The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway.

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.

[Discussion on calculation method of detection limit and quantitative limit of occupational health biological monitoring method].

Objective: To discuss calculation method of detection limit and quantitative limit of occupational health biological monitoring. Methods: The detection limit and the quantitative limit of phenyl glyoxylic acid and Mandelic acid were calculated by using three different methods of IUPAC, NIOSH and OSHA respectively. Results: The IUPAC, NIOSH and OSHA methods were used to calculate the detection limit and the quantitative limit of the phenyl glyoxylic acid and Mandelic acid, and the results are different. Conclusion: To calculate the detection limit and quantitative limit of occupational health biological monitoring methods, the standard curve method is adopted to ensure that the rate of detection in the vicinity of detection limit and more than 75% of the quantitative limits are used.

Betamethasone-based chiral electrochemical sensor coupled to chemometric methods for determination of mandelic acid enantiomers.

A chiral biosensing platform was developed using betamethasone (BMZ) as chiral recognition element through multilayered electrochemical deposition of BMZ, overoxidized polypyrrole, and nanosheets of graphene (OPPy-BMZ/GR), for enantio-recognition of mandelic acid (MA) enantiomers. The deposited film was characterized by scanning electron microscopy, differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy. It was shown that the chiral sensing platform can discriminate R- and S-MA differential pulse voltammetry signals, at the voltages of 1.35 and 1.33 V (vs Ag/AgCl), respectively. To tackle the problem of highly overlapping peaks of these enantiomers, the partial least squares (PLS) regression and genetic algorithm-PLS (GA-PLS) were used for simultaneous quantification of MA enantiomers. Generally, variable selection by genetic algorithm provided an improvement in prediction results when compared to full-voltammogram PLS. Good analytical performances were obtained despite the inherent complexity of the simultaneous determination.

Integrating a post-column makeup pump into preparative supercritical fluid chromatography systems to address stability and recovery issues during purifications.

Purification of many pharmaceutical compounds by supercritical fluid chromatography (SFC) has always been challenging because of degradation of compound during the isolation step in the presence of acidic or basic modifiers in the mobile phase. Stability of such acid or base-sensitive compounds could be improved by post-column addition of a solvent containing base or acid modifier as counter ion through a make-up pump respectively to neutralize the compound fraction without affecting the resolution. One such case study has been presented in this work where the stability of a base-sensitive compound was addressed by the addition of acidic co-solvent through the make-up pump. Details of this setup and the investigation of degradation of the in-house base-sensitive compound are discussed in this paper. In addition, poor retentivity and low recovery of many non-polar compounds in SFC eluting under low co-solvent percentage is another major concern. Even though the desired separation could be achieved with low percentage of co-solvent, it's difficult to get the proper recovery after purification due to precipitation of the sample and significant aerosol formation inside the cyclone. We have demonstrated the first-time use of a post-column make-up pump on SFC 350 system to introduce additional solvent prior to cyclone to avoid the precipitation, reduce the aerosol formation and thus improve the recovery of non-polar compounds eluting under less than 10% of co-solvent.

Engineering Escherichia coli for production of 4-hydroxymandelic acid using glucose-xylose mixture.

BACKGROUND: 4-Hydroxymandelic acid (4-HMA) is a valuable aromatic fine chemical and widely used for production of pharmaceuticals and food additives. 4-HMA is conventionally synthesized by chemical condensation of glyoxylic acid with excessive phenol, and the process is environmentally unfriendly. Microbial cell factory would be an attractive approach for 4-HMA production from renewable and sustainable resources. RESULTS: In this study, a biosynthetic pathway for 4-HMA production was constructed by heterologously expressing the fully synthetic 4-hydroxymandelic acid synthase (shmaS) in our L-tyrosine-overproducing Escherichia coli BKT5. The expression level of shmaS was optimized to improve 4-HMA production by fine tuning of four promoters of different strength combined with three plasmids of different copy number. Furthermore, two genes aspC and tyrB in the competitive pathway were deleted to block the formation of byproduct to enhance 4-HMA biosynthesis. The final engineered E. coli strain HMA15 utilized glucose and xylose simultaneously and produced 15.8 g/L of 4-HMA by fed-batch fermentation in 60 h. CONCLUSIONS: Metabolically engineered E. coli strain for 4-HMA production was designed and constructed, and efficiently co-fermented glucose and xylose, the major components in the hydrolysate mixture of agricultural biomass. Our research provided a promising biomanufacturing route to produce 4-HMA from lignocellulosic biomass.

