Journal of Chromatography B (v.895-896, #C)

Validation of an on-line solid-phase extraction method coupled to liquid chromatography–tandem mass spectrometry detection for the determination of Indacaterol in human serum by Corinne Emotte; Olivier Heudi; Fanny Deglave; Adrien Bonvie; Laurence Masson; Franck Picard; Animesh Chaturvedi; Tapan Majumdar; Ashish Agarwal; Ralph Woessner; Olivier Kretz (1-9).
Indacaterol has been recently approved in Europe for the treatment of chronic obstructive pulmonary disease (COPD). In the present study, we have developed and validated a rapid and sensitive on-line solid phase extraction (SPE) method coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection for the determination of Indacaterol in human serum. The sample preparation involves the serum dilution with a 0.2% acetic acid solution prior to the on-line SPE on a mixed-mode cationic (MCX) polymer based sorbent. The samples were then eluted on a reversed phase column with a mobile phase made of acidified water and methanol and detection was performed by MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.0 min. Standard curves were linear over the range of 10.0 pg/mL (LLOQ) to 1000 pg/mL with correlation coefficient (r 2) greater than 0.990. The method specificity was demonstrated in six different batches of human serum. Intra-run and inter-run precision and accuracy within ±20% (at the LLOQ) and ±15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). The stability at room temperature (38 h) was determined and reported. In addition, the comparison between an off-line SPE procedure and our method gave equivalent results. The results of the present work demonstrated that our on-line SPE–LC–MS/MS method is rapid, sensitive, specific and could be applied to the quantitative analysis of Indacaterol in human serum samples. Our method effectively eliminated the tedious conditioning and rinsing steps associated with conventional off-line SPE and reduced the analysis time. The on-line SPE approach appears attractive for supporting the analysis of several hundreds of clinical samples.
Keywords: Indacaterol; On-line SPE; LC–MS/MS; Validation; Human serum;

► The method only needed a one-step protein precipitation procedure. ► The cycle time was 5.5 min allowing a sample throughput of 120–150 samples per day. ► The method was successfully used to analyze palonosetron in human plasma and urine. ► A two-compartment model was obtained after administrations. ► Palonosetron was eliminated at a slow rate in volunteers.The new analytical method for the determination of palonosetron in human plasma and urine has been developed based on liquid chromatography–mass spectrometry. The method utilized tramadol as the internal standard (IS). Separation was carried out on a Zorbax Eclipse TC-C18 column using methanol–1 mM ammonium formate in water (containing 0.1% formic acid, v/v, pH = 2.8) as mobile phase for gradient elution. Detection is carried out by multiple reaction monitoring (MRM) on 3200Qtrap™ mass spectrometry. The method has a chromatographic run time of 5.5 min and is linear within the concentration range 0.01–5.00 ng/mL for plasma and 0.10–30.00 ng/mL for urine both with a LOD of 0.003 ng/mL. Intra- and inter-day RSD of the concentration was 3.66–6.60%, 1.29–7.71% for plasma and 2.39–5.76%, 2.06–7.13% for urine. The relative error (RE) was −4.58% to 3.26% for plasma and −1.47% to 2.53% for urine. The recovery rates of palonosetron and IS both for plasma and urine were more than 90%. Palonosetron was stable under all the conditions tested. The method was successfully used to analyze palonosetron in human plasma and urine over a period of 168 h after intravenously pumping a single dose of 0.25 mg to volunteers. No significant differences were found between the pharmacokinetic parameters and urine accumulated excretory rate for male and female volunteers (P  > 0.05). A two-compartment model was obtained after administrations. Palonosetron was eliminated at a slow rate in volunteers. The mean urine accumulated excretory rate was 25.97 ± 12.87%. Inter-individual differences could not be neglected due to the high coefficient of variety in several pharmacokinetic parameters and the urine accumulated excretion.
Keywords: Palonosetron; LC–MS/MS; Pharmacokinetics;

Simultaneous determination of 45 pesticides in fruit and vegetable using an improved QuEChERS method and on-line gel permeation chromatography–gas chromatography/mass spectrometer by Dasheng Lu; Xinlei Qiu; Chao Feng; Yu’e Jin; Yuanjie Lin; Libei Xiong; Yimin Wen; Dongli Wang; Guoquan Wang (17-24).
► We improve efficiency of QuEChERS and method automation using online GPC–GC/MS. ► Modified salting-out will improve recovery of pesticides with high water solubility. ► GPC will greatly improve peak shape of pesticides and method accuracy and precision. ► 68–118% of recoveries and <14% of RSDs were obtained at spiked level of 0.01 μg/g. ► International inter-laboratory proficiency tests show satisfactory results.In this study, a method was developed to determine 45 selected pesticides (of different chemical families) in fruit and vegetable (including apple, spinach and cucumber). Samples were extracted using an improved QuEChERS method with salting out and phase separation in two steps. The target pesticides in concentrated extracts were analyzed by an on-line gel permeation chromatography–gas chromatography/mass spectrometer (online-GPC–GC/MS). Online GPC effectively removed matrix interferences and greatly improved the method sensitivity, recoveries and automation. Method limits of quantification were 10 ng/g for uniconazole and metalaxyl, and 5 ng/g for other 43 target analytes. In three fruit and vegetable matrices each spiked with 45 pesticides (0.01 μg/g), mean recoveries ranged from 80 to 118% for most of the tested pesticides except for profenofos (77% in apple) and chlorpyrifos (68% in apple and 75% in cucumber), with relative standard deviations (RSDs) of less than 14%. The results of the proficiency testing showed that the method is very successful in measuring the certified pesticides with less than 1.3 of the absolute value of Z-score. This method has been applied for routinely monitoring pesticides in fresh fruit and vegetable.
Keywords: QuEChERS; On-line GPC–GC/MS; Pesticides; Fruit and vegetable;

