Chromatography-tandem Mass Spectrometric Assay Research Articles

A Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Telmisartan and Chlorthalidone in Rat Plasma and its Application to a Pharmacokinetic Study

A selective, sensitive, rapid, and stable liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed for the simultaneous quantification of chlorthalidone (CLT) and telmisartan (TEL) in rat plasma. Sample preparation involved liquid-liquid extraction by ethyl acetate. Analytes and internal standard (IS) were separated on Gemini C18 (50 x 4.6 mm, 5 μm) using a mixture of methanol-2 mM ammonium acetate (80:20 v/v) as the mobile phase at a flow rate of 0.4 mL/min. Hydrochlorothiazide (HCTZ) was used as an internal standard. Detection was carried out using Electrospray positive and negative ionization mass spectrometry using multiple reaction monitoring of the transition at m/z 515.90→276 for TEL and m/z 337.1→190.05 for CLT, m/z 296.1→205.3 for HCTZ respectively. For TEL and CLT, the method was linear (R2 ≥ 0.995) within the range of 6-1200 ng/mL and 3-600 ng/mL respectively. The retention time for TEL, CLT, and IS was found to be 3.65±0.03 min, 1.83±0.02 min, and 1.76±0.01 min. The mean recovery for both analytes and IS was greater than 70 %. The developed method was validated according to the USFDA guideline. The quantitative method was validated for specificity, linearity, accuracy, precision, recovery, dilution integrity, matrix effect, and analyte stability. The method was successfully applied to a pharmacokinetic study in rat plasma following oral administration.

Liquid chromatography–tandem mass spectrometric assay for the quantification of CDK4/6 inhibitors in human plasma in a clinical context of drug-drug interaction

The CDK4/6 inhibitors palbociclib and ribociclib are kinase inhibitors used in association with hormonal therapy for the management of patients with metastatic breast cancer. Like most kinase inhibitors, therapeutic drug monitoring may be used for personalize their dosage. To this aim, we developed and validated a sensitive and specific HPLC-MS/MS method for palbociclib and ribociclib quantification in blood samples. We then quantified exposure to palbociclib (plasma trough concentration; Ctrough) in a real-life cohort of patients with locally invasive or metastatic breast cancer (n = 18) at day 15 of the first cycle of palbociclib treatment to characterize palbociclib concentration at steady state (Clinicaltrials.gov identifier NCT04025541, IdRCB n° 2018-A00064-51, 03/07/2018). The geometric mean (± standard deviation [min-max]) of palbociclib plasma Ctrough was 88.58 ng/mL (± 26.4 [46.5 ng/mL – 133 ng/mL]) at day 15. Some covariates, such as drug-drug interactions, could explain the concentration variations observed in our Caucasian cohort. These first results in real-life settings obtained with our HPLC-MS/MS method give important information on palbociclib monitoring and pharmacokinetic variability.

Biosynthesis of morin-3-O-rhamnopyranoside in a genetically engineered Escherichia coli

Flavonoids are significant secondary metabolites of vascular plants. They have been proven to possess numerous types of anti-bacterial, anti-tumor, anti-oxidant and anti-inflammatory bioactivities etc. Thereby, there are many flavonoids extracted and purified from plants and have been used for biological tests. Nevertheless, the traditional extraction methods require a large amount of initial sample, tedious and professional techniques and low yield of target compound. Until present, the advanced development of genetic engineerings and recombinant protein as platform allow synthesis of high amount of different types of flavoid in short time. Escherichia coli is one of the most popular host for whole-cell biotransformation of flavonoids and their derivatives due to its simple genetical, physiological system and fast growth. In this paper, we described the biosynthetic method of morin-3-O-rhamnopyranoside from a genetically engineered E. coli using morin as substrate. In particular, the E. coli harboring biosynthetic gene cluster of activated TDP-L-rhamnose and gene encoding for glycosyltransferase was used as host for biotransformation. Morin was fed into the culture broth of recombinant E. coli for rhamnosylation. The formation of morin-3-O-rhamnopyranoside was then quantitied and qualified using Thin Layer Chromatography (TLC), Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) assays. The highest yield of product (56.5 µM) was achieved after 48h, 32 oC and pH = 7.2-8.0. This result showed that morin-3-O-rhamnopyranoside was succesfully synthesized in the genetically engineered E. coli. Furthermore, metabolic engineering of intra-cellular system for improving absorption of substrate as well as excretion of product or enhancement of glycosyltransferase activity may increase the final yield of biotransformation process.

