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Publication year:
2012 Source:Analytica Chimica Acta, Volume 748 Jun Haginaka, Hiromi
Tabo, Hisami Matsunaga Monodisperse molecularly imprinted polymers (MIPs) for
diphenyl phosphate (DPP) and 1-naphthyl phosphate (1-NapP) have been prepared by
a multi-step swelling and polymerization method using 4-vinylpyridine as a
functional monomer, glycerol dimethacrylate as a crosslinker and cyclohexanol or
1-hexanol as a porogen. The retention and molecular-recognition properties of
these MIPs for organophosphorus compounds were evaluated by HPLC using a mixture
of phosphate buffer and acetonitrile as an eluent. In addition to shape
recognition, hydrogen bonding and hydrophobic interactions could play an
important role in the retention and molecular recognition of DPP and 1-NapP.
Furthermore, the MIPs were applied to the separation of adenosine and adenosine
phosphates (AMP, ADP and ATP). These phosphates were retained on the MIPs
according to the number of phosphate groups in the molecule and were well
separated from one another. Hydrogen bonding and hydrophobic interactions seemed
to affect the retention and recognition of adenosine phosphates in low
acetonitrile content, while hydrophilic interactions affected these properties
in high acetonitrile content. Finally, the MIPs were applied to the trapping of
phosphopeptides. The MIPs non-selectively trapped phosphopeptides, which have
phosphorylated tyrosine, serine or threonine in the sequences, and successfully
trapped four phosphopeptides in tryptic digests of bovine α-casein.
Graphical abstract
Graphical abstract Highlights
►
Monodisperse MIPs for organophosphates by multi-step swelling and
polymerization. ► Application of MIPs to the separation of adenosine phosphates.
► Application of MIPs to the trapping of phosphopeptides in tryptic
protein-digests.
Publication year:
2012 Source:Analytica Chimica Acta, Volume 748 Marek Roszko,
Małgorzata Rzepkowska, Arkadiusz Szterk, Krystyna Szymczyk, Renata Jędrzejczak,
Marcin Bryła Polychlorinated biphenyls (PCBs) and polybrominated diphenyl
ethers (PBDEs) are hazardous food contaminants, their maximum legally allowable
levels in food and environment are in the low pgg−1 range. Therefore
some highly selective and sensitive analytical methods must be used to determine
them. The 96/23/EC Directive implemented by EC Decision of 12 August 2002
requires recovery rate of an analyte at a concentration below 1ngg−1
within the 50–120% range at relative standard deviation (RSD) as low as
possible. A method to determine low level PCBs and PBDEs in milk fat based on
the semi-permeable membrane dialysis/ion trap GC MS technique was developed.
Validation experiments proved that the method performance was within bounds set
by the currently standing UE regulations. Recovery rates calculated on the basis
of labeled internal standards for majority of the studied indicator PCB
congeners and PBDE congeners were close to 100% at RSD below 20%. Also,
dioxin-like PCBs recovery rates were compatible with the 1883/2006 EC Regulation
(80–120%, RSD below 15%). The developed method turned out to be linear within a
far broader concentration range than the studied 0.0025–10pgμL−1
range entirely sufficient for analyses of PCB and PBDE in milk fat. Within that
range coefficient of linear correlation (R 2) of calibration curves
exceeded 0.98.
Graphical abstract
Graphical abstract Highlights
► We
have investigated the usefulness of SPM dialysis for determination of PCB/PBDE.
► The factors affecting the dialysis process were evaluated. ► The ion trap MS
was optimized for determination of PCB/PBDE in milk fat. ► Described
extraction/clean up method proves usefulness for the milk fat
analysis.
Publication year:
2012 Source:Analytica Chimica Acta, Volume 748 M.M. Parrilla
Vázquez, P. Parrilla Vázquez, M. Martínez Galera, M.D. Gil García An
ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction
(US-IL-DLLME) procedure was developed for the extraction of eight
fluoroquinolones (marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin,
danofloxacin, enrofloxacin, oxolinic acid and nalidixic acid) in groundwater,
using high-performance liquid chromatography with fluorescence detection
(HPLC-FD). The ultrasound-assisted process was applied to accelerate the
formation of the fine cloudy solution using a small volume of disperser solvent
(0.4mL of methanol), which increased the extraction efficiency and reduced the
equilibrium time. For the DLLME procedure, the IL 1-octyl-3-methylimidazolium
hexafluorophosphate ([C8MIM] [PF6]) and methanol (MeOH) were used as extraction
and disperser solvent, respectively. By comparing [C8MIM] [PF6] with
1-hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM] [PF6]) and
1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM] [PF6]) as extraction
solvents, it was observed that when using [C8MIM] [PF6] the cloudy solution was
formed more readily than when using [C6MIM] [PF6] or [C4MIM] [PF6]. The factors
affecting the extraction efficiency, such as the type and volume of ionic
liquid, type and volume of disperser solvent, cooling in ice-water, sonication
time, centrifuging time, sample pH and ionic strength, were optimised. A slight
increase in the recoveries of fluoroquinolones was observed when an ice-water
bath extraction step was included in the analytical procedure (85–107%) compared
to those obtained without this step (83–96%). Under the optimum conditions,
linearity of the method was observed over the range 10–300ngL−1 with
correlation coefficient >0.9981. The proposed method has been found to have
excellent sensitivity with limit of detection between 0.8 and 13ngL−1
and precision with relative standard deviation values between 4.8 and 9.4% (RSD,
n =5). Good enrichment factors (122–205) and recoveries (85–107%) were obtained
for the extraction of the target analytes in groundwater samples. This simple
and economic method has been successfully applied to analyse real groundwater
samples with satisfactory results.
Graphical abstract
Graphical abstract Highlights
► A
novel method by US-IL-DLLME-LC-FD for fluoroquinolones determination. ► Simple,
rapid and efficient method for water samples. ► Advantages over conventional
methods. ► Low detection limits.
Publication year:
2012 Source:Analytica Chimica Acta, Volume 748 Dayana Rubio Gouvea,
Fernando Meloni, Arthur de Barros Bello Ribeiro, João Luis Callegari Lopes,
Norberto Peporine Lopes Lychnophora salicifolia Mart., which occurs in the
Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the
southeast of the state of Goiás, is the most widely distributed and also the
most polymorphic species of the genus. This plant is popularly known to have
anti-inflammatory and analgesic activities. In this work, we have studied the
variation in terms of polar metabolites of ninety-three Lychnophora salicifolia
Mart. specimens collected from different regions of the Brazilian Cerrado.
Identification of the constituents of this mixture was carried out by analysis
of the UV spectra and MS data after chromatographic separation. Twenty
substances were identified, including chlorogenic acid derivatives, a flavonoid
C-glucoside, and other sesquiterpenes. The analytical method was validated, and
the reliability and credibility of the results was ensured for the purposes of
this study. The concentration range required for analysis of content variability
within the analyzed group of specimens was covered with appropriate values of
limits of detection and quantitation, as well as satisfactory precision and
recovery. A quantitative variability was observed among specimens collected from
the same location, but on average they were similar from a chemical viewpoint.
In relation to the study involving specimens from different locations, there
were both qualitative and quantitative differences among plants collected from
different regions of Brazil. Statistical analysis revealed that there is a
correlation between geographical localization and polar metabolites profile for
specimens collected from different locations. This is evidence that the pattern
of metabolites concentration depends on the geographical distribution of the
specimens.
Graphical abstract
Graphical abstract Highlights
Statistical analysis revealed
that there is a correlation between geographical localization of Lychnophora
salicifolia samples and polar metabolites profile for specimens collected from
different locations. ► A new HPLC-DAD-MS method for
analysis of the crude extract of L. salicifolia leaves. ► Twenty substances were
identified. ► Ninety-three wild specimens were analyzed. ► A correlation between
geographic isolation and decrease of the chemical similarity. ► The pattern of
metabolites depends on the geographical distribution of the
specimens.
