Validation and application of an HPLC-EC method for analysis of coenzyme Q10 in blood platelets

Previous studies have indicated that analysis of coenzyme Q10 (CoQ10) in platelets may be clinically useful. The study objectives are to describe, validate and provide application of an HPLC-EC method for platelet CoQ10 analysis. This method analyzes oxidized (ubiquinone-10) and reduced (ubiquinol-10) forms of CoQ10 using two separate injections with the electrochemical analytical cell set at neutral and oxidizing potentials. Results showed that chromatograms were free of interfering peaks. Calibration curves were constructed over a concentration range 116-2317 nmol/L (r(2) = 0.99). The extraction recovery was >95%. The within-run precision CV% was < or =4.2%, and the day-to-day precision was < or =9.9%. Platelets were isolated by differential centrifugation, and frozen at -70 degrees C until analysis. The application of the method was used to compare accumulation of CoQ10 in platelets vs plasma in eight adult volunteers during a 28 day supplementation period (5 mg/kg/day of ubiquinol-10). Mean platelet total CoQ10 was 164 pmol/10(9) cells, and ubiquinol-10:total CoQ10 ratio was 0.56. During supplementation platelet CoQ10 levels were more consistent and predictable than plasma CoQ10 levels. The results indicate that this validated method for platelet ubiquinol-10 and ubiquinone-10 analysis is acceptable for use in the clinical laboratory, and that platelet CoQ10 may have important advantages over plasma during CoQ10 supplementation.     

Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography

High-speed counter-current chromatography (HSCCC) is applied to the purification of coenzyme Q(10) (CoQ(10)) for the first time. CoQ(10) was obtained from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile-dichloromethane (12:7:3.5, v/v/v) was selected by analytical HSCCC and used for purification of CoQ(10) from 500 mg of the crude extract. The separation yielded 130 mg of CoQ(10) at an HPLC purity of over 99%. The overall results of the present studies show the advantages of HSCCC over an alternative of silica gel chromatography followed by recrystallization. These advantages extend to higher purity (97.8% versus 93.3%), recovery (88% versus 74.3%) and yield (26.4% versus 23.4%). An effort to avoid the toxic, expensive solvent CH(2)Cl(2) was unsuccessful, but at least its percentage is low in the solvent system.     

Determination of coenzyme Q10 in over-the-counter dietary supplements by high-performance liquid chromatography with coulometric detection

A rapid and sensitive method is described for the determination of coenzyme Q10 (Q10) in over-the-counter dietary supplements by automated high-performance liquid chromatography (HPLC) with coulometric detection. Sample solutions of powder-filled capsules, oil-based softgels, and tablets were prepared by serial dilution with 1-propanol. After dilution, a known volume of sample solution containing Q10 and the internal standard, coenzyme Q9 (Q9), was directly injected into the HPLC system. Most of electrochemically active compounds in the injection were oxidized at the precolumn conditioning cell and postcolumn guard cell. Q9 and Q10 were monitored at an analytical cell that contained 2 coulometric electrodes, where Q9 and Q10 were reduced to the corresponding ubiquinol-9 and -10 and then oxidized to produce currents. This method produced a linear detector response for peak height measurements over the concentration range of 0.05-8 microg/mL (r > 0.999). The lower limit of detection was 5 ng/mL (signal-to-noise ratio, > or =3). The mean recovery was 98.9 +/- 0.6%; coefficients of variation for intra- and interday precisions were 1.8-4.0%. The proposed method was successfully applied to the determination of Q10 in marketed products.     

Determination of coenzyme Q10 in human seminal plasma by high-performance liquid chromatography and its clinical application

