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In this reported study, a direct high-performance thin-layer chromatographic (HPTLC) method was developed to qualitatively detect and quantitatively determine glycerol in Antarctic krill for the first time. This procedure was based on the extraction of glycerol by ultrasonic solvent extraction with anhydrous ethanol, silica-gel column chromatographic separation, HPTLC detection and quantification using methylene chloride–methanol (5:1, v/v) as the developing solvent and alkaline potassium permanganate as chromogenic agent. The content of glycerol was 1.3725 ± 0.218 mg/g in freeze-dried Antarctic krill. The structure of glycerol in the Antarctic krill was subsequently determined by gas chromatography–mass spectrometry (GC–MS) which verified the presence of the material in the krill. The HPTLC method exhibited excellent accuracy with a recovery of 90.1–103.3% and good precision with a relative standard deviation (RSD) of 1.59–4.84%. The results clearly exhibited the applicability of the proposed for quantifying glycerol in Antarctic krill.

Open access

Agaricus bisporus and Imleria in vitro cultures were cultivated on modified Oddoux medium, and Oddoux medium was enriched with serine or anthranilic acid. Serine or anthranilic acid was used at the concentrations of 0.1, 0.25, 0.5, and 0.75 g/L of medium. Determination of indole compounds in the obtained biomass was carried out using thin-layer chromatography (TLC) with densitometric detection. In every analyzed sample, presence of serine or anthranilic acid was studied. Comparison of the results obtained for the treatment and control samples allowed us to determine the optimum concentration of serine or anthranilic acid in the medium in order to obtain biomass with increased content of indole compounds. A. bisporus with addition of anthranilic acid or serine to the medium at the concentration of 0.5 g/L was the most beneficial. In the case of Imleria badia, anthranilic acid at the concentration of 0.5 g/L was the most optimal. This is the first report demonstrating the content of indole derivatives in biomass affected by their precursors (serine or anthranilic acid). The study indicates that modification of the medium can provide satisfactory results, and it is worth to search for its new, improved compositions.

Open access

A validated high-performance liquid chromatography (HPLC) method has been developed to analyze the (±)-gossypol in the selection of strains of Candida tropicalis culture. Since gossypol was easily degraded and oxidized, the addition of antioxidant NADPH-Na4 and acetone extraction was chosen to prevent gossypol degradation and gradient elution assay was applied to obtain gossypol resolution. Concentrations of gossypol in C. tropicalis ZD-3 culture 20 μg/mL were determined, and concentration–time profiles were observed. Linearity of the gossypol standard curve by HPLC area method was ranged from 0.1 to 20 μg/mL with Y = 26.954 × X − 29.547, R 2 = 0.9991, and n = 3, with limit of detection (LOD) of 50 ng/mL and lower limit of quantification (LLOQ) of 500 ng/mL. The recovery rate is dose-dependent and ranged from 85.3% to 103.5%. It is a rapid and reliable HPLC method for gossypol quantization in microorganism culture which could be applied in solid fermentation in the feed industry.

Open access

Stability-indicating High-Performance Thin-Layer Chromatography (HPTLC) method for simultaneous estimation of cefixime trihydrate and azithromycin dihydrate was developed. Both the drugs were subjected to different stress conditions recommended by International Conference on Harmonization (ICH) guideline Q1A (R2). Forced degradation was carried out for hydrolytic, oxidative, photolytic, and thermal degradation conditions. Cefixime was susceptible for degradation under all stress conditions showing four degradation products (CI–IV). However, azithromycin formed only one degradation product (AI) under acid hydrolysis. Aluminum plates precoated with silica gel 60F254 were used as the stationary phase while mixture of ethyl acetate–methanol–acetone–toluene–ammonia (1:5:7:0.5:0.5, v/v) was used as mobile phase. Detection wavelength used was 235 nm for CEFI and CI–IV. AZI and AI were detected by post development derivatization, spraying with sulfuric acid–ethanol (1:4, v/v) followed by heating at 100 °C for 5 min. Degradation products were isolated by preparative HPTLC and characterized by MS/MS. The developed method was validated for linearity, precision, accuracy, specificity, and robustness and has been successfully applied in the analysis of these drugs in tablet dosage form.

Open access

Herbal products, which comprise a wide variety of bioactive molecules, have been used as remedies for different diseases throughout history. Lagenaria siceraria, a fruit vegetable, is employed in folk medicine as a treatment for various disorders including diabetes, hyperlipidemia, and heart and liver ailments. In the present work, a number of compounds were isolated and characterized from the ethyl acetate fraction of the methanolic extract of its peel, including β-sitosterol, vanillin, quercetin, rutin, 3-tert-butyl-4-hydroxyanisole, stearic acid, 2,4-bis(1,1-dimethylethyl)phenol, 2,2′-methylenebis[6-(1,1-dimethylethyl)-4-methylphenol], 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester, hexadecanoic acid and its methyl ester, (Z,Z)-9,12-ocatdecadienoic acid and its ester, and (Z,Z,Z)-9,12,15-ocatdecatrienoic acid methyl ester. Separation of the phytochemicals was done using column and thin-layer chromatography, while gas chromatography–mass spectrometry (GC–MS), liquid chromatography-mass spectrometry (LC–MS), and high-performance liquid chromatography (HPLC) were employed for their identification. These compounds are being reported for the first time from the peel of the fruit of L. siceraria. The results provide a possible chemical rationale for the medicinal applications of this fruit.

Open access
Acta Chromatographica
Authors: Yun Zhou, Guifeng Huang, Xiaolan Li, Feng Chen, Hong Liu, Ying Yang, Zhong Fan, Jinghui Jiang and Jun Yang

A credible method for determination of the aglycon moieties of glycosidically bound aroma compounds in Flos Chrysanthemi by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC × GC–TOFMS) has been proposed. The aglycon moieties of glycosidically bound aroma compounds were isolated using methyl-tert-butyl ether (MTBE) extraction following enzymatic hydrolysis. The GC × GC–TOFMS analysis was performed to comprehensively identify different forms of the released aroma components in Flos Chrysanthemi. The result shows that the limit of detection of the released aglycon moieties ranged from 0.3 to 3.1 ng/mL, the recovery of the released 1-octanol was better than 98.3%, and the intra-day and inter-day precisions of this method were 0.2 to 8.9% and 1.3 to 9.1%, respectively. The proposed method was applied to the analysis of four types of Flos Chrysanthemi (Chuju, Boju, Hangju, and Gongju). A total of 60 aglycon moieties of interest were identified in the four types of Flos Chrysanthemi. These aglycones mainly consisted of aliphatic, aromatic, monoterpene, C13-norisoprenoids, and miscellaneous compounds.

Open access
Acta Chromatographica
Authors: D. Trbović, T. Polak, L. Demšar, N. Parunović, M. Dimitrijević, D. Nikolić and V. Đorđević

With the aim to reinforce laboratory competence in the field of testing the quality of fish from aquaculture, a study on the precision of fatty acid (FA) analyses in fish meat and fish feed was undertaken. Different methods were performed in laboratories. In situ transesterification method and extraction of lipids from the fish were followed by capillary gas chromatography with flame ionization detection. The reproducibility (R) values of the majority of FAs were less than 3% of their absolute values. Differences in calculating ionization detector response factors and/or autoxidation caused by faulty sample-handling could lead to variation in quantification of FAs in fish, especially for FA C22:6n-3. Statistical analysis showed a significant correlation between the two laboratories' quantifications of FAs in fish and fish feed (Pearson's correlation coefficient; r = 0.987, r = 0.994, and r = 0.997; for fish Z [trout], fish Š [rainbow trout], and fish feed, respectively). Overall, adequate accuracy was obtained in this study. The proposed method provides a fast and efficient means of identifying fish and feed for quality control purposes.