Publication year:
2012 Source:Analytica Chimica Acta, Volume 748 M. Kraiem, S.
Richter, N. Erdmann, H. Kühn, M. Hedberg, Y. Aregbe Uranium and plutonium
particulate test materials are becoming increasingly important as the
reliability of measurement results has to be demonstrated to regulatory bodies
responsible for maintaining effective nuclear safeguards. In order to address
this issue, the Institute for Reference Materials and Measurements (IRMM) in
collaboration with the Institute for Transuranium Elements (ITU) has initiated a
study to investigate the feasibility of preparing and characterizing a uranium
particle reference material for nuclear safeguards, which is finally certified
for isotopic abundances and for the uranium mass per particle. Such control
particles are specifically required to evaluate responses of instruments based
on mass spectrometric detection (e.g. SIMS, TIMS, LA-ICPMS) and to help ensuring
the reliability and comparability of measurement results worldwide. In this
paper, a methodology is described which allows quantifying the uranium mass in
single micron particles by isotope dilution thermal ionization mass spectrometry
(ID-TIMS). This methodology is characterized by substantial improvements
recently achieved at IRMM in terms of sensitivity and measurement accuracy in
the field of uranium particle analysis by TIMS. The use of monodisperse uranium
oxide particles prepared using an aerosol generation technique developed at ITU,
which is capable of producing particles of well-characterized size and isotopic
composition was exploited. The evidence of a straightforward correlation between
the particle volume and the mass of uranium was demonstrated in this study.
Experimental results have shown that the uranium mass per particle can be
measured via the ID-TIMS method to a relative expanded uncertainty of about 10%
(coverage factor k =2). The availability of reliable and validated methods for
the characterization of uranium particles is considered to be essential for the
establishment of SI-traceable measurement results. It is therefore expected that
the method developed in this study is valuable for the certification of
particulate materials in which the isotopic composition and the content of
uranium must be accurately known.
Graphical abstract
Graphical abstract Highlights
► A
method to quantify the U mass in single micron particles by ID-TIMS was
developed. ► Well-characterized monodisperse U-oxide particles produced by an
aerosol generator were used. ► A linear correlation between the mass of U and
the volume of particle(s) was found. ► The method developed is suitable for
determining the amount of U in a particulate reference material.
Publication year:
2012 Source:Analytica Chimica Acta, Volume 748 Shunsuke Moriya,
Kaori Iwasaki, Keijiro Samejima, Koichi Takao, Kohfuku Kohda, Kyoko Hiramatsu,
Masao Kawakita An analytical method for the determination of three polyamines
(putrescine, spermidine, and spermine) and five acetylpolyamines
[N1-acetylspermidine (N1AcSpd),
N8-acetylspermidine (N8AcSpd),
N1-acetylspermine, N1,N8-diacetylspermidine,
and N1,N12-diacetylspermine] involved in the polyamine
catabolic pathway has been developed using a hybrid tandem mass spectrometer.
Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal
standards labeled with stable isotopes were analyzed simultaneously by TOF MS,
based on peak areas appearing at appropriate m/z values. The isomers,
N1AcSpd and N8AcSpd were determined from their fragment
ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using
MS/MS with 13C2-N1AcSpd and
13C2-N8AcSpd which have the 13C2-acetyl group
as an internal standard. The TOF MS method was successfully applied to measure
the activity of enzymes involved in polyamine catabolic pathways, namely
N1-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and
spermidine/spermine N1-acetyltransferase (SSAT). The following
natural substrates and products labeled with stable isotopes considering the
application to biological samples were identified; for APAO,
[4,9,12-15N3]-N1-acetylspermine and
[1,4,8-15N3]spermidine (15N3-Spd), respectively; for SMO,
[1,4,8,12-15N4]spermine and 15N3-Spd, respectively; and
for SSAT, 15N3-Spd and
[1,4,8-15N3]-N1-acetylspermidine, respectively.
Graphical abstract
Graphical abstract Highlights
►
Compounds in polyamine catabolic pathway were determined by a column-free
ESI-TOF MS. ► N1- and N8-acetylspermidine were determined
by a column-free ESI-MS/MS. ► The method was applied to determine activities of
APAO, SMO, and SSAT in the pathway. ► The assay method contained stable
isotope-labeled natural substrates. ► It is applicable to biological samples
containing natural substrate and product.
AMSBIO has expanded its range of products for cell growth and behaviour with a
suite of Cell Adhesion Assays, designed to evaluate optimal cell attachment to
extracellular matrices and assess factors that influence cell-matrix
interactions. Cell adhesion assays measure adhesion between cells or between a
cell and a surface or extracellular matrix; and have applications in many areas
of disease research, including neuroscience, cancer, and inflammation, among
others.
AMSBIO CultreCoat® Cell Adhesion Assays are pre-coated and ready-to-use in a
black stripwell microplate format that minimizes background, allows greater
sensitivity and affords flexibility in the number of samples assessed. The
stripwell feature also allows multiple experiments to be conducted
simultaneously using the same kit. Calcein labeling allows direct comparisons
between the number of cells that are loaded and the number that adhere.
Controls are provided for determining background and non-specific binding.
The new Adhesion protein array kit enables researchers on a single microplate,
to assess adhesion on Basement Membrane Extract (equivalent to Matrigel),
Laminin, Collagen I, Collagen IV, Fibronectin and Vitronectin. The adhesion
protein array kit is ideal for research applications that involve screening
multiple different extracellular matrices.
For research applications focusing on just one extracellular matrix, AMSBIO has
introduced a new range of Basement Membrane Extract, Laminin, Collagen I,
Collagen IV, Fibronectin and Vitronectin Cell Adhesion Assay Kits. These kits
are supplied as one 96-well stripwell microplate pre-coated with the
extracellular matrix of your choice and all the reagents required to conduct
the assay.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Jingya Zhou,
Shouhong Gao, Feng Zhang, Bo Jiang, Qin Zhan, Fei Cai, Jingxian Li, Wansheng
Chen This paper describes the development and validation of a novel, general
liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the
simultaneous determination of cyclophosphamide, ifosfamide, irinotecan,
etoposide, gemcitabine, carboplatin and pemetrexed concentrations in human
plasma. Samples were prepared by two kinds of extraction method and analyzed
using a gradient separation over an Atlantis T3-C18 column (2.1mm×100mm, 3μm,
Waters). Positive electrospray ionization was employed as the ionization source.
The mobile phase consisted of acetonitrile–water (0.1% formic acid and 10mM
ammonium acetate) at a flow rate of 0.25mL/min. Linear coefficients of
correlation were >0.992 for all analytes. The intra- and inter-day relative
standard deviation across three validation runs over the entire concentration
range was less than 9.2%, while the accuracy was within ±10.5%. The mean
recovery of all the analytes ranged from 50.0 to 81.0%. This method was
successfully applied to clinical samples from cancer patients.
Highlights
► We developed a novel, sensitive, reproducible,
and accurate LC–MS/MS method. ► This method could simultaneous determination of
seven anticancer drugs in human plasma. ► This new method was successfully
applied to clinical samples from cancer patients.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Fabio Fuselli,
Chiara Guarino, Alessandro La Mantia, Lucia Longo, Angelo Faberi, Rosa Maria
Marianella The incorrect use of preservatives in cheeses may compromise food
safety and damage consumers. According to the law, more than one preservative
may be contemporarily used in cheeses. So a method for their contemporary
detection may be useful for both manufacturers and control agencies quality
control. In this research a liquid chromatography–tandem mass spectrometric with
electrospray ionization method for the multi-determination of seven
preservatives (benzoic acid, citric acid, hexamethylenetetramine, lysozyme,
natamycin, nisin and sorbic acid) in cheese was developed. The preservatives
were contemporarily extracted from cheese by a single procedure, and analyzed by
RP-LC/ESI-MS/MS (Ion Trap) in positive ionization mode, with single reaction
monitoring (SRM) acquisition. Three sample types (hard, pasta filata and fresh
cheese) were used for method evaluation. Recoveries were mostly higher than 90%;
MDLs ranged from 0.02 to 0.26mgkg−1, and MQLs were included between
0.07 and 0.88mgkg−1. Due to matrix effect, quantitation was performed
by referring to a matrix matched calibration curve, for each cheese typology.
This method was also applied to commercial cheese samples, with good results. It
appears fast, reliable and suitable for both screening and confirmation of the
presence and quantitation of the preservatives in a single, multi-detection
analysis.
