TECHNIQUES FOR ANALYSIS OF COENZYME Q 10

CoQ₁₀ (see Figure 7.2) has many physiological roles; therefore, being able to analyze its levels in plasma could possibly aid in the treatment and prediction of diseases. Not only this, the new HMG-CoA reductase inhibitors (cholesterol lower-ing drugs) affect the synthesis of CoQ₁₀ and, therefore, have the potential to cause adverse effects such as cardiac myopathy. This is thought to be attributable to a decrease in ubiquinone plasma levels, meaning that a determination of CoQ₁₀ is important in early drug development to predict and avoid adverse effects [Teshima and Kondo 2005].

Finally, with the ever-increasing use of nutraceutical products, it is important to be able to determine levels of these compounds within commercially available products to ensure public safety.

The quantifi cation of CoQ₁₀ plasma levels is, therefore, important from a medi-cal and epidemiologimedi-cal point of view [Karpin´ska et al. 2006]. The analysis of CoQ₁₀ presents several challenges, and numerous techniques have been investigated for potential use [Jiang et al. 2004; Zu et al. 2006].

The average level of coenzyme in a healthy subject is 0.8 ± 0.2 mg/L [Grossi et al. 1992]. Thus, the main analytical challenge was to develop a method selec-tive enough and sensiselec-tive enough to quantify levels in human fl uids and commercial products, which was fast enough to be used routinely in quality-control laboratories.

This would allow for useful information on the action, bioavailability, and fate of CoQ₁₀ products to be collected.

The challenge is made more diffi cult by the chemical nature of CoQ₁₀. First, CoQ₁₀ contains isopentenyl, which makes the compound susceptible to photo- oxidation.

This means that all experiments should be undertaken in particular conditions to

Figure 7.2 The structure of coenzyme Q10. O O

CH₃ H₃CO

H₃CO (CH₂CH=CCH₂)₁₀––H CH₃

avoid the photodegradation, specifi cally in the absence of light. Second, biologi-cal samples of CoQ₁₀ will be accompanied by a complex matrix. This means that any sample will require intensive and time-consuming sample preparation before an assay can be undertaken [Karpin´ska et al. 2006]. This is often a factor that limits the value of a technique if it is to be widely applied.

HPLC

HPLC is probably the most popular technique used in the analysis of CoQ₁₀ [Li et al. 2006]. Several detection methods have been used, including MS, UV, and electrochemical detection. UV and electrochemical detection are perhaps the most popular techniques used because of the wider accessibility of the technology [Karpin´ska et al. 1998]. Each method is associated with certain disadvantages and advantages that will be discussed below.

UV or Electrochemical Detection?

It has been shown that determination of CoQ₁₀ in human plasma can be achieved by HPLC with UV detection after TLC, liquid/liquid [Katayama et al. 1980], or solid-phase extraction [Jiang et al. 2004]. These techniques are used to remove com-plex components that are found within a biological sample. The usual method used is an alcohol-hexane extraction, in which the extract is dried and redissolved before injection. This precolumn manipulation of the sample is time consuming, which makes the methods unsuitable for routine use in clinical chemistry laboratories [Jiang et al. 2004]. Karpin´ska et al. [2006] developed such a method. Before sample injec-tion, a lengthy preparation took place in which 0.25 ml of sample was deproteinized using 0.5 ml of methanol. After that, 0.75 ml of hexane was added. The mixture was then vortexed for 5 min and centrifuged at 5000 rpm for 15 min. Next, the clear hexane layer was transferred to another tube, and extraction of plasma vitamins was repeated with a new 0.75 ml portion of n-hexane. The plasma extracts were combined and evaporated to dryness under a stream of nitrogen. Finally, the dry residue was dissolved in 0.25 ml of mobile phase and chromatographed by HPLC.

Separation took place on a RP C18 125 × 4 mm (5 μm) column with a guard column, 4 × 4 mm (5 μm), with a mobile phase of methanol and hexane 72:28 (v/v). The fl ow rate used was 1 ml/min, with detection achieved by a UV detector at 276 nm. This gave a retention time of 7.47 ± 0.12 min and an LOD and LOQ of 0.87 and 2.98 μM, respectively, for CoQ₁₀.

The use of an electrochemical detector has been shown to be 10–20 times more sensitive than a UV detector. For this reason, the method was used by Tang et al.

[2006] for detection of CoQ₁₀ in human breast milk, in which the levels are even lower than that of the plasma. The analytical column used was a Microsorb-MV RP C18 column (150 × 4.6 mm; 5 μm bead size). The mobile phase used consisted of a mixture of sodium acetate anhydrous (4.2 g), 15 ml of glacial acetic acid, 15 ml of 2-propanol, 695 ml of methanol, and 275 ml of hexane. The mobile phase was fi ltered through a 0.2 μm pore-sized nylon fi lter, and the fl ow rate was 1 ml/min. An

electrochemical detector was used and was shown to give an LOQ of 60 nmol/L, with a time of analysis of approximately 8 min per sample. With the basal concen-tration of CoQ₁₀ in breast milk being 0.166 ± 0.002 μmol/L, this method achieves high sensitivity [Tang et al. 2006]. The other main advantage of electrochemical detection over UV methods is that it allows for the detection of the reduced form of ubiquinone, ubiquinol. This allows for estimation of the total plasma CoQ₁₀. To accomplish this, all CoQ₁₀ in a sample is converted into the reduced form by precol-umn treatment and then detected using such methods [Grossi et al. 1992]. However, as with UV detection, this method still requires manual sample preparation before chromatographing, which has the disadvantage of being time consuming.

