C18 Separation Research Articles

Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.

We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs. These pairs were generated by spontaneous degradation, chemical or enzymatic treatment, analysis of synthetic peptides, or the cotranslational incorporation of noncanonical proline analogues. In addition, these measurements were validated using external data acquired for synthetic peptides and enzymatically induced citrullination. Working in units of hydrophobicity index (HI, % ACN) and evaluating the average retention shifts (ΔHI) represent the simplest approach to describe the effect of modifications from a didactic point of view. Plotting HI values for modified (y-axis) vs nonmodified (x-axis) counterparts generates unique slope and intercept values for each modification defined by the chemistry of the modifying moiety: its hydrophobicity, size, pKa of ionizable groups, and position of the altered residue. These composition-dependent correlations can be used for coarse incorporation of PTMs into models for prediction of peptide retention. More accurate predictions would require the development of specific sequence-dependent algorithms to predict ΔHI values.

Acetic Acid Ion Pairing Additive for Reversed-Phase HPLC Improves Detection Sensitivity in Bottom-up Proteomics Compared to Formic Acid.

Despite the general acceptance of formic acid as the additive of choice for peptide reversed-phase LC-MS/MS applications, some still argue that the selection of acetic acid represents a better option. To settle this debate, we investigated both the difference in MS sensitivity and chromatographic behavior of peptides between these two systems. This interlaboratory study was performed using different MS setups and C18 separation media employing both 0.1% formic and 0.5% acetic acid as ion pairing modifiers. Relative to formic acid, we find an overall ∼2.2-2.5× increase in MS signal and a slight decrease in RP LC retention (-0.7% acetonitrile on average) for acetic acid conditions. While these two features have opposing effects on peptide detectability, we find that acetic acid produces up to 60% higher peptide ID output depending on the type of sample. The drop in RPLC retention increases with peptide net charge at acidic pH. MS signal is dependent on the difference between the charge of the precursor ion and the charge of the peptide in solution, favoring species with a low pI. Lower peptide retention under acetic acid conditions demonstrates its higher hydrophilicity and, as expected, leads to composition and sequence-dependent character of the observed retention shift.

A highly sensitive and selective method for the development and validation of potential genotoxic impurity in Raltegravir by high-throughput UPLC-MS/MS

Raltegravir is a human immunodeficiency virus (HIV) integrase inhibitor, a novel anti-AIDS drug. To quantify genotoxic impurities (GTIs), conventional analysis techniques are inadequate as a result, there is a high need to develop critical analytical methods for drug analysis using the hyphenated analytical technique. A highly sensitive and selective method was developed and validated for the estimation of genotoxic impurity in Raltegravir Active pharmaceutical ingredient (API) by using UPLC-MS/MS. The UPLC-MS/MS analysis for the quantification of GTI was performed on the symmetry C18 (150 mm×4.6 mm,3.5μm) analysis column and separation of genotoxic impurity was achieved under a gradient mode system using a mobile phase comprising 1% acetic acid as mobile phase (A) and acetonitrile as mobile phase (B) with a column temperature of 40°C and flow rate of 0.5 mL/min. A triple mass quadrupole detector was used to measure GTI by combining with the negative-electrospray ionization used in multi-response monitoring mode (MRM). The newly developed method was validated as per ICH guidelines and was able to measure GTI at 2.0 ppm for about 10mg/mL solution of Raltegravir. The %RSD obtained for GTI for six preparations was 1.74 in method precision and 2.56 during the variation of analyst, system and column on a different day. The overall %RSD of 2.73 showed the ruggedness of the method. The GTI is linear from 0.039 to 2.9ppm (LOQ to 150%) and R= 0.9998 showed good linearity. The % recovery obtained for genotoxic impurity was in the range 103.0-109.6% with %RSD 1.51-3.19 from 0.42 to 2.5ppm. The %RSD in precision and accuracy at LOQ is 1.63 and 3.19 indicating the sensitivity of the method. The newly proposed and developed high-sensitive method can be used to detect GTI in Raltegravir API and formulation.

