Chromatographic Signals Research Articles

Chromographic Analysis and Cytotoxic Effects of Chlorhexidine and Sodium Hypochlorite Reaction Mixtures

IntroductionThe literature reveals controversies regarding the formation of para-chloroaniline (PCA) when chlorhexidine (CHX) is mixed with sodium hypochlorite (NaOCl). This study aimed to investigate the stability of PCA in the presence of NaOCl and to examine the in vitro cytotoxic effects of CHX/NaOCl reaction mixtures. MethodsDifferent volumes of NaOCl were added to CHX (mix 1) or PCA (mix 2). Upon centrifugation, the supernatant and precipitate fractions collected from samples were analyzed using high-performance liquid chromatography. The cytotoxic effects of both fractions were examined on human periodontal ligament and 3T3 fibroblast cell lines. ResultsHigh-performance liquid chromatographic analysis showed no PCA signal when NaOCl was mixed with CHX (mix 1). In mix 2, the intensity of PCA was decreased when NaOCl was added to PCA, and chromatographic signals, similar to that of CHX/NaOCl, were also observed. The mortality of precipitates exerted on both cell lines was lower compared with that of supernatants. ConclusionsThe discrepancy in the data from the literature could be caused by the instability of the PCA in the presence of NaOCl. The CHX/NaOCl reaction mixture exhibits a wide range of cytotoxic effects.

New approaches to the calculation and interpretation of asymmetry factors of chromatographic peaks

The suitability of the determination of the asymmetry factor of chromatographic peaks by the ratio of areas of two components separated by a perpendicular dropped from the maximum of the peak to the base-line, A s * = S b /S a , where symbol a corresponds to the leading edge of the peak and b is for its tailing slope, is discussed. It is demonstrated that this method enables the estimation of the asymmetry of even partially separated chromatographic signals, including those eluted “in the tail” of intense peaks of solvents. The concepts of the asymmetry index I(A s *) and its increment ΔI(A s *) = (A s *)–I(A s *) are introduced, which ensures the characterization of the asymmetry of peaks of polar analytes with respect to the asymmetry of nonpolar reference components, that is, the separation of the effects of the polarity of analytes and their quantities injected into the chromatographic column on this parameter. For the first time we revealed a correlation of the asymmetry factors of compounds of different chemical nature with such a characteristic of their polarity as the difference in chromatographic separation temperature and the normal boiling point of analytes.

A real-time wavelet algorithm and its application in chromatographic signal analysis based on finite impulse response filter

A real-time wavelet algorithm and its application in chromatographic signal analysis based on finite impulse response filter

Longest distance shifting: A simple and efficient approach for the alignment of shifted chromatographic peaks.

The preprocessing of chromatograms, such as the alignment of retention time shifts, is often a crucial step in the proper data analysis chain. Here, an efficient approach to align shifted chromatographic signals, longest distance shifting, is presented and highlighted. The performance of this novel strategy was demonstrated by using both simulated chromatograms that covered the different kinds of retention time shifts and the real experimental chromatograms of Pudilan Xiaoyan Tablets obtained by high-performance liquid chromatography with photodiode array detection. The averaged correlation coefficient for experimental chromatograms were in the range of 0.9517-0.9840 and the peak factor was 0.9989. As a comparison, all the chromatograms have also been aligned using correlation optimized warping and Interval Correlation Optimized Shifting algorithms. The obtained results indicate that the longest distance shifting algorithm is simpler, faster and more effective, and will be potentially suitable for the alignment of other types of signals.

