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Simultaneous quantitation of azole antifungals, antibiotics, imatinib, and raltegravir in human plasma by two-dimensional high-performance liquid chromatography–tandem mass spectrometry
11 February 2013,
08:56:38
1 March 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 919–920
High-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) is a standard analytical technique for therapeutic drug monitoring (TDM). A rapid LC–MS/MS method was developed for simultaneous quantitation of 3 antifungals and one active metabolite (posaconazole, voriconazole, itraconazole, and hydroxy-itraconazole), 5 antibiotics (daptomycin, ciprofloxacin, oxacillin, levofloxacin, and rifampicin), an antineoplastic agent (imatinib), and an antiretroviral (raltegravir) in human plasma. Protein precipitation of 10μL of plasma with acetonitrile was used as a single-extraction procedure. After 2-dimensional LC, all drugs were quantified by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated per FDA recommendations including the study of extraction recovery (from 79.3% to 105.9%) and matrix effect via ion suppression/enhancement phenomenon. This method is precise (intra- and inter-assay coefficients of variation of 1.95–12.77%, 2.56–8.16% and 2.12–11.38% for low, medium and high levels of internal quality controls respectively) and accurate (intra- and inter-assay biases of 0.19–12.67%, 0.04 to −12.17% and 0.22–12.98% respectively). This method is an efficient tool for routine TDM and optimization of laboratory resource utilization.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 919–920
High-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) is a standard analytical technique for therapeutic drug monitoring (TDM). A rapid LC–MS/MS method was developed for simultaneous quantitation of 3 antifungals and one active metabolite (posaconazole, voriconazole, itraconazole, and hydroxy-itraconazole), 5 antibiotics (daptomycin, ciprofloxacin, oxacillin, levofloxacin, and rifampicin), an antineoplastic agent (imatinib), and an antiretroviral (raltegravir) in human plasma. Protein precipitation of 10μL of plasma with acetonitrile was used as a single-extraction procedure. After 2-dimensional LC, all drugs were quantified by electrospray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated per FDA recommendations including the study of extraction recovery (from 79.3% to 105.9%) and matrix effect via ion suppression/enhancement phenomenon. This method is precise (intra- and inter-assay coefficients of variation of 1.95–12.77%, 2.56–8.16% and 2.12–11.38% for low, medium and high levels of internal quality controls respectively) and accurate (intra- and inter-assay biases of 0.19–12.67%, 0.04 to −12.17% and 0.22–12.98% respectively). This method is an efficient tool for routine TDM and optimization of laboratory resource utilization.
Highlights
► Fast and simultaneous quantitation of different classes of drugs in human plasma. ► Simple sample preparation step by protein precipitation. ► LC–MS/MS analysis with online sample clean up. ► Efficient tool for optimization of laboratory resource utilization.Determination of gemcitabine and its metabolite in extracellular fluid of rat brain tumor by ultra performance liquid chromatography–tandem mass spectrometry using microdialysis sampling after intralesional chemotherapy
11 February 2013,
08:56:38
1 March 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 919–920
The cytotoxic agent Gemcitabine (2′,2′-difluoro-2′-deoxycytidine) has been proved to be effective in the treatment of malignant gliomas. A rapid, sensitive and specific ultra performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) assay using microdialysis sampling was developed and validated to quantify gemcitabine and its major metabolite 2′,2′-difluoro-2′-deoxyuridine (dFdU) in Sprague–Dawley rat bearing 9L glioma. Microdialysis probes were surgically implanted into the area of rat brain tumor in the striatal hemisphere, and artificial cerebrospinal fluid was used as a perfusion medium. The samples were analyzed directly by UPLC–MS/MS after the addition of 5-bromouracil as an internal standard (IS). Separation was achieved on Agilent SB-C18 (50mm×2.1mm I.D., 1.8μm) column at 40°C using an isocratic elution method with acetonitrile and 0.1% formic acid (4:96, v/v) at a flow rate of 0.2mL/min. Detection was performed using electrospray ionization in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 264.0→112.0 (gemcitabine), m/z 265.1→113.0 (dFdU) and m/z 190.9→173.8 (IS). The calibration curves of gemcitabine and dFdU were linear in the concentration range of 0.66–677.08ng/mL and 0.31–312.00ng/mL, respectively. The lower limit of quantification of gemcitabine and dFdU were 0.66ng/mL and 0.31ng/mL, respectively. The lower limit of detection of gemcitabine and dFdU were calculated to be 0.2ng/mL and 0.1ng/mL, respectively. