Validation and application of a liquid chromatography‐tandem mass spectrometric method for the determination of GDC‐0152 in human plasma using solid‐phase extraction

A liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0152 in human plasma to support clinical development. The method consisted of a solid‐phase extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₇‐GDC‐0152 was used as the internal standard. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 0.02–10.0 ng/mL for GDC‐0152. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 99.3% with a precision (%CV) of 13.9%. For quality control samples at 0.0600, 2.00 and 8.00 ng/mL, the between‐run %CV was ≤8.64. Between‐run percentage accuracy ranged from 98.2 to 99.6%. GDC‐0152 was stable in human plasma for 363 days at ?20°C and for 659 days at ?70°C storage. GDC‐0152 was stable in human plasma at room temperature for up to 25 h and through three freeze–thaw cycles. In whole blood, GDC‐0152 was stable for 12 h at 4°C and at ambient temperature. This validated LC‐MS/MS method for determination of GDC‐0152 was used to support clinical studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid LC–MS/MS method for quantification of vinorelbine and 4‐O‐deacetylvinorelbine in human whole blood suitable to monitoring oral metronomic anticancer therapy

A rapid and sensitive LC–MS/MS method for therapeutic drug monitoring oral vinorelbine (VRL) metronomic anticancer chemotherapy has been developed and validated. Analysis of VRL and its main active metabolite 4‐O‐deacetylvinorelbine (M1) was performed in whole blood matrix. Both analytes were extracted by protein precipitation and separated on an Onyx monolith C₁₈, 50 × 2 mm column then quantified by positive electrospray ionization and multiple reaction monitoring mode. The LLOQ was 0.05 ng/mL for both VRL and M1. Linearity was up to 25ng/mL with R² ≥ 0.994. The intra‐ and inter‐assay precisions were ≤ 11.6 and ≤ 10.4% while the ranges of accuracy were [−8.7%; 10.3%] and [−10.0; 7.4%] for VRL and M1, respectively. The clinical suitability of the method has been proved by the determination of the C^Trough blood concentrations of VRL and M1 in 64 nonsmall cell lung cancer elderly patients. The analytical performance of the assay was suitable for pharmacokinetic monitoring of VRL and M1, allowing the personalization of the VRL metronomic treatments.     

Validation of a liquid chromatography‐triple quadrupole mass spectrometric method for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma and its application to microdose clinical trial

A liquid chromatography–triple quadrupole mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma to support a microdose clinical trial. The method consisted of a liquid–liquid extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₃‐AGM‐130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10–2000 pg/mL for AGM‐130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between‐run accuracy ranged from 98.1 to 101.0%. AGM‐130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM‐130 was also stable in human plasma at room temperature for 6 h and through three freeze–thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC‐MS/MS method for determination of AGM‐130 was used to support a phase 0 microdose clinical trial. Copyright © 2015 John Wiley & Sons, Ltd.     

A supported liquid extraction LC–MS/MS method for determination of concentrations of GDC‐0425, a small molecule Checkpoint kinase 1 inhibitor,in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of GDC‐0425 concentrations in human plasma has been developed and validated. Supported liquid extraction was used to extract plasma samples (50 μL) and the resulting samples were analyzed using reverse‐phase chromatography and mass spectrometry coupled with a turbo‐ionspray interface. The mass analysis of GDC‐0425 was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range of 1.00–1000 ng/mL using linear regression and 1/x² weighting. Within‐run relative standard deviation ranged from 0.8 to 5.1%, while between‐run RSD varied from 1.9 to 4.7% for QCs. The accuracy ranged from 90.0 to 101.0% of nominal for within‐run and from 94.0 to 100.0% of nominal for between‐run. Overall extraction recovery was 87.4% for GDC‐0425 and 87.9% for GDC‐0425‐d₉. Stability of GDC‐0425 was established in human plasma for 374 days at ?20 and ?70 °C and established in reconstituted sample extracts for 88 h when stored at 2–8 °C. Stable‐labeled internal standard was used to minimize matrix effects. This assay was used to characterize the pharmacokinetics of GDC‐0425 in cancer patients.     

