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Phospholipids covalently attached to silica particles as stationary phase in nano-liquid chromatography
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Clara-Eugenia Baños, Susanne K. Wiedmer, Jan-Henrik Smått, Motolani Sakeye, Jana Lokajová, Marja-Liisa Riekkola
Silica particles were covalently modified with phospholipids and used as packing material for nano-liquid chromatography (nano-LC). This modification involved aminopropylsilylation of the raw silica particles using 3-(aminopropyl)-triethoxysilane, covalent binding of glutaraldehyde molecules to the aminopropylsilylated particles, and finally covalent binding of different phospholipid vesicles containing primary amino groups to the iminoaldehyde silica particles. Capillaries with an inner diameter of 100μm were packed with phospholipid-coated silica particles using a slurry packing method. The packed capillaries were tested in nano-LC with UV-detection for the separation of acidic, neutral, and basic model analytes. The effect of the buffer ion on the retention factor of the analytes was evaluated using buffer solutions with constant ionic strength and pH. In addition, the effect of the volume of methanol in the mobile phase was studied. The calculated distribution coefficients (log K D) of the model compounds were in agreement with those reported in the literature. A good correlation between log K D values and octanol/water partitioning coefficients (P o/w) for neutral hydrophobic analytes was obtained proving the applicability of the method for predicting partitioning of the compounds with the biomembranes.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Clara-Eugenia Baños, Susanne K. Wiedmer, Jan-Henrik Smått, Motolani Sakeye, Jana Lokajová, Marja-Liisa Riekkola
Silica particles were covalently modified with phospholipids and used as packing material for nano-liquid chromatography (nano-LC). This modification involved aminopropylsilylation of the raw silica particles using 3-(aminopropyl)-triethoxysilane, covalent binding of glutaraldehyde molecules to the aminopropylsilylated particles, and finally covalent binding of different phospholipid vesicles containing primary amino groups to the iminoaldehyde silica particles. Capillaries with an inner diameter of 100μm were packed with phospholipid-coated silica particles using a slurry packing method. The packed capillaries were tested in nano-LC with UV-detection for the separation of acidic, neutral, and basic model analytes. The effect of the buffer ion on the retention factor of the analytes was evaluated using buffer solutions with constant ionic strength and pH. In addition, the effect of the volume of methanol in the mobile phase was studied. The calculated distribution coefficients (log K D) of the model compounds were in agreement with those reported in the literature. A good correlation between log K D values and octanol/water partitioning coefficients (P o/w) for neutral hydrophobic analytes was obtained proving the applicability of the method for predicting partitioning of the compounds with the biomembranes.
Evaluation of metal concentrations in mentha herbal teas (Mentha piperita, Mentha pulegium and Mentha species) by inductively coupled plasma spectrometry
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
C. Rubio, J.R.D. Lucas, A.J. Gutiérrez, D. Glez-Weller, B. Pérez Marrero, J.M. Caballero, C. Revert, A. Hardisson
Phytopharmaceuticals are gaining popularity worldwide; however, cases of adverse effects and drug interactions have also increased. One reason is in the high metal content both as ingredients but also as contaminants. Metal monitoring in food, like herbal teas, provides basic information on safety aspects in regulatory processes as well as nutritional values. In the present work, Cd, Pb, K, Na, Ca, Mg, Al, B, Ba, Co, Cr, Cu, Fe, Mn, Zn, Li, Ni, and Mo were determined by inductively coupled plasma spectrometry (ICPS) in 36 samples of Mentha sp. Mint tea bags and loose leaves were randomly obtained from supermarkets, traditional markets, herbal stores, and pharmacies in Tenerife (Canary Islands, Spain). Metal contents varied significantly, dependent on the stores the products were purchased in and on tea packaging (loose leaves versus tea bags). Pb analyses revealed levels (0.65±0.71mg/kg) below legal limits. The maximum permissible limit for Cd, 0.3mg/kg, set by the WHO for medicinal plants, was exceeded by 19.44% of the samples (0.22±0.13mg/kg), but all values were below the limit given in the European Pharmacopoeia for this metal (4mg/kg). We observed high Al (151.24±162.73mg/kg) and Li (5.46±3.94mg/kg) levels. B, Ba, Co, Cr, Cu, Fe, Mn, Ni, Zn, and Mo mean levels were 20.51, 14.15, 0.26, 1.65, 10.65, 406.00, 55.05, 1.72, 33.67, and 0.73mg/kg, respectively. Mean Ca, Mg, K, and Na were detected in concentrations of 10.32, 3.83, 7.23 and 1.17g/kg, respectively. In conclusion, metal exposure through herbal mint teas does not seem to be of health concern, as to most of the studied metals, but regulatory limits for Al contents should be imposed.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
C. Rubio, J.R.D. Lucas, A.J. Gutiérrez, D. Glez-Weller, B. Pérez Marrero, J.M. Caballero, C. Revert, A. Hardisson
Phytopharmaceuticals are gaining popularity worldwide; however, cases of adverse effects and drug interactions have also increased. One reason is in the high metal content both as ingredients but also as contaminants. Metal monitoring in food, like herbal teas, provides basic information on safety aspects in regulatory processes as well as nutritional values. In the present work, Cd, Pb, K, Na, Ca, Mg, Al, B, Ba, Co, Cr, Cu, Fe, Mn, Zn, Li, Ni, and Mo were determined by inductively coupled plasma spectrometry (ICPS) in 36 samples of Mentha sp. Mint tea bags and loose leaves were randomly obtained from supermarkets, traditional markets, herbal stores, and pharmacies in Tenerife (Canary Islands, Spain). Metal contents varied significantly, dependent on the stores the products were purchased in and on tea packaging (loose leaves versus tea bags). Pb analyses revealed levels (0.65±0.71mg/kg) below legal limits. The maximum permissible limit for Cd, 0.3mg/kg, set by the WHO for medicinal plants, was exceeded by 19.44% of the samples (0.22±0.13mg/kg), but all values were below the limit given in the European Pharmacopoeia for this metal (4mg/kg). We observed high Al (151.24±162.73mg/kg) and Li (5.46±3.94mg/kg) levels. B, Ba, Co, Cr, Cu, Fe, Mn, Ni, Zn, and Mo mean levels were 20.51, 14.15, 0.26, 1.65, 10.65, 406.00, 55.05, 1.72, 33.67, and 0.73mg/kg, respectively. Mean Ca, Mg, K, and Na were detected in concentrations of 10.32, 3.83, 7.23 and 1.17g/kg, respectively. In conclusion, metal exposure through herbal mint teas does not seem to be of health concern, as to most of the studied metals, but regulatory limits for Al contents should be imposed.
