Reversed-phase Column Chromatography Research Articles

Comparison of reversed-phase, hydrophilic interaction, and porous graphitic carbon chromatography columns for an untargeted toxicometabolomics study in pooled human liver microsomes, rat urine, and rat plasma

IntroductionUntargeted metabolomics studies are expected to cover a wide range of compound classes with high chemical diversity and complexity. Thus, optimizing (pre-)analytical parameters such as the analytical liquid chromatography (LC) column is crucial and the selection of the column depends primarily on the study purpose.ObjectivesThe current investigation aimed to compare six different analytical columns. First, by comparing the chromatographic resolution of selected compounds. Second, on the outcome of an untargeted toxicometabolomics study using pooled human liver microsomes (pHLM), rat plasma, and rat urine as matrices.MethodsSeparation and analysis were performed using three different reversed-phase (Phenyl-Hexyl, BEH C18, and Gold C18), two hydrophilic interaction chromatography (HILIC) (ammonium-sulfonic acid and sulfobetaine), and one porous graphitic carbon (PGC) columns coupled to high-resolution mass spectrometry (HRMS). Their impact was evaluated based on the column performance and the size of feature count, amongst others.ResultsAll three reversed-phase columns showed a similar performance, whereas the PGC column was superior to both HILIC columns at least for polar compounds. Comparing the size of feature count across all datasets, most features were detected using the Phenyl-Hexyl or sulfobetaine column. Considering the matrices, most significant features were detected in urine and pHLM after using the sulfobetaine and in plasma after using the ammonium-sulfonic acid column.ConclusionThe results underline that the outcome of this untargeted toxicometabolomic study LC-HRMS metabolomic study was highly influenced by the analytical column, with the Phenyl-Hexyl or sulfobetaine column being the most suitable. However, column selection may also depend on the investigated compounds as well as on the investigated matrix.Graphical abstract

Effects of tetramethylpyrazine on pharmacokinetics in plasma and brain dialysate and neuropathic pain in rat model of spared sciatic nerve injury

This study aims to explore the effects of tetramethylpyrazine(TMP) on pharmacokinetics in plasma and brain dialysate and neuropathic pain in the rat model of partial sciatic nerve injury(SNI), and to investigate the correlation between the analgesic effect of TMP and its concentrations in the plasma and brain dialysate. Male SD rats were randomized into Sham, SNI, and SNI+TMP groups. Mechanical stimulation with von frey filaments and cold spray method were employed to evaluate the mechanical sensitivity and cold sensitivity of rats. Another two groups, Sham+TMP and SNI+TMP, were used to intubate the common jugular vein and implant microdialysis probes into the anterior cingulate gyrus(ACC), respectively.After intraperitoneal injection of TMP at a dose of 80 mg·kg~(-1), automatic blood collection and intracerebral microdialysis(perfusion rate of 1 μL·min~(-1)) systems were used to collect the blood and brain dialysate for 24 h. HSS T3 C_(18) reversed-phase chromatographic column(2.1 mm×50 mm, 2.5 μm) was used for liquid chromatographic separation. Gradient elution was carried out with the mobile phase of methanol-water(containing 0.005% formic acid) at a flow rate of 0.25 mL·min~(-1). Electrospray ion source was used for mass spectrometry, and the scanning mode was multi-reaction monitoring under the positive ion mode. The ion pairs for quantitative analysis were TMP m/z 137/122 and aspirin m/z 179/137, respectively. DAS 2.11 was used to calculate the pharmacokinetic parameters. The optimal time of TMP to exert the analgesia effect and inhibit cold pain sensitivity was 60 min after treatment. The TMP in the plasma and brain dialysate of SNI rats showed the T_(max) of 15 min and 30 min, the C_(max) of(2 866.43±135.39) and(1 462.14±197.38) μg·L~(-1), the AUC_(0-t) of(241 463.30±28 070.31) and(213 115.62±32 570.07) μg·min·L~(-1), the MRT_(0-t) of(353.13±47.73) and(172.16±12.72) min, and the CL_Z of 0.73 and 0.36 L·min·kg~(-1), respectively. The analgesic effect of TMP had a significant correlation with the blood drug concentration in the ACC, which indicated that this method was suitable for the detection of TMP in rat plasma and brain dialysate. The method is accurate, reliable, and sensitive and can realize the important value of the application of correlation analysis theory of "automatic blood collection-microdialysis/PK-PD" in the research on neuropathic pain.

