Quantification of isoflavonoids and triterpene saponins in Astragali Radix, the root of Astragalus membranaceus, via reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Seung-Min Lee, Ji-Seon Jeong, Ha-Jeong Kwon, Seon-Pyo Hong
Astragali Radix, the root of Astragalus membranaceus Bunge, is one of the most frequently used crude drugs in Asian medicine. We developed a quantification method for 6 components (calycosin, formononetin, astragaloside I–IV) of Astragali Radix and Hwanggi-gyeji-omul-tang (HGOT) using reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection (RP-HPLC-IPAD). The plants were extracted in 80% ethanol for 2h. All target components were detected with good sensitivity using sodium hydroxide (as a post-column eluent). The limit of detection (S/N=3) and limit of quantification (S/N=10) of the target components ranged from 0.10–1.00ng and from 0.30–3.00ng, respectively. The coefficients of linear regression ranged from 0.9993–1.0000, all interday and intraday precision values were <3.64%, and the average recovery ranged from 99.00–102.97% for Astragali Radix and 97.73–102.57% for HGOT. This method exhibited good selectivity, sensitivity, and reproducibility and can be used directly without any pretreatment steps. Our method will therefore be useful as a quality control measure for Astragali Radix.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Seung-Min Lee, Ji-Seon Jeong, Ha-Jeong Kwon, Seon-Pyo Hong

Astragali Radix, the root of Astragalus membranaceus Bunge, is one of the most frequently used crude drugs in Asian medicine. We developed a quantification method for 6 components (calycosin, formononetin, astragaloside I–IV) of Astragali Radix and Hwanggi-gyeji-omul-tang (HGOT) using reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection (RP-HPLC-IPAD). The plants were extracted in 80% ethanol for 2h. All target components were detected with good sensitivity using sodium hydroxide (as a post-column eluent). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) of the target components ranged from 0.10–1.00ng and from 0.30–3.00ng, respectively. The coefficients of linear regression ranged from 0.9993–1.0000, all interday and intraday precision values were <3.64%, and the average recovery ranged from 99.00–102.97% for Astragali Radix and 97.73–102.57% for HGOT. This method exhibited good selectivity, sensitivity, and reproducibility and can be used directly without any pretreatment steps. Our method will therefore be useful as a quality control measure for Astragali Radix.





Micro-QuEChERS extraction coupled to GC–MS for a fast determination of Bisphenol A in human urine

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Luísa Correia-Sá, Sónia Norberto, Cristina Delerue-Matos, Conceição Calhau, Valentina F. Domingues
Bisphenol A (BPA) is considered an endocrine disruptor and public concern over BPA exposure has been raised. Several studies have assessed human exposure to this plasticizer, confirming its ubiquitous presence and highlighting children as a public of special concern. A simple, efficient, cheap and green analytical procedure is reported within this paper. This paper reports, for the first time, the development of a modified Micro-QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method coupled to gas chromatography-mass spectrometry (GC–MS) as a new strategy for the efficient extraction and determination of Bisphenol A in human urine samples. Several parameters that are known to influence extraction were optimized. Good linearity was achieved at the studied concentration range (1–50μg/L), with a correlation coefficient (R2) of 0.998. The optimized method proved to be accurate (≥74% recovery), reproducible (<11% relative standard deviation) and sensitive for BPA determination (detection limit of 0.13μg/L and quantification limit of 0.43μg/L). The analytical procedure was applied to the analyses of 12 urine samples collected from children living in the North/Center region of Portugal. BPA was detected in all the analyzed samples in concentrations ranging from 1.5μg/L to 48.9μg/L. The proposed methodology is suitable for the determination of BPA in urine samples in the framework of biomonitoring studies and bioanalytical analyses, applying GC–MS detection.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Luísa Correia-Sá, Sónia Norberto, Cristina Delerue-Matos, Conceição Calhau, Valentina F. Domingues

Bisphenol A (BPA) is considered an endocrine disruptor and public concern over BPA exposure has been raised. Several studies have assessed human exposure to this plasticizer, confirming its ubiquitous presence and highlighting children as a public of special concern. A simple, efficient, cheap and green analytical procedure is reported within this paper. This paper reports, for the first time, the development of a modified Micro-QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method coupled to gas chromatography-mass spectrometry (GC–MS) as a new strategy for the efficient extraction and determination of Bisphenol A in human urine samples. Several parameters that are known to influence extraction were optimized. Good linearity was achieved at the studied concentration range (1–50μg/L), with a correlation coefficient (R2) of 0.998. The optimized method proved to be accurate (≥74% recovery), reproducible (<11% relative standard deviation) and sensitive for BPA determination (detection limit of 0.13μg/L and quantification limit of 0.43μg/L). The analytical procedure was applied to the analyses of 12 urine samples collected from children living in the North/Center region of Portugal. BPA was detected in all the analyzed samples in concentrations ranging from 1.5μg/L to 48.9μg/L. The proposed methodology is suitable for the determination of BPA in urine samples in the framework of biomonitoring studies and bioanalytical analyses, applying GC–MS detection.





A new reliable, transposable and cost-effective assay for absolute quantification of total plasmatic bevacizumab by LC–MS/MS in human plasma comparing two internal standard calibration approaches

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Rachel Legeron, Fabien Xuereb, Stephane Chaignepain, Alain-Pierre Gadeau, Stephane Claverol, Jean-William Dupuy, Sarah Djabarouti, Thierry Couffinhal, Jean-Marie Schmitter, Dominique Breilh
The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC–MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%.This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC–MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Rachel Legeron, Fabien Xuereb, Stephane Chaignepain, Alain-Pierre Gadeau, Stephane Claverol, Jean-William Dupuy, Sarah Djabarouti, Thierry Couffinhal, Jean-Marie Schmitter, Dominique Breilh

The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC–MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%. This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC–MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies.





