HPLC–UV method for temozolomide determination in complex biological matrices: Application for in vitro,ex vivo and in vivo studies

A high‐performance liquid chromatography method for temozolomide (TMZ) determination in complex biological matrices was developed and validated for application in in vitro, ex vivo and in vivo studies of new nanotechnology‐based systems for TMZ nasal delivery. The method was able to quantify TMZ in nanoemulsions, following cellular uptake, in the porcine nasal mucosa and in mouse plasma and brain. Analyses were performed on a C₁₈ column at 35°C, under UV detection at 330 nm. The mobile phase was methanol–acetic acid 0.5% (30:70, v/v), eluted at an isocratic flow rate of 1.1 mL/min. The method was found to be specific, precise, accurate, robust and linear (0.05 to 5 μg/mL) for TMZ determination in all matrices. No interference of TMZ degradation products was found under various stress conditions such as acidic, alkaline, oxidative, light and thermal exposure, demonstrating stability. The method was applied for the quantification of TMZ in different matrices, i.e. the efficiency of nanoemulsions in vitro in increasing TMZ cellular uptake, ex vivo TMZ permeation and retention in the porcine nasal mucosa tissue, and for in vivo TMZ quantification in mouse brain following intranasal nanoemulsion administration compared with free TMZ.     

A simple and sensitive high‐performance liquid chromatography–electrochemical detection assay for the quantitative determination of monoamines and respective metabolites in six discrete brain regions of mice

A rapid, sensitive, and reproducible assay is described for the quantitative determination of the monoamine neurotransmitters dopamine, norepinephrine and serotonin, their metabolites, and the internal standard 3,4‐dihydroxybenzlyamine hydro‐bromide in mouse brain homogenate using high‐performance liquid chromatography with electrochemical detection. The method was validated in the following brain areas: frontal cortex, striatum, nucleus accumbens, hippocampus, substantia nigra pars compacta and ventral tegmental area. Biogenic amines and relevant metabolites were extracted from discrete brain regions using a simple protein precipitation procedure, and the chromatography was achieved using a C₁₈ column. The method was accurate over the linear range of 0.300–30 ng/mL (r = 0.999) for dopamine and 0.300–15 ng/mL (r = 0.999) for norepinephrine, 3,4‐dihydroxybenzlyamine hydro‐bromide, homovanillic acid and 5‐hydroxyindolacetic acid, with detection limits of ~0.125 ng/mL (5 pg on column) for each of these analytes. Accuracy and linearity for serotonin were observed throughout the concentration range of 0.625–30 ng/mL (r = 0.998) with an analytical detection limit of ~0.300 ng/mL (12 pg on column). Relative recoveries for all analytes were approximately ≥90% and the analytical run time was <10 min. The described method utilized minimal sample preparation procedures and was optimized to provide the sensitivity limits required for simultaneous monoamine and metabolite analysis in small, discrete brain tissue samples.     

The effect of N‐acetylcysteine on amphetamine‐mediated dopamine release in rat brain striatal slices by ion‐pair reversed‐phase high performance liquid chromatography

The amphetamine (AMPH)‐induced alteration in rat brain dopamine levels modified by N‐acetylcysteine (NAC) administration has been examined using isocratic ion‐pair reversed‐phase high‐performance liquid chromatography with electrochemical detection. The aim of the development of a novel validated evaluation scheme implying a double AMPH challenge was to enhance the efficiency of AMPH‐triggered dopamine release measurements in rat brain striatal slices by improving the reproducibility of the results. The proposed experimental protocol was tested in vivo and proved to be capable of fast and reliable drug screening for tracing the effect of NAC as a model compound in AMPH‐mediated dopaminergic response. The subcellular localization of the dopamine mobilizing effect of NAC has been established indirectly by the use of an irreversible dopamine vesicular depletor, reserpine. The antioxidant NAC at 10 mm plays an important role in the complete suppression of acute AMPH‐elicited dopamine release. The possible role of this quenching effect is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of 2‐arachidonoylglycerol, 1‐arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry

Endocannabinoids (ECs), such as anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, 2‐AG. Identification and quantification of 2‐AG, 1‐AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of 2‐AG. We have developed a sensitive and specific quantification method for simultaneous determination of 2‐AG, 1‐AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparation method. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N = 3) at 50, 25 and 65 fmol for 2‐AG, 1‐AG and AA, respectively. The methods were selective, precise (%R.S.D. < 10%) and sensitive over a range of 0.02–20, 0.01–10 and 0.05–50 ng/mg tissue for 2‐AG, 1‐AG and AA, respectively. The quantification method was validated with consideration of the matrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of 2‐AG from mouse brain extracts containing membrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of 2‐AG, 1‐AG and AA in mouse brain and adipose tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 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.     