Preparation and investigation of two-component silica-modified hydrophobic layer for minimizing retention loss of reversed-phase chromatographic columns using fully aqueous mobile phases

Chromatographers often employ fully aqueous mobile phases to retain highly polar compounds in reversed-phase liquid chromatography (RPLC). However, when the flow rate is interrupted, either accidentally or intentionally, a substantial loss in retention occurs due to the spontaneous dewetting of water from the hydrophobic surface of conventional RPLC-C18 particles. Previous studies have shown that maintaining a low C18 surface coverage (approximately 1.5 μmol/m2) can mitigate water dewetting by increasing chain disorder, facilitating the intercalation of water clusters between the C18-bonded chains, and keeping the mesopores wetted. In this research, we explore the potential and additional benefits of using two-component surface bonding materials (C8/C18 and PhenylHexyl (PhHx)/C18) at a constant and low total surface coverage of 1.51 ± 0.15 μmol/m2. We synthesized seven one- and two-component modified silica particles with a volume average particle size of 5.22 μm and an average mesopore size of 104 Å. The surface coverage was increased from 0 to 0.54, 1.00, and to 1.66 μmol2 for C8 chains and from 0 to 0.52, 0.70, and to 1.65 μmol2 for PhHx ligands. To prevent interactions between water and any unreacted silanols, all seven derivatized particles were heavily endcapped with trimethylsilane (TMS) reagent. The fraction of the surface area remaining in contact with water was determined by measuring the retention times of weakly (thiourea) and strongly (thymine) retained compounds at intervals of 1, 2, 4, 8, 16, 32, and 64 minutes following the cessation of flow. Two distinct column temperatures, 24°C and 60°C, were employed in the experiments. Retention losses were found to be minimized in the presence of a small quantity of C8 chains (less than 40% of the total surface coverage). Additionally, it is essential to consider substantial fractions of PhHx chains, as long as the presence of the PhHx ligand does not significantly impact retention and selectivity. Combining mixed RPLC bondings with a low total surface coverage of approximately 1.5 μmol/m2 emerges as a viable solution for further minimizing retention loss in standard C18-bonded RPLC columns, particularly within the surface coverage range of 2.5-3.0 μmol/m2.

Determination of 2,4-dimethylhydroxybenzene by chromatographic methods in forensic toxicological research of biological material

Is to develop a method for determining 2.4-dimethylhydroxybenzene (2.4-DMHOB) in biological material. The analytical methods used in the experiments were extraction, column chromatography of normal pressure, TLC, GC-MS and HPLC. To extract the analyte from the bioactive matrix, maceration with the binary insulating agent acetone-ethyl acetate (3:7) was used, observing the 2:1 mass ratio of the «insulating agent-matrix». Optimal conditions of semi-preparative analyte chromatography were achieved in column (150×10 mm) of «Silasbor» S-18 sorbent in elution with a mixture of acetonitrile-water (7:3), which was used in the proposed cleaning scheme, combining extraction and reversed-phase column chromatography. The application of the mobile phase of tetrachloromethane-dioxane (9.5:0.5) has been substantiated for the selective determination of 2.4-DMHOB by TLC («Sorbfil» plates). The expediency of confirming identification of the analyte in the form of 2.4-dimethyltrymethylsilylphenol using GC-MS (DB-5MS EVIDEX column (25.000×0.2 mm), stationary phase (5%-phenyl)-methylpolysiloxane, carrier gas - helium) has been shown. The group of characteristic particles in the mass spectrum of trimethylsilyl analyte derivative was represented by 45; 59; 73; 82; 91; 105; 119; 135; 149; 163; 179; 194 m/z ions. HPLC (Discovery C18 250×4.6 mm column, eluting liquid - acetate buffer solution with pH 5.5 - acetonitrile, 50:50) was used to confirm the identification and quantification of 2.4-DMHOB. A method for determining 2.4-DMHOB by the HPLC method in biological material (liver tissue) is proposed, which corresponds to the criteria of linearity, selectivity, accuracy, precision and stability. The minimum detectable quantity of 2.4-DMHOB in the bioactive matrix is 0.5 µg/g, the minimum determined quantity is 1.2 ug/g.

