Journal of Chromatography B (v.904, #C)

Application of ionic liquid for extraction and separation of bioactive compounds from plants by Baokun Tang; Wentao Bi; Minglei Tian; Kyung Ho Row (1-21).
► The unique properties of ionic liquids were highlighted. ► IL or IL-based materials were used to extract bioactive compounds in plants. ► Advantages of IL and IL-based materials were in applications.In recent years, ionic liquids (ILs), as green and designer solvents, have accelerated research in analytical chemistry. This review highlights some of the unique properties of ILs and provides an overview of the preparation and application of IL or IL-based materials to extract bioactive compounds in plants. IL or IL-based materials in conjunction with liquid–liquid extraction (LLE), ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), high performance liquid chromatography (HPLC) and solid-phase extraction (SPE) analytical technologies etc., have been applied successfully to the extraction or separation of bioactive compounds from plants. This paper reviews the available data and references to examine the advantages of IL and IL-based materials in these applications. In addition, the main target compounds reviewed in this paper are bioactive compounds with multiple therapeutic effects and pharmacological activities. Based on the importance of the targets, this paper reviews the applications of ILs, IL-based materials or co-working with analytical technologies. The exploitation of new applications of ILs on the extraction of bioactive compounds from plant samples is expected to increase.
Keywords: Ionic liquid; Extraction; Separation; Bioactive compound; Plant;

► We identified three sesquiterpenoids, solavetivone, aristolone and nootkatone from the rhizomes of Cyperus rotundus. ► The sesquiterpenoid solavetivone, a vetispirane sesquiterpenoid, has been identified for the first time from the species. ► HPTLC and HPLC quantification was done for the isolated sesquiterpenoids. ► In vitro antioxidant and radical scavenging potential were screened. ► Nootkatone possessed the highest antioxidant potential among them.Three sesquiterpenoids solavetivone, aristolone and nootkatone were isolated from the acetone extract of Cyperus rotundus by silica gel column chromatography and identified by spectral studies. Solavetivone has been isolated for the first time from the species. Simple, sensitive and selective HPTLC and HPLC methods with ultraviolet detection (245 nm) were developed and validated for the simultaneous quantification. HPTLC method was validated in terms of their linearity, LOD, LOQ, precision, accuracy and compared with RP-HPLC-UV method. Among the three sesquiterpenoids isolated, nootkatone possessed the highest radical scavenging potential (IC50 4.81 μg/ml) followed by aristolone (IC50 5.28 μg/ml) and solavetivone (IC50 6.82 μg/ml) by DPPH radical scavenging assay. Total antioxidant activity against phosphomolybdenum reagent was also studied. The methods described in this paper were able to identify and quantify sesquiterpenoids from the complex mixtures of phytochemicals and could be extended to the marker based standardization of polyherbal formulations containing C. rotundus.
Keywords: Solavetivone; Aristolone; Nootkatone; NMR; HPTLC; HPLC;

► Simultaneous analysis of hydrophobic and hydrophilic analytes using mixed mode SPE. ► Bile differs from plasma affecting the choice of extraction procedure. ► The extraction method was fully automated. ► The cycle time was reduced by a high flow rate on a fused core LC column. ► The method was successfully validated for all three analytes.A method for the determination of AZD0837 and its two metabolites AR-H069927 and AR-H067637 in human bile was developed and validated. All three analytes and their stable isotope-labeled internal standards were isolated from bile using solid phase extraction on a mixed mode reversed phase/anion exchange column. Elution was done at high ionic strength with 0.125 M ammoniumacetate in 50% methanol. The extraction recoveries were >75%. Due to the high concentration of AR-H067637 a portion of the extract was diluted before injection on to the LC column, while undiluted extract was directly injected for the analysis of AZD0837 and AR-H069927. Chromatographic separation of all three analytes was achieved in a single system utilizing a C18 column based on fused core particle technology at high flow rate. The two metabolites were eluted when a gradient from 30 to 57% methanol was applied while the more hydrophobic pro-drug, AZD0837, eluted during a steeper second gradient from 57 to 80% methanol with the ammonium acetate concentration and acetic acid concentration kept constant at 3.8 mmol/L and 0.1%, respectively. The total cycle time was 3.2 min. Detection was performed using positive electrospray ionization tandem mass spectrometry. The linearity range was 0.02–20 μmol/L for AZD0837 and AR-H069927, and 1–1000 μmol/L for AR-H067637. The repeatability and the overall precision were less than 15% (RSD) and the accuracy was within the interval 93–100%.
Keywords: Thrombin inhibitor; Human bile; Mixed mode solid phase extraction; Fused core liquid chromatography;

