Rapid,sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of topically applied azithromycin in rabbit conjunctiva tissues

A simple, rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of azithromycin in rabbit conjunctiva tissues using roxithromycin as internal standard. Following a deproteinization procedure, the samples were eluted isocratically at a flow rate of 0.3 mL/min utilizing a mobile phase containing of 10 mM ammonium acetate (adjusted pH to 5.2 with 0.1% acetic acid)–methanol (18:82, v/v) and a SHISEIDO CAPCELL PAK C₁₈ (3.0 mm × 75 mm, 3 μm). Azithromycin and its internal standard were measured by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode with precursor-to-product qualifier transition m/z 375 [M+2H]²⁺ → 591 and m/z 837 [M+H]⁺ → 679 respectively. The method demonstrated that good linearity ranged from 10 to 2000 ng/mL with r = 0.9998. The lower limit of quantification for azithromycin in conjunctiva tissues was 10 ng/mL with good accuracy and precision. The intra- and inter-day precision (RSD) values were below 15% and accuracy (%) ranged from 90% to 110% at all QC levels. The method was applicable to ocular pharmacokinetic studies of azithromycin.     

Blood lead level association with lower body weight in NHANES 1999–2006

Background

Lead exposure is associated with low birth-weight. The objective of this study is to determine whether lead exposure is associated with lower body weight in children, adolescents and adults.

Methods

We analyzed data from NHANES 1999–2006 for participants aged ≥ 3 using multiple logistic and multivariate linear regression. Using age- and sex-standardized BMI Z-scores, overweight and obese children (ages 3–19) were classified by BMI ≥ 85th and ≥ 95th percentiles, respectively. The adult population (age ≥ 20) was classified as overweight and obese with BMI measures of 25–29.9 and ≥ 30, respectively. Blood lead level (BLL) was categorized by weighted quartiles.

Results

Multivariate linear regressions revealed a lower BMI Z-score in children and adolescents when the highest lead quartile was compared to the lowest lead quartile (β (SE) = − 0.33 (0.07), p < 0.001), and a decreased BMI in adults (β (SE) = − 2.58 (0.25), p < 0.001). Multiple logistic analyses in children and adolescents found a negative association between BLL and the percentage of obese and overweight with BLL in the highest quartile compared to the lowest quartile (OR = 0.42, 95% CI: 0.30–0.59; and OR = 0.67, 95% CI: 0.52–0.88, respectively). Adults in the highest lead quartile were less likely to be obese (OR = 0.42, 95% CI: 0.35–0.50) compared to those in the lowest lead quartile. Further analyses with blood lead as restricted cubic splines, confirmed the dose-relationship between blood lead and body weight outcomes.

Conclusions

BLLs are associated with lower body mass index and obesity in children, adolescents and adults.     

Hemoglobin adducts as biomarkers of estrogen homeostasis: Elevation of estrogenquinones as a risk factor for developing breast cancer in Taiwanese Women

The aim of this study was to establish a methodology to analyze estrogen quinone-derived adducts, including 17β-estradiol-2,3-quinone (E₂-2,3-Q) and 17β-estradiol-3,4-quinone (E₂-3,4-Q), in human hemoglobin (Hb). The methodology was then used to measure the levels of these adducts in Hb derived from female breast cancer patients (n = 143) as well as controls (n = 147) in Taiwan. Our result confirmed that both E₂-2,3-Q- and E₂-3,4-Q-derived adducts, including E₂-2,3-Q-4-S-Hb and E₂-3,4-Q-2-S-Hb, were detected in all breast cancer patients with median levels at 434 (215–1472) and 913 (559–2384) (pmol/g), respectively. Levels of E₂-2,3-Q-4-S-Hb correlated significantly with those of E₂-3,4-Q-2-S-Hb (r = 0.622–0.628, p < 0.001). By contrast, median levels of these same estrogen quinone-derived adducts in healthy controls were 71.8 (35.7–292) and 139 (69.1–453) (pmol/g). This translated to ∼6-fold increase in mean values of E₂-2,3-Q-4-S-Hb and E₂-3,4-Q-2-S-Hb in breast cancer patients compared to those in the controls (p < 0.001). Our findings add further support to the theme that cumulative body burden of estrogen quinones is an important indicator of breast cancer risk. We hypothesize that combination of genetic events and environmental factors may modulate estrogen homeostasis and enhance the production of estrogen quinones which lead to subsequent generation of pro-mutagenic DNA lesions in breast cancer patients.     

