Characterization of isochlorogenic acid A metabolites in rats using high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Isochlorogenic acid A is widely present in fruits, vegetables and herbal medicines, and is characterized by anti-inflammatory, hepatoprotective and antiviral properties. However, little is known about its metabolic fate and pharmacokinetic properties. This study is thus designed to investigate the metabolic fate of isochlorogenic acid A. An analytical method based on high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) was established to characterize the metabolites of isochlorogenic acid A in the plasma, urine and feces of rats. A total of 32 metabolites were identified. The metabolic pathways mainly include hydrolyzation, dehydroxylation, hydrogenation and conjugation with methyl, glucuronic acid, glycine, sulfate, glutathione and cysteine. Moreover, the pharmacokinetic profiles of all the circulating metabolites were investigated. M11 resulting from hydrolyzation, dehydroxylation and hydrogenation was the dominant circulating metabolite after the intragastric administration of isochlorogenic acid A. The results obtained will be useful for further study of elucidating potential bioactive metabolites which can provide better explanation of the pharmacological and/or toxicological effects of this compound.

Low Impact of Urinary Cortisol in the Assessment of Hydrocortisone Replacement Therapy.

Hydrocortisone replacement therapy is a cornerstone in the treatment of adrenal insufficiency (AI). While urinary cortisol has been used as a diagnostic tool for AI, it remains unclear whether it is a useful parameter to monitor hydrocortisone replacement therapy. Aim of this study was to evaluate possible differences in cortisol metabolism between adrenal insufficient patients and healthy subjects and to assess the value of urinary cortisol in AI management. In a case-control study, urinary cortisol excretion was determined in 14 patients with primary and secondary AI receiving hydrocortisone infusions from midnight to 8:00 AM. Results were correlated with serum cortisol levels and compared to urinary values obtained from 53 healthy volunteers. Urinary cortisol excretion in healthy subjects was 14.0±7.8 µg/8 h (range: 0.24-35.4), levels did not differ between 3 groups aged 20-34 years, 35-49 years, and ≥50 years. Patients with AI receiving hydrocortisone infusions demonstrated significantly higher rates of urinary cortisol excretion (51.6±37.8 µg/8 h; range 17.1-120.0, p<0.001); the values correlated with serum cortisol levels (r(2)=0.98). Of interest, patients with secondary AI showed significantly higher serum cortisol levels after hydrocortisone infusion than those with primary AI, conceivably due to residual adrenal function. In conclusion, we showed that: (i) there is a wide inter-individual variability in urinary cortisol excretion rates; (ii) cortisol metabolism in adrenal insufficient patients differs when compared to controls; (iii) there is a strong correlation between urinary and serum cortisol levels; and (iv) urinary cortisol levels despite their variability may help to discriminate between secondary and primary adrenal insufficiency.

Electrospun titania sol-gel-based ceramic composite nanofibers for online micro- solid-phase extraction with high-performance liquid chromatography.

Titanium(IV) tetraisopropoxide was employed as a metal oxide sol-gel precursor to prepare ceramic composite nanofibers by the electrospinning system. To facilitate this process and obtain the desired nanofibers with higher aspect ratios and surface area, poly(vinylpyrrolidone) was added to the sol of titania. Four ceramic nanofibers sheets based on titania were prepared while each sheet contained different transition metals such as Fe-Mn, Fe-Ni, Fe-Co, and Fe-Mn-Co-Ni. The scanning electron microscope images showed good homogeneity for all the prepared ceramic composites with a diameter range of 100-250 nm. The sorption efficiency was investigated by a micro-solid-phase extraction setup in online combination with high-performance liquid chromatography for the determination of naproxen and clobetasol. All the prepared composites exhibited comparable efficiencies for the desired analytes and the type of metal showed insignificant effect. For the selected composite with Fe-Mn, the linearity of the analytes was in the range of 1-1000 µg/L and the limit of detection values were found to be 2 and 0.3 µg/L for naproxen and clobetasol, respectively. The developed method was extended to the analysis of urine and blood plasma samples and acceptable relative standard deviations were obtained at two concentration levels.

