Resolution Of Chromatographic Peaks Research Articles

Utilizing derivatizing agents for the differentiation of cannabinoid isomers in complex food, beverage and personal-care product matrices by ambient ionization mass spectrometry

Recent legalization and decriminalization of marijuana at the state level has not only contributed to a rise in the recreational use of Cannabis sativa, also known simply as Cannabis, but also to an increase in the range of matrices into which cannabinoids derived from it are infused. Traditional methods for analyzing these products, which are typically chromatography-based, are often matrix-dependent and demand time-consuming and resource-intensive sample preparation protocols that are highly nuanced and not readily applicable to multiple matrix types. Furthermore, the differentiation of cannabinoids can be troublesome without implementing lengthy run times to achieve resolution of chromatographic peaks. With complex samples such as edibles, beverages, personal-care products, and plant materials, a method that can be more universally applied to rapidly detect and differentiate between cannabinoids is highly desirable. In this study, foods and personal-care products under the categories of sweets, spreads, condiments/toppings, beverages, oils, and commercial body products were spiked with cannabinoids including Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabicitran (CBT), and cannabigerol (CBG). Chemical derivatization of the samples with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) followed by direct analysis in real time – high-resolution mass spectrometry (DART-HRMS) analysis readily revealed the presence of cannabinoids in the products despite the matrix complexity (i.e., the contributions from matrix-derived peaks did not interfere with the differentiation of cannabinoids). When analyzed under ambient soft ionization conditions, CBT, THC and CBD are indistinguishable because they are isomers with a molecular formula of C21H30O2 and a protonated monoisotopic mass of 315.2324. However, due to the different number of hydroxyl (-OH) groups (zero in CBT, one in THC, and two in CBD) that engage with the derivatizing agent, the cannabinoids are differentiated based on the mass disparities of the protonated adducts formed (m/z 315.2324, 387.2719 and 459.3119 for CBT, THC, and CBD, respectively), which is readily revealed by DART-HRMS. This approach circumvents some of the challenges currently encountered by forensic laboratories in the analysis of highly complex and diverse cannabinoid-infused matrices. The results show proof-of-concept for an approach that can aid in the differentiation of cannabinoid isomers by DART-HRMS that accommodates high matrix diversity and complexity, and demonstrates the potential for the approach to be integrated into current workflows for the forensic analysis of Cannabis-related materials and evidence.

Beyond helium: hydrogen as a carrier gas in multiresidue pesticide analysis in fruits and vegetables by GC-MS/MS.

In this comprehensive study, we evaluated the feasibility of using hydrogen instead of helium as a carrier gas in a GC-MS/MS system for pesticide residue analysis, spanning three matrices: pepper, tomato, and zucchini. Initial assessments focused on the ion source's chemical inertness, employing nitrobenzene as a benchmark to monitor the hydrogenation process. A method with a duration of less than 12 minutes was developed, achieving good chromatographic peak resolution attributable to the enhanced chromatographic performance of hydrogen as a carrier gas. The study emphasized the optimization of system parameters, testing various ion source temperatures, detector voltages, and injection volumes. Sensitivity assessments, based on the DG-SANTE criteria, indicated that the majority of compounds were identifiable at a concentration of 5 μg kg-1 (81% in tomato, 84% in pepper and 73% in zucchini). Detailed validation for reproducibility, matrix effects, and linearity across 150 pesticides unveiled generally favorable outcomes, with a notable majority of compounds displaying low matrix effects, satisfactory linearity ranges and good reproducibility with most compounds returning a relative standard deviation (RSD) below 10%. When applied to 15 real samples, the hydrogen-based system's performance was juxtaposed against a helium-based counterpart, revealing that results are very comparable between both systems. This comparative approach highlights hydrogen's potential as a reliable and efficient carrier gas in pesticide residue analysis for routine food control laboratories, overcoming difficulties resulting from the lack of helium supplies.

