Authors:Gül Fidan Yenel Avci, Beril Anilanmert and Salih Cengiz
The analysis of trace levels of explosives in post-blast debris is critical in homeland security, environmental analysis, and crime scene forensic investigations. A fast and a selective determination method with high recovery was developed for the common explosives 2,4,6-trinitrotoluene (TNT), 3,5-trinitro-1,3,5-triazacyclohexane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in soil, using liquid chromatography—tandem mass spectrometry (LC—MS/MS). An easy and practical sample preparation method was developed using 4.00 mL acidified acetone with 0.25% HCl. After the easy evaporation of acetone extract, 10 min LC—MS/MS analysis provided a clear separation in column. Short duration of the whole procedure allows the use of this method in routine analysis. As a result of the analysis performed in spiked soils in 50.0, 100.0, and 250.0 ng g−1 concentrations, high recoveries such as 100.4 (±8.8)% for RDX, 96.9 (±10.5)% for HMX, and 97.6 (±13.9)% for TNT were obtained. Limit of detection (LOD) and limit of quantification (LOQ) values obtained from the analysis of the spiked soils were 4.3 ng g−1 and 7.00 ng g−1 for RDX, 6.8 ng g−1 and 10.0 ng g−1 for HMX, and 18.9 ng g−1 and 38.0 ng g−1 for TNT, respectively. The Horwitz Ratio (HorRat) calculation was used to evaluate if the inter-day and inter-analyst precisions were in the acceptable limits. The method was successfully applied to three artificial explosion samples for detection of explosives.
Selection of adsorbent for the development of purification process for biomolecules is crucial due to the requirement of large number of binding sites and adsorption area. Considering this, porous structure with high charge density is selected as an adsorbent for macromolecule purification. Such selection may provide high static binding capacity but causes loss of separation performance due to improper porosity of adsorbent in comparison to solute sizes involved. To address this problem for the screening of adsorbent, this work reports adsorbent selection procedure on the basis of adsorbent pore diameter (dp), solute hydrodynamic dimensions (RH), and flow velocity in support of binding capacity. Towards that end, this study evaluated the pore accessibility performance of varying characteristics adsorbents using tracers like acetone, lysozyme, and bovine serum albumin (BSA) by designing nonbinding conditions. All screened adsorbents showed certain loss of total surface area depending on the solute dimensions and pore size. Sepharose type adsorbents showed accessible area loss up to 25% for lysozyme and 50% for BSA. Sepabeads type showed 30% loss, while macroporous UNO type showed only 7% loss of surface area for lysozyme. The study correlates accessibility with size ratio β (dp/RH). The value of β > 38 is found to be required for the accessibility of total pore area and optimum separation performance of ion exchangers investigated. Accessibility and β provide useful information for the selection of suitable adsorbent for the purification of macromolecules.
Authors:Aida Begic, Ana Djuric, Borko Gobeljic, Ivana Stevanovic, Vera Lukic, Ivan Stanojevic, Milica Ninkovic, Luciano Saso, Danilo Vojvodic and Mirjana Djukic
The aim of our work was to optimize and apply simple high-performance liquid chromatography method with ultraviolet detection (HPLC—UV) for simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in biological matrix (specifically, the rat liver tissue was used herein), since the ratio between oxidized and reduced glutathione forms (GSSG—GSH) has been recognized as an important biological marker of oxidatively depleted GSH in oxidative stress (OS)-associated diseases and poisonings. An isocratic chromatographic separation of GSH and GSSG (2.8 min and 6.3 min, respectively) was performed with the mobile phase consisted of sodium perchlorate solution (pH adjusted to 2.8) at flow rate of 1 mL min−1, detection set at 215 nm, and column temperature of 40 °C. The method offers short run time, linearity in the range of 0.01—200 μM concentration for both compounds (R2 = 1), low limits of detection and quantification (GSH: 0.18 μM and 0.56 μM, GSSG: 0.52 μM and 1.58 μM, respectively), precision, accuracy (bias < 2%), and high reproducibility.
Through suitable sample handling, an overestimation of GSSG was prevented. High recovery (>99%) was achieved. The method was successfully applied for the analysis of GSH and GSSG in liver homogenates of Wistar rats intraperitoneally exposed to cadmium (Cd) (1 mg kg−1 CdCl2/21 days). Regardless of other Cd-mediated hepatotoxicity mechanisms, herein, we have exclusively interpreted/emphasized oxidative GSH depletion.
The presented method is acceptable for a routine analysis of GSH and GSSG in biological matrix, while the calculated ratio GSSG—GSH is considered as a valuable OS marker.
Authors:Gui-Hua Gu, Da-Jian Yang, Shi-Yun Wang, Wei Zeng, Wei Wang and Jing-Song Yuan
A high-performance liquid chromatography—diode-array detection method was developed and validated to determine simultaneously eleven major alkaloids in Corydalis decumbens (Thunb.) Pers. The alkaloids detected were corlumidine, protopine, coptisine, tetrahydrojatrorrhizine, palmatine, berberine, sanguinarine, papaverine hydrochloride, tetrahydropalmatine, bicuculline, and corydaline. Chromatographic separation was achieved using a C-18 column with a mobile phase composed of A (0.2% acetic acid solution, adjusted with triethylamine to pH 5.0) and B (acetonitrile), with stepwise gradient elution. Ultraviolet diode-array detection was used; chromatograms were examined at the wavelength of 280 nm. The regression equations showed a good linear relationship between the peak area of each marker and concentration (r = 0.9994–0.9999). The recovery values ranged between 93.66% and 100.54%. The method was fully validated with respect to detection and quantification limits, precision, reproducibility, and accuracy. The described high-performance liquid chromatography (HPLC) method was successfully used for the differentiation and quantification of the eleven major alkaloids in C. decumbens (Thunb.) Pers. and can be considered an effective procedure for the analyses of this important class of natural compounds.
Authors:Y. Zhou, Y. Yang, X.L. Li, Z.Y. Chen, Q.B. Liu, X.L. Zhu and J. Yang
An efficient and sensitive analytical method based on precolumn derivatization and gas chromatography—mass spectrometry—selected ion monitoring (GC—MS—SIM) was proposed and validated for analysis of two cembrenediols (CBDs) which are α-cembrenediol and β-cembrenediol in tobacco samples. CBDs in tobacco samples were extracted by sonication with 50 mL dichloromethane for 10 min before derivatized with 2:3 (v/v) bis(trimethylsilyl)trifluoroacetamide (BSTFA)—pyridine at 20 °C for 100 min. CBDs’ level in tobacco samples was analyzed by GC—MS—SIM and quantified by the internal standard method. The linear range for α-CBD and β-CBD was 13.6–554.6 μg mL−1 and 4.11–162.6 μg mL−1, and the correlation coefficients of both were 0.9998. The limit of detection (LOD) and limit of quantification (LOQ) of α-cembrenediol and β-cembrenediol were 0.40 μg g−1 and 1.34 μg g−1, and 0.27 μg g−1 and 0.90 μg g−1, respectively. Average recoveries of α-CBD and β-CBD were 94.4–99.9% and 91.9–98.2% while the relative standard deviations (RSDs, n = 5) were ranged from 2.67 to 5.6% and 2.04 to 4.22%, respectively. This proposed analytical method has been successfully applied to analyze CBDs in tobacco samples.
Authors:Camila C. Pires, Moacir Kaiser, Lauren D. Grünspan, Fabiano Barreto, Adrine Innocente, Simone Gnoatto, João V. Laureano, Bibiana V. Araujo, Teresa Dalla Costa and Leandro Tasso
An accurate and reliable LC—MS/MS assay was firstly developed and validated for quantitative determination of a new antimalarial prototype drug, 3β-hydroxyurs-12-en-28-oic acid (LAFIS 01), in rat plasma. Dexamethasone was employed as internal standard. Simple protein precipitation by acetonitrile for the sample preparation was used. Effective separation was achieved with Phenomenex Luna C18 (50 × 2 mm, 5 μm) column. The mobile phase consisted of (A) water and (B) acetonitrile, both containing 0.1% acetic acid, delivered by gradient elution. The column temperature was maintained at 40 °C. The LAFIS 01 was monitored by electrospray ionization interface, operating in the negative mode (ESI−) in multiple reactions monitoring (MRM), checking the transitions 455 > 455 for LAFIS 01 and 451 > 361 for the IS. Once LAFIS 01 demonstrated low fragmentation by collision-induced dissociation (CID) nonpresenting abundant high-intensity fragments to meet the desired concentration levels quantification, only pseudomolecular ion was monitored. The flow rate was 500 μL min−1. The lower limit of quantitation achieved was 10 ng mL−1 and linearity was observed from 10 to 500 ng mL−1. The relative standard deviation (RSD) values of the intra- and inter-assay precisions of the method were below 8.42 and 7.94%, respectively. The accuracy ranged from 92.05 to 102.94%. The extraction recovery of LAFIS 01 and IS was up to 90%. The method showed linearity, precision, accuracy, sensitivity, and stability required to quantify LAFIS 01 in preclinical pharmacokinetic study.
