Comparison of release and penetration of tacrolimus ointment reference and trial formulation after dermal application to pigs by liquid chromatography–electrospray ionization tandem mass spectrometry

To investigate the consistency and bioequivalence of tacrolimus ointment reference and trial formulation, the tacrolimus concentrations in blood and skin were determined by HPLC‐ESI‐MS/MS following topical application of two kinds of ointment in porcine skin in a parallel, cross‐over trial. The plasma protein of blood was precipitated by acetonitrile and the tacrolimus in skin was extracted by acetonitrile before HPLC‐ESI‐MS/MS analysis. The internal calibration method (diazepam was the internal standard) was used for quantification analysis (R² > 0.9999), with linear range from 0.05 to 5 ng/mL for blood samples and from 1 to 200 ng/mL for skin samples. The limits of detection for the porcine blood and skin were 0.005 and 0.5 ng/mL, respectively. The average recoveries for the porcine blood and skin spiked at three levels were 97.56–109.53 and 96.48–103.57%, respectively. The precision expressed in RSDs was from 3.43 to 10.83% for porcine blood and from 3.10 to 8.69% for porcine skin. For the same pig, the tacrolimus concentrations and variation with time of the two kinds of ointment in porcine skin were similar, although variation occurred with different individuals. These results showed that the release and penetration of tacrolimus from the reference and trial formulation are similar. Copyright © 2013 John Wiley & Sons, Ltd.     

Highly sensitive and rapid determination of tacrolimus in peripheral blood mononuclear cells by liquid chromatography–tandem mass spectrometry

After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC–MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS‐TDM^PREP. To each sample we added 30 μL lysis buffer, 20 μL reconstitution buffer containing ¹³C²H₄‐tacrolimus as internal standard, 40 μL MagSiMUS‐TDM^PREP Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol‐based protein precipitation. A 10 μL aliquot of the supernatant was injected into the UPLC–MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r² = 0.997) over the range 5.0–1250 pg/1 × 10⁶ PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.     

Simultaneous determination of the bioactive components in rat plasma by UPLC‐MS/MS and application in pharmacokinetic studies after oral administration of radix Scutellariae extract

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantification of the four main bioactive compounds, i.e. baicalin, baicalein, wogonoside and wogonin, in rat plasma after oral administration of Radix Scutellariae extract. Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 447.5 → 270.1 for baicalin, 270.1 → 168.1 for baicalein, 461.2 → 284.0 for wogonoside, 284.2 → 168.1 for wogonin and 748.5 → 158.1 for IS. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 1.13 ng/mL for baicalin, 1.23 ng/mL for baicalein, 0.82 ng/mL for wogonoside and 0.36 ng/mL for wogonin. The calibration curves obtained were linear (r > 0.99) over the concentration range ~ 1–1000 ng/mL. The intra‐ and inter‐day precision was <15% and the accuracy was within ±14.7%. After validation, this method was successfully applied to a pharmacokinetic study of Radix Scutellariae extract.     

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.     

Targeted metabolomic analysis of 33 amino acids and biogenic amines in human urine by ion‐pairing HPLC‐MS/MS: Biomarkers for tacrolimus nephrotoxicity after renal transplantation

Calcineurin inhibitor nephrotoxicity, especially for the widely used tacrolimus, has become a major concern in post‐transplant immunosuppression. Multiparametric amino acid metabolomics is useful for biomarker identification of tacrolimus nephrotoxicity, for which specific quantitative methods are highlighted as a premise. This article presents a targeted metabolomic assay to quantify 33 amino acids and biogenic amines in human urine by high‐performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was carried out on an Agilent Zorbax SB‐C₁₈ column (3.0 × 150 mm, 5 μm) with addition of an ion‐pairing agent in the mobile phase, and MS/MS detection was achieved in both the positive and negative multiple reaction monitoring modes. Good correlation coefficients (r² > 0.98) were obtained for most analytes. Intra‐ and inter‐day precision, stability, carryover and incurred sample reanalysis met with the acceptance criteria of the guidance of the US Food and Drug Administration. Analysis on urine from healthy volunteers and renal transplantation patients with tacrolimus nephrotoxicity confirmed symmetric dimethylarginine and serine as biomarkers for kidney injury, with AUC values of 0.95 and 0.81 in receiver operating characteristic analysis, respectively. Additionally, symmetric dimethylarginine exhibited a tight correlation with serum creatinine, and was therefore indicative of renal function. The targeted metabolomic assay was time and cost prohibitive for amino acid analysis in human urine, facilitating the biomarker identification of tacrolimus nephrotoxicity.     

