Search Results (10)

Midazolam is a benzodiazepine that is utilized for the induction of anesthesia and the facilitation of procedural sedation. Despite the absence of stereogenic centers, the non-planar seven-membered ring devoid of reflection symmetry elements confers planar stereogenicity to the molecule. Due to the rapid conformational inversion of the Rp and Sp enantiomers, which occurs via a simple ring flip, high-performance liquid chromatography (HPLC) enantiomeric separation is restricted to sub-room temperature conditions. In this study, the energy barriers for the racemization of midazolam at five distinct temperatures and in acetonitrile/water mixtures were determined by monitoring the decay of the circular dichroism signal at a specific wavelength over time. The kinetic and thermodynamic data obtained were compared with those determined by dynamic enantioselective high-performance liquid chromatography using the Chiralpak IG-3 chiral stationary phase, which contains the amylose tris(3-chloro-5-methylphenylcarbamate) as the selector. The temperature-dependent dynamic HPLC of midazolam was carried out at the same temperatures and with the same aqueous mixtures used in parallel kinetic off-column experiments. To simulate dynamic chromatographic profiles, a lab-made computer program based on a stochastic model was utilized. The results indicated that the moderate influence of the stationary phase resulted in a slight increase in the activation barriers, which was more pronounced as the time spent in the column increased. This phenomenon was found to be mitigated when switching from a 250 mm × 4.6 mm, 3 µm, Chiralpak IG-3 column to a 50 mm × 4.6 mm, 1.6 µm, Chiralpak IG-U UHPLC column. The outcomes obtained under UHPLC conditions were found to be more closely aligned with those obtained through the ECD technique, with a discrepancy of only 0.1 kcal/mol or less, indicating a high degree of concordance between the two methods. Full article

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of the potential impurities of dexketoprofen, including the distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversed-phase modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition and temperature for enantio- and chemoselectivity showed many unexpected observations. It was observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity was also observed in reversed-phase mode using methanol/water mixtures on amylose-type columns. This could indicate that the three-dimensional structure of the amylose column can change by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled enantioseparation in the operative temperature range were also observed in reversed-phase mode. To find the best methodological conditions for the determination of dexketoprofen impurities, a full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2 column with water/acetonitrile/acetic acid 50/50/0.1 (v/v/v) at a 1 mL/min flow rate at 20 °C), baseline separations were achieved between all compounds within 15 min. Our newly developed HPLC method was validated according to the current guidelines, and its application was tested on commercially available pharmaceutical formulations. According to the authors’ knowledge, this is the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode. Full article

A simple, selective, rapid, sensitive and less costly green automated solid phase extraction bio-analytical high-performance liquid chromatographic-based technique with fluorescence detection (Aut-SPE-BA-HPLC-FL) for the quantification of levofloxacin in human serum samples has been developed and validated. The serum samples were loaded into the chromatographic system without prior treatment and then injected into short (20 mm × 4.6 mm, 20 µm) protein-coated (PC) µBondapak CN (µBCN) silica pre-column (PC-µBCN-pre-column). Levofloxacin was retained and pre-concentrated on the head of the PC-µBCN-pre-column, while proteins and other polar components were eliminated using phosphate buffer saline (PBS), pH 7.4, as the first mobile phase in the extraction step. Levofloxacin is then transferred to the analytical column; ZORBAX Eclipse XDB-C18 (150 mm × 46 mm, 5 µm), through the aid of a column-switching valve technique, on-throughs the elution mode using the second mobile phase containing a methanol and phosphate buffer (0.05 M, pH 5) in a ratio of 70:30 (v/v). Levofloxacin signals were detected using a fluorescence detector operated at excitation/emission wavelengths of 295/500 nm. The proposed Aut-SPE-BA-HPLC-FL methodology showed linearity over a levofloxacin concentration range of 10–10,000 ng/mL (r² = 0.9992), with good recoveries ranging from 87.12 to 97.55%. Because of the validation qualities in terms of linearity, recovery, precision, accuracy, selectivity and robustness, the Aut-SPE-BA-HPLC-FL method has been used in some clinical trials for therapeutic drug monitoring and the pharmacokinetic study of levofloxacin in human serum. Full article

