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This work describes the development of two methods involving supported liquid extraction (SLE) sample treatment followed by ultra-high performance liquid chromatography or ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS and UHPSFC–MS/MS) for the screening of 43 anabolic agents in human urine. After evaluating different stationary phases, a polar-embedded C18 and a diol columns were selected for UHPLC–MS/MS and UHPSFC-MS/MS, respectively. Sample preparation, mobile phases and MS conditions were also finely tuned to achieve highest selectivity, chromatographic resolution and sensitivity. Then, the performance of these two methods was compared to the reference routine procedure for steroid analyses in anti-doping laboratories, which combines liquid–liquid extraction (LLE) followed by gas chromatography coupled to tandem mass spectrometry (GC–MS/MS). For this purpose, urine samples spiked with the compounds of interest at five different concentrations were analyzed using the three analytical platforms. The retention and selectivity of the three techniques were very different, ensuring a good complementarity. However, the two new methods displayed numerous advantages. The overall procedure was much faster thanks to high throughput SLE sample treatment using 48-well plates and faster chromatographic analysis. Moreover, the highest sensitivity was attained using UHPLC–MS/MS with 98% of the doping agents detected at the lowest concentration level (0.1 ng/mL), against 76% for UHPSFC–MS/MS and only 14% for GC–MS/MS. Finally, the weakest matrix effects were obtained with UHPSFC–MS/MS with 76% of the analytes displaying relative matrix effect between −20 and 20%, while the GC–MS/MS reference method displayed very strong matrix effects (over 100%) for all of the anabolic agents. [ABSTRACT FROM AUTHOR]

In recent years, the popularity of hydrophilic interaction chromatography (HILIC) in the analysis of small polar and ionizable molecules increased substantially. A large number of new stationary phases with sub-2-µm and superficially porous particles were developed and used in various applications, including pharmaceuticals, metabolites, biomarkers, amino acids and peptides. Applications of HILIC in ultra-high performance liquid chromatography (UHPLC) mode constitute only about 10% of all current HILIC applications. This review presents an overview of UHPLC in HILIC mode, including the importance of the final sample diluent, a discussion on available stationary phases and approaches to detection, and a comparison of performance with other chromatographic modes. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the selectivity, retention properties, peak shape and loading capacity for bases were practically evaluated using two UHPLC mixed-mode hybrid CSH stationary phases modified by C18 or Phenyl group. The data were compared with the data obtained on other UHPLC hybrid stationary phases (BEH C18, BEH C8, BEH Phenyl and BEH Shield RP18) at both basic and acidic conditions using conventional HPLC buffers (50mM ammonium formate/acetate) as well as low ionic-strength additives such as, e.g. 0.1–0.01% formic/acetic acid and 1mM solution of ammonium formate/acetate, which are widely used in LC–MS applications. Ten pharmaceutically important compounds encompassing acids, bases and neutral were included into the study. Due to properties of CSH sorbent (which possess positively charged surface besides RP group), much improved peak shapes and weaker retention was obtained for bases even at very low concentration of acidic additives. Such conditions are ideally suited for LC–MS analysis of bases, where typical RP chromatographic separation (retention and good selectivity at basic pH) and LS–MS conditions (efficient ionization at acidic pH) are not in agreement. On the other hand, acids were more strongly retained and for some compounds the peak shape was influenced negatively due to ion-exchange mechanism. Further, the behavior of acidic, basic and neutral solutes is discussed using various additives at both basic and acidic pH for all above stated columns. The robustness of retention times after pH change from basic to acidic was also evaluated. The new CSH stationary phases represent an interesting selectivity tool preferably for separation of basic compounds. [Copyright &y& Elsevier]

Abstract: In theory, with identical stationary phase chemistry, the transfer of an HPLC method to UHPLC conditions is straightforward and necessitates the calculation of new conditions based on column and instrument geometries. Occasionally, undesirable changes in selectivity, retention or efficiency have been reported and have been attributed to a frictional heating phenomenon that is due to the elevated generated pressure drop. In the present study, the frictional heating in a UHPLC system was evaluated experimentally under gradient elution conditions (acetonitrile/buffer at pH 3 and 9) with generated pressure drops in the range of 100–1000bar on both 1.0mm and 2.1mm I.D. columns using a mixture of 10 representative basic, acidic and neutral pharmaceutical compounds. Under adiabatic conditions (i.e., still-air oven), the longitudinal temperature gradient was estimated at +4°C, +8°C and +16°C at 300, 600 and 1000bar, respectively, on a 2.1mm I.D. column using an empirical measurement procedure. With the 1.0mm I.D. column, these values were reduced to +3°C, +6°C and +12°C, respectively. Finally, various approaches to eliminate or at least to reduce the effect of frictional heating are briefly discussed. [Copyright &y& Elsevier]