A high-performance liquid chromatographic (HPLC) method for the analysis of coenzyme Q10 (CoQ10) in human seminal plasma was developed and applied to investigate its clinical significance as a reference index relating to oxidative stress and infertile status of spermatozoa. After precipitation of proteins in seminal plasma with methanol, CoQ10 and coenzyme Q9 (CoQ9; internal standard) were extracted with hexane. The supernatant after centrifugation was evaporated to dryness with nitrogen at 45 degrees C. The residue was re-dissolved in isopropanol. HPLC separation of the sample solution was performed on a Lichrospher C(18) column with a mobile phase composed of isopropanol-methanol-tetrahydrofuran in the ratio of 55:39:6 (v/v/v) at a flow rate of 1.0 mL/min. Under the chromatographic conditions described, the CoQ10 and CoQ9 had retention times of approximately 5.83 and 4.97 min, respectively. The peaks were detected at UV 275 nm. Good separation and detectability of CoQ10 in human seminal plasma were obtained. The method was linear in the range 0.01-10.00 microg/mL. The relative standard deviations within- and between-assay for CoQ10 analysis were 0.85 and 1.86%, respectively. The average recoveries were 94.1-99.0% for the human seminal plasma samples. The CoQ10 levels in seminal plasma of 195 patients and 23 control subjects were studied. CoQ10 concentrations in the two populations were: 37.1 +/- 12.2 ng/mL in the fertile group and 48.5 +/- 20.4 ng/mL in the infertile group. The large difference (p < 0.01) between the fertile and infertile populations is evident.     

High-performance liquid chromatography of coenzyme Q-related compounds and its application to biological materials

A convenient and precise method for the separation and determination of coenzyme Q (CoQ)-related compounds (CoQ homologues, plastoquinone-9, ubichromenol-9, etc.) was developed using high-performance liquid chromatography (HPLC). All compounds tested were separated using a reverse-phase column with a suitable mobile phase and detected at a wavelength of 275 nm. CoQ extracts in plasma and erythrocytes were purified by thin-layer chromatography prior to HPLC analysis, but such purification was not necessary when determining CoQ in urine and tissues. Hydroquinone forms of CoQ existing in animal tissues were oxidized to the corresponding quinone forms with potassium hexacyanoferrate(III). This HPLC method was applied satisfactorily to the determination of the contents of CoQ homologues in human and animal samples. CoQ10 was the only homologue detected in human samples, and CoQ8, CoQ9 and CoQ10 were native homologues of CoQ in rat tissues. Ubichromenol-9 and plastoquinone-9 were not detected in these samples.     

Voltammetric determination of coenzyme Q10 in pharmaceutical dosage forms

A simple and rapid voltammetric method has been developed for the quantitative determination of coenzyme Q(10) (CoQ(10)) in pharmaceutical preparations. Studies with differential pulse voltammetry (DPV) were carried out using a glassy carbon electrode (GCE) in a mixed solvent containing 80 vol.% acetic acid and 20 vol.% acetonitrile. A well-defined reduction peak of CoQ(10) was obtained at -20 mV vs. Ag/AgCl. The voltammetric technique applied provides a precise determination of CoQ(10) using the multiple standard addition method. The statistical parameters and the recovery study data clearly indicate good reproducibility and accuracy of the method. The accuracy of the results assessed by recovery trials was observed to be within the range of 101.1% to 102.5%. The detection and quantification limits were found to be 0.014 mM (12 mg L(-1)) and 0.046 mM (40 mg L(-1)), respectively. An analysis of real samples containing CoQ(10) showed no interferences with common additives and excipients, such as unsaturated fatty acids and soya lecithin. The method proposed does not require any pretreatment of the pharmaceutical dosage forms. A spectrophotometric determination of CoQ(10) in real samples diluted in mixtures containing ethanol and n-hexane was also performed for comparison.     

Sensitive and selective analysis of coenzyme Q10 in human serum by negative APCI LC-MS

Two new sensitive and selective LC-MS methods have been developed for the quantification of the total coenzyme Q(10) concentration in human blood serum. The sensitivity of the methods is based on the very efficient formation of the radical anions of CoQ(10)[M(-)[radical dot]] by negative atmospheric pressure ionisation, APCI(-). The mass detection of the [M(-)[radical dot]] ions, m/z= 862.6, was performed either in selective ion monitoring (SIM) or in MS(2) mode (m/z= 862.6 [rightward arrow]m/z= 847.6) using an LCQ-deca ion-trap mass spectrometer. Two standard serum samples with medium (0.73 [micro sign]g ml(-1)) and high (1.96 [micro sign]g ml(-1)) total CoQ(10) concentrations were analysed by LC-APCI(-)-SIM and LC-APCI(-)-MS(2) and the results compared with a HPLC literature procedure with electrochemical detection (ECD). Both the LC-MS methods were shown to be more selective and with comparable or better sensitivity than the HPLC-ECD method. The LC-MS-SIM and LC-MS(2) chromatograms of the medium concentration sample showed CoQ(10) signal to noise ratios of 25 and 625, respectively. In addition, a simple and fast serum pre-treatment procedure was developed, in which the serum CoQ(10)H(2) content was quantitatively oxidised quantitatively to CoQ(10) in less than 15 min by 1,4-benzoquinone.     