Disposition of methylprednisolone acetate in plasma, urine, and synovial fluid following intra-articular administration to exercised thoroughbred horses.

Methylprednisolone acetate (MPA) is commonly administered to performance horses, and therefore, establishing appropriate withdrawal times prior to performance is critical. The objectives of this study were to describe the plasma pharmacokinetics of MPA and time-related urine and synovial fluid concentrations following intra-articular administration to sixteen racing fit adult Thoroughbred horses. Horses received a single intra-articular administration of MPA (100 mg). Blood, urine, and synovial fluid samples were collected prior to and at various times up to 77 days postdrug administration and analyzed using tandem liquid chromatography-mass spectrometry (LC-MS/MS). Maximum measured plasma MPA concentrations were 6.06 ± 1.57 at 0.271 days (6.5 h; range: 5.0-7.92 h) and 6.27 ± 1.29 ng/mL at 0.276 days (6.6 h; range: 4.03-12.0 h) for horses that had synovial fluid collected (group 1) and those that did not (group 2), respectively. The plasma terminal half-life was 1.33 ± 0.80 and 0.843 ± 0.414 days for groups 1 and 2, respectively. MPA was undetectable by day 6.25 ± 2.12 (group 1) and 4.81 ± 2.56 (group 2) in plasma and day 17 (group 1) and 14 (group 2) in urine. MPA concentrations in synovial fluid remained above the limit of detection (LOD) for up to 77 days following intra-articular administration, suggesting that plasma and urine concentrations are not a good indicator of synovial fluid concentrations.

Optimization of microwave-assisted extraction of analgesic and anti-inflammatory drugs from human plasma and urine using response surface experimental designs.

The performance of microwave-assisted extraction and HPLC with photodiode array detection method for determination of six analgesic and anti-inflammatory drugs from plasma and urine, is described, optimized, and validated. Several parameters affecting the extraction technique were optimized using experimental designs. A four-factor (temperature, phosphate buffer pH 4.0 volume, extraction solvent volume, and time) hybrid experimental design was used for extraction optimization in plasma, and three-factor (temperature, extraction solvent volume, and time) Doehlert design was chosen to extraction optimization in urine. The use of desirability functions revealed the optimal extraction conditions as follows: 67°C, 4 mL phosphate buffer pH 4.0, 12 mL of ethyl acetate and 9 min, for plasma and the same volume of buffer and ethyl acetate, 115°C and 4 min for urine. Limits of detection ranged from 4 to 45 ng/mL in plasma and from 8 to 85 ng/mL in urine. The reproducibility evaluated at two concentration levels was less than 6.5% for both specimens. The recoveries were from 89 to 99% for plasma and from 83 to 99% for urine. The proposed method was successfully applied in plasma and urine samples obtained from analgesic users.

Analysis of anti-inflammatory dehydrodiisoeugenol and metabolites excreted in rat feces and urine using HPLC-UV.

Dehydrodiisoeugenol (DDIE) is a lignan in the fruit of Myristica fragrans. It can be converted into several metabolites in in vitro and in vivo metabolism. In this study, the excretion of DDIE in urine and feces was investigated after intravenous (i.v.) and intragastric (i.g.) administration to rats. DDIE and its metabolites (M-1 and M-2) were measured using HPLC. The amount of DDIE and its metabolites excreted was higher in feces than in urine, suggesting that DDIE and its metabolites are eliminated primarily in the feces. Significant differences in the excretion levels of DDIE and its metabolites were seen between i.v. and i.g. administration. Greater amounts of DDIE and its metabolites were excreted following i.v. administration, suggesting that DDIE can exert a longer period of anti-inflammatory activity following i.g. administration. The accuracy, precision, recovery and stability of the analytical method in this study were satisfactory for the measurement of DDIE and its metabolites in rat urine and feces. Observations made in this study will contribute to understanding of the absorption, distribution, metabolism and excretion pathway of DDIE and will aid decision-making regarding the best mode of DDIE administration during treatment to maximize its anti-inflammatory effects.

