Octadecylsilica Column Research Articles

Evaluation of analytical greenness metric for an eco-friendly method developed through the integration of green chemistry and quality-by-design for the simultaneous determination of Nebivolol hydrochloride, Telmisartan, Valsartan, and Amlodipine besylate

In recent years, the field of analytical chemistry has witnessed a notable shift towards the adoption of greener chromatographic methods, aiming to minimize the environmental impact. An effective strategy involves substituting conventional harmful organic solvents with environmentally friendly alternatives, reducing the use of hazardous chemicals that contribute to environmental concerns. However, separating drug substances without the use of buffers and organic solvents presence is a big challenge. To overcome this challenge, a combination of quality-by-design (QbD) and green analytical chemistry (GAC) was employed in this study for method development. A high-performance liquid chromatography (HPLC) method was successfully developed and validated for the simultaneous determination of Nebivolol hydrochloride, Telmisartan, Valsartan, and Amlodipine besylate. The method utilized a mobile phase composed of a mixture of 0.1 % formic acid in water (pH: 2.5) and ethanol. A regular octadecyl silica (ODS) column was employed, and UV detection at 220 nm was utilized. The method exhibited linearity within the concentration range of 25–75 μg/mL for Telmisartan and 150–450 μg/mL for Nebivolol Hydrochloride, Valsartan, and Amlodipine besylate and the correlation coefficient was greater than 0.999 for all the analytes. Limits of detection (LOD) and quantification (LOQ) were determined as 0.01 and 0.04 μg/mL for Telmisartan, 0.06 and 0.20 μg/mL for Nebivolol Hydrochloride, 0.08 and 0.25 μg/mL for Amlodipine besylate, and 0.14 and 0.46 μg/mL for Valsartan, respectively. The developed method underwent thorough validation, encompassing various parameters such as linearity, accuracy, precision, LOD, LOQ, robustness, and ruggedness. The mean recovery values were observed to range between 98.86 % and 99.89 %. The accuracy demonstrated was consistently above 98.98 % for both intra-day and inter-day precisions were with the relative standard deviations less than 2 %. To establish its robustness, a quality-by-design-based experimental design (DoE) approach was implemented. Additionally, the method's environmental friendliness was evaluated using the Analytical Greenness metric (AGREE) an analytical eco scale, both confirming its alignment with sustainable practices and reduced ecological impact. The sustainability of the solvent used in the current study was evaluated by Green Solvents Selecting Tool (GSST) Further, the developed method greenness was evaluated with the green analytical tools such as Analytical method greenness score (AMGS) and using the recently released White Analytical Chemistry (WAC) using RGB assessment tool. By employing this greener approach to chromatography method, this study contributes to the ongoing efforts in analytical chemistry to promote sustainable practices and minimize the environmental footprint of analytical methods.

Analyzing Citramalic Acid Enantiomers in Apples and Commercial Fruit Juice by Liquid Chromatography-Tandem Mass Spectrometry with Pre-Column Derivatization.

Optically active citramalic acid (CMA) is naturally present as an acidic taste component in fruits, such as apples. The absolute configuration of CMA in such fruits was investigated by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) following pre-column derivatization with a chiral reagent, benzyl 5-(2-aminoethyl)-3-methyl-4-oxoimidazolidine-1-carboxylate. The developed LC-MS/MS method successfully separated the enantiomers of CMA using an octadecylsilica column with a resolution and separation factor of 2.19 and 1.09, respectively. Consequently, the R-form of CMA was detected in the peel and fruit of three kinds of apple at concentrations in the 1.24-37.8 and 0.138-1.033 mg/wet 100 g ranges, respectively. In addition, R- CMA was present in commercial apple juice, whereas no quantity was detected in commercial blueberry, perilla, or Japanese apricot juice.

