Pressurized CEC coupled with QTOF‐MS for urinary metabolomics

Pressurized CEC (pCEC) coupled with ESI‐QTOF‐MS using a sheathless interface was applied for metabolomics to develop an alternative analytical method for metabolic profiling of complex biofluid samples such as urine. The hyphenated system was investigated with mixed standards and pooled urine samples to evaluate its precision, repeatability, linearity, sensitivity, and selectivity. The applied voltage, mobile phase, and gradient elution were optimized and applied for the analysis of urinary metabolites. Multivariate data analysis was subsequently performed and used to distinguish lung cancer patients from healthy controls successfully. High separation efficiency has been achieved in pCEC due to the EOF. For metabolite identification, the pCEC‐MS separation mechnism was helpful for discriminating the fragment ions of glutamine conjugates from co‐eluted metabolites. Three glutamine conjugates, including phenylacetylglutamine, acylglutamine C8:1, and acylglutamine C6:1 were identified among 16 differential urinary metabolites of lung cancer. Receiver‐operating‐characteristic analysis of acylglutamine C8:1 resulted in an area‐under‐curve value of 0.882. Overall, this work suggests that this pCEC‐ESI‐QTOF‐MS method may provide a novel and useful platform for metabolomic studies due to its superior separation and identification.     

CE with on‐line detection by ICP‐MS for studying the competitive binding of zinc against cadmium for glutathione

Development of a feasible method for studying the competitive interaction between a pair of antagonists is essential for understanding the antagonism of trace metals in biological systems. Herein, we report the application of CE on‐line coupled with ICP mass spectroscopy (CE‐ICP‐MS) to investigate the competitive binding of Zn²⁺ against Cd²⁺ for glutathione (GSH), which is related to the detoxification of Cd²⁺ in biological system, and introduce a method to evaluate the kinetics and thermodynamics for the competitive binding of Zn²⁺ against Cd²⁺ for GSH. The CE‐ICP‐MS hybrid technique allows easy and sensitive probing of the competitive binding of Zn²⁺ against Cd²⁺ for GSH and quantitative determination of the important thermodynamic and kinetic parameters of the competitive binding of Zn²⁺ against Cd²⁺ for GSH. Owing to the high sensitivity and element selectivity with multi‐elements detection capacity of ICP‐MS, we detailed the evaluation of the kinetics and thermodynamics describing the competition of Zn²⁺ against Cd²⁺ for GSH from the systematic data obtained by CE‐ICP‐MS. The competitive binding of Zn²⁺ against Cd²⁺ for GSH was demonstrated exothermic and thermodynamically favorable (ΔG=?7.2 kJ/mol) and driven entirely by a large favorable enthalpy decrease (ΔH=?15.1 kJ/mol) but with an unfavorable entropy decrease (ΔS=?25.6 J/mol/K). The kinetic data were fit to a second‐order equation with the reaction rate constant (k) of (2.18±0.10)×10² L/(mol·s) under the simulated physiological condition.     

LC‐MS/MS method for the determination of pitolisant: application to rat pharmacokinetic and brain penetration studies

A simple and sensitive LC‐MS/MS method was developed and validated for the quantitation of pitolisant, an H₃ receptor antagonist/inverse agonist. Acetonitrile protein precipitation technique was used to prepare rat blood and brain tissue homogenate samples by using aripiprazole as internal standard (IS). Chromatographic separation was performed by using Xbridge column (2.1 × 50 mm, 3.5 µm) with a gradient elution program. The mobile phase consists of ammonium formate (10 mm ) with 0.2% formic acid and acetonitrile. Multiple reaction monitoring mode was used in positive polarity with a transition of m/z 296.3 → 98.2 for the pitolisant and m/z 448.2 → 285.3 for the IS. The calibration curves were linear in the range of 0.1–100 ng/mL in both the blood and brain homogenate samples. This method was applied to quantify samples obtained from the pharmacokinetic and brain penetration studies in male wistar rats. Mean maximum concentration, area under the curve from zero to infinity and half‐life of the pitolisant were found to be 3.4 ± 1.7 ng/mL, 5 ± 4 ng h/mL and 1.9 ± 0.3 h, respectively, after a 3 mg/kg oral dose. The mean calculated concentrations in the brain were found to be 38, 60 and 52 ng/g at 0.5, 1 and 2 h, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Separation of tryptic peptides of native and glycated BSA using open‐tubular CEC with salophene–lanthanide–Zn2+ complex as stationary phase