Quality evaluation of the artemisinin-producing plant Artemisia annua L. based on simultaneous quantification of artemisinin and six synergistic components and hierarchical cluster analysis

Artemisia annua L. (A. annua) is one of the most widely used traditional Chinese Medicine, and is also an exclusive natural origin of artemisinin for the industrial production. Several sesquiterpenes, flavonoids and coumarin contained in A. annua could enhance the pharmacological effects of artemisinin. However, the contents of these components vary significantly in different places of origin and different parts of A. annua. To evaluate and differentiate its quality, an accurate and rapid high-performance liquid chromatography tandem mass spectrometric (HPLC–MS/MS) assay has been developed and validated for the simultaneous determination of artemisinin and six synergistic components in A. annua. The run time of analyzing one sample is only 6.0 min. The established method could serve as an accurate and rapid assay for the quality evaluation of A. annua from different places of origin and different parts of the plant. The hierarchical cluster analysis results indicated that the content of scopoletin and/or all seven components (artemisinin and six synergistic components) might be utilized to differentiate different parts and different plucking times of A. annua.

PH gradient-liquid chromatography tandem mass spectrometric assay for determination of underivatized polyamines in cancer cells

Altered levels of polyamines in biological specimens have been suggested as potential biomarkers for cancer. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase) is reported to modulate polyamines to potentially attenuate proliferation of neuroblastoma cells. A clinical trial is being conducted to evaluate DFMO in various cancers. To determine the pharmacodynamics effect of DFMO, an analytical assay is needed to accurately measure the changes in polyamines in cancer cells. In this study, a novel pH gradient LC-ESI-MS/MS method was developed and validated for the quantitation of polyamines (putrescine, spermidine and spermine) in cancer cells. To separate polar and basic polyamines, a multi-mode column composed of ODS and weak ionic ligands was used. The pH gradient was generated from pH 5.3 to pH 2.7 with 2 mM ammonium acetate and 0.4% acetic acid in 10% acetonitrile as mobile phase. The detection of polyamines was performed utilizing multiple reaction monitoring on electrospray ionization mass spectrometry operated in positive ion mode. A pH gradient method increased resolution and decreased peak width of conventional analytical assays, resulting in 10–250-fold higher detection limits. Mobile phases without ion-pairing reagents were LC-MS compatible and eliminated possible signal suppression and MS contamination. The developed method was successfully applied to the analysis of polyamines in neuroblastoma and leukemia cells treated with DFMO. Putrescine levels were significantly (p < 0.001) decreased in CCRF-CEM (3.68 vs 1.23 ng/mg protein), SK-N-BE(2) (1.98 vs 1.31 ng/mg protein) and CHLA-20 (2.06 vs 0.90 ng/mg protein) cells treated with DFMO relative to vehicle control. The assay will provide a useful tool in determining the pharmacodynamic effect of DFMO in cancer clinical trials.

Liquid chromatography–tandem mass spectrometric assay for the quantitation of the novel radiation protective agent and radiation mitigator JP4-039 in murine plasma

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC–MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50μL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1–107.6%) and precision (3.7–12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2–136.1% with moderate ionization enhancement (10.9–39.5%). Plasma freeze-thaw stability (98.8–104.2%), stability for 13.5 months at −80°C (93.1–105.6%), and stability for 4h at room temperature (94.2–97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.

Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid-liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 0.5-500ng/ml calibration range with r(2)=0.995±0.002 (n=6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n=18) were 2.7-11.7% and between-run (3 runs; n=18) precisions 3.0-14.5%. Accuracies were between 96-109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.

Analytical challenges in quantitative analysis (LC/MS/MS) of abiraterone: A validated assay to determine abiraterone in human plasma.

329 Background: Abiraterone acetate is rapidly hydrolyzed to the active abiraterone in vivo. In order to study the relation between drug exposure and therapeutic effect an assay to determine plasma concentrations of abiraterone is required. A quantitative bioanalytical liquid chromatography tandem mass spectrometric (LC-MS/MS) assay with a BEH C18(2.1 × 50 mm, 1.7 µm) column was developed. Several analytical challenges were encountered. Methods: Protein precipitation with acetonitrile was used to pretreat the EDTA plasma samples. A stable isotope (D4-abiraterone) was used as the internal standard. During validation five quality controls were used (LLOQ, LQ, MQ, HQ, ULOQ). The method was validated according to EMA guidelines. Results: The LC-MS/MS method was validated over a concentration range from 1-500 ng/mL. Within – and between day precisions and accuracies were &lt; 5%. The LLOQ was 1 ng/ml and the ULOQ was 499 ng/mL. Abiraterone is stable in plasma for at least 7 days at ambient temperature and at 2-8°C. Stability at -40°C was demonstrated for at least 6 months. Abiraterone is only stable for 3 hours in whole blood at ambient temperature. Stock solution stability was demonstrated for at least 1 year. The concentration levels of the LQ, MQ and HQ were 5, 300 and 400 ng/mL, respectively. During method development several analytical challenges were encountered and solved: abiraterone adsorption on glass containers, limited stability in citrate plasma and whole blood, carry-over issues, very limited stability of abiraterone acetate in whole blood was demonstrated, curiously with proven stability in plasma. Throughout the method polypropylene vials and utilities were used. EDTA plasma was used for development and validation. The needle wash was adjusted and the runtime was prolonged with a longer time at 100% acetonitrile to address the carry over issues. Whole blood samples were treated on ice. The quantification of abiraterone acetate was abandoned. Conclusions: After different hurdles the method is successfully validated and applied to determine plasma concentrations of abiraterone in clinical studies and in regular patient care in patients with prostate cancer.

Ultra-performance liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma: application to a pharmacokinetic study.

A rapid, simple and sensitive UHPLC-MS/MS method was developed and validated for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma using huperzine A as an internal standard (IS) after protein precipitation with methanol. The analytes were separated on a Purospher® STAR RP18 UHPLC column (2 µm, 2.1 × 100 mm) by gradient elution using a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Brucine, strychnine, brucine N-oxide and IS were detected in positive ion multiple reaction monitoring mode by means of an electrospray ionization interface (m/z 395.2 → 324.1, m/z 335.2 → 184.1, m/z 411.2 → 394.2, m/z 243.1 → 226.1). The calibration curve was linear over the range of 1-500 ng/mL for brucine and strychnine and 0.2-50 ng/mL for brucine N-oxide. The intra- and inter-day precisions of these analytes were all within 15% and the accuracy ranged from 85 to 115%. The stability experiment indicated that the plasma samples at three concentration levels were stable under different conditions. The developed method was successfully applied for the first time to pharmacokinetic studies of brucine, strychnine and brucine N-oxide following a single oral and intravenous administration of modified total alkaloid fraction in rats. Copyright © 2016 John Wiley & Sons, Ltd.