Highlights
► An RP-HPLC–ESI-MS/MS based method was developed
for the multi-determination of seven preservatives in three typologies of
cheeses. ► The analytical strategy was evaluated in terms of accuracy,
precision, limit of detection, and limit of quantification. ► Our method was
applied to commercial cheese samples with good results.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Yuran Zhang, Yu
Xin, Hailin Yang, Ling Zhang, Xiaole Xia, Yanjun Tong, Yi Chen, Li Ma, Wu
Wang An affinity protocol for purification of xanthine oxidase (XOD) from
Arthrobacter M3 was developed. The isolation procedure consisted of only three
steps, ammonium sulfate precipitation, affinity extraction to exclude the major
impurities, and the final refining procedure with DEAE ion-exchange
chromatography for removal of minor contaminants. In this affinity preparation,
guanine, an analogue of xanthine, was chosen as the affinity ligand, and was
coupled with Sepharose 4B through spacers composed of epichlorohydrin and
ethylenediamine. Crude protein has been run through ammonium sulfate
precipitation and the affinity column, 99.1% of proteins were removed. After
DEAE ion-exchange chromatography, the purity of the refined XOD was 97.5% by
Native-PAGE analysis. The activity recovery of purified XOD (36.1%) was almost
higher than that of other methods reported. Reducing SDS-PAGE analysis showed
that the purified XOD (one band in Native-PAGE analysis) showed two polypeptides
with the molecular weights ∼35kDa and ∼100kDa, respectively. The desorption
constant K d and the theoretical maximum absorption Q max on the affinity medium
were 3.0μg/ml and 2.2mg/g medium in absorption analysis.
Highlights
► A novel affinity protocol for the purification of
xanthine oxidase is developed. ► Only three steps successfully purified xanthine
oxidase. ► The most important step of this protocol is affinity chromatography.
► The mechanism relies on the affinity interaction between ligand and the
enzyme. ► This method has advantage of fewer steps, better recoveries, and
higher purity.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Zufan Debebe,
Sergei Nekhai, Meseret Ashenafi, David B. Lovejoy, Danuta S. Kalinowski, Victor
R. Gordeuk, W. Malcolm Byrnes, Des R. Richardson, Pradeep K. Karla In the
current study, we developed a HPLC method to quantitatively measure the
permeability of the BpT-based chelators, 2-benzoylpyridine
4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine
4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma
(Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and
Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak
area was linear with respect to chelator concentration. Acceptable within-day
and between-day precision (<22%) and accuracy (85–115% of true values) were
obtained over a range of 1.0–100μM for Bp4eT and 1.5–300μM for Bp4aT. Limits of
detection were 0.3μM and 1μM for Bp4eT and Bp4aT, respectively, while
corresponding limits of quantification were 1μM and 5μM. Both chelators showed
significant ability to chelate iron in THP-1 cells using a calcein-based assay
and no apparent cytotoxicity was observed within 24h. Ratios of the apical to
basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at
100μM and 300μM respectively, indicating equal bi-directional movement of the
compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This
study demonstrates that Bp4eT and Bp4aT can be efficiently transported through
Caco-2 cells and can potentially be formulated for oral delivery.
Highlights
► We developed a sensitive HPLC method for
assessment of BpT-E/Z isomers. ► We demonstrated effective iron chelation of
BpT-iron chelators that inhibit HIV-1. ► We estimated the permeability profile
of BpT-E/Z isomers in Caco2 monolayers. ► We anticipate that BpT chelators could
hold promise as orally effective agents.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Soo Hyun Lee,
Hyun Ok Yang, Hak Cheol Kwon, Byung Hwa Jung Glionitrin A (GN A) is a new
diketopiperazine disulfide with an aromatic nitro group, which is isolated from
the coculture of an Aspergillus fumigatus fungal strain and a Sphingomonas
bacterial strain. After intravenous administration of GN A in rats, 13 urinary
metabolites of GN A were identified using ultra-performance liquid
chromatography/quadrupole time-of-flight mass spectroscopy (UPLC–QTOP-MS)
analysis in conjunction with data processing programs such as MetaboLynx™ and
MassFragnent™. Reduction, nitro-reduction and hydration were the primary
metabolic processes affecting GN A in vivo, followed by demethylation or
oxidative deamination to alcohol, as well as cysteine, glycine, glucuronide or
sulfate conjugation. The metabolite resulting from reduction was found to be a
molecule with a dithiol group, and the metabolite made by nitro reduction was
found to be an aromatic amine corresponding to GN A. Both of these products may
have pharmacological or toxicological activity, which is valuable information in
terms of using GN A as a lead compound. In addition, this work showed that
UPLC–QTOP-MS analysis coupled with efficient data processing programs is useful
for rapid and reliable characterization of GN A metabolites in vivo.
Highlights
► After intravenous administration of GN A in rats,
urinary metabolites of GN A were identified. ► Metabolite identification was
performed using UPLC–QTOP-MS analysis in conjunction with data processing
programs. ► Reduction, nitro-reduction and hydration were the primary metabolic
processes affecting GN A in vivo. ► Demethylation, oxidative deamination to
alcohol and conjugations were also included in GN A metabolism.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Shuaihua Zhang,
Ruiyang Ma, Xiumin Yang, Chun Wang, Zhi Wang A sensitive method for the
determination of three cationic alkaloids (berberine, palmatine and
jatrorrhizine) from human plasma samples was developed by micelle to solvent
stacking (MSS) in capillary zone electrophoresis (CZE). In MSS, the sample
preconcentration mainly relies on the reversal in the effective electrophoretic
mobility of the analytes at the boundary zone between the sample and CZE
background solution (BGS). Under the optimized conditions, the sensitivity
enhancement factors achieved in terms of corrected peak area were in the range
from 47 to 53 for the alkaloids. The limits of detection (LODs) (S/N=3) for
berberine, palmatine and jatrorrhizine were 0.01, 0.01 and 0.02μg/mL,
respectively. The intraday (n =6) and interday repeatabilities (n =12) expressed
as the relative standard deviations (RSDs) were less than 6.9% in terms of peak
height and less than 7.3% in terms of corrected peak area, respectively. The
recoveries of the method for the three alkaloids were in the range of
95.9–101.5% with peak height as the quantitative signal, and 92.6–103.6% with
corrected peak area as the quantitative signal, respectively. The MSS-CZE method
proved to be suitable for the analysis of the alkaloids in human plasma samples.
Highlights
► On-line sample concentration and analysis of some
cationic alkaloids in human plasma. ► Micelle to solvent stacking in capillary
zone electrophoresis. ► High sensitivity and good recovery.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Jia Zhan,
Xue-jun Yu, Ying-ying Zhong, Zai-ting Zhang, Xiao-mei Cui, Jin-feng Peng, Rui
Feng, Xiao-tao Liu, Yan Zhu A generic, rapid and simple analytical method
able to identify 255 veterinary drug residues and other contaminants in raw milk
had been developed. The method was based on two-step simple precipitation and
ultra performance liquid chromatography coupled with electrospray ionization and
tandem mass spectrometry (UPLC–ESI–MS/MS) operating both in positive and
negative multiple reaction mode (MRM). For most of the target analytes, the
optimized pretreatment processes led to no significant interference on analysis
from complicated sample matrix. For quantification, matrix-fortified calibration
curves were performed to compensate for the matrix effect and loss in sample
preparation. Competent linearity was found for over 90% of target compounds with
linear regression coefficients (R) higher than 0.99. Detection limits ranged
from 0.05 to 10μg/kg. Average recoveries spiked into raw milk were in the range
from 63% to 141% with associated RSD values from 1% to 29% under the selected
conditions. The method had been validated for its extraction sensitivity,
linearity, recoveries and precision. The results clearly demonstrated the
feasibility of the approach proposed. Application of this method, which improved
efficiency and coverage of residues, would imply a drastic reduction of both
effort and time in routine monitoring programs.
Highlights
► First, the proposed method was generic for a wide
range polarity compounds. ► Second, the method, with only two steps, was rapid
and straightforward. ► Third, the way to remove water in milk for concentration
was novelty. ► Finally, this kind of method is urgently needed in dairy
plants.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Jing Ge,
Fengmao Liu, Eric H. Holmes, Gary K. Ostrander, Qing X. Li An aqueous normal
phase (ANP) liquid chromatography coupled with a hybrid quadrupole
time-of-flight mass spectrometry (ANP-LC-micrOTOFQ) method was used for the
determination of zanamivir in human serum. Zanamivir was extracted with methanol
from protein-precipitated human serum samples and further purified with SCX
solid-phase extraction cartridges. Scherzo SM-C18, Agilent Zorbax SB-Aq, Cogent
Diamond Hydride, Cogent Bidentate and Luna HILIC columns were compared and
optimized for the retention and separation of zanamivir and the Luna HILIC and
Diamond Hydride columns exhibited the best retention of zanamivir. The former
provided a shorter retention time, a sharper peak and relatively high
sensitivity, whereas the latter exhibited a longer retention time and less
matrix interference. The analytical range of the calibration curve was between 5
and 1000ng/mL.