► Monitoring of pesticides with their metabolites is essential in food quality. ► This method is the first validated method to quantify CP and CPO in wine sample. ► The signal of CP and CPO was improved by pulsed splitless technique. ► Low detection limit for CPO compared to the literature. ► QuEChERS method was tested for the separation of CP and CPO.A reliable method has been developed for the determination of Chlorpyrifos (CP) and its metabolite Chlorpyrifos-oxon (CPO) in wine sample using pulsed splitless technique coupled with gas chromatography by using electron capture detector. In this study, a quick, easy and cheap sample preparation method (QuEChERS) based on liquid extraction with acetonitrile, followed by dispersive solid phase extraction using primary secondary amine was tested for the separation and quantification of CP and CPO in wine samples. The accuracy of the developed method was tested upon recovery studies and it was calculated as (92.3 ± 18.2)% for CP and (96.6 ± 16.1)% for CPO. LOD and LOQ values of CP were found as 0.04 and 0.15 ng/mL and 0.49 and 1.62 ng/mL for CPO respectively. By using the pulsed splitless injection mode, the sensitivity of the determination of CP and its metabolite CPO in wine samples was improved compared to splitless technique. CP content of analyzed wine sample was found as 2.05 ± 0.15 ng/mL with a RSD of 7.6% and CPO content was found as 4.99 ± 0.15 ng/mL with a RSD of 3.0% (n  = 3). The expanded measurement uncertainties were calculated as 17% and 6% for CP and CPO, respectively.
Keywords: Chlorpyrifos oxon; Metabolite; Pulsed splitless; Pesticide; Gas chromatography; Wine; Uncertainty;

► The quantification of acetaminophen (APAP) and two of its metabolites by LC–MS/MS. ► Comparison of high-resolution MS/MS versus SRM/MS for quantitative analysis. ► QUAL/QUAN acquisition strategies for LC–MS.The quantification of acetaminophen (APAP) and two of its metabolites, i.e. acetaminophen-glucuronide (APAP-GLUC) and acetaminophen-cysteine (APAP-CYS), is described in human plasma using ultra-high performance liquid chromatography coupled to a triple quadrupole linear ion trap mass spectrometer operating in the selected reaction monitoring (SRM/MS) mode and to a high resolution quadrupole time-of -flight mass spectrometer operating in the MS/MS (HR-SRM/MS) mode. Starting with a 50 μL plasma aliquot, a generic sample preparation was performed using protein precipitation with methanol/ethanol. Both methods were found to be linear over 2.5 orders of magnitude. Similar performances to the SRM/MS assay were obtained for APAP, APAP-CYS and APAP-GLUC using high resolution-selected reaction monitoring mode with LLOQ of 20, 50 and 50 ng/mL, respectively. For all analytes, precision was found to be better than 12% and accuracy in the range 90.3–109%. The present study demonstrates the ability of QqTOF platforms for accurate and precise quantification in MS/MS mode using short duty cycle with similar sensitivity to LC–SRM/MS. Additionally, as full scan data MSALL are available qualitative and quantitative information on metabolites can also be obtained in a single LC–MS run.
Keywords: Acetaminophen; SRM/MS; High resolution mass spectrometry; Metabolism; UHPLC; QUAL/QUAN;

► Flavonoids exhibited biphasic pharmacokinetics in plasma concentration–time profiles. ► Wogonin, oroxylin A and chrysin were absorbed rapidly. ► Multiple plasma concentration peaks of the four alkaloids were observed. ► Pharmacokinetics of nine compounds were investigated in a run time of 7.0 min.A rapid, sensitive and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of five active flavonoids (wogonin, chrysin, oroxylin A, naringenin, hesperetin) and four major alkaloids (berberine, coptisine, jatrorrhizine, palmatine) from Tang-Min-Ling-Pill in rat plasma. Plasma samples (100 μL) were spiked with internal standards daidzein (for flavonoids) and tetrahydropalmatine (for alkaloids), acidified with HCl and extracted by liquid–liquid extraction with acetone. Chromatographic separation was performed on a Zorbax SB-C18 column with the mobile phase of water (containing 0.1% of formic acid)–acetonitrile (30:70, v/v) at a flow rate of 0.3 mL/min in a run time of 7.0 min. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. All analytes showed good linearity over the investigated concentration range (r  > 0.9900). The validated lower limit of quantification was 1.01 ng/mL for wogonin and oroxylin A, 0.238 ng/mL for chrysin, 1.01 ng/mL for naringenin, 0.998 ng/mL for hesperetin, 0.0505 ng/mL for berberine, 0.0996 ng/mL for coptisine, 0.0501 ng/mL for jatrorrhizine, 0.0889 ng/mL for palmatine, respectively. Intra- and inter-day precision (RSD%) was less than 15% and accuracy (RE%) ranged from −7.5% to 4.5%. The validated method was successfully applied to investigate the pharmacokinetics of the major flavonoids and alkaloids of Tang-Min-Ling-Pill after oral administration to rats.
Keywords: Determination of flavonoids and alkaloids; Pharmacokinetics; Tang-Min-Ling-Pill (TMLP); Rat plasma;