Abstract: Tea catechins are an important group of natural compounds associated with health promoting effects and desired commodities for the growing market of dietary supplements and functional foods. Consequently these compounds attract more interest of research groups worldwide. A reliable quantitative analysis of tea catechins is essential for human intervention studies, manufacturers of dietary supplements and quality control by authorities. UHPLC–ESI-MS/MS analytical method was chosen due to rapid runtime, high sensitivity and selectivity. The chromatographic separation of eight tea catechins was achieved within 2.5min on C18 BEH analytical column (100mm× 2.1mm i.d.; 1.7μm), whilst the gradient elution mode was employed using water:methanol mobile phase with addition of volatile organic acid. The concentration of organic acids in the mobile phase was optimised within the range of 0.01–0.1% (v/v). High sensitivities were achieved in positive (10.2-16.8fmol/inj.) and negative ion detection mode (102.1-168.1fmol/inj.), through accurate and complex tuning of MS parameters. The UHPLC-ESI-MS/MS method was validated in terms of linearity (>0.9997; >0.9990), range (0.02–2.40mgL−1; 0.15-24.00mgL−1), LOD (3.0–4.8μgL−1; 30.1–48.0μgL−1), LOQ(9.9–15.8μgL−1; 150.5-240.0μgL−1), intra-day precision (4.4-7.1% RSD; 3.3-5.1% RSD), accuracy (94.06-113.7%; 89.5-108.4%), retention time repeatability (0.0-0.5% RSD; 0.0-0.6% RSD), and peak area repeatability (1.2-4.0% RSD; 2.4-3.5% RSD) for positive and negative ion detection modes, respectively. The statistical comparison of the quantitative results obtained in positive and negative ion detection mode was performed. [Copyright &y& Elsevier]

Abstract: UHPLC–MS/MS method using BEH C18 analytical column was developed for the separation and quantitation of 12 phenolic compounds of Chamomile (Matricaria recutita L.). The separation was accomplished using gradient elution with mobile phase consisting of methanol and formic acid 0.1%. ESI in both positive and negative ion mode was optimized with the aim to reach high sensitivity and selectivity for quantitation using SRM experiment. ESI in negative ion mode was found to be more convenient for quantitative analysis of all phenolics except of chlorogenic acid and kaempherol, which demonstrated better results of linearity, accuracy and precision in ESI positive ion mode. The results of method validation confirmed, that developed UHPLC–MS/MS method was convenient and reliable for the determination of phenolic compounds in Chamomile extracts with linearity >0.9982, accuracy within 76.7–126.7% and precision within 2.2–12.7% at three spiked concentration levels. Method sensitivity expressed as LOQ was typically 5–20nmol/l. Extracts of Chamomile flowers and Chamomile tea were subjected to UHPLC–MS/MS analysis. The most abundant phenolic compounds in both Chamomile flowers and Chamomile tea extracts were chlorogenic acid, umbelliferone, apigenin and apigenin-7-glucoside. In Chamomile tea extracts there was greater abundance of flavonoid glycosides such as rutin or quercitrin, while the aglycone apigenin and its glycoside were present in lower amount. [ABSTRACT FROM AUTHOR]

Abstract: Twenty nine phenolic compounds comprising nine phenolic acids, sixteen flavonoids (including eight tea catechins, glycosides and aglycones), four coumarins plus caffeine were analysed within 20min using ultra high performance liquid chromatography (UHPLC) with PDA detection. UHPLC system was equipped with C18 analytical column (100mm×2.1mm, 1.7μm), utilising 0.1% formic acid and methanol mobile phase in the gradient elution mode. The developed method was tested for the system suitability: resolution, asymmetry factor, peak capacity, retention time repeatability and peak area repeatability. The method was fully validated in the terms of linearity (r 2 >0.9990 for all 30 compounds), range (typically 1–100mgL−1), LOD, LOQ, inter/intra-day precision (<3% and <9% respectively) and inter/intra-day accuracy (typically 100±10%). Subsequently the method was applied to the identification (spectral information and peak purity calculations were profited) and quantification of phenolic compounds and caffeine present in tea infusions and extracts. [Copyright &y& Elsevier]

