Just Published: Journal of Pharmaceutical and Biomedical Analysis

A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:

Journal of Pharmaceutical and Biomedical Analysis
http://rss.sciencedirect.com/publication/science/5266

Selected papers from the latest issue:

Application of the near-infrared spectroscopy in the pharmaceutical technology

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Marzena Jamrógiewicz
Near-infrared (NIR) spectroscopy is currently the fastest-growing and the most versatile analytical method not only in the pharmaceutical sciences but also in the industry. This review focuses on recent NIR applications in the pharmaceutical technology. This article covers monitoring, by NIR, of many manufacturing processes, such as granulation, mixing or drying, in order to determine the end-point of these processes. In this paper, apart from basic theoretical information concerning the NIR spectra, there are included determinations of the quality and quantity of pharmaceutical compounds. Some examples of measurements and control of physicochemical parameters of the final medicinal products, such as hardness, porosity, thickness size, compression strength, disintegration time and potential counterfeit are included. Biotechnology and plant drug analysis using NIR is also described. Moreover, some disadvantages of this method are stressed and future perspectives are anticipated.

Highlights

► This work presents NIR spectroscopy application in the pharmaceutical manufacturing. ► The physical point of view on NIR is shown also with the spectra interpretation. ► A collection of multi-thematic data, also contains aspects of bio-manufacturing. ► NIR quality controlling system of pharmaceuticals and biopharmaceuticals is verified. ► Implementation of NIR method on the contrary to scientific knowledge is pointed out.

HPTLC methods to assay active ingredients in pharmaceutical formulations: A review of the method development and validation steps

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
D.H. Shewiyo, E. Kaale, P.G. Risha, B. Dejaegher, J. Smeyers-Verbeke, Y. Vander Heyden
High-performance thin-layer chromatography (HPTLC) is still increasingly finding its way in pharmaceutical analysis in some parts of the world. With the advancements in the stationary phases and the introduction of densitometers as detection equipment, the technique achieves for given applications a precision and trueness comparable to high-performance liquid chromatography (HPLC). In this review, the literature is surveyed for developed and validated HPTLC methods to assay active ingredients in pharmaceutical formulations published in the period 2005–2011. Procedures and approaches for method development, validation and quantitative assays are compared with the standard ways of conducting them. Applications of HPTLC in some other areas are also briefly highlighted.

The development and application of a system for simultaneously determining anti-infectives and nasal decongestants using on-line solid-phase extraction and liquid chromatography–tandem mass spectrometry

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Ghazanfar Ali Khan, Richard Lindberg, Roman Grabic, Jerker Fick
A method for the simultaneous analysis of antibiotics, antiviral and nasal decongestants in treated sewage effluent and surface water has been developed and validated. The method uses on-line solid phase extraction (SPE) of injected high-volume samples in conjunction with liquid chromatography–tandem mass spectrometry (LC–MS/MS). This method includes a range of antibiotics (Trimethoprim, Oxytetracycline, Ofloxacin, Norfloxacin, Ciprofloxacin, Azithromycin, Doxycycline, Sulfamethoxazole, Erythromycin and Clarithromycin), an antiviral (Oseltamivir) and nasal decongestants (Naphazoline, Oxymetazoline and Xylometazoline). The method's detection limits (MDLs) ranged from (0.2ngL⁻¹) to (3.1ngL⁻¹), based on a 1mL extraction volume. Its intra-day precision was determined by performing nine runs with 200ngL⁻¹ samples; the intra-day relative standard deviation (RSD) ranged from 1% to 19%. Inter-day precision was determined by analyzing samples in triplicate over the course of three days, yielding relative standard deviations ranging from <5% to <26%. The linearity (R ²) for all compounds tested was >0.90. Spike relative recoveries ranged from 40% to 157% and 40% to 152% for STP effluent and surface water samples, respectively. Finally, the method was used to analyze real effluent and surface water.

HPTLC method for the simultaneous determination of four indole alkaloids in Rauwolfia tetraphylla: A study of organic/green solvent and continuous/pulse sonication

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Shikha Gupta, Karuna Shanker, Santosh K. Srivastava
A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of four antipsychotic indole alkaloids (IAs), reserpiline (RP, 1), α-yohimbine (YH, 2), isoreserpiline (IRP, 3) and 10-methoxy tetrahydroalstonine (MTHA, 4) as markers in the leaves of Rauwolfia tetraphylla. Extraction efficiency of the targeted IAs from the leaf matrix with organic and ecofriendly (green) solvents using percolation, ultrasonication and microwave techniques were studied. Non-ionic surfactants, viz. Triton X-100, Triton X-114 and Genapol X-80 were used for extraction and no back-extraction or liquid chromatographic steps were used to remove the targeted IAs from the surfactant-rich extractant phase. The optimized cloud point extraction was found a potentially useful methodology for the preconcentration of the targeted IAs. The separation was achieved on silica gel 60F254 HPTLC plates using hexane-ethylacetate-methanol (5:4:1, v/v/v) as mobile phase. The quantitation of IAs (1–4) was carried out using the densitometric reflection/absorption mode at 520nm after post chromatographic derivatization using Dragendorff's reagent. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD) and quantitation (LOQ). Method specificity was confirmed using retention factor (R f ) and visible spectral (post chromatographic scan) correlation of marker compounds in the samples and standard tracks.

Highlights

► Development of a rapid HPTLC analytical method for the quality assessment of R. tetraphylla using ecofriendly (green) solvents. ► Extraction efficiency of the targeted indole alkaloids from the leaf matrix with organic and ecofriendly (green) solvents have been discussed. ► Non-ionic surfactants, Triton X-100, Triton X-114 and Genapol X-80 used as ecofriendly solvents for the extraction of targeted indole alkaloids. ► No back-extraction or liquid chromatographic steps were used to remove the targeted indole alkaloids from the surfactant-rich extractant phase. ► The optimized cloud point extraction was found a potentially useful methodology for the preconcentration of the targeted indole alkaloids.

Structural characterization of a novel degradant of the antifungal agent posaconazole

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Wendy Zhong, Xi Yang, Weidong Tong, Gary E. Martin
A number of degradants of the antifungal agent posaconazole have been isolated and characterized. Degradation chemistry occurs primarily in the piperazine moiety located near the center of the molecule and typically involves oxidative processes. At least one ring contraction product has also been fully characterized by MS and NMR methods. We report the isolation and characterization of a novel degradant of posaconazole in which remnants of the piperazine ring have been incorporated into a 1,4-benzodiazepine moiety. The structure of the novel degradant was unequivocally established through accurate mass measurements, MS/MS studies, H/D exchange, and a range of 2D NMR experiments that included 1,1-ADEQUATE and covariance calculated HSQC-1,1-ADEQUATE spectra.

