Patent Application: US-9034702-A

Abstract:
this invention relates to an apparatus and method for the simultaneous and rapid determination of coq 10 and coq 10 h 2 concentrations in human samples using hplc - ec . the electrochemical reactions are monitored at electrodes that measure the current produced by the reduction of the hydroquinone group of coq 10 or by the oxidation of the hydroquinol group of coq 10 h 2 .

Description:
this invention relates to an apparatus for electrochemically analyzing an aqueous sample solution comprising electrochemically reversible materials in solution . in its broadest sense , electrochemically reversible materials are those which can be oxidized and / or reduced by the transfer of electrons . there may also be the transfer of protons ( h + ) as well in order to form a chemically neutral species as the final product of the electron transfer reaction . generally , the apparatus comprises an hplc instrument , which includes a solvent delivery module ( pump ), an injector , inline filters , guard column and an analytical hplc column for separation of the materials . in addition , immediately preceding the analytical column is at least one coulometric guard cell that can be used to oxidize or reduce the electrochemically active species . following the analytical column is a series of in - line electrochemical cells for reduction and / or oxidation as well as analysis of the electrochemically active species . the coulometric guard cell preceding the analytical column and the series of post - column in - line electrochemical cells define collectively at least one flow channel for the sample solution . coq 9 and coq 10 are obtained from sigma , st . louis , mo . hplc grade methanol , ethanol , 1 - propanol , 2 - propanol and hexane are obtained from fisher , and used without further purification . sodium acetate and analytical grade acetic acid are also obtained from fisher . the preparation work is carried out under a dim light to avoid photochemical decomposition of coq 10 and coq 9 . to prepare a 5 ug / ml of a coq 10 working solution , 10 mg of coq 10 are dissolved in 10 ml of hexane and diluted to 100 ml with 1 - propanol . the solution is thoroughly vortexed until dissolution is complete . a working solution is then prepared by diluting with 1 - propanol to 5 ug / ml . the concentration of the working solution is then calculated by reading the absorbance at the spectrophotometer ( 275 nm , 1 cm light path quartz cuvette using epsilon = 14 , 200 ). a series of calibration and control solutions is then prepared with the appropriate volume of 1 - propanol to have final concentrations of 10 , 100 , 500 , 1000 , 2000 , and 4000 ng / ml for coq 10 calibration solution , and the control solutions have final concentrations of 75 , 750 , and 1500 ng / ml coq 10 . the coq 9 is chosen as an internal standard . to prepare a coq 9 solution , 2 mg of coq 9 are dissolved in 100 ml of 1 - propanol . the coq 9 solution is thoroughly vortexed until dissolution is complete . a working solution of coq 9 is then prepared by diluting with 1 - propanol to a concentration of 2 ug / ml . all the solutions are stored in 1 . 8 ml polypropylene tubes ( sarstedt , newton , n . c ., usa ) at − 20 ° c . and used throughout the study . the hplc - ec system is depicted in fig2 . for chromatography with coulometric analyses , the hplc system consists of an esa model 582 solvent delivery module ( bedford , mass .) equipped with a double plunger reciprocating pump , an as3000 variable - loop autosampler ( thermo separation products , freemont , calif ., usa ), an analytical column , an esa coulochem ii model 5200a electrochemical detector , and a dell pentium ii 350 mz computer / controller with chromquest software ( thermo separation products ). the system also comprises two coulometric guard cells ( e 1 and e 2 , pre - and post - column , with respect to the analytical column , see fig2 ) and an analytical cell , e 3 and e 4 . there are also two in - line filters , wherein one in - line filter is placed before the guard cell e 1 and the second in - line filter is placed between the analytical column and the guard cell ( e 2 ). the analytical column used is a reverse - phase microsorb - mv column ( 5 μm , 4 . 6 mm × 15 cm ). a reverse phase c 18 guard column ( 5 - μm , 10 × 4 . 6 mm ) is used to protect the analytical column . the analytical column temperature is at room temperature . the as3000 injector is set at needle height of 1 . 