Simultaneous determination and pharmacokinetic study of six flavonoids from Fructus Sophorae extract in rat plasma by LC–MS/MS by Lu Chang; Yanping Ren; Liang Cao; Yingguang Sun; Qian Sun; Ning Sheng; Lin Yuan; Xuran Zhi; Lantong Zhang (59-64).
► We developed an LC–MS/MS method for the pharmacokinetic study of Fructus Sophorae. ► We also accomplished the separation of isomeride pair sophoricoside and genistin. ► The total run time was 8.0 min by LC–ESI-MS.In this study, a new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of six flavonoids including sophoricoside, genistin, genistein, rutin, quercetin and kaempferol in rat plasma after oral administration of Fructus Sophorae extract using sulfamethalazole as internal standard (IS). The plasma samples were pretreated and extracted by liquid–liquid extraction. Chromatographic separation was accomplished on a C18 column with a simple linear gradient elution. The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.1/267.9 for sophoricoside and genistin, 269.0/133.0 for genistein, 609.2/300.0 for rutin, 301.0/150.9 for quercetin, 284.9/93.0 for kaempferol and 252.0/155.9 for IS. The total run time was 8.0 min. Full validation of the assay was implemented including specificity, linearity, accuracy, precision, recovery and matrix effect. This is the first report on determination of the major flavones in rat plasma after oral administration of Fructus Sophorae extract. The results provided a meaningful basis for the clinical application of this herb.
Keywords: LC–MS/MS; Flavonoids; Fructus Sophorae; Pharmacokinetics; MRM;

► We simultaneously purify HupA and HupB by reversed C18 column from Chinese herbs. ► We compare four kinds of acids of their influence to the separation of HupA and HupB. ► Macroporous resins were tested and SP850 was found best for initial purification. ► Trifluoroacetic acid performed best to eliminate the trailing problem.Huperzine A (HupA) and Huperzine B (HupB) are natural alkaloids existed in Lycopodium plants. They both have potential clinical application for treating Alzheimer's Disease (AD). For the purpose of better utilizing the limited plant resources, a quick and low cost method to separate and purify HupA and HupB from Huperzia serrata (Thunb. ex Murray) was established in this paper. Low polarity macroporous resin SP850 was selected from eight kinds of resins during initial purification. Trifluoroacetic acid (TFA) was proved to be the best acid modifier reagent among all acids used in our experiment for improving separation. HupA and HupB were baseline separated on a C18 column by preparative high performance liquid chromatography (Preparative HPLC), the optimal gradient mobile phase system contained methanol increasing from 15% (v/v) to 35% (v/v) and 0.1% (v/v) TFA within the water. The purity of HupA and HupB obtained was 99.1% and 98.6%, respectively, and the total recovery for them was 83.0% and 81.8%, respectively.
Keywords: Huperzine A; Huperzine B; Huperzia serrata; Macroporous resin; Preparative HPLC; Trifluoroacetic acid;