In situ surface-enhanced Raman scattering and X-ray photoelectron spectroscopic investigation of coenzyme Q10 on silver electrode

In this study, coenzyme Q(10) (CoQ(10)) has been investigated by in situ near-infrared Fourier transform surface-enhanced Raman scattering (NIR-FT-SERS) spectroelectrochemistry and angle-resolved X-ray photoelectron spectroscopy (AR-XPS) on silver surface. The surface adsorption behavior of the coenzyme Q(10) radical intermediate could be monitored by potential-dependent SERS technique. At the applied potential lower than -0.30 V vs. SCE, the radical intermediate CoQ(10)H˙ stands perpendicularly on the silver surface with both oxygen atoms of the aromatic ring and isoprenoid side chains. When the applied potential is more positive than -0.30 V vs. SCE or at open circuit potential, the quinone ring (benzene ring) of reduced form of coenzyme Q(10) (CoQ(10)H(2)) adopts a face-on surface configuration on the surface. The responsible mechanism for the potential-dependent SERS spectra is presented. Moreover, the adsorption conformation of CoQ(10) has been further confirmed by AR-XPS at the silver surface.     

Determination of coenzyme Q10 content in raw materials and dietary supplements by high-performance liquid chromatography-UV: collaborative study

An international collaborative study was conducted of a high-performance liquid chromatographic (HPLC)-UV method for the determination of coenzyme Q10 (CoQ10, ubidecarenone) in raw materials and dietary supplements. Ten collaborating laboratories determined the total CoQ10 content in 8 blind duplicate samples. Sample materials included CoQ10 raw material and 4 finished product dietary supplements representing softgels, hardshell gelatin capsules, and chewable wafers. In addition, collaborating laboratories received a negative control and negative control spiked with CoQ10 at low and high levels to determine recovery. Materials were extracted with an acetonitrile-tetrahydrofuran-water mixture. Ferric chloride was added to the test solutions to ensure all CoQ10 was in the oxidized form. The HPLC analyses were performed on a C18 column using UV detection at 275 nm. Repeatability relative standard deviations (RSDr) ranged from 0.94 to 5.05%. Reproducibility relative standard deviations (RSDR) ranged from 3.08 to 17.1%, with HorRat values ranging from 1.26 to 5.17. Recoveries ranged from 74.0 to 115%. Based on these results, the method is recommended for Official First Action for determination of CoQ10 in raw materials and dietary supplement finished products containing CoQ10 at a concentration of >100 mg CoQ10/g test material.     

Formation of CoQ10 reduced form by mixing CoQ10 oxidized form γCD complex and vitamin C in powder

We have already reported the enhancement of the stability and bioavailability of coenzyme Q10 (CoQ10) oxidized form by γ-cyclodextrin (γCD) complexation. In a series of the studies, we investigated an easy and economical conversion of CoQ10 oxidized form to its reduced form in complex powder, using inexpensive vitamin C (VC) as the reductant. CoQ10 oxidized form or its γCD complex and VC were physically mixed at the molar ratio of 1:0 to 1:50. The mixtures were stored at 60 °C and 75% RH. The sampling was made at certain interval, and both CoQ10 oxidized and reduced form contents were measured by high performance liquid chromatography (HPLC). The result shows that the conversion ratio to CoQ10 reduced form in γCD complex was significantly higher than that of non-inclusion compound (ca. 80% versus ca. 30% at the maximum). It was also confirmed that CoQ10 reduced form in γCD complex remains as stable as its oxidized form in γCD complex. Free radical scavenging potential of partially reduced CoQ10–γCD complex was assayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).     

