Chromatographic Mobile Phase Research Articles

Design and evaluation of a liquid chromatographic column oven with adaptive and precise temperature control

High performance liquid chromatography (HPLC) is a key analytical technique that is used in a number of fields. Improving the separation efficiency, stability, and universality of HPLC has been a continuing analytical-chemistry focus. In chromatographic separation, factors such as the composition and ratio of the mobile phase, the type of stationary phase, and the dimensions of the chromatographic column significantly affect the separation efficiency. In addition, the temperatures of the chromatographic column and mobile phase are also important for achieving separation. The column oven is usually used to stably control the column temperature in the HPLC separation system. Indeed, highly accurate temperature control ensures superior separation performance, short analysis times, and repeatability. In this study, we innovatively improved the traditional column oven by combining a variety of temperature-control algorithms to deliver continuous and highly accurate temperature control in the wide 4-90 ℃ range, and by exploring a new chromatographic-method development route. Instead of focusing on the complex hardware system, we optimized the software to achieve highly stable and accurate (±0.1 ℃) temperature control. Temperature-control performance was further improved by optimizing the structure of the thermal insulation and employing reliable and environmentally friendly thermal-insulation materials. Additionally, the thermal conduction of the heat-source device is discussed based on the heat-transfer principle with the aim of improving the performance of the column oven. The improved column oven delivered significantly enhanced chromatographic-separation repeatability and stability thereby reliably guaranteeing the development of highly efficient chromatographic analysis methods.

Evaluation of Short-Term Stability of Different Nitazenes Psychoactive Opioids in Dried Blood Spots by Liquid Chromatography-High-Resolution Mass Spectrometry.

Nitazenes represent a new synthetic opioids sub-class belonging to new psychoactive substances (NPSs). Their high pharmacological potency has led to numerous intoxications and fatalities, even at minimum doses. The aim of this study was to assess the stability of four nitazenes (etazene, flunitazene, isotonitazene and protonitazene) in dried blood spot (DBS) samples at different storage temperatures (room temperature and 4 °C) and determine the optimal storage conditions. Moreover, we developed and validated a new and fast liquid chromatography-high-resolution mass spectrometry method by the optimization of chromatographic conditions with the use of a different chromatographic column and mobile phases. Two concentrations, 1 and 5 ng/mL, were chosen based on the available data on nitazenes-related intoxications and their stability was evaluated at days 0 (control), 1, 7 and 30. The results showed that all analytes at 1 ng/mL were not detectable after 30 days at room temperature; a similar pattern was observed for 1 ng/mL etazene and isotonitazene samples when stored at 4 °C, whereas flunitazene and protonitazene decreased to a mean of 66% and 69% initial concentrations, respectively, at day 30. Differently, all analytes at 5 ng/mL were quantified above 44% and 41% initial concentrations at room temperature and 4 °C, respectively, showing a higher stability. The study of nitazenes stability in DBSs represents an important tool to determine the optimal sample storage conditions, such as temperature and time between sample collection and analysis. In contrast to another study, our study showed distinct stability behaviors for every investigated analyte, which also depended on the concentration. Therefore, it is difficult to define an optimal storage condition acceptable for all nitazenes. Room temperature proved to be the best medium- and long-term storage conditions for the highest concentrations, but the stability of low levels of flunitazene and protonitazene improved at 4 °C.

Determination of eight neonicotinoid pesticides in wastewater by solid phase extraction combined with liquid chromatography-tandem mass spectrometry

Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E-nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients (r) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

Harnessing an elastic flow instability to improve the kinetic performance of chromatographic columns

Despite decades of research and development, the optimal efficiency of slurry-packed HPLC columns is still hindered by inherent long-range flow heterogeneity from the wall to the central bulk region of these columns. Here, we show an example of how this issue can be addressed through the straightforward addition of a semidilute amount (500 ppm) of a large, flexible, synthetic polymer (18 MDa partially hydrolyzed polyacrylamide, HPAM) to the mobile phase (1% NaCl aqueous solution, hereafter referred to as “brine”) during operation of a 4.6 mm × 300 mm column packed with 10μm BEHTM 125 Å particles. Addition of the polymer imparts elasticity to the mobile phase, causing the flow in the interparticle pore space to become unstable above a threshold flow rate. We verify the development of this elastic flow instability using pressure drop measurements of the friction factor versus Reynolds number. In prior work, we showed that this flow instability is characterized by large spatiotemporal fluctuations in the pore-scale flow velocities that may promote analyte dispersion across the column. Axial dispersion measurements of the quasi non-retained tracer thiourea confirm this possibility: they reveal that operating above the onset of the instability improves column efficiency by greater than 100%. These experiments thereby suggest that elastic flow instabilities can be harnessed to mitigate the negative impact of trans-column flow heterogeneities on the efficiency of slurry-packed HPLC columns. While this approach has its own inherent limitations and constraints, our results lay the groundwork for future targeted development of polymers that can impart elasticity when dissolved in commonly used liquid chromatography mobile phases, and can thereby generate elastic flow instabilities to help improve the resolution of HPLC columns.

Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (Tm) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (Tm was 67.1 – 78.2 °C for 10–100 mM [Na+] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li+], 68.1 °C in 10 mM [NH4+], 65.6 °C in 10 mM [Cs+], and 56.6 °C in 10 mM [triethylammonium+] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1–3 °C, and denaturation power increases in the order MeOH<EtOH<MeCN. Next, we investigated the RNA duplex melting using selected chromatographic techniques and within the 10–90 °C column temperature range. Melting temperature obtained with size exclusion chromatography in 25 mM sodium phosphate buffer was ∼ 70 °C, about 5 °C lower compared to DSC values. The apparent melting temperature for a reversed-phase chromatography experiment was < 10 °C for 10 mM ammonium acetate mobile phase with acetonitrile eluent. Ion-pair reversed-phase with 10 mM triethylamine, 100 mM hexafluoroisopropanol and MeCN as eluent yielded Tm 49.3–54.9 °C. Hydrophilic interaction chromatography experiments with 10 mM ammonium acetate and ∼ 50 % acetonitrile mobile phase showed high duplex stability (Tm ∼ 80 °C). The observed chromatographic Tm values suggest that the formation of an organic/aqueous solvent layer on the chromatographic sorbent surface affects the duplex stability more significantly than the composition of the mobile phase alone.

A batch screening technique for the calculation of chromatographic separability

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

Establishing carbon dots assisted high speed countercurrent chromatography and its application for efficient separation of rare earth element ions

Rare earth elements with unique magnetic properties and optical properties, known as the ‘industrial vitamin’, has a very high commercial value. As a secondary resource of rare earth elements, low-concentration solution includes mixed rare earth ions, which need to realize efficient separation and recovery urgently. High speed countercurrent chromatography is suitable for the separation and purification of rare earth element ions due to its advantages of large loading, good tolerance to samples, and simple pretreatment. In this study, a carbon dots assisted high speed countercurrent chromatography method was designed and established, the carbon dots were applied to the mobile phase of high speed countercurrent chromatography for the first time. The low concentration of REEs solution was enriched, and the effective separation of La (III), Ce (III), Gd (III) and Er (III) was successfully achieved. The complete separation of La (III), Ce (III), Gd (III) and Er (III) was achieved with a solvent system of 0.05 mol L−1 P507 (PE), 0.05 mol L−1 HNO3, and 0.1 mol L−1 CDs2 carbon dots (1:1:0.01, v/v/v), the upper phase as stationary phase, the lower phase as mobile phase. Density functional theory result showed that the binding energy of REEs (III)-CDs2 was larger than that of REEs (III)-P507, so the affinity of CDs2 to REEs (III) was stronger than that of P507. Therefore, with the addition of CDs2, the ability of mobile phase to elute REEs from the stationary phase was enhanced, and the separation effect was improved.

Elucidating important factors and corresponding method optimization for sensitive detection of small peptide drugs in human urine by solid-phase extraction and UPLC-HRMS: The influence of MS scan modes, protein precipitants, and ammonium formate.

