Liquid chromatography/mass spectrometry analysis of PHY906, a Chinese medicine formulation for cancer therapy

PHY906 is a Chinese medicine formulation prepared from four medicinal herbs for adjuvant cancer chemotherapy. In this paper, liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) was used to clarify the chemical composition of PHY906. The aqueous extract of PHY906 was separated on a Waters Atlantis C(18) column, and was eluted with acetonitrile/0.05% (v/v) formic acid. The separated compounds were identified with pure standards, or tentatively characterized by analyzing their mass spectra recorded in both negative and positive ion polarity modes. Further structural information was obtained from in-source fragmentation. Based on the LC/MS analysis, we proposed the structures for 64 bioactive compounds, including flavonoids, triterpene saponins, and monoterpene glycosides. All the compounds identified from PHY906 were further assigned in the four individual herbs, and some of them are reported for the first time.     

Analysis of the constituents in rat plasma after oral administration of Shexiang Baoxin pill by HPLC‐ESI‐MS/MS

A valid method using liquid chromatography coupled with electrospray ionization (ESI) and ion trap mass spectrometry was established for the study of the absorbed components in rat plasma after oral administration of a traditional Chinese medicine (TCM) Shexiang Baoxin pill. The plasma was deproteinated by adding methanol prior to liquid chromatography, in which separation was carried out on a Symmetry C₁₈ column (5 µm, 250 × 4.6 mm). A linear gradient with 0.5% formic acid–water–acetonitrile was used as mobile phase. Mass spectra were acquired in both negative and positive modes. Twenty‐one components including 17 components from Shexiang Baoxin pill and four metabolites were observed from a comprehensive analysis of the chromatography of Shexiang Baoxin pill, controlled plasma and dosed plasma. All of the 17 prototype compounds and three of the metabolites were identified by comparing their retention behaviors and MS and MS/MS spectra with reference compounds and literature data. This study developed an integrated method for screening the bioactive constituents in plasma after oral adminstration of Chinese herbal medicine and provided helpful chemical information for further pharmacology and active mechanism research on TCM. Copyright © 2009 John Wiley & Sons, Ltd.     

LC/MS/MS for identification of in vivo and in vitro metabolites of jatrorrhizine

The in vivo and in vitro metabolism of jatrorrhizine has been investigated using a specific and sensitive LC/MS/MS method. In vivo samples including rat feces, urine and plasma collected separately after dosing healthy rats with jatrorrhizine (34 mg/kg) orally, along with in vitro samples prepared by incubating jatrorrhizine with rat intestinal flora and liver microsome, respectively, were purified using a C(18) solid-phase extraction cartridge. The purified samples were then separated with a reversed-phase C(18) column with methanol-formic acid aqueous solution (70:30, v/v, pH3.5) as mobile phase and detected by on-line MS/MS. The structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, seven new metabolites were found in rat urine, 13 metabolites were detected in rat feces, 11 metabolites were detected in rat plasma, 17 metabolites were identified in intestinal flora incubation solution and nine metabolites were detected in liver microsome incubation solution. The main biotransformation reactions of jatrorrhizine were the hydroxylation reaction, the methylation reaction, the demethylation reaction and the dehydrogenation reaction of parent drug and its relative metabolites. All the results were reported for the first time, except for some of the metabolites in rat urine.     

Quantitative determination of metformin,glyburide and its metabolites in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of an LC‐MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87–99%, and that from urine samples was 85–95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra‐day and inter‐day assays in plasma and urine samples, and the accuracy was 86–114% in plasma, and 94–105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolic profile and pharmacokinetics of polyphyllin I,an anticancer candidate,in rats by UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla , is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo . However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.     