Rapid and sensitive assay for trantinterol,a novel β2-adrenoceptor agonist,in human plasma using liquid chromatography–tandem mass spectrometry

A rapid and sensitive assay for trantinterol, a novel β₂-adrenoceptor agonist, in human plasma has been developed. Samples containing the analyte and internal standard, clenbuterol, were analyzed by liquid chromatography–tandem mass spectrometry after liquid–liquid extraction with diethyl ether:dichloromethane (60:40, v/v). Separation was performed on a Venusil MP C₁₈ column (50 mm × 4.6 mm, 5 μm) using methanol:1% formic acid (50:50, v/v) as mobile phase and monitored by multiple reaction monitoring of the precursor-to-product ion transitions of trantinterol at m/z 311.2 → 238.1 and clenbuterol at m/z 277.2 → 203.1. The total run time was only 1.5 min and the method was linear over the concentration range 1–1000 pg/mL with a lower limit of quantitation of 1 pg/mL. Intra- and inter-day precisions (relative standard deviation) were below 7% and 12%, respectively, with accuracy (relative error) below 8%. The method was successfully applied to a pharmacokinetic study involving oral administration of a 50 μg trantinterol tablet to healthy volunteers.     

Preventing addiction related suicide: A pilot study

Persons addicted to alcohol and drugs are at 5–10 times higher risk for suicide as compared to the general population. To address the need for improved suicide prevention strategies in this population, the Preventing Addiction Related Suicide (PARS) module was developed. Pilot testing of 78 patients demonstrated significant post-treatment changes in knowledge [t(66) = 12.07, p = .000] and attitudes [t(75) = 6.82, p = .000] toward suicide prevention issues. Significant gains were maintained at 1-month follow-up for changes in knowledge [t(55) = 6.33, p = .000] and attitudes [t(61) = 3.37, p = .0001], with changes in positive help seeking behaviors in dealing with suicidal issues in friends [χ²(1) = 10.49, p = .007], family [χ²(1) = 9.81, p = .015], and self [χ²(1) = 19.62, p = .008] also observed. The PARS was also highly rated by treatment staff as feasible within their standard clinical practice.     

Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC–MS/MS method

The purpose of this research was to develop a sensitive and reproducible UPLC–MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC–MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1 ± 5.4% at a dose of 2 mg/kg matrine. Matrine at 10 μM was shown to have good permeability (42.5 × 10⁻⁶ cm/s) across the Caco-2 cell monolayer, and the ratio of P_A–B to P_B–A was approximately equal to 1 at two different concentrations (1 and 10 μM). Perfusion study showed that matrine displayed significant differences (P < 0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P_w = 6.18), followed by colon (P_w = 2.07), duodenum (P_w = 0.61) and jejunum (P_w = 0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.     

Simultaneous measurement of amiodarone and desethylamiodarone in human plasma and serum by stable isotope dilution liquid chromatography–tandem mass spectrometry assay

A stable isotope dilution liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) assay to measure amiodarone, the most frequently used agent for maintaining sinus rhythm in patients with atrial fibrillation, and its major metabolite desethylamiodarone in human plasma and serum was developed. Measurement of amiodarone and desethylamiodarone was performed during a 4.0-min run-time using amiodarone-D₄ and desethylamiodarone-D₄ as internal standards. Calibration curves covering 12 calibrators measured in four replicates each for the analysis of both amiodarone and desethylamiodarone were linear and reproducible in the range of 0.01–40.0 mg/L (r > 0.999). Limits of detection in plasma matrix were 2.7 μg/L for amiodarone and 1.9 μg/L for desethylamiodarone, and lower limits of quantification in plasma matrix were 7.5 μg/L for amiodarone and 2.5 μg/L for desethylamiodarone. Interassay imprecision and inaccuracy were <8% and <9% for both substances. Mean extraction yield was 99.6% (range 92.6–107.7%) for amiodarone and 90.2% (range 80.0–94.7%) for desethylamiodarone. Agreement was moderate for amiodarone (n = 162) and desethylamiodarone (n = 117), respectively, between the present method and a HPLC method with UV detection using a commercially available reagent set for the HPLC analysis of these drugs. The Passing–Bablok regression line was HPLC = 0.98 (LC–MS/MS) + 0.10 [mg/L]; r = 0.94 for amiodarone and HPLC = 1.05 (LC–MS/MS) + 0.02 [mg/L]; r = 0.90 for desethylamiodarone. This sensitive and interference-free LC–MS/MS assay permits rapid and accurate determination of amiodarone and desethylamiodarone in human plasma and other body fluids.     

