Journal of Chromatography B (v.985, #C)

An UPLC-MS/MS method for determination of solasonine in rat plasma and its application of a pharmacokinetic and bioavailability study by Yuanyuan Chen; Shuangshuang Zhang; Dahui Chen; Mi Zhou; Jing Zheng; Zheng Xiang (1-5).
Solasonine, a known glycoalkaloid, is a potential anti-cancer agent. In this work, a simple, sensitive and fast ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitative determination of solasonine in rat plasma. Plasma samples were processed with a protein precipitation. The separation was achieved by an ACQUITY HSS T3 (100 × 2.1 mm, 1.8 μm) column with a gradient mobile phase consisting of 0.1% formic acid and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM). The validated method had an excellent linearity in the range of 0.1–500 ng/mL (R 2  > 0.994) with a low limit of detection (0.1 ng/mL) and lower limit of quantification (0.5 ng/mL). The extraction recovery was in the range of 92.4–94.9% for solasonine and 91.9% for dendrobine (internal standard, IS). The intra- and inter-day precision was below 9.8% and accuracy was from 86.0% to 94.3%. No notable matrix effect and astaticism was observed for solasonine. The method has been successfully applied to a pharmacokinetic and bioavailability study of solasonine in rats for the first time, which provides the basis for the further development and application of solasonine.
Keywords: Solasonine; Pharmacokinetics; UPLC-MS/MS; Bioavailability;

Development and validation of a LC–MS/MS method for the quantitation of lumefantrine in mouse whole blood and plasma by Katya Govender; Liezl Gibhard; Lissinda Du Plessis; Lubbe Wiesner (6-13).
A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantitation of the antimalarial drug, lumefantrine (LF), in mouse whole blood and plasma. The analyte was extracted using a protein precipitation method followed by chromatographic separation on a Phenomenex Luna, PFP (50 mm × 2.0 mm, 5 μm) analytical column with a mobile phase consisting of acetonitrile and 0.1% formic acid (formic acid:water, 1:1000, v/v) at a ratio of 3:7 (v/v), delivered at a constant flow rate of 0.5 ml/min. Stable isotope labeled lumefantrine (D9-LF) was used as the internal standard. Multiple reaction monitoring was performed using the transitions m/z 530.1 →  m/z 347.9 and m/z 539.1 →  m/z 347.9 for the quantification of LF and D9-LF, respectively. Calibration curves were constructed over the concentration range 15.6–4000 ng/ml. The mean intra- and inter-assay accuracy values for the analysis of LF in WB was 103% (%CV = 5.5) and 99.5% (%CV = 5.5), respectively. The mean intra- and inter-assay accuracy values for the analysis of LF in plasma was 93.7% (%CV = 3.5) and 93.9% (%CV = 5.5), respectively. No significant matrix effect was observed during the method validation. The validated method was applied to an absorption study in mice, to determine and compare LF concentrations in whole blood and plasma samples. Results of the statistical analysis using a linear mixed effects growth curve model concluded that there was no significant difference (p-value = 0.668) between WB and plasma LF concentrations. This method utilizes a small sample volume of 20 μl, facilitating low blood collection volumes and a short chromatographic run time of 3 min which allows for high sample throughput analysis.
Keywords: Antimalarial; Lumefantrine; LC–MS/MS; Method validation;

Fangji Huangqi Tang (FHT) is a classical formula widely used in Chinese clinical application. In this paper, a novel and advanced strategy has been developed for the multiple constituent identification of FHT in rats, which was basing on an ultra-high performance liquid chromatography equipped with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS) method combined with dynamic background subtract (DBS) data acquisition and enhance peak list (EPL) data processing techniques. Firstly, a total of 58 potential bioactive compounds including alkaloids, flavonoids, saponins, saccharides and terpenoids were detected from FHT. Their chemical structures were identified by comparing the retention time and mass spectrometry data, as well as retrieving the reference literatures. Based on the same instrumental conditions, 33 compounds were found in rat serum after oral administration of FHT. After a considerate comparison with the former chemical identification results of FHT, 33 compounds were found, which turned out to be 8 original compounds of FHT as well as 25 metabolites, including 20 phase I and 5 phase II metabolites. The results indicated that the metabolic reactions included hydroxylation, hydrogenation, demethylation, tarine conjugation and acetylation. This study firstly reported the metabolism description of fangchinoline and tetrandrine in vivo, which could be very useful for further pharmacological and clinical studies of FHT. Meanwhile, it provided a practical strategy for rapid screening and identifying of multiple constituents and their metabolites of complex traditional Chinese medicine in biological matrix.
Keywords: Fangji Huangqi Tang; UHPLC-ESI-Q-TOF-MS; Metabolite; Dynamic background subtract; Enhance peak list;