Small peptide hormones are widely used in sports as performance-enhancing substances, making it crucial to develop sensitive analytical methods for their detection in doping control analysis. Various factors significantly affect analytical sensitivity, such as the selection of ultra-performance liquid chromatography (UPLC) mobile phase, high-resolution mass spectrometry (HRMS) scanning modes, and extraction solvents for pretreatment. Herein, comparative study approach was utilized to investigate the sensitivity of each peptide analyte under both full scan and parallel reaction monitoring (PRM) modes of HRMS and assess the effects of some protein precipitants as a part of extraction solvents on solid-phase extraction (SPE). The results showed that full scan should be selected as the primary scan mode of HRMS, and the combination with PRM mode could effectively compensate for the limitations of full scan, and the addition of protein precipitants would adversely affect the detection of certain small peptide analytes. Meanwhile, influences of ammonium formate in reverse UPLC mobile phase on the charge state distribution of small peptides were investigated and elucidated. Based on these findings, a sensitive and reliable UPLC-HRMS analytical method combining full scan and PRM mode was validated for screening and confirmation of 63 small peptide analytes after SPE, with limits of detection (LODs) ranging between 0.010 and 0.473 ng/ml and limits of identification (LOIs) ranging between 0.015 and 1.512 ng/ml. Additionally, suggestions were provided for the detection of [Arg8]-vasopressin, dermorphin, and its analogues.

Research progress on the detection of water-soluble vitamins in food using liquid chromatography

Currently, liquid chromatography is widely used for the analysis and determination of water-soluble vitamins (WSVs) in functional foods. However, due to the diversity of sample characteristics and the requirement for purification accuracy, the chromatographic conditions and specific methods used vary. This paper reviews the chromatographic conditions of liquid chromatography techniques for detecting WSVs in food, as well as the selection of liquid chromatography methods in different application fields. The chromatographic conditions include chromatographic columns, column temperature, mobile phase, flow rate, and elution gradient, and the application scope of different chromatographic conditions is compared. Liquid chromatography methods include high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry, and discussions are made on the applications and prospects of these two methods under different chromatographic conditions. Therefore, this review provides research insights into different chromatographic conditions and methods for food quality inspection and biochemical analysis research.

Fast analysis of short-chain and ultra-short-chain fluorinated organics in water by on-line extraction coupled to HPLC-HRMS

A rapid on-line solid-phase extraction liquid chromatography high-resolution mass spectrometry (on-line SPE-LC-HRMS) method was developed to analyze 11 ultra-short and short-chain PFAS in surface water. Analytical optimization involved screening 7 chromatographic columns and 5 on-line SPE columns, as well as evaluating SPE loading conditions, filters, sample acidification, chromatographic mobile phases, and SPE loading mobile phases. The optimized method was then applied to 44 river water samples collected in Eastern Canada, including sites near airports with fire-training areas. Among the 11 targeted PFAS, the most frequently detected were trifluoroacetic acid (TFA, 4.6–220 ng/L), perfluorobutanoic acid (PFBA, 0.85–33 ng/L), perfluoropentanoic acid (PFPeA, 1.2–2100 ng/L), trifluoromethane sulfonic acid (TMS, 0.01–4.3 ng/L), and perfluorobutane sulfonic acid (PFBS, 0.07–450 ng/L). Levels of C3-C5 perfluoroalkyl carboxylic acids (PFCAs), C2-C4 perfluoroalkyl sulfonates (PFSAs) and n:3 polyfluoroalkyl acids (n = 2,3; n:3 acids) were significantly higher in water bodies near fire-training area sites compared with rivers in urban areas. In contrast, TFA, TMS, and 1:3 acid were not significantly elevated, likely reflecting atmospheric deposition or other diffuse sources for these compounds. Nontarget and suspect screening analysis revealed an abundance of other ultra-short and short-chain PFAS in AFFF-impacted water bodies. Perfluoroalkyl sulfonamides (FASA, C2, C3, and C5), perfluoroalkyl sulfonamide propanoic acids (FASA-PrA, C1-C2) and n:3 acids (n = 1, 4, and 5) were detected for the first time in environmental surface waters.

Exploring Negative Chemical Ionization of Per- and Polyfluoroalkyl Substances via a Liquid Electron Ionization LC-MS Interface.