Determination of perfluoroalkyl acid isomers in biosolids, biosolids-amended soils and plants using ultra-high performance liquid chromatography tandem mass spectrometry

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Hongna Zhang, Bei Wen, Wen Wen, Yibing Ma, Xiaoyu Hu, Yali Wu, Lei Luo, Shuzhen Zhang
Isomer-specific analysis of perfluoroalkyl acids (PFAAs) is important to accurately assess their environmental source, fate, and human risks. In this study, a method was developed for the determination of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorohexane sulfonate (PFHxS) isomers in biosolids, biosolids-amended soils and plants using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS). The separation efficiencies of two chromatographic columns and extraction capacities of different methods were tested. Compared with the C18 column (ACQUITY UPLC BEH Shield RP18 column), the column with an alkyl perfluorinated C8 stationary phase (Epic FO LB column), in combination with the distinct MS/MS transitions of analytes, allowed better separation of most isomers. The ion-pair extraction method showed more effective matrix separation than that of the alkaline digestion method, with recoveries ranging from 79.6-105% for biosolids, 80.4-116% for soils, and 68.0-114% for plant tissues. The method detection limits ranged from 10 to 55, 3–13, and 8–58pg/g dry weight for biosolids, soil, and plants, respectively. This method was applied successfully to quantify individual isomers in biosolids, biosolids-amended soils and plants. Six PFOA, eight PFOS, and two PFHxS isomers were found in the samples, with linear isomers being the dominant species. Further analysis revealed that the translocation potentials of branched isomers within plants were higher than those of linear isomers.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Hongna Zhang, Bei Wen, Wen Wen, Yibing Ma, Xiaoyu Hu, Yali Wu, Lei Luo, Shuzhen Zhang

Isomer-specific analysis of perfluoroalkyl acids (PFAAs) is important to accurately assess their environmental source, fate, and human risks. In this study, a method was developed for the determination of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorohexane sulfonate (PFHxS) isomers in biosolids, biosolids-amended soils and plants using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS). The separation efficiencies of two chromatographic columns and extraction capacities of different methods were tested. Compared with the C18 column (ACQUITY UPLC BEH Shield RP18 column), the column with an alkyl perfluorinated C8 stationary phase (Epic FO LB column), in combination with the distinct MS/MS transitions of analytes, allowed better separation of most isomers. The ion-pair extraction method showed more effective matrix separation than that of the alkaline digestion method, with recoveries ranging from 79.6-105% for biosolids, 80.4-116% for soils, and 68.0-114% for plant tissues. The method detection limits ranged from 10 to 55, 3–13, and 8–58pg/g dry weight for biosolids, soil, and plants, respectively. This method was applied successfully to quantify individual isomers in biosolids, biosolids-amended soils and plants. Six PFOA, eight PFOS, and two PFHxS isomers were found in the samples, with linear isomers being the dominant species. Further analysis revealed that the translocation potentials of branched isomers within plants were higher than those of linear isomers.





Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Abdelbasset Chafik, Abdelkhalid Essamadi, Safinur Yildirim Çelik, Ahmet Mavi
Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al3+, Cd2+ and Mg2+, whereas Ca2+, Co2+ and Ni2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The Km and Vmax of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with Ki value of 14.43μM, the IC50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Abdelbasset Chafik, Abdelkhalid Essamadi, Safinur Yildirim Çelik, Ahmet Mavi

Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al3+, Cd2+ and Mg2+, whereas Ca2+, Co2+ and Ni2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The Km and Vmax of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with Ki value of 14.43μM, the IC50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert.





Quantification of fluorescent dyes in organ tissue samples via HPLC analysis

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): S. Morsbach, A. García-Bardon, J. Kamuf, B. Müller, N. Beghersa, K. Mohr, K. Landfester
The determination of regional blood flow via the accumulation of fluorescent microspheres is a concept regularly used in medical research. Typically, the microbeads get extracted from the tissue of interest and are then quantified by measuring the absorption or fluorescence of the incorporated dyes without further separation from the medium. However, in that case the absorption spectra of different dyes can overlap when used simultaneously, leading to an overestimation of the concentration. Additionally, background absorption from the medium can be problematic. Therefore, a high performance liquid chromatography method for the simultaneous detection of four dyes (orange, crimson, yellow-green and red) incorporated in different microbeads in samples from biological media such as organ tissue (brain, heart and kidneys) was developed. Since for biological samples often a large sample size is required for sufficient statistics, the method was optimized to yield very short run times. With this method it was possible to detect very low concentrations of only one microsphere per gram of organ tissue. By applying this sensitive quantification technique, it was demonstrated that the application of microbeads for perfusion measurements might not be reliable due to different organ distributions in each animal.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): S. Morsbach, A. García-Bardon, J. Kamuf, B. Müller, N. Beghersa, K. Mohr, K. Landfester

The determination of regional blood flow via the accumulation of fluorescent microspheres is a concept regularly used in medical research. Typically, the microbeads get extracted from the tissue of interest and are then quantified by measuring the absorption or fluorescence of the incorporated dyes without further separation from the medium. However, in that case the absorption spectra of different dyes can overlap when used simultaneously, leading to an overestimation of the concentration. Additionally, background absorption from the medium can be problematic. Therefore, a high performance liquid chromatography method for the simultaneous detection of four dyes (orange, crimson, yellow-green and red) incorporated in different microbeads in samples from biological media such as organ tissue (brain, heart and kidneys) was developed. Since for biological samples often a large sample size is required for sufficient statistics, the method was optimized to yield very short run times. With this method it was possible to detect very low concentrations of only one microsphere per gram of organ tissue. By applying this sensitive quantification technique, it was demonstrated that the application of microbeads for perfusion measurements might not be reliable due to different organ distributions in each animal.





Separation of aflatoxin B1 from synthetic physiological fluids using talc and diatomite: Kinetic and isotherm aspects

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Myroslav Sprynskyy, Iwona Krzemień-Konieczka, Renata Gadzała-Kopciuch, Bogusław Buszewski
The objective of the study was to examine adsorption of the aflatoxin B1 from synthetic gastric fluid and synthetic intestinal fluid by talc, raw and calcined diatomite. The kinetic and equilibrium adsorption processes were studied in the batch adsorption experiments applying high performance liquid chromatography for the aflatoxin B1 determination. The kinetic study showed a very fast adsorption of the aflatoxin B1 onto the selected adsorbents from the both physiological fluids with reaching equilibrium within 1–15min. The aflatoxin B1 was almost completely adsorbed in initial linear step of the kinetic process that can be described well by the zero-order kinetics model. The experimental data of the equilibrium adsorption were characterized using the Langmuir and Freundlich isotherm models. The high adsorption effectiveness was found in a range of 90%–100% and 60%–100% for the diatomite samples and the talc respectively at the initial concentrations of the aflatoxin B1 as 31–300ng/mL. The possible mechanisms of the aflatoxin adsorption onto the used mineral adsorbents are also discussed in the work.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Myroslav Sprynskyy, Iwona Krzemień-Konieczka, Renata Gadzała-Kopciuch, Bogusław Buszewski