Abstract: Any bio-analytical method includes several steps, all of them being important in order to achieve reliable results. The first step is taking aliquots of samples for the analysis, followed by the extraction procedure and sample clean-up, chromatographic analysis and detection. Chromatographic methods, particularly liquid chromatography, are the methods of choice in bio-analytical laboratories. Current trends in fast liquid chromatographic separations involve monolith technology, fused core columns, high temperature liquid chromatography and ultra-high performance liquid chromatography (UHPLC). UHPLC has recently become a wide-spread analytical technique in many laboratories which focus on fast and sensitive bio-analytical assays. The key advantages of UHPLC are the increased speed of analysis, higher separation efficiency and resolution, higher sensitivity and much lower solvent consumption as compared to other analytical approaches. This is all enabled by specially designed instruments and sub-2-microne particle packed analytical columns. There is a great contrast between ultra-fast chromatographic analysis and conventional sample preparation, which remains highly labor-intensive and time-consuming. Conventional sample preparation techniques including SPE, solid phase extraction; LLE, liquid–liquid extraction; PP, protein precipitation and many modern approaches (RAM, restricted access material; MIP, molecularly imprinted polymers; SPME, solid phase microextraction; LLME, liquid–liquid microextraction; MEPS, microextraction by packed sorbent and many others) have also been featured as fundamental and critical step of bio-analytical methods. [Copyright &y& Elsevier]

Abstract: The widespread occurrence of antibiotics as contaminants in the aquatic environment has increased attention in the last years. The concern over the release of antibiotics into the environment is related primarily to the potential for the development of antimicrobial resistance among microorganisms. This article presents an overview of analytical methodologies for the determination of quinolone (Qs) and fluoroquinolone (FQs), macrolide (MLs), tetracycline (TCs), sulfonamide (SAs) antibiotics and trimethoprim (TMP) in different environmental waters. The analysis of these antibiotics has usually been carried out by high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) and to a lesser extent by ultraviolet (UV) or fluorescence detection (FD). A very important step before LC analysis is sample preparation and extraction leading to elimination of interferences and prevention of matrix effect and preconcentration of target analytes. [Copyright &y& Elsevier]

Abstract: Simvastatin and atorvastatin belong to the group of hypolipidemic drugs, more exactly to the second generation of inhibitors of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. They induce a significant reduction in total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides, therefore they are widely used in the treatment of hypercholesterolemia even of its severe form-familiar hypercholesterolemia. Simvastatin and atorvastatin as the most widely used statins in clinical treatment and their hydroxy-acid/lactone forms were determined by means of UPLC in connection with triple quadrupole mass spectrometer. Deuterium labeled reference standard compounds were used as internal standards for the quantitation. Separation was performed on Acquity BEH C18 (100mm×2.1mm, 1.7μm) using gradient elution by mobile phase containing acetonitrile and ammonium acetate pH 4.0, which is convenient in order to prevent interconversion of analytes. ESI in positive mode was used for the ionization of all compounds. Two SRM (selected reaction monitoring) transitions were carefully optimized for each analyte in order to get high sensitivity and selectivity. SPE on Discovery DSC-18 was used as a sample preparation step. Intra-day precision was generally within 10% RSD, while inter-day precision within 15% RSD. Method accuracy expressed as recovery ranged from 75 to 100%. The method was validated with the sensitivity reaching LOQ 0.08–5.46nmol/l and LOD 0.01–1.80nmol/l in biological samples. Atorvastatin, simvastatin, its metabolites and hydroxy-acid/lactone forms were monitored in human serum and in lipoprotein fractions (LDL, HDL and VLDL) at patients with end stage renal diseases. [Copyright &y& Elsevier]

Abstract: CAD (charged aerosol detector) has recently become a new alternative detection system in HPLC. This detection approach was applied in a new HPLC method for the determination of three of the major statins used in clinical treatment—simvastatin, lovastatin and atorvastatin. The method was optimized and the influence of individual parameters on CAD response and sensitivity was carefully studied. Chromatography was performed on a Zorbax Eclipse XDB C18 (4.6mm×75mm, 3.5μm), using acetonitrile and formic acid 0.1% as mobile phase. The detection was performed using both CAD (20pA range) and DAD (diode array detector—238nm) simultaneously connected in series. In terms of linearity, precision and accuracy, the method was validated using tablets containing atorvastatin and simvastatin. The CAD is designated to be a non-linear detector in a wide dynamic range, however, in this application and in the tested concentration range its response was found to be perfectly linear. The limits of quantitation (0.1μg/ml) were found to be two times lower than those of UV detection. [Copyright &y& Elsevier]

