A water-soluble preparation for intravenous administration of isorhamnetin and its pharmacokinetics in rats.

Flavonoids are considered as health-protecting food constituents. The testing of their biological effects is however hampered by their low oral absorption and complex metabolism. In order to investigate the direct effect(s) of unmetabolized flavonoid, a preparation in a biologically friendly solvent for intravenous administration is needed. Isorhamnetin, a natural flavonoid and a human metabolite of the most frequently tested flavonoid quercetin, has very low water solubility (<3.5 µg/mL). The aim of this study was to improve its solubility to enable intravenous administration and to test its pharmacokinetics in an animal model. By using polyvinylpyrrolidone (PVP10) and benzalkonium chloride, we were able to improve the solubility approximately 600 times to 2.1 mg/mL. This solution was then administered intravenously at a dose of 0.5 mg/kg of isorhamnetin to rats and its pharmacokinetics was analyzed. The pharmacokinetics of isorhamnetin corresponded to two compartmental model with a rapid initial distribution phase (t1/2α: 5.7 ± 4.3 min) and a slower elimination phase (t1/2ß: 61 ± 47.5 min). Two sulfate metabolites were also identified. PVP10 and benzalkonium did not modify the properties of isorhamnetin (iron chelation and reduction, and cell penetration) substantially. In conclusion, the novel preparation reported in this study is suitable for future testing of isorhamnetin effects under in vivo conditions.

Effect of P-glycoprotein and Cotreatment with Sofosbuvir on the Intestinal Permeation of Tenofovir Disoproxil Fumarate and Tenofovir Alafenamide Fumarate.

PURPOSE: We aimed to compare the effects of P-glycoprotein (ABCB1) on the intestinal uptake of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), and metabolites, tenofovir isoproxil monoester (TEM) and tenofovir (TFV), and to study the molecular mechanism of drug-drug interaction (DDI) between sofosbuvir (SOF) and TDF/TAF. METHODS: Bidirectional transport experiments in Caco-2 cells and accumulation studies in precision-cut intestinal slices prepared from the ileal segment of rodent (rPCIS) and human (hPCIS) intestines were performed. RESULTS: TDF and TAF were extensively metabolised but TAF exhibited greater stability. ABCB1 significantly reduced the intestinal transepithelial transfer and uptake of the TFV(TDF) and TFV(TAF)-equivalents. However, TDF and TAF were absorbed more efficiently than TFV and TEM. SOF did not inhibit intestinal efflux of TDF and TAF or affect intestinal accumulation of TFV(TDF) and TFV(TAF)-equivalents but did significantly increase the proportion of absorbed TDF. CONCLUSIONS: TDF and TAF likely produce comparable concentrations of TFV-equivalents in the portal vein and the extent of permeation is reduced by the activity of ABCB1. DDI on ABCB1 can thus potentially affect TDF and TAF absorption. SOF does not inhibit ABCB1-mediated transport of TDF and TAF but does stabilise TDF, albeit without affecting the quantity of TFV(TDF)-equivalents crossing the intestinal barrier. Our data thus suggest that reported increases in the TFV plasma concentrations in patients treated with SOF and TDF result either from a DDI between SOF and TDF that does not involve ABCB1 or from a DDI involving another drug used in combination therapy.

Columns in analytical-scale supercritical fluid chromatography: From traditional to unconventional chemistries.

Within this review, we thoroughly explored supercritical fluid chromatography (SFC) columns used across > 3000 papers published from the first study carried out under SFC conditions in 1962 to the end of 2022. We focused on the open tubular capillary, packed capillary, and packed columns, their chemistries, dimensions, and trends in used stationary phases with correlation to their specific interactions, advantages, drawbacks, used instrumentation, and application field. Since the 1990s, packed columns with liquid chromatography and SFC-dedicated stationary phases for chiral and achiral separation are predominantly used. These stationary phases are based on silica support modified with a wide range of chemical moieties. Moreover, numerous unconventional stationary phases were evaluated, including porous graphitic carbon, titania, zirconia, alumina, liquid crystals, and ionic liquids. The applications of unconventional stationary phases are described in detail as they bring essential findings required for further development of the supercritical fluid chromatography technique.

Carbon dioxide expanded liquid: an effective solvent for the extraction of quercetin from South African medicinal plants.