Water-holding capacity of enzymatic protein hydrolysates: A study on the synergistic effects of peptide fractions

The synergistic effects of peptides in bovine skin gelatin hydrolysate (BSGH) on the water-holding capacity (WHC), measured as cooking loss, were studied. These peptides were isolated from the hydrolysate in fractions with different hydrophobicities by reversed-phase chromatography on C18 separation columns. Each peptide fraction was included in the meat matrix at various concentrations for the study of water retention. The fraction with the highest WHC (F5) was obtained. The experimental design consisted of a central compound design with axial points; the independent variables were X1 (ingredient concentration in the meat sample) and X2 (concentration of fraction F5 in the ingredient). The dependent variable was cooking loss. The WHC studies showed substantial synergistic effects occurred between the fractions within the BSGH, and the peptide fractions reduced the cooking loss by 62.96% with respect to a sample without the ingredient. However, a decrease of 42.26% was observed for the F5 fraction. This fraction corresponds to the highest WHC applied for the concentration of 3 g/100 g. The results suggest a synergistic WHC effect of the peptides present in the BSGH since this effect was significantly reduced (p ≤ 0.05) when the peptide fractions were separated.

Development of a novel nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based method for quantification of angiotensin peptides in HUVEC cultures.

BackgroundThis study aimed to develop an analytical method using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of angiotensin (Ang) I, Ang (1-9), Ang II, Ang (1-7), Ang (1-5), Ang III, Ang IV in human umbilical vein endothelial cell (HUVEC) culture supernatant.MethodsHUVEC culture supernatant was added with gradient concentrations (0.05–1,000 ng/ml) of standard solutions of the Ang peptides. These samples underwent C18 solid-phase extraction and separation using a preconcentration nano-liquid chromatography mass spectrometry system. The target peptides were detected by a Q Exactive quadrupole orbitrap high-resolution mass spectrometer in the parallel reaction monitoring mode. Ang converting enzyme (ACE) in HUVECs was silenced to examine Ang I metabolism.ResultsThe limit of detection was 0.1 pg for Ang II and Ang III, and 0.5 pg for Ang (1-9), Ang (1-7), and Ang (1-5). The linear detection range was 0.1–2,000 pg (0.05–1,000 ng/ml) for Ang II and Ang III, and 0.5–2,000 pg (0.25–1,000 ng/ml) for Ang (1-9) and Ang (1-5). Intra-day and inter-day precisions (relative standard deviation) were <10%. Ang II, Ang III, Ang IV, and Ang (1-5) were positively correlated with ACE expression by HUVECs, while Ang I, Ang (1-7), and Ang (1-9) were negatively correlated.ConclusionThe nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based methodology established in this study can evaluate the Ang peptides simultaneously in HUVEC culture supernatant.

Purification and Characterization of Novel Collagen Peptides against Platelet Aggregation and Thrombosis from Salmo salar.

Collagen is a rich source of bioactive peptides and is widely distributed in the skin and bone tissue. In this study, collagen from Salmo salar skin was hydrolyzed with Alcalase or Protamex followed by simulated digestion, YMC ODS-A C18 separation, and ESI-MS/MS analysis. A total of 19 peptides were identified and synthesized for investigation of their antiplatelet activities. Hyp-Gly-Glu-Phe-Gly (OGEFG) and Asp-Glu-Gly-Pro (DEGP) exhibited the most potent activity against ADP-induced platelet aggregation among them with IC50 values of 277.17 and 290.00 μM, respectively, and inhibited the release of β-TG and 5-HT in a dose-dependent manner significantly. Single oral administration of OGEFG and DEGP also inhibited thrombus formation in a ferric chloride-induced arterial thrombosis model at a dose of 200 μmol/kg body weight and did not prolong the bleeding time or cause an immune response in mice. Therefore, our findings indicated that collagen peptides had a potential to be developed into an effective specific medical food in the prevention of thrombotic diseases.

Studies on Analytical Methods of Robenidine Residues in Chicken Meat and Viscera

A high performance liquid chromatographic (HPLC) method for the determination of robenidine residues in chicken meat and viscera has been developed.Samples were extracted with ethylacetate,cleaned up with Alumina B SEP PAK and C18 SEP PAK cartridges, and finally determined by HPLC. Recovery studies were performed using chicken meat, liver, gizzard and skin samples each fortified with robenidine at 0.05,0.25 and 0.5ppm separately.The recovery data were varied from 80.0-92.3% with coefficients of variation which ranged from 0.5-7.0%. The detection limit for the fortified samples was found to be 0.025 ppm. Twenty-five samples of chicken meat, liver, gizzards and skin taken from the market were analyzed. It was found that no robenidine residue was detected in these samples.