Abstract: Two novel chromatographic methods both based on utilization of sub-2-micron particle columns were developed for the analysis of phenolic compounds in this work. An HPLC system was equipped with C18 silica-based analytical column (50mm×4.6mm, 1.8μm) and a UPLC system with ethylene-bridged hybrid C18 analytical column (100mm×2.1mm, 1.7μm). In total 34 phenolic substances were divided into groups of phenolic acids, flavonoids, catechins and coumarins and were analysed in sequence using different gradient methods. System suitability test data, including repeatability of retention time and peak area, mean values of asymmetry factor, resolution, peak capacity and the height equivalent of a theoretical plate were determined for each gradient method by 10 replicate injections. The developed methods were applied in the analysis of real samples (grape wines, teas). [Copyright &y& Elsevier]

This paper deals with the development of a novel method for simultaneous determination of estradiol, its degradation product estrone, and two preservatives, methylparaben and propylparaben, in the topical preparation Estradiol HBF. After optimization of the analytical conditions the method was validated and applied in studies of the stability of the topical preparation Estrogel HBF. Separation of all these compounds was performed on a Supelco Discovery C18 (250 mm×3.0 mm, 5 µm) analytical column. A mixture of acetonitrile, methanol, and water (23:24:53v/v) was chosen as mobile phase. UV absorbance at 225 nm was used for detection and quantitation of analytes. The total analysis time was less than 12 min at a flow rate of 0.9 mL min-1. All the compounds were isolated from the topical gel by simple extraction with an acetonitrile solution of hydrocortisone, as internal standard, and using sonication and centrifugation thereafter. The supernatant was injected directly on to the analytical column. The recovery of the procedure was from 96.9 to 100.4%. Validation of method according international guidelines was successfully performed. [ABSTRACT FROM AUTHOR]

A complete analytical procedure combining optimized tea infusion preparation and validated UHPLC–MS/MS method was developed for routine quantification of eight naturally occurring catechin derivatives in various tea samples. The preparation of tea infusions was optimized in terms of temperature, time and water-to-tea ratio in green, white and black teas. The catechins were analyzed using ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry in a run of only 4 min including equilibration of the system. The UHPLC–MS/MS method was fully validated in terms of inter/intra-day precision, accuracy, linearity ( r 2 > 0.9991), range (50–5000 ng/ml), LOD (1.5–7.5 ng/ml) and LOQ (5–25 ng/ml). Validation of the method included also the determination of the matrix effects that were evaluated in both flavored and unflavored green, white and black teas. Dilution of the resulting tea infusions appeared to be crucial for the matrix effects and also for subsequent catechin quantification in real tea samples in order to fit into the linear range of the UHPLC–MS/MS method. This complete procedure for catechin quantification was finally applied to real sample analysis represented by 70 commercial tea samples. [ABSTRACT FROM AUTHOR]

Fast, selective, and sensitive ultra-high performance liquid chromatography method with tandem mass spectrometry detection for the determination of quercetin and its metabolites with various physico-chemical properties such as molecular weight, lipophilicity, and acid-base properties has been developed. These compounds included small hydrophilic phenolic acids and more lipophilic metabolites with preserved flavonoid structure in small amount of rat plasma. The developed method enables selective separation of phenolic acids and a pair of isomers tamarixetin and isorhamnetin with satisfactory peak shapes and a high sensitivity using mass spectrometry detection. In addition, two sample preparation procedures including protein precipitation and microextraction in packed sorbent (MEPS) were optimized. The sample acidification included in protein precipitation as well as optimizing of MEPS sorbents and elution solvents improved isolation of quercetin and related compounds from rat plasma. Finally, both methods developed for sample preparation were fully validated to demonstrate sufficient accuracy and precision and acceptable matrix effects. Both sample preparation approaches combined with mass spectrometry-based quantification allowed the simultaneous determination of quercetin and its metabolites from a small amount of biological samples of only 50 μL. Due to the fast and non-selective parallel sample preparation, the protein precipitation was eventually applied to plasma samples derived from pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