Highlights
► Aqueous normal phase chromatography was studied
for the separation of zanamivir in human sera. ► Five different columns were
compared for the capability to retain zanamivir. ► Effects of mobile phase
constituents on the retention time were examined. ► The Diamond Hydride column
was the most effective to retain zanamivir and avoid matrix
effects.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Ying Shi, Zhen
Li, Yuanbiao Qiao, Jun Lin This work aimed to develop a rapid capillary zone
electrophoresis (CZE) method to provide abundant purity and identity information
of monoclonal antibodies. The CZE running buffer system was optimized to be 20mM
acetate–acetic acid (pH 6.0) together with the co-addition of 0.3% polyethylene
oxide (PEO) and 2mM triethylenetetramine (TETA), which was further tested with
advantages on the peak resolution improvements. The conditioning period was
scheduled to 1min for both 0.1M HCl and CZE running buffer to reduce total
separation time. Additionally, the applied voltage and effective separation
length were optimized at 30kV and 20cm separately. Compared with the method
reported by Yan [1], this newly developed method showed a higher resolution in
separating the two unknown basic peaks by testing monoclonal antibody sample
(mAb1). The further validation results showed that for all five of charge
isoform peaks of test mAb1, repeatability, intraday and interday precision had a
RSD less than 0.58% for migration time and less than 3.18% for corrected area
percent. The correlation coefficients of more than 0.98 for all peaks also
demonstrated the good linearity for the method. In addition to the application
of distinguishing intact antibody from C-terminal Lys variants, the method also
has advantage in separating the Fab, Fc and intact antibody-relevant substances
quickly, which facilitated the rough evaluation of papain induced digestion.
Highlights
► We prepared a simple CZE running buffer. ► We
developed and optimized a CZE method in dynamically coated capillary. ► We
performed fast separation in less than 5min with high
resolution.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Wuyi Zha,
Linyee Shum A selective and sensitive high performance liquid
chromatography–tandem mass spectrometric method was developed for the analysis
of azelastine and its major metabolite, desmethylazelastine, in human plasma.
Azelastine-13C, d3 was used as internal standard. Azelastine,
desmethylazelastine and the internal standard were extracted by a liquid–liquid
extraction method and separation was performed under isocratic chromatographic
condition. An abnormal signal loss issue for desmethylazelastine during method
development was investigated and resolved. The developed method was precise and
reproducible as shown by good intraday assay and interday assay precision
(CV%≤12.8%). The calibration curve was linear over a range of
10.0/10.0–1000/200pg/mL for azelastine/desmethylazelastine. The method was
successfully applied to a pilot bioequivalence study subsequently.
Highlights
► LC–MS/MS method for simultaneous quantitation of
azelastine and desmethylazelastine. ► Low LOQ of 10pg/mL for both compounds were
achieved. ► An abnormal signal loss issue for desmethylazelastine was
investigated and resolved.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Ádám Tölgyesi,
Virender K. Sharma, Szabolcs Fekete, Dóra Lukonics, Jenő Fekete This paper
describes a newly developed method for the simultaneous determination of eight
corticosteroid residues in bovine muscle, liver and kidney samples using liquid
chromatography–tandem mass spectrometry (LC–MS/MS). The determination of
methylprednisone, the main metabolite of methylprednisolone, in bovine tissues
using LC–MS/MS is carried out for the first time. The method development
demonstrates that the pH is important in optimizing the sample preparation.
Tests performed using different solid-phase extraction (SPE) cartridges were
enabled to produce conditions for reducing the matrix effects (ion suppression
and enhancement) of analysis. Acidic condition and mixed-mode cation exchange
SPE columns resulted in the most suitable clean-up for muscle and liver, and
also yielded acceptable results for kidney. The enhanced sample clean-up
resulted in excellent clear baselines of ion transitions, and therefore, a
higher delta electron multiplier voltage (ΔEMV) could be set in the MS/MS
detector. The application of 500V of ΔEMV improved the signal responses,
however, the noise level did not change, and consequently, the overall
sensitivity and analytical limits (limit of detection, limit of quantification)
could be enhanced. In the HPLC separation, the recently introduced Kinetex
phenyl-hexyl core–shell type column was used that enabled baseline separation
for dexamethasone and its β-epimer, betamethasone. Dexamethasone and
betamethasone were eluted within 12min and such reduced retention, obtained with
core–shell HPLC type column, further enhanced the sensitivity. The method was
validated according to the European Union (EU) 2002/657/EC Decision; the studied
parameters met the EU standards. The decision limits and limit of detections
were calculated in each matrix for all corticosteroids and varied from 0.01 to
13.3μg/kg and from 0.01 to 0.1μg/kg, respectively.
Highlights
► A new LC–MS/MS method is developed for
corticosteroids in tissue samples. ► Acidic pH control and mixed-mode SPE
cartridges are essential for sample clean-up. ► Corticosteroid epimers can be
separated simultaneously using Kinetex HPLC column. ► The enhanced clean-up and
LC–MS/MS separation improved the analytical limits. ► The method is successful
in analyzing dexamethasone in incurred samples.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Meixia Chen, Yu
Xia, Zhiyu Ma, Liang Li, Dafang Zhong, Xiaoyan Chen Pantoprazole (PAN), a
selective proton pump inhibitor, is used clinically as a racemic mixture for the
treatment of acid-related gastrointestinal disorders. To investigate its
stereoselective pharmacokinetics, a chiral liquid chromatography–tandem mass
spectrometry method was developed and validated to determine the pantoprazole
enantiomers in dog plasma. After liquid–liquid extraction, a baseline resolution
of enantiomers was achieved on an ovomucoid column using the mobile phase of
methanol:acetonitrile:10mM ammonium formate (pH 7) (10.4:2.6:87, v/v/v) at 30°C
within 10min. Stable isotopically labeled (+)-d3-pantoprazole and
(−)-d3-pantoprazole were used as internal standards. Acquisition of mass
spectrometric data was performed in multiple reaction monitoring mode via
positive atmospheric pressure chemical ionization. The method was linear in the
concentration range of 20.0–10,000ng/mL for each enantiomer using 25μL of dog
plasma. The lower limit of quantification (LLOQ) for each enantiomer was
20.0ng/mL. Intra- and inter-day precision ranged from 3.2% to 10.3% for
(+)-pantoprazole and 3.7–10.0% for (−)-pantoprazole. Accuracy varied from −1.4%
to −0.2% for (+)-pantoprazole and −1.6% to 0.8% for (−)-pantoprazole. The
validated method was applied successfully for stereoselective pharmacokinetic
studies of racemic pantoprazole.
Highlights
► A chiral LC–MS/MS method was validated to
quantify pantoprazole enantiomers. ► Separation was performed on an ovomucoid
protein column using MS compatible mobile phases. ► Baseline resolution within
10min leads to a reduction in the overall analysis time.
Publication year:
2012 Source:Journal of Chromatography B, Volume 906 Xiaoli Zhang,
Tong Zhao, Ting Cheng, Xiaoyan Liu, Haixia Zhang A rapid resolution liquid
chromatography (RRLC) method was developed for the simultaneous determination of
23 amino acids in rat serum after pre-column derivatization with
2,4-dinitrofluorobenzene (DNFB). The amino acid derivatives were separated on an
Agilent Zorbax Eclipse Plus C18 (4.6mm×50mm, 1.8μm) column at 45°C. Ultraviolet
(UV) detection was set at 360nm. Good separation of 23 amino acids was achieved
within 10min with a ternary gradient elution of mobile phase at a flow rate of
1.5mLmin−1. Calibration curves were linear over the range from 1 to
500μmolL−1 with coefficients 0.9962 or better for each amino acid.
The lower limits of quantification (LLOQ) of all 23 amino acids were
1μmolL−1 with signal-to-noise (S/N) ratio ≥4. Intra- and Inter-day
precisions, expressed as relative standard deviation (RSD) percentages, were
ranged from 0.32% to 3.09% and 0.67% to 5.82%, respectively. Finally, it was
successfully applied to the determination of amino acids in rat serum with
recoveries ranged from 90.8% to 106.0% and RSD percentages ranged from 1.78% to
4.68%, respectively. The results showed that the proposed method provided a
shorter elution time, better resolution and sharper peak shapes for all amino
acids. Compared with the conventional high performance liquid chromatography
(HPLC) methods, even some ultra-performance liquid chromatography tandem mass
spectrometry (UPLC–MS/MS), the established RRLC method was superior performance.
Highlights
► The RRLC method was the first time used for amino
acids detection. ► Compared with common HPLC and some UPLC, the RRLC was
superior performance. ► The reduced solvent consumption was friendly to
environment protection. ► The RRLC based on UV detection was economic and
available in common laboratories.
