The Quality Control Assessment of Commercially Available Coenzyme Q₁₀-Containing Dietary and Health Supplements in Japan

Abstract

Coenzyme Q₁₀ (CoQ₁₀) has been widely commercially available in Japan as a dietary and health supplement since 2001 and is used for the prevention of lifestyle-related diseases induced by free radicals and aging. We evaluated CoQ₁₀ supplements to ensure that these supplements can be used effectively and safely. Commercially available products were selected and assessed by the quality control tests specified in the Japanese Pharmacopoeia XV. When the disintegration time of CoQ₁₀ supplements was measured, a few tested supplements did not completely disintegrate even after incubation in water for an hour at 37°C. In the content test, many samples were well controlled. However, a few supplements showed low recovery rates of CoQ₁₀ as compared to manufacturer’s indicated contents. Among soft capsule and liquid supplements, the reduced form of CoQ₁₀ (H₂CoQ₁₀), as well as the oxidized form, was detected by HPLC with electrochemical detector. The results for experimental formulated CoQ₁₀ supplements demonstrated that H₂CoQ₁₀ was produced by the interaction of CoQ₁₀ with vitamins E and/or C. From these results, we concluded that quality varied considerably among the many supplement brands containing CoQ₁₀. Additionally, we also demonstrated that H₂CoQ₁₀ can be detected in some foods as well as in CoQ₁₀ supplements.

Introduction

It is well known that coenzyme Q (CoQ) serves as an essential carrier for electron transport and proton translocation in the mitochondrial respiratory chain [1]. Besides its role in electron-transfer reactions, CoQ₁₀ serves as a free radical scavenger, thereby preventing oxidative damage in the human body. Several researchers have pointed out that the reduced form of CoQ₁₀ (H₂CoQ₁₀) is an efficient scavenger of lipid radicals and inhibitors of lipid peroxidation in low-density lipoprotein [2, 3], biomembrane [4], and liposomes [5, 6]. CoQ in the bodies of human beings is thought to be provided by both dietary intake, from foods or dietary supplements [7, 8], and de novo biosynthesis [9, 10]. Therefore, decreases in the biosynthesis or intake of CoQ₁₀ might affect the physiological action of CoQ₁₀. In fact, decreased serum levels of CoQ₁₀ were observed in patients undergoing total parenteral nutrition therapy without dietary intake [11], and in patients [12, 13] and animals [14, 15] receiving HMG-CoA reductase inhibitor (statin) administration.

CoQ₁₀ was introduced into clinical therapy in 1974 in Japan. Its generic name is ubidecarenone. Many clinical trials showed that oral administration to patients was effective for mild congestive heart failure symptoms such as edema, lung congestion, and swollen liver. On the basis of these clinical findings, CoQ₁₀ was classified in the group of cardiovascular drugs for metabolic disturbances. In 1991, CoQ₁₀, whose generic name is ubiquinone-10, was also made available as an over-the-counter drug in pharmacies.

While sales of dietary and health supplement products have been rapidly increasing in Japan, it is vital to supply quality-controlled products for consumers. In 2001, the Ministry of Health, Labour and Welfare in Japan permitted the use of CoQ₁₀ as a food additive as long as no claims were made about its pharmacological effectiveness and application. Currently, it is estimated that more than 200 kinds of CoQ₁₀-containing dietary and health supplements (CoQ₁₀ supplements), produced by more than 100 different manufacturers, are available. In particular, CoQ₁₀ supplements are used for the prevention of lifestyle-related diseases induced by free radicals and aging. However, the products may vary in quality. Because CoQ₁₀ has a rather complicated chemical structure and possesses several physical properties, such as low melting point, hydrophobic nature, and light sensitivity, that do not favor large-scale commercial production, highly sophisticated techniques should be employed at all production stages to obtain a satisfactory product [16]. Moreover, many Japanese CoQ₁₀ supplements also contain other components, e.g., vitamins, minerals, amino acids, antioxidative compounds, and enzymes, in the same tablet, soft capsule, or other product. Therefore, the chemical stability of CoQ₁₀ supplements is also an important point to be considered.