Chiral LC-MS/MS method for the simultaneous determination of (R,S)-ketamine, (R,S)-norketamine, and (2R,6R;2S,6S)-hydroxynorketamine in mouse plasma and brain

A convenient LC-MS/MS assay method to simultaneously and sensitively determine (R,S)-ketamine (Ket), (R,S)-norketamine (NK), and (2R,6R;2S,6S)-hydroxynorketamine (HNK) enantiomers in plasma and brain from mice was developed. This method enables the chiral separations of these six enantiomers in one analysis by constructing a column-switching system composed of one achiral column and two chiral columns with a relatively short analysis time (17 min). The chromatography involves the separation of (2R,6R;2S,6S)-HNK from (R,S)-Ket and (R,S)-NK on an octadecyl-silica column, followed by chiral separations on a CHIRALPAK AY-RH column for (2R,6R;2S,6S)-HNK or on a CHIRALPAK AS-RH column for the other analytes. The calibration curves for plasma and brain showed a good linearity in the range of 3–1000 ng/mL and 1.5–500 ng/g, respectively. The accuracy ranged from 90.0% to 104.0% in within-run and between-run. This validated method was applicable to determine the stereoselective pharmacokinetic profiles of (R,S)-Ket, (R,S)-NK, and (2R,6R;2S,6S)-HNK in plasma and brain collected from individual mice after a single intraperitoneal dosing of racemic Ket at an antidepressant dose. It is hoped that this assay will greatly help for understanding the relationship between the antidepressant actions of (R,S)-Ket enantiomers or their metabolites and their pharmacokinetics.

Electrochemical determination of emodin in acidic media by high-performance liquid chromatography and its application to Polygoni Multiflori Radix samples.

Electrochemical reduction of emodin under acidic media occurs at a less negative potential when compared with that under neutral media. When emodin is electrochemically detected at a less negative potential, a decrease in background noise and improvement in specificity benefit the development of high-performance liquid chromatography with electrochemical detection (HPLC-ECD) for its determination. HPLC-ECD was performed using an octadecyl silica column, acetonitrile-water (60:40, v/v) containing 5mmolL-1 hydrochloric acid and 10mmolL-1 lithium perchlorate, as a mobile phase, and an applied potential at -0.4V vs. Ag/AgCl. Under these optimal HPLC-ECD conditions, the detection limit (signal-to-noise ratio, S/N = 3) of emodin was 0.61μgL-1. When this HPLC-ECD system was applied to the determination of emodin in Polygoni Multiflori Radix (PMR) samples, other peaks did not appear close to the emodin peak on a chromatogram. The emodin contents in PMR samples were determined with relative standard deviations (RSDs, n = 6) of less than 3.9%, and their recoveries ranged from 92 to 106%. We have shown that our HPLC-ECD system performed an accurate, precise, and specific determination of emodin in PMR samples.

Preparation of imidazolidinone compounds as derivatization reagent for diastereomerization and chromatographic separation of chiral organic acids

A chiral derivatization reagent for application in LC-tandem mass spectrometry (LC-MS/MS)-based detection, benzyl 5-(2-aminoethyl)-3-methyl-4-oxoimidazolidine-1-carboxylate (CIM-C2-NH2), which can react with the carboxyl group, was synthesized. Both chiral and non-chiral organic acids such as lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, acetic acid, succinic acid, tartaric acid, malic acid, and citramalic acid were derivatized with CIM-C2-NH2; their derivatives were analyzed by LC-MS/MS. We investigated the enantioseparation of chiral organic acids on an octadecylsilica column and obtained the resolution values within 1.31–2.19 with a mobile phase of H2O-CH3CN with the exception of malic acid. In contrast, no enantioseparation of the chiral organic acids was observed when benzyl 5-(aminomethyl)-3-methyl-4-oxoimidazolidine-1-carboxylate (CIM-C1-NH2), which bears fewer methylene units than CIM-C2-NH2, was used. In addition, the mass spectra of malic acid, tartaric acid, and succinic acid, which have two carboxyl groups, showed product ions of m/z 278, while those of other organic acids showed product ions of m/z 91, corresponding to the benzyl moiety. The proposed method was applied to analyze organic acids in commercial wine, and some organic acids such as d- and l-lactic acid were successfully detected.

Validation method for determining enrofloxacin and tylosin levels in broiler liver, kidney, and muscle using high-performance liquid chromatography.