Characterization of metizolam, a designer benzodiazepine, in alternative biological specimens

Designer benzodiazepines provide an attractive alternative to prescribed benzodiazepines for abuse purposes as they are readily available over the Internet at a low price. Although these compounds have been developed by pharmaceutical companies, at this time, they do not have a legal status. Their detection in biological fluids is challenging owing to the very few available data. For instance, there is no information about metizolam (also known as desmethyletizolam) analysis, distribution and metabolism in the scientific literature. Metizolam was ordered via Internet and a 2 mg blue tablet was orally administered to a 54-year-old healthy man (85 kg). Oral fluid (saliva) was collected over 8 hours using the NeoSal™ device. Sweat was collected with the PharmChek™ sweat patch technology over 72 hours. Exhaled breath was collected with the ExaBreath ® DrugTrap device over 15 hours. Beard hair was collected 4 and 10 days after administration. Finally, head hair was collected 3 weeks after administration. Metizolam was detectable in saliva during 8 hours, with concentrations lower than 1 ng/mL (max at 0.77 ng/mL after 2 hours). Increasing concentrations up to 186 pg/patch (after 72 hours) of metizolam were detected in the sweat patches. Metizolam produced for 4 hours a very low but significant chromatographic signal in exhaled breath, with concentrations always lower than 10 pg/filter. Finally, metizolam tested positive in beard hair (0.73 and 0.28 pg/mg after 4 and 10 days, respectively) and head hair (0.27 pg/mg). From the data obtained from one subject, metizolam showed very low concentrations in various alternative matrices, attesting that the potential forensic implications (drug-facilitated crime, addiction, DUID…) of metizolam abuse must be considered as a new challenge for toxicologists.

Using nanometer TiO2 modified with cetyl trimethyl ammonium bromide for separation and preconcentration of Parathion in water sample

In this work, nanometer TiO2 modified by cetyl trimethyl ammonium bromide (CTAB) was used as adsorbent for solid-phase extraction (SPE) of Parathion in environmental water samples. Adsorbed Parathion was then desorbed with different eluents and determined by gas chromatography (GC)/flame ionization detection. Greater selectivity, resolution, and sensitivity have been seen by GC compared with other methods. Parameters that might influence the extraction efficiency, such as the eluent type and its volume, adsorbent amount, sample volume, sample pH and sample flow rate, were optimized. Under the optimized extraction conditions with toluene as the eluent, the experimental results showed the excellent linearity of Parathion (R2 > 0.99) over the range of 0.01–0.8 μg/mL, and the relative standard deviation was 6.3% (n = 5). The detection limit of the proposed method could reach 0.024 ng/mL based on the ratio of chromatographic signal to base line noise (S/N = 3). Recovery of 93% was achieved with spiked water samples. The method was successfully applied to the analysis of surface water samples.

Studies of pattern recognition of fingerprint profile of cattle bile powder and determination of multi-component in it

An HPLC-ELSD method with good specificity and good accuracy was used for the studies of fingerprint and quantification of multi-components for cattle bile powder. The chromatographic analysis was carried out on a Phenomenex Gemini C₁₈ column (4.6 mm×250 mm, 5 μm) with a column temperature of 40 ℃ and a liquid flow-rate of 1.0 mL•min⁻¹ using 10 mmol ammonium acetate solution and acetonitrile as the mobile phase with a linear gradient. An ELSD was used with a nitrogen flow-rate of 2.8 L•h⁻¹, at a drift tube temperature of 110 ℃. The average contents of glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid were (25.2±17.0)%, (4.1±3.4)%, (24.5±20.0)% and (5.2±3.8)% respectively, and the total content of the four bile acids was (59.0±26.0)%. Beyond that, the preprocessing and pattern recognition analysis of the chromatographic fingerprints of samples were applied with chemometric method. The results of this chemometric analysis indicated that the samples from market and self-made samples were different signally, and four regions were noteworthy due to their great impact with poor chromatographic signal. All in one, because this HPLC-ELSD method was simple and accurate, it was suitable for the quality assessment and quality control of cattle bile powder and could be the technological base for its standard perfection.

