extração de coenzima Q 10 em plasma humano : otimização do uso de surfactantes e outras variáveis Determination of a method for extraction of coenzime Q 10 in human plasma : optimization of the use of surfactantes and others 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.


INTRODUCTION
Coenzyme Q 10 (CoQ 10 ), also known as ubiquinone, is a lipid molecule essential for aerobic organisms.It participates in the production of ATP through the oxidative phosphorylation process by transferring electrons from respiratory complexes I and II to complex III in the respiratory chain, located in the inner mitochondrial membrane.Additionally, CoQ 10 participates in many other vital functions within the cell: it acts as an antioxidant of lipoproteins and cell Ferreiro-Barros CC, Sugawara EK, Sanches LR membranes; it is required for the biosynthesis of pyrimidine affecting replication and repair of cellular DNA; it modulates the process of apoptosis through the regulation of the membrane transition pores, and aids in maintenance of body temperature resulting from its function in uncoupling proteins (1) .
CoQ 10 is composed of a ring of benzoquinone associated with a polyprenyl chain derived from the mevalonate pathway, the same synthesis route of cholesterol.The size of this polyprenyl chain varies among organisms; the human species has 10 repetitions (CoQ 10 ), while mice have 9 (CoQ 9 ), and Saccharomyces cerevisiae have 6 (CoQ 6 ).
The endogenous synthesis of CoQ 10 occurs in the mitochondria (1) and it is expressed in all tissues, but in greater concentration in the heart, kidneys, liver and muscles, since they are organs that require a greater quantity of energy (ATP) for their function, and decrease their expression during the process of cellular aging (2) .
The primary deficiency of CoQ 10 is autosomal recessive and is included in the group of mitochondrial diseases.It is caused by a defect in the biosynthetic route of ubiquinone which is not yet completely elucidated (6) .Among the mitochondrial diseases described, to date, CoQ 10 deficiency is the only one that has chances of effective treatment through the exogenous CoQ 10 administration (9) .
Much of what is known about the biosynthesis of coenzyme Q comes from studies carried out with Escherichia coli and S. cerevisiae (10) .There are at least a dozen genes named COQ1, COQ2, COQ3... COQ10 in Saccharomyces cerevisiae, necessary for the respiratory function of coenzyme Q (10)(11)(12)(13) .
The secondary deficiency in the metabolism of CoQ 10 was discussed in the pathophysiology of myopathy by statins, toxicity by anthracyclin and Parkinson's disease, but the relative contribution of CoQ 10 in triggering these diseases is still unknown (21,22) .
However, supplementation with CoQ 10 is amply recommended at high doses (30mg/kg in children and at least 600mg daily in adults) for all patients with mitochondrial diseases, since it is a supplement devoid of side effects and it brings many benefits to cellular metabolism (23,24) .Since it is lipophilic, CoQ 10 is transported in circulation by particles of lipoproteins, and its concentration in the plasma has been correlated with the total levels of cholesterol, especially LDL, in patients who take statins (25,26) .
Considering this effective possibility of treatment, interest in determining the levels of CoQ 10 in the plasma and other types of cells or tissues were the target of various studies.The diagnosis of CoQ 10 deficiency is performed through the analysis of the enzymatic activity of mitochondrial complexes I+II and II+III (1) , as well as by quantification of the total levels of the enzyme directly in the muscle or fibroblasts.The levels of CoQ 10 determined in the blood are not used for diagnosis, since CoQ 10 is present in certain foods and is absorbed through the diet.However, it is used for therapeutic monitoring in patients with confirmed deficiency of the coenzyme.
There are various methods described in scientific literature for the quantification of CoQ 10 in plasma, cells and tissues.The use of reverse phase high performance liquid chromatography (HPLC) equipment coupled with electrochemical or ultraviolet (UV)-type detectors is very frequent.Currently, most of the methods involve the preparation of the sample using a single stage of dilution with 1-propanol, followed by the direct injection of the sample into an HPLC system (27,28) .However, this method is more commonly used in the electrochemical-type detector that displays greater sensitivity, allowing the detection of minimal concentrations of CoQ 10 .Due to the lower level of sensitivity of ultraviolet detectors, their use requires a more elaborate sample processing, with extraction stages using organic solvents such as hexane (29,30) , hexane/methanol (31,32) or ethanol (33) , followed by the concentration of the sample in order to then inject it into the HPLC system.
In this study, two CoQ 10 extraction methods in human plasma were tested: methanol:hexane, and 1-propanol, both with UV detection.In the attempt to improve the methods of CoQ 10 extraction by UV detection, the addition of some surfactants such as Determination of a method for extraction of coenzime Q 10 einstein.2012;10(2):203-8 SDS (sodium dodecyl sulfate), Triton X-100 and Tween-20 was tested as to the possibility of obtaining a better recovery at the end of the extraction process.Other variables that may directly influence the result of CoQ 10 quantification were also studied: temperature (4°C and 19°C) and types of tubes (glass and polypropylene).

