Determination of coenzyme Q10 in human breast milk by high-performance liquid chromatography

An isocratic HPLC method was developed for the determination of coenzyme Q(10) (CoQ(10)) in human breast milk. After a single-step liquid-liquid extraction, the milk extract was injected directly into the HPLC system. The analytical method is based on pre-column inline treatment of CoQ(10). Chromatographic separation of CoQ(10) and coenzyme Q(9) (CoQ(9)) internal standard was achieved using a reversed-phase Microsorb-MV C(18) analytical column. CoQ(10) and CoQ(9) were monitored by an electrochemical detector (ECD). An excellent linearity (r = 0.999) was observed for CoQ(10) in the concentration range 0.06-2.5 micromol L(-1) in breast milk. The limit of quantitation (LOQ) was 60 nmol L(-1). Coefficients of variations (CVs) for intra-day and inter-day assay precisions were less than 5%. A total of 194 breast milk samples were analyzed for the CoQ(10) concentration; the mean value was 0.32 +/- 0.21 micromol L(-1).     

Determination of coenzyme Q10 by in situ EPR spectroelectrochemistry

A rapid and sensitive method is developed for the determination of coenzyme Q₁₀ (CoQ₁₀) on the basis of the measurement of the radical intermediate (ubisemiquinone) formed during the reduction reaction of CoQ₁₀ at a silver electrode by in situ EPR spectroelectrochemical techniques. At the potential of −0.55 V (versus SCE), the ubisemiquinone is formed and is stable in ethanol+water. Under optimal conditions, it was found that the proposed method provided a linear response over the CoQ₁₀ concentration range 5–100 μmol l⁻¹ with a detection limit of 3 μmol l⁻¹. The relative standard deviation of the results was 7.5% for six successive determinations at 10 μmol l⁻¹ CoQ₁₀. This method is a useful tool for improving the selectivity when other chemicals present in the sample do not interfere in the assay.     

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.     

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 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.     

Simultaneous analysis of ellagic acid and coenzyme Q(10) by derivative spectroscopy and HPLC

Antioxidants are gaining tremendous interest as chemopreventive as well as chemotherapeutic agents. Ellagic acid (EA) is a plant derived compound with very poor solubility in water and very low octanol/water partition coefficient and coenzyme Q₁₀ (CoQ₁₀) is a highly lipophilic compound, which is synthesized in the body and can be derived from food supplements as well. The new insights in the combination therapy are promising a better future in many challenging diseases. Synergism is among the key advantages of combination therapy apart from decreased intensity of unwanted effects of a compound, increased patient compliance and reduction in cost of therapy. EA and CoQ₁₀ supplementation in combination will be beneficial in strengthening the weakened antioxidant defense system in many diseases related to oxidative stress. Here we report first derivative UV spectroscopic and HPLC methods for the simultaneous analysis of these two agents in pharmaceutical preparations. Results obtained indicate that the derivative spectroscopy is as efficient as HPLC method in quantitative analysis. Retention of ellagic acid can be increased using PEG bonded column which is poorly retained on C₁₈ column. PEG column can be used for rapid simultaneous analysis of EA and CoQ₁₀, which are having diverse physicochemical properties.     

Stability of ubiquinol‐10 (reduced form of coenzyme Q10) in human blood

The ratio of ubiquinol‐10 in total coenzyme Q₁₀ (TQ₁₀) in human plasma has been proposed as a useful biomarker of oxidative stress. Since ubiquinol‐10 is easily oxidized in air, it is necessary to perform suitable processing at medical institutions prior to analysis. To establish stable storage conditions for blood to determine the ubiquinol‐10/TQ₁₀ ratios properly, the effects of temperature conditions on the stability of ubiquinol‐10 were studied. Blood samples were collected from nine male Japanese volunteers. Changes in ubiquinol‐10/TQ₁₀ ratios in blood samples were evaluated under three temperature conditions (room temperature, refrigerated and ice‐cooled). Plasma levels of ubiquinol‐10 and ubiquinone‐10 were determined by an HPLC system with electrochemical detection and the ubiquinol‐10/TQ₁₀ ratios were calculated. We found that the ubiquinol‐10/TQ₁₀ ratio was stable up to 8 or 4 h when blood samples were stored in refrigerator or ice‐cold container, respectively, and its decreases during these periods were <1.0%. We conclude that, in order to evaluate ubiquinol‐10/TQ₁₀ ratios, blood samples should be stored in a refrigerator or an ice‐cold container, and processed for plasma separation within 4 h. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

反相高效液相色谱法测定化妆品中辅酶Q10的含量

建立一种测定化妆品中辅酶Q10含量的反相高效液相色谱方法.样品用异丙醇振荡萃取后,经C18固相萃取小柱净化.以Hypersil ODS2(5 μm,150 mm×4.6 mm i.d)作分析柱,以辅酶Q9为内标,异丙醇-甲醇(V(异丙醇)V(甲醇)=23)作流动相,在波长275 nm处进行检测.在0.5～100mg/L范围内,峰高比与质量浓度比呈良好的线性关系,化妆品中辅酶Q1o的检出限为0.76ng.方法的RSD《5%.     