A novel di-peptide Met-Glu from collagen hydrolysates inhibits platelet aggregation and thrombus formation via regulation of Gq-mediated signaling.

Increasing evidence has shown that collagen peptides had various biological activities. In this study, a novel antiplatelet peptide Met-Glu (ME) was separated and identified from silver carp skin by YMC ODS-A C18 separation and ESI-MS/MS analysis. Peptide ME inhibited platelet aggregation and secretion of platelet granules induced by ADP, thrombin and collagen, and significantly attenuated ferric chloride-induced thrombus formation in rats. It did not prolong the bleeding time in mice even at the dose of 300μmol/kg body weight that showed potent anti-thrombosis effects. Additionally, peptide ME targeted at Gq-protein to downregulate the phosphorylation of PLCβ, an important upstream effector of PI3K/Akt and Erk/MAPK signaling to inhibit intracellular calcium ion mobilization. These results suggest that peptide ME inhibited thrombosis in vivo and inhibited Gq-mediated signaling in platelets, indicating the possibility that ME could potentially be developed as a novel therapeutic agent in the prevention and treatment of thrombotic diseases. PRACTICAL APPLICATIONS: Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity worldwide. The proximal cause of CVDs is intravascular thrombosis formation, which mostly results from platelet activation, aggregation, and granules secretion. Traditional drugs in the prevention of thrombotic disease, such as aspirin and clopidogrel, are still limited for their side effects, especially bleeding complications. Collagen is a natural source for bioactive peptides and our previous study has shown that collagen peptides could inhibit platelet aggregation in vitro. Understanding the mechanism of collagen peptides on regulation of platelet activation and their in vivo anti-thrombosis activities were important for the development of novel-specific medical food in the prevention of thrombotic diseases.

Study on Differential Protein Expression in Natural Selenium-Enriched and Non-Selenium-Enriched Rice Based on iTRAQ Quantitative Proteomics.

This work was designated to scrutinize the protein differential expression in natural selenium-enriched and non-selenium-enriched rice using the Isobaric-tags for relative and absolute quantification (iTRAQ) proteomics approach. The extracted proteins were subjected to enzyme digestion, desalting, and identified by iTRAQ coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology. High pH C18 separation analysis was performed, and the data were then analyzed by Protein PilotTM (V4.5) search engine. Protein differential expression was searched out by comparing relatively quantified proteins. The analysis was conducted using gene ontology (GO), cluster of orthologous groups of proteins (COG) and Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathways. A total of 3235 proteins were detected and 3161 proteins were quantified, of which 401 were differential proteins. 208 down-regulated and 193 up-regulated proteins were unveiled. 77 targeted significant differentially expressed proteins were screened out for further analysis, and were classified into 10 categories: oxidoreductases, transferases, isomerases, heat shock proteins, lyases, hydrolases, ligases, synthetases, tubulin, and actin. The results indicated that the anti-stress, anti-oxidation, active oxygen metabolism, carbohydrate and amino acid metabolism of natural selenium-enriched rice was higher than that of non-selenium rice. The activation of the starch synthesis pathway was found to be bounteous in non-selenium-enriched rice. Cysteine synthase (CYS) and methyltransferase (metE) might be the two key proteins that cause amino acid differences. OsAPx02, CatC, riPHGPX, HSP70 and HSP90 might be the key enzymes regulating antioxidant and anti-stress effect differences in two types of rice. This study provides basic information about deviations in protein mechanism and secondary metabolites in selenium-enriched and non-selenium-enriched rice.

