Journal of Chromatography B (v.1052, #C)

The potential of immobilized metal/chelate affinity (IMA) in a continuous fashion, referred as conjoint approach, to pre-fractionate plasma proteins (in their native state) prior to LC–MS analysis was investigated in this study. Four transition metal-ions (Co (II), Zn (II), Ni (II) and Cu (II)) were individually chelated with IDA (iminodiacetic acid) coated CIM (Convective Interaction Media) disks and placed in a single housing in the following sequential order: IDA-Co (II) → IDA-Zn (II) → IDA-Ni (II) → IDA-Cu (II). The rationale behind this order is to retain proteins based on their specific requirement for surface exposed histidine topography. This structural pre-fractionation hypothesis was successfully proven using four human plasma proteins (fibrinogen, IgG, transferrin, and albumin) with varying histidine topographies. This conjoint IMA pre-fractionation strategy not only fractionated proteins (from plasma) based on their native surface histidine topography, but also identified 157 proteins from human plasma. The advantage of our conjoint IMA is its ability to fractionate proteins in their native state and reduce plasma complexity in a single step by employing single buffer system.
Keywords: CIM disk; Conjoint IMA; Pre-fractionation; Proteins; Plasma;

Phosphorylated-tyrosine based pseudobioaffinity adsorbent for the purification of immunoglobulin G by Gisele Luiza Pavan; Igor Tadeu Lazzarotto Bresolin; Angélica Grespan; Sonia Maria Alves Bueno (10-18).
The present study evaluated the phosphorylated-tyrosine (P-Tyr) based pseudobioaffinity adsorbent for the purification of human immunoglobulin G (IgG). P-Tyr was selected as a ligand to mimic the natural interactions that occur between the immunoreceptor tyrosine-based activation motif and the IgG. The ligand was coupled to bisoxirane-activated agarose gel and the effect of buffer system, pH, and conductivity was performed to elucidate the nature of IgG-P-Tyr interactions. P-Tyr-agarose was able to purify IgG from human plasma solution in HEPES buffer at pH 7.0 exhibiting a purification factor of 9.1 with IgG purity of 91% (based on ELISA analysis of albumin, transferrin, and immunoglobulins A, G, and M). The evaluation of different functional groups of P-Tyr on the adsorption of human IgG indicated the predominance of electrostatic interactions with phosphate groups, although the contributions of aromatic and carboxylic groups also play a role. The thermodynamic parameters (ΔH°, ΔS°, ΔG°) for IgG adsorption onto P-Tyr-agarose were determined from the temperature dependence. The maximum IgG binding capacity at 20 °C was 273.51 ± 12.63 mg g−1 and the dissociation constant value of the complex IgG-P-Tyr was in the order of 10−5  mol L−1 indicating low-affinity.
Keywords: O-Phospho-tyrosine; Human immunoglobulin G; Adsorption; Purification; Human plasma;

Plasma and brain pharmacokinetics of ganoderic acid A in rats determined by a developed UFLC–MS/MS method by Fang-Rui Cao; Bing-Xin Xiao; Li-Sha Wang; Xue Tao; Ming-Zhu Yan; Rui-Le Pan; Yong-Hong Liao; Xin-Min Liu; Qi Chang (19-26).
Ganoderic acid A (GAA), an active triterpenoid of the traditional Chinese herbal medicine Lingzhi, has been reported to exhibit antinociceptive, antioxidative, and anti-cancer activities. The present study aims to establish a sensitive and rapid UPLC–MS/MS method for studying the plasma and brain pharmacokinetics of GAA in rats. The analytes were separated on a C18 column eluted with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at 0.3 mL/min. The eluate was monitored by a mass detector using an MRM (m/z, 515.3-285.1) model in negative electrospray ionization. The calibration curve showed good linearity (r2  > 0.99), with limits of detection and quantification of 0.25 and 2.00 nmol/L, respectively. The intra- and inter-day precision and accuracy were less than 9.99% and ranged from 97.45% to 114.62%, respectively. The extraction recovery from plasma was between 92.89% and 98.87%. GAA was found to be stable in treated samples at room temperature (22 °C) for 12 h and in plasma at −20 °C for 7 d. The developed method was successfully applied to a pharmacokinetic study of GAA in rats. GAA could be rapidly absorbed into the circulation (Tmax, 0.15 h) and eliminated relatively slowly (t1/2, 2.46 h) after orally dosing, and could also be detected in the brain lateral ventricle (Tmax, 0.25 h and t1/2, 1.40 h) after intravenously dosing. The absolute oral bioavailability and brain permeability of GAA were estimated to be 8.68% and 2.96%, respectively.
Keywords: Ganoderic acid A; UFLC–MS/MS; Pharmacokinetics; Plasma; Brain microdialysis;