Open‐tubular CEC (OT‐CEC) with a new stationary phase, salophene–lanthanide–Zn²⁺ complex, has been applied to the separation of tryptic peptides of native BSA and BSA glycated by glucose and ribose. Glycation of proteins (non‐enzymatic modification by sugars) significantly affects their properties and it is of great importance from a physiological point of view. Separation of tryptic peptides of glycated BSA by CZE was poor because of their strong adsorption to the bare fused silica capillary. An improved separation of tryptic peptides of both native and glycated BSA was achieved by OT‐CEC in the fused silica capillary non‐covalently coated with salophene–lanthanide–Zn²⁺ complex, which suppressed the adsorption of peptides to the capillary and via specific interactions with some (glyco)peptides enhanced selectivity of the separation. Significant differences have been found in OT‐CEC analyses of tryptic hydrolysates of native and glycated BSA. In OT‐CEC‐UV profile of tryptic peptides of native BSA, 44 peaks could be resolved, whereas a reduced number of 38 peaks were observed in the profile of tryptic peptides of glucose‐glycated BSA and only 30 peaks were found in the case of ribose‐glycated BSA. The developed OT‐CEC can be potentially used for monitoring of protein glycation.     

Novel UHPLC‐MS/MS method for the determination of rotigotine in the plasma of patients with Parkinson's disease

A novel ultra‐high‐pressure liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of the dopamine receptor agonist rotigotine in human plasma. Following liquid–liquid extraction with tert‐ butyl methyl ether from 500 μL plasma, the chromatographic analysis was performed on a Gemini NX3 column using 5 mm pH 5.0 ammonium acetate–5 mm ammonium acetate in methanol as binary gradient mobile phase, at a flow rate of 0.3 mL/min. The MS/MS ion transitions were 316.00 → 147.00 for rotigotine and 256.10 → 211.00 for the internal standard (lamotrigine). The lower limit of quantitation was 50 pg/mL and the linearity was determined from 50 to 2500 pg/mL. The mean recovery was 96.9%. Both intra‐ and interassay imprecision and inaccuracy were ≤15% at all quality control concentrations. The method was successfully applied to measure morning trough plasma rotigotine concentrations in a series of patients with Parkinson's disease on chronic treatment. The present study describes the first fully validated method for rotigotine determination in human plasma.     

Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l ‐glutamine and N‐acetyl‐d ‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.     

Ultrastiff Hydrogels Prepared by Schiff's Base Reaction of Bis(p‐Formylphenyl) Sebacate and Pillar[5]arene Appended with Multiple Hydrazides

Herein a facile method is reported to prepare polymer gels based on the formation of acylhydrazone bond under mild conditions. A pillar[5]arene derivative appended with ten hydrazide groups provides multiple sites for the reaction with the aldehyde groups of bis(p‐formylphenyl) sebacate in the presence of a small amount of HCl as the catalyst in dimethyl sulfoxide (DMSO), producing transparent polymer organogels. The mechanical properties of gels can be easily tuned by the molar ratio of the reactant compounds. After solvent exchange from DMSO to water, translucent polymer hydrogels with dramatically enhanced strength and stiffness are obtained. The tensile breaking stress and Young's modulus of hydrogels are 20−60 and 1.2–2.7 MPa, respectively, 100 and 20 times those of the corresponding organogels. These robust hydrogels with ultrahigh stiffness should find applications such as in load‐bearing artificial organs. This work should merit designing functional materials using other macrocycles.

     

Development and validation of an LC‐ESI‐MS/MS quantification method for a potential γ‐aminobutyric acid transporter 3 (GAT3) marker and its application in preliminary MS binding assays

Binding assays for the γ‐aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry‐based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC‐ESI‐MS/MS quantification method for DDPM‐1007 {(RS)‐1‐[4,4,4‐Tris(4‐methoxyphenyl)but‐2‐en‐1‐yl]piperidine‐3‐carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C₈ column in combination with a mobile phase composed of 10 mm ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 μL/min DDPM‐1007 could be analyzed in the positive multiple reaction monitoring mode [(m/z) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM‐1007 [(²H₉)DDPM‐1007] was synthesized and employed as internal standard. This way DDPM‐1007 could be quantified in a range from 100 pm to10 nm in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra‐ and inter‐batch accuracy. Based on this LC‐ESI‐MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM‐1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM‐1007 at GAT3 could be unambiguously detected. Additionally, the established LC‐MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM‐1007, as exemplified for its logD determination. Copyright © 2012 John Wiley & Sons, Ltd.     

