Journal of Chromatography B (v.927, #C)

Trends and applications of fast liquid chromatography in bioanalysis by Constantinos K. Zacharis; Paraskevas D. Tzanavaras (1-2).

State-of-the-art in fast liquid chromatography–mass spectrometry for bio-analytical applications by Oscar Núñez; Héctor Gallart-Ayala; Claudia P.B. Martins; Paolo Lucci; Rosa Busquets (3-21).
► State-of-the-art in fast LC–MS bioanalytical applications is presented. ► Main problems in the sample treatment and LC–MS analysis are discussed. ► On-line SPE, MIPs, RAM and TFC are addressed with relevant applications. ► Novel applications of sub-2 μm and porous shell columns are discussed. ► Novelties in HRMS for bioanalysis are presented.There is an increasing need of new bio-analytical methodologies with enough sensitivity, robustness and resolution to cope with the analysis of a large number of analytes in complex matrices in short analysis time. For this purpose, all steps included in any bio-analytical method (sampling, extraction, clean-up, chromatographic analysis and detection) must be taken into account to achieve good and reliable results with cost-effective methodologies. The purpose of this review is to describe the state-of-the-art of the most employed technologies in the period 2009–2012 to achieve fast analysis with liquid chromatography coupled to mass spectrometry (LC–MS) methodologies for bio-analytical applications. Current trends in fast liquid chromatography involve the use of several column technologies and this review will focus on the two most frequently applied: sub-2 μm particle size packed columns to achieve ultra high pressure liquid chromatography (UHPLC) separations and porous-shell particle packed columns to attain high efficiency separations with reduced column back-pressures. Additionally, recent automated sample extraction and clean-up methodologies to reduce sample manipulation, variability and total analysis time in bio-analytical applications such as on-line solid phase extraction coupled to HPLC or UHPLC methods, or the use of other approaches such as molecularly imprinted polymers, restricted access materials, and turbulent flow chromatography will also be addressed. The use of mass spectrometry and high or even ultra-high resolution mass spectrometry to reduce sample manipulation and to solve ion suppression or ion enhancement and matrix effects will also be presented. The advantages and drawbacks of all these methodologies for fast and sensitive analysis of biological samples are going to be discussed by means of relevant applications.
Keywords: Bio-analysis; UHPLC column technology; MIP and RAM technology; Turbulent flow chromatography; On-line SPE; High resolution mass spectrometry;

► Screening of steroid profiling for anti-doping controls and for diagnostic tests. ► UHPLC/MS determination of prohibited steroids and their metabolites. ► Identification of unknown species by UHPLC/MS. ► Overview about the biological matrices suitable for steroid determination.The use of doping agents, once restricted to professional athletes, has nowadays become a problem of public health, since it also concerns young people and non-competing amateurs in different sports. The use is also diffused in social life for improving physical appearance and enhancing performance and even dietary supplements assumed to improve performance often contain anabolic steroids. While decades ago the so-called “classical doping agents” (like stimulants and narcotics) were used, to-day anabolic steroids are more widely diffused. Anabolic steroids are synthetic substances prepared by introducing modifications in the molecular structure of testosterone, the main natural androgenic anabolic steroid that forms in testes interstitial cells. The first report concerning the use of anabolic steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports was banned by the International Olympic Committee and control tests were implemented in 1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a limited number of exogenous steroids. Over the years, always new doping substances are synthesized and, as a consequence, the list of prohibited compounds is continuously updated and new suitable analytical methods for their detection and determination in biological matrices are continuously required. In doping control analysis the knowledge of steroid metabolism pathway in human body is of primary importance and the analytical methods must permit the simultaneous detection and determination not only of the forbidden precursor agents but also of their metabolites. In addition, the potential presence and amount in the biological samples of species that can interfere in the analysis should be evaluated. Also the several anabolic steroids, specifically designed to circumvent doping control, put on the market have been incorporated in the list of the prohibited substances of the World Anti-Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly reminded that assumption of doping agents not only leads to athletes the possible failing of doping tests but causes important health risk and WADA prohibited list establishes criteria to highlight the alteration of the natural steroid profile caused by exogenous administration. Doping control analyses are generally performed in urine, a matrix that provides a prolonged detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To identify the chemical structures of anabolic steroids the use of mass spectrometry detection is very advantageous. Gas chromatography–mass spectrometry (GC–MS) techniques allowed for the development of comprehensive screening methods. GC–MS methods are sensitive and robust but present the disadvantages of time-consuming sample pretreatment, that is often based on hydrolysis and derivatisation reactions. Liquid chromatography–mass spectrometry (LC–MS) methods have been successfully used to identify and determinate steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has become the technique of choice for steroid analysis. Due to its generally higher speed, sensitivity, reproducibility and specificity with respect to HPLC, it can be used to simultaneously separate and determinate multi component steroid mixtures. The technique is of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency enhancement due to the small particle (sub-2 μm) column packing, which provides high peak capacity within analysis times even 5–10 fold shorter than conventional HPLC methods. Modern multiplex instruments can analyze thousands of samples per month so that, notwithstanding the generally high instrumental costs, the cost of the individual assay is affordable. In addition, the improved specificity and resolution offered by time-of-flight or quadrupole time-of-flight mass spectrometry allow their application in doping control analysis or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of the present review is to consider, compare and discuss the applications of the UHPLC/MS methods present in literature for the identification and determination of forbidden steroids and their metabolites in human biological matrices.
Keywords: Prohibited steroids; Doping; Steroid profiling; UHPLC; LC–MS; Biological matrix;