The objective of the study was to examine adsorption of the aflatoxin B1 from synthetic gastric fluid and synthetic intestinal fluid by talc, raw and calcined diatomite. The kinetic and equilibrium adsorption processes were studied in the batch adsorption experiments applying high performance liquid chromatography for the aflatoxin B1 determination. The kinetic study showed a very fast adsorption of the aflatoxin B1 onto the selected adsorbents from the both physiological fluids with reaching equilibrium within 1–15min. The aflatoxin B1 was almost completely adsorbed in initial linear step of the kinetic process that can be described well by the zero-order kinetics model. The experimental data of the equilibrium adsorption were characterized using the Langmuir and Freundlich isotherm models. The high adsorption effectiveness was found in a range of 90%–100% and 60%–100% for the diatomite samples and the talc respectively at the initial concentrations of the aflatoxin B1 as 31–300ng/mL. The possible mechanisms of the aflatoxin adsorption onto the used mineral adsorbents are also discussed in the work.





Homogenization-assisted cavitation hybrid rotation extraction and macroporous resin enrichment of dihydroquercetin from Larix gmelinii

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Yu Xia, Yinhang Wang, Wei Li, Chunhui Ma, Shouxin Liu
Cavitation hybrid rotation, which was and is still looked upon as an unavoidable nuisance in the flow systems, for extraction processing intensification of active chemical compounds from natural products. In this study, a homogenization-assisted cavitation hybrid rotation extraction method was applied to extract dihydroquercetin (DHQ) from larch (Larix gmelinii) wood root. The extraction parameters were optimized in single factor experiments with the DHQ extraction yields as the response values. The optimum conditions were as follows: number of extractions, three; ethanol volume fraction for the extraction, 60%; liquid–solid ratio for homogenization, 10mL/g; homogenization time, 8min; liquid–solid ratio for cavitation extraction, 9mL/g, and cavitation extraction time, 35min. Under these conditions, the DHQ content in extract was 4.50±0.02mg/g, and the extraction efficiency was higher than those of traditional techniques. Cavitation can be effectively used to improve the extraction rate by increasing the mass transfer rates and possible rupture of cell wall due to formation of microcavities leading to higher product yields with reduced processing time and solvent consumption. After the extraction process, macroporous resin column chromatography was used to concentrate and purify the DHQ. Three resins were selected from fifteen macroporous resins for further investigation of their performance. Among these resins, AB-8 resin exhibited relatively better adsorption capacities and desorption ratios for DHQ. The ethanol volume fraction of the solutions for sample loading and desorption, and flow rates for loading and desorption were optimized for the macroporous resin column chromatography.

Graphical abstract

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Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Yu Xia, Yinhang Wang, Wei Li, Chunhui Ma, Shouxin Liu

Cavitation hybrid rotation, which was and is still looked upon as an unavoidable nuisance in the flow systems, for extraction processing intensification of active chemical compounds from natural products. In this study, a homogenization-assisted cavitation hybrid rotation extraction method was applied to extract dihydroquercetin (DHQ) from larch (Larix gmelinii) wood root. The extraction parameters were optimized in single factor experiments with the DHQ extraction yields as the response values. The optimum conditions were as follows: number of extractions, three; ethanol volume fraction for the extraction, 60%; liquid–solid ratio for homogenization, 10mL/g; homogenization time, 8min; liquid–solid ratio for cavitation extraction, 9mL/g, and cavitation extraction time, 35min. Under these conditions, the DHQ content in extract was 4.50±0.02mg/g, and the extraction efficiency was higher than those of traditional techniques. Cavitation can be effectively used to improve the extraction rate by increasing the mass transfer rates and possible rupture of cell wall due to formation of microcavities leading to higher product yields with reduced processing time and solvent consumption. After the extraction process, macroporous resin column chromatography was used to concentrate and purify the DHQ. Three resins were selected from fifteen macroporous resins for further investigation of their performance. Among these resins, AB-8 resin exhibited relatively better adsorption capacities and desorption ratios for DHQ. The ethanol volume fraction of the solutions for sample loading and desorption, and flow rates for loading and desorption were optimized for the macroporous resin column chromatography.

Graphical abstract

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Development and validation of modified QuEChERS method coupled with LC–MS/MS for simultaneous determination of cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite in various types of honey and royal jelly

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Weijia Zheng, Jin-A. Park, A.M. Abd El-Aty, Seong-Kwan Kim, Sang-Hyun Cho, Jeong-min Choi, Hee Yi, Soo-Min Cho, Amer Ramadan, Ji Hoon Jeong, Jae-Han Shim, Ho-Chul Shin
Over the past few decades, honey products have been polluted by different contaminants, such as pesticides, which are widely applied in agriculture. In this work, a modified EN – quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method was developed for the simultaneous quantification of pesticide residues, including cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite 2,4-dimethylaniline (2,4-DMA), in four types of honey (acacia, wild, chestnut, and manuka) and royal jelly. Samples were buffered with 0.2M dibasic sodium phosphate (pH 9), and subsequently, acetonitrile was employed as the extraction solvent. A combination of primary secondary amine (PSA) and C18 sorbents was used for purification prior to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) analysis. The estimated linearity measured at six concentration levels presented good correlation coefficients (R2)≥0.99. The recovery, calculated from three different spiking levels, was 62.06–108.79% in honey and 67.58–106.34% in royal jelly, with an RSD<12% for all the tested compounds. The matrix effect was also evaluated, and most of the analytes presented signal enhancement. The limits of quantification (LOQ) ranged between 0.001 and 0.005mg/kg in various samples. These are considerably lower than the maximum residue limits (MRL) set by various regulatory authorities. A total of 43 market (domestic and imported) samples were assayed for method application. Among the tested samples, three samples were tested positive (i.e. detected and quantified) only for cymiazole residues. The residues in the rest of the samples were detected but not quantified. We concluded that the protocol developed in this work is simple and versatile for the routine quantification of cymiazole, 2,4-DMA, fipronil, coumaphos, amitraz, and fluvalinate in various types of honey and royal jelly.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Weijia Zheng, Jin-A. Park, A.M. Abd El-Aty, Seong-Kwan Kim, Sang-Hyun Cho, Jeong-min Choi, Hee Yi, Soo-Min Cho, Amer Ramadan, Ji Hoon Jeong, Jae-Han Shim, Ho-Chul Shin