OBJECTIVE
To establish a routine for extraction of the total levels of CoQ 10 in human plasma through the ultra high performance liquid chromatography (UHPLC) analysis.

Standard solution
The standard solution of CoQ 10 was prepared at the concentration of 1mg/mL using 1-propanol as dilution solvent, and storing it at -20°C protected from light.

Sample preparation
For initial optimization and standardization studies of the CoQ 10 extraction process, we used a 4% human albumin solution with the objective of simulating plasma samples, which then received the addition of the standard solution of CoQ 10 resulting in the final concentration of 1000mg/mL.After definition of the best method for extraction, the blood collection protocol was followed as described below.
Samples of blood were collected from the same control individual by peripheral venous puncture into glass tubes containing anticoagulant citric acid, sodium citrate and dextrose, and maintained at 4°C.The plasma was obtained after centrifugation at 894.2g for 10 minutes at 4°C, transferred to Eppendorftype tubes and stocked at -80°C for further analysis.Sample collection was performed after obtaining the signature on the informed consent form approved by the Research Ethics Committee of the Hospital Israelita Albert Einstein (No.10/1465).

Methods for extraction of CoQ 10
CoQ 10 extraction with methanol:hexane The initial protocol was used (34) , with the following modifications: 700mL of plasma, 100mL of detergents (SDS, Triton X-100 and Tween-20) at concentrations of 1%, 3%, 5% and 10% separately, 1400mL of methanol and 1500mL of hexane were added to a polypropylene tube and a glass tube.The samples were submitted to mechanical shaking for one minute and centrifuged at 1752.8g for 10 minutes at 10°C.The supernatants were transferred to a new tube and evaporated without heating under a flow of nitrogen for 20 minutes.Next, the residues were resuspended in 60mL of mobile phase of methanol:hexane (85:15, v/v), homogenized under vortex mechanical shaking for 15 seconds and orbital shaking for 15 minutes.For the chromatographic analysis, 20mL were used.Extractions were performed in triplicate at 19°C (controlled temperature) and at 4°C (ice bath).

CoQ 10 extraction with 1-propanol
The initial protocol was used (27) with the following modifications: 700mL of plasma, 100mL of detergents (SDS, Triton X-100 and Tween 20) at the concentrations of 1%, 3%, 5% and 10% separately, 1400mL of 1-propanol were added to a polypropylene tube and a glass tube.The samples were submitted to shaking for one minute and were centrifuged at 894.2g for 10 minutes at 10°C, then transferred to an "amber vial" with the help of a filtrating unit with 0.22mm, 13mm.The chromatographic analysis was performed by means of a 20mL injection of the sample.The extractions were performed in triplicate at 19°C (controlled temperature) and at 4°C (ice bath).

Chromatographic analysis and detection of CoQ 10
The equipment used for Ultra High Performance Liquid Chromatography (UHPLC) was HP1290 (Hewlett-Packard) constituted by an automatic sampler, binary pump, and UV detector with variable wavelength.The detection was made at 275nm.For the chromatographic separation, an analytical column was used, Zorbax Eclipsy C18 ® (50 x 2.1mm, 1.8µm), with an equivalent guard column, both obtained from Hewlett-Packard and maintained at 45°C during the analysis.The CoQ 10 displayed a retention time of 1.5 minutes and the total time of chromatographic analysis was 3.5 minutes.The Ferreiro-Barros CC, Sugawara EK, Sanches LR mobile phase used was methanol-hexane (85:15; v/v) with a flow rate of 0.45mL/min.