In this study, we evaluated the quality of commercially available CoQ₁₀ supplements to ensure that these supplements can be used effectively and safely. In addition, to investigate the distribution of CoQ₁₀ in foods from a nutritional point of view, we also measured both CoQ₁₀ and H₂CoQ₁₀ contents in selected foods.

Materials and Methods

Reagents

Authentic CoQ homologues from CoQ₇ to CoQ₁₁ were kindly supplied by Nisshin Pharma Inc., Tokyo. High performance liquid chromatography (HPLC) solvents and ethanol were purchased (HPLC grade) from Wako Pure Chemical Industries, Ltd., Osaka, Japan. All other chemicals used were of analytical grade, available from commercial suppliers.

CoQ₁₀ supplements

CoQ₁₀ supplements were commercially purchased from pharmacies, health food stores, department stores, convenience stores, and sport goods shops in the Kobe city area. Forms tested in this study included tablet (TB), hard capsule (HCP), soft capsule (SCP), granule (GN), liquid (LQ), jelly (JL), and inclusion complex with κ-dextrin (ICD). The quality control tests of CoQ₁₀ supplements were completed within 6 months before the product’s consume-by date. CoQ₁₀ supplements obtained for testing were preserved in tight, light-resistant containers at room temperature until the quality control tests were conducted.

Experimental manufacturing preparations for SCPs, a LQ, and an HCP of “Formulas 1 to 5”

Experimental manufacturing products for 3 SCP forms, a LQ form, and an HCP form of formulas 1 to 5 were kindly provided by Shiseido Medical, Co., Ltd., Tokyo, Japan. The detailed formulas of their components are shown in Table 1.

Table 1.

Experimental formulas of CoQ₁₀ supplement forms of soft capsule, liquid, and hard capsule

Contents	“Formula 1”	“Formula 2”	“Formula 3”	“Formula 4”	“Formula 5”
Intake form	Soft capsule	Soft capsule	Soft capsule	Liquid	Hard capsule
Based Oil	Safflower oil	Safflower oil	Olive oil	—	—
CoQ10	30 mg	10 mg	30 mg	30 mg	30 mg
Vitamin E	10 mg	30 mg	10 mg	10 mg	10 mg
Vitamin C	30 mg	—	30 mg	30 mg	30 mg

Quality control tests

Disintegration test—This test was performed using the apparatus (Model TMB-81, Toyama Sangyo Co., Ltd., Osaka, Japan) and test conditions specified in the Japanese Pharmacopoeia XV (J.P.XV). Water and the first (pH 1.2) and the second (pH 6.8) fluids of the J.P.XV method were used as the immersion solution at 37 ± 2°C. The time required for each TB, HCP, and SCP to completely disintegrate was recorded, and the mean disintegration time was calculated. Complete disintegration is defined by the J.P.XV as that state in which any residue of the unit, except fragments of insoluble coating or capsule shell, remaining on the screen (0.25–0.31 mm) of the test apparatus is a soft mass having no palpably firm core.

Content test—All procedures for the content test of CoQ₁₀ supplements were carried out in the dark.

i) Pre-preparation of CoQ₁₀ supplements

TB and ICD products were crushed carefully in a mortar to obtain a homogenous fine powder. This powder was transferred completely to a 50-ml brown volumetric flask, and about 30 ml of ethanol was added to the flask.

HCP and SCP products were carefully opened, the contents were transferred completely to a brown 50-ml volumetric flask, and about 30 ml of ethanol was added to the flask. If ethanol-insoluble contents and/or fragments were observed, these were sonicated on ice for 1 min with output 3 to 5 (Ultrasonic Distruptor, Model UR-200P, Tomy Seiko Co, Ltd., Tokyo, Japan).

