Indirect fluorimetric detection of proteins via postcolumn mixing with fluorescence probe in size-exclusion chromatography

Summary Proteins were visualized by postcolumn mixing with 2-p-toluidinyl-6-naphthalene sulfonate or 1-anilino-8-naphthalene sulfonate in size-exclusion chromatography. The indirect detection is based on fluorescence enhancement of the fluorescence probe owing to hydrophobic interaction with proteins. Bovine serum albumin gave the highest signal intensity among the proteins examined.     

Indirect Fluorimetric Detection of Cyclodextrins via Postcolumn Mixing with 2-p-Toluidinyl-6-naphthalenesulfonate in Liquid Chromatography

Cyclodextrins (CD's) were visualized by postcolumn mixing with 2-p-toluidinylnaphthalene sulfonate (TNS) in liquid chromatography. The indirect detection is based on fluorescence enhancement due to the formation of an inclusion complex between TNS and CD's. β-CD gave larger signal intensity than α- and γ-CD. Different selectivities were observed for alkyl-bonded silica stationary phases with different chain length.     

Direct fluorescence detection of non-steroidal anti-inflammatory agents separated by TLC with 9-isothiocyanatoacridine derivatives

Summary Non-steroidal anti-inflammatory agents were separated by thin-layer chromatography, utilizing precoated plates with silica gel R as the stationary phase and chloroform:methanol:25% ammonia (80∶15∶5), as the mobile phase. The spots were visualized by spraying with 0.1% solution of 9-isothiocyantoacridine derivatives in methylene chloride or benzene and irradiating with UV at 254 and 366nm. The visual detection limit of a spot was 0.1μg.     

Characterization of dextrans by size-exclusion chromatography on unmodified silica gel columns, with light-scattering detection, and capillary electrophoresis with laser-induced fluorescence detection

Unmodified silica gel size-exclusion columns were used in an on-line combination with light-scattering detection for a size characterization of dextrans. The influence of electrostatic interactions on analyte retention was briefly investigated. Size-exclusion chromatography was also used for evaluation of the fluorescence labeling procedure for dextrans with 8-aminonaphthalene-1,3,6-trisulfonic acid. The derivatives obtained through this procedure were used for electrophoretic measurements with laser-induced fluorescence detection. A comparison between the size-exclusion data and capillary electrophoresis indicates that the effectiveness of fluorescent labeling decreases with molecular mass of the dextran analytes.     

Synthesis of platinum colloids sterically stabilized by poly(N-vinylformamide) or poly(N-vinylalkylamide) and their stability towards salt

Colloidal dispersions of nanometer-sized platinum colloids were prepared by ethanol reduction of PtCl₆ ²⁻ in the presence of poly(N-vinylformamide) (PNVF), poly(N-vinylacetamide) (PNVA) or poly(N-vinylisobutyramide) (PNVIBA) and analyzed by UV-vis spectroscopy and transmission electron microscopy. The dispersion stability of each colloid to the presence of added KCl was determined by a stirring and centrifugation procedure. The platinum colloid stabilized by PNVF (PNVF-Pt) was the most stable and its critical flocculation concentration was not observed up to the highest electrolyte concentration employed (4.0 M). The stability of the platinum colloids stabilized by poly(N-isopropylacrylamide) (PNIPAAm) and poly(vinylpyrrolidone) (PVP) was also examined. The sequence of polymer-stabilized platinum colloids in increasing order of dispersion stability was found to be PNIPAAm-Pt < PNVIBA-Pt < PVP-Pt < PNVA-Pt < PNVF-Pt. Received: 25 August 1998 Accepted in revised form: 14 January 1999     

Fast quantitation of 5-hydroxymethylfurfural in honey using planar chromatography