► A LC–MS/MS method for simultaneously analyzing 5 phthalate monoester metabolites and BPA in human urine is developed. ► The method was validated and demonstrated to be sensitive, accurate and precise using 200 μl sample volume with a run time of 12 min. ► This method has been used successfully in assessing the exposure of phthalates and BPA in 50 humans.Phthalates and bisphenol A are environmental endocrine-disrupting chemicals used widely in common consumer products. There is increasing concern about human exposure to phthalates and bisphenol A due to the potential adverse effects related to the anti-androgenic activity of phthalates and estrogenic activity of bisphenol A. In assessing environmental exposure to phthalates and bisphenol A, it is essential to have a validated analytical method that can quantify trace concentrations of phthalate metabolites and bisphenol A in humans. In this study, we developed and validated an accurate, sensitive, and robust LC–MS/MS method to simultaneously quantify 5 phthalate monoester metabolites, including mono-methyl phthalate, mono-ethyl phthalate, mono-butyl phthalate, mono-benzyl phthalate, mono-2-ethylhexyl phthalate, and bisphenol A in human urine. In this method, the phthalate metabolites and bisphenol A, along with their isotope labeled internal standards, were extracted from 200 μl of human urine using automated off-line solid phase extraction. The analytes were quantitatively determined using LC–MS/MS operated in negative electrospray ionization multiple reaction-monitoring mode. The limit of quantification was 0.3 ng/ml for mono-methyl phthalate, mono-ethyl phthalate, mono-benzyl phthalate and bisphenol A, and 1 ng/ml for mono-butyl phthalate and mono-2-ethylhexyl phthalate. The precision and accuracy were well within the acceptable 15% range. This validated method has been used successfully in assessing exposure to phthalates and bisphenol A in humans.
Keywords: Phthalate metabolite; Bisphenol A; SPE; LC–MS/MS;

► Berenil pseudo-affinity chromatography to purify two different sized plasmids from clarified E. coli lysate. ► Optimized clarification of pDNA solutions. ► Plasmids are completely purified using small amounts of salt in the eluent. ► Impurity levels in accordance to FDA requirements. ► Promising alternative to purify pDNA for therapeutic use.In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3 M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0 M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use.
Keywords: Plasmid purification; Small DNA ligands; Berenil; Pseudo-affinity chromatography;

Determination of celiprolol in human plasma using high performance liquid chromatography with fluorescence detection for clinical application by Akiko Itohda; Kimiko Tsutsumi; Hiromitsu Imai; Miyuki Iwao; Tsutomu Kotegawa; Kyoichi Ohashi (88-92).
► A simple and sensitive HPLC method for determination of celiprolol in plasma was developed. ► The plasma samples were prepared by solid phase extraction. ► The calibration curves were linear over the range of 1–1000 ng/ml (LLOQ, 1 ng/ml). ► This validated method was successfully applied to an interaction study in humans.A new method of analysis has been developed and validated for the determination of plasma celiprolol concentration. Plasma samples (1 ml) were pre-purified by solid-phase extraction with Bond Elut® C18. The separation was achieved with XBridge™ C18 column (150 mm × 3.0 mm i.d., 3.5 μm) at 35 °C using a mixture of acetonitrile and 10 mM ammonium acetate buffer (pH 10.5) (34:66, v/v) under isocratic conditions at a flow rate of 0.4 ml/min. The peak was detected using a fluorescence detector at excitation 250 nm and emission 482 nm. Retention times for the internal standard (acebutolol) and celiprolol were 4.2 min and 6.3 min, respectively. Calibration curves were linear over the range of 1.0–1000 ng/ml (r  > 0.999), with a limit of quantification at 1.0 ng/ml. Intra- and inter-assay precision (relative standard deviation) were less than 13.3% and the accuracy (relative error) was −5.1% to 11.5% at four different concentrations. This proposed method was successfully applied to a study of pharmacokinetic interactions between celiprolol and apple juice in humans.
Keywords: Celiprolol; HPLC; Solid phase extraction; Fluorescence detection; Apple juice;

► IGFBP-3 in patients with diabetes mellitus is glycated and additionally sialylated. ► Glycation increases the affinity of IGFBP-3 for IGF-I, sialylation decreases it. ► Altered glycation/glycosylation may contribute to diabetic complications. ► Non-glycosylated and glycosylated/glycated IGFBP-3 undergo specific fragmentation.Structural and ligand-binding properties of the insulin-like growth factor-binding protein (IGFBP)-3 in patients with poorly controlled diabetes mellitus type 2 were investigated using boronic acid- and lectin-affinity chromatography. IGFBP-3 species separated by chromatography were analyzed by immunoblotting and surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Increased IGFBP-3 binding to boronic acid in patients was shown to be accompanied by the increased ligand-binding. Increased binding of IGFBP-3 forms to lectins from Sambucus nigra (SNA) and Canavalia ensiformis (ConA) in patients, on the other hand, was either not accompanied by altered ligand-binding (in the case of ConA) or it was reduced (in the case of SNA). Strong and opposite effects of glycation and additional sialylation on ligand binding qualify them as factors that may be involved in the regulation of the amount of free, physiologically active IGFs, and modulation of processes that accompany development and progression of diabetes. SELDI-TOF MS analysis revealed a fragment of 13.9 kDa as representative for the non-glycosylated form of IGFBP-3, whereas a fragment of 28.0 kDa profiled as typical for the glycosylated/glycated IGFBP-3 species. The same fragmentation pattern found in healthy persons and in patients indicates that the same degradation process predominantly occurs in both groups of individuals.
Keywords: Diabetes mellitus type 2; Fragmentation pattern; Glycation/glycosylation; IGFBP-3; Ligand-binding;

