Multivariate approaches for efficient detection of potential metabolites from liquid chromatography/mass spectrometry data

This work describes a novel method for rapid screening of unknown metabolites in urine samples that narrows down the list of potential metabolites. Prior to analysis by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS), urine samples were prepared using solid-phase extraction (SPE). Automatic curve resolution was used for deconvolution of the LC/MS data, followed by peak alignment. Preprocessed data were then used for metabolite pattern recognition using principal component analysis (PCA), parallel factor analysis (PARAFAC), and multilinear partial least squares (N-PLS). This approach enabled the rapid detection of metabolites of citalopram in urine by maximizing the information extracted. The metabolites thus identified were compared with earlier studies on the metabolism of citalopram. In addition, new, unreported metabolites were found and characterized by LC/MS/MS and accurate mass measurements. A combination of data from positive and negative ionization enhanced the identification of metabolites.     

Studies on the metabolism of gomisin A (TJN-101). II. Structure determination of biliary and urinary metabolites in rat

After oral administration of gomisin A (1) to rats, the bile and urine were collected and treated with beta-glucuronidase and arylsulfatase. Seven metabolites, met B (2), met A-III (3), met E (4), met D (5), met F (6), met G (7), and met H (8) were isolated from the bile treated with the enzymes. Eight metabolites 2-8, and met A-II (9) were isolated from the urine treated with the enzymes. A major metabolite 2, and two minor metabolites 3 and 9 were identified as met B, met A-III, and met A-II, respectively, which are oxidative products of 1 formed by rat liver S9 mix. The structures of five new metabolites 4-7, and 8 were determined on the basis of chemical and spectral studies.     

Urinary metabolites of cinobufagin in rats and their antiproliferative activities

Cinobufagin was one of the important cardenolidal steroids and a major component of Chan'Su, a famous traditional Chinese medicine. The urinary metabolites of cinobufagin after single oral doses of 25?mg?kg?1 in rats were investigated. Eleven metabolites were isolated and purified by liquid-liquid extraction, open-column chromatography, medium-pressure liquid chromatography, as well as semi-preparative high-performance liquid chromatography. Their structures were elucidated by chemical and various spectroscopic methods, which were identified as desacetylcinobufagin (M-1), 3-oxo-desacetylcinobufagin (M-2), 3-oxo-cinobufagin (M-3), 3-epi-desacetylcinobufagin (M-4), 3-epi-12β-hydroxyl desacetylcinobufagin (M-5), 5β-hydroxyl cinobufagin (M-6), 5β-hydroxyl desacetylcinobufagin (M-7), 12β-hydroxyl cinobufagin (M-8), 1β,12β-dihydroxyl cinobufagin (M-9), 12β-hydroxyl desacetylcinobufagin (M-10) and 1β,12β-dihydroxyl desacetylcinobufagin (M-11), respectively. Among them, M-1 was the main urinary metabolite of cinobufagin with a yield of 17.7%. Most metabolites were hydroxylated products of cinobufagin at C-1β, 5β and 12β positions, as well as deacetylated products at C-16. Except M-1, M-4 and M-7, the other eight metabolites were novel in vivo metabolites of cinobufagin. Some metabolites showed potential cytotoxicity against human hepatoma cells (HepG2) and human leukaemia (K562, HL-60) cells; however, their cytotoxicities generally decreased after metabolic conversion.     

Metabolic studies on the total phenolic acids from the roots of Salvia miltiorrhiza in rats

Phenolic acids are the main active constituents of Salvia miltiorrhiza Bunge. The metabolism of total phenolic acids from the roots of Salvia miltiorrhiza in rats was investigated. A sample preparation method combining the solid-phase extraction with liquid-liquid extraction was established to separate metabolites from the biological matrix. HPLC-UV and HPLC-MS methods were employed to analyze the metabolites. Five metabolites (M1-M5) were identified by HPLC-MS analysis and comparison with those of the reference standards. The fi ve metabolites were characterized as danshensu (M1), caffeic acid (M2), ferulic acid (M3), isoferulic acid (M4) and methylized ferulic acid (M5), respectively. The possible metabolic pathway of the phenolic acids is proposed.     