An approach for rapid quantitation of 5-hydroxymethylfurfural (HMF) in honey using planar chromatography is suggested for the first time. In high-performance thin-layer chromatography (HPTLC) the migration time is approximately 5 min. Detection is performed by absorbance measurement at 290 nm. Polynomial calibration in the matrix over a range of 1:80 showed correlation coefficients, r, of ≥ 0.9997 for peak areas and ≥ 0.9996 for peak heights. Repeatability in the matrix confirmed the suitability of HPTLC–UV for quantitation of HMF in honey. The relative standard deviation (RSD, %, n = 6) of HMF at 10 ng/band was 2.9% (peak height) and 5.2% (peak area); it was 0.6% and 1.0%, respectively, at 100 ng/band. Other possible detection modes, for example fluorescence measurement after post-chromatographic derivatization and mass spectrometric detection, were also evaluated and can coupling can be used as an additional tool when it is necessary to confirm the results of prior quantitation by HPTLC–UV. The confirmation is provided by monitoring the HMF sodium adduct [M + Na]⁺ at m/z 149 followed by quantitation in TIC or SIM mode. Detection limits for HPTLC–UV, HPTLC–MS (TIC), and HPTLC–MS (SIM) were 0.8 ng/band, 4 ng/band, and 0.9 ng/band, respectively. If 12 μL honey solution was applied to an HPTLC plate, the respective detection limits for HMF in honey corresponded to 0.6 mg kg⁻¹. Thus, the developed method was highly suitable for quantitation of HMF in honey at the strictest regulated level of 15 mg kg⁻¹. Comparison of HPTLC–UV detection with HPTLC–MS showed findings were comparable, with a mean deviation of 5.1 mg kg⁻¹ for quantitation in SIM mode and 6.1 mg kg⁻¹ for quantitation in TIC mode. The mean deviation of the HPTLC method compared with the HPLC method was 0.9 mg kg^-1 HMF in honey. Re-evaluation of the same HPTLC plate after one month showed a deviation of 0.5 mg kg⁻¹ HMF in honey. It was demonstrated that the proposed HPTLC method is an effective method for HMF quantitation in honey.      

Statistical Optimization Applied to Simultaneous Determination of Maprotiline, Desipramine, and Moclobemide by Capillary Zone Electrophoresis

Summary. A method of Capillary Zone Electrophoresis (CZE) for separation of three most-frequently prescribed antidepressants in the market: maprotiline, desipramine, and moclobemide was developed. The proposed method is fully validated for a ternary laboratory mixture but it is also suitable for individual determination of the investigated components in pharmaceutical dosage forms. Since the preliminary investigations did not show complete separation because of close migration time of desipramine and maprotiline, a complete set, 2³ experimental design was applied. All the factors that affect separation as well as their mutual interactions were investigated. Voltage, temperature of capillary and the pH of phosphate buffer were independent variables or factors to be investigated in two levels. Applying response surface methodology, from experimental points the appropriate graphs were constructed and optimal chromatographic conditions for the separation were defined. The optimum conditions were: running voltage of 20 kV, phosphate buffer (pH = 2.35), temperature 25°C, and UV detection at 200 nm. An uncoated (fused silica) capillary (50 μm i.d.) with a total length of 50 cm and a distance of 47 cm between the injection and detection points was used.     

The development of a rapid method for the isolation of four azaspiracids for use as reference materials for quantitative LC–MS–MS methods

The azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 after consumption of contaminated shellfish (Mytilus edulis). Levels of azaspiracids in shellfish for human consumption are monitored in accordance with EU guidelines: only shellfish with less than 160 μg kg⁻¹ are deemed safe. The limited availability of commercially available standards for azaspiracids is a serious problem, because validated LC–MS methods are required for routine analysis of these toxins in shellfish tissues. The procedure described herein has been used for the separation and the isolation of four azaspiracid (AZA) toxins from shellfish, for use as LC–MS–MS reference materials. Five separation steps have been used to isolate azaspiracids 1, 2, 3, and 6. The purity of the toxins obtained has been confirmed by multiple mass spectrometric methods using authentic azaspiracid standards. The same techniques have been used for quantification of the toxins extracted. The isolation procedure involves several chromatographic purification techniques: solid-phase extraction (diol sorbent, 90% mass reduction, and 95 ± 1% toxin recovery); Sephadex size-exclusion chromatography (87% mass reduction and up to 95 ± 2% toxin recovery), Toyopearl HW size-exclusion chromatography (90% mass reduction and up to 92.5 ± 2.5% toxin recovery), and semi-preparative LC (78 ± 3% toxin recovery). The procedure effectively separates the toxins from the sample matrix and furnishes azaspiracid toxins (AZA1, AZA2, AZA3 and AZA6) of sufficient purity with an average yield of 65% (n = 5). Triple-quadrupole mass spectrometry was used for qualitative and quantitative monitoring of the isolation efficiency after each stage of the process. High-resolution mass spectrometric evaluation of the toxic isolated material in both positive and negative modes suggests high purity.     

Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)₂ and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[α]-carbazol-11-yl) ethyl chloroformate (BCEC–Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[α]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC–Cl. The new reagent BCEC–Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC–amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)₂ and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I _BCEC/I _BCEOC = 1.17–3.57, I _BCEC/I _FMOC = 1.13–8.21, and UV_BCEC/UV_BCEOC = 1.67–4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C₁₈ column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)₂. Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)₂ and Try from bee-collected pollen (bee pollen) samples.     

"Dilute-and-shoot" triple parallel mass spectrometry method for analysis of vitamin D and triacylglycerols in dietary supplements

A method is demonstrated for analysis of vitamin D fortified dietary supplements that eliminates virtually all chemical pretreatment prior to analysis, which is referred to as a “dilute-and-shoot” method. Three mass spectrometers, in parallel, plus a UV detector, an evaporative light-scattering detector (ELSD), and a corona charged aerosol detector (CAD) were used to allow a comparison of six detectors simultaneously. Ultraviolet data were analyzed using internal standard, external standard, and response factor approaches. The contents of gelcaps that contained 2,000 IU (50 μg) vitamin D₃ in rice bran oil, diluted to 100 mL, were analyzed without the need for lengthy saponification and extraction. Vitamin D₃ was analyzed using UV detection, extracted ion chromatograms, selected ion monitoring (SIM) atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and two transitions of multiple reaction monitoring (MRM) APCI-MS. The internal standard, external standard, and response factor methods gave values of 0.5870 ± 0.0045, 0.5893 ± 0.0041, and 0.5889 ± 0.0045 μg/mL, respectively, by UV detection. The values obtained by MS were 0.6117 ± 0.0140, 0.6018 ± 0.0244, and 0.5848 ± 0.0146 μg/mL by SIM and two transitions of MRM, respectively. The triacylglycerols in the oils were analyzed using full-scan APCI-MS, electrospray ionization (ESI) MS, up to MS⁴, an ELSD, and a CAD. The method proved to be very sensitive for vitamin D₃, as well as triacylglycerols (TAGs), allowing identification of intact TAGs containing fatty acids up to 28 carbons in length. LC-ESI-MS of glycerin polymers is also demonstrated.     

Development of a HPLC Method for Analysis of Linear Alkylbenzene Sulphonates and Detection by UV and FTIR Spectroscopy Using Thermospray Interface

 A reversed-phase high performance liquid chromatography (RP-HPLC) method, with acetic acid and sodium perchlorate phase modifiers, was developed to separate a mixture of linear alkylbenzene sulphonates (LAS), containing 10 to 13 carbon atoms. The effects of methanol-water compositions and concentrations of used modifiers were investigated and compared. The separation achieved with 50 mg L⁻¹ acetic acid was found satisfactory whereas a concentration of 10 g L⁻¹ sodium perchlorate was preferred. Chromatograms obtained with UV and Fourier transform infrared (FTIR) detection showed almost similar features and HPLC-FTIR interface spectra of LAS components exhibited excellent agreement of absorption features to those of standard FTIR spectrum and no thermal degradation was found to occur. Received May 20, 1998 Revision March 25, 1999.     

