Journal of Chromatography B (v.879, #9-10)

A novel, precise, accurate and rapid isocratic reversed-phase high performance liquid chromatographic/ultraviolet (RP-HPLC/UV) method was developed, optimized and validated for simultaneous determination of rosuvastatin and atorvastatin in human serum using naproxen sodium as an internal standard. Effect of different experimental parameters and various particulate columns on the analysis of these analytes was evaluated. The method showed adequate separation for rosuvastatin and atorvastatin and best resolution was achieved with Brownlee analytical C18 column (150 × 4.6 mm, 5 μm) using methanol–water (68:32, v/v; pH adjusted to 3.0 with trifluoroacetic acid) as a mobile phase at a flow rate of 1.5 ml/min and wavelength of 241 nm. The calibration curves were linear over the concentration ranges of 2.0–256 ng/ml for rosuvastatin and 3.0–384 ng/ml for atorvastatin. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for rosuvastatin were 0.6 and 2.0 ng/ml while for atorvastatin were 1.0 and 3.0 ng/ml, respectively. All the analytes were separated in less than 7.0 min. The proposed method could be applied for routine laboratory analysis of rosuvastatin and atorvastatin in human serum samples, pharmaceutical formulations, drug–drug interaction studies and pharmacokinetics studies.
Keywords: Atorvastatin; Rosuvastatin; RP-HPLC/UV; Optimization; Validation;

Potential application of hydrogel-based strong anion-exchange membrane for plasmid DNA purification by Luyang Zhong; Jeno Scharer; Murray Moo-Young; Drew Fenner; Lisa Crossley; C. Howie Honeyman; Shing-Yi Suen; C. Perry Chou (564-572).
The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid recovery yield of ∼90%. The effect of the inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance was systematically investigated. Plasmid DNAs with smaller tertiary structure tended to have a better recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other. Membrane pre-treatment significantly improved pore distribution and increased membrane porosity resulting in a better adsorption, recovery, and higher flux. The selection of proper operating pH led to further improvement. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH.
Keywords: Plasmid DNA; Hydrogel; Anion-exchange membrane chromatography; Membrane pre-treatment; Environmental Scanning Electron Microscopy (ESEM);

A comparative analytical assessment of iodides in healthy and pathological human thyroids based on IC-PAD method preceded by microwave digestion by Anna Błażewicz; Grażyna Orlicz-Szczęsna; Piotr Szczęsny; Andrzej Prystupa; Anna Grzywa-Celińska; Marcin Trojnar (573-578).
The aim of the study was to examine correlations between the content of iodides in 66 nodular goiters and 100 healthy human thyroid tissues (50- frozen and 50 formalin-fixed). A fast, accurate and precise ion chromatography method on IonPac AS11 chromatographic column (Dionex, USA) with a pulsed amperometric detection (IC-PAD) followed by alkaline digestion with tetramethylammonium hydroxide (TMAH) in a closed system and with the assistance of microwaves was developed and used for the comparative analysis of two types of human thyroid samples. Statistical analysis revealed over eightfold reduction of iodine concentration in the pathological tissues (the mean value was 77.13 ± 14.02 ppm) in comparison with the control group (622.62 ± 187.11 ppm for frozen samples and 601.49 ± 192.11 ppm for formalin-fixed ones). A good correspondence (for 10 additional determinations) between the certified (3.38 ± 0.02 ppm with variation coefficient (V.C.) of 0.59% for Standard Reference Material (SRM) NIST 1549-non-fat milk powder) and the measured iodine concentrations (3.52 ± 0.29 ppm; V.C. = 10%) was achieved. It was pointed out that the way of tissue preservation (either in formalin or by freezing) had no significant effect on the iodine determination result (α  = 0.1). Significantly lower iodide content was found in nodular goiter thyroid samples. The applied conditions of digestion, reinforced by the action of microwaves, brought about a decidedly shorter (less than 20 min) sample preparation time. Suitability of the developed IC method was supported by validation results.
Keywords: Iodine; Iodine-measurement; Ion chromatography; Thyroid diseases; Nodular goiter;

