Journal of Chromatography B (v.1057, #C)

Sensitive bromine-based screening of potential toxic furanoids in Dioscorea bulbifera L. by Zhengyu Zhang; Dongju Lin; Weiwei Li; Huiyuan Gao; Ying Peng; Jiang Zheng (1-14).
Numerous furanoids have been reported to be toxic, and many of them were found in herbal medicines. Toxicities of furanoids are suggested to result from the generation of cis-enedials via biotransformation. The detection of the electrophilic metabolic intermediates is a challenge. Earlier, we developed a selective approach to screen potential toxic furanoids, through which we found two major furanoids, diosbulbin B and 8-epidiosbulbin E acetate, in Dioscorea bulbifera L., a known furanoid-containing and hepatotoxic herbal medicine. In the present study, we improved the approach to analyze furanoids in D. bulbifera L., which allowed us to detect additional six potential furanoids, including diosbulbin A, diosbulbin D, diosbulbin E, diosbulbin F, diosbulbin M, and diosbulbin D glycoside. The achievements of this study enhanced the sensitivity to screen potential toxic furanoids through elevating S/N values by approximately 3 times. This will facilitate the understanding of mechanisms of toxic actions of D. bulbifera L. and other furanoid-containing toxic herbal medicines.
Keywords: Furanoids; Reactive intermediates; Dioscorea bulbifera L.; LC–Q-TOF MS; LC–Q-q-Q MS;

Simultaneous determination of multi-class veterinary drugs in chicken processed foods and muscle using solid-supported liquid extraction clean-up by Souichi Yoshikawa; Chieko Nagano; Maki Kanda; Hiroshi Hayashi; Yoko Matsushima; Takayuki Nakajima; Yumi Tsuruoka; Marie Nagata; Hiroshi Koike; Kotaro Sekimura; Tsuneo Hashimoto; Ichiro Takano; Tetsuya Shindo (15-23).
We developed a simultaneous determination method for 37 veterinary drugs in two chicken processed foods (deep-fried chicken and non-fried chicken cutlet) and muscle via liquid chromatography–mass spectrometry. The veterinary drugs belong to 7 different classes, including 4 antifolics, 4 benzimidazoles, 5 macrolides, 7 polyethers, 2 quinolones, 7 sulfonamides, and 8 other classes. The samples were extracted with ethyl acetate followed by acetonitrile with salt and buffers extraction. The two-step extraction enabled analyte extraction from highly lipid samples. The clean-up procedure, a solid-supported liquid extraction clean-up using a diatomaceous earth mini-cartridge, eliminated lipid co-extraction. The prepared sample matrix did not have an effect on the 36 analytes. The method was validated in accordance with the requirements of Japanese validation guidelines. Almost all targeted veterinary drugs successfully satisfied the guideline criteria in the three types of food matrices. The method exhibited recoveries of 70–105%, and the precision of repeatability and within-laboratory reproducibility ranged from 1 to 11% and 1 to 15%, respectively. The limits of quantification were estimated to range from 0.2 to 1.0 μg/kg. Applying this method to samples commercially available in Tokyo, residues were detected in 3 out of 26 deep-fried chickens, 5 out of 20 non-fried chicken cutlets, and 17 out of 39 chicken muscles.
Keywords: Veterinary drugs; Chicken processed foods; Solid-supported liquid extraction clean-up; Two-step extraction; LC–MS/MS;