Open access

A simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of zinc pyrithione (ZnPT) and pyrithione (PT) in shampoos. The method consisted of a liquid–liquid extraction for sample preparation. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode via the positive electrospray ionization interface. A linear regression (weighted 1/x) was used to fit calibration curves over the concentration range of 50–2000 ng/mL for both ZnPT and PT. Excellent linearity (r 2 ≥ 0.9996) was achieved for all. The method was validated and found to be accurate (95.9–108.2% for ZnPT and 94.9–110.4% for PT), precise, and selective. Analytes in shampoos were found to be stable in the autosampler (6 °C for 6 h), in room temperature (for 6 h), and after three freeze–thaw cycles, and recovery of analytes was reproducible (90.8–94.6% for ZnPT and 90.2–96.3% for PT).

Open access

In this research, a novel method was developed for the matrix solid phase dispersion (MSPD) followed by high-performance liquid chromatography (HPLC) quantification of four marker constituents (vitamin C, gallic acid, rutin, and ellagic acid) in the freeze-dried pomegranate fruit juice. Various MSPD parameters like type of dispersant, sample–dispersant ratio, solvents, its volume, and time of extraction have been optimized after many trials. Furthermore, HPLC method has been developed and optimized for the analysis of all four components. The HPLC separation was achieved using a 250 × 4.6 mm column, particle size of 5 μm, C18 reverse phase column, with a mobile phase consisting of acetonitrile and 0.05% H3PO4, in gradient elution mode with a mobile phase flow rate of 1 mL/min, using ultraviolet (UV)–visible detection at 254 nm. All calibration curves showed good linear regression (r 2 ≥ 0.9925) within test ranges. The extraction recoveries of the marker constituents analyzed by MSPD methods were found as ranging from 97.5% to 103.5%. From comparing the chromatograms, validation data and other parameters like time, labor, and feasibility, we found that MSPD technique was most suitable for the analysis as compared to conventional liquid–liquid extraction technique.

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We have developed a strategy to analyze the components absorbed in the plasma and brain tissue of rats after intragastric administration of Terminalia chebula Retz extracts by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC–QTOF-MS). Nine components (gallic acid, methyl gallate, ferulic acid, ethyl gallate, brevifolin carboxylic acid, ellagic acid, galloflavin, arjugenin, and arjunic acid) and four metabolites were identified in plasma, and five components (ethyl gallate, brevifolin carboxylic acid, ellagic acid, arjugenin, and arjunic acid) were identified in the rat brain based on their fragmentation behaviors. The components present in the plasma were associated with the antioxidant activity of T. chebula Retz, and the components absorbed in the brain were associated with its neuro-protective effects. This approach allowed us to rapidly determine the active components of T. chebula Retz and develop a method for its quality control. This analysis method showed good resolution and high sensitivity, and is a potentially powerful tool for the determination of effective components of natural products.

Open access

Krebs buffer is considered one of the most used physiological buffers in biomedical research. In the current work, a rapid reversed-phase high-performance liquid chromatographic (RP-HPLC) method with ultraviolet (UV) detection at 214 nm was developed and validated according to European Medicines Evaluation Agency (EMEA) guidelines for the determination and quantification of propranolol in Sprague–Dawley rat's serum and in Krebs buffer. This method can be applied for both in vivo and in vitro studies with short run time of 7.0 min . Isocratic elution with a flow rate of 1.0 mL/min was employed. BDS Hypersil C-18 column (150 mm × 4.6 mm and 5 μm) was used to obtain satisfactory resolution. The mobile phase used contained a mixture of acetonitrile, methanol, and triethylammonium phosphate solution (15.0:32.5:52.5, v/v). Best separation between propranolol and the internal standard (I. S.) sildenafil was obtained at 4.2 and 5.5 min, respectively. Propranolol was linear over a concentration range of 50.00–3000 ng/mL with acceptable accuracy, and intra- and inter-day precision. Dilution integrity was assessed and was found to be within the acceptable range for both serum and Krebs buffer. Sample stability tests were studied at different storage conditions, and all the analytes were found to be stable. The mean percentage of recovery of propranolol was found to be 97.06% and 98.57% for serum and Krebs buffer, respectively.

Open access

A reliable and rapid high-performance liquid chromatography coupled with diode array detector method (HPLC–DAD) was established and validated to determine eight gingerol simultaneously in the rhizomes of Zingiber offcinale Rosc. The separation of eight compounds (4-hydroxy-3-methoxy-benzenebutanol,3,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl) decane, 3,5-dihydroxy-1-(3,4-dimethoxyphenyl) decane, 6-gingerol, 8-gingerol, 6-shogaol, 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1,4-decadien-3-one, and 10-gingerol) were performed on an Agilent TC(2) C18 (250 mm × 4.6 mm, 5 μm) at 30 °C using acetonitrile (A) and 1% formic acid aqueous solution (B) as the mobile phase with gradient elution (0–10 min, 20%–35% A; 10–28 min, 35%–55% A; 28–35 min, 55%–60% A; 35–55 min, 60%–70% A; 55.01–60 min, 100%–100% A). The detection wavelength was set at 280 nm, and the flow rate was 0.8 mL/min. Validation of the analytical method was performed by linearity, precision, and accuracy test. All compounds were quantified with good linear calibration curves (coefficient of determination R 2, >0.9999). The method showed good precision with overall coefficients of variation between 0.56% and 0.84%. The range of recovery was from 95.50% to 104.14% for the analytes. This method was successfully applied to quantify eight gingerols in Z. offcinale Rosc from different regions in China, so it can provide quality assessment for this medicine.

Open access

An effective, reliable, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) with diode array detector (DAD) method was investigated for simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in Tetrastigma hemsleyanum. The chromatographic separation of the four compounds was carried out on a Welchrom ODS column (4.6 mm × 250 mm, 5 μm) by gradient elution with phosphoric acid (H3PO4) aqueous solution (0.4%)–methanol as the mobile phase, at the temperature of 30 °C and a flow rate of 1.0 mL/min. The detection wavelength was set at 270 nm. Under optimum conditions, the baseline separation of these four compounds can be performed within 30 min. The developed method was validated in terms of detection limit, quantification limit, linearity, precision, and recovery tests. Eventually, the established HPLC–DAD method was successfully applied to the simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in the extract of herb T. hemsleyanum.

Open access

The objective of the current research is to understand the degradation behavior of avanafil under different stress conditions and to develop a stability-indicating high-performance liquid chromatography (HPLC) method for simultaneous determination of degradants observed during degradation. Avanafil tablets were exposed to acid, base, water, oxidative, thermal, and photolytic degradation conditions. In acid, oxidative, thermal, and humidity degradation, significant degradation was observed. All the degradants observed during degradation were separated from known impurities of avanafil by using reverse-phase (RP)-HPLC. Mobile phase A, 0.1% trifluoro acetic acid and triethylamine in water, and mobile phase B, water and acetonitrile in the ratio of 20:80 (v/v), were used at a flow rate of 1.2 mL/min in gradient elution mode. Separation was achieved by using Inertsil ODS 3 column (3 μm, 4.6 mm × 250 mm) at 45 °C. Peak responses were recorded at 245 nm. Method capability for detecting and quantifying the degradants, which can form during stability, was proved by demonstrating the peak purity of avanafil peak in all the stressed samples. Mass balance was established by performing the assay of stressed sample against reference standard. Mass balance was found >97% for all the stress conditions. The developed analytical method was validated as per International Conference on Harmonization (ICH) guidelines. The method was found specific, linear, accurate, precise, rugged, and robust.

Open access

Elsholtzia densa Benth. var. densa (Lamiaceae) is a famous medicinal herb which has been widely used for treatment of colds, headaches, pharyngitis, fever, diarrhea, digestion disorder, rheumatic arthritis, nephritises, and nyctalopia in China. In this study, fraction of the ethyl alcohol extract of E. densa (aerial part) by different polarity solvents indicated that the ethyl acetate soluble fraction exhibited a potent 1,1-diphenyl-2-picryhydrazyl (DPPH) radical scavenging activity with the IC50 value of 148.2 μg/mL. Under the target guidance of DPPH experiment, isoquercitrin, trachelogenin, ethyl caffeate, and arctigenin were separated with purities 95.98%, 92.98%, 96.07%, and 88.83%, respectively, by a dual-mode high-speed counter-current chromatography (HSCCC) method using n-hexane–ethyl acetate–methanol–water (4.5:5:3:4, v/v/v/v) as the solvent system. In order to evaluate the scientific basis, antioxidant activity of four isolated compounds was assessed by the radical scavenging effect on DPPH radical; isoquercitrin and ethyl caffeate showed stronger antioxidant activities with IC50 values of 9.4 μg/mL and 9.2 μg/mL, respectively, while trachelogenin and arctigenin showed weak antioxidant activities with IC50 values of >500 μg/mL and 72.8 μg/mL, respectively. Results of the present study indicated that the combinative method using DPPH antioxidant assay and dual-mode HSCCC could be widely applied for rapid screening and isolating of antioxidants from complex traditional Chinese medicine extract.

