ALBERT

Description: Microwave-assisted ionic liquid extraction (MAILE) has been investigated for the extraction of n -alkanes and isoprenoid hydrocarbons from petroleum source rock and the conditions for maximum yield of the analytes were determined. An aqueous solution of an ionic liquid, 1-butyl-3-methylimidazolium bromide (BmimBr), was employed as the extracting medium. The results showed that the concentration of ionic liquid, extraction time and extraction temperatures have effects on extraction yields of aliphatic hydrocarbons with optimal conditions at: 3.0 mol L −1 ionic liquid solution, 30 min and 120 °C, respectively. The extraction yields of all the n -alkanes and acyclic isoprenoid hydrocarbons were much higher using microwave-assisted ionic liquid extraction than with Soxhlet extraction and accelerated solvent extraction. There were good correlations of the diagnostic geochemical ratios calculated from the gas chromatographic (GC) data of MAILE, Soxhlet extraction and accelerated solvent extraction. The results of this study show that MAILE is an efficient and green analytical preparatory technique for geochemical evaluation of petroleum source rock.

Description: Zeolite NaY modified with cetyltrimethylammonium bromide (CTAB) was considered for extraction/preconcentration of carbamate pesticides using an on-line SPE-HPLC system. The simultaneous determination of carbamate pesticides, including aldicarb, carbofuran, carbaryl, isoprocarb, methiocarb and promecarb, was performed by HPLC–UV using a LichroCART RP-18 column with gradient elution of methanol and 0.1 % acetic acid. The sorbent presented admicelles of CTAB on its surfaces and exhibited a sorption capacity of 180–18,600 mg kg −1 sorbent, which could be re-modified for at least five extraction cycles. The quantitative retention of target pesticides on the admicellar sorbent involved hydrophobic and π -cation interaction, while pesticides were eluted from the admicellar SPE column using only 750 μL of methanol. LODs and LOQs of the proposed method were 0.005–140 and 0.02–600 μg L −1 , respectively. The analytes were effectively concentrated with the enrichment factors between 5 and 551. The developed on-line admicellar SPE-HPLC system was successfully applied to the determination of carbamate pesticides in ten environmental water samples from different sources. Recoveries of spiked samples at a concentration of 0.1–5 mg L −1 ranged from 77 to 111 %.

Description: Isoflavones are a very important group of natural products. This study investigated the separation of eight isoflavones, namely ononin, daidzin, genistin, biochanin A, formononetin, puerarin, genistein, and daidzein, from pueraria by micellar electrokinetic chromatography (MEKC) with different surfactants. The following micellar systems of MEKC were systematically compared for the analysis of these isoflavones: (1) a single surfactant comprising the anionic surfactant sodium dodecyl sulfate (SDS), the cationic surfactant hexadecyltrimethylammonium bromide, the neutral surfactant polyoxyethylene sorbitan monolaurate (Tween 20), and the ionic liquid-type surfactant (also a cationic surfactant) 1-dodecyl-3-methylimidazolium tetrafluoroborate (C 12 MIMBF 4 ); (2) different single surfactants with 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF 4 ) as an additive (modifier); and (3) mixed micelles of SDS + Tween 20 and C 12 MIMBF 4  + Tween 20. Both SDS with BMImBF 4 as additive and mixed micelles of SDS + Tween 20 had the highest separation efficiency for the eight investigated compounds. Furthermore, the SDS with BMImBF 4 as additive was more stable (good repeatability of retention time and peak shape of analytes) than mixed micelles of SDS + Tween 20, which may be the result of a stabilizing effect of BMImBF 4 . Therefore, the final analytical conditions were 15 mM SDS added with 50 mM BMImBF 4 in 30 mM sodium tetraborate (STB, pH 9.5) as running buffer; applied voltage, 20 kV; injection, 50 mbar for 5 s; cartridge temperature, 25 °C; compounds were detected at 260 nm. The developed method was fully validated (limit of detection, limit of quantification, intraday precision, inter-day precision, and recovery) and successfully applied to determine the eight analytes in three Radix Puerariae samples. The present study indicated that SDS with ionic liquids as additive in MEKC was suitable for the analysis of isoflavones.

Description: A novel and sensitive solid phase extraction method based on the adsorption of cetyltrimethylammonium bromide (CTAB) on the surface of Fe 3 O 4 nanoparticles was developed for the extraction and preconcentration of trace amounts of UV absorbers and light stabilizers in plastic packaging. The remarkable properties of Fe 3 O 4 @CTAB including strong magnetization and high surface area were utilized in the magnetic solid phase extraction procedure, and satisfactory extraction recoveries (84.5–98.9 %) were obtained by using only 12 mg of magnetic adsorbent (200 µL, 60 mg mL −1 ). Five parameters on the extraction recovery were investigated, such as the amount of surfactant, pH value, the amount of Fe 3 O 4 @CTAB, eluent and sorption time. Under the optimum conditions, the method was linear in the range of 0.5–25 μg mL −1 and good linearity ( R 2  〉 0.996) was obtained for all calibration curves. The limits of detection in the plastic packaging were 0.09 and 0.17 μg mL −1 and the relative standard deviations of the analytes ( n  = 5) were in the range from 0.37 to 2.40 %.

Description: The use of amino phases induced a significant increase of the bioluminescence for the high-performance thin-layer chromatography (HPTLC)- Aliivibrio fischeri bioassay. By this, the detectability of alkaloids was improved. Detection limits at the low ng per zone level were obtained for the five alkaloids berberine, palmatine, ephedrine, norephedrine, and methylephedrine. The influence of other parameters on this effect, such as fluorescence indicators and pH milieu, was proven to be insignificant. This implied that propyl amino groups linked to the silica gel layer might play a pronounced role in the improved visualization mechanism. The optimized bioassay was applied for profiling of alkaloid-rich herbal drugs such as Philodendron , Coptis , Tinospora , and Ephedra , leading to information-rich biofingerprints. The direct coupling of HPTLC to electrospray ionization mass spectrometry enabled unambiguous and straightforward confirmation of the bioactive ingredients.

Description: Decomposition odour analysis involves the chemical profiling of volatile organic compounds produced by decomposing remains. This is important for areas of forensic science that rely on the detection of decomposition odour such as insect attraction to carrion, positive alerts of cadaver dogs to decomposing remains, and the development of field instrumentation for search and recovery procedures. Traditionally decomposition odour analysis has been performed using gas chromatography–quadrupole mass spectrometry (GC–qMS); however, the use of comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–TOFMS) is rapidly becoming more prevalent. The objective of this study was to compare GC–qMS and GC×GC–TOFMS for decomposition odour profiling based on inter-year replicate field studies using decomposing porcine remains. The increased peak capacity, sensitivity and selectivity afforded by GC×GC–TOFMS allowed peak co-elutions, chromatographic artefacts, and dynamic range to be more easily addressed and managed. Furthermore, the software associated with GC×GC–TOFMS provided several additional benefits including improved peak alignment between samples and increased consistency of reported results, overall allowing for additional statistical tests to be applied following data processing. Future GC–qMS results could be improved by implementing some of these software-associated procedures, potentially reducing the magnitude of variation observed between GC–qMS and GC×GC–TOFMS studies. One-dimensional GC analysis may also benefit substantially from coupling with TOFMS detection to provide an indirect increase in peak capacity using deconvolution. However, the wealth of information gained by using GC×GC–TOFMS in decomposition odour profiling is undoubtedly an asset in this field of research.

Description: Nine impurities in amikacin sulfate made in China were separated and identified by HPLC–MS n for the further improvement of official monographs in pharmacopoeias. The mass fragmentation patterns and structural assignment of these impurities were studied. The column was Acchrom Click XIon (250 × 4.6 mm, 5 μm). The mobile phase was 250 m mol L −1 ammonium formate and 1.4 % formic acid aqueous solution–acetonitrile–water (30:48:22). In positive mode, full scan LC–MS was first performed in order to obtain the m/z value of the protonated molecules, LC–MS–MS was then carried out on the compounds of interest on AB SCIEX 4000 Q TRAP™ composite triple quadrupole/linear ion trap tandem mass spectrometer. The complete fragmentation patterns of nine impurities were studied and used to obtain information about the structure of these impurities. The structures of nine impurities in amikacin sulfate were deduced based on the HPLC–MS n data, in which three impurities were novel impurities. Three novel impurities were 1- N -( l -4-amino-2-hydroxybutyryl) derivative of 4-O-(6-AG)DS, 1- N -( l -4-amino-2-hydroxybutyryl) derivative of 6-O-(3-AG)DS and 1- N -( l -4-amino-2-hydroxybutyryl) derivative of kanamycin D.