Pharmacokinetics of TAK‐875 and its toxic metabolite TAK‐875‐ acylglucuronide in rat plasma by liquid chromatography tandem mass spectrometry

TAK‐875 is a selective partial agonist of human GPR40 receptor, which was unexpectedly terminated at phase III clinical trials owing to its severe hepatotoxicity. The purpose of this study was to investigate the pharmacokinetics of TAK‐875 and its toxic metabolite TAK‐875‐acylglucuronide in rat plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS). Plasma samples were extracted with ethyl acetate and chromatographic separations were achieved on a C₁₈ column with water and acetonitrile containing 0.05% ammonium hydroxide as mobile phase. The sample was detected in selected reaction monitoring mode with precursor‐to‐product ion transitions being m/z 523.2 → 148.1, m/z 699.3 → 113.1 and m/z 425.2 → 113.1 for TAK‐875, TAK‐875‐acylglucuronide and IS, respectively. The assay showed good linearity over the tested concentration ranges (r > 0.9993), with the LLOQ being 0.5 ng/mL for both analytes. The extraction recovery was >78.45% and no obvious matrix effect was detected. The highly sensitive LC–MS/MS method has been further applied for the pharmacokinetic study of TAK‐875 and its toxic metabolite TAK‐875‐acylglucuronide in rat plasma. Pharmacokinetics results revealed that oral bioavailability of TAK‐875 was 86.85%. The in vivo exposures of TAK‐875‐acylglucuronide in terms of AUC_0–t were 17.54 and 22.29% of that of TAK‐875 after intravenous and oral administration, respectively.     

A liquid chromatography‐tandem mass spectrometry method for the simultaneous determination of exemestane and its metabolite 17‐dihydroexemestane in human plasma

A simple and sensitive liquid chromatography‐tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantitation of exemestane (Exe) and its main metabolite 17‐dihydroexemestane (DhExe) in human plasma. The analytes were extracted by protein precipitation with acetonitrile, containing stable ¹³C‐labelled Exe (¹³C₃‐Exe) as internal standard, and measured by LC–MS/MS. The best chromatographic separationof the analytes from the interferences was achieved by using a Phenyl column operating under isocratic regime conditions. The total chromatographic runtime was 5.0 min and the elution of Exe and DhExe occurred at 2.5 min and 2.9 min, respectively. Quantitation was performed by employing the positive electrospray ionization (ESI) technique and multiple reaction monitoring mode (MRM). The monitored precursor to product‐ion transitions for Exe, DhExe and ¹³C₃‐Exe internal standard were m/z 297.0 → 120.8, m/z 299.1 → 134.9 and m/z 300.0 → 123.2, respectively. The lower limit of quantitation (LLOQ) was 0.1 ng/ml for DhExe and 0.2 ng/ml for Exe. The method was linear up to 36–51 ng/ml with r² ≥ 0.998. The intra‐ and inter‐assay precision were ≤7.7% and 5.1% for Exe and ≤8.1 and 4.9% for DhExe while deviations from nominal values were in the 1.5–13.2% and ? 9.0–5.8% ranges for Exe and DhExe, respectively. The analytical method resulted robust and suitable for pharmacokinetic monitoring of Exe and its main metabolite during adjuvant therapy in patients with breast cancer. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid and sensitive LC‐MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study

A rapid, simple and fully validated LC‐MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one‐step liquid–liquid extraction with methyl‐tert‐butyl‐ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C₁₈ column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)–methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal‐to‐noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1–2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra‐ and inter‐day precision and accuracy, recovery, matrix effect and stability. The validated LC‐MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace?) to five healthy Korean male volunteers under fed conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of metformin,glyburide and its metabolites in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of an LC‐MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87–99%, and that from urine samples was 85–95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra‐day and inter‐day assays in plasma and urine samples, and the accuracy was 86–114% in plasma, and 94–105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.     

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

A very accurate and selective LC‐MS/MS method was developed and validated for the quantification of 2′‐C‐modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96‐well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm×2.0 mm, 3 μm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 μL/min flow rate. The 2′‐C‐modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05–50 μM concentration range. Satisfying results, confirming the high reliability of the established LC‐MS/MS method, were obtained for intraday precision (CV = 2.5–9.1%) and accuracy (92.6–94.8%) and interday precision (CV = 9.6–11.5%) and accuracy (94.4–102.4%) as well as for recovery (82.0–112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2′‐C‐methyl‐cytidine‐triphosphate in liver tissue samples.     