Lipoxins as biomarkers of lupus and other inflammatory conditions.

Inflammatory events persist in systemic lupus erythematosus (lupus) despite the use of anti-inflammatory (both steroidal and non-steroidal) and immunosuppressive drugs leading to delay in the healing/repair process and so tissue/organ damage continues. The continuation of inflammation in lupus could be attributed to failure of the resolution process due to deficiency of potent endogenous pro-resolution-inducing molecules such as lipoxin A4 (LXA4). It is likely that progression and flares of lupus and lupus nephritis are due to decreased formation and release of LXA4. Hence, administration of LXA4 and its analogues could be of benefit in lupus. Furthermore, plasma and urinary measurement of lipoxins may be used to predict prognosis and response to therapy. It is likely that lipoxins and other bioactive anti-inflammatory lipids such as resolvins, protectins, maresins and nitrolipids play a significant role in other auto-immune diseases such as rheumatoid arthritis, type 1 diabetes mellitus and multiple sclerosis and hence, could be of significant benefit in these diseases.

Pharmacokinetic study of the novel phosphocholine derivative 3-dibutylaminopropylphosphonic acid by LC-MS coupling.

CRP is an important mediator of the inflammatory response. Pro-inflammatory CRP effects are mediated by pCRP* and mCRP, dissociation products of the native pCRP. The concentration of pCRP during inflammation may rise up to concentrations 1000-fold from baseline. By prevention of the conformational change from pCRP to pCRP*, pro-inflammatory immune responses can be inhibited and local tissue damage reduced. 3-(Dibutylamino)propylphosphonic acid (C10m) is a new substance that can suppress ischemic-reperfusion injury by targeting CRP in the complement cascade. It hampers dissociation of pCRP into its monomers, thus preventing exacerbation of tissue inflammation subsequent to reperfusion injury. In this study, the pharmacokinetics and metabolism of the new drug candidate C10m was investigated. A sensitive and selective method for detection of C10m and its metabolites from plasma and urine was developed with LC-MS and LC-MS/MS coupling. The LLOQ is at 0.1 µg mL-1 and recovery at 87.4% ± 2.8%. Accuracy and precision were within 15% coefficient of variation and nominal concentrations, respectively. Concentration time profile after i.v. bolus injection of C10m was analyzed by LC-MS/MS. Bioavailability has shown to be below 30%. Most likely due to the compounds' very polar chemical properties, no phase-I or phase-II metabolism could be observed. Absence of phase-I metabolism was cross-checked by performing microsomal incubations. Our study revealed that C10m is rapidly eliminated via urine excretion and that half-times appear to be increased with coadministration of the target pCRP.

Comparison of the tissue distribution and metabolism of AN1284, a potent anti-inflammatory agent, after subcutaneous and oral administration in mice.

This study is to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Anti-inflammatory activity of AN1284 and its metabolites was detected in lipopolysaccharide (LPS) activated RAW 264.7 macrophages. Mice were given AN1284 by injection or gavage, 15 min before LPS. IL-6 protein levels were measured after 4 h. Using a liquid chromatography/mass spectrometry method we developed, we showed that AN1284 is rapidly metabolized to the indole (AN1422), a 7-OH derivative (AN1280) and its glucuronide. AN1422 has weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. AN1284 (0.5 mg/kg) caused maximal reductions in IL-6 in the plasma, brain, and liver when injected subcutaneously and after gavage only in the liver. Similar reductions in the plasma and brain required a dose of 2.5 mg/kg, which resulted in 5.5-fold higher hepatic levels than after injection of 0.5 mg/kg, but 7, 11, and 19-fold lower ones in the plasma, brain, and kidneys, respectively. Hepatic concentrations produced by AN1284 were 2.5 mg/kg/day given by subcutaneously implanted mini-pumps that were only 12% of the peak levels seen after acute injection of 0.5 mg/kg. Similar hepatic concentrations were obtained by (1 mg/kg/day), administered in the drinking fluid. These were sufficient to decrease hepatocellular damage and liver triglycerides in previous experiments in diabetic mice. AN1284 can be given orally by a method of continuous release to treat chronic liver disease, and its preferential concentration in the liver should limit any adverse effects.

