Chromatography-linear Ion Trap Mass Research Articles

Discrimination between the Triglyceride Form and the Ethyl Ester Form of Fish Oil Using Chromatography-Mass Spectrometry.

Although the triglyceride form is the natural form of fish oil found in fish, the ethyl ester form of fish oil, which is used during processing to save costs, is also present on the market. In this study, fatty acids and lipids were determined using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-linear ion trap mass spectrometry (LC-LIT/MS), respectively, according to developed methods. The identification of fatty acids was based on the mass spectral characteristics and equivalent chain lengths. However, the fatty acid contents of both forms of fish oils are quite similar. The application of the LC-LIT/MS method for the structural characterization of triacylglycerols (TAGs) and the mechanism of LIT/MS fragmentation are also discussed. Neutral losses of CH2=CH2 (m/z 28) and CH3CH2OH (m/z 46), which are LIT/MS characteristics of ethyl ester from fish oil, were found for the first time. The triglyceride form of fish oils was easily and accurately identified using fingerprint chromatography. In conclusion, lipid analysis combined with LC-LIT/MS showed an improved capability to distinguish between types of fish oil.

Development of a portable gas chromatography linear ion trap mass spectrometer (GC-LIT-MS) for VOCs analysis in water

The detection of volatile organic compounds (VOCs) in water faces challenges due to the presence of complex matrix interferences and low concentration. This paper presented a portable gas chromatography linear ion trap mass spectrometer (GC-LIT-MS) coupled with dynamic headspace sampling (DHS) system for sensitive and rapid on-site analysis of VOCs in water. The instrument weighs 18 kg and has compact dimensions of 43 cm × 35 cm x 20 cm, offering portability and convenience. A low thermal mass (LTM) chromatographic column, utilizing resistive heating, enables rapid heating and cooling within the runtime of GC. The linear ion trap, for the first time, has been integrated as a mass analyser in portable gas chromatography-mass spectrometer (GC-MS). Its ability to store a greater number of ions within the trap expands the dynamic range of the instrument. The influence of dynamic headspace injection conditions on the purging efficiency of VOCs was investigated and the equilibrium temperature was optimized. The GC-LIT-MS method was demonstrated to be capable to identify and separate 55 VOCs in water within 4 min, with most of them exhibiting detection limits of less than 1 μg L−1. The calibration of 4 VOCs with concentrations ranged from 2 parts per million (ppm) to 200 ppm showed excellent linearity (R2 > 0.99) and dynamic range more than five orders of magnitude. Resolution superior to unit mass was achieved within the range of 15 AMU to 550 AMU.

Phenolic Composition and Antioxidant Activity of Purple Sweet Potato (Ipomoea batatas (L.) Lam.): Varietal Comparisons and Physical Distribution.

The outer layer of purple sweet potato is removed during processing; however, this layer serves as a potential source of phenolics, especially anthocyanins. Herein, the phenolic composition and antioxidant activity were determined for the inner and outer layers of five purple sweet potato cultivars (‘Sinjami’, ‘Jami’, ‘Danjami’, ‘Yeonjami’, and ‘Borami’) harvested in Korea. Anthocyanins were identified using ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometer (UHPLC-(ESI)-qTOF-MS) and ultra-high-performance liquid chromatography-linear ion trap mass spectrometer (UHPLC-Ion trap-MS), and their composition was quantified using HPLC-coupled with diode array detector (DAD). Non-anthocyanin phenolic compounds (phenolic acids and flavonols) were quantified using UHPLC-(ESI)-triple quadrupole (QqQ). A total of 20 anthocyanins, including non-acylated or acylated peonidin, cyanidin, and pelargonidin glycosides, were identified. Peonidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside was the major anthocyanin, with the highest level in the ‘Sinjami’ cultivar (outer; 12,366 mg/kg DW, inner; 14,832 mg/kg DW). Additionally, 12 phenolic acids and 6 flavonols (quercetin derivatives) were identified, with the outer layers of all cultivars displaying higher total levels than the inner layers. ‘Sinjami’ and ‘Jami’ had higher phenolic acid and quercetin derivative content and antioxidant activities than the other three cultivars (p < 0.05). Thus, the outer layers of ‘Sinjami’ and ‘Jami’ cultivars could be potential sources of anthocyanins and other phenolics.

Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in Tac1−/− mouse spinal cords reveals significant lower concentration of opioid peptides

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1−/− spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1−/− mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1−/− spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p<0.0001). No significant concentration differences were observed in mouse Tac1−/− spinal cords for Met-Enk and CGRP. The analysis of Tac1−/− mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Simultaneous qualitative and quantitative determination of seven anticoagulant rodenticides in whole blood and urine samples using on-line solid phase extraction with liquid chromatography-linear ion trap mass spectrometry

A new and sensitive analytical method has been developed for the simultaneous determination of seven anticoagulant rodenticides in whole blood and urine samples by liquid chromatography-linear ion trap mass spectrometry (LC-LIT/MS) with on-line solid phase extraction (on-line SPE). The samples were treated with acetonitrile, followed by dilution, centrifugation, and filtration. The resulting solution was injected into the LC system directly and processed by on-line SPE column for enrichment and purification. Separation was performed on a C18 column with mixed mobile phases of methanol and 0.02 mol/L ammonium acetate aqueous solution for gradient elution. The analytes were detected by the mass spectrometer with electrospray ionization (ESI) in negative mode. MS2 full scan signals of the target parent ions within the locked retention time window were recorded. Self-built database searching was performed for qualitative confirmation, and MS2 fragment ions with high sensitivity and specificity were selected for quantification. Simultaneous qualitative and quantitative analyses of the seven rodenticides were achieved in this way. Good linearities were obtained within the investigated mass concentration ranges of the seven rodenticides, with r2 ≥ 0.9958 in blood and r2 ≥ 0.9946 in urine. The LODs varied from 0.02 ng/mL to 1.00 ng/mL, and the LOQs varied from 0.10 ng/mL to 4.00 ng/mL. The recoveries at three spiked levels in blood and urine samples ranged from 81.0% to 113.9%, with RSDs of 0.1%-6.2% (n = 6). The developed method is simple, sensitive, and can be used for the rapid detection and accurate quantification of the seven anticoagulant rodenticides in whole blood and urine samples.

Identification of apolipoprotein C-I as a potential Wilms' tumor marker after excluding inflammatory factors.

Wilms’ tumor is one of the most common malignant tumors observed in children, and its early diagnosis is important for late-stage treatment and prognosis. We previously screened and identified protein markers for Wilms’ tumor; however, these markers lacked specificity, and some were associated with inflammation. In the current study, serum samples from children with Wilms’ tumors were compared with those of healthy controls and patients with systemic inflammatory response syndrome (SIRS). After exclusion of factors associated with inflammation, specific protein markers for Wilms’ tumors were identified. After comparing the protein peak values obtained from all three groups, a protein with a m/z of 6438 Da was specified. Purification and identification of the target protein using high-pressure liquid chromatography (HPLC) and two-dimensional liquid chromatography-linearion trap mass spectrometry(2D-LC-LTQ-MS) mass spectrometry, respectively, revealed that it was apolipoprotein C-I (APO C-I). Thus, APO C-I is a specific protein marker for Wilms’ tumor.

Determination of eight pesticides of varying polarity in surface waters using solid phase extraction with multiwalled carbon nanotubes and liquid chromatography-linear ion trap mass spectrometry

We describe a MWCNT-based method for the solid-phase extraction of eight pesticides from environmental water samples. The analytes are extracted from 100 mL samples at pH 5.0 (containing 5 mmol L−1 of KCl) by passing the solution through a column filled with 20 mg of multiwalled carbon nanotubes. Following elution, the pesticides were determined by LC and electrospray ionization hybrid quadrupole linear ion trap MS. Two selected reaction monitoring transitions were monitored per compound, the most intense one being used for quantification and the second one for confirmation. In addition, an information-dependent acquisition experiment was performed for unequivocal confirmation of positive findings. Matrix effect was not found in real waters and therefore the quantitation was carried out with calibration graphs built with solvent based standards. Except for cymoxanil, the detection and quantitation limits in surface waters are in the range from 0.3 to 9.5 ng L−1 and 1.6 to 45.2 ng L−1, respectively. Recoveries from spiked ultrapure water are ~100 %, except for the most polar pesticides methomyl and cymoxanil. The same behavior is found for real water samples (except for phosalone). The relative standard deviation is <10 % in all cases.