Liquid Chromatographic Separation of MM 302 Da Polycyclic Aromatic Hydrocarbons in Synthetic Mixtures Using Chiral Stationary Phase Material

High molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) are an example of a family of compounds that exist with isomeric mixtures. While multi-instrument techniques may isolate many isomeric PAHs at qualitative and quantitative levels, complete separation of this class of environmental pollutant has not yet been attained, leaving ample room for method improvement. In this research, the separation of 21 commercially available isomers of MM 302 Da was studied using polysaccharide-based chiral immobilized materials as stationary phase. Isocratic, normal phase runs were performed using mixed concentrations and combinations of 302 Da PAH isomers. Retention times and calibration data were determined using a chiral chromatographic setup. Selectivity values are given for all the evaluated HMW-PAH isomers, retention and intensity factors were also calculated based on the directions outlined in Environmental Protection Agency Method 550.1, along with chromatographic peak resolution values. With the use of chiral separation, a total of 16 HMW-PAH peaks are visible on the chromatogram of the 21 MM 302 Da PAH synthetic mixture. This group of molecules does not possess chiral centers in the classical sense; however, larger molecules such as these may possess pseudochiral characteristics, providing a little-researched opportunity for separation strategy of formerly considered achiral molecules to be separated using chiral stationary phases. The resolution of this method makes it suitable for the identification and quantitation of MM 302 Da PAH isomers fractionated from a larger complex sample. The potential for this approach with other classes of high molecular weight achiral molecules is yet to be explored.

Single-Standard Quantification Strategy for Lignin Dimers by Supercritical Fluid Chromatography with Charged Aerosol Detection.

The increased interest in utilizing lignin as a feedstock to produce various aromatic compounds requires advanced chemical analysis methods to provide qualitative and quantitative characterization of lignin samples along different technology streamlines. However, due to the lack of commercially available chemical standards, routine quantification of industrially relevant lignin oligomers in complex lignin samples remains a challenge. This study presents a novel method for universal quantification of lignin dimers based on supercritical fluid chromatography with charged aerosol detection (CAD). A series of lignin-derived dimeric compounds that have been reported from reductive catalytic fractionation (RCF) were synthesized and used as standards. The applicability of using linear regression instead of quadratic calibration curves was evaluated over a concentration range of 15-125 mg/L, demonstrating that the former calibration method is as appropriate as the latter. The response factors of lignin dimeric compounds were compared to assess the uniformity of the CAD signal, revealing that the CAD response for the tested lignin dimers did not differ substantially. It was also found that the response factors were not dependent on the number of methoxy groups or linkage motifs, ultimately enabling the use of only one calibrant for these compounds. The importance of chromatographic peak resolution in CAD was stressed, and the use of a digital peak sharpening technique was adopted and applied to address this challenge. The developed method was verified and used for the quantification of lignin dimers in an oil obtained by a RCF of birch sawdust.

Detection of four biogenic amines by liquid chromatography based on aptamer signal replacement combined with cyclic amplification

Biogenic amines (BAs) represent a class of potentially harmful substances in foods and medicines. Their content is thus an important indicator of proper hygiene in food preparation, and purity of medicines. It is of great practical significance to establish accurate and sensitive detection of BAs in food and drugs. In this study, a high performance liquid chromatography (HPLC) method was developed for the simultaneous detection of multiple BAs in fish, pork and antibiotics based on aptamer signal replacement and cyclic amplification strategy. First, non-fluorescent targets were converted into fluorescent nucleic acid probes using a two-step replacement process. Subsequently, a large number of nucleic acid probes with different lengths and base sequences were generated using a double-stranded specific nuclease-assisted signal amplification strategy. Finally, various BAs in real samples were accurately identified using an HPLC platform. The influence of base sequence and nucleic acid probe length on separation via HPLC was studied to improve discrimination among fluorescent signals. Four different sequences were selected as tails to the DNA probe, and their retention times increased in turn. Experimental conditions, including column temperature, flow rate, gradient elution process, reaction temperature, and incubation time, were optimized by orthogonal experiments to further improve signal separation efficiency. Specifically, the methanol gradient was changed from 10% to 20% during 0-20 min, 35 ℃ of column temperature and 1.0 mL/min of flow rate were chosen as the HPLC conditions. The final resolution of chromatographic peaks was 3.44, 3.59 and 2.37, indicating complete separation between peaks. Optimal incubation time for BA capture by aptamer was 120 min, and optimal dosage of duplex specific nuclease (DSN) and Mg2+were 0.9 U and 30 mmol/L. The optimal pH, incubation temperature, and DSN incubation time were 7.0, 40 ℃ and 210 min, respectively. The proposed method exhibited high sensitivity towards BAs, with a linear range of 1 pmol/L-1 μmol/L, and the limits of detection of tyramine, histamine, spermine, and tryptamine were 0.25, 0.21, 0.27 and 0.19 pmol/L, respectively. The feasibility of this method was verified, and contrast experiments indicated that it could achieve highly selective detection of four BAs in one run. The applicability of this integrated method was also investigated for the detection of real samples (gentamycin sulfate, fish and pork). To assess the matrix effect, each BA with different concentrations were spiked into real fish and pork samples. Relative recoveries and relative standard deviations (RSDs) ranged from 101.2% to 104.5% and from 1.5% to 4.3%, respectively. The above detection results for real samples showed that this method could accurately capture, separate, and identify BAs in complex matrix samples. This strategy can effectively improve analyte selectivity and reduce the matrix effect. This assay is thus expected to provide a new approach for food and drug analyses.