Centrifugal partition chromatography elution gradient for isolation of sesquiterpene lactones and flavonoids from Anvillea radiata by Emilie Destandau; Meryem Alaoui Boukhris; Sandrine Zubrzycki; Mohamed Akssira; Lhoucine El Rhaffari; Claire Elfakir (29-37).
An innovative procedure coupling pressurized solvent extraction and centrifugal partition chromatography (CPC) used in linear gradient elution mode was developed to isolate two pure germacranolides (9α-hydroxyparthenolide and 9β-hydroxyparthenolide) and to separate flavonoids (nepetin, isorhamnetin and jaceosidin) and chlorophyll pigments from aerial parts of Anvillea radiata (Coss.&Durieu). The two main germacranolides recovered using this method represent 2 and 5% of the dried plant material respectively. These molecules were extracted using accelerated solvent extraction with chloroform. After optimization of the CPC method, a two-phase solvent system composed of heptane/ethyl acetate/methanol/water (1:5:1:5 v/v/v/v) was employed in descending mode to isolate the germacranolides. Then the lower phase of a heptane/ethyl acetate/methanol/water (6:5:6:5 v/v/v/v) system was pumped in descending mode to generate a linear elution gradient, progressively decreasing the mobile phase polarity, that enabled the flavonoid compounds to be separated in the same run. The efficiency of the preparative separation was controlled through RP-HPLC analysis of the obtained fractions using UV, evaporative light scattering and mass spectrometry detection. The structural identification of the two germacranolides purified over 99% was established by 1H NMR and 13C NMR. The least abundant flavonoids were identified by mass spectrometry.
Keywords: Anvillea radiata (Coss.&Durieu); Asteraceae; Hydroxyparthenolides; Flavonoids; Mass Spectrometry; CPC gradient elution;

Curcumin, a yellow pigment derived from the rhizomes of Curcuma longa Linn, is a natural antioxidant that exhibits a variety of pharmacological activities and therapeutic properties. However, as curcumin is generally conjugated when absorbed through the intestine, free curcumin is present at extremely low levels in the body. Thus, curcumin metabolites are presumed to be responsible for curcumin bioactivity. In this study, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF MS) with automated data analysis software (MetaboLynxXS) for rapid analysis of the metabolic profile of curcumin in human intestinal flora. The results show that curcumin undergoes extensive phase I and phase II metabolism. A total of 23 curcumin metabolites were detected and identified in vitro. Furthermore, we identified a number of novel metabolic pathways of curcumin in the human intestinal microflora system.
Keywords: Extrahepatic metabolism; Human intestinal microflora system; Curcumin; UPLC–Q-TOF MS; MetaboLynxXS; Metabolite;

Iron (Fe) is an essential element for higher plants, which take it up from the soil at the root surface and transport it to shoots through the xylem. Fe(III) chelators, such as organic acids and phytosiderophores, play important roles in the acquisition and transportation of Fe(III). Therefore, a selective and sensitive method for analyzing Fe(III) chelators is required to study the many Fe-related physiological mechanisms in plants. A novel analytical approach employing a high-performance liquid chromatography post-column method with fluorescence detection was developed to separate and detect Fe(III) chelators. This method takes advantage of the quenching of the fluorescence of Calcein Blue (CB) that occurs with the formation of an Fe(III)–CB complex and the dequenching that occurs with the release of CB as a result of competition for Fe(III) between CB and an Fe(III) chelator. This simple experimental method does not require complicated pretreatments and can selectively detect Fe(III) chelators according to their Fe(III)-chelating ability. The detection limit for citric acid using this method was 72 pmol. Furthermore, this method can also detect unknown Fe(III) chelators that exhibit a high affinity for Fe(III). The method was evaluated with xylem sap of barley, which was shown to contain several Fe(III) chelators.
Keywords: Amino acids; Carboxylic acids; Fluorescence quenching; HPLC; Organic acids; Phytosiderophores;

Protective effects of ginsenosides Rg1 and Rb1 on an Alzheimer's disease mouse model: A metabolomics study by Naijing Li; Ling Zhou; Wei Li; Ying Liu; Jiahe Wang; Ping He (54-61).
Alzheimer's disease (AD) is a neurodegenerative disease that affects aging populations, with no effective method for its treatment to date. Elucidating the changes in biofluid small metabolite concentration during AD could improve both diagnosis and treatment. Ginsenosides Rg1 (GRg1) and Rb1 (GRb1), the pharmacologically active ingredients in ginseng, reportedly offer protection against AD symptoms, but the underlying mechanisms are unclear. These were investigated in the present study by examining the metabolite profiles of AD mice. The cognitive function of mice was tested in the Morris water maze and pathological changes to the brain were assessed by immunohistochemistry. Global metabolite profiling by ultra performance liquid chromatography/mass spectrometry and principal component analysis were carried out to identify metabolites that are differentially expressed in AD animals with or without ginsenosides treatment. A total of 10 potential biomarkers were identified that were associated with the metabolism of lecithin, amino acids, and sphingolipids in AD mice. The peak intensities of lysophosphatidylcholine, tryptophan, and dihydrosphingosine were lower, while that of phenylalanine was higher, in AD than in control mice. GRg1 treatment affected all three metabolic pathways, while GRb1 treatment affected lecithin and amino acid but not sphingolipid metabolism. These results provide the first evidence of a link between metabolite imbalance and AD, and reveal a molecular basis for the therapeutic benefits of ginsenosides in AD treatment.
Keywords: Alzheimer's disease; Metabolomics; UPLC–MS; Ginsenosides Rg1 and Rb1;