► UHPLC-MS is a highly suitable technology for the discovery of biomarkers for diseases. ► Combining RP- and HILIC-UHPLC separations gives comprehensive covering of metabolome. ► UHPLC-MS/MS methods tailored to a class of compounds allow to identify many new metabolites. ► UHPLC-MS targeted analysis allows ultra-fast and highly selective monitoring of biomarkers.The development and use of UHPLC-based methods for the identification, validation and analysis of biomarkers for diseases is reviewed. The currents trends in types of stationary phases and modes of detection are discussed. Afterwards, examples are provided on the use of UHPLC-MS for finding novel biomarkers in samples from in vitro or in vivo animal models of human diseases, as well as in biofluid samples (mainly urine and plasma) obtained from patients. Molecular profiling and targeted analysis are considered, providing an overview of recent experimental or clinical works carried out using UHPLC analysis of compounds from various chemical classes, such as low molecular weight metabolites, hormones, lipids, peptides and proteins.
Keywords: UHPLC; UHPLC-MS; Biomarkers; Metabolomics; Animals models; Human; Pathology;

► Review was aimed at presenting of the progress in methods for the drug analysis. ► Trends in fast liquid chromatographic were described. ► Liquid chromatography is an important tool for monitoring drugs and metabolites. ► Sample preparation, column types, mobile phases, types of detection are discussed. ► Advantages of modern fast liquid chromatography methods in clinical application.One of the major challenges facing the medicine today is developing new therapies that enhance human health. To help address these challenges the utilization of analytical technologies and high-throughput automated platforms has been employed; in order to perform more experiments in a shorter time frame with increased data quality. In the last decade various analytical strategies have been established to enhance separation speed and efficiency in liquid chromatography applications. Liquid chromatography is an increasingly important tool for monitoring drugs and their metabolites. Furthermore, liquid chromatography has played an important role in pharmacokinetics and metabolism studies at these drug development stages since its introduction. This paper provides an overview of current trends in fast chromatography for the analysis of cardiovascular drugs and their metabolites in clinical applications. Current trends in fast liquid chromatographic separations involve monolith technologies, fused-core columns, high-temperature liquid chromatography (HTLC) and ultra-high performance liquid chromatography (UHPLC). The high specificity in combination with high sensitivity makes it an attractive complementary method to traditional methodology used for routine applications. The practical aspects of, recent developments in and the present status of fast chromatography for the analysis of biological fluids for therapeutic drug and metabolite monitoring, pharmacokinetic studies and bioequivalence studies are presented.
Keywords: Fast liquid chromatography; UHPLC; Drugs; Metabolites; Biological sample; Review;

Fast separation of large biomolecules using short monolithic columns by Aleš Podgornik; Shuichi Yamamoto; Matjaž Peterka; Nika Lendero Krajnc (80-89).
► Application of short monolithic columns for analytics of macromolecules and nanoparticles – recent overview. ► Impact of macromolecule interactions on peak spreading and resolution in IEX. ► Overview of binding sites number for different class of macromolecules and nanoparticles.Chromatographic monoliths have already penetrated in many different areas of separation sciences. This is due to their properties, especially advantageous for fast separation and purification of large biologic macromolecules, even at low pressure drop. Probably the most outstanding features are flow unaffected binding capacity and resolution, later resulting in very short analysis times. Furthermore, since large biomolecules interact with the matrix via many binding sites, efficient separation can be achieved with the monolithic columns of a very short length, further reducing pressure drop over matrix. In this review brief introduction to the monoliths is given with the emphasize on the theory of separation of large molecules, particularly on a linear gradient elution and estimation of peak broadening. As an outcome of this analysis the most efficient separation is expected when short monolithic column with accordingly adjusted gradient is implemented, especially for macromolecules interacting with the monolith functionalities via over 10 binding sites. This is experimentally demonstrated by several recent examples of short monolithic column applications for analysis of antibodies, viruses, virus like particles (VLPs) and polynucleotides like plasmid DNA (pDNA) and RNA, indicating their potential for process monitoring, control and optimization but also for product final formulation and quality control.
Keywords: Monoliths; Analytics; Peak spreading; Convective Interaction Media (CIM); Short columns; Biomolecues;

Evaluation of different column chemistries for fast urinary metabolic profiling by Dick-Paul Kloos; Henk Lingeman; Wilfried M.A. Niessen; André M. Deelder; Martin Giera; Oleg A. Mayboroda (90-96).
► Comprehensive column chemistry evaluation for urinary metabolomics. ► Evaluation based on relevant analyte standards and pooled urine samples. ► Application of automated peak score evaluation and feature detection. ► Application to small sample set of urine specimen. ► Conclusion: superior behaviour of diol-HILIC material under specified conditions.Fast analytical methodologies are mandatory for large scale metabolic profiling. Here, we present a thorough evaluation of different column chemistries in combination with different mobile phases for fast LC–MS urinary metabolic profiling. Three porous HILIC materials were investigated, next to core–shell C18-, XB-C18- and PFP-RPLC material. The performance of the selected column chemistries was tested in a non-targeted manner with pooled urine samples and in a targeted manner with a set of 54 common urinary metabolites. In order to evaluate the differential behaviour of the tested columns in a targeted manner, we applied a peak scoring algorithm. This algorithm takes into account several quality criteria such as retention time, dead time, peak height and peak shape. In general, HILIC columns generate more retention for polar metabolites. Our results show that the diol-HILIC column outperforms the RPLC columns. However, because of their opposite nature, comprehensive behaviour is observed as well, which was shown by investigating gender differences in a small urinary sample set. All applied column chemistries enabled sufficient peak capacity within a short gradient time.
Keywords: Metabolomics; Urinary metabolic profiling; LC–MS; Core–shell; Reversed-phase; HILIC;