Simultaneous Determination of the Five Constituents in Maiwei Dihuang Pills by the HPLC-DAD Method.

Objective The purpose of study is to establish an HPLC-DAD method for determination of the five constituents (deoxyschizandrin, γ-schizandrin, loganin, paeoniflorin, and paeonol) in Maiwei Dihuang Pills. Methods An Agilent ZORBAX SB-C18 chromatographic column was carried out to determine the five constituents of 50% methanol extract of Maiwei Dihuang Pills. Results It was found the chromatographic peak resolution of each component in the study sample solution was 1.5 higher than that of other peaks and no peaks appeared in the blank control solution during the same time, suggesting specificity of HPLC-DAD was well established. The linearity test indicated that deoxyschizandrin, γ-schizandrin, loganin, paeoniflorin, and paeonol were 11.6–72.3 μg/mL, 6.4–45.2 μg/mL, 35.2–237.6 μg/mL, 18.1–114.2 μg/mL, and 32.2–215.3 μg/mL, respectively, suggesting each component has a good linear relationship within its own range. Additionally, the precision of HPLC-DAD was confirmed by a precision test; the stability of the study sample solution was confirmed by a stability test; and good reproducibility of HPLC-DAD was proved by a reproducible test. The recovery rate test showed that relative standard deviation (RSD) of recovery rate in deoxyschizandrin, γ-schizandrin, loganin, paeoniflorin, and paeonol was 100.26% (1.80%), 101.39% (1.74%), 101.19% (1.76%), 102.50% (1.65%), and 102.30% (1.58%), respectively. Conclusions HPLC-DAD used to determine the five constituents in Maiwei Dihuang Pills, and it was easier and faster to operate, showing good condition in repeatability, precision, stability, and recovery, which is a great option for quality control.

Simultaneous determination of methanol, ethanol and isopropanol in human blood and white spirit samples

A simple and cost-effective method for simultaneous determination of methanol, ethanol, and isopropanol in human blood and white spirit samples using headspace gas chromatography coupled with flame ionization detection (HS-GC-FID) was developed and validated for clinical and toxicological purposes. In this study, the headspace sampling procedure was investigated, indicating optimized temperature of 70 °C in 20 min. Concentrations of target compounds were determined by internal standard (IS) method with acetonitrile as IS compound. A good resolution of chromatographic peaks was achieved. The linear ranges for all the compounds were from 1 to 500 mg/dL. Our method was validated with adequate accuracy (recovery > 98%) and precision (RSD < 0.1%) in whole human blood and white spirit samples. The limits of detection were below 0.5 mg/L for the three compounds. This method is easy to perform, making it suitable for routine analysis in clinical biochemistry and forensic laboratories.

Improving peak capacities over 100 in less than 60 seconds: operating above normal peak capacity limits with signal processing.

