Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry

Publication date: Available online 20 February 2018
Source:Journal of Chromatography B
Author(s): Valentina D’Atri, Szabolcs Fekete, Dwight Stoll, Matthew Lauber, Alain Beck, Davy Guillarme
Brentuximab vedotin (Adcetris) is a cysteine-linked antibody-drug conjugate (ADC) used in the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). In this study, the drug payload and glycan modifications of this ADC were simultaneously characterized using a unique LC-MS middle-up analysis, involving hydrophilic interaction chromatography (HILIC). This work demonstrates that HILIC is an effective and complementary analytical technique to reversed phase liquid chromatography (RPLC) for subunit-level characterization of immuno-conjugates.

Publication date: Available online 20 February 2018
Source:Journal of Chromatography B

Author(s): Valentina D’Atri, Szabolcs Fekete, Dwight Stoll, Matthew Lauber, Alain Beck, Davy Guillarme

Brentuximab vedotin (Adcetris) is a cysteine-linked antibody-drug conjugate (ADC) used in the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). In this study, the drug payload and glycan modifications of this ADC were simultaneously characterized using a unique LC-MS middle-up analysis, involving hydrophilic interaction chromatography (HILIC). This work demonstrates that HILIC is an effective and complementary analytical technique to reversed phase liquid chromatography (RPLC) for subunit-level characterization of immuno-conjugates.





Targeted and untargeted-metabolite profiling to track the compositional integrity of ginger during processing using digitally-enhanced HPTLC pattern recognition analysis

Publication date: Available online 19 February 2018 Source:Journal of Chromatography B Author(s): Reham S. Ibrahim, Hoda Fathy Tracking the impact of commonly applied post-harvesting and industrial processing practices on the compo…

Publication date: Available online 19 February 2018
Source:Journal of Chromatography B

Author(s): Reham S. Ibrahim, Hoda Fathy

Tracking the impact of commonly applied post-harvesting and industrial processing practices on the compositional integrity of ginger rhizome was implemented in this work. Untargeted metabolite profiling was performed using digitally-enhanced HPTLC method where the chromatographic fingerprints were extracted using ImageJ software then analysed with multivariate Principal Component Analysis (PCA) for pattern recognition. A targeted approach was applied using a new, validated, simple and fast HPTLC image analysis method for simultaneous quantification of the officially recognized markers 6-, 8-, 10-gingerol and 6-shogaol in conjunction with chemometric Hierarchical Clustering Analysis (HCA). The results of both targeted and untargeted metabolite profiling revealed that peeling, drying in addition to storage employed during processing have a great influence on ginger chemo-profile, the different forms of processed ginger shouldn't be used interchangeably. Moreover, it deemed necessary to consider the holistic metabolic profile for comprehensive evaluation of ginger during processing.





A high-throughput UPC2-MS/MS method for the separation and quantification of C19 and C21 steroids and their C11-oxy steroid metabolites in the classical, alternative, backdoor and 11OHA4 steroid pathways

Publication date: Available online 20 February 2018
Source:Journal of Chromatography B
Author(s): Therina du Toit, Maria A. Stander, Amanda C. Swart
In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical method was developed and validated for the determination of 17 C19 and 14 C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately.Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C19 and C21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C19 and C21 steroids, together with their C11-hydroxy and C11-keto metabolites –one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients.

Publication date: Available online 20 February 2018
Source:Journal of Chromatography B

Author(s): Therina du Toit, Maria A. Stander, Amanda C. Swart

In the present study an ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical method was developed and validated for the determination of 17 C19 and 14 C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids. The limit of detection and limit of quantification ranged from 0.01 to 10 ng/mL and from 0.01 to 20 ng/mL, respectively, and the method shows the recovery, matrix effect and process efficiency of steroids isolated from a serum matrix to be within acceptable limits. Good accuracy, repeatability and reproducibility were also shown and the method provided excellent sensitivity and selectivity as stereoisomers and regioisomers were also resolved and quantified accurately. Clinical conditions such as congenital adrenal hyperplasia, polycystic ovary syndrome in females and disorders of sex development in neonates and in children, amongst others, are characterized by abnormal steroid levels. Steroid profiling is essential to accurately diagnose steroid levels in the above settings as well as in androgen excess or deficiency in adrenal-linked endocrine diseases. Our method, separating C19 and C21 steroids in a single chromatographic step, offers a reduced sample turnover rate in the clinical setting, while providing comprehensive steroid profiles of in vivo steroids in the nmol/L range. This is, to our knowledge, the first method reported to simultaneously separate C19 and C21 steroids, together with their C11-hydroxy and C11-keto metabolites –one which may hold promise in the identification of new steroid markers in steroid-linked endocrine diseases, in addition to profiling steroid metabolism and abnormal enzyme activity in patients.





Evaluation of a method for measuring the radioprotective metabolite WR-1065 in plasma using chemical derivatization combined with UHPLC-MS/MS

Publication date: Available online 20 February 2018
Source:Journal of Chromatography B
Author(s): Eric S. Simon, Dawn Reyna, Richard J. Lister, Cheryl Harteg, Elke Lipka
Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4 nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150 nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10 mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.

Publication date: Available online 20 February 2018
Source:Journal of Chromatography B

Author(s): Eric S. Simon, Dawn Reyna, Richard J. Lister, Cheryl Harteg, Elke Lipka

Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4 nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150 nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10 mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.