A simple and efficient method, ionic liquid-based ultrasound-assisted liquid–liquid microextraction, has been developed for the determination of three biogenic amines including octopamine (OCT), tyramine (TYR) and phenethylamine (PHE). Fluorescence probe 2, 6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester was applied for derivatization of biogenic amines and high-performance liquid chromatography coupled with fluorescence detection was used for the determination of the derivatives. The factors affecting the extraction efficiency, such as the type and volume of ionic liquid, ultrasonication time and centrifugation time have been investigated in detail. Under the optimum conditions, linearity of the method was observed in the range of 0.5–50 μg mL−1 for OCT and TYR, and 0.025–2.5 μg mL−1 for PHE, respectively, with correlation coefficients (γ) > 0.996. The limits of detection ranged from 0.25–50 ng mL−1 (S/N = 3). The spiked recoveries of three target compounds in beer samples were in the range of 90.2–114%. As a result, this method has been successfully applied for the sensitive determination of OCT, TYR and PHE in beer samples.
Keywords: Ionic liquids; Ultrasound-assisted liquid–liquid microextraction; Biogenic amines; High-performance liquid chromatography–fluorescence detection;

Development and validation of a highly sensitive LC–MS/MS method for organic cation transporter (OCT) substrate tetraethylammonium (TEA) in rabbits by Jayabalan Nirmal; Thirumurthy Velpandian; Sundararajan Baskar Singh; Nihar Ranjan Biswas; Vasantha Thavaraj; Rajvardhan Azad; Supriyo Ghose (585-590).
Tetraethylammonium is widely used as a probe in organic cation transporters studies. A simple, highly sensitive, and specific method using direct protein precipitation was developed using Hydrophilic Interaction Liquid Chromatography coupled with positive electrospray ionization tandem mass spectrometry for the determination of tetraethylammonium (TEA) in rabbit plasma. Isocratic separation was achieved using a ZIC-HILIC column with acetonitrile and 5 mM ammonium acetate in the ratio of 8:2 containing 0.1% formic acid. Acquisition was performed in multiple reaction monitoring mode with the transitions: m/z 130 → 100 and 130 → 86 for TEA and m/z 276.1 → 142.2 for internal standard (homatropine). This method was validated to determine selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. A good linearity was found within a range of 1.53–784.6 ng/mL. The above method has been demonstrated for its capability to estimate the plasma levels of TEA after its topical instillation in rabbit eyes. This method provides an accurate, precise and sensitive tool for determining TEA levels for transporter studies.
Keywords: Tetraethylammonium; LC–MS/MS; HILIC; OCT; Tandem mass spectroscopy;

Fathead minnows (Pimephales promelas) comprise a species-of-choice for the hazard assessments of various environmental contaminants, including compounds capable of disrupting endocrine function. Towards this end, the use of liquid chromatography coupled with mass spectrometry (LC–MS) and/or tandem mass spectrometry (MS/MS) is gaining common use for the quantification of steroid hormones as biomarkers of endocrine stress in small-fish toxicological studies. In this work, 2-hydrazinopyridine (2-HP) was used to derivatize and quantify the physiologically relevant steroid hormones of: 17α-hydroxypregnenolone, progesterone, 11-ketotestosterone, 11-deoxycortisol and 17α,20β-dihydroxypregnenone, in the blood plasma of male and female fathead minnows. Liquid chromatographic separation was achieved using a Waters™ Sunfire C18 column (2.1 mm × 50 mm with a 3.5 μm particle size) and Milli-Q water:methanol (both with 0.1% formic acid) mobile phase over a gradient of 15 min. All mass analyses were conducted using electrospray ionization in the positive mode with tandem mass spectrometry (ESI+/MS/MS). This is the first such application of 2-HP derivatization for the quantifications of the structurally and functionally diverse C19 androgen of 11-ketotestosterone; C21 progestogens of 17α-hydroxypregnenolone, progesterone and17α,20β-dihydroxypregnenone; and C21 corticosteroid of 11-deoxycortisol, in fathead minnow blood plasma. The limits of detection (LOD) were set to the lowest calibration standard that gave a signal-to-background response of ≥3, and were: 0.16 ng/ml for progesterone, 0.63 ng/ml for 17α-hydroxypregnenolone, 11-deoxycortisol and 17α,20β-dihydroxypregnenone, and 1.25 ng/ml for 11-ketotestosterone. This study demonstrates the application of 2-HP derivatization for the analysis of a variety of steroid hormones representative of endocrine function in a species of fish commonly used in toxicological studies.
Keywords: Androgen; Progestogens; Corticosteroid; Fathead minnow; Plasma; LC-ESI+/MS/MS;