Herein, we developed a novel integrated device to perform phase separation based on ultrasound-assisted salt-induced liquid–liquid microextraction for determination of five fluoroquinones (FQs) in human body fluids. The integrated device consisted of three simple HDPE components used to separate the extraction solvent from the aqueous phase prior to retrieving the extractant. A series of extraction parameters were optimized using the response surface method based on central composite design. Optimal conditions consisted of 945 μL acetone extraction solvent, pH 2.1, 4.1 min stir time, 5.9 g Na2SO4, and 4.0 min centrifugation. Under optimized conditions, the limits of detection (at S/N = 3) were 0.12–0.66 μg L−1, the linear range was 0.5–500 μg L−1 and recoveries were 92.6–110.9% for the five FQs extracted from plasma and urine. The proposed method has several advantages, such as easy construction from inexpensive materials, high extraction efficiency, short extraction time, and compatibility with HPLC analysis. Thus, this method shows excellent prospects for sample pretreatment and analysis of FQs in human body fluids.
Keywords: Integrated microextraction device; Phase separation; Fluoroquinolones; Central composite design; Ultrasound-assisted salt-induced liquid–liquid microextraction; Plasma and urine testing;

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine ruscogenin in rat plasma using midazolam as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate to 0.2 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. Ruscogenin and IS were eluted at 1.74 and 1.11 min, respectively. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 431.2 → 287.0 for ruscogenin and m/z 326.2 → 291.1 for IS. The linearity of this method was found to be within the concentration range of 2–1000 ng/mL with a lower limit of quantification of 2 ng/mL. Only 2.0 min was needed for an analytical run. The matrix effect was 92.4–107.3% for ruscogenin. The intra- and inter-day precision (RSD%) were less than 11.2% and accuracy (RE%) was within ±9.8%. The recovery ranged from 75.4% to 86.3%. Ruscogenin was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of ruscogenin in rats.
Keywords: Ruscogenin; UPLC-MS/MS; Rat plasma; Pharmacokinetics;

Although mercury (Hg) is an important environmental and occupational pollutant, its toxicological effects, especially in serum and red blood cells (RBCs), have been scarcely studied. A toxicometabolomics workflow based on high resolution mass spectrometry approaches has been applied to investigate the toxicological effects of Hg in Mus musculus mice after subcutaneous injection for 10 days, which produced inflammation and vacuolization, steatosis and karyolysis in the hepatic tissue. To this end, direct infusion mass spectrometry (DIMS) of polar and lipophilic extracts from serum and RBCs, using positive and negative mode of acquisition (ESI+/ESI−), and gas chromatography–mass spectrometry were used. A quantitative analysis of reversible oxidized thiols in serum proteins demonstrated a strong oxidative stress induction in the liver of Hg-exposed mice. Endogenous metabolites alterations were identified by partial least squares-discriminant analysis (PLS-DA). Mercury-exposed mice show perturbations in energy metabolism, amino acid metabolism, membrane phospholipid breakdown and oxidative stress-related metabolites in serum along the exposure. This work reports for the first time the effects of Hg-exposure on RBCs metabolic pathways, and reveals disturbances in glycolysis, membrane turnover, glutathione and ascorbate metabolisms.
Keywords: Toxicometabolomics; Red blood cells; Serum; Mercury; Mus musculus; Mass spectrometry;

A new method based on ultra high performance liquid chromatography (UPLC) with photometric and fluorometric detection for the determination of acetylsalicylic acid and its main metabolites, namely gentisic, salicylic and salicyluric acids, in bovine urine samples is reported. Photometric detection was used for acetylsalicylic acid determination, whereas the native fluorescence of the metabolites was monitored using fluorometric detection. The separation was performed under isocratic conditions, using acetonitrile–phosphate solution (3.5 mM, pH 3.5) (26:74, v/v) as the mobile phase. The retention times of the four compounds were lower than 2 min, which are shorter than those achieved using conventional HPLC. Under the optimum separation conditions, the dynamic ranges and detection limits (ng mL−1) were: 0.2–2500, 0.09 for gentisic acid; 0.2–2500, 0.08 for salicylic acid and 2.5–15,000, 1.1 for salicyluric acid, using fluorescence detection, and 10–25,000, 2.2 for acetylsalicylic acid, using UV detection. Intra-day and inter-day precision data were assessed at two levels of concentration of each analyte using both detection systems. The selectivity of the method was checked by assaying different drugs of veterinary use showing that most of them did not interfere with the determination of the analytes. The method has been applied to the analysis of bovine urine samples, which only required a simple clean up step of the samples prior to injection in the UPLC system. A recovery study was performed, which provided values in the range of 80–100%. This fact proves the practical usefulness of this method as an ultrafast analytical tool for the therapeutic control of acetylsalicylic acid administration in bovine animals intended for food production.
Keywords: Acetylsalicylic acid; Major metabolites; Ultraperformance liquid chromatography; Photometric and fluorometric detection; Bovine urine samples;