A rapid, simple and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA), 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human urine. The urine samples were mixed with buffer solutions (pH 8) and subsequently cleaned up by solid supported liquid/liquid extraction (SLE). The target analytes were efficiently separated with a Waters Atlantis T3 column (150 mm × 4.6 mm, 5 μm), ionized with electrospray ion source in positive mode, and quantitatively determined by tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. In order to minimize matrix effects, the matrix-matched calibration curves of eight analytes were adopted with correlation coefficients (R2) above 0.99. The method were further validated by determining the limits of detection (LODs, 0.3–0.6 ng/mL), the limits of quantitation (LOQs, 1.0–2.0 ng/mL) and recoveries (89.1%–108.4%) with intra-day and inter-day relative standard deviation (RSD, <11%). The established method was applied and demonstrated in a real case by assaying a urine sample from a female poisoned by formetanate. The achieved results proved this method to be rapid, sensitive and accurate for simultaneous quantitation of eight analytes in human urine for intended forensic cases of human poisoning.
Keywords: Formamidine pesticides; Metabolites; Human urine; Solid supported liquid/liquid extraction (SLE); HPLC–MS/MS;

High-resolution mass spectrometry as an alternative detection method to tandem mass spectrometry for the analysis of endogenous steroids in serum by Federico Ponzetto; Julien Boccard; Norbert Baume; Tiia Kuuranne; Serge Rudaz; Martial Saugy; Raul Nicoli (34-42).
Recently, steroid hormones quantification in blood showed a promising ability to detect testosterone doping and interesting complementarities with the urinary module of the Athlete Biological Passport (ABP). In this work, an ultra-high pressure liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) method was developed for the quantification of eleven endogenous steroids in serum. The performance of the full scan and targeted SIM acquisition modes was evaluated and compared to the performance of tandem mass spectrometry (MS/MS). Passing-Bablok regressions and Bland-Altman plots were assessed for each analyte of interest, and concentration values measured by HRMS showed high correlation with the ones obtained by MS/MS for all target hormones, with low absolute differences in the majority of cases. A slight decrease in terms of sensitivity was observed with HRMS in both acquisition modes, but performing an analysis of variance multiblock orthogonal partial least squares (AMOPLS) on the dataset obtained with all three methods revealed that only 0.8% of the total variance was related to instrumentation and acquisition methods. Moreover, the evaluation of the testosterone administration effect over time highlighted testosterone itself and dihydrotestosterone as the most promising biomarkers of exogenous testosterone administration. This conclusion suggests that HRMS could provide suitable performance for blood steroid analysis in the anti-doping field.
Keywords: Testosterone; Doping; Serum; UHPLC-HRMS; AMOPLS;