Determination of some sex hormones in health supplements by liquid chromatography with tandem mass spectrometry

The trend of using dietary supplements in daily life is increasingly popular. However, sex hormones are illegally mixed to improve product effectiveness such as promoting hair growth, developing chest muscles, preventing skin aging, promoting growth, etc. In this study, a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and applied to quantify sex hormones: progesterone (PG), testosterone (TS), and 17β-estradiol (E2) in health supplements. A Symmetry C18 reversed-phase chromatography column (150 mm x 3.0 mm x 3.5 μm) using a mobile phase system consisting of A: 10 mM HCOONH4/0.1% HCOOH, B: ACN was used.  Mass spectrometry conditions were set at positive ionization mode, precursor ion and two corresponding daughter ions were selected for each analyte. Quantification limits of PG and TS were 0.4 mg/kg and E2 was 4 mg/kg in liquid matrix, hard and soft capsules. The validated method was applied to detect sex hormones in 16 health supplement samples purchased at pharmacies in Hanoi, Vietnam and some online websites. Three samples were detected to be positive for sex hormones, including one sample containing a sex hormone (testosterone) banned from use in health supplements at a low content (about 0.5 mg/kg).

Study of micelles and surface properties of triterpene saponins with improved isolation method from Hedera helix

Triterpene saponin fractions were extracted from Hedera helix, and in-depth analysis of their physicochemical properties was conducted. Hederasaponin B and hederacoside C were extracted from Hedera helix leaves, and their purification was carried out using reverse phase column chromatography with a modified method, providing an affordable alternative to HPLC. Structurally, hederacoside C differs from hederasaponin B only by the presence of a hydroxyl group at the carbon 23 of the aglycon. The critical micelle concentration (cmc) measurement confirmed hydrophilic nature of hederacoside C that led to a higher cmc value compared to hederasaponin B and alpha-hederin. Therefore, the cmc value of hederasaponin B is nearly an order of magnitude lower compared to hederacoside C. Additionally, the study of the surface tension revealed that the more lipophilic alpha-hederin displayed a greater surface tension value (γcmc = 39.8 mN·m−1) compared to hederasaponin B and hederacoside C. Measurements of the surface tension dependence on the concentration in water were enabled to determine the area corresponding to a single saponin molecule at the water/air phase interface (Acmc). Notably, structural changes had negligible effects, as Acmc values remained practically identical. Particle size determination further indicated that hederacoside C forms only micelles compared to the remaining substances that showed signs of vesicles formation. Alpha-hederin, as the only measured molecule capable of ionization, showed a negative zeta potential.

“Ghost peaks” in reversed-phase liquid chromatography separation of an electron-rich aniline compound: Mechanism and solution for this phenomenon

Two “ghost peaks” appeared in the process of developing an impurities analytical method for an electron-rich aniline compound by RP-HPLC. Surprisingly, the “ghost peaks” disappeared when the mobile phase was re-prepared. The "ghost peak" slowly began to reappear and gradually increased after increasing the injection volume or changing different instruments and columns. In this paper, the structures of two “ghost peaks” were analyzed by LC-MS/MS, which were identified as the dimer of the main peak and the oxidation products of the dimer. The mechanism was confirmed by studying the “ghost peaks” appearance phenomenon in the mobile phase with different pH values, different types of buffer salts, and different brand chromatographic columns. The potential chemical behavior of this type of compound on the column was clearly revealed in the process of RP-HPLC separation. The coupling reaction of electron-rich aniline compounds can be induced by partial or complete dissociation of the silanol group to Si-O−in the stationary phase of a reversed-phase chromatographic column under neutral and basic conditions of the mobile phase. Adding inorganic salts to the mobile phase can inhibit the reaction, and the higher salt concentration, the stronger the effect.