5 mm and injection volume of 20 μl for each sample . the cooler temperature of the autosampler is at 0 - 4 ° c . each guard cell ( e 1 and e 2 ) is a single coulometric cell ( esa model 5020 ). the guard cells are installed before and after the analytical column ( fig2 ). the analytical cell ( e 3 and e 4 , esa model 5010 ) is connected in series to the postcolumn guard cell e 2 . the analytical cell consists of a series of two coulometric electrodes . the first electrode is operated in the reduction mode ( about − 650 mv or lower ) for reduction of coq 10 and he second electrode is operated in the oxidation mode ( about + 500 mv or higher ) for detection of coq 10 h 2 . the mobile phase for the isocratic elution of coq 10 is prepared as follows : sodium acetate trihydrate ( 6 . 8 g ), 15 ml of glacial acetic acid and 15 ml of 2 - propanol are added to 695 ml of methanol and 275 ml of hexane . mobile phase is filtered by 0 . 2 μm pore - sized , 47 mm nylon filter or analogous filter . mobile phase has a ph value of 6 . the flow rate is 1 . 0 ml / min . this system can be operated at three different modes for three different purposes : 1 ) precolumn reduction mode for measuring total coq 10 h 2 ; 2 ) precolumn oxidation mode for measuring total coq 10 ; and 3 ) precolumn off mode for simultaneous determination of coq 10 h 2 and coq 10 . in order to determine the optimal applied - voltage for ec detection , a hydrodynamic voltammogram is obtained by analyzing a solution of coq 10 ( 4000 ng / ml ) at different voltage settings . anodic currents and cathodic currents reach maximum responses at applied voltages of + 500 mv and − 600 mv , respectively . hence , the detection potential is maintained at + 500 mv or higher vs . the hydrogen / palladium reference electrode . for measuring total coq 10 h 2 , the precolumn guard cell is operated in the reduction mode (− 800 mv or lower ) in order to transform coq 10 to coq 10 h 2 . the reduction mode is also used to establish a calibration curve of coq 10 h 2 . for measuring total coq 10 , the precolumn guard cell is operated in the oxidation mode (+ 700 mv or higher ) in order to oxidize coq 10 h 2 to coq 10 . for measuring coq 10 h 2 and coq 10 simultaneously , the precolumn guard cell is operated in the off mode ( no current flows into the guard cell ). the postcolumn guard cell is operated at oxidation mode (+ 700 mv or higher ) to oxidize any electrochemically active elutes . according to the present invention , venous blood is collected into a vacutainer containing heparin as anticoagulant and mixed gently by inversion 5 - 6 times . at this point the blood - heparin tube is securely capped , and is then placed in ice and / or kept in refrigeration prior to processing . the blood - heparin tube should be processed within 4 hours . blood samples should not be collected in tubes containing metal chelators , such as ethylenediaminetetraacetic acid ( edta ). the blood - heparin tube is subject to centrifugation at an appropriate speed such as 3000 rpm for 10 minutes at 4 ° c . heparinized plasma is separated from red blood cells and placed in a capped polypropylene tube and immediately stored at − 75 ° c . or below until analysis . each frozen sample is thawed at room temperature , and 100 μl of this sample is placed in a 1 . 5 - ml capped polypropylene tube containing 50 μl of internal standard solution ( coq 9 , 2 μg / ml in 1 - propanol ). all the tubes are kept in an ice - bath . this is mixed with 850 μl of cold 1 - propanol . the tubes are vortexed for 2 minutes on a mechanical vortexer and centrifuged for 10 min at 0 - 4 ° c . the resulting supernatant is separated from the precipitate and transferred to an autosampler glass vial . the sample vial is immediately placed in the autosampler tray at 0 - 4 ° c . a batch of up to 20 samples can be extracted and processed at the same time . an aliquot of 20 μl of 1 - propanol extract from a vial is injected immediately onto an automated hplc . twenty samples can be analyzed sequentially , within 4 hours . if an error has occurred in the system , these sample vials can be resealed and immediately restored at − 75 ° c . or below for further investigation . standard solutions of coq 10 are prepared as described above . coulometric analysis and subsequent measurements of current responses are performed as described above . two oxidation peaks are observed for coq 9 and coq 10 at approximately 5 . 6 min . and 6 . 9 min , respectively ( fig3 ). peak height measurements for each calibration curve are obtained by using the chromquest software . the peak height ratios of coq 10 / coq 9 are used to obtain least squares linear regression equations , which are used to calculate the coq 10 concentrations of the frozen control samples and patient samples . a linear response up to a concentration of 4 μg / ml of coq 10 is obtained when the peak height ratios are plotted versus coq 10 concentration ( fig4 ). the same solutions of coq 10 used in the section describing quantitation of coq 10 are reduced electrochemically for coq 10 h 2 measurements as described above . coulometric analysis and subsequent measurements of current responses are performed as described above . two reduction peaks are observed for coq 9 h 2 and coq 10 h 2 at approximately 3 . 