Authors:Narendra A. Gajbhiye, Jayanti Makasana, Tushar Dhanani and Raju Saravanan
Aegle marmelos Correa (Bael tree) is a medicinal fruit tree, widely used for healing purposes in various systems of medicines. Coumarins and alkaloids present in various parts of bael tree including roots and fruit pulp are the primary active constituents implicated for its biological activities. An efficient liquid chromatography–electrospray ionization—tandem mass spectrometry (LC—ESI—MS/MS) method was developed for identification and simultaneous determination of four coumarin derivatives, namely, umbelliferone, psoralene, marmin, and imperatorin, and an alkaloid, skimmianine, in root and stem bark of A. marmelos. The chromatographic separation of analytes was performed on Altima C18 (50 × 4.6 mm, 3 μm) column using methanol and 0.1% acetic acid in water (54:46, v/v) as the mobile phase under isocratic conditions. The LC–MS/MS parameters were optimized in the positive ionization mode using electrospray ionization source. The quantification of the analytes was performed using multiple reaction monitoring (MRM) transitions, umbelliferone (m/z 163.1 → 107.1), psoralene (m/z 187.2 → 131.1), marmin (m/z 333.5 → 163.2), imperatorin (m/z 271.1 → 203.1), and skimmianine (m/z 260.1 → 227.0). The extraction method was standardized for optimum yield of coumarin derivatives and the alkaloid in different extraction solvents. Higher yield of the analytes was found in methanolic extracts in comparisons to petroleum ether, chloroform, ethyl acetate, ethanol, and water. The method was validated for linear range, intra- and inter-batch precision and accuracy. The distribution of coumarin derivatives and an alkaloid was found to vary significantly in different plant samples, and their concentration was much higher in roots as compared to stem bark.
Authors:C.M.M. Silveira, C.M. Della Lucia, M.R. Pirozi, T.A. Montini and H.M. Pinheiro-Sant’Ana
This study aimed to optimize and validate methods for the analysis of thiamin and folic acid in fortified rice, pure and mixed to the milled rice (raw and cooked). The analysis was performed by high-performance liquid chromatography coupled to a diode array detector (HPLC—DAD). Different mobile phases were tested. Different ratios of organic modifier, pH ranges, triethylamine concentrations, and flow rates were used. For the validation, tests of recovery, repeatability, linearity, limit of detection (LOD), and limit of quantification (LOQ) were performed. The optimized methods showed good resolution of vitamins’ peaks, excellent recovery (82.6 to 104%), repeatability with relative standard deviation of peak areas, and retention times less than 10% and high coefficients of determination (0.9998 for thiamin and 0.9997 for folic acid). The LOD and LOQ were 0.00193 μg and 0.0193 μg for thiamin and 0.000934 μg and 0.00934 μg for folic acid. The optimized methods demonstrated reliability and sensitivity in the detection and quantification of these vitamins in fortified rice, pure and mixed to milled rice (raw and cooked). Furthermore, the methods were performed in isocratic mode, with short run time (<13 min), reflecting positively on the economy of reagents and analysis times.
Authors:P. Zhu, S. Wang, J. Wang, L. Zhou and P. Shi
In order to assess the contribution of adenosine triphosphate and its metabolites to the cellular metabolism process in Saccharomyces cerevisiae, it is very important to simultaneously determine the relative concentrations of ATP and its metabolites. In this study, a fast, simple reversed-phase high-performance liquid chromatography with high selectivity was developed to simultaneously measure adenosine triphosphate and its metabolites (adenosine diphosphate, adenosine monophosphate, and cyclic adenosine monophosphate) in yeast. The method was performed under the gradient grogram, and the detection was monitored at 254 nm. Analysis was achieved within 25 min. The four components can be detected with linear response over the concentration range from 1 to 100 mg L−1 with excellent correlation coefficients (r2) > 0.999. The recovery of the four analytes was 92.9%, 90.4%, 99.1%, and 105.1%, respectively. To demonstrate the good analysis of yeast samples, changes in the four adenine nucleotides levels caused by caloric restriction in yeast were determined. It is expected that the current method may contribute to further metabolomics and system biology investigations of yeast.
Authors:Y.T. Kamal, Mhaveer Singh, Shahana Salam and Sayeed Ahmad
The alternative system of medicines like Unani and Ayurveda is preferred worldwide nowadays due to its therapeutic efficacy, lower side effects, holistic approach, psychological dimensions, and qualitative action of weather and seasonal requirement. A simple procedure is described for the simultaneous extraction and estimation of piperlongumine and piperine in a well-known Unani polyherbal formulation using reversed-phase high-performance liquid chromatography (HPLC). The chromatography was carried out on reversed-phase C18 (250 × 4.6 mm) column with a mobile phase containing acetonitrile—water (50:50 v/v). Detection was accomplished with ultraviolet (UV) detection at λ = 325 nm. The flow rate was kept as 1.0 mL−1. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines for accuracy (94.4–105.0%), precision (0.37–2.17% RSD), and robustness (0.14–2.11% RSD). The limit of detection (LOD) values were found as 30 and 10 ng mL−1, while limit of quantification (LOQ) was 100 and 30 ng mL−1 for piperlongumine and piperine, respectively, which proved the sensitivity of the method satisfactory enough for accurate analysis of the both piperlongumine and piperine.
Authors:Ankita Misra, Amit Srivastava, Sharad Srivastava and A.K.S. Rawat
Commelina benghalensis (Commelinaceae) is widely used as traditional and folklore medicine in India. In the present study, a reverse-phase high-performance liquid chromatography—photodiode array detection (RP-HPLC—PDA) method was developed for the separation, identification, and quantification of bioactive phenolics. Antioxidant potential was also accessed to validate the presence of identified markers. Method was developed on C18 column with 1% formic acid (in water) and acetonitrile as solvent system, and data acquisitions were achieved at wavelength of 285 nm. The developed method was also validated for accuracy, precision, robustness, limit of detection and quantification (LOD and LOQ), repeatability, and recovery according to International Conference on Harmonization (ICH) guidelines. In this method, five phenolics, viz., protocatechuic acid (0.033%), vanillic acid (0.262%), ferulic acid (0.365%), apigenin (0.126%), and kaempferol (0.544%), were quantified in linearity range of 0.2–1.0 μg with correlation coefficient of more than 0.9949. Relative standard deviation (RSD) (%), LOD, LOQ, and recovery (%) are within the acceptable limit. Besides that, methanolic extract shows the inhibition (%) range from 24.45 to 68.75% at 0.02–0.12 mg mL−1. IC50 of extract was observed at 46.75 μg mL−1, suggesting the promising activity in methanol extract. Hence, the proposed method for simultaneous quantification of five bioactive phenolics in the tuber of C. benghalensis using HPLC–PDA detection under the specified conditions is specific and accurate, and validation proves its selectivity and reproducibility.
Authors:Panadda Phattanawasin, Jankana Burana-Osot, Uthai Sotanaphun and Anchalee Kumsum
A simple and rapid thin-layer chromatographic (TLC)–image analysis
method was developed for stability-indicating studies and determination of andrographolide in bulk drug and in Andrographis paniculata formulations. Good chromatographic separation of andrographolide and the degradation products under various stress conditions was achieved on a silica gel G60 F254 TLC plate with the use of two mobile phases, dichloromethane—toluene—ethanol (6:3:1, v/v/v) and toluene—ethyl acetate—formic acid (5:3.5:1.5, v/v/v) and p-anisaldehyde as visualization reagent. Image analysis of the scanned TLC plate was performed by Sorbfil TLC Videodensitometer, UN-SCAN-IT, JustTLC, and ImageJ software to measure the quantity of the dark bluecolored band of andrographolide on a TLC plate. The TLC—image analysis method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, and robustness and was also applied for determination of the amount of andrographolide in A. paniculata formulations and the content of andrographolide remained in the samples under forced degradations. The analytical results determined by the TLC—image analysis method, TLC—densitometry, and high-performance liquid chromatography (HPLC) methods were compared and found to be not significantly different.
Dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography equipped with flame photometric detection (GC—FPD) is introduced to extract and determine the fifteen organophosphorus pesticides (OPPs) in hawthorn (Crataegus pinnatifida var. major) juice samples. Some parameters affecting the DLLME efficiency, such as the type and volume of extraction and dispersive solvents, extraction time, and salt concentration, were studied and optimized. The optimized extraction and dispersive solvents are trichloroethane and acetonitrile, respectively. Good linearity was ranged from 0.5 to 100 ng mL−1 with correlation coefficients from 0.9991 to 0.9999. The limits of quantification (LOQs) varied from 0.15 to 0.3 ng mL−1, and the limits of detections (LODs) ranged from 0.05 to 0.1 ng mL−1. The relative standard deviations (RSDs) varied from 1.0% to 2.8% (n = 6) with the relative recoveries in the range of 85.6–119.1%. The method was successfully applied in the determination of OPPs in ten commercial hawthorn juice samples. The chlorpyrifos was detected in one sample.
Authors:Georg J. Lichtenegger, Manuel Maier, Johannes G. Khinast and Heidrun Gruber-Wölfler
An integrated process including continuous-flow syntheses directly coupled to product isolation via continuous crystallization is presented. For the synthesis part, Ce0.495Sn0.495Pd0.01O2—δ was used as heterogeneous catalyst in a custom-made packed-bed reactor (the so-called “Plug and Play” reactor) for continuous Suzuki—Miyaura crosscouplings of various para- and ortho-substituted bromoarenes with phenylboronic acid using environmentally friendly aqueous ethanolic mixtures as reaction solvents. The reactions were stable for up to 30 h without any detectable catalyst deactivation. The desired biaryl products were obtained in gram scale with good to excellent yields and high selectivity. For three methyl-, ketyl-, and nitrile-functionalized biphenyl products, isolation was done using water as antisolvent in an integrated crystallization process as continuous downstream protocol. The desired products could be isolated with high purity and with yields of up to 95% for the overall process.
Authors:Q. Bui, J. Sherma, B. Fried and J.K. Hines
Prions of the baker’s yeast Saccharomyces cerevisiae allow for the inheritance of complex traits based solely on the acquisition of cytoplasmic protein aggregates and confer distinctive phenotypes to the cells which harbor them, creating heterogeneity within an otherwise clonal cell population. These phenotypes typically arise from a loss-of-function of the prion-forming protein that is unable to perform its normal cellular function( s) while sequestered in prion amyloid aggregates, but the specific biochemical consequences of prion infection are poorly understood. To begin to address this issue, we initiated a direct investigation into the potential control that yeast prions exert over fungal lipid content by utilizing the prions [URE3] and [PSI+], the first two prions discovered in yeast. We utilized silica gel high-performance thin-layer chromatography (HPTLC)—densitometry to conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether—diethyl ether—acetic acid (80:20:1) mobile phase, phosphomolybdic acid (PMA) detection reagent]; steryl esters and squalene (hexane—petroleum ether—diethyl ether—acetic acid (50:20;5:1), PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform– diethyl ether—acetic acid (65:25:4.5), cupric sulfate—phosphoric acid) in otherwise clonal prion-infected ([PSI+] or [URE3]) and prion-free ([psi−] or [ure-o]) cells in two growth phases: log-phase and stationary phase. Our analysis revealed multiple statistically significant differences (p < 0.00625) between prion-infected and prion-free cells. Interestingly, prion-induced changes varied dramatically by growth phase, indicating that prions exert differential influences on cell physiology between log and stationary growth. Further experimental replication and extension of the analysis to other prions is expected to resolve additional physiological effects of prion infection. This investigation demonstrates that HPTLC—densitometry is an effective method for studying prion-induced alterations in lipid content in yeast.
Authors:E.M. Yilmaz, Z. Aydoğmuş and H.Y. Aboul-Enein
A precise and sensitive reversed phase high-performance thin-layer chromatography (RP-HPLC) method was developed for the determination of nilotinib (NTB) in spiked plasma, urine, and pharmaceutical capsule formulation. The method was based on derivatization NTB with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in the borax buffer (pH 9). The method employs an isocratic elution using acetonitrile and 10 mM orthophosphoric acid (40:60 v/v) as a mobile phase and an C18 column (4.6 mm × 250 mm, 5 μm, Waters Symmetry), with a fluorescence detector (λex: 447 nm, λem: 530 nm). The method validation was performed with respect to linearity, recovery, accuracy, precision, and stability. The linear ranges were 100–600 ng mL−1 in standard solution, plasma, and urine. Correlation coefficients (r2) were higher than 0.9997 for all of the analytes, indicating good linear relationship. The percentage recovery was 87.89% for plasma, 95.35% for urine, and 96.07% for capsules.
Authors:J. Wang, L. Jia, W. Wei, S. Lang, P. Shao and X. Fan
This paper develops an instrumental analytical approach for detection of fourteen polycyclic aromatic hydrocarbons (PAHs) in edible oil samples using gel permeation chromatography (GPC) and ultra-high performance liquid chromatography (UHPLC) coupled with diode array detector (DAD), and fluorescence detector (FLD). The GPC was used to remove triglycerides from edible oil samples. The extracted samples were then detected using UHPLC—DAD—FLD. In order to obtain good separation and high reproducibility, the UHPLC—DAD—FLD experimental condition was optimized. The PAHs including three groups of isomeric PAHs can be separated completely in 12 min using BEH Shield RP 18 column with a suitable gradient elution program. The mean recoveries were in the range of 73–110% with an acceptable reproducibility (RSD < 10%, n = 3). During real sample analysis, the method can decrease the chance of false positives with both DAD and FLD being used simultaneously. The results indicate that the approach is simple, easy, and acceptably reproducible, thereby showing great potential as a method for detection of fourteen PAHs contained in edible oil samples.
Authors:Effat Souri, Siavash Mottaghi, Mohammad Zargarpoor, Reza Ahmadkhaniha and Hassan Jalalizadeh
Rivaroxaban is an inhibitor of factor Xa, which is used as an oral anticoagulant for the prevention of thromboembolism. The objective of this study was to develop a stability-indicating high-performance liquid chromatographic method for the quantitative determination of rivaroxaban in pharmaceutical dosage forms. Rivaroxaban was subjected to acidic, basic, oxidative, photolytic, and thermal conditions for forced stress degradation studies. Considerable degradation was observed in all stress degradation tests. Rivaroxaban and its degradation products were separated on a Nova-Pak C8 column utilizing a mixture of acetonitrile and KH2PO4 50 mM (pH 3.0) (40:60, v/v) as the mobile phase, and the chromatogram was recorded at 270 nm using a general ultraviolet (UV) detector.
The developed method was linear over the concentration range of 1–50 μg mL−1 showing acceptable within-day and between-day precision and accuracy values (CV <2% and Error <2%). The dissolution profile of rivaroxaban tablets was also studied in the presence of a surfactant using optimized conditions. The validated method was successfully used for the determination of rivaroxaban in dosage forms and also in dissolution medium indicating the specificity of the assay method.
Authors:M. Crevar-Sakač, Z. Vujić, Z. Vujčić, B. Marković and D. Vasiljević
A simple and sensitive liquid chromatography—tandem mass spectrometry method was developed for the quantification of atorvastatin, ortho-hydroxyatorvastatin, para-hydroxyatorvastatin, and atorvastatin lactone in rat plasma. Solid-phase extraction was used for preparation of samples. Rosuvastatin was chosen as an internal standard. Chromatographic separation was achieved on ZORBAX Eclipse C18 Analytical, 4.6 × 100 mm (3.5 μm) column with a gradient mobile phase composed of acetonitrile and 0.1% acetic acid, at a flow rate of 400 μL min−1. The column was kept at constant temperature (25 °C), and autosampler tray temperature was set at 4 °C. The following selected reaction monitoring (SRM) transitions were selected: (m/z, Q1 → Q3, collision energy) atorvastatin (559.47 → 440.03, 22 eV), atorvastatin lactone (541.36 → 448.02, 19 eV), ortho-ohydroxyatorvastatin (575.20 → 440.18, 20 eV), para-hydroxyatorvastatin (575.54 → 440.18, 20 eV), and rosuvastatin (482.25 with selected combination of two fragments 257.77, 31 eV, and 299.81, 35 eV) in positive ion mode. The method was validated over a concentration range of 0.5–20 ng mL−1 for ortho-hydroxyatorvastatin and para-hydroxyatorvastatin and 0.1–20 ng mL−1 for atorvastatin and atorvastatin lactone with excellent linearity (r2 ≥ 0.99). This method demonstrated acceptable precision and accuracy at four quality control concentration levels. The detection limits were 0.1 and 0.13 ng mL−1 for orth-ohydroxyatorvastatin and para-hydroxyatorvastatin, respectively, and 0.05 ng mL−1 for atorvastatin and atorvastatin lactone. All analytes were found to be stable at examined conditions. Validated method was applied for determination of atorvastatin and its metabolites in plasma of experimental animals.