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. The validated method was simple, precise and accurate, which was successfully employed to determinate the concentrations of gemcitabine and dFdU in the extracellular fluid of rat brain tumor.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 919–920
The cytotoxic agent Gemcitabine (2′,2′-difluoro-2′-deoxycytidine) has been proved to be effective in the treatment of malignant gliomas. A rapid, sensitive and specific ultra performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) assay using microdialysis sampling was developed and validated to quantify gemcitabine and its major metabolite 2′,2′-difluoro-2′-deoxyuridine (dFdU) in Sprague–Dawley rat bearing 9L glioma. Microdialysis probes were surgically implanted into the area of rat brain tumor in the striatal hemisphere, and artificial cerebrospinal fluid was used as a perfusion medium. The samples were analyzed directly by UPLC–MS/MS after the addition of 5-bromouracil as an internal standard (IS). Separation was achieved on Agilent SB-C18 (50mm×2.1mm I.D., 1.8μm) column at 40°C using an isocratic elution method with acetonitrile and 0.1% formic acid (4:96, v/v) at a flow rate of 0.2mL/min. Detection was performed using electrospray ionization in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 264.0→112.0 (gemcitabine), m/z 265.1→113.0 (dFdU) and m/z 190.9→173.8 (IS). The calibration curves of gemcitabine and dFdU were linear in the concentration range of 0.66–677.08ng/mL and 0.31–312.00ng/mL, respectively. The lower limit of quantification of gemcitabine and dFdU were 0.66ng/mL and 0.31ng/mL, respectively. The lower limit of detection of gemcitabine and dFdU were calculated to be 0.2ng/mL and 0.1ng/mL, respectively. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. The validated method was simple, precise and accurate, which was successfully employed to determinate the concentrations of gemcitabine and dFdU in the extracellular fluid of rat brain tumor.
Highlights
► Gemcitabine is effective in the treatment of malignant gliomas. ► Gemcitabine's metabolite dFdU may contribute to gemcitabine's toxicity. ► A microdialysis technique with UPLC–MS/MS method was developed. ► All the validation data are within the required limits. ► The method was applied to determine gemcitabine and dFdU in rat tumor.
11 February 2013,
08:56:38
15 February 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
Clopidogrel has been applied in antiplatelet therapy since 1998 and is the thienopyridine with the largest clinical experience. By 2011, clopidogrel (Plavix®) was the second top-selling drug in the world. Following complete patent expiry in 2012/2013 its use is expected to grow even further from generics entering the market. Prefaced by a brief description of clopidogrel metabolism, this review analyzes analytical methods addressing the quantification of clopidogrel and its metabolites in biological samples. Techniques that have been applied to analyze human plasma or serum are predominantly LC–MS and LC–MS/MS. The lowest level of clopidogrel quantification that has been achieved is 5pg/mL, the shortest runtime is 1.5min and almost 100% recovery has been reported using solid-phase extraction for sample preparation.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
Clopidogrel has been applied in antiplatelet therapy since 1998 and is the thienopyridine with the largest clinical experience. By 2011, clopidogrel (Plavix®) was the second top-selling drug in the world. Following complete patent expiry in 2012/2013 its use is expected to grow even further from generics entering the market. Prefaced by a brief description of clopidogrel metabolism, this review analyzes analytical methods addressing the quantification of clopidogrel and its metabolites in biological samples. Techniques that have been applied to analyze human plasma or serum are predominantly LC–MS and LC–MS/MS. The lowest level of clopidogrel quantification that has been achieved is 5pg/mL, the shortest runtime is 1.5min and almost 100% recovery has been reported using solid-phase extraction for sample preparation.