A quantitative LC–MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue

Compound 27 {1, 12‐bis[4‐(4‐amino‐6,7‐dimethoxyquinazolin‐2‐yl)piperazin‐1‐yl]dodecane‐1,12‐dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC–MS/MS) has been developed for the quantification of this compound. Liquid–liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse‐phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2–200 ng/mL with the intra‐ and inter‐day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.     

Flow‐Injection Amperometric Method for Indirect Determination of Dopamine in the Presence of a Large Excess of Ascorbic Acid

A simple flow injection amperometric system for fast and indirect quantification of dopamine (DA) in the presence of a large excess of ascorbic acid (AA) is reported. The method consists of the application of three sequential potential pulses to an unmodified glassy carbon‐working electrode positioned in a wall‐jet cell. DA is indirectly detected at +0.35 V through the reduction of the oxidation product (o‐dopaminoquinone) electrochemically generated at +0.80 V. The third potential pulse (0.00 V) is applied for the regeneration (cleaning) of the unmodified working electrode. The limits of detection (LOD) and quantification (LOQ) were calculated as 50 and 170 nmol L^?1, respectively. Considering the LOQ value, the present methodology allows DA quantification in the presence up to 5000‐fold more of AA (1.0 mmol L^?1).     

Long‐Lived Charge Carriers in Mn‐Doped CdS Quantum Dots for Photoelectrochemical Cytosensing

Photoelectrochemical (PEC) biosensing with semiconductor quantum dots (QDs) has received great attention because it integrates the advantages of both photo‐excitation and electrochemical detection. During the photon‐to‐electricity conversion in PEC processes, electron–hole (charge) separation competes with electron–hole recombination, and the net effect essentially determines the performance of PEC biosensors. Herein, we propose a new approach for slowing down electron–hole recombination to increase charge separation efficiency for PEC biosensor development. Through doping with Mn²⁺, a pair of d bands (⁴T₁ and ⁶A₁) is inserted between the conduction and valence bands of CdS QDs, which alters the electron–hole separation and recombination dynamics, allowing the generation of long‐lived charge carriers with ms‐scale lifetime that decay about 10⁴–10⁵‐fold more slowly than in the case of undoped QDs. Photocurrent tests indicated that Mn²⁺ doping resulted in an approximately 80 % increase in photocurrent generation compared with undoped CdS QDs. For application, the Mn‐doped CdS QDs were coated on the surface of a glassy carbon electrode and functionalized with a cell surface carbohydrate‐specific ligand (3‐aminophenylboronic acid). In this way, a sensitive cytosensor for K562 leukemia cells was constructed. Moreover, the sugar‐specific binding property of 3‐aminophenylboronic acid allowed the electrode to serve as a switch for the capture and release of cells. This has been further explored with a view to developing a reusable PEC cytosensing platform.     

Analysis of ketone‐based neurosteroids by reactive low‐temperature plasma mass spectrometry

Rationale

Neurosteroids are important signalling molecules that modulate neuronal activity. Their low concentrations and low volatility make neurosteroid detection and quantification by ambient mass spectrometry challenging. Here we develop a reactive low‐temperature plasma mass spectrometry (LTP‐MS) method and demonstrate its potential for fast screening and quantification of neurosteroids in mouse brain.

Methods

Ketone‐based neurosteroids were analysed with the LTP‐MS method. The plasma of the LTP was heated in order to improve the desorption efficiency of low‐volatility neurosteroids. Methylamine with a concentration of 500 ppbv was employed as the reactive reagent. Neurosteroids in mouse brain tissue extracts were detected in 70 s with mass errors less than ±3 ppm due to coupling of the ion source with a high‐performance mass spectrometer.

