Development of a high-performance liquid chromatography-tandem mass spectrometric method for the determination of Methimazole in human blood matrices.

Methimazole (MMI, 1-methyl-2-mercaptoimidazole) is widely used for the treatment of hyperthyroidisms. There are methods available for the measurement of MMI concentration in human serum or plasma from the past, but none meet the current regulatory standards for bioanalytical method validations. In this paper, we developed and validated a total MMI measurement method using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technique that conforms to current bioanalytical method validation. To form a free sulfhydryl group on MMI, sodium bisulfite was added to 50 µl of plasma or serum samples containing MMI before the derivatization step. The internal standard (MMI-D3) was spiked into samples, then these samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole. After derivatization, these samples were extracted by supported liquid extraction. Then, the organic solvent was evaporated and the residue was dissolved in 50% methanol and injected into the LC-MS/MS system. A calibration curve was plotted over the concentration range 1-1000 ng/mL (r2 = 0.999). The intra-day and inter-day precisions were less than 10.2% and 9.8%, respectively. The intra-day and inter-day accuracies were between 89.5% and 101.1%, and 96.0% and 99.7%, respectively. The long-term stability of samples showed good precision and accuracy. The validated method was successfully applied to determine serum total MMI concentration in Graves' disease patients after oral administration of 5, 10 or 15 mg MMI. The range of circulating total MMI concentrations was found to be between 2.69 and 304.27 ng/mL in this study. It was shown that the measured serum total MMI concentrations changed in a dose-dependent manner.

Unconjugated bile acids in rat brain: Analytical method based on LC/ESI-MS/MS with chemical derivatization and estimation of their origin by comparison to serum levels.

Although some studies have revealed the implication of bile acids (BAs) and neurological diseases, the levels and origin of the BAs in the brain are not fully understood. In this study, we first developed and validated a sensitive and specific method for the determination of three unconjugated BAs [cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)] in the rat brain by liquid chromatography/electrospray ionization-tandem mass spectrometry combined with chemical derivatization. The measured brain concentrations (mean±standard deviation, n=10) of normal rats were 58.7±48.8, 14.2±11.7 and 13.2±8.7ng/g tissue for CA, CDCA and DCA, respectively. For their origin, we developed the hypothesis that they might be mostly derived from the periphery. To test this hypothesis, the brain BA levels were compared with the serum levels. The brain levels had high correlations with the serum levels, and were always lower than the serum levels for the three unconjugated BAs. Furthermore, the higher brain-to-serum concentration ratios were found for the BAs with higher logD values (higher lipophilicity). Moreover, the brains of the rats intraperitoneally administered with deuterium-labeled CA and CDCA were also analyzed; the deuterium-labeled BAs were detected in the brain of the rats administered with these compounds. Based on all the results, we concluded that the BAs found in the brain are mostly derived from the periphery and the major mechanism for the transportation of the unconjugated BAs to the brain is by passive diffusion.

Correction: Correlation between Serum Levels of 3,3',5'-Triiodothyronine and Thyroid Hormones Measured by Liquid Chromatography-Tandem Mass Spectrometry and Immunoassay.

[This corrects the article DOI: 10.1371/journal.pone.0138864.].

Correlation between Serum Levels of 3,3',5'-Triiodothyronine and Thyroid Hormones Measured by Liquid Chromatography-Tandem Mass Spectrometry and Immunoassay.