Over the past few decades, honey products have been polluted by different contaminants, such as pesticides, which are widely applied in agriculture. In this work, a modified EN – quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method was developed for the simultaneous quantification of pesticide residues, including cymiazole, fipronil, coumaphos, fluvalinate, amitraz, and its metabolite 2,4-dimethylaniline (2,4-DMA), in four types of honey (acacia, wild, chestnut, and manuka) and royal jelly. Samples were buffered with 0.2M dibasic sodium phosphate (pH 9), and subsequently, acetonitrile was employed as the extraction solvent. A combination of primary secondary amine (PSA) and C18 sorbents was used for purification prior to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) analysis. The estimated linearity measured at six concentration levels presented good correlation coefficients (R2 )0.99. The recovery, calculated from three different spiking levels, was 62.06–108.79% in honey and 67.58–106.34% in royal jelly, with an RSD<12% for all the tested compounds. The matrix effect was also evaluated, and most of the analytes presented signal enhancement. The limits of quantification (LOQ) ranged between 0.001 and 0.005mg/kg in various samples. These are considerably lower than the maximum residue limits (MRL) set by various regulatory authorities. A total of 43 market (domestic and imported) samples were assayed for method application. Among the tested samples, three samples were tested positive (i.e. detected and quantified) only for cymiazole residues. The residues in the rest of the samples were detected but not quantified. We concluded that the protocol developed in this work is simple and versatile for the routine quantification of cymiazole, 2,4-DMA, fipronil, coumaphos, amitraz, and fluvalinate in various types of honey and royal jelly.





An UPLC-MS/MS method for quantifying tetrandrine and its metabolite berbamine in human blood: Application to a human pharmacokinetic study

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Guangyi Yang, Chenning Zhang, Pei Hu, Meiling Zhu, Ming Hu, Song Gao
Tetrandrine (TET) was approved by the China Food and Drug Administration (CFDA) for the treatment of silicosis. However, patients can’t use this effective drug chronically due to side effects such as hypersomnia, asthenia, etc. The purpose of this study is to develop an UPLC–MS/MS method to quantify TET and its major metabolite and apply the method in a single dose human pharmacokinetic study. A Restek UItra BiPh column (100×2.1mm, 5μm) was used with acetonitrile and 0.1% formic acid in water as the mobile phases. The mass analysis was performed in a Waters Xevo TQ mass spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A one-step protein precipitation by acetonitrile was used to extract the analytes from blood sample. The method showed linearity in the concentration ranges of 2.05–1050.00ng/mL for TET and 1.27–650.00ng/mL for berbamine. The intra/inter-day precisions were less 15% for these two analytes. The extraction recoveries of these two analytes were from 75.6% to 107.8% and the matrix effects ranged from 92.4% to 110.4%. The stabilities of these compounds in plasma were evaluated by analyzing three different concentrations following storage at 25°C for 6h, and −80°C for 30days. All the samples displayed less than 15.0% variations. The validated method was applied to PK study in human and the PK parameters of TET and berbamine were determined. In conclusion, a robust and sensitive LC–MS/MS method was developed and validated. In addition, the results of human PK experiment showed that TET and berbamine could be accumulated and more study is needed to establish a reasonable dose segment.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Guangyi Yang, Chenning Zhang, Pei Hu, Meiling Zhu, Ming Hu, Song Gao

Tetrandrine (TET) was approved by the China Food and Drug Administration (CFDA) for the treatment of silicosis. However, patients can’t use this effective drug chronically due to side effects such as hypersomnia, asthenia, etc. The purpose of this study is to develop an UPLC–MS/MS method to quantify TET and its major metabolite and apply the method in a single dose human pharmacokinetic study. A Restek UItra BiPh column (100×2.1mm, 5μm) was used with acetonitrile and 0.1% formic acid in water as the mobile phases. The mass analysis was performed in a Waters Xevo TQ mass spectrometer via multiple reaction monitoring (MRM) with positive scan mode. A one-step protein precipitation by acetonitrile was used to extract the analytes from blood sample. The method showed linearity in the concentration ranges of 2.05–1050.00ng/mL for TET and 1.27–650.00ng/mL for berbamine. The intra/inter-day precisions were less 15% for these two analytes. The extraction recoveries of these two analytes were from 75.6% to 107.8% and the matrix effects ranged from 92.4% to 110.4%. The stabilities of these compounds in plasma were evaluated by analyzing three different concentrations following storage at 25°C for 6h, and −80°C for 30days. All the samples displayed less than 15.0% variations. The validated method was applied to PK study in human and the PK parameters of TET and berbamine were determined. In conclusion, a robust and sensitive LC–MS/MS method was developed and validated. In addition, the results of human PK experiment showed that TET and berbamine could be accumulated and more study is needed to establish a reasonable dose segment.





Determination of tropane alkaloids by heart cutting reversed phase – Strong cation exchange two dimensional liquid chromatography

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Zhen Long, Yanhai Zhang, Paul Gamache, Zhimou Guo, Frank Steiner, Nana Du, Xiaoda Liu, Yan Jin, Xingguo Liu, Lvye Liu
Current Chinese Pharmacopoeia (ChP) standards apply liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pretreatments lead to a low analytical efficiency and possible component loss. In this study, a heart cutting reversed phase – strong cation exchange two dimensional liquid chromatography (RP – SCX 2DLC) approach was optimized for simultaneously quantifying tropane alkaloids (anisodine, scopolamine and hyoscyamine) in herbal medicines and herbal medicine tablets without further treatment of the filtered extract. The chromatographic conditions were systematically optimized in terms of column type, mobile phase composition and flow rate. To improve peak capacity and obtain symmetric peak shape of alkaloids, a polar group embedded C18 column combined with chaotropic salts was used in the first dimension. To remove the disturbance of non-alkaloids, achieve unique selectivity and acquire symmetric peak shape of alkaloids, an SCX column combined with phosphate buffer was used in the second dimension. Method validation was performed in terms of linearity, precision (0.54–0.82%), recovery (94.1–105.2%), limit of detection (LOD) and limit of quantification (LOQ) of the three analytes varied between 0.067–0.115mgL−1 and 0.195–0.268mgL−1, respectively. The method demonstrated superiority over 1DLC method in respect of resolution (less alkaloid co-eluted), sample preparation (no pretreatment procedure) and transfer rate (minimum component loss). The optimized RP – SCX 2DLC approach was subsequently applied to quantify target alkaloids in five herbal medicines and herbal medicine tablets from three different manufactures. The results demonstrated that the developed heart cutting RP – SCX 2DLC approach represented a new, strategically significant methodology for the quality evaluation of tropane alkaloid in related herbal medicines that involve complex chemical matrix.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Zhen Long, Yanhai Zhang, Paul Gamache, Zhimou Guo, Frank Steiner, Nana Du, Xiaoda Liu, Yan Jin, Xingguo Liu, Lvye Liu