A One-Step Sample Processing Method in Combination with HPLC-MS/MS for the Simultaneous Quantification of Atorvastatin, Ezetimibe and Three Metabolites including o-Hydroxyl Atorvastatin, p-Hydroxyl Atorvastatin, and Ezetimibe-Glucuronide in Human Plasma

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of atorvastatin (ATOR), ezetimibe (EZM), and their three metabolites, including o-hydroxyl atorvastatin (o-OH ATOR), p-hydroxyl atorvastatin (p-OH ATOR), and ezetimibe–glucuronide (EZM-G) in human plasma using benzyl paraben (BP) as the internal standard (IS). The analytes and IS were ionized using ESI positive ion mode (ATOR, o-OH ATOR, and p-OH ATOR), ESI negative ion mode (EZM, EZM-G, and BP), and operated in multiple reaction monitoring (MRM) mode. They were then extracted via salting-out assisted liquid–liquid extraction with acetonitrile and analyzed via liquid chromatography on a reversed-phase chromatographic column (50 mm × 4.6 mm; 3.5 µm) using a mixture of acetonitrile and an acetic acid solution (0.5%) as the mobile phase, showing high extraction efficiency (>70%), and a minimized matrix effect. The method was satisfactorily validated, and it showed excellent linearity over wide concentration ranges of 0.06–15 ng/mL, 0.6–150 ng/mL, 0.4–100 ng/mL, 0.12–30 ng/mL, and 0.05–3 ng/mL for EZM, EZM-G, ATOR, o-OH ATOR, and p-OH ATOR, respectively.

Isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus using centrifugal partition chromatography

Replacement of synthetic colorants with natural ones is a current marketing trend. Nevertheless, the naturally occurring blue color is rare compared to other colours. In this work, centrifugal partition chromatography (CPC) process was developed as a more efficient and sustainable alternative to reversed phase column chromatography (RP-CC) for the preparative-scale purification of portisins. The strategy began with the extraction of anthocyanins from blueberry surplus and hemi-synthesis of respective portisins. Then, the CPC method development started with the biphasic solvent system selection followed by the optimization of the operating parameters and ended up with a comparison with RP-CC. Aiming at maximizing the portisin content, process throughput, efficiency, and minimizing the environmental risk factor, the effect of sample load (100–500 mg/100 mL of column volume), mobile phase flow rate (10–20 mL/min), and rotation speed (1000–1600 rpm) was evaluated. The two-phase solvent system consisted of tert‑butyl‑methyl ether, n-butanol, acetonitrile, and water (volume ratio 2:2:1:5) acidified with 0.1 vol.% of HCl was selected. The best conditions were 464 mg of sample/100 mL of column volume, 20 mL/min of mobile phase flow rate, and 1600 rpm of rotation speed at reversed phase mode, allowing the purification of portisins by 5-fold. Compared to the RP-CC, the CPC process efficiency was 2.4 times higher, while the CPC process environmental risk factor was 5.5 times lower. Overall, this study suggests that CPC can be considered an effective, and sustainable alternative process for the preparative isolation of portisins. With this purification approach, the blueberry surplus has been valorized and a naturally derived product has been prepared, allowing its subsequent use as a natural blue colorant.

