Journal of Chromatography B (v.926, #C)
Editorial Board (i).
LC–MS/MS quantification of a neuropeptide fragment kisspeptin-10 (NSC 741805) and characterization of its decomposition product and pharmacokinetics in rats by Zhongfa Liu; Chen Ren; William Jones; Ping Chen; Stephanie B. Seminara; Yee-Ming Chan; Nicola F. Smith; Joseph M. Covey; Jeffrey Wang; Kenneth K. Chan (1-8).
► First LC–MS/MS method to measure kisspeptin-10 (Metastin) in rat plasma. ► The LLOQ is 0.5 ng/mL with CVs (<16%) and accuracy (85–115%). ► Capable of characterizing the pharmacokinetics of metastin up to 30 min in rats. ► A N-terminal tyrosine deleted kisspeptin-10 46NWDSFGLRF-NH2 54 metabolite characterized.The kisspeptins are critical regulators of mammalian reproduction. Kisspeptin-10 (45YNWNSFGLRF-NH2 54, kisspeptin-112–121 or metastin 45–54, NSC 741805), an active fragment of kisspeptin, has been shown to be a potent stimulator of gonadotropin-releasing hormone and secretion of luteinizing hormone in both rodents and primates. This shorter peptide fragment may have clinical utility potential and it is important to characterize its pharmacokinetic property. Recently, the pharmacokinetics of both kisspeptin-54 and kisspeptin-10 were characterized in humans using a radioimmunoassay (RIA), which measures only the immunoreactive kisspeptin (kisspeptin-IR). In this study, a highly sensitive and specific LC–MS/MS assay was developed to quantify kisspeptin-10 levels in rat plasma. The lower limit of quantitation (LLOQ) was 0.5 ng/mL, the within-day and between-day coefficient of variations (CVs) ranged from 5.2 to 15.4% and 1.3 to 14.2%, and the accuracy values ranged from 98 to 114% and 99 to 105%, respectively. With this method, stability studies demonstrated that kisspeptin-10 degraded rapidly with decomposition half-lives of 6.8 min, 2.9 min and 1.7 min at 4 °C, 25 °C, and 37 °C, respectively. The principal decomposition product was characterized as the N-terminal tyrosine deleted kisspeptin-10 46NWDSFGLRF-NH2 54. Pharmacokinetic study in rats showed that low ng/mL kisspeptin-10 was detected in the first few minutes, and eliminated rapidly and became undetectable 30 min after intravenous (i.v.) bolus administration of 1.0 mg/kg kisspeptin-10.
Keywords: Kisspeptin-10; LC–MS/MS; Pharmacokinetics; Metabolites;
Simultaneous determination of tramadol, O-desmethyltramadol and N-desmethyltramadol in human urine by gas chromatography–mass spectrometry by Abdel-Aziz Y. El-Sayed; Khaled M. Mohamed; Ahmed Y. Nasser; Jennifer Button; David W. Holt (9-15).
► Simultaneous determination of tramadol and its main metabolites using GC–MS was recommended. ► The presented extraction procedure offers a rapid way to isolate tramadol and its main metabolites from the urine matrix. ► The procedure was successfully applied for the analysis of tramadol and its main metabolites in urine samples. ► The procedure was easily incorporated into routine testing for a laboratory.Analytical procedures for the determination of tramadol (T), O-desmethyltramadol (ODT), and N-desmethyltramadol (NDT) in human urine have been developed and validated using gas chromatography–mass spectrometry (GC/MS). Sample preparation involved liquid–liquid extraction with methyl-tert-butyl ether (MTBE) and followed by back extraction with 0.1 M hydrochloric acid. Proadifen (SKF525A) was selected as internal standard (IS). Extraction efficiencies of T, ODT and NDT were 102.12, 101.30, and 98.21%, respectively. The calibration curves were linear (r 2 > 0.99) in the concentration range 10–1000 ng/mL for all compounds. Limits of quantification (LOQ) were 10, 10 and 20 ng/mL for T, ODT and NDT, respectively. Intra-assay precision was within 1.29–6.48% and inter-assay precision was within 1.28–6.84% for T, ODT and NDT. Intra-assay accuracy was within 91.79–106.89% for all analytes. This method detected urine concentrations of T, ODT and NDT in six healthy volunteers for 7 days after administration of 50 mg oral doses of tramadol.