Open access
Acta Chromatographica
Authors: Peiwu Geng, Jing Zhang, Bingbao Chen, Qianqian Wang, Shuanghu Wang and Congcong Wen

Dauricine is the major bioactive component isolated from the roots of Menispermum dauricum D.C., a bisbenzylisoquinoline alkaloid derivative, and has shown multiple pharmacological properties. In this work, a sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed for determination of dauricine in rat plasma and its application to pharmacokinetic study of dauricine after intravenous and oral administration in rats. After addition of daurisoline as an internal standard (IS), protein precipitation by acetonitrile was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reactions monitoring (MRM) mode was used for quantification. Calibration plots were linear throughout the range 2–600 ng mL−1 for dauricine in rat plasma. Relative standard deviation (RSD) of intra-day and inter-day precision was less than 13%. The accuracy of the method was between 95.8% and 105.9%. Matrix effect of dauricine in rat plasma ranged from 88.0% to 90.3%. Mean recoveries of dauricine in rat plasma ranged from 91.5% to 95.1%. The method was successfully applied to pharmacokinetic study of dauricine after intravenous and oral administration in rats. The bioavailability of dauricine was found to be 55.4% for the first time.

Open access
Acta Chromatographica
Authors: Yunfang Zhou, Bingbao Chen, Junyan Chen, Yanwen Dong, Shuanghu Wang, Congcong Wen, Xianqin Wang and Xiaomin Yu

In this work, a sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and fully validated for determination of jaceosidin in rat plasma. Avicularin was used as the internal standard (IS), and protein precipitation by acetonitrile was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization (ESI) source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification. Calibration plots were linear throughout the range 2–500 ng mL−1 for jaceosidin in rat plasma. Relative standard deviation (RSD) of intra-day and inter-day precision was less than 12%. The accuracy of the method was between 88.7% and 109.7%. Mean recoveries of jaceosidin in rat plasma ranged from 65.4% to 77.9%. The developed UPLC–MS/MS method was successfully applied to pharmacokinetic study of jaceosidin after intravenous administration of 2 mg kg−1 in rats. We could find that the jaceosidin rapidly eliminated, the t 1/2 was 0.7 ± 0.3 h, and clearance (CL) was 22.4 ± 3.0 L h−1 kg−1.

Open access

A newly proposed method for detecting content of adriamycin in pectin–adriamycin conjugate has been developed and evaluated. The content of adriamycin was detected by selective degradation of adramycin to adriamycinone. It was realized by a two-phase reaction system (water–chloroform reaction system), in which adriamycin was quantitatively converted to adriamycinone. Therefore, the latter can be used to calculate the precise content of adramycin in the polymer drug. To develop the method, the catalyst for degradation, the extraction solvent for adriamycinone, the temperature and time of degradation, and the ratio of pectin–adriamycin conjugate were investigated. The optimal reaction condition was as follows: 30 mg of pectin–adriamycin conjugate dissolved in 25 mL of water was added to a mixture of 25 mL of hydrochloric acid (1.5 mol/L) and 50 mL of chloroform; the mixture was heated to 40 °C to react for 1.5 h; after that, the mixture was extracted with chloroform for three times, and then the organic layer was combined and, subsequently, evaporated to remove solvent. Under this condition, adriamycinone generation rate reached 99.87%. The quantitative method was evaluated for linearity, the limit of detection (LOD) and limit of quantitation (LOQ), recovery, accuracy, robustness, and precision. The recoveries were between 99.47% and 101.07% with relative standard deviation <1.23%. The LOD and LOQ were 0.06 and 0.17 μg/mL, respectively. Compared to the traditional ultraviolet (UV) detection, this method is considered to be more precise for detecting content of adriamycin in its polymer conjugate.

Open access

A novel multi-walled carbon nanotubes (MWCNTs) dispersive solid phase extraction (d-SPE) method which combined with gas chromatography (GC) coupled with electron capture detector (ECD) was developed for the determination of five pyrethroid pesticides in liquid milk for the first time. The effect of d-SPE conditions on the kinds of sorbent, MWCNTs and magnesium sulfate anhydro mass ratio, and extraction condition were researched, and then, the suitable method was found. Under the optimal conditions, the linear range was from 20 to 500 μg kg−1. The recoveries were from 81.8% to 112.1%, with the corresponding relative standard deviations (RSDs) less than 6%, correlation coefficients from 0.9978 to 0.9990, and limits of detection and quantification from 2.62 to 4.86 μg kg−1 and 8.73 to 16.2 μg kg−1. The proposed method is simple, fast, safe, and has high recovery and sensitivity applicable to analyze pyrethroid pesticides in liquid milk sample.

Open access

A new method for simultaneous extraction and quantification of 6 nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) and 16 parent polycyclic aromatic hydrocarbons (PAHs) in water matrices was optimized and validated.

The extraction procedure was based on dispersive liquid-liquid microextraction technique, followed by gas chromatography-mass detection. The optimum conditions of extraction (volume of the extraction solvent, dispersive solvents and amount of salt) were selected using central composite design. The best results were found by using 200 μL of acetonitrile as dispersive solvent, 60 μL of chloroform as extraction solvent, and 10% (w/v) NaCl. Excellent linearity was observed in the range of 10–150 ng L−1 with correlation coefficients (r 2) ranging between 0.9996 and 0.9999 for nitro-PAHs and in the range of 5–150 ng L−1 with r 2 ranging from 0.9998 to 1.000 for PAHs. The limits of detection for the nitro-PAHs studied ranged from 0.82 to 3.37 ng L−1, whereas for PAHs ranged from 0.62 to 3.48 ng L−1. The intra- and inter-day precisions for nitro-PAHs were in the range of 0.45 to 19.54% and 0.43 to 19.62%, respectively, and for PAHs ranged between 0.45 to 17.42% and 0.38 to 18.97%, respectively. The proposed method was successfully applied in analyses of groundwater, sea, rain water and river water, being appropriate for routine analyses.

Open access

A high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection for simultaneous determination of metronidazole, methylparaben, and propylparaben in vaginal gel formulation was described. The chromatography was carried out on a C18 (250 mm × 4.6 mm, 5 μm) column with acetonitrile and 0.3% phosphoric acid solution (20:80 v/v) modified by 0.1% triethylamine as mobile phase, at a flow rate of 1.0 mL min−1, with detection at 260 nm. Under these chromatographic conditions, the obtained retention times were approximately 3.43 min for metronidazole, 5.17 min for propylparaben, and 15.12 min for methylparaben.

Analytical parameters specificity, linearity, accuracy, and precision were determined by validation procedure and found to be satisfactory. Overall, the proposed method was found to be simple, precise, and accurate for quality control of metronidazole in the presence of preservatives in gel formulation.

Open access

Shuganjieyu (SGJY) capsule is a classical formula widely used in Chinese clinical application. In this paper, an ultra-performance liquid chromatography coupled with electrospray ionization and ion trap mass spectrometry has been established to separate and identify the chemical constituents of SGJY and the multiple constituents of SGJY in rats. The chromatographic separation was performed on a C18 RRHD column (150 × 2.1 mm, 1.8 μm), while 0.1% formic acid–water and 0.1% formic acid–acetonitrile was used as mobile phase. Mass spectral data were acquired in both positive and negative modes. On the basis of the characteristic retention time (R t) and mass spectral data with those of reference standards and relevant references, 73 constituents from the SGJY and 15 ingredients including 10 original constituents and 5 metabolites from the rat plasma after oral administration of SGJY were identified or tentatively characterized. This study provided helpful chemical information for further pharmacology and active mechanism research on SGJY.