超高效液相色谱-质谱联用技术在药物肝损伤代谢组学研究中的应用

药物引起的肝损伤是全世界关注的健康问题,药源性肝损伤的早期诊断仍然是临床治疗肝损伤的一大难题。本研究中,首先利用对乙酰氨基酚、四氯化碳、大黄素、雷公藤甲素和马兜铃酸构建不同类型的肝细胞损伤模型,利用超高效液相色谱-质谱联用技术(UPLC-MS)分别得到正常组和损伤组的细胞代谢轮廓谱。然后利用模式识别构建分类模型,筛选肝损伤相关的差异代谢物。结果显示,不同类型肝损伤在肝细胞代谢谱上可以被区分开,最终鉴定出14种差异代谢物。损伤组肝细胞经保肝阳性药联苯双酯干预后,差异代谢物水平均趋向于正常组,在一定程度上验证了差异标志物与肝损伤的相关性。实验结果表明细胞代谢组学是研究药物肝损伤的有效工具之一。     

Structural elucidation of in vitro metabolites of emodin by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was employed to investigate the in vitro metabolism of emodin. Emodin was incubated with rat liver microsomes in the presence of a NADPH-generating system, followed by extraction with ethyl acetate. After separation on a reversed-phase C18 analytical column with a linear gradient elution of methanol and 0.1% formic acid in water, negative electrospray ionization tandem mass spectrometry experiments were performed. As a result, the parent drug and its six metabolites were detected from rat liver microsomal incubations. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. Besides three mono-hydroxylated metabolites (omega-hydroxyemodin, 2-hydroxyemodin, 4-hydroxyemodin), three other metabolites were identified, which were emodic acid, 3-carbomethoxy-6-methoxy-1,8-dihydroxyanthraquinone and physcion, respectively.     

An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection

A new strategy using a hybrid linear ion trap/Orbitrap mass spectrometer and multiple post-acquisition data mining techniques was evaluated and applied to the detection and characterization of in vitro metabolites of indinavir. Accurate-mass, full-scan MS and MS/MS data sets were acquired with a generic data-dependent method and processed with extracted-ion chromatography (EIC), mass-defect filter (MDF), product-ion filter (PIF), and neutral-loss filter (NLF) techniques. The high-resolution EIC process was shown to be highly effective in the detection of common metabolites with predicted molecular weights. The MDF process, which searched for metabolites based on the similarity of mass defects of metabolites to those of indinavir and its core substructures, was able to find uncommon metabolites not detected by the EIC processing. The high-resolution PIF and NLF processes selectively detected metabolites that underwent fragmentation pathways similar to those of indinavir or its known metabolites. As a result, a total of 15 metabolites including two new indinavir metabolites were detected and characterized in a rat liver S9 incubation sample. Overall, these data mining techniques, which employed distinct metabolite search mechanisms, were complementary and effective in detecting both common and uncommon metabolites. In summary, the results demonstrated that this analytical strategy enables the high-throughput acquisition of accurate-mass LC/MS data sets, comprehensive search of a variety of metabolites through the post-acquisition processes, and effective structural characterization based on elemental compositions of metabolite molecules and their product ions.     

Urinary metabolites of cannabidiol in dog, rat and man and their identification by gas chromatography-mass spectrometry

Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography-mass spectrometry as trimethylsilyl (TMS), [2H9]TMS, methyl ester-TMS and methyloxime-TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4"- and 5"-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of beta-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2"-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the delta-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.     

Metabolism of 14C-miloxacin in rats--metabolites in urine, bile and feces (author's transl)

Isolation and characterization of metabolites of miloxacin, a new antimicrobial agent, were undertaken with rats. 14C-Miloxacin was orally administered to Sprague-Dawley rats at a dose of 50 mg/kg, and urine, bile and feces were collected. The metabolites extracted from the biological samples were isolated by column and thin-layer chromatographies. Characterization of the isolated metabolites was carried out by comparison with the authentic materials in various physicochemical analyses. Eight metabolites together with intact miloxacin were identified; containing the metabolites of N-demethoxy (M-1), catechol (M-3) and 6-methoxy (M-2 and M-4) types and their conjugates with glucuronic acid.     