Determination of N-methyl carbamate pesticides in diet samples by high performance liquid chromatography-linear ion trap mass spectrometry with gel permeation chromatography cleanup

A method for the determination of N-methyl carbamate (NMC) pesticides in diet samples was developed by high performance liquid chromatography-linear ion trap mass spectrometry (HPLC-LIT-MS). The samples with the isotope internal standard were ultrasonically extracted with acetonitrile saturated with cyclohexane, and then cleaned-up by passing through gel permeation chromatographic (GPC) column. The LC separation was performed on a CAPCELL PAK CR column (100 mm x 2.1 mm, 5 microm; SCX-C18 (1 : 4)) using acetonitrile and 5 mmol/L ammonium formate/0.1% (v/v) formic acid aqueous solution as mobile phases (gradient elution) at a flow rate of 0.2 mL/min. The ionization of the analytes was performed by positive electrospray mode. Selective reaction monitoring (SRM) was used for the monitoring of MS3 transitions for each compound. The internal standard was used for quantitation. The average recoveries were in the range of 60.4% - 114%, and the relative standard deviations (RSDs) were all not more than 16.2%. The limits of detection (LODs) were 0.001-0.010 mg/kg. The method was successfully applied to the determination of 15 compounds of interest in nine foodstuffs from the fourth Chinese Total Diet Study (TDS) performed in 2007, and aldicarb and carbofuran were found in several samples.

A qualitative/quantitative approach for the detection of 37 tryptamine-derived designer drugs, 5 β-carbolines, ibogaine, and yohimbine in human urine and plasma using standard urine screening and multi-analyte approaches

The first synthetic tryptamines have entered the designer drug market in the late 1990s and were distributed as psychedelic recreational drugs. In the meantime, several analogs have been brought onto the market indicating a growing interest in this drug class. So far, only scarce analytical data were available on the detectability of tryptamines in human biosamples. Therefore, the aim of the presented study was the development and full validation of a method for their detection in human urine and plasma and their quantification in human plasma. The liquid chromatography-linear ion trap mass spectrometry method presented covered 37 tryptamines as well as five β-carbolines, ibogaine, and yohimbine. Compounds were analyzed after protein precipitation of urine or fast liquid-liquid extraction of plasma using an LXQ linear ion trap coupled to an Accela ultra ultra high-performance liquid chromatography system. Data mining was performed via information-dependent acquisition or targeted product ion scan mode with positive electrospray ionization. The assay was selective for all tested substances with limits of detection in urine between 10 and 100ng/mL and in plasma between 1 and 100ng/mL. A validated quantification in plasma according to international recommendation could be demonstrated for 33 out of 44 analytes.

Simultaneous determination of 19 triazine pesticides and degradation products in processed cereal samples from Chinese total diet study by isotope dilution–high performance liquid chromatography–linear ion trap mass spectrometry

A selective and sensitive isotope dilution–high performance liquid chromatography–linear ion trap mass spectrometry (Isotope Dilution–HPLC–LIT-MS3) method was developed for the simultaneous determination of 19 triazine pesticides and their degradation products in processed cereal samples from Chinese total diet study (TDS). The method integrated the addition of isotope internal standards, liquid–liquid extraction (LLE), clean-up with MCX solid-phase extraction (SPE) cartridges and HPLC–LIT-MS3 analysis with selected reaction monitoring (SRM) mode. Matrix-matched calibration curves showed good linearity (R2≥0.9940) verified by applying the Mandel's fitting test (p>0.087) performed at the 95% confidence level. Decision limits (CCαs) and detection capabilities (CCβs) of the 19 triazine pesticides and their degradation products fell in the ranges of 0.0020–0.4200μgkg−1 and 0.0024–0.4500μgkg−1, respectively. Recoveries ranged from 70.1% to 112.8%, with the relative standard deviations (RSDs) ranging from 1.5% to 13.5%. Furthermore, the proposed method was applied to analyzing the proposed cereal samples from the fourth Chinese TDS. Eleven triazines were detected in six cereal samples with the concentrations ranging from 0.013 to 0.987μg kg−1. This method can also be used for the further determination of the triazines in other food group composites, and ultimately served as a methodological foundation for assessing the triazines in the average Chinese diet in the general population.