Open access

By application of preparative high-speed counter-current chromatography (HSCCC) to the crude quinolone alkaloids (1.1 g) from the fruit of Tetradium ruticarpum, 1-methyl-2((6Z,9Z)-pentadecadienyl)-4(1H)-quinolone (1, 8.4 mg), dihydroevocarpine (2, 27.0 mg), and 1-methyl-2-pentadecyl-4(1H)-quinolone (3, 18.8 mg) were isolated in one step with sufficient purity using the solvent system composed of hexane–ethyl acetate–methanol–water (Hex–EtOAc–MeOH–H2O, 5:2:5:3). Further purification of the subfraction was performed by amending the solvent composition and achieved another three quinolone alkaloids, i.e., 1-methyl-2-undecylquinolin-4(1H)-one (4, 13.7 mg), (Z)-1-methyl-2-(tridec-5-en-1-yl) quinolin-4(1H)-one (5, 14.0 mg) from subfraction FR3-A3-85 using Hex–EtOAc–MeOH–H2O (5:3.5:8.75:8.25), and 1-methyl-2-nonylquinolin-4(1H)-one (6, 15.1 mg) from subfraction FR3-A3-36 using Hex–EtOAc–MeOH–H2O (5:3.8:5:4.8). The relationship between the structure of the six alkaloids and their affinities for bovine serum albumin (BSA) was investigated using fluorescence titration analysis. The length and the presence of double bond of the side chain affected their binding process with BSA. The binding behavior might influence their other biological activities.

Open access

A method was developed for the preparative separation of two alkaloids from the crude extract of the radix of Rauvolfia verticillata (Lour.) Baill. in a single run. The two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (5:5:2:8, v/v), where triethylamine (40 mmol/L) was added to the upper organic phase as the stationary phase and hydrochloric acid (10 mmol/L) was added to the lower aqueous phase as the mobile phase, was selected for this separation by pH-zone-refining counter-current chromatography (PZRCCC). For the preparative separation, the apparatus was rotated at a speed 850 rpm, while the mobile phase was pumped into the column at 2 mL/min. As a result, 112 mg of reserpine and 21 mg of yohimbine were obtained from 3 g of crude extract in a single run. The analysis of the isolated compounds was determined by high-performance liquid chromatography (HPLC) at 230 nm with purities of over 91.0%, and the chemical identification was carried out by the data of electrospray ionization–mass spectrometry (ESI–MS) and nuclear magnetic resonance (NMR) spectroscopy. The technique introduced in this paper is an efficient method for preparative separation of reserpine and yohimbine from devil pepper radix. It will be beneficial to utilize medicinal materials and also useful for the separation, purification, and pharmacological study of Chinese herbal ingredients.

Open access

Remimazolam is a new chemical entity belonging to the benzodiazepine class of sedative drugs. A sensitive and rapid method based on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) has been developed and validated for the determination of remimazolam and its major carboxylic acid metabolite (M1) in human urine. Urine samples were prepared by dilution and analyzed using an isocratic chromatographic separation. Inter- and intra-batch results for remimazolam were within 10.7% for accuracy and 5.5% for precision, and for M1, within 5.8% for accuracy and 4.2% for precision, respectively. This study represents the first reported example for the quantification of remimazolam and its main metabolite in human urine. Furthermore, this method has been successfully applied for the urine recovery study of remimazolam in Chinese healthy subjects. Only about 0.01% of the administered remimazolam dose was eliminated in the urine over the 24 h period in the form of unchanged remimazolam, and more than 75.1% of the administered dose was eliminated in the form of M1. Remimazolam is excreted mainly in the form of M1 in urine after intravenous administration, and there is no excessive accumulation in vivo after administration of remimazolam.

Open access

A novel, simple, robust, and rapid reversed-phased high-performance liquid chromatographic method has been developed for the separation and quantitative determination of the related substances of ezetimibe and simvastatin in combined dosage forms. Successful separation of the drug from the process-related impurities and degradation products formed under stress conditions was achieved on Inertsil ODS-3V (150 × 4.6 mm, 5.0 μm) column. The gradient liquid chromatography (LC) method employs solution A and solution B as mobile phase. The solution A contains 0.1% orthophosphoric acid solution in water, and solution B contains 0.1% orthophosphoric acid solution in acetonitrile. Flow rate was monitored at 2.0 mL/min, and the ultraviolet (UV) detection, at 238 nm. In forced degradation studies, the effect of acid, base, oxidation, UV light, and temperature was investigated, showing that good resolution between the peaks corresponds to process-related impurities and degradation products from both analyte. The performance of the method was validated according to the present International Conference on Harmonization (ICH) guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness, and robustness. To the best of our knowledge, a rapid LC method, which separates all the impurities of ezetimibe and simvastatin in combined dosage forms, disclosed in this investigation was not published elsewhere.

Open access
Acta Chromatographica
Authors: Zhaojun Sheng, Ruhan Ye, Siyuan Ge, Chenggang Wang, Xuetao Xu, Guangwen Zhang and Ping Luo

An efficient and convenient reversed-phase high-performance liquid chromatography method has been developed and validated for the quantitative determination of cholic acid bulk drugs and their related impurities. Chromatographic separation was performed on a YMC-Pack ODS-AQ column (250 mm × 4.6 mm, S-5 μm, 12 nm), and the mobile phase consisted of acetonitrile, methanol, and diluted formic acid solution (pH 2.5) at a flow rate of 1.0 mL/min. The analytes were monitored using a refractive index detector at 30 °C, and the column temperature was 30 °C. Under the above chromatographic conditions, the method has good specificity and specified impurities can be effectively separated. The proposed method is found to have linearity in the 2.0–80.0 μg/mL concentration range with correlation coefficients of not less than 0.9999. The compounds analyzed in the solutions are stable for at least 7 days, and spike recoveries for all specified impurities range from 91.3% to 109.3% with relative standard deviations (RSDs) not more than 7.3%. The limit of detection and the limit of quantification for the analytes are 0.060 μg/mL and 2.0 μg/mL, respectively. The proposed method can be applied in the quality control assay of cholic acid bulk drugs, with the advantages of simplicity, accuracy, robustness, good selectivity, and high sensitivity.

Open access

A reliable isotope dilution method for the determination of chloramphenicol (CAP) in drinking water was developed by using an evaporation preparative step. Each sample was monitored by ultrahigh-pressure liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS/MS) using an electrospray ionization interface (ESI) in negative ion modes. Recoveries of spiked samples were in the range from 93.2% to 95.7% with intra-day relative standard deviation lower than 6.7% and inter-day relative standard deviation lower than 8.2%. Limit of quantification (LOD) was 0.002 ng/mL. The developed method was successfully applied to the analysis of CAP in drinking water of Shannan region of Tibet.

Open access

A new molecularly imprinted polymer (MIP) was prepared by using catechin (C) as the template molecule. The polymer was characterized by swelling tests, scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET), and Fourier transform infrared (FTIR) spectroscopy. The MIP with high recovery was selected as a solid-phase extraction (SPE) sorbent in this work. The standard solutions were directly applied onto the SPE cartridges following loading, washing, and elution procedures. A solution of the collected fractions was analyzed by high-performance liquid chromatography–diode-array detection (DAD) and fluorescence detector. The optimization of the method and validation was achieved on a C18 column (5 μm, 250 × 4.6 mm) with methanol–water (35:65, v/v) mixture adjusted pH 2.5 as the mobile phase at a flow rate of 1 mL min−1 at room temperature. The selectivity coefficient (k) of imprinted p(HEMA–MAH) cryogel was 5.1-fold that of non-imprinted cryogel. It showed good selectivity and affinity for C molecule. A comparison was made between the results obtained with the MIP cartridges and a traditional C18 reversed-phase cartridge. It was observed that 2.3 times higher recovery of C can be obtained on catechin-MIP cryogel. The results of the presented work showed that the prepared MIP can be used as SPE sorbent for extracting of C from red wines.

