An efficient high-speed countercurrent chromatography method for preparative isolation of highly potent anti-cancer compound antroquinonol from Antrodia camphorata after experimental design optimized extraction.

To avoid irreversible stationary phase adsorption and tedious and time-consuming separation steps, high-speed countercurrent chromatography was employed for the preparative separation of anti-tumor compound antroquinonol from solid fermentation culture of Antrodia camphorata for the first time. A Box-Behnken experimental design, based on three parameters including liquid-to-solid ratio, extraction time, and extraction temperature, was applied to optimize the ultrasonic extraction procedure. The optimal extraction condition was set as follows: liquid-to-solid ratio: 49.57:1; extraction time: 55.76 min; extraction temperature was arranged as 44.21°C. Meanwhile, an optimized solvent system containing petroleum ether, ethyl acetate, methanol, and water (4:1:4:1, v/v/v/v) was selected for the preparative separation of antroquinonol at a flow rate of 2.0 mL/min. The yield of isolated antroquinonol was determined to be 6.0 mg from 0.67 g of ethyl acetate extracts. The isolated antroquinonol was elucidated by ultra-high-performance liquid chromatography-tandem mass spectrometry, and NMR spectroscopy, and by comparison with literature data. The purity of isolated antroquinonol was determined to be 97.12%. This study confirmed that high-speed countercurrent chromatography was powerful and cost-effective for the preparative separation of the high-potently anti-tumor compound antroquinonol from solid fermentation culture of A. camphorata.

4-Acetylantrocamol LT3, a New Ubiquinone from Antrodia cinnamomea, Inhibits Hepatocellular Carcinoma HepG2 Cell Growth by Targeting YAP/TAZ, mTOR, and WNT/ß-Catenin Signaling.

4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from the mycelium of Antrodia cinnamomea (Polyporaceae), has been recently shown to possess anticancer activity. However, the detailed mechanisms of such action remain unclear. In this study, the molecular mechanisms of 4AALT3 on hepatocellular carcinoma cells (HCC) were investigated. Human hepatocellular carcinoma cell line HepG2 cells were treated with concentrations of 4AALT3. Cell viability, colony formation, and the underlying mechanisms were then analyzed by CCK-8, colony formation, qPCR, and Western blotting assays. We found that 4AALT3 significantly decreased cell viability and colony formation in a dose-dependent manner. Accordingly, 4AALT3 significantly decreased protein levels of cyclin B, E1, D1, and D3, thereby facilitating cell cycle arrest. In addition, 4AALT3 significantly suppressed the nuclear localization of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), mammalian target of rapamycin (mTOR), and WNT/[Formula: see text]-catenin signaling pathways, all of which are well-known signaling pathways that contribute to the malignant properties of HCC. These effects are associated with activation of 5' AMP-activated protein kinase (AMPK) and autophagy. Our findings indicate that 4AALT3 exerts inhibitory effects on HepG2 cell growth via multiple signaling pathways and may be a potential agent for HCC therapy.

Enhancement of antroquinonol and antrodin C productions via in situ extractive fermentation of Antrodia camphorata S-29.

This study describes the application of in situ extractive fermentation (ISEF) to increase the yields of antroquinonol (AQ) and antrodin C (AC) from Antrodia camphorata S-29. In initial screening experiments, nine solvents were tested to identify the most suitable extractant for the in situ extraction of AQ and AC. These solvents included n-tetradecane, n-dodecane, n-decane, heavy paraffin, light paraffin, oleyl alcohol, oleic acid, butyl oleate, and isopropyl myristate. Of these, oleic acid was the most suitable solvent for the in situ extraction of AQ and AC. The use of oleic acid as an in situ extractant significantly improved AQ and AC productions, which were approximately 5-fold and 8-fold that of the control, respectively. The recovered oleic acid was treated with a silica gel solid-phase extraction column, which was able to rapidly adsorb the bioactive metabolites. The separated solvent hardly contained fermentation products and could be directly reused in ISEF. AQ and AC were obtained with purities of over 75% by silica gel column chromatography. The recoveries of AQ and AC reached 70.7 ± 0.8% and 81.5 ± 1.2%, respectively.

Miniaturized imprinted solid phase extraction to the selective analysis of Coenzyme Q10 in urine.