6 and 4 . 1 min , respectively ( fig3 ). the peak height measurements for each calibration are obtained by using the chromquest software . peak height measurements of coq 9 obtained in the section of “ quantitation of coq 10 ” are used herein . the peak height ratios of coq 10 h 2 / coq 9 are used to obtain a least squares linear regression equation , which is then used to calculate the coq 10 h 2 concentrations of the frozen control samples and patient samples . a linear response - up to a concentration of 4 μg / ml of coq 10 h 2 is obtained when the peak height ratios are plotted versus coq 10 h 2 concentration ( fig4 ). for coq 10 h 2 analysis , many previous studies have wrongly used plasma samples anticoagulated with edta . the choice of anticoagulant has an effect on oxidation of coq 10 h 2 ( fig5 ). each patient &# 39 ; s blood specimen is drawn into two paired vacutainers containing heparin or edta . although the time of sample handling and delivery may be varied from patient to patient , each pair of blood - containing tubes are handled and processed identically . the mean values of coq 10 h 2 and tq 10 from the heparinized plasma samples were 748 (± 346 ) and 770 (± 353 ) ng / ml , respectively , but somewhat poorer for those in edta - plasma with the mean values of 643 (± 321 ) and 696 (± 326 ) ng / ml , respectively . for the ratio of coq 10 h 2 : tq 10 the mean values of heparinized plasma , 97 . 0 (± 1 . 4 )%, were better than those in edta - plasma , 91 . 5 (± 4 . 4 )%. these results clearly indicate that ratios and levels of coq 10 h 2 and tq 10 in heparinized plasma are consistently higher than in plasma anticoagulated with edta ( p & lt ; 0 . 001 for difference in paired samples ). when blood samples in vacutainers are opened and kept refrigerated , the coq 10 h 2 : tq 10 ratios ( n = 30 ) in edta samples decreased by ˜ 30 % over 7 h , whereas the coq 10 h 2 : tq 10 ratios in heparinized samples were stable over the same period with variation of less than 3 % ( median 0 . 949 ; range 0 . 927 - 0 . 970 ; n = 30 ). there was as significant difference of coq 10 h 2 : tq 10 ratios in heparin in edta ( p = 0 . 028 ). the observed findings therefore reflect the effect of air - exposure on edta samples during the blood sampling procedure . the results also indicate that delays , in processing and analyzing edta samples would result in lowered and variable coq 10 h 2 : tq 10 ratios . using the presently described procedure , the coq 10 h 2 : tq 10 ratio was & gt ; 95 % in the plasma specimens of normal volunteers . heparin seems to possess antioxidant properties , but a previous study has shown that heparin has no direct antioxidant properties even at concentrations far higher than those usually used therapeutically . thus , other mechanisms not strictly antioxidant - type may be involved in heparin - mediated protection on coq 10 h 2 . while not being bound by theory , it is thought that either heparin is interacting with a lipoprotein to enhance antioxidant protection perhaps through release of superoxide dismutase , or the chelation of metal ions by edta is limiting the activity of antioxidant metalloenzymes . coq 10 h 2 is not stable and is oxidized in air at room temperature . coq 10 h 2 is unstable in whole blood , plasma and n - propanol extract when edta is used as anticoagulant . edta - anticoagulated blood loses approximately 15 % of coq 10 h 2 within 4 hours when stored in refrigeration ( 4 ° c . ), whereas only & lt ; 1 % loss of coq 10 h 2 occurs in heparin - anticoagulated blood under the same conditions ( fig6 ). when fresh blood samples in closed heparin vacutainers are kept refrigerated , no significant difference in coq 10 h 2 : tq 10 ratios was observed between samples stored for 0 h and 24 h ( p = 0 . 052 ). the median coq 10 h 2 : tq 10 ratio stored for 24 h was 0 . 955 ( range 0 . 923 - 0 . 972 ; n = 8 ) in comparison to the initial value of 0 . 964 ( range 0 . 935 - 0 . 