Triprotic site-specific acid–base equilibria and related properties of fluoroquinolone antibacterials

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Aura Rusu, Gergő Tóth, Levente Szőcs, József Kökösi, Márta Kraszni, Árpád Gyéresi, Béla Noszál
The complete macro- and microequilibrium analyses of six fluoroquinolone drugs – ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, ofloxacin and moxifloxacin – are presented. Previous controversial literature data are straightened up, the protonation centers are unambiguously identified, and the protonation macro- and microconstant values are reported. The macroconstants were determined by ¹H NMR-pH titrations while the microconstants were determined by a multi-modal spectroscopic-deductive methodology, in which methyl ester derivatives were synthesized and their NMR-pH titration data contributed to the evaluation of all the microconstants. The full ¹H, ¹³C and ¹⁵N NMR assignments, NMR-pH profiles, macro- and microprotonation schemes and species-specific diagrams are included. Our studies show that the fluoroquinolones have three protonation centers: the carboxylate group, the N-1′ and N-4′ piperazine nitrogens and concentration of the uncharged microspecies is way below the values published earlier. The results could be well interpreted in terms of structural properties. The protonation macro- and microconstant values allow the pre-planned method development in techniques such as capillary zone electrophoresis and also, the interpretation of fluoroquinolone mechanism of biological action, including the pharmacokinetic properties, and antibacterial activities that are all heavily influenced by the states of protonation.

Highlights

► The protonation centers of fluoroquinolone antibacterials are unambiguously identified. ► The protonation macro- and microconstants of fluoroquinolones were determined. ► The results could be well explained in terms of their structural properties. ► Protonation states of these drugs are crucial for binding of bacterial DNA gyrase.

Usefulness of reversed-phase HPLC enriched with room temperature imidazolium based ionic liquids for lipophilicity determination of the newly synthesized analgesic active urea derivatives

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Jolanta Flieger, Anna Czajkowska-Żelazko, Marzena Rządkowska, Elżbieta Szacoń, Dariusz Matosiuk
Lipophilicity of several novel analgesic active 1-(1-arylimidazolidyn-2-ylidyn)-3-arylalkyl urea derivatives has been estimated by the use of chromatographic method. The investigated compounds were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) using mixtures of methanol or acetonitrile and water with addition of imidazolium based room temperature ionic liquids varying in an anion chaotropicity as the mobile phases. The relationships between log k values vs. concentration of organic solvent was used for determination of the log k w values by extrapolation technique. The partition coefficients (log P) values were calculated by means of the Pallas 3.1.1.2. and Spartan 10.0 softwares and further correlated with log k w measured experimentally in classical organic–aqueous eluent system and systems modified with ionic liquids addition. It was found that log k w values measured in eluent system modified with butyl-methyl imidazoilum chloride correlate the best with the logarithm of partition coefficient calculated by Pallas software (log P calc.). Furthermore, it was found that the examined compounds form H-bonding with imidazoilum cation of modifiers improving the chromatographic peak parameters (the symmetry factor, the theoretical plates number) especially when ionic liquid's anion was more chaotropic. Amphiphilic ionic liquid possessing longer alkyl chain substituent (OMIM BF4) can be considered as a new cationic surfactant. Micellar conditions improved separation selectivity of chloro- and methoxy substituted derivatives.

Quantification of main bioactive metabolites from saffron (Crocus sativus) stigmas by a micellar electrokinetic chromatographic (MEKC) method

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Sándor Gonda, Péter Parizsa, Gyula Surányi, Gyöngyi Gyémánt, Gábor Vasas
Saffron is an expensive spice, cultivated in many regions of the world. Its chief metabolites include crocins, which are responsible for the coloring ability, safranal, which is the main essential oil constituent, and picrocrocin which is the main bitter constituent of the spice. A simple micellar capillary electrochromatographic (MEKC) method capable of quantifying all three types of main constituents was established. The pH, sodium dodecyl sulphate (SDS) content and electrolyte concentration of the background electrolyte was optimized. A simple extraction protocol was developed which can extract all metabolites of different polarity from the saffron stigmas. Optimal background electrolyte composed of 20mM disodium phosphate, 5mM sodium tetraborate, 100mM SDS, pH was set 9.5. Optimal extracting solvent was the background electrolyte, incubated with the sample for 60min. The proposed method allows quantification of picrocrocin, safranal, crocetin- Di-(β-d-gentiobiosyl) ester and crocetin (β-d-glycosyl)-(β-d-gentiobiosyl) ester within 17.5min, with limit of detection values ranging from 0.006 to 0.04mg/ml, from a single stigma.

A high pH based reversed-phase high performance liquid chromatographic method for the analysis of aminoglycoside plazomicin and its impurities

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Li Tan, Kenneth B. Wlasichuk, Donald E. Schmidt, Robert L. Campbell, Pam Hirtzer, Lisa Cheng, Dane E. Karr
A reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the aminoglycoside (AG) plazomicin (ACHN-490). This method employed a high pH mobile phase (pH>11) with a gradient of 0.25M ammonium hydroxide in water and acetonitrile, an XBridge C18 column and UV detection at 210nm. Although the molar UV absorption of plazomicin is weak, the high pH conditions of this method allow for higher loadings, which compensates for the inherent low UV sensitivity. Under these high pH conditions, impurities and degradants were base line separated from plazomicin. The mobile phases used for this method allowed for on-line mass detection for the impurities and degradants. The RP-HPLC method has been validated in terms of specificity, linearity and range, accuracy, and precision. The analytical method met specificity requirements of a homogenous peak with no interferences from the blank or from the known impurities in plazomicin. The linearity of the method for the plazomicin impurity determination was excellent, with a coefficient of determination (r ²) of 0.9993, over the freebase (FB) concentration range of 0.0025–3.0mg/mL. The method is capable of detecting impurities down to 0.1% of the peak area of plazomicin. A single point standard at a concentration of 1.0mg/mL FB was validated over the range of 50–150% for quantitation of the freebase content (the assay) in bulk drug substance. The mean recoveries of FB are in the range 98.6–102.0% with a mean RSD (relative standard deviation) <1.0%. The study also examined the method precision for purity, impurities and the assay with two instruments on two different days. The method showed adequate accuracy and precision for the intended use. This high pH method was successfully used to determine the impurity and measure the drug content in the final plazomicin drug substance. In addition, the method with an on-line mass spectrometry detector has been used to characterize the structures of the impurities in plazomicin.

Non-destructive detection of adulterated tablets of glibenclamide using NIR and solid-phase fluorescence spectroscopy and chemometric methods

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Rafael da Silva Fernandes, Fernanda Saadna Lopes da Costa, Patrícia Valderrama, Paulo Henrique Março, Kássio Michell Gomes de Lima
This study describes a method for non-destructive detection of adulterated glibenclamide tablets. This method uses near infrared spectroscopy (NIRS) and fluorescence spectroscopy along with chemometric tools such as Soft Independent Modeling of Class Analogy (SIMCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Unfolded Partial Least Squares with Discriminant Analysis (UPLS-DA). Both brand name (Daonil) and generic glibenclamide tablets were used for analysis. The levels of glibenclamide in each type of tablet were evaluated by derivative spectrophotometry in the ultraviolet region. The results obtained from the NIR and fluorescence spectroscopy along with those obtained from multivariate data classification show that this combined technique is an effective way to detect adulteration in drugs for the treatment of diabetes. In the future, this method may be extended to detect different types of counterfeit medications.