Coenzyme Q10 (CoQ10) is an important cofactor in the mitochondrial respiratory chain and a potent endogenous antioxidant. CoQ10 deficiency is currently associated with numerous diseases like mitochondrial and neurodegenerative pathologies, in which the earliest diagnosis and treatment with CoQ10 supplementation becomes paramount for patient's treatment. Consequently, the determination of CoQ10 levels in different biological matrices positions as a fundamental tool. Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for CoQ10 analysis. However, it poses an analytical challenge, as it generally requires a complex sample preparation, with multiple steps. In this work we developed and validated a molecularly imprinted polymer solid phase extraction (MIP-SPE) followed by a HPLC-MS/MS method for the analysis of CoQ10 in urine. The MIP-SPE method developed is simple and fast compared to previously traditional reported methods, with reduced processing time, improved sample cleaning and excellent recovery values, along with its inherent high selectivity. The developed chromatographic method was validated according to FDA guidelines, and demonstrated to be suitable for the analysis of CoQ10 in urine samples with LOQ and LOD values of 0.6 ng/mL and 0.2 ng/mL of CoQ10 in urine respectively. Recovery values at three concentration levels were higher than 90.0%.The proposed method is amenable to be applied in pediatric patients due to the low sample requirement and useful for diagnosis and post-treatment control.

Pharmacological activities of antroquinonol- Mini review.

Antrodia camphorata is an expensive mushroom that grows on the inner cavity of an endangered native tree of Taiwan namely Cinnamomum kanehirai Hayata. It is used as a traditional medicine in Taiwan and has several health benefits including free radical scavenging, anti-inflammatory, antimicrobial, hepatoprotective, neuroprotective, antidiabetic, and free radical-induced DNA damage protecting activities. Antroquinonol is a tetrahydro ubiquinone derivative found predominately in the mycelium of Antrodia camphorata, and is characterized by numerous biological and pharmacological activities. Several studies have revealed potential anticancer effects of antroquinonol in various carcinogenic models. Moreover, a phase II clinical trial is ongoing in the US and Taiwan to treat the lung cancer patients with this active compound. The present review aims at depicting a detailed view of the synthetic procedures of antroquinonol as well as deciphering its potential health benefits with a special emphasis on anticancer properties.

Identification of Ubiquinones in Honey: A New View on Their Potential Contribution to Honey's Antioxidant State.

Honey is composed of macromolecules arranged into multicomponent colloidal particles dispersed in a supersaturated sugar solution. The core part of colloidal particles in honey is made up of high-molecular weight protein-polyphenol complexes. We designed a multi-step extraction process to gain better insight into the phenolic compounds strongly bound to proteins in honey. Honeys were sequentially extracted by solvents of reduced polarities and the extraction process was monitored by LC-ESI-MS/MS. Unexpectedly, the results revealed ubiquinone-like compounds that partitioned to both, soluble supernatants and protein-bound insoluble residues from which they were released after the pronase-digestion of proteins. The accurate mass measurement and MS/MS fragmentation patterns using UPHLC-MS/MS coupled to quadrupole orbitrap confirmed their identification as ubiquinones. Distribution of ubiquinone-bound proteins was further investigated by the fractionation of honey protein-polyphenol complexes by size-exclusion chromatography followed by LC-ESI-MS analysis. Mass spectra revealed the presence of ubiquinones (UQs) in fractions of high polyphenol to protein ratio. The dominant mass peaks observed in these fractions were identified as UQ-3, UQ-5, and UQ-7. Since the quinone group of UQs is involved in redox reaction, we discuss the possibility that UQs may contribute to the antioxidant/proxidant activity of these complexes.

Meroterpenoids from a Medicinal Fungus Antrodia cinnamomea.

Antrodia cinnamomea, a medicinal fungus indigenous to Taiwan, has been shown to exhibit a broad spectrum of bioactivities for the treatments of alcoholic intoxication, diarrhea, abdominal pain, and fatigue, and a number of active principles have been identified. Among the bioactive entities, clinical trials of antroquinonol and 4-acetyl antroquinonol B are being carried out for curing cancer, hypercholesterolemia, and hyperlipidemia. The total synthesis of antroquinonol has been achieved; however, investigating the structure-activity relationship of this class of compounds remained difficult due to the lack of available analogues. Twenty antroquinonols isolated from A. cinnamomea IFS006 are reported herein. Their structures were elucidated using spectral analysis and by comparison with literature values. Of these, 11 antroquinonol analogues, namely, antroquinonols N-X (1-11), were previously unreported. The growth inhibitory activity of all the antroquinonol analogues was evaluated against human A549 and PC-3 cancer cell lines, and antroquinonol A exhibited the most potent activity, with GI50 values of 5.7 ± 0.2 and 13.5 ± 0.2 µM, respectively. Antroquinonols V (9) and W (10) also showed growth inhibitory activity against A549 cells with GI50 values of 8.2 ± 0.8 and 7.1 ± 2.1 µM, respectively, compared to 5-fluorouracil (GI50 = 4.2 ± 0.2 µM).