Metabonomic analysis of urine from rats after low-dose exposure to 3-chloro-1,2-propanediol using UPLC–MS by Liyan Liu; Yujie He; Huimin Lu; Maoqing Wang; Changhao Sun; Lixin Na; Ying Li (97-104).
► We studied metabolite profiles of the low-dosed exposure to 3-MCPD by metabolomics. ► We found a change in urinary NAG and GAL on 90 days after 3-MCPD treatment. ► Some biomarkers such as N-acetylneuraminic acid were detected on 30 days. ► These biomarkers changed more sensitively and early than conventional parameters.To study the toxic effect of chronic exposure to 3-chloro-1,2-propanediol (3-MCPD) at low doses, a metabonomics approach based on ultrahigh-performance liquid chromatography and quadruple time-of-flight mass spectrometry (UPLC–Q-TOF-MS) was performed. Two different doses of 3-MCPD (1.1 and 5.5 mg/kg bw/d) were administered to Wistar rats for 120 days (1.1 mg/kg bw/d: lowest observed adverse effect level [LOAEL]). The metabolite profiles and biochemical parameters were obtained at five time points after treatment. For the 3-MCPD-treated groups, a significant change in urinary N-acetyl-β-d-glucosaminidase and β-d-galactosidase was detected on day 90, while some biomarkers based on the metabonomics, such as N-acetylneuraminic acid, N-acetyl-l-tyrosine, and gulonic acid, were detected on day 30. These results suggest that these biomarkers changed more sensitively and earlier than conventional biochemical parameters and were thus considered early and sensitive biomarkers of exposure to 3-MCPD; these biomarkers provide more information on toxicity than conventional biochemical parameters. These results might be helpful to investigate the toxic mechanisms of 3-MCPD and provide a scientific basis for assessing the effect of chronic exposure to low-dose 3-MCPD on human health.
Keywords: Metabonomics; UPLC–MS; Biomarker; 3-Chloro-1,2-propanediol;

A validated, rapid UPLC–MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples by Alexander A. Zoerner; Christoph Schroeder; Arslan Arinc Kayacelebi; Maria T. Suchy; Frank-Mathias Gutzki; Dirk O. Stichtenoth; Jens Tank; Jens Jordan; Dimitrios Tsikas (105-111).
► UPLC–MS/MS measurement of ivabradine, reboxetine and metoprolol in human plasma. ► Use of deuterium labeled internal standard for each analyte. ► Validation according to the EMA guideline on bioanalytical method validation. ► Clinical trial samples. ► Measuring patient adherence.A recent clinical trial assessing human autonomic cardiovascular regulation applied pacemaker channel inhibition with ivabradine, norepinephrine transporter blockade with reboxetine, and beta-adrenoreceptor blockade with metoprolol. To verify patient adherence, we developed and validated a fast UPLC–MS/MS assay measuring all three compounds simultaneously. Deuterium-labeled drugs, d3-ivabradine, d5-reboxetine and d7-metoprolol, served as internal standards. Sample preparation of 200 μL human plasma consisted of a single liquid–liquid extraction step by means of ethyl acetate. Chromatographic separation was performed on a 50-mm long BEH C18 column with gradient elution using a mixture of water and methanol each containing 2 mM ammonium acetate over 4.5 min. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode. Characteristic product ions resulting from collision-induced dissociation of unlabeled and deuterium-labeled drugs with argon were used for quantification in the selected-reaction monitoring mode. We validated the method according to the European Medicines Agency (EMA) guideline on bioanalytical method validation over the range from 1 ng/mL to 500 ng/mL for all three analytes. Linear responses with correlation coefficients > 0.99 over that range were acquired. The LOQ value was 1 ng/mL for each drug. Regulatory criteria for accuracy (80–120%) and precision (RSD < 15%) were met for all drugs. The internal standard-normalized matrix factor was close to 1 for low and high analyte concentrations. We successfully measured ivabradine, reboxetine, and metoprolol concentrations in 107 human plasma samples from a clinical trial. Quality control samples processed in parallel confirmed the method's reliability in a clinical setting.
Keywords: Human plasma; Matrix effect; EMA guideline; Solvent extraction; Tandem mass spectrometry; Validation;