Comparison of solid phase microextraction versus spectroscopic techniques for binding studies of carbamazepine

28 May 2012, 09:52:59

Publication year: 2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 66
Barbara Bojko, Dajana Vuckovic, Janusz Pawliszyn
The binding of carbamazepine to human serum albumin was studied in vitro using solid-phase microextraction (SPME) with liquid chromatography–ultraviolet detection (LC–UV), as well as spectroscopic fluorescence and nuclear magnetic resonance (¹H NMR) techniques. We were able to recognize one high affinity binding site with both fluorescence and SPME methods. Additionally, SPME experiment showed the existence of one lower affinity binding site for carbamazepine at the range of concentrations studied with fluorescence. The analysis of Hill's plot indicated positive cooperativity between drugs located in these two binding sites. Two low affinity-binding sites have been found with SPME–LC–UV analysis performed in parallel to ¹H NMR study, which does not show any complex formation. In conclusion, the results of the studies with carbamazepine as a model drug showed the advantages of simultaneous use of solid phase microextraction and spectroscopic methods in protein binding studies and indicated complementary information, which can be obtained with the use of SPME. Furthermore, we show that SPME in combination with liquid chromatography–mass spectrometry permitted direct in vitro determination of plasma–protein binding and direct in vivo evaluation of inter-animal variability in free concentrations of carbamazepine at physiologically relevant concentrations, the type of experiments typically inaccessible by spectroscopic techniques due to poor sensitivity and different mode of implementation. 

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Just Published: Talanta

A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:

Talanta

http://rss.sciencedirect.com/publication/science/5288

Selected papers from the latest issue:

Stability-indicating capillary zone electrophoresis method for the assessment of recombinant human granulocyte-macrophage colony-stimulating factor and its correlation with reversed-phase liquid chromatography method and bioassay

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Sergio Luiz Dalmora, Cairo dos Santos Butzge, Francine Trevisan Machado, Maurício Elesbão Walter, Maria Elisabeth de Ávila Dalmora, Ricardo Bizogne Souto
A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using leuprorelin acetate (LA), as internal standard (IS). A fused-silica capillary (75μm i.d.; effective length, 72cm) was used at 25°C; the applied voltage was 12kV. The background electrolyte solution consisted of 50mM di-sodium hydrogen phosphate solution at pH 8.8. Injections were performed using a pressure mode at 50mbar for 9s, with detection by photodiode array detector set at 200nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 2.5–200μgmL⁻¹ (r ² =0.9995) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.79μgmL⁻¹ and 2.5μgmL⁻¹, respectively. The accuracy was 99.14% with bias lower than 1.40%. The method was applied to the quantitative analysis of biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC), and an in vitro bioassay, showing non-significant differences (p >0.05).

Highlights

► Stability-indicating capillary zone electrophoresis method for rhGM-CSF. ► Correlation between physicochemical methods and bioassay for rhGM-CSF. ► Content/potency assessment of rhGM-CSF in biotechnology-derived product.

On the chemical identification and determination of flavonoids in solid-state

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Bojidarka B. Ivanova, Michael Spiteller
The paper presented with qualitative and quantitative analysis of alkaloids in solid-state, using the excitations within the THz-spectroscopic region of 300–30cm⁻¹ (9.00–0.9THz). Series of nine plant flavonoids (FLs) and their mixtures were analyzed both qualitatively and quantitatively. For first time in the literature was reported the quantitative analysis of alkaloids and their mixtures within the THz-region using the solid-state Raman spectroscopy, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The calibration and validation, concentration limit of detection (LOD), limit of quantification (LOQ), and linearity limit (LL) were obtained. The chemometric nonlinear and linear approaches for analysis and interpretation of the quantities were applied. The results obtained were compared with a parallel QA, using the calibrated and validated HPLC electrospray ionization (ESI) mass spectrometric method, electronic absorption (EAs) and CD spectroscopy. The metrology, including accuracy, measurement repeatability (intra-serial precision condition of measurement), measurement precision, trueness of the measurement, and reproducibility of the measurement, measurement bias and errors of the measurements were discussed.

Highlights

► Forensic chemistry. ► Raman spectroscopy. ► Mass spectrometry. ► Natural products. ► Alkaloids.

An LC method for the analysis of phosphatidylcholine hydrolysis products and its application to the monitoring of the acyl migration process

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Grzegorz Kiełbowicz, Damian Smuga, Witold Gładkowski, Anna Chojnacka, Czesław Wawrzeńczyk
An assay for quantitative analysis of phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and its hydrolysis products: 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, sn-glycero-3-phosphocholine and palmitic acid using high-performance liquid chromatography with charge aerosol detector (CAD) was developed. The separation of the compounds of interest was achieved on a reversed-phase/hydrophilic interaction stationary phase with a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate solution. The method was applied to control the acyl migration process of LPC regioisomers in the most common solvents used in the synthesis or modification of PC.

Highlights

► Phosphatidylcholine (PC) hydrolysis product formation is a serious problem especially in the enzyme-catalyzed acyl exchange of PC. ► Quantitative HPLC analysis of PC and its hydrolysis products is presented. ► The method was applied to examine the stability of the lysophosphatidylcholine (LPC) isomers in 16 different solvents. ► 1-Palmitoyl LPC isomer is more stable than 2-palmitoyl LPC isomer. ► The acyl migration and hydrolysis processes are prefer at alkaline pH.

A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Raquel Obregon, Yoshiko Horiguchi, Toshiharu Arai, Shihomi Abe, Yuanshu Zhou, RyosukeTakahashi, Akiko Hisada, Kosuke Ino, Hitoshi Shiku, Tomokazu Matsue
A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E 1/2) for [Fe(CN)6]⁴⁻ oxidation and O2 reduction measured versus the internal Pt pseudo reference was shifted by about −0.2V, compared with the E 1/2 measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at −0.7V versus the Pt pseudo reference and compared with those monitored at −0.5V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O2)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R ² =0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E 1/2 for PAP oxidation measured versus the Pt layer was not shifted, compared with the E 1/2 versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3V versus the Pt layer and compared with those monitored at +0.3V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier.

Graphical abstract

Graphical abstract Highlights

► An integrated probe was fabricated, with working/pseudo reference electrode. ► We measured oxygen consumption and alkaline phosphatase activity of a single mouse embryoid body. ► The consumption pattern observed agreed well with the conventional SECM method.