Determination of nutlin-3a in murine plasma by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS)

A sensitive and precise LC–ESI-MS/MS method for determination of nutlin-3a in murine plasma using ketoconazole as an internal standard was developed and validated. Plasma nutlin-3a samples were prepared by either a simple protein precipitation (PP) for the high concentration range (10–20,000 ng/mL) or by liquid–liquid extraction (LLE) for the low concentration range (0.25–300 ng/mL). Nutlin-3a and ketoconazole were separated on a modified C18 analytical column (4 μm, 75 mm × 2 mm) with an isocratic mobile phase (acetonitrile/5 mM HCOONH₄ = 70/30, v/v). The retention times of nutlin-3a and ketoconazole were 1.14 and 1.45 min. Detection was achieved by a tandem MS system, monitoring m/z 582/99 and m/z 532/82 for nutlin-3a and ketoconazole, respectively. The PP method was linear in a range of 10–20,000 ng/mL (R² ≥ 0.993) and the LLE method was linear in a range of 0.25–300 ng/mL (R² ≥ 0.992). The mean recoveries for PP and LLE were 24% and 78%, respectively. Within-day and between-day precisions were ≤4.5% for PP and were ≤4.9% for LLE. Within-day and between-day accuracies (% error) ranged from 4.8 to −7.9 for PP, and from −0.2 to −8.4 for LLE. The two extraction methods produced equivalent results, allowing use of both within the same study. This method has been applied to the measurement of nutlin-3a concentrations in murine plasma samples obtained from a preclinical pharmacokinetic study.     

A case-control study of polymorphisms in xenobiotic and arsenic metabolism genes and arsenic-related bladder cancer in New Hampshire

Arsenic is associated with bladder cancer risk even at low exposure levels. Genetic variation in enzymes involved in xenobiotic and arsenic metabolism may modulate individual susceptibility to arsenic-related bladder cancer. Through a population-based case-control study in NH (832 cases and 1191 controls), we investigated gene-environment interactions between arsenic metabolic gene polymorphisms and arsenic exposure in relation to bladder cancer risk. Toenail arsenic concentrations were used to classify subjects into low and high exposure groups. Single nucleotide polymorphisms (SNPs) in GSTP1, GSTO2, GSTZ1, AQP3, AS3MT and the deletion status of GSTM1 and GSTT1 were determined. We found evidence of genotype-arsenic interactions in the high exposure group; GSTP1 Ile105Val homozygous individuals had an odds ratio (OR) of 5.4 [95% confidence interval (CI): 1.5–20.2; P for interaction = 0.03] and AQP3 Phe130Phe carriers had an OR = 2.2 (95% CI: 0.8–6.1; P for interaction = 0.10). Bladder cancer risk overall was associated with GSTO2 Asn142Asp (homozygous; OR = 1.4; 95% CI: 1.0–1.9; P for trend = 0.06) and GSTZ1 Glu32Lys (homozygous; OR = 1.3; 95% CI: 0.9–1.8; P for trend = 0.06). Our findings suggest that susceptibility to bladder cancer may relate to variation in genes involved in arsenic metabolism and oxidative stress response and potential gene-environment interactions requiring confirmation in other populations.     

In vitro and in vivo antibacterial activities of garenoxacin against group G Streptococcus dysgalactiae subsp. equisimilis

In this study, garenoxacin showed potent in vitro activity against clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis [minimum inhibitory concentration for 90% of the organisms (MIC₉₀) = 0.125 μg/mL] and was superior to levofloxacin (MIC₉₀ = 1 μg/mL) and moxifloxacin (MIC₉₀ = 0.25 μg/mL). In experimental pneumonia caused by group G S. dysgalactiae subsp. equisimilis in mice, the effective dose for 50% survival (ED₅₀) of garenoxacin following single oral administration was 1.87 mg/kg, >10.7-fold and 4.6-fold less than the ED₅₀ values of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively. The area under the free serum concentration-time curve from 0-24 h (fAUC_0-24)/MIC ratio of garenoxacin in serum following oral administration of 20 mg/kg was 73.2, which was 8.7-11.4-fold and 1.4-fold greater than that of levofloxacin (6.44-8.46) and moxifloxacin (51.4), respectively. These results suggest that garenoxacin has potential for the treatment of infectious diseases caused by S. dysgalactiae subsp. equisimilis.     

In vitro activity of S-(3,4-dichlorobenzyl)isothiourea hydrochloride and novel structurally related compounds against multidrug-resistant bacteria, including Pseudomonas aeruginosa and Burkholderia cepacia complex