Description: In the biopharmaceutical industry, protein aggregation and/or degradation has profound pathological implications and is encountered routinely during production, shipping, storage and administration. Lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) was subjected to stress conditions, namely, oxidation, pH, temperature, agitation and repeated freeze–thaw to generate all possible degradation products. An orthogonal stability-indicating testing protocol (RP-HPLC, SE-HPLC, ELISA and SDS-PAGE) was developed and validated for assessment of the pattern and kinetics of aggregation/degradation, under the studied experimental conditions. Results indicated clearly that Lenograstim is susceptible to degradation induced by the studied stress conditions. However, Lenograstim was found relatively more stable than Filgrastim (non-glycosylated recombinant human granulocyte colony-stimulating factor) which was attributed to the effect of glycosylation. Oxidized forms and high molecular weight aggregates of Lenograstim and Filgrastim were detected in all samples subjected to stress conditions to different degrees. ELISA assay and SDS-PAGE results were generally in agreement to those obtained using SE-HPLC assay which confirmed its selectivity to the intact drug. However, formation of soluble aggregates of both drugs was found to occur via physical adsorption and formation of intermolecular disulfide bonds. Results confirmed the need for an orthogonal testing protocol since it was impossible to reveal all types of degradation products using a single technique. Results raised a concern about the efficacy and safety of such sensitive products and highlighted the need for simple tools to inspect biologics for soluble aggregates and sub-visible particles before administration.

Description: In this work flour samples of various cereal species, a group of different cultivars of small grain species (wheat, Triticum spp.; barley, Hordeum vulgare ; oat, Avena sativa ; rye, Secale cereale ; triticale, Triticosecale ) and cultivars of corn species, Zea Mays , growing in the same period and in the same geographical area, were selected to establish differences between each other, using a new rapid method: a comparison of derivatized hexane extracts by GC–MS and multivariate analysis, using the characteristic fragmentation ion m/z 74, without performing qualitative and quantitative analysis of eluting components. Obtained results were compared with the results obtained using a common electron microscopy method. Flour samples made of every corn and oat cultivar showed complete differences compared to flour samples of each wheat, barley, rye and triticale cultivar investigated in this study. The GC–MS approach combined with multivariate analysis outperforms the standard electron microscopy method in a faster and easier way, and may be used to verify flour types in the market.

Description: A quality by design approach utilizing experimental design methodologies was applied to develop a CE method to evaluate the enantiomeric purity of ( S )-ambrisentan, a selective endothelin receptor antagonist used for the treatment of pulmonary arterial hypertension. Initial method scouting was performed by screening native cyclodextrins (CDs) as well as neutral CD derivatives and a positively charged derivative at pH 5 and pH 9, identifying γ-CD as suitable selector at pH 5. Upon defining the critical quality attributes and the critical process parameters, i.e., chiral resolution and run time, method development was performed by application of a screening design for the identification of the significant variables and, subsequently, by a response surface methodology for obtaining the design space. A Plackett–Burman design was employed for robustness testing. The final working point conditions used a background electrolyte composed of a 50-mM sodium acetate buffer, pH 4.0, containing 30 mM γ-CD in a 75-μm ID fused-silica capillary with an effective length of 40 cm at an applied voltage of 25 kV and a capillary temperature of 25 °C. The method was validated according to the ICH Q2(R1) guideline and allowed the determination of a relative concentration of the ( R )-enantiomer of 0.1 %.

Description: A high-performance liquid chromatography (HPLC) screening method with a photodiode array detector (PDAD) was established for the simultaneous determination of residues of 13 benzimidazoles (BZDs) and metabolites in sheep and cattle muscle and liver. Samples were extracted by ultrasonication in ethyl acetate and purified over DVB-NVP-SO 3 Na sorbent. Under the optimized conditions, good linearities were obtained for BZDs and metabolites with correlation coefficients ( R 2 ) greater than 0.9911. The recoveries of the 13 BZDs and metabolites from spiked samples were 72.0–119.3 %, with intraday and interday relative standard deviations (RSDs) below 22.8 %. The limits of detection (LODs) and quantitation (LOQs) were 0.8–4.9 and 2.6–18.2 μg kg −1 , respectively. The results clearly demonstrated that the developed approach enables reliable screening of 12 BZDs and metabolites except flubendazole (FLU) and could be used as a regulatory tool for the screening of BZD and metabolite residues in the muscle and liver of sheep and cattle.

Description: Bosentan monohydrate (4- tert -butyl- N -[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]benzene-1-sulfonamide monohydrate) is a dual endothelin receptor antagonist (ERA) applied in the treatment of pulmonary arterial hypertension. To achieve effective process control of the bosentan monohydrate synthesis, it was necessary to develop a selective and not highly time-consuming method for ultra-high performance liquid chromatography (UHPLC). The method is characterized by adequate sensitivity, reproducibility and selectivity for the determination of bosentan monohydrate and related compounds from all synthetic stages. The UHPLC separation was carried out by reversed phase chromatography on the Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm) with a mobile phase composed of solvent A (0.1 %, v/v, acetic acid in water) and solvent B (methanol), in the gradient mode at the flow rate of 0.4 mL min −1 . Limits of detection and quantification for the compounds were ≤0.1 µg mL −1 and 0.3 µg mL −1 , respectively. The linearity for all related compounds was investigated as in the range for the active pharmaceutical ingredient (API) and as in the range for the in-process control. The developed method was validated according to the current guidelines, proving the suitability of the method for its intended purpose.

Description: In this work, a simple and rapid high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) method was first developed for the quantitation of toosendanin, the major constituent of the dried fruit of Melia toosendan Sieb. Et Zucc. Samples were well separated on an Agilent ZOBAX SB C18 column (4.6 mm × 250 mm, 5 μm) by isocratic elution using 33 % acetonitrile and 67 % water containing 0.1 % formic acid (v/v) at the flow rate of 1.0 mL min −1 . The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35 °C. The established method was well validated. Satisfactory linearity was achieved ( r 2  〉 0.9997) in a relatively wide concentration range (5–500 μg mL −1 ). The intra- and inter-day precisions, repeatability, and stability of the method were good with relative standard deviations (RSDs) of 1.05, 2.23, 2.39, and 2.03 %, respectively. The method also showed excellent accuracy with recovery rates of 97.42–101.87 %. Particularly, CAD showed much better sensitivity (LOQ 4 μg mL −1 ) than evaporative light scattering detector (LOQ 100 μg mL −1 ) for toosendanin’s determination. The established method was further applied in the quantitation of toosendanin in 39 batches of raw and stir-fried toosendan fructus. The HPLC-CAD method was rapid and accurate, and could be used for the routine analysis and quality control of toosendan fructus and its preparations.

Description: A simple, rapid and robust enantioselective method was developed and validated for the quantitation of OTX015 enantiomers [(−)-OTX015 and (+)-OTX015] with ultrahigh-performance liquid chromatography (UHPLC) as per ICH guidelines. The active [(−)-OTX015] and inactive [(+)-OTX015] enantiomers were resolved on a Chiralpak-IA column using methanol consisting of 0.1 % diethyl amine at a flow rate of 1.0 mL min −1 . The resolution between the enantiomers was found to be more than 3.7 in the optimized method. The developed method was extensively validated and proven to be robust. The calibration curve for (+)-OTX015 showed excellent linearity over the concentration range of 10–100 µg mL −1 . The limit of detection and the limit on quantitation for (+)-OTX015 were 5 and 10 µg mL −1 , respectively. The recovery for (+)-OTX015 ranged between 100.7 and 102.5 % in the bulk drug sample of (−)-OTX015. The proposed method was found to be suitable and accurate for quantitative determination of (+)-OTX015 in bulk drug.

Description: In this study, a metabonomics analysis of heart homogenates from myocardial ischemic rats was performed by LC–TOF–MS. Hydrophilic interaction chromatography (HILIC) was used to separate the endogenous metabolites in heart homogenates. Partial least squares to latent structure-discriminant analysis (PLS-DA) was used for data analysis. Good separations were observed between the normal and model groups and 15 potential biomarkers were identified. The major disturbed metabolic pathways were purine metabolism, pyrimidine metabolism, urea cycle, and energy metabolism. The results demonstrated that a metabonomics approach based on HILIC-MS was useful for studying metabolic mechanism on target tissue of the myocardial infarction rat.

