C30 Chromatographic Packing Separates High-Purity Coenzyme Q10 Test

 Coenzyme Q10 has the functions of improving human immunity, enhancing antioxidant, delaying aging and increasing human vitality, and is also widely used in health products or food additives. According to the requirements of the 2020 edition of the Chinese Pharmacopoeia [1] and the United States Pharmacopoeia USP28-NF23 [2], the total content of coenzyme Q10 should not be less than 98%; and the separation of coenzyme Q10 can meet the requirements after extraction, silica gel chromatography and crystallization, and the current annual production can reach the level of several hundred tons. However, the subsequent European Pharmacopoeia 8.8 and later editions, in addition to the main content of not less than 98.0%, increased the level of single heterogeneous content of not less than 0.1% [3].

 

In the present study, we tried to isolate the impurities by the traditional method, and found that the other impurities could meet the requirements after two silica gel chromatography and crystallization, but one of the impurities was difficult to be reduced to the level; the relative retention time of the impurity was about 1.45 by using the method of 2020 edition of the Chinese Pharmacopoeia, and the literature reported that this impurity was called coenzyme Q11 [4].

 

The aim of this paper is to establish a safe and energy-saving method for the purification of coenzyme Q10 by preparative liquid chromatography. Combined with the chromatographic analysis method of Pharmacopoeia, the most probable process for the separation and removal of coenzyme Q11 is reversed-phase chromatography (RPCC). The patent "A purification method of coenzyme Q10" (Patent No. 201810233454.1) reported that octadecyl- or octyl-bonded silica gel was used as the stationary phase, and acetone/methanol or acetone/ethanol was used as the mobile phase, and the whole system was separated in a heated state, with the volume of the sample above 10% and the recovery rate above 80%. The whole system must be separated at 45 ℃, which consumes too much energy for large-scale industrial production, increases the cost of equipment and the complexity of the system, and raises the risk of explosion or fire of organic solvents under pressure.

 

After analysis, the sample in acetone solubility is good, but in the C18 packing or C8 retention is poor, after adding a certain amount of methanol, the solubility of the sample is significantly reduced, so the use of heating to solve the problem of solubility. In this study, it was concluded that if the length of the carbon chain was increased, the retention of the sample in the stationary phase would be relatively increased, and the retention could be reduced by decreasing the proportion of methanol in the mobile phase, so that the separation of coenzyme Q11 could be realized at ambient temperature or a little higher than ambient temperature.

 

1 Materials and Methods

1.1 Reagents and Consumables    

Coenzyme Q10 crude (Batch No. 20210603C, provided by North China Pharmaceutical); Coenzyme Q10 control (Batch No. 20220112, homemade); Methanol (Batch No. 20200403, analytically pure, Komeo); Acetone (Batch No. 20200105, analytically pure, Concord); Chromatographic packing C30, 30 μm, 105Å (Batch No. NS30A12B210903, Bojun Bio); Biorain C18 (5 μm, 100Å, 4.6 × 150 mm, Bojun Bio); Chromatographic column Biorain C18 (5 μm, 4.6 × 150 mm, Bojun Bio). NS30A12B210903, Biorain biological); chromatographic column Biorain C18 (5 μm, 100 Å, 4.6 × 150 mm, Biorain biological).

 

1.2 Instruments   

An Agilent 1200-phase liquid chromatograph (with degasser, high-pressure dual-pump system, autosampler, column oven, DAD detector); TF-Prep liquid chromatography (100 mL-min-1 dual-pump system, 10 mL-min-1 high-pressure injection pump, 6-channel fraction collector); and a 100,000-part balance (EX125DZH, Ohaus Instruments Co., Ltd.) were used for the analysis of the analytes.

 

1.3 Experimental Methods

1.3.1 Sample Dissolution

Weighing coenzyme Q10 crude about 20 g, with about 160 mL of acetone ultrasonic dissolution, and then add about 40 mL of methanol, mixing, and then placed at room temperature away from light, the sample concentration of 100 g - L -1 .