The association between coenzyme Q10 concentrations in follicular fluid with embryo morphokinetics and pregnancy rate in assisted reproductive techniques.

PURPOSE: This study seeks to evaluate the association between follicular fluid (FF) coenzyme Q10 (CoQ10) levels, embryo morphokinetics, and pregnancy rate. METHODS: Sixty infertile patients who underwent intracytoplasmic sperm injection (ICSI) cycles were included in the study. For each patient, CoQ10 level of the follicular fluid was measured by high-performance liquid chromatography system. After the ICSI of each oocyte, the relationship between the level of CoQ10 content of each follicular fluid, the subsequent embryo quality, and embryo morphokinetics was investigated. The relationship between the level of CoQ10 content of each follicle and optimal time-lapse parameters for the embryos of these follicles including t5, s2, and cc2 was also analyzed. The embryos were further classified into four categories, namely, grades A, B, C, and D, according to morphokinetic parameters using t5-t2 and t5-t3 (cc3). Each follicular fluid analysis was performed for a single oocyte of a single embryo which was transferred to the patients. Additionally, follicular fluid CoQ10 levels and pregnancy rates were evaluated. RESULTS: Follicular fluid CoQ10 levels were significantly higher in grades A and B than grades C and D embryos (p < 0.05). The concentration of CoQ10 levels was significantly higher in the pregnant group (p < 0.05). There was no significant correlation between optimal t5 and s2 morphokinetic parameters and CoQ10 levels. However, CoQ10 levels were significantly higher in follicular fluid of embryos which had optimal cc2 (p < 0.05). CONCLUSION: High follicular fluid CoQ10 level is associated with optimal embryo morphokinetic parameters and higher pregnancy rates.

Induction of antroquinonol production by addition of hydrogen peroxide in the fermentation of Antrodia camphorata S-29.

BACKGROUND: Antroquinonol have significantly anti-tumour effects on various cancer cells. There is still lack of reports on regulation of environmental factors on antroquinonol production by Antrodia camphorata. RESULTS: An effective submerged fermentation method was employed to induce antroquinonol with adding H2 O2 . The production of antroquinonol was 57.81 mg L-1 after fermentation for 10 days when adding 25 mmol L-1 H2 O2 at day 4 of the fermentation process. Then, antroquinonol was further increased to 80.10 mg L-1 with cell productivity of 14.94 mg g-1 dry mycelium when the feeding rate of H2 O2 was adjusted to 0.2 mmol L-1 h-1 in the 7 L fermentation bioreactor. After inhibiting the generation of reactive oxygen species with the inhibitor diphenyleneiodoium, the synthesis of antroquinonol from A. camphorata was significantly reduced, and the yield was only 3.3 mg L-1 . CONCLUSION: The results demonstrated that addition of H2 O2 was a very effective strategy to induce and regulate the synthesis of antroquinonol in submerged fermentation. Reactive oxygen species generated by H2 O2 during fermentation caused oxidative stress, which induced the synthesis of antroquinonol and other chemical compounds. Moreover, it is very beneficial process to improve production and diversity of the active compounds during liquid fermentation of A. camphorata mycelium. © 2016 Society of Chemical Industry.

Synthesis and characterization of molecularly imprinted polymer nanoparticles for coenzyme Q10 dispersive micro solid phase extraction.