Per- and polyfluoroalkyl substances (PFASs) are a class of aliphatic manufactured compounds comprising fluoro-chemicals with varied functional groups and stable carbon-fluorine bonds. They are defined as "forever chemicals" due to their persistent and bioaccumulative character. These substances have been detected in various environmental samples, including water, air, soil, and human blood, posing significant health hazards. High-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) is typically employed for the analysis of PFASs. Negative chemical ionization (NCI) is generally coupled to gas chromatography (GC) and offers high selectivity and sensitivity for compounds containing electronegative atoms, such as PFASs. The liquid electron ionization (LEI) interface is an efficient mechanism developed to robustly couple a liquid flow rate from an LC system to an EI or a CI source. This interface has been successfully utilized for pesticide determination in UHPLC-LEI-CI in negative ion mode (NCI). This work aims to evaluate different parameters involved in the ionization of PFASs analyzed in LC-LEI-NCI and subsequently develop a method for their detection in real samples. The parameters considered for this study include (i) a comparison of different CI reagent gases (methane, isobutane, and argon); (ii) the use of acetonitrile as both the chromatographic solvent and CI reagent gas; (iii) the presence of water and formic acid as chromatographic mobile phase components; and (iv) the mobile phase flow rate. The optimal combination of these parameters led to promising results. Tentative fragmentation pathways of PFASs in NCI mode are proposed based on the dissociative electron capture mechanism.

A green reversed phase HPLC and HPTLC methods and their validation for simultaneous estimation of remogliflozin, vildgliptin and metformin in fixed dose combination formulation

To estimate remogliflozin, vildagliptin and metformin in a new fixed dose combination two chromatographic methods were developed and validated. The initial method is a green RP- HPLC method which uses a (150 × 4.6) mm, 2.7 mm C18 column, Ascentis as stationary phase for chromatographic separation and mobile phase made up of acetonitrile (ACN) and phosphate buffer in a ratio of 35:65 by volume. A PDA detector was used for quantification at 220 nm. 1.5 mL/min is the flow rate. Linearity was observed at 62.5–375 µg/mL, 6.25–37.5 µg/mL and 12.5–75 µg/mL for remogliflozin, vildagliptin and metformin respectively. A subsequent technique developed is HPTLC densitometric method which was also found as greener method for the separation and quantification of the three analytes in which pre-coated silica gel 60F254 aluminum plates were taken as stationary phase and the mobile phase contain phosphate buffer: ACN: glacial acetic acid in the ratio of 3: 7: 0.2 by volume. The densitometric measurements were achieved at a wavelength of 205 nm. Linearity was observed at concentrations 200–600 ng/spot, 20–60 ng/spot, 40–120 ng/spot for metformin, vildagliptin and remogliflozine respectively. Mean % recovery was 98.92–100.41% for remogliflozin, 99.92–100.33% for vildagliptin and 99.17–100.19% for metformin. The method was tested precise, specific and robust.

Insight into the photolytic degradation products of Rosuvastatin: Full chiral and structural elucidation and conversion kinetics by a combined chromatographic, spectroscopic and theoretical approach

Rosuvastatin (RSV) is a well-established lipid-lowering drug. RSV is susceptible to degradation under various stress conditions and forms two cyclic derivatives by a radical-mediated photolytic mechanism. On a structural basis, these epimeric compounds (reported as FP-B in the European Pharmacopeia monograph Rosuvastatin tablets) retain the configuration of the stereogenic carbons of RSV (3R,5S) and have opposite absolute configurations at the third stereogenic center. Herein, we report the kinetics of formation and the complete structural characterization, including the assignment of the absolute configuration, of each epimer collected after HPLC separation on a chiral stationary phase. The stereochemistry of the epimers was determined by comparison of the experimental circular dichroism data with the corresponding theoretical values. Kinetic studies revealed that RSV degrades completely to FP-B within 3 h at room temperature. Furthermore, through a multi-disciplinary approach involving chromatography (HPLC and UHPLC), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), it was demonstrated that FP-B in turn degrades to the lactones under the mild acidic conditions of the chromatographic mobile phase. The ability of RSV to form multiple degradation products may affect the quantification of RSV-related substances and draw attention to potentially toxic RSV-like species in the environment.

Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers.

Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.

Method development and validation of nifedipine and lignocaine by RP-HPLC

For the validation of the nifedipine and lignocaine assay by reverse phase high performance liquid chromatography, in both pure form and tablet dosage form, a straightforward, quick, and exact approach has been established. ACN, Methanol, and perchloric acid were used as the mobile phase in chromatography on a Kromasil 100-5-C18 (4.6 x 150 mm, 5 m) column at a flow rate of 1.0 ml/min. 210 nm was used for detection. Nifedipine and lignocaine had retention times of 3.30 and 5.820.02 min, respectively. In the concentration range of 10–50 mg/ml of nifedipine and 20–100 mg/ml of lignocaine, the approach yields linear responses. The method precision for the assay result was less than 2.0%RSD, and the Nifedipine and lignocaine individual assays should be between 98% and 102.0%, respectively. The technique is helpful for pharmaceutical and bulk formulation quality control.