A convenient and sensitive method for haloacetic acid analysis in tap water by on‐line field‐amplified sample‐stacking CE–ESI‐MS

In this study, we propose a simple strategy based on flow injection and field‐amplified sample‐stacking CE–ESI‐MS/MS to analyze haloacetic acids (HAAs) in tap water. Tap water was passed through a desalination cartridge before field‐amplified sample‐stacking CE–ESI‐MS/MS analysis to reduce sample salinity. With this treatment, the signals of the HAAs increased 300‐ to 1400‐fold. The LODs for tap water analysis were in the range of 10 to 100 ng/L, except for the LOD of monochloroacetic acid (1 μg/L in selected‐ion monitoring mode detection). The proposed method is fast, convenient, and sensitive enough to perform on‐line analysis of five HAAs in the tap water of Taipei City. Four HAAs, including trichloroacetic acid, dichloroacetic acid, dibromoacetic acid, and monobromoacetic acid, were detected at concentrations of approximately 1.74, 1.15, 0.16, and 0.15 ppb, respectively.     

Preparation and application of trimethylamine amination polychloromethyl styrene nanolatex coated capillary column for the determination of bromate by field‐amplified sample stacking open‐tubular capillary electrochromatography

A new trimethylamine amination polychloromethyl styrene nanolatex (TMAPL) and TMAPL coated capillary column (ccc‐TMAPL) were successfully prepared. The TMAPL coating was characterized with reversed steady EOF values of ca. ?16.8 × 10^?5 cm² V^?1 s^?1. It was applied to establish open‐tubular (OT) CEC and field‐amplified sample stacking (FASS) OT‐CEC methods for the determination of bromate in tap water. Compared to OT‐CEC, the LOD with FASS‐OT‐CEC was improved from 80 to 8 ng/mL. The developed FASS‐OT‐CEC method was practically used for the analysis of bromate in tap water samples with recoveries ranging from 93.6 to 103.5%.     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of Cefalothin and Cefazolin in Human Plasma,Urine and Peritoneal Dialysate by UHPLC‐MS/MS: application to a pilot pharmacokinetic study in humans

An ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) method for the analysis of cefazolin and cefalothin in human plasma (total and unbound), urine and peritoneal dialysate has been developed and validated. Total plasma concentrations are measured following protein precipitation and are suitable for the concentration range of 1–500 µg/mL. Unbound concentrations are measured from ultra‐filtered plasma acquired using Centrifree^® devices and are suitable for the concentration range of 0.1–500 µg/mL for cefazolin and 1–500 µg/mL for cefalothin. The urine method is suitable for a concentration range of 0.1–20 mg/mL for cefazolin and 0.2–20 mg/mL for cefalothin. Peritoneal dialysate concentrations are measured using direct injection, and are suitable for the concentration range of 0.2–100 µg/mL for both cefazolin and cefalothin. The cefazolin and cefalothin plasma (total and unbound), urine and peritoneal dialysate results are reported for recovery, inter‐assay precision and accuracy, and the lower limit of quantification, linearity, stability and matrix effects, with all results meeting acceptance criteria. The method was used successfully in a pilot pharmacokinetic study with patients with peritoneal dialysis‐associated peritonitis, receiving either intraperitoneal cefazolin or cefalothin. Copyright © 2015 John Wiley & Sons, Ltd.     

CE‐MS with electrokinetic supercharging and application to determination of neurotransmitters

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI‐MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI‐MS. Using EKS, the sensitivity of the method was improved 5000‐fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10 pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE‐ESI‐MS/MS to analyze biological samples.     

Development of an LC‐MS/MS method for quantification of kadsurenone in rat plasma and its application to a pharmacokinetic study

A sensitive and rapid LC‐MS/MS method was developed and validated for the determination of kadsurenone in rat plasma using lysionotin as the internal standard (IS). The analytes were extracted from rat plasma with acetonitrile and separated on a SB‐C₁₈ column (50 × 2.1 mm, i.d.; 1.8 µm) at 30 °C. Elution was achieved with a mobile phase consisting of methanol–water–formic acid (65:35:0.1, v/v/v) at a flow rate of 0.30 mL/min. Detection and quantification for analytes were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 357.1 → 178.1 for kadsurenone, and m/z 345.1 → 315.1 for IS. Calibration curves were linear over a concentration range of 4.88–1464 ng/mL with a lower limit of quantification of 4.88 ng/mL. The intra‐ and inter‐day accuracies and precisions were <8.9%. The LC‐MS/MS assay was successfully applied for oral pharmacokinetic evaluation of kadsurenone using the rat as an animal model. Copyright © 2013 John Wiley & Sons, Ltd.     