Keywords: Tramadol; O-Desmethyltramadol; N-Desmethyltramadol; Urine; GC/MS; Validation;
Large scale purification of the SERCA inhibitor Thapsigargin from Thapsia garganica L. roots using centrifugal partition chromatography by Anthony Ollivier; Raphaël Grougnet; Xavier Cachet; Djamila Meriane; Janick Ardisson; Sabrina Boutefnouchet; Brigitte Deguin (16-20).
► Thapsigargin is a very useful tool to study calcium-dependent signaling pathways. ► Isolation of Thapsigargin from Thapsia garganica L. is highly hazardous. ► We developed a two steps procedure coupling accelerated solvent extraction to CPC. ► Thapsigargin amount in different specimens of Thapsia garganica L. was quantified. ► Highly pure Thapsigargin could be obtained using a fast and safe procedure.Thapsigargin (Tg) is a selective and irreversible inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-dependent pump at subnanomolecular concentrations. As such, it has become a powerful tool in the study of Ca2+ signaling pathway. Purification of Tg from Thapsia species requires repeated chromatographic steps with normal-phase alumina or silica and reverse phase chromatography. We thus developed an innovative procedure coupling high pressure automatized extraction with centrifugal partition chromatography allowing a fast and safe large-scale isolation of highly pure Tg, in two steps from Thapsia garganica L. roots. Comparison of influence of extraction procedures, storage conditions and harvesting areas on Tg content in different Algerian specimens of Thapsia garganica L. roots has been precised by mean of HPLC quantification procedure. Highest Tg content were found in the fresh material of the sample from Setif area.
Keywords: Thapsigargin; Thapsia garganica; Apiaceae; Centrifugal partition chromatography; Accelerated solvent extraction;
Reversed phase High Performance Liquid Chromatography used for the physicochemical and thermodynamic characterization of piceatannol/β-cyclodextrin complex by Hanane Messiad; Habiba Amira-Guebailia; Omar Houache (21-27).
► The first attempt to study the complexation of piceatannol with β-cyclodextrin. ► HPLC with UV–vis detection was used for characterization of complex in solution. ► Decreasing retention times with increasing β-cyclodextrin concentration confirmed the complex formation.Piceatannol is one of resveratrol derivatives having health promoting potential. However, its low water-solubility and bioavailability could limit its use in both food and pharmaceutical fields. The aim of this work is the study of piceatannol complexation by β-cyclodextrin (β-CD) in aqueous media. The complex formed could improve the bioavailability, the solubility and the stability of piceatannol. The method used was based on RP-HPLC in which, β-CD was added to methanol/water mixtures mobile phases. The apparent formation constant of piceatannol/β-cyclodextrin complex was determined. Within the concentration interval studied (0–4 mM), whenever the concentration of β-CD increased, retention time of piceatannol decreased; indicating an enhancement of solubility due to the formation of the piceatannol/β-CD complex. The formation constants (K F) of the piceatannol/β-CD complex varied significantly with both the methanol concentration in the water–methanol mixture and the temperature. Moreover, in all the physicochemical conditions tested, the stoichiometry of piceatannol/β-CD complex was 1:1. Finally, the thermodynamic parameters were determined: ΔG° = -13.123 kJ mol−1, ΔH° = −33. 265 kJ mol−1 and ΔS° = −67.559 J mol−1 K−1.
Keywords: Piceatannol; β-Cyclodextrin; Mobile phase; Solubility; Apparent formation constant; Thermodynamic parameters;
Comparative pharmacokinetics of timosaponin B-II and timosaponin A-III after oral administration of Zhimu–Baihe herb-pair, Zhimu extract, free timosaponin B-II and free timosaponin A-III to rats by Zhirui Liu; Xin Dong; Xuan Ding; Xiaofei Chen; Lei Lv; Yueyue Li; Yifeng Chai (28-35).