Bisphenols (BPs) are a class of endocrine disruptors widely existing in the environment. They have a great impact on human health owing to their environmental endocrine disrupting effects, chronic toxicity, neurotoxicity, cytotoxicity and genetic toxicity. In this paper, an on-line packed fiber solid phase extraction (PFSPE) coupling with column-switching HPLC-FLD determination method was developed for the determination of eight BPs in drinking water. The poly (dibenzo-18-crown-6-ether)/polystyrene composite nanofibers (PDB18C6/PS) were prepared by electrospinning and used as an adsorbent for the on-line PFSPE column. The on-line PFSPE-HPLC equipment contained a dual ternary pump and a switching valve to enable enrichment, purification, and analysis directly in the system. The results showed that the proposed on-line PFSPE-HPLC-FLD method realized the simultaneous separation and detection of eight BPs: BPF, BPE, BPA, BPB, BPAF, BPAP, BPC and BPZ. The curves of the target analytes were prepared with good correlation coefficient values (r² > 0.998) in the range of 50–1000 pg/mL. The limit of detection (S/N = 3) was 20 pg/mL, the limit of quantitation (S/N = 10) is 50 pg/mL. The recoveries of eight BPs were 94.8–127.3%, and the intra-day precisions (RSD) were less than 10%. The PFSPE column made of the PDB18C6/PS composite nanofibers has stable properties and can be reused at least 200 times. In the detection of drinking water samples, BPZ was detected in nearly 80% of drinking water samples, and BPA, BPAP, BPF and BPAF were also detected in some water samples. This high level of integration and automation was achieved in pretreatment of eight BPs from water samples. The proposed simple, rapid, and practical method has been successfully applied to the detection of eight BPs in drinking water, which can provide powerful technical support for drinking water quality and safety monitoring. Full article

A novel, validated, reversed-phase (RP), chiral high performance liquid chromatography (HPLC) method was developed for the enantiopurity control analysis of naproxen, a frequently used non-steroidal anti-inflammatory agent using polysaccharide-type chiral stationary phase (CSP). In the screening phase of method development, seven columns were tested in polar organic (PO) mode using mobile phases consisting of 0.1% acetic acid in methanol, ethanol, 2-propanol, and acetonitrile. Enantiorecognition was observed only in five cases. The best enantioseparation was observed on a Lux Amylose-1 column with 0.1% (v/v) acetic acid in ethanol with a resolution (R_s) of 1.24. The enantiomer elution order was unfavorable, as the distomer eluted after the eutomer. When the ethanolic mobile phase was supplemented with water, enantiomer elution order reversal was observed, indicating a difference in the enantiorecognition mechanism upon switching from PO to RP mode. Furthermore, by changing ethanol to methanol, not only lower backpressure, but also higher resolution was obtained. Subsequent method optimization was performed using a face-centered central composite design (FCCD) to achieve higher chiral resolution in a shorter analysis time. Optimized parameters offering baseline separation were as follows: Lux Amylose-1 stationary phase, thermostated at 40 °C, and a mobile phase consisting of methanol:water:acetic acid 85:15:0.1 (v/v/v), delivered with 0.65 mL/min flow rate. Using these optimized parameters, a R_s = 3.21 ± 0.03 was achieved within seven minutes. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of different pharmaceutical preparations, such as film-coated tablets and gel, as well as fixed-dose combination tablets, containing both naproxen and esomeprazole. Full article

Application of the poly-ɛ-caprolactone composite sorbent consisting of the micro- and nanometer fibers for the on-line extraction of non-steroidal anti-inflammatory drugs from a biological matrix has been introduced. A 100 μL human serum sample spiked with ketoprofen, naproxen, sodium diclofenac, and indomethacin was directly injected in the extraction cartridge filled with the poly-ɛ-caprolactone composite sorbent. This cartridge was coupled with a chromatographic instrument via a six-port switching valve allowing the analyte extraction and separation within a single analytical run. The 1.5 min long extraction step isolated the analytes from the proteinaceous matrix was followed by their 13 min HPLC separation using Ascentis Express RP-Amide (100 × 4.6 mm, 5 µm) column. The recovery of all analytes from human serum tested at three concentration levels ranged from 70.1% to 118.7%. The matrix calibrations were carried out in the range 50 to 20,000 ng mL⁻¹ with correlation coefficients exceeding 0.996. The detection limit was 15 ng mL⁻¹, and the limit of quantification corresponded to 50 ng mL⁻¹. The developed method was validated and successfully applied for the sodium diclofenac determination in real patient serum. Our study confirmed the ability of the poly-ɛ-caprolactone composite sorbent to remove the proteins from the biological matrix, thus serving as an alternative to the application of restricted-access media. Full article