Micelle formation of polystyrene-<Emphasis Type="Italic">block</Emphasis>-poly(4-<Emphasis Type="Italic">tert</Emphasis>-butoxystyrene) in hexane

A new type of hexane-soluble polymeric surfactant based on poly(4-tert-butoxystyrene) (P ^t BSt) was prepared by the nitroxide-mediated living radical polymerization, and their self-assemblies in hexane were explored. Polystyrene-block-P ^t BSt diblock copolymers with six different P ^t BSt block lengths were obtained by the sequential living radical polymerization of styrene followed by 4-tert-butoxystyrene using 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl as the mediator; Mn(P ^t BSt block) = 13,500, 21,700, 26,600, 47,500, 91,300, and 108,000 at the constant length of the PSt block (Mn = 12,900). Dynamic light-scattering studies demonstrated that the copolymers self-assembled into monodispersed spherical micelles in hexane. The hydrodynamic diameter of the micelles increased with an increase in the P ^t BSt block length. The micellar size also increased as the copolymer concentration increased. However, the size decreased as a result of the increasing temperature due to a decrease in the aggregation number. The ¹H NMR analysis confirmed that the copolymers formed micelles with PSt cores.     

Development of an HPLC–UV–ELSD Method for Quantification of Multiple Components of a Chinese Medicine Made from Radix salvia miltiorrhiza and Panax notoginseng

‘Fufang Danshen tablet’ (FDT), made from Radix salvia miltiorrhiza and Panax notoginseng, is a widely used botanical drug derived from traditional Chinese medicine. Quantification of the active components of Radix salvia miltiorrhiza and Panax notoginseng is very important for regulation of FDT products. In this study HPLC hyphenated with ultraviolet (UV) detection and evaporative light-scattering detection (ELSD) was used for simultaneous determination of nine active components (three salvianolic acids, three tanshinones, and three saponins) of FDT products. Separation was performed on a 250 mm × 4.6 mm i.d., 5.0 μm particle-size, C₁₈ column with linear gradient elution. UV detection at 280 and 254 nm was used for detection of the three salvianolic acids and the three tanshinones, respectively. ELSD was used for detection of the three saponins, which were difficult to analyze by use of UV detection. The linearity of the calibration plots was excellent over the concentration ranges investigated (values of R ² were >0.99 for all the analytes) and recovery measured at three concentrations was between 92.2 and 107.7%. The validated method was successfully used for simultaneous determination of these components in FDT products.     

Degradation kinetics of the Alternaria mycotoxin tenuazonic acid in aqueous solutions

The degradation kinetics of the Alternaria mycotoxin tenuazonic acid (l-TA) in aqueous buffer were studied over a period of 4 months at different pH levels (3.5 and 7.0) and temperatures (4, 25 and 40 °C). l-TA and its degradation products were quantified by newly developed high-performance liquid chromatography methods with UV or electrospray multistage mass spectrometry detection. At pH 3.5, significant degradation occurred at 25 and 40 °C, the respective l-TA half-lives being 73.8 ± 0.4 and 14.0 ± 0.1 days. Two degradation processes, epimerization and hydrolysis, were evaluated kinetically. The hydrolytically formed iso-deacetyl TA (iso-DTA, epimeric mixture) was found to be the stable end product of l-TA degradation under the conditions of this study. This indicates that iso-DTA as well as the l-TA epimer u-TA are formed in aqueous beverage matrices.     

Development and validation of an HPLC-UV detection assay for the determination of rufinamide in human plasma and saliva

The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min^-1, with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [r ² = 0.998 ± 0.002 for plasma (n = 10) and r ² = 0.999 ± 0.001 for saliva (n = 9)] over the range of 0.25–20.0 μg ml^-1, with a limit of quantification at 0.25 μg ml^-1. Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring.     