Description: Polyethylenimine (PEI) and 2,4,6,8-tetramethyl-2,4,6,8-tetrakis(propyl glycidyl ether)cyclotetrasiloxane (POSS–epoxy) were used as precursors for the preparation of organic-silica hybrid monolithic columns (PEI–POSS monolith) via epoxy–amine ring-opening polymerization (ROP). The high density of amine groups in PEI provides rich chromatographic interaction sites for the polar or acidic analytes in hydrophilic interaction (HILIC) and weak anion exchange (WAX) mechanisms. The column preparation conditions, such as the porogens, solvent and reaction temperature, were systematically investigated according to the morphology, permeability and column efficiency. The separation mechanisms of HILIC and WAX were evaluated with neutral polar compounds and halogen benzoic acids. Owing to the existence of reactive amine groups on the matrix surface, the PEI–POSS monolith is also an ideal starting material for the preparation of HILIC or strong anionic exchange (SAX) stationary phases by modification. The modification of PEI–POSS monoliths with iodomethane or bromoacetic acid via the nucleophilic substitution reaction could achieve the retention mechanisms of SAX or zwitterionic HILIC, respectively.

Description: A quantitative structure–retention relationship (QSRR) study was performed to correlate descriptors representing molecular structures to the Kováts retention indices of polycyclic aromatic hydrocarbon compounds. The complete set of 37 compounds was divided randomly into a training set of 26 compounds and a test set of 11 compounds. In the pool of descriptors calculated using Dragon and Hyperchem software, the modified Harary index ( \( H^{\prime\prime} \) ) developed by our team was added. The best selected descriptors by genetic algorithm to build the three linear models were: the first-order Randić connectivity index ( \( {}^{1}\chi \) ), \( H^{\prime\prime} \) and the third-order valence connectivity index ( \( {}^{3}\chi_{p}^{v} \) ). The selected model was compared with the one built with \( {}^{1}\chi \) , the first Harary index (implanted in Dragon H ) and \( {}^{3}\chi_{p}^{v} \) . Statistical analysis showed that the model with modified Harary index ( R 2  = 99.25, SD 5.82, F  = 1517.74) produces better correlation with Kováts retention indices than the one implanted in Dragon ( R 2  = 98.20, SD 9.02, F  = 626.09). The final QSRR was internally and externally validated. The leave-one-out, cross-validation, bootstrapping, and y -randomization test indicated that the final model is robust and has no chance correlation. The external validations indicated that the model with modified Harary index showed a good predictive power. The mechanistic interpretation of QSRR model was carried out according to the definition of descriptors. Therefore, the result meets the five principles recommended by the Organization for Economic Co-operation and Development for validation of QSRR model.

Description: Head space sorptive extraction (HSSE) has been evaluated for the analysis of volatile compounds from oak wood in wines. The extraction conditions have been optimized using a two-level factorial design. After optimization, the final extraction conditions were: PDMS stir bars (20 mm length × 0.5 mm film thickness), sample volume of 5 mL, extraction time 120 min, stirring speed 2000 rpm, and NaCl until saturation. The HSSE optimized method was further validated and showed high linearity ( r 2  〉 0.9960), adequate detection and quantification limits and linear ranges for the analysis of these compounds in aged wines. Moreover, it provided good values of analytical parameters such as precision and recovery (3.49–11.22 % for intra-assay precision, 4.35–8.77 % for inter-assay precision, and 70.6–124 % for recovery). The method was finally applied to different types of fortified wines (Mistelle, Fino and Oloroso wine) from Condado de Huelva Denomination of Origin submitted to different periods of aging in oak wood.

Description: A performant method for the simultaneous quantification of daidzein, genistein, formononetin, and biochanin A in forages using an UPLC ® -MS/MS was developed and fully validated. The ultrasound-assisted extraction and enzymatic hydrolysis used in the sample preparation step were optimized using the Box–Behnken experimental design. The optimal extraction conditions used for a representative mix of forage plants were 80 °C, 10 min, and 55 % methanol, and for hydrolysis, they were 20 °C, 18 h, and pH = 6. The chromatographic separation was achieved using an Acquity UPLC ® HSS T3 column, with a water/methanol linear gradient containing 0.01 % of formic acid at a 0.55 mL min −1 flow rate. The four isoflavones were detected by ESI mass spectrometry in positive ion MRM mode. The method allows high throughput analyses of samples and showed an adequate linear regression model for all isoflavones over a range from 5 to 125 ng mL −1 . There were good intra- and inter-day precisions (≤8.2 and ≤7.6 %) and accuracy (≤11.4 and ≤7.1 %). The recovery rates were in an acceptable range of 70–120 %, except for biochanin A, where the rate was about 50 %. Good method repeatability was also observed, and there was no matrix effect or carryover problem. The sample extracts were stable for at least 6 days of storage at -21 and 6 °C. The method proved to be sensitive, precise, and accurate for discriminating a wide variety of forages likely to be grazed by ruminants according to their isoflavone contents and to observe the impact of storage process on isoflavone content in forages.

Description: A rapid and sensitive UFLC–MS/MS method was developed for the simultaneous determination of liguzinediol and its four primary metabolites (M1, M2, M3, and M4) in rat plasma using antipyrine as internal standards. The analytes were separated on an XR-ODS column (50 mm × 2.0 mm, 2.2 μm) using 0.1 % formic acid–methanol gradient elution at a flow rate of 0.5 mL·min −1 . The detection was performed on a triple quadrupole tandem mass spectrometer in a multiple reaction monitoring mode with positive electrospray ionization. Method validation was performed as per the Food and Drug Administration guidelines. The resulting calibration curves offered satisfactory linearity ( r  〉 0.9993) with the set ranges. The limits of quantification for liguzinediol, M1, M2, M3, and M4 were 20, 20, 21, 27, and 10 ng mL −1 , respectively. The recovery rates in different matrices ranged from 91.2 to 114.1 %, and the inter-day and intra-day precisions were all less than 13.4 % for the target analytes. After validation, this method was successfully applied to further study pharmacokinetics profiles of liguzinediol and its metabolites after intravenous administration of 10 mg·kg −1 in male and female rats.

Description: The vascular endothelial growth factor receptor-2 (VEGFR-2) in some tumor cells is a significant target for drug discovery. In this work, a modified model of VEGFR-2 cell membrane stationary phase (CMSP) was prepared by immobilizing U251 cell membrane onto the surface of chitosan-silica (CTS-SiO 2 ) hybrid carrier. The surface and chromatographic characteristics of VEGFR-2 CMSP were studied. We have developed modified VEGFR-2 cell membrane chromatography for screening drugs and sunitinib malate was used as a positive control. The interaction between the new compounds and membrane receptor was determined by the capacity factors ( k ʹ). The in vitro cytotoxicity of 10 new compounds on U251 cell viability was determined by MTT test separately to verify the potential pharmacological activity. The modified VEGFR-2 cell membrane chromatographic system demonstrated fast and effective characteristics for screening leading compounds.

Description: We describe a method for the quantification of proteins in a biological matrix through digestion with pepsin. Pepsin is a gastric protease that efficiently cleaves proteins in an acidic environment. In this study, it has been used to generate peptides used for the quantification of physiologically relevant thioredoxin proteins in a lysate of Bacillus anthracis —the causative agent of anthrax. Carefully selected signature peptides for proteins that were digested with pepsin were immobilized on agarose gel. Filtered samples were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) and by two-dimensional liquid chromatography tandem mass spectrometry (LC/LC–MS/MS) when additional selectivity was needed. Some important incubation parameters were adjusted to get the highest possible peptide yield. Escherichia coli was used as a surrogate matrix for the method development. The method was validated at a low nM range for selectivity, accuracy and precision. Validation showed that signature peptides were selective for the proteins, and that the method accuracy varied between 89 and 115 % with a precision of less than 12 %. The results from using pepsin in analyzing samples from Bacillus anthracis were similar to those previously obtained using western blot, and they validate pepsin as a suitable protease to generate signature peptides in a complex biological matrix as an alternative to trypsin.