Automated resolution of chromatographic signals by independent component analysis–orthogonal signal deconvolution in comprehensive gas chromatography/mass spectrometry-based metabolomics

Comprehensive gas chromatography–mass spectrometry (GC×GC–MS) provides a different perspective in metabolomics profiling of samples. However, algorithms for GC×GC–MS data processing are needed in order to automatically process the data and extract the purest information about the compounds appearing in complex biological samples. This study shows the capability of independent component analysis–orthogonal signal deconvolution (ICA–OSD), an algorithm based on blind source separation and distributed in an R package called osd, to extract the spectra of the compounds appearing in GC×GC–MS chromatograms in an automated manner. We studied the performance of ICA–OSD by the quantification of 38 metabolites through a set of 20 Jurkat cell samples analyzed by GC×GC–MS. The quantification by ICA–OSD was compared with a supervised quantification by selective ions, and most of the R2 coefficients of determination were in good agreement (R2>0.90) while up to 24 cases exhibited an excellent linear relation (R2>0.95). We concluded that ICA–OSD can be used to resolve co-eluted compounds in GC×GC–MS.

Amplified Chromatographic Signal and UV-Absorbance Spectroscopy to Evaluate Lysozyme after Thermal Denaturation and in Contact Lens Extracts

Objective: Amplify the absorbance signal related to the lysozyme extracted from contact lenses as measured by high-performance liquid chromatography (HPLC); examine the purity of lysozyme and how much thermal denaturation affects its elution profile. Methods: In trial chromatographic runs during which contact lens extracts had been injected into the system, the fractions collected between 4 and 5.5 minutes pointed to lysozyme as the eluting protein as indicated by a Western blot. Proteins were extracted from contact lenses in a 50:50 solution of 0.2% trifluoroacetic acid (TFA): acetonitrile (ACN). Each contact lens extract was separated into 2 aliquots. After vacuum evaporation, aliquots no 1 were dissolved into the initial mobile phase to produce an enrichment factor of 8. Aliquots no 2 were left untreated in the regular extraction solution. Calibration of the absorbance signal at 220 nm allowed measuring the lysozyme levels in chromatograms. Parallel injections of both aliquots into the HPLC allowed comparing their lysozyme content. Purity of the lysozyme extracted was evaluated by viewing its absorbance spectrum across peaks. Lysozyme solutions, previously heated to 80 and 100°C were injected and compared with control solutions. Differences in median lysozyme content between aliquots no 1 and 2 and in peak area of lysozyme heated compared to control were tested with non-parametric methods. Results: Median lysozyme level measured in enriched and regular extracts differed significantly (39.8 and 21.5 μg, respectively). However, once corrected for different injection volume and concentrating factor, the mean enriched lysozyme level (21.4 μg) was close to the one found in the regular extract. Observation of spectral absorbance suggests that eluting lysozyme is free from contaminants. Median ratios of peak areas of heated lysozyme over peak area of control lysozyme differed significantly from 1 at the temperature of 100°C (0.91), but not at the one of 80°C (0.95) with the signed rank test. Even when heated at 80°C, the elution profile of lysozyme appeared less symmetrical compared to control and presented an additional inflection point. These subtle changes were increased at 100°C. Conclusion: Enrichment of the lysozyme extracted from contact lenses and solubilized into the initial mobile phase improves the sensitivity of this chromatographic procedure. This chromatographic protocol coupled with UV absorbance spectroscopy can detect thermal denaturation at 80 and 100°C.

Band target entropy minimization for retrieving the information of individual components from overlapping chromatographic data

Band target entropy minimization (BTEM) is a self-modeling curve resolution (SMCR) approach relying on non-negative criterion and minimization of Shannon entropy. In this study, BTEM algorithm was applied to retrieving the information of individual components from overlapping gas chromatography–mass spectrometry (GC–MS) data. The algorithm starts with dividing the whole data into bands along the retention time. In each band, singular value decomposition (SVD) is used to decompose the data into scores and loadings. Because the pure chromatographic signal possesses the lowest Shannon entropy, the chromatographic signal of each component can be constructed by optimizing the combination of the loadings with minimal Shannon entropy under non-negative criterion. To show the efficiency of the algorithm, a simulated four-component overlapping GC–MS data and an experimental GC–MS data of 18 organophosphorus pesticide mixture are investigated. The results show that both the chromatographic profiles and mass spectra of the components can be successfully extracted from the overlapping signals.