Authors:Eric Mielke, Dominique M. Roberge and Arturo Macchi
Based on previous work studying complex microreactors, it was desired to further improve the mixing efficiency by varying the mixing unit design for fast liquid—liquid reactions. Different flow regimes were studied, including slug flow, parallel flow, and drop flow. The two-phase hydrolysis of 4-nitrophenyl acetate in sodium hydroxide solution was used to evaluate the overall volumetric mass transfer coefficients (Korga) as a function of the average rate of energy dissipation (ε) for each microreactor design and all flow regimes. The liquid—liquid systems investigated used n-butanol or toluene as the organic phase solvent and a 0.5-M NaOH aqueous solution. The use of surfactant was also investigated with the toluene—water system. All microreactor geometry designs were based on contraction—expansion repeating units with asymmetric obstacles to aid the breakup of slugs and desynchronize the recombination of split streams. The investigated designs were chosen to avoid the formation of the parallel flow regime, contrary to curvature-based mixing-unit designs. The microreactor design can then be optimized to reduce the ε required to reach drop flow, since Korga has been found to be constant at equal ε for a given solvent system in this flow regime, regardless of the reactor selection. Additionally, the “3/7th” scaleup rule was applied and confirmed with the LL-Triangle mixer. It was found that, for low interfacial-tension systems (i.e., n-butanol—water), the onset of drop flow occurred at a lower ε for the LL-Triangle mixer when compared with the Sickle or LL-Rhombus mixers.
Authors:A. Ochoa, J. Fechine, J.C. Escalona, J. García, S.G. dos Santos and M. Sobral da Silva
Excoecaria lucida Sw. is an evergreen shrub widely distributed in Cuba and throughout the Caribbean region. In spite of its extended traditional use as antiasthmatic and antimicrobial by the local population, scientific reports on the species are almost nonexistent. This paper focuses on the isolation and characterization of compounds present in the crude extract of E. lucida Sw. leaves through the combined use of chromatographic and spectroscopic techniques (medium pressure liquid chromatography, thin-layer chromatography, gas chromatography, nuclear magnetic resonance 1H, and mass spectrometry). A total of 15 nonpolar substances were identified in the four main fractions obtained; some of these substances could be related with the antimicrobial properties attributed to the species.
An accurate and rapid liquid chromatography–electrospray ionizaion– tandem mass spectrometry (LC—ESI—MS/MS) analytical method was developed and validated for the simultaneous determination of antcins A, B, C, H, and K, dehydroeburicoic acid, and 4,7-dimethoxy-5-methyl-1,3-benzodioxole in the extract and capsule of Antrodia cinnamomea (AC) fruiting body. These seven signature compounds were ionized using an electrospray ion source and analyzed by a triple-quadrupole mass analyzer under a multiple reaction monitoring (MRM) mode. The MRM transitions of m/z 453/409 (antcin A), m/z 467/408 (antcin B), m/z 469/425 (antcin C), m/z 485/413 (antcin H), m/z 487/407 (antcin K), m/z 467/337 (dehydroeburicoic acid), and m/z 197/139 (4,7-dimethoxy-5-methyl-1,3-benzodioxole) were used to quantify these seven components, respectively. Their calibration curves presented good linear regressions (R2 > 0.997) within the tested concentration range. The intra- and inter-day precisions were less than 1.97% and 2.53%, respectively. The overall recovery was in the range of 87.55%–95.41%. This validated high-performance liquid chromatography (HPLC)—MS/MS method offers promising applications for the accurate and rapid quantification of signature compounds in the fruiting body and its commercial products.
Authors:Rasha M. Youssef, Heba H. Abdine, Magda A. Barary and Mira M. Wagih
Incidence of suicidal attempts presents an explanation for the high prevalence of quetiapine (QTI) in forensic cases. Thus, the interpretation of its concentrations in biological specimens is needed, but in forensic toxicology, potential postmortem changes such as instability of the target drugs should be taken in consideration. High-performance liquid chromatography (HPLC) method has been developed for determination of QTI. This method was based on reversed phase (RP)-HPLC separation of QTI on a C-18 column (150 mm × 4.6 mm, 5 μm) with elution system of acetonitrile—methanol—0.025 M phosphate buffer (pH 2.5), containing 1 mL TEA in each 250 mL, in a ratio of 40:30:30%, v/v, at the flow rate of 1.2 mL min−1 using mirtazapine as internal standard (IS). The proposed method was applied to the determination of QTI in plasma in presence of coadministered drugs. The application of the proposed method was extended for long-term stability study of two different concentration levels of QTI in the whole blood.
Authors:R. Głowacki, J. Stachniuk and K. Borowczyk
A new and simple method based on high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for the determination of cysteine (Cys) and cysteinylglycine (CysGly) in plasma and urine has been developed. The method involves reduction of disulfide bonds with tris(2-carboxyethyl)phosphine, derivatization of the analytes with 2-chloro-1-methylquinolinium tetrafluoroborate, and separation on Aeris PEPTIDE XB-C18 column (150 mm × 4.6 mm, 3.6 μm, Phenomenex) with UV detection at 355 nm. The calibration lines, obtained with human plasma and urine spiked with Cys- Gly and Cys, were linear in the range of 2.5–50 μmol L−1 and 20–300 μmol L−1, respectively. The intra- and inter-day precision values of the method, expressed as a relative standard deviation, were 0.25–11.1% and 0.71–12.3%, respectively. The analytical recovery varied from 89.7 to 112.3%. The LOQs for total Cys and CysGly were 1.5 pmol and 2.3 pmol in peak, respectively. The method was successfully applied to samples donated by apparently healthy individuals. Concentrations of Cys and CysGly in human plasma from 18 subjects varied from 141.6 to 217.8 μmol L−1 and from 21.1 to 50.9 μmol L−1, respectively. Their concentrations in urine samples (n = 14) ranged from 137.3 to 426.8 μmol L−1 and from 1.6 to 4.9 μmol L−1, respectively.
A simple and convenient chromatographic method of simultaneous separation, identification, and quantitative determination of thimerosal (TM) (preservative) and aluminum (adjuvant) in vaccines and pharmaceuticals by reversed phase highperformance liquid chromatography (RP-HPLC) with visible (VIS) detection was developed and validated. Due to postcolumn derivatization with dithizone, any interference from matrix was excluded. Similarly, a possibility of on-column decomposition of dithizonates was eliminated. Evaluated detection limits were 0.3 μg TM and 3.0 μg Al, which correspond to the smallest, but possible to recognize, visible peak.
Authors:Razwan Baber, Luca Mazzei, Nguyen T.K. Thanh and Asterios Gavriilidis
Synthesis of silver nanoparticles (NPs) in an impinging jet reactor (IJR) was investigated due to its unique properties of efficient mixing and lack of channel walls which avoid fouling. Silver NPs were formed at room temperature by reducing silver nitrate with sodium borohydride in the presence of sodium hydroxide. Two types of ligand were used to stabilize the NPs, trisodium citrate, and polyvinyl alcohol (PVA). Weber number, the ratio between inertial forces and surface tension forces, is used to characterize flow in impinging jets. Flow regimes were investigated forWeber numbers in the range of 13–176. A liquid sheet/chain regime was identified at lowerWeber numbers (<90), and an unstable rim structure was identified at higherWeber numbers (>90). Mixing time was found to be in the range 1–7ms, using theVillermaux—Dushman reaction system and interaction by exchange with the mean mixing (IEM) model. Fastest mixing occurred at Weber number ca. 90. Using trisodium citrate as a ligand, NP size decreased from 7.9 ± 5.8 nm to 3.4 ± 1.4 nm when flow rate was increased from 32 mL/min to 72 mL/min using 0.5 mm jets, and from 6.4 ± 3.4 nm to 5.1 ± 4.6 nm when flow rate was increased from 20 mL/min to 32 mL/min using 0.25 mm jets. Using PVA as a ligand, NP size decreased from 5.4 ± 1.6 nm to 4.2 ± 1.1 nm using 0.5 mm jets and stayed relatively constant between 4.3 ± 1 nm and 4.7 ± 1.3 nm using 0.25 mm jets. In general, the size of the NPs decreased when mixing was faster.