A primary focus in liquid chromatography analysis of complex samples is high peak capacity separations. Using advanced instrumentation and optimal small, high-efficiency columns, complex multicomponent mixtures can now be analyzed in relatively short times. Despite these advances, chromatographic peak overlap is still observed. Recently, attention has shifted from improvements in chromatographic efficiency and selectivity to enhancing data processing after collection. Curve fitting methods can be used to trace underlying peaks, but do not directly enhance chromatographic resolution. Methods based on the properties of derivatives and power transform were recently shown to enhance chromatographic peak resolution while maintaining critical peak information (peak areas and retention times). These protocols have been extensively investigated for their fundamental properties, advantages, and limitations, but they have not been evaluated with complex chromatograms. Herein, we evaluate the use of deconvolution via Fourier transform (FT), even-derivative peak sharpening, and power law with the fast separation (< 60s) of a 101-component mixture using ultra-high-pressure liquid chromatography. High noise and peak overlap are present in this gradient separation, which is representative of fast chromatography. Chromatographic resolution enhancement is demonstrated and described. Further, accurate quantitation is maintained and shown with representative examples. Enhancements in peak capacity and peak-to-peak resolutions are discussed. Finally, the statistical theory of overlap is used for 101 peaks and predictions are made for the number of singlet, doublet, and multiplets analyte peaks. The effect of increasing peak capacity by FT even derivative sharpening and power laws leads to a decrease in the number of peak overlaps and an increase in total peak number. Graphical abstract.

Efficient synthesis and characterisation of the amyloid beta peptide, Aβ1-42, using a double linker system.

The amyloidogenic Aβ42 peptide was efficiently prepared using a double linker system, markedly improving solubility and chromatographic peak resolution, thus enabling full characterisation using standard techniques. The tag was readily cleaved with sodium hydroxide and removed by aqueous extraction, affording Aβ42 in high purity and yield for biophysical characterisation studies.

Separation of β-Blocker Enantiomers on Silica Modified with Gold Nanoparticles with Immobilized Macrocyclic Antibiotic Vancomicin

A new chiral sorbent based on mercaptosilica modified by gold nanoparticles, then treated by 3-mercaptopropionic acid and macrocyclic antibiotic vancomycin, was obtained. The enantioseparation of isomeric β-blockers (nadolol, atenolol, metoprolol, alprenolol, oxprenolol, and pindolol) by HPLC was studied on the synthesized sorbent. The effect of the composition of the mobile phase (nature and concentration of the organic solvent, concentration and pH of the buffer solution) on retention times of β-blocker enantiomers, selectivity of separation, and resolution of chromatographic peaks were studied. The best separation was achieved for pindolol and metoprolol. The procedure was used to determine the pindolol enantiomers in the preparation “Visken,” and metoprolol in the “Vazocardin” preparation.

Effect of MnCl2 deposition content on the textural properties of activated alumina and its elution performance for hydrogen isotopes

By a solution impregnation method, three modified stationary phases were prepared by contacting activated alumina with MnCl2 at different MnCl2:Al2O3 molar ratios = 1:10, 1:5, 1:2.5, respectively. N2 sorption isotherms at 78 K were examined for the unmodified and modified samples, from which pore size distributions (PSD) of textural properties were derived with density function theory. Elution chromatographic tests of hydrogen isotopes were carried out at 78 K. The results indicated that both the microporous specific surface area (SSA) and the mesoporous SSA decline after modification, while the microporous SSA remains nearly unvaried among the modified ones. The chromatographic retention times as well as the bandwidths decline as the Mn content rises. The resolution of chromatographic peaks increases after modification, it remains unvaried and then decreases as the Mn content increases. The PSD of alumina SSA changed by the modification significantly influences the hydrogen isotopic elution chromatography.

Evaluation of changes induced in rice metabolome by Cd and Cu exposure using LC-MS with XCMS and MCR-ALS data analysis strategies.