2-Methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling "research chemicals." The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors' well-established gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MS(n)) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography-high-resolution linear ion trap mass spectrometry (LC-HR-MS(n)) and the phase II metabolites by LC-HR-MS(n). The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user's dose, 2-MPA and its metabolites were identified in rat urine using the authors' GC-MS and the LC-MS(n) screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.

Glyphosate Exposure in a Farmer’s Family

We tested the presence of glyphosate in the urines of a farmer who sprayed a glyphosate based herbicide on his land, and in his family, as his children were born with birth defects that could be due to or promoted by pesticides. Glyphosate residues were measured in urines a day before, during, and two days after spraying, by liquid chromatography-linear ion trap mass spectrometry. Glyphosate reached a peak of 9.5 µg/L in the farmer after spraying, and 2 µg/L were found in him and in one of his children living at a distance from the field, two days after the pulverization. Oral or dermal absorptions could explain the differential pesticide excretions, even in family members at a distance from the fields. A more detailed following of agricultural practices and family exposures should be advocated together with information and recommendations.

Qualitative studies on the metabolism and the toxicological detection of the fentanyl-derived designer drugs 3-methylfentanyl and isofentanyl in rats using liquid chromatography–linear ion trap–mass spectrometry (LC-MSn)

The opioid 3-methylfentanyl, a designer drug of the fentanyl type, was scheduled by the Controlled Substance Act due to its high potency and abuse potential. To overcome this regulation, isofentanyl, another designer fentanyl, was synthesized in a clandestine laboratory and seized by the German police. The aims of the presented study were to identify the phase I and phase II metabolites of 3-methylfentanyl and isofentanyl in rat urine, to identify the cytochrome P450 (CYP) isoenzymes involved in their initial metabolic steps, and, finally, to test their detectability in urine. Using liquid chromatography (LC)-linear ion trap-mass spectrometry (MS(n)), nine phase I and five phase II metabolites of 3-methylfentanyl and 11 phase I and four phase II metabolites of isofentanyl could be identified. The following metabolic steps could be postulated for both drugs: N-dealkylation followed by hydroxylation of the alkyl and aryl moiety, hydroxylation of the propanamide side chain followed by oxidation to the corresponding carboxylic acid, and, finally, hydroxylation of the benzyl moiety followed by methylation. In addition, N-oxidation of isofentanyl could also be observed. All hydroxy metabolites were partly excreted as glucuronides. Using recombinant human isoenzymes, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were found to be involved in the initial metabolic steps. Our LC-MS(n) screening approach allowed the detection of 0.01 mg/L of 3-methylfentanyl and isofentanyl in spiked urine. However, in urine of rats after the administration of suspected recreational doses, the parent drugs could not be detected, but their common nor metabolite, which should therefore be the target for urine screening.

Metabolism studies of the Kratom alkaloids mitraciliatine and isopaynantheine, diastereomers of the main alkaloids mitragynine and paynantheine, in rat and human urine using liquid chromatography–linear ion trap-mass spectrometry