In the case of LQ products, an aliquot of 5 ml of drink solution was pipetted into a 50-ml brown volumetric flask directly, and about 30 ml of ethanol was added to the flask. For JL and GN products, an equivalent of 5 mg was weighed accurately and dissolved in 10 ml of water and about 20 ml of ethanol in a mortar to obtain a homogenous solution. The solution was completely transferred to a brown 50-ml volumetric flask.

ii) Preparation of CoQ₁₀ supplements

After the ethanol solution was prepared as described above, it was incubated for 10 min at 50°C and allowed to stand for 10 min at room temperature. Then, the ethanol solution was added to the flask again to scale up to 50 ml. The solution was filtered with filter paper (No. 3) and diluted with ethanol to adjust to a 10 µg/ml CoQ₁₀-containing ethanol solution. Finally, except for SCP products, an aliquot of 10 µl of the ethanol solution was injected into the column to determine H₂CoQ₁₀.

In the case of SCP products only, an aliquot of 0.5 ml of 10 µg/ml CoQ₁₀-containing ethanol solution was pipetted into a brown glass-stoppered centrifuged tube, and then 0.5 ml of distilled water, 1.5 ml of ethanol, and 5 ml of n-hexane were added in turn. The mixture was shaken vigorously reciprocally at a rate of 80 times per minute for 10 min and centrifuged at 500 g for 10 min. This extraction procedure was repeated three times. Subsequently, the combined n-hexane layer was concentrated in vacuo under a stream of nitrogen. The resulting residue was dissolved in 0.5 ml of ethanol, and an aliquot of 10 µl of the ethanol solution was injected into the column to determine H₂CoQ₁₀.

Separately, an aliquot of 10 µl of 0.25% sodium borohydride solution (0.25 mg/ml water) was added to 0.4 ml of the ethanol and the mixture allowed to stand for 10 min at room temperature. An aliquot of 10 µl of the solution was injected into the column to determine total CoQ₁₀, the sum of reduced and oxidized CoQ₁₀.

iii) HPLC conditions for determination of CoQ₁₀ and H₂CoQ₁₀

The content of CoQ₁₀ supplements was determined by an HPLC method with electrochemical detection (ECD), as previously described [17]. Authentic CoQ homologues from CoQ₇ to CoQ₁₁ were prepared by dissolution in ethanol to yield concentrations of 10 µg/ml. Authentic CoQ homologues were freshly prepared from the corresponding CoQ, adding 25 µg of sodium borohydride (10 µl of a 0.25% solution of sodium borohydride in water) to give a concentration of 1 µg/ml prior to HPLC-ECD analysis.

Food samples

Food samples were obtained from local food stores or supermarkets in the Kobe city area. All food items were analyzed raw, without freezing. In some cases, samples of the same food were collected on separate days.

Measurement of CoQ homologue contents in foods

Food samples were homogenized with distilled water at 4°C using a Polytron homogenizer (Type PT 10/35; Kinematica, Lucerne, Switzerland) at a setting of 7 to 30 seconds. The final volume of the homogenate was adjusted so as to contain about 1 to 2 µg of H₂CoQ₁₀. An aliquot of 0.5 ml of the homogenate was pipetted into a brown glass-stoppered centrifuged tube, and then 2 ml of ethanol and 5 ml of n-hexane were added. The solution was extracted as described above for SCP products, and the contents of H₂CoQs and CoQs were determined by HPLC-ECD [17].

Results

Internal profile for SCP forms of CoQ₁₀ supplements

Among CoQ₁₀ supplements, SCP is the most common commercially available intake form in Japan. According to the United States Pharmacopoeia, SCPs (sometimes called soft gelatin capsules or softgels) are filled with liquid contents in most cases. Typically, CoQ₁₀ is dissolved or suspended in a liquid vehicle such as vegetable oil or the lower-molecular-weight polyethylene glycols.

First, we assessed the internal profile of different SCP CoQ₁₀ supplement products. Most samples showed the typical internal profile of SCPs. However, as shown in Fig. 1, in a few SCPs, CoQ₁₀ crystallized itself inside and existed in the solid state but not the lipid-soluble form. Moreover, a certain SCP did not show the uniform distribution of CoQ₁₀ in the capsule.

Fig. 1.

Internal appearance of soft capsule forms of CoQ₁₀-supplements. (A) Crystallized CoQ₁₀; the yellowed compound is crystallized CoQ₁₀. (B) Typical soft capsule appearance; CoQ₁₀ distributes uniformly in the soft capsule.