A simple and selective fluorometric assay for dopamine using a calcein blue–Fe2+ complex fluorophore

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Daisuke Seto, Tomoharu Maki, Nobuaki Soh, Koji Nakano, Ryoichi Ishimatsu, Toshihiko Imato
A novel fluorimetric assay for dopamine using calcein blue (CB) complexed with Fe²⁺ ion as a chemical sensor is described. The fluorescence arising from CB of the CB–Fe²⁺ complex is quenched by the Fe²⁺ ion. When dopamine is added to a solution of the CB–Fe²⁺ complex, a dopamine–Fe²⁺ complex is formed as the result of a ligand exchange reaction between CB and dopamine which permits the fluorescence from CB to be recovered. The fluorescence intensity at the wavelength of 440nm (at the excitation wavelength of 340nm) was found to be proportional to the concentration of the dopamine added to the CB–Fe²⁺ complex solution, which permits dopamine to be quantitatively determined. The selectivity for dopamine in the presence of other catecholamines and related compounds was good. The calibration curve for dopamine, determined using experimental data was successfully simulated based on the equilibrium of the ligand exchange reaction between CB and dopamine. The working range is from 50μM to 1mM and the limit of detection and limit of quantization are ca 10μM and 50μM, respectively. The assay is simple and economical, compared with conventional methods such as an enzyme-linked immunosorbent assay (ELISA).

Highlights

► A fluorimetric determination of dopamine using calcein blue (CB) complexed with Fe²⁺ ion based on ligand exchange mechanism is the first report. ► The method is simple just mixed the CB–Fe²⁺ complex solution with a dopamine solution. ► The method is with good selectivity to dopamine.

Simultaneous analysis of organochlorinated pesticides (OCPs) and polychlorinated biphenyls (PCBs) from marine samples using automated pressurized liquid extraction (PLE) and Power Prep™ clean-up

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Murad I.H. Helaleh, Amal Al-Rashdan, A. Ibtisam
An automated pressurized liquid extraction (PLE) method followed by Power Prep™ clean-up was developed for organochlorinated pesticide (OCP) and polychlorinated biphenyl (PCB) analysis in environmental marine samples of fish, squid, bivalves, shells, octopus and shrimp. OCPs and PCBs were simultaneously determined in a single chromatographic run using gas chromatography–mass spectrometry-negative chemical ionization (GC–MS-NCI). About 5g of each biological marine sample was mixed with anhydrous sodium sulphate and placed in the extraction cell of the PLE system. PLE is controlled by means of a PC using DMS 6000 software. Purification of the extract was accomplished using automated Power Prep™ clean-up with a pre-packed disposable silica column (6g) supplied by Fluid Management Systems (FMS). All OCPs and PCBs were eluted from the silica column using two types of solvent: 80mL of hexane and a 50mL mixture of hexane and dichloromethane (1:1). A wide variety of fish and shellfish were collected from the fish market and analyzed using this method. The total PCB concentrations were 2.53, 0.25, 0.24, 0.24, 0.17 and 1.38ngg⁻¹ (w/w) for fish, squid, bivalves, shells, octopus and shrimp, respectively, and the corresponding total OCP concentrations were 30.47, 2.86, 0.92, 10.72, 5.13 and 18.39ngg⁻¹ (w/w). Lipids were removed using an SX-3 Bio-Beads gel permeation chromatography (GPC) column. Analytical criteria such as recovery, reproducibility and repeatability were evaluated through a range of biological matrices.

Determination of metal impurities in advanced lead zirconate titanate ceramics by axial view mode inductively coupled plasma optical emission spectrometry

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
M.E. Villanueva Tagle, M.T. Larrea Marín, O. Martin Gavilán, M.D. Durruthy Rodríguez, F. Calderón Piñar, M.S. Pomares Alfonso
An inductively coupled plasma optical emission spectrometry quantification method for the determination of Al, Ca, Cr Cu, Fe, Mn, Mg, Ni, Zn, Ba, K, In and Co in lead zirconate-titanate (PZT) ceramics, modified with strontium and chromium, was developed. Total digestion of ceramics was achieved with a HNO3, H2O2 and HF (ac) mixture by using a microwave furnace. The sensitivity of the net signal intensity respect to radiofrequency power (P) and nebulizer argon flow (F N) variations was strongly dependent of the total excitation energy of line (TEE). For lines with TEE near metastable atoms and ions of argon, an increment of the sensitivities to P and F N variation was observed. At robust plasma conditions the matrix effect was reduced for all matrices and analytes considered. The precision of analysis ranged from 3 to 13%, whereas the analytes recoveries in the spiked samples varied, mostly, from 90 to 110%. The detection limits of studied elements were from 0.004 to 10mgkg⁻¹.

Highlights

► ICP-OES method has been developed for analysis of PZT ceramics. ► Influence of operating parameters on SBR, net signal intensity and matrix effect was studied. ► Some lines with an anomalous behaviour were observed. ► The precision of analysis ranged from 3 to 13%. ► The accuracy was from 90 to 110%.

An integrated electrochemical device based on immunochromatographic test strip and enzyme labels for sensitive detection of disease-related biomarkers

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Zhe-Xiang Zou, Jun Wang, Hua Wang, Yao-Qun Li, Yuehe Lin
A novel electrochemical biosensing device that integrates an immunochromatographic test strip and a screen-printed electrode (SPE) connected to a portable electrochemical analyzer was presented for rapid, sensitive, and quantitative detection of disease-related biomarker in human blood samples. The principle of the sensor is based on sandwich immunoreactions between a biomarker and a pair of its antibodies on the test strip, followed by highly sensitive square-wave voltammetry (SWV) detection. Horseradish peroxidase (HRP) was used as a signal reporter for electrochemical readout. Hepatitis B surface antigen (HBsAg) was employed as a model protein biomarker to demonstrate the analytical performance of the sensor in this study. Some critical parameters governing the performance of the sensor were investigated in detail. Under optimal conditions, this sensor was capable of detecting a minimum of 0.3ngmL⁻¹ (S/N=3) HBsAg with a wide linear concentration range from 1 to 500ngmL⁻¹. The sensor was further utilized to detect HBsAg spiked in human plasma with an average recovery of 91.3%. In comparison, a colorimetric immunochromatographic test strip assay (ITSA) was also conducted. The result shows that the SWV detection in the electrochemical sensor is much more sensitive for the quantitative determination of HBsAg than the colorimetric detection, indicating that such a sensor is a promising platform for rapid and sensitive point-of-care testing/screening of disease-related biomarkers in a large population.

Highlights

► A test strip based biosensor for rapid screening of HBsAg is proposed. ► Enzymatic electrochemical measurement allows sensitive and quantitative detection. ► The enzyme label simplifies the fabrication of test strip and detection procedure.

