Drugs possessing aryloxypropanamine pharmacophore, duloxetine, dapoxetine and propranolol, increase allopregnanolone in rat brain: Possible involvement of allopregnanolone in their central nervous system effects.

Allopregnanolone (AP) is a neurosteroid synthesized in the brain and a positive allosteric modulator of γ-aminobutyric acid (GABA) type A receptors. Some drugs possessing the aryloxypropanamine (AOPA) pharmacophore, such as fluoxetine, exert their central nervous system (CNS) effects by increasing the brain AP. Although duloxetine (DLX), dapoxetine (DPX), atomoxetine (ATX) and propranolol (PRL) also possess the AOPA pharmacophore and are used to treat some psychiatric disorders, the capabilities of these drugs to increase the brain AP and the possible involvement of AP in their CNS effects remain to be fully elucidated. To clarify these points, we first developed a method for quantifying AP in the rat brain by liquid chromatography/electrospray ionization-tandem mass spectrometry. Analysis of the changes in the brain AP levels using this method revealed that the intraperitoneal administration of DLX (10 mg/kg), DPX (10 mg/kg) and PRL (20 mg/kg) significantly increased the brain AP (DLX: < 0.40-2.74 ng/g tissue, DPX: 1.48-3.83 ng/g tissue and PRL: < 0.40-2.09 ng/g tissue) compared to the saline administration (<0.40 ng/g tissue). These results suggested the possible involvement of the GABAergic neurosteroid, AP, in the central actions of DLX, DPX and PRL. In contrast, ATX (10 mg/kg) did not affect the AP levels in the brain. In addition, the brain and serum AP levels had a remarkably high positive correlation after the administration of DLX, DPX and PRL. Thus, this study proposed the AP-related novel mechanism of actions of DLX, DPX and PRL in the CNS.

Evaluation of thumbnail clipping as a specimen for retrospectively assessing average production of testosterone.

BACKGROUND: The chronic abnormal production of testosterone (T) is associated with many disorders in men. Fingernail clippings might be more suited for the diagnosis and medium-to-long term therapeutic monitoring for the T-related chronic disorders than the blood-derived specimens. The objective of this study was to characterize a thumbnail clipping as the specimen for assessing the several months-old T status. METHODS: Thumbnail clippings from various subjects were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry to evaluate the gender difference, and changes caused by aging and androgen deprivation therapy (ADT) in the thumbnail T concentration. RESULTS: There was an evident gender difference in the thumbnail T concentrations [male; 2.55 ± 0.85 ng/g and female; 0.48 ± 0.29 ng/g, mean ± SD (n = 25 each), Welch t-test]. The thumbnail T concentrations significantly decreased with age in men (n = 268, Scheffé F-test), which was similar to those of the free or bioavailable T in serum/plasma. The thumbnail T concentrations sharply decreased by a 6-months ADT (especially the effect of the luteinizing hormone-releasing hormone agonist/antagonist) for patients with prostate cancer (n = 10). CONCLUSIONS: The thumbnail clipping can be a specimen to retrospectively assess the average T production.

23,25-Dihydroxyvitamin D₃ is liberated as a major vitamin D₃ metabolite in human urine after treatment with ß-glucuronidase: Quantitative comparison with 24,25-dihydroxyvitamin D₃ by LC/MS/MS.

The complete understanding of the excretion of surplus 25-hydroxyvitamin D₃ [25(OH)D₃] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] 24-glucuronide was identified as a major urinary vitamin D₃ metabolite, while the glucuronide of 23,25-dihydroxyvitamin D₃ [23,25(OH)₂D₃] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH)₂D₃ liberated in urine by the treatment with ß-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH)₂D₃ in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH)₂D₃ and 24,25(OH)₂D₃ liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH)₂D₃ was almost as much as that of 24,25(OH)₂D₃, in contrast to the fact that the plasma concentration of 23,25(OH)₂D₃ was much lower than that of 24,25(OH)₂D₃. These results strongly suggested that 23,25(OH)₂D₃ is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH)₂D₃. Furthermore, the amount ratios of 23,25(OH)₂D₃ to 24,25(OH)₂D₃ in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin D₃ supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)D₃.

An LC/MS/MS method for quantifying testosterone and dehydroepiandrosterone sulfate in four different serum samples during a single run.