Baicalin (baicalein 7-O-glucuronide), which is one of the major bioactive constituents isolated from Scutellariae Radix, possesses many biological activities, such as antiallergic, antioxidation, and anti-inflammatory activities. In the present study, an efficient strategy was established using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap MS) to profile the in vivo metabolic fate of baicalin in rat plasma, urine, and various tissues. A combination of post-acquisition mining methods including extracted ion chromatogram (EIC) and multiple mass defect filters (MMDF) was adopted to identify the common and uncommon baicalin metabolites from the full mass scan data sets. Their structures were elucidated based on the accurate mass measurement, relevant drug biotransformation knowledge, the characteristic collision induced fragmentation pattern of baicalin metabolites, and bibliography data. Based on the proposed strategy, a total of 32 metabolites were observed and characterized. The corresponding reactions in vivo such as methylation, hydrolysis, hydroxylation, methoxylation, glucuronide conjugation, sulfate conjugation, and their composite reactions, were all discovered in the study. The results demonstrated that the rat liver and kidney are the most important organs for the baicalin metabolites presence. Six metabolites might play an important role in exerting pharmacological effects of baicalin in vivo. The newly discovered baicalin metabolites significantly expanded our understanding on its pharmacological effects, and could be targets for future studies on the important chemical constituents from herbal medicines.
Keywords: Baicalin; UPLC-ESI-MS/MS; Metabolic profile; Distribution; Multiple data processing method;

A gas chromatography/mass spectrometric method was developed for the simultaneous determination of eight phthalic acid esters (PAEs) in toys and child care articles that are made of plasticized plastic. The novel method was used to determine the total concentration of the PAEs in addition to the migrated PAEs values into artificial saliva, under conditions that simulate real life situations. The extraction method, which was developed for the first time to determine the total concentration of PAEs, utilized a novel optimization of four parameters involving the solvent, time, temperature and weight of sample. The PAEs were extracted with tetrahydrofuran, as extraction the solvent, and using the ultrasonic water bath shaker for 30 min, at room temperature. Another extraction method was developed to determine the migrated PAEs into artificial saliva at pH 6.2 and 37 °C, implementing a liquid–liquid extraction with chloroform. Both methods were subjected to validation steps in terms of linearity, precision, accuracy and recovery, which ensured that all obtained results were well within the norms of acceptable limits and specifications. The analytes were separated at the following retention times: 4.99, 5.21, 5.31, 6.63, 7.41, 9.05 min for di-“isobutyl” phthalate (DIBP), dibutyl phthalate (DBP), bis(2-methoxyethyl)phthalate (DMEP), benzyl butyl phthalate (BBP), dibutyl phthalate (DEHP) and di-n-octyl phthalate (DnOP), respectively. The chromatographic peaks corresponding to di-“isononyl” phthalate (DINP) and di-“isononyl” phthalate (DIDP), were separated, using the extracted ion chromatogram (EIC) mode within the time ranges of 8.05–12.10 min for DINP and 8.50–14.50 min for DIDP. The instrument detection limits for DIBP, DBP, DMEP, BBP, DEHP, DnOP, DINP and DIDP were determined at 0.100, 0.100, 0.045, 0.035, 0.015, 0.370, 0.320, 0.260 μg/ml, respectively. The calibration curve working ranges were determined at 0.5–25 μg/ml for DIBP, DBP, DMEP, BBP and DEHP, 2–100 μg/ml for DnOP and 5–100 μg/ml for DINP and DIDP. The coefficients of variation (precision) for the total PAEs method were in the range 0.55–12.10% and for the migration method were in the range 0.72–7.70%. Recovery values for total PAEs and for the migrated PAEs were ranged within 90.6–111.7% and 86.3–117.2%; respectively. Both developed methods were used to survey the Jordanian market, where twenty seven samples were collected and tested for plastic type, total PAEs and migrated PAEs. Seventeen of those samples were found to be polyvinyl chloride (PVC), and all of them containing total PAEs having concentrations in excess of the allowed limit of 0.1% (w/w). The rest of the samples were non-PVC and only one of them containing PAEs, but below the allowed limit. In contrast, all the migration results were found to be well within the allowed limits. It was also found that as the PAEs total concentration increase, the corresponding migrated value increase, while the opposite trend holds for in the PAEs molecular weight.
Keywords: PAEs plasticizers; Toys; Migration; GC–MS;