Modeling the retention mechanism for high-performance liquid chromatography with a chiral ligand mobile phase and enantioseparation of mandelic acid derivatives.

The chromatographic retention mechanism describing relationship between retention factor and concentration of Cu(2+) (l-phenylalanine)2 using chiral ligand mobile phase was investigated and eight mandelic acid derivatives were enantioseparated by chiral ligand exchange chromatography. The relationship between retention factor and concentration of the Cu(2+) (l-phenylalanine)2 complex was proven to be in conformity with chromatographic retention mechanism in which chiral discrimination occurred both in mobile and stationary phase. Different copper(II) salts, chiral ligands, organic modifier, pH of aqueous phase, and conventional temperature on retention behavior were optimized. Eight racemates were successfully enantioseparated on a common reversed-phase column with an optimized mobile phase composed of 6 mmol/L of l-phenylalanine or N,N-dimethyl-l-phenylalanine and 3 mmol/Lof copper(II) acetate or copper(II) sulfate aqueous solution and methanol.

A pair of chiral fluorescent sensors for enantioselective recognition of mandelate in water.

A pair of chiral compounds S-1 and R-1 derived from (1S, 2S) or (1R, 2R)-1, 2-diphenylethane-1, 2-diamine were designed and synthesized, the interactions of S-1 and R-1 with mandelate were studied in H2O (0.01 M HEPES buffer, pH=7.4) by fluorescence titration experiments. The sensors S-1 and R-1 were found to present enantioselective fluorescent sensing ability to mandelate. The results indicated that the sensors S-1 and R-1 were very promising to be used as fluorescent sensors in determining the enantiomeric composition of mandelate in H2O.

Effect of chromatographic conditions on enantioseparation of bovine serum albumin chiral stationary phase in HPLC and thermodynamic studies.

Twelve chiral compounds were enantiomerically resolved on bovine serum albumin chiral stationary phase (BSA-CSP) by high-performance liquid chromatography (HPLC) in reversed-phase modes. Chromatographic conditions such as mobile phase pH, the percentage of organic modifier, and concentration of analyte were optimized for separation of enantiomers. For N-(2, 4-dinitrophenyl)-serine (DNP-ser), the retention factors (k) greatly increase from 0.81 to 6.23 as the pH decreasing from 7.21 to 5.14, and the resolution factor (R(s)) exhibited a similar increasing trend (from 0 to 1.34). More interestingly, the retention factors for N-(2, 4-dinitrophenyl)-proline (DNP-pro) decrease along with increasing 1-propanol in mobile phase (3%, 5%, 7% and 9% by volume), whereas the resolution factor shows an upward trend (from 0.96 to 2.04). Moreover, chiral recognition mechanisms for chiral analytes were further investigated through thermodynamic methods.

Separation of mandelic acid and its derivatives with new immobilized cellulose chiral stationary phase.

A new liquid chromatographic method has been developed for the chiral separation of the enantiomers of mandelic acid and their derivatives 2-chloromandelic acid, 4-hydroxymandelic acid, 4-methoxymandelic acid, and 3,4,5-trismethoxymandelic acid. The enantiomers were separated by a CHIRALPAK(®) IC (250 mm×4.6 mm, 5 µm). Mandelic acid, 4-methoxymandelic acid, and 3,4,5-trismethoxymandelic acid were baseline resolved (resolution factor (RS)=2.21, RS=2.14, and RS=3.70, respectively). In contrast, the enantioselectivities between CHIRALPAK(®) IC and 2-chloromandelic acid and 4-hydroxymandelic acid investigated were low. By comparing the chromatographs of mandelic acid enantiomers and mandelic acid spiked with (R)-mandelic acid, it was determined that the first effluent was (R)-mandelic acid.