Publication year:
2012 Source:Journal of Chromatography B Sergey Vshivkov, Egor
Pshenichnov, Zamira Golubenko, Alik Akhunov, Shadman Namazov, Robert D.
Stipanovic Gossypol is a toxic compound that occurs as a mixture of
enantiomers in cotton plant tissues including seed and flower petals. The
(-)-enantiomer is more toxic to non-ruminant animals. Efforts to breed
cottonseed with a low percentage of (-)-gossypol requires determination of the
(+)- to (-)-gossypol ratio in seed and flower petals. We report a method to
quantitatively determine the total gossypol and percent of its enantiomers in
cotton tissues using high performance capillary electrophoresis (HPCE). The
method utilizes a borate buffer at pH 9.3 using a capillary with internal
diameter of 50μm, effective length of 24.5cm, 15kV and cassette temperature of
15°C. This method provides high accuracy and reproducible results with a limit
of detection of the individual enantiomers of less than 36ng/mL providing base
line separation in less than 6minutes.
Highlights
► Gossypol occurs as a mixture of enantiomers in
cottonseed. ► (-)-Gossypol is more toxic to non-ruminant animals. ► Plant
breeders are developing plants with a low percent of (-)gossypol. ► A capillary
electrophoresis method was developed to quantitate the enantiomers. ► Breeders
can use this method to select plants with the low (-)gossypol seed
trait.
Publication year:
2012 Source:Journal of Chromatography B Jinmei Fu, Jacob Bongers,
Li Tao, Dan Huang, Richard Ludwig, Yunping Huang, Yueming Qian, Jonathan Basch,
Joel Goldstein, Ramji Krishnan, Li You, Zheng Jian Li, Michael J. Grace, Reb J.
Russell Low levels of alanine to serine sequence variants were identified in
an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and
tandem mass spectrometry. The levels of the identified sequence variants A183S
and A152S, both in the light chain, have been determined to be 7.8-9.9% and
0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14,
respectively. The A183S variant was confirmed through tryptic map spiking
experiments using synthetic peptide, SDYEK, which incorporated Ser at the
position of native Ala in the tryptic peptide L16. Both mutations were also
observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was
also quantified by LC-UV with detection at 280nm and fluorescence detection of
tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and
fluorescent detection are in close agreement with each other. The levels of the
sequence variants are comparable among the antibody samples manufactured at
different scales as well as locations, indicating that the variants’ levels are
not affected by manufacture scale or locations. DNA sequencing of the master
cell bank revealed the presence of mixed bases at position 183 encoding both
wild and mutated populations, whereas bases encoding the minor sequence variant
at position152 were not detected. The root cause for A152S mutation is not yet
clearly understood at this moment.
Highlights
► Alanine to serine sequence variants were
identified in an IgG4 monoclonal antibody by LC/MS/MS. ► The sequence variants
were confirmed by use of synthetic peptide. ► DNA sequencing of the mater cell
bank revealed one variant was caused by mutation at the DNA
level.
Publication year:
2012 Source:Journal of Chromatography B Ruina Gao, Dafang Zhong, Ke
Liu, Yu Xia, Rongwei Shi, Hua Li, Xiaoyan Chen Morinidazole is a new
third-generation 5-nitroimidazole antimicrobial drug. To investigate the
pharmacokinetic profiles of morinidazole and its major metabolites in humans, a
liquid chromatography–tandem mass spectrometry method was developed and
validated for simultaneous determination of morinidazole, its N-oxide metabolite
(M4-1), a sulfate conjugate (M7), and two diastereoisomeric N
+-glucuronides (M8-1 and M8-2) in human plasma. A simple
acetonitrile-induced protein precipitation was employed to extract five analytes
and internal standard metronidazole from 50 μL human plasma. To avoid the
interference from the in-source dissociation of the sulfate and achieve the
baseline-separation of diastereoisomeric N +-glucuronides, all the
analytes were separated from each other with the mobile phase consisting of 10mM
ammonium formate and acetonitrile using gradient elution on a Hydro-RP C18
column (50 mm × 2 mm, 4 μm) with a total run time of 5 min. The API 4000 triple
quadrupole mass spectrometer was operated under the multiple reaction-monitoring
mode using the electrospray ionization technique. The developed method was
linear in the concentration ranges of 10.0ng/mL to 12000ng/mL for morinidazole,
1.00ng/mL to 200ng/mL for M4-1, 2.50ng/mL to 500ng/mL for M7, 3.00ng/mL to
600ng/mL for M8-1, and 10.0ng/mL to 3000ng/mL for M8-2. The intra- and inter-day
precisions for each analyte met the accepted value. Results of the stability of
morinidazole and its metabolites in human plasma were also presented. The method
was successfully applied to the clinical pharmacokinetic studies of morinidazole
injection in healthy subjects, patients with moderate hepatic insufficiency, and
patients with severe renal insufficiency, respectively.
Highlights
► Simultaneously determine morinidazole and its
four metabolites in human plasma. ► Gradient elution was used to obtain the
resolution of two N+-glucuronide isomers. ► The potential
interference from the in-source dissociation of the conjugates was avoided. ►
The method was applied to clinical pharmacokinetic studies of morinidazole
injection.
Publication year:
2012 Source:Journal of Chromatography B Thomas P. Mechtler, Thomas
F. Metz, Hannes G. Müller, Katharina Ostermann, Rene Ratschmann, Victor R. De
Jesus, Bori Shushan, Joseph M. Di Bussolo, Joseph L. Herman, Kurt R. Herkner,
David C. Kasper The interest in early detection strategies for lysosomal
storage disorders (LSDs) in newborns and high-risk population has increased in
the last years due to the availability of novel treatment strategies coupled
with the development of diagnostic techniques. We report the development of a
short-incubation mass spectrometry-based protocol that allows the detection of
Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease
within four hours including sample preparation from dried blood spots. Optimized
sample handling without the need of time-consuming offline preparations, such as
liquid-liquid and solid-phase extraction, allows the simultaneous quantification
of five lysosomal enzyme activities using a cassette of substrates and
deuterated internal standards. Applying incubation times of 3h revealed in intra
- day CV% values ranging from 4% to 11% for all five enzyme activities,
respectively. In a first clinical evaluation, we tested 825 unaffected newborns
and 16 patients with LSDs using a multiplexed, turbulent flow
chromatography–ultra high performance liquid chromatography–tandem mass
spectrometer assay. All affected patients were identified accurately and could
be differentiated from non-affected newborns. In comparison to previously
published two-day assays, which included an overnight incubation, this protocol
enabled the detection of lysosomal enzyme activities from sample to first result
within half a day.
Highlights
► We report a short-incubation mass
spectrometry-based protocol for lysosomal enzymes. ► Optimized sample handling
and online clean-up allowed a 4h analysis. ► Up to 5 lysosomal enzyme activities
are analyzed simultaneously from dried blood spots. ► Method validation was
performed under routine clinical laboratory environment.
Publication year:
2012 Source:Journal of Chromatography B Núria Monfort, Rosa
Ventura, Georgina Balcells, Jordi Segura Di-(2-ethylhexyl)phthalate (DEHP) is
the most commonly used plasticizer for polyvinyl chloride, which is found in a
large variety of products, including most of the bags used for blood storage
because of its protective role on erythrocytes survival. DEHP metabolites have
been recently proposed as markers of the misuse of blood transfusion in
athletes. In this study, a method to quantify the main five DEHP metabolites in
urine has been developed: mono-(2-ethylhexyl)phthalate (MEHP),
mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP),
mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP),
mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP), and
mono-(2-carboxymethylhexyl)phthalate (2cx-MMHP). The method involved an
enzymatic hydrolysis with β-glucuronidase from Escherichia coli followed by an
acidic extraction with ethyl acetate. The hydrolyzed extracts were analysed by
ultraperformance liquid chromatography tandem mass spectrometry. Isotope
labelled MEHP, MEOHP and 5cx-MEPP were used as internal standards. Analysis of
all the metabolites was achieved in a total run time of 10min, using a C18
column and a mobile phase containing deionized water and acetonitrile with
formic acid, with gradient elution at a flow-rate of 0.6mLmin−1.
Detection of the compounds was performed by multiple reaction monitoring, using
electrospray ionization in positive and negative ion modes. The method was
validated for quantitative purposes. Extraction recoveries were greater than 90%
and the limits of quantitation ranged from 1.2 to 2.6ngmL−1.