A rational route to the development of a competitive capillary electrophoresis immunoassay: Assessment of the variables affecting the performances of a competitive capillary electrophoresis immunoassay for human serum albumin

25 May 2012, 10:06:28

Publication year: 2012
Source:Talanta, Volume 94
Cristina Giovannoli, Claudio Baggiani, Cinzia Passini, Flavia Biagioli, Laura Anfossi, Gianfranco Giraudi
Affinity capillary electrophoresis is a powerful analytical tool to extract quantitative information about the binding properties of different interacting systems. The use of LIF detection makes the technique suitable for screening strong binding interactions. The non-equilibrium electrophoretic separations of pre-equilibrated mixtures of ligand and receptor are generally used for such strong molecular interactions allowing the assessment of capillary electrophoresis immunoassays, mostly in competitive formats. As the analytical performances of the assay strongly depend on the preservation of the binding properties during the separation, a rational route to assay development has to be followed to get the best conditions. The paper describes the steps followed to set-up a competitive immunoassay for human serum albumin (HSA) by using a labeled protein (HSA-FITC) and an anti-HSA polyclonal antiserum. A labeling degree of around 1 of the HSA-FITC conjugates is needed to get narrow electrophoretic peak while the titration curve is used to define the optimal antiserum dilution. An antiserum-labeled protein affinity constant of 1.34×10⁷ M⁻¹ was measures in the selected separation conditions. Furthermore, in order to maximize the assay competition between the labeled and unlabelled HSA a short pre-incubation step of the antiserum with the unlabelled HSA (the analyte) was introduced to promote a sharp increase in assay sensitivity.

Highlights

► A planning strategy for the development of a competitive capillary electrophoresis immunoassay is described. ► The approach is similar to that used in the conventional microplate immunoassay. ► It allows us to get the full optimization of the assay performance. ►The sensitivity is increased by incubating the HSA with the antiserum prior to addition of labeled HSA.

New Ureaplasma parvum Monoclonal Antibodies

Ureaplasma are very small free-living bacteria that, like the Mycoplasmas, have no cell walls.
Named for its metabolic utilization of urea, it possesses a small simple genome. Ureaplasma parvum is the genus found in the human urogenital tract. Its presence has been associated with several conditions including urethritis, pelvic inflammatory disease, congenital pneumonia and neonatal meningitis.
ViroStat introduces a set of new monoclonal antibodies to U. parvum that have applications in ELISA and IFA.
To download the data sheet for these NEW Antibodies and to obtain pricing, go to the ViroStat website at www.virostat-inc.com or call 207-856-6620

Just Published: Sensors & Actuators B: Chemical

A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:

Sensors & Actuators B: Chemical
http://rss.sciencedirect.com/publication/science/5283

Selected papers from the latest issue:

Increasing sensitivity of complementary metal–oxide–semiconductor (CMOS) based fluorescence detection

23 May 2012, 09:24:28

Publication year: 2012
Source:Sensors and Actuators B: Chemical
Joshua Balsam, Hugh Alan Bruck, Yordan Kostov, Avraham Rasooly
Optical technologies are important for biological analysis. Current biomedical optical analyses rely on high-cost, high-sensitivity optical detectors such as photomultipliers, avalanched photodiodes or cooled CCD. In contrast, Webcams, mobile phones and other popular consumer electronics use lower-sensitivity, lower-cost optical components such as photodiodes or CMOS. In order for consumer electronics devices to be useful for biomedical analysis, they must have increased sensitivity. We combined two strategies to increase the sensitivity of CMOS based fluorescence detector. 1. We captured hundreds of low sensitivity images using a Webcam in video mode, instead of a single image typically used in cooled CCD devices. 2. Then we used a computational approach, with an image stacking algorithm, to remove the noise from the many images and combine them into a single image. While video mode is widely used for dynamic scene imaging (e.g. movies or time-lapse photography), it is not used to capture still single static images, removing the images noise increased sensitivity by more than thirty fold. The portable, battery-operated Webcam-based fluorometer system developed here consists of five modules: (1) a low cost CMOS Webcam to monitor light emission, (2) a plate to perform assays, (3) filters and multi-wavelength LED illuminator for fluorophore excitation, (4) a portable computer to acquire and analyze images, and (5) image stacking software for image enhancement. The samples were various concentrations of fluorescein, ranging from 30 uM to 1000 uM, in a 36-well miniature plate. In the single frame mode, the fluorometer's limit-of-detection (LOD) for fluorescein is ∼1000 uM, which is relatively insensitive. However, when used in video mode combined with image stacking enhancement, the LOD is dramatically reduced to 30 uM, sensitivity which is similar to that of state-of-the-art ELISA plate photomultiplier based readers. Numerous medical diagnostics assays rely on optical and fluorescence readers, Our novel combination of detection technologies which is new to biodetection may enable the development of new low cost optical detectors based on an inexpensive Webcam (<$10) has the potential to form the basis of high sensitivity, low cost medical diagnostics for resource-poor settings.

A novel biosensor based on Hafnium oxide: Application for early stage detection of human interleukin-10

23 May 2012, 09:24:28

Publication year: 2012
Source:Sensors and Actuators B: Chemical
Michael Lee, Nadia Zine, Abdellatif Baraket, Miguel Zabala, Francesca Campabadal, Raffaele Caruso, Maria Giovanna Trivella, Nicole Jaffrezic-Renault, Abdelhamid Errachid
Measurement of interleukin-10 (IL-10) has subsequently become a crucial tool to identify end-stage heart failure (ESHF) patients prone to adverse outcomes during the early phase of left ventricular assisted device (LVAD) implantation. In this context, label-free detection using a novel substrate based on Hafnium oxide (HfO2) grown by Atomic Layer Deposition (ALD) on silicon was applied. Here, we studied the interaction between recombinant human (rh) IL-10 with the corresponding monoclonal antibody (mAb) for early cytokine detection of an anti-inflammatory response due to LVAD implantation. For this purpose, HfO2 has been functionalized using an aldehyde-silane ((11-(Triethoxysilyl) undecanal (TESUD)) self-assembled monolayer (SAMs), to directly immobilize the anti-human IL-10 mAb by covalent bonding. The interaction between the antibody-antigen (Ab-Ag) was characterized by fluorescence patterning and electrochemical impedance spectroscopy (EIS). Confirmation for the bio-recognition of the protein was achieved by fluorescence patterning, while Nyquist plots have shown a stepwise variation due to the polarization resistance (Rp) between the Ab activated surfaces with the detection of the protein. For early expression monitoring, commercial proteins of rh IL-10 were analyzed between 0.1pg/mL to 50ng/mL. Protein concentrations within the linear range of 0.1–20pg/mL were detected, and these values formulated a sensitivity of 0.49 (ng/mL)⁻¹. These preliminary results demonstrated that the developed biosensor was sensitive to the detection of rh IL-10, and the measured limit of 0.1pg/mL in phosphate buffered saline (PBS) was clearly detectable, which displays the high sensitivity of EIS. On analysis of an interference attributable to non-specific binding of other cytokine biomarkers; tumour necrosis factor-α (TNF-α), and IL-1β were analyzed without causing an interference to the IL-10 mAb. This established that selective sensitivity was responsive only to rh IL-10. To our knowledge, this is the first biosensor that has been based on HfO2 for Ag detection by EIS. In time, the HfO2 insulator will be incorporated into the gate of silicon-based ion-sensitive field-effect transistors (ISFETs) and developed as a portable real time detection system for the IL family of biomarkers in human serum.