Establishment of the CoQ 10 extraction protocol
For the establishment of the CoQ 10 extraction protocol, initially performed were tests using 4% human albumin solution with added CoQ 10 solution of 1000mg/mL.The extraction methods methanol:hexane and 1-propanol were tested in the presence and absence of surfactants such as SDS (anionic), Triton X-100 (non-ionic) and Tween-20 (non-ionic) at concentrations of 1%, 3%, 5% and 10%, at 4°C and 19°C.The first results indicated that the extraction method using methanol:hexane in the presence of the surfactants Triton X-100 and Tween-20 showed better rates of CoQ 10 recovery, while with the use of SDS, the reproducibility and efficiency were much lower (data not shown).
The same extraction protocol used in the tests with CoQ 10 added to albumin was applied to samples of plasma from control individuals without the addition of CoQ 10 to verify the reproducibility of the method in real samples.The samples were analyzed as to temperature of extraction (controlled at 19°C and at 4°C, ice bath)  and as to the composition of the tubes used for the extraction (polypropylene and glass), as per table 1.
The results demonstrate that the extractions performed in real samples of plasma presented a similar response as that shown with a solution of albumin in which the use of the detergent Tween-20 at 3%, at 4°C, using methanol:hexane resulted in the best response obtained for the detection of CoQ 10 (Figure 1).In addition to presenting the best recovery, there was also good reproducibility between the replicates with a CV (coefficient of variation) of 4.3%.Thus, the option of using a surfactant such as Tween-20 resulted in an improvement (51.2%) in efficiency.
The chromatograms demonstrate the chromatographic quality obtained with the mobile phase composed of methanol:hexane (85:15, v/v) with a flow of 0.45mL/min.(Figure 2).The retention time of CoQ 10 was 1.45 minutes and the peak purity (99%) was obtained through the analysis of its spectrum (ChemStation, Agilent).

DISCUSSION
The application of chromatographic techniques has been widely used in studies of enzymatic determination and pharmacokinetics for therapeutic monitoring.Recently, the interest in determining the levels of CoQ 10 in plasma and other types of cells or tissues has been the target of various studies, given the possibility of effective treatment in neurological patients or those with metabolic diseases through the oral supplementation (35) .
CoQ 10 is present in circulating plasma associated with lipoproteins, and its concentration is directly related to the concentration of cholesterol present in the plasma (36) .Due to this characteristic binding of CoQ 10 to lipophilic molecules, we chose to use surfactants as aids in the extraction process with the intent of breaking this type of bond and thus increasing the levels of detection of the coenzyme.Some authors demonstrated that the action of SDS in plasma significantly increases the recovery of CoQ 10 after the extraction process (37) .Conversely, the effect of Triton X-100 on the solubilization of membranes was observed both in proteins and in lipids (38) .Indeed, when initiating the first experiments, low rates of CoQ 10 were attained without the addition of surfactants during the process of extraction, observed by the small chromatographic peak area (mUA).
In this study, the best results were achieved with the methanol:hexane extraction method when compared to 1-propanol.Some authors have used 1-propanol as a solvent for precipitation of proteins due to its greater lipophilic character, but the success of this technique only occurs when used in electrochemicaltype detectors, since they are more sensitive than what was used in this study (UV).This occurs because this technique does not allow the concentration of the sample in the process of extraction, and the addition of 1-propanol merely acts by precipitating the proteins and diluting the sample, making it more difficult to quantify CoQ 10 with the use of UV detectors.It was also observed that the use of surfactants Triton X-100 and Tween-20 resulted in better rates of recovery of CoQ 10 , while the use of SDS showed lower reproducibility and efficiency.Additionally, even when used at low concentrations, SDS promoted the formation of a "cloud point" that hindered the separation of the organic and aqueous phases.
When the established protocol using the human albumin solution was applied for the extraction of CoQ 10 in real samples of plasma, the response was similar to that with the use of surfactant Tween 20 at 3%, at 4°C in methanol:hexane, resulting in a better recovery, good reproducibility among the replicates, a CV of 4.3%, and an increase of 51.2% in efficiency of the extraction process.
Temperature is also a relevant parameter to be evaluated when the substance studied is an enzyme, which since it is thermolabile, may easily suffer denaturation at higher temperatures.
The best results of this study showed that the control of the temperature (4°C) is of vital importance during the extraction process.It was also noted that there was better separation of the organic and aqueous phases when polypropylene tubes were used, contributing, in fact, to an improvement in the efficiency of the extraction.

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 CoQ 10 by the methanol:hexane extraction method.This method showed good reproducibility, with a low CV and high sensitivity, since CoQ 10 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.
Since this stage of method optimization was concluded, this extraction protocol is in the validation phase which will allow its applicability for future clinical tests with a guarantee of reliable results.

Figure 1 .Figure 2 .CD
Figure 1.Result of the extraction of CoQ 10 in plasma samples: comparison of the effect of Triton X-100 at 10% and Tween-20 at 3%

Table 1 .
Analysis of the variants that interfere in the process of CoQ 10 extraction: addition of detergents, tubes and temperature conditions