Interactions of three iridoid glycosides extracted from Cornus officinalis Sieb. et Zucc. (CIG) with protein were simultaneously explored by on-line dialysis sampling coupled with high-performance liquid chromatography (DS–HPLC). Three main compounds in CIG were unequivocally identified as loganin, sweroside and cornuside by comparing their t R , MS data and UV spectra with those of reference compounds. Dialysis recoveries and quantitative characteristics of DS–HPLC for three iridoid glycosides were determined. Recoveries of dialysis sampling ranged from 73.9 to 91.7% with the RSD below 3.0%. Based on the determination of concentrations before and after interaction with human serum albumin (HSA), the binding parameters of loganin, sweroside and cornuside with HSA were obtained and the binding mechanisms were investigated.
Keywords: Drug–protein interaction; Iridoid glycosides; Cornus officinalis Sieb. et Zucc.; On-line dialysis sampling; Binding parameter;

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) by Berit Packert Jensen; Jane Winifred Ann Vella-Brincat; Evan James Begg (605-609).
A rapid and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) assay was developed and validated for quantification of cyclizine and its main metabolite norcyclizine in human plasma. Samples were prepared by protein precipitation with acetonitrile and cinnarizine was used as internal standard (recovery >87%). The analytes were eluted from a C8 50 mm × 2.0 mm analytical column using a linear gradient of methanol and 0.05% formic acid with a total analysis time of 4 min. Analytes were detected by MS/MS using electrospray ionisation in the positive mode with multiple reactions monitoring (MRM) of the precursor ion/product ion transitions 267.2/167.2 for cyclizine and 253.2/167.2 for norcyclizine. Matrix effects were negligible. Standard curves for cyclizine and norcyclizine were linear (r 2  ≥ 0.996) over the range 2–200 ng/mL, with 2 ng/mL representing the lower limit of quantification. Relative standard deviations were <14% for intra- and inter-day precision and the accuracy was within ±8%. The assay was successfully applied to a clinical study.
Keywords: Cyclizine; Norcyclizine; Cinnarizine; LC–MS/MS; Plasma; Human;

Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC–MS by Emine Gokce; Genna L. Andrews; Ralph A. Dean; David C. Muddiman (610-614).
Fractionation prior to mass spectrometry is an indispensable step in proteomics. In this paper we report the success of performing offline reversed phase high pressure liquid chromatography (HPLC) fractionation on a C18 2.0 mm × 150 mm column at the peptide level with microliter per minute flow rates prior to online nano-flow reversed phase liquid chromatography mass spectrometry (nanoLC–MS) using the well-studied fungus Saccharomyces cerevisiae. A C18 75 μm × 150 mm column was used online and the online elution gradients for each fraction were adjusted in order to obtain well resolved separation. Comparing this method directly to only performing nanoLC–MS we observed a 61.6% increase in the number of identified proteins. At a 1% false discovery rate 1028 proteins were identified using two dimensions of RPLC versus 636 proteins identified in a single nano-flow separation. The majority of proteins identified by one dimension of nano-LC were present in the proteins identified in our two dimensional strategy. Although increasing analysis time, this non-orthogonal and facile pre-fractionation method affords a more comprehensive examination of the proteome.
Keywords: RP HPLC; Fractionation methods; Proteomics; Mass spectrometry;