Chlorinated briarane-type diterpenoids from the gorgonian coral Ellisella robusta (Ellisellidae)

Four chlorinated metabolites featuring briarane carbon skeletons have been isolated from the gorgonian coral Ellisella robusta, which was collected off the coast of southern Taiwan: two new natural products, robustolides D (1) and E (2), and two known metabolites, robustolides F (3) and G (4). The structures of metabolites 1–4 were determined by spectroscopic methods, using 1D and 2D NMR in particular. The structures and absolute stereochemistry of robustolides D (1), F (3), and G (4) were directly established by X-ray diffraction analysis. Robustolide D (1) is the first metabolite of briarane-related natural products found to possess two halogen atoms.     

Beta-orcinol metabolites from the lichen Hypotrachyna revoluta

Four new beta-orcinol metabolites, hypotrachynic acid (1), deoxystictic acid (2), cryptostictinolide (3) and 8'-methylconstictic acid (4) along with the metabolites 8'-methylstictic acid (5), 8'-methylmenegazziaic acid (6), stictic acid (7), 8'-ethylstictic acid (8) and atranorin (9), that have been previously described, were isolated for the first time from the tissue extracts of the lichen Hypotrachyna revoluta (Fl?rke) Hale. The structures of the new metabolites were elucidated on the basis of extensive spectroscopic analyses. Radical scavenging activity (RSA) of the metabolites isolated in adequate amounts, was evaluated using luminol chemiluminescence and comparison with Trolox.     

超高效液相色谱-四极杆-飞行时间质谱检测和鉴定猪尿中氯丙那林的主要代谢产物

采用超高效液相色谱-四极杆-飞行时间质谱(UPLC/Q-TOF MS)检测和鉴定了猪尿中氯丙那林的主要代谢产物,并讨论了氯丙那林在猪体内的主要代谢途径。按10 mg/kg(b. w.)的剂量口服灌食氯丙那林,分别采集给药前及给药后的猪尿液样品。采用UPLC/Q-TOF MS对样品进行分析,并应用质量亏损过滤和离子色谱峰提取等数据处理技术,在给药后24 h内的猪尿中检测和鉴定了9种氯丙那林的代谢产物,其中,Ⅰ相代谢产物2种,Ⅱ相代谢产物7种。然后,根据氯丙那林原形和代谢产物的碎片离子特征,对代谢产物的结构进行鉴定。最后,根据所鉴定的代谢产物,推测氯丙那林在猪体内的代谢途径包括苯环羟基化、β -羟基和仲氨基的葡萄糖醛酸轭合、羟基化后的葡萄糖醛酸和硫酸轭合等。研究结果表明,羟基化氯丙那林及其轭合产物的相对含量大于60%,明显高于氯丙那林原形及其轭合产物,是尿液中的主要代谢产物。本研究将为确定氯丙那林在动物体内的残留标示物及加强对氯丙那林非法使用的监控提供科学依据。     

New Metabolites of Aconitine in Rabbit Urine

A sensitive analytical method to identify and determine aconitine and its metabolites in rabbit urine was developed by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS^n). In this method,aconitine and its four metabolites in rabbit urine were isolated and deduced as 16-O-demethylaconine (M1), benzoylaconine (M2),16-O-demethylbenzoylaconine (M3) and aconine(M4).M1 and M3 are new metabolites of aconitine and M2 and M4 are first identified in rabbit urine.     