Synthesis and characterization of Ag+-decorated poly(glycidyl methacrylate) microparticle design for the adsorption of nucleic acids

Publication date: Available online 18 February 2018
Source:Journal of Chromatography B
Author(s): Kadir Erol, Aytekin Uzunoglu, Kazım Köse, Büşra Sarıca, Emre Avcı, Dursun A. Köse
In this study, we report on the adsorption of RNA and DNA molecules by exploiting the high binding affinity of these nucleic acids to Ag+ ions anchored on magnetic poly(glycidyl methacrylate) (PGMA) microparticles. PGMA microparticles were synthesized and modified with nicotinamide which enabled to anchor Ag+ ions on the surface. The successful preparation of PGMA was confirmed by the presence of characteristic FTIR peaks. The ESR results showed that the incorporation of FeNi salt to the polymeric structure provided a magnetic property to the microparticles. The amount of nicotinamide and Ag+ ions used to modify the surface of the particles were found to be 1.79 wt% and 52.6 mg Ag/g microparticle, respectively. The high affinity of nucleic acids to Ag+ ions were exploited for the adsorption studies. At the optimum working conditions, the adsorption capacity of microparticles was found to be 40.1 and 11.48 mg nucleic acid/g microparticle for RNA and DNA, respectively. Our study indicated that the use of novel Ag+-decorated magnetic PGMA particles can be successfully employed as adsorbents for fast, easy, and cost-friendly adsorption of nucleic acids with high purity as well as high in quantity.

Publication date: Available online 18 February 2018
Source:Journal of Chromatography B

Author(s): Kadir Erol, Aytekin Uzunoglu, Kazım Köse, Büşra Sarıca, Emre Avcı, Dursun A. Köse

In this study, we report on the adsorption of RNA and DNA molecules by exploiting the high binding affinity of these nucleic acids to Ag+ ions anchored on magnetic poly(glycidyl methacrylate) (PGMA) microparticles. PGMA microparticles were synthesized and modified with nicotinamide which enabled to anchor Ag+ ions on the surface. The successful preparation of PGMA was confirmed by the presence of characteristic FTIR peaks. The ESR results showed that the incorporation of FeNi salt to the polymeric structure provided a magnetic property to the microparticles. The amount of nicotinamide and Ag+ ions used to modify the surface of the particles were found to be 1.79 wt% and 52.6 mg Ag/g microparticle, respectively. The high affinity of nucleic acids to Ag+ ions were exploited for the adsorption studies. At the optimum working conditions, the adsorption capacity of microparticles was found to be 40.1 and 11.48 mg nucleic acid/g microparticle for RNA and DNA, respectively. Our study indicated that the use of novel Ag+-decorated magnetic PGMA particles can be successfully employed as adsorbents for fast, easy, and cost-friendly adsorption of nucleic acids with high purity as well as high in quantity.





A novel technique for determination of the fructose, glucose and sucrose distribution in nectar from orchids by HPLC-ELSD

Publication date: Available online 18 February 2018
Source:Journal of Chromatography B
Author(s): Dan Nybro Lindqvist, Henrik Ærenlund Pedersen, Lars Holm Rasmussen
The dominant components in floral nectar is fructose, glucose and sucrose. The concentration and the ratio between the sugars are indicative for plant species and play an important part in the interplay between plants and pollinators. In this paper we present a novel HPLC-ELSD based analytical method for sugar characterization of nectar from orchids. Nectar was collected on Whatman No. 1 paper and preserved in the field by 70 v/v% ethanol. The analytical method had a linear range up to at least 3000 mg L−1 for all 3 sugars with a precision of 1.5–1.7%. Correlation coefficients were 0.9999 to 1.0000. The LOD of all sugars were 5–7 mg L−1 and the LOQ were 17–19 mg L−1. Field samples were stable for min. 7 weeks at −18 °C. The technique was applied to two species of Platanthera (Orchidaceae) in order to test whether species-related differences in sugar composition could be observed. No differences were found between the two species, which were sucrose-dominant (53.5–100%) though with high variation within species and between individual flowers.

Publication date: Available online 18 February 2018
Source:Journal of Chromatography B

Author(s): Dan Nybro Lindqvist, Henrik Ærenlund Pedersen, Lars Holm Rasmussen

The dominant components in floral nectar is fructose, glucose and sucrose. The concentration and the ratio between the sugars are indicative for plant species and play an important part in the interplay between plants and pollinators. In this paper we present a novel HPLC-ELSD based analytical method for sugar characterization of nectar from orchids. Nectar was collected on Whatman No. 1 paper and preserved in the field by 70 v/v% ethanol. The analytical method had a linear range up to at least 3000 mg L−1 for all 3 sugars with a precision of 1.5–1.7%. Correlation coefficients were 0.9999 to 1.0000. The LOD of all sugars were 5–7 mg L−1 and the LOQ were 17–19 mg L−1. Field samples were stable for min. 7 weeks at −18 °C. The technique was applied to two species of Platanthera (Orchidaceae) in order to test whether species-related differences in sugar composition could be observed. No differences were found between the two species, which were sucrose-dominant (53.5–100%) though with high variation within species and between individual flowers.





A UPLC-ESI-Q-TOF method for rapid and reliable identification and quantification of major indole alkaloids in Catharanthus roseus

Publication date: Available online 17 February 2018
Source:Journal of Chromatography B
Author(s): Won Tae Jeong, Heung Bin Lim
We developed a novel ultra performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) mass spectrometry method that allows sensitive, rapid, and reliable detection and identification of six representative indole alkaloids (vincristine, vinblastine, ajmalicine, catharanthine, serpentine, and vindoline) that exhibit physiological activity in Catharanthus roseus (C. roseus). The alkaloids were eluted on a C18 column with acetonitrile and water containing 0.1% formic acid and 10 mM ammonium acetate, and separated with good resolution within 13 min. Electrospray ionization-Q-TOF (ESI-Q-TOF) analysis was performed to characterize the molecules and their fragment ions, and the characteristic ions and fragmentation patterns were used as to identify the alkaloids. The proposed analytical method was verified in reference to the ICH guidelines and the results showed excellent linearity (R2 > 0.9988), limit of detection (1 ng/mL to 10 ng/mL), limit of quantification (3 ng/mL to 30 ng/mL), intra-day and inter-day precisions, and extraction recovery rates (92.8% to 104.1%) for all components. The validated UPLC-Q-TOF method was applied to the analysis of extracts from the root, stem, and leaves of C. roseus, allowing the identification of six alkaloids by comparison of retention times, molecular ions, and fragmentation patterns with those of reference compounds. Sixteen additional indole alkaloids were tentatively identified by comparison of chromatograms to chemical databases and literature reports. The contents of bis-indole alkaloids (vincristine and vinblastine) were high in the aerial parts, while the contents of mono-indole alkaloids (ajmalicine, catharanthine, serpentine, and vindoline) were high in the roots. The present results demonstrate that the proposed UPLC-Q-TOF method can be useful for the investigation of phytochemical constituents of medicinal plants.