Qualitative screening for adulterants in weight-loss supplements by ion mobility spectrometry
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Jamie D. Dunn, Connie M. Gryniewicz-Ruzicka, Daniel J. Mans, Laura C. Mecker-Pogue, John F. Kauffman, Benjamin J. Westenberger, Lucinda F. Buhse
Ion mobility spectrometry (IMS) served as a rapid, qualitative screening tool for the analysis of adulterated weight-loss products. We have previously shown that sibutramine extracted into methanol from dietary supplements can be detected at low levels (2ng) using a portable IMS spectrometer, and have adapted a similar method for the analysis of additional weight-loss product adulterants. An FDA collaborative study helped to define the limits for fluoxetine with a limit of detection of 2ng. We also evaluated more readily available, less toxic extraction solvents and found isopropanol and water were comparable to methanol. Isopropanol was favored over water for two reasons: (1) water increases the analysis time and (2) aqueous solutions were more susceptible to pH change, which affected the detection of sibutramine. In addition to sibutamine and fluoxetine, we surveyed 11 weight-loss adulterants; bumetanide, fenfluramine, furosemide, orlistat, phenolphthalein, phentermine, phenytoin, rimonabant, sertraline and two sibutramine analogs, desmethylsibutramine and didesmethylsibutramine, using portable and benchtop ion mobility spectrometers. Out of these 13 active pharmaceutical ingredients (APIs), portable and benchtop ion mobility spectrometers were capable of screening products for 10 of these APIs. The developed procedure was applied to two weight-loss dietary supplements using both portable and benchtop instruments. One product contained didesmethylsibutramine while the other contained didesmethylsibutramine and phenolphthalein.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Jamie D. Dunn, Connie M. Gryniewicz-Ruzicka, Daniel J. Mans, Laura C. Mecker-Pogue, John F. Kauffman, Benjamin J. Westenberger, Lucinda F. Buhse
Ion mobility spectrometry (IMS) served as a rapid, qualitative screening tool for the analysis of adulterated weight-loss products. We have previously shown that sibutramine extracted into methanol from dietary supplements can be detected at low levels (2ng) using a portable IMS spectrometer, and have adapted a similar method for the analysis of additional weight-loss product adulterants. An FDA collaborative study helped to define the limits for fluoxetine with a limit of detection of 2ng. We also evaluated more readily available, less toxic extraction solvents and found isopropanol and water were comparable to methanol. Isopropanol was favored over water for two reasons: (1) water increases the analysis time and (2) aqueous solutions were more susceptible to pH change, which affected the detection of sibutramine. In addition to sibutamine and fluoxetine, we surveyed 11 weight-loss adulterants; bumetanide, fenfluramine, furosemide, orlistat, phenolphthalein, phentermine, phenytoin, rimonabant, sertraline and two sibutramine analogs, desmethylsibutramine and didesmethylsibutramine, using portable and benchtop ion mobility spectrometers. Out of these 13 active pharmaceutical ingredients (APIs), portable and benchtop ion mobility spectrometers were capable of screening products for 10 of these APIs. The developed procedure was applied to two weight-loss dietary supplements using both portable and benchtop instruments. One product contained didesmethylsibutramine while the other contained didesmethylsibutramine and phenolphthalein.