► TAHS and FOSF provide poorer chromatographic separation and smaller dynamic linear range. ► TAHS and FOSF provide lower LoD/LoQ values than DNS, FMOC-Cl and DEEMM. ► DNS, FMOC-Cl and DEEMM have good chromatographic separation and wide dynamic linear range. ► DEEMM is most optimal, with good LoQ values, chromatographic separation and wide linear range.Amino acid analysis with high performance liquid chromatography with electrospray ionization mass spectrometry (LC–ESI-MS) is an emerging method. For more sensitive analysis, derivatization is used and next to commercially available derivatization reagents such as dansyl chloride (DNS), 9-fluorenylmethyl chloroformate (FMOC-Cl) and diethyl ethoxymethylenemalonate (DEEMM), new derivatization reagents are designed specially for LC–ESI-MS, like p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide (TAHS) which provides very low limits of detection. In this work, a novel phosphazene based derivatization reagent (FOSF) that provides comparable limits of quantitation (LoQ) to TAHS is introduced. Moreover, a thorough comparison between FOSF, TAHS, DNS, FMOC-Cl and DEEMM is carried out for 7 different amino acids – Arg, Asp, Gly, β-Ala, Pro, Trp and Phe. This is a first time that thorough comparison is carried out on the same instrument for amino acid derivatization reagents. Results on the same instrument for five amino acid derivatization reagents show that novel reagents are sensitive with LoQ values around 80 fmol but have disadvantages such as problematic chromatographic separation. Next to novel reagents, DEEMM offers very good LoQ-s (average of 150 fmol) and wide dynamic linear range.
Keywords: Amino acids; Derivatization reagents; LC–ESI-MS;

Simultaneous quantification of niacin and its three main metabolites in human plasma by LC–MS/MS by Man Liu; Dan Zhang; Xiaolin Wang; Lina Zhang; Jing Han; Man Yang; Xue Xiao; Yanan Zhang; Huichen Liu (107-114).
► A LC–MS/MS method for simultaneous quantification of four analytes was developed. ► Simple protein precipitation was employed for the sample preparation. ► The concentrations of endogenous NAM and 2-Pyr were determined. ► The rLLOQ of endogenous NAM and 2-Pyr was calculated. ► The interferences from isotope effect or endogenous substances were avoided.A sensitive and specific LC–MS/MS method for the simultaneous quantification of niacin (NA) and its three main metabolites nicotinamide (NAM), nicotinuric acid (NUA) and N-methyl-2-pyridone-5-carboxamide (2-Pyr) in human plasma has been developed and validated. Plasma samples (200 μL) were prepared by deproteinization with acetonitrile (500 μL), then the supernatant after centrifugation was evaporated and reconstituted. Chromatography was performed on a phenomenex synergi hydro-RP column with an isocratic elution of methanol-0.1% formic acid (5:95, v/v). The full separation of all analytes was achieved within 9 min. Multiple-reaction monitoring (MRM) using the fragmentation transitions of m/z 124.1 → 80.1, 123.1 → 80.0, 181.0 → 79.0 and 153.1 → 110.2 in positive electrospray ionization (ESI) mode was performed to quantify NA, NAM, NUA and 2-Pyr, respectively. The calibration curves were linear over the concentration range of 2.0–3000 ng/mL for NA and NUA, 10.0–1600 ng/mL for NAM and 50.0–5000 ng/mL for 2-Pyr. This method has been validated in accordance with the US FDA guidelines for bioanalytical method development and applied to the determination of NA and its three main metabolites in Chinese subjects following a single oral dose of niacin extended-release and simvastatin 1000 mg/20 mg. In particular, because of the endogenous NAM and 2-Pyr in human plasma, the concentrations of NAM and 2-Pyr in human plasma after dosing were determined by subtracting blank values of them.
Keywords: Niacin; Nicotinamide; Nicotinuric acid; N-methyl-2-pyridone-5-carboxamide; Pharmacokinetics;