Temperature-Modulated Differential Scanning Calorimetry in a MEMS Device

23 May 2012, 09:24:28

Publication year: 2012
Source:Sensors and Actuators B: Chemical
Bin Wang, Qiao Lin
We present a MEMS-based approach to temperature-modulated differential scanning calorimetry (AC-DSC) for biomolecular characterization. Based on a MEMS device integrating microfluidic handling with highly sensitive thermoelectric detection as well as on-chip AC heating and temperature sensing, we perform, for the first time, MEMS-based AC-DSC detection of liquid biological samples, demonstrated by the measurements of protein unfolding. The specific heat capacity and the melting temperature of the protein during the unfolding process are obtained and found to be consistent with published data. This MEMS AC-DSC approach has potential applications to label-free characterization of biomolecules.

Influences of Al, Pd and Pt additives on the conduction mechanism as well as the surface and bulk properties of SnO2 based polycrystalline thick film gas sensors

23 May 2012, 09:24:28

Publication year: 2012
Source:Sensors and Actuators B: Chemical
M. Hübner, N. Bârsan, U. Weimar
The reasons of the effect of Pd, Pt and Al additives on the sensing and conduction mechanism of SnO2 based thick film porous gas sensing layers are studied by a combination of DC-resistance, work function changes and catalytic conversion measurements. This is done by analyzing the dependence of the DC resistance on the corresponding band bending changes over a large range and the use of previously reported conduction models. The gained information deals with the surface band bending in the absence of ambient atmosphere oxygen, the position of the Fermi level, the concentration of free charge carriers, the Debye length and the width of the surface charge layer in various ambient conditions. Very interestingly, we found that in all cases the “doping” had an impact on both surface and bulk properties even if the additives and the technology of “doping” were targeted towards surface activation, in the case of Pt and Pd, and bulk compensation of donors, in the case of Al. Besides that, the catalytic conversion experiments indicated that the presence of Pt is associated with the reduction of the material in the absence of ambient atmosphere oxygen.

Development of gas sensors coatings by polyaniline using pressurized fluid

23 May 2012, 09:24:28

Publication year: 2012
Source:Sensors and Actuators B: Chemical
Clarice Steffens, Marcos L. Corazza, Elton Franceschi, Fernanda Castilhos, Paulo S.P. Herrmann,Jr, J. Vladimir Oliveira
The objective of this work was to develop gas sensors using three different techniques for the deposition of polyaniline film: a) precipitation of polyaniline particles doped with dodecylbenzenesulfonic acid (DBSA) using supercritical carbon dioxide (SC-CO2), b) rapid expansion with SC-CO2 (modified RESS) of polyaniline doped with DBSA, and c) in-situ polymerization polyaniline doped with chloridric acid. The sensors responses were evaluated at moisture and volatile organic compounds (VOCs). All of the sensors presented a satisfactory sensitivity and reversibility when exposed to VOCs. The films morphology was characterized by scanning electronic microscopy for field emission (FE-SEM). Results showed a difference regarding phase formation and behavior for the sensors obtained using SC-CO2 coatings with polyaniline and by in-situ polymerization, indicating that techniques the use of supercritical fluid techniques are promising in manufacturing such electronic devices.

Impedance-activated microseparator based on amplitude modulation sensing and dielectrophoretic switching methods

23 May 2012, 09:24:28

Publication year: 2012
Source:Sensors and Actuators B: Chemical
Song-I Han, Young-Don Joo, Ki-Ho Han
We present an impedance-activated microseparator that can detect and isolate microparticles according to size. Using the amplitude modulation sensing method, the impedance-activated microseparator precisely detected the size of 5-, 8-, and 10-μm polystyrene beads. To obtain high sensitivity, sensing electrodes were designed in a parallel overlap electrode structure, including two excitation electrodes patterned on the roof and a sensing electrode patterned on the bottom floor. The peak output voltage was proportionally correlated with particle volume; thus, the measured peak output values were used both to calculate microparticle size and to count the number of microparticles. Experimental results showed that volume resolution of the impedance-activated microseparator is less than 30μm³ and the minimum detectable particle size is approximately 2μm in diameter. By using the dielectrophoresis-based track switching method, the proposed impedance-activated microseparator could isolate microparticles according to their size with greater than 90% separation efficiency. Consequently, the proposed impedance-activated microseparator may facilitate the identification and separation of microparticles according to size from a heterogeneous admixture.

Theoretical optimizations of acoustic wave gas sensors with high conductivity sensitivities

23 May 2012, 09:24:28

Publication year: 2012
Source:Sensors and Actuators B: Chemical
Li Fan, Shu-yi Zhang, Huan Ge, Hui Zhang
The frequency shift and electric loss induced by the variation of the surface conductivity caused by the sorbed matter in the acoustic wave (AW) gas sensor are evaluated with the transfer matrix method. It is found that the high conductivity sensitivity is always accompanied by the high electric loss, which increases the insert losses and the minimum detectable masses of the sensors. However, with the same electric perturbation, the AW sensors with longer operating wavelengths have smaller electric losses, which provides the opportunities to achieve the high conductivity sensitivity and reduce the minimum detectable mass simultaneously in the gas sensor. 

Just Published: Journal of Chromatography A

A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:

Journal of Chromatography A

http://rss.sciencedirect.com/publication/science/5248

Selected papers from the latest issue:

Combined quantification of faecal sterols, stanols, stanones and bile acids in soils and terrestrial sediments by gas chromatography–mass spectrometry

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Jago Jonathan Birk, Michaela Dippold, Guido L.B. Wiesenberg, Bruno Glaser
Faeces incorporation can alter the concentration patterns of stanols, stanones, Δ⁵-sterols and bile acids in soils and terrestrial sediments. A joint quantification of these substances would give robust and specific information about the faecal input. Therefore, a method was developed for their purification and determination via gas chromatography–mass spectrometry (GC–MS) based on a total lipid extract (TLE) of soils and terrestrial sediments. Stanols, stanones, Δ⁵-steroles and bile acids were extracted by a single Soxhlet extraction yielding a TLE. The TLE was saponified with KOH in methanol. Sequential liquid–liquid extraction was applied to recover the biomarkers from the saponified extract and to separate the bile acids from the neutral stanoles, stanones and Δ⁵-steroles. The neutral fraction was directly purified using solid phase extraction (SPE) columns packed with 5% deactivated silica gel. The bile acids were methylated in dry HCl in methanol and purified on SPE columns packed with activated silica gel. A mixture of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and pyridine was used to silylate the hydroxyl groups of the stanols and Δ⁵-sterols avoiding a silylation of the keto groups of the stanones in their enol-form. Silylation of the bile acids was carried out with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing N-trimethylsilylimidazole (TSIM). TLEs from a set of soils with different physico-chemical properties were used for method evaluation and for comparison of amounts of faecal biomarkers analysed with saponification and without saponification of the TLE. Therefore, a Regosol, a Podzol and a Ferralsol were sampled. To proof the applicability of the method for faecal biomarker analyses in archaeological soils and sediments, additional samples were taken from pre-Columbian Anthrosols in Amazonia and an Anthrosol from a site in central Europe settled since the Neolithic. The comparison of the amounts of steroids in combination with and without saponification of the TLE showed that high amounts of faecal biomarkers occur bound to other lipids and were liberated by saponification. The method was evaluated by standard addition. The standard contained 5β-stanols, 5β-stanones and their 5α-isomers together with Δ⁵-sterols and bile acids (19 substances). The standard addition revealed mean recoveries of individual substances ≥85%. The recoveries of biomarkers within each biomarker group did not differ significantly. Precisions were ≤0.22 (RSD) and quantification limits were between 1.3 and 10ngg⁻¹ soil. These data showed that the method can be applied for quantification of trace amounts of faecal steroids and for the analyses of steroid patterns to detect enhanced faeces deposition in soils and sediments.