Electrochemical recognition for carboxylic acids based on multilayer architectures of ß-cyclodextrin and methylene blue/reduce-graphene interface on glassy carbon electrodes.

A chiral interface has been designed for specific recognition of carboxylic acids using multilayer architectures of ß-cyclodextrin (ß-CD) and methylene blue/reduce-graphene (MB@rGO) on glassy carbon electrodes. The advantages of ß-CD as a chiral selector and MB@rGO composite as an electrochemical indicator were perfectly presented in this novel interface. It displayed good redox signal for sensing chiral target with high sensitivity and conductivity. Enormous signal differences were obtained after adsorption of target L isomer, due to strong blocking of the electron transfer process of methylene blue. Meanwhile mandelic acid was found to be the best chiral guest and obtained more effective chiral recognition.

Enantioselective recognition of mandelic acid based on γ-globulin modified glassy carbon electrode.

A new chiral biosensor has been fabricated by immobilizing γ-globulin on gold nanoparticles modified glassy carbon electrodes, which could recognize and detect mandelic acid (MA) enantiomers. Differential pulse voltammetry, quartz crystal microbalance, ultraviolet-visible spectroscopy, and atomic force microscopy were used to characterize the enantioselectivity. The results exhibited that γ-globulin modified electrode could enantioselectively recognize MA enantiomers, and larger response signals were obtained from R-MA. The factors influencing the performance of the resulting biosensor were investigated. The enantiomeric composition of R- and S-MA enantiomer mixtures could be determined by measuring the current responses of the sample. The developed electrodes have the advantages of simple preparation, good stability, and rapid detection.

Enantioselective recognition of mandelic acid by a 3,6-dithiophen-2-yl-9H-carbazole-based chiral fluorescent bisboronic acid sensor.

We have prepared chiral fluorescent bisboronic acid sensors with 3,6-dithiophen-2-yl-9H-carbazole as the fluorophore. The thiophene moiety was used to extend the π-conjugation framework of the fluorophore in order to red-shift the fluorescence emission and, at the same time, to enhance the novel process where the fluorophore serves as the electron donor of the photoinduced electron transfer process (d-PET) of the boronic acid sensors; i.e., the background fluorescence of the sensor 1 at acidic pH is weaker compared to that at neutral or basic pH, in stark contrast to the typical a-PET boronic acid sensors (where the fluorophore serves as the electron acceptor of the photoinduced electron transfer process). The benefit of the d-PET boronic acid sensors is that the recognition of the hydroxylic acids can be achieved at acidic pH. We found that the thiophene moiety is an efficient π-conjugation linker and electron donor; as a result, the d-PET contrast ratio of the sensors upon variation of the pH is improved 10-fold when compared to the previously reported d-PET sensors without the thiophene moiety. Enantioselective recognition of tartaric acid was achieved at acid pH, and the enantioselectivity (total response K(D)I(F)(D)/K(L)I(F)(L)) is 3.3. The fluorescence enhancement (I(F)(Sample)/I(F)(Blank)) of sensor 1 upon binding with tartaric acid is 3.5-fold at pH 3.0. With the fluorescent bisboronic acid sensor 1, enantioselective recognition of mandelic acid was achieved for the first time. To the best of our knowledge, this is the first time that the mandelic acid has been enantioselectively recognized using a chiral fluorescent boronic acid sensor. Chiral monoboronic acid sensor 2 and bisboronic acid sensor 3 without the thiophene moiety failed to enantioselectively recognize mandelic acid. Our findings with the thiophene-incorporated boronic acid sensors will be important for the design of d-PET fluorescent sensors for the enantioselective recognition of α-hydroxylic acids such as mandelic acid, given that it is currently a challenge to recognize these analytes with boronic acid fluorescent molecular sensors.