Description: In the proposed work, the simultaneous analysis of amlodipine–rosuvastatin and amlodipine–atorvastatin in their dosage forms was achieved. Simultaneous dissolution profiles of the amlodipine–rosuvastatin and amlodipine–atorvastatin tablets are realized using Apparatus II with a simple, accurate and precise RP-LC method. The mobile phase consisting of 0.2 % H 3 PO 4 and pH 5:methanol:acetonitrile (46:27:27) was used. The samples of 10 µL were injected onto a Zorbax SB C18 (100 mm, 4.6 mm, 3.5 µm particle size) column with 1.2 µL min −1 flow rate. The samples were detected at 236 nm. By plotting peak area ratios vs. concentration, the linearity for amlodipine–rosuvastatin and amlodipine–atorvastatin was determined. With the developed RP-LC method, AML, ROS and ATOR were detected within the range of 0.25–10, 0.5–10 and 0.25–25 µg mL −1 , respectively. LOD and LOQ values were also calculated as 0.028, 0.058, 0.021 and 0.095 µg mL −1 , 0.195 µg mL −1 , 0.070 µg mL −1 for AML, ROS and ATOR, respectively. System suitability tests parameters, such as capacity factor, selectivity to previous peak, selectivity to next peak, resolution to previous peak, resolution to next peak, tailing factor, theoretical number of plates, were performed and found coherent with the ICH guideline parameters. The proposed method has been extensively validated in terms of recovery, and recovery results were between 99 and 101 %. For proving the precision, between-day and within-day repeatability results of the method were proposed. The method can be used for the simultaneous determination of amlodipine–rosuvastatin and amlodipine–atorvastatin.

Description: A quick, easy, cheap, rugged, effective, and safe (QuEChERS)-based method has been validated for the extraction of 42 pesticides and herbicides including organophosphorus pesticides (OPPs), carbamate pesticides (CBs), herbicides (HBs), organochlorine pesticides (OCPs), and synthetic pyrethroid pesticides (PYRs) from chicken eggs. The QuEChERS-based extraction procedure was followed by cleanup steps using C18 and primary secondary amine sorbents. The supernatant was analyzed by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and gas chromatography–mass spectrometry (GC–MS). The OPPs, CBs, and HBs were quantified by UHPLC–MS/MS, while the OCPs and PYRs were detected by GC–MS. The limits of quantification ranged from 0.01 to 8.5 μg kg −1 , and the analyte recoveries were in the range of 64.9–123.2 %. Furthermore, the repeatabilities (intra-day and inter-day) were good, and linear matrix-matched calibration curves were obtained. Acetochlor was identified in concentrations ranging from 0.27 to 0.44 μg kg −1 in four samples from 80 chicken eggs. The method was successfully demonstrated for the fast and reliable analysis of pesticides and herbicides in chicken egg samples.

Description: In this paper, polythiophene/chitosan magnetic nanocomposite as a novel adsorbent is proposed for the preconcentration of triazines in aqueous samples prior to gas chromatography. The synthesized nanoparticles, magnetic chitosan and polythiophene–chitosan magnetic nanocomposite were characterized by scanning electron microscopy. The magnetic polythiophene–chitosan nanocomposite containing analytes could be removed from the sample solution by applying a permanent magnet. The major factors influencing the extraction efficiency including desorption conditions, nanocomposite components ratio, sorbent amount, extraction time, ionic strength and sample pH were optimized. The developed method proved to be rather convenient and offers sufficient sensitivity and good reproducibility. The limit of detection ( S / N  = 3) and limit of quantification ( S / N  = 10) of the method under optimized conditions were 10–30 and 100 ng L −1 , respectively. Under the optimum conditions, good linearity was obtained within the range of 100–5000 ng L −1 for all triazines with correlation coefficients 〉0.9994. The relative standard deviation at a concentration level of 150 ng L −1 was 7–12 %. Furthermore, the method was successfully applied to the determination of triazines in real samples, where relative recovery percentages of 96–102 % were obtained. Compared with other methods, the current method is characterized by easy, fast separation and low detection limits.

Description: New chiral silica adsorbents with well-defined peracetylated thiosaccharide (a.k.a. thiosaccharide) surfaces containing 1, 3, 7, and 13 glucose units have been synthesized through the reaction of trimethoxysilyl-derivatives of thiosaccharides with bare silica gel. As determined by FTIR, nitrogen adsorption, thermo-gravimetric analysis, and chemical analysis, the adsorbents prepared contained closely packed uniform monolayer surfaces of thiosaccharide groups grafted to silica. The chromatographic behavior of the thiosaccharide-silicas prepared has been evaluated in the HPLC separations of stereoisomers in both normal and reversed phase modes. With the exception of low grafted thiomaltodecatriose-silica, there was no effect of the size of thiosaccharide on the selectivity of the separations.

Description: Lowered plasma concentrations of the endogenous amino acid l -homoarginine have been recently identified as an independent risk factor for cardiovascular and all-cause mortality in patients referred for coronary angiography in the LURIC study. To support further investigations into this matter, we describe here a fast and easy LC–MS–MS method for the detection of l -homoarginine in human plasma. The sample preparation consisted only of the addition of the stable isotope-labeled internal standard d 4 - l -homoarginine and protein precipitation. The analytes were separated isocratically on an HILIC silica column. Detection took place by tandem mass spectrometry. The calibration function was linear in the range of 0.1–10 μmol L −1 . The intra-day precision was better than 2 % RSD and the inter-day precision better than 4 % RSD in plasma. The accuracy was always better than 5 % deviation. The method was matrix independent owing to the usage of the analogous stable isotope-labeled internal standard.

Description: Pressurized planar electrochromatography (PPEC) is a separating technique in which an electric field is applied to force the mobile phase movement through a porous media (electroosmotic effect). High separation efficiency, fast separations and changes in separation selectivity in comparison to liquid chromatography, especially thin layer chromatography (planar chromatography, TLC), are features of this technique. Constructional methodological challenges to PPEC are obstacles to its development and application in laboratory practice. In this article, an attempt to overcome the challenges related to device construction and sample application/injection is described. The introduced device enables both prewetting of the adsorbent layer and electrochromatogram development with a single PPEC device. It also enables simultaneous application/injection of six samples on a chromatographic plate in a stream of the mobile phase (on-line application/injection). In addition, the PPEC chamber was equipped with a thermostat. The device is characterized by an impressive throughput in comparison to the other planar technique, TLC/HPTLC. Although the developed device still needs improvement, it is, in our opinion, a considerable step toward possible automation of this planar separation technique.

Description: We present a video-densitometric quantification method for benzocaine in lozenges. The quantification is based on a derivatisation reaction with 4-dimethylaminobenzaldehyde. Measurements were carried out using a 16-bit flatbed scanner. Benzocaine was separated to a distance of 50 mm in a vertical developing chamber without vapour saturation. We present an RP-18 phase separation on a cyanopropyl plate (Merck, Darmstadt, Germany) using water, CH 3 CN, dioxane, ethanol, and NH 3 (25 %) (8 + 2 + 1 + 1 + 0.05, v/v) as the mobile phase. We also separated benzocaine in a normal phase system on silica gel 60 LiChrospere ® plates (Merck, Darmstadt, Germany) with the mobile phase MTBE /cyclohexane (1 + 1, v/v). The calibration functions for benzocaine in both separations were linear in the range from 1 to 1,000 ng per spot. The range of linearity covers two magnitudes of power because the Kubelka–Munk expression was used for data transformation. In the cyanopropyl-system, the benzocaine amount was quantified as 242.5 ± 18.2 ng in a spot or 6.86 ± 0.52 mg in a single lozenge. The amount of 7.0 mg benzocaine per lozenge was labelled. The combined uncertainty of sample and calibration measurements was statistically calculated using a significance level of α  = 0.05 to a total relative uncertainty of 7.49 %. The separation method is inexpensive, fast and reliable.

Description: In this work we demonstrated analytical capability of micro-planar (micro-TLC) technique comprising one and two-dimensional (2D) separation modes to generate fingerprints of environmental samples originated from sewage and ecosystems waters. We showed that elaborated separation and detection protocols are complementary to previously invented HPLC method based on temperature-dependent inclusion chromatography and UV-DAD detection. Presented 1D and 2D micro-TLC chromatograms of SPE (solid-phase extraction) extracts were optimized for fast and low-cost screening of water samples collected from lakes and rivers located in the area of Middle Pomerania in northern part of Poland. Moreover, we studied highly organic compounds loaded in the treated and untreated sewage waters obtained from municipal wastewater treatment plant “Jamno” near Koszalin City (Poland). Analyzed environmental samples contained number of substances characterized by polarity range from estetrol to progesterone as well as chlorophyll-related dyes previously isolated and pre-purified by simple SPE protocol involving C18 cartridges. Optimization of micro-TLC separation and quantification protocols of such samples were discussed from the practical point of view using simple separation efficiency criteria including total peaks number, log(product Δ hR F ), signal intensity and peak asymmetry. Outcomes of the presented analytical approach, especially using detection involving direct fluorescence (UV366/Vis) and phosphomolybdic acid (PMA) visualization are compared with UV-DAD HPLC-generated data reported previously. Chemometric investigation based on principal components analysis revealed that SPE extracts separated by micro-TLC and detected under fluorescence and PMA visualization modes can be used for robust sample fingerprinting even after long-term storage of the extracts (up to 4 years) at subambient temperature (−20 °C). Such approach allows characterization of wide range of sample components that are present in given extract in high and middle concentration range. Due to protocol simplicity and low cost of analysis this method can be useful for preliminary sample screening.