Publication date: Available online 17 February 2018
Source:Journal of Chromatography B

Author(s): Won Tae Jeong, Heung Bin Lim

We developed a novel ultra performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) mass spectrometry method that allows sensitive, rapid, and reliable detection and identification of six representative indole alkaloids (vincristine, vinblastine, ajmalicine, catharanthine, serpentine, and vindoline) that exhibit physiological activity in Catharanthus roseus (C. roseus). The alkaloids were eluted on a C18 column with acetonitrile and water containing 0.1% formic acid and 10 mM ammonium acetate, and separated with good resolution within 13 min. Electrospray ionization-Q-TOF (ESI-Q-TOF) analysis was performed to characterize the molecules and their fragment ions, and the characteristic ions and fragmentation patterns were used as to identify the alkaloids. The proposed analytical method was verified in reference to the ICH guidelines and the results showed excellent linearity (R2 > 0.9988), limit of detection (1 ng/mL to 10 ng/mL), limit of quantification (3 ng/mL to 30 ng/mL), intra-day and inter-day precisions, and extraction recovery rates (92.8% to 104.1%) for all components. The validated UPLC-Q-TOF method was applied to the analysis of extracts from the root, stem, and leaves of C. roseus, allowing the identification of six alkaloids by comparison of retention times, molecular ions, and fragmentation patterns with those of reference compounds. Sixteen additional indole alkaloids were tentatively identified by comparison of chromatograms to chemical databases and literature reports. The contents of bis-indole alkaloids (vincristine and vinblastine) were high in the aerial parts, while the contents of mono-indole alkaloids (ajmalicine, catharanthine, serpentine, and vindoline) were high in the roots. The present results demonstrate that the proposed UPLC-Q-TOF method can be useful for the investigation of phytochemical constituents of medicinal plants.





Comparisons of the pharmacokinetic and tissue distribution profiles of withanolide B after intragastric administration of the effective part of Datura metel L. in normal and psoriasis guinea pigs

Publication date: Available online 17 February 2018
Source:Journal of Chromatography B
Author(s): Lianrong Yang, Xin Meng, HaixueKuang
A simple, highly sensitive ultra-performance liquid chromatography- electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed to quantify of withanolide B and obakunone (IS) in guinea pig plasma and tissues, and to compare the pharmacokinetics and tissue distribution of withanolide B in normal and psoriasis guinea pigs. After mixing with IS, plasma and tissues were pretreated by protein precipitation with methanol. Chromatographic separation was performed on a C18 column using aqueous (0.1% formic acid) and acetonitrile (0.1% formic acid) solutions at 0.4 mL/min as the mobile phase. The gradient program was selected (0–4.0 min, 2–98% B; 4.0–4.5 min, 98–2% B; and 4.5–5 min, 2% B). Detection was performed on a 4000 QTRAP UPLC–ESI-MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Withanolide B and obakunone (IS) were monitored under positive ionization conditions. The optimized mass transition ion-pairs (m/z) for quantitation were 455.1/109.4 for withanolide B and 455.1/161.1 for obakunone.

Publication date: Available online 17 February 2018
Source:Journal of Chromatography B

Author(s): Lianrong Yang, Xin Meng, HaixueKuang

A simple, highly sensitive ultra-performance liquid chromatography- electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed to quantify of withanolide B and obakunone (IS) in guinea pig plasma and tissues, and to compare the pharmacokinetics and tissue distribution of withanolide B in normal and psoriasis guinea pigs. After mixing with IS, plasma and tissues were pretreated by protein precipitation with methanol. Chromatographic separation was performed on a C18 column using aqueous (0.1% formic acid) and acetonitrile (0.1% formic acid) solutions at 0.4 mL/min as the mobile phase. The gradient program was selected (0–4.0 min, 2–98% B; 4.0–4.5 min, 98–2% B; and 4.5–5 min, 2% B). Detection was performed on a 4000 QTRAP UPLC–ESI-MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Withanolide B and obakunone (IS) were monitored under positive ionization conditions. The optimized mass transition ion-pairs (m/z) for quantitation were 455.1/109.4 for withanolide B and 455.1/161.1 for obakunone.





Supercritical fluid chromatographic-tandem mass spectrometry method for monitoring dissipation of thiacloprid in greenhouse vegetables and soil under different application modes

Publication date: Available online 16 February 2018
Source:Journal of Chromatography B
Author(s): Runan Li, Zenglong Chen, Fengshou Dong, Jun Xu, Xingang Liu, Xiaohu Wu, Xinglu Pan, Yan Tao, Yongquan Zheng
A rapid, sensitive and effective supercritical fluid chromatographic-tandem mass spectrometry (SFC-MS/MS) method was developed to analyze thiacloprid for the first time. The SFC-MS/MS conditions were optimized with the ultra-performance convergence chromatography (UPC2) BEH column (100 mm × 3.0 mm, 1.7 μm particle size) and thiacloprid was eluted at 1.22 min in gradient mode with CO2/methanol as mobile phase. The 0.1% formic acid in methanol (v/v) was used as postcolumn compensation solution to improve sensitivity. The ABPR pressure, flow rate of mobile phase and flow rate of compensation pump were set at 1800 psi, 1.8 mL/min, and 0.1 mL/min, respectively. The average recoveries of thiacloprid in soil at four spiking levels (5, 10, 100, 1000 μg/kg) ranged between 78.8% and 107.1% with relative standard deviations (RSDs) lower than 12.2% and the limit of quantitation (LOQ) was 5 μg/kg. The proposed method can distinctly improve the analysis efficiency by 2–12 times and reduce the solvent consumption by 5%–95% compared with reported methods. It was applied to investigate the dissipation rates of thiacloprid in greenhouse vegetables and soil under different application modes. The half-lives of thiacloprid in cucumber and soil were 9.55–20.44 days and 3.74–9.14 days separately under different application modes, 10.60 days in tomato under foliar spraying. The residues in vegetables under root irrigation were all less than that under foliar spraying. The results could offer useful data for risk assessment of thiacloprid in agricultural production.

