Preparation of a new benzylureido-ß-cyclodextrin-based column and its application for the determination of phenylmercapturic acid and benzylmercapturic acid enantiomers in human urine by LC/MS/MS.

A benzylureido-ß-cyclodextrin was synthesized by the reaction of 6-amino-ß-cyclodextrin with an active benzyl isocyanate. Then, it was bonded to silica gel by a thiol-ene addition reaction, obtaining a new benzylureido-ß-cyclodextrin-based chiral stationary phase (BzCDP). Its chemical structure was characterized by infrared spectroscopy, elemental analysis, and solid-state nuclear magnetic resonance spectroscopy. The BzCDP was successfully used to separate phenylmercapturic acid (PMA) and benzylmercapturic acid (BMA) enantiomers, which were confirmed as biomarkers of exposure to benzene and toluene in human urine. The enantiomeric separations were also optimized through the investigation of related factors. The resolutions of PMA and BMA enantiomers could be up to 2.25 and 2.14, respectively, within 30 min under reversed-phase chromatography. Based on the optimal chromatographic and mass spectrometry conditions, a new LC-MS/MS quantitative method for the PMA and BMA enantiomers was established by negative ion multiple reaction monitoring (MRM) and an isotope-labeled PMA (d2-PMA) as an internal standard. The limits of detection (LODs) of enantiomers were less than 0.17 µg/L for PMA and 0.14 µg/L for BMA, and the averaged recoveries of enantiomers were in the range of 86~100% for PMA and 86~113% for BMA. The method had good reproducibility levels with the RSDs (3.5~11.3% for intra-day and 3.9~13.1% for inter-day). The method was successfully applied to urine testing of 60 painting and printing workers. The results showed that only L-PMA was detected in the urine of the Printers, while a high content of L-PMA (27.5~106 µg/L) and D-PMA (19.9~82.8 µg/L) can be detected simultaneously in the urine of the Painters, indicating that benzene pollution was more serious in this group. The positive rate of BMA was rather higher, indicating that toluene pollution was more common than benzene. BMA also existed in the form of two enantiomers (L-BMA and D-BMA), but the difference between the two types of occupational groups was small. It is a meaningful work to deeply study the existence and content of chiral markers in human urine, which will help to better understand and evaluate the harmful effects of benzene series on human beings. Graphical abstract.

Simultaneous enantiomeric determination of multiple triazole fungicides in fruits and vegetables by chiral liquid chromatography/tandem mass spectrometry on a bridged bis(ß-cyclodextrin)-bonded chiral stationary phase.

A LC-MS/MS method for simultaneous determination of twelve triazole enantiomers (hexaconazole, tebuconazole, triticonazole, flutriafol, diniconazole, paclobutrazol) in six fruits and vegetables was established based on a stable and self-made bridged bis(ß-cyclodextrin)-bonded chiral stationary phase. Simultaneous enantio-separation of multiple analytes was achieved with resolution ca. 1.67-2.14. Magnetically assisted QuECHERS was used to simplify and optimize sample pre-treatment. The new method was validated (accuracy, precision, matrix effect, etc.). Good linearity (0.5-20 µg/L, R2 > 0.99) and high recoveries (76.1-103.4%) based on intra- and inter-day relative standard deviation (RSDs) (2.6-11.9%), were obtained. Furthermore, a total of 90 samples were analyzed using this method and enantiomeric fractions (EF) for tebuconazole in strawberry and cucumber (0.63 and 0.43, respectively) were determined as well as 0.57 for flutriafol in tomato. This high-throughput detection method supported a convenient enantiomeric monitoring for chiral pesticides in fruits and vegetables.

Preparation of a New ß-Cyclodextrin-bonded Chiral Stationary Phase with Thiocarbamated Benzamide Spacer for HPLC.

A new ß-cyclodextrin-bonded chiral stationary phase (TCDP) with a thiocarbamated benzamide spacer was prepared and evaluated on HPLC by separating a series of chiral pesticides and drugs. TCDP resolved all 24 analytes (11 triazoles, 8 flavanones and 5 ß-blockers), and 18 of them were completely separated. The enantioresolution ranges were 1.45 - 3.33 for triazoles, 0.35 - 2.45 for flavanones and 1.26 - 1.58 for ß-blockers. Among them the best resolution of tebuconazole was up to 3.33 within 13 min. Bitertanol (two chiral centers) and triticonazole with cis/trans isomers were separated into four peaks, respectively. TCDP could also completely resolve myclobutanil (Rs = 1.68), which was difficult to be separated on other CD-CSPs. Common CDSP only resolved a few analytes. The results showed that the thiocarbamated benzamide spacer of TCDP may provide a synergistic effect of hydrogen bonding and coordination. The spacer deserved full utilization, by which may avoid troublesome derivatization at low cost.

[Preparation and evaluation of diureido bridged ß-cyclodextrin-bonded chiral stationary phase by high-performance liquid chromatography].