Intra-day precisions were better than 8% for all metabolites while inter-assay
precisions were better than 12%. Concentrations of DEHP metabolites were
measured in a control group (n=30, subjects reflecting the common environmental
DEHP exposure), and in sportsmen (n=464), to evaluate population distribution
exposure to DEHP. Additionally, threshold concentrations indicating outliers of
common exposure for DEHP metabolites are proposed.
Highlights
► Di-(2-ethylhexyl)phthalate (DEHP) is the most
commonly used plasticizer for PVC ► DEHP metabolites have been proposed as
markers of the misuse of blood transfusion ► A method to quantify five DEHP
metabolites has been developed and validated ► Cutoff concentrations were
proposed to determine suspicions of a possible transfusion ► The method is
proposed as screening test for transfusion detection in doping control
Olympus
has released the innovative new IX3 series of inverted research microscope
systems for effortless, intuitive live cell imaging and clinical analysis. This
includes the fully automated IX83 for high-end research applications, the
flexible IX73, which can be configured in manual, semi-motorised or motorised
modes, and the easy-to-use IX53 with fluorescent capabilities, which is
optimised for the routine examination of tissue samples. Built using worldwide
customer feedback and designed to meet the needs of a wide range of users, the
new systems offer exceptional ease-of-use and unprecedented optical flexibility
via a new, customisable light path. New components can be easily slid into the
light path using a series of swappable decks. This opens up many new avenues
for exploration, allowing researchers to follow their imaginations. The new
systems also utilise the latest Olympus innovations in frame design, optics and
software, providing exceptional stability, optical quality, accuracy and
reliability.
The new IX3
inverted microscope systems are optimised for live cell imaging. In particular,
the IX73 and IX83 are built using a new swappable deck design, which allows
optical modules to be easily and rapidly slipped in and out as needed,
including magnification changers, filter turrets, a right side port and more.
This means that the systems are truly ‘open access’ and can be fully customised
to meet the requirements of a wide range of applications, even allowing the
utilisation of user-designed custom modules to provide the ultimate level of
flexibility. This design also allows the systems to grow alongside the evolving
demands of your life science research projects. This is especially true of the
IX73, which is highly configurable and capable of integrating with a range of
computer-coded or motorised components.
The new
frame design of the IX3 systems provides an excellent level of mechanical and
thermal stability, and includes an ultrasonic motorised stage for quiet, smooth
and precise movement. Olympus UIS2 optics deliver sharp, bright images, while
the proprietary Olympus fly-eye fluorescence illuminator generates even, vivid
illumination across the specimen. This provides a much wider field of view than
previously possible, with even illumination that fills the detectors of most
cameras, even those with larger chips. IX3 systems are also compatible with the
Olympus Real-Time Controller, offering high-precision imaging via microsecond
synchronisation for advanced studies using high-speed light sources and
triggered cameras.
Optimised
for ease-of-use, the IX3 systems also help streamline complex imaging and
measurement tasks. The systems integrate seamlessly with the powerful Olympus
cellSens imaging software to allow advanced analysis at the click of a button,
while the IX83 also offers a touch-panel interface that can be located in the
most ergonomic position for each user, providing simple, quick and comfortable
operation. With live cell imaging in mind, the IX83 is also compatible with the
new Olympus ZDC Z-Drift Compensation system, which automatically ensures that
every image you capture is in perfect focus. In particular, the ZDC can work
independently of software control, and uniquely combines a one-shot and continuous
mode in the same unit, ensuring your system can be easily optimised for a wide
range of different applications.
Built to
provide the highest-level of quality, flexibility and reliability, the IX3
series of microscope systems will meet all your research and examination needs,
now and in the future.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Suling Zhang,
Zhuo Du, Gongke Li A graphene-supported zinc oxide (ZnO) solid-phase
microextraction (SPME) fiber was prepared via a sol–gel approach. Graphite oxide
(GO), with rich oxygen-containing groups, was selected as the starting material
to anchor ZnO on its nucleation center. After being deoxidized by hydrazine, the
Zn(OH)2/GO coating was dehydrated at high temperature to give the ZnO/graphene
coating. Sol–gel technology could efficiently incorporate ZnO/graphene
composites into the sol–gel network and provided strong chemical bonding between
sol–gel polymeric SPME coating and silica fiber surface, which enhanced the
durability of the fiber and allowed more than 200 replicate extractions. Results
indicated that pure ZnO coated fiber did not show adsorption selectivity toward
sulfur compounds, which might because the ZnO nanoparticles were enwrapped in
the sol–gel network, and the strong coordination action between Zn ion and S ion
was therefore blocked. The incorporation of graphene into ZnO based sol–gel
network greatly enlarged the BET surface area from 1.2m2/g to
169.4m2/g and further increased the adsorption sites. Combining the
superior properties of extraordinary surface area of graphene and the strong
coordination action of ZnO to sulfur compounds, the ZnO/graphene SPME fiber
showed much higher adsorption affinity to 1-octanethiol (enrichment factor, EF,
1087) than other aliphatic compounds without sulfur-containing groups
(EFs<200). Also, it showed higher extraction selectivity and sensitivity
toward sulfur compounds than commercial polydimethylsiloxane (PDMS) and
polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fibers. Several most
abundant sulfur volatiles in Chinese chive and garlic sprout were analyzed using
the ZnO/graphene SPME fiber in combination with gas chromatography–mass
spectrometry (GC–MS). Their limits of detection were 0.1–0.7μg/L. The relative
standard deviation (RSD) using one fiber ranged from 3.6% to 9.1%. The
fiber-to-fiber reproducibility for three parallel prepared fibers was 4.8–10.8%.
The contents were in the range of 1.0–46.4μg/g with recoveries of 80.1–91.6% for
four main sulfides in Chinese chive and 17.1–122.6μg/g with recoveries of
73.2–80.6% for three main sulfides in garlic sprout.
Highlights
► A graphene-supported zinc oxide SPME fiber via a
sol–gel approach was originally developed. ► The selectivity and sensitivity of
ZnO/graphene SPME coating to sulfur compounds were excellent. ► The ZnO/graphene
SPME was successfully applied to determine sulfur volatiles in complex
samples.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Karsten
Müller, Andreas Seubert A method for the ultra trace analysis of 21
fluorobenzoic acids (FBAs) via GC–MS based on solid-phase extraction (SPE) and
derivatization with BF3·MeOH is described. All fluorobenzoic acids were enriched
and determined simultaneously. Solid-phase extraction on
hydrophilic–lipophilic-balanced reversed-phase cartridges containing a
poly(divinylbenzene-co-N-vinylpyrrolidone) polymer allowed a 250-fold enrichment
of the acids if 100mL sample volume is used with extraction efficiencies between
71% and 94%. The method enables the determination of fluorobenzoic acid methyl
esters (FBAMEs) down to the range of 6–44ngL−1 combined with a fast
and easy sample-preparation (pH-adjusting prior to SPE and derivatization within
24h at 64°C directly in the vial). It uses low amounts of chemicals and is
adaptable to larger and smaller sample volumes. Simultaneous extraction and
determination of 21 fluorinated aromatic acids in reservoir samples with high
salinity confirmed the applicability and reproducibility of the method.
Highlights
► New method for the derivatization of
fluorobenzoic acids using BF3·MeOH. ► New SPE method using a material never used
before for this application. ► Quantitative extraction of all commercially
available 23 FBAs. ► Determination of all 23 fluorobenzoic acid methyl esters in
the ngL−1 range. ► Capability of the method for highly saline
reservoir water samples is shown.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Xiangju Mao,
Bin Hu, Man He, Wenying Fan In this study, novel off/on-site stir bar
sorptive extraction (SBSE) approaches with a home-made portable electric stirrer
have been developed for the analysis of polycyclic aromatic hydrocarbon
compounds (PAHs). In these approaches, a miniature battery-operated electric
stirrer was employed to provide agitation of sample solutions instead of the
commonly used large size magnetic stirrer powered by alternating current in
conventional SBSE process, which could extend the SBSE technique from the
conventional off-site analysis to the on-site sampling. The applicability of the
designed off/on-site SBSE sampling approaches was evaluated by
polydimethylsiloxane (PDMS) coating SBSE-high performance liquid
chromatography-fluorescence detection (HPLC-FLD) analysis of six target PAHs in
environmental water. The home-made portable electric stirrer is simple,
easy-to-operate, user friendly, low cost, easy-to-be-commercialized, and can be
processed in direct immersion SBSE, headspace sorptive extraction (HSSE) and
continuous flow (CF)-SBSE modes. Since the stir bar was fixed onto the portable
device by magnetic force, it is very convenient to install, remove and replace
the stir bar, and the coating friction loss which occurred frequently in
conventional SBSE process could be avoided. The parameters affecting the
extraction of six target PAHs by the home-made portable SBSE sampling device
with different sampling modes were studied. Under the optimum extraction
conditions, good linearity was obtained by all of three SBSE extraction modes
with correlation coefficient (R) higher than 0.9971. The limits of detection
(LODs, S/N=3) were 0.05–3.41ngL−1 for direct immersion SBSE,
0.03–2.23ngL−1 for HSSE and 0.09–3.75ngL−1 for CF-SBSE,
respectively. The proposed portable PDMS-SBSE-HPLC-FLD method was applied for
the analysis of six target PAHs in East Lake water, and the analytical results
obtained by on-site SBSE sampling were in good agreement with that obtained by
off-site SBSE sampling. The accuracy of the developed method was evaluated by
recovery test and the recoveries for the spiked sample were found to be in the
range of 87.1–122.8% for off-site CF-SBSE, 88.8–114.3% for on-site sampling, and
87.7–123.6% for off-site SBSE, respectively. The developed method is one of the
most sensitive methods for PAHs determination and the home-designed SBSE system
is feasible for the field sampling.