Column Switching HPLC

Because of the complexity of biological fl uid specimens, most samples normally require some sort of purifi cation before injection into the HPLC system, such as solid-phase extraction, liquid/liquid, or TLC. Column switching avoids this by allowing online sample purifi cation. This results in the method being suitable for automation and decreased handling time, which can result in artifi cial error and poor reproducibility. Jiang et al. [2004] used such a method for the determination of CoQ₁₀ in human plasma. In this technique, a short precolumn (20 × 4.6 mm inner diameter) was packed with 5 μm Hypersil C18. This was used to eliminate both polar com-pounds, including reagents and strongly retained solutes. The mobile phase used for the precolumn was pure methanol at a fl ow rate of 0.5 ml/min. The analyte was then transferred to the analytical column (150 × 4.6 mm inner diameter packed with 5 μm of Hypersil ODS₂). The mobile phase through the analytical column con-tained 10% (v/v) isopropanol in methanol at a fl ow rate of 1.5 ml/min. The detec-tion was performed with UV set at 275 nm. The lower limit of detecdetec-tion achieved was approximately 1 μg/ml in human plasma, which is comparable with methods using solid-phase extraction to purify samples [Jiang et al. 2004]. The disadvan-tage noted by Grossi et al. [1992] is that column-switching technology may not be available in many laboratories. Whether this comment still applies 15 years on is debatable.

Mass Spectrometry Detection

As technology develops, researchers are adopting the use of HPLC coupled with MS as a useful method for the identifi cation and determination of compounds [Teshima and Kondo 2005; Zu et al. 2006]. The use of LC-MS for the detection of CoQ10 in complex samples is effective because of the high sensitivity and selectiv-ity. Strazisar et al. [2005] demonstrated that an LC-MS method was more sensitive than LC-UV. Hansen et al. [2004] showed that HPLC-MS has comparable or better sensitivity than HPLC-electrochemical detection.

Zu et al. [2006] used an LC-MS/MS method for the determination of CoQ₁₀ in tobacco leaves. Chromatographic analysis was performed on an RP C18 column, with a mobile phase consisting of a mixture of acetonitrile and isopropanol (8:7 v/v)

containing 0.5% formic acid at a fl ow rate of 0.3 ml/min. ESI-MS, operating in the positive ion mode, was used for detection and analysis. CoQ₁₀ was monitored using the multiple reaction monitoring mode. Under these conditions, a retention time of 2.91 ± 0.1 min and an LOQ and LOD of 4.0 and 1.2 ng/ml, respectively, were estab-lished for CoQ₁₀. This method was therefore able to give an LOD 205.8, 71.9, 41.7, and 208.6 times lower than the LOD obtained by spectrophotometric, voltammetric, and chromatographic methods with UV and fl uorescence detection. It has been noted in lit-erature that matrix components do have the potential to cause erroneous results unless optimization of sample preparation and the chromatographic system takes place.

Another LC-MS/MS technique using turbo-spray ionization was investigated by Teshima and Kondo [2005] for analysis of CoQ₁₀ levels in rat heart and thigh muscle.

Teshima and Kondo looked at existing methods using LC-MS/MS with a turbo-spray ionization source for the analysis of ubiquinones and looked to improve their sensitivity. Separation was achieved on a YMC Pack Pro (75 × 2 mm inner diameter) analytical column, and a mixture of methanol/2-propanol/formic acid (45:55:0.5) containing 5 mmol/L methylamine was selected as the mobile phase. The addition of the methylamine to the mobile phase led to 12.5-fold higher signal intensity than without. A triple-stage quadrapole mass spectrometer with a turbo-spray ionization source was used for the detection of CoQ₁₀. Under these conditions, CoQ₁₀ had the observed retention time of 4 min, and the method was able to quantify CoQ₁₀ over a wide concentration range in rat thigh and heart muscle, 1–500 and 10–10,000 μg/g, respectively [Teshima and Kondo 2005].

All of the above HPLC methodologies are suitable for analysis of CoQ₁₀ in prod-ucts and biological samples and are routinely used. Intuitively, the column-switching method seems the most sensible for use in clinical investigations of CoQ₁₀ because of its fast, accurate, and automated properties.