Drug release into hydrogel-based subcutaneous surrogates studied by UV imaging
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Fengbin Ye, Susan Weng Larsen, Anan Yaghmur, Henrik Jensen, Claus Larsen, Jesper Østergaard
Upon subcutaneous administration, the distribution of drug between the delivery vehicle and the biological tissue critically affects the absorption of drug substances. Utilization of physical models resembling the native tissues appears promising for obtaining a detailed understanding of the performance of drug delivery systems based on in vitro experiments. The objective of this study was to evaluate a UV imaging-based method for real-time characterization of the release and transport of piroxicam in hydrogel-based subcutaneous tissue mimics/surrogates. Piroxicam partitioning from medium chain triglyceride (MCT) into 0.5% (w/v) agarose or 25% (w/v) F127-based hydrogels was investigated by monitoring the concentration profiles of the drug in the gels. The effect of pH on piroxicam distribution and diffusion coefficients was studied. For both hydrogel systems, the diffusion of piroxicam in the gels was not affected significantly by the pH change from 4.0 to 7.4 but a considerable change in the oil–gel distribution coefficients was found (24 and 34 times less at pH 7.4 as compared those observed at pH 4.0 for F127 and agarose gels, respectively). In addition, the release and transport processes of piroxicam upon the injection of aqueous or MCT solutions into an agarose-based hydrogel were investigated by UV imaging. The spatial distribution of piroxicam around the injection site in the gel matrix was monitored in real-time. The disappearance profiles of piroxicam from the injected aqueous solution were obtained. This study shows that the UV imaging methodology has considerable potential for characterizing transport properties in hydrogels, including monitoring the real-time spatial concentration distribution in vitro after administration by injection.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Fengbin Ye, Susan Weng Larsen, Anan Yaghmur, Henrik Jensen, Claus Larsen, Jesper Østergaard
Upon subcutaneous administration, the distribution of drug between the delivery vehicle and the biological tissue critically affects the absorption of drug substances. Utilization of physical models resembling the native tissues appears promising for obtaining a detailed understanding of the performance of drug delivery systems based on in vitro experiments. The objective of this study was to evaluate a UV imaging-based method for real-time characterization of the release and transport of piroxicam in hydrogel-based subcutaneous tissue mimics/surrogates. Piroxicam partitioning from medium chain triglyceride (MCT) into 0.5% (w/v) agarose or 25% (w/v) F127-based hydrogels was investigated by monitoring the concentration profiles of the drug in the gels. The effect of pH on piroxicam distribution and diffusion coefficients was studied. For both hydrogel systems, the diffusion of piroxicam in the gels was not affected significantly by the pH change from 4.0 to 7.4 but a considerable change in the oil–gel distribution coefficients was found (24 and 34 times less at pH 7.4 as compared those observed at pH 4.0 for F127 and agarose gels, respectively). In addition, the release and transport processes of piroxicam upon the injection of aqueous or MCT solutions into an agarose-based hydrogel were investigated by UV imaging. The spatial distribution of piroxicam around the injection site in the gel matrix was monitored in real-time. The disappearance profiles of piroxicam from the injected aqueous solution were obtained. This study shows that the UV imaging methodology has considerable potential for characterizing transport properties in hydrogels, including monitoring the real-time spatial concentration distribution in vitro after administration by injection.
Comparative analysis of zaleplon complexation with cyclodextrins and hydrophilic polymers in solution and in solid state
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Jasna Jablan, Gábor Szalontai, Mario Jug
The aim of this work was to investigate the potential synergistic effect of water-soluble polymers (hypromellose, HPMC and polyvinylpyrrolidone, PVP) on zaleplon (ZAL) complexation with parent β-cyclodextrin (βCD) and its randomly methylated derivative (RAMEB) in solution and in solid state. The addition of HPMC to the complexation medium improved ZAL complexation and solubilization with RAMEB (K ZAL/RAMEB =156±5M−1 and K ZAL/RAMEB/HPMC =189±8M−1; p <0.01), while such effect was not observed for βCD (K ZAL/βCD =112±2M−1 and K ZAL/βCD/HPMC =119±8M−1; p >0.05). Although PVP increased the ZAL aqueous solubility from 0.22 to 0.27mg/mL, it did not show any synergistic effects on ZAL solubilization with the cyclodextrins tested. Binary and ternary systems of ZAL with βCD, RAMEB and HPMC were prepared by spray-drying. Differential scanning calorimetry, X-ray powder diffraction and scanning electron microscopy demonstrated a partial ZAL amorphization in spray-dried binary and ternary systems with βCD, while the drug was completely amorphous in all samples with RAMEB. Furthermore, inclusion complex formation in all systems prepared was confirmed by solid-state NMR spectroscopy. The in vitro dissolution rate followed the rank order ZAL/RAMEB/HPMC>ZAL/RAMEB=ZAL/βCD/HPMC>ZAL/βCD≫ZAL, clearly demonstrating the superior performance of RAMEB on ZAL complexation in the solid state and its synergistic effect with HPMC on drug solubility. Surprisingly, when loaded into tablets made with insoluble microcrystalline cellulose, RAMEB complexes had no positive effect on drug dissolution, because HPMC and RAMEB acted as a binders inside the tablets, prolonging their disintegration. Oppositely, the formulation with mannitol, a soluble excipient, containing a ternary RAMEB system, released the complete drug-dose in only 5min, clearly demonstrating its suitability for the development of immediate-release oral formulation of ZAL.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Jasna Jablan, Gábor Szalontai, Mario Jug
The aim of this work was to investigate the potential synergistic effect of water-soluble polymers (hypromellose, HPMC and polyvinylpyrrolidone, PVP) on zaleplon (ZAL) complexation with parent β-cyclodextrin (βCD) and its randomly methylated derivative (RAMEB) in solution and in solid state. The addition of HPMC to the complexation medium improved ZAL complexation and solubilization with RAMEB (K ZAL/RAMEB =156±5M−1 and K ZAL/RAMEB/HPMC =189±8M−1; p <0.01), while such effect was not observed for βCD (K ZAL/βCD =112±2M−1 and K ZAL/βCD/HPMC =119±8M−1; p >0.05). Although PVP increased the ZAL aqueous solubility from 0.22 to 0.27mg/mL, it did not show any synergistic effects on ZAL solubilization with the cyclodextrins tested. Binary and ternary systems of ZAL with βCD, RAMEB and HPMC were prepared by spray-drying. Differential scanning calorimetry, X-ray powder diffraction and scanning electron microscopy demonstrated a partial ZAL amorphization in spray-dried binary and ternary systems with βCD, while the drug was completely amorphous in all samples with RAMEB. Furthermore, inclusion complex formation in all systems prepared was confirmed by solid-state NMR spectroscopy. The in vitro dissolution rate followed the rank order ZAL/RAMEB/HPMC>ZAL/RAMEB=ZAL/βCD/HPMC>ZAL/βCD≫ZAL, clearly demonstrating the superior performance of RAMEB on ZAL complexation in the solid state and its synergistic effect with HPMC on drug solubility. Surprisingly, when loaded into tablets made with insoluble microcrystalline cellulose, RAMEB complexes had no positive effect on drug dissolution, because HPMC and RAMEB acted as a binders inside the tablets, prolonging their disintegration. Oppositely, the formulation with mannitol, a soluble excipient, containing a ternary RAMEB system, released the complete drug-dose in only 5min, clearly demonstrating its suitability for the development of immediate-release oral formulation of ZAL.