Current Chinese Pharmacopoeia (ChP) standards apply liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pretreatments lead to a low analytical efficiency and possible component loss. In this study, a heart cutting reversed phase – strong cation exchange two dimensional liquid chromatography (RP – SCX 2DLC) approach was optimized for simultaneously quantifying tropane alkaloids (anisodine, scopolamine and hyoscyamine) in herbal medicines and herbal medicine tablets without further treatment of the filtered extract. The chromatographic conditions were systematically optimized in terms of column type, mobile phase composition and flow rate. To improve peak capacity and obtain symmetric peak shape of alkaloids, a polar group embedded C18 column combined with chaotropic salts was used in the first dimension. To remove the disturbance of non-alkaloids, achieve unique selectivity and acquire symmetric peak shape of alkaloids, an SCX column combined with phosphate buffer was used in the second dimension. Method validation was performed in terms of linearity, precision (0.54–0.82%), recovery (94.1–105.2%), limit of detection (LOD) and limit of quantification (LOQ) of the three analytes varied between 0.067–0.115mgL−1 and 0.195–0.268mgL−1, respectively. The method demonstrated superiority over 1DLC method in respect of resolution (less alkaloid co-eluted), sample preparation (no pretreatment procedure) and transfer rate (minimum component loss). The optimized RP – SCX 2DLC approach was subsequently applied to quantify target alkaloids in five herbal medicines and herbal medicine tablets from three different manufactures. The results demonstrated that the developed heart cutting RP – SCX 2DLC approach represented a new, strategically significant methodology for the quality evaluation of tropane alkaloid in related herbal medicines that involve complex chemical matrix.





Separation and purification of four phenolic compounds from persimmon by high-speed counter-current chromatography

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Jinming Peng, Kaikai Li, Wei Zhu, Xiangyi Deng, Chunmei Li
An efficient method was established by high-speed counter-current chromatography (HSCCC) for preparation of four phenolic compounds from the depolymerization products of persimmon tannin. Using the two solvent systems of n-hexane/ethyl acetate/water (3:17:20, v/v/v) and ethyl acetate/methanol/water (50:1:50, v/v/v), the preparative isolation was successfully performed by a two-step separation. The yields of one run (150mg crude sample) for gallic acid, methyl gallate, and epigallocatechin-3-gallate-(4β→8, 2β→O→7)-epigallocatechin-3-gallate dimer (A-type EGCG dimer) were 4.7, 44.2 and 5.9mg, respectively. In addition, 4.6mg epicatechin-3-gallate-(4β→8, 2β→O→7)-epicatechin-3-gallate dimer (A-type ECG dimer) was obtained by further preparative high-performance liquid chromatography (prep-HPLC). The purities of these compounds were all above 95.0% and their structures were identified by HPLC/ESI-MS. We found that HSCCC had definite advantages for the preparation of dimeric procyanidins compared with previous methods. Furthermore, it was shown that the four phenolic compounds possessed greater antioxidant activities than Trolox.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Jinming Peng, Kaikai Li, Wei Zhu, Xiangyi Deng, Chunmei Li

An efficient method was established by high-speed counter-current chromatography (HSCCC) for preparation of four phenolic compounds from the depolymerization products of persimmon tannin. Using the two solvent systems of n-hexane/ethyl acetate/water (3:17:20, v/v/v) and ethyl acetate/methanol/water (50:1:50, v/v/v), the preparative isolation was successfully performed by a two-step separation. The yields of one run (150mg crude sample) for gallic acid, methyl gallate, and epigallocatechin-3-gallate-(4β8, 2βO7)-epigallocatechin-3-gallate dimer (A-type EGCG dimer) were 4.7, 44.2 and 5.9mg, respectively. In addition, 4.6mg epicatechin-3-gallate-(4β8, 2βO7)-epicatechin-3-gallate dimer (A-type ECG dimer) was obtained by further preparative high-performance liquid chromatography (prep-HPLC). The purities of these compounds were all above 95.0% and their structures were identified by HPLC/ESI-MS. We found that HSCCC had definite advantages for the preparation of dimeric procyanidins compared with previous methods. Furthermore, it was shown that the four phenolic compounds possessed greater antioxidant activities than Trolox.





Non-targeted metabolomics-guided sildenafil metabolism study in human liver microsomes

Publication date: 1 January 2018 Source:Journal of Chromatography B, Volume 1072 Author(s): Ju-Hyun Kim, Jun Hyun Jo, Kyung-ah Seo, Hayoung Hwang, Hye Suk Lee, Sangkyu Lee Metabolomics combined with high-resolution mass spe…

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Ju-Hyun Kim, Jun Hyun Jo, Kyung-ah Seo, Hayoung Hwang, Hye Suk Lee, Sangkyu Lee

Metabolomics combined with high-resolution mass spectrometry (HR-MS) and multivariate data analysis has broad applications in the study of xenobiotic metabolism. Although information about xenobiotic metabolism is essential to understand toxic mechanisms, pharmacokinetic parameters and excretion pathways, it is limited to predict all generated metabolites in biological fluids. Here, we revisited sildenafil metabolism in human liver microsomes using a metabolomics approach to achieve a global picture of sildenafil phase 1 metabolism. Finally, 12 phase 1 metabolites were identified in human liver microsomes; M1-M5 were previously known metabolites. The chemical structures of the novel metabolites were elucidated by MS2 fragmentation using an HR-MS system as follows: M6, reduced sildenafil; M7, N,N-deethylation and mono-oxidation; M8, demethanamine, N,N-deethylation and mono-hydroxylation; M9, demethanamine and N,N-deethylation; M10 and M11, mono-oxidation in the piperazine ring after N-demethylation; and M12, mono-oxidation. All metabolites, except M1, were formed by CYP3A4 and CYP3A5. In conclusion, we successfully updated the metabolic pathway of sildenafil in human liver, including 7 novel metabolites using metabolomics combined with HR-MS and multivariate data analysis.





