Determination of raloxifene in urine by liquid chromatography-tandem mass spectrometry for doping.

Raloxifene is one of the selective estrogen receptor modulators and is often used to prevent and treat osteoporosis in postmenopausal women. Because of the indirect impact on serum testosterone levels and the potential ability for performance enhancement, it is banned by the World Anti-Doping Agency (WADA). This study established a fast, sensitive and selective liquid chromatography-tandem mass spectrometry method to quantify total raloxifene (unchanged and glucuronidated) in human urine for doping analysis. Urines from six healthy volunteers were collected 240 h after taking a single dose of raloxifene. The concentrations of urinary raloxifene were analyzed by the established method after sample preparation, including hydrolysis with ß-glucuronidase. The lowest limit of quantification was 0.5 ng/mL. Linearity was observed for raloxifene concentrations ranging from 0.5 to 100 ng/mL, with a correlation coefficient of 0.999. The recoveries were >92.81%. Inaccuracies were below ±5%, and precisions varied from 2.18 to 5.37%. The results showed that urinary raloxifene was immediately detectable within 4 h after the administration of only a single dose of raloxifene. Such a result indicates a violation of the WADA rules. Furthermore, ingesting raloxifene would be detectable after 6 days in the urine of males or >10 days in the urine of female.

Validated LC-MS-MS method for the determination of quetiapine in human plasma: application to a pharmacokinetic study.

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of quetiapine was developed and validated over the linearity range 1-1500 ng/mL with 0.1 mL of plasma using clozapine as the internal standard. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive electrospray ionization and quantification was performed by selected reaction monitoring mode. The MS-MS ion transitions monitored were m/z 384.1 → 253.1 and 327.0 → 270.0 for quetiapine and clozapine, respectively. The between- and within-run precision was less than 7.44% and accuracy was less than 10.2%. The lower limit of quantification was 1 ng/mL. The extraction recoveries of quetiapine were over 90%. The method is proved to be accurate and specific, and was applied to the pharmacokinetic study in healthy Chinese volunteers.

Essentiality of HIV testing and education for effective HIV control in the national pilot harm reduction program: the Taiwan experience.

In 2005, a national pilot harm reduction program (PHRP), which mainly included a methadone maintenance treatment program (MMTP) and a needle/syringe exchange program (NSP), was implemented in Taiwan. We conducted this study to evaluate the effectiveness of harm reduction measures on HIV control among injecting drug users (IDUs) between PHRP and nonPHRP. The data on HIV, collected from incumbent Taiwanese authorities, were analyzed for their associations, risk and protective factors with PHRP measures. While the monthly HIV incidences did not show significant differences before and after PHRP in the four areas with PHRP (Taipei City, Taipei County, Taoyuan County and Tainan County), a significant increase in the HIV incidence was found in the 21 areas without PHRP. Hence, the implementation of the PHRP did result in a significant difference in the monthly HIV incidence between areas with and without the PHRP. Mandatory HIV testing was significantly associated with the HIV incidence according to the generalized estimation equations (GEE) model. With adjustments of time period and area with PHRP, and urban area, protective factors associated with HIV incidence were: educational materials, condoms, dilution water, and alcohol sponges/swabs. MMTP contributed to a higher HIV incidence, probably due to the concurrent HIV testing upon admission. Since HIV testing was not required in the NSP, the HIV testing-dependent MMTP may explain the association of the PHRP intervention and an increased HIV incidence. In summary, HIV testing and education were essential for effective HIV control upon implementing the PHRP.

Simultaneous determination of sulphur metabolites in Arabidopsis thaliana via LC-ESI-MS/MS and ³4S-metabolic labelling.