Publication date: Available online 16 February 2018
Source:Journal of Chromatography B

Author(s): Runan Li, Zenglong Chen, Fengshou Dong, Jun Xu, Xingang Liu, Xiaohu Wu, Xinglu Pan, Yan Tao, Yongquan Zheng

A rapid, sensitive and effective supercritical fluid chromatographic-tandem mass spectrometry (SFC-MS/MS) method was developed to analyze thiacloprid for the first time. The SFC-MS/MS conditions were optimized with the ultra-performance convergence chromatography (UPC2) BEH column (100 mm × 3.0 mm, 1.7 μm particle size) and thiacloprid was eluted at 1.22 min in gradient mode with CO2/methanol as mobile phase. The 0.1% formic acid in methanol (v/v) was used as postcolumn compensation solution to improve sensitivity. The ABPR pressure, flow rate of mobile phase and flow rate of compensation pump were set at 1800 psi, 1.8 mL/min, and 0.1 mL/min, respectively. The average recoveries of thiacloprid in soil at four spiking levels (5, 10, 100, 1000 μg/kg) ranged between 78.8% and 107.1% with relative standard deviations (RSDs) lower than 12.2% and the limit of quantitation (LOQ) was 5 μg/kg. The proposed method can distinctly improve the analysis efficiency by 2–12 times and reduce the solvent consumption by 5%–95% compared with reported methods. It was applied to investigate the dissipation rates of thiacloprid in greenhouse vegetables and soil under different application modes. The half-lives of thiacloprid in cucumber and soil were 9.55–20.44 days and 3.74–9.14 days separately under different application modes, 10.60 days in tomato under foliar spraying. The residues in vegetables under root irrigation were all less than that under foliar spraying. The results could offer useful data for risk assessment of thiacloprid in agricultural production.





Comparative study of strong cation exchangers: Structure-related chromatographic performances

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B
Author(s): Vignesh Narasimhan Janakiraman, Marion Solé, Sophie Maria, Jérome Pezzini, Charlotte Cabanne, Xavier Santarelli
Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure, thereby allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we independently evaluated cation exchangers to facilitate media selection and investigated the relationship between surface modification and chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained high capacities even with high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions with minimal residence/contact time. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity, and could contribute to cost reduction.In this work, we evaluated the dynamic binding capacities of various new ion exchange resins at different binding conductivities for different residence times, and observed that: Up to 7.5 mS/cm, Cellufine Max S-h and Max S-r have the highest dynamic binding capacity, which decreased with conductivity increasing beyond 10 mS/cm; From 10 mS/cm, Nuvia S, GiGaCap S and Eshmuno S exhibit better dynamic binding capacity; At higher conductivities, only Nuvia S shows a dynamic binding capacity of 100 mg/ml (at 12.5 mS/cm) and 80 mg/ml (at 15 mS/cm).

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B

Author(s): Vignesh Narasimhan Janakiraman, Marion Solé, Sophie Maria, Jérome Pezzini, Charlotte Cabanne, Xavier Santarelli

Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure, thereby allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we independently evaluated cation exchangers to facilitate media selection and investigated the relationship between surface modification and chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained high capacities even with high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions with minimal residence/contact time. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity, and could contribute to cost reduction. In this work, we evaluated the dynamic binding capacities of various new ion exchange resins at different binding conductivities for different residence times, and observed that: Up to 7.5 mS/cm, Cellufine Max S-h and Max S-r have the highest dynamic binding capacity, which decreased with conductivity increasing beyond 10 mS/cm; From 10 mS/cm, Nuvia S, GiGaCap S and Eshmuno S exhibit better dynamic binding capacity; At higher conductivities, only Nuvia S shows a dynamic binding capacity of 100 mg/ml (at 12.5 mS/cm) and 80 mg/ml (at 15 mS/cm).





Micellar electrokinetic chromatography with laser induced fluorescence detection shows increase of putrescine in erythrocytes of Parkinson’s disease patients

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B
Author(s): L. Betancourt, P. Rada, L. Hernandez, H. Araujo, G.A. Ceballos, L.E. Hernandez, P. Tucci, Z. Mari, M. De Pasquale, D.A. Paredes
A highly sensitive method was developed to measure putrescine by micellar electrokinetic chromatography with laser induced fluorescence detection with excellent linearity in the 1 nM to 3 μM range. The technique was tested on a drop of blood from Parkinson’s disease patients obtained by finger prick. The results showed a statistically significant increase of putrescine in the erythrocytes compared to controls and a non-significant increase in plasma. This high level of putrescine does not constitute by itself proof that putrescine and polyamines are directly related to Parkinson’s disease. However, the present results and several others addressed in the discussion suggest that these compounds might be causally involved in the pathophysiology of Parkinson’s disease. In addition, the analytical method reported here may help to find new biomarkers for many diseases including Parkinson’s disease.

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B

Author(s): L. Betancourt, P. Rada, L. Hernandez, H. Araujo, G.A. Ceballos, L.E. Hernandez, P. Tucci, Z. Mari, M. De Pasquale, D.A. Paredes

A highly sensitive method was developed to measure putrescine by micellar electrokinetic chromatography with laser induced fluorescence detection with excellent linearity in the 1 nM to 3 μM range. The technique was tested on a drop of blood from Parkinson's disease patients obtained by finger prick. The results showed a statistically significant increase of putrescine in the erythrocytes compared to controls and a non-significant increase in plasma. This high level of putrescine does not constitute by itself proof that putrescine and polyamines are directly related to Parkinson's disease. However, the present results and several others addressed in the discussion suggest that these compounds might be causally involved in the pathophysiology of Parkinson's disease. In addition, the analytical method reported here may help to find new biomarkers for many diseases including Parkinson's disease.