Identification of ace inhibitory cryptides in Tilapia protein hydrolysate by UPLC–MS/MS coupled to database analysis by Ben Henda Yesmine; Bonnet Antoine; Nunes Gonzalez da Silva Ortência Leocádia; Boscolo Wilson Rogério; Arnaudin Ingrid; Bridiau Nicolas; Maugard Thierry; Piot Jean-Marie; Sannier Frédéric; Bordenave-Juchereau Stéphanie (43-50).
An ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry method was developed and applied to identify short angiotensin-I-converting enzyme (ACE) inhibitory cryptides in Tilapia (Oreochromis Niloticus) protein hydrolyzate.A database was created with previously identified ACE-inhibitory di- and tripeptides and the lowest molecular weight fraction of Tilapia hydrolysate was analysed for coincidences. Only VW and VY were identified. Further analysis of collected fractions conducted to the identification of 51 different peptides in major fractions. 19 peptides selected were synthesised and tested for their ACE inhibitory potential. TL, TI, IK, LR, LD, IQ, DI, AILE, ALLE, ALIE and AIIE were identified as new ACE inhibitors.The findings from this study point UPLC–MS/MS combined with the creation of a database as an efficient technique to identify specific short peptides within a complex hydrolysate, in addition with de novo sequencing. This efficient characterisation of bioactive factors like cryptides in protein hydrolysates will extend their use as functional foods.
Keywords: Cryptides; ACE; UPLC; MS/MS; Database; Tilapia protein hydrolysate;

Simple and rapid determinations of some psychotropic drugs in some pharmaceutical wastewater and human plasma samples were successfully accomplished via the tandem dispersive liquid–liquid microextraction combined with high performance liquid chromatography-ultraviolet detection (TDLLME-HPLC-UV). TDLLME of the three psychotropic drugs clozapine, chlorpromazine, and thioridazine was easily performed through two consecutive dispersive liquid–liquid microextractions. By performing this convenient method, proper sample preconcentrations and clean-ups were achieved in just about 7 min. In order to achieve the best extraction efficiency, the effective parameters involved were optimized. The optimal experimental conditions consisted of 100 μL of CCl4 (as the extraction organic solvent), and the pH values of 13 and 2 for the donor and acceptor phases, respectively. Under these optimum experimental conditions, the proposed TDLLME-HPLC-UV technique provided a good linearity in the range of 5–3000 ng mL−1 for the three psychotropic drugs with the correlation of determinations (R2s) higher than 0.996. The limits of quantification (LOQs) and limits of detection (LODs) obtained were 5.0 ng mL−1 and 1.0-1.5 ng mL−1, respectively. Also the proper enrichment factors (EFs) of 96, 99, and 88 for clozapine, chlorpromazine, and thioridazine, respectively, and good extraction repeatabilities (relative standard deviations below 9.3%, n = 5) were obtained.
Keywords: Tandem dispersive liquid–liquid microextraction; Psychotropic drugs; High performance liquid chromatography-ultraviolet detection;

A selective HPLC–MS/MS method for quantification of SND-117 in rat plasma and its application to a pharmacokinetic study by Bei-Hua Bao; An Kang; Yang Zhao; Qi Shen; Jun-Song Li; Liu-Qing Di; Jian-Xin Li (60-65).
Rheumatoid arthritis (RA), a chronic systemic inflammatory disorder affects many adults. Sinomenine, a natural product, has been clinically available for the treatment of RA in China. SND-117, a sinomenine derivative with much more potent activity, might serve as a candidate for anti-arthritis. The aim of the present study was to develop a sensitive and rapid high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method for quantification of SND-117 in rat plasma and to understand its absolute bioavailability. The HPLC–MS/MS method was developed and fully validated for determination of SND-117 in rat plasma, and the pharmacokinetic differences were investigated after different administration routes. The pharmacokinetics parameters were calculated by non-compartment model with DAS 3.0 software. After the oral or intravenous administration of different doses of SND-117, the time to peak is 1.5 h, half-life time is 8–10 h. The absolute oral bioavailability of SND-117 in rats was 9.60%. The results showed that SND-117 in rats was quickly absorbed, slowly eliminate, and the kinetics were linear. This method was suitable for pharmacokinetic studies of SNA-117 in rats.
Keywords: SND-117; Pharmacokinetics; Anti-arthritis; HPLC–MS/MS;