 

1.3.2 Column Packing

Take about 50 g of C30 bonded packing and fill it into the 21.2 × 250 mm column tube by squatting: add the packing into the column tube one by one with a medicine spoon, 1 ~ 1.5 g at a time, squatting, and then add packing until the packing surface no longer falls; hang off the excess packing with a scraper, clean off the excess packing on the outer edge of the column tube, fasten the sieve plate with a polytetrafluoroethylene washer, weigh the weight of the packed material, and tighten the stainless steel screw threads with a wrench. Stainless steel threads, chromatographic column loading is complete, labeled mobile phase inflow direction for the opposite direction of filling.

 

1.3.3Column Equilibrium

The column was connected to a preparative liquid chromatography system and equilibrated with acetone/methanol (4/1, v/v) at a flow rate of 15 mL-min-1 for about 20 min.

 

1.3.4 Coenzyme Q10 Isolation

The sample was injected with the injection pump at a flow rate of 13~20 mL - min - 1, and the injection volume was set so that the sample volume was in the range of 5%~7%. The injection volume was recorded and the sample ratio was calculated; then acetone/methanol (4/1, v/v) was used as the mobile phase, and the flow rate was set at 15 mL - min - 1, and the detection wavelength was set at 275 nm for the DAD detector; the fractions were collected in full collection mode, and one fraction was collected every 5 min; the analysis was performed according to the conditions of Coenzyme Q10 in the 2020 Chinese Pharmacopoeia [4], and the percentage of impurity Coenzyme Q11 was determined by area normalization.

 

Fractions were collected in full collection mode, one fraction every 5 min; the chromatographic conditions of coenzyme Q10 in the 2020 edition of the Chinese Pharmacopoeia [4] were used to determine the percentage of the impurity coenzyme Q11 by area normalization, and the fractions with the content of coenzyme Q11 less than 0.1% were combined. Under the same operation mode, the preparative chromatographic separation was carried out with different sample volumes, and the qualified fractions were collected.

 

1.3.5 Post-processing

The qualified fractions were combined, and then concentrated to about 25% by volume under reduced pressure spinning at 30~35 ℃. The solution was transferred to a beaker, cooled at room temperature and allowed to stand for 18~24 h. The solid was then filtered through a sand-core funnel and allowed to air dry at room temperature for 18~24 h. The recoveries were calculated by weighing the solid, and solvent consumption was calculated according to the separation times of different sample volumes.

 

2.Results

After the above process, the typical separation liquid chromatogram was shown in Figure 1; the analytical analysis of the purified coenzyme Q10 fractions was carried out, and the typical analytical analysis of the qualified samples was shown in Figure 2; the separation recoveries and solvent consumptions of coenzyme Q10 under different sample sizes were shown in Table 1.

 

Table 1 Recovery and solvent consumption of coenzyme Q10 at different sample volumes.

 

Packing volume Sample volume Sample ratio Recovery rate				Coenzyme Q11 ( % )	Runtime solvent consumption
( g)	( g)	Example ( % )	( % )
					( min)	( L)
46.6	2.71	5.82	87. 31	0.04	160	2.40
	3.05	6.55	72.44	0.07	168	2.52
	2.45	5.26	88. 65	0.05	157	2.36

 

3.Discussion

3.1 Sample Analysis

Fractions were analyzed with reference to the 2020 edition of the Chinese Pharmacopoeia for Coenzyme Q10 chromatographic conditions. The received fraction contained acetone, which had strong absorption at 275 nm, so it was necessary to take the fraction and dissolve it with anhydrous ethanol after blowing dry under nitrogen gas to a concentration of about 0.5 mg -mL-1, and injected into the sample for 20 μL; the mobile phase was methanol-anhydrous ethanol (1∶1); the column temperature was set at 35 ℃; the detection wavelength was 275 nm; the flow rate was 1.5 mL - min-1; the method could separate the main components from the key impurities, and the separation degree was more than 2.0; in addition, the impurities were all related to coenzyme Q10, and the separation degree was more than 2.0, and the impurities were all related to coenzyme Q10. This method can separate the main components and key impurities, and the separation degree is more than 2.0; in addition, the impurities are similar to the structure of coenzyme Q10, only the number of polymerized isoprene is different, so the corresponding intensity of UV is not much different, and it can be used as a method for the detection of the impurity content.