Development of an immunoaffinity column for the highly sensitive analysis of bisphenol A in 14 kinds of foodstuffs using ultra-high-performance liquid chromatography tandem mass spectrometry

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B
Author(s): Kai Yao, Kai Wen, Wenchong Shan, Sanlei Xie, Tao Peng, Jianyi Wang, Haiyang Jiang, Bing Shao
An immunoaffinity clean-up material based on a monoclonal antibody (mAb) has been prepared for concentrating and purifying bisphenol A (BPA) in 14 kinds of foodstuffs at trace level. Haptens and immunogen of bisphenol A have been synthesized and comprehensively characterized. An mAb towards BPA were prepared and cross-reactivities with 14 BPA analogues were below 5%. The prepared antibody was coupled to N-hydroxysuccinimide-activated Sepharose 4B to manufacture an immunoaffinity column (IAC), which was applied to purify BPA in 14 kinds of foodstuffs. The analyte was then detected by means of ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Under the optimized conditions, compared with two traditional SPE clean-up methods, the IAC showed better selectivity (matrix effect <16.8%) and higher sensitivity. The limits of detection for BPA in 14 kinds of foodstuffs ranged from 0.001 μg L−1 to 0.01 μg kg−1, and the limits of quantification were in the range from 0.003 μg L−1 to 0.04 μg kg−1. The recoveries of BPA from spiked samples ranged from 82.0% to 104.9%, with RSDs below 13.8%. Besides, the IAC exhibited good reusability, with 40% column capacity remaining and no significant loss of recovery after 25 application cycles in real sample detection. These results demonstrated that the developed IAC-UPLC-MS/MS approach has wide applicability for purifying and detecting BPA in various foodstuffs.

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B

Author(s): Kai Yao, Kai Wen, Wenchong Shan, Sanlei Xie, Tao Peng, Jianyi Wang, Haiyang Jiang, Bing Shao

An immunoaffinity clean-up material based on a monoclonal antibody (mAb) has been prepared for concentrating and purifying bisphenol A (BPA) in 14 kinds of foodstuffs at trace level. Haptens and immunogen of bisphenol A have been synthesized and comprehensively characterized. An mAb towards BPA were prepared and cross-reactivities with 14 BPA analogues were below 5%. The prepared antibody was coupled to N-hydroxysuccinimide-activated Sepharose 4B to manufacture an immunoaffinity column (IAC), which was applied to purify BPA in 14 kinds of foodstuffs. The analyte was then detected by means of ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Under the optimized conditions, compared with two traditional SPE clean-up methods, the IAC showed better selectivity (matrix effect <16.8%) and higher sensitivity. The limits of detection for BPA in 14 kinds of foodstuffs ranged from 0.001 μg L−1 to 0.01 μg kg−1, and the limits of quantification were in the range from 0.003 μg L−1 to 0.04 μg kg−1. The recoveries of BPA from spiked samples ranged from 82.0% to 104.9%, with RSDs below 13.8%. Besides, the IAC exhibited good reusability, with 40% column capacity remaining and no significant loss of recovery after 25 application cycles in real sample detection. These results demonstrated that the developed IAC-UPLC-MS/MS approach has wide applicability for purifying and detecting BPA in various foodstuffs.





Identification of five Mitragyna alkaloids in urine using liquid chromatography-quadrupole/time of flight mass spectrometry

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B
Author(s): Stephanie Basiliere, Kelsie Bryand, Sarah Kerrigan
Mitragyna speciosa (Kratom) is a psychoactive plant that has recently emerged as a recreational drug. Mitragyna alkaloids are not within the scope of traditional forensic toxicology screening methods, which may contribute to under-reporting. Solid phase extraction (SPE) and liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS) were used to identify five alkaloids in urine. Target analytes included the two known psychoactive compounds, mitragynine and 7-hydroxymitragynine, in addition to speciociliatine, speciogynine, and paynantheine. Two deuterated internal standards (mitragynine-D3 and 7-hydroxymitragynine-D3) were employed. Using traditional reversed phase chromatography all compounds and isomers were separated in 10 min. The procedure was validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation. Extraction efficiencies were 63–96% and limits of quantitation were 0.5–1 ng/mL. Precision, bias and matrix effect were all within acceptable thresholds, with the exception of 7-hydroxymitragynine, which is notably unstable and unsuitable for quantitative analysis. In this paper we present a simultaneous quantitative analytical method for mitragynine, speciociliatine, speciogynine and paynantheine, and a qualitative assay for 7-hydroxymitragynine in urine using high resolution mass spectrometry (HRMS).

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B

Author(s): Stephanie Basiliere, Kelsie Bryand, Sarah Kerrigan

Mitragyna speciosa (Kratom) is a psychoactive plant that has recently emerged as a recreational drug. Mitragyna alkaloids are not within the scope of traditional forensic toxicology screening methods, which may contribute to under-reporting. Solid phase extraction (SPE) and liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS) were used to identify five alkaloids in urine. Target analytes included the two known psychoactive compounds, mitragynine and 7-hydroxymitragynine, in addition to speciociliatine, speciogynine, and paynantheine. Two deuterated internal standards (mitragynine-D3 and 7-hydroxymitragynine-D3) were employed. Using traditional reversed phase chromatography all compounds and isomers were separated in 10 min. The procedure was validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation. Extraction efficiencies were 63–96% and limits of quantitation were 0.5–1 ng/mL. Precision, bias and matrix effect were all within acceptable thresholds, with the exception of 7-hydroxymitragynine, which is notably unstable and unsuitable for quantitative analysis. In this paper we present a simultaneous quantitative analytical method for mitragynine, speciociliatine, speciogynine and paynantheine, and a qualitative assay for 7-hydroxymitragynine in urine using high resolution mass spectrometry (HRMS).