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Novel magnetic solid-phase extraction using carboxylated multiwalled carbon nanotubes was proposed with ultra high-performance liquid chromatography–tandem mass spectrometry for the determination of silodosin in biological samples. The effects of various experimental parameters including adsorbent amount, pH, adsorption time, desorption conditions, and adsorbent reusability were systematically validated. Under the optimized conditions, the calibration curve was linear within the concentration range of 1.0–800 ng mL−1 with the correlation coefficient of 0.9997 and the lower limit of detection was 0.3 ng mL−1. The extraction recoveries were over 90.0% with relative standard deviation (RSD) of less than 5.0%. All these results suggested that magnetic extraction method can be used for enrichment and quantification of silodosin in biological samples.

Open access
Acta Chromatographica
Authors: Liyi Li, Liming Hu, Bingbao Chen, Yanwen Dong, Zixia Lin, Zhiyi Wang, Congcong Wen, Xianqin Wang and Shuanghu Wang

In this study, we developed a urine metabolomic method by gas chromatography–mass spectrometry (GC–MS) combination with biomedical results to evaluate the effect of activated carbon on methomyl poisoning rats. The rats were divided into four groups, methomyl group, two activated carbon treatment group, and control group. According to the biochemical results, it indicated that activated carbon treated rats could cause liver and kidney function changes. According to the urine metabolomics results, activated carbon treatment group (10 min) and activated carbon treatment group (30 min) could be distinguished from methomyl group, and activated carbon treatment group (10 min) could be separated from activated carbon treatment group (30 min) rats, which indicated that the treatment of rats by activated carbon in different time had a different effect. The results indicate that metabolomic method by GC–MS may be useful to elucidate activated carbon treated on methomyl poisoning rats.

Open access
Acta Chromatographica
Authors: Shuanghu Wang, Zixia Lin, Ke Su, Jing Zhang, Lijing Zhang, Zhimou Gao, Zhiyi Wang, Jianshe Ma and Xianqin Wang

The rats were randomly divided into paraquat group, curcumin treatment group, and pirfenidone treatment group. The concentration of paraquat in rat plasma was determined by an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method over the range of 10–2000 ng mL−1. Chromatographic separation was achieved on a BEH HILIC (2.1 mm × 100 mm, 1.7 μm) column. The mobile phase was consisted of acetonitrile and 10 mm ammonium formate buffer (containing 0.1% formic acid) with gradient elution pumped at a flow rate of 0.4 mL min−1. Protein precipitation with acetonitrile was used as sample preparation. Compared with the paraquat group, there is statistical toxicokinetic difference for curcumin treatment group and pirfenidone treatment group, AUC(0 − t) decreased (P < 0.05), clearance (CL) increased (P < 0.05) for curcumin or pirfenidone treatment group, and C max decreased (P < 0.05) for curcumin treatment group. The results showed that treatment by curcumin and pirfenidone could relieve acute paraquat poisoning in rats.

Open access
Acta Chromatographica
Authors: Yuping Shen, Minhui Xu, Peipei Deng, Qinying Gu, Huawu Yin, Guohua Xia, Xiaobin Jia, Huan Yang and James Tam

Red Toon is a popular vegetable of favorable health benefits over Asia and Russia regions. In this study, isolation and identification of chemical constituents were performed to assess the quality of this functional food cultivated in various origins or harvested in different months. As a result, eight flavonol glycosides including rutin (I), myricitrin (II), quercetin-3-O-β-d-galactoranoside (III), quercetin-3-O-β-d-glucopyranose (IV), quercetin-3-O-α-l-arabinopyranoside (V), astragalin (VI), quercetin-3-O-α-l-rhamopyranoside (VII), and kaempferol-3-O-α-l-rhamopyranoside (VIII) were obtained. Among these, compounds III and V were isolated from Toona genus for the first time. Importantly, a rapid and convenient ultra-performance liquid chromatography (UPLC) method was developed to quantify the flavonol glycosides in Red Toon and validated for linearity, precision, stability, repeatability, and accuracy successively. In addition, it was found that total flavonoid glycosides (about 2.6%) in the food were kept at a higher level from April to June than other months of the year. Furthermore, their content in the Red Toon collected from ten different origins was also determined and compared, and the results suggested that the total flavonoid glycosides from Shandong Yantai were the highest, followed by Shandong Ximou, supporting a well-recognized viewpoint that Red Toon cultivated in Shandong Province, China, is considered genuine due to the best health benefits and flavor.

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The levels of persistent organic pollutants (POPs) from industrial by-products were determined in beach sand and marine sediments from different sites along the Jordanian coast of the Gulf of Aqaba. Seventeen polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans (PCDD/Fs) compounds were identified. Automatic Soxhlet (Soxtec) extraction method was used for PCDD/Fs extractions using toluene as a solvent. Pre-washed multilayer silica gel column was used for cleanup step. Samples were analyzed using high-resolution gas chromatography and mass spectroscopy detector (HRGC–MS). Low levels of POPs were found in all sand and sediment samples. Concentrations of PCDD/Fs ranged between 3.91 and 8.91 ng kg−1 dw with an average of 6.49 ng kg−1 dw for beach sand samples and between 6.560 and 45.95 ng kg−1 dw with an average of 28.70 ng kg−1 dw for marine sediment samples. Concentrations of PCDD/Fs for soil and sediment were mostly less than other sites worldwide. PCDD/Fs concentrations measured in this study can be considered as a baseline for future monitoring and control of PCDD/Fs as requested by Stockholm Convention.

Open access
Acta Chromatographica
Authors: Aneta Cernikova, Pavel Bobal, Janette Bobalova, Jiri Dohnal and Josef Jampilek

The investigation deals with the affection of the permeation of acyclovir through full-thickness pig ear skin using a Franz diffusion cell from the donor vehicles of phosphate buffer (pH 7.4) and propylene glycol–water (1:1) using synthesised (S)-8-methyl-6,9-diazaspiro[4.5]decane-7,10-dione, alaptide as a transdermal permeation enhancer. Alaptide was applied in ratio 1:10 (w/w) relative to the amount of acyclovir. At the first hour after application, the permeated amount of acyclovir from propylene glycol–water system, simulating semisolid dosage forms, was ca. four-fold higher than from the formulation without alaptide. Despite that the enhancement ratio of alaptide in a steady state was 1.7, the pseudo-enhancement ratio of alaptide in the time range of first to third hour was 2.3. Both enhancement ratios indicate that alaptide modifies skin structure, while the short-term application of the alaptide formulation seems to be more advantageous.

Open access

A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method and ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) were optimized and validated for 16 antibiotics belonging to three families (macrolides, quinolones, and sulfonamides) that were found in preserved eggs. Samples were extracted in 4 mL water and 10 mL acetonitrile with 1% acetic acid and subjected to a cleanup procedure using dispersive solid-phase extraction with C18 and primary secondary amine sorbents, prior to detection by UHPLC–MS/MS. Matrix-matched calibration was used for quantification to reduce the matrix effect with limits of quantification in the range of 0.3–3.0 μg/kg. Validation of the method was conducted by recovery and precision experiments. Recoveries of the spiked samples ranged from 73.8% to 127.4%, and the intra- and inter-day relative standard deviations were lower than 21.2% and 22.3%, respectively. This method was successfully applied to the analysis of antibiotics in preserved egg samples.

Open access

The number of new drugs launched to the market is constantly increasing; however, the metabolism of many of them is still not fully established. The knowledge of drug metabolism pathways is crucial for the efficacy and safety of therapies and, in classical approach, requires the use of animals as well as human volunteers, but this kind of research is expensive and time-consuming. Therefore, nowadays, more and more biological and chemical in vitro methods are developed for the drug metabolism study. This review is focused on the photocatalytic degradation of chemicals and the application of this process in chromatographic methods of drug metabolism research. A theoretical background of photocatalysis and all its applications in a drug metabolism study were reviewed, and other in vitro methods that are actually used were summarized and discussed. Other analytical methods used in this area were also discussed and compared.

