Urinary excretion study of coenzyme Q10 in rats by ultra-performance liquid chromatography-mass spectrometry.

A method based on ultra-performance liquid chromatography mass spectrometry (UPLC-MS) applying atmospheric pressure chemical ionization in the positive ion mode is developed for the determination of coenzyme Q10 (CoQ10) in rat urine. The assay involves the extraction of crude urine, fast liquid chromatography on a Waters Acquity UPLC BEH C18 column (1.7 microm, 1.0 x 50 mm), and selected ion monitoring detection using mass transition. The calibration range is found to be 0.05-25 microg/mL, with the lower limit of quantitation of 0.05 microg/mL. Intra- and inter-day precision (relative standard deviation) for CoQ10 in rat urine range from 0.7% to 15%, and accuracy expressed in recovery rates in urine is between 83% and 118%. The recovery of this method is found to be between 80% and 95% at three concentrations. The total cumulative recovery of CoQ10 is 1.16 +/- 1.05% (percentage of dose intake, n = 4) from rat urine collected over 30 h after oral administration of the drug. The UPLC-MS method described allows the quick determination of CoQ10 in rat urine with good precision and accuracy. It is suitable for further excretion studies of CoQ10 in animals.

Safety assessment of PureSorb-Q40 in healthy subjects and serum coenzyme Q10 level in excessive dosing.

PureSorb-Q40 (water-soluble type CoQ10 powder, CoQ10 content is 40 w/w%; hereinafter referred to as P40) is reported in the single-dose human and rat studies to have a greater absorption rate and absorbed volume of CoQ10 even taken postprandially, than those of regular CoQ10, which is lipid-soluble and generally taken in the form of soft-gel capsules. Thus, it was anticipated that the serum CoQ10 level might be higher with P40 tablets than with soft-gel capsules, even for the same dose of CoQ10. In the present study, in order to confirm the safety and measure the serum CoQ10 level for the case of an excessive dose of P40, a double-blinded Placebo controlled comparative study was conducted on 46 healthy volunteers and they were randomly divided into two groups. The P40 tablets or placebo were repeatedly taken by the volunteers. As the result of the study, for the group of taking 2250 mg/d of P40 (that is, 900 mg/d of CoQ10) for 4 consecutive wk, the serum CoQ10 level peaked at 2 wk after the start of intake at 8.79 +/- 3.34 microg/mL, and at 4 wk, it was at the level of 8.33 +/- 4.04 microg/mL. At 2 wk from withdrawal of intake, the serum CoQ10 level decreased to 1.30 +/- 0.49 microg/mL. The serum CoQ10 levels at these three points were significantly higher than those of the first day of intake and the Placebo group, which had no significant change throughout the study. Furthermore, P40 intake did not cause any significant changes in symptoms or clinical laboratory results as assessed by physical, hematological, blood biochemical or urinalysis tests. Physician examinations also did not reveal any abnormalities. These results confirm that P40 is an extremely safe material and it can produce better absorption of CoQ10.

Mass spectrometric method for detecting carbon 13 enrichment introduced by folate coenzymes in uric acid.

Using [2-13C]uric acid as a test material, we developed a mass spectrometric procedure that detects and estimates the difference in 13C enrichment at the positions of carbons 2 and 8 of the purine ring. This method could replace radiochemical methods and could trace the incorporation of carbon fragments into the purine ring from 13C-labeled metabolites in humans.

Detection of urinary tract infections by reduction of nitroblue tetrazolium.

BACKGROUND: Nitroblue tetrazolium (NBT) reduction to formazan has been used as a marker for nitric oxide synthase (NOS). Since inducible NOS activity is elevated in urine from patients with urinary tract infections (UTIs), we investigated the accuracy of NBT reduction as an early predictor of UTIs and quantified the relationship between inducible NOS and NBT. METHODS: Urine samples from 434 patients were screened for the presence of UTIs with leukocyte-esterase and nitrite dipsticks and with NBT reduction. The rapid screening results from each test were compared to urine culture results. In addition, NBT reduction parameters were measured in urine pellet at 595 nm after incubation with one of four factors: NOS cofactors, NOS inhibitors, NADH, or superoxide dismutase/catalase. RESULTS: As a urine screening test for UTIs, NBT reduction was more sensitive with a higher negative predictive accuracy than the nitrite dipstick. NBT reduction also was more specific with a higher positive predictive accuracy and negative predictive accuracy than the leukocyte-esterase dipstick. In infected urine pellet, both NADPH, a NOS cofactor, and NADH increased NBT reduction. Superoxide dismutase/catalase decreased NBT reduction. CONCLUSIONS: Although NOS may not be the only NBT reducing enzyme, rapid, visible reduction of NBT is induced in urine from patients with UTIs.

Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry.

A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallop's group for human plasma, adrenal and urine.