The aim of this study was to compare the pharmacokinetics of timosaponin B-II and timosaponin A-III in rat plasma after oral administration of Zhimu–Baihe herb-pair, Zhimu extract, free timosaponin B-II and free timosaponin A-III. After addition of internal standard (IS) ginsenoside Rh1, plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on a SHISEIDO CAPCELL PAK C18 column (100 mm × 3 mm i.d., 3.0 μm) with the mobile phase consisting of acetonitrile and 0.05% (v/v) formic acid by linear gradient elution. The detection was performed on an Agilent G1946D single quadrupole mass spectrometer with negative electrospray ionization (ESI) interface in select-ion-monitoring (SIM) mode. The following ions: m/z 919 for timosaponin B-II, m/z 739 for timosaponin A-III and m/z 683 for the IS were used for quantitative determination. Good linearity was achieved over the concentration ranged from 4.0–793.3 ng/mL to 3.9–781.3 ng/mL for the two saponins. The precision of the in vivo study was evaluated by intra- and inter-day assays and the percentages of relative standard deviation were all within 15%. The plasma concentrations of timosaponin B-II and timosaponin A-III in rats at designated time periods were successfully determined using this fully validated method, and statistically significant differences (p < 0.5) in pharmacokinetic parameters including AUC0−t , AUC0−∞ and MRT (mean residence time) were obtained. Compared to these pharmacokinetic parameters after oral administration of Zhimu extract and monomer solution, higher peak concentration (C max is higher), slower elimination (MRT is longer) and larger AUC values could be observed after giving Zhimu–Baihe herb-pair in our study. Therefore, this result not only elucidated the steady and long-lasting pharmacological properties but also revealed the practical value of the compatibility of herb-pair remedy.
Keywords: Timosaponin B-II; Timosaponin A-III; Liquid chromatography–mass spectrometry; Comparative pharmacokinetics;
A validated HPLC–MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: Application to a clinical pharmacokinetic study by Michael T. Furlong; Ishani Savant; Moucun Yuan; Laura Scott; William Mylott; Thomas Mariannino; Pathanjali Kadiyala; Vikram Roongta; Mark E. Arnold (36-41).
► Assay to quantify four chemically reactive thiol metabolites of clopidogrel. ► Stabilization of the thiol group by alkylation with 2-bromo-3′-methoxyacetophenone. ► Separation of the four derivatized diastereomeric metabolites by HPLC. ► Application of the assay to a clinical pharmacokinetic study.Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3′-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC–MS/MS assay was validated over concentration ranges of 0.125–125 ng/mL for metabolites H1–H3 and 0.101–101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at −20 °C and −70 °C, and following at least five freeze–thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study.
Keywords: Clopidogrel; Thiol metabolite; HPLC–MS/MS; Assay; Derivatization; Pharmacokinetics;
Determination of dapoxetine in rat plasma by ultra-performance liquid chromatography–tandem mass spectrometry by Tae Kon Kim; In Sook Kim; Seok Hyun Hong; Yun Kyoung Choi; Hohyun Kim; Hye Hyun Yoo (42-46).
In this study, we describe and validate a rapid and sensitive method for quantitation of dapoxetine in rat plasma by using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI/MS/MS). Plasma samples were prepared by protein precipitation with acetonitrile, and sildenafil was used as an internal standard (IS). The mobile phase consisted of 0.5% formic acid/acetonitrile (60:40, v/v); a C18 reversed-phase column (2.0 × 50 mm, 1.7 μm) was used for chromatographic separation. Multiple reaction monitoring (MRM) was used in the positive ion mode for mass spectrometric detection. The calibration curve for dapoxetine was linear (r 2 = 0.999) in the concentration range of 1–500 ng/mL. The intra- and inter-day precision was between 3.8% and 8.3%, and the intra- and inter-day accuracy was between 101.1% and 109.0%. Dapoxetine was found to be stable in various conditions with the recoveries >87.0% (RSD <7.2%). The method was found to be specific, precise, and accurate, and no matrix effect was observed. Our results suggest that this method can be successfully applied in pharmacokinetic studies of dapoxetine in rat plasma.