Highlights
► A portable stir bar sorptive extraction system
was designed for on-site sampling. ► It can be used for off/on-site sampling
with different extraction modes. ► The portable SBSE system was applied for HPLC
analyzing six PAHs in lake water. ► The portable SBSE system shows a great
application potential in field sampling.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Xiang-Dang Du,
Yin-Liang Wu, Hong-Ju Yang, Ting Yang A simple and inexpensive pretreatment
procedure was developed for 10 β2-agonists (clenbuterol, ractopamine,
salbutamol, bambuterol, penbuterol, tulobuterol, clorprenaline, mabuterol,
cimaterol and terbutaline) in swine urine using dispersive solid phase
extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was
analysed after purification by ultra high performance liquid
chromatography–positive electrospray ionisation tandem mass spectrometry
(UHPLC-ESI–MS/MS). The pH value of the swine urine, extraction time, type and
amount of MWCNTs and type of eluent were optimised to increase the sample
throughput and sensitivity. The samples were quantified using clenbuterol-D9,
ractopamine-D6 and salbutamol-D3 as internal standards. The recoveries of the
target compounds from swine urine samples at pH 10.0 were most efficient when
using 20mg of MWCNTs with a 30–50nm outer diameter and a length of 10–30μm,
while a mixture of water/methanol (90:10, v/v) with 0.5% formic acid was shown
to be the most suitable solvent for desorbing the compounds from the MWCNTs. The
proposed method was validated according to the European Commission Decision
2002/657/EC, which determines linearity, specificity, decision limit (CCα),
detection capability (CCβ), recovery, precision and stability.
Highlights
► A simple and cheap method was developed for
β2-agonists using dSPE with MWCNTs. ► The pH, extraction time, the type and
amount of MWCNTs were optimised. ► The proposed method was validated according
to 2002/657/EC. ► The whole method was fast, reliable, convenient and
sensitive.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Emmanuela
Prado de Paiva, Clayton Anderson de Azevedo Filho, Sabrina Gomes Ferreira, Tânia
Lucia Montenegro Stamford, Jose Almiro da Paixão The main protocols of
extraction were investigated for the six folate forms in vegetable matrices,
treated in two fractions, liquor and fiber. In a pilot study, it was used
ammonium acetate added of 2-mercaptoetanol and ascorbic acid as extraction
solution. The condition of use of protease and folate conjugase was evaluated,
besides alternative treatments without enzyme use. Based on the results of this
stage, it was built the factorial design 24, with three replications
at the central point, using the following variables: temperature, time for
reaction, molar concentration of the extraction solution and ratio
sample/solution as independent variables and dependent variable, the amount of
each folate form extracted as well as spectral and chromatographic parameters.
In the pilot study it was verified that the enzyme use can cause an increase in
the variability of the folate content, which enabled to build the factorial
design without the enzyme use. The binomial time and temperature showed greatest
impact on the extraction profile, besides high concentrations of ammonium
acetate resulting in bifurcation of some peaks. 5-Methyltetrahydrofolate was
extracted primordially in the liquor fraction, indicating that this treatment on
the matrix provoked suitable extraction condition to this folate.
Highlights
► Statistic factorial design confirms the binomial
impact of time and temperature. ► Low temperature and short time are indicated
as the best extraction conditions. ► 5-MTHF was suitability extracted from
liquor fraction of all vegetable matrices.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Claudia
Oellig, Wolfgang Schwack Efficient clean-up is indispensable for preventing
matrix effects in multi-residue analysis of pesticides in food by liquid and gas
chromatography (LC and GC) coupled to mass spectrometry (MS). High-throughput
planar solid phase extraction (HTpSPE) was recently introduced as a new clean-up
concept in residue analysis of pesticides in fruit and vegetables (C. Oellig, W.
Schwack, 2011 [45]). Thin-layer chromatography (TLC) was used to completely
separate pesticides from matrix compounds and to focus them into a sharp zone,
followed by extraction of the target zone by the TLC–MS interface. As rather
challenging matrices, tea samples were chosen in this study. Besides
chlorophylls and polyphenols, high amount of caffeine is co-extracted resulting
in strong matrix effects both in LC–MS and GC–MS. The former HTpSPE procedure
was adapted to initial extracts of green and black tea resulting in colorless
extracts nearly free of matrix effects and interferences, as shown for seven
chemically representative pesticides (acetamiprid, penconazole, azoxystrobin,
chlorpyrifos, pirimicarb, fenarimol, and mepanipyrim). LC–MS/MS calibration
curves obtained in the range of 0.002–0.5mg/kg from matrix-matched standards and
solvent standards were nearly identical and demonstrated the effectiveness of
clean-up by HTpSPE. Mean recoveries determined by LC–MS/MS against solvent
standards at spiking levels of 0.01 and 0.1mg/kg ranged between 72 and 114% with
relative standard deviations (RSDs) of 0.7–4.7% (n =4), while LC–MS measurements
of tea samples spiked at 1mg/kg provided recoveries of 81–104% with RSDs of
1.2–4.9% (n =6). Using LC–MS/MS, the method showed high sensitivity with
signal-to-noise ratios >10 for concentrations below 0.002mg/kg. HTpSPE of one
sample was done in a few minutes, while numerous samples were cleaned in
parallel at minimal costs with very low sample and solvent consumption.
Highlights
► A new clean-up concept for pesticide residue
analysis in tea by LC–MS is introduced. ► Planar solid phase extraction was
proven to be the highly efficient clean-up method. ► Nearly matrix-free tea
extracts generally avoided matrix effects in LC–MS(/MS). ► Applying pure solvent
standards, method validation showed impressive results.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Weiqiang Zhan,
Janusz Pawliszyn Various needle trap devices (NTDs) with different designs
have been developed during the past decade. A theoretical model on the
fundamentals of the NTD was recently proposed, which employed the theory of
frontal (gas–solid) chromatography to describe the sampling process. In the
current work, different types of sorbent particles with different dimensions
were packed into the needle as the adsorbent. The effects of particle
dimensions, which would affect the packing density and consequently affect the
capacity, the extraction efficiency and desorption efficiency of the NTD were
experimentally investigated and the proposed theory was validated. The results
demonstrated that NTDs packed with small particles possess higher extraction
capacity and efficiency but much higher resistances to flow as well. The higher
resistance did not necessarily result in poor desorption efficiency. The
observed relationships among those physical parameters provide valuable guidance
on how to design a NTD with high performance for future applications.
Highlights
► Frontal chromatography process is experimentally
validated to describe the sampling process of a needle trap device. ► The
trapping efficiency and desorption efficiency of a needle trap device in regard
to particle dimensions are investigated and optimized. ► Effects of particles on
the extraction of toluene, ethylbenzene and o-xylene are
presented.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Amith D. Naik,
Stefano Menegatti, Hannah R. Reese, Patrick V. Gurgel, Ruben G. Carbonell The
production of therapeutic proteins using transgenic plants offers several
advantages, including low production cost, absence of human pathogens, presence
of glycosylation mechanisms, and the ability to fold complex therapeutic
proteins into their proper conformation. However, impurities such as phenolic
compounds and pigments encountered during purification are quite different from
those faced during purification from mammalian cell culture supernatants. This
paper deals with the development of a pretreatment and affinity separation
process for the purification of a monoclonal antibody from transgenic Lemna
plant extract. A pretreatment step is described using dextran-coated charcoal
for the removal of pigments and phenolic compounds without reducing the antibody
concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial
polymethacrylate resin is used for the capture and purification of MAb from the
pretreated plant extract. The final yield and purity of the MAb obtained were
90% and 96% respectively. The performance of the hexamer peptide resin after the
pretreatment step was found to be similar to that obtained with a commercial
Protein A resin.