► Simultaneously determine flumatinib and its metabolites in CML patient plasma. ► Flumatinib and its metabolites have great differences in physicochemical properties. ► Each analyte was retained on the C18 column by using strong acidic mobile phase. ► A simple one-step protein precipitation increased throughput and efficiency. ► The method was applied to clinical studies of flumatinib mesylate in CML patients.Flumatinib is an antineoplastic tyrosine kinase inhibitor used for the treatment of chronic myelogenous leukemia (CML). Its major metabolites in the circulation are N-desmethyl flumatinib (M1) and amide hydrolysis product (M3). To investigate the pharmacokinetics of flumatinib in CML patients, a simple, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of flumatinib and its two major metabolites in patient plasma. After a simple, one-step protein precipitation with methanol, flumatinib, its two metabolites, and internal standard (HHGV-E) were separated on a C18 column using an isocratic mobile phase of methanol:5 mM ammonium acetate:formic acid (60:40:0.4, v/v/v). A total chromatographic run time of 4.2 min was achieved. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 563 →  m/z 463 for flumatinib, m/z 549 →  m/z 463 for M1, m/z 303 →  m/z 175 for M3, and m/z 529 →  m/z 429 for HHGV-E. The method was linear over the concentration ranges of 0.400–400 ng/mL for flumatinib, 0.100–100 ng/mL for M1, and 0.200–200 ng/mL for M3, using only 50 μL of plasma. The intra- and inter-day precisions were less than 8.5% for flumatinib, 9.8% for M1, and 10.6% for M3 in terms of the relative standard deviation. The accuracy was within ±2.2% for flumatinib, ±6.0% for M1, and ±9.9% for M3 in terms of relative error. The validated method was successfully applied to clinical pharmacokinetic studies of flumatinib mesylate in CML patients following oral administration at all dosage regimens.
Keywords: Flumatinib mesylate; N-Demethylated flumatinib; Amide hydrolysis product; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics;

► Microdialysis is used for measuring protein-unbound aspirin and salicylic acid in rat. ► The HPLC system was applied to assay the blood and brain microdialysates. ► The herbal formulation has no significant effect on the pharmacokinetics of aspirin. ► These results are practical information for clinical practice of herb–drug interactions.Aspirin is commonly used for the prevention of myocardial infarction and ischemic stroke; whereas the Chinese people employ the bu-yang-huan-wu-tang (BYHWT) as a routine herbal formulation for the treatment and prevention of transient ischemic stroke. The current study develops a microdialysis technique coupled to a validated liquid chromatography system to measure free-form aspirin and salicylic acid for herbal–drug interaction in rat blood and brain. The intra- and inter-day precisions in biological dialysates were within 0.1–9.4% in the concentration ranges of 0.1–50 μg/mL and the accuracies ranged from −4.7 to 6.1%. The pharmacokinetic data demonstrate that the area under the concentration time curve (AUC) of the aspirin was 2031 ± 266 min μg/mL after aspirin administration (100 mg/kg, i.v.). The AUC of salicylic acid was 12660 ± 1799 min μg/mL, which suggests that aspirin is quickly hydrolyzed to salicylic acid in blood and the metabolite can also be detected within 15 min in brain dialysate. The herbal–drug pharmacokinetic interaction showed no significant effect in blood and brain. The results of pharmacodynamics for the bleeding time suggested that there were no significant differences between the aspirin alone group and the BYHWT pretreated group. However, the bleeding time has been prolonged when compared aspirin alone or the group pretreated with BYHWT to the blank control. The conclusion provides practical information for clinical practice for the herbal formulation BYHWT and aspirin used concurrently.
Keywords: Aspirin; Bu-yang-huan-wu-tang; Herbal medicine; Pharmacokinetics; Stroke; Traditional Chinese medicine;

Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra high performance liquid chromatography coupled to tandem mass spectrometry by Renata Pereira Lopes; Rocío Cazorla Reyes; Roberto Romero-González; José Luis Martínez Vidal; Antonia Garrido Frenich (39-47).
► Simultaneous analysis of several classes of veterinary drugs in fish. ► QuEChERS methodology approach has been coupled to UHPLC–MS/MS. ► The proposed method minimizes sample handling and increases sample throughput. ► The validation procedure allows a reliable determination of target analytes. ► The proposed method can be applied in routine analysis for multirresidue determination.A simple, selective and fast multiresidue method was developed for the determination of 32 veterinary drug residues belonging to several families, in gilthead sea bream (Sparus aurata) by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). The extraction was based on modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure, using as extraction solution a mixture of acetonitrile and methanol (75:25, v/v), and it reduces sample handling, increasing sample throughput in relation to current methodologies. The developed method was validated and mean recovery ranged from 69% to 125% (at 10, 25, 50 and 100 μg/kg). Intra and interday precision, estimated as the same levels and expressed as relative standard deviation, RSD, were lower than 20% and 30%, respectively. Limits of detection (LODs) and quantification (LOQs) were lower than 7.5 and 25 μg/kg, respectively, except for danofloxacin, oxytetracycline and tetracycline (LOD and LOQ of 15.0 and 50 μg/kg, respectively). Decision limit (CCα) and detection capability (CCβ) were also calculated and ranged from 16.7 μg/kg (levamisole) to 605.0 (flumequine) μg/kg and from 23.5 μg/kg (levamisole) to 611.5 μg/kg (flumequine), respectively. The expanded uncertainty, U, was also evaluated ant it was below 25% at 100 μg/kg level, except for tetracycline (28%). Finally, the method was applied to ten samples obtained from local supermarkets in Almería (Spain) and traces of some compounds were detected.
Keywords: Aquaculture products; Multiclass; Veterinary drug residues; QuEChERS; UHPLC–MS/MS;