Results

Reaction between neurosteroids and methylamine, seeded into the LTP gas stream, resulted in the formation of protonated methylamine–neurosteroid adducts with 5‐ to 100‐fold abundances, compared to [M + H]⁺ ions detected in non‐reactive LTP‐MS. The lowest detectable concentrations of neurosteroid standards were in the range of ng/mL. Concentrations of neurosteroids in male and female mouse brain extracts as determined with reactive LTP‐MS were on the level of ng/g, comparable to results obtained with high‐performance liquid chromatography–tandem mass spectrometry.

Conclusions

The developed reactive LTP‐MS is capable of providing sensitive identification and quantification of ketone‐based neurosteroids in mouse brain extracts with minimal sample treatment, and showcases the potential of reactive LTP‐MS as a tool for fast screening of neurosteroid levels in brain.

     

An HPLC‐ECD method for monoamines and metabolites quantification in cuttlefish (cephalopod) brain tissue

The cuttlefish belongs to the mollusk class Cephalopoda, considered as the most advanced marine invertebrates and thus widely used as models to study the biology of complex behaviors and cognition, as well as their related neurochemical mechanisms. Surprisingly, methods to quantify the biogenic monoamines and their metabolites in cuttlefish brain remain sparse and measure a limited number of analytes. This work aims to validate an HPLC‐ECD method for the simultaneous quantification of dopamine, serotonin, norepinephrine and their main metabolites in cuttlefish brain. In comparison and in order to develop a method suitable to answer both ecological and biomedical questions, the validation was also carried out on a phylogenetically remote species: mouse (mammals). The method was shown to be accurate, precise, selective, repeatable and sensitive over a wide range of concentrations for 5‐hydroxyindole‐3‐acetic acid, serotonin, dopamine, 3,4‐dihydroxyphenylacetic acid and norepinephrine in the both extracts of cuttlefish and mouse brain, though with low precision and recovery for 4‐hydroxy‐3‐methoxyphenylethylene glycol. Homovanillic acid, accurately studied in rodents, was not detectable in the brain of cuttlefish. Overall, we described here the first fully validated HPLC method for the routine measurement of both monoamines and metabolites in cuttlefish brain. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and full validation of an HPLC methodology to quantify atorvastatin and curcumin after their intranasal co‐delivery to mice

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C₁₈ column, at 35°C, using a mobile phase composed of acetonitrile–methanol–2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r² ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.     

Development of a PDMS‐based microchip electrophoresis device for continuous online in vivo monitoring of microdialysis samples

A PDMS‐based microfluidic system for online coupling of microdialysis sampling to microchip electrophoresis with fluorescence detection for in vivo analysis of amino acid neurotransmitters using naphthalene‐2,3‐dicarboxaldehyde and sodium cyanide as the derivatization reagents is described. Fabricating chips from PDMS rather than glass was found to be simpler and more reproducible, especially for chips with complex designs. The microchip incorporated a 20‐cm serpentine channel in which sample plugs were introduced using a “simple” injection scheme; this made fluid handling and injection on‐chip easier for the online system compared with gated or valve‐based injection. The microchip was evaluated offline for the analysis of amino acid standards and rat brain microdialysis samples. Next, precolumn derivatization was incorporated into the chip and in vivo online microdialysis‐microchip electrophoresis studies were performed. The system was employed for the continuous monitoring of amino acid neurotransmitters in the extracellular fluid of the brain of an anesthetized rat. Fluorescein was dosed intravenously and monitored simultaneously online as a marker of in vivo blood–brain barrier permeability. The microdialysis‐microchip electrophoresis system described here will be employed in the future for simultaneous monitoring of changes in blood–brain barrier permeability and levels of amino acid neurotransmitters in the rat stroke model.     