Supported liquid extraction in combination with LC‐MS/MS for high‐throughput quantitative analysis of hydrocortisone in mouse serum

A high‐throughput LC–MS/MS bioanalytical method was developed and validated for the determination of hydrocortisone in mouse serum via supported liquid extraction (SLE) in a 96‐well plate format. Although sample extracts from SLE result in similar matrix effects compared with conventional liquid–liquid extraction (LLE), greater analyte extraction recovery and much higher analysis throughput for the quantitative analysis of hydrocortisone in mouse serum were obtained. The current LC‐MS/MS method was validated for a concentration range of 2.00–2000 ng/mL for hydrocortisone using a 0.100 mL volume of mouse serum. The intra‐ and inter‐day precision and accuracy of the quality control samples at low, medium and high concentration levels showed ≤12.9% CV and ?3.4–6.2% bias for the analyte in mouse serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of GDC‐0980 (apitolisib), a small molecule dual phosphatidylinositide 3‐kinase/mammalian target of rapamycin inhibitor in dog plasma by LC‐MS/MS to support a GLP toxicology study

An LC‐MS/MS method for the determination of GDC‐0980 (apitolisib) concentrations in dog plasma has been developed and validated for the first time to support pre‐clinical drug development. Following protein precipitation with acetonitrile, the resulting samples were analyzed using reverse‐phase chromatography on a Metasil AQ column. The mass analysis was performed on a triple quadruple mass spectrometer coupled with an electrospray interface in positive ionization mode. The selected reaction monitoring transitions monitored were m/z 499.3 → 341.1 for GDC‐0980 and m/z 507.3 → 341.1 for IS. The method was validated over the calibration curve range 0.250–250 ng/mL with linear regression and 1/x² weighting. Relative standard deviation (RSD) ranged from 0.0 to 10.9% and accuracy ranged from 93.4 to 113.6% of nominal. Stable‐labeled internal standard GDC‐0980‐d₈ was used to minimize matrix effects. This assay was used for the measurement of GDC‐0980 dog plasma concentrations to determine toxicokinetic parameters after oral administration of GDC‐0980 (0.03, 0.1 and 0.3 mg/kg) to beagle dogs in a GLP toxicology study. Peak concentration ranged from 3.23 to 84.9 ng/mL. GDC‐0980 was rapidly absorbed with a mean time to peak concentration ranging from 1.3 to 2.4 h. Mean area under the concentration–time curve from 0 to 24 hours ranged from 54.4 to 542 ng h/mL. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of complanatoside A in rat plasma using LC‐MS/MS and its application to a pharmacokinetic study

Complanatoside A is a flavonol glycoside isolated from Astragalus complanatus, and currently it is used as a quality control index for A. complanatus in the 2010 edition of the Chinese Pharmacopoeia. For the first time, a simple and sensitive LC‐MS/MS method was developed for the determination of complanatoside A in rat plasma over the range of 2.3–575 ng/mL. Complanatoside A was extracted from plasma by a protein precipitation procedure, separated by LC and detected by MS/MS in positive electrospray ionization mode. The method was validated for selectivity, carryover, sensitivity, linearity, extraction recovery, matrix effect, accuracy, precision and stability studies. The lower limit of quantification was established at 2.3 ng/mL. Intra‐ and inter‐day precisions (LLOQ, low‐QC, med‐QC and high‐QC) were <7.9%, and accuracies were between 94.0 and 105.1%. Matrix effect was acceptable (97.9–103.0%) and extraction recovery was reproducible (88.5–94.4%). Complanatoside A was stable in the investigated conditions. The method was applied to the pharmacokinetics of complanatoside A in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification and validation of HPLC‐UV and LC‐MS assays for therapeutic drug monitoring of ertapenem in human plasma

Rapid and simple HPLC‐UV and LC‐MS methods were developed and validated for the quantification of ertapenem (Invanz?) in human plasma. Ertapenem is a unique drug in that current dosing recommendations call for a 1 g dose for normal renal function patients, despite body weight. These assays, which involve a protein precipitation followed by liquid–liquid extraction, allow for fast therapeutic drug monitoring of ertapenem in patients, which is especially useful in special populations. Both methods were sufficient to baseline resolve meropenem (internal standard) and ertapenem, and were validated over 3 days using a six‐point calibration curve (0.5–50 µg/mL). Validation was collected using four different points on the calibrations curve yielding acceptable precision (<15% inter‐day and intra‐day; <20% for lower limit of quantitation, LLOQ) as well as accuracy (<15% inter‐day and intra‐day; <20% for LLOQ). The lower limit of detection (LOD) was determined to be 0.1 and 0.05 µg/mL for the HPLC‐UV and LC‐MS methods, respectively. The developed HPLC‐UV and LC‐MS methods for ertapenem quantification are fast, accurate and reproducible over the calibration range and can be used to determine ertapenem plasma concentrations for monitoring clinical efficacy. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) technique was developed and validated for the determination of sibutramine and its N‐desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t‐butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse‐phase Luna C₁₈ column with a mobile phase of acetonitrile–10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI‐MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05–20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra‐ and inter‐day validation for sibutramine, M1 and M2 were acceptable. This LC‐MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.     