Fig. 1 Preparative liquid chromatogram of coenzyme Q10 separated by C30 at 275 nm (the red box is the qualified fraction collection window).

Fig. 2 Chromatogram of qualified fraction samples

(14.606 min and 20.186 min for coenzyme Q10 and coenzyme Q11, respectively, with area percent contents of 99.82% and 0.043%, respectively)

 

3.2 Selection of Preparation Conditions   

Based on the patent "a method of purification of coenzyme Q10" (invention patent No. 201810233454.1), the separation conditions of reversed-phase chromatography (RPCC) are C18-bonded silica gel as the chromatographic packing material, methanol-acetone (4:1) as the mobile phase and dissolved samples as the solvent, and the separation is carried out under the condition of about 45 ℃, and the separation effect of the process has been measured, and the effect of the process can be achieved, and coenzyme Q11 can be separated from the sample.

 

In this study, the separation effect of this process was also tested, and it was found that the separation and removal of coenzyme Q11 could be achieved. However, the heating method requires additional electric power and heat source, which is a safety hazard in general laboratory operation, and the same problem exists in industrial production.

 

Considering that coenzyme Q10 is soluble in acetone but poorly soluble in methanol, and that the increase of acetone content increased the solubility but the retention of coenzyme Q10 in C18 was relatively weak, we used to increase the carbon chain of the bonded silica gel from C18 to C30, which is because C30 is more nonpolar and the retention of coenzyme Q10 is relatively better, and the increase of the acetone content could achieve the isolation of coenzyme Q10 in the ambient temperature. This is because C30 is more non-polar and retains coenzyme Q10 better. After adjustment, it was found that the retention time of coenzyme Q10 was 45~120 min when the ratio of acetone to methanol was 4∶1; this retention time was very favorable to increase the sample volume and improve the separation effect.

 

3.3 Selection of Sample Volume   

With reference to the patent requirements, we compared the sample volume of 5.2%, 5.8% and 6.6% of the filler volume, and found that the recovery of qualified samples decreased with the increase of the sample volume, this is due to the overloading of the sample volume sample retention will be significantly reduced, the enrichment of coenzyme Q11 in the immobilization of coenzyme Q10 in a competitive relationship between coenzyme Q11 and coenzyme Q10 has become more obvious, that is, the peaks of Q11 broadened, resulting in a reduction in the qualified sample fraction.

 

Considering that the increase of sample volume from 5.8% to 6.6% resulted in a significant decrease of recovery from 87% to 72%, while the increase of sample volume from 5.2% to 5.8% only resulted in a decrease of recovery from 89% to 87%, we considered that the optimal volume of sample was 5.8%; although the volume of sample was only 50% compared with 10% of the volume of sample of the method reported in the patent, we still considered that this method of separation was relatively easy to operate at room temperature. Although the sample size is only 50% compared with the 10% sample size of the patent method, we still think that this method is relatively safe and energy-saving at room temperature, and it is also an alternative separation process.

 

References:

[1] National Pharmacopoeia Commission, ed. Chinese Pharmacopoeia (2020 Edition, Part II) [S]. Beijing: China Pharmaceutical Science and Technology Press ,2020 :1459.

[2] European Pharmacopoeia, ed. Ubidecarenone [S]:USP38 NF33 :6243.

[3] European Pharmacopoeia Editorial Committee Ubidecarenone [S]: EUROPEAN PHARMCO- POEIA 8.8 :6065.

[4] Huang Yuqing. Research on the extraction process of coenzyme Q10 [D]. Master's thesis of Zhejiang University ,2017.