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.     

Determination of diclofenac in plasma by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitative determination of diclofenac potassium in plasma was developed. Naproxen was used as the internal standard. The drug and internal standard were isolated from plasma by extraction with dichloromethane and 2 M hydrochloric acid. Chromatographic separation was performed on a C18 column with methanol-water (68:32, v/v) adjusted to pH 3.2 with phosphoric acid as mobile phase. The oxidation potential for detection was established by constructing a voltammogram for diclofenac. The quantification limit for diclofenac in plasma was 5 ng mL(-1). Linearity of the method was confirmed in the range 5-2000 ng mL(-1), correlation coefficient 0.9998. Within-day relative standard deviations (RSDs) ranged from 0.66 to 14.00% and between-day RSDs from 0.59 to 15.78%. The method was successfully applied for the determination of pharmacokinetic parameters after ingestion of a 50 mg dose of diclofenac. Studies were performed on 18 healthy volunteers of both sexes.     

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%.     

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.     

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 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.     

辅酶Q10的合成方法研究进展

介绍了采用侧链直接引入法、侧链延长法和母体与侧链同时增碳法合成辅酶Q10的研究进展。参考文献18篇。     

血浆辅酶Q10的高效液相色谱快速测定

建立了一种简单快速测定血浆辅酶Q10(CoQ10)的反相高效液相色谱方法.血浆经正丙醇萃取,上层清液直接进样分析.色谱柱为Hypersil ODS2 5μm,150 mm ×4.6 mm i.d,以异丙醇-甲醇(体积比19)作流动相,275 nm作检测波长,外标法定量.在0.05～20 mg/L范围内,峰高与质量浓度呈良好的线性关系(r=0.999 8),血浆中辅酶Q10的检出限为0.03 mg/L(S/N=3).该方法简单、快速、精密度高(RSD＜5%),适宜于血浆辅酶Q10含量的检测.     

高效液相色谱法测定奶粉和液态奶中的三聚氰胺

建立了奶粉和液态奶中三聚氰胺的高效液相色谱（HPLC）检测方法。经阳离子交换固相萃取柱净化后的样品采用HPLC测定。优化的色谱条件：C18柱（4.6 mm ×200 mm,5 μm）,流动相为乙腈-0.01 mol/L庚烷磺酸钠（pH 3.3）（体积比为10∶90）,流速为1.0 mL/min,检测波长为236 nm,柱温为40 ℃,进样量为20 μL。方法的线性范围为1~500 mg/L,检出限为0.2 mg/kg （S/N=3）,定量限为1 mg/kg （S/N=15）,回收率为81.4%~83.7%,相对标准偏差为3.3%~8.5%（n=6）。     

Fabricating hierarchical ZSM-5 to induct long chain antioxidant coenzyme Q10 for biomedical application

The study investigates the hydrophobic antioxidant Coenzyme Q10 (CoQ10) adsorption over industrially relevant hierarchical ZSM-5 for potential therapeutic applications. The designing of hierarchical nanocomposites was optimized with two different NaOH/CTAB ratios (0.18 and 0.62) over ZSM-5 (SiO₂/Al₂O₃ = 22–27) of different crystal sizes (0.5 µm, 2.0 µm and 3.0 µm) and different SiO₂/Al₂O₃ ratios (23, 80, 280 and 1500) through top-down approach. Hydrothermal stability of samples was studied using 100% water steam at 700 °C and 750 °C for 2 h, respectively. Reassembling nano ZSM-5 into hierarchical composite was found to exhibit ordered/disordered hexagonal mesophase depending on alkaline treated ZSM-5 crystal sizes and silica to alumina ratios. The phase changes were analyzed using different textural characterizations such as X-ray diffraction, BET surface area, ammonia desorption technique, ²⁷Al MAS NMR, thermogravimetric analysis and transmission electron microscope. 21 different nanoformulations were screened for CoQ10 adsorption and subsequently selected samples were applied for curcumin and ascorbic acid adsorption. Hierarchical ZSM-5 with SiO₂/Al₂O₃ ratio 80 (ZSM-5-80 (0.62)) was found to be steam stable and optimum with highest Q10 adsorption (55%). The adsorption influence over different crystal size and silica to alumina ratios, clearly showed the dependency over synergistic action of textural characteristics such as external surface area, pore volume, and weak acidity. The structured nanosupports with pore sizes between 3 and 4.0 nm were found to exhibit highest CoQ10 adsorption.     