The Tannins from Sanguisorba officinalis L. (Rosaceae): A Systematic Study on the Metabolites of Rats Based on HPLC–LTQ–Orbitrap MS2 Analysis

Sanguisorba tannins are the major active ingredients in Sanguisorba ofJicinalis L. (Rosaceae), one of the most popular herbal medicines in China, is widely prescribed for hemostasis. In this study, three kinds of tannins extract from Sanguisorba officinalis L. (Rosaceae), and the metabolites in vivo and in vitro were detected and identified by high-pressure liquid chromatography, coupled with linear ion trap orbitrap tandem mass spectrometry (HPLC–LTQ–Orbitrap). For in vivo assessment, the rats were administered at a single dose of 150 mg/kg, after which 12 metabolites were found in urine, 6 metabolites were found in feces, and 8 metabolites were found in bile, while metabolites were barely found in plasma and tissues. For in vitro assessment, 100 μM Sanguisorba tannins were incubated with rat liver microsomes, liver cytosol, and feces, after which nine metabolites were found in intestinal microbiota and five metabolites were found in liver microsomes and liver cytosol. Moreover, the metabolic pathways of Sanguisorba tannins were proposed, which shed light on their mechanism.     

Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.     

Repeatability of the measurement of exhaled volatile metabolites using selected ion flow tube mass spectrometry

Selected ion flow tube mass spectrometry, SIFT-MS, has been used to determine the repeatability of the analysis of volatile metabolites within the breath of healthy volunteers, with emphasis on the influence of sampling methodology. Baseline instrument specific coefficients of variability for examined metabolites were as follows: acetone (1%), ammonia (1%), isoprene (2%), propanol (6%), ethanol (7%), acetic acid (7%), and hydrogen cyanide (19%). Metabolite concentration and related product ion count rate were identified as strong determinants of measurement variation. With the exception of ammonia, an orally released metabolite, variability in repeated on-line breath analysis tended to be lower for metabolites of systemic origin. Standardization of sampling technique improved the repeatability of the analysis of selected metabolites. Off-line (bag) alveolar breath sampling, as opposed to mixed (whole) breath sampling, likewise improved the repeatability of the analysis of all metabolites investigated, with the exception of acetic acid. We conclude that SIFT-MS analysis of common volatile metabolites within the breath of healthy volunteers is both reliable and repeatable. For selected metabolites, the finding that repeatability is improved through modification of sampling methodology may have implications in terms of future recommended practices.     

Detection of sibutramine administration: a gas chromatography/mass spectrometry study of the main urinary metabolites

A gas chromatographic/mass spectrometric (GC/MS) study aimed at identifying the metabolites of sibutramine (1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl)cyclobutanemethanamine) in urine is described. Urinary excretion of sibutramine metabolites following the oral administration of a single dose of sibutramine was followed by GC/MS analysis. After identification of the chromatographic signals corresponding to the six main urinary metabolites, the fragmentation pattern was studied in electron ionization (EI) mode after derivatization to the corresponding methyl and trimethylsilyl derivatives. Urine samples were pretreated according to a reference procedure (liquid/liquid separation, enzymatic hydrolysis, pre-concentration under a stream of nitrogen and derivatization, either under thermal incubation and by microwave irradiation). All sibutramine metabolites were excreted as glucuroconjugates, and retain the chiral carbon present in the sibutramine skeleton. The metabolites identified included mono-desmethylsibutramine (nor-sibutramine), bi-desmethylsibutramine (nor-nor-sibutramine), and the corresponding hydroxylated compounds, the hydroxylation taking place either on the cyclobutane or on the isopropyl chain. The excretion profiles of the different metabolites were also evaluated. From an analytical point of view, the method can be applied to different fields of forensic analytical toxicology, including anti-doping analysis. Although the lack of certified reference materials does not allow a precise determination of the limits of detection (LODs) of all the sibutramine metabolites, an estimation taking into account the response factor of similar compounds ensures that all metabolites are still clearly detectable in a range of concentrations between 10 and 50 ng/mL, thus satisfying the minimum required performance limits (MRPLs) of the World Anti-Doping Agency (WADA).     