Description: Ultrathin-layer chromatography (UTLC) differs from high-performance thin-layer chromatography (HPTLC) and from thin-layer chromatography (TLC) in two basis things: the layer thickness, and the migration distances of the analytes. UTLC has a monolithic or a nanostructured stationary silica gel phase bound directly to the glass plates. Layer thickness in UTLC is 10 μm, instead of 100–250 μm in HPTLC. Migration distances are in the range of 1–3 cm for UTLC, instead of 8–10 cm for HPTLC. Therefore, the major advantages of UTLC over HPTLC and TLC are the shorter development times and higher separation efficiency and sensitivity. Moreover, separations on UTLC plates require smaller reagent and sample volumes. However, the UTLC plates are very difficult to manage with the TLC and HPTLC equipment currently available. Therefore, the next challenge in this area is the development of an inexpensive solution with appropriate instrumentation (sensitive optical scanners and sample application systems). UTLC had been used for separations of many compounds, e.g., pharmaceutically active ingredients, pesticides, plasticisers, natural products, and other chemical substances.

Description: Planar chromatography is a very useful tool for analysis of wide range of different mixtures. Thanks to its possibility for rapid separation of large number of samples simultaneously, low solvent consumption and ability to analyse rough material allow to receive precise and reliable results in short time and low cost. Miniaturization of planar techniques brings a lot of advantages, such as shortening distance and time of chromatogram development, and further lowering of solvent consumption. Besides, it often allows to improve separation parameters and raise efficiency of chromatographic system. In this paper, ability of analysis of tropane alkaloids mixture from Datura Inoxia Mill. extract using conventional TLC technique with five micro TLC techniques (short distance TLC, HPTLC, UTLC, OPLC and ETLC) in maximally closed chromatographic conditions was compared in order to present abilities of micro TLC techniques in plant material analysis.

Description: A liquid chromatography–tandem mass spectrometry method has been developed and validated for confirmation and quantification of three amphetamine-type stimulants (ATS) in oral fluid (OF), namely fenproporex (FEN), diethylpropione (DIE) and methylphenidate (MPH), which are misused by some Brazilian drivers and have potentially dangerous consequences in road traffic. In this method, the OF was extracted by a simple and fast liquid–liquid extraction using acetonitrile. Calibration curves were linear throughout the concentration range from 2.5 to 90 ng mL −1 in OF, using internal standard. The lower limit of quantification and the limit of detection were 2.5 and 0.5 ng mL −1 , respectively, for all ATS. Intra- and inter-assay precision and accuracy values were within regulatory limits. The analyte extraction presented poor recoveries, but was considered appropriated for analyses of ATS in OF due to the low limits of detection. Matrix effects and carry over were not detected. All analytes were stable during the whole analytical procedure. The present analytical method was considered simple and sensitive for simultaneous determination of FEN, DIE, and MPH in OF and suitable for clinical and forensic toxicology.

Description: Biomarkers in amniotic fluid (AF) include both non-modified and phosphorylated proteins and can be used in the diagnosis of pregnancy-associated pathologic conditions. In this work, an integrated LC–MS method for selective, sensitive and reproducible analysis of phosphorylation in proteins has been applied to AF. Online digestion of (phospho) proteins was coupled with the selective enrichment on a TiO 2 trap, and separated by RPLC–MS n of both normal and phosphorylated produced peptides. First, an AF-pooled sample was analyzed and a general map of contained proteins and biomarkers was derived in a single run. Then, individual AF samples were analyzed with a downscaled platform with improved sensitivity. On purpose, a trypsin-based CIM ® minidisk was used for online digestion of AF. The obtained protein profile was highly consistent with the one obtained with traditional off-line digestions. Moreover, the use of a specific phospho-enrichment tool followed by LTQ-Orbitrap, enhanced the confidence in the determination of protein phosphorylation state and phosphorylation sites. The phosphorylation sites of IGFBP-1 and osteopontin present in the AF of two individual samples were monitored with a total of 24 and 17 phosphopeptides, respectively, encoding for 12 putative novel phosphorylation sites in addition to known sites.

Description: For the analysis of protein digests, the peak capacity in reversed-phase liquid chromatography is of paramount importance. A univariate method to maximize the peak capacity as developed by Wang et al. (Anal Chem 78:3406–3416, 20 ) has been applied and tested for a monolithic RP-18 silica capillary column. In their method, using model peptides representing a bovine serum albumin digest, the gradient time and temperature are kept constant while the flow rate and eluent strength are varied. Despite our criticism on the fixed starting conditions, a long gradient time leading to an unnecessary long analysis time and a high temperature leading to possible degradation products in the chromatogram, and the peak capacity as the only optimization parameter this fast and simple optimization strategy turns out to be applicable to capillary monolithic columns. Furthermore, the influence of the peak capacity on a second optimization parameter, the MS protein identification score, is examined. The procedure is also used to enhance the performance of two popular types of monolithic capillary LC columns (silica-C18 and poly(styrene–divinylbenzene)) of the same length for the analysis of protein digests. Comparison of both columns show that the calculated chromatographic parameters, like productivity and peak capacity, and identification score for both columns are about the same. For a more complicated nine-protein digest the performance of the silica monolith is slightly better.

Description: This article describes a rapid LC–MS/MS target screening method based on an automated extraction of 5 μL dried blood spots (DBS), two 5 min chromatographic runs on orthogonal phase columns (RP and Hilic) and a data dependent acquisition (DDA) of product ions spectra for the reliable identification of the detected compounds. The extraction step was performed in 2 min by using the LC autosampler itself in 96-well plates. This procedure was evaluated using 22 model compounds frequently encountered in forensic investigations, i.e., cocaine, benzodiazepines, amphetamines, opioids, antidepressants and antipsychotics. These investigations showed that even if the extraction step was reduced to a minimum, the extraction recoveries were satisfactory (median value of 40 %) and allowed for the detection of the model compounds in their therapeutic ranges, with the exception of morphine. Moreover, the use of two different chromatographic columns broadened the number of screening targets to those that behaved poorly under RP conditions, such as amphetamines or glucuronides, while keeping chromatographic gradients very short. This procedure was applied to 34 authentic post-mortem cases. It allowed the detection of 89 % of the compounds that were quantified in the routine procedures and the formal identification of 77 % of the compounds using their product ions spectra. These results were considered more than satisfactory compared to routine screening alone (GC–MS and LC-DAD, 55 % compound identification). The method described in this article is therefore a powerful approach for a fast, reliable and efficient target screening of drugs in forensic and clinical investigations.

Description: An acetonitrile–salt stacking method was established for the assay of lipoic acid (LA) in biological samples. Samples were deproteinized with acetonitrile at a final concentration of 60 % (v/v) and then injected hydrodynamically at 3.45 × 10 3  Pa for 180.0 s. The optimum background electrolyte was found to be 90.0 mmol L −1 pH 9.1 borate buffer. LA could be detected within 35 min at +7.0 kV with satisfactory repeatability (relative standard deviations, RSDs, of migration times and peak areas were both below 10 % for intraday and interday; n  = 6/9) and a relatively low limit of detection of ca. 0.5 μmol L −1 .

Description: To support real biological sample application, a simple, selective and rapid LC–MS method has been developed and validated for the sensitive determination of metoclopramide in rabbit blood, ex vivo permeation studies and pharmaceutical dosage form. LC–MS analysis was performed isocratically on a Zorbax SB-C 18 column with a mobile phase consisting of methanol:ammonium acetate buffer (pH 3.0) 75:25 (v/v) at a flow rate of 0.70 mL min −1 . The outlet of the column was connected to a single quadrupole mass spectrometer with positive mass spectrometric detection. Ions were detected in the positive multiple reaction monitoring mode. The assay was linear over the concentration range of 1.25–200 pg μL −1 with a limit of detection of 0.077 pg μL −1 for standard solutions and 2.5–200 pg μL −1 with a limit of detection of 0.42 pg μL −1 for serum samples. The method is applicable, covering a variety of pharmaceutical and biological studies. Metoclopramide was extracted from rabbit blood by liquid–liquid extraction using ether as the extraction solvent. The reproducibility of the method was found to be between 0.96 and 1.98 % (RSD) values. The proposed method has been extensively validated for the determination of metoclopramide in all working media. The sample preparations, flow rate and run time of the analytical systems are not time consuming. Moreover, for the stability of metoclopramide, the effect of temperature, UV light, H 2 O 2 , HCl and NaOH were also investigated.