Transfer of four thin-layer chromatography (TLC) Global Pharma Health Fund Minilab mobile kit protocols for detecting fake pharmaceutical products to quantitative high-performance TLC (HPTLC)—densitometry methods was carried out using a model process published earlier. The developed and validated methods were for the drugs quinine sulfate, mefloquine, dihydroartemisinin, and piperaquine phosphate. EMD Millipore Premium Purity silica gel 60 F254 glass plates, automated standard and sample solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification were used. Sample peak identity and purity validation were carried out by spectral comparison checks available in the winCATS software, and accuracy was estimated by the standard addition approach. HPTLC gives better efficiency, selectivity, and resolution than TLC, and the new methods overcome the deficiencies in technology related to manual application and visual zone comparison that do not allow the Minilab TLC procedures to support regulatory compliance actions. These new methods should be fully validated according to International Conference on Harmonization guidelines or by interlaboratory studies if required by their applications. In addition, a previously reported transferred simultaneous HPTLC–densitometry method for lumefantrine and artemether was used to analyze a new combination tablet to demonstrate its applicability.
Authors:B. Muszyńska, K. Sułkowska-Ziaja, A. Maślanka, J. Rojowski, W. Opoka and M. Łojewski
The analysis demonstrated that biomass of Agaricus bisporus, Boletus badius, and Cantharellus cibarius contains non-hallucinogenic indole compounds. Addition of l-tryptophan to the in vitro cultures raised the total content of indole compounds. l-Tryptophan became metabolized, causing an increase of the concentration of some indole compounds. The compounds found in the tested biomass from in vitro culture on Oddoux medium without and with addition of l-tryptophan were l-tryptophan, 5-hydroxy-l-tryptophan, serotonin, melatonin, tryptamine, and 5-methyltryptamine (ranged from 4.28 to 132.51 mg/100 g dry weight). l-Tryptophan is an amino acid exogenous to the human body, and therefore, it must be supplied to the body with food. The highest amount of 5-hydroxy-l-tryptophan was found in the extracts from biomass of B. badius cultured on medium with addition of l-tryptophan (132.51 mg/100 g dry weight). Also, in this case, the highest total content of examined indole compounds (168.00 mg/100 g dry weight) was determined. Melatonin was found only in biomass of A. bisporus cultured on medium with addition of l-tryptophan but in smaller amount (4.28 mg/100 g dry weight).
Authors:M. Orłowska, K. Pytlakowska, A. Mrozek-Wilczkiewicz, R. Musioł, M. Waksmundzka-Hajnos, M. Sajewicz and T. Kowalska
In this study, six numerical data sets are presented valid for eighteen thyme (Thymus L.) species and characterizing three biological properties of these herbs, i.e., antioxidant, antibacterial, and anticancer activity. Four data sets characterize antioxidant properties, one data set characterizes antibacterial property, and one data set characterizes anticancer activity. Antioxidant properties were measured with two free radical standards (DPPH and ABTS), two free radical scavenger standards (trolox and gallic acid), and three analytical techniques (EPR spectroscopy, ultraviolet–visible [UV–vis] spectrophotometry, and the dot blot test with bioautographic detection). Antibacterial activity was tested upon the Gram-positive Bacillus subtilis (ATCC 6633) strain, and anticancer activity was evaluated upon the human colon adenocarcinoma cells (HCT116). It was found out that the thyme extracts characterize with all three biological activities (yet with anticancer activity not very strongly pronounced) and that in quantitative terms, each activity strongly depends on the thyme species considered. An ultimate goal of this study was to investigate if any quantitatively confirmed correlation exists among these three biological activities, which might point out to a common mechanism of their action. To this effect, six sets of numerical data underwent hierarchical clustering and Principal Component Analysis. Based on the results obtained, no quantitative correlation was established among antioxidant, antibacterial, and anticancer activity of the thyme species, which seems indicative of different molecular mechanisms of these three actions.
Authors:A. Kumar, R.K. Dubey, K. Kant, D. Sasmal, M. Ghosh and N. Sharma
Deltamethrin, a well-known type 2 synthetic pyrethroid insecticide, is a widespread environmental toxicant. It has potential to accumulate in body fluids and tissues due to its lipophilic characteristics. The immune system is among the most sensitive targets regarding toxicity of environmental pollutants. Various methods are available in the literature to analyze deltamethrin (DLM) concentration in plasma and tissues, but regarding the immune organs, only one gas chromatography–tandem mass spectrometry (GC–MS/MS) method (on spleen tissues) has been reported. In the present investigation, a rapid and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated to determine DLM concentration in plasma, thymus, and spleen using zaleplone as an internal standard. Liquid chromatography (LC) separation is performed on an Agilent Zorbax® C8 column (250 mm × 4.6 mm, i.d., 5 μm) with isocratic elution using a mobile phase consisting of acetonitrile–5 mM KH2PO4 (70:30, v/v) at a flow rate of 1 mL min−1. The lower limit of quantification (LLOQ) for DLM is 10 ng mL−1 (plasma, thymus, and spleen). The method has been validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy, and precision (intra- and inter-day) and stabilities study. This validated method was successfully applied to a pharmacokinetic and tissue distribution study of DLM in mice.
Authors:Longyuan Wang, Xiaoxiao Hu, Hong Zhu, Xiaotian Zhang, Chuan Wang and Zhiwu Han
A rapid, selective, sensitive, and simple method for simultaneous determination of tigecycline and its epimer in human plasma samples was developed and validated by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Sample preparation involved one-step protein precipitation by adding 0.1% formic acid–methanol and phosphate buffer (PB) solution to the plasma. Chromatographic separation was obtained with XBridge BEH C18 column (3.5 μm, 50 × 4.6 mm) through a 9.5-min gradient mobile phase at the flow rate of 0.6 mL min−1 at 4 °C. The calibration curves were linear over concentration 5.00–2000 ng mL−1 with correlation coefficient greater than 0.998. Intra-batch and inter-batch accuracy of the assay were in the ranges of −2.90% to 3.00%, and the corresponding precision was less than 6.97%. The extraction recovery of tigecycline and its epimer with the current method were 87.2% and 76.9%, respectively. The applied LC–MS/MS method was shown to be sufficiently sensitive and will be suitable for pharmacokinetic studies.
Authors:F. K. Hurtado, M. Kaiser, L. Tasso and T. Dalla Costa
A fast, simple, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and fully validated for the determination of moxifloxacin (MXF) in rat plasma. MXF and gatifloxacin (internal standard, I.S.) were extracted from plasma by single-step protein precipitation with acidified acetonitrile. Chromatographic separation was accomplished in less than 8 min on an Atlantis ® T3 column with 0.4% aqueous triethylamine–methanol–acetonitrile (60:35:5, v/v/v) solution as mobile phase. Detection was achieved by fluorescence (λexcitation = 295 nm, λemission = 500 nm), and the calibration curves were found to be linear over the plasma concentration range of 10–2,500 ng mL−1 with a mean correlation coefficient (r) of 0.9946 (n = 6). The intra- and inter-assay imprecision (% CV) was less than 2.4 and 3.3%, respectively, and the accuracy was >90%. The mean extraction recoveries for MXF and I.S. from plasma were 77 and 82%, respectively. The method was also validated for specificity, sensitivity, and stability; all the results were within the acceptable range. The proposed method was then successfully applied to the quantitative analysis of MXF in rat plasma samples, being a valuable and high-throughput assay to support ongoing pharmacokinetic studies on this promising anti-infective agent.
Authors:S.V. Mulgund, S. Anbazhegan and S.Y. Gabhe
In the present study, the degradation behavior of Fenofibrate under different International Conference on Harmonization (ICH) suggested conditions was studied. Characterization of degradation products by liquid chromatography–tandem mass spectrometry (LC–MS/MS) studies in solution form was done, and the possible mechanism for the formation of degradants is discussed. Fenofibrate was subjected to different hydrolytic stress conditions and thermal stress condition (in solid form). Successful separation of drug from degradants was achieved on a C18 column using water–acetonitrile (25:75 v/v) as the mobile phase. Other high-performance liquid chromatography (HPLC) parameters were: flow rate, 1 mL min−1; detection wavelength, 286 nm; column temperature, 25 °C; and injection volume, 20 μL. The method was validated for linearity, precision, accuracy, robustness, and specificity and was stability-indicating one, based on the specificity studies. The drug degraded under acidic, basic, and oxidative hydrolytic stress while it was relatively stable towards neutral hydrolysis and thermal stress. The stressed samples were subjected to LC–MS/MS analysis. On the basis of spectral data, the structures of four degradation products and one interaction product were suggested. Degradation products were characterized to be isopropyl acetate, 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl propanoic acid, 4-hydroxy benzoic acid, and benzoic acid. The structure of one interaction product was proposed as methyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate.