Exploratory metabolomics of biomarker identification for the internet gaming disorder in young Korean males by Yeo Ul Cho; Deokjong Lee; Jung-Eun Lee; Kyoung Heon Kim; Do Yup Lee; Young-Chul Jung (24-31).
The main aim of the current research is to characterize the molecular dynamics related to internet gaming disorder (IGD) using non-targeted plasma metabolite profiling based on gas-chromatography time-of-flight mass spectrometry (GC-TOF MS). IGD is a psychiatric disorder instigated by excessive and prolonged internet gaming, which shared many pathological symptoms with attention deficit hyperactivity disorder (ADHD). The prevalence of the disorder has been rapidly increased particularly in East Asia countries (5.9% in South Korea) compared to Europe or North America (0.3–1.0% in United States and 1.16% in Germany). Thus we comparably explored the correlation between plasma metabolites and internet addiction severity in IGD patients, and potential biomarker composite in combination with clinical parameters. The systematic metabolite profiling of 54 blood samples (normal user, N  = 28 and IGD, N  = 24) identified a total of 104 metabolites out of 1212 metabolic feature, and revealed unique relation of co-linearly regressed set of plasma metabolites (arabitol, myo-inositol, methionine, pyrrole-2-carboxylic acid, and aspartic acid) with internet addiction severity scale (R = 0.795). In addition, orthogonal partial least squared discriminant analysis (OPLS-DA) and receiver operating characteristic (ROC) analysis identified the potential biomarker cluster that simultaneously discriminated the different types of the psychiatric status. The potential biomarker re-composite was comprehensively evaluated by a receiver operating characteristic (ROC) analysis where the AUCs were 0.890, 0.880, 1.000, and 0.935 for control, IGD, AD and IGD + AD, respectively (N  = 18, 19, 5, and 10) against the others. This exploratory method may provide robustness of predictive diagnosis in population screening of IGD. The identified metabolic features, the relatedness with clinical parameters, and the putative biochemical linkage will hopefully aid future pathological studies in IGD.
Keywords: Biomarker cluster; Plasma metabolite; Internet addiction severity; Gas chromatography time-of-flight mass spectrometry (GC-TOF MS); Internet gaming disorder (IGD);

An RP-HPLC-PDA method for the simultaneous analysis of 9 compounds deriving from the phytohormones class was developed and optimized, namely indoleacetic acid (IAA), indolebutyric acid (IBA), phenyleacetic acid (PAA), naphtyleacetic acid (NAA), trans-zeatin (TZ), kinetin (KA), isopentenyladenine (IA), 6-benzylaminopurine (6-BA) and abscisic acid (ABA). Validation of the method was performed on the SFE-CO2 extract made out of the mixture of Baltic algae. The regression coefficients for plant hormones were in the range from 0.997 to 0.999. The LOD and LOQ were on the levels from 0.05–0.29 and 0.15–0.88 mg/L, respectively. Developed method was used for the separation and determination plant hormones in extracts obtained by SFE-CO2 (supercritical fluid extraction) made out of the mixture of Baltic algae, Cladophora glomerata and Spirulina sp. In the extract of Baltic seaweed 2 of tested compounds were present in the concentration of 154,45 ± 20,63 μg/g for TZ and 362,47 ± 13,00 μg/g for PAA, whereas in Cladophora glomerata extract contained PAA and IAA in the concentration of 229,30 ± 7,90 μg/g and 23,91 ± 0,80 μg/g, respectively (all values per g of extract). The differences in the hormones levels may occur due to the different scale of extract preparation (laboratory or industrial) and other factors like the place of algae collection, year of collection or the way of biomass preparation.
Keywords: Phytohormones; Seaweed extracts; RP-HPLC-PDA; SFE-CO2 seaweed extract;