Mitragyna speciosa ( Kratom in Thai), native in Southeast Asia, is increasingly misused as a herbal drug of abuse. During metabolism studies on the Kratom alkaloids mitragynine, its diastereomers speciogynine and speciociliatine as well as paynantheine in rats and humans, further isomeric compounds were detected in Kratom users’ urine. The question arose whether these compounds were formed from the low abundant, isomeric alkaloids mitraciliatine (MC) and isopaynantheine (ISO-PAY). Therefore, the aim of the presented study was to identify using liquid chromatography–linear ion trap-mass spectrometry their phase I and II metabolites in rat urine after administration of pure MC or ISO-PAY, to confirm their formation in humans, and finally to confirm whether the above-mentioned isomeric compounds in human urine represent MC and ISO-PAY and/or their metabolites. The metabolic pathways of both alkaloids in rats were found to be comparable to those of their corresponding diastereomers. In the human urines tested, not all metabolites found in rats could be detected because of the much lower amounts of MC and ISO-PAY in Kratom. However, all the above-mentioned so far unknown isomeric compounds could be identified in the human urine samples as MC, ISO-PAY and/or their metabolites. The used LC separation was also suitable for the differentiation of all other Kratom alkaloids and their metabolites in human urine.

The determination of vitamin D 3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D 3) and ergocalciferol (D 2). The main source of vitamin D 3 is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D 3, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MS n) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D 3 in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MS n and LC–MS/MS, respectively. Recoveries of vitamin D 3 added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D 3 was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).

Determination of 23 β2-agonists and 5 β-blockers in animal muscle by high performance liquid chromatography-linear ion trap mass spectrometry

A high performance liquid chromatography-linear ion trap mass spectrometry method using isotope dilution technique has been developed for the simultaneous determination of 23 β2-agonists and 5 β-blockers in animal muscle tissues. Pork and chicken muscle samples were acid hydrolyzed and extracted with 5% trichloracetic acid in water, and then cleaned up using MCX solid phase extraction (SPE) cartridge. Methanol and 0.1% formic acid were used as mobile phases for gradient elution. A Waters Atlantis®T3 column was used for separation. ESI positive ion scan mode was used with selective reaction monitoring. 9 β2-Agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 23 β2-agonists and 5 β-blockers were 5–200 μg/L, the coefficient of correlation was not less than 0.995, and the limit of detection for each compound in the muscle tissue was below 0.2 μg/kg. The recoveries of each compound in the spiked samples at three levels 5, 10, 20 μg/kg were in the range of 47.3%–123.7%, and the relative standard deviations were in the range of 3.2%–25.7%. The developed method is sensitive and specific for the determination of β2-agonists and β-blockers in pork and chicken muscle samples.

Determination of malachite green residues in fish using molecularly imprinted solid-phase extraction followed by liquid chromatography–linear ion trap mass spectrometry

This paper describes the development of an analytical procedure to determine malachite green (MG) residues in salmon samples using molecularly imprinted polymers (MIPs) as the extraction and clean-up material, followed by liquid chromatography–linear ion trap mass spectrometry (LC–QqQLIT-MS/MS). MG and two structurally related compounds, crystal violet (CV) and brilliant green (BG) were employed for the selectivity test. The imprinted polymers exhibited high binding affinity for MG, while CV and BG showed less binding capacity: 47% and 34%, respectively. The recovery values of MG in salmon samples fortified with leucomalachite green (LMG) were determined by measuring the amount of MG in the sample, after carrying out the oxidation reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which converts the LMG back into chromic-form. The average recovery of MG in spiked salmon muscle over the concentration range 1–100 ng g −1 was 98% with a relative standard deviation value (R.S.D.) below 12%. The method detection limits (MDLs) obtained for MG, CV, BG and their leuco-metabolites were in the range of 3–20 ng kg −1 (ppt).

Screening of pesticides in blood with liquid chromatography–linear ion trap mass spectrometry

In clinical or forensic toxicology, general unknown screening procedures are used to identify as many xenobiotics as possible, belonging to numerous chemical classes. We present here a general unknown screening procedure based on liquid chromatography coupled with use of a single linear ion trap mass spectrometer, and focus on the identification of pesticides and/or metabolites in whole blood. After solid-phase extraction (SPE), the compounds of interest were separated using a reversed-phase column and identified by the mass spectrometer operated first in the full-scan mass spectrometry (MS) mode, in the positive and negative polarities, followed by MS(2) and MS(3) scanning of ions selected in data-dependent acquisition. The total scan time was 2.45 s. Two mass spectral libraries (MS(2) and MS(3)), each of 450 spectra, were created for the 320 pesticides and metabolites detected after injection of pure solutions. Robustness of the spectra and matrix effects were studied and were satisfactory for the present application. Detection limits for the 320 compounds were studied by extracting 1 mL spiked blood at concentrations between 10 microg/L and 10 mg/L. If necessary, it was possible to decrease the detection limits of some compounds by 10-100-fold by scanning MS(2) in only one polarity, owing to a shorter total scan time. However, at the same time, the detection specificity decreased as no confirmation could be recorded in the following MS(3) scan and no information could be registered in the other polarity. So, in these rare cases, confirmation by another method was required.