Development and validation of a method for the quantitative determination of rotenone in the homogenate of the cerebral cortex of rats by high-performance liquid chromatography (HPLC)

Introduction. Rotenone is a neurotoxin that causes damage of dopaminergic neurons in the substantia nigra and is used as a model for experimental parkinsonian syndrome. The development of a technique for the quantitative determination of rotenone in the brain will allow testing new strategies for the pharmacotherapy of parkinsonism by reducing the penetration of toxic substances into the brain.
 The aim of the study was to develop and validate an HPLC method for the quantitative determination of rotenone in the cerebral cortex of rats.
 Material and methods. Quantitative determination of rotenone was carried out using a Stayer chromatographic system (Аквилон, Russia) with a UV spectrophotometric detector UVV 104 at a wavelength of 296 nm in isocratic mode. A reverse-phase chromatographic column Luna C18 100Å (250*4.6) with a grain size of 5 μm at a temperature of 37°C was used. The composition of the mobile phase was deionized water, acetonitrile in a ratio of 70:30. Determination of rotenone concentration was carried out by the method of absolute calibration by the area of the peaks.
 Sample preparation consisted in homogenization of 500 mg of crushed frontal lobe of the rat cerebral cortex in 500 μl of purified water, followed by centrifugation (1750 g), collection of the supernatant and sedimentation of the proteins by acetonitrile. The liquid layer was evaporated on a rotary vacuum. 250 µl of the mobile phase was added to the dry residue, and 100 µl was injected into the chromatograph.
 Results. The method was validated for the following parameters: selectivity, linearity, accuracy, precision, limits of detection and determination, sample transfer, sample stability. The analytical range was 62.5−1000.0 ng/g brain with a correlation coefficient of more than 0.99. The limits of detection and quantification of rotenone were 25.0 and 62.5 ng/g, respectively. The calculation of intra- and inter-cycle accuracy and precision showed that these parameters do not exceed 20% for the concentration corresponding to the lower limit of quantitative determination, and 15% for higher concentrations. The stability of the technique was demonstrated during short-term storage at room temperature, three freeze-thaw cycles at –80°C, and storage at –80°C for 60 days. There was no sample transfer.
 Limitations. The chromatographic technique makes it possible to analyze the content of rotenone in the cerebral cortex of rats in the concentration range of 62.5–1000.0 ng/g.
 Conclusion. A method for the quantitative determination of rotenone in the homogenate of the cerebral cortex of rats has been developed and validated.

Towards non-target proactive food safety: identification of active compounds in convenience tomato products by ten-dimensional hyphenation with integrated simulated gastrointestinal digestion

Current strategies for non-target food screening focus mainly on known hazardous chemicals (adulterants, residues, contaminants, packaging migrants, etc.) instead of bioactive constituents in general and exclude the biological effect detection. To widen the perspective, a more proactive non-target effect-directed strategy is introduced to complement food safety in order to detect not only known but also unknown bioactive compounds. The developed 10-dimensional hyphenation included on-surface digestion (1D), planar chromatographic separation (2D), visualization using white light (3D), UV light (4D), fluorescence light (5D), effect-directed assay analysis (6D), heart-cut zone elution to an orthogonal reversed phase column chromatography including online desalting (7D) with subsequent diode array detection (8D), high-resolution mass spectrometry (9D), and fragmentation (10D). Metabolism, i.e., intestinal digestion of each sample, was simulated and integrated on the same adsorbent surface to study any changes in the compound profiles. As proof of principle, nine convenience tomato products and a freshly prepared tomato soup were screened via five different planar assays in a non-targeted mode. Non-digested and digested samples were compared side by side. In their effect-directed profiles, 14 bioactive compounds from classes of lipids, plant hormones, spices, and pesticides were identified. In particular, bioactive compounds coming from the lipid class were increased by gastrointestinal digestion, while spices and pesticides remained unaffected. With regard to food safety, the determination of the two dinitrophenol herbicides dinoterb and dinoseb in highly processed tomato products should be given special attention. The hyphenation covered a broad analyte spectrum and showed robust and reliable results.Graphical

Extraction of natural blue emissive pigments from Fraxinus lanuginosa by column chromatography

We investigated an extraction method of blue fluorescent pigments from Fraxinus lanuginosa (Japanese ash, aodamo in Japanese) and found that aodamo contains several fluorescent pigments: not only blue but also red. Reversed-phase column chromatography was performed, and the mushed aodamo’s solution was separated into multiple layers in which the pigments were isolated. Absorbance and photoluminescence measurements indicated that the brown solution first removed from the column contains blue fluorescent pigments and is free of red fluorescent pigments. In addition, the brown solution’s nuclear magnetic resonance (NMR) spectrum clearly suggests that it contains the blue fluorescent pigments of esculin and fraxetin.