Disintegration tests for TB, HCP, SCP, and ICD forms of CoQ₁₀ supplements

According to the disintegration test specified in the J.P.XV, TB supplements should disintegrate with water as the immersion fluid within 30 min after operating the apparatus. For both HCP and SCP supplements, the time limit is 20 min. Although ICD is the inclusion complex form of CoQ₁₀ and γ-dextrin, the disintegration time is not specified by J.P.XV.

When we measured the disintegration time of 2 kinds of CoQ₁₀-containing over-the-counter (OTC) drugs as controls, their disintegration times were 3.8 min for OTC-TB and 4.6 min for OTC-SCP, respectively. OTC drugs were well controlled and met the necessary requirements specified in the J.P.XV (Table 2).

Table 2.

The disintegration time of CoQ₁₀ supplements, tablets, hard capsules, soft capsules, and inclusion complex with γ-dextrin

Samples*	Disintegration Time (min)
	Water	First fluid (pH 1.2, J.P.XV)	Second fluid (pH 6.8, J.P.XV)
OTC-TB	3.8 ± 0.2	4.1 ± 0.4	6.0 ± 0.4
OTC-SCP	4.6 ± 0.1	6.8 ± 0.1	9.8 ± 0.5
TB-1	11.3 ± 0.9	16.5 ± 0.6	11.1 ± 0.7
TB-2	3.2 ± 0.4	3.6 ± 1.0	2.4 ± 0.1
TB-3	10.7 ± 0.4	12.8 ± 0.6	13.1 ± 0.3
TB-4	11.1 ± 0.1	12.1 ± 0.1	13.4 ± 0.1
TB-5	>60	>60	>60
TB-6	55.2 ± 0.9	56.4 ± 0.4	55.9 ± 0.8
TB-7	4.6 ± 0.6	3.3 ± 0.0	4.1 ± 0.8
TB-8	31.5 ± 2.6	32.1 ± 13.1	33.7 ± 3.9
TB-9	23.4 ± 0.7	32.8 ± 0.5	21.4 ± 0.4
TB-10	11.8 ± 3.2	11.8 ± 1.9	14.8 ± 4.2
TB-11	17.5 ± 2.8	19.6 ± 1.3	16.5 ± 2.0
TB-12	>60	>60	>60
TB-13	18.3 ± 0.2	18.6 ± 0.5	18.8 ± 0.6
TB-14	56.7 ± 1.3	53.4 ± 0.8	58.7 ± 1.6
TB-15	19.6 ± 1.4	18.9 ± 1.5	18.7 ± 1.6
TB-16	9.3 ± 1.0	11.3 ± 0.3	11.6 ± 0.5
TB-17	18.8 ± 1.0	18.6 ± 0.8	25.1 ± 1.9
HCP-1	6.9 ± 1.9	7.2 ± 1.5	17.6 ± 0.4
HCP-2	10.8 ± 0.6	9.9 ± 0.3	11.9 ± 0.7
HCP-3	3.2 ± 0.2	4.0 ± 0.4	3.6 ± 0.3
HCP-4	23.8 ± 3.0	22.5 ± 3.8	13.9 ± 0.9
HCP-5	4.9 ± 0.9	3.2 ± 0.1	5.3 ± 0.9
SCP-1	10.2 ± 1.0	12.9 ± 1.0	11.2 ± 1.0
SCP-2	5.6 ± 0.2	5.6 ± 0.5	10.7 ± 0.5
SCP-3	47.2 ± 1.5	54.9 ± 2.8	57.2 ± 0.9
SCP-4	>60	>60	>60
SCP-5	>60	44.0 ± 2.5	52.7 ± 2.0
SCP-6	>60	>60	>60
SCP-7	>60	>60	>60
SCP-8	41.0 ± 0.9	40.7 ± 2.5	32.6 ± 1.9
SCP-9	19.0 ± 1.5	21.5 ± 0.5	13.8 ± 0.9
SCP-10	>60	>60	>60
SCP-11	14.8 ± 0.7	16.9 ± 1.5	14.0 ± 1.6
SCP-12	>60	>60	>60
SCP-13	11.4 ± 0.7	7.8 ± 0.3	8.0 ± 0.7
ICD-1	32.5 ± 3.4	30.8 ± 2.7	41.6 ± 2.1
ICD-2	29.9 ± 2.1	41.3 ± 1.1	28.1 ± 1.5
ICD-3	12.1 ± 0.8	15.6 ± 1.9	11.6 ± 0.9
ICD-4	30.0 ± 2.2	29.8 ± 0.7	31.4 ± 2.4
ICD-5	26.4 ± 3.3	36.4 ± 2.7	25.1 ± 2.8
ICD-6	8.8 ± 0.7	12.1 ± 0.9	7.6 ± 0.6