Echinacea (Echinacea purpurea), green tea (Camellia sinensis) and yaylacayi (Thymus praecox OPIZ subsp. grossheimii (Ronniger) Jalas) are herbs used in traditional medicine because of their beneficial health effects. A high performance liquid chromatography (HPLC) post-column method was developed by coupling HPLC with on-line antioxidant assays using diode array (DAD) and ultraviolet (UV) detectors. Thus antioxidant compounds and fifteen phenolic standards were detected in echinacea, green tea and yaylacayi methanol extracts simultaneously. Later on, the developed on-line HPLC coupled system was used to compare three different antioxidant methods, ABTS [2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], DPPH (2,2-diphenyl-1-picrylhydrazyl and FRAP (Ferric reducing antioxidant power). Eight compounds with antioxidant activity were identified as gallic acid, protocatechuic aldehyde, p-OH benzoic acid, chlorogenic acid, caffeic acid, vanillin, ferulic acid, and rosmarinic acid. For validation purposes, LOD (limit of detection) (0.36–14.68 μg/mL) and LOQ (limit of quantification) (1.35–48.92 μg/mL) values were calculated for six standards. The results showed that while the post-column ABTS and FRAP assays were repeatable, sensitive and supportive of each other, the post-column DPPH assay did not provide reliable results. Also due to the base-line noise and drifts in post-column DPPH assay, identification and quantitation of analytes could not be done accurately.
Keywords: Echinacea purpurea; Camellia sinensis; Thymus praecox; Antioxidants; On-line HPLC-ABTS/DPPH/FRAP;

Deciphering the influence of column chemistry and mass spectrometry settings for the analyses of geometrical isomers of L-chicoric acid by Keabetswe Masike; Fidele Tugizimana; Nombuso Ndlovu; Elize Smit; Louis du Preez; Ian Dubery; Edwin Madala (73-81).
Resolving the chemo-diversity of plant extract samples is an essential step for in-depth analyses of natural products which often exhibit promising biological activities. One of the challenges in this endeavor has been the confident differentiation of geometrical isomers. In this study, we investigated these aspects in chromatography (column chemistry and mobile phase composition) and mass spectrometry settings with regards to better differentiation of geometrical isomers. A standard of a hydroxycinnamic acid (HCA) derivative, L-chicoric acid (L-CA) – a di-acylated caffeoyltartaric acid ester found in a number of plant families – was used. Geometrical isomers of L-CA were formed by exposing the compound to ultraviolet (UV) radiation, to mimic the natural environment. The high performance liquid chromatography photo-diode array (HPLC-PDA) and ultra-high performance liquid chromatography mass spectrometry (UHPLC–MS) platforms were used to analyze the trans and cis geometrical isomers of L-CA. The HPLC-PDA results confirmed the generation of two cis geometrical isomers following UV exposure of the authentic trans-L-CA standard. Furthermore, the HPLC-PDA analyses demonstrated that the changes in both column chemistry (reverse-phase: C18, biphenyl, phenyl-hexyl and pentafluorophenyl propyl) and mobile phase composition (aqueous acetonitrile and aqueous methanol) affect the chromatographic elution profiles of the L-CA isomers. The MS results, on the other hand, revealed undisputed fragmentation differences between the geometrical isomers of L-CA. Thus, this study demonstrates that the identification of the L-CA isomers can be achieved more efficiently and confidently with good chromatography coupled to well-optimized mass spectrometry conditions, a requirement which has been proven impossible with other types of HCA derivatives. Moreover, differences in the binding modes of L-CA geometrical isomers to the HIV type 1 integrase enzyme were observed, suggesting a synergistic anti-HIV-1 activity of these isomers.
Keywords: Chicoric acid (di-caffeoyltartaric acid); Fragmentation; Geometrical isomers; cis/trans isomerization; Mass spectrometry; Photochemical reaction; Ultra-violet (UV) light;