► An on-line comprehensive Ag+  × RP liquid chromatography system was constructed. ► The second dimensional column was optimized to get better LC × LC separations. ► 28 TAGs from peanut oil and 44 TAGs from mouse liver were identified. ► Identifications were based on TAGs’ retention behaviors and their MS fragments.Triacylglycerides (TAGs) are a large class of complex neutral lipids that naturally occur in both plants and animals. In the present work, an on-line comprehensive silver-ion liquid chromatography (silver-ion LC) × reversed-phase liquid chromatography (RPLC) system was constructed to analyze these compounds. A micro bore silver-ion modified column was employed in the first dimension with the commonly used hexane-based mobile phase. After a series of C18 columns were assessed, a wide bore column packed with 1.5 μm particles was selected as the second dimension column to reduce the negative effect caused by the large volume and strong solvent injection in the second dimension. The system coupled with mass spectrometry was applied to the analysis of an edible peanut oil and a mouse liver extract. Twenty-eight TAGs from the peanut oil and forty-four from the mouse liver were identified based on the TAGs’ retention behaviors on the comprehensive two-dimensional LC system and their APCI MS fragments.
Keywords: Comprehensive two dimensional liquid chromatography; Trigacylglyceride; Mouse liver; Silver-ion LC; APCI MS;

► We develop simultaneous quantitation tool of 5 CYP probe drugs and their metabolites. ► High recovery of all analytes was achieved by dual solvent extraction from plasma. ► LC/MS/MS method afforded high sensitivity to detect pg level of the drugs. ► This method is suitable for mini-dose cocktail clinical trials for drug interactions.A sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method with electrospray ionization was developed for the simultaneous quantitation of five probe drugs and their metabolites in human plasma for assessing the in vivo activities of cytochrome P450 (CYP). CYP isoform specific substrates and their metabolites of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan) and CYP3A (midazolam) were all simultaneously analyzed using LC–MS/MS after administration of a mixture of five drugs (i.e., a “cocktail approach”) to healthy volunteers. The assay uses propranolol as an internal standard; dual liquid extraction; a Xbridge MS C18 (100 mm × 2.1 mm, 3.5 μm) column; a gradient mobile phase of 0.1% formic acid/acetonitrile (7/3 → 3/7); mass spectrometric detection in positive ion mode. The method was validated from 5 to 500 ng/mL for caffeine and paraxanthine, 0.1–40 ng/mL for losartan and EXP3174, 0.05–20 ng/mL for omeprazole and 5-hydroxyomeprazole, 0.008–0.8 ng/mL for dextromethorphan and dextrorphan, 0.01–1.0 ng/mL for midazolam, and 0.04–4 ng/mL for 1′-hydroxymidazolam. The intra- and inter-day precision over the concentration ranges for all analytes were lower than 12.5% and 13.8% (relative standard deviation, %RSD), and accuracy was between 86.5% and 108.4% and between 87.0% and 107.0%, respectively. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10–100 times lower than typical therapeutic doses.
Keywords: LC–MS/MS; Cocktail dosing; CYP; Phenotyping; Plasma; Simultaneous analysis;

► Low density solvent based dispersive-liquid–liquid microextraction for cypermethrin in rat tissues and blood samples. ► High enrichment factors for each matrix. ► DLLME procedure requires no centrifugation. ► Useful for various toxicological studies of cypermethrin in blood and tissue samples.A simple and rapid method to determine the cypermethrin (CYP) insecticide in rat tissues (kidney, liver and brain) and blood has been developed for the first time using low density solvent-dispersive liquid–liquid microextraction (LDS-DLLME) followed by gas chromatography–electron capture detector (GC–ECD) analysis. Initially, tissue samples containing CYP were homoginized in acetone. Subsequently, homogenate was mixed with n-hexane (extraction solvent) and the mixture was rapidly injected into water. The upper n-hexane layer was collected in a separate microtube and injected into GC–ECD for analysis. Blood samples were diluted with ultrapure water and subjected to DLLME through similar procedure. Parameters such as type and volume of disperser and extraction solvent, salting out effect and extraction time, which can affect the extraction efficiency of DLLME, were optimized. Method was validated by investigating linearity, precision, recovery, limit of detection (LOD) and quantification (LOQ). LODs in tissue were in the range of 0.043–0.314 ng mg−1 and for blood it was 8.6 ng mL−1 with a signal to noise ratio of 3:1. LOQs in tissue were in the range of 0.143–1.03 ng mg−1 and for blood it was 28.3 ng mL−1 with a signal to noise ratio of 10:1. Mean recoveries of CYP at three different concentation levels in all the matrices were found to be in the range of 81.6–103.67%. The results show that, LDS-DLLME coupled with GC–ECD offers a simple, rapid and efficient technique for extraction and determination of CYP in rat tissues and blood samples, which in turn would be useful for toxicological studies of CYP.
Keywords: LDS-DLLME; Cypermethrin; Rat tissue; Blood; GC–ECD;

Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping by Li Zang; Tyler Carlage; David Murphy; Ruth Frenkel; Peter Bryngelson; Mark Madsen; Yelena Lyubarskaya (71-76).
► We studied the variability of LC-UV/MS method for analysis of methionine oxidation. ► The variability is mainly due to the residual heavy metals occurred to protein. ► Inclusion of EDTA in the digestion buffer suppresses the artificial oxidation. ► Residual heavy metals in LC columns also induce extra oxidation of peptides. ► Careful control of the heavy metals minimized the variability of LC-UV/MS method.Methionine oxidation has been demonstrated to play an important role in protein stability in vitro and in vivo. It may also cause changes in biological activity and immunogenicity profile of therapeutic proteins. Therefore, it is critical to monitor methionine oxidation in biopharmaceuticals during process and formulation development, as well as long-term stability studies. A common analytical method for methionine oxidation determination is peptide mapping analysis of protein enzymatic digests using UV detection with or without mass spectrometric detection. The quantitation of oxidation is performed based on the UV or extracted ion chromatographic peak areas of the oxidized and non-oxidized peptides. This method was found to be susceptible to significant variability over long-term use. Major factors leading to this variability included presence of low levels of metal ions, especially iron, in the digestion buffer, chromatographic column, LC injector, and other sample contact surfaces. Careful control of metal ion levels generally leads to less variability and long-term consistency of peptide mapping methods for oxidation determination.
Keywords: Methionine oxidation; LC-UV/MS; Protein digestion; Residual metal;