Liquid chromatography–tandem mass spectrometric assay for the simultaneous determination of the irreversible BTK inhibitor ibrutinib and its dihydrodiol-metabolite in plasma and its application in mouse pharmacokinetic studies

A validated simple, fast and sensitive bio-analytical assay for ibrutinib and its dihydrodiol metabolite in human and mouse plasma was set up. Sample preparation was performed by protein precipitation, and addition of the respective deuterated internal standards, followed by LC–MS/MS analysis. Separation was performed on a 3.5μm particle-size, bridged ethylene hybrid column with gradient elution by 0.1% v/v formic acid and acetonitrile. The full eluate was transferred to an electrospray interface in positive ionization mode, and subsequently analyzed by a triple quadrupole mass spectrometer by selected reaction monitoring. The assay was validated in a 5–5000 ng/ml calibration range. Both ibrutinib and dihydrodiol-ibrutinib were deemed stable under refrigerated or frozen storage conditions. At room temperature, ibrutinib showed a not earlier described instability, and revealed rapid degradation at 37°C. Finally, the assay was used for a pharmacokinetic study of plasma levels in treated FVB mice.

A liquid chromatography-tandem mass spectrometric assay for the antihypertensive agent azelnidipine in human plasma with application to clinical pharmacokinetics studies.

A robust and sensitive high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS/MS) assay for the high-throughput quantification of the antihypertensive drug azelnidipine in human plasma was developed and validated following bioanalytical validation guidelines. Azelnidipine and internal standard (IS), telmisartan, were extracted from human plasma by precipitation protein and separated on a C18 column using acetonitrile-methanol-ammonium formate with 0.1% formic acid as mobile phase. Detection was performed on a turbo-spray ionization source (ESI) and mass spectrometric positive multiple reaction monitoring mode (+MRM) using the respective transitions m/z 583.3 → 167.2 for azelnidipine and m/z 515.3 → 497.2 for IS. The method has a wide analytical measuring range from 0.0125 to 25 ng/mL. For the lowest limit of quantitation, low, medium and high quality controls, intra- and interassay precisions (relative standard deviation) were 3.30-7.01% and 1.78-8.09%, respectively. The drug was sufficiently stable under all relevant analytical conditions. The main metabolite of azelnidipine, M-1 (aromatized form), was monitored semiquantitatively using the typical transition m/z 581.3 → 167.2. Finally, the method was successfully applied to a clinical pharmacokinetic study in human after a single oral administration of azelnidipine 8 mg. The assay meets criteria for the analysis of samples from large research trials.

Simultaneous determination of rosuvastatin and amlodipine in human plasma using tandem mass spectrometry: Application to disposition kinetics

The liquid chromatography–tandem mass spectrometric assay method for the simultaneous determination of rosuvastatin and amlodipine in human plasma using deuterated analogs as internal standards has been developed and validated. The analytes were extracted from 100μL aliquots of human plasma via liquid–liquid extraction using a mixture of ethyl acetate and n-hexane (80:20, v/v) as an extraction solvent. The optimized mobile phase was composed of 0.1% formic acid in 5mM ammonium acetate, methanol, and acetonitrile (20:20:60, v/v/v) and delivered at a flow rate of 0.75mL/min. The calibration curve obtained was linear (R2⩾0.999) over the concentration range of 0.52–51.77ng/mL for rosuvastatin and 0.10–10.07ng/mL for amlodipine. A sample turnover rate of less than 2.5min makes it an attractive procedure in high-throughput bioanalysis of rosuvastatin and amlodipine. The present method was found to be applicable to clinical studies and the results were authenticated by incurred sample reanalysis.