Determination of the ubiquinol-10 and ubiquinone-10 (coenzyme Q10) in human serum by liquid chromatography tandem mass spectrometry to evaluate the oxidative stress

A method for the rapid and simultaneous determination of ubiquinone-10 (coenzyme Q10, CoQ(10)) and the reduced form ubiquinol-10 (CoQ(10)H(2)) in human serum by LC-MS-MS with electrospray ionization (ESI) in the positive mode is here proposed. High selective identification and sensitive quantitation of both analytes have been carried out by monitoring the transition from the corresponding precursor ion to the product ion. Prior to the chromatographic analysis, serum samples (100 microl) were subject to a conventional pre-treatment based on protein precipitation, liquid-liquid extraction, evaporation to dryness and reconstitution with 95:5 methanol/hexane (v/v). The overall method has enabled to achieve low detection limits--5.49 and 15.8 ng/ml for CoQ(10) and CoQ(10)H(2), respectively--which were estimated with serum. The accuracy and potential matrix effects have been studied with spiked serum resulting recoveries between 92.82 and 106.97%. The proposed method has been applied to serum samples from healthy middle-age women, in which the CoQ(10)H(2)/CoQ(10) ratio has been used as marker of oxidative stress.     

液相色谱-电化学检测法测定小鼠血浆和组织线粒体中的辅酶Q和维生素E

 建立了同时测定氧化型和还原型辅酶Q以及维生素E的液相色谱 电化学检测方法。样品中氧化型和还原型辅酶Q9和Q10 以及维生素E混合物经过液相色谱分离柱分离 ,在 - 5 5 0mV的电化学调节池中将氧化型辅酶Q还原为还原型 ,再经过 15 0mV分析池将样品中原有的还原型辅酶Q和经过调节池还原的辅酶Q以及维生素E一同氧化。该方法用于小鼠组织线粒体和血浆样品中氧化型和还原型辅酶Q9和Q10 以及维生素E的同时检测 ,灵敏度高 ,选择性好 ,结果令人满意。     

The use of coenzyme Q0 as a template in the development of a molecularly imprinted polymer for the selective recognition of coenzyme Q10

In this work, a novel molecularly imprinted polymer (MIP) for use as a solid phase extraction sorbent was developed for the determination of coenzyme Q10 (CoQ10) in liver extract. CoQ10 is an essential cofactor in mitochondrial oxidative phosphorylation and a powerful antioxidant agent found in low concentrations in biological samples. This fact and its high hydrophobicity make the analysis of CoQ10 technically challenging. Accordingly, a MIP was synthesised using coenzyme Q0 as the template, methacrylic acid as the functional monomer, acetonitrile as the porogen, ethylene glycol dimethacrylate as the crosslinker and benzoyl peroxide as the initiator. Various parameters affecting the polymer preparation and extraction efficiency were evaluated. Morphological characterisation of the MIP and its proper comparison with C18 as a sorbent in solid phase extraction were performed. The optimal conditions for the molecularly imprinted solid phase extraction (MISPE) consisted of 400 μL of sample mixed with 30 mg of MIP and 600 μL of water to reach the optimum solution loading. The loading was followed by a washing step consisting of 1 mL of a 1-propanol solution (1-propanol:water, 30:70,v/v) and elution with 1 mL of 1-propanol. After clean-up, the CoQ10 in the samples was analysed by high performance liquid chromatography. The extraction recoveries were higher than 73.7% with good precision (3.6–8.3%). The limits of detection and quantification were 2.4 and 7.5 μg g⁻¹, respectively, and a linear range between 7.5 and 150 μg g⁻¹ of tissue was achieved. The new MISPE procedure provided a successful clean-up for the determination of CoQ10 in a complex matrix.     

Determination of ubidecarenone (coenzyme Q10, ubiquinol-10) in raw materials and dietary supplements by high-performance liquid chromatography with ultraviolet detection: single-laboratory validation

A method based on high-performance liquid chromatography with ultraviolet detection has been developed to quantify ubidecarenone [coenzyme Q10 (CoQ10)] in raw materials and dietary supplements. Single-laboratory validation has been performed on the method to determine repeatability, accuracy, selectivity, limits of detection and quantification (LOQ), ruggedness, and linearity for CoQ10. As CoQ10 can exist as the biologically active reduced form, the application of an oxidizing agent, ferric chloride, drives the equilibrium mechanics to the fully oxidized state and allows for exact quantification of total CoQ10 in the sample. This method was found to be fit and linear for the testing of materials containing CoQ10 in the range of approximately equal 50-1000 mg/g. Repeatability precision for CoQ10 was between 2.15 and 5.00% relative standard deviation. Observed recovery of CoQ10 was found to be between 93.8 and 100.9%. LOQ was found to be 9 microg/mL. Further, limited studies showed that some adulterants and degraded material could be satisfactorily separated from CoQ10 and identified.     