Combining human periodontal ligament cell membrane chromatography with online HPLC/MS for screening osteoplastic active compounds from Coptidis Rhizoma by Jin Liu; Jin Yang; Sicen Wang; Junyi Sun; Jianfeng Shi; Guozhou Rao; Ang Li; Jianzhong Gou (115-120).
► We developed an hPDLC/CMC system combined with an HPLC/MS analytical method. ► Berberine from Coptidis Rhizoma was screened and identified as the active component which could act on the hPDLCs using the method. ► The effective of berberine were verified by MTT assay, and osteoplastic activity tests.We have developed an online analytical method that combines human periodontal ligament cell membrane chromatography (hPDLC/CMC) with high-performance liquid chromatography and mass spectrometry (LC/MS) for recognizing and identifying osteoplastic active components from Coptidis Rhizoma. Retention fractions on hPDLC/CMC were enriched onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using simvastatin (SIM) as a positive control, berberine from Coptidis Rhizoma was identified as the active component which could act on the hPDLC. The MTT colorimetric assay, alkaline phosphatase (ALP) activity, and staining tests revealed that berberine could promote hPDLC growth, increase the secretion of ALP in the culture medium, and enhance the formation of mineralized nodule, thus it is a potential osteoplastic ingredient. This hPDLC/CMC–online-LC/MS method can be applied for screening active components acting on hPDLC from traditional Chinese medicines exemplified by Coptidis Rhizoma and will be of great utility in drug discovery using natural medicinal herbs as a source of leading compounds.
Keywords: Human periodontal ligament cell (hPDLC); Cell membrane chromatography (CMC); High performance liquid chromatography/mass spectrometry (LC/MS); Coptidis Rhizoma; Berberine; Simvastatin;

► A dual preconcentration method combined in an on-line mode. ► Simple and rapid operation without time-consuming matrix-transfer steps. ► High enrichment efficiencies are got in trace analysis of analytes in urine matrix. ► The method is used for pharmacokinetic of chlorpheniramine in human urine.A rapid and efficient dual preconcentration method of on-line single drop liquid–liquid–liquid microextraction (SD-LLLME) coupled to sweeping micellar electrokinetic chromatography (MEKC) was developed for trace analysis of three antihistamines (mizolastine, chlorpheniramine and pheniramine) in human urine. Three analytes were firstly extracted from donor phase (4 mL urine sample) adjusted to alkaline condition (0.5 M NaOH). The unionized analytes were subsequently extracted into a drop of n-octanol layered over the urine sample, and then into a microdrop of acceptor phase (100 mM H3PO4) suspended from a capillary inlet. The enriched acceptor phase was on-line injected into capillary with a height difference and then analyzed directly by sweeping MEKC. Good linear relationships were obtained for all analytes in a range of 6.25 × 10−6 to 2.5 × 10−4  g/L with correlation coefficients (r) higher than 0.987. The proposed method achieved limits of detections (LOD) varied from 1.2 × 10−7 to 9.5 × 10−7  g/L based on a signal-to-noise of 3 (S/N = 3) with 751- to 1372-fold increases in detection sensitivity for analytes, and it was successfully applied to the pharmacokinetic study of three antihistamines in human urine after an oral administration. The results demonstrated that this method was a promising combination for the rapid trace analysis of antihistamines in human urine with the advantages of operation simplicity, high enrichment factor and little solvent consumption.
Keywords: Antihistamines; On-line; Single drop liquid–liquid–liquid microextraction; Sweeping micellar electrokinetic chromatography; Urine analysis;