Determination of mycophenolic acid in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry and its pharmacokinetic application in kidney transplant patients

To implement and validate an analytical method by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC MS/MS) to quantify mycophenolic acid (MPA) in kidney transplant patients. Quantification of MPA was performed in an ACQUITY UPLC H Class system coupled to a Xevo TQD detector and it was extracted from plasma samples by protein precipitation. The chromatographic separation was achieved through an ACQUITY HSS C₁₈ SB column with 0.1% formic acid and acetonitrile (60:40 vol/vol) as mobile phase. The pharmacokinetic parameters were calculated by non‐compartmental analysis of MPA plasma concentrations from 10 kidney transplant patients. The linear range for MPA quantification was 0.2–30 mg/L with a limit of detection of 0.07 mg/L; the mean extraction recovery was 99.99%. The mean intra‐ and inter‐day variability were 2.98% and 3.4% with a percentage of deviation of 8.4% and 6.6%, respectively. Mean maximal concentration of 10 mg/L at 1.5 h, area under the concentration–time curve of 36.8 mg·h/L, elimination half‐life of 3.9 h, clearance of 0.32 L/h/kg and volume of distribution of 1.65 L/kg were obtained from MPA pharmacokinetics profiles. A simple, fast and reliable UPLC–MS/MS method to quantify MPA in plasma was validated and has been applied for pharmacokinetic analysis in kidney transplant patients.     

Detection of trace amounts of citrinin in dried orange peel by using an optimized extraction method coupled with ultra‐performance liquid chromatography–tandem mass spectrometry

A fast and sensitive method involving ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was introduced to detect citrinin in dried orange peel. A series of extraction, purification and chromatographic conditions was also systematically examined. With the proposed method, the obtained calibration graph was linear, with an R of 0.9996 within a concentration range of 0.5–10 ng/mL. The estimated limits of detection and quantification were 0.05 and 0.17 ng/mL, respectively. Under the selected conditions, the relative recoveries in different citrus products spiked with 1–10 ng/mL citrinin were 89.4–98.7% with RSDs of <2.5%. Compared with previously reported analytical methods, the newly developed UPLC–MS/MS method showed excellent sensitivity and good precision in detecting citrinin. The results indicated that it is a reliable and effective technique for the detection of trace citrinin in dried orange peel.     

Pharmacokinetic study of dendrobine in rat plasma by ultra‐performance liquid chromatography tandem mass spectrometry

Dendrobine, considered as the major active alkaloid compound, has been used for the quality control and discrimination of Dendrobium which is documented in the Chinese Pharmacopoeia. In this work, a sensitive and simple ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method for determination of dendrobine in rat plasma is developed. After addition of caulophyline as an internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C₁₈ (2.1 ×100 mm, 1.7 µm) column with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 264.2 → 70.0 for dendrobine and m/z 205.1 → 58.0 for IS. Calibration plots were linear throughout the range 2–1000 ng/mL for dendrobine in rat plasma. The RSDs of intra‐day and inter‐day precision were both <13%. The accuracy of the method was between 95.4 and 103.9%. The method was successfully applied to pharmacokinetic study of dendrobine after intravenous administration. Copyright © 2015 John Wiley & Sons, Ltd.     

A highly sensitive and efficient UPLC‐MS/MS assay for rapid analysis of tedizolid (a novel oxazolidinone antibiotic) in plasma sample

Tedizolid (TDZ) is a novel oxazolidinone class antibiotic, indicated for the treatment of acute bacterial skin and skin structure infections in adults. In this study a highly sensitive UPLC‐MS/MS assay was developed and validated for the determination of TDZ in rat plasma using rivaroxaban as an internal standard (IS). Both TDZ and IS were separated on an Acquity UPLC BEH? C₁₈ column using an isocratic mobile phase comprising of acetonitrile–20 mm ammonium acetate (85:15, v/v), eluted at 0.3 mL/min flow rate. The plasma sample was processed by liquid liquid extraction technique using ethyl acetate as an extracting agent. The analyte and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.09 > 343.10 for TDZ and m/z 435.97 > 144.94 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 0.74–1500 ng/mL and the lower limit of quantification was 0.74 ng/mL only. The developed assay was validated following standard guidelines for bioanalytical method validation (US Food and Drug Administration) and all the validation results were within the acceptable limits. The developed assay was successfully applied into a pharmacokinetic study in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

A simple and sensitive assay for eflornithine quantification in rat brain using pre‐column derivatization and UPLC‐MS/MS detection

Eflornithine (α‐difluoromethylornithine) has been used to treat second‐stage (or meningoencephalitic‐stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS)‐based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse‐phase C₁₈ UPLC column with a 6‐min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra‐ and interday accuracy and precision over a wide dynamic range (5000‐fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 μL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a sensitive LC‐MS/MS method for determination of tacrolimus on dried blood spots