A rapid-resolution ultra high-performance liquid chromatography separation method (UHPLC) for the simultaneous determination of the following β-blockers: milrinone, sotalol, metoprolol, propranolol and carvedilol, and their metabolites: 5′-hydroxylphenyl-carvedilol, O-desmethylcarvedilol, 4-hydroxypropranolol, α-hydroxy-metoprolol, O-desmethyl-metoprolol; the following isoflavones: genistein, daidzein, glycitin, glycitein, puerarin and biochanin A; as well as their metabolites: dihydrogenistein, desmethylglycitein, 8-hydroxygenistein, daidzein-7,4′-diglucoside, 8-hydroxydaidzein, dihydrobiochanin A in human urine was optimized. The analysed compounds were extracted from human urine by means of solid phase extraction (SPE). The effective UHPLC separation of the examined compounds was applied on a Hypersil GOLD™ (50 mm × 2.1 mm, 1.9 μm) column with a gradient mobile phase system and a UV detector. The complete separation of all analytes was achieved within 8.0 min. The method was validated for the determination of the aforementioned substances in human urine. The linear ranges, limits of detection (LOD) and limits of quantification (LOQ) for β-blockers, isoflavones and their metabolites were determined. The intra- and inter-day precision (%C.V.) was less than 4.48%, and the intra-day and inter-day accuracy was less than 4.74%. The tested SPE sorbent proved that appropriate absolute recoveries can be obtained for Oasis HLB (Waters). The mean recovery of the analytes, using the new SPE procedure, amounted from 70.14% to 99.85%. The present paper reports, for the first time, the method for the determination of β-blockers, isoflavones and their metabolites in human urine samples. The newly developed method was suitably validated and successfully applied for the analysis of the certain of the aforementioned analytes in human urine samples obtained from the patients suffering cardiovascular disease.
Keywords: β-Blockers; Isoflavones; Metabolites; UHPLC;

Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis by Ryan A. Picou; Indu Kheterpal; Amber D. Wellman; Madhavi Minnamreddy; Ginger Ku; S. Douglass Gilman (627-632).
A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aβ) peptide preparations at different aggregation states. Aggregation of Aβ (1-40) and Aβ (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aβ peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aβ species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aβ (1-40) and Aβ (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aβ for some CE–UV peaks. The CE–UV method reported here clearly indicates that monomeric and aggregated Aβ were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aβ (1-40) and Aβ (1-42) were observed. This CE–UV method can differentiate between Aβ monomer, oligomeric intermediates, and mature fibrils.
Keywords: Capillary electrophoresis; UV absorbance; Amyloid beta peptide; Protein aggregation; Mass spectrometry;

This paper evaluates the performances of reversed-phase (RPLC) and ion-pairing chromatography (IPLC) coupled with UV detection for the analysis of a set of 12 catecholamines and related compounds. Different chromatographic columns (porous C18-silica, perfluorinated C18-silica, porous graphitic carbon, monolithic and fused-core silica-based C18 columns) were tested using semi-long perfluorinated carboxylic acids as volatile ion-pairing reagents. Much more promising results were obtained by IPLC than by RPLC and important improvements in analytes peak symmetry and separation resolution were observed when using the “fast chromatography” columns (monolithic and fused-core C18) under IPLC conditions. For UV detection, a satisfactory separation of the 12 selected analytes was achieved in less than 20 min by using a fused-core particles column (Halo C18) and a mobile phase composed of a 1.25 mM nonafluoropentanoic acid aqueous solution and methanol under gradient elution mode. The chromatographic method developed can be directly coupled with electrospray ionization tandem mass spectrometry (ESI–MS/MS) in positive ionization mode and 10 solutes among those selected can be observed. The presence of the acidic ion-pairing reagent in the mobile phase makes this system incompatible with negative ionization mode and thus unable to detect the two acidic compounds that only responded in negative mode. In terms of MS detection, Monolithic C18 column proved to be the best one to reach the lowest detection limits (LODs) (from 0.5 ng mL−1 to 10 ng mL−1 depending on the neurotransmitter). The applicability of the optimized LC–MS/MS method to a “real world” sample was finally evaluated. The presence of the matrix leads to signal suppression for several solutes and thus to higher LODs.
Keywords: Catecholamine; Fused-core; Monolithic; PGC; Ion pairing chromatography; Mass spectrometry;