OBJECTIVE: For measuring serum 3,3',5'-triiodothyronine (rT3) levels, radioimmunoassay (RIA) has traditionally been used owing to the lack of other reliable methods; however, it has recently become difficult to perform. Meanwhile, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been attracting attention as a novel alternative method in clinical chemistry. To the best of our knowledge, there are no studies to date comparing results of the quantification of human serum rT3 between LC-MS/MS and RIA. We therefore examined the feasibility of LC-MS/MS as a novel alternative method for measuring serum rT3, thyroxine (T4), and 3,5,3'-triiodothyronine (T3) levels. METHODS: Assay validation was performed by LC-MS/MS using quality control samples of rT3, T4, and T3 at 4 various concentrations which were prepared from reference compounds. Serum samples of 50 outpatients in our department were quantified both by LC-MS/MS and conventional immunoassay for rT3, T4, and T3. Correlation coefficients between the 2 measurement methods were statistically analyzed respectively. RESULTS: Matrix effects were not observed with our method. Intra-day and inter-day precisions were less than 10.8% and 9.6% for each analyte at each quality control level, respectively. Intra-day and inter-day accuracies were between 96.2% and 110%, and between 98.3% and 108.6%, respectively. The lower limit of quantification was 0.05 ng/mL. Strong correlations were observed between the 2 measurement methods (correlation coefficient, T4: 0.976, p < 0.001; T3: 0.912, p < 0.001; rT3: 0.928, p < 0.001). CONCLUSIONS: Our LC-MS/MS system requires no manual cleanup operation, and the process after application of a sample is fully automated; furthermore, it was found to be highly sensitive, and superior in both precision and accuracy. The correlation between the 2 methods over a wide range of concentrations was strong. LC-MS/MS is therefore expected to become a useful tool for clinical diagnosis and research.

Development of analytical method for simultaneous determination of five rodent unique bile acids in rat plasma using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry.

Bile acids (BAs) are crucial for the diagnosis, follow-up, and prognostics of liver injuries and other BA metabolism related diseases. In particular, rodent unique BAs, α-muricholic acid (α-MCA), ß-MCA, ω-MCA, tauro-α-MCA (α-TMCA), and ß-TMCA, are valuable biomarkers for preclinical drug development. To the best of our knowledge, however, a simple, selective, sensitive, and robust analytical method for ω-MCA and taurine-conjugated MCAs has never been reported. We have developed a simple, selective, and sensitive analytical method for measurement of 16 BAs including the five rodent unique BAs in rat plasma using an ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method. Activated charcoal was utilized to prepare BA-free plasma, which served as the surrogate matrix for the preparation of calibration standards and quality control (QC) samples. Results of matrix effects evaluation suggested that the BA-free plasma could be adequate as a surrogate matrix for BAs determination. Three stable isotope labelled internal standards were separated by reverse phase UPLC using gradient elution and were detected by TOF-MS in negative ion mode. The calibration curve was linear for all BAs over a range of 10-25ng/mL to 1000-10,000ng/mL, with overall imprecision below 15% and 20% at lower limit of quantification (LLOQ), respectively. This analytical method was used to determine BA concentrations in more than 300 plasma samples from rats with liver injuries induced using α-naphthylisocyanate, carbon tetrachloride, or flutamide. The alteration of BA concentrations was most evident for necrosis, and cholestasis hepatotoxins, with more subtle effects by steatosis and idiosyncratic hepatotoxins. In conclusion, we have developed a simple, selective, and sensitive analytical method to measure plasma 16 BAs including 5 rodent unique BAs, α-MCA, ß-MCA, ω-MCA, α-TMCA, and ß-TMCA. Our data suggested that α-TMCA and ß-TMCA could be useful for identification or prediction of liver injuries, a currently unmet need in preclinical toxicity. Our method using TOF-MS is useful to determine BAs in rat plasma and of use in structural analyses of metabolites in early stage of drug development.

A Trial of Nursing Cost Accounting using Nursing Practice Data on a Hospital Information System.

Hospital administration is very important and many hospitals carry out activity-based costing under comprehensive medicine. However, nursing cost is unclear, because nursing practice is expanding both quantitatively and qualitatively and it is difficult to grasp all nursing practices, and nursing cost is calculated in many cases comprehensively. On the other hand, a nursing information system (NIS) is implemented in many hospitals in Japan and we are beginning to get nursing practical data. In this paper, we propose a nursing cost accounting model and we simulate a cost by nursing contribution using NIS data.

Effects of triiodothyronine on turnover rate and metabolizing enzymes for thyroxine in thyroidectomized rats.