INTRODUCTION: Sulphur-containing metabolites play an important role in metabolism and homeostasis. Determination of these metabolites is challenging owing to their low concentrations and the interference in mass spectrometry analysis. OBJECTIVE: To develop a sensitive and accurate method based on liquid chromatography, electrospray ionisation, tandem mass spectrometry (LC-ESI-MS/MS) and ³4S-metabolic labelling for quantification of methionine, reduced glutathione, oxidised glutathione in Arabidopsis thaliana. METHODOLOGY: A hydroponic set-up was used for the in vivo ³4S-metabolic labelling of A. thaliana. The ³4S-labelled metabolites biosynthesised in plant were extracted and used as internal standards. Tissue was extracted with perchloric acid (PCA) or PCA containing a known amount of the analytes for recovery analysis. Tissue extract mixed with extract of ³4S-labelled A. thaliana in an appropriate ratio was subjected to a LC system and electrospray ionisation-mass spectrometric (ESI-MS) analysis. Quantification of metabolites was measured by comparing the ³4S/³4S ratios obtained for samples with the calibration curves. RESULTS: Calibration curves showed linearity with regression coefficients in the range of 0.9994-0.9999. Analyte recoveries were approximately 100%. The coefficients of variation of intra-assay and inter-assay were less than 4.2% and 5%, respectively. The ranges for the limits of detection determined for Met, GSSG and GSH were 10 fmol, < 10 fmol and 1.12 fmol and the limits of quantification determined for Met, GSSG and GSH were 0.44 pmol, 0.16 pmol and 34 fmol, respectively. CONCLUSION: The validated method for determination of methionine, reduced glutathione and oxidised glutathione was effectively applied to measure metabolite dynamics of sulphur-containing metabolites at the whole-plant level.

Novel terpenoids from Calocedrus macrolepis var. formosana.

Calocetriol (1), diacetylcalocediol (2), and ferrugimenthenol (3) were isolated from the bark of Calocedrus macrolepis var. formosana. Among them, 1 and 2 are secoabietane-type diterpenoids, and 3, with a novel C(20)-C(10) skeleton, is classified as a meroterpenoid. The structures of 1-3 were elucidated by spectroscopic analyses, and their biological activities were also evaluated. Compound 3 exhibited significant cytotoxic activity against human oral epidermoid carcinoma KB cells with an IC(50) value of 9.0±0.1 µM.

Simultaneous quantification of triazolam and its metabolites in human urine by liquid chromatography-tandem mass spectrometry.

A simple, simultaneous, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of triazolam and its metabolites, α-hydroxytriazolam (α-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), in human urine was developed and validated. Triazolam-d4 was used as the internal standard (IS). This analysis was carried out on a Thermo(®) C(18) column, and the mobile phase was composed of acetonitrile/H(2)O/formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem MS using positive ion mode electrospray ionization, and quantification was performed by multiple reaction monitoring mode. The MS-MS ion transitions monitored were m/z 343.1 → 308.3, 359.0 → 331.0, 359.0 → 111.2, and 347.0 → 312.0 for triazolam, α-OHTRZ, 4-OHTRZ, and triazolam-d(4), respectively. The lower limits of quantification of the analytical method were 0.5 ng/mL for triazolam, 5 ng/mL for α-OHTRZ, and 0.5 ng/mL for 4-OHTRZ. The within- and between-run precisions were less than 15%, and accuracy was -12.33% to 9.76%. The method was proved to be accurate and specific, and it was applied to a urinary excretion study of triazolam in healthy Chinese volunteers.

Effects of tremella-alginate-liposome encapsulation on oral delivery of inactivated H5N3 vaccine.

In this study, we evaluated a system for oral vaccine delivery, consisting of liposomes coated first with a layer of tremella and then with an outer layer of acid-induced alginate. In vitro release studies showed that the triple layer of alginate-tremella-liposomes was more resistant to an acidic pH and modulated the release profiles at an alkaline pH. Transepithelial electrical resistance (TEER) studies revealed that liposomes or tremella-coated liposomes were able to open tight junctions of the Caco-2 cell monolayer. In mice, although serum immunoglobulin G (IgG) was not expected to increase and haemagglutination inhibition showed that antibody levels were still too low to provide sufficient protection, alginate-tremella-liposomes encapsulated virus-induced intestinal secretory immunoglobulin A (s-IgA) production to provide protection against virus infection. In conclusion, an oral virus vaccine entrapped in alginate-tremella-liposomes improved the mucosal antiviral s-IgA response. This system may have potential use as a carrier for oral vaccine delivery.

Sub-maximal exercise altered the prednisolone absorption pattern.