Application of high-speed counter-current chromatography coupled with a reverse micelle solvent system to separate three proteins from Momordica charantia by Yingnan Li; Lianhong Yin; Lingli Zheng; Lina Xu; Youwei Xu; Yanyan Zhao; Yan Qi; Jihong Yao; Xu Han; Kexin Liu; Jinyong Peng (77-82).
► Proteins from M. charantia were separated by HSCCC using reverse micelle system. ► Separation conditions were optimized and three proteins were isolated. ► The products showed with high purities and two of them were identified. ► One un-known protein with significant anticancer activity was found.High-speed counter-current chromatography (HSCCC) coupled with a reverse micelle solvent system was successfully developed to separate three proteins from Momordica charantia. Suitable HSCCC conditions were carefully optimized as follows: the stationary phase was a reverse micellar phase composed of isooctane and 50 mM bis-(2-ethylhexyl)-1-sulfosuccinate sodium (AOT). The mobile phase contained mobile phase A (50 mM Tris–HCl buffer containing 50 mM KCl at pH 7.0) for forward-extraction and mobile phase B (50 mM Tris–HCl buffer containing 0.5 M KCl at pH 10.0) for back-extraction. The flow rate, detection wavelength and column temperature were set at 1.5 ml/min, 280 nm and 4 °C, respectively. Under these conditions, three fractions (I, II and III) were separated, which showed high purity when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The structures of these proteins were then identified by MALDI-TOF/TOF-MS/MS and compared with the NCBInr database. Fractions I and III were identified as resistance-like protein P-B and pentatricopeptide repeat-containing protein, respectively, which were found in M. charantia for the first time. However, fraction II, which is thought to be a new protein, was not identified, and further investigations on this fraction are required. The anticancer activities of these three proteins on the human gastric cancer cell line SGC-7901 were evaluated in vitro. The results indicated that fraction II has excellent anticancer activity (IC50  = 0.116 mg/ml for 48 h treatment). This is the first report on the use of HSCCC to isolate proteins from M. charantia.
Keywords: Anticancer activity; HSCCC; Momordica charantia; Protein separation and purification; Reverse micelles;

► A rapid on line SPE-HPLC method was developed to determine cefdinir in dog plasma. ► On line SPE following 96-Well protein precipitation could reduce more interference. ► Application of 96-well format improved the extraction efficiency. ► The method was applied to the pharmacokinetic study of cefdinir in beagle dogs. ► This method is economic.The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining cefdinir in beagle dog plasma. After simple pretreatment for plasma with 6% perchloric acid, a volume of 100 μL upper layer of the plasma sample was injected into the self-made on-line SPE column. The analytes were retained on the trap column (Lichrospher C18, 4.6 mm × 37 mm, 25 μm), and the biological matrix was washed out with the solvent (20 mM KH2PO4 adjusted pH 3.0) at flow rate of 2 mL/min. By rotation of the switching valve, the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (methanol–acetonitrile–20 mM KH2PO4 adjusted pH 3.0, 11.25:6.75:82, v/v/v) at flow rate of 1.5 mL/min, and then separated on the analytical column (Ultimate™ XB-C18, 4.6 mm × 50 mm, 5 μm). The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4 min. The UV detection was performed at 286 nm. The calibration curves showed excellent linear relationship (R 2  = 0.9995) over the concentration range of 0.05–50 μg/mL. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This method was successfully applied to quantify cefdinir in beagle dog plasma to support the pre-clinical pharmacokinetic trial.
Keywords: Cefdinir; On-line solid-phase extraction; HPLC; Pharmacokinetic study; 96-Well protein precipitation;

Antibody affinity purification using metallic nickel particles by Jun Gao; Zhijun Li; Thomas Russell; Zhiyu Li (89-93).
► Solid nickel ferromagnetic particles for rapid, scalable, and efficient antibody affinity purification. ► Immobilizing Protein A on the surface of nickel particles for chromatographic and diagnostic applications. ► Minimizing Protein A leaching during antibody affinity purification. ► Isolating IgG antibodies directly from mouse serum in less than 5 min. ► Magnetic separation methods combining the pre-cleaning step and the affinity capture into one single step.Functionalized magnetic particles are emerging as a reliable and convenient technique in the purification of biomacromolecules (proteins and nucleic acids) and cell separation. In this study, we used novel solid nickel ferromagnetic particles coated with Protein A for the affinity purification of antibody. The study demonstrated that IgG can be purified from undiluted mouse serum in as few as 5 min using Protein A-coated nickel particles. Further, protein crosslinking was shown to stabilize the Protein A on the nickel particle surfaces to minimize Protein A leaching during the affinity purification and elution of IgG. The separation procedure is gentle, scalable, automatable, efficient and economical. By modifying the functional groups of amino acids in the protein coating, crosslinked nickel particles can be used not only for protein affinity purification but for other biological sample preparation and chromatographic applications as well. Methods proposed and tested in this study can be easily modified for small and medium scale antibody purification in lab and pre-clinical research.
Keywords: Antibody; Affinity purification; Ferromagnetic particle; Nickel particle; Protein A; IgG;

► Determination of metformin by reverse-phase chromatography. ► Chromatographic determination of metformin and four novel antidiabetic drugs. ► Applicability to wastewater, river water and tap water tested. ► Metformin, sitagliptin and vildagliptin detected in wastewater and river water.Antidiabetic compounds are among the most prescribed pharmaceuticals. Nevertheless, their presence in the environment has been scarcely evaluated as there is no method for their determination in environmental samples. This paper reports the development of an analytical method for the determination of traditionally used antidiabetics (metformin and glibenclamide) and novel antidiabetics (vildagliptin, sitagliptin and pioglitazone). The method is based on solid-phase extraction and determination by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. The method was applied to effluent wastewater, river water and tap water. Mean recoveries of glibenclamide, vildagliptin, sitagliptin and pioglitazone in the matrices evaluated were in the range 78–83%; limits of quantification were in the range 0.4–4.3 ng L−1; and precision values were in the range 2.2–13%. The high hydrophilicity and polarity of metformin complicated its simultaneous extraction. Chromabond Tetracycline cartridges and sample pH 8.5 were applied to the extraction of glibenclamide, vildagliptin, sitagliptin and pioglitazone. Oasis HLB cartridges, neutral sample pH and SDS as ion-pair reagent were used for the extraction of metformin. Validation results of metformin were not as favorable as those of the other antidiabetic drugs but were comparable with others previously reported. The developed method was applied to the first-time determination of the concentrations of the five antidiabetic drugs in wastewater, river water and tap water. Metformin was the antidiabetic drug at the highest concentration in wastewater and surface water (up to 253 ng L−1 and 104 ng L−1, respectively). Two of the antidiabetic drugs of recent prescription, sitagliptin and vildagliptin, were found in effluent wastewater at concentrations of 117 ng L−1 and 12 ng L−1, respectively, and in river water at concentrations of 35 ng L−1 and 6 ng L−1, respectively, whereas the classic antidiabetic drug glibenclamide and the novel drug pioglitazone were not detected.
Keywords: Antidiabetic drugs; Solid-phase extraction; Liquid chromatography quadrupole time-of-flight mass spectrometry; Wastewater; River water; Tap water;