The comprehensive analysis of untargeted metabolomics data acquired using LC-MS is still a major challenge. Different data analysis tools have been developed in recent years such as XCMS (various forms (X) of chromatography mass spectrometry) and multivariate curve resolution alternating least squares (MCR-ALS)-based strategies. In this work, metabolites extracted from rice tissues cultivated in an environmental test chamber were subjected to untargeted full-scan LC-MS analysis, and the obtained data sets were analyzed using XCMS and MCR-ALS. These approaches were compared in the investigation of the effects of copper and cadmium exposure on rice tissue (roots and aerial parts) samples. Both methods give, as a result of their application, the whole set of resolved elution and spectra profiles of the extracted metabolites in control and metal-treated samples, as well as the values of their corresponding chromatographic peak areas. The effects caused by the two considered metals on rice samples were assessed by further chemometric analysis and statistical evaluation of these peak area values. Results showed that there was a statistically significant interaction between the considered factors (type of metal of treatment and tissue). Also, the discrimination of the samples according to both factors was possible. A tentative identification of the most discriminant metabolites (biomarkers) was assessed. It is finally concluded that both XCMS- and MCR-ALS-based strategies provided similar results in all the considered cases despite the completely different approaches used by these two methods in the chromatographic peak resolution and detection strategies. Finally, advantages and disadvantages of using these two methods are discussed. Graphical Abstract Summary of the workflow for untargeted metabolomics using the compared approaches.

Development of a novel pyrolysis-gas chromatography/mass spectrometry method for the analysis of poly(lactic acid) thermal degradation products

Thermal degradation of PLA is a complex process since it comprises many simultaneous reactions. The use of analytical techniques, such as differential scanning calorimetry (DSC) and thermogravimetry (TGA), yields useful information but a more sensitive analytical technique would be necessary to identify and quantify the PLA degradation products. In this work the thermal degradation of PLA at high temperatures was studied by using a pyrolyzer coupled to a gas chromatograph with mass spectrometry detection (Py-GC/MS). Pyrolysis conditions (temperature and time) were optimized in order to obtain an adequate chromatographic separation of the compounds formed during heating. The best resolution of chromatographic peaks was obtained by pyrolyzing the material from room temperature to 600°C during 0.5s. These conditions allowed identifying and quantifying the major compounds produced during the PLA thermal degradation in inert atmosphere. The strategy followed to select these operation parameters was by using sequential pyrolysis based on the adaptation of mathematical models. By application of this strategy it was demonstrated that PLA is degraded at high temperatures by following a non-linear behaviour. The application of logistic and Boltzmann models leads to good fittings to the experimental results, despite the Boltzmann model provided the best approach to calculate the time at which 50% of PLA was degraded. In conclusion, the Boltzmann method can be applied as a tool for simulating the PLA thermal degradation.

Development and Validation of a Simple and Efficient RPLC Method for Analysis of Captopril, Metformin, Pioglitazone and Glibenclamide in API, Formulations and Human Serum

The association between the use of ACE inhibitors and the incidence of hypoglycemia is controversial. A recent study reported that 14% of all hospital admissions for hypoglycemia might be attributable to ACE inhibitors. In this paper, a novel, precise, specific, accurate and rapid reversed-phase high performance liquid chromatographic method was developed, optimized and validated for determining captopril and hypoglycemic (metformin, pioglit azone and glibenc lamide) in bulk, pharmaceutical formulations and human serum with the best chromatographic peak resolution, reduced run time and low cost of analysis. The method was validated according to the US Food and Drug Administration (FDA) and ICH guidelines for the parameters: specificity, stability, limits of detection (LLOD), limits of quantification (LLOQ), linearity, accuracy, precision and recovery. This method showed the best resolution by using Hypersil ODS,C18 (150×4.6 mm, 5 micron) column using mobile phase, methanol: water (70: 30 v/v) adjusted to pH 3 via ortho phosphoric acid 85% with flow rate of 1 mLmin -1 at ambient temperature and wavelength of 230 nm. The signal-to-noise ratio (S/N) was employed as a quality measurement. This tool permits to establish the influence of some selected factors (methanol: water ratio, pH, and flow rate) on two responses (peak areas and retention time). The LLOD and LLOQ values for CAP, MET, PGL and GLB were found to be 2.3, 1.5, 2.3 and 2.3 and 0.7, 0.4, 0.7, and 0.7 μgmL-1 respectively. Calibration curves were linear in the concentration range of 2.5-100 μgmL -1 for hypoglycemic and captopril with regression coefficient (r 2 ) value of 0.999 for all drugs. The data for accuracy, precision and recovery were within the FDA limits. Intra ad inter-day precision and accuracy results were 98.0 to 102%. Retention time for captopril was found to be 3.3 minute and for metformin, pioglitazone and glibenclamide 2.4, 2.8, 7.2 minutes respectively. Proposed method was selective, precise and accurate short time analysis therefore can be used for routine, quality control and clinical study. This is the first full report of a method for the simultaneous determination of these four drugs: captopril , metformin, pioglita zone and glibenclamide in API, formulations and serum. The newly developed method is useful for future routine analysis of these drugs and could be used in therapeutic drug monitoring and adherence to medicine studies, which would be helpful in decision making regarding treatment change in combination therapies.