Molecularly imprinted polymer nanoparticles (MIPNPs) with the ability to recognize coenzyme Q10 (CoQ10) were synthesised in order to be employed as sorbent in a dispersive micro-solid phase extraction (DMSPE) for the determination of CoQ10 in a liver extract. CoQ10 is a redox-active, lipophilic substance integrated in the mitochondrial respiratory chain which acts as an electron carrier, shuttling electrons from complex I (NADH-ubiquinone oxidoreductase) and II (succinate-ubiquinone oxidoreductase) to complex III (ubiquinol-cytochrome c reductase), for the production of cellular energy. The MIPNPs were synthesised by precipitation polymerization using coenzyme Q0 as the dummy template, methacrylic acid as the functional monomer, an acetonitrile: water mixture as the porogen, ethylene glycol dimethacrylate as the crosslinker and potassium persulfate as initiator. The nanoparticles were characterized by microscopy, capillary electrophoresis, dynamic light scattering, N2 adsorption-desorption isotherms, and infrared spectroscopy. The MIPNPs demonstrated the presence of selective cavities complementary to the quinone nucleus of CoQ10, leading to a specific recognition of CoQ10 compared with related compounds. In the liver extract the relative CoQ10 peak area (CoQ10 area/total peak area) increased from 4.6% to 25.4% after the DMSPE procedure. The recovery percentage of CoQ10 from the liver matrix was between 70.5% and 83.7% quantified against CoQ10 standard processed under the same conditions. The DMSPE procedure allows the elution of almost all the CoQ10 retained (99.4%) in a small volume (200µL), allowing the sample to be concentrated 2.5 times (LOD: 1.1µgg(-1) and LOQ: 3.7µgg(-1) of tissue). The resulted clean up of the sample, the improvement in peak shape and baseline and the reduction of interferences, evidence that the MIPNPs could potentially be applied as sorbent in a DMSPE with satisfactory results and with a minimum amount of sorbent (1mg).

Coenzyme Q10 analytical determination in biological matrices and pharmaceuticals.

In recent years, the analytical determination of coenzyme Q10 (CoQ10) has gained importance in clinical diagnosis and in pharmaceutical quality control. CoQ10 is an important cofactor in the mitochondrial respiratory chain and a potent endogenous antioxidant. CoQ10 deficiency is often associated with numerous diseases and patients with these conditions may benefit from administration of supplements of CoQ10. In this regard, it has been observed that the best benefits are obtained when CoQ10 deficiency is diagnosed and treated early. Therefore, it is of great value to develop analytical methods for the detection and quantification of CoQ10 in this type of disease. The methods above mentioned should be simple enough to be used in routine clinical laboratories as well as in quality control of pharmaceutical formulations containing CoQ10. Here, we discuss the advantages and disadvantages of different methods of CoQ10 analysis.

Anti-cancer agents derived from solid-state fermented Antrodia camphorata mycelium.

Three new ubiquinone derivatives, antrocamol LT1, antrocamol LT2, and antrocamol LT3, along with two known compounds, were isolated from Antrodia camphorata (Polyporaceae) mycelium. The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analyses. These ubiquinones exhibited selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 1.79µΜ.

Acinetobacter apis sp. nov., isolated from the intestinal tract of a honey bee, Apis mellifera.

A novel Gram-negative, obligate aerobic, non-motile, and both coccobacillus- and bacillus-shaped bacterium, designated strain HYN18(T), was isolated from the intestinal tract of a honey bee (Apis mellifera). The isolate was oxidase-negative and catalase-positive. Strain HYN18(T) showed optimum growth at 25°C, pH 6-7, and in the presence of 1% (w/v) NaCl in trypticase soy broth medium. The isolate was negative for hydrolyses of starch, casein, gelatin and urea, indole production from tryptone and hemolysis on sheep blood agar. A phylogenetic analysis based on the 16S rRNA gene and rpoB gene sequence showed that strain HYN18(T) was most closely related to Acinetobacter nectaris SAP 763.2(T) and A. boissieri SAP 284.1(T) with 98.3% and 98.1% similarity (16S rRNA gene), respectively, and 84.4% similarity with Acinetobacter nectaris SAP 763.2(T) (rpoB gene). The major cellular fatty acids were summed features 3 (comprising C16:1ω7c /C16:1ω6c ), C12:0 and C16:0. The main isoprenoid quinone was ubiquinone-9 (Q-9). The polar lipids of strain HYN18(T) were phosphatidylethanolamine, three unidentified lipids, an unidentified phospholipid and an unidentified glycolipid. The DNA G+C content was 40.6 mol%. DNA-DNA hybridization experiments indicated less than 33 ± 10% relatedness to the closest phylogenetic species, Acinetobacter nectaris SAP 763.2(T). Thus, the phenotypic, phylogenetic and genotypic analyses indicate that strain HYN18(T) is a novel species within the genus Acinetobacter, for which the name Acinetobacter apis is proposed. The type strain is HYN18(T) (=KACC 16906(T) =JCM 18575(T)).

Efficient preparation of pseudoalteromone A from marine Pseudoalteromonas rubra QD1-2 by combination of response surface methodology and high-speed counter-current chromatography: a comparison with high-performance liquid chromatography.