Keywords: Dapoxetine; UPLC–MS/MS; Rat plasma; Pharmacokinetic study;
Development and validation of a simple and sensitive method for quantification of levodopa and carbidopa in rat and monkey plasma using derivatization and UPLC–MS/MS by Venkatraman Junnotula; Hermes Licea-Perez (47-53).
A simple, selective, and sensitive quantitative method has been developed for the simultaneous determination of levodopa and carbidopa in rat and monkey plasma by protein precipitation using acetonitrile containing the derivatizing reagent, flourescamine. Derivatized products of levodopa and carbidopa were separated on a BEH C18 column (2.1 mm × 50 mm; 1.7 μm particle size) using ultra high performance liquid chromatography (UHPLC) without any further purification. Tandem mass spectrometry (MS/MS) was used for detection. The method was validated over the concentration range of 5–5000 ng/mL and 3–3000 ng/mL for levodopa and carbidopa, respectively in rat and monkey plasma. Due to the poor stability of the investigated analytes in biological matrices, a mixture of sodium metabisulfite and hydrazine dihydrochloride was used as a stabilizer. This method was successfully utilized to support pharmacokinetic studies in both species. The results from assay validations and incurred samples re-analysis show that the method is selective, sensitive and robust. To our knowledge, this is the first UHPLC–MS/MS based method that utilizes derivatization with fluorescamine and provides adequate sensitivity for both levodopa and carbidopa with 50 μL of sample and a run time of 3.5 min.
Keywords: Levodopa; Carbidopa; Fluorescamine; Sodium metabisulfite; Hydrazine dihydrochloride; Derivatization; Mass spectrometry; UHPLC; Rat plasma and monkey plasma;
Analysis of tacrolimus and creatinine from a single dried blood spot using liquid chromatography tandem mass spectrometry by Dennis R. Koop; Lisa A. Bleyle; Myrna Munar; Ganesh Cherala; Amira Al-Uzri (54-61).
► Methods developed to determine both tacrolimus and creatinine from a single dried blood. ► The method correlated well with current clinical laboratory tests. ► The method can be used to only monitor renal function if needed.Long term therapeutic drug monitoring and assessment of renal function are required in renal transplant recipients on immunosuppressant therapy such as tacrolimus. Dry blood spots (DBS) have been used successfully in the clinic for many years and offers a convenient, simple and non-invasive method for repeated blood tests. We developed and performed a preliminary validation of a method for the analysis of tacrolimus and creatinine from a single DBS using liquid chromatography-tandem mass spectrometric (LC–MS/MS). Tacrolimus and creatinine were extracted from a 6 mm punch with a mixture of methanol/acetonitrile containing ascomycin and deuterated creatinine as internal standards. A 10 μl aliquot of the extract was analyzed directly after dilution for creatinine with normal phase high performance liquid chromatography and multiple reaction monitoring. The remainder of the extract was processed and analyzed for tacrolimus. The lower limit of quantification for tacrolimus was 1 ng/ml with accuracy of 0.34% bias and precision (CV) of 11.1%. The precision ranged from 1.33% to 7.68% and accuracy from −4.44% to 11.6% bias for the intra- and inter-day analysis. The lower limit of quantification of creatinine was 0.01 mg/dL with precision of 7.94%. Accuracy was based on recovery of additional creatinine spiked into whole blood samples and ranged from −2.45% bias at 5 mg/dL to 3.75% bias at 0.5 mg/dL. Intra- and inter-day precision was from 3.48 to 4.11%. The assay was further validated with DBS prepared from pediatric renal transplant recipients. There was excellent correlation between the levels of tacrolimus and creatinine obtained from the clinical laboratory and the DBS method developed. After additional validation, this assay may have a significant impact on compliance with medication intake as well as potentially lowering the cost associated with intravenous blood draws in clinical laboratories.