Graphical abstract
Highlights
► Overview of analytical techniques for the quantification of clopidogrel and clopidogrel carboxylic acid in biological samples. ► New developments addressing the determination of the active clopidogrel metabolite. ► Thorough comparison of validation data obtained by the reviewed literature reports.Development of ultrasound-assisted emulsification microextraction for determination of thiocynate ion in human urine and saliva samples
11 February 2013,
08:56:38
15 February 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
Ultrasound-assisted emulsification microextraction (USAE-ME) procedure coupled with UV–vis spectrophotometric measurement has been developed for determination of thiocyanate ion (SCN−) in water and biological fluids samples. The method is based on protonation of SCN− ions in acidic medium and extraction of thiocyanic acid into fine droplets of chloroform as an extraction solvent contains rhodamine B (RhB). The RhB was protonated in presence of thiocynanic acid to form highly colored ion-pair complex of [thiocynate][RhBH+] in chloroform, which used for subsequent spectrophotometric determination of SCN− ions. Experimental parameters for both spectrophotometric reaction and USAE-ME procedure have been optimized. Under optimized conditions the calibration curve for SCN− showed good linearity in the range of 38.0–870.0ngmL−1 (R 2 =0.9967). The limit of detection (S/N=3) and preconcentration factor were 5.0ngmL−1 and 40, respectively. Relative standard deviation for determination of 200ngmL−1 of SCN− was 2.8% (n =5). The proposed method has been successfully applied for determination of SCN− ion in tap water, mineral bottled water and human saliva and urine samples with an average recovery of 99.2%.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
Ultrasound-assisted emulsification microextraction (USAE-ME) procedure coupled with UV–vis spectrophotometric measurement has been developed for determination of thiocyanate ion (SCN−) in water and biological fluids samples. The method is based on protonation of SCN− ions in acidic medium and extraction of thiocyanic acid into fine droplets of chloroform as an extraction solvent contains rhodamine B (RhB). The RhB was protonated in presence of thiocynanic acid to form highly colored ion-pair complex of [thiocynate][RhBH+] in chloroform, which used for subsequent spectrophotometric determination of SCN− ions. Experimental parameters for both spectrophotometric reaction and USAE-ME procedure have been optimized. Under optimized conditions the calibration curve for SCN− showed good linearity in the range of 38.0–870.0ngmL−1 (R 2 =0.9967). The limit of detection (S/N=3) and preconcentration factor were 5.0ngmL−1 and 40, respectively. Relative standard deviation for determination of 200ngmL−1 of SCN− was 2.8% (n =5). The proposed method has been successfully applied for determination of SCN− ion in tap water, mineral bottled water and human saliva and urine samples with an average recovery of 99.2%.
Highlights
► For first time, USAE-ME coupled with UV–vis spectrophotometry for determination of an inorganic spicies (thiocyanate ion). ► The method was developed for analysis of drinking mineral water, urine and saliva samples. ► High enrichment factor and ease of operation are the main advantages of proposed method. ► Hyphenation of USAE-ME with ordinary UV–vis spectrophotometry improved the sensitivity significantly.High-performance liquid chromatography using pressurized liquid extraction for the determination of seven tetracyclines in egg, fish and shrimp
11 February 2013,
08:56:38
15 February 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A simple and especially rapid method, pressurized liquid extraction, has been developed and applied to the quantitative determination of oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, demeclocycline and doxycycline in egg, fish and shrimp. The procedure consisted of a trichloracetic acid/methanol extraction conducted at elevated temperature (60°C) and pressure (65bar), without further clean-up, the extraction solution was concentrated and finally for high-performance liquid chromatography analysis. The limits of detection were 5.0–10.0μg/kg and the limits of quantification were 10.0–15.0μg/kg for tetracyclines in egg, fish and shrimp using UV detection. The analytical limits CCα and CCβ were also calculated. The recoveries of tetracyclines spiked at levels of 15–300μg/kg, averaged 75.6–103.5% with the relative standard deviation values less than 11%. The optimized procedure has been successfully applied to real samples in our laboratories. It demonstrated that the new method was robust and useful for monitoring and quantification of 7 tetracycline residues in food of animal origin.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A simple and especially rapid method, pressurized liquid extraction, has been developed and applied to the quantitative determination of oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, demeclocycline and doxycycline in egg, fish and shrimp. The procedure consisted of a trichloracetic acid/methanol extraction conducted at elevated temperature (60°C) and pressure (65bar), without further clean-up, the extraction solution was concentrated and finally for high-performance liquid chromatography analysis. The limits of detection were 5.0–10.0μg/kg and the limits of quantification were 10.0–15.0μg/kg for tetracyclines in egg, fish and shrimp using UV detection. The analytical limits CCα and CCβ were also calculated. The recoveries of tetracyclines spiked at levels of 15–300μg/kg, averaged 75.6–103.5% with the relative standard deviation values less than 11%. The optimized procedure has been successfully applied to real samples in our laboratories. It demonstrated that the new method was robust and useful for monitoring and quantification of 7 tetracycline residues in food of animal origin.