Evaluation of matrix effects using a spike recovery approach in a dilute-and-inject liquid chromatography–tandem mass spectrometry opioid monitoring assay

BackgroundLiquid chromatography–tandem mass spectrometry has become the gold standard for quantitative analysis of compounds in human matrices. Introduction of these assays into clinical practice, where false positive and false negative results have substantial implications, requires careful attention to matrix effects. We describe an evaluation of matrix effects in human urine from a dilute-and-inject liquid chromatography–tandem mass spectrometric assay for the quantitative analysis of opioids and metabolites. MethodsA spike-recovery approach was employed for each analyte in each sample. We examined the impact of spike-recovery for the 6 glucuronides measured in this assay and compared the analytes for which conventional stable isotope-labeled internal standards were used with the analytes for which analog internal standards were used. ResultsFor analytes that had analog internal standards, up to 1.5% of negative samples failed our requirement of recovering at least 80% of the expected spiked concentration while passing all other quality control parameters. ConclusionsUsing spike-recovery as a quality control parameter decreases the rate of false negatives for compounds using analog internal standards, but does not have benefit for compounds with conventional stable isotope-labeled internal standards.

High performance liquid chromatography-tandem mass spectrometric assay of dexmedetomidine in plasma, urine and amniotic fluid samples for pregnant ewe model.

Dexmedetomidine (DEX; Precedex(®)), approved by the Food and Drug Administration (FDA) in 1999 as a sedative for use in the intensive care unit, is a potent and highly selective α2-adrenoceptor agonist with significant sedative, analgesic and anxiolytic effects. However, the research of DEX use during pregnancy is limited and the impact of DEX on the fetal development is unclear. This article describes a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay suitable for various biomatrices of plasma, urine and amniotic fluid, as a prerequisite for pharmacokinetic characterization of DEX in the pregnant ewe model. DEX and testosterone (internal standard; IS) were extracted from 200μL of plasma, urine or amniotic fluid with ethyl acetate. The HPLC resolution was achieved on an Agilent ZORBAX SB-CN column with a gradient elution at a flow rate of 0.5mL/min using a mobile phase of 5-100% of acetonitrile with 0.5% formic acid (mobile phase B) in water (mobile phase A). The detection was performed by a triple quadrupole tandem mass spectrometer with positive electrospray ionization. The precursor/product transitions (m/z) in the positive ion mode [M+H](+) were m/z 201.5→95.4 for DEX and m/z 289.2→109.1 for IS. The method was validated in the concentration range of 25 (lower limit of quantification; LLOQ)-5000pg/mL for both maternal and fetal plasma, and of 50 (LLOQ)-5000pg/mL for urine and amniotic fluid, respectively. The intra- and inter-day precision and accuracy were within ±9%. The overall recoveries of DEX were 82.9-87.2%, 85.7-88.4%, 86.2-89.7% and 83.7-88.1% for maternal plasma, urine, fetal plasma and amniotic fluid, respectively. The percentage matrix factors in different biomatrices were less than 120%. Stability studies demonstrated that DEX was stable after three freeze/thaw cycles, in the autosampler tray at 20°C for 24h and during the 3h sample preparation at room temperature. The validated HPLC-MS/MS method has been successfully employed for pharmacokinetic evaluation of DEX in pregnant ewes and fetuses.

Abstract P298: Relationship Of Plasma Asymmetrical Dimethylarginine To Carotid Intima-media Thickness In General Japanese Population: The ERA-JUMP And The SESSA