Pseudoalteromone A (PA) is a cytotoxic and anti-inflammatory ubiquinone discovered recently from a marine bacterium Pseudoalteromonas sp. CGH2XX. In order to meet its sample supply for further in vivo pharmacological investigation, an efficient method was developed for the preparation of PA by combination of response surface methodology (RSM) and high-speed counter-current chromatography (HSCCC) from marine bacterium P. rubra QD1-2. First, optimization of culture conditions was studied by the RSM to enhance PA production. The results indicated that the optimal cultivation condition was peptone (2.21 g/l), yeast extract (3.125 g/l), glucose (0.125 g/l), KBr (0.02 g/l), inoculum size (6.5 %), medium volume (595 ml), initial pH value (7.0), temperature (28 °C). Under the optimized fermentation condition, PA production was 1.04 mg/l with 14.8-fold increase comparing to 0.07 mg/l under original standard fermentation condition. The PA production was further investigated using a 14-l jar fermenter. Compared to the flask culture, P. rubra QD1-2 offered 45 % increase of PA production at 1.51 mg/l. Then, a rapid and efficient method for the separation and purification of PA from crude culture extract was developed using HSCCC. The two-phase solvent system used for HSCCC separation was composed of n-hexane-ethyl acetate-methanol-water (5:5:9:5, v/v/v/v). The isolation was accomplished within 100 min, and the purity of PA was over 95 %. The recovery of the process was 93 %.

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.

Simultaneous analysis of circulating 25-hydroxy-vitamin D3, 25-hydroxy-vitamin D2, retinol, tocopherols, carotenoids, and oxidized and reduced coenzyme Q10 by high performance liquid chromatography with photo diode-array detection using C18 and C30 columns alone or in combination.

Circulating lipid-phase micronutrients (LPM) such as 25-hydroxylated D vitamers, retinol, tocopherols, carotenoids including their isomers, and coenzyme Q10 play important roles in health maintenance and disease prevention and can serve as useful biomarkers. We developed fast, affordable, and accurate HPLC assays that simultaneously measured all above LPM in a single run using UV/VIS detection at 265nm, 295nm, and 480nm with (1) a C18 column alone; (2) a C30 column alone; or (3) each of these columns connected in series. The C18 column alone could separate all major LPM of interest in less than 17min but insufficiently resolved the lycopene isomers, the 25-hydroxylated D vitamers, lutein from zeaxanthin and ß- from γ-tocopherol. The C30 column alone separated all LPM of interest including many isomeric analytes but failed to resolve the Q10 compounds, which co-eluted with carotenoids. Connecting the C18 and C30 columns in series with a detector after the C30 column and a pressure resistant detector between the columns resulted in ideal resolution and accurate quantitation of all LPM of interest but required software capable of processing the acquired data from both detectors. Connecting the C18 and C30 columns in series with exclusively one detector after the C30 column resulted in carotenoid-Q10 interferences, however, this was remedied by heart-cutting 2D-LC with a 6-port valve between the columns, which resolved all analytes in 42min. Faster run times led to some analytes not being resolved. Many variations of these methods are possible to meet the needs of individual requirements while minimizing sample material and turn-around-times.

The rice bacterial pathogen Xanthomonas oryzae pv. oryzae produces 3-hydroxybenzoic acid and 4-hydroxybenzoic acid via XanB2 for use in xanthomonadin, ubiquinone, and exopolysaccharide biosynthesis.

Xanthomonas oryzae pv. oryzae, the causal agent of rice bacterial blight, produces membrane-bound yellow pigments, referred to as xanthomonadins. Xanthomonadins protect the pathogen from photodamage and host-induced perioxidation damage. They are also required for epiphytic survival and successful host plant infection. Here, we show that XanB2 encoded by PXO_3739 plays a key role in xanthomonadin and coenzyme Q8 biosynthesis in X. oryzae pv. oryzae PXO99A. A xanB2 deletion mutant exhibits a pleiotropic phenotype, including xanthomonadin deficiency, producing less exopolysaccharide (EPS), lower viability and H2O2 resistance, and lower virulence. We further demonstrate that X. oryzae pv. oryzae produces 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic acid (4-HBA) via XanB2. 3-HBA is associated with xanthomonadin biosynthesis while 4-HBA is mainly used as a precursor for coenzyme Q (CoQ)8 biosynthesis. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis in PXO99A. These findings suggest that the roles of XanB2 in PXO99A are generally consistent with those in X. campestris pv. campestris. The present study also demonstrated that X. oryzae pv. oryzae PXO99A has evolved several specific features in 3-HBA and 4-HBA signaling. First, our results showed that PXO99A produces less 3-HBA and 4-HBA than X. campestris pv. campestris and this is partially due to a degenerated 4-HBA efflux pump. Second, PXO99A has evolved unique xanthomonadin induction patterns via 3-HBA and 4-HBA. Third, our results showed that 3-HBA or 4-HBA positively regulates the expression of gum cluster to promote EPS production in PXO99A. Taken together, the results of this study indicate that XanB2 is a key metabolic enzyme linking xanthomonadin, CoQ, and EPS biosynthesis, which are collectively essential for X. oryzae pv. oryzae pathogenesis.