A bioanalytical method for the quantification of tacrolimus (TAC) on dried blood spots (DBS) using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC‐ESI‐MS/MS) was developed and validated. It involves solvent extraction of a punch disk of DBS followed by liquid–liquid extraction. The analyte and the internal standard (IS, ascomycin) were separated on a phenyl column using an isocratic mobile phase elution at a flow rate of 0.3 mL/min. The assay was linear from 1 to 80 ng/mL. The mean recovery of TAC was 76.6%. Intra‐assay, inter‐assay imprecision and biases were all less than 15%. TAC on DBS was stable for at least 10 days at room temperature, and at least 24 h at 50°C. A chromatographic effect of the filter paper (Whatman 903) was not detected. The volume of blood (15–50 μL) and hematocrit of blood (ranging from 23.2 to 48.6%) did not show a significant influence on detection of TAC concentration by DBS‐LC‐MS/MS. Fifty samples from patients were detected by both DBS‐LC‐MS/MS and microparticle enzyme‐linked immunoassay (MEIA). TAC concentrations measured by DBS‐LC‐MS/MS method tended to be lower than those by MEIA. Copyright © 2012 John Wiley & Sons, Ltd.     

A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody,antibody‐conjugated drug and free payload of antibody–drug conjugates

A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65–613.00 ng/mL with an equation y = ax² + bx + c for the antibody‐conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with an equation y = ax² + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01–2200 ng/mL with an equation y = ax² + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐captured‐LC‐TOF‐MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine–citrulline linker at the early drug discovery stage.     

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.     

Validated UPLC‐MS/MS method for determination of moclobemide in human brain cell supernatant and its application to bidirectional transport study

A simple and sensitive analytical method based on ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) has been developed for determination of moclobemide in human brain cell monolayer as an in vitro model of blood–brain barrier. Brucine was employed as the internal standard. Moclobemide and internal standard were extracted from cell supernatant by ethyl acetate after alkalinizing with sodium hydroxide. The UPLC separation was performed on an Acquity UPLC^TM BEH C₁₈ column (50 × 2.1 mm, 1.7 µm, Waters, USA) with a mobile phase consisting of methanol–water (29.5:70.5, v/v); the water in the mobile phase contained 0.05% ammonium acetate and 0.1% formic acid. Detection of the analytes was achieved using positive ion electrospray via multiple reaction monitoring mode. The mass transitions were m/z 269.16 → 182.01 for moclobemide and m/z 395.24 → 324.15 for brucine. The extraction recovery was 83.0–83.4% and the lower limit of quantitation (LLOQ) was 1.0 ng/mL for moclobemide. The method was validated from LLOQ to 1980 ng/mL with a coefficient of determination greater than 0.999. Intra‐ and inter‐day accuracies of the method at three concentrations ranged from 89.1 to 100.9% for moclobemide with precision of 1.1–9.6%. This validated method was successfully applied to bidirectional transport study of moclobemide blood–brain barrier permeability. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid and robust confirmation and quantification of 11‐nor‐Δ9‐tetrahydrocannabinol‐9‐carboxylic acid (THC‐COOH) in urine by column switching LC‐MS‐MS analysis

A method for the rapid and robust confirmation of 11‐nor‐?9‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in urine involving basic hydrolysis with NaOH and direct injection of the hydrolysate in a column‐switching LC‐MS‐MS system was developed and validated. THCA‐d3 was used as internal standard. Detection was performed in negative‐ion mode by monitoring the transitions from the [M‐CO₂]‐ ion m/z 299.2→245.2 and and m/z 299.2→191.1 that were found to provide a better signal‐to‐noise ratio than the transition from the pseudomolecular ion at m/z 343. The high sensitivity of detection enabled the injection of a small volume (10 µl) of the NaOH hydrolysate which, together with the applied column switching system, proved to confer ruggedness to the method and to avoid the deterioration of the instrumental apparatus despite the large amount of inorganic ions in the hydrolysate. The LLOQ was established at 5 ng/ml, and the LLOD was calculated as 0.2 ng/ml (S/N =3). The method was submitted to thorough validation including evaluation of the calibration range (5–500 ng/ml), accuracy and precision, matrix effects, overall process efficiency, autosampler stability, carryover and cross‐talk, and 10‐times reduction of sample volume (0.1 ml). Proof of applicability was obtained by direct comparison with the reference GC‐MS method in use in the lab (the R² between the two methods was 0.9951). Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative analysis of costunolide and dehydrocostuslactone in rat plasma by ultraperformance liquid chromatography–electrospray ionization–mass spectrometry

