OBJECTIVE: To determine the blood concentration of coenzyme Q 10 by high performance liquid chromatography (HPLC) and to compare the bioavailability of two types of coenzyme Q 10 tablets in humans. Methods: Ten healthy male subjects were orally administered with 20 mg of Coenzyme Q 10 tablets 3 times a day for 7 days, and 60 mg of Coenzyme Q 10 tablets were administered on the morning of d8 in a crossover test. Serum Coenzyme Q 10 concentration was measured in venous blood before, 1, 2, 3, 4, 6, 8, and 12 hours after dosing on the morning of d 8. A second test was performed 2 wk later, in which the same dose of the control or the test product was administered as a crossover.
RESULTS: The main pharmacokinetic parameters of the test article were AUC = ( 5.9 ± 1.5) and AUC = ( 5.5 ± 1.5), respectively. 9 ± 1 . 78) μg -h -ml 1 1, cm ax = . 66 ± 0 . (17) μg - ml 1 1 , T peak = . 00 ± 1 . (25) h; the parameters for the control were AUC = (6 . 30 ± 2 . 09) μg - h - ml i 1 , cm ax = . 70 ± 0 . (20) μg - ml i 1 , T peak = . 60 ± 1 . 58) h . There were no significant differences in AUC, cm ax and T peak between the two preparations.
CONCLUSION: The relative bioavailability of Coenzyme Q 10 test tablets and control tablets was 93.9%. The relative bioavailability of Coenzyme Q 10 test tablets and control tablets was 93.9%, and the equivalence analysis showed that the two preparations were bioequivalent.
Coenzyme Q 10 is found in the inner membrane of the mitochondria of plants and animals, mainly humans, and acts as a hydrogen transporter in the respiratory chain, activates many enzymes, and has been widely used as a metabolic cardiotonic agent. Coenzymes have been used in acute and chronic hepatitis, mild and moderate congestive heart failure and so on. In accordance with the requirements for the declaration of new drugs in China, we investigated the pharmacokinetics of the two coenzyme Q 10 tablets in humans according to the crossover protocol, conducted comparative bioavailability tests, and referred to the relevant domestic and foreign literature [1~3] to establish the HPLC method for the determination of coenzyme Q 10 in serum, and to carry out the bioequivalence study of the two tablets.
1 Materials and Methods
1 . 1 Drugs and Instruments
Coenzyme Q 10 tablets (test product): provided by Tianjin Zhengan Pharmaceutical Co., Ltd, each tablet contains 10 mg of Coenzyme Q 10, batch no. 980401; Nengqilang tablets (control product): produced by Shenyang Eisai Pharmaceutical Co., Ltd, each tablet contains 10 mg of Coenzyme Q 10, batch no. 980701; methanol, anhydrous ethanol was excellent pure, n-hexane, lithium perchlorate were analytically pure, and the water was ultrapure water. Shimadzu LC-10A high performance liquid chromatography system, equipped with full auto-sampler, column thermostat, chromatographic workstation and ultraviolet detector, Mill-Q Ⅱ ultrapure water purifier, Milli-pore Company, Japan. LD5-2A medical centrifuge, Beijing Medical Instrument Factory, KH-851 oscillator, Shanghai Huanyu Instrument Factory.
1 . 2 Methods of Blood Concentration Determination
1 . 2 . 1 Chromatographic conditions
Column : C18, ODS, 10 μm, ( 250 × 4.6 ) mm ( L × ID ); mobile phase : methanol : anhydrous ethanol ( 1 : 9 ) ( v /v ) with 0.5 μm 6) mm ( L × ID) ; mobile phase : methanol : anhydrous ethanol ( 1 : 9) (v /v) containing 0.02 mol . 02 mol . L I 1 lithium perchlorate; flow rate .
1 . 0 ml . m in 1; column temperature: 30 ℃; detection: Uv 275 nm. The retention time (tR) of Coenzyme Q 10 was 8.5 m in, and the chromatogram is shown in Fig. 1. The chromatogram is shown in Fig. 1.