► UPLC–MS/MS method for the determination of 15 pharmaceuticals. ► Fully validated method using a high pH mobile phase. ► Extraction from post-mortem whole blood by LLE.An ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for the determination of fifteen basic pharmaceuticals, for analysis of post- and ante-mortem whole blood samples. The following compounds were included: amitriptyline and its metabolite nortriptyline, trimipramine, mianserin, mirtazapine, citalopram, paroxetine, sertraline, and venlafaxine (all antidepressants), levomepromazine and quetiapine (antipsychotics), ketobemidone and tramadol (analgesics), alimemazine (sedative antihistamine), and metoprolol (beta-blocker). The sample pretreatment consisted of liquid–liquid extraction (LLE) using ethylacetate:n-heptane (80:20, v/v). Six deuterated analogues were used as internal standards (IS). The compounds were separated using a reversed phase C18-column (2.1 mm × 100 mm, 1.7 μm), a flow rate of 0.5 mL/min, and gradient elution with 5 mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode, using two transitions for the compounds and one transition for the IS. The run time of the method was 8 min including equilibration time. The calibration curves had R 2 values above 0.995 for all the compounds. The intermediate precision had a relative standard deviation (RSD, %) ranging between 2.0 and 16%. Recoveries of the compounds were ≥81%. The lower limits of quantifications (LLOQs) for the compounds varied from 5.0 nmol/L to 0.10 μmol/L (1.3–26 ng/mL) and the limits of detections (LODs) from 1.0 to 20 nmol/L (0.24–5.3 ng/mL). LLOQ corresponds to 0.28–5.5 pg injected on column. Matrix effects (ME) were between 91 and 113% when calculated against an IS. A comparison with former confirmation LC–MS methods at the Norwegian Institute of Public Health, Division of Forensic Medicine and Drug Abuse Research (NIPH) was performed during method validation. Good correlation was seen for all compounds except sertraline, where the old LC–MS method was showing 33% higher results. The method has been running on a routine basis for more than a year, and has proven to be very robust and reliable with results for external quality samples, including sertaline, corresponding well to consensus mean or median.
Keywords: Antidepressants; Antipsychotics; Post-mortem whole blood; Quantitative analysis; Liquid–liquid extraction; Ultra high performance liquid chromatography–tandem mass spectrometry;

► Green bioanalytical approach by elimination of ACN from all analytical stages is presented. ► ACN based protein precipitation was replaced by liquid–liquid extraction in 1-octanol. ► Large volume injection from the 1-octanol layer in the chromatographic column (RP) was achieved. ► Acetonitrile in the mobile phase was replaced by propylene carbonate/ethanol (7/3, v/v). ► LC/MS–MS assay of enalapril and enalaprilat in human plasma was used as application.Green bioanalytical approaches are oriented toward minimization or elimination of hazardous chemicals associated to bioanalytical applications. LC/MS–MS assay of enalapril and enalaprilat in human plasma was achieved by elimination of acetonitrile from both sample preparation and chromatographic separation stages. Protein precipitation (PP) by acetonitrile addition was replaced by liquid–liquid extraction (LLE) in 1-octanol followed by direct large volume injection of the organic layer in the chromatographic column operated under reversed phase (RP) separation mechanism. At the mean time, acetonitrile used as organic modifier in the mobile phase was successfully replaced by a mixture of propylene carbonate/ethanol (7/3, v/v). Three analytical alternatives ((I) acetonitrile PP + acetonitrile based chromatographic elution; (II) 1-octanol LLE + acetonitrile based chromatographic elution; (III) 1-octanol LLE + propylene carbonate/ethanol based chromatographic elution) were validated and the quality characteristics were compared. Comparison between these alternative analytical approaches was also based on results obtained on incurred samples taken during a bioequivalence study, through application of the Bland–Altman procedure.
Keywords: Green bioanalytical application; Large volume injection; 1-Octanol as diluent; Propylene carbonate/ethanol/water mobile phases; Enalapril; Enalaprilat; LC/MS–MS;

► Oral fluid represents an important alternative to blood. ► Detection of pharmaceutical and illicit drugs in oral fluid is crucial. ► We developed a UHPLC–MS/MS method to detect 44 compounds in oral fluid. ► The method proved simple, accurate, rapid and highly sensitive.A simple and extremely fast procedure for the quantitative determination in oral fluid samples of 44 substances, including the most common drugs of abuse and several pharmaceutical drugs, was developed and fully validated. Preliminary sample treatment was limited to protein precipitation. The resulting acetonitrile solution was directly injected into an ultra-high performance liquid chromatograph (UHPLC) equipped with a C18 column (100 mm × 2.1 mm, 1.7 μm). The mobile phase eluted with linear gradient (water/formic acid 5 mM: acetonitrile/formic acid 5 mM; v:v) from 98:2 to 0:100 in 5.0 min, followed by isocratic elution at 100% B for 1.0 min. The flow rate was 0.6 mL/min and the total run time was 9.0 min including re-equilibration at the initial conditions. The analytes were revealed by a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The method proved to be simple, accurate, rapid and highly sensitive, allowing the simultaneous detection of all compounds. The ease of sample treatment, together with the wide range of detectable substances, all with remarkable analytical sensitivity, make this procedure ideal for the screening of large populations in several forensic and clinical contexts, whenever oral fluid sampling has to be preferred to blood sampling, as for example in short retrospective investigations.
Keywords: Oral fluid; Ultra-high performance liquid chromatography; Multianalyte; Validation;

