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.

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.

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.

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.

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.

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.

Enantiomer self-disproportionation of chiral compounds on achiral ordered mesoporous silica M41S and regular silica gel as a stationary phase.

Chromatographic behavior of nonracemic mixtures, viz., mandelic acid and stilbene oxide as analytes has been studied in detailed by enantiomer self-disproportionation on achiral ordered mesoporous material M41S and regular silica gel as stationary phases. Enantiomer self-disproportionation gave enhanced separation of analytes. The extent and magnitude of enantiomer self-disproportionation is dependent on the optical purity of the starting non-racemic molecules, presence of intermolecular hydrogen bonding/pi-pi interactions and the nature of eluents used. The present study and previous literature data suggest that percentage ee of a nonracemic mixture needs to be determined before any chromatographic purification is taken up as enantiomer self-disproportionation phenomenon could occur during purification. The data show that enantiomer self-disproportionation of nonracemic mixtures can be harnessed for its enantioenrichment on inexpensive achiral stationary phases.

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.

Numerical determination of competitive adsorption isotherm of mandelic acid enantiomers on cellulose-based chiral stationary phase.

The use of inverse method for the determination of competitive adsorption isotherm of mandelic acid enantiomers on cellulose tris(3,5-diethylphenyl carbamate) stationary phase is proposed in this work. Non-dominated sorting genetic algorithm with jumping genes (NSGA-II-JG) was applied to acquire the isotherm parameters by minimizing the sum of square deviations of the model predictions from the measured elution profiles. Three different competitive isotherm models, i.e., Langmuir, biLangmuir and Tóth, combined with transport-dispersive chromatographic model were used in predicting the elution profiles. Orthogonal collocation on finite element (OCFE) method was applied to obtain the calculated elution profiles. Results indicate that biLangmuir isotherm and Tóth isotherm give remarkably similar equilibrium isotherms within the investigated liquid concentration range. Band profiles calculated from both isotherm models are in good agreement with the experimental data. The validity of the determined parameters was verified by comparing the model predictions with experimental elution profiles at various experimental conditions.

Enantioselective analysis of oxybutynin and N-desethyloxybutynin with application to an in vitro biotransformation study.

An enantioselective method using liquid-phase microextraction (LPME) followed by HPLC analysis was developed for the determination of oxybutynin (OXY) and its major metabolite N-desethyloxybutynin (DEO) in rat liver microsomal fraction. The LPME procedure was optimized using multifactorial experiments. Under the optimal extraction conditions, the mean recoveries were 61 and 55% for (R)-OXY and (S)-OXY, respectively, and 70 and 76% for (R)-DEO and (S)-DEO, respectively. The validated method was employed to an in vitro biotransformation study using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of OXY.

Polymeric matrix membrane sensors for stability-indicating potentiometric determination of oxybutynin hydrochloride and flavoxate hydrochloride urogenital system drugs.

Four polyvinyl chloride (PVC) matrix membrane electrodes responsive to 2 drugs affecting the urogenital system--oxybutynin hydrochloride (OX) and flavoxate hydrochloride (FX)--were developed, described, and characterized. A precipitation-based technique with tungstophosphate (TP) and ammonium reineckate (R) anions as electroactive materials in a PVC matrix with an OX cation was used for electrode 1 and 2 fabrication, respectively. Electrode 3 and 4 fabrication was based on use of the precipitation technique of FX cation with tetrakis (4-chlorophenyl) borate and R anions as electroactive materials. Fast and stable Nernstian responses in the range 1 x 10(-2)-1 x 10(-6) M for the 2 drugs over the pH range 5-8 revealed the performance characteristics of these electrodes, which were evaluated according to International Union of Pure and Applied Chemistry recommendations. The method was applied to FX and OX in their pharmaceutical formulations and in human plasma samples. The 4 proposed sensors were found to be specific for the drugs in the presence of up to 60% of their degradation products. Validation of the method according to the quality assurance standards showed suitability of the proposed electrodes for use in the quality control assessment of these drugs. The recoveries for determination of the drugs by the 4 proposed selective electrodes were 99.5 +/- 0.5, 100.0 +/- 0.4, 99.9 +/- 0.4, and 100.1 +/- 0.4% for sensors 1-4, respectively. Statistical comparison between the results obtained by this method and the official method of the drugs was done, and no significant difference found.

Catecholamine turnover in normotensive and hypertensive man: effects of antiadrenergic drugs.

Intravenous administration of tritium-labeled 3,4-dihydroxyphenylalanine (dopa) to human subjects resulted in the labeling of endogenous catecholamines and vanillylmandelic acid (VMA). Determination of the changes in specific activity of these compounds with time in fractional collections of urine and in cardiac biopsies from patients undergoing corrective cardiac surgery permitted estimation of apparent turnover rates. The average half-time of the exponential decline in specific activity of labeled urinary norepinephrine was about 8 hr and that of VMA was 11-16 hr in five normal subjects. No significant differences from normal were observed in eight patients with essential hypertension. The average half-life of norepinephrine was only 5 hr in cardiac patients undergoing surgery, and the levels and rate of decline of cardiac norepinephrine specific activity correlated well with the exponential phase of the urinary disappearance curve. There were significant effects of treatment with alpha-methyltyrosine, reserpine, and pargyline hydrochloride on the labeling and apparent turnover rates of norepinephrine and VMA; the effects noted were consistent with known actions of these three drugs. It is suggested that the technique used is a suitable means of assessing "over-all" catecholamine metabolism in man, particulary if combined with quantitative assay of urinary catecholamine metabolites.

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.

Determination of methenamine, methenamine mandelate and methenamine hippurate in pharmaceutical preparations using ion-exchange HPLC.

An ion-exchange column high-performance liquid chromatography (HPLC) method has been developed for the determination of methenamine in methenamine and methenamine hippurate pharmaceutical preparations. The HPLC method uses a Zorbax SCX-300 column with acetonitrile-0.1M sodium perchlorate monohydrate (pH 5.8) (70:30, v/v) as the mobile phase at the flow rate of 1 mL/min. UV-detection was at 212 nm. The linear concentration plots for methenamine were linear over the concentration range of 0.25-50mM for methenamine and methenamine mandelate standards. The intra-day RSD precision was <1.25%, and for inter-day, <1.85%. The peaks for mandelic acid, hippuric acid and the other ingredients from placebo tablets do not interfere with the analysis for methenamine. The accuracy of this method was shown to be 99-101% by measuring the recovery of methenamine from spiked placebo tablets. The assay of methenamine from methenamine hippurate tablets and from a urinary antiseptic tablet containing methenamine were in the range of 98-102%. This HPLC method is a fast, simple and straightforward method for the analysis of methenamine in pharmaceutical preparations.

Quantification of propiverine by liquid chromatography/electrospray tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy subjects.

Here we report on the development and validation of a sensitive and rapid reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of propiverine in human plasma. After adding an internal standard (oxybutynin chloride) to human plasma, samples were extracted using n-hexane/ethylacetate (8:2, v/v). Compounds extracted were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with multiple reaction monitoring (MRM) mode for analyte detection. This method for determination of propiverine proved accurate and reproducible, with a limit of quantitation of 0.5 ng/ml in human plasma. The standard calibration curve for propiverine was linear (r2=0.9988) over the concentration range 0.5-1000.0 ng/ml in human plasma. The intra- and inter-day precision over this concentration range was lower than 8.66% (relative standard deviation, %R.S.D.), and accuracy was between 99.46 and 109.41%, respectively. This method was successfully applied to a bioequivalence study of two propiverine hydrochloride tablet formulations (20 mg) in 24 healthy subjects after a single administration.