Objective: Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is known as a mediator of endothelial cell dysfunction. Some clinical studies have found evidence that increased plasma ADMA levels are associated with a higher risk of cerebrovascular events. However, studies on the relationship of plasma ADMA to subclinical atherosclerosis have been scarce. We evaluated the association of ADMA with carotid intima-media thickness (IMT) in population-based samples in Japan. Methods: Population-based Japanese samples composed of 313 men aged 40-49 (40s men) from the ERA-JUMP and 732 men aged 60-79 (60s+70s men) from the SESSA were examined with physical examinations, life-style questionnaires, laboratory assessment and IMT using standardized methods. Plasma samples were stored at -80°C, and concentration of ADMA was determined using a validated high-throughput liquid chromatographic-tandem mass spectrometric assay. The IMTs of the right and left common carotid arteries, the carotid bulbs, and the internal carotid arteries were examined. Statistical associations were evaluated using multiple linear regression models with adjustment for age, body mass index, systolic blood pressure, LDL-cholesterol, HDL-cholesterol, fasting blood glucose, smoking, alcohol intake, medications for hypertension, hyperlipidemia, or diabetes, and estimated glomerular filtration rate (eGFR) . Results: The average concentrations and standard deviation (SD) of plasma ADMA in men aged 40-49 and 60-79 were 0.39 ± 0.06 and 0.43 ± 0.06 μmol/L, respectively. The mean and SD of IMT in men aged 40-49 and 60-79 were 0.61 ± 0.07 and 0.91 ± 0.20 mm, respectively. Higher level of ADMA was significantly and positively associated with proportion of current smoking both in 40s and 60s+70s men (correlation coefficient: both p &lt;0.001). It was also significantly and positively associated with eGFR in 60s+70s men (p &lt;0.001) but not associated with eGFR in 40s men (p = 0.429). In full adjusted linear regression models, 1 SD increase in ADMA was associated with 0.14mm increase in IMT in 40s men (p=0.01) but only 0.04mm increase in IMT in 60s+70s men (p=0.26). Conclusions: Higher level of ADMA was associated with more IMT independently of traditional risk factors and renal function in young adult men, but not in elderly men.

Pharmacokinetics and Safety of Tenofovir in HIV-Infected Women During Labor and Their Infants During the First Week of Life

Data describing the pharmacokinetics and safety of tenofovir in neonates are lacking. The HIV Prevention Trials Network 057 protocol was a phase 1, open-label study of the pharmacokinetics and safety of tenofovir disoproxil fumarate (TDF) in HIV-infected women during labor and their infants during the first week of life with 4 dosing cohorts: maternal 600 mg doses/no infant dosing; no maternal dosing/infant 4 mg/kg doses on days 0, 3, and 5; maternal 900 mg doses/infant 6 mg/kg doses on days 0, 3, and 5; maternal 600 mg doses/infant 6 mg/kg daily for 7 doses. Pharmacokinetic sampling was performed on cohort 1 and 3 mothers and all infants. Plasma, amniotic fluid, and breast milk tenofovir concentrations were determined by liquid chromatographic-tandem mass spectrometric assay. The pharmacokinetic target was for infant tenofovir concentration throughout the first week of life to exceed 50 ng/mL, the median trough tenofovir concentration in adults receiving standard chronic TDF dosing. One hundred twenty-two mother-infant pairs from Malawi and Brazil were studied. Tenofovir exposure in mothers receiving 600 and 900 mg exceeded that in nonpregnant adults receiving standard 300 mg doses. Tenofovir elimination in the infants was equivalent to that in older children and adults, and trough tenofovir plasma concentrations exceeded 50 ng/mL in 74%-97% of infants receiving daily dosing. A TDF dosing regimen of 600 mg during labor and daily infant doses of 6 mg/kg maintains infant tenofovir plasma concentration above 50 ng/mL throughout the first week of life and should be used in the studies of TDF efficacy for HIV prevention of mother-to-child transmission and early infant treatment.

Liquid chromatography–tandem mass spectrometric assay for the PARP inhibitor rucaparib in plasma

A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the poly(ADP-ribose) polymerase-1 inhibitor rucaparib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing gefitinib as internal standard. Diluted extract was directly injected into the reversed-phase chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer.The assay was validated in a 1.25–2000ng/ml calibration range with r2=0.9958±0.0012 for linear regression with quadratic weighting (n=6). Within day precisions (n=18) were 2.0–5.4%, between day (3 days; n=18) precisions 3.2–8.0% and accuracies (n=18) were 89.7–93.2%. At the lower limit of quantification (1.25ng/ml) these parameters were 9.6%, 13.7% and 85.3%, respectively. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully used to determine drug pharmacokinetics in female FVB wild type mice.