UPLC–MS/MS determination of ractopamine residues in retinal tissue of treated food-producing pigs by Ana Vulić; Jelka Pleadin; Nina Perši; Dinka Milić; Wolfgang Radeck (102-107).
► The present study assessed persistence of ractopamine in retina as a pigmented tissue. ► Results showed mean residue concentrations from 67.11 μg/kg to 110.36 μg/kg. ► These data indicated high accumulation of ractopamine despite a low dose applied. ► Results supported the use of retina in the monitoring of this β2-adrenergic agonist.Ractopamine is a β2-adrenergic agonist, which reduces fat deposition and promotes muscle growth in animals for meat production. In the European Union countries, systematic monitoring and control of this contaminant residue is regularly performed by use of validated analytical methods of detection in different biological materials. The aim of the present study was to assess persistence of ractopamine in retina as a pigmented tissue by determination of its residues using UPLC–MS/MS as a quantitative confirmatory method after pig exposure to a ractopamine dose of 0.51 mg/kg b.w. Experimental group (n  = 9) of pigs were orally administered ractopamine for 28 days and then randomly sacrificed (n  = 3) on days 1, 3 and 8 of treatment discontinuation, whereas control animals (n  = 3) were left untreated. Study results showed mean ractopamine residue concentrations of 110.36 μg/kg, 67.11 μg/kg and 89.93 μg/kg on days 1, 3 and 8 after withdrawal, respectively, indicating high accumulation of ractopamine in retina despite a low dose applied. These data pointed to high affinity of ractopamine for binding to the pigmented segment of the eye, thus supporting the use of pigmented tissues as matrices in the regulatory monitoring of this β2-adrenergic agonist.
Keywords: Ractopamine; Retinal tissue; Food-producing pigs; UPLC–MS/MS;

► Simultaneously determine apatinib and its four major metabolites in human plasma. ► Apatinib and its metabolites have great different physicochemical properties. ► A simple protein precipitation and short chromatographic run time were achieved. ► The method shows advantages of high selectivity and reproducibility. ► The method was successfully applied to clinical study of apatinib mesylate.Apatinib, also known as YN968D1, is a novel antiangiogenic agent that selectively inhibits vascular endothelial growth factor receptor-2. Currently, apatinib is undergoing phase II/III clinical trials in China for the treatment of solid tumors. Apatinib is extensively metabolized in humans, and its major metabolites in circulation include cis-3-hydroxy-apatinib (M1-1), trans-3-hydroxy-apatinib (M1-2), apatinib-25-N-oxide (M1-6), and cis-3-hydroxy-apatinib-O-glucuronide (M9-2). To investigate the pharmacokinetics of apatinib and its four major metabolites in patients with advanced colorectal cancer, a sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of apatinib, M1-1, M1-2, M1-6, and M9-2 in human plasma. After a simple protein precipitation using acetonitrile as the precipitation solvent, all the analytes and the internal standard vatalanib were separated on a Zorbax Eclipse XDB C18 column (50 mm × 4.6 mm, 1.8 μm, Agilent) using acetonitrile: 5 mmol/L ammonium acetate with 0.1% formic acid as the mobile phase with gradient elution. A chromatographic total run time of 9 min was achieved. Mass spectrometry detection was conducted through electrospray ionization in positive ion multiple reaction monitoring modes. The method was linear over the concentration range of 3.00–2000 ng/mL for each analyte. The lower limit of quantification for each analyte was 3.00 ng/mL. The intra-assay precision for all the analytes was less than 11.3%, the inter-assay precision was less than 13.8%, and the accuracy was between −5.8% and 3.3%. The validated method was successfully applied to a clinical pharmacokinetic study following oral administration of 500 mg apatinib mesylate in patients with advanced colorectal cancer.
Keywords: Apatinib; YN968D1; Metabolite; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics; Human plasma;

► A fast and sensitive radio-LC method have developed and validated for a wide array of PET radioligands. The method has an LOD of 1 Bq for 11C-labeled radioligands with good temporal resolution (3.5 min) and improved sample loadability (1.5–2.0 mL plasma). ► This characteristic enables higher numbers of samples (up to 24 samples per PET measurement) to be analyzed compared to conventional method. ► This method can successfully be applied to study the metabolism of PET radioligands in human and monkey plasma.A fast and sensitive liquid chromatographic (fast-LC) method with radiometric detection was developed and validated to analyze positron emission tomography (PET) radioligands in plasma during PET studies. The plasma samples were deproteinized with acetonitrile and the extracts were injected into the fast-LC system coupled to an on-line radioactivity detector. Under the optimum conditions, complete separation of target PET radioligands from their radioactive metabolites was achieved within the short run time of only 3.5-min. The limits of detection were 1.0–1.2 Becquerel (Bq) for 11C and 18F-labeled compounds. This method can successfully be applied to study the metabolism of a wide variety of PET radioligands in human and monkey plasma with higher numbers of samples to be analyzed compared to the traditional LC method.
Keywords: Positron emission tomography (PET); Fast liquid chromatography (fast-LC); Metabolite analysis; Short-lived radioligand; Radioactive metabolite;