Agomelatine is one of the newest antidepressants. Due to a different mechanism of action it offers a completely new approach in the treatment of depressive disorders. Two chromatographic methods for determination of agomelatine and its impurities were developed. The separations on UHPSFC system were accomplished using stationary phase based on BEH 2-EP and gradient elution with CO 2 and methanol containing 20 mM ammonium formate and 5% of water. The UHPLC separations were performed on stationary phase BEH Shield RP18. The mixture of acetonitrile and methanol in ratio 1:1 and ammonium acetate buffer pH 9.5 were used as mobile phase. Both developed methods were properly validated in terms of linearity, sensitivity (LOD, LOQ), accuracy and precision according to ICH guidelines. The UHPSFC method was linear in the range 0.25–70 μg/ml for all analytes with method accuracy ≥97.4% and ≥100.2% and method intra-day precision RSD ≤2.4 and ≤0.8 for impurities and API (active pharmaceutical ingredient), respectively. The UHPLC method was linear in the range 0.1–10 μg/ml for all analytes except three impurities for which the linear range was larger 0.1–25 μg/ml. Method accuracy ≥95.7% and ≥95.2% and method intra-day precision RSD ≤2.6 and ≤1.5 were found for impurities and API, respectively. The measurement of tablet samples was performed and the selected parameters of the methods were compared. In conclusion, both methods were appropriate for the determination of agomelatine and its impurities in pharmaceutical quality control (QC), although the UHPSFC method was found as more convenient especially in the method development phase. The advantages of newly developed UHPSFC-PDA (photo diode array detector) method were its environmental friendliness due to the mobile phase used, a very good resolution, selectivity and high speed of analysis (total time of separation was 4.1 min). [ABSTRACT FROM AUTHOR]

Highlights: [•] Two new impurities were determined using HPLC method with UV detection in retigabine. [•] The impurities were identified as dimeric impurities of retigabine using LC–MS, NMR and IR study. [•] Reference standards of impurities were prepared via organic synthesis and HPLC purification. [Copyright &y& Elsevier]

Abstract: 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 1.7μm 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 7ngmL−1 to 50μgmL−1 with method limit of detection (MLOD) as low as 0.2ngmL−1 for most of the analytes. MS detection showed even higher sensitivity reaching MLOD of 0.003ngmL−1, which is the highest sensitivity reported so far in analysis of TCs. Matrix matched calibration curves in the range of 0.01–50ngmL−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. [Copyright &y& Elsevier]

Abstract: A simple and reproducible method for the determination of pravastatin and pravastatin lactone in rat plasma and urine by means of ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) using deuterium labeled internal standards for quantification is reported. Separation of analytes was performed on BEH C18 analytical column (50mm×2.1mm, 1.7μm), using gradient elution by mobile phase consisting of acetonitrile and 1mM ammonium acetate at pH 4.0. Run time was 2min. Quantification of analytes was performed using the SRM (selected reaction monitoring) experiment in ESI negative ion mode for pravastatin and in ESI positive ion mode for pravastatin lactone. Sample treatment consisted of a protein precipitation by ACN and microextraction by packed sorbent (MEPS) for rat plasma. Simple MEPS procedure was sufficient for rat urine. MEPS was implemented using the C8 sorbent inserted into a microvolume syringe, eVol hand-held automated analytical syringe and a small volume of sample (50μl). The analytes were eluted by 100μl of the mixture of acetonitrile: 0.01M ammonium acetate pH 4.5 (90:10, v:v). The method was validated and demonstrated good linearity in range 5–500nmol/l (r 2 >0.9990) for plasma and urine samples. Method recovery was ranged within 97–109% for plasma samples and 92–101% for the urine samples. Intra-day precision expressed as the % of RSD was lower than 8% for the plasma samples and lower than 7% for the urine samples. The method was validated with sensitivity reaching LOD 1.5nmol/l and LOQ 5nmol/l in plasma and urine samples. The method was applied for the measurement of pharmacokinetic plots of pravastatin and pravastatin lactone in rat plasma and urine samples. [Copyright &y& Elsevier]

Abstract: Candida albicans is generally one of the most commonly isolated fungal pathogen from human body. It is a frequent cause of nosocomial infections, bloodstream infections, urinary infections and mucosal infections of oral cavity and vagina C. albicans can grow as hyphae, pseudohyphae, or budding yeast. Morphological conversion of a yeast form to pseudohyphal or hyphal one is often characterized by the change of commensal status to an invasive form. Farnesol and tyrosol can participate in these transformation processes as quorum sensing molecules together with some physical–chemical factors. A new analytical method for identification and quantification of biologically active substances farnesol and tyrosol using ultra high performance liquid chromatography (UHPLC) in connection with tandem mass spectrometry was developed. The analytes were separated on Acquity BEH C18 analytical column using binary mobile phase consisting of acetonitrile and formic acid 0.075% (75:25) at flow-rate 0.20ml/min. SRM (selected reaction monitoring) mode was applied in order to ensure sufficient selectivity and sensitivity using the first most intensive transition as a quantitative (121>77 and 205>121) and second one for the confirmation purposes (121>93 and 205>109). The method was validated in terms of linearity (>0.9994), precision (0.5–3.8% RSD), accuracy (78.9–106.0%), LOD (limit of detection) and LOQ (limit of quantitation). The method can serve as an analytical tool for the detection and determination of quorum-sensing molecules in biological samples. [Copyright &y& Elsevier]