Determination of coenzyme Q10 tissue status via high-performance liquid chromatography with electrochemical detection in swine tissues (Sus scrofa domestica).

Swine tissues were used as surrogates for human tissues with coenzyme Q10 (CoQ10) as the primary endogenous quinoid to establish a reliable method for the analysis of total CoQ10 concentration and redox status using the reduced and oxidized forms of CoQ9 as internal standards. Specimens of frozen swine tissues were disrupted by bead milling using 2-propanol as the homogenization medium supplemented with the internal standards. After hexane extraction, CoQ10 was analyzed via high-performance liquid chromatography with electrochemical detection. The method is linear (12-60 mg fresh muscle tissue/sample), sensitive (~200 pmol CoQ10/sample), and reproducible (coefficients of variation of 6.0 and 3.2% for total CoQ10 and 2.4 and 3.2% for the redox status of within-day and day-to-day precision, respectively), with analytic recoveries for ubiquinone-10, ubihydroquinone-10, and total Q10 of 91, 104, and 94%, respectively. The concentration and redox status were stable for at least 3 months at -84°C. The total CoQ10 concentrations (pmol/mg fresh tissue) in swine tissues were as follows: lung (17.4±1.42), skeletal muscle (26.7±2.57), brain (40.7±4.02), liver (62.1±31.0), kidney (111.7±37.08), and heart muscle (149.1±36.78). Significant tissue-specific variations were also found for the redox status (% oxidation of total): swine liver (~28), lung (~36), kidney (~37), heart muscle (~57), skeletal muscle (~61), and brain (~67).

Analytical problems with the determination of coenzyme Q10 in biological samples.

The article discusses analytical problems related to the determination of coenzyme Q10 in biological samples. The assaying of coenzyme Q10 in complex samples, such as plasma, tissues, or food items requires meticulous sample preparation prior to final quantification. The process typically consists of the following steps: deproteinization, extraction, and ultimately reduction of extract volumes. At times drying under a gentle stream of neutral gas is applied. In the case of solid samples, a careful homogenization is also required. Each step of the sample preparation process can be a source of analytical errors that may lead to inaccurate results. The main aim of this work is to point to sources of analytical errors in the preparation process and their relation to physicochemical properties of coenzyme Q10. The article also discusses ways of avoiding and reducing the errors.

Determination of a method for extraction of coenzyme Q10 in human plasma: optimization of the use of surfactants and other variables.

OBJECTIVE: To establish a routine for the extraction of the total levels of CoQ10 in human plasma through the Ultra High Performance Liquid Chromatography (UHPLC). METHODS: Two extraction protocols were tested: a) methanol: hexane and b) 1-propanol. The following parameters were analyzed: extraction temperature (19ºC and 4ºC), extraction tubes (glass and polypropylene), and surfactants (SDS, Triton X-100, Tween-20) at different concentrations, i.e., 1%, 3%, 5% and 10%. RESULTS: The results showed that the method of extraction of CoQ10 in a sample of human plasma at 4ºC, using solvents methanol: hexane (85:15, v/v) in the presence of surfactant Tween-20 at 3% and polypropylene tubes showed better efficiency and reproducibility when compared to the method with 1-propanol. CONCLUSION: By the analyses performed, it was possible to observe that the addition of the surfactant Tween-20 promoted an increase in the recovery of CoQ10 by the methanol:hexane extraction method. This method showed good reproducibility, with a low coefficient of variation and high sensitivity, since CoQ10 was detected in samples of plasma of a control individual using a UV-type detector. The use of UHPLC equipment allowed a total analysis with total run time of 3.5 minutes, enabling the rapid achievement of results, considered mandatory for laboratory routines.