Open access

A simple and rapid high-performance liquid chromatographic (HPLC) method was established for simultaneous determination of butorphanol tartrate and ondansetron hydrochloride in analgesic mixture samples used for patient-controlled analgesia (PCA). The separation of butorphanol tartrate and ondansetron hydrochloride in PCA solution was carried out on phenomenex C18 column (4.6 mm × 150 mm, 5 μm) using 50 mM sodium acetate (pH 4.0) buffer and acetonitrile (72:28, v/v). Flow rate was 1.0 mL min−1 with a column temperature of 30 °C, and detection wavelength was carried out at 280 nm and 306 nm. Validation of the method was made in terms of specificity, linearity, accuracy, and intra- and inter-day precision, as well as quantification and detection limits. The developed method was successfully used to evaluate the chemical stability of butorphanol tartrate and ondansetron hydrochloride in analgesic mixtures at the usual concentration used for PCA.

Open access

The use of polyphenols in food fortification is a common custom generally carried out to increase its nutritional value. In this paper, ground chili pepper was proposed as a potential functional coffee additive. Various phenolic compounds present in this spice were analyzed by a new, sensitive, and selective ultrahigh-performance liquid chromatography combined with mass spectrometry (UPLC–MS). Separation was done on a column filled with a modified silica gel RP-18, in gradient solvent systems A (1% H3PO4 in water) and B (40% CH3CN in solution A). The capsaicin was found as the main phenolic compound of ground chilli pepper, which concentration was 295.95 mg g−1. It was demonstrated that quercetin is present in this spice also in different forms: not only as dihydrocapsaicin but also as quercetin-3-O-deoxyhexoside-glucuronide and quercetin-3-O-deoxyhexoside, whereas luteolin in the form of three compounds: luteolin-7-O-dihexoside, luteolin-6-C-hexoside-8-C-pentoside, and luteolin-7-O-malonyl-dihexosyl-pentoside. We have also identified apigenin-6-C-hexoside-8-C-pentoside. Furthermore, this paper, for the first time, evaluates the potential bioaccessibility of and interactions between compounds with multidirectional antioxidant properties from coffee and ground chili pepper. All samples, coffee, chili, and a mixture of the two showed ability to scavenge free radicals and chelate iron ions and were characterized by reducing power. The level of these activities changed after simulated gastrointestinal digestion. In the mixtures of water extracts, phytochemicals acted synergistically in the case of five from six tested methods. Interestingly, after digestion, in vitro chili extract lost ability to scavenge O2 radicals; that is why it was impossible to determine the interactions between coffee and chili in this case. Moreover, an antagonism in the action was observed for those cases, where, in water extracts, we have identified synergistic interaction.

Open access

The important role of fruits in human health and nutrition has been better understood with the recent studies on biochemical contents of fruits having antioxidant properties. Being one of the similar studies, in this study, total antioxidant capacity (TAC), phenolic compound, organic acid, and vitamin C contents of three plum species (Prunus domestica L., Prunus cerasifera Ehrh., and Prunus spinosa L.) grown in Van locality (Turkey) were identified, and the correlation between the measured values was investigated. Phenolic compound, organic acid, and vitamin C contents were determined by high-performance liquid chromatography (HPLC) method. Analysis of phenolic compound indicated that chlorogenic acid was the predominant phenolic compound, and the highest value was measured in P. spinosa L. as 12.985 mg kg−1. Malic acid was the predominant organic acids and the highest value was measured in P. spinosa L. as 1.245 g 100 g−1. The highest TAC and vitamin C contents were also measured in P. spinosa L. as 1.021 mmol TE kg−1 and 25.492 mg 100 g−1, respectively. P. spinosa L. was found to be superior to the other two species with respect to antioxidant capacity and other biochemical contents. A significant (P ≤ 0.01) and positive correlation was reported between antioxidant capacity and vitamin C content.

Open access
Acta Chromatographica
Authors: Stefano Dugheri, Alessandro Bonari, Ilenia Pompilio, Marco Colpo, Nicola Mucci, Manfredi Montalti and Giulio Arcangeli

World consumption of formaldehyde (FA) is forecast to grow at an average annual rate of about 4% from 2015 to 2020 with world production to exceed 52 million tons in 2017. From the first day of January 2016, the Commission Regulation No. 91/2015 established the FA classification through an indication from European Chemical Agency as category 2 mutagenic and category 1B carcinogen. A novel method for the determination of gaseous FA in air is presented herewith. The sampling was carried out using a miniaturized cartridge by means of a medium-flow pumping system (1.0 L min-1, 5–60 min) and absorption of FA vapors on 2,4-dinitrophenylhydrazine. Cartridge desorption removing the excess derivatizing agent based upon solid-phase extraction was performed by an innovative xyz robotic system on-line with fast gas chromatography (GC)—mass spectrometry (MS). Through the generation of standard atmospheres of known concentration of FA, we evaluated the precision (relative standard deviation for n = 10, 8.8%), lower limit of quantification (0.072 µg/cartridge), and linearity (from 0.125—64 µg/cartridge with correlation coefficient of 0.99) of the method. The described procedure combines the efficiency of fast GC—MS systems with both the high throughput of autosampler and the quantitative accuracy of FA-dinitrophenylhydrazone for measuring American Conference of Governmental Industrial Hygienists TLV Ceiling.

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Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund Minilab kit protocols for detecting fake or markedly substandard drugs in pharmaceutical products in the field to quantitative high-performance TLC (HPTLC)–densitometry methods was carried out using a model process published earlier. The developed and validated methods for tablets or capsules containing cefixime, cefuroxime axetil, cephalexin·H2O, ciprofloxacin HCl, levofloxacin, and metronidazole involved use of EMD Millipore Premium Purity silica gel 60 F254 plates, automated sample and standard solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification.

Open access

In this study, we developed a highly sensitive, robust method for determining 12 congeners of two to ten chlorinated polychlorinated biphenyls (PCBs) in serum samples using gas chromatography (GC)–mass spectrometry (MS) operating in selected ion monitoring mode (SIM: m/z 35) with negative ion chemical ionization (NICI), and the results were compared with those from GC coupled with high-resolution MS (HRMS) with electron impact (EI). The recovery rates of the surrogate PCB congeners were 97.2%–112% (coefficient of variation: 5.3%–8.5%), and the method detection limits for PCBs in each matrix obtained by GC–NICI–quadrupole mass spectrometry (qMS) were 1.9–20 pg g−1 wet wt. The analytical values of the target compounds in the samples analyzed by GC–NICI–qMS and GC–EI–HRMS were comparable (Passing–Bablok regression: R = 0.888–0.967), and the analytical values obtained via GC–NICI–qMS were almost comparable with those of the certified serum samples from National Institute of Standards and Technology (NIST: SRM1957), indicating that GC–NICI–qMS is suitable for the analysis of tetra- to hepta-chlorinated PCBs in serum samples.

Open access

10-Methoxycamptothecin (MCPT) and 10-hydroxycamptothecin (HCPT) are the indole alkaloids isolated from a Chinese tree, Camptotheca acuminata, and have a wide spectrum of anticancer activity in vitro and in vivo mainly through inhibitory effects on topoisomerase I. HCPT is a major metabolite of MCPT in rats; the pharmacokinetic analysis and tissue distribution of MCPT and HCPT in rats have also been determined after i.v. injection of MCPT, but the excretion of MCPT and its metabolite HCPT has not been assessed up to now. In the present study, the excretion study of MCPT and its metabolite HCPT in rat bile, feces, and urine after i.v. administration of MCPT (5 mg kg−1) was performed by high-performance liquid chromatography (HPLC) method coupled with a fluorescence detector. The results showed that MCPT mainly biotransformed to HCPT and excreted in the form of HCPT and MCPT in bile, urine, and feces after i.v. administration of MCPT. It was excreted about 1.24 ± 0.07% as MCPT and 5.49 ± 0.40% as HCPT in bile within 6 h after i.v. administration. The cumulative excretions of MCPT and HCPT were mainly within 24 h after i.v. administration, which were 0.41 ± 0.10% and 7.66 ± 1.43% of the dosage in urine and about 0.16 ± 0.04% and 20.30 ± 3.35% of the dosage in feces. The total excretion of MCPT in urine, bile, and feces was 1.81 ± 0.09% in the form of original MCPT and 33.45 ± 1.57% detected as the metabolite HCPT in urine, bile, and feces, suggesting that MCPT might undergo other biotransformation.