Highlights
► Zaleplon (ZAL) complexation with RAMEB and HPMC enhanced its solubility about 9 times. ► βCD was less effective in ZAL solubilization, not forming ternary complexes with HPMC or PVP. ► CP-MAS confirmed the inclusion complexation for all spray dried binary and ternary samples. ► βCD complexes contained 39.8–31.1% of crystalline ZAL; RAMEB complexes were amorphous. ► ZAL/RAMEB/HPMC complex tabletted with mannitol gained immediate-release formulation.Analysis of synthetic cannabinoids in herbal blends by means of nano-liquid chromatography
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Gustavo Merola, Zeineb Aturki, Giovanni D’Orazio, Rossella Gottardo, Teodora Macchia, Franco Tagliaro, Salvatore Fanali
In this study, a rapid and simultaneous separation of 12 synthetic cannabinoids and Δ9-tetrahydrocannabinol (Δ9-THC) in herbal blends was obtained by means of nano-liquid chromatography (nano-LC). The nano-LC experiments were performed in a 100μm i.d. capillary column packed with Cogent® bidentate C18 silica particles for 25.0cm. All compounds were resolved using an isocratic elution mode in less than 30min. A mobile phase containing ACN/MeOH/H2O/formic acid 69/5/25/1 (v/v/v/v) was employed for the chromatographic separation. The developed analytical method was validated in terms of precision, linearity, sensitivity and accuracy. Under optimal nano-LC–UV conditions, the resulting RSD percentages for intra-day and inter-day repeatability, related to retention time and peak area, were below 2.98 and 6.40%, respectively. Limits of detection and quantification were 0.2 and 0.5μg/mL, respectively, for all the studied compounds. Linearity was assessed in the concentration range of interest for all analytes with determination coefficients r 2 ≥0.9975. The method was then applied to the determination of synthetic cannabinoids in herbal blends. Quantitative analyses of the cannabimimetic compounds in six products showed that there was a wide difference in the concentration of the studied compounds among different products. Further, the nano-LC system was coupled with a mass spectrometer measuring the MS and MS–MS spectra to unequivocally identify the cannabinoids present in smoking mixtures.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Gustavo Merola, Zeineb Aturki, Giovanni D’Orazio, Rossella Gottardo, Teodora Macchia, Franco Tagliaro, Salvatore Fanali
In this study, a rapid and simultaneous separation of 12 synthetic cannabinoids and Δ9-tetrahydrocannabinol (Δ9-THC) in herbal blends was obtained by means of nano-liquid chromatography (nano-LC). The nano-LC experiments were performed in a 100μm i.d. capillary column packed with Cogent® bidentate C18 silica particles for 25.0cm. All compounds were resolved using an isocratic elution mode in less than 30min. A mobile phase containing ACN/MeOH/H2O/formic acid 69/5/25/1 (v/v/v/v) was employed for the chromatographic separation. The developed analytical method was validated in terms of precision, linearity, sensitivity and accuracy. Under optimal nano-LC–UV conditions, the resulting RSD percentages for intra-day and inter-day repeatability, related to retention time and peak area, were below 2.98 and 6.40%, respectively. Limits of detection and quantification were 0.2 and 0.5μg/mL, respectively, for all the studied compounds. Linearity was assessed in the concentration range of interest for all analytes with determination coefficients r 2 ≥0.9975. The method was then applied to the determination of synthetic cannabinoids in herbal blends. Quantitative analyses of the cannabimimetic compounds in six products showed that there was a wide difference in the concentration of the studied compounds among different products. Further, the nano-LC system was coupled with a mass spectrometer measuring the MS and MS–MS spectra to unequivocally identify the cannabinoids present in smoking mixtures.