A rapid ultra HPLC–MS/MS method for the quantitation and pharmacokinetic analysis of 3-deazaneplanocin A in mice by Cody J. Peer; Mahadev Rao; Shawn D. Spencer; Shandiz Shahbazi; Patricia S. Steeg; David S. Schrump; William D. Figg (142-146).
► We developed and validated a novel uHPLC–MS/MS assay for the quantification of 3-deazaneplanocin A (DZNep). ► This method has a faster run time and is more sensitive than previous methods. ► This method is accurate, precise, and a simple liquid extraction procedure recovers 90% of drug from mouse plasma. ► Successfully applied to preclinical pharmacokinetic study in mice.3-Deazaneplanocin A (DZNep) has been shown to have anti-cancer activity in numerous cancer types and its continued preclinical, and eventual clinical, drug development will require rapid and sensitive bioanalytical methods in order to quantitate this drug for pharmacokinetic analyses. The ultra HPLC with positive thermospray tandem mass spectrometric (LC–MS/MS) detection affords the most sensitive (limit of quantitation 5 ng/mL) and rapid (3 min run time) bioanalytical method to date for DZNep. Due to the polar nature of this drug and the internal standard (tubercidin), a hydrophilic-interaction column (HILIC) was used. The method was accurate, with less than 10% deviation from nominal values, as well as precise, where both within-day and between-day precisions were less than 15%. A liquid–liquid extraction procedure was able to recover ∼90% of drug from a small volume (50 μL) of mouse plasma. This method was successfully applied to a pharmacokinetic study in mice intravenously injected with DZNep.
Keywords: 3-Deazaneplanocin; Ultra HPLC–MS/MS; Pharmacokinetics;

UPLC–MS/MS measurement of S-nitrosoglutathione (GSNO) in human plasma solves the S-nitrosothiol concentration enigma by Dimitrios Tsikas; Mario Schmidt; Anke Böhmer; Alexander A. Zoerner; Frank-Mathias Gutzki; Jens Jordan (147-157).
► UPLC–MS/MS measurement of S-nitrosoglutathione (GSNO) in human plasma. ► Use of S-[15N]nitrosoglutathione (GS15NO) as internal standard. ► Stabilization of GSNO by EDTA, NEM, serine/borate. ► Selected-reaction monitoring. ► This method solves the S-nitrosothiols enigma.We developed and validated a fast UPLC–MS/MS method with positive electrospray ionization (ESI+) for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. We used a published protocol for the inactivation of plasma γ-glutamyltransferase (γGT) activity by using the γGT transition inhibitor serine/borate and the chelator EDTA for the stabilization of GSNO, and N-ethylmaleimide (NEM) to block SH groups and to avoid S-transnitrosylation reactions which may diminish GSNO concentration. S-[15N]Nitrosoglutathione (GS15NO) served as internal standard. Fresh blood was treated with NEM/serine/borate/EDTA, plasma spiked with GS15NO (50 nM) was ultrafiltered (cut-off 10 kDa) and 10 μL aliquots of the ultrafiltrate were analyzed by UPLC–MS/MS. Five HILIC columns and an Acquity UPLC BH amide column were tested. The mobile phase was acetonitrile–water (70:30, v/v), contained 20 mM ammonium formate, had a pH value of 7, and was pumped isocratically (0.5 mL/min). The Nucleoshell column allowed better LC performance and higher MS sensitivity. The retention time of GSNO was about 1.1 min. Quantification was performed by selected-reaction monitoring the mass transition m/z 337 ([M+H]+) →  m/z 307 ([M+H― 14NO]• +) for GSNO (i.e., GS14NO) and m/z 338 ([M+H]+) →  m/z 307 ([M+H― 15NO]• +) for GS15NO. NEM/serine/borate/EDTA was found to stabilize GSNO in human plasma. The method was validated in human plasma (range, 0–300 nM) using 50 nM GS15NO. Accuracy and precision were in generally acceptable ranges. A considerable matrix effect was observed, which was however outweighed by the internal standard GS15NO. In freshly prepared plasma from heparinized blood donated by 10 healthy subjects, no endogenous GSNO was determined above 2.8 nM, the limit of quantitation (LOQ) of the method. This study challenges previously reported GSNO plasma concentrations being far above the present method LOQ value and predicts that the concentration of low-molecular-mass and high-molecular-mass S-nitrosothiols are in the upper pM- and lower nM-range, respectively.
Keywords: Artefacts; Fast-liquid chromatography; Nitric oxide; Quantification; S-Nitrosothiols; Tandem mass spectrometry; Validation;

► UHPLC is a highly sensitive and quick technique for the determination of sex steroids. ► Seven steroids were separated during 5 min in synthetic urine. ► Octadecyl UHPLC columns allow complete separation of estradiol isomers.The main aim of the study was to develop a simple, fast, sensitive and inexpensive method for the separation and quantification of various steroid hormones in urine. Ultra high performance liquid chromatography was used to analyze estrone, estriol, 17-α-estradiol, 17-β-estradiol, progesterone, pregnenolone, and testosterone. Three columns were chosen for the present study: two octadecyl columns and one octyl column. The best results of separation were obtained for the octadecyl columns. Complete separation of all sex steroids was impossible when methanol was used during the chromatographic studies. The most interesting and valuable result was obtained with regard to the complete separation of isomers. All seven steroids were successfully separated in 10 min, next the time of single analysis was reduced to 5.5 min with gradient elution. Linearity was evaluated over a range of concentrations of 0.08–12.11 ng ml−1. The correlation coefficient ranged from 0.9987 to 0.9998. The LOD values were between 0.02 and 0.33 ng ml−1 and LOQ ranged between 0.10 and 1.10 ng ml−1. The developed method is suitable for routine analysis of these compounds in urine.
Keywords: Sex steroids; Ultra high performance liquid chromatography; Stationary phase; Resolution; Urine;