Open access

An ionic liquid-based cloud-point extraction (IL-CPE) method was developed for the extraction of quercetin in juice samples before its determination by high-performance liquid chromatography (HPLC). 1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) was used as the ionic liquid. The cloud-point extraction parameters such as sample pH, extraction temperature, extraction time, amount of ionic liquid, extraction volume, and salt concentration were carefully studied and optimized for the achievement of maximum extraction recovery. Under the optimized conditions, i.e., 20 min heating at 40 °C, 100 μL IL volume, pH 2.0, and no salt addition, a mean recovery of 92.5% and an enrichment factor of 20 were obtained for quercetin. Relative standard deviation of the method was 3.76% for 6 replicates, and the calculated detection limit (3σ) of quercetin was 0.002 mg L−1. The method, coupled to HPLC was successfully applied to the sensitive determination of quercetin in apple and gapes juice samples with quantitative recoveries.

Open access

Bee pollen is a health food with a wide range of nutritional and therapeutic properties. However, the bioactive compounds of bee pollen have not been extensively revealed due to low efficacy in separation. High-speed counter-current chromatography (HSCCC) and solvent extraction were applied to separate tyrosinase inhibitors from camellia pollen in this study. The camellia pollen extracts prepared with petroleum ether, ethyl acetate, and n-BuOH have tyrosinase inhibitory activity. Acidic hydrolysis could promote the tyrosinase inhibitory activity of crude sample. Three fractions with tyrosinase inhibitory activity were separated from the hydrolysate by a one-step HSCCC procedure. Among the fractions, two chemicals were sufficiently purified and identified to be levulinic acid (LA) and 5-hydroxymethylfurfural (5-HMF). The recovery was 0.80 g kg−1 pollen for LA and 1.75 g kg−1 pollen for 5-HMF; and their purity was all over 98%. The study demonstrates that HSCCC method is powerful for preparative separation of tyrosinase inhibitors from camellia pollen.

Open access

New, simple, selective, and sensitive liquid chromatography–ultraviolet (LC–UV) methods have been developed and subsequently validated for simultaneous determination of linagliptin–empagliflozin combination and simultaneous determination of alogliptin benzoate–pioglitazone hydrochloride combination. Linearity was found to be acceptable over the concentration ranges of 2–50 μg mL−1, 4–100 μg mL−1, 0.5–25 μg mL−1, and 1–25 μg mL−1 for linagliptin (LNG), empagliflozin (EMG), alogliptin (ALG), and pioglitazone (PGN), respectively. All the methods were applied successfully to the analysis of the pharmaceutical dosage forms. The optimized methods were validated and proved to be robust and accurate for the quality control of the mentioned drugs in their different pharmaceutical dosage forms.

Open access

A simple, sensitive, and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for determination of phloretin in dog plasma using darunavir as internal standard. The phloretin was separated by the Inertsil® ODS3 C18 column (150 mm × 4.6 mm, 5 μm) and determined by LC–MS/MS. The electrospray ionization (ESI) source was operated in negative ionization mode for phloretin and positive ionization mode for darunavir (internal standard, IS). The multiple reaction monitoring (MRM) transitions were chosen to be m/z 273.0 → m/z 148.9 for phloretin, m/z 443.2 → m/z 401.0 for 2′,4′,6′,4-tetra-acetylphloretin and m/z 548.1 → m/z 69.1 for IS. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect. All validation parameters met the acceptance criteria according to regulatory guidelines. 2′,4′,6′,4-Tetra-acetylphloretin, as a prodrug of phloretin, is more stable than phloretin (PH) in vitro, protecting phenolic hydroxy from being oxygenated. The method had been successfully applied to a pharmacokinetic study of administration of phloretin and 2′,4′,6′,4-tetra-acetylphloretin in beagle dogs. Significant differences of t max, C max, and area under the plasma concentration curve (AUC) were observed between phloretin and 2′,4′,6′,4-tetra-acetylphloretin.

Open access

A rapid and simple analytical method based on matrix solid-phase dispersion combined with liquid chromatography coupled with tandem mass spectrometry has been developed by using bamboo charcoal as a dispersive adsorbent to simultaneously determine tetrabromobisphenol A (TBBPA) and hexabromocyclododecane diastereoisomers (HBCDs) in soil. The factors influencing the performance of the proposed method were investigated and optimized in detail, and the matrix effects were evaluated. Under optimum conditions, the proposed method showed good linearity within the range of 0.8–80 ng g−1 and limits of detection of 4–75 pg g−1 (S/N = 3). The satisfactory recoveries of TBBPA ranging from 72.8% to 92.5% and HBCDs ranging from 76.8% to 102.2% were obtained with relatively standard deviation (RSD) ranging from 3.4% to 9.8%. The proposed method has been successfully applied to analyze TBBPA and HBCDs in actual soil samples from the Yellow River Delta in China.

Open access

A stability-indicating capillary electrophoresis method coupled to a diode array detector (DAD) was developed and validated for the simultaneous determination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) in combined tablets. This proposed method utilized a fused silica capillary (effective length, 62 cm; internal diameter [ID], 75 μm) and a background electrolyte (BGE) consisting of phosphate solution (pH 9.5, 50 mM). The separation was achieved at a voltage of 25 kV and a temperature of 21 °C using paracetamol as an internal standard. The described method was linear over the range of 5–200 μg/mL for both drugs (r = 0.9992). Intra- and inter-day relative standard deviation (RSD) (n = 9) was 0.41%. The limits of detection for FTC and TDF were 1.25 and 1.00 μg/mL, respectively. The average percentage recoveries of FTC and TDF from their tablet formulations were 99.66 ± 0.73 and 99.48 ± 0.33, respectively. The two drugs were subjected to thermal, photolytic, hydrolytic, and oxidative stress conditions, and then the stressed samples were analyzed by the proposed method. Degradation products produced as a result of stress studies did not interfere with the detection of FTC and TDF. The assay can thus be considered stability indicating.

Open access

The off-line two-dimensional supercritical fluid chromatography (SFC)–ultrahigh-performance liquid chromatography (UHPLC) was selected to separate the triterpene saponins from Panax notoginseng. The separation by SFC was performed on an Atlantis® HILIC silica column. Methanol was selected as a modifier, and the most time-saving gradient was developed. The decrease of the column temperature and the increase of the back pressure could shorten the retention time but had no effect on the separation selectivity. Then, the back pressure, column temperature, and flow rate were set as 131 bar, 45 °C, and 4.0 mL min−1, respectively. The retention behavior of the saponins from P. notoginseng was different between SFC and reversed-phase liquid chromatography (RPLC), which facilitated to construct an off-line SFC/RPLC–mass spectrometry (MS) system. In first dimension, a total of eight fractions were collected under SFC and further analyzed by RPLC–MS in second dimension. The result indicated that the retention behavior of triterpene saponins was mainly controlled by the hydrogen bonding interactions which were affected by the number and types of sugars, as well as the aglycone in the structure of triterpene saponins. Thus, the presence of “clustering effect” under SFC was observed, namely, one SFC peak always contained several saponins with same number of sugars and similar structure of aglycone. The clustering effect of triterpene saponins promised SFC to be used as first dimension to complete the preliminary crude separation in the two-dimensional liquid chromatography.

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The particle size distribution (PSD) values obtained for a soil database representing the main Hungarian soil types using the Hungarian standard (MSZ-08-0205-78) and the international standard (ISO/DIS 11277:1994) were compared with the pipette method. The relationship between these PSDs and other physical soil characteristics (upper limit of plasticity according to Arany, water vapour adsorption according to Sík) was also analysed, and a suggestion was made of how these results could be converted into each other.