LC–MS/MS quantification of felinine metabolites in tissues, fluids, and excretions from the domestic cat (Felis catus)

Publication date: 1 January 2018 Source:Journal of Chromatography B, Volume 1072 Author(s): Ayami Futsuta, Wataru Hojo, Tamako Miyazaki, Tetsuro Yamashita, Masao Miyazaki Domestic cat urine contains large concentrations of th…

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Ayami Futsuta, Wataru Hojo, Tamako Miyazaki, Tetsuro Yamashita, Masao Miyazaki

Domestic cat urine contains large concentrations of the unusual amino acid, felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid). A felinine derivative, 3-mercapt-3-methylbutanol is a potential scent signal for letting other animals know that the scent owners are cats. 3-Methylbutanol-glutathione (MBG) is an upstream precursor of the biosynthetic pathway of felinine that may be produced in hepatocytes by conjugation of glutathione with isopentenyl pyrophosphate, an intermediate for cholesterol biosynthesis. However, little evidence exists to support the biosynthesis of MBG in the liver. This study examined the distribution of metabolites of the felinine biosynthetic pathway in multiple tissues, body fluids, and excretions of cats. MBG, the felinine precursor, 3-methylbutanol-cysteinylglycine (MBCG), felinine, and felinine N-acetyl derivative were quantified by liquid chromatography-electron spray ionization-tandem mass spectrometry. All compounds were detected in cat serum. In males, MBG and MBCG contents were significantly higher than felinine and N-acetylfelinine contents. MBG was detected in multiple tissues, including the salivary gland, heart, liver, spleen, gut, kidney, bladder, adipose tissue, and muscle. Sex differences in MBG levels were observed in the liver and other tissues. Felinine and N-acetylfelinine were also detected in those tissues. Furthermore, we detected all compounds in cat bile and fecal samples, indicating that felinine is excreted into the feces via bile from the liver. We conclude that MBG is synthesized in several tissues in a sex-dependent manner. These findings improve our understanding of felinine metabolism and function in cats.





Coupling of on-column trypsin digestion–peptide mapping and principal component analysis for stability and biosimilarity assessment of recombinant human growth hormone

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Sara M. Shatat, Basma M. Eltanany, Abeer A. Mohamed, Medhat A. Al-Ghobashy, Faten A. Fathalla, Samah S. Abbas
Peptide mapping (PM) is a vital technique in biopharmaceutical industry. The fingerprint obtained helps to qualitatively confirm host stability as well as verify primary structure, purity and integrity of the target protein. Yet, in-solution digestion followed by tandem mass spectrometry is not suitable as a routine quality control test. It is time consuming and requires sophisticated, expensive instruments and highly skilled operators. In an attempt to enhance the fuctionality of PM and extract multi-dimentional data about various critical quality attributes and comparability of biosimilars, coupling of PM generated using immobilized trypsin followed by HPLC-UV to principal component analysis (PCA) is proposed. Recombinant human growth hormone (rhGH); was selected as a model biopharmaceutical since it is available in the market from different manufacturers and its PM is a well-established pharmacopoeial test. Samples of different rhGH biosimilars as well as degraded samples: deamidated and oxidized were subjected to trypsin digestion followed by RP-HPLC-UV analysis. PCA of the entire chromatograms of test and reference samples was then conducted. Comparison of the scores of samples and investigation of the loadings plots clearly indicated the applicability of PM-PCA for: i) identity testing, ii) biosimilarity assessment and iii) stability evaluation. Hotelling’s T2 and Q statistics were employed at 95% confidence level to measure the variation and to test the conformance of each sample to the PCA model, respectively. Coupling of PM to PCA provided a novel tool to identify peptide fragments responsible for variation between the test and reference samples as well as evaluation of the extent and relative significance of this variability. Transformation of conventional PM that is largely based on subjective visual comparison into an objective statiscally-guided analysis framework should provide a simple and economic tool to help both manufacturers and regulatory authorities in quality and biosimilarity assessment of biopharmaceuticals.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Sara M. Shatat, Basma M. Eltanany, Abeer A. Mohamed, Medhat A. Al-Ghobashy, Faten A. Fathalla, Samah S. Abbas

Peptide mapping (PM) is a vital technique in biopharmaceutical industry. The fingerprint obtained helps to qualitatively confirm host stability as well as verify primary structure, purity and integrity of the target protein. Yet, in-solution digestion followed by tandem mass spectrometry is not suitable as a routine quality control test. It is time consuming and requires sophisticated, expensive instruments and highly skilled operators. In an attempt to enhance the fuctionality of PM and extract multi-dimentional data about various critical quality attributes and comparability of biosimilars, coupling of PM generated using immobilized trypsin followed by HPLC-UV to principal component analysis (PCA) is proposed. Recombinant human growth hormone (rhGH); was selected as a model biopharmaceutical since it is available in the market from different manufacturers and its PM is a well-established pharmacopoeial test. Samples of different rhGH biosimilars as well as degraded samples: deamidated and oxidized were subjected to trypsin digestion followed by RP-HPLC-UV analysis. PCA of the entire chromatograms of test and reference samples was then conducted. Comparison of the scores of samples and investigation of the loadings plots clearly indicated the applicability of PM-PCA for: i) identity testing, ii) biosimilarity assessment and iii) stability evaluation. Hotelling’s T2 and Q statistics were employed at 95% confidence level to measure the variation and to test the conformance of each sample to the PCA model, respectively. Coupling of PM to PCA provided a novel tool to identify peptide fragments responsible for variation between the test and reference samples as well as evaluation of the extent and relative significance of this variability. Transformation of conventional PM that is largely based on subjective visual comparison into an objective statiscally-guided analysis framework should provide a simple and economic tool to help both manufacturers and regulatory authorities in quality and biosimilarity assessment of biopharmaceuticals.