Pharmacokinetic study of representative anti-oxidative compounds from Denshen-Chuanxiong-Honghua following oral administration in rats by Xianhua Zhang; Wan Zheng; Huali Xu; Xi Huang; Ping Ren; Hui Zou; Guihua Liu; Jian Wang; Xinliang Ma (82-90).
Almost no pharmacokinetic compounds to date have been precisely linked with the activity of their herbal or Traditional Chinese Medicine (TCM) formula. This creates challenges for pharmacokinetic significance and application of the TCM. In our study, a sensitive and selective liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed to quantitatively or qualitatively determine multiple-components (tanshinol, ferulic acid, protocatechuic acid, rosmarinic acid, salvianolic acid B, baicalin and 9′-methyl lithospermate B) in rat plasma following the oral administration of Denshen-Chuanxiong-Honghua (DCH) extract (20 g/kg). Chromatographic separation was carried out on a 300SB-C18 column using a gradient elution with a mobile phase composed of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 1.0 mL/min. Determination by mass spectrometry (MS) was conducted in multiple reaction monitoring (MRM) mode with negative electrospray ionization. The validated method exhibited good linearity, with correlation coefficients greater than 0.9949 over a wide concentration range, and the lower limits of quantification were 2.09–12.2 ng/mL for the 5 analytes. This assay was successfully applied to investigate the pharmacokinetics of 5 compounds in rat plasma after the oral administration of DCH extracts. In addition, the anti-oxidant capacities of the 5 active ingredients of DCH extract in vitro and the total absorbed DCH extract in vivo were investigated at different concentrations during pharmacokinetic studies.
Keywords: DCH extract; Herb; Pharmacokinetic; LC/MS/MS; Anti-oxidative;

Although much is known about the pharmacological activities of Dragon's Blood (DB, a traditional Chinese herb), its metabolism in human liver microsomes (HLMs) and the cytochrome P450 (CYP) enzymes has not been studied. This study aims to identify the metabolic profile of five flavonoids (loureirin A, loureirin B, loureirin C, 7,4′-dihydroxyflavone and 5,7,4′-trihydroxyflavanone) from DB in HLMs as well as the CYP enzymes that are involved in the metabolism of them. High-resolution mass spectrometry was used to characterize the structures of their metabolites and 10 cDNA-expressed CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) were used to verify which isozymes mediate in the metabolism of the metabolites. Totally, 29 metabolites including 10 metabolites of loureirin A, 10 metabolites of loureirin B, 4 metabolites of loureirin C, 2 metabolites of 7,4′-dihydroxyflavone and 3 metabolites of 5,7,4′-trihydroxyflavanone were elucidated and identified on the basis of the high-resolution MS n data. The metabolic profile of the five flavonoids in HLMs involved hydroxylation, oxidation and demethylation. Among them, hydroxylation was the predominant biotransformation of the five flavonoids in HLMs, occurring in combination with other metabolic reactions. Assay with recombinant P450s revealed that CYP2C9 and CYP2C19 played an important role in the hydroxylation of flavonoids in HLMs. To the best of our knowledge, this is the first in vitro evaluation of the metabolic profile of loureirin A, loureirin B, loureirin C, 7,4′-dihydroxyflavone and 5,7,4′-trihydroxyflavanone in HLMs.
Keywords: Dragon's Blood; Human liver microsomes; HPLC-HR-MS; Metabolites;

Ultra-performance liquid chromatography tandem mass spectrometry for determination of Direct Acting Antiviral drugs in human liver fine needle aspirates by Andrew J. Ocque; Colleen E. Hagler; Robin DiFrancesco; Gene D. Morse; Andrew H. Talal (103-109).
An ultra-performance liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of direct acting antiviral drug concentrations in human liver fine needle aspirates. Liver fine needle aspirate (FNA) biopsy samples were homogenized in acetonitrile to stabilize the analytes and precipitate protein. The acetonitrile supernatants were diluted with internal standards and mobile phase. Separation was achieved with a Waters Acquity BEH C18 column (50 × 2.1 mm, 1.7 um) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 4.25 min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 12.5 to 5000 ng/mL for dasabuvir and the m1 metabolite of dasabuvir, 1.25 to 2500 ng/mL for ombitasvir and ritonavir, and 5.00 to 5000 ng/mL for paritaprevir. The intra- and inter-day accuracy and precision were less than 13.7% in low, medium, and high quality control samples. The validated method was applied to the analysis of a liver fine needle aspirate of a patient undergoing direct acting antiviral therapy for hepatitis C virus.
Keywords: Dasabuvir; Ombitasvir; Paritaprevir; Liver; Fine needle aspirate; UPLC–MS/MS;