Semisynthetic Sesquiterpene Lactones Generated by the Sensibility of Glaucolide B to Lewis and Brønsted-Lowry Acids and Bases: Cytotoxicity and Anti-Inflammatory Activities.

Sesquiterpene lactone (SL) subtypes including hirsutinolide and cadinanolide have a controversial genesis. Metabolites of these classes are either described as natural products or as artifacts produced via the influence of solvents, chromatographic mobile phases, and adsorbents used in phytochemical studies. Based on this divergence, and to better understand the sensibility of these metabolites, different pH conditions were used to prepare semisynthetic SLs and evaluate the anti-inflammatory and antiproliferative activities. Therefore, glaucolide B (1) was treated with various Brønsted-Lowry and Lewis acids and bases-the same approach was applied to some of its derivatives-allowing us to obtain 14 semisynthetic SL derivatives, 10 of which are hereby reported for the first time. Hirsutinolide derivatives 7a (CC50 = 5.0 µM; SI = 2.5) and 7b (CC50 = 11.2 µM; SI = 2.5) and the germacranolide derivative 8a (CC50 = 3.1 µM; SI = 3.0) revealed significant cytotoxic activity and selectivity against human melanoma SK-MEL-28 cells when compared with that against non-tumoral HUVEC cells. Additionally, compounds 7a and 7c.1 showed strongly reduced interleukin-6 (IL-6) and nitrite (NOx) release in pre-treated M1 macrophages J774A.1 when stimulated with lipopolysaccharide. Despite the fact that hirsutinolide and cadinanolide SLs may be produced via plant metabolism, this study shows that acidic and alkaline extraction and solid-phase purification processes can promote their formation.

HPTLC AND FIRST DERIVATIVE UV-SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF VILAZODONE IN PHARMACEUTICALS

A new HPTLC method and a sensitive UV spectrophotometric method were established for estimating vilazodone hydrochloride in its pharmaceutical formulations. An environment-friendly chromatographic mobile phase consisting of ethyl acetate: toluene: formic acid (5:4:1, V/V/V) along with ultraviolet densitometric detection at 240 nm using pre-coated silica gel plates was used. First derivative UV spectrophotometric determinations were performed using methanol as solvent, and amplitudes were measured for quantification purposes. Validation studies were compliant with ICH guidance. Vilazodone shows an optimum Rf value of 0.73 ± 0.02 by HPTLC method. First derivative spectrophotometric measurements were done at 230 nm (peak) and 247 nm (valley), respectively. Best method linearity was observed in the range of 100 - 600 ng band-1 and 100 - 600 ng mL-1 for the chromatographic and spectrophotometric methods, respectively. Method detection limits were 61.4 ng band-1 and 36.03 ng mL-1, for chromatography and spectrophotometry, respectively. Higher % recovery (&gt; 99 %) of vilazodone from the in-house formulation satisfied study intent as well as instituted selectivity of both the methods. Both the newly developed methods have the potential of application in routine quantification of vilazodone in pharmaceuticals.

Method development for the acquisition of adsorption isotherm of ion pair reagents Tributylamine and Triethylamine in ion pair chromatography

Tributylamine (TBuA) and triethylamine (TEtA) are the most commonly used ion pair reagents in ion pair chromatography especially for the analysis of oligonucleotides. In order to improve the understanding of the retention and separation mechanism of oligonucleotides in ion pair chromatography, it is important to understand the retention mechanism and the nature of interaction of these ion pair reagents with the stationary phase in the chromatographic column. Adsorption isotherm is helpful in evaluating such interactions, and subsequently predicting the retention mechanism. Alkylamines are very polar molecules which lack suitable chromophore and are commonly present in charged forms. Therefore, their determination and the subsequent acquisition of their adsorption isotherms using traditional liquid chromatography is very difficult. In this study, we first developed an analytical method for the determination of TBuA and TEtA in a typical chromatographic mobile phase (acetonitrile-water) and then used the same method to acquire the adsorption isotherms for tributylammonium acetate (TBuAA) and triethylammonium acetate (TEtAA). This method started with the conversion of the alkylammonium ions to free neutral forms by treating the sample with a strong base, followed by pentane-mediated extraction and finally the analysis of the extracts using gas chromatography-flame ionization detector (GC-FID). This three-step method was validated for parameters like range, linearity, intra-day and inter-day precision and accuracy, limit of detection and limit of quantitation. For the adsorption isotherms, the C18 column was first equilibrated with the solutions having different concentrations of alkylammonium ions and then stripped with eluent devoid of alkylammonium ions. Several stripping eluents were investigated and it was discovered that the eluent requirement could be decreased by the addition of sodium chloride. The effluents from the stripping phase were collected and analyzed using the developed analytical method to acquire the adsorption data. Under the investigated conditions, adsorption of TBuAA and TEtAA showed type III and type I isotherm behavior respectively.