Highly Sensitive Dopamine Detection Using a Bespoke Functionalised Carbon Nanotube Microelectrode Array

Understanding how the brain works requires developing advanced tools that allow measurement of bioelectrical and biochemical signals, including how they propagate between neurons. The introduction of nanomaterials as electrode materials has improved the impedance and sensitivity of microelectrode arrays (MEAs), allowing high quality recordings of single cells in situ using electrode diameters of ≤20 μm. MEAs also have the potential to measure electroactive biological molecules in situ, such as dopamine, a neurotransmitter in the nervous system. Thus, this work focused on fabricating a functionalised carbon nanotube (CNT)‐based MEA to demonstrate its potential for future measurement of small signals generated from excitable cells. To this end, the functionalised CNT MEA has recorded one of the lowest electrochemical interfacial impedances available in the literature, 2.8±0.2 kΩ, for an electrode of its geometric surface area. Electrochemical detection of dopamine revealed again one of the best sensitivity values per area available in the literature, 9.48 μA μM⁻¹ mm⁻². Additionally, a limit of detection of 7 nM was recorded for dopamine using the functionalised CNT MEA, with selectivity against common electrochemical interferents such as ascorbic acid. These results indicate improvement beyond currently available MEAs, along with the feasibility of using these devices for multi‐site detection of physiologically relevant electroactive biomolecules.     

A New Signal‐On Photoelectrochemical Biosensor Based on a Graphene/Quantum‐Dot Nanocomposite Amplified by the Dual‐Quenched Effect of Bipyridinium Relay and AuNPs

A new photoelectrochemical (PEC) biosensor was developed by using carboxyl‐functionalized graphene and CdSe nanoparticles. This sensitive interface was then successfully applied to detection of thrombin based on the dual‐quenched effect of PEC nanoparticle, which relied on the electron transfer of a bipyridinium relay and energy transfer of AuNPs. After recognition with an aptamer, the PEC nanoparticle was removed and a signal‐on PEC biosensor was obtained. Moreover, the bio‐barcode technique used in the preparation of PEC nanoparticle could avoid cross‐reaction and enhances the sensitivity. Taking advantages of the various methods mentioned above, the sensitivity could be easily enhanced. In addition, in this work we also investigated graphene that was modified with different functional groups and AuNPs of different particle sizes. Under optimal conditions, a detection limit of 5.9×10^?15 M was achieved. With its simplicity, selectivity, and sensitivity, this strategy shows great promise for the fabrication of highly efficient PEC biosensors.     

Development of a new UPLC‐MSMS method for the determination of temozolomide in mice: application to plasma pharmacokinetics and brain distribution study

A sensitive and accurate liquid chromatography method with mass spectrometry detection was developed and validated for the quantification of temozolomide in mouse plasma and brain. Theophyllin was used as the internal standard. A single‐step protein precipitation was used for plasma and brain sample preparation. The method was validated with respect to selectivity, extraction recovery, linearity, intra‐ and inter‐day precision and accuracy, limit of quantification and stability. The method has a limit of quantification of 50 ng/mL for temozolomide in plasma and 125 ng/g in brain. This method was used successfully to perform brain and plasma pharmacokinetic studies of temozolomide in mice after intraperitoneal administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a rapid HPLC method for quantitation of SP‐141, a novel pyrido[b]indole anticancer agent,and an initial pharmacokinetic study in mice

There is an increasing interest in targeting the MDM2 oncogene for cancer therapy. SP‐141, a novel designed small molecule MDM2 inhibitor, exerts excellent in vitro and in vivo anticancer activity. To facilitate the preclinical development of this candidate anticancer agent, we have developed an HPLC method for the quantitative analysis of SP‐141. The method was validated to be precise, accurate, and specific, with a linear range of 16.2–32,400 ng/mL in plasma, 16.2–6480 ng/mL in homogenates of brain, heart, liver, kidneys, lungs, muscle and tumor, and 32.4–6480 ng/mL in spleen homogenates. The lower limit of quantification was 16.2 ng/mL in plasma and all the tissue homogenates, except for spleen homogenates, where it was 32.4 ng/mL. The intra‐ and inter‐assay precisions (coefficient of variation) were between 0.86 and 13.39%, and accuracies (relative errors) ranged from ?8.50 to 13.92%. The relative recoveries were 85.6–113.38%. SP‐141 was stable in mouse plasma, modestly plasma bound and metabolized by S9 microsomal enzymes. We performed an initial pharmacokinetic study in tumor‐bearing nude mice, demonstrating that SP‐141 has a short half‐life in plasma and wide tissue distribution. In summary, this HPLC method can be used in future preclinical and clinical investigations of SP‐141. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterisation of a Platinum‐based Electrochemical Biosensor for Real‐time Neurochemical Analysis of Choline