Highlights
► Twelve synthetic cannabinoids and tetrahydrocannabinol were separated by nano-LC. ► Capillary columns packed with a C18 stationary phase were used for the separations. ► Analytes of forensic interest were separated by isocratic elution mode. ► Nano-LC was coupled with mass spectrometry for compounds characterization.Characterization of physalins and fingerprint analysis for the quality evaluation of Physalis alkekengi L. var. franchetii by ultra-performance liquid chromatography combined with diode array detection and electrospray ionization tandem mass spectrometry
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Yunliang Zheng, Lianjun Luan, Yong Chen, Yiping Ren, Yongjiang Wu
Physalins are important bioactive compounds from genus Physalis. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior and UV characteristics of seven physalins from genus Physalis were firstly investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and diode array detection (DAD). Combined with ultra-performance liquid chromatography (UPLC) and DAD, the established approach to the structural identification of physalins by ESI-MS/MS was then applied to the analysis of Physalis alkekengi L. According to the UPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by MS/MS spectra, about 19 fingerprint peaks were identified, including 14 physalins and 5 other compounds. Finally, the established fingerprint method was applied to the analysis of 31 P. alkekengi L. samples collected from different locations, which reflected their similar chemical constituent properties. The proposed method provides a scientific and technical platform to the herbal industry for quality control and safety assurance of herbal preparations that contain this class of physalins.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Yunliang Zheng, Lianjun Luan, Yong Chen, Yiping Ren, Yongjiang Wu
Physalins are important bioactive compounds from genus Physalis. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior and UV characteristics of seven physalins from genus Physalis were firstly investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and diode array detection (DAD). Combined with ultra-performance liquid chromatography (UPLC) and DAD, the established approach to the structural identification of physalins by ESI-MS/MS was then applied to the analysis of Physalis alkekengi L. According to the UPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by MS/MS spectra, about 19 fingerprint peaks were identified, including 14 physalins and 5 other compounds. Finally, the established fingerprint method was applied to the analysis of 31 P. alkekengi L. samples collected from different locations, which reflected their similar chemical constituent properties. The proposed method provides a scientific and technical platform to the herbal industry for quality control and safety assurance of herbal preparations that contain this class of physalins.
Analysis of total and unbound hydromorphone in human plasma by ultrafiltration and LC–MS/MS: Application to clinical trial in patients undergoing open heart surgery
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Teijo I. Saari, Jörg Fechner, Harald Ihmsen, Jürgen Schüttler, Christian Jeleazcov
A method for a sensitive and specific analysis of hydromorphone total and unbound drug concentrations in human plasma was developed and validated. Sample preparation was preceded with an ultrafiltration step to separate the unbound drug from the protein bound fraction of hydromorphone. Both the ultrafiltrate and plasma samples were extracted with solid-phase extraction and substituted with stable isotope-labeled hydromorphone that was used as internal standard. Chromatographic separation was performed by gradient elution with UPLC-like system and eluates were analyzed by tandem mass spectrometry equipped with an electrospray ionization source. Sample preparation was optimized for good recovery of hydromorphone and the results were consistent. Calibration curves demonstrated linearity in the concentration range of 78–5000pg/ml for analysis of both total and unbound concentrations of hydromorphone. The limit of detection was 1pg and the lower limit of quantification was 78pg/ml for both total and unbound hydromorphone plasma drug concentrations. Intra- and interassay reproducibility and inaccuracy did not exceed 10%. Hydromorphone was on the average 14% bound to plasma proteins, supporting the previously published unreferenced statements that the protein binding of hydromorphone is low. Method was applied to a clinical trial in patients undergoing open heart surgery to generate a target controlled infusion model for the postoperative patient controlled analgesia with hydromorphone.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Teijo I. Saari, Jörg Fechner, Harald Ihmsen, Jürgen Schüttler, Christian Jeleazcov
A method for a sensitive and specific analysis of hydromorphone total and unbound drug concentrations in human plasma was developed and validated. Sample preparation was preceded with an ultrafiltration step to separate the unbound drug from the protein bound fraction of hydromorphone. Both the ultrafiltrate and plasma samples were extracted with solid-phase extraction and substituted with stable isotope-labeled hydromorphone that was used as internal standard. Chromatographic separation was performed by gradient elution with UPLC-like system and eluates were analyzed by tandem mass spectrometry equipped with an electrospray ionization source. Sample preparation was optimized for good recovery of hydromorphone and the results were consistent. Calibration curves demonstrated linearity in the concentration range of 78–5000pg/ml for analysis of both total and unbound concentrations of hydromorphone. The limit of detection was 1pg and the lower limit of quantification was 78pg/ml for both total and unbound hydromorphone plasma drug concentrations. Intra- and interassay reproducibility and inaccuracy did not exceed 10%. Hydromorphone was on the average 14% bound to plasma proteins, supporting the previously published unreferenced statements that the protein binding of hydromorphone is low. Method was applied to a clinical trial in patients undergoing open heart surgery to generate a target controlled infusion model for the postoperative patient controlled analgesia with hydromorphone.