Comparative metabolomics analysis for the compatibility and incompatibility of kansui and licorice with different ratios by UHPLC-QTOF/MS and multivariate data analysis by Juan Shen; Zong-Jin Pu; Jun Kai; An Kang; Yu-Ping Tang; Li-Li Shang; Gui-Sheng Zhou; Zhen-Hua Zhu; Er-Xin Shang; Shao-Ping Li; Yu-Jie Cao; Wei-Wei Tao; Shu-Lan Su; Li Zhang; Huiping Zhou; Da-Wei Qian; Jin-Ao Duan (40-45).
Kansui, the root of Euphorbia kansui T.N. Liou ex T.P. Wang (Euphorbiaceae), is a well-known poisonous traditional Chinese medicine (TCM). However, many monographs of TCM indicated that it cannot be co-used with licorice, as kansui-licorice is a typical “eighteen incompatible” medicaments. Our previous studies have indicated that kansui was effective in treating malignant pleural effusion (MPE), and the efficacy could be weakened by the co-use of licorice, even causing serious toxicity at the given ratio. Nevertheless, the actual mechanisms of their dosage-toxicity-efficacy relationship need to be well clarified. The present study aimed to investigate the effect of individual and combined use of kansui and licorice on MPE rats, and explain the underlying mechanisms from a metabolomic perspective. Urine samples were analyzed by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the interaction between kansui and licorice. Seven potential biomarkers contribute to the separation of model group and control group were tentatively identified. And selenoamino acid metabolism and nicotinate and nicotinamide metabolism with the impact-value 0.31 and 0.24, respectively, were filtered out as the most important metabolic pathways. Kansui and kansui-licorice at a ratio of 4:1 can treat MPE rats by adjusting abnormal metabolic pathways to the normal state, while it may have opposite result with kansui-licorice 1:4. The different influences to the two metabolic pathways may partially explain the dosage-toxicity-efficacy relationship of kansui-licorice with different ratios. The results could offer valuable insights into the compatibility property changes for the two herbs.
Keywords: Kansui; Licorice; Malignant pleural effusion; Incompatibility; Metabolomics; UHPLC–QTOF/MS;

This study aims to establish a fast and sensitive LC–MS/MS method for simultaneous determination of seven alkaloids from Rhizoma Corydalis Decumbentis in rabbit aqueous humor. Aqueous humor samples were processed by protein precipitation and then separated on a Thermo Syncronis C18 column (50 mm × 2.1 mm, 5 μm) with a mobile phase using acetonitrile-0.05% formic acid (28:72, v/v). Detection of the analytes and the internal standard (coptisine) were performed in positive electrospray ionization with selected reaction monitoring. The method showed good linearity (r  > 0.9931) for all the seven alkaloids. This fully validated method was applied to the studies of aqueous humor pharmacokinetics of seven alkaloids from Rhizoma Corydalis Decumbentis and the effects of borneol on corneal penetration of these alkaloids into aqueous humor. This is the first work that presents a reliable LC–MS/MS method for simultaneous determination of seven alkaloids in rabbit aqueous humor and its application of ocular pharmacokinetics of seven alkaloids from Rhizoma Corydalis Decumbentis.
Keywords: Alkaloids; LC–MS/MS; Pharmacokinetics; Rabbit aqueous humor; Rhizoma Corydalis Decumbentis;

Mass spectral characterization of tabun-labeled lysine biomarkers in albumin by Fengjuan Sun; Junjie Ding; Xiaogang Lu; Runli Gao; Xiaojing Lu; Enxue Shi; Hongmei Wang; Chengxin Pei (54-61).
Tabun has been shown to form phosphylated adducts on tyrosine residues in albumin in vivo and in vitro. However, in this work, tabun-labeled lysine adducts were found in albumin. Three types of albumin were treated with overdose of tabun in vitro and 17 tabun-labeled lysine residues were found: K4, K12, K224, K377, and K524 in bovine albumin, K186, K188, K212, K329, K414, and K525 in leporine albumin, and K79, K186, K188, K212, K376, and K525 in rat albumin. To investigate the modification of tabun in vivo, three leporines were injected with 0.8 × LD50 dose of tabun. The results showed that the labeled lysine residues in vivo, were consistent with modified lysines in vitro. Structure characteristics and the binding mode of 6 tabun-labeled lysines of leporine albumin were further analyzed using theory simulation and molecular docking in Discovery Studio. For the first time, we show that tabun-labeled lysine peptides are found in vivo and in vitro. These modified lysine peptides are good biomarkers for exposure to tabun in albumin of leporine and rat.
Keywords: Tabun; Labeled-lysine adducts; Biomarker; Molecular modeling;