Rapid reversed-phase liquid chromatography separation of cyclolinopeptides with monolithic and microparticulate columns by Clara M. Olivia; Peta-Gaye G. Burnett; Denis P. Okinyo-Owiti; Jianheng Shen; Martin J.T. Reaney (128-134).
► Comparison of reverse phase monolithic and perfusion HPLC columns. ► An optimized gradient was developed to separate flaxseed cyclolinopeptides. ► Gradient elution with total analysis time of 1.5 min was achieved. ► Monolithic columns offer constant resolution and back pressure over 2000 samples. ► Monolithic column is suitable for high-throughput screening of cyclolinopeptides.Three monolithic C18-bonded silica gel columns i.e. Chromolith® SpeedROD (CSR), Chromolith® Performance (CP), and Chromolith® High Resolution (CHR), MerckKGaA Darmstadt, Germany and two particle-based columns i.e. ZORBAX Eclipse XDB-C18 (ZEX), Agilent and POROS R1/20 (POR), Applied Biosystems were compared for their performance in separating a mixture of flaxseed cyclolinopeptides (CLs). Gradient mobile phases of acetonitrile and water were optimized for each column. The performance of CHR column in profiling CL standards, measured as the resolution of individual CL, selectivity, and peak asymmetry exceeded the performance of traditional particle-packed columns and the other monolithic columns. The profiling of CLs in aqueous methanolic flaxseed extract was optimized for high-throughput analysis. A total analysis time of 1.5 min at a flow rate of 3.0 mL min−1 was achieved on a CSR column. Injection of over 2000 methanol extracts of flaxseed on a CSR column had no impact on backpressure or resolution of a standard CL mixture.
Keywords: Monolithic columns; Cyclolinopeptides; Liquid chromatography;

Determination of selected biogenic amines in red wines by automated on-line combination of capillary isotachophoresis–capillary zone electrophoresis by Pavlína Ginterová; Jozef Marák; Andrea Staňová; Vítězslav Maier; Juraj Ševčík; Dušan Kaniansky (135-139).
► cITP–CZE–UV method was developed for the determination of selected biogenic amines. ► The analytical characteristics of the proposed method were evaluated. ► Method was successfully applied on the analysis of red wine samples.A screening analytical method based on an automated on-line combination of capillary isotachophoresis–capillary zone electrophoresis (cITP–CZE) in hydrodynamically closed separation system, equipped with photometric detection at 280 nm, was developed for a routine determination of the selected biogenic amines, namely histamine, 2-phenylethylamine and tyramine, in red wines. The evaluated limits of detection (LODs) were 0.35 mg L−1 for histamine, 0.33 mg L−1 for 2-phenylethylamine and 0.37 mg L−1 for tyramine. The repeatability of the migration time and peak area for histamine were 1.1% and 2.6%, respectively, for 2-phenylethylamine 0.7% and 2.0%, respectively, and for tyramine 0.8% and 2.1%, respectively. The method recoveries were 92.1% for histamine, 96.4% for 2-phenylethylamine and 95.5% for tyramine. The developed automated cITP–CZE–UV method was applied for the determination of histamine, 2-phenylethylamine and tyramine in seven red wine samples originating from Czech Republic.
Keywords: Capillary isotachophoresis; Capillary zone electrophoresis; Histamine; Tyramine; 2-Phenylethylamine; Red wine;

► A quantification method for l-arginine and its metabolites ADMA and SDMA is presented. ► A very easy and fast sample preparation is proposed. ► Stable isotope labeled I.S.(s) are used for each l-arginine, ADMA and SDMA. ► Very good performance regarding precision and accuracy is achieved due to the I.S.(s). ► Matrix independency is achieved due to the I.S.(s).The amino acid l-arginine and its metabolites ADMA and SDMA are important markers for a range of diseases in humans. Increased levels of ADMA and SDMA in plasma point to endothelial dysfunction, hypertension, renal impairment and other pathological states. We present here a method to quantify l-arginine, ADMA and SDMA in human plasma, which is suitable to support clinical research in this field. Sample preparation consisted only of protein precipitation and the analytes were separated using a silica based HILIC column. The analytes were detected by ESI MS/MS, providing high selectivity and sensitivity. The calibration functions were linear in the ranges of 7.5–150 μmol/l for l-arginine, 0.15–3 μmol/l for ADMA and 0.2–4 μmol/l for SDMA. These ranges cover the concentrations encountered in healthy and pathological human plasma. The method employs 13C6-arginine, D7-ADMA and, for the first time in LC–MS/MS, D6-SDMA as internal standards for l-arginine, ADMA and SDMA. Therefore, matrix independency and a high intra-day precision of 0.82% for l-arginine, 2.12% for ADMA and 2.83% for SDMA, were achieved at basal plasma concentrations. The respective inter-day precision values were 4.01% for l-arginine, 3.77% for ADMA and 3.86% for SDMA.
Keywords: l-Arginine; ADMA; SDMA; HILIC; LC–MS/MS; Stable isotope dilution;