Quantitative determination of chromium picolinate in animal feeds by solid phase extraction and liquid chromatography-tandem mass spectrometry

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Miaomiao Han, Ying Tian, Zhen Li, Yiqiang Chen, Wenjun Yang, Liying Zhang
Chromium picolinate is one of the important Cr3+ resources and is widely used in animal production. A convenient, reliable and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative determination of chromium picolinate in animal feeds. Feed samples were extracted with acetonitrile and subsequently cleaned up by solid phase extraction cartridges Supelclean™ LC-18. Chromium picolinate was efficiently separated with a Waters ACQUITY UPLC® BEH C18 column, ionized with electrospray ion source in positive mode (ESI+), and quantitatively determined by tandem mass spectrometry in multiple reaction monitoring mode. Standard calibration curve of chromium picolinate in the concentration range from 0.5 to 1000ng/mL was obtained with good linearity correlation coefficient (R2=0.9982). Average recoveries ranged from 95.37%∼105.54%, as detected by spiking 0.02∼640mg/kg of chromium picolinate in complete feed, concentrated feed and premix. Intra-day and inter-day coefficient of variation were 0.59%∼6.67% and 2.36%∼6.97%, respectively. The limits of quantitation were 0.02mg/kg, 0.025mg/kg, and 2mg/kg for complete feed, concentrated feed, and premix, respectively. Actual sample analysis indicated that the developed method can be an effective tool to monitoring CrPic content in animal feed.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Miaomiao Han, Ying Tian, Zhen Li, Yiqiang Chen, Wenjun Yang, Liying Zhang

Chromium picolinate is one of the important Cr3+ resources and is widely used in animal production. A convenient, reliable and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative determination of chromium picolinate in animal feeds. Feed samples were extracted with acetonitrile and subsequently cleaned up by solid phase extraction cartridges Supelclean™ LC-18. Chromium picolinate was efficiently separated with a Waters ACQUITY UPLC® BEH C18 column, ionized with electrospray ion source in positive mode (ESI+), and quantitatively determined by tandem mass spectrometry in multiple reaction monitoring mode. Standard calibration curve of chromium picolinate in the concentration range from 0.5 to 1000ng/mL was obtained with good linearity correlation coefficient (R2 =0.9982). Average recoveries ranged from 95.37%105.54%, as detected by spiking 0.02640mg/kg of chromium picolinate in complete feed, concentrated feed and premix. Intra-day and inter-day coefficient of variation were 0.59%6.67% and 2.36%6.97%, respectively. The limits of quantitation were 0.02mg/kg, 0.025mg/kg, and 2mg/kg for complete feed, concentrated feed, and premix, respectively. Actual sample analysis indicated that the developed method can be an effective tool to monitoring CrPic content in animal feed.





Carbamazepine, lamotrigine, levetiracetam and valproic acid in dried blood spots with liquid chromatography tandem mass spectrometry; method development and validation

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Camilla Linder, Anna Hansson, Sara Sadek, Lars L. Gustafsson, Anton Pohanka
Monitoring of antiepileptic drugs in children with epilepsy require multiple visits at a clinic for blood collection. Dried blood spot sampling is an alternative way of collection, performed at home by self-collection and can save time and costs for patients and family members. The aim was to develop and validate an LC–MS/MS dried blood spot method for carbamazepine, lamotrigine, levetiracetam and valproic acid with the requirements of using standard equipment and material in a routine laboratory setting.Whatman-903 filter paper was utilized, and discs were punched into a 96 well plate with an automated puncher and barcode reading. Extraction with methanol/water solution including internal standards on an orbital shaker was followed by a vacuum centrifuge step and reconstitution in mobile phase. Bioanalytical validation was performed according to guidelines from European Medicines Agency and additional dried blood spot specific validation.Calibration curves of the four included drugs had R2 values ≥0.994. Therapeutic relevant concentrations were well within measuring ranges. Within and −between run precision had %CV:s of 2.9–10.5%. Accuracy (%bias) was between −16.5% (lower limit of quantification) to +7.4%. Blood spots in a volume range of 15–50μL with hematocrit in expected ranges for this patient group were within precision and accuracy limits. To test the method, concentrations from dried blood spot venous and capillary patient samples (n=50) were compared with plasma concentrations. Good correlations for all four drugs with R2 of >0.92 was shown.In summary, a fast method for dried blood spots based on a 96 well format was developed for four commonly prescribed antiepileptic drugs. This validated method with traceability in sample preparation by bar code reading makes it suitable for the clinical laboratory.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Camilla Linder, Anna Hansson, Sara Sadek, Lars L. Gustafsson, Anton Pohanka

Monitoring of antiepileptic drugs in children with epilepsy require multiple visits at a clinic for blood collection. Dried blood spot sampling is an alternative way of collection, performed at home by self-collection and can save time and costs for patients and family members. The aim was to develop and validate an LC–MS/MS dried blood spot method for carbamazepine, lamotrigine, levetiracetam and valproic acid with the requirements of using standard equipment and material in a routine laboratory setting. Whatman-903 filter paper was utilized, and discs were punched into a 96 well plate with an automated puncher and barcode reading. Extraction with methanol/water solution including internal standards on an orbital shaker was followed by a vacuum centrifuge step and reconstitution in mobile phase. Bioanalytical validation was performed according to guidelines from European Medicines Agency and additional dried blood spot specific validation. Calibration curves of the four included drugs had R2 values ≥0.994. Therapeutic relevant concentrations were well within measuring ranges. Within and −between run precision had %CV:s of 2.9–10.5%. Accuracy (%bias) was between −16.5% (lower limit of quantification) to +7.4%. Blood spots in a volume range of 15–50μL with hematocrit in expected ranges for this patient group were within precision and accuracy limits. To test the method, concentrations from dried blood spot venous and capillary patient samples (n=50) were compared with plasma concentrations. Good correlations for all four drugs with R2 of >0.92 was shown. In summary, a fast method for dried blood spots based on a 96 well format was developed for four commonly prescribed antiepileptic drugs. This validated method with traceability in sample preparation by bar code reading makes it suitable for the clinical laboratory.