Liquid chromatography–tandem mass spectrometric assay for the cyclin-dependent kinase inhibitor AT7519 in mouse plasma

A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the cyclin-dependent kinase inhibitor AT7519 in mouse plasma was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing rucaparib as internal standard. After dilution with water, the extract was directly injected into the reversed-phase LC system. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer.The assay was validated in a 5–10,000ng/ml calibration range using double logarithmic calibration, 5ng/ml was the lower limit of quantification. Within day precisions (n=6) were 2.9–5.6%, between day (3 days; n=18) precisions 3.2–7.2%. Accuracies were between 95.9 and 99.0% for the whole calibration range. The drug was stable under all relevant analytical conditions. Finally, the assay was successfully used to determine plasma pharmacokinetics after intraperitoneal administration of AT7519 in mice with neuroblastoma xenografts.

Validation of a rapid liquid chromatography–tandem mass spectrometric assay for the determination of octreotide plasma concentrations

ObjectivesThe aim of this study was to develop and validate a fast liquid chromatography–tandem mass spectrometric (LC–MSMS) assay to determine circulating concentrations of octreotide (a somatostatin analog used in acromegaly and neuroendocrine tumors) in patients' plasma samples. Design and methods500μL of heparin–plasma was used to extract the drug on a cation exchanger SPE column, and the eluate was injected into a LC–MSMS system. Reversed phase chromatography was performed on a phenyl grafted column in isocratic mode. Octreotide and internal standard (triptorelin) were identified in positive electrospray ionization mode using ion transitions of m/z 512>120 and 890>249, respectively. ResultsThe assay was linear in the concentration range of 0.5–25ng/mL, with intra- and inter-assay imprecisions of <10.6% and <12.2%, respectively. The limits of quantification and detection were 0.5 and 0.25ng/mL. The recovery and ion suppression effect ranged between 79.7 and 84.5% and between −8.1 and −21.3%, respectively. A subcutaneous injection of 0.1mg octreotide induced a time- and patient-dependent surge of peptide concentrations peaking at 2h. ConclusionThis fast, sensitive, and selective method for quantification of plasma octreotide by LC–MSMS might be used to investigate the pharmacokinetic–pharmacodynamic relationship, with potential contribution to treatment optimization and therapeutic drug monitoring application.

Effect of CYP2D6*10 and APOE Polymorphisms on the Efficacy of Donepezil in Patients With Alzheimer’s Disease

BackgroundThe aim of this study was to evaluate the effect of CYP2D6*10 and APOE polymorphisms on both steady-state plasma concentrations (Cp) and clinical response of donepezil in patients with mild-to-moderate Alzheimer’s disease (AD). MethodsA total of 110 Chinese AD patients participated in this study. Patients were treated with 5 to 10 mg of donepezil daily for 6 months. The genotypes of CYP2D6*10 and APOE were analyzed by polymerase chain reaction–restriction fragment length polymorphism. The steady-state Cp of donepezil was measured by high-performance liquid chromatography–tandem mass spectrometric assay method. The cognition of patients was evaluated at baseline and at 6-month follow-up by Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subscale. ResultsAt 6-month follow-up, 56 of 96 patients (58.3%) were evaluated as responders and 40 patients (41.7%) as nonresponders to donepezil treatment. A significantly higher frequency of patients with genotypes CYP2D6*1/*10 and *10/*10 were found in responders than in nonresponders (P<0.05). Besides, patients with CYP2D6*1/*10 and *10/*10 genotypes had higher Cp of donepezil and improved cognition scores than those with CYP2D6*1/*1 genotype (P<0.05). However, the frequency of APOE ε4 carriers and noncarriers showed no difference between the 2 groups (P>0.05). ConclusionsAD patients with mutant allele (*10) in CYP2D6 gene may respond better to donepezil than those with wild allele (*1). We did not find the relationship between APOE ε4 status and the efficacy of donepezil in our study.