Determination of oxybutynin in pharmaceuticals via reaction with mixed acids anhydrides: application to content uniformity testing.

Sensitive and simple spectrophotometric (Method I) and spectrofluorimetric (Method II) methods were developed and validated for the determination of oxybutynin HCl (OXB) in its dosage forms. The method was based on the reaction of OXB with malonic acid anhydride in acetic acid anhydride to form a highly yellow colored product that was measured at 375 nm spectrophotometrically. The same reaction product exihibits strong fluorescence that was measured at 440 nm after excitation at 390 nm. The factors affecting formation and stability of the reaction product were carefully studied and optimized, and the reaction mechanism was postulated. The absorbance-concentration plot is rectilinear over the range 4-40 µg/mL with LOD of 1.12 µg/mL and LOQ of 3.39 µg/mL. The fluorescence-concentration plot is rectilinear over the range 0.5-6 µg/mL with LOD of 0.11 µg/mL and LOQ of 0.33 µg/mL. The method was applied to the analysis of commercial tablets Detronin® and Uripan®. Statistical comparison of the results with those of the reference method revealed good agreement and proved that there were no significant difference in the accuracy and precision between the two methods respectively. The study was extended to content uniformity testing.

Application of oxybutynin selective sensors for monitoring the dissolution profile and assay of pharmaceutical dosage forms.

Two ion-selective sensors of the plastic membrane type were prepared for the determination of oxybutynin hydrochloride (OxCl). They depend on the incorporation of the ion-associates with phosphotungestic acid or phosphomolybdic acid in a PVC matrix. A comparative study is made between their performance characteristics in batch and FIA conditions. The sensors have nearly the same usable concentration, temperature and pH range. They have a wide range of selectivity and can be applied for the determination of the relevant drug with nearly the same precision and accuracy in vitro. Dissolution testing was applied using the sensors; this offers a simple, rapid, cheap way out of sophisticated and high cost instruments used in the pharmacopeial method using HPLC. The investigated drug was determined in its pure and pharmaceutical preparations. The results were accurate and precise, as indicated by the recovery values and coefficients of variation.

Quantitative determination of oxybutynin hydrochloride by spectrophotometry, chemometry and HPTLC in presence of its degradation product and additives in different pharmaceutical dosage forms.

Simple, accurate, sensitive and validated UV spectrophotometric, chemometric and HPTLC-densitometric methods were developed for determination of oxybutynin hydrochloride (OX) in presence of its degradation product and additives in its pharmaceutical formulations. Method A is the first derivative of ratio spectra (DD(1)) which allows the determination of OX in presence of its degradate in pure form and tablets by measuring the peaks amplitude at 216 nm. Method B and C are principal component regression (PCR) and partial least-squares (PLS) for determination of OX in presence of its degradate in pure form, tablets and syrup. While, the developed high performance thin layer chromatography HPTLC-densitometric method was based on the separation of OX from its degradation product, methylparaben and propylparaben followed by densitometric measurement at 220 nm which allows the determination of OX in pure form, tablets and syrup. The separation was achieved using HPTLC silica gel F(254) plates and chloroform:methanol:ammonia solution:triethylamine (100:3:0.5:0.2, v/v/v/v) as the developing system. The accuracy, precision and linearity ranges of the developed methods were determined. The results obtained were statistically compared with each other and to that of a reported HPLC method, and there was no significant difference between the proposed methods and the reported method regarding both accuracy and precision.

Characterization of binding affinities in a chromatographic system by suspended state HR/MAS NMR spectroscopy.

In the current work a racemate of (R)- and (S)-benzylmandelate was separated with a stereoselective polysaccharide-based chiral stationary phase by HPLC. To elucidate the occurring chiral molecular recognition processes in the heterogeneous system used, NMR spectroscopy was chosen under high resolution/magic angle spinning (HR/MAS) NMR conditions in the suspended state. Therefore, and as a proof of concept, a combination of several NMR methods such as spin-lattice relaxation time (T(1)) measurements (T(1)), the saturation transfer difference, and the 2D experiment of the transferred nuclear overhauser enhancement spectroscopy technique were applied. With HR/MAS NMR it is feasible to combine NMR and chromatography to achieve further insights into the separation process.