Determination of the endocannabinoid anandamide in human plasma by high-performance liquid chromatography

Anandamide (N-arachidonylethanolamine) is an endogenous cannabinoid receptor ligand that has been implicated in various physiological and pathophysiological functions. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with fluorometric detection was validated and applied for the analysis of anandamide in human plasma. Following derivatization with the fluorogenic reagent 4-(N,N-dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methyl-amino)-2,1,3-benzoxadiazole (DBD-COCl), the analyte was separated using an acetonitrile-water gradient at a flow rate of 0.8 mL/min, and spectrophotometric detection at 560 nm with an excitation wavelength of 450 nm. The retention times for anandamide and R+-methanandamide (internal standard) were 27.1 and 30.7 min, respectively. The validated quantification range was 1-15 ng/mL. The developed procedure was applied to determine anandamide levels in human plasma following a 24 h incubation of human whole blood at 37 degrees C in the presence or absence of phenylmethylsulfonyl fluoride, an inhibitor of the anandamide-degrading enzyme fatty acid amide hydrolase. Anandamide levels determined under both conditions were within the validated concentration range with anandamide levels being 2.3-fold higher in plasma from PMSF-treated blood.     

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.     

Determination of nifedipine in human plasma by high performance liquid chromatography using column-switching technique

A highly sensitive reversed-phase HPLC method has been developed for the determination of nifedipine in human plasma with electrochemical detection. A liquid-liquid extraction procedure is used in sample preparation with an average extraction recovery of 75%. Removing the highly lipophilic plasma components using a special column switching technique reduced the duration of the HPLC measurement from 30 to 9 min. The method is applicable for the pharmacokinetic characterization and bioavailability study of a sustained-release (retard) formulation of nifedipine and for human drug monitoring as it is indicated by the validation of the analysis method. The assay gave a linear response over the concentration range 2.5–50 ng/ml. All the validation parameters are within the internationally required limits.     

Investigation of Coenzyme Q10 by Voltammetry

A simple, accurate and rapid voltammetric method has been developed for the quantitative determination of coenzyme Q₁₀. Studies with direct current voltammetry were carried out using a glassy carbon electrode (GCE) in a phosphate buffer solution (pH 6.86). A well-defined oxidation peak of CoQ₁₀ was obtained at -0.600 V vs Ag/AgCl. The magnitude of the oxidation peak current has been found to be related to the concentration of the coenzyme over the range of (2.010^-5 to 2.010^-4 M) (r = 0,991). Antioxidant activity of CoQ₁₀ was investigated.     

Determination of polyamines by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the determination of polyamines (PA) by high-performance liquid chromatography with chemiluminescence detection. It is based on the unsaturated complex of PA with Cu(II) which had a strong catalytic effect on the luminol-H₂O₂ chemiluminescence reaction. The separation of PA was carried out on a reveres phase C18 column using methanol/water (25/75, v/v) as a mobile phase. The method was applied to the analysis of putrescine and the total amount of spermine and spermidine in apple leaves and strawberry fruit. The results indicated that the method is practical and useful.     

高效液相色谱法检测液态奶中的苯酚类和水杨酸苯胺类抗蠕虫药

采用高效液相色谱法建立了硝碘酚腈、氯羟柳胺、氯氰碘柳胺、碘醚柳胺在液态奶中的多残留检测方法。取5 g样品,用含1%(v/v)三乙胺的乙腈提取,经MAX柱净化。以乙腈和0.02 mol/L乙酸铵水溶液(pH 4.0)作为流动相,经C18反相色谱柱分离后用紫外检测器检测,外标法定量。结果表明:空白加标奶样中4种药物在5~500 μg/kg范围内均呈现良好的线性关系,相关系数均大于0.99。空白加标奶样品的检出限(LOD)为3 μg/kg,定量限(LOQ)为5 μg/kg。硝碘酚腈、氯羟柳胺、氯氰碘柳胺、碘醚柳胺在1/2最高残留限量(MRL)、1倍MRL、2倍MRL添加水平下的平均回收率和相对标准偏差分别是92.20%~96.13%和5.55%~16.30%; 87.40%~94.74%和5.40%~12.21%; 86.97%~91.09%和2.67%~8.17%; 77.86%~95.36%和5.02%~13.15%。表明该检测方法简单,灵敏,适用于液态奶中水杨酸苯胺类多残留的定量分析检测。     

高效液相色谱法测定硫酸沙丁胺醇

利用高效液相色谱仪-电子捕获检测器(HPLC-ECD)测定了市售片剂硫酸沙丁胺醇的含量.结果表明,利用HPLC-ECD技术测定硫酸沙丁胺醇的线性范围为0.944~33.8mg·L-1(r=0.999 6);回收率为99.0%~107.8%(RSD≤4.6%).该法可用于测定片剂的硫酸沙丁胺含量.     

Determination of coenzyme Q by non-aqueous reversed- phase liquid chromatography.

A non-aqueous reversed-phase liquid chromatographic method for determination of coenzyme Q10 in pharmaceutical formulations has been developed. The reversed-phase system provides better reproducibility and better selectivity for the separation of coenzyme Q10 analogues and degradation products than studied normal-phase systems. Furthermore, the non-aqueous mobile phase showed a very good solubility and provides a greater variety of work-up solvents for the lipophilic formulations than aqueous mobile phases. Validation studies showed detector response linearity over a concentration range of 0.2-100 micrograms/ml. The lower limit of detection was 2 ng on-column. The intra-assay precision (relative standard deviation) for a soybean oil formulation was 2.0% (n = 11).     

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.