In the present study, the impact of the extraction solvent on the accuracy of endogenous progesterone assay in human serum has been investigated using two selective reaction monitoring (SRM) transitions (315 > 97 & 315 > 109). Higher levels of noise and more interference were observed when more polar solvents were used for extraction, thus resulting in serious bias of the measured values of progesterone in serum. This is confirmed by monitoring the ion ratio of 315 > 97–315 > 109. This issue could not be easily resolved by changes in MS/MS transitions or chromatography conditions. More bias was observed with the SRM transition 315 > 109 for the polar solvent extraction. Hexane and 1-chlorobutane (polarity index of 0 and 1, respectively) did provide the cleanest samples with a lower noise level in the chromatograms. Moreover, the measured values of progesterone were not changed with different SRM transitions or longer retention time in search of an improved separation. Recovery tests of progesterone have been performed with 1-chlorobutane in matrices with phosphate buffered saline (PBS) 1x, PBS 1 × 3% bovine serum albumin (BSA), stripped serum/H2O (1:1) and unstripped serum. The recovery (70% ∼ 80%) consistency is observed not only at different levels but also in different matrices. The equivalent recovery between PBS 1x, PBS 1 × 3% BSA and unstripped serum shows that the impact of progesterone binding to serum proteins on the measurement accuracy can be avoided with this sample preparation procedure. No significant matrix effect on the determination of progesterone was observed with 1-chlorobutane. Within the range of 12.5–2000 pg/mL, a good linearity is observed with R > 0.99 and weighting factor 1/X. Bias and covariance efficiency of QCs are within 10%. With 1-chlorobutane as the extraction solvent, the concentration of progesterone was measured where the range for postmenopausal serum is 5.74 ∼ 91.7 pg/mL, which is well below the reported concentrations of 314 pg/mL ∼ 942 pg/mL in postmenopausal serum by immunoassay-based techniques, while the range in premenopausal serum is 12.8 pg/mL ∼ 18.6 ng/mL.
Keywords: Progesterone; LC–MS/MS; Liquid-liquid extraction; 1-Chloro-butane; Hexane; Methyl tertiary-butyl ether; Ethyl acetate;

Affinity binding of proteins to the modified bacterial cellulose nanofibers by Monireh Bakhshpour; Emel Tamahkar; Müge Andaç; Adil Denizli (121-127).
The potential of the modified bacterial cellulose (BC) nanofibers was determined bearing metal ion coordination interactions to enhance the protein adsorption and binding capacity. Thus, a household synthesized metal chelating monomer, namely N-methacryloyl-l-histidine methylester (MAH), and a commercial metal chelating monomer, namely 4-vinylimidazole (VIm), were used to complex with metal ions Cu(II) and Ni(II) respectively for the synthesis of the modified BC nanofibers. The modified nanofibers were characterized by FT-IR, SEM and EDX measurements. The protein adsorption tests were carried out using hemoglobin as a model protein and it was determined that the maximum adsorption capacity of hemoglobin onto the modified BC nanofibers was found as 47.40 mg/g. The novel strategy for the preparation of metal chelated nanofibers was developed.
Keywords: Protein recognition; Metal ion coordination; Bacterial cellulose nanofibers;