Use of liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry for studying the metabolism of paynantheine, an alkaloid of the herbal drug Kratom in rat and human urine

The Thai medicinal plant Mitragyna speciosa (Kratom in Thai) is misused as a herbal drug of abuse. During studies on the main Kratom alkaloid mitragynine (MG) in rats and humans, several dehydro analogs could be detected in urine of Kratom users, which were not found in rat urine after administration of pure MG. Questions arose as to whether these compounds are formed from MG only by humans or whether they are metabolites formed from the second abundant Kratom alkaloid paynantheine (PAY), the dehydro analog of MG. Therefore, the aim of the presented study was to identify the phase I and II metabolites of PAY in rat urine after administration of the pure alkaloid. This was first isolated from Kratom leaves. Liquid chromatography-linear ion trap mass spectrometry provided detailed structure information of the metabolites in the MS(n) mode particularly with high resolution. Besides PAY, the following phase I metabolites could be identified: 9-O-demethyl PAY, 16-carboxy PAY, 9-O-demethyl-16-carboxy PAY, 17-O-demethyl PAY, 17-O-demethyl-16,17-dihydro PAY, 9,17-O-bisdemethyl PAY, 9,17-O-bisdemethyl-16,17-dihydro PAY, 17-carboxy-16,17-dihydro PAY, and 9-O-demethyl-17-carboxy-16,17-dihydro PAY. These metabolites indicated that PAY was metabolized via the same pathways as MG. Several metabolites were excreted as glucuronides or sulfates. The metabolism studies in rats showed that PAY and its metabolites corresponded to the MG-related dehydro compounds detected in urine of the Kratom users. In conclusion, PAY and its metabolites may be further markers for a Kratom abuse in addition of MG and its metabolites.

The Role of Human UDP-Glucuronyltransferases on the Formation of the Methylenedioxymethamphetamine (Ecstasy) Phase II MetabolitesR- andS-3-Methoxymethamphetamine 4-O-Glucuronides

Different pharmacokinetic properties have been observed for the two enantiomers of the entactogen 3,4-methylendioxy-methamphetamine, most probably a result of enantioselective metabolism. The aim of the present work was to study the involvement of human UDP-glucuronyltransferase (UGT) isoforms in the glucuronidation of the enantiomers of its major metabolite 4-hydroxy-3-methoxymethamphetamine (HMMA). First, the reference standards of R- and S-HMMA-O-glucuronide were synthesized semipreparatively using the enzymes of rat liver microsomes, followed by isolation with semipreparative high-performance liquid chromatography and identification using mass spectrometry and NMR. Racemic HMMA was then incubated using heterologously expressed human UGTs and pooled human liver microsomes (HLMs), and the glucuronides were quantified by liquid chromatography-linear ion trap-mass spectrometry. UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were involved in the glucuronidation of HMMA. UGT2B15, UGT2B17, and HLM revealed classic Michaelis-Menten kinetics, whereas UGT1A9 and UGT2B7 showed sigmoidal curves and the respective Eadie-Hofstee plots indicated autoactivation kinetics. UGT2B15 showed the highest affinity and activity. UGT2B15, UGT2B17, and HLMs were not considerably enantioselective but showed slight preferences for S-HMMA. Marked enantioselectivity could only be observed for UGT1A9 with respect to the S-enantiomer and for UGT2B7 with respect to the R-enantiomer. In conclusion, the O-glucuronidation of HMMA in vivo should not be expected to be enantioselective, and the different pharmacokinetic properties may not be caused directly by glucuronidation.