Development of suitable method for large-scale urinary glucocorticoid analysis by liquid chromatography–mass spectrometry by Akiko Matsumoto; Chisato Shimanoe; Keitaro Tanaka; Masayoshi Ichiba; Megumi Hara (62-69).
Levels of urinary glucocorticoids and their concentration ratios have been analyzed as potential markers for various pathological statuses. Large-scale studies may possibly accelerate the investigations; however, a suitable method needs to be established. Analytical conditions for measurement of urinary glucocorticoids with LCMS were examined. Electrospray ionization in the positive ion mode was applied for detection of cortisol (precursor > product ion: 363.3 > 121.0), cortisol-d4 (internal standard, IS, 367.4 > 121.1), and cortisone (361.2 > 163.2). To maximize ionization, acetic acid-ammonium acetate buffer (18 mM) at pH 5.3 was employed as eluent A. A C18 column (100 mm × 2.1 mm, 2.7 μm) at 50 °C was used for the 9.5 min binary gradient separation starting with 60% eluent A with methanol being eluent B. Linear correlations were observed between the concentrations and the peak areas in the concentration range of 1–300 ng/mL with correlation coefficients (r) of 0.998 and 0.997 for cortisol and cortisone, respectively, without IS adjustment, and 0.999 with IS adjustment for both cortisol and cortisone. Solid-phase extraction (SPE) using a 2 mL centrifuge column was performed for the urine samples, with the original and final volumes being 100 μL. The SPE of 12 urine specimens could be performed within 30 min. The effect of the sample matrix on the quantification of endogenous compounds present in the urine extract was limited (coefficient of variation (CV) of IS-adjusted matrix factor: 4.4–8.1%; urine extracts of 8 individuals); however, substantial peak reduction of cortisol was observed at low concentrations. Exogenous contaminants originating from the SPE centrifuge column seemed to be a main cause for this phenomenon because the pure-water extract showed similar peak reduction. A recovery of ∼50% was obtained for both cortisol and cortisone. Adjustment with the IS improved the apparent recovery, with ∼100% being obtained for both cortisol and cortisone. The recovery rate decreased when the urine samples were concentrated in the SPE step; the reduction was greater for cortisol than for cortisone. The lower limit of quantification (LLOQ) was set at 2.5 ng/mL when the injection volume was 10 μL, based on the reproducibility of the standards which were measured (CV of 12 repetitions: 10.1% for 0.5 ng/mL cortisol and 19.6% for 1 ng/mL cortisone), the matrix effect (−55% at 2 ng/mL concentrations of cortisol), and the recovery rate (∼50%). Furthermore an alternative approach for preparation of the cortisol standards was required for low concentration range (2.5–20 ng/mL) because of the effect of the matrix. Degradation of original urine specimens at room temperature was minimal during the first 24 h. The extracted urine samples degraded over time; however, their concentrations were corrected with the IS, allowing for analysis up to 5 days after extraction. In conclusion, an analytical method for urinary glucocorticoids was established, which is fast, sensitive, and well suited for practical application to large-scale study.
Keywords: Cortisol; Cortisone; Cortisol/cortisone ratio; LCMS; Solid phase extraction; Urine;