Nano-sized molecularly imprinted polymer (nMIP) was synthesized through precipitation polymerization method and used it as solid phase extraction (SPE) sorbent for the selective and simultaneous extraction of hydroxylated metabolites of polycyclic aromatic hydrocarbons (PAHs) from urine followed by ultra-high performance liquid chromatography (UHPLC) analysis coupled with the fluorescent detector (FLD). Multi-template imprinting approach was used in the synthesis of nMIP by taking 1-naphthol, 9-phenanthrol and 9-hydroxyfluorene as templates, methacrylic acid (MAA) as a monomer, ethyleneglycoldimethacrylate (EGDMA) as a crosslinker and AIBN as an initiator. The synthesized nMIP exhibit the highest degree of binding affinity in comparison to non-imprinted polymer (NIP), and its binding affinity found to be in the range of 50–90% for all five metabolites tested. Method exhibits good linearity over a range of concentrations of metabolites with a R 2 value ranges from 0.9789 to 0.9921. Limit of detection (LOD) and limit of quantitation (LOQ) in urine samples were found to be in the range of 0.33–2.6 and 0.99–8 ng mL−1, respectively. Precision study shows that intra and inter-day precision was found to be less than 10%. The application of nMIP as SPE sorbent offers an effective and selective affinity towards the PAH metabolite's and found to be an alternative to the conventional sorbent for PAH metabolite's extraction from the biological samples. The developed nMIP offers wide advantages like simultaneous determination of PAH metabolites with improved sensitivity and found to be cost-effective for the routine analysis.
Keywords: Polycyclic aromatic hydrocarbon metabolites; Multi-template imprinting approach; Nano-sized molecularly imprinted polymer; Precipitation polymerization; Ultra-high performance liquid chromatography;

An LC-MS/MS method for the quantitation of cabozantinib in rat plasma: Application to a pharmacokinetic study by Qinghong Su; Jian Li; Xiwei Ji; Jingyun Li; Tianyan Zhou; Wei Lu; Liang Li (119-123).
A simple, rapid and sensitive high-performance liquid chromatography tandem mass-spectrometric method (LC-MS/MS) for the novel multiple tyrosine kinase inhibitor (TKI) cabozantinib was developed and validated using erlotinib as internal standard (IS). Plasma samples were pre-treated by liquid–liquid extraction with ethyl acetate. Separation was achieved on a reversed phase C18 column (50 × 2 mm, 5 μm) at ambient temperature using isocratic elution with acetonitrile-water (45:55, v/v) containing 5 mM ammonium formate buffer (finally adjusted to apparent pH* = 5.0 with formic acid) at a flow rate of 0.4 mL/min. The analytes were monitored by a triple quadrupole tandem mass spectrometer with electrospray ionization source in the positive ion mode. Calibration curve was linear (r  > 0.99) in a concentration range of 0.5–1000 ng/mL with the lower limit of quantification (LLOQ) of 0.5 ng/mL. Intra- and inter-day accuracy and precision were validated by relative error values (RE) and relative standard deviation values (RSD), respectively, which were both lower than the limit of 15%. This method was successfully applied to a pharmacokinetic study of cabozantinib in rats.
Keywords: Cabozantinib; LC-MS/MS; Rat plasma; Pharmacokinetics;

A specific and reliable HPLC–MS/MS method was developed and validated for simultaneously determination of sanguinarine, chelerythrine and their metabolites (dihydrosanguinarine and dihydrochelerythrine) in chicken tissue for the first time. This is important because these compounds are related to the use of a naturally occurring and novel feed additive with many benefits, but the levels of these compounds must be strictly controlled. The compounds were extracted by acetonitrile and 1% HCl–methanol solution successively and then separated on a C18 column. A triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was used for detection. Quantification was performed using multiple reaction monitoring with positive mode. The method was validated in terms of specificity, linearity, precision, accuracy and stability. The calibration curves were linear over the concentration range of 0.5–100.0 ng/g for sanguinarine, 0.5–100.0 ng/g for chelerythrine, 0.2–100.0 ng/g for dihydrosanguinarine and 0.1–100 ng/g for dihydrochelerythrine, respectively. All of the recovery rates of the four analytes were over 85%. The RSD of intra-day and inter-day precision was less than 5.0%, and the relative error was all within 12.0%. This validated method has been successfully applied to assess the drug residue and metabolite residue characteristics of sanguinarine and chelerythrine in chicken tissue after oral administration of the extracts of Macleaya cordata (Willd.) R. Br, and to investigate the pharmacokinetic parameters of sanguinarine and dihydrosanguinarine in chicken plasma.
Keywords: HPLC–MS/MS; Sanguinarine; Chelerythrine; Dihydrosanguinarine; Dihydrochelerythrine; Metabolite;

Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: Clinical and animal model applications by Sarah F. Cook; Amber D. King; Yan Chang; Gordon J. Murray; Hye-Ryun K. Norris; Richard C. Dart; Jody L. Green; Steven C. Curry; Douglas E. Rollins; Diana G. Wilkins (131-141).
The aims of this study were to develop, validate, and apply a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra- and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10 μM, and intra- and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24 h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at −20 °C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma collected from APAP-treated mice, a common animal model of APAP-induced hepatotoxicity.
Keywords: Acetaminophen-cysteine; Protein adduct; Hepatotoxicity; Biomarker; Human serum; HPLC-ESI-MS/MS;