Non-targeted metabolomics reveals alterations in liver and plasma of gilt-head bream exposed to oxybenzone

The extensive use of the organic UV filter oxybenzone has led to its ubiquitous occurrence in the aquatic environment, causing an ecotoxicological risk to biota. Although some studies reported adverse effects, such as reproductive toxicity, further research needs to be done in order to assess its molecular effects and mechanism of action. Therefore, in the present work, we investigated metabolic perturbations in juvenile gilt-head bream (Sparus aurata) exposed over 14 days via the water to oxybenzone (50 mg/L). The non-targeted analysis of brain, liver and plasma extracts was performed by means of UHPLC-qOrbitrap MS in positive and negative modes with both C18 and HILIC separation. Although there was no mortality or alterations in general physiological parameters during the experiment, and the metabolic profile of brain was not affected, the results of this study showed that oxybenzone could perturb both liver and plasma metabolome. The pathway enrichment suggested that different pathways in lipid metabolism (fatty acid elongation, α-linolenic acid metabolism, biosynthesis of unsaturated fatty acids and fatty acid metabolism) were significantly altered, as well as metabolites involved in phenylalanine and tyrosine metabolism. Overall, these changes are signs of possible oxidative stress and energy metabolism modification. Therefore, this research indicates that oxybenzone has adverse effects beyond the commonly studied hormonal activity, and demonstrates the sensitivity of metabolomics to assess molecular-level effects of emerging contaminants.

Polymeric monolith column composited with multiwalled carbon nanotubes-β-cyclodextrin for the selective extraction of psoralen and isopsoralen.

A polymeric column that contains multiwalled carbon nanotubes-β-cyclodextrin composite was developed. The composite was wrapped into the poly(butyl methacrylate-ethylene dimethacrylate) monolith column (0.76mm id and 10cm in length). The column was then applied for the online solid-phase microextraction of psoralen and isopsoralen from Fructus Psoraleae. Following microextraction, the coumarins were quantified by high-performance liquid chromatography with C18 separation column and UV detection. The effects of sample flow rate, sample volume, and pH value were optimized. The method showed low limits of detection (20pg/mL, S/N=3) for both psoralen and isopsoralen. Finally the method was successfully applied to the determination of psoralen and isopsoralen in spiked herb extracts and rat plasma where it gave recoveries that ranged between 93.2 and 102.1%. The empty hydrophobic cavities of β-cyclodextrin and the hydrophobicity of multiwalled carbon nanotubes provided specific extraction capability for psoralen and isopsoralen.

Parabens determination in cosmetic and personal care products exploiting a multi-syringe chromatographic (MSC) system and chemiluminescent detection

Parabens are widely used in dairy products, such as in cosmetics and personal care products. Thus, in this work a multi-syringe chromatographic (MSC) system is proposed for the first time for the determination of four parabens: methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) in cosmetics and personal care products, as a simpler, practical, and low cost alternative to HPLC methods. Separation was achieved using a 5mm-long precolumn of reversed phase C18 and multi-isocratic separation, i.e. using two consecutive mobile phases, 12:88 acetonitrile:water and 28:72 acetonitrile:water. The use of a multi-syringe buret allowed the easy implementation of chemiluminescent (CL) detection after separation. The chemiluminescent detection is based on the reduction of Ce(IV) by p-hydroxybenzoic acid, product of the acid hydrolysis of parabens, to excite rhodamine 6G (Rho 6G) and measure the resulting light emission. Multivariate designs combined with the concepts of multiple response treatments and desirability functions have been employed to simultaneously optimize and evaluate the responses. The optimized method has proved to be sensitive and precise, obtaining limits of detection between 20 and 40µgL−1 and RSD <4.9% in all cases. The method was satisfactorily applied to cosmetics and personal care products, obtaining no significant differences at a confidence level of 95% comparing with the HPLC reference method.