Background and Aim:Enrofloxacin and tylosin can be combined into an antibiotic formulation which is expected to have a broader range of antibacterial activity against various infections in broilers. Validation method analysis of the levels of these two active compounds needs to be done for future use in pharmacokinetic or residual studies. The present study aims to determine a suitable validation method of isocratic high-performance liquid chromatography (HPLC) to measure the concentration of antibiotic combinations in the broiler liver, kidney, and muscles.Materials and Methods:The combination of enrofloxacin and tylosin in the liver, kidney, and muscle was validated by HPLC method to find the procedures, processes, equipment, and systems used, consistently provides the appropriate results. The chromatography system consisted of an Octadecyl-silica column of 5 mm in diameter and 150 mm in length with a mobile phase of a mixture of 0.05 M monobasic sodium phosphate (pH 2.5) and acetonitrile (65:35 v/v). The solution was detected at a wavelength of 280 nm, 30°C, a flow rate of 1 mL/min, and an injection volume of 20 mL. The combination antibiotics powder was produced from PT Tekad Mandiri Citra, Bandung, Indonesia, and broiler tissues obtained from day-old chick broilers maintained for 30 days with free antibiotic feed.Results:Validation of a combination solution of enrofloxacin and tylosin shows the linearity values of enrofloxacin and tylosin in the liver, kidney, and muscles as r2=0.9988, r2=0.9999, r2=0.9997, r2=0.9989, r2=0.9978, and r2=0.9962. The accuracy and precision values of enrofloxacin in the liver, kidney, and muscles were 5.53, 6.23, and 6.93, respectively. The values of accuracy and precision of tylosin in the liver, kidney, and muscles were 10.43, 4.63, and 7.16%, respectively. The retention times for enrofloxacin and tylosin were 1.945-2.000 min and 4.175-4.342 min. The limit of detection (LOD) and limit of quantity (LOQ) values for enrofloxacin were 3.03 and 10.1 μg/g, respectively. In contrast, the LOD and LOQ values for tylosin were 9.05 and 30.17 μg/g, respectively.Conclusion:The value of linearity, accuracy, precision, specificity, and sensitivity of the combined solution of enrofloxacin and tylosin showed promising results.

Determination Method for Pyrroloquinoline Quinone in Food Products by HPLC-UV Detection Using a Redox-Based Colorimetric Reaction.

We have developed an HPLC-UV method for the determination of pyrroloquinoline quinone (PQQ), which utilizes a redox-based colorimetric reaction. In the proposed colorimetric reaction, the redox reaction between PQQ and dithiothreitol generates superoxide anion radicals that can convert 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to formazan dye. After PQQ separation on an octadecyl silica column, it was mixed online with dithiothreitol and INT, and the formed formazan dye was monitored by absorbance at 490 nm. The detection limit (S/N = 3) of the proposed method was 7.6 nM (152 fmol/injection). The proposed method could selectively detect PQQ in food products without any clean-up procedures.

Application of Linear Gradient Solvent System in Centrifugal Partition Chromatography Facilitating Bioassay-Guided Fractionation of Yongdamsagan-Tang, Traditional Oriental Decoction.

As important pharmaceutical resources, traditional herbal medicines retain continuous attention. To do that, isolation and identification of bioactive molecules from traditional herbal decoction are important. However, conventional fractionation through octadecyl silica column faces irreversible sample adsorption that causes a bias in bioactivity assessment. However, liquid-liquid chromatographic system suffers tedious K value calculation as well as insufficient capacity in separation power when crude extract composed of widely ranging polarities. Here, we developed a comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) to aid bioassay-guided isolation. The lower aqueous phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary, whereas its upper organic phase followed by the upper phase of ethyl acetate-acetonitrile-water and water-saturated n-butanol-acetonitrile-water in the same ratio were eluted in a linear gradient mode, thereby increasing polarity in the mobile phase. The HPLC profiling of CPC fraction showed that proposed gradient CPC was suitable to separate metabolites from Yongdamsagan-Tang, a traditional medicinal decoction made of ten herbal plants. Exhibiting a high recovery yield of 98.3%, antioxidant response element (ARE) luciferase-inducing assay in HepG2 cells indicated that the fractions composed of baicalein and wogonin, the marker natural products of Scutellaria baicalensis, were to be the most effective molecules from Yongdamsagan-Tang. The presented results demonstrated that bioassay-guided separation that assisted with a linear gradient CPC is an incomparable alternative to HPLC and biphasic CPC in terms of higher yield rate and redundant K value calculation, respectively, which led to an unbiased/time-saving separation and identification of bioactive molecules from the complex crude extract of natural products.