From molecular model to sparse representation of chromatographic signals with an unknown number of peaks.

Analysis of a fluid mixture using a chromatographic system is a standard technique for many biomedical applications such as in-vitro diagnostic of body fluids or air and water quality assessment. The analysis is often dedicated towards a set of molecules or biomarkers. However, due to the fluid complexity, the number of mixture components is often larger than the list of targeted molecules. In order to get an analysis as exhaustive as possible and also to take into account possible interferences, it is important to identify and to quantify all the components that are included in the chromatographic signal. Thus the signal processing aims to reconstruct a list of an unknown number of components and their relative concentrations. We address this question as a problem of sparse representation of a chromatographic signal. The innovative representation is based on a stochastic forward model describing the transport of elementary molecules in the chromatography column as a molecular random walk. We investigate three methods: two probabilistic Bayesian approaches, one parametric and one non-parametric, and a determinist approach based on a parsimonious decomposition on a dictionary basis. We examine the performances of these 3 approaches on an experimental case dedicated to the analysis of mixtures of the micro-pollutants Polycyclic Aromatic Hydrocarbons (PAH) in a methanol solution in two cases of high and low signal to noise ratio (SNR).

Broadening of polymer chromatographic signals: Analysis, quantification and correction through effective diffusion coefficients

Average molecular weights and polydispersity indexes are some of the most important parameters considered in the polymer characterization. Usually, gel permeation chromatography (GPC) and multi angle light scattering (MALS) are used for this determination, but GPC values are overestimated due to the dispersion introduced by the column separation. Several procedures were proposed to correct such effect usually involving more complex calibration processes.In this work, a new method of calculation has been considered including diffusion effects. An equation for the concentration profile due to diffusion effects along the GPC column was considered to be a Fickian function and polystyrene narrow standards were used to determine effective diffusion coefficients. The molecular weight distribution function of mono and poly disperse polymers was interpreted as a sum of several Fickian functions representing a sample formed by only few kind of polymer chains with specific molecular weight and diffusion coefficient.Proposed model accurately fit the concentration profile along the whole elution time range as checked by the computed standard deviation. Molecular weights obtained by this new method are similar to those obtained by MALS or traditional GPC while polydispersity index values are intermediate between those obtained by the traditional GPC combined to Universal Calibration method and the MALS method.Values for Pearson and Lin coefficients shows improvement in the correlation of polydispersity index values determined by GPC and MALS methods when diffusion coefficients and new methods are used.

A Novel Approach for Quantitation of Nonderivatized Sialic Acid in Protein Therapeutics Using Hydrophilic Interaction Chromatographic Separation and Nano Quantity Analyte Detection

This paper describes a novel approach for the quantitation of nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, and detection by Nano Quantity Analyte Detector (NQAD). The detection technique of NQAD is based on measuring change in the size of dry aerosol and converting the particle count rate into chromatographic output signal. NQAD detector is suitable for the detection of sialic acid, which lacks sufficiently active chromophore or fluorophore. The water condensation particle counting technology allows the analyte to be enlarged using water vapor to provide highest sensitivity. Derivatization-free analysis of glycoproteins using HPLC/NQAD method with PolyGLYCOPLEX™ amide column is well correlated with HPLC method with precolumn derivatization using 1, 2-diamino-4, 5-methylenedioxybenzene (DMB) as well as the Dionex-based high-pH anion-exchange chromatography (or ion chromatography) with pulsed amperometric detection (HPAEC-PAD). With the elimination of derivatization step, HPLC/NQAD method is more efficient than HPLC/DMB method. HPLC/NQAD method is more reproducible than HPAEC-PAD method as HPAEC-PAD method suffers high variability because of electrode fouling during analysis. Overall, HPLC/NQAD method offers broad linear dynamic range as well as excellent precision, accuracy, repeatability, reliability, and ease of use, with acceptable comparability to the commonly used HPAEC-PAD and HPLC/DMB methods.