1 . 2 . 2 Serum processing methods
Referring to the literature method [1 , 2 ] and improving it, the extraction condition of coenzyme Q 10 in serum was determined as follows: take 1 ml of serum containing coenzyme Q 10, add 1.8 ml of anhydrous ethanol and n-hexane, and then add 1.8 ml of coenzyme Q 10 into the serum. 8 ml of anhydrous ethanol and n-hexane
4 . 4.5 ml, shake 3 times for 30 s, centrifuge at 3 000 rpm for 5 m in, remove 3.6 ml of the organic phase, and dry in a water bath at 30 ℃ with high purity nitrogen. 6 ml of the organic phase was evaporated with high purity nitrogen in a water bath at 30 °C. The residue was dissolved in 200 μl of anhydrous ethanol. The residue was dissolved in 200 μl of anhydrous ethanol and analyzed in 25 μl of sample.
1 . 3 Bioavailability determination in healthy humans
1 . 3 . 1 Subjects
Healthy male subjects, age (29 ± 5) years, height (172 ± 3) cm, weight (66 ± 5) kg. Subjects underwent a complete physical examination with normal liver and renal function and electrocardiogram, and met the test requirements. They had not taken any other medication for 2 weeks prior to the trial, had a standardized meal, were non-smokers, and did not consume ethanol-containing beverages. In accordance with the principles of the Declaration of Helsinki, all subjects signed a volunteer informed consent form with the trial administrator and witnesses before the trial in order to protect the rights of the subjects.
1 . 3 . 2 Test program
Due to the presence of a certain amount of coenzyme Q 10 in the body, the increase in blood coenzyme Q 10 after a single dose is limited. In order to accurately determine the change in body concentration of oral coenzyme Q 10, a crossover study was conducted in which a low dose was administered for 7 days, and then a higher dose was administered after the body concentration of coenzyme Q 10 had stabilized.
1 . 3 . 3 Administration and sampling
Ten healthy male subjects were randomly divided into two groups of 5 each. In the first trial, 5 persons in group 1 took the test product, d 1~7, 20 mg three times a day, 60 mg per day, d 8, 60 mg at 8:00 a.m.; 5 persons in group 2 took the same dose of the control product in the same way. 2 wk later, the second trial was carried out, and they crossed over to take the same dose of the control product or the test product. Each subject should take the drug with 200 ml of warm water after meal every day. 4 ml of venous blood should be collected before and 1, 2, 3, 4, 6, 8, and 12 hours after taking the drug on the morning of D8, and then centrifuged to separate the serum, frozen at -20℃, and then the concentration of Coenzyme Q 10 in serum should be measured within 1 wk. Observe and record the adverse reactions of each subject during the test.
1 . 4 Data processing
The mean and standard deviation of the blood concentration data from 10 subjects at different times were determined. The main pharmacokinetic parameters were calculated for each subject (T Peak and cm ax were calculated from measured values, and AUC was calculated using the trapezoidal method). Relative bioavailability was calculated for each subject based on the cumulative concentration-time curve (AUC) of the test and control cross-dosed to each subject. Fr = AUC test/ AUC control × 100%. In accordance with the FDA and WHO guidelines for bioequivalence testing, the new drug statistic procedure (NDST 4.1) compiled by Prof. Sun Ruiyuan et al. was used for AUC and cm ax. 1) two-way one-sided t-test for AUC and cm ax, and non-parametric method (Wilcox on method) for T Peak, and the equivalence analysis results of these three parameters were used to determine whether the test product and the control product were equivalent.
2 Results
2 . 1 Certification of assay methods
2 . 1 . 1 Preparation of calibration curves
Take 20 μg · ml-1 coenzyme Q10 standard solution 5, 10, 25, 50, 100, 125 μl, evaporate to dryness under nitrogen, add 1 ml serum each, and you have0 . 1、0 . 2、0 . 5、 1 . 0 . 1, 0 . 2, 0 . 5, 1 . 0, 2 . 0.1, 0.2, 0.5, 1.0, 2.0, and 2.0. 5 μg ml-1 solution was processed and analyzed according to the proposed serum treatment method. The concentrations were regressed on the peak area of coenzyme Q 10 to obtain a calibration curve:
Y = - 505188 + 842109X ( r = 0 . 9998)
The concentration range of this method is 0 . 1 ~ 2 . 5 μg -ml- 1, sensitivity 0.1 g -ml- 1. 1 g -ml- 1.