A choline biosensor was characterised in detail to determine the effects of physiologically relevant parameters on the ability of the sensor to reliably detect neurochemical changes in choline. This first generation Pt‐based polymer enzyme composite sensor displayed excellent shelf‐life and biocompatibility with no significant decrease in choline sensitivity observed following 14 days of storage dry, or in ex‐vivo rodent brain tissue. However, subjecting the sensor to repeated calibrations and storage over the same period resulted in significant decreases (20–70 %) due to enzyme denaturation associated with the repeated calibration and storage cycles. Potential interference signals generated by the principal electroactive interferents present in the brain were minimal; typically <1 % of the choline current response at in vivo levels. Additionally, changing temperature over the physiologically relevant range of 34–40 °C had no effect on sensitivity, while increasing pH between 7.2 and 7.6 produced only a 5 % increase in signal. The limit of detection of the sensor was in the low μM range (0.11±0.02 μM), while the in vitro response time was determined to be less than the solution mixing time and within ca. 5 s, suggesting potential sub‐second in vivo response characteristics. Finally, the sensor was implanted in the striatum of freely moving rats and demonstrated reliable detection of physiological changes in choline in response to movement, and pharmacological manipulation by injection of choline chloride.     

Validation of a new high‐throughput method to determine urinary S‐phenylmercapturic acid using low‐temperature partitioning extraction and ultra high performance liquid chromatography–mass spectrometry

A new highly sensitive and environmentally friendly analytical method, using low‐temperature partition extraction and ultra‐high‐performance liquid chromatography with tandem mass spectrometry, without the use of a labeled analyte, was developed and validated to determine and quantify urinary S‐phenylmercapturic acid in urine samples. The World Health Organization, in its guidelines for air quality in Europe, recognizes that benzene is carcinogenic to humans and there is no safe level of exposure. Urinary S‐phenylmercapturic acid is a sensitive and specific biological marker of exposure to benzene. The new analytical method, extraction, and analysis, were linear in the working range between 0.1 and 200.0 μg/L, precise (relative standard deviation lower than 6.0%), accurate (97.0–105.0%), and sensitive. The method's limits of detection and quantification were 0.02 and 0.084 μg/L, respectively. The recovery with the low‐temperature partition extraction was 96.1%, with relative standard deviation less than 3.8%. The method is simple, accurate, and reproducible, and has been successfully applied in the evaluation of nonoccupational exposure to benzene, by urinary S‐phenylmercapturic acid in urine samples.     

Development and validation of a single HPLC method for determination of α‐tocopherol in cell culture and in human or mouse biological samples

A straightforward and common analytical method for α‐tocopherol (αT) determination in various biological samples, including plasma, red blood cells (RBC), tissues and cultured cell lines, was developed and validated, using a reverse phase‐chromatographic method (RP‐HPLC). Even though many chromatographic methods for αT determination have been reported, most of them require readjustment when applied to different types of samples. Thus, an effective and simple method for αT determination in different biological matrices is still necessary, specifically for translational research. This method was applied using a C₁₈ column (250 × 4.6 mm, 5 µm particle size) under isocratic elution with MeOH:ACN:H₂O (90:9:1 v/v/v) at a flow rate of 1 mL/min and detected using photodiode array at 293 nm. Linearity (r >0.9997) was observed for standard calibration with inter‐ and intraday variation of standard <4%. Lower limits of detection and quantification for αT in this assay were 0.091 and 0.305 µg/mL respectively. Validation proved the method to be selective, linear, accurate and precise. The method was successfully applied in great variety of biological samples, that is, human and mouse plasma, RBCs, murine tissues and human/mouse/rat cultured cell lines. More importantly, a single protocol of extraction and detection can be applied, making this method very convenient for standardization of different types of samples. Copyright © 2014 John Wiley & Sons, Ltd.