Ultra high performance liquid chromatography–tandem mass spectrometry method for cyclosporine a quantification in biological samples and lipid nanosystems by M. Guada; E. Imbuluzqueta; A. Estella-Hermoso de Mendoza; H. Lana; M.C. Dios-Viéitez; M.J. Blanco-Prieto (164-172).
► A new sensitive UHPLC–MS/MS method was developed and validated to quantify CyA in lipid nanosystems, whole blood and eight tissue samples. ► A commercially available and inexpensive substance (amiodarone) was used as internal standard. ► This method was successfully applied for quality control of lipid nanosystems and in vivo studies.Cyclosporine A (CyA) is an immunosuppressant cyclic undecapeptide used for the prevention of organ transplant rejection and in the treatment of several autoimmune disorders. An ultra high performance liquid chromatography–tandem mass spectrometry method (UHPLC–MS/MS) to quantify CyA in lipid nanosystems and mouse biological matrices (whole blood, kidneys, lungs, spleen, liver, heart, brain, stomach and intestine) was developed and fully validated. Chromatographic separation was performed on an Acquity UPLC® BEH C18 column with a gradient elution consisting of methanol and 2 mM ammonium acetate aqueous solution containing 0.1% formic acid at a flow rate of 0.6 mL/min. Amiodarone was used as internal standard (IS). Retention times of IS and CyA were 0.69 min and 1.09 min, respectively. Mass spectrometer operated in electrospray ionization positive mode (ESI+) and multiple reaction monitoring (MRM) transitions were detected, m/z 1220.69 → 1203.7 for CyA and m/z 646 → 58 for IS. The extraction method from biological samples consisted of a simple protein precipitation with 10% trichloroacetic acid aqueous solution and acetonitrile and 5 μL of supernatant were directly injected into the UHPLC–MS/MS system. Linearity was observed between 0.001 μg/mL–2.5 μg/mL (r  ≥ 0.99) in all matrices. The precision expressed in coefficient of variation (CV) was below 11.44% and accuracy in bias ranged from −12.78% to 7.99% including methanol and biological matrices. Recovery in all cases was above 70.54% and some matrix effect was observed. CyA was found to be stable in post-extraction whole blood and liver homogenate samples exposed for 6 h at room temperature and 72 h at 4 °C. The present method was successfully applied for quality control of lipid nanocarriers as well as in vivo studies in BALB/c mice.
Keywords: Cyclosporine A; UHPLC–tandem mass spectrometry; Lipid nanocarriers; Pharmacokinetics; Biodistribution;

Separation and identification of phenolic compounds in canned artichoke by LC/DAD/ESI-MS using core–shell C18 column: A comparative study by Jianbing Wu; Yongsheng Qian; Peipei Mao; Linyao Chen; Yanbin Lu; Huizhong Wang (173-180).
► The performance of core–shell column was compared with porous sub-2 μm column. ► The comparison was performed in both isocratic and gradient elution modes. ► The van Deemter, Knox, Poppe plots and back pressures of both columns were studied. ► The peak capacities of both columns were measured and compared. ► 10 phenolic compounds of canned artichoke head identified by core–shell-LC/DAD/MS.Core–shell silica stationary phase was considered as a breakthrough in column technology in HPLC world. In this work, the chromatographic performance of core–shell column, made by fusing a 0.5 μm porous silica layer onto 1.7 μm nonporous silica cores, was compared with sub-2 μm fully porous particle materials for separation and identification of phenolic compounds in canned artichoke heads. The anti-oxidant caffeoylquinic acids of artichoke extract was taken as representative for calculating the plate heights in a wide flow rate range and analyzed on the basis of the van Deemter and Knox equations. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Both phases gave similar minimum plate heights when using non-reduced coordinates. Meanwhile, the flat C-term of core–shell column provided the possibilities for applying high flow rates without significant loss in efficiency. In addition, the peak capacities of both columns were measured, at constant chromatographic linear velocity and intrinsic gradient steepness, in order to generate comparable retention window for the least and the most retained compounds. Finally, the core–shell column was successfully applied for separation and identification of 10 phenolic compounds in canned artichoke extracts by liquid chromatography–diode array detection–tandem mass spectrometry, exhibiting great potential in the field of food analysis.
Keywords: Core–shell particles; Porous particles; LC/DAD/ESI-MS; Artichoke; Food analysis;