Identification of seven metabolites of oxyresveratrol in rat urine and bile using liquid chromatography/tandem mass spectrometry

Oxyresveratrol (trans‐2,4,3′,5′‐tetrahydroxystilbene) is a major compound isolated from Smilax china, a Chinese herbal medicine. The rat urine and bile samples were pretreated by solid‐phase extraction method after oral administration at a dose of 100 mg/kg of oxyresveratrol. Seven metabolites were identified by LC‐MS/MS method with electrospray ionization in negative ion mode. The results indicated that main metabolites of oxyresveratrol were monoglucuronided and monosulfated oxyresveratrol. Based on the results, the metabolic pathway of oxyresveratrol in rat urine and bile was proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of an LC‐MS/MS method for determining the pharmacokinetics of clonidine following oral administration of Zhenju antihypertensive compound

Zhenju antihypertensive compound (ZJAHC) is a combined Chinese–Western medicine formula including clonidine (CLO), hydrochlorothiazide (HCT), rutin, Chrysanthemum indicum extract and pearl powder. Compared with CLO preparations, ZJAHC shows improved activities and decreased adverse effects. It is believed that the side effects of CLO are caused by its high peak plasma concentration. Hence, study of the influence of ZJAHC on the pharmacokinetic behaviors of clonidine seems essential. In present study, the plasma concentrations of CLO were determined with a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method. The MS/MS transitions monitored for clonidine and internal standard were 230.2 → 213.1 and 152.2 → 110.2, respectively. The analyte was quantified in a single run within 3 min. The pharmacokinetic study showed that the area under the plasma concentration–time curve of CLO in ZJAHC (60 µg/kg CLO) was similar to that of CLO‐HCT‐high (120 µg/kg CLO) but the peak concentration was much lower than that in CLO‐HCT‐high. ZJAHC could enhance the bioavailability without greatly increasing peak concentration of clonidine. This comprehensive effect of enhancing the bioavailability and avoiding the high peak plasma concentration for CLO might mainly result from the co‐contribution of Western medicine and traditional Chinese medicine (TCM), while the effect of TCM was stronger than that of Western medicine. Copyright © 2014 John Wiley & Sons, Ltd.     

Qualitative and quantitative evaluation of Oroxylum indicum (L.) Kurz by HPLC and LC‐qTOF‐MS/MS

Oroxylum indicum, as a popular functional Chinese herbal medicine for reducing hyperactivity, relieving sore throat, smoothing the liver and adjusting stomach, mainly contains flavonoids. In this study, we aimed to establish a fast and sensitive method that enables to analyze the chemical components in O. indicum qualitatively and quantitatively. First, a total of 42 components were characterized by LC‐quadrupole time‐of‐flight (qTOF)‐tandem mass spectrometry (MS/MS), including 23 flavonoid glycosides, 13 flavonoids and six other types of compounds. Then, 17 characteristic components of the 19 common peaks in the chromatographic fingerprints of O. indicum were confirmed. Fifty samples were classified into two groups by hierarchical clustering analysis and orthogonal partial least squares‐discriminant analysis, which also identified the 10 main chemical markers responsible for differences between samples. Last, the quantitative analysis of multiple components with a single marker method was established for simultaneous determination of six main active components in O. indicum by LC‐UV with oroxin B was chosen as internal reference substance. Finally, a rapid and efficient method integrating HPLC with LC‐electrospray ionization‐qTOF‐MS/MS analysis was established to comprehensively discriminate and assess the quality of O. indicum samples.     

Integrated LC/MS and GC/MS Metabolomics Data for the Evaluation of Protection Function of Fructus Ligustri Lucidi on Mouse Liver

A comprehensive metabolomic strategy, integrating GC/MS and LC/MS data, had been developed to study the protection function of herbal medicine, Fructus Ligustri Lucidi, on mouse liver. Mouse plasma samples were analyzed by GC/MS and LC/MS in conjunction with multiblock multivariate analysis method, multiblock partial least squares discriminant analysis (MBPLS-DA), to supply more important and distinct information of metabolomic biomarkers. Then, the biological pathway analysis was carried out to help further understanding the mechanism of liver protection of Fructus Ligustri Lucidi.     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.     