Keywords: Creatinine; Tacrolimus; Therapeutic drug monitoring; Liquid chromatography tandem mass spectrometry (LC–MS/MS); Dried blood spot (DBS);
Ultrasound-assisted surfactant-enhanced emulsification microextraction with solidification of floating organic droplet followed by high performance liquid chromatography for the determination of strobilurin fungicides in fruit juice samples by Pei Liang; Guojiao Liu; Fang Wang; Wanting Wang (62-67).
► UASEME-SFOD with HPLC was developed for the determination of strobilurin fungicides. ► The enrichment factors of this method are from 95 to 135. ► The extraction recoveries are in the range of 82.6–97.5%. ► The limits of detection are 2–4 ng mL−1.A novel method, ultrasound-assisted surfactant-enhanced emulsification microextraction with solidification of floating organic droplet (UASEME-SFOD), has been developed for the extraction of four strobilurin fungicides (kresoxim-methyl, picoxystrobin, pyraclostrobin and trifloxystrobin) in fruit juices. In the UASEME-SFOD technique, Tween 80 was used as emulsifier, and 1-undecanol was used as extraction solvent without using any organic dispersive solvent. Several parameters that affect the extraction efficiency, such as the kind and volume of extraction solvent, the type and concentration of the surfactant, extraction time, extraction temperature and salt addition were investigated and optimized. Under the optimum extraction condition, the method yields a linear calibration curve in the concentration range from 5 to 10,000 ng mL−1 for the targeted analytes with the correlation coefficient ranging from 0.9991 to 0.9998. The enrichment factors were in the range between 95 and 135, and the limits of detection of the method were 2–4 ng mL−1. The fruit juice samples were successfully analyzed using the proposed method, and the relative recoveries at fortified levels of 50 and 100 ng mL−1 were in the range of 82.6–97.5%.
Keywords: Ultrasound-assisted surfactant-enhanced emulsification microextraction; Solidification of floating organic droplet; High performance liquid chromatography; Strobilurin fungicides; Fruit juice samples;
Liquid chromatography/tandem mass spectrometry method for quantitative estimation of polyethylene glycol 400 and its applications by V. Vijaya Bhaskar; Anil Middha; Sudhir Tiwari; Savithiri Shivakumar (68-76).
► An MRM based bioanalytical method was developed for the quantitative estimation of PEG 400 in rat plasma. ► Developed methods applicability was proved by performing a rodent PK study and characterised the PK parameters of PEG 400. ► Formulation excipients can be analysed as qualifier in the analysis of NCEs to address the spiky profiles. ► Differences in pharmacokinetics of PEG 400 oligomers was established.A rapid sensitive and selective MRM based method for the determination of polyethylene glycol 400 (PEG 400) in rat plasma was developed using liquid chromatography/tandem mass spectrometry (LC–MS/MS). PEG 400 and telmisartan (Internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (Waters Xbridge, 50 × 4.6 mm, 3.5 μm) with the mobile phase (A – 0.1% formic acid in water; B – methanol). A generic gradient method with a short run time of 3.5 min was developed for the analysis of PEG 400. A total of nine oligomers were identified for PEG 400. The most abundant ions corresponding to PEG 400 oligomers at m/z 327, 371, 432, 476, 520, 564, 608, 652 and 696 with daughter ion at m/z 89 were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation. Analyte peak area of the oligomers was summed up to calculate the plasma concentrations of total PEG 400. The standard curve was linear (0.9954) over the concentration range of 1.01–1013.40 μg/mL. The lower limit of quantitation for PEG 400 was 1.01 μg/mL using 50 μL plasma. The coefficient of variation and relative error for inter and intraassay at three QC levels were 2.31–13.34 and −7.99 to 0.37, respectively. The method was validated for various parameters such as extraction recovery, matrix effect, autosampler stability, benchtop stability, freeze thaw stability, long term stability and was proved to be consistent across three QC levels with overall %CV less than 15. The developed method was successfully applied to the absolute bioavailability study of PEG 400 in male Sprague Dawley rats. Plasma concentrations of PEG 400 was measured after administration through oral and intravenous routes in male Sprague Dawley rats at a dose of 3.38 g/kg. Pharmacokinetic (PK) parameters were characterised by performing the analysis using Phoenix Winnonlin software (v 6.3). PEG 400 has good oral bioavailability with mean absolute bioavailability of 47.23%. Plasma concentration profile/PK parameters of PEG 400 was established in both intravenous and oral routes, which helps to qualify the analytical batch of NCEs having spiky plasma concentration profiles/erratic results. Purity of the PEG 400 oligomers was estimated using ELSD detection. Differences in pharmacokinetics of oligomers was studied. It was found that with increase in molecular weight of the oligomer, a decrease in absolute bioavailability was observed.