Determination of emodin in L-02 cells and cell culture media with liquid chromatography–mass spectrometry: Application to a cellular toxicokinetic study
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Cui-Li Li, Jiang Ma, Li Zheng, Hui-Jun Li, Ping Li
The emodin-involved hepatotoxicity has been gaining increasing attention. The purpose of the present study was to evaluate the cytotoxicity of emodin on cultured human liver cells (L-02) and predict the possible relation between its cytotoxicity and cellular toxicokinetics. Cell viability and cell damage were assessed by Cell Counting Kit-8 (CCK-8) assay and phase-contrast microscopy, respectively. Cytotoxicity tests demonstrated a concentration- and time-dependent toxic effect of emodin on L-02 cells. Furthermore, emodin at concentration of 30μM led to a significant apoptosis in a time-dependent manner supported by the morphological changes of drug-treated cells. In addition, to elucidate the toxicokinetic characteristics of emodin, a highly sensitive and selective liquid chromatography–mass spectrometry (LC–MS) method was employed and validated for detecting the dynamic alteration of emodin in cells and cell culture media. The proposed method appeared to be suitable for the analysis of emodin with desirable linearity (r 2 >0.99), and satisfying precision being less than 8.7%. The range of recoveries of this method was 90.2–101.9%. The preliminary cellular toxicokinetic study revealed a time-dependent intracellular accumulation of emodin, which was consistent with its in vitro toxic effects. These findings confirmed the cytotoxicity of emodin against L-02 cells and displayed the cytotoxic manner of emodin in terms of its cellular uptake and accumulation in L-02 cells.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Cui-Li Li, Jiang Ma, Li Zheng, Hui-Jun Li, Ping Li
The emodin-involved hepatotoxicity has been gaining increasing attention. The purpose of the present study was to evaluate the cytotoxicity of emodin on cultured human liver cells (L-02) and predict the possible relation between its cytotoxicity and cellular toxicokinetics. Cell viability and cell damage were assessed by Cell Counting Kit-8 (CCK-8) assay and phase-contrast microscopy, respectively. Cytotoxicity tests demonstrated a concentration- and time-dependent toxic effect of emodin on L-02 cells. Furthermore, emodin at concentration of 30μM led to a significant apoptosis in a time-dependent manner supported by the morphological changes of drug-treated cells. In addition, to elucidate the toxicokinetic characteristics of emodin, a highly sensitive and selective liquid chromatography–mass spectrometry (LC–MS) method was employed and validated for detecting the dynamic alteration of emodin in cells and cell culture media. The proposed method appeared to be suitable for the analysis of emodin with desirable linearity (r 2 >0.99), and satisfying precision being less than 8.7%. The range of recoveries of this method was 90.2–101.9%. The preliminary cellular toxicokinetic study revealed a time-dependent intracellular accumulation of emodin, which was consistent with its in vitro toxic effects. These findings confirmed the cytotoxicity of emodin against L-02 cells and displayed the cytotoxic manner of emodin in terms of its cellular uptake and accumulation in L-02 cells.
The effect of anionic surfactant on poliovirus particles during capillary electrophoresis
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Iuliana Oita, Hadewych Halewyck, Bert Thys, Bart Rombaut, Yvan Vander Heyden
Because of its essential role in SDS-PAGE, sodium dodecylsulphate (SDS) is generally associated with protein denaturation. However, for SDS-PAGE, proteins are linearized in the presence of SDS, following the exposure to high temperatures and reducing agents. In comparison, the conditions employed during a capillary electrophoretic (CE) separation involve only a limited exposure to SDS, at much lower temperatures. As the outer surface of the non-enveloped viruses consists of proteins, virus interaction with SDS can be judged from the perspective of SDS–protein interaction. Several studies have indicated that proteins have a different susceptibility to SDS, depending on their secondary structure and number of subunits. Therefore it is not straightforward to estimate what should be expected when intact polioviruses and subviral particles obtained by thermal conversion of the poliovirions, are exposed to SDS during CE separation. In this study it is shown that, during CE separations, SDS has no effect on the integrity of the poliovirion, but the presence of SDS in the separation system influences the poliovirus peak height and shape. The implication of SDS in the CE separation of poliovirus is discussed in detail. On the contrary, the proteinaceous subviral particles, such as the empty capsids, are less stable in the presence of SDS during the CE separation, and aggregates between the individual poliovirus capsid proteins and SDS are formed. Finally, we have proposed an alternative separation approach, involving an SDS gradient, for an improved separation of the subviral particles.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Iuliana Oita, Hadewych Halewyck, Bert Thys, Bart Rombaut, Yvan Vander Heyden
Because of its essential role in SDS-PAGE, sodium dodecylsulphate (SDS) is generally associated with protein denaturation. However, for SDS-PAGE, proteins are linearized in the presence of SDS, following the exposure to high temperatures and reducing agents. In comparison, the conditions employed during a capillary electrophoretic (CE) separation involve only a limited exposure to SDS, at much lower temperatures. As the outer surface of the non-enveloped viruses consists of proteins, virus interaction with SDS can be judged from the perspective of SDS–protein interaction. Several studies have indicated that proteins have a different susceptibility to SDS, depending on their secondary structure and number of subunits. Therefore it is not straightforward to estimate what should be expected when intact polioviruses and subviral particles obtained by thermal conversion of the poliovirions, are exposed to SDS during CE separation. In this study it is shown that, during CE separations, SDS has no effect on the integrity of the poliovirion, but the presence of SDS in the separation system influences the poliovirus peak height and shape. The implication of SDS in the CE separation of poliovirus is discussed in detail. On the contrary, the proteinaceous subviral particles, such as the empty capsids, are less stable in the presence of SDS during the CE separation, and aggregates between the individual poliovirus capsid proteins and SDS are formed. Finally, we have proposed an alternative separation approach, involving an SDS gradient, for an improved separation of the subviral particles.