Short-chain fatty acids (SCFAs) of intestine microbial have caught accumulating attention for their beneficial effects on human health. Botanic compounds with low bioavailability such as berberine (BBR) and resveratrol might interact with intestinal microbial ecosystem and promote gut bacteria to produce SCFA, which contribute to their biological effects. In the present study, a comprehensive assay system was built to detect SCFAs production in intestinal bacteria, in which stringent anaerobic culture was applied for in vitro bacterial fermentation, followed by direct-injection GC detection (chemical detection) in combination with real time polymerase chain reaction (RT-PCR, biological detection). BBR was used as positive reference. The direct injection GC method was calibrated and successfully applied to analyze the concentration of SCFAs in gut microbiota and BBR was proved to be effective in the dose- and time-dependent up-regulation of SCFAs production. As compared to the saline group, the concentration of acetic acid, propionate acid and butyric acid (the main SCFAs in gut microbiota) were increased by 17.7%, 11.1% and 30.5%, respectively, after incubating intestinal bacteria with 20 μg/mL BBR for 24 h. The increase reached to 34.9%, 22.4% and 51.6%, respectively when the BBR was 50 μg/mL. Additionally, consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) were designed for the detection of acetate kinase (ACK), Methylmalonyl-CoA decarboxylase (MMD) and butyryl-CoA: acetate-CoA transferase (BUT), as they are the key enzymes in the synthetic pathway for acetic acid, propionate acid and butyric acid, respectively. After 24hr’s incubation, BBR was shown to promote the gene expression of ACK, MMD and BUT significantly (86.5%, 27.2% and 60.4%, respectively, with 20 μg/mL BBR; 130.2%, 84.2% and 98.4%, respectively, with 50 μg/mL BBR), showing a solid biological support for the chemical detection. This comprehensive assay system might be useful in identifying SCFAs promoting agents with information on their mechanism.
Keywords: Intestinal bacteria; In vitro fermentation; Short-chain fatty acids; Gas chromograph; Berberine;

A high sensitive and reliable multiresidue method was developed and validated for determination of twelve sulfonamides and five acetylated metabolites in baby foods by using UHPLC-Orbitrap-MS. The sample preparation included two extraction methods as QuEChERS and ASE. The original QuEChERS method was performed, whereas ASE method was optimized to achieve the best recovery for all analytes. UHPLC-Orbitrap-MS parameters were optimized to obtain good retention and separation in LC and high sensitivity and selectivity in MS. The performance of the method was evaluated according to the European Commision Decision 2002/657/EC. Matrix matched calibration curves showed good r2 (≥0.999) and LOQ was ranged from 0.10 to 0.55 μg/kg. ASE was significantly more effective for extraction of the analytes (recovery ranged from 75.5 to 96.6% with RSD ≤ 10.1%) than QuEChERS (recovery ranged from 60.9 to 85.9% with RSD ≤ 19.1%). This method was applied to the analysis of 47 different baby foods and no positive samples were found.
Keywords: Sulfonamide; Orbitrap-exactive; ASE; QuEChERS; Baby foods; Validation;

Effects of breviscapine on amyloid beta 1-42 induced Alzheimer’s disease mice: A HPLC-QTOF-MS based plasma metabonomics study by Hongjun Xia; Lingling Wu; Mengying Chu; Huimin Feng; Chunliang Lu; Qinghe Wang; Minghai He; Xiaoqun Ge (92-100).
Herba Erigerontis has long been used to cure apoplexy hemiplegia and precordial pain in China. In addition, the bioactivities of its total flavonoids-breviscapine included inhibiting amyloid beta (Aβ) fibril formation, antioxidation and metal chelating, which are beneficial to treat Alzheimer’s disease (AD). Hence, A HPLC-QTOF-MS based plasma metabonomics approach was applied to investigate the neuroprotective effects of breviscapine on intracerebroventricular injection of aggregated Aβ 1-42 induced AD mice for the first time in the study. Ten potential biomarkers were screened out by multivariate statistical analysis, eight of which were further identified as indoleacrylic acid, C16 sphinganine, LPE (22:6), sulfolithocholic acid, LPC (16:0), PA (22:1/0:0), taurodeoxycholic acid, and PC (0:0/18:0). According to their metabolic pathways, it was supposed that breviscapine ameliorated the learning and memory deficits of AD mice predominantly by regulating phospholipids metabolism, elevating serotonin level and lowering cholesterols content in vivo.
Keywords: Breviscapine; Alzheimer’s disease; Mice; Amyloid beta 1-42; Metabonomics; HPLC-QTOF-MS;