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.     

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.     

A rapid and sensitive LC-MS/MS method for determination of coenzyme Q10 in tobacco (Nicotiana tabacum L.) leaves

A rapid and sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring has been proposed for the analysis of coenzyme Q10 in (CoQ10) tobacco leaves. The method used electrospray ionization with detection in positive ion mode. Sample pretreatment involved ultrasonic extraction of fresh tobacco leaves with anhydrous ethanol for 15 min and followed by extraction of the supernatant with hexane. The separation of CoQ10 was performed on a Symmetry Shield RP18 column with a mixture of acetonitrile and isopropanol (8:7, v/v) containing 0.5% formic acid as mobile phase. Quantification of CoQ10 was performed by the standard addition method. The limit of detection and limit of quantitation of CoQ10 were, respectively, 1.2 ng/mL (S/N = 3) and 4.0 ng/mL (S/N = 10). The relative standard deviations of peak area were 0.91% and 1.21% for intra-day and inter-day, respectively. The recoveries of CoQ10 ranged from 98.2 to 99.3% and the corresponding RSDs were less than 2.4%. Analysis took 5 min, making the method suitable for rapid determination of CoQ10 in tobacco leaves. The proposed method has been successfully applied to the analysis of CoQ10 in the leaves from eight varieties of tobacco.     

Urinary excretion study of coenzyme Q10 in rats by ultra-performance liquid chromatography-mass spectrometry

A method based on ultra-performance liquid chromatography mass spectrometry (UPLC-MS) applying atmospheric pressure chemical ionization in the positive ion mode is developed for the determination of coenzyme Q10 (CoQ10) in rat urine. The assay involves the extraction of crude urine, fast liquid chromatography on a Waters Acquity UPLC BEH C18 column (1.7 microm, 1.0 x 50 mm), and selected ion monitoring detection using mass transition. The calibration range is found to be 0.05-25 microg/mL, with the lower limit of quantitation of 0.05 microg/mL. Intra- and inter-day precision (relative standard deviation) for CoQ10 in rat urine range from 0.7% to 15%, and accuracy expressed in recovery rates in urine is between 83% and 118%. The recovery of this method is found to be between 80% and 95% at three concentrations. The total cumulative recovery of CoQ10 is 1.16 +/- 1.05% (percentage of dose intake, n = 4) from rat urine collected over 30 h after oral administration of the drug. The UPLC-MS method described allows the quick determination of CoQ10 in rat urine with good precision and accuracy. It is suitable for further excretion studies of CoQ10 in animals.     

Pseudorotaxane-like supramolecular complex of coenzyme Q10 with gamma-cyclodextrin formed by solubility method

The purpose of this study is to reveal whether Coenzyme Q10 (CoQ10) forms pseudorotaxane-like supramolecular complex with gamma-cyclodextrin (gamma-CyD). The poorly soluble complex of CoQ10 with gamma-CyD in water was prepared by the solubility method. The X-ray diffraction pattern of the CoQ10/gamma-CyD complex was different from that of the physical mixture, but almost the same as that of polypropylene glycol (PPG)/gamma-CyD polypseudorotaxane. Also, the differential scanning calorimetrical study and FT-IR study demonstrated the interaction between CoQ10 and gamma-CyD in the solid state. The 1H-NMR study and the yield study of the supramolecular complex of CoQ10 with gamma-CyD demonstrated that the stoichiometry was 5 : 1 (gamma-CyD : CoQ10). The dispersion rate of CoQ10 was markedly increased by the formation of the supramolecular complex with gamma-CyD, possibly due to submicron-ordered particle formulation. In fact, CoQ10 was found to form submicron-sized supramolecular particles with gamma-CyD, when prepared by the solubility method. Consequently, the present study showed that CoQ10 forms the pseudorotaxane-like supramolecular complex with gamma-CyD in water.     

Calcium binding and transport by coenzyme Q

Coenzyme Q10 (CoQ10) is one of the essential components of the mitochondrial electron-transport chain (ETC) with the primary function to transfer electrons along and protons across the inner mitochondrial membrane (IMM). The concomitant proton gradient across the IMM is essential for the process of oxidative phosphorylation and consequently ATP production. Cytochrome P450 (CYP450) monoxygenase enzymes are known to induce structural changes in a variety of compounds and are expressed in the IMM. However, it is unknown if CYP450 interacts with CoQ10 and how such an interaction would affect mitochondrial function. Using voltammetry, UV-vis spectrometry, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), fluorescence microscopy and high performance liquid chromatography-mass spectrometry (HPLC-MS), we show that both CoQ10 and its analogue CoQ1, when exposed to CYP450 or alkaline media, undergo structural changes through a complex reaction pathway and form quinone structures with distinct properties. Hereby, one or both methoxy groups at positions 2 and 3 on the quinone ring are replaced by hydroxyl groups in a time-dependent manner. In comparison with the native forms, the electrochemically reduced forms of the new hydroxylated CoQs have higher antioxidative potential and are also now able to bind and transport Ca(2+) across artificial biomimetic membranes. Our results open new perspectives on the physiological importance of CoQ10 and its analogues, not only as electron and proton transporters, but also as potential regulators of mitochondrial Ca(2+) and redox homeostasis.     