An analytical method for catecholamines and related compounds using hydrophilic interaction liquid chromatography (HILIC) with native fluorescence detection has been developed. We found that ZIC-cHILIC with phosphorylcholine was suitable for the separation of catechol compounds with good peak shapes among six different HILIC columns (Inertsil SIL, Inertsil Amide, Inertsil Diol, TSKgel NH2-100, ZIC-HILIC, and ZIC-cHILIC). Using ZIC-cHILIC, eight catechol compounds (dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylglycol, 3,4-dihydroxymandelic acid, and internal standard 3,4-dihydroxybenzylamine) were separated within 15 min. The limit of detection at a signal to noise ratio of 3 was 3–28 nM. An improved sensitivity was obtained as compared to that of reversed-phase liquid chromatography. This was partly attributed to the increase in the fluorescence intensity of the catechol compounds in the acetonitrile-rich mobile phase. Solid phase extraction using a monolithic silica disk-packed spin column with phenylboronate moieties, which have affinity to catechol compounds, was performed for the selective extraction of catechol compounds from mouse urine. Dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were successfully quantified in mouse urine.
Keywords: Catecholamines; ZIC-cHILIC; Monolithic silica; Phenylboronate;

Although Polygonatum odoratum has been widely used as medicinal plant and food supplement for treating diabetes, little is known regarding its bioactive components. In this study, ultrafiltration–HPLC based ligand screening was developed to screen α-glucosidase inhibitors from P. odoratum for the first time. Then bioactive components were target-guided separated by combining stepwise high-speed counter-current chromatography (HSCCC) using petroleum ether–ethyl acetate–methanol–water (1:4:0.8:4.2, v/v/v/v), (1:4:1.8:3.2, v/v/v/v) and (1:4:2.3:2.7, v/v/v/v) as solvent systems with Sephadex LH-20 chromatography eluted by MeCN–MeOH (1:1, v/v). Five phenethyl cinnamides, N-cis-feruloyloctopamine (1); N-trans-p-coumaroyloctopamine (2), N-trans-feruloyloctopamine (3), N-trans-p-coumaroyltyramine (4) and N-trans-feruloyltyramine (5), and four homoisoflavanones, (3R)-5,7-dihydroxyl-3-(2′,4′-dihydroxylbenzyl)-chroman-4-one (6), (3R)-5,7-dihydroxyl-6-methyl-3-(4′-hydroxylbenzyl)-chroman-4-one (7), (3R)-5,7-dihydroxyl-6-methyl-8-methoxyl-3-(4′-hydroxylbenzyl)-chroman-4-one (8); and (3R)-5,7-dihydroxyl-6,8-dimethyl-3-(4′-hydroxylbenzyl)-chroman-4-one) (9), with purity over 98.5% were purified, and their structures were identified by UV, MS, and 1H NMR. Notably, compounds 2 and 4 were first reported in genus Polygonatum, while compound 1 was first obtained from family Liliaceae. In addition, α-glucosidase inhibitory activities of compounds 19 were evaluated, and compounds 2 and 4 exhibited stronger α-glucosidase inhibitory activity with IC50 values of 2.3 and 2.7 μM. The results suggested the potential medicinal use of P. odoratum, and the technology could be widely applied for rapid screening and preparative separation of a group of bioactive compounds from complex matrix.
Keywords: Polygonatum odoratum; Phenethyl cinnamide; α-Glucosidase inhibitor; Ultrafiltration; HSCCC; Sephadex LH-20;

Evaluation of a LC–MS method for everolimus preclinical determination in brain by using [13C2D4]RAD001 internal standard by Stefano Giovagnoli; Tommaso Cassano; Lorenzo Pace; Alessandro Magini; Alice Polchi; Brunella Tancini; Marzia Perluigi; Federico De Marco; Carla Emiliani; Diego Dolcetta (155-163).
Isotopic internal standards are increasingly frequent in LC–MS analysis to control biological matrix effects in the quantitation of immunosuppressant drugs, such as everolimus (RAD001). Here we present the evaluation of a LC–MS method, exploiting [13C2D4]RAD001 as internal standard, for preclinical determination of RAD001 in mice brain tissue. Samples were purified by solid phase extraction. Brain and blood were collected from vehicle-treated and RAD001-treated mice. The QTOF MS detector was set to select RAD001 ammonium adducts (m/z 975.6152) and [13C2D4]RAD001 (m/z 981.6481). Two different UHPLC columns were preliminarily tested. The method showed linear behavior between 4 and 100 ng/mL (r 2  = 0.99943) and linearity was preserved in the presence of blood (r 2  = 0.99107) and brain (r 2  = 0.99098) matrix components. Intra-day and inter-day precision (3–19%) and accuracy (82–109%) were comparable between standards and spiked blood and brain samples. As resulting from recovery comparison (82–98%), [13C2D4]RAD001 compensated ion suppression phenomena maintaining method performance over a wide range of consecutive analytical runs. The comparison with a HPLC-UV method showed reliability of the method with good correlation between blood (r 2  = 0.94319) and brain (r 2  = 0.97773) samples and acceptable biases (<15%). This validation suggests that the investigated method could be useful for the preclinical monitoring of RAD001 brain therapeutic concentrations in animal models.
Keywords: Biological samples; LC–MS; Everolimus; Isotopically labeled internal standard; Brain levels;