Highlights
► Pressurized liquid extraction has been developed to the HPLC quantitative determination of seven tetracyclines in egg, fish and shrimp. ► The extraction procedure is simple and rapid. ► The optimized procedure has been successfully applied to real samples. ► The method is robust and useful for monitoring and quantification of tetracycline residues.Development of a sensitive and selective LC–MS/MS method for the determination of urea in human epithelial lining fluid
11 February 2013,
08:56:38
15 February 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A sensitive, selective, and quantitative method for the determination of urea has been developed and validated in human epithelial lining fluid (ELF; the supernatant from bronchoalveolar lavage). The method employs a simple derivatization of urea with camphanic chloride to improve the chromatographic retention and separation. The derivatization was performed after drying an aliquot of ELF (20μL) without prior sample clean-up. Ultra High Performance Liquid Chromatography (UHPLC) on a HSS-T3 stationary phase column with 1.8μm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration range of 8.78–103.78μg/mL, however the dynamic range can be further lowered if needed. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support analysis of urea in ELF samples. In addition, the relatively small sample volume (20μL) and a run time of 1.5min facilitate automation and allow for high-throughput analysis. This derivatization method was compared to a commercially available colorimetric assay kit, and it was used in a preclinical non-GLP mouse study where urea measurements were used as marker of bronchoalveolar lavage fluid dilution.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A sensitive, selective, and quantitative method for the determination of urea has been developed and validated in human epithelial lining fluid (ELF; the supernatant from bronchoalveolar lavage). The method employs a simple derivatization of urea with camphanic chloride to improve the chromatographic retention and separation. The derivatization was performed after drying an aliquot of ELF (20μL) without prior sample clean-up. Ultra High Performance Liquid Chromatography (UHPLC) on a HSS-T3 stationary phase column with 1.8μm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration range of 8.78–103.78μg/mL, however the dynamic range can be further lowered if needed. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support analysis of urea in ELF samples. In addition, the relatively small sample volume (20μL) and a run time of 1.5min facilitate automation and allow for high-throughput analysis. This derivatization method was compared to a commercially available colorimetric assay kit, and it was used in a preclinical non-GLP mouse study where urea measurements were used as marker of bronchoalveolar lavage fluid dilution.
Highlights
► Derivatization with improved chromatographic retention and separation. ► The derivatization was performed without prior sample clean-up. ► The method was validated over the concentration range of 8.78–103.78μg/mL. ► This method was compared to a commercially available colorimetric assay kit. ► The method can also be adapted for determination of urea in plasma samples.Purification of recombinant EGFP by fusion with L2 (252–273) from ribosomal protein L2 using magnetic particles
11 February 2013,
08:56:38
15 February 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A basic polypeptide L2 (252–273) derived from Escherichia coli ribosomal protein L2 was used as a purification tag. In order to develop faster, less expensive methods for expression and purification of proteins, the L2 (252–273)-small ubiquitin like modifier (SUMO) fusion expression system was constructed. We comparatively analyzed the adsorption properties of the deleted protein of L2 (L2 (252–273)) on diatomite and superparamagnetic carboxymethyl chitosan nanoparticles. The time required to reach adsorption equilibrium of L2 (252–273) fusion protein on diatomite was shorter than that of L2 (252–273) fusion protein on magnetic particles. The maximum adsorption capacity of L2 (252–273) fusion protein on magnetic particles was about 5 times larger than that of L2 (252–273) fusion protein on diatomite. SUMO was introduced as a specific protease cleavage site between the target protein and the purification tags. The enhanced green fluorescent protein (EGFP) as a model protein was fused with the L2 (252–273)-SUMO fusion protein and purified by a simple method which involves the electrostatic adsorption of L2 (252–273) fusion proteins on superparamagnetic carboxymethyl chitosan nanoparticles and the L2 (252–273)-SUMO fusion partner was removed based on the robust cleavage by the poly lysine tagged SUMO protease. The high purity of tag-free EGFP (>93%) was obtained. Our results preliminary proved that the system was an effective fusion expression system for the production of recombinant proteins in E. coli.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A basic polypeptide L2 (252–273) derived from Escherichia coli ribosomal protein L2 was used as a purification tag. In order to develop faster, less expensive methods for expression and purification of proteins, the L2 (252–273)-small ubiquitin like modifier (SUMO) fusion expression system was constructed. We comparatively analyzed the adsorption properties of the deleted protein of L2 (L2 (252–273)) on diatomite and superparamagnetic carboxymethyl chitosan nanoparticles. The time required to reach adsorption equilibrium of L2 (252–273) fusion protein on diatomite was shorter than that of L2 (252–273) fusion protein on magnetic particles. The maximum adsorption capacity of L2 (252–273) fusion protein on magnetic particles was about 5 times larger than that of L2 (252–273) fusion protein on diatomite. SUMO was introduced as a specific protease cleavage site between the target protein and the purification tags. The enhanced green fluorescent protein (EGFP) as a model protein was fused with the L2 (252–273)-SUMO fusion protein and purified by a simple method which involves the electrostatic adsorption of L2 (252–273) fusion proteins on superparamagnetic carboxymethyl chitosan nanoparticles and the L2 (252–273)-SUMO fusion partner was removed based on the robust cleavage by the poly lysine tagged SUMO protease. The high purity of tag-free EGFP (>93%) was obtained. Our results preliminary proved that the system was an effective fusion expression system for the production of recombinant proteins in E. coli.
