Development and Validation of a Simple Method for Simultaneously Measuring the Concentrations of BCR-ABL and Bruton Tyrosine Kinase Inhibitors in Dried Blood Spot (DBS): A Pilot Study to Obtain Candidate Conversion Equations for Predicting Plasma Concentration Based on DBS Concentration.

BACKGROUND: Dried blood spots (DBSs) are promising candidates for therapeutic drug monitoring. In this study, a simple method for the simultaneous measurement of tyrosine kinase inhibitors (TKIs), including bosutinib, dasatinib, ibrutinib, imatinib, nilotinib, and ponatinib, using DBS was developed and validated. The prediction of the plasma concentration of TKIs based on the TKI concentrations in the DBS was assessed using the developed measurement method. METHODS: DBS was prepared using venous blood on Whatman 903 cards. One whole DBS sample containing the equivalent of 40 µL of blood was used for the analysis. The analytical method was validated according to the relevant guidelines. For clinical validation, 96 clinical samples were analyzed. The regression equation was derived from a weighted Deming regression analysis, and correction factors for calculating the estimated plasma concentrations (EPCs) of the analytes from their concentrations in the DBS and the predictive performance of EPC were evaluated using 2 conversion equations. RESULTS: This method was successfully validated. Hematocrit had no significant effect on the method's accuracy or precision. Ibrutinib was stable in the DBS for up to 8 weeks at room temperature, whereas all BCR-ABL TKIs were stable for 12 weeks. All BCR-ABL TKIs exhibited similar predictive performance for EPCs using both calculation methods. Good agreement between EPCs and the measured plasma concentrations of bosutinib, imatinib, and ponatinib was observed with both conversion equations. However, Bland-Altman analysis showed that blood sampling time affected the EPC accuracy for dasatinib and nilotinib. CONCLUSIONS: A simple method for the simultaneous determination of BCR-ABL and Bruton TKI concentrations in DBS was developed and validated. Owing to the small clinical sample size, further clinical validation is needed to determine the predictive performance of EPCs for the 6 TKIs.

Simultaneous Quantification of BCR-ABL and Bruton Tyrosine Kinase Inhibitors in Dried Plasma Spots and Its Application to Clinical Sample Analysis.

BACKGROUND: Recent reports highlight the importance of therapeutic drug monitoring (TDM) of BCR-ABL and Bruton tyrosine kinase inhibitors (TKIs); thus, large-scale studies are needed to determine the target concentrations of these drugs. TDM using dried plasma spots (DPS) instead of conventional plasma samples is a promising approach. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of BCR-ABL and Bruton TKIs for further TDM studies. METHODS: A 20-µL aliquot of plasma was spotted onto a filter paper and dried completely. Analytes were extracted from 2 DPS using 250 µL of solvent. After cleanup by supported liquid extraction, the sample was analyzed by LC-MS/MS. Applicability of the method was examined using samples of patients' DPS transported by regular mail as a proof-of-concept study. The constant bias and proportional error between plasma and DPS concentrations were assessed by Passing-Bablok regression analysis, and systematic errors were evaluated by Bland-Altman analysis. RESULTS: The method was successfully validated over the following calibration ranges: 1-200 ng/mL for dasatinib and ponatinib, 2-400 ng/mL for ibrutinib, 5-1000 ng/mL for bosutinib, and 20-4000 ng/mL for imatinib and nilotinib. TKI concentrations were successfully determined for 93 of 96 DPS from clinical samples. No constant bias between plasma and DPS concentrations was observed for bosutinib, dasatinib, nilotinib, and ponatinib, whereas there were proportional errors between the plasma and DPS concentrations of nilotinib and ponatinib. Bland-Altman plots revealed that significant systematic errors existed between both methods for bosutinib, nilotinib, and ponatinib. CONCLUSIONS: An LC-MS/MS method for the simultaneous quantification of 6 TKIs in DPS was developed and validated. Further large-scale studies should be conducted to assess the consistency of concentration measurements obtained from plasma and DPS.

An Liquid Chromatography-Tandem Mass Spectrometry Method for the Simultaneous Determination of Afatinib, Alectinib, Ceritinib, Crizotinib, Dacomitinib, Erlotinib, Gefitinib, and Osimertinib in Human Serum.

BACKGROUND: Routine therapeutic drug monitoring is a promising approach for the rational use of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase (ALK) inhibitors. The purpose of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 5 EGFR-TKIs (afatinib, dacomitinib, erlotinib, gefitinib, and osimertinib) and 3 ALK inhibitors (alectinib, ceritinib, and crizotinib). METHODS: A 100-mL aliquot of serum was diluted with 100 µL of 1% aqueous ammonia containing internal standards and then purified using the supported liquid extraction method. LC-MS/MS was conducted in positive ionization mode, and the method was validated according to published guidelines. RESULTS: Calibration curves were linear across concentration ranges examined. The intra- and interassay accuracies were 90.7%-110.7% and 94.7%-107.6%, respectively. All intra- and interassay imprecision values were ≤10.1%. The EGFR-TKIs and ALK inhibitors examined in this study, except osimertinib, which could be stored on ice for at least 5 hours, were stable at room temperature for 3 hours. For the internal standard-normalized matrix factors, the mean recovery and percent coefficient of variation values ranged between 54%-112% and 1.7%-11.7%, respectively. This method successfully determined serum concentrations of afatinib, alectinib, erlotinib, gefitinib, and osimertinib in clinical samples. Serum levels of kinase inhibitors consistently reflected those reported in previous studies. CONCLUSIONS: An LC-MS/MS method suitable for the simultaneous determination of 5 EGFR-TKIs and 3 ALK inhibitors in serum was developed and validated. The newly developed method enabled the determination of 5 of 8 target drugs examined in clinical samples. However, a large number of clinical samples need to be analyzed to verify the usefulness of the method.