Experience showed that the pre-treatments applied as part of the ISO/DIS method may change the ratio of particle size fractions: there was a significant increase in the clay content, while the silt content decreased to a lesser and the sand content to a greater extent, possibly because some of the particles remain in microaggregate form when the MSZ method is used. The results confirmed the greater accuracy of the ISO/DIS method: the clay contents measured with the ISO/DIS method exhibited stronger correlations with the upper limit of plasticity according to Arany and with hygroscopicity values than those measured with the MSZ method.

The estimated ISO/DIS fractions became much closer to the measured ones when the suggested pedotransfer functions were applied. The conversion method proved to be more reliable for the prediction of clay and sand content than for silt content. In its present form the estimation method is not suitable for replacing the ISO/DIS method, but it could be of good service in research and comparative analysis in cases where only the MSZ method can be used or where only old MSZ PSD data exist.

Open access
Acta Chromatographica
Authors: Jong-Woo Jeong, Yun-Hwan Seol, Hun-Chan Hyun, Hye-Rim Kim, Jong-Hwa Lee, Young-Dae Gong, Nam Sook Kang and Tae-Sung Koo

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of an anticancer drug, supinoxin (RX-5902), in rat plasma. Following precipitation pretreatment using 0.1% formic acid in acetonitrile, separation was performed using a reverse phase liquid chromatography column packed with C18 (3.5 μm, 2.1 × 50 mm) along with a mobile phase of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL min−1. Detection was achieved using MS/MS by multiple reaction monitoring via an electrospray ionization source at mass/charge transitions of m/z 442.30 → 223.30 for supinoxin and m/z 430.08 → 223.20 for the internal standard DGG-200064. This method demonstrated a linear standard curve (r = 0.9980) over a supinoxin concentration range of 0.0005–1 μg mL−1, as well as intra- and inter-assay precisions below 7.08% and 13.74%, respectively, and an accuracy of 1.15–4.50%. The matrix effect, recovery, and process efficiency were 93.63%, 99.70%, and 93.33%, respectively. Thus, a sensitive and reliable LC–MS/MS method was developed and validated for the quantification of supinoxin in rat plasma. This method was successfully applied to the evaluation of pharmacokinetic studies after single intravenous and oral administration of 1 mg kg−1 supinoxin in rats.

Open access

A highly sensitive analytical tool for the fast quantification of irsogladine in human plasma was developed. Cleanup using a solid-phase extraction technique is a simple method for extracting both irsogladine and lamotrigine (internal standard) spiked into human plasma. The resolvable separation of both analytes through reversed-phase high-performance liquid chromatography (HPLC) was carried out within 5 min. The HPLC–electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) method, which was operated in a selected reaction monitoring mode specific to the target analytes, was verified for use in the quantification of irsogladine. The inter- and intra-day precision (relative standard deviation, RSD) of irsogladine spiked into quality control samples were <7%, and their accuracies were between 96.6% and 102.1%. The calibration curve for irsogladine spiked into human plasma was linear over the range from 1.8 to 100 ng mL−1 with lower limit of quantification at 1.8 ng mL−1. The established method was successfully applied for a bioequivalence study of irsogladine.

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Thin-layer chromatography—direct bioautography (TLC—DB) followed by liquid chromatography—tandem mass spectrometry (LC—MS/MS) was used for screening and tentative identification of the antibacterial constituents of Salvia officinalis L. ethanol extract. Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, that is, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, luminescence gene-tagged Pseudomonas syringae pv. maculicola, and naturally luminescent marine bacterium Aliivibrio fischeri. Eight fractions with the widest antimicrobial spectrum were detected using TLC—DB, isolated by semi-preparative TLC, and subjected to LC—MS/MS analyses. Finally, five bioactive components were tentatively identified, based on their fragmentation pattern, such as salvigenin, cirsimaritin, rosmanol, carnosic acid, and 12-O-methyl carnosic acid.

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Scutellaria barbata D. Don has been used as a traditional Chinese medicine for antitumor and anti-inflammatory. However, there were just a few investigations about S. barbata D. Don according to bioactivity-directed isolation and online identification for the chemical constituents. In this work, eight compounds were isolated from S. barbata D. Don. The three flavonoids indicated the cytotoxic activity against human leukemic Reh cell lines. In addition, the constituents of S. barbata D. Don were further characterized and identified by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The UHPLC- Q-TOF-MS method was in negative ion mode. HPLC separation was performed on a Tosoh TSK gel ODS-100V (4.6 × 150 mm, 3.0 μm) column by gradient elution using water containing 0.3% formic acid and acetonitrile as mobile phase at a flow rate of 0.8 mL min−1. A total of 18 compounds, including 4 phenolic acids and 14 flavonoids were tentatively characterized and identified by means of the retention time, accurate mass, and characteristic fragment ions.

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Ionic liquid-based ultrasonic-assisted extraction (ILUAE) was successfully applied to the extraction of the four chromones (prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside, and sec-O-glucosylhamaudol) from Saposhnikovia divaricata (Radix Saposhnikoviae) for the first time. A series of l-alkyl-3-methylimidazolium ILs differing in anion and cation compositions was evaluated for extraction efficiency, and [C3MIM]Br was selected as the optimal solvent. In addition, ultrasound extraction parameters were optimized, and the chromones were directly quantified and analyzed by rapid resolution liquid chromatography-electrospray ionization/mass spectrometry (RRLC-ESI/MS). The optimal conditions were as follows: 0.4 M concentration of [C3MIM]Br, 20:1 solvent to solid ratio, and ultrasonic time, temperature, and frequency of 5 min, 40 °C, and 50 kHz, respectively. This approach obtained the highest extraction yield of 10.188 ± 0.473 mg g−1 for total chromones. Compared with regular UAE, the proposed approach exhibited a higher efficiency (61.56% increase) and shorter extraction time (nine times shorter). Also, ILUAE was an efficient, rapid, and simple sample preparation technique for extraction of chromones, and the established RRLC-DAD method could serve as a rapid and effective technique for extracting chromones from Radix Saposhnikoviae.

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Continuous-flow processing in the manufacturing of modern biotherapeutics represents a great potential and could significantly improve productivity and product quality as well as reduce operating costs. Microfluidic perfusion systems are not only capable for producing therapeutic proteins but also suitable for organ-on-a-chip based drug testing and toxicology studies. Integrating modular unit operations for protein purification in the microfluidic cell culture device can lead to point-of-care therapeutic protein production. The multi-organ microfluidic platforms that integrate several organ-on-a-chip microfluidic units will help in preclinical testing of drug substances and toxicological studies by producing highly reliable preclinical pharmacokinetic data. In this perspective, the current state of the art and future trends of continuous flow systems are summarized for biopharmaceutical production and organ-on-a-chip drug testing.

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While continuous-flow chemistry is steadily increasing its footprint in academic research and in the manufacturing of pharmaceutical intermediates and fine chemicals, the attention for flow chemistry in educational programs is on average rather limited. This account is meant to provide a personal overview of the possibilities to address the involvement of flow chemistry in the various stages of chemical education.

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Acta Chromatographica
Authors: K. Wróblewski, A. Petruczynik, B. Buszewski, M. Szultka-Młyńska, H. Karakuła-Juchnowicz and M. Waksmundzka-Hajnos

Vortioxetine is a new drug against major depressive disorder with high affinity for a range of different serotonergic targets in the central nervous system. Therapeutic drug monitoring is an important tool for the clinical management of patients receiving a pharmacotherapy, particularly in psychiatry. For this reason, determination of drug concentration in biological fluids is important for a rational dosage of drugs. Rapid and reliable analytical assays are also required to detect and identify drugs of toxicological importance. For analysis of vortioxetine by high-performance liquid chromatography (HPLC), no procedures for its determination in saliva have been reported and there are only a few ones for its determination in serum. A sensitive and selective highperformance liquid chromatography with diode array detector (HPLC-DAD) or mass spectrometer (HPLC-MS) method was developed for the fast quantification of vortioxetine in human saliva and serum. The determination was performed on a Synergi Polar RP column in isocratic mode under the optimal mobile phase containing 70% methanol, 20% acetate buffer at pH 3.5, 10% double distilled water, and 0.025 M L−1 diethylamine.

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