In-vivo metabolite profiling of chicoric acid in rat plasma, urine and feces after oral administration using liquid chromatography quadrupole time of flight mass spectrometry

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B
Author(s): Zhijun Diao, Jing Li, Qian Liu, Yutang Wang
Chicoric acid (CA) is an active derivative of caffeic acid, which is naturally present in many medicinal plants and vegetables. In the present study, the metabolic profile of CA was determined in rat plasma, urine and feces and was subsequently used to propose the metabolic pathways of CA. CA (100 mg/kg) was orally administered to rats by gastric intubation. Then, the plasma, urine and feces samples were collected and treated with methanol and acetonitrile (1:1, V/V) to precipitate the proteins. The pretreated samples were separated by ultra performance liquid chromatography (UPLC) equipped with an HSS T3 column (2.1 mm × 100 mm I.D., 1.7 μm) and with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) as the detection method. A total of nineteen metabolites were detected and identified based on the characteristics of their deprotonated ions in the plasma, urine and feces samples. The results revealed that the metabolism of CA followed a number of known in-vivo mammalian biotransformation pathways including hydrolysis, reduction, methylation, sulfation, glucuronidation, acetylation, isomerization and deoxygenation.

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B

Author(s): Zhijun Diao, Jing Li, Qian Liu, Yutang Wang

Chicoric acid (CA) is an active derivative of caffeic acid, which is naturally present in many medicinal plants and vegetables. In the present study, the metabolic profile of CA was determined in rat plasma, urine and feces and was subsequently used to propose the metabolic pathways of CA. CA (100 mg/kg) was orally administered to rats by gastric intubation. Then, the plasma, urine and feces samples were collected and treated with methanol and acetonitrile (1:1, V/V) to precipitate the proteins. The pretreated samples were separated by ultra performance liquid chromatography (UPLC) equipped with an HSS T3 column (2.1 mm × 100 mm I.D., 1.7 μm) and with quadrupole time-of-flight mass spectrometry (Q-TOF-MS) as the detection method. A total of nineteen metabolites were detected and identified based on the characteristics of their deprotonated ions in the plasma, urine and feces samples. The results revealed that the metabolism of CA followed a number of known in-vivo mammalian biotransformation pathways including hydrolysis, reduction, methylation, sulfation, glucuronidation, acetylation, isomerization and deoxygenation.





LC-MS determination of steroidal glycosides from Dioscorea deltoidea Wall cell suspension culture: Optimization of pre-LC-MS procedure parameters by Latin Square design

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B
Author(s): Boris Sarvin, Elizaveta Fedorova, Oleg Shpigun, Maria Titova, Mikhail Nikitin, Dmitry Kochkin, Rodin Igor, Andrey Stavrianidi
In this paper, the ultrasound assisted extraction method for isolation of steroidal glycosides from D. deltoidea plant cell suspension culture with a subsequent HPLC-MS determination was developed. After the organic solvent was selected via a two-factor experiment the optimization of extraction parameters was carried out using Latin Square 4 × 4 experimental design for the following parameters: extraction time, organic solvent concentration in extraction solution and the ratio of solvent to sample. It was also shown that the ultrasound-assisted extraction method is not suitable for isolation of steroidal glycosides from the D. deltoidea plant material. The results were confirmed using the multiple successive extraction method and refluxing extraction. Optimal conditions for the extraction of steroidal glycosides by the ultrasound-assisted extraction method were: extraction time, 60 min; acetonitrile (water) concentration in extraction solution, 50% (50%); the ratio of solvent to sample, 400 mL/g. Also, the developed method was tested on D. deltoidea cell suspension cultures of different terms and conditions of cultivation. The completeness of the extraction was confirmed using the multiple successive extraction method.

Publication date: Available online 13 February 2018
Source:Journal of Chromatography B

Author(s): Boris Sarvin, Elizaveta Fedorova, Oleg Shpigun, Maria Titova, Mikhail Nikitin, Dmitry Kochkin, Rodin Igor, Andrey Stavrianidi

In this paper, the ultrasound assisted extraction method for isolation of steroidal glycosides from D. deltoidea plant cell suspension culture with a subsequent HPLC-MS determination was developed. After the organic solvent was selected via a two-factor experiment the optimization of extraction parameters was carried out using Latin Square 4 × 4 experimental design for the following parameters: extraction time, organic solvent concentration in extraction solution and the ratio of solvent to sample. It was also shown that the ultrasound-assisted extraction method is not suitable for isolation of steroidal glycosides from the D. deltoidea plant material. The results were confirmed using the multiple successive extraction method and refluxing extraction. Optimal conditions for the extraction of steroidal glycosides by the ultrasound-assisted extraction method were: extraction time, 60 min; acetonitrile (water) concentration in extraction solution, 50% (50%); the ratio of solvent to sample, 400 mL/g. Also, the developed method was tested on D. deltoidea cell suspension cultures of different terms and conditions of cultivation. The completeness of the extraction was confirmed using the multiple successive extraction method.