PURPOSE: Prednisolone has been widely used due to its relatively short pituitary inhibition and its moderate potency. Exercise is one of the non-pharmacological interventions for glucocorticoid- induced osteoporosis prevention. However, the effects of exercise on the pharmacokinetics of prednisolone were unclear. The purpose of this study was to evaluate the effects of sub-maximal exercise on the pharmacokinetics of prednisolone. METHODS: A randomized, double-blind, cross-over experimental design was used. Subjects needed to undertake two trials: a non-exercise trial (NE) and an exercise trial (E). After the first blood sampling, the subjects consumed 5 mg of prednisolone with 100 mL of water and then took a rest for 30 min. In the E trial, subjects cycled at 70% VO(2max) intensity until exhaustion. When subjects underwent the NE trial, they remained seated for the duration of the experiment. Serial blood samples were collected at 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 4, 5, 6, 8, 10 and 12 hr after prednisolone administration. Prednisolone and cortisol concentration were analyzed with a validated high performance liquid chromatographic method. RESULTS: This study indicated that the maximum concentration of the E trial was significantly lower than the NE trial (p<.05). However, the area under the plasma concentration-time, half life, the time-averaged total body clearance, and the apparent volume of the distribution showed no significant difference between two trials. The mean percentage change of cortisol of E trail was significantly higher than NE trail (p<.05). CONCLUSIONS: The results suggested that sub-maximal exercise altered the prednisolone absorption pattern.

The influence of liposomal adjuvant on intranasal vaccination of chickens against Newcastle disease.

The adjuvant effect of liposomes formulated with three phospholipids including phosphatidylcholine-liposomes (PC-Lip), phosphatidylserine-liposomes (PS-Lip), and stearylamine-liposomes (SA-Lip) was compared with virus alone using inactivated Newcastle disease virus (NDV) as a model antigen. The difference in adjuvanticity was evaluated using the haemagglutination-inhibition (HI) test, enzyme-linked immunosorbent assay, and a challenge study following intranasal inoculation of specific pathogen-free chickens. After two inoculations, a liposomal vaccine consisting of NDV in PC-Lip resulted in a significant increase in HI titre, up to 32-fold higher than a vaccine containing virus alone and 320-fold higher than a vaccine containing NDV in SA-Lip. PC-Lip also elicited a significant mucosal secretary immunoglobulin A response (P<0.05) in tracheal lavages and a serum IgG response (P<0.05). In response to viral challenge, all control animals died, whereas 90% of animals which received PC-Lip survived. The results suggest that PC-Lip may be suitable as an adjuvant for mucosal vaccination against NDV in chickens.

Evaluation of encapsulated Newcastle disease virus liposomes using various phospholipids administered to improve chicken humoral immunity.

We propose the adjuvant effects of phospholipid liposome compositions using intranasal inoculation of a liposomal-Newcastle disease virus (NDV) vaccine in chickens. The immunogenicity of three liposome formulations was determined in chickens using the hemagglutination-inhibition (HI) test, nasal secretory immunoglobulin A and serum immunoglobulin A (IgG) antibody titers using the enzyme-linked immunosorbent assay. The immune response against NDV antigens was determined after immunization with neutral charged liposomes composed of egg phosphatidylcholine (EPC) (60 micromol), cholesterol (Chol) (15 micromol), and EPC-liposomes (EPC-Lip), which elicited strong systemic (serum) and local (nasal) humoral responses. However, the intranasal administration with cationic charged liposomes composed of EPC (30 micromol), stearylamine (SA) (15 micromol), Chol (15 micromol), and SA-liposomes (SA-Lip) induced poor humoral immune responses. Only the vaccine formulated with anionic charged liposomes composed of EPC (30 micromol), dipalmitoylphosphatidylserine (15 micromol), Chol (15 micromol), and phosphatidylserine-liposomes (PS-Lip) elicited the highest titers of HI antibodies. These are the first results to suggest that antigen delivery using EPC-Lip is very useful in enhancing antibody production at the mucosal site and in serum.

Urinary elimination of ephedrines following administration of the Traditional Chinese Medicine preparation Kakkon-to.