Determination and pharmacokinetic study of two triterpenoid saponins in rat plasma after oral administration of the extract of Aralia elata leaves by UHPLC–ESI–MS/MS by Zhibin Wang; Qiong Wu; Yonghai Meng; Yichun Sun; Qi Wang; Chunjuan Yang; Qiuhong Wang; Bingyou Yang; Haixue Kuang (164-171).
Aralia elata (Miq.) Seems (A. elata) grow in Northeast China and the total saponins of A. elata is used to auxiliary treatment for the acute hepatitis, chronic hepatitis and the transaminase on the high side. Aralia-saponinV and Aralia-saponinVI are the major bioactive saponins in A. elata leaves. A selective and sensitive UHPLC–MS/MS method was developed and validated for the determination and pharmacokinetic study of Aralia-saponinV and Aralia-saponinVI indwelling the extract in rat plasma in this article. The sample pretreatment involved a one-step extraction of 0.2 mL plasma with methanol. Shengmaxinside C was used as internal standard (I.S.). The separation was carried out on an Agilent SB-C18 column (1.8 μm, 50 mm × 2.1 mm) at 30 °C with a mobile phase of acetonitrile–5 mM ammonium acetate (90:10, v/v) at a flow rate of 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion-pairs (m/z) monitored for Aralia-saponinV, Aralia-saponinVI and I.S. were 1103.2/941.2, 1119.2/957.0 and 707.0/647.1, respectively. The current UHPLC–MS/MS assay method was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability, and it was suitable for the pharmacokinetic studies of the two saponins after oral administration of extract of A. elata leaves. The lower limits of quantification were 5.70 ng/mL for Aralia-saponinV and 6.15 ng/mL for Aralia-saponinVI. Intra-day and inter-day precisions were less than 7.4% and the accuracy range was from 1.19% to 8.60%. The mean extraction recoveries of analytes and I.S. from rat plasma were all more than 89.5%. This paper described a simple, sensitive and validated UHPLC–MS/MS method for simultaneous determination of Aralia-saponinV and Aralia-saponinVI in rat plasma after oral administration of the extract of A. elata leaves, and investigated on their pharmacokinetic studies as well.
Keywords: Aralia elata (Miq.) Seem; UHPLC–ESI–MS/MS; Aralia-saponinV; Aralia-saponinVI; Pharmacokinetics;

Quantitation of pilsicainide in microscale samples of human biological fluids using liquid chromatography–tandem mass spectrometry by Mikiko Shimizu; Masayuki Hashiguchi; Tsuyoshi Shiga; Koichi Nakamura; Hiro-omi Tamura; Mayumi Mochizuki (172-179).
This paper describes a sensitive, reliable method to determine pilsicainide (PLC) levels in microscale sample volumes of human biological fluids using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI). PLC and quinidine as an internal standard were extracted with diethylether from 0.1 mL of alkalinized biological fluids. The extract was injected into an analytical column (l-column 2 ODS, 75 mm × 2.1 mm i.d.). The mobile phase for separation consisted of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) and was delivered at a flow rate of 0.2 mL/min. The drift voltage was 100 V. The sampling aperture was heated at 120 °C and the shield temperature was 260 °C. The ion transitions used to monitor analytes were m/z 273 →  m/z 110 for PLC and m/z 325 →  m/z 79 for quinidine. The total time for chromatographic separation was less than 8 min. The validated concentration ranges of this method for PLC were 5–2000 ng/mL in plasma, 5–500 ng/mL in ultrafiltered plasma solution, and 25–2000 ng/mL in urine. Mean recoveries of PLC in plasma, ultrafiltered plasma solution, and urine were 93.2–99.7%, 91.4–100.6%, and 93.9–104.7%, respectively. Intra- and interday coefficients of variation for PLC were less than 6.0% and 4.3% in plasma, 6.1% and 3.7% in ultrafiltered plasma solution, and 5.4% and 2.5% in urine at the above concentration ranges, respectively. The lower limit of quantification for PLC in plasma, ultrafiltered plasma solution, and urine were 5 ng/mL, 5 ng/mL, and 25 ng/mL, respectively. This method can be applied to pharmacokinetic study and therapeutic drug monitoring in special populations such as neonates, infants, and the elderly by making effective use of residual samples used for general clinical laboratory testing.
Keywords: Pilsicainide; LC–MS/MS; Plasma; Urine; Pharmacokinetics; Therapeutic drug monitoring;