A selective and sensitive analytical method for the simultaneous determination of cucurbitacin B, E, I and E-glucoside in plant material and body fluids by HPLC–MS was developed. After liquid–liquid extraction with dichlormethane, separation was achieved on a Phenomenex Luna Pentafluorophenyl Column (150 mm × 2 mm, 5 μm) using acetonitrile-water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis Q1 Scans with negative ionisation were chosen. The method was validated for serum as the matrix of choice. Limits of detection are in the picogram range, limits of quantification are between 0.05 and 0.42 ng/mL, recoveries are above 50%. The assay was linear in the calibration range from 1.0 to 50 ng/mL for cucurbitacin E and from 0.10 to 50 ng/mL for the cucurbitacins B, I and E-glucoside. The applicability of the method was demonstrated by the determination of cucurbitacins in zucchini plant material and body fluids from intoxication cases.
Keywords: Cucurbitacins; Cucurbitaceae; Zucchini; High performance liquid chromatography-mass-spectrometry (HPLC–MS); Intoxication;

Simultaneous quantification of 11 active constituents in Shexiang Baoxin Pill by ultraperformance convergence chromatography combined with tandem mass spectrometry by Chao Lv; Liqing Chen; Peng Fu; Niao Yang; Qun Liu; Yongwei Xu; Qinglong Sun; Rui Li; Changsen Zhan; Weidong Zhang; Runhui Liu (135-141).
On account of the complexity of chemical constituents of Shexiang Baoxin Pill (SBP), a famous traditional Chinese medicine (TCM) formula, a novel and effective UPC2-MS/MS method was developed to simultaneously determine the content of 11 active compounds of SBP with outstanding separation ability. Eleven components in SBP, including 2 ginsenosides, 2 bile acids, 3 bufadienolides and 4 volatiles were detected by electrospray ionization tandem mass spectrometry in positive and negative ion modes with multiple reaction monitor (MRM). The analysis was performed at 30 °C using an Acquity UPC2 Diol (3.0 × 50 mm, 1.7 μm) column with linear gradient elution (eluent A, CO2; eluent B, methanol containing 20 mM ammonium acetate), back pressure of 2000 psi, flow rate of 1.2 mL/min and the injection volume of 1.0 μL. The method was extensively validated regarding the linearity (r ≥ 0.9974), precision (≤3.11%), recovery (93.34–104.50%), repeatability (≤2.00%) and stability (≤4.20%). Using this method, 11 active compounds of SBP with different polarity were simultaneously quantified in one chromatography analysis within 8 min. Statistical analysis of the effects of 11 compounds on the quality of SBP revealed that the content of cinnamaldehyde varied widely in different batches. This work presents an exemplary study for quality control of complex samples, especially for TCMs.
Keywords: Shexiang Baoxin Pill; UPC2-MS/MS; Quantification; Non-volatiles; Volatiles;

Optimized conversion of antiproliferative lignans pinoresinol and epipinoresinol: Their simultaneous isolation and identification by centrifugal partition chromatography and high performance liquid chromatography by Anna Sólyomváry; Ágnes Alberti; András Darcsi; Rita Könye; Gergő Tóth; Béla Noszál; Ibolya Molnár-Perl; László Lorántfy; Judit Dobos; László Őrfi; Szabolcs Béni; Imre Boldizsár (142-149).
High amount of the valuable lignan pinoresinol (PR) was determined in Carduus nutans fruit (7.8 mg/g) for the first time. A preparative separation method using two consecutive, identical steps of centrifugal partition chromatography (CPC) was developed in order (i) to isolate PR and (ii) to subsequently isolate PR and its 7′ epimer epipinoresinol (EPR) simultaneously after an optimized acid treatment which resulted in PR epimerization forming equal amounts of PR and EPR, from C. nutans fruit. As optimal conditions, a two-phase solvent system consisting of methyl tert-butyl ether:acetone:water (4:3:3, v/v/v) for CPC separation, and an acid treatment performed at 50 °C for 30 min for the epimerization were applied. Thus, 33.7 mg and 32.8 mg PR and EPR, in as high as 93.7% and 92.3% purity, were isolated from 10.0 g  C. nutans fruit, representing 86.4% and 84.1% efficiency, respectively. Conversion characteristic of PR and EPR in acidic medium, determined as a function of time and temperature of acid treatment provides their unambiguous identification by on-line high performance liquid chromatography (HPLC). Antiproliferative assay of isolated PR and EPR in two different types of colon cancer cell lines (HCT116 and SW480) confirmed that both epimers caused a more significant decrease of viability in HCT116 cells than in SW480 cells, suggesting their similar mechanism of antiproliferative action.
Keywords: Centrifugal partition chromatography; HPLC; Isolation; Pinoresinol; Epipinoresinol; Colon cancer;