Rapid quantification of tryptophan and tyrosine in chemically defined cell culture media using fluorescence spectroscopy
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Amandine Calvet, Boyan Li, Alan G. Ryder
The rapid and inexpensive analysis of the complex cell culture media used in industrial mammalian cell culture is required for quality and variance monitoring. Excitation-emission matrix (EEM) spectroscopy combined with multi-way chemometrics is a robust methodology applicable for the analysis of raw materials, media, and bioprocess broths. We have shown that the methodology can identify compositional changes and predict the efficacy of media in terms of downstream titer [1]. Here we describe how to extend the measurement methodology for the quantification of tryptophan (Trp), tyrosine (Tyr) in complex chemically defined media. The sample type is an enriched basal RDF medium in which five significant fluorophores were identified: Trp, Tyr, pyridoxine, folic acid, and riboflavin. The relatively high chromophore concentrations and compositional complexity lead to very significant matrix effects which were assessed using PARAllel FACtor analysis2 (PARAFAC2). Taking these effects into account, N-way partial least squares (NPLS) combined with a modified standard addition method was used to build calibration models capable of quantifying Trp and Tyr with errors of ∼4.5 and 5.5% respectively. This demonstrates the feasibility of using the EEM method for the rapid, quantitative analysis of Trp and Tyr in complex cell culture media with minimal sample handling as an alternative to chromatographic based methods.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Amandine Calvet, Boyan Li, Alan G. Ryder
The rapid and inexpensive analysis of the complex cell culture media used in industrial mammalian cell culture is required for quality and variance monitoring. Excitation-emission matrix (EEM) spectroscopy combined with multi-way chemometrics is a robust methodology applicable for the analysis of raw materials, media, and bioprocess broths. We have shown that the methodology can identify compositional changes and predict the efficacy of media in terms of downstream titer [1]. Here we describe how to extend the measurement methodology for the quantification of tryptophan (Trp), tyrosine (Tyr) in complex chemically defined media. The sample type is an enriched basal RDF medium in which five significant fluorophores were identified: Trp, Tyr, pyridoxine, folic acid, and riboflavin. The relatively high chromophore concentrations and compositional complexity lead to very significant matrix effects which were assessed using PARAllel FACtor analysis2 (PARAFAC2). Taking these effects into account, N-way partial least squares (NPLS) combined with a modified standard addition method was used to build calibration models capable of quantifying Trp and Tyr with errors of ∼4.5 and 5.5% respectively. This demonstrates the feasibility of using the EEM method for the rapid, quantitative analysis of Trp and Tyr in complex cell culture media with minimal sample handling as an alternative to chromatographic based methods.
A full validated hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the quantification of oxaliplatin in human plasma ultrafiltrates
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Hajime Ito, Hiroaki Yamaguchi, Asuka Fujikawa, Nobuaki Tanaka, Ayako Furugen, Kazuaki Miyamori, Natsuko Takahashi, Jiro Ogura, Masaki Kobayashi, Takehiro Yamada, Nariyasu Mano, Ken Iseki
Oxaliplatin is a platinum agent that is used for treatment of colorectal cancer. A sensitive and selective hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the quantification of oxaliplatin was developed. Human plasma ultrafiltrates were precipitated by acetonitrile containing carboplatin as an internal standard and further diluted with acetonitrile. Chromatographic separation of oxaliplatin and the internal standard was achieved with a column modified with phosphorylcholine and an isocratic mobile phase (acetonitrile/water/acetic acid=90:10:0.1, v/v/v) at the flow rate of 0.2mL/min. The lower limit of quantification for oxaliplatin was 25ng/mL. The linearity range of the method was from 25 to 5000ng/mL. The intra-day precision and inter-day precision (RSD) ranged from 0.8 to 6.1%, and the accuracy (RE) was within ±4.5%. The extraction recoveries from human plasma ultrafiltrates were 83.6–91.6%, and ion suppression caused by matrix components was 86.7–88.5% at three different levels, respectively. This method was applied to a clinical pharmacokinetic study of oxaliplatin in a cancer patient. The maximum concentration of colorectal cancer patient administered oxaliplatin was 1650ng/mL.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Hajime Ito, Hiroaki Yamaguchi, Asuka Fujikawa, Nobuaki Tanaka, Ayako Furugen, Kazuaki Miyamori, Natsuko Takahashi, Jiro Ogura, Masaki Kobayashi, Takehiro Yamada, Nariyasu Mano, Ken Iseki
Oxaliplatin is a platinum agent that is used for treatment of colorectal cancer. A sensitive and selective hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the quantification of oxaliplatin was developed. Human plasma ultrafiltrates were precipitated by acetonitrile containing carboplatin as an internal standard and further diluted with acetonitrile. Chromatographic separation of oxaliplatin and the internal standard was achieved with a column modified with phosphorylcholine and an isocratic mobile phase (acetonitrile/water/acetic acid=90:10:0.1, v/v/v) at the flow rate of 0.2mL/min. The lower limit of quantification for oxaliplatin was 25ng/mL. The linearity range of the method was from 25 to 5000ng/mL. The intra-day precision and inter-day precision (RSD) ranged from 0.8 to 6.1%, and the accuracy (RE) was within ±4.5%. The extraction recoveries from human plasma ultrafiltrates were 83.6–91.6%, and ion suppression caused by matrix components was 86.7–88.5% at three different levels, respectively. This method was applied to a clinical pharmacokinetic study of oxaliplatin in a cancer patient. The maximum concentration of colorectal cancer patient administered oxaliplatin was 1650ng/mL.