苯丙酮尿症新生儿血斑中辅酶及葡萄糖等物质的分析

苯丙酮尿症(PKU)是新生儿先天性苯丙氨酸羟化酶缺陷所引起的苯丙氨酸代谢障碍疾病.本研究采用超高效液相色谱-质谱联用技术, 测定了5例PKU新生儿出生3天和出生30天后的血斑与20例年龄相仿的正常新生儿血斑中辅酶Q10的绝对含量和辅酶Q9的相对含量,其中,健康新生儿血斑中辅酶Q10的含量为(122.1±24.9) ng/mL,PKU新生儿组的含量为(59.0±12.0) ng/mL.采用气相色谱-质谱联用技术测定了胆固醇和葡萄糖的相对含量.研究结果表明,与对照组相比,PKU新生儿血斑中辅酶Q10、Q9、胆固醇和葡萄糖的含量均显著降低,辅酶Q10的降低与血斑中苯丙氨酸含量升高呈现显著反向相关.本研究结果为PKU患儿的饮食治疗方案提供了依据.     

Electrokinetic injection across supported liquid membranes: New sample pretreatment technique for online coupling to capillary electrophoresis. Direct analysis of perchlorate in biological samples

A simple and sensitive method for quantifying perchlorate in biological samples using CE and capacitively coupled contactless conductivity detection was developed. An online combination of a supported liquid membrane, an inert polypropylene membrane impregnated with 1-hexanol, and electrokinetic injection of perchlorate across the supported liquid membrane directly into the separation capillary reduced the need for laborious sample pretreatment procedures, resulting in a cheap and rapid method with low LODs capability. Baseline separation of perchlorate and other anions in biological samples was achieved in background electrolyte solution consisting of 15 mM nicotinic acid and 1 mM 3-(N,N-dimethylmyristylammonio)propanesulfonate at pH 3.3. The analytical method showed excellent parameters in terms of reproducibility; RSD values for peak areas and corrected migration times at a spiked concentration of 100 μg/L of perchlorate were below 10 and 0.4%, respectively. Linear calibration curves were obtained for perchlorate in the concentration range 10-1000 μg/L (r(2) >0.999) with LODs between 2 and 5 μg/L for human urine, breast milk, serum, cow's milk, and red wine. Recoveries at 25 μg/L of perchlorate were between 97 and 106% for all biological samples. The low LODs rivaling those of presently used analytical methods support the use of this method for quantification of perchlorate in biological samples in the future.     

A capillary electrophoretic system based on a novel microemulsion for the analysis of coenzyme Q10 in human plasma by electrokinetic chromatography

A new analytical method for determination of coenzyme Q10 (2,3‐dimethoxy‐5‐methyl‐6‐decaprenyl‐1,4‐benzoquinone, CoQ10) in human plasma was developed based on CE using a double tensioactive microemulsion. CoQ10 was quantitatively extracted into 1‐propanol/hexane and quantified by MEEKC. The microemulsion was prepared by mixing 1.4% w/w sodium bis(2‐ethylhexyl) sulfosuccinate, 4% w/w cholic acid, 1% w/w octane, 8.5% w/w butanol, 0.1% w/w PVA and 85% w/w 10 mM Tris buffer at pH 9.0. The optimized electrophoretic conditions included the use of an uncoated silica capillary of 60 cm total length and 75 μm id, an applied voltage of 20 kV, room temperature and 214 nm ultraviolet detection. Selectivity, linearity, LOD, LOQ, precision and accuracy were evaluated as the parameters of validation. Owing to its simplicity and reliability, the proposed method can be an advantageous alternative to the traditional methodology for the quantitation of CoQ10 in human plasma with good accuracy and precision.     