Description: Molecular shape selectivity for polycyclic aromatic compounds on a core–shell-type octadecylsilica (ODS) phase at subambient column temperatures was studied in reversed-phase liquid chromatography. The plate height on the core–shell ODS column was relatively stable at subambient column temperatures when compared with that of a conventional ODS column. In order to compare the sample diffusivities in the conventional and core–shell ODS columns, van Deemter plots were prepared. The plate height of the core–shell column was significantly lower than that of conventional column, suggesting an advantageous feature of the core–shell-type stationary phase especially at a high flowrate of the mobile phase. An enhanced molecular shape recognition capability of the core–shell ODS phase was also confirmed at subambient column temperature. The result could be consistent with an improved shape selectivity as normally observed on conventional ODS phases at low temperatures, however, the enhanced molecular shape recognition capability of the core–shell phase enables a good separation between benz[ a ]anthracene and chrysene at subambient column temperatures. Similar improved shape selectivities for terphenyl isomers were also confirmed on the core–shell phase.

Description: As part of increasing research in the field of separation science, there have been many efforts to undertake planar chromatography with more efficient separation and better resolution in the shortest period of time, together with a specificity and a capability to identify more precisely an unknown compound present in a mixture. Ultra-thin layer chromatography (UTLC) is a modern technique which gives separation within 10–30 mm and development in just 1–6 min, with the consumption of less solvent. The stationary phase of UTLC is made up of a silica gel monolithic layer of 10 μm thickness having 3- to 4-nm mesopores and 1- to 2-μm macropores. Glancing angle deposition (GLAD)-UTLC is a modification of UTLC which gives separation within 15 mm distance and in less than 2 min. Anisotropic media of GLAD UTLC gives a unique migration direction effect. UTLC atmospheric pressure–matrix-assisted laser desorption ionizer–mass spectrometery (UTLC-AP-MALDI-MS) is a choice of technique for the identification of an unknown compound in a mixture or an impure form. ULTC-AP-MALDI-MS allows the fast changing of plates, produces more intact protonated molecules, less fragmentation and less entry of chromatographic material, and yielding less complicated spectra than the vacuum condition. Thus, UTLC is a useful technique for very rapidly giving the separation and identification of new components present in mixtures. This review provides a brief overview of UTLC, the stationary phases used for UTLC, and the detection options and applications of UTLC.

Description: The retention of aliphatic hydrocarbons with polar groups has been compared in respect to the separation selectivity changes in reversed-phase high-performance liquid chromatography with C18 stationary phase type and binary water eluent composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The changes in separation selectivity when one modifier is replaced by another in the eluent is explained, taking into consideration molecular interactions of the solutes with components of the stationary phase region, i.e., extracted modifier, and ordering of the stationary phase by the modifier.

Description: A simple, reliable, and rapid RP-LC method has been developed for the determination of some anticancer drugs (daunorubicin, doxorubicin and vincristine sulfate) in their dosage forms and human urine. These compounds are well separated on a C18 column using the mobile phase consisting of a mixture of acetonitrile (50:50; v/v) at a flow rate of 1.5 mL min −1 . The analyte peaks were detected at 235 nm for doxorubicin and daunorubicin, and 220 nm for vincristine. Linearity was obtained in different concentration ranges between 0.10 and 12 μg mL −1 for all compounds. Good sensitivity for all analytes was observed with DAD detection. LOD and LOQ of the method were found satisfying. The proposed method has been extensively validated in accordance with ICH guidelines and obtained results proved that the proposed method was precise, accurate, selective, and sensitive for simultaneous analysis of studied compounds. All analytical procedures including sample preparation, flow rate, and run time were at low levels. Also, p K a values were determined using the dependence of the retention factor on the pH of the mobile phase. The effect of the mobile phase composition on the ionization constant was studied by measuring the p K a at different methanol–water mixtures, ranging between 45 and 60 % (v/v).

Description: Cell membrane chromatography (CMC) is a useful method for the simultaneous isolation and identification of active compounds from natural products. However, it suffers from high cell membrane consumption and is time-consuming to operate. In this study, CMC was performed for the first time with a silica capillary, termed cell membrane capillary chromatography (CMCC). Pancreatic islet cell membranes from a mouse were immobilized onto the capillary inner wall functionalized with aldehyde groups. Scanning electron microscopy observation of the prepared column showed that the cell membrane was evenly coated onto the capillary inner wall. Three model analytes with the pharmacological property of hypoglycemic activity including glibenclamide, glipizide and berberine were tested. They were all retained by the prepared column. Furthermore, the retention factors of the analytes in CMCC correlated well with their pharmacological action. The analytical procedure including washing (to obtain a flat baseline), injection and separation was accomplished within 10 min. The CMCC column was also used for screening active compounds from a natural plant ( Coptis chinensis ). The hypoglycemia activity of active components such as berberine was verified using the method. The results indicated that CMCC is a viable alternative method for screening active compounds from natural products.

Description: Paper spray has been developed as a fast sampling ionization method for direct analysis of raw biological and chemical samples using mass spectrometry (MS). Quantitation of therapeutic drugs in blood samples at high accuracy has also been achieved using paper spray MS without traditional sample preparation or chromatographic separation. The paper spray ionization is a process integrated with a fast extraction of the analyte from the raw sample by a solvent, the transport of the extracted analytes on the paper, and a spray ionization at the tip of the paper substrate with a high voltage applied. In this study, the influence on the analytical performance by the solvent–substrate systems and the selection of the elution methods was investigated. The protein hemoglobin could be observed from fresh blood samples on silanized paper or from dried blood spots on silica-coated paper. The on-paper separation of the chemicals during the paper spray was characterized through the analysis of a mixture of the methyl violet 2B and methylene blue. The mode of applying the spray solvent was found to have a significant impact on the separation. The results in this study led to a better understanding of the analyte elution, on-paper separation, as well as the ionization processes of the paper spray. This study also helps in establishing a guideline for optimizing the analytical performance of paper spray for direct analysis of target analytes using mass spectrometry.

Description: A simple and fast method based on magnetic separation for extraction of pyrethroid pesticides including beta-cyfluthrin, cyhalothrin and cyphenothrin from environmental water samples has been established. Magnetic titanium dioxide was used as sorbent, which was synthesized by coating TiO 2 on Fe 3 O 4 in liquid-state co-precipitation method. The sorbent has been characterized by scanning electron microscopy and Fourier-transform infrared spectrometry, and the magnetic properties were investigated with physical property measurement system. Various parameters affecting the extraction efficiency were evaluated to achieve optimal condition and decrease ambiguous interactions. The analytes desorbed from the sorbent were detected by high performance liquid chromatography. Under the optimal condition, the linearity of the method is in the range of 25–2,500 ng L −1 . The detection limits and quantification limits of pyrethroid pesticides are in the range of 2.8–6.1 ng L −1 and 9.3–20.3 ng L −1 , respectively. The relative standard deviations of intra- and inter-day tests ranging from 2.5 to 7.2 % and from 3.6 to 9.1 % were obtained. In all three spiked levels (25, 250 and 2,500 ng L −1 ), the recoveries of pyrethroid pesticides were in the range of 84.5–94.1 %. The proposed method was successfully applied to determine pyrethroids in three water samples. Cyphenothrin was found in one river water near farmlands, and its concentration was 52 ng L −1 .

Description: The use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, for separation and fast purification of the enzyme uridine diphosphate galactopyranose mutase (UGM) from the cell extract of Escherichia coli overproducing the recombinant enzyme is presented in this feasibility study. UGM is required to produce galactofuranose for the cell wall biosynthesis of many pathogenic microorganisms and represents a very attractive candidate for the development of new antimicrobial drugs. CITP separations were carried out under slightly alkaline pH conditions (8.7), in which UGM enzyme is negatively charged. Significantly simplified proteinous matrix isolated in several fractions by employed preparative CITP procedure with the aid of properly selected discrete spacers was subsequently confirmed by SDS PAGE with Coomassie staining. It was shown that preparative CITP is very effective tool for fast purification of the target enzyme from other proteinous matrix constituents when purification and isolation step lasted 20 min. The enzymatic activity of UGM was confirmed in the sample after the preparative CITP purification step, which is a crucial requirement for further biochemical applications.