Circular dichroism thermal lens microscope in the UV wavelength region (UV-CD-TLM) for chiral analysis on a microchip

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 × 10⁻⁴ mol L⁻¹ (ΔA = 5.2 × 10⁻⁶ Abs.) for (+)-CSA and 8.4 × 10⁻⁴ mol L⁻¹ (ΔA = 5.0 × 10⁻⁶ Abs.) for (−)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified. Figure Sensitive chiral analysis by thermal lens microscope (TLM)     

Real-time immuno-PCR assay for detecting PCBs in soil samples

A fast and robust assay, based on immuno-polymerase chain reaction (IPCR) techniques, was developed for the detection of polychlorinated biphenyls (PCBs) in soil samples. Real-time IPCR (rt-IPCR) is a powerful technique that combines enzyme-linked immunosorbent assay (ELISA) with the specificity and sensitivity of PCR. In our assay, indirect ELISAs based on immobilization of PCB37 hapten–ovalbumin conjugates was used for evaluation of the immune response. The effect of optimal reagent concentrations to reduce background fluorescence was also investigated. Using the optimized assay, the linear sensitivity range of the assay covered more than six orders of magnitude, and the minimum detection limits reached 5 fg ml^–1 antigen. Rt-IPCR was tested for its cross-reactivity profiles using four selected congeners and four Aroclor products. The assays were highly specific for congeners but less specific for Aroclor1242. We took four soil samples to validate the method, and the results were confirmed by gas chromatography/mass spectrometry (GC/MS). The rt-IPCR results for soil samples correlated well with the concentrations of PCBs obtained by GC/MS (r = 0.99, n = 6). These data indicate that this highly specific, sensitive, and robust assay can be modified for detecting PCB compounds in the environment.     

Dispersion of silica nano-particles in sol-gel hybrid resins for fabrication of multi-scale hybrid nanocomposite

Multi-scale hybrid nanocomposites containing both ∼15 nm silica colloids and ∼2 nm oligosiloxanes in a methacryl polymer matrix were newly designed and fabricated. Colloidal silica sols were dispersed in methacryl oligosiloxanes nano-hybrid resins synthesized by sol-gel reaction of methacryloxypropylmethoxysilane and diphenylsilanediol. On the basis of TEM and SANS analyses, it was confirmed that the silica colloids were compatibly dispersed and different sizes of colloidal silica and oligosiloxanes co-exist in the solutions. Multi-scale hybrid nanocomposites fabricated by UV and thermal curing with incorporation of silica colloids in the nano-hybrid materials show enhanced mechanical and thermal characteristics.     

Quantitative in silico analysis of the selectivity of graphitic carbon synthesized by different methods

Molecular interaction energy (MI) values calculated by molecular mechanics (MM2) using a model graphitic carbon phase were used for studying the selectivity of different types of graphitic carbon columns. The MI values well correlated with logk values measured on a graphitic carbon synthesized from 100% organic materials (r = 0.961, n = 13) but not with logk values measured on a graphitic carbon synthesized using silica matrix (r = 0.558, n = 17). The latter logk values correlated well with the hydrogen bonding energy values calculated using a model silica phase (r = 0.856, n = 17). The reason for the poor correlation of the logk values measured on the latter graphitic carbon is that the silica matrix might not be completely eliminated in the production process.     

Analysis of commercial ceramides by non-aqueous reversed-phase liquid chromatography with evaporative light-scattering detection

Summary This paper describes the development of a chromatographic system for analysis of commercial ceramides structurally similar to those found in the stratum corneum. The ceramides used in this study contain different amine based (phytosphingosine, sphingosine and dihydrosphingosine) and fatty acids of different chain lengths and with different functional groups (hydroxylated and unsaturated). Non-aqueous reversed-phase (NARP) liquid chromatography with evaporative light-scattering detection (ELSD) were the techniques chosen in accordance with the nature of the ceramides. The eluent strength and the potential selectivity of different organic solvents were investigated. On a C₁₈-bonded silica, the most promising chromatographic conditions employed a gradient from ACN-THF, 95∶5, to ACN-THF-PrOH, 35∶5∶60, in 15 min with a constant concentration of TEA (10 mM) and a stoichiometric amount of formic acid.