AIM: Previous studies in rats have indicated that surgical thyroidectomy represses turnover of serum thyroxine (T4). However, the mechanism of this process has not been identified. To clarify the mechanism, we studied adaptive variation of metabolic enzymes involved in T4 turnover. MAIN METHODS: We compared serum T4 turnover rates in thyroidectomized (Tx) rats with or without infusion of active thyroid hormone, triiodothyronine (T3). Furthermore, the levels of mRNA expression and activity of the metabolizing enzymes, deiodinase type 1 (D1), type 2 (D2), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase were also compared in several tissues with or without T3 infusion. KEY FINDINGS: After the T3 infusion, the turnover rate of serum T4 in Tx rats returned to normal. Although mRNA expression and activity of D1 decreased significantly in both liver and kidneys without T3 infusion, D2 expression and activity increased markedly in the brain, brown adipose tissue, and skeletal muscle. Surprisingly, hepatic UGT mRNA expression and activity in Tx rats increased significantly in comparison with normal rats, and returned to normal after T3 infusion. SIGNIFICANCE: This study suggests that repression of the disappearance of serum T4 in rats after Tx is a homeostatic response to decreased serum T3 concentrations. Additionally, T4 glucuronide is a storage form of T4, but may also have biological significance. These results suggest strongly that repression of deiodination of T4 by D1 in the liver and kidneys plays a major role in thyroid hormone homeostasis in Tx rats, and that hepatic UGT also plays a key role in this mechanism.

Influence of iodine-deficiency on thyroid hormones homeostasis in rats.

Little is known about the kinetics and metabolism of thyroid hormones in the hypothyroid state. In order to optimize hormone replacement therapy, it is important to understand variations in the kinetics and metabolism of thyroid hormones. To investigate these factors, we monitored serum thyroxine (T4) and triiodothyronine (T3) levels in iodine-deficient diet (ID) rats using online solid-phase extraction liquid chromatography-mass spectrometry/mass spectrometry (Online SPE LC-MS/MS). Furthermore, we evaluated supply and turnover rates of T4 in ID rats using a stable isotope-labeled T4 ([¹³C9]T4). Although serum T4 levels gradually declined after beginning ID treatment, T3 levels were unchanged throughout the experimental period. After intravenous administration of [¹³C9]T4 to ID rats, [¹³C9]T4 levels were monitored. We previously reported that significant differences of supply and turnover rates for T4 were observed in surgically thyroidectomized (Tx) rats. Surprisingly, there were no differences of supply and turnover rates for T4 between ID rats and intact rats. In conclusion, there were significant differences of supply and turnover rates for T4 between the hypothyroid states of ID and Tx rats. In ID rats, T3 might be preferentially biosynthesized in the thyroid, and ID treatment might not affect T4 kinetics. Our method, online SPE LC-MS/MS monitoring using a stable isotope tracer, has the potential to be used as a diagnostic tool to investigate the pathogenesis of thyroid disease and is valuable for optimizing the dosage in thyroid hormone replacement therapy.

Influence of thyroidectomy on thyroxine metabolism and turnover rate in rats.

Little is known about the kinetics and metabolism of thyroid hormones in the hypothyroid state. To investigate these factors, we developed a reliable method for measurement of serum thyroxine (T(4)), triiodothyronine (T(3)), reverse-T(3) (rT(3)) and stable isotope-labeled T(4) ([(13)C(9)]T(4)), using online solid-phase extraction liquid chromatography-mass spectrometry/mass spectrometry (online SPE LC-MS/MS). We measured supply and turnover rates of T(4) in thyroidectomized (Tx) rats using [(13)C(9)]T(4) as a tracer. In rats, serum T(4), T(3) and rT(3) were decreased but not completely ablated after surgical Tx. Endogenous T(4) and T(3) levels in Tx rats were maintained at a constant low level throughout the experimental period. [(13)C(9)]T(4) levels declined with a half-life of ∼1.2 days after it was administered to Tx rats intravenously. These findings strongly suggest that serum T(4) levels in Tx rats are maintained by T(4) supplied by extra-thyroidal tissues (e.g. secretion of extra-thyroidal storage, enhancement of enterohepatic recirculation, and production in extra-thyroidal tissues). Moreover, the turnover rate of T(4) in Tx rats was approximately twofold lower than in controls. This finding suggests that degradation of serum T(4) is repressed by Tx. In conclusion, serum T(4) is maintained at a constant low level by T(4) supply from extra-thyroidal tissues and repression of T(4) degradation in Tx rats. The powerful online SPE LC-MS/MS tool can be used to investigate thyroid hormones kinetics and metabolism, and thus has the potential to be used as a diagnostic tool and to investigate the pathogenesis of thyroid disease.