Application of an UHPLC–MS/MS method to tissue distribution and excretion study of 2-(2-hydroxypropanamido) benzoic acid in rats

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Qili Zhang, Jiao Guan, Shasha Li, Yunli Zhao, Zhiguo Yu
2-(2-Hydroxypropanamido) benzoic acid (HPABA) is a potential anti-inflammatory agent isolated from the marine fungus Penicillium chrysogenum. In vivo tissue distribution and excretion of HPABA were unknown. Therefore, the purpose of this paper was to establish an UHPLC–MS/MS method for the determination of HPABA and proceed to apply for the study of tissue distribution and excretion in rats. The results obtained indicated that HPABA in tissues was rapidly distributed and cleared. The highest level was found in intestine and stomach, followed by kidney, liver, lung and heart. These observations showed that HPABA was well distributed into abundant blood-supply tissues, suggesting that the blood flow or perfusion rate of organs plays a major role. A small amount of HPABA was detected in brain indicated that the ability of it in crossing the blood brain barrier (BBB) was very weak in rats. Excretion studies demonstrated that HPABA was mainly excreted by kidney. The total cumulative excretion of HPABA were 54.3% and 44.1% in male and female rats, respectively. In addition, gender-related differences in the excretion and distribution profile of HPABA were observed in rats.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Qili Zhang, Jiao Guan, Shasha Li, Yunli Zhao, Zhiguo Yu

2-(2-Hydroxypropanamido) benzoic acid (HPABA) is a potential anti-inflammatory agent isolated from the marine fungus Penicillium chrysogenum. In vivo tissue distribution and excretion of HPABA were unknown. Therefore, the purpose of this paper was to establish an UHPLC–MS/MS method for the determination of HPABA and proceed to apply for the study of tissue distribution and excretion in rats. The results obtained indicated that HPABA in tissues was rapidly distributed and cleared. The highest level was found in intestine and stomach, followed by kidney, liver, lung and heart. These observations showed that HPABA was well distributed into abundant blood-supply tissues, suggesting that the blood flow or perfusion rate of organs plays a major role. A small amount of HPABA was detected in brain indicated that the ability of it in crossing the blood brain barrier (BBB) was very weak in rats. Excretion studies demonstrated that HPABA was mainly excreted by kidney. The total cumulative excretion of HPABA were 54.3% and 44.1% in male and female rats, respectively. In addition, gender-related differences in the excretion and distribution profile of HPABA were observed in rats.





Direct analysis of ethylene glycol in human serum on the basis of analyte adduct formation and liquid chromatography–tandem mass spectrometry

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Marek Dziadosz
The aim of this work was to develop a fast, cost-effective and time-saving liquid chromatography-tandem mass spectrometry (LC–MS/MS) analytical method for the analysis of ethylene glycol (EG) in human serum. For these purposes, the formation/fragmentation of an EG adduct ion with sodium and sodium acetate was applied in the positive electrospray mode for signal detection. Adduct identification was performed with appropriate infusion experiments based on analyte solutions prepared in different concentrations. Corresponding analyte adduct ions and adduct ion fragments could be identified both for EG and the deuterated internal standard (EG-D4). Protein precipitation was used as sample preparation. The analysis of the supernatant was performed with a Luna 5μm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisting of 95% A (H2O/methanol=95/5, v/v) and 5% B (H2O/methanol=3/97, v/v), both with 10mmolL−1 ammonium acetate and 0.1% acetic acid. Method linearity was examined in the range of 100–4000μg/mL and the calculated limit of detection/quantification was 35/98μg/mL. However, on the basis of the signal to noise ratio, quantification was recommended at a limit of 300μg/mL. Additionally, the examined precision, accuracy, stability, selectivity and matrix effect demonstrated that the method is a practicable alternative for EG quantification in human serum. In comparison to other methods based on liquid chromatography, the strategy presented made for the first time the EG analysis without analyte derivatisation possible.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Marek Dziadosz

The aim of this work was to develop a fast, cost-effective and time-saving liquid chromatography-tandem mass spectrometry (LC–MS/MS) analytical method for the analysis of ethylene glycol (EG) in human serum. For these purposes, the formation/fragmentation of an EG adduct ion with sodium and sodium acetate was applied in the positive electrospray mode for signal detection. Adduct identification was performed with appropriate infusion experiments based on analyte solutions prepared in different concentrations. Corresponding analyte adduct ions and adduct ion fragments could be identified both for EG and the deuterated internal standard (EG-D4). Protein precipitation was used as sample preparation. The analysis of the supernatant was performed with a Luna 5μm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisting of 95% A (H2O/methanol=95/5, v/v) and 5% B (H2O/methanol=3/97, v/v), both with 10mmolL−1 ammonium acetate and 0.1% acetic acid. Method linearity was examined in the range of 100–4000μg/mL and the calculated limit of detection/quantification was 35/98μg/mL. However, on the basis of the signal to noise ratio, quantification was recommended at a limit of 300μg/mL. Additionally, the examined precision, accuracy, stability, selectivity and matrix effect demonstrated that the method is a practicable alternative for EG quantification in human serum. In comparison to other methods based on liquid chromatography, the strategy presented made for the first time the EG analysis without analyte derivatisation possible.





Determination of benzotriazoles and benzothiazoles in human urine by UHPLC-TQMS

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Jiufeng Li, Hongzhi Zhao, Yanqiu Zhou, Shunqing Xu, Zongwei Cai
Benzotriazole (BTR) and benzothiazole (BTH) derivatives are extensively applied in industrial processes and consumer products, and are thus frequently detected in the environmental matrices. Due to their potential estrogenic effects reported in animal studies, the assessment of human exposure to BTRs and BTHs is important. In this study, a method was developed and validated to determine six BTRs and five BTHs in human urine by using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-TQMS) in positive electrospray ionization mode. After de-conjugation by β-glucuronidase, urine samples were liquid-liquid extracted for the measurement of total concentrations of BTRs and BTHs. The linearity, detection limit, precision, recovery, accuracy and matrix effect were evaluated. The limits of detection were less than 0.5ng/mL. The validated method demonstrated good precision (RSD%<15%), linearity (R2>0.99), recovery (>80%) and accuracy (80–100%). The method was successfully applied for the analysis of 22 human urine samples. Four out of eleven targeted compounds were detected in more than half of participants at ng/mL levels.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Jiufeng Li, Hongzhi Zhao, Yanqiu Zhou, Shunqing Xu, Zongwei Cai

Benzotriazole (BTR) and benzothiazole (BTH) derivatives are extensively applied in industrial processes and consumer products, and are thus frequently detected in the environmental matrices. Due to their potential estrogenic effects reported in animal studies, the assessment of human exposure to BTRs and BTHs is important. In this study, a method was developed and validated to determine six BTRs and five BTHs in human urine by using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-TQMS) in positive electrospray ionization mode. After de-conjugation by β-glucuronidase, urine samples were liquid-liquid extracted for the measurement of total concentrations of BTRs and BTHs. The linearity, detection limit, precision, recovery, accuracy and matrix effect were evaluated. The limits of detection were less than 0.5ng/mL. The validated method demonstrated good precision (RSD%<15%), linearity (R2 >0.99), recovery (>80%) and accuracy (80–100%). The method was successfully applied for the analysis of 22 human urine samples. Four out of eleven targeted compounds were detected in more than half of participants at ng/mL levels.