Development and validation of a high performance chromatographic method for determining sumatriptan in niosomes

In this paper, a novel, precise, specific, accurate and rapid reversed-phase high performance liquid chromatographic method was developed, optimized and validated for determining sumatriptan succinate in niosomes with the best chromatographic peak resolution, reduced run time and low cost of analysis. The formulation has been previously optimized in terms of composition and preparation technique to obtain a high drug encapsulation efficiency and adequate vesicle size distribution.This method showed the best resolution by using Spherisorb® OSD2 C18 column (250mm×4.6mm, 5μm) using phosphate buffer (0.05M):acetonitrile (80:20, v/v; pH adjusted to 6.0) as a mobile phase at a flow rate of 1mL/min and wavelength of 214nm.The main objective of this research was to demonstrate the robustness of the reversed-phase HPLC method development by applying the Taguchi robust methodology. The signal-to-noise ratio (S/N) was employed as a quality measurement. This tool permits to establish the influence of some selected factors (acetonitrile:phosphate ratio, pH buffer, oven temperature and flow rate) on two responses (peak areas and retention time). On the basis of the results obtained, we can conclude that this analytical method was robust for all the factors studies, as exception of the flow rate, where the higher quality was obtained for the fewer values (0.8mL/min). Therefore, this parameter must be carefully controlled when this method was employed, to avoid any modification in the peak areas overall.

Biotransformation of flavonols and taxifolin in hepatocyte in vitro systems as determined by liquid chromatography with various stationary phases and electrospray ionization-quadrupole time-of-flight mass spectrometry

Liquid chromatography (LC) on various stationary phases was used for the metabolite profile analysis of quercetin, rutin, isoquercitrin and taxifolin. The metabolites were obtained using an in vitro model system of human and rat hepatocytes in the form of cell suspensions and the primary cultures. For separations of the parent compounds and their metabolites, stationary phases based on C18, C8, cyanopropyl (CNP) or phenyl (PHE) modifications of silica were tested. CNP and PHE stationary phases operating in reversed-phase mode have been shown to be efficient for separation of parent flavonoids and their polar metabolites. Individual metabolites were identified on the basis of an elemental composition determination using electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) on-line connected with an LC system. Detailed analytical parameters such as retention times, selectivity, resolution of chromatographic peaks, MS fragmentation and UV–vis absorption maxima were determined for individual metabolites, namely for phase II biotransformation products. The predominant metabolites were methylated flavonols and flavonol glucuronides. The highest biotransformation rate was found with taxifolin, which was mainly converted to sulfates. The HPLC/ESI-QqTOF MS analyses revealed that quercetin and taxifolin were metabolized more extensively than the studied glycosides, rutin and isoquercitrin.

A method for dextruxin analysis by HPLC-PDA-ELSD-MS

Destruxins (Dtx) are cyclodepsipeptides produced by enthomopathogenic fungi, which are used in biological control of different agricultural insect plagues. The present investigation reports a new approach for analysis of destruxins produced by the fungal strain Beauveria felina, using LC-PDA-ELSD-MS. Compared to previous methods, the new approach uses a clean-up on C18 cartridges, which effectively removes growth media constituents. Moreover, the use of 50:50 (v/v) MeCN/MeOH as the strongest eluting solvent in a gradient system over 0.1% H2O proved to give a better resolution of chromatographic peaks. Simultaneous detection using photodiode array (PDA), evaporative light scattering detector (ELSD) and mass spectrometry (MS) indicated a practically identical response of all detectors for destruxins analysis. Five samples obtained from the culture media of B. felina were analysed, and indicated the presence of twenty known destruxins and six yet unreported cyclodepsipeptides. Considering the reduced use of MeCN, and the effectiveness of ELSD as a detector for destruxins, the method proved to be valuable and cost-effective for quality control analysis of destruxin-producing fungal strains.