Abstract 1646: Synthesis of [11C]onapristone for clinical investigation

Abstract Background: Onapristone is a type I anti-progestin, which prevents the progesterone receptor (PR) monomers from dimerizing, inhibits ligand-induced phosphorylation and prevents association of the PR with its co-activators, thus preventing PR-induced transcription. Onapristone has the potential to treat patients with endometrial cancer, breast cancer, uterine sarcomas, a potential subset of ovarian cancer, prostate cancer and other tumors in which the progesterone receptor plays a role in growth, proliferation and metastasis. [11C]-radiolabeled onapristone and its visualization via PET-scan, coupled with pharmacokinetic (PK) studies, has the potential to determine tissue-specific and blood PK parameters including tumor/tissue and plasma concentrations, whole body distribution and half-life of onapristone. The aim of this study is the development of a rapid Good Manufacturing Product (GMP) synthesis of parentally administered [11C]onapristone. Methods: The production of [11C]-labeled radiopharmaceuticals used a Tracerlab® FX c-Pro (GEMS) synthesis module. Carbon-11 was produced at Institut Curie-Hôpital René Huguenin via the 14N(p,α)11C nuclear reaction using a PETTrace cyclotron (GEMS) equipped with a carbon-11 target. Carbon-11 is delivered from the cyclotron as [11C]CO2 in the synthesis module, and reduced to [11C]CH4. Methane is halogenated to [11C]CH3I and converted to [11C]methyl triflate (CH3OTf). To prepare [11C]onapristone, the module was loaded with 1mg of N-desmethyl-onapristone (Arno Therapeutics) and 500µL of DMSO in the reaction vessel (Sigma®). [11C]CH3OTf was bubbled into the reaction vessel and heated at +50°C for 10 minutes. The reaction mixture was then diluted with 1 ml of mobile phase and purified using a semi-preparative HPLC column: Sunfire C18 5µm 250X10mm (Waters®), mobile phase: acetonitrile (Sigma®)/water (Waters®) 50/50 v/v; flow rate 4 mL/min. The product fraction was collected in 40mL of sterile water and was passed through a C18 SEP PAK (Waters®). This fraction was eluted with isotonic saline (Braun®) and ethanol (Sigma®). The resulting formulation was passed through a 0.22µm sterilizing filter into a sterile dose vial. Results: 10 tests were performed. Time of synthesis was 50 minutes. The amount of carbon-11 delivered was 12-14GBq for the irradiation parameters as follows: 10 min; 10µA. The decay-corrected yield of reducing [11C]CO2 to [11C]CH4 was &amp;gt;99%. The yields of preparing [11C]CH3I were 27-29%. Time retention of [11C]onapristone was 8.5-9.5 minutes and the non-decay-corrected radiolabelling yields were 1-2%. Conclusions: We have successfully developed a fully-automated production of [11C]onapristone ready for use in clinical trials. Citation Format: Olivier Madar, Julien Fouque, Stefan Proniuk, Keyvan Rezai, Samuel Huguet, Alexander Zukiwski, Erard M. Gilles, Alice S. Bexon, François Lokiec. Synthesis of [11C]onapristone for clinical investigation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1646. doi:10.1158/1538-7445.AM2014-1646

Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC–MS/MS

The aim of this study was to develop an LC–MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100μL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5μm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex® API 2000 LC–MS/MS system (ESI+ and −, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC–MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology.

MICRO-SCALE METHOD FOR SEPARATION AND QUANTIFICATION OF ATENOLOL AND HYDROCHLOROTHIAZIDE BY SEQUENTIAL INJECTION CHROMATOGRAPHY

A microscale stopped-flow separation manifold, which is termed sequential injection chromatograph (SIC), was exploited for separating and quantifying atenolol and hydrochlorothiazide. A short C18 separation column (4.6 × 25 mm) with a monolithic structure was used to offer a rapid and sufficient separation. Microscale fiber optic spectrometric devices were coupled with the SIC manifold for UV detection at 220 nm. The proposed SIC method was optimized, validated, and applied to both formulations single and binary mixture. The mobile phase composition (MPC) was optimized using the 23 full-factorial design approach while other conditions were optimized using the univariate approach. The optimum MPC was 40 µmol/L phosphate:methanol:acetonitrile (85:7.5:7.5, v/v/v) at pH 3.0. Other optimum conditions were 40 µL/s for flow rate and 40 µL for sample volume. Satisfactory separation with a resolution of 1.6 and numbers of theoretical plates of more than 630 was achieved. The interesting features of the proposed SIC method are the microliter-scale of the consumed volume of mobile phase (3.50 mL) and high sample frequency (23 samples/hr).