Cyclodextrin-MEEKC for the analysis of oxybutynin and its impurities.

The development of a cyclodextrin-MEEKC method for the analysis of oxybutynin and five related impurities is described. Experimental design strategies were applied in order to reach baseline separation of the compounds in a short analysis time. Mixture design made it possible to find the best composition for the microemulsion acting as pseudostationary phase, which was constituted by 89.1% 10 mM borate buffer pH 9.2, 1.7% n-heptane, 9.2% SDS/n-butanol in 1:2 ratio. The addition of (2-hydroxypropyl)-beta-cyclodextrin to the background electrolyte was found to improve analysis performance. A Doehlert design, for the factors cyclodextrin concentration and voltage, was carried out and Derringer desirability function led to the identification of 18 mM and 29 kV as the optimal values. Applying the optimum conditions, separation of all the compounds, including the enantiomers of impurity 1, was obtained in less than 12 min. The method was validated according to ICH guidelines for drug assay and determination of impurities and was applied to oxybutynin tablet analysis.

Chiral salen Mn(III) complex-based enantioselective potentiometric sensor for L-mandelic acid.

A new enantioselective potentiometric sensor containing chiral salen Mn(III) as the chiral selector was designed for the assay of L-mandelic acid (L-MA). Optimized membrane electrodes displayed linear dynamic range from 1x10(-5) to 1x10(-1) mol L(-1) with a detection limit of 7.2x10(-6) mol L(-1) and a Nernstian response of -58.1+/-0.5 mV decade(-1) towards L-MA within pH range 7.0-10.2. The potentiometric enantioselectivity coefficient (logK(L,D)(Pot)) of this sensor was -4.0, indicating that the chiral salen Mn(III) complex-based electrode exhibited fairly good discrimination toward L-MA over counter isomer D-MA. The mechanism of chiral recognition for L-MA is discussed by using HF/STO-3G calculation method simulation.

Chiral recognition of mandelic acid on quartz crystal microbalance by vapor diffused molecular assembly method.

This study presents a new approach for the highly selective recognition of chiral L/D-mandelic acid (MA) using a quartz crystal microbalance (QCM) with L-phenylalanine (L-Phe) as the selector. The immobilization of L-Phe on the gold surface of the QCM sensor was performed using a four steps layer-by-layer assembling procedure. The modification of the gold surface of the QCM sensor after each modification step was verified by the cyclic voltammetry, contact angle, FTIR, and QCM detection. The chiral recognition ability of L- and D-MA on the chiral surface was checked by sensing using the vapor diffused molecular assembly (VDMA) method with the QCM. The chiral discrimination factor between L- and D-MA, aplha(L-MA/D-MA), was found to be about 9. The L-Phe-modified QCM gold sensor also showed good stability and reusability. The high chiral discrimination ability of the modified surface might be the result of the different hydrogen bonding force between L- or D-MA and L-Phe.

Chiral recognition of mandelic acid by L-phenylalanine-modified sensor using quartz crystal microbalance.

This study presents a new method for the highly selective recognition of chiral mandelic acid (MA) using L-phenylalanine (L-Phe) as the selector. The proposed method is based on quartz crystal microbalance (QCM) detection, integrated with a vapor diffused molecular assembly (VDMA) reaction technique. The construction of the L-Phe-modified QCM sensor involved a two-step assembly procedure. The chiral recognizability of L- and D-MA on the L-Phe-modified surface was then examined using the VDMA method and QCM. The chiral discrimination factor between L- and D-MA detected by QCM, alpha(L-MA/D-MA), was found to be about 8. The VDMA selective sensing of L-MA on the L-Phe-modified surface was also confirmed by the contact angle measurements. The L-Phe-modified QCM sensor showed good stability and reusability. The present chiral recognition results suggest that L-phe is an excellent resolving agent for the resolution of chiral mandelic acid.