Simultaneous determination of selective serotonin reuptake inhibitors and their main metabolites in human breast milk by liquid chromatography-electrospray mass spectrometry by Etienne Weisskopf; Alice Panchaud; Kim An Nguyen; Delphine Grosjean; Jean-Michel Hascoët; Chantal Csajka; Chin B. Eap; Nicolas Ansermot (101-109).
Display OmittedA bioanalytical method by high performance liquid chromatography coupled to electrospray mass spectrometry (HPLC-ESI–MS), adapted from a previously published method in plasma, was validated in breast milk for the simultaneous quantification of all antidepressants belonging to the class of selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their major metabolites (desmethylcitalopram and norfluoxetine). Milk samples (250 μl) first underwent protein precipitation followed by solid-phase extraction on a reversed phase/cation exchange sorbent. Analytes were thereafter separated on a XBridge C18 column (2.1 mm × 100 mm; 3.5 μm) using a mobile phase composed of ammonium acetate buffer (pH 8.1; 50 mM) and acetonitrile in gradient mode. Detection was performed by a single quadrupole mass spectrometer running in selected ion monitoring in positive ionization mode. Method validation covered a wide concentration range of 2–500 ng/ml for citalopram, desmethylcitalopram and paroxetine, 5–500 ng/ml for sertraline, and 2–1000 ng/ml for fluoxetine, norfluoxetine and fluvoxamine. Validation performances such as trueness (90.3–111.6%), repeatability (0.8–9.3%) and intermediate precision (0.9-9.5%) were in agreement with criteria from international guidelines and matrix effects for the analyte/internal standard ratios ranged from 92% to 110% (relative standard deviation <15%). Accuracy profiles (total error of trueness and precision) were lying within the limits of ±30% accepted in bioanalysis. Finally, the method was successfully applied to patient samples collected in a clinical pharmacokinetic study of nursing mothers taking an antidepressant treatment.
Keywords: Antidepressants; SSRI; LC–MS; Breast milk; Quantification;

LC–MS/MS methods to measure ribociclib in mouse plasma and Ringer’s solution were successfully developed and validated. Reverse phase chromatography was performed with gradient elution using C18 (100A, 50 × 4.6 mm, 3 μ) and C8-A (50 × 2.0 mm, 5 μ) columns for plasma and Ringer’s samples, respectively. Mouse plasma samples were extracted using solid phase extraction method, whereas no extraction was required for the Ringer’s solution samples. Analytes were detected using positive ion MRM mode. The precursor to product ions (Q1 → Q3) selected for ribociclib and d6-ribociclib were (m/z) 435.2 → 252.1 and 441.2 → 252.1, respectively. The linear range of quantification of ribociclib was 62.5–10,000 ng/ml for plasma method and 0.1–100 ng/ml for Ringer’s solution method. The results for the inter-day and intra-day accuracy and precision of quality control samples were within the acceptable range. The lower limit of quantitation (LLOQ) for plasma and Ringer’s samples were 62.5 ng/ml (S/N > 30) and 0.1 ng/ml (S/N > 13), respectively, whereas the limit of detection (LOD) was 6.9 ng/ml (S/N > 7) and 0.05 ng/ml (S/N > 3), respectively. The developed methods were successfully applied to the analysis of ribociclib in mouse plasma and dialysate samples collected during a cerebral microdialysis study of ribociclib in a non-tumor bearing mouse.
Keywords: Ribociclib; CDK4/6; LC–MS/MS; Solid phase extraction; Microdialysis; Ringer’s solution;

Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography by Marianne L. Bergmann; Seyed Sadjadi; Anne Schmedes (118-123).
The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography − tandem mass spectrometry (LC–MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC–MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60 mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC–MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns.
Keywords: Catecholamine; Urine; Mass spectrometry; Ion-pairing chromatography; Epinephrine; Norepinephrine;