Simultaneous determination of some antibiotics with coccidiosis effect in feed by LCMS/MS

Liquid chromatography tandem mass spectrophotometry (LC-MS/MS) has been developed for simultaneous determination of four antibiotics which have coccidiicidal effects including monensin, dimetridazole, sulfadimethoxin, and sulfadimidine in feed samples. The research conducted by using mobile phase A (0.1% formic acid solution; 10 mM ammonium formate) and phase B (MeOH; 0.1% formic acid; 10 mM ammonium formate) with C18 reversed-phase chromatography column (2.1 × 1.5 mm, ID 3.5 μm) simultaneously and successfully identified these four antibiotics in a short time about 10 minutes. The method detection limits and quantification limits for all analytes were 15.0 and 45.0 µg/kg, respectively. The calibration curve of the method was in the range of 45 - 1,500 µg/L. The method was validated for high accuracy, efficiency and applied to analyze the feed samples on the Hanoi market.

Two new triterpeneoids from roots of Dolomiaea souliei

The present study analyzed the chemical constituents in dried roots of Dolomiaea souliei. Chromatographic methods, such as normal-phase, and reversed-phase column chromatography, TLC, and preparative HPLC, were applied to separate and purify the petroleum ether extract of D. souliei. The structures of the purified constituents were identified by multiple spectroscopic methods including 1 D NMR, 2 D NMR, IR, UV, and HR-ESI-MS. Fourteen triterpenoids were obtained and identified as bauer-8-ene-3,11-dione-7α-ol(1), bauer-8-ene-3-one-7α,11α-diol(2), 3-oxo-11α-hydroxy-urs-12-ene(3), 3-oxour-12-ene-1β,11α-diol(4), 3β,11α-dihydroxy-urs-12-ene(5), taraxast-20-ene-3β,30-diol(6), 28-hydroxy-3-oxo-12-ursene(7), 3β-hydroxytaraxast-20-ene-30-aldehyde(8), urs-12-ene-2α,3β,28-triol(9), 20-hydroxylupan-3-one(10), monogynol A(11), obtusalin(12), 3-oxo, 11α-hydroxy-olean-12-ene(13), and isocabralealactone(14). Among them, compounds 1 and 2 were new compounds. Compounds 4-10, 12, and 14 were isolated from this genus for the first time. Compounds 3 and 11 were obtained from D. souliei for the first time.

Extraction and Use of Anthocyanins from Radish (Raphanus Sativus L Var Crimson Gigant) as a Natural Colorant in Yogurt

Radish (Raphanus sativus L. var Crimson Gigant) contains significant quantities of glycosylated anthocyanins, it can be used as natural food colorants due to their high stability. These pigments have therapeutic and pharmacological characteristics that are associated to antioxidant activity. Furthermore, the pigments of radish are important for nutritional and medicinal uses, for all the benefits that they provide to human health. The objective of this research was to extract anthocyanins from the shell of radish in pure form, evaluate its antioxidant activity in vitro and then use it as a food coloring in natural yogurt. For sample extraction, the radish shell was separated by finely cutting it from the body of the radish, it was macerated with an acidified solution of methanol-water and evaporated by 80%. The extract was absorbed with an ion exchange resin, subsequently the anthocyanins were released in an acidified solution, finally the extracts were purified by reverse phase column chromatography. 14 fractions were obtained and were monitored by thin layer chromatography, afterwards antioxidant activity was evaluated by methods such as DPPH, ABTS and TBARS. A stable cherry powder was obtained, which was used as a yogurt colorant and was very well accepted by the judges.

REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-ULTRAVIOLET-BASED APPROACH FOR METHOD DEVELOPMENT AND VALIDATION OF LACOSAMIDE ESTIMATION IN HUMAN SERUM

Objective: The main aim of the study was to develop and validate a simple, accurate, and rapid isocratic reverse phase high-performance liquid chromatographic method with UV detection for the determination of lacosamide, an antiepileptic agent, in human serum. Methods: Chromatographic separation was performed using a reverse phase chromatographic column (Zorbax SB-C18, 5 μm 4.6×250 mm) with a mobile phase being a mixture of potassium dihydrogen phosphate buffer (pH adjusted to 3.0 with orthophosphoric acid) and acetonitrile (ratio of 83:17 v/v) at a flow rate of 1.2 mL/min. UV detection was carried out at 210 nm and the sample temperature was maintained at 4°C. Results: Linear calibration curves in the range of 1.012–40.894 μg/ml gave a correlation coefficient of 0.9988. The intra-day (n=6) and inter-day (n=18) precision (expressed as relative standard deviation) were in the range of 0.79–2.485% and from 0.99 to 3.21%, respectively. The retention time (in minutes) of lacosamide and internal standard was approximately 8.785±0.19 and 3.985±0.77, respectively, with no matrix interference. The method was validated for system suitability, specificity, precision, accuracy, robustness, linearity, limit of detection, and limit of quantification following the International Conference on Harmonization guidelines. The method was further validated using sera of epileptic patients consuming lacosamide, and it was observed that the results matched with the patients’ clinical response. Conclusion: Our method developed to estimate serum lacosamide level is simple, cost-effective, and reliable for therapeutic drug monitoring.

Induction of the Prenylated Stilbenoids Arachidin-1 and Arachidin-3 and Their Semi-Preparative Separation and Purification from Hairy Root Cultures of Peanut (Arachis hypogaea L.).

Prenylated stilbenoids such as arachidin-1 and arachidin-3 are stilbene derivatives that exhibit multiple pharmacological activities. We report an elicitation strategy using different combinations of cyclodextrin, hydrogen peroxide, methyl jasmonate and magnesium chloride to increase arachidin-1 and arachidin-3 production in peanut hairy root cultures. The treatment of hairy root cultures with cyclodextrin with hydrogen peroxide selectively enhanced arachidin-1 yield (132.6 ± 20.4 mg/L), which was 1.8-fold higher than arachidin-3. Similarly, cyclodextrin combined with methyl jasmonate selectively enhanced arachidin-3 yield (178.2 ± 6.8 mg/L), which was 5.5-fold higher than arachidin-1. Re-elicitation of the hairy root cultures further increased the levels of arachidin-1 and arachidin-3 by 24% and 42%, respectively. The ethyl acetate extract of the culture medium was consecutively fractionated by normal- and reversed-phase column chromatography, followed by semi-preparative HPLC purification on a C18 column to yield arachidin-1 with a recovery rate of 32% and arachidin-3 with a recovery rate of 39%, both at higher than 95% purity. This study provided a sustainable strategy to produce high-purity arachidin-1 and arachidin-3 using hairy root cultures of peanuts combined with column chromatography and semi-preparative HPLC.

Quantitative Analysis of Fat-Soluble Vitamins in Feed Additives Using an In-House Developed and Validated HPLC Method