Description: An ion-exclusion chromatography (IELC) comparison between a conventional ion-exchange column and an ultra high-performance liquid chromatography (UHPLC) dynamically surfactant modified C18 column for the separation of an aliphatic carboxylic acid and two aromatic carboxylic acids is presented. Professional software is used to optimize the conventional IELC separation conditions for acetylsalicylic acid and the hydrolysis products: salicylic acid and acetic acid. Four different variables are simultaneously optimized including H 2 SO 4 concentration, pH, flow rate, and sample injection volume. Thirty different runs are suggested by the software. The resolutions and the time of each run are calculated and feed back to the software to predict the optimum conditions. Derringer’s desirability functions are used to evaluate the test conditions and those with the highest desirability value are utilized to separate acetylsalicylic acid, salicylic acid, and acetic acid. These conditions include using a 0.35 mM H 2 SO 4 (pH 3.93) eluent at a flow rate of 1 mL min −1 and an injection volume of 72 μL. To decrease the run time and improve the performance, a UHPLC C18 column is used after dynamic modification with sodium dodecyl sulfate. Using pure water as a mobile phase, a shorter analysis time and better resolution are achieved. In addition, the elution order is different from the IELC method which indicates the contribution of the reversed-phase mode to the separation mechanism.

Description: Recently, extracts from the pericarp of mangosteen, Garcinia mangostana L., exhibited various pharmacological properties such as anti-oxidative, anti-inflammatory, anti-bacterial and chemopreventive activities. Albeit it has diverse application, there is little information about its pharmacokinetic aspects. Thus, the present study was undertaken to develop the simultaneous determination of α- and γ-mangostins (α- and γ-MG), major and active compounds, from extracts for the application of pharmacokinetic studies in mice using combined liquid chromatography–tandem mass-spectrometry and microsampling systems. The intra- and inter-validation, precision, accuracy, stability, recovery and matrix effects of α- and γ-MG were conducted in mouse plasma. Based on the developed analytical methods, pharmacokinetic parameters of α- and γ-MG after intravenous and oral administration of mangosteen extract were calculated. In sample preparation steps, the biological samples were deproteinized by acetonitrile and chromatographic separation was accomplished on a C 18 column. The detection was accomplished by multiple-reaction monitoring scanning after electrospray ionization source in the positive ionization mode. The optimized mass transition ion pairs ( m/z ) for quantitation were 411.062 → 354.900, 397.384 → 340.900, and 808.379 → 527.200 for α- and γ-MG and docetaxel (internal standard), respectively. The total run time was 5 min. The results provided a meaningful basis for the preclinical and clinical application of mangosteen extract.

Description: Nineteen impurities in roxithromycin drug substance made in China were separated and identified by HPLC–MS n (TOF and TRAP) for the further improvement of official monographs in Pharmacopoeias. The fragmentation patterns and structural assignment of these impurities were studied. The column was Shim VP-ODS (250 × 4.6 mm, 5 μm). The mobile phase was 10 m mol L −1 ammonium acetate and 0.1 % formic acid aqueous solution-acetonitrile (62.5:37.5). In positive mode, full scan LC–MS was first performed to obtain the m/z value of the protonated molecules and formulas of all detected peaks on Agilent 6538Q TOF high resolution mass spectrometer. LC–MS-MS and LC–MS-MS–MS were then carried out on the compounds of interest on AB SCIEX 4000 Q TRAP™ composite triple quadrupole/linear ion trap tandem mass spectrometer. The complete fragmentation patterns of nineteen impurities were studied and used to obtain information about the structures of these impurities. The structures of nineteen impurities in roxithromycin drug substance were deduced based on the HPLC–MS n data, in which nine impurities were novel impurities.

Description: The decision limit ( CCα ), capability of detection ( CCβ ) and quantification limit ( QL ) are importance performance characteristics in method validation. The TLC-Scanner 3 from Camag provides the possibility to choose the slit dimension of light to determine the peak chromatogram of a substance. The influence of the slit dimension for determination of CCα , CCβ and QL of paracetamol has been carried out. Paracetamol was spotted onto plates of AL-TLC Si G 60 F254 by linomat 4 in the range of 50–400 ng/spot and 10–400 ng/band, then on twin chambers eluted with TAEA (toluene:acetone-ethanol:conc.ammonia, 45 + 45 + 7 + 3 v/v) for 45 mm. Eluted spots were scanned in different slit dimensions at 248 nm. The CCα , CCβ and QL of paracetamol were estimated through the linear regression (LRM) and signal-to-noise (S/N) methods. Slit lengths between 50 and 133 % of the band width of the spots, and with the noise factor of the slit under 2.6, produced good precision measurements of TLC-densitometry between plates, while slit lengths between 50 and 83 % of the band width of the spots introduced a higher sensitivity response of the detector. The estimated CCα , CCβ and QL were determined by how the data were collected, the analytical optical setting, and the usage method for the estimation of both validation parameters.

Description: High-performance thin-layer chromatography (HPTLC) is a separation technique commonly used to identify and quantify compounds in chemical mixtures. Preliminary experiments indicated that Lichrospher silica gel 60 F254s HPTLC sheets were the most suitable for analyzing vitamin B 12 compounds. This study revealed the advantages of miniaturized Lichrospher HPTLC for analyzing authentic vitamin B 12 compounds as short migration distances (5 cm) and short development times (〈45 min) in combination with high separation efficiency and sensitivity (〉25 pmol at 254 nm). The practicability of using miniaturized HPTLC was demonstrated by the separation and identification of vitamin B 12 compounds purified from foods using an immunoaffinity column.

Description: Microfluidic paper-based analytical devices and micro total analysis systems are relatively new group of analytical tools, capable of analyzing complex biochemical samples containing macromolecules, proteins, nucleic acids, toxins, cells or pathogens. Within one analytical run, fluidic manipulations like transportation, sorting, mixing or separation are available. Recently, microfluidic devices are a subject of extensive research, mostly for fast and non-expensive biochemical analysis but also for screening of medical samples and forensic diagnostics. They are used for neurotransmitter detection, cancer diagnosis and treatment, cell and tissue culture growth and amplification, drug discovery and determination, detection and identification of microorganisms. This review summarizes development history, basic fabrication methods, applications and also future development trends for production of such devices.

Description: Thin-film microextraction (TFME) is a format of solid-phase microextraction (SPME) technique which offers improvement of sensitivity without sacrificing time through the increase of available surface area and extractive phase volume. This technique offers significant advantages which make it attractive for many analytical/bioanalytical applications. This review discusses the fundamental principle of TFME and its benefits versus the rod fiber geometry of SPME, and demonstrates the agreements of the experimental data for the available TFME systems with the theoretical concept. The current configurations, coating chemistries, coating preparation methods, and applications for TFME system are reported.

Description: An overview of opportunities of contemporary planar chromatography in pattern recognition and fingerprint analysis is presented. The most used chemometric methods are highlighted and their main advantages and drawbacks are underlined. In addition a cross section of the application of planar chromatographic fingerprinting in food, pharmaceutical, environmental, and forensic analysis is given.

Description: One of the tasks of food law enforcement authorities is to supervise the composition of cosmetics. In the case of mouthwashes, they are likely to contain (labeled or unlabeled) antimicrobial compounds. Conventional analyses, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) only shed light on a compound’s structure, but not on its biological function. In this study, we demonstrate that the task of detecting antimicrobials in mouthwashes can be streamlined using the luminescent bacterium Vibrio fischeri as a biodetector coupled with high-performance thin-layer chromatography (HPTLC) as a pre-separation method. The employment of subsequent conventional techniques could then be restricted to fractions with proven V. fischeri toxicity. Samples were separated in parallel on silica gel and amino layer HPTLC plates, developed with a solvent system containing tertiary butyl methyl ether and n -hexane and dried on a plate heater. After applying V. fischeri onto the HPTLC plate, zones of interest were extracted from a parallel plate and identified by HPLC–UV or GC-mass spectrometry. The reaction of V. fischeri to more than 40 standard substances which might be present in mouthwashes was determined. Based on this information, six commercially available mouthwashes were analyzed. The workflow proved to be viable for an effect-directed screening for antimicrobial compounds. The analysis of mouthwashes revealed that not only declared preservatives are used (sodium benzoate, cetylpyridinium chloride) but also other compounds, especially constituents of essential oils. Because their main purpose is flavoring of the mouthwash, they are summarized as “aroma” (anethole, carvone, menthol, thymol) which is in compliance with legal restrictions.