Phase I and II metabolites of speciogynine, a diastereomer of the main Kratom alkaloid mitragynine, identified in rat and human urine by liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry

Mitragyna speciosa (Kratom in Thai), a Thai medical plant, is misused as herbal drug of abuse. Besides the most abundant alkaloids mitragynine (MG) and paynantheine (PAY), several other alkaloids were isolated from Kratom leaves, among them the third abundant alkaloid is speciogynine (SG), a diastereomer of MG. The aim of this present study was to identify the phase I and II metabolites of SG in rat urine after the administration of a rather high dose of the pure alkaloid and then to confirm these findings using human urine samples after Kratom use. The applied liquid chromatography coupled to low- and high-resolution mass spectrometry (LC-HRMS-MS) provided detailed information on the structure in the MS(n) mode particularly with high resolution. For the analysis of the human samples, the LC separation had to be improved markedly allowing the separation of SG and its metabolites from its diastereomer MG and its metabolites. In analogy to MG, besides SG, nine phase I and eight phase II metabolites could be identified in rat urine, but only three phase I and five phase II metabolites in human urine. These differences may be caused by the lower SG dose applied by the user of Kratom preparations. SG and its metabolites could be differentiated in the human samples from the diastereomeric MG and its metabolites comparing the different retention times determined after application of the single alkaloids to rats. In addition, some differences in MS(2) and/or MS(3) spectra of the corresponding diastereomers were observed.     

基于超高效液相色谱-四极杆飞行时间质谱联用技术的血瘀模型大鼠血浆代谢组学分析

采用盐酸肾上腺素加冰水浴建立急性血瘀大鼠模型，使用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF/MS）检测空白对照组与血瘀模型组中血浆代谢物，用主成分分析（PCA）、有监督偏最小二乘法判别分析（PLS-DA）及正交偏最小二乘法判别分析（OPLS-DA）对代谢组学数据进行多维统计分析，筛选潜在生物标志物。与对照组相比，在血瘀模型组大鼠血浆中检测出46个差异代谢物，血瘀模型组中乙酰胆碱、N6，N6，N6-三甲基-L-赖氨酸、胞嘧啶、乙酰肉碱等21个代谢物显著上调，吲哚丙酸、LysoPC（14：0）等25个代谢物显著下调，可能与脂质代谢、半乳糖代谢、亚油酸代谢、不饱和脂肪酸生物合成、糖酵解、花生四烯酸代谢等通路有关。代谢产物可作为血瘀证研究中的重要标记物，该研究结果有助于揭示血瘀证的发病机制，可为临床血瘀疾病的诊断及选用药物治疗提供思路，为后续治疗手段提供参考依据。     

Exploring the In Vivo Existence Forms (23 Original Constituents and 147 Metabolites) of Astragali Radix Total Flavonoids and Their Distributions in Rats Using HPLC-DAD-ESI-IT-TOF-MSn

Astragali Radix total flavonoids (ARTF) is one of the main bioactive components of Astragali Radix (AR), and has many pharmacological effects. However, its metabolism and effective forms remains unclear. The HPLC-DAD-ESI-IT-TOF-MSⁿ technique was used to screen and tentatively identify the in vivo original constituents and metabolites of ARTF and to clarify their distribution in rats after oral administration. In addition, modern chromatographic methods were used to isolate the main metabolites from rat urine and NMR spectroscopy was used to elucidate their structures. As a result, 170 compounds (23 original constituents and 147 metabolites) were tentatively identified as forms existing in vivo, 13 of which have the same pharmacological effect with ARTF. Among 170 compounds, three were newly detected original constituents in vivo and 89 were new metabolites of ARTF, from which 12 metabolites were regarded as new compounds. Nineteen original constituents and 65 metabolites were detected in 10 organs. Four metabolites were isolated and identified from rat urine, including a new compound (calycoisn-3’-O-glucuronide methyl ester), a firstly-isolated metabolite (astraisoflavan-7-O-glucoside-2’-O-glucuronide), and two known metabolites (daidzein-7-O-sulfate and calycosin-3’-O-glucuronide). The original constituents and metabolites existing in vivo may be material basis for ARTF efficacy, and these findings are helpful for further clarifying the effective forms of ARTF.