Highlights
► The L2 (252–273) from E. coli ribosomal protein L2 was used as a purification tag. ► Magnetic nanoparticles had a higher adsorption loading than the other adsorbents. ► Tag-free target protein purification by L2 (252–273)-SUMO fusion technology. ► The tag-free recombinant EGFP with a purity of greater than 93% was obtained.Simultaneous determination of amlodipine and atorvastatin with its metabolites; ortho and para hydroxy atorvastatin; in human plasma by LC–MS/MS
11 February 2013,
08:56:38
15 February 2013
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A simple liquid chromatography/ion trap mass spectrometry method for the quantification of amlodipine and atorvastatin with its metabolites, ortho and para hydroxy atorvastatin, simultaneously in human plasma was developed. Analytes with internal standard were extracted by protein direct precipitation with acetonitrile. Adequate chromatographic separation was achieved using Phenomenex Synergi 4u polar-RP 80A (150mm×4.6mm, 4μm) column in the isocratic elution mode and the eluent was water/methanol (14:86%, v/v) adjusted by trichloroacetic acid to pH 3.2 which was delivered isocratically at constant flow rate of 0.50mL/min. Standard solutions for the analytes were prepared using amlodipine besylate, atorvastatin calcium, ortho-hydroxy atorvastatin dihydrate monosodium salt, para-hydroxy atorvastatin disodium salt, and pravastatin sodium as an internal standard. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to USFDA guideline. Standard calibration levels were prepared by pooled human plasma to attain final dynamic range of 0.2–20.0ng/mL for amlodipine, 1.5–150ng/mL for atorvastatin, 1.0–100.0ng/mL for ortho-hydroxy atorvastatin and 0.2–20.0ng/mL for para-hydroxy atorvastatin. Clinical bioequivalence study was successfully investigated by the application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products 10mg amlodipine/80mg atorvastatin in a single dose. In this study, 29 healthy volunteers were participated in randomized, two periods, double blend, open label cross over design. Pharmacokinetic parameters of C max, AUC0–t and AUC0–∞ were calculated to compare a test product with CADUET® reference product.
Publication year: 2013
Source:Journal of Chromatography B, Volumes 917–918
A simple liquid chromatography/ion trap mass spectrometry method for the quantification of amlodipine and atorvastatin with its metabolites, ortho and para hydroxy atorvastatin, simultaneously in human plasma was developed. Analytes with internal standard were extracted by protein direct precipitation with acetonitrile. Adequate chromatographic separation was achieved using Phenomenex Synergi 4u polar-RP 80A (150mm×4.6mm, 4μm) column in the isocratic elution mode and the eluent was water/methanol (14:86%, v/v) adjusted by trichloroacetic acid to pH 3.2 which was delivered isocratically at constant flow rate of 0.50mL/min. Standard solutions for the analytes were prepared using amlodipine besylate, atorvastatin calcium, ortho-hydroxy atorvastatin dihydrate monosodium salt, para-hydroxy atorvastatin disodium salt, and pravastatin sodium as an internal standard. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to USFDA guideline. Standard calibration levels were prepared by pooled human plasma to attain final dynamic range of 0.2–20.0ng/mL for amlodipine, 1.5–150ng/mL for atorvastatin, 1.0–100.0ng/mL for ortho-hydroxy atorvastatin and 0.2–20.0ng/mL for para-hydroxy atorvastatin. Clinical bioequivalence study was successfully investigated by the application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products 10mg amlodipine/80mg atorvastatin in a single dose. In this study, 29 healthy volunteers were participated in randomized, two periods, double blend, open label cross over design. Pharmacokinetic parameters of C max, AUC0–t and AUC0–∞ were calculated to compare a test product with CADUET® reference product.
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