Quantification of vitamins in complex matrices such as feed additives is a time-consuming analytical procedure. In this study, a simple and precise in-house High Performance Liquid Chromatography (HPLC) method was developed and validated for the simultaneous detection and quantification of four fat-soluble vitamins such as vitamin A, D3 , E, and K3 in feed additives. The HPLC method was developed and validated using reversed-phase column chromatography. The chromatographic separation of the vitamins was carried out at 25° C temperature on a reverse-phase C18 column using a binary gradient pump mode. Mobile phase constituents were solvent (a): deionized water and (b) methanol. Detection was performed with HPLC ultraviolet/visible detection set at 325, 265, 230, and 254 nm wavelength for vitamin A, D3 , E, and K3 respectively. The flow rate was 1.0mL/min and the total run time was 20min. The method was validated according to the guidelines of the International Conference on Harmonization (ICH) and Food and Drug Administration (FDA), USA, and acceptance criteria for system suitability, specificity, linearity, accuracy, and precision were met in all the cases. The Relative Standard Deviation (RSD) for system suitability and precision was <2% for all the studied vitamins. The linearity of the calibration curves was excellent (R2 >0.999) at concentrations of 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 µg/mL for all vitamins, and the range of linearity of this method was 0.0-50.0 μg/mL with R2 value greater than 0.999. The limits of detection values were 0.0022, 0.0012, 0.0022, and 0.0020 µg/ mL for vitamin A, D3 , E, and K3 , respectively, and the limits of quantification values were 0.0066, 0.0038, 0.0066, and 0.0061 µg/mL for vitamin- A, D3 , E, and K3 respectively. The recovery percentages ranged from 85% to 103%, and the robustness of the method is also high with excellent reproducibility. The overall parameters of the proposed method met the validation criteria and this method could be a precise and highly desirable analytical procedure for accurate quantification of four fat-soluble vitamins such as A, D3 , E, and K3 in feed additives using a single chromatographic run.

Inside Front Cover: A simple and sensitive liquid chromatography–tandem mass spectrometry method for quantification of multi‐residual pesticides in blood

DOI: 10.1002/sscp.202200067 The cover picture shows the extraction of pesticides from blood samples and a developed LC-MS/MS method to determine the levels of 33 different pesticides in human blood. The developed method used a simple extraction method that started with blood collection from farmers in an EDTA tube. A 100 μL of blood was added to a centrifuge tube and then adding cold acetonitrile to remove cell debris and then subjected to cold incubation for pesticides in blood to solubilize into the solvent. Finally, the extracted sample was filtered through 0.2-micron filters to get a clear sample. A 10 μL of the extracted sample was injected into an LCMS/MS system, and the results were recorded. An Agilent zorbax C18 reverse-phase chromatographic column was used in this study. This developed method efficiently separated the 33 pesticides of our interest. The chromatographic run involved a 20 min run with different retention times for each analyte. The developed method was checked by employing validation parameters such as robustness, ruggedness, recovery, and found that the method has passed all the parameters as per the European standard guidelines.

A Fully Automated Online SPE-LC-MS/MS Method for the Determination of 10 Pharmaceuticals in Wastewater Samples.

The increasing use of pharmaceuticals, their presence in the aquatic environment, and the associated toxic effects, have raised concerns in recent years. In this work, a new multi-residue analytical method was developed and validated for the determination of 10 pharmaceuticals in wastewaters using online solid-phase extraction (online SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds included in the method were antineoplastics (cabazitaxel, docetaxel, doxorubicin, etoposide, irinotecan, methotrexate, paclitaxel, and topotecan), renin inhibitors (aliskiren), and antidepressants (maprotiline). The method was developed through several experiments on four online SPE cartridges, three reversed phase chromatography columns, and four combinations of mobile phase components. Under optimal conditions, very low limits of detection (LODs) of 1.30 to 10.6 ng L−1 were obtained. The method was repeatable, with relative standard deviations (RSD, %) for intraday and interday precisions ranged from 1.6 to 7.8 and from 3.3 to 13.2, respectively. Recovery values ranged from 78.4 to 111.4%, indicating the reproducibility of the method. Matrix effects were mainly presented as signal suppression, with topotecan and doxorubicin being the two most affected compounds (31.0% signal suppression). The proposed method was successfully applied to hospital effluents, detecting methotrexate (4.7–9.3 ng L−1) and maprotiline (11.2–23.1 ng L−1). Due to the shorter overall run time of 15 min, including sample preparation, and reduced sample volume (0.9 mL), this on-line SPE-LC-MS/MS method was extremely convenient and efficient in comparison to the classical off-line SPE method. The proposed method was also highly sensitive and can be used for ultratrace quantification of the studied pharmaceuticals in wastewaters, providing useful data for effective environmental monitoring.