BACKGROUND: Quercetin is one of the most important bioflavonoids having positive effects on the biological processes and human health. Typically, it is extracted from plant matrices using conventional methods such as maceration, sonication, infusion, and Soxhlet extraction with high solvent consumption. Our study aimed to optimize the environmentally friendly carbon dioxide-based method for the extraction of quercetin from quince fruit with an emphasis on extraction yield, repeatability, and short extraction time. RESULTS: A two-step design of experiments was used for the optimization of the key parameters affecting physicochemical properties, including CO2/co-solvent ratio, co-solvent type, temperature, and pressure. Finally, gas expanded liquid combining CO2/ethanol/H2O in a ratio of 10/81/9 (v/v/v) provided the best extraction yield. Extraction temperature 66 °C and pressure 22.3 MPa were the most suitable conditions after careful optimization, although both parameters did not significantly affect the process. It was confirmed by experiments in various pressure and temperature conditions and statistical comparison of obtained data. The optimized extraction procedure at a flow rate of 3 mL/min took 30 min. The repeatability of the extraction method exhibited an RSD of 20.8%. CONCLUSIONS: The optimized procedure enabled very fast extraction in 30 min using environmentally friendly solvents and it was successfully applied to 16 different plant samples, including 14 bulbs and 2 fruits from South Africa. The quercetin content in extracts was quantified using ultra-high performance liquid chromatography (UHPLC) with tandem mass spectrometry. UHPLC hyphenated with high-resolution mass spectrometry was used to confirm chemical identity of quercetin in the analyzed samples. We quantified quercetin in 11 samples of all 16 tested plants. The quercetin was found in Agapanthus praecox from the Amaryllidaceae family and its presence in this specie was reported for the first time.

Benefits and Pitfalls of HPLC Coupled to Diode-Array, Charged Aerosol, and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples.

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes.

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.

Featuring ultimate sensitivity of high-resolution LC-MS analysis of phenolics in rat plasma.

Sensitive analysis of very low-molecular weight metabolites using liquid chromatography with quadrupole-time-of-flight mass spectrometry is challenging due to the high losses of ions in a time-of-flight analyzer. Improvement in sensitivity for these analytes via the optimization of advanced parameters, including quadrupole profile, ion guide parameters, and duty cycle, has been achieved. The optimization of the method was carried out using a large spectrum of structurally different compounds including (iso)flavonoids and their known metabolites. These compounds can be categorized into two major groups, that is, compounds with (iso)flavonoid core and low-molecular weight phenolics. The optimization of the duty cycle enabled up to a 15-fold increase in analyte responses while the contribution of tuning ion optics and quadrupole profile was negligible. The limits of quantifications of our new method were assessed using both standard solutions and rat plasma. They were decreased at least 10 times for several low-molecular weight phenolics enabling measurement of their concentrations in a range of 1-50 ng/mL in rat plasma after protein precipitation. Concurrently, the limits of quantifications for compounds with (iso)flavonoid core did not increase distinctly allowing their detection in a range of 0.5-10 ng/mL. The new method was used for the targeting of phenolics in biological samples from pharmacokinetics experiments.

Unambiguous determination of farnesol and tyrosol in vaginal fluid using fast and sensitive UHPLC-MS/MS method.

The new ultra-high performance liquid chromatography method with tandem mass spectrometry detection (UHPLC-MS/MS) has been optimized to allow fast, selective, and high-throughput analysis of two Candida albicans quorum sensing molecules (QSM), farnesol and tyrosol. The problem of the presence of the interference in the samples and system was successfully solved by careful optimization of chromatographic conditions. Charged hybrid stationary phase modified with pentafluorophenyl group and optimized gradient elution provided adequate separation selectivity and peak shapes. The impurity was identified as dibutyl phthalate and had the same m/z ions as farnesol leading to an important interference on selected reaction monitoring channel. Two different types of biological matrices originating from vaginal fluid, supernatant and sediment, were analysed. Micro-solid phase extraction in pipette tips was optimized for the selective isolation of QSM from the supernatant. The insufficient retention of farnesol on the extraction sorbent was improved when 1% of organic solvent was added prior to extraction, while the retention of tyrosol was only possible when using combined C8 and polymer sorbent type. Strong retention of farnesol had to be solved by increasing elution solvent strength and volume up to 600 µL. However, this approach did not allow the pretreatment of sediment samples due to the sorbent clogging. Therefore, our previously developed protein precipitation method was modified and validated to analyse the sediments. New developed UHPLC-MS/MS method provided suitable accuracy and precision for the determination of QSM in vaginal fluid while using only 50 µL sample volume and two different sample preparation methods.