How to extrapolate a withdrawal time from an EHSLC published detection time: a Monte Carlo simulation appraisal.

REASON FOR PERFORMING STUDY: For legitimate medications, veterinarians must advise the owners or trainers of horses on appropriate withholding times after a treatment, to avoid the risk of incurring a positive drug test. OBJECTIVE: To explore the safety span to select that a veterinarian may extrapolate a tailored withdrawal time (WT) from a generic detection time (DT) as published by the European Horserace Scientific Liaison Committee (EHSLC). METHODS: Using Monte Carlo simulations, it was shown that for a low variability of pharmacokinetic parameters (CV=20%), an uncertainty span of about 40% may be selected to transform a mean DT into a WT (i.e. WT=1.4 DT), which covers 90% of the horse population. In contrast for a highly variable drug (CV=40%), an uncertainty factor of about 2.1-2.2 needs to be selected, i.e. a WT that is twice the DT. RESULTS: The relative impact of the different factors of variability on the final WT was documented by a so-called sensitivity analysis. It was shown that the parameters that have the greatest influence on the value of a DT are those that control the terminal half-life of the drug disposition. In contrast, parameters controlling the level of urine (or plasma) concentrations (i.e. the actual administered dose, the urine-to-plasma ratio and the bioavailability) collectively have a minimal influence on the DT. CONCLUSIONS AND POTENTIAL RELEVANCE: In practice, this means that the main sources of uncertainty are of biological origin and cannot be reduced by any managerial options. The influence of the number of experimental horses that are used by EHSLC to establish a DT was shown that with the standard EHLSC protocol of 6 horses, half of the trials lead to a proposed DT that is equal to or higher than the population 90(th) percentile. Increasing the number of investigated horses to 8 and 10 would increase this last probability to 85 and 90%, respectively.

A capillary zone electrophoretic method for simultaneous determination of seven drugs in pharmaceuticals and in human urine.

A simple, sensitive, and novel method has been developed and validated for the separation and simultaneous quantitation of seven structurally different drugs-pipemidic acid and ofloxacin quinolone antibiotics, pseudoephedrine decongestant, piroxicam anti-inflammatory, thiamin, pyridoxine, and cobalamin-in a mixture by capillary zone electrophoresis. Factors affecting the separation were pH, concentration of buffer, and applied voltage. Separation was carried out in < 9 min with a 50 mM sodium tetraborate buffer, pH 10, and an applied voltage of 30 kV in an uncoated silica capillary tube. The carrier electrolyte gave baseline separation with good resolution, reproducibility, and accuracy. Calibration plots were linear over at least three orders of magnitude of analyte concentrations, and the lower LODs were within the range of 1-5 microg/mL. Detection was performed by UV absorbance at 230 nm. The method was validated for the analysis of drugs in pharmaceutical preparations and in urine samples with RSD of 0.5-2.4% and recovery of > 99%.

Pilot study to quantify the time to clear dexamethasone from plasma and urine of adult horses following a single nebulisation.