Authors:S.E. Kepekci Tekkeli, M.V. Kiziltas, D. Dincel, R. Erkoc and G. Topcu
Introduction: In patients with chronic kidney disease (CKD), uremic toxins accumulate in blood and cannot be excreted with urine. Accumulation of these toxins has negative effects on many body functions. Because of the importance of these toxins, we developed and validated a simple, sensitive, accurate, and precise method for the determination of two main uremic toxins: phenol and p-cresol in human urine. Materials and methods: Separation of these analytes in urine samples was achieved by reverse-phase high-performance liquid chromatography (RP-HPLC) with a C18 column at 35 °C using the mobile phase of methanol–water (65:35) at a flow rate of 1.4 mL min−1. Fluorimetric detection was used at 284 nm for excitation and 310 nm for emission. Results: The method is linear over the range of 1.5–35 ng mL−1 and 1–45 ng mL−1 for phenol and p-cresol, respectively. The method was applied to urine samples from 10 healthy subjects and 10 chronic kidney disease patients. Conclusions: This assay appears to be useful in routine analysis of clinical samples for simultaneous determination of phenol and p-cresol levels in urine.
Authors:Š. Jirkal, M. Machovcová and J.G.K. Ševčík
Possibilities have been studied of using a solvation model to predict the retention behavior of solutes in liquid chromatography mobile phases of water-acetonitrile and water-methanol with the organic solvent content varying from 1 to 100 vol%. Twenty-one test solutes, both aliphatic and aromatic compounds, have been selected on the basis of two-level factorial designs. Using the multiple linear regression analysis, regression coefficients, which are characteristics of the stationary and mobile-phase system, were calculated for different mobile phases in the solvation model. Regression coefficients have been used for the prediction of the retention behavior. Unbiased results have been obtained by using two sets, one training and the other the testing set. The predicted retention has been compared with the experimental data. The methanol-water system provided good results at low and medium methanol concentrations; the retention prediction was unsatisfactory for mobile phases containing more than 90% of methanol. The acetonitrile-water system yielded similar results, but the retention prediction ceased to be at acetonitrile concentrations greater than 80%. The retention has primarily been determined by cohesive and acid-base interactions. The dependences of the regression coefficients on the mobile-phase composition were similar for the acetonitrile-water and methanol-water systems.
Authors:Matthew Coblyn, Agnieszka Truszkowska and Goran Jovanovic
Microchannel-based hemodialysis has a potential to improve survival rates and quality of life for end-stage renal disease patients compared to conventional hemodialysis technology. Characterization of hydrodynamic behavior in microchannel geometries is necessary for improving flow uniformity, a critical challenge in realizing a commercial device. A test loop was developed for measuring the impulse response of a tracer dye injected into a dialyzer test article for the purpose of developing residence time distributions (RTD) to characterize lamina design. RTD variance tended to lower for designs that are more dominated, volume-wise, by the microchannel array versus the headers. RTD results also emphasize how defect issues can significantly impact a microchannel device via discrepancies between conceptual and operational devices. A multisegmented CFD model, developed for pairing with the impulse response test loop and dialyzer, showed good agreement between visual observation of the tracer in simulations and experiments, and the shape and peak of the output profiles.
Authors:J. Chen, Q. Chen, F. Yu, H. Huang, P. Li, J. Zhu and X. He
A rapid and sensitive method for the identification and quantification of phillyrin (POG) in Forsythia suspense is described. The phillyrin standard solution was directly infused into the ion trap mass spectrometers (IT-MS) for collecting the MSn spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of phillyrin was proposed, and the ESI-MSn fragmentation behavior of phillyrin was deduced in detail. The major product ion at m/z 355 belongs to furofuran, which was formed by loss the glucopyranoside (180 Da), and the characteristic fragment ions m/z 473, 395, 337, 309, and 249 were observed. The loss of 18 Da could arise from two different fragmentation pathways, and the observed ion was composed of a mixture of two different structural ions. Quantification of phillyrin was assigned in positive-ion mode at a product ion at m/z 557 → 355 by liquid chromatography-mass spectrometry (LC-MS). The LC-MS method was validated for linearity, sensitivity, accuracy, and precision and then used to determine the content of the phillyrin. Lastly, the LC-MS method was successfully applied to determine phillyrin in real sample F. suspense and three of its medicinal preparations in the positive mode at the first time.
Authors:Sujata D. Wangkheirakpam and Warjeet S. Laitonjam
The different separation patterns of petroleum ether extract of leaves of Heiba (Ficus pomifera Wall.) were studied with open column chromatography, highperformance liquid chromatography (HPLC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). A new method of locating and estimating newly discovered compounds by LC-ESI-MS was developed. Compound 1, a triterpenoid of mass 398, was identified by spectral analysis. It was located in the chromatogram by comparison of mass spectrum of the isolated compound to mass spectra of in mass spectral data of LC-ESI-MS. The concentration of the triterpenoid was found to be 8.64 ppm in 2000 ppm of the extract, assuming that all the components are eluted.
Authors:Z. Stefanowicz, M. Sobczak, A. Piętniewicz and W.L. Kołodziejski
Biodegradable and bioresorbable macromolecular conjugates of paclitaxel were prepared. The release of drug from conjugates has been carried out in vitro at 37 °C in phosphate buffered saline solution (PBS, pH 7.4), with 0.1% (w/v) Cremophor® EL. Periodically, all the solution in the samples was removed and replaced with fresh buffer until limit of detection (LOD) of paclitaxel was released. The quantity of released drug was analyzed by means of high-performance liquid chromatography (HPLC) method. Chromatographic separations were conducted using the NUCLEODUR C18 Gravity column with a guard column at 30 °C. Mobile phase consisted of a mixture of acetonitrile, methanol, and deionized water (60:2:38). The flow rate was 1.0 mL min−1, and paclitaxel was detected at 229 nm, retention time of 3.5 min. The applied analytical method was validated according to International Conference on Harmonization (ICH) procedures or recommendations. The chromatographic separation was excellent. The linearity in the range 0.1–4.5 μg mL−1 was found to be very good (R2 = 0.9999). LOD and limit of quantification (LOQ) were calculated to be 0.023 μg mL−1 and 0.068 μg mL−1, respectively. The release profiles were evaluated and compared. The process of paclitaxel release from Paclcon-2 conjugate seems to be the most interesting. Paclitaxel is released the longest and the most evenly.
Authors:M.A. Hawrył, M. Niemiec, K. Słomka, M. Waksmundzka-Hajnos and G. Szymczak
Micro-thin-layer chromatography in two dimensional (2D-mTLC) mode in normal and reversed phase systems by use of diol bonded stationary phase was applied to make fingerprints of 11 species of Mentha genus and two finished pharmaceutical products.
Nonaqueous eluents (propan-2-ol or ethyl acetate dissolved in n-heptane) were used in normal phase systems. Mixtures of acetonitrile with water were used in reversed phase chromatographic systems.
Optimization of one dimensional systems was performed by determining of RF vs. composition of mobile phases dependencies for standards occurring in various species of Mentha. Most selective eluents were chosen to optimize two-dimensional systems by creating RF in normal-phase (NP) systems vs. RF in reversed-phase (RP) systems correlations.
2D-mTLC on diol polar bonded stationary phase were optimized to separate phenolic compounds and make fingerprints of examined plant materials and this method was never applied earlier in the chromatographic analysis.
Authors:G.-F. Shi, Z.-J. Wang, G.-Y. Wang, R.-X. Yao, F.-W. Chen and Z.-R. Shi
High-performance liquid chromatography-mass spectrometry (HPLC-MS) method coupled with radical reaction for screening active ingredients from perennial fujimoto bean whole herb was established. The active ingredients, present in perennial fujimoto bean whole herb, possess scavenging effects towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide, peroxy radical, and hydroxyl radical. The radical scavenging abilities of these active ingredients were evaluated based on the relative peak areas in the HPLC chromatogram. The results indicate that potent antioxidants are present in the anhydrous methanol extract of perennial fujimoto bean whole herb. Based on HPLC-MS analysis, it was found that the scavenging ability can be mostly attributed to the presence of three compounds: cyanidin-3-o-β-d-glucopyranoside, troxerutin, and rutin. The structures were identified based on the MS and nuclear magnetic resonance (NMR) data. Free radical scavenging activity decreased in the following order: troxerutin > rutin > cyanidin-3-o-β-d-glucopyranoside.