Plasma pharmacochemistry combined with microdialysis to screen potential bioactive components and their metabolites in Anemarrhena asphodeloides saponin extract using ultrahigh‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Although various techniques have been employed to analyze drug metabolites, the metabolism of multicomponent herbal medicine has seldom been fully addressed. In contrast to chemical drugs, a number of compounds in herbal medicine could get into circulation and then be metabolized. Metabolism study on active constituents in herbal medicine is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of herbal medicine. The present work aims to elucidate the multicomponent metabolic characteristics of a herbal medicine by the combination of plasma pharmacochemistry and microdialysis sampling. Anemarrhena asphodeloides, a well‐known traditional Chinese medicine, was chosen as a model. After oral administration of A. asphodeloides saponin extract to rats, microdialysis samples were collected continuously in the jugular vein and analyzed by ultrahigh‐performance LC/quadrupole‐TOF MS. The identification of compounds in biosamples was achieved by accurate mass measurement and detailed fragmentation pathway analysis. The results showed that unbound constituents in blood circulation of the rat included seven parent saponins and six metabolites, which might be the potential active components in vivo. Among which, three metabolites have not been previously reported and were identified in this study. It is the first report on systemic metabolism of total saponins of A. asphodeloides in mammalian plasma.     

Metabolite profiling of ginsenosides in rat plasma,urine and feces by LC–MS/MS and its application to a pharmacokinetic study after oral administration of Panax ginseng extract

Panax ginseng is widely consumed as a functional food in the form of tea, powder, capsules, among others, and possesses a range of pharmacological activities including adaptogenic, immune‐modulatory, anti‐tumor, anti‐aging and anti‐inflammatory effects. The aim of this study was to identify and quantify the major ginsenosides and their metabolites in rat plasma, urine and feces after administration of P. ginseng extract using LC–MS/MS. We collected rat plasma samples at 0.5, 1, 2, 4, 8, 12, 24 and 48 h, and the amounts of urine and fecal samples accumulated in 24 h. Fourteen major ginsenosides and their metabolites were observed in fecal samples at high levels; however, low levels of 11 ginsenosides were detected in urine samples. The pharmacokinetics of the major ginsenosides and their metabolites was investigated in plasma. The results indicated that the maximum plasma concentration, time to maximum concentration and area under the curve of compound K were significantly greater than those of other ginsenosides. This study thus provides valuable information for drug development and clinical application of P. ginseng.     

Identification of chemical constituents and rat metabolites of Kangxianling granule by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry method was established to characterize the chemical constituents of Kangxianling granule (KXL), a traditional Chinese medicine formula, and the metabolic profile in rat urine and plasma after oral administration of KXL. A total of 27 compounds in KXL extract and 13 prototype compounds with 12 metabolites in rat urine and plasma were identified. Among the 27 detected compounds, 15 were identified by comparing the retention time and MS data with that of reference compounds and the other 12 compounds were tentatively assigned based on the MS data and reference literature. The main prototype components absorbed in rat were amygdalin, salvianolic acid B, tanshinones and anthraquinones. Hydroxylation, glucuronidation and sulfation were the principal metabolic pathways in rat. The results revealed that the 25 compounds identified in rat urine and plasma were the potential active ingredients of KXL, which provides helpful chemical information for further study of the pharmacology mechanism of KXL. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatography/mass spectrometry‐based plasma metabolic profiling study of escitalopram in subjects with major depressive disorder

Liquid chromatography‐mass spectrometry (LC‐MS) method revealed the plasma metabolite profiles in major depressive disorder patients treated with escitalopram (ECTP) (n = 7). Depression severity was assessed according to the 17‐item Hamilton Depression Rating Scale. Metabolic profiles were derived from major depressive disorder subject blood samples collected after ECTP treatment. Blood plasma was separated and processed in order to effectively extract metabolites, which were then analyzed using LC‐MS. We identified 19 metabolites and elucidated their structures using LC‐tandem MS (LC‐MS/MS) combined with elemental compositions derived from accurate mass measurements. We further used online H/D exchange experiments to verify the structural elucidations of each metabolite. Identifying molecular metabolites may provide critical insights into the pharmacological and clinical effects of ECTP treatment and may also provide useful information informing the development of new antidepressant treatments. These detailed plasma metabolite analyses may also be used to identify optimal dose concentrations in psychopharmacotherapeutic treatment through drug monitoring, as well as forming the basis for response predictions in depressed subjects.     