A sensitive LC–MS/MS method for simultaneous quantification of geniposide and its active metabolite genipin in rat plasma and its application to a pharmacokinetic study

Genipin (GP), an active metabolite of geniposide (GE), exhibits more potent pharmacological effects than its parent compound. In this paper, a sensitive LC‐MS/MS method was developed and fully validated for the simultaneous determination of GE and GP in rat plasma. We found that GP degraded rapidly in rat plasma at room temperature as a result of irreversible binding with the endogenous nucleophiles in plasma. GP was stable when the sample's pH was ≤4.0. The degradation of GP in rat plasma was well prevented by immediate addition of 5% glacial acetic acid to the freshly collected plasma. The detection was performed on a tandem mass spectrometer coupled with electrospray ionization source in negative mode. Quantification was conducted by multiple reaction monitoring of the transitions [M + CH₃COO]⁻ m/z 447.3 → 225.3 for GE and [M − H]⁻ m/z 225.2 → 123.1 for GP. The method exhibited high sensitivity (LLOQ 1 ng/mL for GE and 0.2 ng/mL for GP) by selecting the acetate adduct ions as the precursor ions for GE. The robust developed method was successfully applied to a pharmacokinetic study in rats after oral administration of GE.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

High‐sensitivity simultaneous quantification of tacrolimus and 13‐O‐demethyl tacrolimus in human whole blood using ultra‐performance liquid chromatography coupled to tandem mass spectrometry

Blood concentrations of tacrolimus show large variability among patients and the narrow therapeutic range is related to adverse effects. Therefore, therapeutic drug monitoring is needed for strict management. 13‐O‐Demethyl tacrolimus (13‐O‐DMT) was reported as the major metabolite formed by cytochrome P450 (CYP)3A such as CYP3A5. In previous studies, the best lower limit of quantification (LLOQ) was 0.1 ng/mL for both substances. However, this LLOQ may not be low enough now because the dosage of tacrolimus has decreased in recent years. The purpose of this study was to develop and validate a high‐sensitivity and high‐throughput assay for simultaneous quantification of tacrolimus and 13‐O‐DMT in human whole blood using ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Thirty‐five stable kidney transplant recipients receiving tacrolimus were recruited in this study. The calibration curve range was 0.04–40 ng/mL. All calibration samples and quality control samples fulfilled the requirements of the US Food and Drug Administration and the European Medicines Agency guidelines for assay validation. Trough concentrations of tacrolimus and 13‐O‐DMT in 35 stable kidney transplant recipients receiving tacrolimus were within the range of the respective calibration curve. Our novel UPLC–MS/MS method is more sensitive than previous methods for quantification of tacrolimus and 13‐O‐DMT.     

A robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of 5‐azacytidine

The DNA methyltransferase inhibitor 5‐azacytidine is being evaluated clinically as an oral formulation to treat various solid tumors. A sensitive, reliable method was developed to quantitate 5‐azacytidine using LC‐MS/MS to perform detailed pharmacokinetic studies. The drug of interest was extracted from plasma using Oasis MCX ion exchange solid‐phase extraction 96‐well plates. Chromatographic separation was achieved with a YMC J'sphere M80 C₁₈ column and isocratic elution with a methanol–water–formic acid (15:85:0.1, v/v/v) mobile phase over a 7 min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5‐azacytidine. The assay range was 5–500 ng/mL and proved to be accurate (97.8–109.1%) and precise (CV ≤ 9.8%). Tetrahydrouridine was used to stabilize 5‐azacytidine in blood/plasma samples. With the addition of tetrahydrouridine, long‐term frozen plasma stability for 5‐azacytidine at ?70°C has been determined for at least 323 days. The method was applied for the measurement of total plasma concentrations of 5‐azacytidine in a cancer patient receiving a 300 mg oral daily dose. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous LC‐MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self‐microemulsifying drug‐delivery systems

In this study, a liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid–liquid extraction using methyl tert‐butyl ether–dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB‐C₁₈ column (100 × 2.1 mm, 3 μm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide‐d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra‐ and inter‐day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel‐loaded self‐microemulsifying drug‐delivery systems.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.