Development of 3,5-Dinitrophenyl-Containing 1,2,4-Triazoles and Their Trifluoromethyl Analogues as Highly Efficient Antitubercular Agents Inhibiting Decaprenylphosphoryl-ß-d-ribofuranose 2'-Oxidase.

We report herein the discovery of 3,5-dinitrophenyl 1,2,4-triazoles with excellent and selective antimycobacterial activities against Mycobacterium tuberculosis strains, including clinically isolated multidrug-resistant strains. Thorough structure-activity relationship studies of 3,5-dinitrophenyl-containing 1,2,4-triazoles and their trifluoromethyl analogues revealed the key role of the position of the 3,5-dinitrophenyl fragment in the antitubercular efficiency. Among the prepared compounds, the highest in vitro antimycobacterial activities against M. tuberculosis H37Rv and against seven clinically isolated multidrug-resistant strains of M. tuberculosis were found with S-substituted 4-alkyl-5-(3,5-dinitrophenyl)-4H-1,2,4-triazole-3-thiols and their 3-nitro-5-(trifluoromethyl)phenyl analogues. The minimum inhibitory concentrations of these compounds reached 0.03 µM, which is superior to all the current first-line anti-tuberculosis drugs. Furthermore, almost all compounds with excellent antimycobacterial activities exhibited very low in vitro cytotoxicities against two proliferating mammalian cell lines. The docking study indicated that these compounds acted as the inhibitors of decaprenylphosphoryl-ß-d-ribofuranose 2'-oxidase enzyme, which was experimentally confirmed by two independent radiolabeling experiments.

A validated UHPLC method for the determination of caffeoylquinic and di-caffeoylquinic acids in green coffee extracts using an RP-Amide fused-core column.

The presented work describes the development and validation of a rapid UHPLC-UV method using a fused core particle column with an RP-Amide stationary phase for the separation and quantitative analysis of caffeoylquinic and di-caffeoylquinic acids in green coffee extracts. Three caffeoylquinic acids (3-caffeoylquinic acid, 4-caffeoylquinic acid, and 5-caffeoylquinic acid) and two di-caffeoylquinic acids (1,3-di-caffeoylquinic acid, and 3,5-di-caffeoylquinic acid) were separated and analyzed in 8 min. That was possible due to the unique selectivity of the RP-Amide stationary phase for the analyzed acids. The retention behavior of all analytes under different compositions of the mobile phase on different columns was evaluated in this study. The optimal chromatographic separation was performed using an Ascentis Express RP-Amide (100 × 2.1 mm) fused-core column with a particle size of 2.7 µm at a temperature of 30 °C. For validation of the newly developed method, acetonitrile was used as mobile phase B and 5% formic acid, filtrated through a 0.22 µm filter, was used as mobile phase A. They were delivered at a flow rate of 0.9 mL min-1 according to the elution gradient program. The detection wavelength was set at 325 nm. A solid-liquid extraction with a solution of methanol and a 5% water solution of formic acid (25 + 75 v/v) using an ultrasonic bath was chosen for the preparation of the available commercial samples of food supplements containing a green coffee extract. Recoveries for all analyzed acids were 98.2-101.0% and the relative standard deviation ranged from 0.3% to 1.4% for intra-day and from 0.3% to 3.0% for inter-day repeatability. The limits of detection were in the range of 0.30-0.53 µg mL-1.

Development of water-soluble 3,5-dinitrophenyl tetrazole and oxadiazole antitubercular agents.

In this work, four series of tertiary amine-containing derivatives of 3,5-dinitrophenyl tetrazole and oxadiazole antitubercular agents were prepared, and their in vitro antimycobacterial effects were evaluated. We found that the studied compounds showed lipophilicity-dependent antimycobacterial activity. The N-benzylpiperazine derivatives, which had the highest lipophilicity among all of the series, showed the highest in vitro antimycobacterial activities against Mycobacterium tuberculosis CNCTC My 331/88 (H37Rv), comparable to those of the first-line drugs isoniazid and rifampicin. The presence of two tertiary amines in these N-benzylpiperazine derivatives enabled us to prepare water-soluble dihydrochloride salts, overcoming the serious drawback of previously described 3,5-dinitrophenyl tetrazole and oxadiazole lead compounds. The water-soluble 3,5-dinitrophenyl tetrazole and oxadiazole antitubercular agents described in this work are good candidates for further in vitro and in vivo pharmacokinetic and pharmacodynamic studies.