Keywords: PEG 400; Oligomers; LC–MS/MS; NCE; Method validation; Bioavailability;
Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate by Yu-Jie Zhou; Cheng-Li Hu; Na Wang; Wei-Wei Zhang; Xiao-Qi Yu (77-82).
An aqueous two-phase system (ATPS) was applied for the purification of porcine pancreatic lipase (PPL) from crude PPL using polyethylene glycol (PEG) and potassium phosphate. Phase diagrams for polyethylene glycol (PEG) and potassium phosphate dibasic were determined at room temperature to find an operating region to first form the ATPS. The PPL was preferentially partitioned into the PEG-rich phase in systems with molecular weights of 1000 and 1500 and concentrated in the phosphate-rich phase in systems with PEG of 4000. Moreover, instead of tie line length (TLL), we used a stability ratio without NaCl in the system, and we first applied fluorescence spectroscopy for the protein conformational analysis of the ATPS. The molecular weight of the purified lipase was determined to be approximately 52 kDa by SDS-PAGE. The enzyme was efficiently purified in PEG 1500/potassium phosphate (17/13, %) at a pH of 7.0 at 4 °C. This system obtained an enzyme partition coefficient of 12.7, an extraction efficiency of 94.7% and a purification factor of approximately 4. These results demonstrate that the aqueous two-phase system is a highly efficient method for PPL purification
Keywords: Porcine pancreatic lipase; Aqueous two-phase systems; Purification; PEG;
Simultaneous analysis of anticancer agents bortezomib, imatinib, nilotinib, dasatinib, erlotinib, lapatinib, sorafenib, sunitinib and vandetanib in human plasma using LC/MS/MS by Irina Andriamanana; Ines Gana; Bénédicte Duretz; Anne Hulin (83-91).
► The quantification of nine inhibitors of tyrosine kinase in a single run. ► The improvement of the specificity using LCMSMS and confirmation/quantification ions ratios. ► The ability to add in our method new ITKs that could have an interest in clinical practices. ► The large dynamic ranges of concentrations that allow to carry out some pharmacokinetics studies.A new liquid chromatography–tandem mass spectrometry (LC–MS/MS) method, performed by electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been developed and validated for the simultaneous determination of bortezomib (BORT), dasatinib (DASA), imatinib (IMAT), nilotinib (NILO), erlotinib (ERLO), lapatinib (LAPA), sorafenib (SORA), sunitinib (SUNI) and vandetanib (VAND) in human plasma. Separation is achieved on an Hypersil Gold® PFP column using a gradient elution of 10 mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.3 mL/min. After addition of the internal standard and protein precipitation, the supernatant is diluted 2-fold in a mixture A and B (50/50, v/v). Two selected reaction monitoring transitions are used for each analyte: one is used for quantitation, the second one is used for confirmation. The standard curves are ranged from 2 ng/mL to 250 ng/mL for BORT, DASA and SUNI and from 50 ng/mL to 3500 ng/mL for the others and were fitted to a 1/x weighted linear regression model. The lowest limits of quantification were 2 ng/mL for BORT, DASA and SUNI and 50 ng/mL for the other TKIs. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day RSD from 3.7% to 13.8%), accuracy (from 86.8% to 113.5%), recovery as well as stability of the analytes under various conditions. The method also may contribute to better understand the relationship between pharmacokinetics and pharmacodynamics of TKIs in hematological malignancies and solid tumors.