According to the EU legislation, ochratoxin A contamination is controlled in wines. Tokaj wine is a special type of sweet wine produced from botrytized grapes infected by “noble rot” Botrytis cinerea. Although a high contamination was reported in sweet wines and noble rot grapes could be susceptible to coinfection with other fungi, including ochratoxigenic species, no screening of Tokaj wines for mycotoxin contamination has been carried out so far. Therefore, we developed an analytical method for the determination of ochratoxin A (OTA) and ochratoxin B (OTB) involving online SPE coupled to HPLC-FD using column switching to achieve the fast and sensitive control of mycotoxin contamination. The method was validated with recoveries ranging from 91.6% to 99.1% with an RSD less than 2%. The limits of quantification were 0.1 and 0.2 µg L⁻¹ for OTA and OTB, respectively. The total analysis time of the online SPE-HPLC-FD method was a mere 6 min. This high throughput enables routine analysis. Finally, we carried out an extensive investigation of the ochratoxin contamination in 59 Slovak Tokaj wines of 1959–2017 vintage. Only a few positives were detected. The OTA content in most of the checked wines did not exceed the EU maximum tolerable limit of 2 µg L⁻¹, indicating a good quality of winegrowing and storing. Full article

It is critical to assess the severity of alcohol consumption in certain diseases such as alcohol liver disease and alcohol addiction. Ethyl glucuronide (EtG) is a highly stable metabolite of ethanol in hair; thus, it was proposed as a long-term monitoring marker for alcohol consumption. Therefore, an HPLC-MS/MS method for EtG in hair was developed and applied to a clinical setting to assess the relevance of the EtG concentration and/or the Alcohol Use Disorders Identification Test (AUDIT) score to high-risk alcohol consumption. EtG was extracted from 10 mg of hair using water and analyzed using on-line sample purification coupled to HPLC-MS/MS. The diagnostic performances of the EtG concentration and/or the AUDIT score for detecting high-risk alcohol consumption were statistically evaluated between alcohol addicts (n = 44) and average alcohol users (n = 19). The on-line sample purification resulted in labor-saving with smaller sample amount. Both the EtG concentrations (4.0–587.4 pg/mg vs. 12.9–74.9 pg/mg) and the AUDIT scores (4–40 vs. 5–28) obtained from the alcohol addicts were significantly higher than those from the average alcohol users. The performance evaluation demonstrated that the integration score of the EtG concentration and the AUDIT score increased diagnostic performance for high-risk alcohol consumption. Full article

Column-switching high performance liquid chromatography (HPLC) is extensively used for the critical analysis of radiolabeled ligands and their metabolites in plasma. However, the lack of streamlined apparatus and consequently varying protocols remain as a challenge among positron emission tomography laboratories. We report here the prototype apparatus and implementation of a fully automated and simplified column-switching procedure to allow for the easy and automated determination of radioligands and their metabolites in up to 5 mL of plasma. The system has been used with conventional UV and coincidence radiation detectors, as well as with a single quadrupole mass spectrometer. Full article

Cost-effective isolation methods were developed on preparative HPLC, flash LC, and simulated moving bed (SMB) to prepare the process impurity, 3-(aminomethyl)-5-methylhex-4-enoic acid (4-ene impurity), of pregabalin. By a thorough experimental study on the different isolation techniques available, it was concluded that SMB was the most cost-effective. Hence, it was a continuous chromatography that utilized the advantage of SMB so that a high quantity of the impurity was generated in a short period of time. SMB was equipped with eight reversed-phased columns and was used to separate the process impurity of pregabalin. The effects of flow rate in zone 2 (Q2) and 3 (Q3), as well as switching time, on the operating performance parameters like purity, productivity, and desorbent consumption were studied. Operating conditions leading to more than 90% purity in the raffinate outlet stream were identified, together with those achieving optimal performance. All of these developed methods are novel, cost-effective, and can be applied to the isolation of other process- and stability-related impurities of pregabalin. Full article