Analysis of perfluorinated chemicals in umbilical cord blood by ultra-high performance liquid chromatography/tandem mass spectrometry by Guang-Wen Lien; Ting-Wen Wen; Wu-Shiun Hsieh; Kuen-Yuh Wu; Chia-Yang Chen; Pau-Chung Chen (641-646).
Perfluorinated compounds (PFCs) can cross the placental barrier and enter fetal circulation. This study aimed at developing a fast and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry method for the determination of twelve perfluorinated compounds in cord blood. Samples were processed with protein precipitation using formic acid and methanol, mixed with stable isotope labeled standard, followed by sonication and centrifugation, and were analyzed using a Waters ACQUITY UPLC coupled with a Waters Quattro Premier XE triple-quadrupole mass spectrometer. The instrument was operated in selected reaction monitoring (SRM) with negative electrospray ionization. Using BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with 10-mM N-methylmorpholine/methanol gradient elution provided a fast chromatographic separation (5.5 min) and sharp peaks. Intra- and inter-day calibration bias was less than 7% and intra- and inter-day calibration of relative standard deviations were within 0.02–8.22% for all the analytes and concentrations. The recoveries of PFCs spiked into bovine serum ranged from 85 to 104% with relative standard deviations from 0.02 to 6.37%. The limits of quantitation (LOQs), defined as a signal-to-noise ratio of ten, ranged from 0.15 to 3.1 ng/mL for the twelve PFCs. Perfluorooctanoic acid (PFOA), perfluorooctyl sulfonate (PFOS), perfluoroundecanoic acid (PFUA) and perfluorononanoic acid (PFNA) were detected in up to 68% of umbilical cord plasma (n  = 444) in Taiwan Birth Panel Study and the health effect of these chemicals on children developmental deserves further investigation.
Keywords: Protein precipitation; Taiwan Birth Panel Study;

Alkylresorcinols (AR) are amphiphilic compounds present at high concentrations in the outer parts of wheat and rye kernels. Due to their specificity to whole grain and bran products of these cereals, AR and their metabolites have been proposed as biomarkers for intake of such foods. Two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), have previously been quantified in human urine using two different methodologies: high-performance liquid chromatography coupled to a coulometric electrode array detector (HPLC–CEAD) and gas chromatography in combination with mass spectrometry (GC–MS). In this study, these two methodologies were compared by analysing 114 urine samples from free-living Swedish subjects consuming their habitual diet. Data were evaluated by graphical investigation of difference-plots and statistical inference of agreement was assessed by weighted Deming regression analysis. The median DHBA concentrations were 11 μM (GC–MS) and 13 μM (HPLC–CEAD), respectively. Both difference-plot and regression analysis showed a small but statistically significant additive bias, with HPLC–CEAD resulting in a slightly higher DHBA concentration than GC–MS. The median concentration of DHPPA was 18 μM for both methods. Examination of the difference-plot of DHPPA did not indicate any systematic difference between the methods, but regression analysis showed small but statistically significant constant and proportional biases. The conclusion was that the two methodologies are equally suitable for analysis of alkylresorcinol metabolites in human urine and that any small systematic differences observed are most likely of limited practical importance.
Keywords: Alkylresorcinol; Metabolites; Biomarker; Whole grain; GC–MS; HPLC–CEAD; Method comparison; Coularray;