Micro-SPE in pipette tips as a tool for analysis of small-molecule drugs in serum.

AIM: Micro-SPE in pipette tips (µ-SPE-PT) with particle sorbent has never been used in small-molecule drug analysis. Methodology & results: µ-SPE-PT was used for the extraction of statins from biological materials followed by UHPLC-MS/MS. The commercial and homemade µ-SPE-PT tips filled with particle sorbent were compared. While the homemade tips enabled direct serum sample loading into the sorbent, protein precipitation (PP) had to be implemented before µ-SPE-PT procedure using commercial tips. Three µ-SPE-PT methods were developed and validated: method A: µ-SPE-PT with homemade tips; method B: PP + µ-SPE-PT with homemade tips; and method C: PP + µ-SPE-PT with commercial tips. Method A enabled a simple high-throughput approach (48 samples in 90 min) compared with methods B and C that required three-times longer time. However, PP increased the recoveries of protein-bound analytes and extracts purity in methods B and C. The matrix effects without internal standards correction for method C were significantly higher than those for the methods A and B. CONCLUSION: Compared with commercial tips, homemade tips filled with particles were found to be more suitable for drug analysis. Commercial tips tested in this study were found challenging but the conditions under which they could be applicable were also defined.

Development of MEPS-UHPLC-MS/MS multistatin methods for clinical analysis.

BACKGROUND: Statins are the microsomal 3-hydroxy-3methylglutaryl-coenzyme A reductase inhibitors used for the treatment of hypercholesterolemia. Some recent studies revealed also the extra-lipid effects and anticancer activities. Due to the wide incidence of cancer diseases, the number of studies dealing with anticancer statin activities has grown in recent years. Development of one universal multistatin method will be a very convenient way of providing practical and economical multiple statin analysis. Results/methodology: Fast and sensitive methods for determination of seven clinically relevant statins, their interconversion products and metabolites (17 analytes in total) in biological samples using microextraction by packed sorbent for sample preparation and UHPLC-MS/MS for subsequent analysis were developed and validated. Three MS platforms with different ion sources, transfer optics, collision cell technologies and scan speed parameters were compared. CONCLUSION: Significant differences among the methods were observed in terms of selectivity and sensitivity. Microextraction by packed sorbent was successful in the extraction of all 17 analytes from biological matrix.

Development and validation of UHPLC-MS/MS method for determination of eight naturally occurring catechin derivatives in various tea samples and the role of matrix effects.

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.

Determination of amphetamine and methadone in human urine by microextraction by packed sorbent coupled directly to mass spectrometry: an alternative for rapid clinical and forensic analysis.

Speed and low cost, together with regulatory approval, are the most important requirements of clinical assays. Therefore, a fast and automated on-line sample preparation method is essential for the routine analysis of biological samples. Microextraction by packed sorbent is an option for optimal sample preparation due to its easy automation, minimal requirements for the sample and elution solvent volumes, elimination of evaporation and reconstitution steps, and ability to integrate sample preparation and injection into one step. The use of effective sample preparation steps circumvents the need for chromatographic separation and therefore allows more rapid and less expensive sample analysis in clinical and forensic practice. Two biologically active compounds, amphetamine and methadone, were chosen as representative drugs of abuse for the application of microextraction by packed sorbent coupled directly to mass spectrometry. The developed method was validated, with the results confirming the suitability of the combination of these techniques for the analysis of biological samples. The approach was confirmed to be appropriate for use in clinical and forensic practice with regard to cost and time requirements for analysis.

Study of the retention behavior of small polar molecules on different types of stationary phases used in hydrophilic interaction liquid chromatography.

The retention behavior of a large group of analytes (35) with varied properties (pKa and logP) was studied on eight hydrophilic interaction LC columns with different surfaces, stationary phase chemistries, and types of particles. The acetonitrile content (5-95%), buffer concentration (0.5-200 mM), and pH of the mobile phase (3.8 and 6.8) were evaluated for their effects on the retention behavior. The type of stationary phase had a significant impact on the selectivity and retention time of the tested analytes. Completely different selectivity was observed on the aminopropyl stationary phase. In this study, the influence of the buffer concentration was similar for all tested columns, except for the aminopropyl stationary phase. Increasing the buffer concentration led to decreased retention times for the basic compounds and increased retention times for the acidic compounds, while the inverse behavior was observed on the aminopropyl stationary phase. The selectivity of the individual stationary phases was evaluated at pH 3.8 and 6.8. Much lower selectivity differences between the stationary phases were observed at pH 6.8 than pH 3.8. Bare silica stationary phases were used in the comparison of the particles (fused-core and fully porous particles of 3 and 1.7 µm) and the columns provided by different manufacturers.