OBJECTIVE: To quantify the time to clear dexamethasone from plasma and urine of horses following a single nebulisation. DESIGN: Experimental using six Standardbred mares. METHODS: Dexamethasone sodium phosphate (0.04 mg/kg) diluted in 0.9% sodium chloride was administered as an aerosol using a Flexineb E2® nebuliser. Blood samples (0, 2, 4, 6, 8, 10, 12, 24, 32, 48, 72 and 96 h) and urine samples (0, 1, 4, 8, 24, 32, 48, 72 and 96 h) were collected for analysis using liquid chromatography mass spectrometry. RESULTS: Maximum plasma concentrations (tmax ) were reached by the earliest detection point (2 h) after nebulisation (0.6-1.8 ng/mL), but was no longer detectable at 48 h. However, in one horse 0.1 ng/mL was found at 96 h after three consecutive readings of 0 ng/mL. The tmax in urine was reached by the earliest collection point (1 h) after nebulisation (3.2-23.8 ng/mL), but was no longer present in urine at 72 h in five horses, while detectable levels (0.1 ng/mL) were still present at 96 h in one horse. CONCLUSIONS: A single dose of 0.04 mg/kg of DSP administered as an aerosol through a FlexinebE2® mask was no longer detectable in blood at 48 h in six horses tested, but one horse returned a reading of 0.1 ng/mL at 96 h after having no detectable levels. Dexamethasone was not detectable in urine at 72 h in five horses but was detectable at a low concentration (0.1 ng/mL) at 96 h in one horse.

Elimination profile of triamcinolone hexacetonide and its metabolites in human urine and plasma after a single intra-articular administration.

Triamcinolone hexacetonide (THA) is a synthetic glucocorticoid (GC) used by intra-articular (IA) administration. GCs are prohibited in sports competitions by systemic routes, and they are allowed by other routes considered of local action (IA administration, among others). The aim of the present work was to study the metabolic profile of THA in urine and plasma following IA administration. Eight patients (4 males and 4 females) with knee osteoarthritis received an IA dose of THA (40 mg) in the knee joint. Spot urine and plasma samples were collected before injection and at different time periods up to day 23 and 10 post-administration, respectively. The samples were analysed by liquid chromatography-tandem mass spectrometry. Neither THA nor specific THA metabolites were detected in urine. Triamcinolone acetonide (TA) and 6ß-hydroxy-triamcinolone acetonide were the main urinary metabolites. Maximum concentrations wereobtained between 24 and 48 h after administration. Using the reporting level of 30 ng/mL to distinguish allowed from forbidden administrations of GCs, a large number of false adverse analytical findings would be reported up to day 4. On the other hand, TA was detected in all plasma samples collected up to day 10 after administration. THA was also detected in plasma but at lower concentrations. The detection of plasma THA would be an unequivocal proof to demonstrate IA use of THA. A reversible decrease was observed in plasma concentrations of cortisol in some of the patients, indicating a systemic effect of the drug.

In vitro simulated digestion and in vivo metabolism of chlorogenic acid dimer from Gynura procumbens (Lour.) Merr.: Enhanced antioxidant activity and different metabolites of blood and urine.

Gynura procumbens (Lour.) Merr. is an evergreen edible vine in southern China. The antioxidant activity and metabolites of chlorogenic acid dimer from Gynura procumbens (Lour.) Merr. were evaluated by the model of in vitro digestion and in vivo metabolomics approach, respectively. Moreover, metabolites of chlorogenic acid dimer in blood and urine of Sprague-Dawley rats were determined by HPLC-ESI-QTOF-MS/MS. In vitro digestion results suggested the antioxidant activity of the purified chlorogenic acid dimer was significantly enhanced after simulated digestion. Meanwhile, in vivo metabolism results showed that 7 and 20 new metabolites were observed in blood and urine, respectively, suggesting that hydrolysis along with methylation, glucuronidation and other reactions may all happen when the chlorogenic acid dimer entered the digestive and metabolic systems, which inducing and exhibiting various biological activities through metabolism. PRACTICAL APPLICATIONS: Gynura procumbens (Lour.) Merr. (GPM) is an evergreen edible vine with the effects of anticancer, anti-inflammatory, antiviral, depressurization, and antioxidation. As a health care vegetable, it is not usually eaten in daily life. Our current study shows that chlorogenic acid dimer extracted from GPM has a significant enhanced antioxidant ability after gastro-intestinal digestion in vitro, and their metabolites in vivo of urine is far more than that of blood, which may indicate that the chlorogenic acid dimer can be fully absorbed and decomposed through the gastro-intestinal digestion and metabolism. Thus, GPM could be used as a functional food ingredient for antioxidant enhancement to promote the economic value. The research also provides theoretical data for the intensive processing and utilization of GPM, as well as for the relative research on digestion and metabolism of edible plants.

Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone.

Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4%, respectively, for the LC/IT-MS method and lower than 8.4 and 4.4%, respectively, for the LC/TOF-MS method. Accuracy (bias percentage) was less than 9.7% for both methods.

Urinary metabolomics study on the anti-inflammation effects of flavonoids obtained from Glycyrrhiza.

Rheumatoid arthritis (RA) is a chronic disease with pain, swelling, and limitation in the motion and function of multiple joints thus leading to high disability. Previous studies have shown that flavonoids and saponins are the most abundant and active constituents in Glycyrrhiza, which possess a wide range of pharmacological effects such as anti-inflammatory, antioxidant and anti-bacteria. But the mechanisms of those actions are not entirely clear. In order to clarify the mechanisms of those actions, the pharmacodynamical assessments of extraction of water-soluble components and flavonoids and saponins obtained from Glycyrrhiza were investigated. Combining the pharmacodynamical researches, we found that flavonoids obtained from Glycyrrhiza had more significant therapeutic effects on acute inflammation, chronic inflammation and inflammatory pain than that of extraction of water-soluble components and saponins obtained from Glycyrrhiza. The results indicated that flavonoids are the main medicinal ingredients in Glycyrrhiza. In order to further investigate the mechanism of the action of flavonoids in Glycyrrhiza on treating RA, a urine metabolomics method based on ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was established to observe the metabolic variations in adjuvant-induced arthritis (AIA) rats and investigate the therapeutic effect of flavonoids in Glycyrrhiza on RA. As a result, twenty potential biomarkers were found by comparison with the model group (MG) and flavonoid treated group (FG). We associated these compounds with related metabolic pathways, the results showed that these biomarkers were mainly associated with purine metabolism, taurine and hypotaurine metabolism, tryptophan metabolism, phenylalanine metabolism, tricarboxylic acid cycle (TCA cycle), pantothenate and coenzyme A (CoA) biosynthesis. The results about the pharmacodynamics and metabolomics provided a theoretical basis for clarifying the mechanism of flavonoids in Glycyrrhiza in the treatment of RA.

Phase 1 Randomized, Double-Blind, Placebo-Controlled Study to Determine the Safety, Tolerability, and Pharmacokinetics of a Single Escalating Dose and Repeated Doses of CN-105 in Healthy Adult Subjects.

Spontaneous intracranial hemorrhage (ICH) remains a devastating stroke subtype, affecting as many as 80,000 people annually in the United States and associated with extremely high mortality. In the absence of any pharmacological interventions demonstrated to improve outcome, care for patients with ICH remains largely supportive. Thus, despite advances in the understanding of ICH and brain injury, there remains an unmet need for interventions that improve neurologic recovery and outcomes. Recent research suggesting inflammation and APOE genotype play a role in modifying neurologic outcome after brain injury has led to the development of an APOE-derived peptide agent (CN-105). Preclinical studies have demonstrated that CN-105 effectively downregulates the inflammatory response in acute brain injury, including ICH. Following Investigational New Drug (IND) enabling studies in murine models, this first-in-human single escalating dose and multiple dose placebo-controlled clinical trial was performed to define the safety and pharmacokinetics (PK) of CN-105. A total of 48 subjects (12 control, 36 active) were randomized in this study; all subjects completed the study. No significant safety issues were identified with both dosing regimens, and PK analysis revealed linearity without significant drug accumulation. The median half-life in the terminal elimination phase of CN-105 following a single or repeated dosing regimen did not change (approximately 3.6 hours). With the PK and preliminary safety of CN-105 established, the drug is now poised to begin first-in-disease phase 2 clinical trials in patients with ICH who urgently need new therapeutic options.

Quantification of dimethylsulfoxide (DMSO) in equine plasma and urine using HILIC-MS/MS.