Metabolomic profiling of Campylobacter jejuni with resistance gene ermB by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry and tandem quadrupole mass spectrometry

Publication date: 15 March 2018
Source:Journal of Chromatography B, Volume 1079
Author(s): Qin Fu, Dejun Liu, Yingyu Wang, Xiaowei Li, Lina Wang, Fugen Yu, Jianzhong Shen, Xi Xia
The metabolome changes of Campylobacter jejuni with resistant gene ermB remain unclear. Here, we described an untargeted metabolomic workflow based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to investigate the metabolites perturbations mediated by ermB in C. jejuni. After optimization of extractants and chromatographic conditions, the combination of 100% methanol extraction with a 12 min gradient by C18 column was adopted for untargeted metabolomic profiling in reversed phase separation. Meanwhile, 60% methanol extraction followed by a 14 min separation using hydrophilic interaction chromatography column was suitable to complementally expand the metabolite coverage of C. jejuni. Multivariate statistical analysis was performed by means of orthogonal projection to latent structures−discriminant analysis to select metabolic features. The selected features were further confirmed by ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry. A total of thirty-six differential metabolites between the susceptible strain (C. jejuni NCTC 11168) and resistant stain (C. jejuni NCTC 11168 with ermB) were identified. These pivotal metabolites were primarily participated in biological processes as cell signaling, membrane integrity/stability, fuel and energy source/storage and nutrient. The biofilm formation capability of resistant strain was inferior to that of susceptible strain, confirming the influence of ermB on membrane integrity/stability of C. jejuni. Our findings revealed important metabolic regulatory pathways associated with resistant C. jejuni with ermB.

Publication date: 15 March 2018
Source:Journal of Chromatography B, Volume 1079

Author(s): Qin Fu, Dejun Liu, Yingyu Wang, Xiaowei Li, Lina Wang, Fugen Yu, Jianzhong Shen, Xi Xia

The metabolome changes of Campylobacter jejuni with resistant gene ermB remain unclear. Here, we described an untargeted metabolomic workflow based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to investigate the metabolites perturbations mediated by ermB in C. jejuni. After optimization of extractants and chromatographic conditions, the combination of 100% methanol extraction with a 12 min gradient by C18 column was adopted for untargeted metabolomic profiling in reversed phase separation. Meanwhile, 60% methanol extraction followed by a 14 min separation using hydrophilic interaction chromatography column was suitable to complementally expand the metabolite coverage of C. jejuni. Multivariate statistical analysis was performed by means of orthogonal projection to latent structures−discriminant analysis to select metabolic features. The selected features were further confirmed by ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry. A total of thirty-six differential metabolites between the susceptible strain (C. jejuni NCTC 11168) and resistant stain (C. jejuni NCTC 11168 with ermB) were identified. These pivotal metabolites were primarily participated in biological processes as cell signaling, membrane integrity/stability, fuel and energy source/storage and nutrient. The biofilm formation capability of resistant strain was inferior to that of susceptible strain, confirming the influence of ermB on membrane integrity/stability of C. jejuni. Our findings revealed important metabolic regulatory pathways associated with resistant C. jejuni with ermB.





LC-MS/MS method for the determination of the prodrug aripiprazole lauroxil and its three metabolites in plasma and its application to in vitro biotransformation and animal pharmacokinetic studies

Publication date: Available online 10 February 2018
Source:Journal of Chromatography B
Author(s): Yiying Sun, Xiaoyan Lu, Yunyun Gai, Chunjie Sha, Guangyi Leng, Xiucheng Yang, Wanhui Liu
A sensitive and selective LC-MS/MS method for determination of the prodrug aripiprazole lauroxil (AL) and the three metabolites (N-hydroxymethyl aripiprazole [NHA], aripiprazole [ARP], and dehydro aripiprazole [DHA]) in plasma was developed using ARP-d8 as an internal standard. The analytes were determined on an AB Sciex Triple Quad™ 4500 system using positive ion electrospray ionization and selected multiple reaction monitoring mode. Solid phase extraction was applied for sample preparation for AL, ARP, and DHA, and protein precipitation for NHA. Chromatographic separation was performed on an Agilent Eclipse XDB-CN column (100 × 2.1 mm i.d., 3.5 μm) using the mobile phase of water and acetonitrile (25:75, v/v) containing 0.1% formic acid with a flow rate of 0.5 mL/min. The linear ranges for AL, NHA, ARP, and DHA were 0.5–50 ng/mL, 1.0–50 ng/mL, 0.5–50 ng/mL, and 0.05–5.0 ng/mL, respectively. The average recovery in the plasma sample was stable and reproducible. The precision and accuracy of the intra- and inter-run were within assay variability criteria limits. The developed method was suitable for in vitro biotransformation studies in plasma and animal pharmacokinetic studies after intramuscular injection of AL formulations.

Publication date: Available online 10 February 2018
Source:Journal of Chromatography B

Author(s): Yiying Sun, Xiaoyan Lu, Yunyun Gai, Chunjie Sha, Guangyi Leng, Xiucheng Yang, Wanhui Liu

A sensitive and selective LC-MS/MS method for determination of the prodrug aripiprazole lauroxil (AL) and the three metabolites (N-hydroxymethyl aripiprazole [NHA], aripiprazole [ARP], and dehydro aripiprazole [DHA]) in plasma was developed using ARP-d 8 as an internal standard. The analytes were determined on an AB Sciex Triple Quad™ 4500 system using positive ion electrospray ionization and selected multiple reaction monitoring mode. Solid phase extraction was applied for sample preparation for AL, ARP, and DHA, and protein precipitation for NHA. Chromatographic separation was performed on an Agilent Eclipse XDB-CN column (100 × 2.1 mm i.d., 3.5 μm) using the mobile phase of water and acetonitrile (25:75, v/v) containing 0.1% formic acid with a flow rate of 0.5 mL/min. The linear ranges for AL, NHA, ARP, and DHA were 0.5–50 ng/mL, 1.0–50 ng/mL, 0.5–50 ng/mL, and 0.05–5.0 ng/mL, respectively. The average recovery in the plasma sample was stable and reproducible. The precision and accuracy of the intra- and inter-run were within assay variability criteria limits. The developed method was suitable for in vitro biotransformation studies in plasma and animal pharmacokinetic studies after intramuscular injection of AL formulations.