Pharmacokinetic, bioavailability, metabolism and plasma protein binding evaluation of NADPH-oxidase inhibitor apocynin using LC–MS/MS by Hardik Chandasana; Yashpal S. Chhonker; Veenu Bala; Yarra D. Prasad; Telaprolu K. Chaitanya; Vishnu L. Sharma; Rabi S. Bhatta (180-188).
Apocynin is a major active constituent of Picrorhiza kurroa that exhibits potent anti-inflammatory activity by inhibiting superoxide-generating NADPH oxidase enzyme. To elucidate detailed pharmacokinetic profile of apocynin, high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed in rat and human plasma. To the best of our knowledge, this is the first method for complete validation of apocynin in biological matrix using LC–MS/MS. Apocynin was rapidly absorbed after oral administration at 50 mg/kg in rats and peak plasma level achieved within 5 min. Moreover, plasma levels were observed up to 48 h. The bioavailibity of apocynin was found to be 8.3%. In vitro plasma protein binding was found to be 83.41–86.07% and 71.39–73.34% in rat and human plasma, respectively. Apocynin was found stable in gastric (pH 1.2), intestinal (pH 6.8) and physiological (pH 7.4) fluids including microsomal (rat and human) stability studies. Further, apocynin did not convert to its dimeric form diapocynin in any of these studies. The data presented here provide crucial information about apocynin to support its pharmacological efficacy and further development as a potential anti-inflammatory drug candidate.
Keywords: Apocynin; Diapocynin; NADPH-oxidase inhibitor; Pharmacokinetics; HPLC–MS/MS;

A mass spectrometric method for drugs of abuse testing in exhaled breath employing a sampling device collecting aerosol particles was developed and applied in routine use. Analytes covered were amphetamine, methamphetamine, 6-acetylmorphine, morphine, cocaine, benzoylecgonine, diazepam, oxazepam and tetrahydrocannabinol. The method involved eluting drugs from the collection filter with methanol, quantification using deuterated analogs as internal standards, reversed phase chromatography with gradient elution, positive electrospray ionization and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring range was 6.0–1000 pg/filter. The intra- and inter-assay imprecision expressed as the coefficient of variation was less than 7%. Influence from matrix was noted for most compounds but was compensated for the use of co-eluting internal standards. The LLOQ was 6.0 pg/filter with intra-assay CV <5% and accuracy within 99–102% for all analytes. No chromatographic interference was observed in 20 negative control samples. The LC–MS/MS method was successfully applied for measuring drugs in unknown samples collected for the purpose of drug testing. Among the 1096 analyzed samples analytical findings were made in breath in 39 cases (3.6%). Most frequently found substances were the following: amphetamine (25 cases) methamphetamine (10 cases), THC (8 cases), cocaine (4 cases), benzoylecgonine (2 cases) and diazepam (2 cases). In conclusion, a fully validated and robust screening method suitable for the routine measurement of drugs of abuse in exhaled breath with a simple procedure for specimen collection and sample preparation was successfully developed.
Keywords: Exhaled breath; Drugs of abuse testing; LC–MS/MS;

Development of ELISA for detection of Rh1 and Rg2 and potential method of immunoaffinity chromatography for separation of epimers by Huihua Qu; Yan Wang; Wenchao Shan; Yue Zhang; Huibin Feng; Jiayang Sai; Qingguo Wang; Yan Zhao (197-205).
In this work, hybridomas producing anti-ginsenoside-Rh1 monoclonal antibodies (MAbs) were generated. These MAbs were subsequently used to create indirect competitive enzyme-linked immunosorbent assays (icELISAs). A linear correlation was obtained for G-Rh1 concentrations in the range from 26 to 512 ng/mL. The regression equation was y  = 1.979 − 0.201 Log2(X) with a regression coefficient of 0.9898. Precision and accuracy of the icELISA method were evaluated by the variations between replicates from well to well (intra-assay) and plate to plate (inter-assay). The recovery rates ranged from 93.16% to 108.43%. Testing with the icELISA demonstrated that the MAbs were specific for 20(S)-Rh1 and 20(S)-Rg2 with no cross-reactivity against 20(R)-Rh1 and 20(R)-Rg2. The immunoaffinity chromatography column (IAC) was constructed by covalently coupling monoclonal antibody (MAb) against G-Rh1 to CNBr-activated Sepharose 4B. When 20(R)-type-Rg2 passed through the IAC column, it was adsorbed, but the amount adsorbed was lower than that when 20(S)-type-Rg2 ran through the column. The differences in adsorption between the 20(S) and 20(R) type ginsenosides bring a new approach or method to separate 20(S)-Rg2 and 20(R)-Rg2 by IAC. Our results indicate that the icELISA is a sensitive and efficient approach for the identification of epimers, and the application of IAC using MAbs against small molecules provides a totally new thought and potential method for resolving epimers.
Keywords: Ginsenoside-Rh1; 20(R,S)-ginsenoside-Rg2; Monoclonal antibody; Indirect competitive enzyme-linked immunosorbent assay; Immunoaffinity chromatography;