Quantification of biomarkers of environmental exposure to di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) in urine via HPLC–MS/MS by André Schütze; Claudia Pälmke; Jürgen Angerer; Tobias Weiss; Thomas Brüning; Holger M. Koch (123-130).
► DINCH is a major substitute for classical phthalate plasticizers. ► Secondary oxidized monoesters of DINCH are promising biomarker of DINCH exposure. ► We developed a fast and reliable on-line HPLC–MS/MS method. ► Our LOQs are sufficiently low to detect DINCH metabolites in a general population. ► DINCH metabolites are found in 86% of all samples analyzed.Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) is a major substitute for some high molecular weight phthalates that adversely affect reproductive function. Like for the phthalates a broad exposure of the population has to be expected. We postulated the DINCH monoester (MINCH) and secondary oxidized metabolites (OH-MINCH, cx-MINCH and oxo-MINCH) as human metabolites and possible biomarkers of DINCH exposure. We developed an on-line HPLC–MS/MS method for their determination in human urine. Identification was performed with authentic standard substances and quantification via isotope dilution. The analytical method is highly selective and sensitive with limits of quantification (LOQ) between 0.05 μg/l and 0.1 μg/l. In a pilot study with 22 volunteers from the general German population oxidized DINCH metabolites were found in above 80% of the samples. OH-MINCH was most abundant (mean 0.71 μg/l; maximum 3.69 μg/l) followed by cx-MINCH (0.61 μg/l; 2.82 μg/l) and oxo-MINCH (0.33 μg/l; 1.05 μg/l). All three oxidized metabolites correlated strongly among each other (ρ  ≥ 0.76). MINCH was detected in one sample only and has to be regarded a weak marker of exposure. With this analytical method we are able to perform human metabolism studies to provide metabolic conversion factors and to investigate the extent of DINCH exposure in the general population.
Keywords: Di(isononyl)cyclohexane-1,2-dicarboxylate; DINCH; Plasticizer; Exposure assessment; Urinary metabolites; Human biomonitoring;

► HF-LPME was for the first time reported for extraction of MIT in biological fluids. ► After the extraction, MIT was analyzed using HPLC–UV. ► The sample clean-up offered clean extracts. ► Resulting in significant reduction in analysis time. ► The proposed method is fast, sensitive, consumes minute amounts of extracting solvent.A hollow fiber liquid phase microextraction (HF-LPME) in conjunction with reversed phase HPLC–UV method was developed for the extraction and determination of trace amounts of the antidiabetic drug, mitiglinide (MIT) in biological fluids. The drug was extracted from 10 mL aqueous sample (donor phase (DP)) into an organic phase impregnated in the pores of hollow fiber, followed by the back extraction into a second aqueous solution (acceptor phase (AP)) located in the lumen of the hollow fiber. Parameters influencing the extraction efficiency including the kind of organic solvent, composition of DP and AP, extraction time, stirring rate and salt addition were investigated and optimized. Under the optimized extraction conditions, high enrichment factors (210-fold), good linearity (5–1000 ng mL−1) and detection limit lower than 1.38 ng mL−1 were achieved. Recoveries of spiked samples were in the range (88.3–96.3%) and (92.0–99.3%) for urine and plasma samples, respectively. The percent relative standard deviation (n  = 9) for the extraction and determination of three concentration levels (100, 400 and 800 ng mL−1) of MIT were less than 10.6% and 13.6% for urine and plasma samples, respectively. The developed method is simple, sensitive and has been successfully applied to the analysis of MIT in biological fluids.
Keywords: Hollow-fiber liquid-phase microextraction; Mitiglinide; High performance liquid chromatography; Biological fluids;

► Peptide adsorption-controlled liquid chromatography–tandem mass spectrometric method. ► Quantitation of amyloid β 1–38, 1–40, 1–42 and 1–43 peptides in dog CSF. ► Stable isotope labeled Aβ as the internal standard. ► A simple sample preparation of only dilution. ► Stability of amyloid β 1–38, 1–40, 1–42 and 1–43 peptides.To evaluate the usefulness of the peptide adsorption-controlled liquid chromatography–tandem mass spectrometry (PAC-LC–MS/MS) for reproducible measurement of peptides in biological fluids, simultaneous quantitation of amyloid β 1–38, 1–40, 1–42 and 1–43 peptides (Aβ38, Aβ40, Aβ42 and Aβ43) in dog cerebrospinal fluid (CSF) was tried. Each stable isotope labeled Aβ was used as the internal standard to minimize the influence of CSF matrix on the reproducible Aβ quantitation. To reduce a loss of Aβ during the pretreatment procedures, the dog CSF diluted by water–acetic acid–methanol (2:6:1, v/v/v) was loaded on PAC-LC–MS/MS directly. Quantification of the Aβ in the diluted dog CSF was carried out using multiple reaction monitoring (MRM) mode. The [M+5H5+] and b 5+ ion fragment of each peptide were chosen as the precursor and product ions for MRM transitions of each peptide. The calibration curves were drawn from Aβ standard calibration solutions using PAC-LC–MS/MS. Analysis of dog CSF samples suggests that the basal concentration of Aβ38, Aβ40, Aβ42 and Aβ43 in dog CSF is approximately 300, 900, 200 and 30 pM, respectively. This is the first time Aβ concentrations in dog CSF have been reported. Additionally, the evaluation of intra- and inter-day reproducibility of analysis of Aβ standard solution, the freeze–thaw stability and the room temperature stability of Aβ standard solution suggest that the PAC-LC–MS/MS method enables reproducible Aβ quantitation.
Keywords: Peptide adsorption-controlled liquid chromatography; Tandem mass spectrometry; Amyloid β peptide; Dog cerebrospinal fluid; Simultaneous quantitation;