Kakkon-to is one of the most common Traditional Chinese Medicine preparations for the attenuation of colds. Ephedrae Herba is one of the prescriptions of Kakkon-to. The major ingredients of Ephedrae Herba, ephedrines, are banned substances on the World Anti-Doping Agency (WADA) list. The purpose of this study was to investigate the elimination of urinary ephedrines after administering Kakkon-to and to determine the possibility of urinary positive ephedrine test results. Six healthy volunteers took one single dose of 2.5 g Kakkon-to extract granules. The concentrations of urinary ephedrines were analyzed by high-performance liquid chromatography. The result showed that ephedrine and norpseudoephedrine were excreted in the urine after taking one single dose of Kakkon-to. However, the highest amount of ephedrines in urine was ephedrine and the peak concentration was 4.35 +/- 1.82 microg/mL (mean +/- standard deviation), which was lower than the WADA permitted value (10 microg/mL). The estimated elimination half-lives of ephedrine, norephedrine, pseudoephedrine, and norpseudoephedrine following administration of this preparation were: 5.2 +/- 1.2, 4.2 +/- 1.3, 4.2 +/- 0.9, and 6.5 +/- 2.8 h, respectively. This study concluded that the urine would not violate the rule of doping after administering a single dose of Kakkon-to. Nevertheless, a further study on administering the preparation for 3 times per day for 3 days showed a positive ephedrine result. Athletes should be careful when taking more than a single dose of Kakkon-to.

Simultaneous determination of triazolam and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of triazolam and its metabolites, alpha-hydroxytriazolam (alpha-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), was developed and validated. Triazolam-D4 was used as the internal standard (IS). This analysis was carried out on a Thermo C(18) column and the mobile phase was composed of acetonitrile:H(2)O:formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) and quantification was performed by multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 343.1-->308.3, 359.0-->308.3, 359.0-->111.2 and 347.0-->312.0 for triazolam, alpha-OHTRZ, 4-OHTRZ and triazolam-D4, respectively. LLOQ of the analytical method was 0.05 ng/mL for triazolam and 0.1 ng/mL for alpha-OHTRZ and 4-OHTRZ. The within- and between-run precisions were less than 15.26% and accuracy was -8.08% to 13.33%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of triazolam in healthy Chinese volunteers.

Effects of swimming on the pharmacokinetics and glucose tolerance of metformin in insulin-resistant rats.

The purpose of the study was to investigate the effects of swimming on the pharmacokinetic and insulin sensitivity of metformin in insulin resistant rats. Rats with fructose-induced insulin resistance were assigned into four groups: control group (C, n=8), metformin group (M, n=8), swimming group (S, n=8) and metformin with swimming group (MS, n=8). After 12 h of fasting, the S and MS group swam for 45 min, while the M and C groups were placed in 4 cm deep water for the same time period. The first blood samples were withdrawn from the tail 60 min after the four groups had left the water. An oral glucose loading was performed in all groups and metformin was administered to the M and MS groups after the first blood sample. Blood samples were collected at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10 and 12 h. The results showed that the MS group increased the time to the maximum concentration, the time to half-life concentration and enhanced insulin sensitivity. This study suggests that swimming before administration of metformin significantly improved insulin sensitivity and the rate of metformin absorption.

Simultaneous quantification of six ephedrines in a Mahwang preparation and in urine by high-performance liquid chromatography.

A rapid and reliable high-performance liquid chromatographic method was developed and validated for the simultaneous determination of norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE), methylephedrine (ME) and methylpseudoephedrine (MPE) in both a Mahwang traditional Chinese medicine (TCM) preparation and in urine using alpha-ethylbenzylamine as the internal standard. The method uses a Spherisorb C(18) column for an isocratic elution in a tetraethylammoniumphosphate-methanol mobile phase at a wavelength of 206 nm. The limits of detection of NE, NPE, E, PE, ME and MPE in sample solutions ranged from 0.1 to 0.3 microg[sol ]mL at a signal-to-noise ratio of 3. The within-day precision as calculated from the Mahwang TCM preparation and urine samples was below 6.2 and 1.4% for each analyte. The between-day precision as calculated from the Mahwang TCM preparation and urine samples was below 6.8 and 5.9% for each analyte. The between-day accuracy as determined from the Mahwang TCM preparation and urine samples was below 2.2 and 6.8% for each analyte. The recoveries for six compounds, obtained with compounds spiked into the Mahwang TCM preparation and urine, were found to be more than 93.6%. This method can be successfully applied to doping and excretion rate studies.