Simultaneous determination of amlodipine and bisoprolol in rat plasma by a liquid chromatography/tandem mass spectrometry method and its application in pharmacokinetic study
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Huichao Chang, Jinyin Li, Ji Li, Xiaoduo Guan, Fanlu Sun, Zhongzhi Qian, Kaishun Bi, Guorong Fan
A sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the quantitative determination of amlodipine and bisoprolol, using clenbuterol as the internal standard (IS). The analytes and IS were isolated from 100μL plasma samples by a simple liquid–liquid extraction (LLE). Reverse-phase high performance liquid chromatography (RP-HPLC) separation was accomplished on a Diamonsil C18 column (50mm×4.6mm, 5μm) with a mobile phase composed of methanol–water–formic acid (75:25:0.01, v/v/v) at a flow rate of 0.3mL/min. The method had a chromatographic total run time of 3min. Multiple reacting monitoring (MRM) transitions of m/z [M+H]+ 409.1→237.9 (amlodipine), m/z [M+H]+ 326.2→116.0 (bisoprolol) and m/z [M+H]+ 277.0→203.0 (clenbuterol, IS) were used to quantify amlodipine, bisoprolol and IS, respectively. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.2ng/mL for both amlodipine and bisoprolol, and the linear range was 0.2–50ng/mL for both amlodipine and bisoprolol (r 2 >0.9961). All the validation data, such as accuracy, precision and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic studies of amlodipine and bisoprolol in Sprague–Dawley (SD) rats.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Huichao Chang, Jinyin Li, Ji Li, Xiaoduo Guan, Fanlu Sun, Zhongzhi Qian, Kaishun Bi, Guorong Fan
A sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the quantitative determination of amlodipine and bisoprolol, using clenbuterol as the internal standard (IS). The analytes and IS were isolated from 100μL plasma samples by a simple liquid–liquid extraction (LLE). Reverse-phase high performance liquid chromatography (RP-HPLC) separation was accomplished on a Diamonsil C18 column (50mm×4.6mm, 5μm) with a mobile phase composed of methanol–water–formic acid (75:25:0.01, v/v/v) at a flow rate of 0.3mL/min. The method had a chromatographic total run time of 3min. Multiple reacting monitoring (MRM) transitions of m/z [M+H]+ 409.1→237.9 (amlodipine), m/z [M+H]+ 326.2→116.0 (bisoprolol) and m/z [M+H]+ 277.0→203.0 (clenbuterol, IS) were used to quantify amlodipine, bisoprolol and IS, respectively. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.2ng/mL for both amlodipine and bisoprolol, and the linear range was 0.2–50ng/mL for both amlodipine and bisoprolol (r 2 >0.9961). All the validation data, such as accuracy, precision and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic studies of amlodipine and bisoprolol in Sprague–Dawley (SD) rats.
Direct glutathione quantification in human blood by LC–MS/MS: comparison with HPLC with electrochemical detection
16 October 2012,
13:47:21
Publication year:
2012
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Isabella Squellerio, Donatella Caruso, Benedetta Porro, Fabrizio Veglia, Elena Tremoli, Viviana Cavalca
Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC–MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01–50μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10% for both GSH and GSSG), accuracy (bias% varied between −2.1 and 7.9% for both analytes), quantification limits (LLOQs were 0.5μM and 0.0625μM for GSH and GSSG respectively). Furthermore the LC–MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC–MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.
Source:Journal of Pharmaceutical and Biomedical Analysis, Volume 71
Isabella Squellerio, Donatella Caruso, Benedetta Porro, Fabrizio Veglia, Elena Tremoli, Viviana Cavalca
Glutathione plays a central role in the defence against oxidative damage and in signaling pathways. Upon oxidation the reduced glutathione (GSH) is transformed to glutathione disulfide (GSSG). The concentration of GSH and GSSG in whole blood samples and their ratios is useful indicator of the oxidative stress status and/or disease risk. Here we describe a liquid-chromatographic method coupled with tandem mass spectrometry (LC–MS/MS) and we present the results of its comparison with a high-performance liquid-chromatographic method with electrochemical detection (HPLC-ECD). The method performed well in terms of validation parameters, i.e. linear range (0.01–50μM for both GSH and GSSG), precision (intra- and inter-day coefficients of variation were less than 10% for both GSH and GSSG), accuracy (bias% varied between −2.1 and 7.9% for both analytes), quantification limits (LLOQs were 0.5μM and 0.0625μM for GSH and GSSG respectively). Furthermore the LC–MS/MS method showed a good agreement with the HPLC-ECD assay. However, major benefits of LC–MS/MS are the improved selectivity, precision and accuracy, the higher sensitivity and the unaltered capacity of detection with time in contrast to ECD.
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