*OTC, over-the-counter drug; TB, tablet; HCP, hard capsule; SCP, soft capsule; ICD, inclusion complex with γ-dextrin. Data are expressed as means ± SD (n = 6).

For CoQ₁₀ supplements, as shown in Table 2, the disintegration time was 3.2 min to >60 min for TBs, 3.2 min to 23.8 min for HCPs, 5.6 min to >60 min for SCPs, and 8.8 min to 32.5 min for ICDs; considerable differences were observed among the many supplements. In particular, the disintegration time of about half of the SCPs was more than 60 min. Among the different forms of CoQ₁₀ supplements, the disintegration performance of ICDs was not always faster than that of other forms. Moreover, as tested supplements might be enteric-coated tablets, we also carried out the disintegration tests again for the first fluid, simulated gastric fluid (pH 1.2), and the second fluid, simulated intestinal fluid (pH 6.8). However, the results did not differ from those of water as the immersion fluid.

Content tests for CoQ₁₀ supplements

Although the J.P.XV specifies the HPLC-UV method as the identification and content test for ubidecarenone, we applied the HPLC-ECD method as described in Materials and Methods instead because this method can measure both the reduced and oxidized forms of CoQ₁₀ and shows higher sensitivity than the HPLC-UV method does.

Of course, the content of the over-the-counter drugs used as controls was well controlled within an extremely limited range (99–100%). Most CoQ₁₀ supplements showed a content value of more than 80%. However, a few supplements exhibited low recovery rates of CoQ₁₀ as compared to manufacturers’ indicated contents. Moreover, in several SCP and LQ products, H₂CoQ₁₀ as well as CoQ₁₀ was detected. Interestingly, in LQ-6, as shown in Table 3, CoQ₁₀ was recovered entirely as the reduced form, H₂CoQ₁₀.

Table 3.

Content test of CoQ₁₀ supplements, tablets, hard capsules, soft capsules, liquids, jellies, granules, and inclusion complex with γ-dextrindextrin