Authors:X.R. Zhi, Z.Y. Zhang, R.Y. Li, L. Chang, P.P. Jia, N. Sheng and L.T. Zhang
A new, sensitive, and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) has been developed for the quantification of six flavonoids (sophoricoside, genistin, genistein, rutin, quercetin, and kaempferol) in rat bile and urine. The sample pretreatment was simple by liquid-liquid extraction. Sulfamethalazole was used as internal standard (IS). During method development, the effect of extraction volume, mobile phase composition, column temperature, and injection volume were varied to optimize sensitivity and achieve a run time as short as possible. Chromatographic separation was accomplished on a C18 column with a simple linear gradient elution within 9 min. Full validation of the assay was in accordance with the requirement of the validation of the method in vivo and implemented including specificity, linearity, accuracy, precision, recovery, and matrix effect. This is the first report on determination of the major flavones in rat bile and urine after oral administration of Fructus Sophorae extract. The method has been used successfully in excretion studies of six major flavonoids in rat bile and urine.
Citri Grandis Exocarpium (CGE) is a traditional Chinese medicine with a variety of biological activities. For efficient quality control of CGE, a simple, rapid, and accurate high-performance liquid chromatographic (HPLC) method was developed for simultaneous determination of four main compounds (naringin, rhoifolin, meranzin hydrate, and isoimperatorin) in this herb. These four compounds were separated on a C18 column by gradient elution with methanol and water. The flow rate was 1.0 mL·min−1, and the detection wavelength was 324 nm. The recoveries of the method ranged from 96.32% to 103.71%, and good linear relationships (r2 > 0.9998) over relative wide concentration ranges were obtained. Then this validated method was successfully applied to the analysis of nine batches of CGE samples.
A simple stability-indicating high-performance liquid chromatography-diode array detection (HPLC-DAD) method has been developed for the simultaneous determination of triamterene (TRI) and xipamide (XIP) in presence of the degradation products generated in studies of forced decomposition. Drugs were subjected to stress by hydrolysis (acidic, alkaline, and neutral), oxidation, photolysis (254 and 365 nm), and dry and wet heat treatments. Degradation occurs under acidic and alkaline conditions (TRI only), oxidative stress (TRI and XIP), and by photolysis (XIP only), but both drugs were stable under other stress conditions investigated. Separation of the two drugs from all the degradant peaks was achieved within 11 min using C8 column (250 × 4.6 mm, 5 μm) and mobile phase consisting of acetonitrile and 0.05 M phosphate buffer adjusted to pH 4 delivered at a flow rate of 1 mL min−1 using gradient elution system. The drugs were quantified at 220 nm using photodiode array detector, based on peak area. Peak homogeneity of the two drugs was checked using diode array detector, and the purity angle was within the purity threshold limit in all of the stressed samples. The calibration graphs for each drug were rectilinear in the range of 0.2–50 and 0.1–20 μg mL−1 for TRI and XIP, respectively. The method was validated in compliance with International Conference on Harmonization (ICH) guidelines; in terms of linearity, accuracy, precision, robustness, limit of detection, and limit of quantitation. The proposed method was successfully applied for the determination of the investigated drugs in their tablet without interference from excipients with acceptable accuracy and precision; the label claim percentages were 100.23 ± 0.70% and 100.75 ± 1.11% for TRI and XIP, respectively.
Authors:E. Sieniawska, T. Baj, J. Dudka, T. Mroczek and K. Głowniak
Many phytochemical investigations have been focused recently on the antioxidant activity of herbal extracts which can be used in phytotherapy. The previous study revealed antioxidative properties of Mutellina purpurea extract, but the constituents responsible for this action were not described yet. The aim of this study was activityguided separation and identification of antioxidant compounds from M. purpurea herb. Thin-layer chromatography-2,2-diphenyl-1-picrylhydrazyl (TLC-DPPH•) assay was used to detect compounds of interest; liquid chromatography-diode array detection-mass spectrometry (LC-DAD-MS) analysis allowed to identify antioxidants. The active fractions analyzed with LC-DAD-MS contained as a main compound chlorogenic acid accompanied with p-coumaric acid, ferulic acid, three dicaffeoylquinic acids, and caffeoylferuloylquinic acid. The fast TLC-DPPH• assay with LC-DAD-MS identification enabled the accurate identification of antioxidants in M. purpurea herb, which was done for the first time.
Authors:Q. Liu, K. Qin, B. Shen, H. Cai and B. Cai
To evaluate the quality of Fructus Arctii, an accurate and reliable method of high-performance liquid chromatography/diode array detection—electrospray ionization—mass spectrometry (HPLC/DAD—ESI—MS) was developed. Nine compounds, including chlorogenic acid, caffeic acid, trans-p-hydroxycinnamic acid, arctiin, arctignan A, ethyl caffeate, matairesinol, arctigenin, and lappaol B, were determined simultaneously in 19 batches of Fructus Arctii samples collected from different localities. Nineteen common peaks were identified or tentatively assigned by comparing their mass spectrometric data with reference compounds, self-established compound library, and published literatures. Also, the 19 common peaks were selected as characteristic peaks to assess the similarity of chromatographic fingerprinting of these samples. Moreover, hierarchical clustering analysis (HCA) and principal components analysis (PCA) were successfully applied to demonstrate the variability of samples. The results indicated the content of nine compounds that varied greatly among the samples, and 19 samples collected from different localities could be discriminated. Furthermore, chlorogenic acid, arctiin, and arctigenin were found to be chemical markers for evaluating the quality of Fructus Arctii.
Authors:M. Mori, T. Masuno, D. Kozaki, N. Nakatani, K. Tanaka and H. Itabashi
Ion chromatography of inorganic cations using a phosphate-coated zirconia stationary phase (PZ) was first attempted. The retentions of cations to PZ increased by elevating the column temperature and the reproducibility of the separation could improve at the higher temperature. The PZ functioned as a cation-exchanger from changes in the retention factor of cations as a function of eluent pH. Furthermore, the Gibbs free energies of cations were estimated from enthalpy and entropy using the retention factors of cations as a function of the column temperature. The reaction was based on the endothermic reaction.
Authors:A. Oniszczuk, M. Waksmundzka-Hajnos, R. Podgórski, R. Nowak, W. Żukiewicz-Sobczak and T. Blicharski
The use of matrix solid-phase dispersion (MSPD) was tested to extract some coumarins (umbelliferone, xanthotoxin, isopimpinellin, bergapten, and pimpinellin) from Pimpinella roots. The method was compared with liquid—solid extraction methods (LSE) such as accelerated solvent extraction (ASE), ultrasound-assisted extraction (USAE) and Soxhlet extraction followed by solid-phase extraction (SPE). Several methanol concentrations and temperature conditions were examined during the liquid—solid extraction techniques. The analysis was performed by high-performance liquid chromatography with diode array detector (HPLC—DAD). MSPD extracted furanocoumarins with satisfactory recoveries ranging from 91.65% to 97.55% and relative standard deviations lower than 5,0415%. The results presented in the paper reveal that MSPD is an efficient, fast, simple and easy-to-perform method suitable for the isolation of furanocoumarins from herbs.
A rapid high-performance liquid chromatography (HPLC) method for chiral purity determination of tenofovir disoproxil fumarate in raw material and pharmaceutical formulations was developed. The (S)-enantiomer appears to be as an impurity and pharmacologically inactive. The effects of various stationary phases, mobile phase composition, and column temperature on enantiomeric separation of tenofovir disoproxil were investigated and optimized. Chromatography resolution of tenofovir disoproxil enantiomers was performed on NUCLEOCEL ALPHA-RP S column (250 × 4.6 mm i.d., 5 μm). The elution was achieved by using 95:5% (υ/υ) methanol—acetonitrile, containing 0.1% triethylamine at a flow rate of 0.8 mL min−1. The ultraviolet (UV) detector was set at 260 nm. Calibration curves were linear in the range of 1–100 μg mL−1 and 0.2–20 μg mL−1 for (R)-tenofovir disoproxil and (S)-enantiomer, respectively. Limits of detection and quantitation for (S)-enantiomer were 0.06 and 0.2 μg mL−1. The run time of analysis was less than 7.0 min. The proposed method was used successfully for separation and quantification of tenofovir disoproxil enantiomers in raw material and pharmaceutical formulations.