This paper reports the development and validation of a methodology for the low-level quantification of melamine and cyanuric acid in limited samples of rat serum. The methodology, based upon ion-exchange solid phase extraction (SPE) and ultra-performance liquid chromatography (UPLC) coupled with electrospray tandem mass spectrometry (MS/MS) in multiple reaction monitoring (MRM) mode, relies on the use of stable isotope-labeled internal standards and requires only 15 μL samples of serum. The method provides a recovery of 80–110% of melamine with a signal suppression of ca. 55%, and a recovery of 50–90% of cyanuric acid with a signal suppression ca. 40–60%, affording lower limits of quantification (LLOQ) for melamine or cyanuric acid of, respectively, 5 ppb (mean accuracy 109%; CV = 4.9%) and 10 ppb (mean accuracy 96%; CV = 8.6%). The small sample requirements, excellent sensitivity, accuracy and precision, and high-throughput (5 min of instrument run time) make this methodology optimal for toxicokinetic or exposure assessments studies.
Keywords: Melamine; Cyanuric acid; Rat serum; UPLC–ESI-MS/MS;

In this study, we investigated a novel application of matrix solid-phase dispersion (MSPD) methodology for the extraction of endogenous peptides from porcine hypothalamus tissue samples. Several experimental factors of the MSPD procedure were examined. Finally, silica-based octadecyl was chosen as dispersing material and blended with 0.25 g porcine hypothalamus at a ratio of 5, and 10 mL of 60% acetonitrile with 0.2% formic acid in water was chosen as the extraction and elution solvent. This MSPD extraction method was compared to the classic acid extraction method. More peaks were observed in the MSPD extracts (74 ± 5) by MALDI-TOF MS than in acid extracts (34 ± 5). Moreover, 14 potential endogenous peptides were identified in the MSPD extracts after nanoLC–MS/MS analysis, while only 2 endogenous peptides in the acid extracts. These results indicated that MSPD could be employed as a simple and efficient method for the extraction of endogenous peptides from tissues.
Keywords: Endogenous peptides; Matrix solid-phase dispersion extraction; NanoLC–MS/MS; Peptide extraction; Porcine hypothalamus;

Vinorelbine is a semi-synthetic vinca alkaloid with demonstrated high activities against various types of advanced cancer. To support a clinical pharmacokinetic study, a simple, rapid and sensitive method to determine vinorelbine in human plasma was developed using reversed phase liquid chromatography (LC) coupled with electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). Vinorelbine and vinblastine (the internal standard) were extracted from human plasma by one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether. The chromatographic separation was achieved on a Spursil polar-modified C18 column (50 mm × 2.1 mm, 3 μm, Dikma Technologies) with an isocratic mobile phase of a 75:25 (v/v) acetonitrile–4 mmol/L ammonium formate (pH 3.0) mixture at a flow-rate of 0.4 mL/min. The MS/MS detection was performed in the positive ion multiple reaction monitoring (MRM) mode by monitoring the precursor → product ion transitions at m/z 779.4 → 122.0 and m/z 811.3 → 224.2 for vinorelbine and the internal standard, respectively. The assay was validated in the range 0.1–200 ng/mL (r  > 0.997), the lowest level of this range being the lower limit of quantification (LLOQ) based on 50 μL of plasma. The intra- and inter-day precisions were within 6.0%, while the accuracy was within ±4.7% of nominal values. Detection and quantification of both analytes within 2 min make this method suitable for high-throughput analyses. The method was successfully applied to evaluate the systemic pharmacokinetics of vinorelbine after a 20-min intravenous infusion of 25 mg/m2 of vinorelbine to patients with metastatic breast cancer.
Keywords: Vinorelbine; LC–MS/MS; Human plasma; Liquid–liquid extraction; Pharmacokinetics;