Flow-injection analysis and voltammetric detection of NADH with a poly-Toluidine Blue modified electrode.

Poly-Toluidine Blue film was prepared by electrooxidative polymerization at a glassy carbon electrode in a phosphate buffer solution. The resulting chemically modified electrode (CME) exhibited excellent electrocatalysis toward the oxidation of reduced nicotinamide coenzyme (NADH) with over a 450 mV decrease of the overpotential compared with that at a bare glassy carbon electrode. Two electrochemical determinations of NADH, cyclic voltammetry and flow injection analysis, were established based on the electrocatalytical performance of the resulting modified electrode. Under an identical determinate condition, the voltammetric detection for NADH gave a detection limit of 3.3 micromol L(-1) with a linear concentration range of 9.1 micromol L(-1) to 1.8 mmol L(-1). As a detector in a flow-injection system, the CME gave a detection limit of 0.1 micromol L(-1) for NADH with a linear concentration range of 1.0 micromol L(-1) to 3.2 mmol L(-1). Obviously, flow-injection analysis is superior to voltammetric detection in NADH determination for its lower detection limit and wider detectable linear range.     

Quantitative determination of coenyzme Q10 by liquid chromatography and liquid chromatography/mass spectrometry in dairy products

The dietary sources of CoQ10 and the evaluation of CoQ10 in dairy products were characterized. For quantitation of CoQ10 in food samples, 2 liquid chromatography (LC) methods with UV and mass spectrometry (MS) detections were developed. LC with UV detection was performed at 25 degrees C on a Hyperclone ODS 5 microm 150 x 4.6 mm column with mobile phase consisting of methanol-ethanol-2-propanol (70 + 15 + 15, v/v/v). Flow rate was 1.0 mL/min. Retention time of CoQ10 was 10.9 +/- 0.1 min. The method was sensitive [limit of detection (LOD) = 0.2 mg/kg], reproducible [relative standard deviation (RSD) = 3:0%), and linear up to 25 mg/kg (R > 0.999). LC/MS analysis was performed on a LUNA C18 3 microm, 150 x 4.6 mm column, using mobile phase consisting of ethanol-dioxane-acetic acid (9 + 1 + 0.01, v/v/v), flow rate was 0.6 mL/min, and the retention time of CoQ10 was 4.1 +/- 0.1 min. Identification and quantitation were performed with a Finnigan-LCQ mass detector in positive atmospheric pressure chemical ionization mode. Mass spectra were obtained in selected-ion monitoring mode; molecular mass (M+H)+ m/z 863.4 +/- 1 was used for quantitative determination. MS detection is more sensitive than UV detection (LOD = 0.1 mg/kg), less reproducible (RSD = 4.0%), and linear in selected range. Analytical recoveries are 75-90% and depend on the ratio between the amount of fat in the matrix and the concentration of CoQ10 in the sample. Some soybean milk products were analyzed together with different cow, goat, and sheep milk products. Concentrations obtained with LC and LC/MS were compared with a few accessible results available from the literature. Concentrations varied from 0 ppm in soybean milk to nearly 2 ppm in fresh milk from local farms.     

Determination of bisphenol A in human breast milk by HPLC with column-switching and fluorescence detection

A highly sensitive HPLC method was developed for the determination of xenoestrogenic compound, bisphenol A (BPA) in human breast milk samples. After a two-step liquid-liquid extraction, BPA was derivatized with fluorescent labeling reagent, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). The excess fluorescent reagent could be removed effectively using a column-switching system. The separation of DIB-BPA from endogenous materials in milk was carried out on two C(18) columns and fluorescence intensity was monitored at 475 nm with the excitation of 350 nm. A good linearity (r = 0.994) was observed of BPA in the concentration range of 0.2-5.0 ng mL(-1) in breast milk, and the detection limit was 0.11 ng mL(-1) at a signal-to-noise ratio of 3. Intra- and inter-day precision (RSD, %) were less than 8.7 and 10.4, respectively. Twenty-three breast milk samples of healthy lactating women were analyzed for the BPA concentration; the mean value was 0.61 +/- 0.20 ng mL(-1), with no correlation to the lipid content of milk samples.