CoQ10-supplements*	Content (mg)
	Labeled CoQ10	H2CoQ10 (%)**	Total CoQ10 (%)***
OTC-TB	10	ND****	100.5 ± 0.1 (100)
OTC-SCP	10	ND	99.4 ± 0.2 (99)
TB-1	10	ND	9.6 ± 0.3 (96)
TB-2	6.3	ND	3.4 ± 0.1 (54)
TB-3	11.1	ND	9.4 ± 0.5 (85)
TB-4	1.7	ND	1.6 ± 0.1 (94)
TB-5	5	ND	4.6 ± 0.2 (92)
TB-6	16.7	ND	17.2 ± 0.4 (103)
TB-7	10	ND	9.3 ± 0.4 (93)
TB-8	60	ND	55.6 ± 1.4 (93)
TB-9	20	ND	20.0 ± 0.5 (100)
TB-10	0.34	ND	0.4 ± 0.01 (118)
TB-11	3.75	ND	3.7 ± 0.3 (99)
TB-12	0.83	ND	0.9 ± 0.1 (108)
HCP-1	90	ND	92.3 ± 0.5 (103)
HCP-2	80	ND	4.2 ± 0.1 (5)
HCP-3	30	ND	28.7 ± 1.47 (96)
HCP-4	60	ND	59.3 ± 1.3 (99)
HCP-5	30	ND	30.8 ± 0.9 (103)
SCP-1	33.3	5.8 ± 0.5 (17)	33.8 ± 1.4 (101)
SCP-2	30	14.1 ± 1.0 (46)	30.6 ± 0.9 (102)
SCP-3	30	7.0 ± 0.8 (22)	31.0 ± 1.2 (103)
SCP-4	30	13.5 ± 0.4 (44)	31.1 ± 0.7 (104)
SCP-5	30	13.7 ± 0.7 (52)	26.4 ± 1.0 (88)
SCP-6	35	1.6 ± 0.5 (4)	35.8 ± 0.8 (102)
SCP-7	30	1.8 ± 0.03 (6)	28.0 ± 0.6 (94)
SCP-8	30	25.3 ± 0.7 (88)	28.8 ± 0.6 (96)
SCP-9	150	6.2 ± 0.4 (5)	120.8 ± 3.8 (80)
SCP-10	30	1.4 ± 0.1 (4)	30.7 ± 0.3 (102)
SCP-11	30	3.8 ± 0.7 (12)	30.7 ± 0.9 (102)
SCP-12	50	ND	49.6 ± 1.0 (99)
SCP-13	30	ND	31.1 ± 0.4 (104)
SCP-14	30	5.9 ± 0.7 (19)	31.0 ± 0.5 (103)
SCP-15	10	ND	10.1 ± 0.1 (101)
SCP-16	10	ND	10.0 ± 0.1 (100)
SCP-17	10	1.1 ± 0.2 (14)	8.0 ± 0.6 (80)
SCP-18	30	5.2 ± 0.4 (16)	31.4 ± 0.4 (105)
SCP-19	30	2.6 ± 0.7 (9)	30.4 ± 0.6 (101)
SCP-20	45	3.9 ± 0.2 (8)	46.5 ± 0.7 (103)
SCP-21	15	8.0 ± 0.5 (53)	14.9 ± 0.6 ( 99)
SCP-22	30	3.4 ± 0.2 (12)	27.2 ± 0.9 ( 91)
SCP-23	20	0.9 ± 0.1 (4.2)	20.2 ± 1.1 (101)
SCP-24	60	ND	58.0 ± 4.1 (97)
SCP-25	0.25	0.04 ± 0.1 (16)	0.2 ± 0.1 (96)
LQ-1	40	ND	41.4 ± 1.9 (104)
LQ-2	30	ND	31.7 ± 1.4 (106)
LQ-3	30	ND	32.5 ± 0.3 (108)
LQ-4	1	ND	1.0 ± 0.03 (100)
LQ-5	50	12.3 ± 2.4 (24)	52.1 ± 0.8 (104)
LQ-6	—	5.2 ± 0.1 (—)	5.2 ± 0.1 (—)
JL-1	30	ND	29.3 ± 0.1 (98)
JL-2	50	ND	61.5 ± 4.6 (123)
GN-1	30	ND	28.5 ± 0.3 (95)
GN-2	30	ND	30.4 ± 0.2 (101)
GN-3	30	ND	30.2 ± 0.2 (101)
GN-4	30	ND	30.1 ± 0.1 (100)
ICD-1	3.75 as ICD	ND	1.0 ± 0.01 (27)
ICD-2	84 as ICD	ND	10.4 ± 0.3 (12)
ICD-3	18 as ICD	2.0 ± 0.6 (50)	3.9 ± 0.72 (22)
ICD-4	100 as ICD	ND	21.8 ± 1.4 (22)
ICD-5	12.5 as ICD	ND	0.9 ± 0.1 (7)
ICD-6	100 as ICD	ND	22.8 ± 0.1 (23)

*OTC, over-the-counter drug; TB, tablet; HCP, hard capsule; SCP, soft capsule; LQ, liquid; JL, jelly; GN, granule; ICD, inclusion complex with k-dextrin. Data are expressed as means ± SD (n = 6).

** [H₂CoQ₁₀/total CoQ₁₀ (sum of CoQ₁₀ and H₂CoQ₁₀)] × 100.

*** [Sum of CoQ₁₀ and H₂CoQ₁₀] × 100.

****Not detected.

ICD is the inclusion complex form of CoQ₁₀ and γ-dextrin. The purposes of developing this inclusion complex with CoQ₁₀ seem to be reduction of instability to light and enhancement of bioavailability. However, the manufacturer’s indicated contents were mentioned and listed on the package only as “CoQ₁₀ inclusion complexes with γ-dextrin.” Thus, the CoQ₁₀ content of the product was uncertain. As shown in Table 3, we estimated that the inclusion ratio of CoQ₁₀ to γ-dextrin in the tested ICD will be in the range from 5% to 30%.