This paper describes quantitative methods for the determination of dimethylsulfoxide (DMSO) in equine plasma and urine based on simple precipitation and dilution followed by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS). DMSO is a polar solvent with analgesic and anti-inflammatory properties. Its pharmacological features make it prohibited in horse racing. However, since DMSO is naturally present in the horses' environment, international threshold values have been implemented for plasma and urine (1 and 15 µg/mL, respectively). Previously presented quantitative methods for the determination of DMSO are based on gas chromatography, thus demanding a tedious extraction step to transfer the analyte from the aqueous bodily fluid to an injectable organic solvent. The column used in the presented method was an Acquity BEH HILIC and the mobile phase was a mixture of ammonium acetate buffer and acetonitrile delivered as a gradient. Hexadeuterated DMSO (2 H6 -DMSO) was used as the internal standard. Validation was performed in the range of the international thresholds concerning selectivity, carry-over, linearity, precision, accuracy, stability and inter-individual matrix variation. The results fulfilled the predefined criteria and the methods were considered fit for purpose. Successful applications on real equine doping control samples were carried out with determined DMSO concentrations exceeding the international thresholds. Copyright © 2016 John Wiley & Sons, Ltd.

Detection of budesonide in human urine after inhalation by liquid chromatography-mass spectrometry.

Budesonide, a corticosteroid frequently used in the treatment of asthma, is most often administered via inhalation. Its use in sports is allowed when medically necessary. A fast, sensitive and accurate LC-MS method was developed and validated for the quantification of budesonide and its major metabolite 16alpha-hydroxyprednisolone in urine samples after inhalation of a metered dose (Pulmicort-Turbohaler 200). Sample preparation consists of an alkaline liquid-liquid extraction with ethyl acetate. Analysis was performed using liquid chromatography-tandem mass spectrometry with electrospray ionization (ESI). The method was linear in the range of 5-100 and 0.5-10ng/mL for 16alpha-hydroxyprednisolone and budesonide, respectively. The limits of quantification were 5ng/ml for 16alpha-hydroxyprednisolone and 0.5ng/mL for budesonide. The accuracy ranged from 2.2 to 3.5% for 16alpha-hydroxyprednisolone and from 0.8 to 16.4% for budesonide. After administration of 200microg of budesonide to five healthy volunteers budesonide could not be detected in any urine sample whereas 16alpha-hydroxyprednisolone was detectable up to 12h post-administration.

Spectrophotometric and chemometric methods for determination of imipenem, ciprofloxacin hydrochloride, dexamethasone sodium phosphate, paracetamol and cilastatin sodium in human urine.

New accurate, sensitive and selective spectrophotometric and chemometric methods were developed and subsequently validated for determination of Imipenem (IMP), ciprofloxacin hydrochloride (CIPRO), dexamethasone sodium phosphate (DEX), paracetamol (PAR) and cilastatin sodium (CIL) in human urine. These methods include a new derivative ratio method, namely extended derivative ratio (EDR), principal component regression (PCR) and partial least-squares (PLS) methods. A novel EDR method was developed for the determination of these drugs, where each component in the mixture was determined by using a mixture of the other four components as divisor. Peak amplitudes were recorded at 293.0 nm, 284.0 nm, 276.0 nm, 257.0 nm and 221.0 nm within linear concentration ranges 3.00-45.00, 1.00-15.00, 4.00-40.00, 1.50-25.00 and 4.00-50.00 µg mL(-1) for IMP, CIPRO, DEX, PAR and CIL, respectively. PCR and PLS-2 models were established for simultaneous determination of the studied drugs in the range of 3.00-15.00, 1.00-13.00, 4.00-12.00, 1.50-9.50, and 4.00-12.00 µg mL(-1) for IMP, CIPRO, DEX, PAR and CIL, respectively, by using eighteen mixtures as calibration set and seven mixtures as validation set. The suggested methods were validated according to the International Conference of Harmonization (ICH) guidelines and the results revealed that they were accurate, precise and reproducible. The obtained results were statistically compared with those of the published methods and there was no significant difference.