СОСТАВ ЭКСТРАКТОВ ЛУБА КОРЫ БЕРЕЗЫ

Planned experiment was done with variation of three parameters: the concentration of alcohol KOH and liquid flow module. Extraction was carried out in a microwave field.The effect of alkali consumption and the concentration of ethanol in the extract yield bast birch bark in the alcohol-alkaline treatment. It is noted that reducing the concentration of ethyl alcohol and alkali consumption increases yield of extractives (EW) increases. It is shown that the larger the alkali is used in the processing of the feedstock, the increasing its content in the solid residue formed excessive alkali content in the phloem after extraction. It has been found that the amount of free alkali in the solid phase is significantly less than in the extract. No clear relationship alkali content in the extracts from the amount of the alkali used for the experiments. Get adequate regression equations of the alkali content in the extract and a solid residue.Study the chemical composition of an alcohol-alkali bast birch bark extracts obtained after microwave extraction. A selective analysis of the obtained extracts. Standard chromatograms obtained mixture phenolic compounds and one of the extracts of the samples. Presented calibration dependence of the integral intensity of the chromatographic signal (S) the concentration c (mg / dm3) of analytes. Results of investigation of the compounds detected in the content of the extracts.

Analysis of cave atmospheres by comprehensive two-dimensional gas chromatography (GC×GC) with flame ionization detection (FID)

In this paper, we describe a simple method for sampling, pre-concentrating, and separating volatile and semi-volatile components from two different cave atmospheres. Sampling is performed by capturing a volume of cave atmosphere in a Tedlar bag or Suma canister for sample storage and transport back to the laboratory. Loading a portion of the sample on a multi-bed sorption trap allows for sample pre-concentration prior to separation and detection of components on a comprehensive two-dimensional gas chromatograph (GC×GC). Comparison of two Texas caves reveals the power of comprehensive two-dimensional gas chromatography (GC×GC) for volatile separation and detection, and to our knowledge marks the first use of GC×GC for the analysis of cave atmospheres. Analysis of the results revealed 138 and 146 chromatographic signals over an S/N threshold of 500 and direct comparison of the two samples revealed 50 identical chromatographic signals. This study is a first step toward demonstrating the ability of GC×GC to separate the complex volatiles and semi-volatiles in the cave atmosphere as a fingerprinting tool.

Application of Fourier transform to preprocessing chromatographic fingerprints of traditional Chinese medicine

Retention time shift is a significant impediment against analyzing chromatographic fingerprints of Traditional Chinese Medicine (TCM) with similarity estimation and pattern recognition methods. This work aims at developing a fingerprint preprocessing method that can eliminate the negative effect of retention time shift on similarity estimation and pattern recognition analysis in the quality evaluation of TCM. Both simulated chromatograms and experimental chromatographic fingerprints of Radix Puerariae thomsonii (RPT) were investigated in order to evaluate the practicality of the proposed method. Fourier transform (FT) was used to process these chromatographic signals. The magnitude data of these signals were calculated. In the obtained magnitude data, the negative effect of retention time shift was successfully excluded. Thus, the quality of the investigated RPT samples can be correctly evaluated by using the magnitude data as the input variables of similarity estimation and pattern recognition analysis. It is demonstrated that FT is a practicable and promising method for preprocessing chromatographic fingerprints of TCM prior to similarity estimation and pattern recognition analysis.