Determination of intracellular 2-hydroxyglutarate enantiomers using two-dimensional liquid chromatography

In this study, a heart-cutting two-dimensional liquid chromatography (2D-LC) method for the determination of intracellular 2-hydroxyglutarate (2-HG) enantiomers is proposed. D-2-HG is an oncometabolite produced by tumors harboring isocitrate dehydrogenase (IDH) mutations. Hence, achieving accurate quantification of the 2-HG enantiomers in cancer cells is important. In this study, a fluorescence derivatization reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), was used. We found that 2-HG was successfully derivatized with NBD-PZ after lactonization. The enantiomers of 2-HG derivatized with NBD-PZ were quantified using the heart-cutting 2D-LC method. NBD-PZ-2-HG was stable for 1 week and was separated from endogenous compounds using an octadecyl silica column before enantiomeric separation on a chiral column. The developed heat-cutting 2D-LC method was validated and successfully applied to cell samples. The amount of D-2-HG and L-2-HG in U937 cells were 9.8 and 8.3 pmol/5.0 × 105 cells, respectively. Furthermore, high amounts of D-2-HG were measured in U937 cells expressing mutant IDH proteins as expected. This method can therefore be applied to further elucidate the role of 2-HG in cancer cells, particularly those harboring isocitrate dehydrogenase mutations.

Chemical constituents of triterpenoids from Euphorbia resinifera

The combination of normal-phase silica gel column chromatography, octadecyl silica(ODS) column chromatography, semi-preparative high performance liquid chromatography(HPLC), etc. was employed to isolate and purify the chemical components from Euphorbia resinifera, and 7 triterpenoids were separated from the ethanol extract of the medicinal materials. Their structures were identified by various spectroscopy methods as cycloartan-1,24-diene-3-one(1), cycloartan-1,24-diene-3-ol(2), 3β-hydroxy-lanosta-8,24-diene-11-one(3), lnonotusane C(4), eupha-8,24-diene-3β-ol-7,11-dione(5), eupha-24-methylene-8-ene-3β-ol-7,11-dione(6), and eupha-8,24-diene-3β,11β-diol-7-one(7). Compounds 1 and 2 are new compounds, and compound 3 is obtained from nature for the first time.

Feasibility of supercritical fluid extraction-supercritical fluid chromatography mass spectrometry for the determination of polycyclic aromatic hydrocarbons in particulate matter samples.

There has been an increased interest in the development of green analytical methods for polycyclic aromatic hydrocarbons in environmental samples due to their toxicity and ubiquitous nature. In this work, the feasibility of on-line supercritical fluid extraction-supercritical fluid chromatography-tandem mass spectrometry was investigated for rapid and automated determination of the 16 United States Environmental Protection Agency priority polycyclic aromatic hydrocarbons in particulate matter samples. Three specialty polycyclic aromatic hydrocarbons columns with different stationary phases were screened and an octadecyl-silica column was selected for method development. Enhanced extraction efficiency was achieved using an extraction program with gradient flow rate and gradient concentration of acetonitrile as a modifier. The method was evaluated by analyzing standard reference materials of urban dust and diesel particulate matter from the National Institute of Standards and Technologies. Possible reasons for the unsatisfactory recoveries with certain polycyclic aromatic hydrocarbons are discussed.