Isolation of brefeldin A from Eupenicillium brefeldianum broth using macroporous resin adsorption chromatography by Ya-Jun Wang; Ye-Fei Wu; Feng Xue; Zhi-Xian Wu; Ya-Ping Xue; Yu-Guo Zheng; Yin-Chu Shen (146-153).
► One step isolation of brefeldin A from broth with macroporous resin is developed. ► The static equilibrium adsorption data of brefeldin A fits the Freundlich model. ► One-step column chromatography recovers 92.1% of brefeldin A in a purity of 90.4% from fermentation broth. ► A combination of adsorption chromatography and crystallization produces brefeldin A with purity of >99%.Brefeldin A (BFA) is a macrolide lactone antibiotic, possessing antitumor, antiviral, antifungal activities. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography combined with crystallization was established for BFA purification from Eupenicillium brefeldianum CCTCC M 208113 fermentation broth. Among six macroporous resin adsorbents tested, the non-polar resin HZ830 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Freundlich equation, and the adsorption kinetic followed the pseudo-second order model. Through experimental optimization of column adsorption and desorption, BFA in purity of 90.4% (w/w), 92.1% (w/w) yield was obtained by a one-step macroporous resin adsorption chromatography, using a stepwise elution protocol. Furthermore, high purity (>99%, w/w) of BFA crystals were prepared from E. brefeldianum CCTCC M 208113 fermentation broth in an overall recovery of 67.0% (w/w), using a combination of adsorption chromatography packed with non-polar macroporous adsorbent HZ830 and crystallization in acetone.
Keywords: Brefeldin A; Macroporous resin; Purification; Adsorption chromatography; Crystallization;

► LC–MS/MS method for simultaneous quantitation of six alkaloids and one monterpene. ► Seven active constituents of “Wuji Pill” simultaneously quantitated in rat plasma. ► LLOQs of these seven constituents are below 4.18 ng/mL. ► Efficient one-step liquid–liquid extraction of these constituents in rat plasma. ► Pharmacokinetic of seven compounds in rat plasma after oral dosing of “Wuji Pill”.A simple and sensitive method for simultaneous determination of seven active constituents, jatrorrhizine, berberine, coptisine, palmatine, evodiamine, rutacarpine and paeoniflorin, from a Chinese medicine Wuji Pill in rat plasma was developed based on a liquid chromatography and tandem mass spectrometry method. The separation of these seven compounds was carried out on a Shiseido CAPCELL PAK C18 column using a mobile phase consisting of acetonitrile (containing 0.1% formic acid and water (containing 0.1% formic acid and 10 mmol/L ammonium acetate) and carbamazepine as an internal standard. Electrospray ionization in positive-ion mode and multiple reaction monitoring was used to identify and quantitate active components. All calibration curves gave good linearity (r  > 0.993) over the concentration range from 0.42–208.0 ng/mL to 4.18–418.0 ng/mL for all components. The precision of the in vivo study was evaluated by intra- and inter-day assays and the percentages of relative standard deviation were all within 15%. The method was successfully applied to pharmacokinetic study of all six alkaloids and one monoterpene in rat plasma after oral administration of the Wuji Pill.
Keywords: Evodiae Rutaecarpa; Rhizoma Coptidis; Radix Paeoniae Alba; LC–MS/MS; Herbal medicine;

Analysis of Panax notoginseng metabolites in rat bile by liquid chromatography–quadrupole time-of-flight mass spectrometry with microdialysis sampling by Xiao-Dong Wen; Jie Yang; Rong-Hua Ma; Wen Gao; Lian-Wen Qi; Ping Li; Brent A. Bauer; Guang-Jian Du; Zhiyu Zhang; Jacqueline Somogyi; Chong-Zhi Wang; Chun-Su Yuan (162-168).
► Analysis of hepatic bile excretion is important to understanding drug metabolism. ► Technical difficulties limited notoginseng compound analysis in rat bile. ► A bile duct microdialysis with Q-TOF-MS was developed to obtain its metabolic profile. ► Four parent compounds and 3 metabolites were identified via real-time monitoring. ► One identified active metabolite, compound K, has significant biological activity.A dynamic microdialysis sampling method with liquid chromatography–quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was developed for rapid and sensitive analysis of the metabolite profile of Panax notoginseng extract (PNE) in rat bile. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the bile duct before bile samples were collected from 0 to 12 h. Metabolites of PNE were identified using dynamic adjustment of the fragmentor voltage to produce structure-relevant fragment ions. The mass accuracy of precursor and fragment ions was typically within 5 ppm of the theoretical values. We identified 7 compounds: 4 parent compounds (notoginsenoside R1, ginsenosides Rg1, Rb1, and Rd) and 3 metabolites (ginsenosides Rg2, Rh2, and compound K). Data from this study suggest that this microdialysis technique could be used in notoginseng saponin metabolic animal studies.
Keywords: Microdialysis sampling; Liquid chromatography–quadrupole time-of-flight mass spectrometry; Panax notoginseng; Ginsenosides; Bile; Metabolites;

Liquid chromatography–tandem mass spectrometric assay for the VEGFR inhibitor cediranib and its primary human metabolite cediranib-N+-glucuronide in plasma by Rolf W. Sparidans; Selvi Durmus; Ning Xu; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (169-173).
► The first bioanalytical assay for cediranib-N+-glucuronide has been reported. ► The assay is more simple and faster than the existing assay for cediranib. ► Analytes are stable under all conditions relevant for the assay. ► The assay was successfully validated for both compounds. ► Purification of enzymatically synthesized metabolic reference standard was not required.A quantitative bioanalytical assay for cediranib and its N+-glucuronide metabolite was developed and validated. Human plasma samples were pre-treated using protein precipitation with acetonitrile containing erlotinib and CYT-387 as internal standards for the glucuronide metabolite and parent compound, respectively. The extract was diluted with water and injected into the chromatographic system. This system consisted of sub-2 μm particles, a trifunctional bonded octadecyl silica column with gradient elution using 0.005% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analytes were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 1–290 ng/ml calibration range for cediranib and 0.2–52 ng/ml for its glucuronide metabolite. The lowest levels of these ranges corresponded to the lower limits of quantification for both compounds. Within day precisions were 4.0–6.7% for cediranib and 4.1–11.9% for its glucuronide, between day precisions were 4.2–10.2 and 4.8–14.4% and accuracies were between 99 and 106 and 84 and 94% for cediranib and its metabolite, respectively. Stabilities of both compounds were sufficient under all relevant conditions. Finally, the assay was successfully used to assess drug levels in a pharmacokinetic mouse study.
Keywords: Cediranib; Cediranib-N+-glucuronide; VEGF inhibitor; LC–MS/MS; Human plasma; Mouse plasma;