Highlights

► Method allows quantification of steroids in order to detect faeces deposition. ► Saponification yields higher amounts of steroids than analyses without saponification. ► Standard addition to soils samples was used for method evaluation. ► Method was tested in soils with different physico-chemical properties. ► Mean recoveries of individual steroids were ≥85%.

Hydrophobic interaction chromatography for purification of monoPEGylated RNase A

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Karla Mayolo-Deloisa, Ma. Elena Lienqueo, Barbara Andrews, Marco Rito-Palomares, Juan A. Asenjo
The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ∼85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.

Highlights

► Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is reported. ► Products of PEGylation reaction of RNase A were processed using three resins with different degrees of hydrophobicity. ► This process represents an alternative to separate PEGylated proteins. ► monoPEGylated RNase A was obtained with a yield and purity of 85% and 97%, respectively.

Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Kei Takahashi, Masafumi Tokuoka, Hiromi Kohno, Nobuko Sawamura, Yuka Myoken, Akihiro Mizuno
Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC–MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1–97.2μM in sake. The analysis was validated by dipeptide recovery of 64.0–107.2% (2.5μM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.

Highlights

► A separation method of AQC-derivatized dipeptide was developed by UHPLC using two sub-3μm core-shell type columns. ► Quantification and structural estimation of dipeptide using MS/MS and Q-TOFMS were examined. ► Dipeptide content in alcoholic beverages was quite differ among Japanese sake, beer, and wine. ► Sake contains many species and significant quantities of dipeptides. More than 60 species of dipeptides were found in sake.

Design of countercurrent separation of Ginkgo biloba terpene lactones by nuclear magnetic resonance

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Feng Qiu, J. Brent Friesen, James B. McAlpine, Guido F. Pauli
Terpene lactones such as bilobalide, ginkgolides A, B, C, and J are major bioactive compounds of Ginkgo biloba L. Purification of these compounds is tedious due to their similar chemical properties. For the purpose of developing an effective and efficient method for both analytical and preparative separation of terpene lactones in G. biloba, an innovative orthogonality-enhanced high-speed countercurrent chromatography (HSCCC) method was established. Taking advantage of quantitative ¹H NMR (qHNMR) methodology, partition coefficients (K) of individual terpene lactones were calculated directly from crude G. biloba leaf extract, using their H-12 signals as distinguishing feature. The partitioning experiment assisted the design of a two dimensional (2D) HSCCC procedure using a pair of orthogonal HSCCC solvent systems (SSs), ChMWat +4 and HEMSoWat +3/0.05%. It was surprising that the resolution of ginkgolides A and B was improved by 25% in the HEMWat +3 SS modified with 0.5% DMSO. Consequently, all five terpene lactones could be well separated with qHNMR purity>95% from G. biloba leaf extract. The separation was further evaluated by offline qHNMR analysis of HSCCC fractions associated with Gaussian curve fitting. The results showed less than 2% error in HSCCC retention predicted from the partitioning experiment. This compelling consistency demonstrates that qHNMR-derived K determination (“K-by-NMR”) can be used to predict CCC fractionation and target purification of analytes from complex mixtures. Furthermore, Gaussian curve fitting enabled an accurate prediction of less than 2% impurity in the CCC fraction, which demonstrates its potential as a powerful tool to study the presence of minor constituents, especially when they are beyond the detection limit of conventional spectroscopic detectors.

Highlights

► Novel qHNMR-based approach to countercurrent partition coefficients (K values). ► Enabled targeted countercurrent separation (CS) of Ginkgo biloba terpene lactones. ► Established orthogonal solvent systems (SSs) for separation of Ginkgo lactones. ► Developed DMSO-modified SSs as a new means of resolution enhancement in CS. ► Evaluated ID and residual complexity of eluents by qHNMR with Gaussian fitting.

Preparation and characterization of agarose–nickel nanoporous composite particles customized for liquid expanded bed adsorption

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
F. Asghari, M. Jahanshahi, A.A. Ghoreyshi
Agarose–nickel nanoporous composite matrices with a series of densities, named Ag–Ni, were prepared herein for expanded bed adsorption of nanobioproduct/bioproduct by a water-in-oil emulsification method. The optical microscope (OM), scanning electronic microscope (SEM) and particle size analyzer (PSA) were utilized in order to characterize the structure and morphology of the agarose–nickel composite. The results indicated that the matrices prepared had a spherical appearance, appropriate wet density of 1.73–2.56g/ml, water content of 32.2–58.5% and porosity of 79.4–96.37% and pore size of about 100–150nm. All the Ag–Ni beads follow logarithmic normal size distribution with the range of 60–230μm and average diameter of 133.68–148.4μm. One of the useful properties of the Ag–Ni particles is the high wet density up to 2.56g/ml, which shows a potential for the operation in an expanded bed at high flow rate. The impact of nickel powder addition on the physical and hydrodynamic properties was also investigated. In addition, the fluidization behavior of the Ag–Ni particles under various conditions was characterized by the measurement of bed expansion and axial dispersion coefficients for the liquid phase when operated in a standard fluidized bed contactor. It was observed that the expansion factors were decreased with the increasing matrix density under the same velocity. The bed expansion and fluid velocity were correlated with Richardson–Zaki equation for all particles prepared and the correlation parameters (the terminal settling velocity U t and expansion index n) were investigated. Using measurements of residence time distributions, hydrodynamic properties in the expanded beds were investigated and were compared with reported matrices in other literatures. In addition, the impact of the flow velocity, bed expansion degree and density of adsorbent on hydrodynamic properties in the expanded beds were investigated. The results indicated that the expansion factor showed little effect on the hydrodynamic properties while the fluid velocity was the most essential factor on this regard. Furthermore, the results indicated that the heavy matrices of Ag–Ni-3, Ag–Ni-4 and Ag–Ni-5, were more suited for high operation fluid velocity. In addition, even the light matrices, i.e. Ag–Ni-1 and Ag–Ni-2, seem to be superior to other matrices in hydrodynamic properties, which made them promising adsorbents for further use in EBA processes.

Highlights

► Agarose–nickel nanoporous particles have been fabricated with high wet density of 1.73–2.56g/ml and pore size of 100–150nm. ► The influence of matrix density on the performance of expanded bed adsorption has been studied. ► The results indicated that the matrix with small density can form a better stable expanded bed. ► As compared to HETP, B 0 and D ax analysis, it was observed that D ax is the best parameter for characterizing the bed stability.