Detection and Validated Quantification of the Phosphodiesterase Type 5 Inhibitors Sildenafil, Vardenafil, Tadalafil, and 2 of Their Metabolites in Human Blood Plasma by LC-MS/MS─Application to Forensic and Therapeutic Drug Monitoring Cases

Phosphodiesterase type 5 inhibitors such as sildenafil, vardenafil, and tadalafil are a class of drugs used primarily in the treatment of erectile dysfunction. Sildenafil and tadalafil are also approved for the treatment of pulmonary hypertension. The aim of this study was to develop and validate a procedure for the detection and quantification of these 3 drugs and some of their metabolites in human blood plasma. After liquid-liquid extraction of 0.5 mL of blood plasma using diethyl ether-ethyl acetate (1:1), the analytes sildenafil, norsildenafil, vardenafil, norvardenafil, and tadalafil were separated using a Shimadzu Prominence High-Performance Liquid Chromatography System (C18 separation column, gradient elution, and a total flow of 0.5 mL/min). They were detected using an AB Sciex 3200 Q-Trap LC-MS-MS System (electrospray ionization and multiple reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the tested compounds. It was linear from 5 to 1000 ng/mL for sildenafil, from 2 to 700 ng/mL for norsildenafil, from 0.5 to 350 ng/mL for vardenafil, from 0.5 to 200 ng/mL for norvardenafil, and from 5 to 1000 ng/mL for tadalafil. The recoveries were generally more than 50%. Matrix effects were not observed. Accuracy, repeatability, and intermediate precision were within the required limits (<15% or <20% near the limit of quantification). No instability was observed after repeated freezing and thawing or in processed samples. A liquid chromatography-tandem mass spectrometry assay for the determination of sildenafil, norsildenafil, vardenafil, norvardenafil, and tadalafil in human blood plasma was developed and validated. It has proven to be selective, linear, accurate, and precise for all studied drugs. The method has also proven to be applicable for forensic cases and for therapeutic drug monitoring.

Cinnamon Bark Volatile Oils Separation and Determination Using Solid-Phase Extraction and Gas Chromatography

One of the SPE method is reversed phase separation which involve a polar usually aqueous or moderately polar sample matrix (mobile phase) and a nonpolar stationary phase. The analyte of interest is typically mid-to non polar. Several SPE materials are in the reversed phase category including C18. The hydrophilic silanol groups at the surface of the raw silica packing have been chemically modified with hydrophobic alkyl functional groups by reaction with the corresponding silanes. Retention of organic analytes from polar solutions on to the SPE materials is due primarily to attractive forces between the carbon-hydrogen bonds in the analyte and the functional group in the silica surface. These nonpolar-nonpolar attractive forces are van der waals forces. In this work, at first the aqueous solutions of Cinnamomum zeylanicum Blume bark were obtained by hydrodistillation and superheated water extraction (SWE) methods, then main the volatile compounds were extracted from aqueous solutions by solid phase extraction (SPE) method. This method involves adsorption of the desired components on C18 SEP PAK (Monofunctional -Si (CH3)2C18H37), followed by desorption in the desorption chamber, using minimum volume solvent as the mobile phase. Solid phase extraction conditions optimized on C18 SEP PAK classic cartridge. Different solvent including acetone, hexane, ethyl acetate, ethanol, and mix methanol: water were examined. A good separation of Ecinnamaldehyde was obtained on the adsorbent column. The amount of extracted essential oil composition of C. zeylanicum by SPE, evaluated in different conditions. Gas chromatography/mass spectrometry and gas chromatography were used to identify and quantity the volatile compound composition. GC analysis showed that the maximum yield of E-cinnamaldehyde obtained by methanol: water in (70:30 v/v) ratio by SPE extraction. At the optimum condition, the GC results indicated that E-cinnamaldehyde was the highest area percentage of 79.60% in the volatile oils extracted by hydrodistillation and 88.50% by superheated water extraction.