► A rapid UHPLC-DAD assay to monitor Vitex agnus-castus was developed. ► The assay covers all major classes of non-volatile V. agnus-castus phyto-chemicals. ► Based on UHPLC-DAD-QTOF-MS experiments 62 chromatographic peaks of V. agnus-castus extract could be characterized. ► The assay was validated and can be applied to fruits, tablets, and extracts.A rapid ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method was developed and validated for the simultaneous determination of all classes of non-volatile phytochemicals (iridoids, flavonoids and diterpenes) in Vitex agnus-castus (Lamiaceae) fruits, a traditional medicinal plant used against premenstrual symptoms (PMS) and other disorders. Seven marker compounds, 3,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, agnuside, 5-hydroxykaempferol-3,6,7,4′-tetramethylether, 1,2-dibenzoic acid glucose, methoxy-vitexilactone, and vitetrifolin D were isolated from the methanol extract of V. agnus-castus to be used as reference substances. Chromatographic separation was performed on a Zorbax Eclipse XDB-C18 (50 mm × 2.1 mm) UHPLC column with 1.8 μm particle size, within 20 min. A solvent gradient from 0.5% acetic acid to acetonitrile at a flow rate of 0.6 mL/min was used as mobile phase. Analyte detection and quantification was realized at 210 nm and 260 nm. The UHPLC-DAD assay was validated for the quantitative analysis of agnuside, isovitexin, casticin, 5-hydroxykaempferol-3,6,7,4′-tetramethylether and vitetrifolin D. It was found to be specific, accurate, precise, and reproducible for the quantification of these compound within a concentration range of 0.7–500.0 μg/mL for casticin and 5-hydroxykaempferol-3,6,7,4′-tetramethylether, 1.4–1000.0 μg/mL for isovitexin and agnuside, and 12.4–1000.0 μg/mL for vitetrifolin D. Intra- and inter-day variations showed relative standard deviations (RSD) of less than 3.9% and 6.4%, respectively. Tentatively assignment of 62 chromatographic features found in the UHPLC-DAD assay was carried out by coupling the UHPLC instrument to a quadrupole time-of-flight mass spectrometer via an electrospray ionization interface (ESI-QTOF-MS) operated in positive and negative ion mode. By using the established quantitative UHPLC-DAD assay to asses agnuside, isovitexin, casticin, 5-hydroxykaempferol-3,6,7,4′-tetramethylether and vitetrifolin D in V. agnus-castus derived preparations as extracts, tinctures and tablets, the applicability of the developed assay to phytopharmaceuticals was successfully proven.
Keywords: Vitex agnus-castus; UHPLC-DAD; Validation, LC–MS; Phytochemicals;

► A new UHPLC application for food analyses is proposed. ► Twenty-three amino acids and fifteen biogenic amines were separated in 9 min only. ► Various acid-curd cheeses were profiled for their free amino acid and biogenic amine levels. ► In general, overall biogenic amine contents were in an acceptable range. ► Particular samples showed biogenic amine amounts exceeding 100 mg/100 g.A new UHPLC method for the simultaneous determination of amino acids and biogenic amines in a single run, and its first application to profile ripened acid-curd cheeses was presented. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy succinimidyl carbamate (AQC), 23 amino acids and 15 amines were separated in 9 min only (12 min total run time), and eluates monitored using their UV response at 249 nm. Limits of detection (0.05–0.29 mg/100 g) and quantification (0.16–0.97 mg/100 g), repeatability for sample preparation (1.0–6.1% RSD) and method recoveries (83–120%) were found suitable for cheese analysis. In total, 47 acid-curd cheeses classified into sub-groups like cooked, Quargel-type or grey cheeses were analyzed for their free amino acid and amine (histamine, tyramine, putrescine, cadaverine, and tryptamine) contents, which (as expected) were highlighted by a great variability. Total free amino acid levels ranged between less than 100 and more than 4000 mg/100 g (median 567 mg/100 g), implying that for some cheeses less or not ripened/fresh quark was used for production or, in contrast, a higher degree of proteolysis had occurred. For the sum of biogenic amines, median concentration was determined at 7.0 mg/100 g, while only 5% of all cheeses had levels higher than 161.9 mg/100 g. Thus, the obtained results suggest quite acceptable biogenic amine levels for (mostly underrated) ripened acid-curd cheeses, although partly exceptional high concentrations (>250 mg/100 g) were indeed observed in individual samples.
Keywords: Amino acids; Biogenic amines; 6-Aminoquinolyl-N-hydroxy succinimidyl carbamate (AQC); UHPLC; Acid-curd cheese;

Fast and sensitive UHPLC methods with fluorescence and tandem mass spectrometry detection for the determination of tetracycline antibiotics in surface waters by Karolina Škrášková; Lúcia H.M.L.M. Santos; Dalibor Šatínský; Angelina Pena; Maria Conceição B.S.M. Montenegro; Petr Solich; Lucie Nováková (201-208).
► Development of two methods for determination of tetracyclines in surface waters. ► Speeded up highly sensitive analyses. ► Comparison of the methods in terms of validation parameters. ► Mixed-mode stationary phase for reducing peak tailing of tetracyclines.In this paper two fast and highly sensitive ultra-high performance liquid chromatography (UHPLC) methods for the determination of tetracycline antibiotics (oxytetracycline, tetracycline, doxycycline, demeclocycline, chlortetracycline, minocycline and degradation product epitetracycline) in surface waters have been developed using fluorescence (FL) and mass spectrometry (MS) detection. ACQUITY UPLC BEH C8 and ACQUITY CSH C18 columns were employed for FL and MS detection, respectively, both packed with 1.7 μm particles. Mixed-mode separation mechanism of CSH (charged surface technology) sorbent was found particularly useful in analysis of TCs, which possess problematic amphoteric structures. The FL methodology was based on chelation of tetracyclines with calcium ions to perform on-column derivatisation. The developed methods were compared in the terms of validation parameters including linearity, sensitivity, precision and accuracy. The linearity range for FL detection was within 7 ng mL−1 to 50 μg mL−1 with method limit of detection (MLOD) as low as 0.2 ng mL−1 for most of the analytes. MS detection showed even higher sensitivity reaching MLOD of 0.003 ng mL−1, which is the highest sensitivity reported so far in analysis of TCs. Matrix matched calibration curves in the range of 0.01–50 ng mL−1 were used for quantification to compensate for matrix effects with the correlation coefficients demonstrating good linearity (0.9940–0.9999). The extraction of the antibiotics from surface waters was performed using solid phase extraction with Oasis HLB cartridges. Accuracy was expressed as recovery with values ranging from 96.52% to 127.30% and from 91.66% to 123.70% for FL and MS detection, respectively.
Keywords: UHPLC; Mixed-mode stationary phases; Tetracyclines; MS/MS; Surface waters; Environmental contaminants;