A highly sensitive method for the simultaneous UHPLC–MS/MS analysis of clonidine, morphine, midazolam and their metabolites in blood plasma using HFIP as the eluent additive by Rūta Veigure; Rudolf Aro; Tuuli Metsvaht; Joseph F. Standing; Irja Lutsar; Koit Herodes; Karin Kipper (150-157).
In intensive care units, the precise administration of sedatives and analgesics is crucial in order to avoid under- or over sedation and for appropriate pain control. Both can be harmful to the patient, causing side effects or pain and suffering. This is especially important in the case of pediatric patients, and dose-response relationships require studies using pharmacokinetic-pharmacodynamic modeling. The aim of this work was to develop and validate a rapid ultra-high performance liquid chromatographic-tandem mass spectrometric method for the analysis of three common sedative and analgesic agents: morphine, clonidine and midazolam, and their metabolites (morphine-3-glucuronide, morphine-6-glucuronide and 1′-hydroxymidazolam) in blood plasma at trace level concentrations. Low concentrations and low sampling volumes may be expected in pediatric patients; we report the lowest limit of quantification for all analytes as 0.05 ng/mL using only 100 μL of blood plasma. The analytes were separated chromatographically using the C18 column with the weak ion-pairing additive 1,1,1,3,3,3-hexafluoro-2-propanol and methanol. The method was fully validated and a matrix matched calibration range of 0.05–250 ng/mL was attained for all analytes In addition, between-day accuracy for all analytes remained within 93–108%, and precision remained within 1.5–9.6% for all analytes at all concentration levels over the calibration range.
Keywords: UHPLC–MS/MS; Chromatographic separation; Morphine; Clonidine; Midazolam; Hexafluoroisopropanol;

Chemometrics-enhanced one-dimensional/comprehensive two-dimensional gas chromatographic (GC/GC × GC) technologies, were used to explore the compositions of Chaihu Shugan San essential oils, that were extracted from the herbal formulae by different schemes. We have shown that chemometric resolution using gas chromatographic- mass spectrometry (GC–MS) could be used for the qualitative and quantitative analysis of the majority of Terpenoids or Phthalides from herb formulae and single herbs. A GC × GC system was further optimized to achieve the increased peak capacity and the enhanced signal of the hydro-distillation sample (CSSh). When hardware bottleneck resulted from very complex sample, chemometric tools were once again applied to recover the stained information in the second dimension (2D) matrix data. Heuristic evolving latent projections (HELP) could be used for two dimensional (2D) sub-matrixes Xi at n spectral detection channels, after three dimensional (3D) data splitting. For a real 3D data matrix, alternating trilinear decomposition (ATLD) algorithm could conduct regularization for an iterative trilinear decomposition procedure, by Moore-Penrose pseudoinverse computations based on singular value decomposition. After retention indices (RI) confirmation, 216 target analytes (terpenoids or phthalides) could be elucidated both in CSSh and in supercritical fluid extract (CSSs). Based on the obtained data, some potential quality markers (Q-markers) were identified which may affect the quality of the products. Finally, a “connectivity map” was plotted to describe the unique mechanisms of tradition Chinese medicine (TCM).
Keywords: Chemometrics; Chaihu Sugan San; Essential oils; Terpenoids; Phthalides; GC × GC-qMS;