Liquid chromatography–tandem mass spectrometric assay for the JAK2 inhibitor CYT387 in plasma by Rolf W. Sparidans; Selvi Durmus; Ning Xu; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (174-177).
► The first bioanalytical assay for CYT387 has been reported. ► The LC–MS/MS assay is simple, fast and sensitive. ► The assay has successfully been validated in the 0.25–1000 ng/ml range. ► The drug is stable under all conditions relevant for the assay.A quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for the JAK2 inhibitor CYT387 was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing cediranib as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with a gradient using 0.005% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.25–1000 ng/ml calibration range. Within day precisions were 3.0–13.5%, between day precisions 5.7% and 14.5%. Accuracies were between 96% and 113% for the whole calibration range. The drug was stable under all relevant analytical conditions. Finally, the assay was successfully used to assess drug levels in mice.
Keywords: CYT387; JAK2 inhibitor; LC–MS/MS; Human plasma; Mouse plasma;

Rapid quantitative analysis of clarithromycin in rat plasma by UPLC–MS/MS after intravenous injection of the clarithromycin-loaded ultrafine PLGA nanoparticles by Yu-Jing Wang; Yi-Ting Wu; Jia-Yi Lin; Chih-Hung Chu; Hsin-Ying Huang; Yu-Chao Wang; Jen-Kun Chen; Chung-Shi Yang (178-181).
► A rapid UPLC–MS/MS-based method for analyzing plasma clarithromycin was developed. ► Clarithromycin loaded in ultrafine PLGA nanoparticles was synthesized. ► Rat plasma pharmacokinetic profile for nanoformulated clarithromycin was obtained.Nanoparticles were designed to encapsulate drugs to alter their pharmacological behaviors, therefore, it is very essential to monitor the pharmacokinetic profile of drug encapsulated in nanoparticles in order to clarify and predict their efficacy and side effects. In this paper, we reported a simple, rapid μ-elution 96-well solid phase extraction (μSPE) method combining with ultra high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for determination of nanoformulated drug in rat plasma. This method presented satisfactory results in terms of sensitivity, precision, accuracy, and recovery, for the first time, of quantitatively analyzing clarithromycin (CLA) in rat plasma after intravenous administration CLA-loaded ultrafine PLGA nanoparticles for pharmacokinetic study. This method has been proved to be fast, reliable and reproducible to accurately analyze drug encapsulated in polymeric nanoparticles sample for a pharmacokinetic study.
Keywords: Ultrafine PLGA NP; μ-SPE; Clarithromycin; Pharmacokinetics; Rat plasma; UPLC–MS/MS;

Reverse injection capillary electrophoresis UV detection for serotonin quantification in human whole blood by Angelo Zinellu; Salvatore Sotgia; Luca Deiana; Ciriaco Carru (182-185).
► We describe the first CE-UV detection method to measure serotonin in human whole blood. ► We investigate all parameters such as concentration and pH of run buffer and injection mode. ► The suitability of the method was tested by measuring 5-HT levels in 20 healthy volunteers.We describe the first capillary electrophoresis UV detection method to measure serotonin in human whole blood (WB). Procedural parameters such as concentration and pH of run buffer and injection mode were investigated. The reverse injection allows to decrease the analysis time by injecting samples at the outlet end of the silica capillary close to the detection window, so reducing the migration distance. Thus, when a capillary with an effective length of 10 cm and a 400 mmol/L Tris phosphate as background electrolyte at pH 3.25 was used, the migration time of the serotonin peak was 2.6 min. These conditions gave a good reproducibility of migration times (CV, 0.77%) and peak areas (CV, 2.44%). Intra- and inter-assay CV were 3.85% and 7.32%, respectively, and the analytical recovery was between 96.8% and 99.4%.
Keywords: Serotonin; Capillary electrophoresis; Reverse injection; Tris phosphate;

Ultra sensitive measurement of endogenous epinephrine and norepinephrine in human plasma by semi-automated SPE-LC–MS/MS by Guodong Zhang; Yizhong Zhang; Chengjie Ji; Thomas McDonald; Justin Walton; Elizabeth A. Groeber; Rick C. Steenwyk; Zhaosheng Lin (186-190).
► Measuring epinephrine and norepinephrine in human plasma. ► High throughput with semi-automated alumina-based SPE and derivatization procedure. ► Ultra sensitivity LC–MS/MS clinical biomarker assay.Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC–MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C18 column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00 pg/mL for E and 20.0 pg/mL for NE.
Keywords: Catecholamines; Epinephrine; Norepinephrine; Alumina SPE; Human plasma; Biomarker; LC–MS/MS;

► A new HPLC-FLD method was developed for bile acid and free fatty acid detection. ► The method was validated by linearity, LOD, repeatability, accuracy and precision. ► We compared the proposed method with the reported methods.A sensitive and selective method using 2-(7H-dibenzo[a,g]carbazol-7-yl)ethyl 4-methylbenzenesulfonate (DBCETS) as a new fluorescent labeling reagent has been proposed for simultaneously detecting BA and FFA by HPLC with fluorescence detector. The developed method offered the low detection limits of 0.42–0.70 and 0.28–0.57 ng/mL for BA and FFA, respectively. Compared with the reported methods, the proposed method here is capable of offering higher detection sensitivity and selectivity, with less cost and lower volume of sample preparation. This method was validated to ensure high accuracy and precision, and the reliability of its results. When applied to the serum samples of healthy volunteers and patients with hepatic carcinoma, it showed excellent applicability.
Keywords: Bile acids; Free fatty acids; Human serum; Fluorescent labeling; HPLC-FLD;