Quantification of the next-generation oral anti-tumor drugs dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib and two metabolites in human plasma by liquid chromatography-tandem mass spectrometry

Publication date: Available online 8 February 2018
Source:Journal of Chromatography B
Author(s): Evelina Cardoso, Thomas Mercier, Anna Dorothea Wagner, Krisztian Homicsko, Olivier Michielin, Kim Ellefsen-Lavoie, Laurène Cagnon, Manuel Diezi, Thierry Buclin, Nicolas Widmer, Chantal Csajka, Laurent Decosterd
A sensitive and selective method of high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) has been developed for the simultaneous quantification of six anticancer protein kinase inhibitors, dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib, and two active metabolites (regorafenib-M2 and regorafenib-M5) in human plasma. Plasma protein precipitation with methanol enables the sample extraction of 100 μL aliquot of plasma. Analytes are detected by electrospray triple-stage quadrupole mass spectrometry and quantified using the calibration curves with stable isotope-labeled internal standards. The method was validated based on FDA recommendations, including assessment of extraction yield (74–104%), matrix effects, analytical recovery (94–104%) with low variability (<15%). The method is sensitive (lower limits of quantification within 1 to 200 ng/mL), accurate (intra- and inter-assay bias: −0.3% to +12.7%, and −3.2% to +6.3%, respectively) and precise (intra- and inter-assay CVs within 0.7–7.3% and 2.5–8.0%, respectively) over the clinically relevant concentration range (upper limits of quantification 500 to 100,000 ng/mL). This method is applied in our laboratory for both clinical research programs and routine therapeutic drug monitoring service of PKIs.

Publication date: Available online 8 February 2018
Source:Journal of Chromatography B

Author(s): Evelina Cardoso, Thomas Mercier, Anna Dorothea Wagner, Krisztian Homicsko, Olivier Michielin, Kim Ellefsen-Lavoie, Laurène Cagnon, Manuel Diezi, Thierry Buclin, Nicolas Widmer, Chantal Csajka, Laurent Decosterd

A sensitive and selective method of high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) has been developed for the simultaneous quantification of six anticancer protein kinase inhibitors, dabrafenib, trametinib, vemurafenib, cobimetinib, pazopanib, regorafenib, and two active metabolites (regorafenib-M2 and regorafenib-M5) in human plasma. Plasma protein precipitation with methanol enables the sample extraction of 100 μL aliquot of plasma. Analytes are detected by electrospray triple-stage quadrupole mass spectrometry and quantified using the calibration curves with stable isotope-labeled internal standards. The method was validated based on FDA recommendations, including assessment of extraction yield (74–104%), matrix effects, analytical recovery (94–104%) with low variability (<15%). The method is sensitive (lower limits of quantification within 1 to 200 ng/mL), accurate (intra- and inter-assay bias: −0.3% to +12.7%, and −3.2% to +6.3%, respectively) and precise (intra- and inter-assay CVs within 0.7–7.3% and 2.5–8.0%, respectively) over the clinically relevant concentration range (upper limits of quantification 500 to 100,000 ng/mL). This method is applied in our laboratory for both clinical research programs and routine therapeutic drug monitoring service of PKIs.





Supercritical fluid chromatography-photodiode array detection-electrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances

Publication date: Available online 8 February 2018
Source:Journal of Chromatography B
Author(s): Gregory F. Pirrone, Rose M. Mathew, Alexey A. Makarov, Frank Bernardoni, Artis Klapars, Robert Hartman, John Limanto, Erik L. Regalado
Impurity fate and purge studies are critical in order to establish an effective impurity control strategy for approval of the commercial filing application of new medicines. Reversed phase liquid chromatography-diode array-mass spectrometry (RPLC-DAD-MS) has traditionally been the preferred tool for impurity fate mapping. However, separation of some reaction mixtures by LC can be very problematic requiring combination LC-UV for area % analysis and a different LC-MS method for peak identification. In addition, some synthetic intermediates might be chemically susceptible to the aqueous conditions used in RPLC separations. In this study, the use of supercritical fluid chromatography-photodiode array-electrospray ionization mass spectrometry (SFC-PDA-ESIMS) for fate and purge of two specified impurities in the 1-uridine starting material from the synthesis of a bis-piv 2′keto-uridine, an intermediate in the synthesis of uprifosbuvir, a treatment under investigation for chronic hepatitis C infection. Readily available SFC instrumentation with a Chiralpak IC column (4.6 × 150 mm, 3 μm) and ethanol: carbon dioxide based mobile phase eluent enabled the separation of closely related components from complex reaction mixtures where RLPC failed to deliver optimal chromatographic performance. These results illustrate how SFC combined with PDA and ESIMS detection can become a powerful tool for direct impurity fate mapping across multiple reaction steps.

Publication date: Available online 8 February 2018
Source:Journal of Chromatography B

Author(s): Gregory F. Pirrone, Rose M. Mathew, Alexey A. Makarov, Frank Bernardoni, Artis Klapars, Robert Hartman, John Limanto, Erik L. Regalado

Impurity fate and purge studies are critical in order to establish an effective impurity control strategy for approval of the commercial filing application of new medicines. Reversed phase liquid chromatography-diode array-mass spectrometry (RPLC-DAD-MS) has traditionally been the preferred tool for impurity fate mapping. However, separation of some reaction mixtures by LC can be very problematic requiring combination LC-UV for area % analysis and a different LC-MS method for peak identification. In addition, some synthetic intermediates might be chemically susceptible to the aqueous conditions used in RPLC separations. In this study, the use of supercritical fluid chromatography-photodiode array-electrospray ionization mass spectrometry (SFC-PDA-ESIMS) for fate and purge of two specified impurities in the 1-uridine starting material from the synthesis of a bis-piv 2′keto-uridine, an intermediate in the synthesis of uprifosbuvir, a treatment under investigation for chronic hepatitis C infection. Readily available SFC instrumentation with a Chiralpak IC column (4.6 × 150 mm, 3 μm) and ethanol: carbon dioxide based mobile phase eluent enabled the separation of closely related components from complex reaction mixtures where RLPC failed to deliver optimal chromatographic performance. These results illustrate how SFC combined with PDA and ESIMS detection can become a powerful tool for direct impurity fate mapping across multiple reaction steps.