Simultaneous determination of oxalate and citrate in urine and serum of calcium oxalate kidney stone rats by IP-RP LC-MS/MS

Oxalate and citrate in 24 h urine and serum are considered to be associated with the incidence and recurrence risk of calcium oxalate kidney stones. The quantification of oxalate and citrate contributes to understand the pathological metabolism of kidney stones and guide the early diagnosis and recurrence monitoring. Although simultaneous quantification of oxalate and citrate in urine using liquid chromatography tandem mass spectrometry (LC-MS/MS) have been reported, the optimization of chromatographic column, mobile phase and mass spectrometry (MS) parameters has not been performed. In addition, these is a lack of suitable method for simultaneous detection of oxalate and citrate both in serum and urine. Therefore, we developed a method for the simultaneous determination of oxalate and citrate in urine and serum based on ion-pairing reversed-phase (IP-RP) LC-MS/MS. Herein, five ion-pair reagents, namely, triethanolamine, dimethylbutyl amine, diisopropenyl amine, N,N-dimethylcyclohexylamine and tripropylamine, and three ion-pairing reagent (IPR) buffers, namely, acetic acid, hexafluoro-2-isopropanol, and hexafluoro-2-methyl-2-propanol, were compared in regard to their chromatographic peak abundance and separation of oxalate and citrate. Moreover, MS parameters and the multiple reaction monitoring (MRM) conditions were also evaluated and optimized to obtain the maximum peak abundance. After that, the method was validated in the linear range of 0.25–1000 µM, and the correlation coefficient was ≥ 0.99. The precision and accuracy were < 14.70% and < 19.73%, respectively. The extraction recovery was 80.53–108.79%, and the matrix effect was < 8.79%. The quality control samples were stable at room temperature for 4 h, 4 °C for 24 h, and for 3 freeze–thaw cycles. Finally, this method was applied to the determination of oxalate and citrate in the serum and urine of rats with calcium oxalate kidney stones. The establishment of a stable and effective oxalate and citrate detection method is conducive to the diagnosis and monitoring of kidney stones.

Rapid screening and confirmation of 86 illegally added chemicals in fishery drugs by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry

建立了超高效液相色谱-四极杆-飞行时间质谱法快速筛查与确证渔药中86种非法添加禁限用药物的方法。渔药以80%(v/v)乙腈水溶液进行提取,通过稀释降低基质效应,采用ACQUITY PREMIER HSS T3色谱柱进行分离,以甲醇和0.1%甲酸水溶液作为流动相进行梯度洗脱,采用电喷雾双喷离子源(Dual AJS ESI)正离子模式分析检测。建立了86种药物的一级精确质量数据库和二级碎片质谱库。在全扫描采集模式下,以化合物的色谱保留时间、精确质量数、同位素分布和同位素丰度比定性;在Target MS/MS采集模式下,通过二级碎片离子的匹配进一步确证化合物,以准分子离子峰的峰面积定量,实现渔药样品中多目标药物的快速定性定量分析。86种药物在各自的线性范围内均呈现良好的线性关系,相关系数均大于0.99,中草药制剂和抗生素粉剂的定量限(LOQ)范围分别为1~15 mg/kg和5~75 mg/kg,添加回收率范围为76.8%~112.1%,相对标准偏差(RSD, n=3)小于11.7%。该方法快速、简便、准确、灵敏,适用于不同种类渔药中禁限用非法添加药物的高通量筛查。将该方法应用于浙江省渔用投入品质量安全监督抽检项目中,共筛查60个样品,其中8种中草药制剂筛查出说明书中未明确标明的药物成分,1种抗生素粉剂未检出有效成分。该研究为渔药的质量安全监控提供了有效的技术手段。