How to address the sample preparation of hydrophilic compounds: Determination of entecavir in plasma and plasma ultrafiltrate with novel extraction sorbents.

Two fast, simple, selective and economical sample preparation methods for the determination of entecavir in biological materials available at low amounts are reported. The choice of optimal extraction techniques was performed with regard to analyte hydrophilicity, sample volumes, selectivity, method recovery and rapidity. The compatibility of the eluate with the hydrophilic interaction chromatography (HILIC) mobile phase was crucial to allow the elimination of the evaporation and reconstitution steps and to obtain acceptable peak shapes. Different types of sorbents were employed for the extraction of two biological materials (plasma and plasma ultrafiltrate). The mixed-mode polymeric sorbent MCX was chosen as a suitable one for the solid phase extraction (SPE) of plasma samples. The analytes were eluted with 1ml of the mixture of 5 % ammonium hydroxide in ACN:water (95:5). Protein precipitation (PP) with 1ml of ACN was used to remove proteins from 500µl of plasma sample prior to SPE extraction. The microextraction by packed sorbent (MEPS) was employed for the cleaning up of plasma ultrafiltrate samples due to very small volumes available for the analysis. MEPS implemented a novel sorbent based on porous graphitic carbon, semi-automatic analytical syringe and a small volume of sample (50µl). The elution step was performed using 100µl of the mixture of 5mM ammonium acetate pH 4.0:ACN (25:75). The MEPS eluate was fully compatible with HILIC mobile phase subsequently used for the analysis of entecavir, unlike SPE eluate, which had to be evaporated and reconstituted in mobile phase. Both analytical methods were validated and demonstrated good linearity in a range 1-100ng/ml (r(2)>0.9992) for plasma samples and in a range 0.5-100ng/ml (0.9991) for the plasma ultrafiltrate samples. Intra-day accuracy expressed as recovery was within the range from 80-98% for the plasma samples and 97-106% for the plasma ultrafiltrate samples. Inter-day accuracy ranged within 81-106% for the plasma and 95-101% for the plasma ultrafiltrate samples. The intra-day precision expressed as the % of RSD was lower than 4% for both matrices and inter-day precision was lower than 7% for plasma and lower than 17% for plasma ultrafiltrate. Method sensitivity reached LLOQ of 1ng/ml in plasma and 0.5ng/ml in plasma ultrafiltrate samples. The method was applied for the determination of concentration-time profiles of entecavir in plasma of the perfusate for rat kidney perfusion and for the measurement of concentration of entecavir in plasma ultrafiltrate samples. The results should be helpful in the evaluation of excretion mechanism of entecavir.

HILIC UHPLC-MS/MS for fast and sensitive bioanalysis: accounting for matrix effects in method development.

BACKGROUND: Matrix effects are considered to be a main obstacle of quantitative bioanalytical LC-MS/MS methods. Therefore it is often required to minimize them in order to increase method reliability. HILIC has been referenced as one of possible approaches. However, there is a lack of experimental evidence in scientific literature so far. METHODOLOGY: Matrix effects were evaluated using spiked serum samples after SPE and protein precipitation prior to UHPLC-ESI-MS/MS. Chromatography was performed in both HILIC and reversed-phase mode. The influence of the matrix effects on the signal response was assessed using a set of 34 compounds of pharmaceutical interest and post-extraction addition approach. RESULTS: The advantages and drawbacks of the HILIC and reversed-phase chromatographic modes were compared and discussed in detail. CONCLUSION: HILIC demonstrated the potential to reduce the occurrence of matrix effects when a more thorough sample pretreatment procedure such as SPE was applied.

Highly sensitive fast determination of entecavir in rat urine by means of hydrophilic interaction chromatography-ultra-high-performance liquid chromatography-tandem mass spectrometry.

Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC-UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3 ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC-MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1 ml, 30 mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C(2)(13) N(15), which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87-114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100 pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%).

Evaluation of new mixed-mode UHPLC stationary phases and the importance of stationary phase choice when using low ionic-strength mobile phase additives.

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.