978 ; n = 8 ). the coq 10 h 2 : tq 10 ratios decreased by ˜ 6 % after 48 h and by ˜ 28 % after 72 h . storage of blood specimens in vacutainers at + 4 ° c . ( in refrigeration prior to separation of plasma from blood cells can extend the stability of coq 10 h 2 to 24 hours without a significant loss (& lt ; 3 %, see fig7 ). since blood coq 10 h 2 in closed heparin vacutainers kept refrigerated is stable for at least 24 hours , an overnight shipment of a blood sample on ice pack is acceptable for testing . when plasma is kept frozen at − 75 ° c . or below , coq 10 h 2 is stable for at least 6 months and the coq 10 h 2 : tq 10 ratio does not change significantly during this period . since measurements of coq 10 h 2 from frozen samples is unfeasible , frozen samples must be thawed , extracted , and analyzed quickly to ensure minimal coq 10 h 2 oxidation during the measurement process . okamoto et al [ 10 ] reported the percentage of coq 10 h 2 with respect to the tq 10 was constant in plasma for one day when kept at 2 ° c . and − 10 ° c . when extracted from plasma and prepared in ethanol solution , however , any coq 10 h 2 in solution rapidly oxidized . an optimized hplc system for measuring coq 10 h 2 sequentially within 4 hours shows that 3 . 75 % of coq 10 h 2 in 1 - propanol extract oxidizes in an hour ( at a rate of ˜ 2 nm / min ) at room temperature , whereas , the loss of coq 10 h 2 is only 1 % in 5 hours when the temperature is set at 0 - 4 ° c . ( fig8 ). using 1 - propanol as an extracting solvent allows fast and simple sample processing . the procedure has been simplified to a single extraction and no evaporation process is needed . this allows for efficient sample handling . extraction of coq 10 h 2 and coq 10 from a variety of biological matrices is made facile with 1 - propanol , because it can be mixed with water in any proportion . additionally , coq 10 h 2 and coq 10 have highest solubility in 1 - propanol relative to other alcohols . thus , 1 - propanol is an effective extraction solvent ( table 2 ). the 1 - propanol extract can be directly analyzed by the described hplc - ec method . furthermore , this procedure takes advantage of the use of coq 9 as an internal standard . because the levels of coq 9 in human blood are negligible , it is desirable to spike control samples with this coq 9 to validate the extraction process . using a series of postcolumn guard cells and analytical cells operating within the stated oxidation and reduction potentials provides a high degree specificity to the detector system , and only those compounds capable of undergoing a reversible reduction - oxidation at the low potentials used are capable of being detected by the last electrode . accordingly , as a result of a compromise involving the mobile phase composition , flow - rate and concentration of the supporting electrolyte , an excellent separation of an authentic mixture of coq 9 , coq 10 and coq 10 h 2 is obtained on a microsorb - mv c18 column ( 15 cm × 4 . 6 mm ) using a mobile phase ( ph ˜ 6 ) containing 0 . 05 m sodium acetate at a flow - rate of 1 . 0 ml / min . a patient &# 39 ; s sample was obtained according to the current method , wherein the retention times were 4 . 1 min for coq 10 h 2 , 5 . 6 min for coq 9 and 6 . 9 min for coq 10 ( fig9 ). the procedure described herein is useful for investigating the effect of coq 10 supplementation on patients with metabolic disorders ( fig1 ). coq 10 h 2 is generally obtained by chemical reduction of the commercially available coq 10 . this requires a strong reducing agent such as sodium tetrahydroborate . the reduced form is unstable at room temperature and quickly becomes oxidized , so a fresh solution of coq 10 h 2 must prepared daily . all these procedures are time consuming , labor - intensive , tedious and unsuitable for routine determinations . an alternative procedure using electrochemical reduction to obtain the reduced form relies on a coulometric detector which is able to provide more than 99 % yield of the desired electrochemical reaction ( table 3 ) and thus providing a calibration curve for measuring the coq 10 h 2 concentration . the calibration curves for reduced coq 10 and oxidized coq 10 were linear ( r = 0 . 999 ) over the concentration range 10 - 4000 ng / ml . the detection limits of reduced coq 10 and oxidized coq 10 are about 5 ng / ml ( s / n = 3 ) ( fig4 ). this simplified 1 - propanol extraction procedure is reproducible , allows for a recovery of coq 10 , and allows for the assessment of the coq 10 h 2 content from a standard concentration curve . quantitative recovery of coq 10 ( 100 %) using 1 - propanol solvent has been obtained . the inter - day and intra - day assay variations ( cvs ) are less than 10 % over a range 10 - 4 , 000 ng / ml ( table 4 ). the chromatography run time for each specimen is typically less than 10 minutes ( fig9 ). the total time required for a complete sample analysis is less than 30 minutes . this method allows at least 40 samples to be analyzed in an 8 hour working day , and 24 samples could be completed for overnight analysis . 1 . takada , m ., ikenoya , s . yuzuriha t ., and katayama , k . simultaneous determination of reduced and oxidized ubiquinones . methods enzymol , 1984 , 105 : 147 - 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