基于UPLC-Q-Orbitrap MS/MS技术研究酸枣仁发酵过程中的化学成分转化

利用超高效液相色谱-四极杆-静电场轨道阱串联质谱（UPLC-Q-Orbitrap MS/MS）研究了酸枣仁经茯苓发酵前后的主要化学成分变化，初步探讨了茯苓对酸枣仁发酵的转化规律.鉴定了发酵产物中24种化学成分，整合多元统计方法对酸枣仁发酵0，7和14 d后的差异性化学成分进行研究，采用正交偏最小二乘分析找到7个差异成分.采用t-检验对24种化学成分的相对含量进行分析；并分析了酸枣仁中黄酮苷、皂苷类成分的代谢反应途径.通过茯苓发酵，酸枣仁中化学成分主要发生了脱酰基和脱糖基等水解反应.为酸枣仁发酵产物的化学成分检测提供了快速、直观且准确的方法，为酸枣仁-茯苓共发酵产物的深入开发与利用提供了科学依据.     

Application of LC/MS and ICP/MS for establishing the fingerprint spectrum of the traditional Chinese medicinal preparation Gan-Lu-Yin

We developed a method to analyze the fingerprint spectrum qualitatively and quantitatively for the traditional Chinese herbal medicinal preparation Gan-Lu-Yin with HPLC combined with photodiode array detection, and MS, and to identify the preparation's 14 main components including baicalin, baicalein, oroxylin A-7-O-glucuronide, wogonin-7-O-glucuronide, wogonin, and oroxylin A in Radix Scutellariae; naringin and neohesperidin in Aurantii fructus; liquiritigenin, liquiritin, and glycyrrhizic acid in Radix Glycyrrhizae. In LC/UV assay, a Cosmosil 5C18-MS-II column was used as the stationary phase, and a gradient of potassium dihydrogen phosphate, ACN, and water as the elute solution. The UV detection wavelengths were 250 and 280 nm. In LC/MS assay, a gradient of phosphoric acid, ACN, and water was used as the elute solution, and electrospray positive ion mode ((+)-ESI) as the analytic mode. In order to explore the distribution of trace metal elements effectively in Gan-Lu-Yin, a microwave digestion method was used for sample treatment, and an inductively coupled plasma MS assay was used to analyze fingerprint spectra of the inorganic metals in Gan-Lu-Yin. Combined with fingerprint spectra of organic compounds by LC/UV and LC/MS, it was expected to provide effective quality control in the production of Gan-Lu-Yin.     

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.     

In vivo metabolic investigation of silodosin using UHPLC–QTOF–MS/MS and in silico toxicological screening of its metabolites

Silodosin (SLD) is a novel α1‐adrenoceptor antagonist which has shown promising clinical efficacy and safety in patients with benign prostatic hyperplasia (BPH). However, lack of information about metabolism of SLD prompted us to investigate metabolic fate of SLD in rats. To identify in vivo metabolites of SLD, urine, feces and plasma were collected from Sprague–Dawley rats after its oral administration. The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 13 phase I and six phase II metabolites of SLD have been identified in rat urine which includes hydroxylated, N‐dealkylated, dehydrogenated, oxidative, glucosylated, glucuronide and N‐sulphated metabolites, which are also observed in feces. In plasma, only dehydrogenated, N‐dealkylated and unchanged SLD are observed. The structure elucidation of metabolites was done by fragmentation in MS/MS in combination with HRMS data. The potential toxicity profile of SLD and its metabolites were predicted using TOPKAT software and most of the metabolites were proposed to show a certain degree of skin sensitization and occular irritancy. Copyright © 2016 John Wiley & Sons, Ltd.