The microextraction by packed sorbent (MEPS) was applied to a fractionation of cuticular wax extracts from several solanaceous plant species. A procedure developed requires only 0.6 mL of organic solvents and may be completed in less than 10 min. Hydrocarbons, which are frequently used in chemotaxonomy of Solanaceae, were almost exclusively eluted in one fraction. The amounts of most commonly detected polar wax components (alcohols, sterols, triterpenes) in the same fraction were reduced to ca. 34–46% of the total amounts in the whole extract. Despite the contamination of the hydrocarbon fraction with other wax components, the results obtained using MEPS and standard column chromatography on silica gel were similar when compared using cluster analysis based on the hydrocarbon profiles. However, the method was far less successful in removing the sucrose esters from extracts of Nicotiana rustica leaves. Thus, MEPS fractionation of plant cuticular waxes may be a fast and reliable alternative for the standard liquid chromatography techniques as long as no sucrose esters are present in the extract.
Authors:A. Pavlova, D. Stratiev, M. Mitkova, K. Stanulov, N. Dishovsky and K. Georgiev
Liquid products produced from two different types of waste pyrolysismunicipal wastes and spent tyre wastes are investigated using gas chromatography—mass spectrometry. This method has been applied for detailed identification of composition of the samples. The components were characterized in terms of their Kováts retention indices on a PONA capillary column. The obtained analytical data were successfully used for the characterization of the samples. More than three hundred compounds were detected. The liquid products were complex mixtures, composed mainly of C4—C12 compounds. The examination of the selected m/z values very clearly indicates the existence of the different groups of compounds. With a lot of olefins content (31.9%), followed by aromatics (20.0%), paraffins (17.3%), and naphthenes (7.5%), it is described as the liquid product from pyrolysis of municipal solid wastes. The aromatic compounds in liquid product from pyrolysis of spent tyre wastes have the highest concentration (33.5%), and they are followed by naphthenes (28.6%), olefins (19.2%), and paraffins compounds (7.0%). The present study has shown that the pyrolysis of municipal waste and spent tyres can be used as a means for reduction of environmental pollution and production of liquid product which could be used as a fuel source.
Authors:E.N. Fedorova, A.D. Kivero, N.V. Geraskina and L.R. Ptitsyn
A simple and rapid ultra-performance liquid chromatographic (UPLC) method for analyzing acetoin in bacterial culture fluid was developed and validated for the first time. The samples were separated using an Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm particle size) and isocratic elution with 30 mM phosphoric acid—1% acetonitrile as the mobile phase. A photodiode array detector (PDA) was used. The run time was 6 min, and the detection limit was 2.11 × 10−4 mg mL−1. The UPLC method was compared with high-pressure liquid chromatography (HPLC) for acetoin analysis. The proposed UPLC method is highly sensitive and was successfully applied to the analysis of acetoin in bacterial culture fluid.
Authors:W. Kukula-Koch, J. Widelski, W. Koch and K. Głowniak
The presence of phenolic content in overground extracts of Euonymus verrucosus Scop. — commonly growing in Europe — has been reported recently. The chromatographical and spectral data revealed the presence of several simple phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, vanillic, syringic, caffeic, p-coumaric, ferulic, and m-coumaric acids), both as free and conjugated with other secondary metabolites. The comparison of two-dimensional TLC systems on cellulose stationary phases with HPLC— DAD reversed-phase chromatography was performed to assess a cheap and rapid technique in the identification process of major phenolic constituents. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging tests, expressed as IC50, revealed the most beneficial results for the fraction after alkaline hydrolysis and yielded 205 ± 8 μg mL−1.
This work describes the comparison of five sample extraction methods for the determination of pesticide residues in grapes and vegetables by using gas chromatography—time-of-flight mass spectrometry (GC—TOF-MS). These methods were based on original methods QuEChERS, mini-Luke, ethyl acetate, and DIN EN 15637, and some of these were slightly modified to increase the number of identified and quantified pesticides, to improve their quantification limits, and to be fast and less expensive in terms of material cost. The acceptable performance parameters combined with the properties of easy and quick handling and cost-effectiveness have made mini-Luke modified extraction method as the most favorable in the pesticide residues analysis from grapes and tomatoes by GC—TOF-MS. The efficiency of the chosen extraction method was also verified for lettuce; for this matrix, a cleanup step with graphitized black carbon (GBC) was added. Analysis of extracts was carried out by GC—TOF-MS within 29.2 min run time. The GC method was validated for grapes and tomatoes in terms of linearity, accuracy, limit of detection (LOD), and limit of quantification (LOQ). Good linearity with correlation coefficients (r2) higher than 0.98 was obtained. For most analytes from both matrices, recoveries were in the range of 71–120% and LOQ values in the range of 0.01–0.05 mg kg−1. The GC—TOF-MS and modified mini-Luke extraction methods were successfully tested on real vegetable and fruit samples belonging to the same commodities group as those from validated methods according to European Guide DG SANCO 12571/2013 (tomatoes, pepper, cucumber, potatoes, carrots, eggplants, onion, lettuce and grapes) and in proficiency EU tests.
Authors:P.R. Ravi, S. Joseph, U. S. R. Avula and S. Anthireddy
The objective of this study was to develop and validate a novel, simple, and selective high-performance liquid chromatographic (HPLC) method with photodiode array detector for the estimation of tenofovir in rat plasma, which can be utilized in analyzing the pharmacokinetic samples from rats. Prior to analysis, an optimized protein precipitation technique was used to extract tenofovir from plasma. The mobile phase for this method comprised of 10 mM ammonium acetate buffer (pH 4) and methanol in the ratio of 97:3 υ/υ. Chromatographic separation of tenofovir was achieved using Spincotech C-18G enabled column (250 × 4.6 mm, 5 μm). Tenofovir was monitored at a wavelength of 260 nm, and the calibration curve was linear in the range of 250–4000 ng mL−1 (R2 = 0.999). High recovery obtained after extraction (97%–101%) of plasma samples precluded the use of an internal standard. Validation studies were performed as per the standard guidelines, and the developed method was accurate, precise, and selective for the determination of tenofovir in the rat plasma. The stability studies performed during the sample pretreatment process and sample storage conditions did not show a quantifiable degradation of tenofovir. Further, this method was able to estimate tenofovir and determine its pharmacokinetic parameters, post IV bolus administration in male Wistar rats. The pharmacokinetic profile of tenofovir followed one compartmental open model.
Authors:A. Kwiecień, K. Piątek, P. Żmudzki and J. Krzek
A simple, selective, and sensitive thin-layer chromatographic—densitometric method has been developed for the determination of sulfasalazine besides its possible impurities in pharmaceutical preparations. The mobile phase was composed of ethyl acetate—methanol—ammonia 25% 10:7:3 (υ/υ/υ), and the stationary phase was aluminum plates precoated with silica gel 60 F254 that enabled to obtain well resolved peaks of sulfasalazine and its impurities. The developed chromatograms were analyzed densitometrically at λ = 360 nm. RF values and ultraviolet (UV) spectra were used to identify the compounds. The developed method is highly sensitive (limit of detection [LOD] = 17.11 ng spot−1, limit of quantitation [LOQ] = 51.84 ng spot−1), precise (relative standard deviation [RSD] = 1.43%–4.28%), and accurate (RSD = 1.64%–4.27%). The linearity of the method was checked within the range 20–120 ng spot−1. The method was successfully applied for the determination of sulfasalazine in pharmaceutical preparations besides its impurities. The structures of impurities present in the standard substance and in pharmaceutical preparations were established by ultra-performance liquid chromatography—tandem mass spectrometry (UPLC—MS/MS) technique.
Authors:X. Wang, Y. Wu, Q. Wu, Y. Qian, W. Yue and Q. Liang
The rhizome of Sparganium stoloniferum Buch.-Ham has been used as a traditional Chinese folk medicine for thousands of years. Phenolic compounds are the main bioactive ingredients of the plant. In order to determine the content of phenolic compounds from different major cultivations, a reliable method has been developed using ultra-high performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry. Seven compounds, including rutin, kaempferol, p-hydroxybenzaldehyde, formononetin, ferulic acid, vanillic acid, and p-coumaric acid, were simultaneously measured in 10 min. The established approach was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and successfully applied to determine seven phenolic compounds of Rhizoma Sparganii. This study may be helpful in the quality control of Rhizoma Sparganii and can offer technical support for the pharmacological and clinical study of related drugs.