immunochemical tests. For analysis of urine samples, knowledge of the main metabolites is necessary as the unchanged compounds are usually not found in urine after consumption. In this paper, the identification of the major metabolites of the currently most common seven synthetic cannabinoids is presented. Urine samples from patients of psychiatric facilities known to have consumed synthetic cannabinoids were screened by LC‐MS/MS and HR‐MS/MS techniques, and the major metabolites for each of the following synthetic cannabinoids were identified by their enhanced product ion spectra and accurate mass measurement: JWH‐018, JWH‐073, JWH‐081, JWH‐122, JWH‐210, JWH‐250 and RCS‐4. The major metabolic pathway is monohydroxylation either at the N‐alkyl side chain, the naphthyl moiety or the indole moiety. In addition, metabolites with carboxylated alkyl chains were identified for some of the compounds. These results facilitate the design of urine screening methods for detecting consumption of synthetic cannabinoids. Copyright © 2012 John Wiley & Sons, Ltd.     

The biotransformation of oleuropein in rats

A highly sensitive and specific LC‐MS/MS method was developed to investigate the in vivo bio‐transformation of oleuropein in rat. Rat feces and urine samples collected after oral administration were determined by liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat feces and urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.50 mL/min on an RP‐C₁₈ column with a total run time of 31 min. This method was successfully applied to simultaneous determination of oleuropein and its metabolites in rat feces and urine. De‐glucosylation, hydrolysis, oxygenation and methylation were found to comprise the major metabolic pathway of oleuropein in rat gastrointestinal tract and three metabolites were absorbed into the blood circulatory system within 24 h after oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolic routes along digestive system of licorice: multicomponent sequential metabolism method in rat

This study was conducted to establish the multicomponent sequential metabolism (MSM) method based on comparative analysis along the digestive system following oral administration of licorice (Glycyrrhiza uralensis Fisch., leguminosae), a traditional Chinese medicine widely used for harmonizing other ingredients in a formulae. The licorice water extract (LWE) dissolved in Krebs–Ringer buffer solution (1 g/mL) was used to carry out the experiments and the comparative analysis was performed using HPLC and LC‐MS/MS methods. In vitro incubation, in situ closed‐loop and in vivo blood sampling were used to measure the LWE metabolic profile along the digestive system. The incubation experiment showed that the LWE was basically stable in digestive juice. A comparative analysis presented the metabolic profile of each prototype and its corresponding metabolites then. Liver was the major metabolic organ for LWE, and the metabolism by the intestinal flora and gut wall was also an important part of the process. The MSM method was practical and could be a potential method to describe the metabolic routes of multiple components before absorption into the systemic blood stream. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of metabolites of tanshinone IIA in rats by liquid chromatography/tandem mass spectrometry

The metabolism of tanshinone IIA was studied in rats after a single-dose intravenous administration. In the present study, 12 metabolites of tanshinone IIA were identified in rat bile, urine and feces with two LC gradients using LC-MS/MS. Seven phase I metabolites and five phase II metabolites of tanshinone IIA were characterized and their molecular structures proposed on the basis of the characteristics of their precursor ions, product ions and chromatographic retention time. The seven phase I metabolites were formed, through two main metabolic routes, which were hydroxylation and dehydrogenation metabolism. M1, M4, M5 and M6 were supposedly tanshinone IIB, hydroxytanshinone IIA, przewaquinone A and dehydrotanshinone IIA, respectively, by comparing their HPLC retention times and mass spectral patterns with those of the standard compounds. The five phase II metabolites identified in this research were all glucuronide conjugates, all of which showed a neutral loss of 176 Da. M9 and M12 were more abundant than other identified metabolites in the bile, which was the main excretion path of tanshinone IIA and the metabolites. M12 was the main metabolite of tanshinone IIA. M9 and M12 were proposed to be the glucuronide conjugates of two different semiquinones and these semiquinones were the hydrogenation products of dehydrotanshinone IIA and tanshinone IIA, respectively. This hydrogenized reaction may be catalyzed by the NAD(P)H: quinone acceptor oxidoreductase (NQO). The biotransformation pathways of tanshinone IIA were proposed on the basis of this research.     

Designer drug 2,4,5-trimethoxyamphetamine (TMA-2): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric techniques

Studies are described on the metabolism and the toxicological detection of the amphetamine-derived designer drug 2,4,5-trimethoxyamphetamine (TMA-2) in rat urine using gas chromatographic/mass spectrometric (GC/MS) techniques. The identified metabolites indicated that TMA-2 was metabolized by oxidative deamination to the corresponding ketone followed by reduction to the corresponding alcohol, O-demethylation followed by oxidative deamination, and finally O,O-bis-demethylation. All metabolites carrying hydroxy groups were found to be partly excreted in urine as glucuronides and/or sulfates. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation allowed the detection, in rat urine, of an intake of TMA-2 that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure in human urine should be suitable as proof of an intake of TMA-2.     

Liquid chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study

A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed for the determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile and internal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandem mass spectrometry by the multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear over the concentration range from 0.3 to 1000 ng/mL. The lower limit of quantification was 0.3 ng/mL using 50 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively. The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

An introduction to hybrid ion trap/time‐of‐flight mass spectrometry coupled with liquid chromatography applied to drug metabolism studies

Metabolism studies play an important role at various stages of drug discovery and development. Liquid chromatography combined with mass spectrometry (LC/MS) has become a most powerful and widely used analytical tool for identifying drug metabolites. The suitability of different types of mass spectrometers for metabolite profiling differs widely, and therefore, the data quality and reliability of the results also depend on which instrumentation is used. As one of the latest LC/MS instrumentation designs, hybrid ion trap/time‐of‐flight MS coupled with LC (LC‐IT‐TOF‐MS) has successfully integrated ease of operation, compatibility with LC flow rates and data‐dependent MSⁿ with high mass accuracy and mass resolving power. The MSⁿ and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of uncommon or unexpected metabolites. These features make the LC‐IT‐TOF‐MS a very powerful analytical tool for metabolite identification. This paper begins with a brief introduction to some basic principles and main properties of a hybrid IT‐TOF instrument. Then, a general workflow for metabolite profiling using LC‐IT‐TOF‐MS, starting from sample collection and preparation to final identification of the metabolite structures, is discussed in detail. The data extraction and mining techniques to find and confirm metabolites are discussed and illustrated with some examples. This paper is directed to readers with no prior experience with LC‐IT‐TOF‐MS and will provide a broad understanding of the development and utility of this instrument for drug metabolism studies. Copyright © 2012 John Wiley & Sons, Ltd.