A metabolomics-based approach for predicting stages of chronic kidney disease.

Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.3% accuracy (range, 73.9-87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.

Novel high-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of BCR-ABL and Bruton's tyrosine kinase inhibitors and their three active metabolites in human plasma.

Therapeutic drug monitoring is important in patients taking BCR-ABL and Bruton's tyrosine kinase inhibitors (TKIs). Some TKI active metabolites with long elimination half-lives, such as dihydrodiol ibrutinib (DHI), N-desmethyl imatinib (N-DI), and N-desmethyl ponatinib (N-DP), have been characterized, indicating that these active metabolites should be monitored along with the parent compounds. However, there are currently no methods for the simultaneous quantification of BCR-ABL and Bruton's TKIs and their three active metabolites. The present study aimed to develop and validate a method for the simultaneous quantification of nine pharmacologically active compounds (bosutinib, dasatinib, DHI, ibrutinib, imatinib, N-DI, N-DP, nilotinib, and ponatinib) using high-performance liquid chromatography-tandem mass spectrometry. A 150-µL sample of plasma was analyzed after purification with supported liquid extraction. The method has a run time of 7 min and was successfully validated over the following calibration ranges: 0.25-75 ng/mL for N-DP, 0.5-150 ng/mL for dasatinib and ponatinib, 10-3000 ng/mL for imatinib and nilotinib, and 1-300 ng/mL for the other analytes. Stability of the analytes after short- and long-term storage in the presence of plasma matrix was examined, and all analytes were found to be stable under all tested conditions. The recovery was ≥83%, and the relative standard deviation of internal-standard normalized matrix effects ranged from 3.9 to 13.9%. Dilution integrity up to 4-fold was ensured. The applicability of the method for all analytes was demonstrated using patient samples.

Study of flow rate in pressurized gradient capillary electrochromatography using splitter and separation of peptides using an Amide stationary phase.

A pressurized gradient capillary electrochromatograph using a splitter was constructed. The variation in flow rate during gradient elution was investigated and separations of peptides using an Amide stationary phase were demonstrated. The flow rate, which is one of the important factors to control chromatographic behavior, was increased during the gradient elution, and the mismatching of mobile phase between the column and the resistance tubing derived three variation patterns in the flow rate. The electrophoretic migration in electrochromatography could enhance in separation of peptides. The separated peak number of tryptic digest of bovine serum albumin was increased from 30 to 40 by the application of +5 kV.

Peptide separation by Hydrophilic-Interaction Chromatography: a review.

Recent developments in the separation of peptides by high-performance liquid chromatography (HPLC) using polar sorbents with less polar eluents are summarized in this review. This separation mode is now commonly referred to as Hydrophilic-Interaction Chromatography (HILIC). The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions are studied on TSKgel Amide-80 columns, which consist of carbamoyl groups bonded to a silica gel matrix, using a mixture of acetonitrile (MeCN)-water containing 0.1% trifluoroacetic acid (TFA). Some applications are given in peptide field using Hydrophilic-Interaction Chromatography.

High-speed analyses using rapid resolution liquid chromatography on 1.8-microm porous particles.

The potential of high-speed analyses by rapid resolution liquid chromatography (RRLC) and RRLC/MS on 1.8-microm porous particles packed into short columns operated at high flow-rate was investigated and compared with the performance of 5-microm porous particles packed into conventional columns. Using similar chemistries, the ease of conversion from conventional HPLC to an RRLC method was demonstrated. In order to display the practicality of RRLC separations, the analysis of pesticides in crops and catechins in Japanese green tea was selected. Using the Japanese Food Hygiene Law method, which employs a conventional 5-microm RP column (250 mm x 4.6 mm) for quantification of pesticides in crops, the analysis time was 25 min under isocratic conditions. Using the RRLC method on the short (50 mm x 4.6 mm) column packed with 1.8-microm porous particles, the same separation could be performed in 0.8 min with the RRLC/MS method without a loss in resolution. At the highest flow rate, compared to the conventional method, the time could be reduced by a factor of 31. In gradient elution, the fastest separation of catechins in Japanese green tea was achieved by RRLC on 50-mm x 4.6-mm id or 50-mm x 2.1-mm id RRLC columns packed with 1.8-microm particles. The analysis time at 5 mL/min was less than 1 min. Compared to the conventional HPLC method on a 150-mm column packed with 5-microm particles, time was reduced by a factor of 15. The effect of other experimental parameters such as the column temperature, acquisition rate of the detector and the influence of cell volume on chromatographic performance was also investigated. After the optimization, the analysis precision under the fastest RRLC conditions was examined. RSDs of retention time and peak area were 0.2% and 0.47%, respectively.