Chemical constituents of sesquiterpenes from Chloranthus multistachys

With repeated silica gel, octadecyl silica(ODS), and Sephadex LH-20 column chromatography, normal-phase and reverse-phase high performance liquid chromatography(HPLC), etc., a pair of new enantiomers and 5 known compounds were separated from the 95% ethanol extract of Chloranthus multistachys. These compounds were identified by the nuclear magnetic resonance spectroscopy(including 1 D-NMR and 2 D-NMR), single-crystal X-ray diffraction, circular dichroism(CD) spectroscopy, mass spectrometry(MS), and some other methods as(1R,4R,5R,8S,10R)-chloraeudolide H(1 a),(1S,4S,5S,8R,10S)-chloraeudolide H(1 b), hydroxyisogermafurenolide(2), 4α-hydroxy-5α,8β(H)-eudesm-7(11)-en-8,12-olide(3), chloraniolide A(4), chlorantene D(5), 4α,8β-dihydroxy-5α(H)-eudesm-7(11)-en-8,12-olide(6). Compounds 1 a and 1 b are a pair of new eudesmane-type sesquiterpene enantiomers, and compounds 2-4 were isolated from C. multistachys for the first time.

A Molecular Shape Recognitive HPLC Stationary Phase Based on a Highly Ordered Amphiphilic Glutamide Molecular Gel.

Chiral glutamide-derived lipids form self-assembled fibrous molecular gels that can be used as HPLC organic phases. In this study, HPLC separation efficiency was improved through the addition of branched amphiphilic glutamide lipids to the side chains of a terminally immobilized flexible polymer backbone. Poly(4-vinylpyridine) with a trimethoxysilyl group at one end was grafted onto the surface of porous silica particles (Sil−VP15, polymerization degree = 15), and the pyridyl side chains were quaternized with a glutamide lipid having a bromide group (BrG). Elemental analysis indicated that the total amount of the organic phase of the prepared stationary phase (Sil−VPG15) was 38.0 wt%, and the quaternization degree of the pyridyl groups was determined to be 32.5%. Differential scanning calorimetric analysis of a methanol suspension of Sil−VPG15 indicated that the G moieties formed a highly ordered structure below the phase transition temperature even on the silica surface, and the ordered G moieties exhibited a gel-to-liquid crystalline phase transition. Compared with a commercially available octadecylated silica column, the Sil−VPG15 stationary phase showed high selectivity toward polycyclic aromatic hydrocarbons, and particularly excellent separations were obtained for geometrical and positional isomers. Sil−VPG15 also showed highly selective separation for phenol derivatives, and bio-related molecules containing phenolic groups such as steroids were successfully separated. These separation abilities are probably due to multiple interactions between the elutes and the highly ordered functional groups, such as the pyridinium and amide groups, on the highly ordered molecular gel having self-assembling G moieties.

Development of a Derivatization Reagent with a 2-Nitrophenylsulfonyl Moiety for UHPLC-HRMS/MS and Its Application to Detect Amino Acids Including Taurine.

Taurine (Tau) has some important ameliorating effects on human health and is present in bivalve. For the selective analysis of Tau with other amino acids, we designed a derivatization reagent, 2,5-dioxopyrrolidin-1-yl(4-(((2-nitrophenyl)sulfonyl)oxy)-6-(3-oxomorpholino)quinoline-2-carbonyl)pyrrolidine-3-carboxylate (Ns-MOK-β-Pro-OSu). After derivatization with Ns-MOK-β-Pro-OSu, amino acids with Tau in Japanese littleneck clams were determined through ultra-high-performance-liquid chromatography with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) using an octadecyl silica column. We could detect 18 amino acids within 10 min. Tau, valine, glutamine, glutamic acid, and arginine in the clams were determined in the negative ion mode using the characteristic fragment ion, C6H4N1O5S, which corresponded to the 2-nitrobenzenesulfonylate moiety. The fragment ion, C6H4N1O5S, was recognized as a common feature regardless of the amino acid to be derivatized, and it was convenient for detecting amino acid derivatives with high selectivity and sensitivity. Therefore, highly selective quantification using UHPLC-HRMS/MS was possible using Ns-MOK-β-Pro-OSu.