Simultaneous measurements of the circulating testosterone (TS) and dehydroepiandrosterone sulfate (DHEAS) are deemed to be helpful for the assessment of men's health. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) is the most reliable methodology for this purpose; however, it has room for improvement in analysis throughput. In this study, a quadruplicate of the Girard reagents was used to develop an LC/ESI-MS/MS method capable of quantifying TS and DHEAS in four different serum samples in a single run. The four serum samples were separately pretreated, derivatized with one of four Girard reagents, and then combined. The LC/ESI-MS/MS analysis of the combined sample provided the androgen concentrations of four serum samples in parallel. The method had practical measuring ranges, in which good precision and accuracy, as well as negligible matrix effects were verified. The speed-up capability of the developed method was evaluated through the analysis of ten batches of serum samples (total 40 samples); the method saved a 60% post-pretreatment analysis time compared to the non-derivatization method for 40 samples.

A method for determination of aldosterone concentrations of six adrenal venous serum samples during a single LC/ESI-MS/MS run using a sextet of Girard reagents.

The accurate quantification of aldosterone (ALD) in adrenal tributary venous serum/plasma collected by a super-selective adrenal venous sampling (ssAVS) technique has become a definitive procedure to identify the forms (laterality) of primary aldosteronism (PA) and the location of the affected segment(s), and to subsequently make the treatment decision. In this study, a stable isotope-dilution LC/ESI-MS/MS-based method was developed and validated for the determination of the ALD concentrations of the six ssAVS serum samples during a single run. A sextet of Girard reagents was used to convert ALD to the derivatives having different masses for each sample. Based on the validation tests, satisfactory specificity, precision and accuracy were observed and the matrix effect was found to be acceptable. The developed method was used for the analysis of the ssAVS serum samples from eight PA patients. The batch analysis of the six samples permitted the direct comparison of the ALD concentrations between the samples and enhanced to determine the laterality and location of the affected segment(s). The total analysis time after the sample purification was also reduced to 1/3 for the 24 samples by the sample-sextuplex procedure, which was also an advantage of the developed method.

Simultaneous Determination of Five Bile Acids as Potential Biomarkers for Alzheimer's Disease in Mouse Brain and Plasma.

In this study, we developed an analytical method using LC-MS/MS for the simultaneous determination of five bile acids (BAs) that have been recently reported as candidate diagnostic biomarkers for Alzheimer's disease (AD) or AD related factors in the brain. The measurement of BAs in the brains of healthy mice led to the determination of candidate diagnostic markers for AD, such as cholic acid and deoxycholic acid, and other bile acids, such as chenodeoxycholic acid noted for the ameliorating effect on the symptoms of AD. Significant positive correlations were observed between the brain and plasma concentrations of four BAs in healthy young mice. These results indicate that the BA level in the brain may be estimated by the corresponding BA level in the plasma. Thus, our study suggested that the proposed method for the analysis of the five bile acids would aid in the diagnosis of AD or in studies that use AD model mice.

Derivatization-based quadruplex LC/ESI-MS/MS method for high throughput quantification of serum dehydroepiandrosterone sulfate.

The quantification of the circulating dehydroepiandrosterone sulfate (DHEAS) might be of diagnostic help for several diseases. For the DHEAS quantification, LC/ESI-MS/MS has the advantage of a high specificity compared with immunoassay, whereas LC/ESI-MS/MS has room to improve the analysis throughput. One of the promising solutions to enhance the analysis throughput is sample-multiplexing in the same injection, which can reduce the total LC/ESI-MS/MS run time. In this study, a quadruplex LC/ESI-MS/MS method was developed to quantify DHEAS in four different serum samples in a single run. After the four samples were separately deproteinized and derivatized with one of four Girard reagents (Girard reagent T, P and their isotopologs), the resulting samples were mixed, then injected into the LC/ESI-MS/MS. The applicability and advantage of the developed method were evaluated based on the analysis of nine batches of serum samples from healthy subjects (total 36 samples). The limit of quantitation was 0.050 µg/ml, which was sensitive enough for clinical laboratory use. The method was precise (intra- and inter-assay RSDs ≤ 3.6%), accurate (94.4-108.1%) and robust for the matrix effects. The analysis time was also shortened by about 60% for 36 samples by the introduced method compared with the conventional method.