Quantitative and enantioselective analysis of monoterpenes from plant chambers and in ambient air using SPME

Abstract. A headspace solid-phase microextraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS) system has been developed for quantifying enantiomeric and nonenantiomeric monoterpenes in plant chamber studies and ambient air. Performance of this system was checked using a capillary diffusion system to produce monoterpene standards. The adsorption efficiency, competitive adsorption and chromatographic peak resolution of monoterpene enantiomer pairs were compared for three SPME fibre coatings: 75 μm Carboxen-PDMS (CAR-PDMS), 50/30 μm divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS) and 65 μm divinylbenzene-polydimethylsiloxane (DVB-PDMS). Key parameters such as the linearity and reproducibility of the SPME system have been investigated in this work. The best compromise between the enantiomeric separation of monoterpenes and competitive adsorption of the isoprenoids on the solid SPME fibre coating was found for DVB-PDMS fibres. The optimum conditions using DVB-PDMS fibres were applied to measure the exchange rates of monoterpenes in the emission of Quercus ilex using a laboratory whole plant enclosure under light and dark conditions, as well as in ambient air. With 592 and 223 ng m−2 s−1 respectively, β-myrcene and limonene were the predominant monoterpenes in the emission of Q. ilex. These values were closely comparable to those obtained using a zNose and cartridge GC-FID systems.

Validation of HPLC method for determination of tetracycline residues in chicken meat and liver

High-performance liquid chromatographic method with diode-array detection (HPLC–DAD) was optimised and validated for determination of tetracyclines (TCs) residue in chicken meat and liver through evaluating each step of various methods. The principle steps involved ultrasonic-assisted extraction of TCs from chicken samples by 2 ml of 20% trichloroacetic acid and citrate buffer (pH 4) which gave a clearer supernatant and high recovery, followed by centrifugation and purification on SPE (Strata C18-E cartridge) using 10 ml of 0.01 M methanolic oxalic acid for TCs elution. Separation was on reversed-phase column (Nuclosil 100 C18, 25 cm × 4.6 mm ID, 5 μ) by multisteps gradient elution which provided a better chromatographic peak resolution and the late eluting peaks were as sharp as those eluting earlier. Monitoring was at 351 nm which gave a higher detector response factor. Validity study of the method revealed that all obtained calibration curves showed good linearity ( r 2 > 0.999) over the range of 50–5000 ng. Sensitivity was found to be 1.44, 1.90, 0.95 and 1.23 ng for OTC, TC, CTC and DC, respectively. Accuracy was in the range of 71.88–92.44.3% and 68.88–84.84% for meat and liver, respectively. Precision was lower than 10% in all cases indicating that the method can be used as a validated method. Limit of detection (LOD) was found to be 4.4, 5, 13 and 10 ng for OTC, TC, CTC and DC, respectively. The corresponding values of limit of quantitation (LOQ) were 10, 13, 27 and 22 ng.

Protein-loaded PLGA microparticles engineered by flow focusing: Physicochemical characterization and protein detection by reversed-phase HPLC

In the present study, a novel synthesis technique based on the flow focusing (FF) technology is investigated for the preparation of green fluorescent protein (GFP)-loaded poly( d, l-lactide- co-glycolide) (PLGA) microparticles. To our knowledge, this novel technique has never been applied to the formulation of proteins in polymeric systems. A simple, specific and rapid reversed-phase HPLC (RP-HPLC) method was validated for the determination of GFP in PLGA microparticles with the best chromatographic peak resolution, reduced run time and low cost of analysis. In order to achieve the finest GFP-loaded polymeric particles, experimental parameters mainly associated to the FF device were studied (liquid flow rate and pressure of the focusing air). Very high GFP encapsulation values (>90%) were obtained by this technique, and the electrokinetic characterization of these systems suggested that this protein was incorporated into the polymeric matrix. This study is intended to offer information on which to base the development of high molecular weight protein-loaded polymeric delivery systems prepared by FF.