Comparison of in-silico modelling and reversed-phase liquid chromatographic retention on an octadecyl silica column to predict skin permeability of pharmaceutical and cosmetic compounds

This study focuses on the in-silico modelling of the skin permeability using a test set of pharmaceutical and cosmetic compounds. Two sets of theoretical molecular descriptors, obtained from the E-Dragon and Vega ZZ software programs, were used in the models. Different linear regression methods, i.e. Multiple Linear Regression (MLR) and Partial Least Squares (PLS) regression, were applied for modelling and estimating the skin permeability. The best model was obtained using a stepwise MLR approach on the E-Dragon descriptor set. In a second step, the retention of the test set compounds was measured on a C18 column at two pH levels: pH 5.5 and pH 7. Different organic-modifier fractions were applied in the mobile phase to be able to extrapolate the retention factors to a log kw value, with kw the estimated retention factor in an aqueous mobile phase without organic modifier. Thereafter it was examined whether combining this chromatographic descriptor with the theoretical descriptors could improve the modelling of the skin permeability. The chromatographic descriptor often did not show an added value compared to the models containing only theoretical descriptors. Therefore, the in-silico models were preferred, and these models could be useful to predict the skin permeability of pharmaceutical and cosmetic compounds.

Fluorimetric determination of the enantiomers of vigabatrin, an antiepileptic drug, by reversed-phase HPLC with a novel diastereomer derivatization reagent.

Herein, determination of an antiepileptic drug, (±)-vigabatrin (VB), was performed by reversed-phase HPLC with fluorimetric detection using a newly designed and synthesized fluorescence derivatization reagent, 2,5-dioxopyrrolidin-1-yl (4-{[(2-nitrophenyl)sulfonyl]oxy}-6-(3-oxomorpholino)quinoline-2-carbonyl)prolinate [Ns-MOK-(R)- or (S)-Pro-OSu]. During the derivatization of VB with Ns-MOK-(R)-Pro-OSu at 60°C, the nosyl (Ns) group, which was introduced to protect a phenolic hydroxy group, was released within 30 min to produce MOK-(R)-Pro-VB, which was detected fluorimetrically at 448 nm with an excitation wavelength of 333 nm. The VB enantiomers were separated on an octadecylsilica (ODS) column with a resolution value of 5.57, because Ns-MOK-(R)-Pro-OSu bears an optically active D-proline structure. A complete separation of MOK-(R)-Pro-(R)- and -(S)-VB enantiomers was achieved on the ODS column within 40 min using stepwise gradient elution, and the detection limits were ~0.80 and 0.37 pmol on the column, respectively. The proposed HPLC with fluorimetric detection method was successfully used for determining VB enantiomers in VB-spiked human serum following solid-phase extraction with an anion-exchange cartridge.

Effect of Organic Modifier on the Retention of Low-Molecular-Weight Organic Compounds in Low-Temperature HPLC Using a Liquid CO<sub>2</sub> Mobile Phase and an Octadecyl Stationary Phase

The retention behaviors of various low-molecular-weight compounds in low-temperature HPLC with an ODS (octadecyl silica) column and liquid CO2 mobile phase containing methanol or acetonitrile as modifiers were investigated. The addition of methanol/acetonitrile reduced the retention of xanthone, which was dominated by the liquid CO2 content, independent of the modifier. Additionally, the retention of polycyclic aromatic hydrocarbons (PAHs) was reduced by the addition of methanol/acetonitrile. Because acetonitrile resulted in a rapid reduction in the retention compared with methanol, it was suggested that the CH- interactions have an important role in the retention of PAHs in the C18 stationary phase. Notably, the retention of alkylbenzenes were only slightly affected by the addition of methanol/acetonitrile. Acetonitrile slightly increased the retention of alkylbenzenes with longer alkyl chains than those with shorter alkyl chains. It is suggested that the interaction of the phenyl moiety in alkylbenzenes with C18 chains was reduced by the increase in the acetonitrile content, and enhanced in the case of the alkyl chain moieties. The retentions of alkyl benzenes were approximately compensated, when the alkyl chain length was relatively short.