A diureido bridged ß-cyclodextrin dimer was synthesized by the reaction of 6-deoxy-6-hydroxyethylamino-ß-cyclodextrin with hexamethylene diisocyanate. The dimer was then bonded onto silica to obtain a novel diureido bridged ß-cyclodextrin-bonded stationary phase (UBCDP). The structure of diureido bridged stationary phase was characterized by infrared spectroscopy, mass spectrometry, and elemental analysis. By using racemic drugs and pesticides as probes (flavanones, triazoles, and dansylated amino acids), the chromatographic properties of UBCDP were evaluated. The separation mechanism was elucidated by comparison with a single ß-cyclodextrin chiral stationary phase (CDCSP). The composition, pH value of the mobile phase and column temperature were optimized. The results showed that the UBCDP can resolve 25 chiral compounds, especially 2'-hydroxyflavanone, hexaconazole and dansyl leucine with high resolutions (Rs, 1.52-4.35), and the larger volume hesperidin could also be separated. This trend was related to the synergistic inclusion of bridged cyclodextrins. CDCSP can separate only a small number of enantiomers with low resolutions. UBCDP exhibited better enantio-separation ability and hence had potential application in chiral drugs and chiral pesticide residues analysis.

Preparation and evaluation of an ethylenediamine dicarboxyethyl diamido-bridged bis(ß-cyclodextrin)-bonded chiral stationary phase for high performance liquid chromatography.

An ethylenediamine dicarboxyethyl diacetamido-bridged bis(ß-cyclodextrin) was firstly synthesized through the reaction of 6-deoxy-6-amino-ß-cyclodextrin (NH2-CD) with ethylenediaminetetraacetic dianhydride. Then it was bonded onto the surface of silica gel SBA-15 to obtain an ethylenediamine dicarboxyethyl diacetamido-bridged bis(ß-CD)-bonded chiral stationary phase (EBCDP). The structures of the bridged bis(ß-CD) and EBCDP were characterized by infrared spectroscopy, mass spectrometry, elemental analysis and thermogravimetric analysis, accordingly. The chiral chromatographic performances of EBCDP were systematically evaluated by separating 28 racemic analytes in the reversed-phase or polar organic mode, including eight flavanones, eight bolckers, five dansyl-amino acids, three DL-amino acids and four other common drugs. As a result, the relatively high enantioselectivity of EBCDP was observed in comparison with a native ß-CD-CSP (CDSP). All selected analytes were separated on EBCDP, of which 20 analytes had resolutions up to baseline, 2'-hydroxyflavanone and arotinolol had resolutions up to 4.35 and 2.05 in about 30 min, respectively, whereas CDSP only separated 11 analytes with low resolutions (0.55~1.69). Moreover, EBCDP was able to utilize the complexation of the bridging linker (ethylenediamine dicarboxyethyl diamide group, EDTA-based) to realize direct separations of DL-amino acids with a mobile phase containing copper ion (Cu2+), which was similar to the chiral ligand exchange chromatography. Unlike the native cyclodextrin with small cavity (~242 Å3), the bridged bis(ß-CD) combined two ß-CD units with a bridging linker, having a well-organized "pseudo-cavity" as an organic whole to encapsulate more analytes, which made EBCDP have broad-spectrum applications in chiral separations.

Preparation of a stilbene diamido-bridged bis(ß-cyclodextrin)-bonded chiral stationary phase for enantioseparations of drugs and pesticides by high performance liquid chromatography.

A stilbene diamido-bridged bis(ß-cyclodextrin) was synthesized via the reaction between 4,4'-stilbene dicarboxylic acid and 6-deoxy-6-amino-ß-cyclodextrin. Then it was bonded onto the surface of an ordered mesoporous SBA-15 to obtain a novel bridged bis(ß-cyclodextrin)-bonded chiral stationary phase (SBCDP). The structures of the bridged bis(ß-cyclodextrin) and SBCDP were characterized by the mass spectrometry, nuclear magnetic resonance, infrared spectroscopy, elemental analysis and thermogravimetric analysis. The chromatographic performance of SBCDP was systematically evaluated by separating 23 racemic drugs and pesticides, including trimeprazine, praziquantel, flavanones, ß-blockers and triazole pesticides in the reversed-phase chromatography or the polar organic mode. The chromatographic conditions that affect the enantioselectivity or diasterioselectivity of SBCDP were investigated in detail, including the mobile phase composition, pH value and column temperature. As a result, all tested analytes were resolved on SBCDP with high resolutions (1.51∼5.15) within about 25 min, and the enantioseparation resolutions of flavanone and imazalil were up to 5.15 and 4.38, respectively. Compared with the native ß-cyclodextrin stationary phase (CDCSP), the SBCDP had a better chromatographic performance in enantioselectivity and diasterioselectivity. For example, enantiomers of trimeprazine, praziquantel, flavanone and imazalil those could not be separated by CDCSP, were separated by SBCDP with high resolutions. Unlike the small cavity (0.65 nm) of native CD, the bridging linker of the bridged bis(ß-CD) supplied a well-organized "pseudo-cavity", and combined two native CDs as an organic whole, which could synergistically encapsulate and complex some bulky analytes, making the chiral discrimination of SBCDP more precise. Moreover, we also found that SBCDP possessed high enantioselectivity and diastereoselectivity over a wide range of temperature (30∼60 °C), which made the fast analysis possible. As a new chiral separation material, SBCDP may have wider applications in analysis of chiral compounds.