LC‐MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study

A sensitive and selective LC‐MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid–liquid extraction, the analytes were separated on a Zorbax SB‐C₁₈ column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow‐rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry‐over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet. Copyright © 2013 John Wiley & Sons, Ltd.     

A sensitive LC‐MS/MS method for simultaneous determination of R‐bambuterol and its active metabolite R‐terbutaline in human plasma and urine with application to a clinical pharmacokinetic study

A sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for simultaneous determination of R‐bambuterol and its active metabolite R‐terbutaline in human plasma and urine was established. The inhibition for the biotransformation of R‐bambuterol in plasma was fully investigated. Plasma samples were prepared on ice and neostigmine metilsulfate added as a cholinesterase inhibitor immediately after sample collection. All samples were extracted with ethyl acetate and separated on a C₁₈ column under gradient elution with a mobile phase consisting of methanol and water containing 5 mm ammonium acetate at a flow rate of 0.6 mL/min. The analytes were detected by an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte in plasma. In urine samples, the LLOQs were 20.00 and 500.0 pg/mL for R‐bambuterol and R‐terbutaline, respectively. The intra‐ and inter‐day precisions were <12.7 and <8.6% for plasma and urine, respectively. The analytical runtime within 6.0 min per sample made this method suitable for high‐throughput determination. The validated method has been successfully applied to the human pharmacokinetic study of R‐bambuterol involving 10 healthy volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

An LC‐MS/MS method for determination of calceorioside B with cardiomyocyte protective activity in rat plasma and application to a pharmacokinetic study

A simple, sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the determination of calceorioside B (CLB) in rat plasma. Detection was performed on a Thermo Scientific Hypersil Gold chromatography column using isocratic elution with a mobile phase of methanol–5 m m ammonium acetate–formic acid (70:30:0.1, v/v/v). Mass spectrometry was performed in selection reaction monitoring mode using a positive electrospray ionization interface. Good linearity was found for CLB in plasma in the linear range of 1.00–500 ng/mL (r > 0.9960). The validated method was successfully applied to the pharmacokinetic study of CLB in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a fast LC‐MS/MS assay for the determination of deferiprone in human plasma and application to pharmacokinetics

A fast and accurate liquid chromatography/tandem mass spectrometric (LC‐MS/MS) assay was first developed and validated for the determination of deferiprone in human plasma. The analytes were extracted with acetonitrile from only 50 μL aliquots of human plasma to achieve the protein precipitation. After extraction, chromatographic separation of analytes in human plasma was performed using a Synergi Fusion‐RP 80A column at 30 °C. The mobile phase consisted of methanol and 0.2% formic acid containing 0.2 mM EDTA (60:40, v/v). The flow rate of the mobile phase was 0.8 mL/min. The total run time for each sample analysis was 4 min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor‐to‐parent ion transitions m/z 140.1 → 53.1 for deferiprone and m/z 143.1 → 98.1 for internal standard. A linear range was established from 0.1 to 20 µg/mL. The limit of detection was determined as 0.05 µg/mL. The validated method was estimated for linearity, recovery, stability, precision and accuracy. Intraday and interday precisions were 4.3–5.5 and 4.6–7.3%, respectively. The recovery of deferiprone was in the range of 80.1–86.8%. The method was successfully applied to a pharmacokinetic study of deferiprone in six thalassemia patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of automated SPE‐LC‐MS/MS method for determination of indapamide in human whole blood and its application to real study samples

A fast and simple liquid chromatography–electrospray ionization tandem mass spectrometry method for determination of indapamide in human whole blood was developed and validated. The sample extraction of indapamide from human whole blood was achieved using automated solid‐phase extraction. Chromatographic separation was performed on Kinetex C₁₈ column (100 × 2.1 mm, 1.7 µm particle size) using acetonitrile and 2 mm ammonium formate in ratio 90:10 (v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode using positive electrospray ionization for indapamide and the internal standard (zolpidem tartarate). The total run time was 2.5 min. The present method was found to be linear in the concentration range of 1–50 ng/mL with the coefficient of determination 0.9987. The absolute recoveries of indapamide were 90.51–93.90%. The method was validated according the recommendations for validation of bioanalytical methods of European Medicines Agency guideline and was successfully used to analyze human whole blood samples for application in a pharmacokinetic study. Copyright © 2013 John Wiley & Sons, Ltd.