Description: Chromatographic separation, analysis of in situ radical-scavenging activity, and quantitative analysis of green sea urchin shell pigments were carried out by online HPTLC-DAD and HPTLC-DPPH methods. Methanol:chloroform:acetic acid:water (11:50:5:2, v/v) was selected as the best mobile phase on silica gel plates for the separation of the pigments. Two pigment spots, S1 and S2, were observed on the TLC plates, while echinochrome A was not detected in the pigment extract of green sea urchin shells. Based on the estimated ID 50 values, the antiradical activity was S2 (0.043 μg) 〉 S1 (0.058 μg) 〉 echinochrome A (0.134 μg).

Description: Dried blood spot (DBS) samples are already successfully used in newborn screening and pharmacological analyses. The application of DBS matrix to further metabolomic methods will considerably extend the analytical options for the diagnostics of metabolic diseases. We present an MS/MS based method for the simultaneous extraction and quantification of 188 metabolites from dried blood spots. We provide a sensitive and reproducible method that adapts the Absolute IDQ ™ p180 kit of Biocrates to the DBS matrix for the quantification of metabolites of different substance classes including amino acids, biogenic amines, free carnitine, acylcarnitines, hexoses, glycerophospholipids, lysophosphatidylcholines, phosphatidylcholines, and sphingolipids.

Description: A simple, sensitive and environmentally friendly capillary electrophoresis method for the determination of free sulfate anions in heparin and low-molecular-weight heparin with indirect UV detection is reported. The background electrolyte consists of 20 mM nitrate (pH 5.5) containing 0.2 mM CTAB. The use of nitrate as the probe anion is based on the consideration that nitrate has a very close electrophoretic mobility to that of sulfate, leading to a very narrow peak shape. Moreover, the apparent molar absorptivity of nitrate at 202 nm (8413 L mol −1 cm −1 ) is much higher than that of the commonly used probe anion chromate at 254 nm (2400 L mol −1 cm −1 ). The combination of the narrow peak shape with the high apparent molar absorptivity of the probe ion improved the limit of detection by 2.4 times and the limit of quantitation by 3.2 times. The effect of various CE parameters on the separation of sulfate from chloride was investigated and optimized. The method displays linearity in the range from 0.05 to 20 mM. The RSD % for intraday ( n  = 6), and interday ( n  = 3) repeatability in terms of migration times and peak areas were all less than 3 %. The limit of detection ( S / N  = 3) and limit of quantitation ( S / N  = 10) for sulfate were determined as 18 and 53 µM, respectively. The method was successfully applied for the quantification of sulfate anions in heparin and low-molecular-weight heparins.

Description: In this work, a new immobilized enzyme reactor (IMER) containing gelatinase enzyme (a mixture of MMP-2 and -9) was developed and then inserted into a HPLC system to evaluate the inhibitory potency of novel compounds towards gelatinase. The gelatinase was immobilized on an epoxy-silica column by in situ technique. To monitor the performance of the newly developed method, the kinetic constant ( K m ) of immobilized gelatinase as well as the inhibition constant (IC 50 ) of a known inhibitor, i.e., LY52, were determined and these results were compared with those obtained by classical succinyl-gelatin assay. The IMER-HPLC system was subsequently applied to the screening of 39 synthetic compounds. The inhibitory potential of these inhibitors (expressed in IC 50 ) was found to be in agreement with the literature.

Description: Based on the successful graft of P( N -isopropylacrylamide-4-vinylphenylboronic acid) [P(NIPAAm-co-VPBA)] on glass substrates by surface-initiated atom transfer radical polymerization, a microfludic chip with the performance of thermoresponsive boronate affinity was fabricated and was used for capture–release of the cis-diol biomolecule adenosine. The copolymers modified successfully on the surface of glass substrate were characterized by Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. The results demonstrated that, by simply adjusting the temperature from 4 to 55 °C, the microchip grafted with P(NIPAAm-co-VPBA) was specific to the cis-diol-containing molecules, and can selectively and quickly realize the capture and release of the cis-diol biomolecule adenosine from the mixture of adenosine and deoxyadenosine, which was validated by high performance liquid chromatography/electronic spray ion–ion trap mass spectrometry (HPLC/ESI–ion trap MS). Moreover, less than 10 min was taken from injection to the collection of the sample at the end of the channel. With further development, the P(NIPAAm-co-VPBA)-grafted microchip with high selectivity and repeatability may be chosen as a promising tool for the enrichment of the cis-diol-containing biomolecules, such as glycoproteins, carbohydrates, glycosylated peptides, nucleosides, etc.

Description: A simple and accurate HPLC–DAD method has been developed for simultaneous determination of tocopherols (α, (β + γ), and δ-tocopherols), phytosterols (campesterol, β-sitosterol, and stigmasterol), and squalene in vegetable oil deodorizer distillates (VODD). Chromatographic separation was performed on a C 18 column with a mobile phase gradient prepared from 98:2 ( v / v ) methanol–water and isopropanol, at a flow rate of 1.0 mL min −1 , in 33 min, at 30 °C. Tocopherols, phytosterols, and squalene were detected at 296, 210, and 210 nm, respectively. Recoveries ranged from 92.4 to 103.8 %. Limits of quantification were in the range 0.50–2.50 μg mL −1 . The proposed method was successfully used for simultaneous analysis of tocopherols, phytosterols, and squalene in VODD.

Description: A simple chiral liquid chromatographic method for the enantiomeric purity of methyl (2S)-(2-chlorophenyl)-(2-(thiophen-2-yl)ethylamino) acetate—a clopidogrel intermediate—is proposed. Chromatographic separations are carried out isocratically on a chiral amylose-based column (ChiralPAK AD-H) using a mobile phase consisting of hexane/isopropanol/diethylamine, 90/10/0.1, v/v/v. Under the optimum conditions, the resolution between the two isomers was found to be more than 4.5. The method was developed and validated in terms of linearity, precision and accuracy, limit of detection (LOD) and quantitation (LOQ) and robustness. The proposed approach has been successfully applied to bulk samples for evaluating enantiomeric purity.

Description: A novel stability-indicating high-performance liquid chromatographic (HPLC) method has been developed and validated for the analysis of the impurities A–G in cabazitaxel. Chromatographic separation was achieved on a Welch Xtimate™ C18 (250 × 4.6 mm; 5 μm) column, using the mixture of 0.02 mol L −1 sodium dihydrogen phosphate buffer pH 3.0 (pH value was adjusted with phosphoric acid) and acetonitrile as mobile phase by gradient elution with a flow rate of 1.0 mL min −1 , and UV detection was performed at 230 nm. The column temperature was maintained at 40 °C by column oven. The method was validated according to the International Conference on Harmonization (ICH) guidelines. Linearity ( r  〉 0.9990) was observed over the concentration ranges of 25.0–1500.0, 31.5–1518.0, 74.9–1796.8, 65.6–1573.2, 59.4–1425.6, 22.2–1332.0, 1.3–1570.8, 30.8–1476.0 ng mL −1 of cabazitaxel and its impurities A–G, respectively. The RSD value of the recovery for each impurity was 〈5.0 % ( n  = 9). The method was found simple and rapid with good specificity and robustness, which can be suitable for the determination of the impurities in cabazitaxel.

Description: A highly selective, sensitive, and reliable high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N -methyl metabolite in mouse sera. LE300, its N -methyl metabolite, and verapamil (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis using a deproteinization procedure was performed by injecting an aliquot of the supernatant into the chromatographic system. Chromatographic separation was achieved on a reversed-phase Spherisorb Cyano (CN) column with a mobile phase consisting of acetonitrile:50 mM phosphate buffer pH 3.5 (70:30, v/v) pumped at a flow rate of 1.0 mL min −1 . Regression analyses showed excellent linearity ( r  = 0.999) for concentrations of LE300 ranging from 4 to 500 ng mL −1 and for concentrations of its N -methyl metabolite of 6–600 ng mL −1 . The HPLC-FLD method had limits of detection of 1.6 ng mL −1 for LE300 and 2.4 ng mL −1 for its N -methyl metabolite in mouse sera. The precision results, expressed as the intraday and interday relative standard deviation (RSD) values, ranged from 0.65 to 2.85 % (repeatability) and from 0.37 to 2.62 % (intermediate precision) for LE300 and its N -methyl metabolite, respectively; these values are in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study. The mean values of T max and C max were 2 h and 25.03 ± 5.60 ng mL −1 for LE300 and 3 h and 19.92 ± 2.88 ng mL −1 for its N -methyl metabolite, respectively.