Preparation and Evaluation of a Cholesterol Derivatized ß-Cyclodextrin-bonded Phase for Achiral and Chiral HPLC.

A cholesterol mono-derivatized ß-cyclodextrin was synthesized and bonded onto silica gel (SBA-15) to obtain a cholesterol mono-derivatized ß-cyclodextrin-bonded stationary phase (CHCDP). The chemical structures of mono-derivatized ß-cyclodextrin and CHCDP were characterized by infrared spectroscopy, mass spectrometry, elemental analysis and thermogravimetric analysis, correspondingly. Furthermore, the separation ability of CHCDP in terms of achiral compounds was systematically evaluated by separating benzene homologs, polycyclic aromatic hydrocarbons (PAHs) and some positional isomers. As a result, CHCDP completely separated five benzene homologs and nine PAHs within 30 min under the reversed-phase. In addition, the chiral chromatographic property of CHCDP was also evaluated by separating some racemic compounds including flavanones, triazoles, ß-blockers, etc. The results showed that the CHCDP exhibited high enantioselectivities towards most of selected analytes. The enantioresolutions were in the range from 1.43 to 2.51 on CHCDP. Especially the resolutions of 2'-hydroxyflavanone, hexaconazole, Dns-serine and atenolol were as high as 1.94, 1.91, 2.15 and 1.57, respectively. Obviously, the CHCDP was a versatile stationary phase with chiral and achiral separation capabilities in multi-mode chromatography, which was related to the introduction of cholesterol to the port of cyclodextrin, enhancing the hydrophobic interaction of cyclodextrin with achiral compounds, while maintaining the inclusion complexation of it with chiral compounds as well.

The preparation of a new 3,5-dichlorophenylcarbamated cellulose-bonded stationary phase and its application for the enantioseparation and determination of chiral fungicides by LC-MS/MS.

A new 3,5-dichlorophenylcarbamated cellulose-bonded silica gel stationary phase (CELCSP) was prepared by a "thiol-ene" addition reaction. The alkenyl group was first introduced onto the cellulose, and then the cellulose was completely isocyanated with 3,5-dichlorophenyl isocyanate. Finally, the alkenyl group was reacted with the 3-mercaptopropyl silica gel to obtain the cellulose-bonded stationary phase. The structures of the ligand and the stationary phase were characterized by infrared spectroscopy, nuclear magnetic resonance spectroscopy and elemental analysis. The newly prepared cellulose-bonded phase was successfully used for the enantio-resolution of six common chiral fungicides including triticonazole, hexaconazole, tebuconazole, triadimefon, metalaxyl and benalaxyl under reversed-phase mode, respectively. The resolution (Rs) and selectivity factor (α) of the above fungicide enantiomers on CELCSP could reach 3.46 and 1.27, respectiviely, by using the common 0.1% formic acid-acetonitrile as the mobile phase. Base on the CELCSP column, a new LC-MS/MS method for the quantitative determinations of all six chiral fungicide enantiomers in ten kinds of fruits and vegetables such as cucumber, grape, etc. was established within 30 min. After the sample pretreatment with Fe3O4 magnetic particles, the enantiomers were separated by LC and determined in positive ion multi-reaction monitoring (MRM) by mass spectrometry. The good linear relationship between the response and the concentration of the enantiomers were observed in a range from 0.10 µg/L to 100 µg/L with the relative standard deviation (RSDs, correlation coefficient (0.9965-0.9982)). The averaged recoveries from fruits and vegetables were between 65% and 110% (n = 3). The detection limit (LODs) and limit of quantitation (LOQs) for the enantiomers were 0.05-0.61 µg/kg and 0.18-2.01 µg/kg, respectively. The RSDs of the repeated determination in the samples were 1.2%-6.0% (intra-day, n = 5) and 2.5%-13.0% (inter-day, n = 10), respectively. The mild condition of the thiol-ene addition reaction was beneficial to maintain the order stereostructure of cellulose, while its high yield of the oriented synthesis could also provide sufficient binding amount of chiral ligand, which made the new stationary phase have good chiral chromatographic performance. The newly prepared stationary phase has strong solvent resistance, which is a guarantee for establishing a reliable food safety analysis method for monitoring pesticide enantiomer residues by LC-MS/MS.