Profiling of polyunsaturated fatty acids (PUFAs) and their oxidized metabolites, mainly eicosanoids, in human plasma by fast liquid chromatography–mass spectrometry is described. Sample preparation involved protein precipitation of 200 μL plasma followed by on-line solid-phase extraction. 7 PUFAs and 94 oxidized metabolites were separated utilizing a C-18 column packed with 2.6 μm core–shell particles in 7 min. The analytes and deuterium-labeled standards were detected via scheduled multiple reaction monitoring transitions (123 sMRM). Simultaneously, linear ion trap fragment spectra were acquired for confirmation, if necessary. The lower limit of quantitation ranged between 200 and 1000 ng/mL for the PUFAs and 10–1000 pg/mL for the metabolites. The method was applied to a study on plasma samples from 50 healthy subjects.
Keywords: Mass spectrometry; Fast liquid chromatography; Linear ion trap; On-line solid-phase extraction; Eicosanoids; Polyunsaturated fatty acid metabolism;

Amino acid analysis using core–shell particle column by Yanting Song; Takashi Funatsu; Makoto Tsunoda (214-217).
► The minimum theoretical plate height of core–shell particle column reached 5.1 μm. ► The analysis of NBD-amino acids was performed within 7 min with core–shell particle column. ► The developed method was proved to be valid for analyzing biological sample.In this study, the separation efficiency of a core–shell particle column was compared with particle-packed and monolithic silica columns, which showed that the core–shell particle column had a smaller theoretical plate height and that its separation efficiency was not affected significantly by the increase in flow rate. A fast HPLC method using a core–shell particle column was developed for the determination of amino acids. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was used as a fluorescence derivatization reagent for amino acids, followed by separation on a core–shell Kinetex C18 column. The analysis time for 21 NBD-amino acids was within 7 min, which was faster than that in our previous studies with conventional particle-packed columns or monolithic silica columns. The linearities of the calibration curves for all the amino acids were found to be good over a range of injection amounts from 40 fmol to 40 pmol. The accuracies for the amino acid determinations were 90.9–107%. The method was proved to have potential for the fast determination of amino acids in biological samples.
Keywords: 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F); van Deemter equation; Biological sample; Fluorescence;

Development and validation of a rapid ultra high pressure liquid chromatographic method for the determination of methylxanthines in herbal infusions by Constantinos K. Zacharis; Fotini S. Kika; Paraskevas D. Tzanavaras; Konstantinos Fytianos (218-222).
► Simultaneous determination of methylxanthines. ► Quantitative analysis of herbal infusions. ► Fast chromatographic separation by UHPLC.An ultra high pressure liquid chromatographic method coupled with diode array detector (UHPLC–DAD) has been developed and validated for the fast separation and determination of three major methylxanthines, i.e., caffeine, theophylline and theobromine, in various herbal beverages. Isocratic elution using 0.1 vol% formic acid/CH3OH (92.5:7.5, v/v) enabled the completion of the separation cycle in less than 3 min using a flow rate of 0.7 mL/min and a column temperature of 50 °C. Validation of the method included linearity (0.5–50 mg/L), limits of detection (12–35 μg/L) and quantification (40–120 μg/L), precision, matrix effect and accuracy. The percent recoveries ranged between 90 and 108%.
Keywords: Theobromine; Theophylline; Caffeine; Herbal beverages; Ultra high pressure liquid chromatography; Determination;

Determination of 2-methylimidazole, 4-methylimidazole and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole in caramel colours and cola using LC/MS/MS by Claudia Schlee; Mariya Markova; Julia Schrank; Fanette Laplagne; Rüdiger Schneider; Dirk W. Lachenmeier (223-226).
► LC/MS/MS allows the rapid separation and quantification of three imidazoles. ► No sample preparation besides dilution is needed. ► A survey of about 100 samples was conducted. ► Only 4-methylimidazole (4-MI) was detected in caramel colours and cola beverages. ► The human exposure of 4-MI is judged as being below levels of concern.Substituted imidazoles recently came under scrutiny as they may be indirectly introduced into cola beverages via the use of class IV (E150d) caramel colours and may pose health hazards. A LC/MS/MS method was developed for determining 2- and 4-methylimidazole (2-MI, 4-MI) and 2-acetyl-4-(1,2,3,4)-tetrahydroxybutylimidazole (THI) in beverages and caramel colours. The method is very rapid and easy to conduct as it requires only dilution in eluent for sample preparation. For 4-MI, the recovery was between 94 and 102% for spiked cola samples. The limit of detection was 2 μg/L in the measuring solution (corresponding to 40 μg/L for cola samples diluted 1:20 during sample preparation). 97 cola samples and 13 caramel colours from Germany and France were analysed. From the 3 analytes, only 4-MI was found in the samples with very varying concentrations (non quantifiable traces to 0.6 mg/L in colas and 175–658 mg/kg in E150d). The exposure for cola drinkers in worst case scenarios is estimated to be 2–5 μg/kg bodyweight/day, which is judged as being only a low risk for public health.
Keywords: 2-Methylimidazole; 4-Methylimidazole; 2-Acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole; Soft drinks; Caramel colours; HPLC–MS;