Costunolide and dehydrocostuslactone are well‐known sesquiterpene lactones contained in many plants used as popular herbs, such as Saussurea lappa and Laurus novocanariensis, and have been considered as potential candidates for the treatment of various types of tumor. In the present work, a sensitive UPLC‐MS/MS for the quantification of costunolide and dehydrocostuslactone in biological matrices has been developed. The method is based on protein precipitation with acetonitrile followed by isocratic ultraperformance liquid chromatographic separation using methanol–formic acid (0.1% in water; 70:30, v/v) mobile phase. Detection was performed by ESI mass spectrometry in MRM mode with the precursor‐to‐product ion transitions m/z 233–187 and m/z 231–185, respectively. The calibration curves of analytes showed good linearity within the established range 0.19–760 ng/mL for costunolide and 0.23–908 ng/mL for dehydrocostuslactone. The lower limits of quantification of costunolide and dehydrocostuslactone were found to be 0.19 and 0.23 ng/mL, respectively. The intra‐day and inter‐day presicions of this method for the entire validation were less than coefficient of variation of 7% and the accuracy was within ±8% (relative error). The mean extraction recoveries were 73.8 and 75.3%, respectively. The method was found to be precise, accurate and specific during the study, and was successfully used to analyze the pharmacokinetics of costunolide and dehydrocostuslactone. Copyright © 2010 John Wiley & Sons, Ltd.     

Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a LC‐MS/MS method for simultaneous determination of metoprolol and its metabolites, α‐hydroxymetoprolol and O‐desmethylmetoprolol,in rat plasma: application to the herb–drug interaction study of metoprolol and breviscapine

A simple, specific and sensitive LC‐MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), α‐hydroxymetoprolol (HMT) and O‐desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC‐C₁₈ column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, and post‐column splitting (1:4) was used to give optimal interface flow rates (0.2 mL/min) for MS detection; the total run time was 8.5 min. Mass spectrometric detection was achieved using a triple‐quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42–7000 ng/mL for MET, 2.05‐4200 ng/mL for HMT and 1.95‐4000 ng/mL for DMT. The analytical method was successfully applied to herb–drug interaction study of MET and breviscapine after administration of breviscapine (12.5 mg/kg) and MET (40 mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

A UPLC–MS/MS method for analysis of vancomycin in human cerebrospinal fluid and comparison with the chemiluminescence immunoassay

Vancomycin (VCM) is clinically used in treating patients with postoperative intracranial infections. The cerebrospinal fluid (CSF) concentration of VCM varies greatly among patients. To guide the dosage regimens, monitoring of VCM in CSF is needed. However a method for analysis of VCM in human CSF is lacking. An ultraperformance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated for analysis of VCM in human CSF, and the agreement of UPLC–MS/MS and chemiluminescence immunoassay (CLIA) in the analysis of CSF VCM was evaluated. The ion transitions were m/z 725.5 > 144.1 for VCM and m/z 455.2 > 308.2 for methotrexate (internal standard). The agreement between UPLC–MS/MS and CLIA was evaluated by Bland–Altman plot in 179 samples. The calibration range of the UPLC–MS/MS method was 1–400 mg/L. The inaccuracy and imprecision were −0.69–10.80% and <4.95%. The internal standard normalized recovery and matrix factor were 86.14–99.31 and 85.84–92.07%, respectively. The measurements of CLIA and UPLC–MS/MS were strongly correlated (r > 0.98). The 95% limit of agreement of the ratio of CLIA to UPLC‐MS/MS was 61.66–107.40%. Further studies are warranted to confirm the results.     

A quantitative determination of fluorochloridone in rat plasma by UPLC‐MS/MS method: application to a pharmacokinetic study

A precise, high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed for the determination of fluorochloridone (FLC) in rat plasma. The extraction of analytes from plasma samples was carried out by protein precipitation procedure using acetonitrile prior to UPLC‐MS/MS analysis. Verapamil was proved as a proper internal standard (IS) among many candidates. The chromatographic separation based on UPLC was well optimized. Multiple reaction monitoring in positive electrospray ionization was used with the optimized MS transitions at: m/z 312.0 → 292.0 for FLC and m/z 456.4 → 165.2 for IS. This method was well validated with good linear response (r² > 0.998) observed over the investigated range of 3–3000 ng/mL and with satisfactory stability. This method was also characterized with adequate intra‐ and inter‐day precision and accuracy (within 12%) in the quality control samples, and with high selectivity and less matrix effect observed. Total running time was only 1.5 min. This method has been successfully applied to a pilot FLC pharmacokinetic study after oral administration. Copyright © 2016 John Wiley & Sons, Ltd.