Keywords: LC–MS/MS; Anticancer targeted therapy; Tyrosine kinase inhibitor;
Bio-generation of stable isotope labeled internal standards for absolute and relative quantitation of drug metabolites in plasma samples by LC–MS/MS by Pei Li; Yong Gong; Heng-Keang Lim; Wenying Jian; Richard W. Edom; Rhys Salter; Jose Silva; Naidong Weng (92-100).
In order to achieve a better understanding of the toxicity of drug candidates, quantitative characterization of circulatory drug metabolites has been of increasing interest in current pharmaceutical research. Stable isotope labeled (STIL) internal standards (IS) are ideally used to simplify drug metabolite quantitation via liquid chromatography and tandem mass spectrometry (LC–MS/MS) analysis, primarily due to their capability to compensate matrix effects, thereby leading to faster method establishment by using generic assay conditions. However, chemical synthesis of STIL metabolites can often be resource intensive, requiring lengthy exploratory synthesis route development and/or extensive optimization to achieve the required stability for some metabolites. To overcome these challenges, we developed a general method that could generate STIL metabolites in a matter of hours from STIL parent drugs through the utilization of an appropriate in vitro metabolic incubation. This methodology can potentially save valuable synthesis resources, as well as provide timely availability of STIL IS. The following work demonstrates the proof-of-concept that multiple STIL metabolites can be generated simultaneously to provide satisfactory performance for both absolute quantitation of drug metabolites and for potential use in assessment of relative exposure coverage across species in safety tests of drug metabolites (MIST).
Keywords: Stable isotope labeled internal standard; Microsomal incubation; Metabolite; MIST; Quantitation; Relative exposure coverage; LC–MS/MS analysis;
Development and validation of a rapid and sensitive UPLC–MS/MS method for determination of total docetaxel from a lipid microsphere formulation in human plasma by Ping Du; Ning Li; Hongyu Wang; Sheng Yang; Yuanyuan Song; Xiaohong Han; Yuankai Shi (101-107).
► Docetaxel lipid microsphere is a novel formulation of docetaxel without Tween 80. ► A rapid and sensitive UPLC–MS/MS method is firstly developed. ► Shorter analytical time of only 2.5 min. ► The LLOQ of this assay was 2 ng/ml using 200 μl human plasma. ► This method could be applied to a clinical pharmacokinetic study.Docetaxel lipid microsphere (DT-LM), an intravenous lipid emulsion for docetaxel without Tween 80, has demonstrated significant advantage over other conventional docetaxel formulations with respect to keeping sustained release, reducing irritation or toxicity of drug, sterile for intravenous injection and presenting targeting. A rapid, sensitive and reproducible ultra high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for determination of total docetaxel from a lipid microsphere formulation in human plasma using paclitaxel as internal standard (IS) has been developed and validated. The analytes and IS were extracted from plasma by simple liquid–liquid extraction and separated on ACQUITY UPLC BEH C18 column at a flow rate of 0.3 ml/min using gradient elution mode. The total analytical time was only 2.5 min. Detection and quantitation were performed by electrospray ionization (ESI) in the positive ionization mode by multiple reaction monitoring (MRM) of the transitions at m/z 808.3 → 527.1 for docetaxel and 854.0 → 285.9 for IS. The assay was linear over the concentration range of 2–5000 ng/ml (r 2 > 0.99) with the lower limit of quantification (LLOQ) of 2 ng/ml. The intra- and inter-day precision in terms of relative standard deviation (RSD%) was within 9% and accuracy in terms of relative error (RE%) was within 12%. The rapid, sensitive and reproducible UPLC–MS/MS method is now used to support clinical pharmacologic studies with DT-LM injection in patients with advanced cancer.
Keywords: Docetaxel lipid microsphere; UPLC–MS/MS; Human plasma; Pharmacokinetics;
Retraction notice to “Pilot production of recombinant human clotting factor IX from transgenic sow milk” [J. Chromatogr. B 898 (2012) 78–89] by Yu-ling Sun; Yuo-sheng Chang; Yin-shen Lin; Chon-ho Yen (108).