Derivative Spectrophotometry

Although HPLC methods are highly sensitive, it has been noted that they can be time consuming and complicated. An alternative method was therefore sought by Karpin´ska et al. [2006]. They investigated the use of spectrophotometry because it is a rapid, specifi c, simple, and reliable analytical technique. Unfortunately, classical UV spectrophotmetric methods could not be used because of spectra interference from the complex matrix of plasma samples of CoQ₁₀. This made determination of CoQ₁₀ impossible. To avoid this problem, Karpin´ska et al. used a mathematical technique to eliminate the infl uences of the matrix/background, namely deriva-tive spectrophotometry (derivaderiva-tive of the spectra is taken using the Savitzky–Golay algorithm). This technique enhances the sensitivity and selectivity of the analysis.

Karpin´ska used a HP-8452A diode array spectrophotometer, and the spectrophoto-metric measurements were performed at 284 nm. This technique was able to deter-mine the presence of CoQ₁₀ in pharmaceuticals in the range of 0.5–10 ppm. Because levels of CoQ₁₀ do not exceed 1.5 ppm, the method was applicable. Karpin´ska et al.

concluded that the technique could be used as an alternative to HPLC analysis of

CoQ₁₀ in commercial formulations; however, the method has limited application for analysis of biological samples, and a relatively large sample volume is required.

Voltammetric Determination

Voltammetry is an electroanalytical technique that is suitable for use with CoQ₁₀ because of its redox and adsorptive behavior. Two methods for the analysis of CoQ₁₀ have been used in the literature.

Square-Wave Voltammetry with Glassy Carbon Electrode

Litescu et al. [2001] analyzed commercially available CoQ₁₀ formulations using a standard addition voltammetry method on a glassy carbon electrode. This is made possible because CoQ₁₀ is electroactive at the glassy carbon electrode. This tech-nique is said to be a simple, rapid, sensitive, and accurate means for CoQ₁₀ analy-sis via a reduction process. Litescu et al. recommended the technique as a means of analysis for quality-control laboratories because it does not require any lengthy sample preparation steps except for previous extraction with hexane.

Mercury Hanging Electrode

Emons et al. [1992] developed a method for determination of ubiquinones with dif-ferential pulse voltammetry on a mercury hanging electrode. They concluded that this method was only suitable for determination of CoQ₁₀ in samples with a less complex matrix, meaning that it has limited application for routine analysis of biological samples.

Both of the above methods have been said to lack analytical sensitivity and have a disadvantage of needing to be operated in dim light because of the sensitivity of CoQ₁₀ to photodegradation. The application of these methods above HPLC, or spec-trophotometry, would be debatable.

Electron Paramagnetic Spectroelectrochemistry

Electron paramagnetic spectroscopy is a powerful tool for characterization of radical ion intermediates in reactions. If these intermediates are suffi ciently stable, it is possible to determine relative concentrations from the relative resonance intensities.

The absolute concentrations can then be found by comparing the relative resonance intensities with those of a standard. Long [1999] used this methodology for the deter-mination of CoQ₁₀ in commercial products. In this case, the intermediate radical ubisemiquinone is measured that is formed as part of the reduction reaction of CoQ₁₀ at a silver electrode by in situ electron paramagnetic spectroelectrochemical tech-niques. Long concluded that this method showed good agreement to data produced from HPLC methods and with that of the manufacturers’ labeling. The technique was stated as useful for enhancing selectivity of other methods when other chemicals present in the sample do not interfere with the assay [Long 1999].

CONCLUSIONS

This review describes a number of techniques used for the analysis of glu-cosamine and CoQ₁₀, including radiolabeling, HPLC with both precolumn and post-column derivatization reagents, using UV, fl uorescence, and ESI-MS detection, GC, high-performance thin-layer chromatography, CE with derivatization reagents, IR with chemometrics, derivative spectrophotometry, voltammetric determination, and electron parametric spectroelectrochemistry. In addition to the techniques described here, high-speed counter-current chromatography, micellar electrokinetic capillary chromatography, microemulsion electrokinetic chromatography, ¹H nuclear mag-netic resonance, and near IR spectroscopy have also been used recently for analysis of other nutraceuticals. A number of novel alternative techniques have also been devised, such as the use of biosensors [Kirrane and Lockwood 2008]. The plethora of methods available include almost the whole gamut of techniques available for organic molecules.

With the ever-increasing use of nutraceuticals, the need to have analytical tech-niques capable of attaining accurate and reliable information is of growing impor-tance, gaining more insight as to their distribution. If the traditional paradigm for drug development continues to fail, pharmaceutical companies will pay an ever-increasing amount of attention to the potential use of nutraceuticals. Central to this are reliable, reproducible, sensitive, selective, and quantitative analytical techniques.

With respect to glucosamine and CoQ₁₀, many challenges are presented in terms of analysis, and every technique available has its reported drawbacks.

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Determination of glucosamine sulfate in human plasma by precolumn derivatization using high performance liquid chromatography with fl uorescence detection: Its

Determination of glucosamine sulfate in human plasma by precolumn derivatization using high performance liquid chromatography with fl uorescence detection: Its