Sensitive determination of haloperidol in human serum by radioimmunoassay

An improved procedure for the sensitive determination of the neuroleptic drug, haloperidol, in human serum is described. The method is based on a single hexane extraction procedure and a radioimmunoassay. Antiserum was elicited in guinea pigs immunized with haloperidol hemisuccinate derivative coupled to bovine serum albumin. Any appreciable cross-reactivity was observed neither with known metabolites of haloperidol nor with haloperidol decanoate (KD-136). Some devices in the extraction procedure, e.g. the use of 10 ml of extraction solvent, made it possible to measure haloperidol levels as low as 0.05 ng/ml. The accuracy and reproducibility of the method were shown to be sufficient. The present method can be used to study the pharmacokinetics of haloperidol in the phase 1 study of KD-136. In addition, it is simple enough for use in clinical laboratories that are monitoring haloperidol concentrations in the blood of psychiatric patients.     

Redox state of coenzyme Q10 determines its membrane localization

The interaction between oxidized (ubiquinone-10) and reduced (ubiquinol-10) coenzyme Q 10 with dimyristoylphosphatidylcholine has been examined by differential scanning microcalorimetry, X-ray diffraction, infrared spectroscopy, and (2)H NMR. Microcalorimetry experiments showed that ubiquinol-10 perturbed considerably more the phase transition of the phospholipids than ubiquinone-10, both forms giving rise to a shoulder of the main transition peak at lower temperatures. Small angle X-ray diffraction showed an increase in d-spacing suggesting a thicker membrane in the presence of both ubiquinone-10 and ubiquinol-10, below the phase transition and a remarkable broadening of the peaks indicating a loss of the repetitive pattern of the lipid multilamellar vesicles. Infrared spectroscopy showed an increase in wavenumbers of the maximum of the CH 2 stretching vibration at temperatures below the phase transition, in the presence of ubiquinol-10, indicating an increase in the proportion of gauche isomers in the gel phase, whereas this effect was smaller for ubiquinone-10. A very small effect was observed at temperatures above the phase transition. (2)H NMR spectroscopy of perdeuterated DMPC showed only modest changes in the spectra of the phospholipids occasioned by the presence of coenzyme Q 10. These small changes were reflected, in the presence of ubiquinol-10, by a decrease in resolution indicating that the interaction between coenzyme Q and phospholipids changed the motion of the lipids. The change was also visible in the first spectral moment (M1), which is related with membrane order, which was slightly decreased at temperatures below the phase transition especially with ubiquinol-10. A slight decrease in M 1 values was also observed above the phase transition but only for ubiquinol-10. These results can be interpreted to indicate that most ubiquinone-10 molecules are localized in the center of the bilayer, but a considerable proportion of ubiquinol-10 molecules may span the bilayer interacting more extensively with the phospholipid acyl chains.     

Analysis of pentacyclic triterpenes by LC-MS. A comparative study between APCI and APPI

The analytical performances of three atmospheric-pressure sources, electrospray (ESI), atmospheric-pressure chemical ionization (APCI), and atmospheric-pressure photoionization (APPI), were evaluated for the analysis of pentacyclic triterpenes in liquid chromatography-mass spectrometry (LC-MS). Among these sources, APPI and APCI are particularly well adapted to sensitive analyses of pentacyclic triterpenes by LC-MS. Detection parameters were optimized for both the sources, and the effects of three dopants (toluene, acetone and anisole) on the detection (sensitivity and ion fingerprints in MS spectra) were studied in detail for APPI-MS.The limits of quantification were measured under selected ion monitoring conditions, in the range of 0.005-0.015 mg l(-1) and 0.002-0.84 mg l(-1) in APPI and APCI, respectively, depending on the studied pentacyclic triterpene. Overall, APPI was found more sensitive than APCI in positive ion mode, whereas APCI shows the greatest sensitivity for acidic triterpenes in negative ion mode.Following this study, the developed LC-MS method was used for the characterization of pentacyclic triterpenes in three plant extracts. High amounts of betulinic acid, betulinic aldehyde and betulinic aldehyde acetate were observed in plane bark. The main component of birch bark is betulin and extracts of okoume resin exhibit high amounts of alpha- and beta-amyrin.