H₂CoQ₁₀ production in the SCP and LQ forms of CoQ₁₀ supplements

We recognized that all CoQ₁₀ supplements in which H₂CoQ₁₀ was detected contained vitamin E (α-tocopherol) and/or vitamin C (ascorbic acid). Therefore, we presumed that H₂CoQ₁₀ might be produced by the interaction of CoQ₁₀ with vitamin E and/or vitamin C. To confirm this possibility, we prepared the experimental formulas listed in Table 1.

As shown in Fig. 2, co-existence of CoQ₁₀ and vitamin E and/or vitamin C produced reduction of CoQ₁₀ to H₂CoQ₁₀ in SCPs and a LQ. The content ratio of vitamin E to CoQ₁₀ was also important. The existence of safflower oil in SCPs was also an essential condition for production of H₂CoQ₁₀ from CoQ₁₀ in SCPs. In particular, a LQ form of CoQ₁₀ that co-existed with vitamin E and vitamin C exhibited a high ratio of the reduced form to total CoQ₁₀ in a time-dependent manner. In an HCP, however, the reduction of CoQ₁₀ to H₂CoQ₁₀ was not observed even if CoQ₁₀ co-existed with vitamin E and C.

Fig. 2.

H₂CoQ₁₀ produced by interaction of CoQ₁₀ with vitamins C and/or E in soft capsule and liquid. Each formula is shown in Table 1.

H₂CoQ₁₀ and CoQ₁₀ contents in foods

To determine the dietary intake of H₂CoQ₁₀ as well as CoQ₁₀ in human beings, we analyzed the CoQ₁₀ contents of raw foods. Table 4 shows both the CoQ₁₀ and H₂CoQ₁₀ contents of meats and vegetables. The greater part of the CoQ homologues in meats and vegetables was CoQ₁₀. Moreover, H₂CoQ₁₀ as well as CoQ₁₀ was detected in all examined samples. In particular, squid and eel meats showed high ratios of the reduced form, H₂CoQ₁₀, as compared to other animal products. H₂CoQ₉ and CoQ₉ were also detected in small amounts in beef, pork, and chicken.

Table 4.

H₂CoQs and CoQs contents in foods

Samples	CoQ9	(µg/g wet weight)	CoQ10	(µg/g wet weight)
	H2CoQ9 (%)*	Total CoQ9**	H2CoQ10 (%)*	Total CoQ10**
Meats:
Beef
sirloin	0.1 ± 0.1 (17)	0.6 ± 0.1	1.8 ± 0.2 (6)	30.6 ± 1.4
tenderloin	ND***	ND	1.8 ± 0.2 (7)	26.5 ± 1.2
Pork
sirloin	0.1 ± 0.1 (14)	0.7 ± 0.2	0.6 ± 0.2 (4)	14.0 ± 0.6
heart	ND	ND	6.7 ± 1.9 (6)	118.1 ± 12.2
liver	0.3 ± 0.1 (17)	1.8 ± 0.3	11.1 ± 1.6 (21)	54.0 ± 5.7
Chicken
chest	ND	ND	0.9 ± 0.1 (5)	16.6 ± 1.6
heart	ND	ND	5.0 ± 1.2 (4)	123.2 ± 7.2
liver	0.2 ± 0.1 (12)	1.7 ± 0.4	18.9 ± 3.0 (16)	116.2 ± 6.2
Salmon	ND	ND	1.2 ± 0.3 (16)	7.6 ± 0.9
Eel	ND	ND	4.6 ± 1.6 (62)	7.4 ± 1.8
Squid	ND	ND	2.3 ± 0.2 (60)	3.8 ± 0.8
Vegetables:
Chinese cabbage	ND	ND	0.4 ± 0.1 (15)	2.7 ± 0.4
Eggplant	ND	ND	0.2 ± 0.1 (9)	2.2 ± 0.6
Parsley	ND	ND	7.5 ± 0.8 (28)	26.4 ± 1.9

Data are expressed as means ± SD (n = 5).