Evaluation of mobile phase characteristics on three zwitterionic columns in hydrophilic interaction liquid chromatography mode for liquid chromatography-high resolution mass spectrometry based untargeted metabolite profiling of Leishmania parasites

It has been reported that HILIC column chemistry has a great effect on the number of detected metabolites in LC–HRMS-based untargeted metabolite profiling studies. However, no systematic investigation has been carried out with regard to the optimisation of mobile phase characteristics. In this study using 223 metabolite standards, we explored the retention mechanisms on three zwitterionic columns with varied mobile phase composition, demonstrated the interference from poor chromatographic peak shapes on the output of data extraction, and assessed the quality of chromatographic signals and the separation of isomers under each LC condition. As expected, on the ZIC-cHILIC column the acidic metabolites showed improved chromatographic performance at low pH which can be attributed to the opposite arrangement of the permanently charged groups on this column in comparison with the ZIC-HILIC column. Using extracts from the protozoan parasite Leishmania, we compared the numbers of repeatedly detected LC–HRMS features under different LC conditions with putative identification of metabolites not amongst the standards being based on accurate mass (±3ppm). Besides column chemistry, the pH of the mobile phase plays a key role in not only determining the retention mechanisms of solutes but also the output of the LC–HRMS data processing. Fast evaporation of ammonium carbonate produced less ion suppression in ESI source and consequently improved the detectability of the metabolites in low abundance in comparison with other ammonium salts. Our results show that the combination of a ZIC-pHILIC column with an ammonium carbonate mobile phase, pH 9.2, at 20mM in the aqueous phase or 10mM in both aqueous and organic mobile phase components, provided the most suitable LC conditions for LC–HRMS-based untargeted metabolite profiling of Leishmania parasite extracts. The signal reliability of the mass spectrometer used in this study (Exactive Orbitrap) was also investigated.

Chemometric strategy for automatic chromatographic peak detection and background drift correction in chromatographic data

Peak detection and background drift correction (BDC) are the key stages in using chemometric methods to analyze chromatographic fingerprints of complex samples. This study developed a novel chemometric strategy for simultaneous automatic chromatographic peak detection and BDC. A robust statistical method was used for intelligent estimation of instrumental noise level coupled with first-order derivative of chromatographic signal to automatically extract chromatographic peaks in the data. A local curve-fitting strategy was then employed for BDC. Simulated and real liquid chromatographic data were designed with various kinds of background drift and degree of overlapped chromatographic peaks to verify the performance of the proposed strategy. The underlying chromatographic peaks can be automatically detected and reasonably integrated by this strategy. Meanwhile, chromatograms with BDC can be precisely obtained. The proposed method was used to analyze a complex gas chromatography dataset that monitored quality changes in plant extracts during storage procedure.

Rapid determination of amino acids in ginseng by high performance liquid chromatography and chemometric resolution

Ginseng is one of the most important traditional Chinese medicines and functional foods. A method for the fast determination of amino acids in ginseng samples using high performance liquid chromatography(HPLC) was developed, in which strong isocratic elution was employed for simplifying the separation and speeding up the analysis. All amino acids were eluted within 3 min with the chromatogram composed of overlapped peaks from the interferences. Then, non-negative immune algorithm(NNIA) was adopted to resolve the chromatographic signals of the components from the chromatogram measured. The results show that the signals of the amino acids can be correctly extracted by NNIA and the signal extracted can be used for the quantitative analysis. The method was validated via determining six amino acids of four different samples of ginseng. The recoveries of the spiked samples are in a range of 96.6%–106.3%.

The Importance of Detector Acquisition Rate in Comprehensive Two-Dimensional Gas Chromatography (GC×GC)

The use of comprehensive two-dimensional gas chromatography (GC×GC) is becoming more popular for complex sample analysis and the optimization of this technique is paramount for accurate qualitative and quantitative reporting. In this work, the effect of detector data acquisition rate (or detector sampling speed) for GC×GC is investigated and summarized. Effects on chromatographic signal such as peak area, height, and width, as well as analytical figures of merit such as resolution and limit of detection, are investigated at four data acquisition rates using a multi-component test mixture. Adjusting the data acquisition rate of the detector has a profound effect on peak areas, heights, and peak widths. In addition, the increase in detected signal and decreased peak widths led to improved chromatographic resolution and limits of detection (LOD) under optimized conditions.