Discovering the major metabolites of the three novel fentanyl analogues 3-methylcrotonylfentanyl, furanylbenzylfentanyl, and 4-fluorocyclopropylbenzylfentanyl for forensic case work

The highly potent opioid analgesic fentanyl and its analogues are involved in an increasing number of overdose deaths worldwide. New fentanyl analogues are continuously emerging, and there is a lack of knowledge concerning the metabolism of these compounds. The determination of fentanyl analogues can be challenging due to their low circulating concentrations and rapid and extensive metabolism, making metabolite identification necessary for confirming drug intake. The aim of this study was to discover and elucidate the structures of the major metabolites of the three novel fentanyl analogues 3-methylcrotonylfentanyl (3-MCF), furanylbenzylfentanyl (FBF), and 4-fluorocyclopropylbenzylfentanyl (4-FCBF), which were all seized at European borders in 2018. 3-MCF, FBF, or 4-FCBF was incubated with human liver microsomes and human hepatocytes for up to 4 h. The metabolites formed were separated by ultra-high-performance liquid chromatography using an octadecyl silica column employing solvent gradient elution with a mobile phase consisting of ammonium formate and methanol. The compounds were detected by quadrupole time-of-flight mass spectrometry. The major metabolites of 3-MCF were formed by N-dealkylation, carboxylation, oxidation, or hydroxylation of the 3-methyl-2-butene, and hydroxylation of both the 3-methyl-2-butene and the piperidine ring. FBF was metabolized through N-dealkylation, amide hydrolysis with/without subsequent hydroxylation at the N-phenyl, and dihydrodiol formation at the furan ring. 4-FCBF metabolism was dominated by N-dealkylation and N-oxidation at the piperidine ring. In the present study, we successfully discovered and elucidated the structures of the major metabolites of 3-MCF, FBF, and 4-FCBF which could be used as markers to confirm intake of these compounds in forensic case work.

Qualitative analysis of zolpidem and its metabolites M-1 to M-4 in human blood and urine using liquid chromatography–tandem mass spectrometry

The aim of this study was to develop the method for the qualitative analysis of zolpidem and its metabolites M-1 to M-4 using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in human blood and urine. To obtain analytical standards, a method for synthesizing zolpidem metabolites M-1 to M-4 was developed. A combination of a copper-catalyzed three-component coupling reaction and pyrrolidine-catalyzed imine formation was adopted to synthesize the methyl esters of M-1 (M-1-Me) and M-2 (M-2-Me), which were hydrolyzed to afford M-1 and M-2, respectively. M-3 and M-4 were also synthesized. The LC–MS/MS conditions were optimized using authentic standards. M-1 to M-4 were analyzed in human blood and urine. M-1 to M-4 were successfully synthesized. The mass spectra of M-1 and M-2 were almost identical, but their peaks were chromatographically separated using a octadecyl silica column. Likewise, mass spectra of M-3 and M-4 were similar, but their peaks were chromatographically separated. The peak intensities of the metabolites in human blood and urine were M-1 > M-2, and M-4 > M-3; M-3 was detected only in urine. The presence of other hydroxyzolpidems was also revealed. Chromatographic separation of M-1 and M-2 and that of M-4 and other hydroxyzolpidems is necessary to analyze zolpidem metabolites in biological matrices because their mass spectra are quite similar. This study is the first to analyze zolpidem and M-1 to M-4 simultaneously using LC–MS/MS, which can provide more compelling evidence of zolpidem intake.

LC-HRMS based on mixed-mode chromatography for the separation of teicoplanin and the unravelment of its composition

Liquid chromatography – high resolution mass spectrometry (LC-HRMS) based on mixed-mode chromatography has proven to be a useful tool in pharmaceutical analysis for the characterization of very polar antibiotics such as teicoplanin. This compound is a semisynthetic glycopeptide antibiotic thought to be a mixture of five major compounds (named A2-1 through A2-5) and four minor ones (RS-1 to RS-4), all sharing the same glycopeptide core structure (dubbed A3-1) and differing only on the length of a hydrocarbon side chain. These nine compounds have been fully characterized in the past thirty years by means of HPLC coupled to UV and MS detectors. However, HPLC separations were all based on octadecylsilica columns (C18), which nowadays may not be the best choice for the chromatographic separation of such polar and charged compounds. In this work, several different chromatographic alternatives are tested for the separation of teicoplanin, including mixed-mode chemistries. It has been demonstrated that a C18-PFP mixed-mode column provides the best chromatographic resolution, furthermore, confirming the existence of several minor compounds in the composition of teicoplanin. Up to fourteen minor components were characterized by means of High-resolution MS/MS and confirmed by the analysis of a commercial teicoplanin product (Targocid®).