* [H₂CoQn/total CoQn (sum of CoQn and H₂CoQn)] × 100 (%).

**Sum of CoQn and H₂CoQn.

***Not detected.

Discussion

Although CoQ₁₀ supplements have not been required to meet the same rigorous product quality performance standards as medicines do, impaired product performance, such as failure to disintegrate in the gastrointestinal tract, might limit the absorption of CoQ₁₀. However, standardized guidelines and methods for assessing the quality control of dietary and health supplements have not been established in Japan. So, we applied the Japanese Pharmacopoeial Convention General Tests to evaluate the quality of CoQ₁₀ supplements in this study.

Externally administered CoQ₁₀ appears to be converted to H₂CoQ₁₀ in the human body and then to act as an antioxidant against lipid peroxidation. In fact, a CoQ₁₀ supplement given to a human increases the serum level of H₂CoQ₁₀ as well as CoQ₁₀ [18]. The enzymes responsible for the reduction of CoQ₁₀ to H₂CoQ₁₀ have been reported to be not only mitochondrial respiratory enzymes but also NADPH-CoQ reductase [19–21], NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) [22, 23], thioredoxin reductase [24], and lipoamide dehydrogenase [25, 26]. CoQ in the human body is thought to be provided by both dietary intake from foods and/or dietary supplements and biosynthesis de novo.

In this study, we tested two quality control tests, disintegration and content tests. The disintegration profile and content test are thought to be essential parameters relative to absorption and intake efficacy. Many commercially available CoQ₁₀ supplements in Japan were well controlled and met the requirements specified by J.P.XV. However, a few products exhibited insufficient quality performance. In particular, the disintegration of many SCPs required more than 60 min. The gelatin shell of an SCP is somewhat thicker than that of an HCP shell and is plasticized by the addition of a polyol such as sorbitol or glycerol. The ratio of dry plasticizer to dry gelatin determines the hardness of the shell and may be varied to accommodate environmental conditions as well as the nature of the contents. Therefore, the disintegration time of SCPs may have a higher score than that of HCPs. In this study, we demonstrated that the quality of the many supplement products containing CoQ₁₀ in Japan varies considerably, and thus, we recommend introducing simple quality control tests for CoQ₁₀ supplements.

Among CoQ₁₀ supplements, in SCPs and LQs, H₂CoQ₁₀ as well as CoQ₁₀ was detected by the HPLC-ECD method. The results for experimentally formulated CoQ₁₀ supplements suggested that H₂CoQ₁₀ was produced by the interaction of CoQ₁₀ with vitamins E and/or C. Moreover, although H₂CoQ₁₀ is unstable when exposed to air, and can easily be oxidized into oxidized CoQ, chemically unstable H₂CoQ₁₀ is thought to exist in the body to serve as an antioxidant. Recently, Yan et al. have reported [27] that dietary supplementation with H₂CoQ₁₀ decreased the degree of senescence in middle-aged SAMP1 mice. H₂CoQ₁₀ produced by the interaction of CoQ₁₀ with vitamins E and/or C, therefore, may exert beneficial effects in the human body.

Many investigators have reported that exogenous CoQ₁₀ exhibits useful health effects. If humans consume H₂CoQ₁₀ as well as CoQ₁₀ in meals every day, foods containing H₂CoQ₁₀ and CoQ₁₀ might have some physiological activities. Therefore, it is important to investigate the distribution of these substances in foods. Some investigators have reported the CoQ homologue contents in foods. However, they could not analyze reduced forms of CoQ homologues because of the HPLC-UV method and insufficient sensitivity. In our study, we measured the distribution of both CoQ₁₀ and H₂CoQ₁₀ in food items. The results clarify that humans consume not only CoQ₁₀ but also H₂CoQ₁₀ from foods. In other words, the present study confirms the human intake of H₂CoQ₁₀ in daily foods and dietary CoQ₁₀ supplements.