2 . 1 . 2 Recovery and precision
The results of Coenzyme Q 10 recovery and precision tests after extraction from serum are shown in Table 1 . The recoveries of Coenzyme Q 10 in the range of 0 . 1 -2 . 2.5 μg ml-1 , the relative standard deviations (SD) of the intra- and inter-day measurements were within 10%. Table 1 Absolute recovery and precision (n = 5)
Labeled concentration/μg -ml - 1 | Relative error/% | Recovery rate/% | |
in a few days | daytime | ||
0 . 1 | 3 . 9 | 3 . 3 | 95 . 3 |
0 . 5 | 8 . 5 | 8 . 0 | 93 . 6 |
2 . 5 | 3 . 2 | 7 . 2 | 99 . 6 |
2.2 Serum drug concentration and bioavailability
3.The blood concentration-time curves of the two preparations in 10 healthy male subjects after a single dose of the test and control are shown in Figure 2, and the main pharmacokinetic parameters are summarized in Table 2.
Table 2 Pharmacogenetic parameters after oral administration of Coenzyme Q10tablets test and control to subjects (x ± s , n = 10)
parameters | test item | control product |
cm ax /μg . ml- 1 | 0 . 66 ± 0 . 17 | 0 . 70 ± 0 . 20 |
AUC /μg . h . ml- 1 | 5 . 91 ± 1 . 78 | 6 . 30 ± 2 . 09 |
Tpeak/h | 4 . 00 ± 1 . 25 | 4 . 60 ± 1 . 58 |
F /% | 93 . 9 ± 13 . 4 |
2.3 Bioequivalence analysis
2. 3. 1 Statistical analysis of AUC The AUC data of the two preparations were analyzed by ANOVA and two-way one-sided t-test, and the results of the low side one-sided t-test t 1 = 2. 958, the high side one-sided t-test t 2 = 5. 571, and the t0 .05 = 1. 858 showed that the low side one-sided and the high side one-sided were significant (p < 0.05) and proved the equivalence of the AUC of the two preparations.
2. 3. Statistical analysis of 2 cm ax
ANOVA and two-way one-sided t-test were performed on the cm ax data of the two formulations, and the results of the low side one-sided t-test t 1 = 2. 845, high side one-sided t-test t 2 = 5. 192, t0 .05 = 1. 858 indicated that both the low side one-sided and high side one-sided were significant (p < 0.05), which proved that the cm axes of the two formulations were equivalent.
2. 3. 3 Statistical analysis of T peaks
The T peak data of the two preparations were statistically analyzed by the nonparametric-Wilcox on method. 8673) < u 0 .05 ( 1. 96), indicating that the difference in the time to peak between the two preparations was not significant, and it was concluded that the parameters were equivalent.
The equivalence of AUC, cm ax and T peak of the two preparations was evaluated, and it was proved that Coenzyme Q10 tablets and Nengqilang tablets were equivalent.
3 Discussion
The ultraviolet absorption of coenzyme Q 10 has two peaks at 213 nm and 275 nm, in which the absorption value at 213 nm is three times of that at 275 nm. However, at the wavelength of 213 nm, there are many interfering factors in the blood, which affects the determination of the samples, and therefore, the wavelength of 275 nm was chosen as the detection wavelength. When choosing the mobile phase, the chromatographic behaviors of coenzyme Q 10 were compared with those of methanol and ethanol, methanol and n-hexane, methanol and water in different ratios, and the final mobile phase was determined to be methanol : anhydrous ethanol (1:9) (v/v) containing 0.02 mol. 02 mol . L-1 lithium perchlorate.
In addition, Coenzyme Q 10 is poorly photostable and should be protected from light in the assay. The bioavailability of two Coenzyme Q 10 tablets at the same dose in multiple administration showed that the relative bioavailability of the test product was 93.9%. The relative bioavailability of the test product was 93.9%. The blood concentrations of both the test and control reached the peak at 4 h after administration, both were (0.62 ± 0.62%), and the blood concentration of the test and control reached the peak at 4 h after administration, both were (0.62 ± 0.62%). 62 ± 0 . 21) μg.ml-1 . The concentration-related parameters were analyzed by ANOVA, which showed that there was no significant difference in the bioavailability of AUC, cm ax and T peak between the two preparations, and it was proved that CoQ 10 tablets and its control product were equivalent by the equivalence evaluation of AUC, cm ax and T peak of the two preparations.
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