Validated method for the determination of ethylglucuronide and ethylsulfate in human urine

Detection of the alcohol metabolites ethylglucuronide (EtG) and ethylsulfate (EtS) has become routine in many forensic laboratories over the last few years. Most previously published methods using liquid chromatography coupled with electrospray tandem mass spectrometry require a post-chromatographic addition of solvent and/or extensive sample preparation prior to analysis. The aim of the study was to develop a simplified method. To 20 μL urine, internal standard containing EtG-d5 and EtS-d(5) was added and the mixture was treated with elution buffer internal standard. EtG and EtS were separated using a Shimadzu Prominence high performance liquid chromatography (HPLC) system with a C18 separation column (Restek Ultra Aqueous C18, 4.6 × 150 mm, 5 μm), using isocratic elution with a mobile phase consisting of 10 mM ammonium acetate buffer pH 7 (total run time, 6 min). The compounds were detected using an Applied Biosystems API 5000 liquid chromatography tandem mass spectrometry system (atmospheric pressure chemical ionization, multiple-reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the compounds of interest. It was linear from 0.1 to 10 mg/L for all analytes (R(2) > 0.99). Matrix effects studies showed the presence of a slight but consistent ion enhancement (n = 10 different urine samples) at low concentrations and no effects at higher concentrations. Accuracy data were between 0.75% and 8.1% bias for EtG and between -5.0% and -11.3% bias for EtS. Precision data were between 4.3% and 6.9% relative standard deviations (RSD) for EtG and between 6.0% and 7.5% RSD for EtS. No instability was observed after repeated freezing and thawing. This fast, reliable, and accurate method enables the detection and quantification of alcohol metabolites in urine. The method is easier to use and more sensitive than previously published methods.

Comprehensive Analytical Methods of the Synthetic Cannabinoids Appearing In the Illicit Drug Market

Comprehensive analytical method to identify 11 kinds of synthetic cannabinoids has been investigated by thin layer chromatography (TLC), gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analytes used in this study have already been detected from various herbal-type designer drugs: 8 kinds of aminoalkylindoles (AAIs) (JWH-015, JWH-018, JWH-073, JWH-081, JWH-200, JWH-250, JWH-251 and JWH-398), two kinds of cyclohexylphenols (CPs) (CP 47,497 and Cannabicyclohexanol), and a Δ9-tetrahydrocannabinol analog (HU-210). Although specific color changes were observed for the cannabinoids using Marquis reagent, identification of each analyte based on Rf values was difficult to be obtained by TLC. On the other hand, GC/MS and LC/MS/MS were appropriate for their qualitative analyses because of their chromatographic and mass spectral differentiation. A semi-polar capillary column DB-5MS showed the best separation and retention properties of the targeted cannabinoids among the tested GC column phases. Also, characteristic fragment ions were observed in each electron ionization-mass spectrum. The observed fragment ions were mainly derived from α-cleavage of ketone and α-cleavage of amine for AAIs, simple cleavage for CPs, and McLafferty rearrangements for HU-210. Based on the ionization efficiency of the target analytes using LC/MS/MS, electrospray ionization positive mode was selected for AAIs, and negative mode for CPs and HU-210. All analytes were completely separated by gradient elution of ammonium formate aqueous solution-acetonitrile mobile phase on a C18 (ODS) separation column. In addition, characteristic fragment ions were observed in product ion spectra of AAIs and second generation product ion spectra of CPs and HU-210, enabling reliable confirmation. These results provide useful information not only for simultaneous analyses of the targeted cannabinoids but also for structural assignment of future cannabimimetic compounds that may appear in the illicit drug market.

Selectively preparative purification of aristolochic acids and aristololactams from Aristolochia plants

A novel method has been developed for the purification of aristolochic acids and aristololactams compounds from Aristolochia plants, a kind of typically toxic traditional Chinese medicine. In this method, Oligo (ethylene glycol) separation column which has “clustering function” for compounds in TCMs was used to produce the fractions containing the compounds with similar structures. A four-channel parallel preparative HPLC with C18 separation column was employed to purify the target compounds. The extraction sample of the blending of Radix Aristolochiae, Fructus Aristolochiae and Caulis Aristolochiae Manshuriensis was used to develop the method. Then, four aristolochic acids and three aristololactams were obtained using this method and the chemical identification was confirmed by Q-TOF-MS, 1H NMR and 13C NMR. Thus, this method can deal with more than one traditional Chinese medicine simultaneously. Additionally, the results demonstrated that this method was an effective way to purify target compounds selectively from TCMs.