Highlights
► This paper deals with the purification of a
monoclonal IgG from a plant extract. ► We used a two-step approach, with a
charcoal treatment and affinity chromatography step. ► The final product shows
high purity and recovery, competitive to commercial products.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Lieven Van
Meulebroek, Julie Vanden Bussche, Kathy Steppe, Lynn Vanhaecke Phytohormones
are key signalling biomolecules and are of particular interest because of their
regulating role in numerous physiological and developmental plant processes.
Since the plant response to a given stimulus results amongst others from the
complex interaction between phytohormones, there is a mounting interest for
multiple phytohormone analysis. Therefore, with the primary aim of profiling the
hormonal status of the tomato plant, a generic extraction protocol and an
U-HPLC–Orbitrap-MS analysis were developed and validated for both tomato fruit
and leaf tissue. To this end, eight phytohormones were considered, i.e.
gibberellic acid, indol-3-acetic acid, abscisic acid, jasmonic acid, salicylic
acid, zeatin, N6-benzyladenine and epibrassinolide, representing the
major hormonal classes. The sample pre-treatment involved liquid extraction with
a buffer of methanol, ultrapure water and formic acid (75:20:5, v/v/v), after
which the extract was purified by means of an Amicon® Ultra
centrifugal unit. Subsequently, analytes were chromatographically separated on a
sub-2μm particles Nucleodur Gravity C18 column and detected by an Exactive™
high-resolution mass spectrometer. Validation of the analytical method
demonstrated that linearity (≥0.99), precision (CV≤15%) and mean corrected
recovery (between 80% and 110%) performed well for the majority of the eight
targeted phytohormones. In addition, the generic nature of the extraction
protocol and the full scan approach of the Orbitrap mass spectrometer allowed
metabolomic profiling of the hormonal status of the tomato plant.
Highlights
► Development of a generic extraction for
phytohormones from tomato plant tissue. ► Development of a full scan
U-HPLC–Orbitrap-MS method for phytohormone analysis. ► Validation of the
developed methods for both tomato leaf and fruit tissue. ► Demonstration of the
metabolomic character of the developed methods.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Carla G.A. da
Silva, Carol H. Collins Endcapped stationary phases were prepared after
thermal immobilization of poly(methyloctadecylsiloxane) (PMODS) onto zirconized
and titanized silica supports. These new stationary phases have lower densities
of residual hydroxyl groups, according to infrared spectroscopy and
29Si CP-MAS NMR and as shown by the symmetrical peaks of basic
compounds from the Tanaka, Engelhardt and SRM 870 test mixtures. Stability tests
for the endcapped stationary phases, measured using severe alkaline conditions
(70:30 (v/v) methanol:0.05mol/L K2CO3/KHCO3, pH 10, 50°C), revealed that the
stabilities of these phases are greater than the stabilities of similar
nonendcapped phases. The stationary phases showed good performance for the
separation of basic pharmaceuticals.
Highlights
► HPLC support prepared by metallization of silica
and thermal immobilization of PMODS onto the support. ► Endcapping reaction with
hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). ► Evaluation with
several standardized test mixtures. ► Improvement of the chemical stability in
severe alkaline conditions. ► Potential application for separation of basic
pharmaceuticals.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Tobias K.H.
Müller, Matthias Franzreb The effect of temperature in the range from 10°C to
40°C and comparatively low ammonium sulfate (AS) concentrations of up to 0.5M on
the adsorption of bovine serum albumin (BSA) on four different commercially
available sepharose-based stationary phases was investigated. The determined
isotherms were fitted by the Langmuir equation, and thermodynamic values were
calculated by van’t Hoff analysis. The adsorption of BSA onto the
chromatographic resin Butyl Sepharose 4FF showed the strongest temperature
influence; however, protein unfolding effects occurred when characterizing this
system by dynamic column experiments, with an unfolded BSA fraction strongly
attached to the sorbent. The percentage of the unfolding fraction was determined
for different operating conditions and found to increase with the concentration
of the cosmotropic salt, but even stronger with increasing temperature.
Temperature-induced cyclic adsorption and desorption experiments were carried
out to investigate the long-term performance of Butyl Sepharose 4FF by applying
purely temperature-controlled regeneration. Over a period of five cycles, the
working capacity remained stable, but BSA also started to accumulate on the
column due to incomplete regeneration. Finally, the possibility to fractionate
different proteins with a single temperature shift was shown by the complete
separation of lysozyme and BSA. The results presented indicate that
temperature-induced binding and elution may offer a possibility to shift the
operation conditions of HIC resins toward reduced salt concentrations, thus
saving chemicals and facilitating salt removal in further downstream processing
stages.
Highlights
► Temperature-dependent adsorption behavior of
model proteins on HIC gels. ► Temperature as advantageous parameter regarding
purification applications using HIC. ► Non-isothermal HIC resin regeneration. ►
Temperature-mediated protein fractionation. ► Protein unfolding in dependence of
ionic strength, temperature and pulse injections.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Miloš
Netopilík The SEC separation of a randomly branched polymer, in particular
local dispersity due to branching, are theoretically examined. A model of the
SEC separation of randomly branched polymer with tetrafunctional branch points
enabling the estimation of local dispersity was developed. Measurable quantities
(branching indices) were compared with real data. Local dispersity due to
branching is demonstrated to depend on elution volume and degree of branching
and in the area of the beginning of the elution curve it can reach
non-negligible values.
Highlights
► SEC analysis of randomly branched polymer was
modeled. ► Results were compared with experiment. ► Local dispersity in the
frontal area of the elution curve was found.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Pedro Araujo,
Steve Janagap, Elisabeth Holen A protocol for the analysis of multiple
prostaglandins and leukotrienes in cell culture media by using multiple internal
standards was validated. A two-factor Doehlert design was used to determine the
behaviour of the relationship analyte/internal standard (namely: PGE2/PGE2-d 4,
PGE3/PGE2-d 4, LTB4/LTB4-d 4 and LTB5/LTB4-d 4) and to select the optimal
amounts of deuterated internal standards for quantifying simultaneously the
prostaglandins and leukotrienes in cell culture media by LC–MS/MS. The selection
of optimal amounts of internal standards was based on mathematical models that
allow visualizing concentration regions where the response factors remain
constant over a wide range of analytical concentrations. The linearity of the
calibration curves for each analyte at the optimal levels suggested by the
mathematical models was statistically confirmed by means of the ratio
lack-of-fit to pure error. The validated protocol was successfully applied in
the simultaneous quantification of pro- and anti-inflammatory eicosanoids in
stimulated cod head kidney cell culture media. The two-factor Doehlert design
has permitted to estimate the experimental response as a function of six
variables (PGE2, PGE3, LTB4, LTB5, PGE2-d 4 and LTB4-d 4) which represents a
substantial reduction of resources, time and experiments of approximately 84%
(7×3 experiments) when compared with the full six-factor Doehlert design (43×3
experiments).
Highlights
► Rational design for selecting optimal amounts of
multiple internal standards. ► Study of six variables with a Doehlert design
intended for two variables. ► 84% reduction in resources compared to a
six-factor Doehlert design.
Publication year:
2012 Source:Journal of Chromatography A, Volume 1260 Jeong-Heui
Choi, Marc Lamshöft, Sebastian Zühlke, Ki Hun Park, Jae-Han Shim, Michael
Spiteller The detection of veterinary drugs in blood meal is needed since it
is used as an environment-friendly agricultural material despite its origination
from animal blood. A method using accelerated solvent extraction and liquid
chromatographic linear ion trap quadrupole Orbitrap mass spectrometry was
developed to determine sedatives and adrenergic blockers in blood meal. The
determination method was established following optimizations of accelerated
solvent extraction, dispersive solid-phase extraction and high resolution mass
spectrometric detection. Linearity, sensitivity, accuracy, repeatability and
reproducibility of the method were fully validated. The method was applied to
commercial blood meal products.
Highlights
► Combination of ASE and LC–Orbitrap MS for
sedatives and α-, β-blockers in blood meal. ► Dispersive solid-phase extraction
as an efficient cleanup method. ► Good results of the sensitivity, linearity,
accuracy and precision. ► Finding a metabolite of xylazine via the high
resolution MS detection.