Simultaneous metabolite fingerprinting of hydrophilic and lipophilic compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Federica Pellati, Giulia Orlandini, Stefania Benvenuti
In this study, a detailed phytochemical characterization of Echinacea pallida (Nutt.) Nutt. root extracts and dietary supplements was carried out for the first time by developing advanced chromatographic techniques, based on HPLC with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection (with ion trap and triple quadrupole mass analyzers), for the simultaneous analysis of hydrophilic and lipophilic secondary metabolites. The HPLC analyses were carried out on an Ascentis C18 column (250mm×4.6mm I.D., 5μm), with a mobile phase composed by H2O and ACN both containing 0.1% formic acid, under gradient elution. The UV spectra, in combination with MS and MS/MS data, allowed the identification of fourteen compounds, including caffeic acid derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in detail and the typical fragmentation patterns of each class of secondary metabolites were identified. For the first time, a hydroperoxide intermediate was characterized as an oxidation product of one of E. pallida monocarbonylic acetylenes, providing a confirmation of the mechanism that leads to the generation of hydroxylated derivatives. The HPLC method was fully validated in agreement with ICH guidelines and then applied to real samples. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the dietary supplements containing E. pallida root extracts: the content of total caffeic acid derivatives ranged from 2.31 to 11.45mg/g and the amount of total polyacetylenes and polyenes from 6.38 to 30.54mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be tetradec-(8Z)-ene-11,13-diyn-2-one, pentedeca-(8Z,11Z)-dien-2-one and pentadec-(8Z)-en-2-one. The developed method can be considered suitable for metabolite fingerprinting and quality control of E. pallida plant material and natural products.

Highlights

► Echinacea pallida plant material and dietary supplements were analyzed. ► Analysis of both hydrophilic and lipophilic secondary metabolites was performed. ► HPLC-UV/DAD, ESI-MS and MS/MS allowed the identification of fourteen compounds. ► The typical fragmentation patterns of each class of constituents were identified. ► Great variability was observed in the content of E. pallida dietary supplements.

Chemical anchoring of lauryl methacrylate-based reversed phase monolith to 1/16″ o.d. polyetheretherketone tubing

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Shin Shu, Hiroharu Kobayashi, Masaki Okubo, Akhmad Sabarudin, Michio Butsugan, Tomonari Umemura
In this paper, we describe a method for the preparation of easy-to-use reversed-phase monolithic microbore columns. Polyetheretherketone (PEEK) tubing with an outer diameter of 1/16″ and an inner diameter of 1.0mm was used as a column housing (empty column), and in it lauryl methacrylate (LMA) was copolymerized with ethylene dimethacrylate (EDMA). In order to chemically anchor the polymer monolith to the tube wall, the inner wall surface was pretreated by the following two-step procedure. (1) 50% sulfuric acid was filled into the PEEK tubing and left to stand for 6h to generate sulfonate groups on the surface. (2) After washing with Milli-Q water, the sulfonated PEEK surface was brought into contact with 1M glycidyl methacrylate in dichloromethane (or acetone) at 40°C for 4h to introduce methacryloyl groups via the reaction of sulfonate groups and epoxy groups. Mechanical strength and column efficiency of the resulting monoliths were evaluated through the separation of a series of alkylbenzenes in acetonitrile–water (50:50, v/v) eluent over the flow rate range of 50–750μL/min (corresponding to 1.7–25.5mm/s). The poly(LMA-co-EDMA) monolith provided acceptable column efficiency of 2000 theoretical plates/10cm (HETP value of 50μm) for amylbenzene (separation factor k =40) and low flow resistance of 0.5MPa/10cm at a normal flow rate of 50μL/min. The methacryloylated PEEK tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 15MPa, allowing rapid separation at a 15–20 fold higher flow rate than normal.

Highlights

► The inner wall of a PEEK tubing was sulfonated by exposing to sulfuric acid. ► The sulfonated PEEK tube wall was subsequently methacryloylated by exposing to GMA. ► Polymer monolith was in situ synthesized in the methacryloylated PEEK tubing. ► The monolithic column exhibited good pressure resistance.

Comprehensive two-dimensional HepG2/cell membrane chromatography/monolithic column/time-of-flight mass spectrometry system for screening anti-tumor components from herbal medicines

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Xiaofei Chen, Yan Cao, Diya Lv, Zhenyu Zhu, Junping Zhang, Yifeng Chai
Cell membrane chromatography (CMC) is a biological affinity chromatographic method using specific cell membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and membrane receptors. In this study, a novel comprehensive two-dimensional (2D) chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p <0.05). The result demonstrated that the proposed comprehensive 2D HepG2/CMC/monolithic column/TOFMS system has the advantages of strong recognition and rapid analysis abilities for the total screening procedure, which will be selectable and practical in drug discovery from complex HM samples and can also be applied to other biochromatography models.

Highlights

► A new HepG2 cell membrane model was developed. ► Novel comprehensive two-dimensional chromatography approach was established and optimized for fast screening procedure. ► Four anti-tumor active components from herbal medicines were screened and reliably identified by TOFMS. ► EGFR was preliminary selected as the target membrane receptor via competitive displacement assays. ► BBR and THP revealed inhibiting effects on cell proliferation in vitro.

Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for a lipidomic analysis of Leishmania donovani

23 May 2012, 08:19:04

Publication year: 2012
Source:Journal of Chromatography A, Volume 1242
Laurent Imbert, Mathieu Gaudin, Danielle Libong, David Touboul, Sonia Abreu, Philippe M. Loiseau, Olivier Laprévote, Pierre Chaminade
A comparison of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) for the analysis of a wide range of lipids has been performed on standard mixtures and extracts of Leishmania donovani promastigotes resistant to Amphotericin B (AmB). Calibration model, precision, limits of detection and quantification (LOD and LOQ) were assessed for each source. APPI provided the highest signal, signal-to-noise (S/N), and sensitivity for non-polar and low-polarity lipids, while ESI and APCI gave better results for the most polar ones. The linear model was valid for all lipids, except for one class with APPI, six classes with ESI, and eleven classes with APCI. LODs ranged from 0.2 to 20μgmL⁻¹ for ESI, from 0.1 to 10μgmL⁻¹ for APCI, and from 0.02 to 9.5μgmL⁻¹ for APPI. LOQs ranged from 0.2 to 61μgmL⁻¹ for ESI, from 0.4 to 31μgmL⁻¹ for APCI, and from 0.1 to 29μgmL⁻¹ for APPI. Each source provided similar lipid composition and variations in a comparison of three different L. donovani samples: miltefosine-treated, miltefosine-resistant and treated miltefosine-resistant parasites. A treated miltefosine-resistant sample was finally analyzed with each ion source in order to verify that the same lipid molecular species are detected.

Highlights

► We coupled a normal-phase chromatographic separation of lipids to a mass spectrometer. ► We compared the performance of the three atmospheric pressure ion sources. ► Atmospheric pressure photoionization is interesting for non-polar and low polarity lipids. ► Polar lipids are better analyzed through electrospray ionization.