The aim of this study was to establish the antimicrobial activities of S-(3,4-dichlorobenzyl)isothiourea hydrochloride (A22) and a series of structurally related compounds against multidrug-resistant (MDR) bacteria. The minimum inhibitory concentrations (MICs) of 21 compounds were determined against 18 strains of pathogenic bacteria in addition to Pseudomonas aeruginosa (n = 19) and Burkholderia cepacia complex (BCC) (n = 20) isolated from the sputa of cystic fibrosis patients. Selected compounds were tested against further isolates, including P. aeruginosa (n = 100), BCC (n = 12) and Stenotrophomonas maltophilia (n = 19). The interaction of S-(4-chlorobenzyl)isothiourea hydrochloride (C2) in combination with conventional antimicrobials was examined against 10 P. aeruginosa strains. Selected compounds were also tested against Enterobacteriaceae producing NDM-1 carbapenemase (n = 64) and meticillin-resistant Staphylococcus aureus (MRSA) (n = 37). Of the 21 compounds, 14 showed antimicrobial activity that was generally more pronounced against Gram-negative bacteria. Against P. aeruginosa, the most active compound was C2 [MIC for 50% of the organisms (MIC₅₀) = 32 μg/mL]. This compound was also the most active against BCC, with all isolates inhibited by 64 μg/mL. For all ten strains of P. aeruginosa subjected to combination testing with C2 and conventional antimicrobials, a bactericidal effect was achieved with at least one combination. C2 and A22 both showed strong activity [MIC for 90% of the organisms (MIC₉₀) = 4 μg/mL] against Enterobacteriaceae that produced NDM-1 carbapenemase. Finally, S-(4-chlorobenzyl)-N-(2,4-dichlorophenyl)isothiourea hydrochloride showed good activity (MIC₉₀ = 8 μg/mL) against MRSA. This work establishes the activity of isothiourea derivatives against a broad range of clinically important MDR bacteria.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.     

Quantitative determination of flavonoids and cycloartanol glycosides from aerial parts of Sutherlandia frutescens (L.) R. BR. by using LC-UV/ELSD methods and confirmation by using LC–MS method

This paper describes the first analytical method for the determination of four flavonoids (sutherlandins A–D) and four cycloartanol glycosides (sutherlandiosides A–D) from the aerial parts of Sutherlandia frutescens (L.) R. Br. A separation by HPLC was achieved by using a reversed phase (RP-18) column, PDA with ELS detection, and a water/acetonitrile gradient as the mobile phase. The wavelength used for quantification of four flavonoids with the diode array detector was 260 nm. Owing to their low UV absorption, the cycloartanol glycosides were detected by evaporative light scattering. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). The limits of detection and limits of quantification of eight compounds were found to be in the range from 0.1 to 7.5 μg/mL and 0.5 to 25 μg/mL, respectively. The analysis of products showed considerable variation of 1.099–5.224 mg/average weight for the major compound, sutherlandioside B. The eight compounds in plant sample and products of S. frutescens were further confirmed by LC–ESI-TOF. This method involved the use of the [M+H]⁺ and [M+Na]⁺ ions in the positive ion mode with extractive ion monitoring (EIM).     

HPLC method validation for Digitalis and its analogue by pulsed amperometric detection

We developed a highly sensitive and selective reversed-phase HPLC-pulsed amperometric detection (RP-HPLC-PAD) method for cardiac glycoside detection. Eight cardiac glycosides were completely separated within 45 min on a reversed-phase column using a water–acetonitrile gradient, and were detected using a PAD under NaOH alkaline conditions. The detection (S/N = 3) and quantification (S/N = 10) limits for the cardiac glycosides were 0.1–0.3 and 0.3–0.8 ng, respectively. The linear regression coefficient was 0.9962–0.9998 for concentrations of 1–25 μg/mL. Cardiac glycosides in the Digitalis purpurea leaf displayed intra- and inter-day precisions (RSDs) of <9.30% and average recoveries of 98.63–99.94%. The contents of gitoxin, digitonin, and digitoxin in the D. purpurea were 0.197, 0.11, and 0.379 mg/g for leaf dried at 60 °C, 0.058, 0.11, and 0.090 mg/g for leaf dried at ambient temperature, and N.D. (not detected), and 18.379 mg/g, N.D. for seed, respectively. We conclude that our method shows good precision and accuracy.     

Development and validation of an HPLC-DAD method for bis(12)-hupyridone and its application to a pharmacokinetic study

A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of bis(12)-hupyridone in rat blood has been developed and validated. Chromatographic separation was carried out in an Agilent Extend C₁₈ 5 μm column (length, 250 mm; inner diameter, 4.6 mm) using a mixture of water–acetonitrile–trifluoroacetic acid (81:19:0.04, v/v/v) as the mobile phase at a flow rate of 1 mL/min, with detection at 229 nm. The method used for the bis(12)-hupyridone quantification showed linearity for concentration range of 0.1–7.5 μg/mL with r² = 0.9991. The limit of detection and quantification of this method were 0.05 μg/mL and 0.1 μg/mL, respectively. The intra- and inter-day variations of the analysis were less than 4.22% with standard errors less than 13.3%. The developed method was successfully applied to the pharmacokinetic study of bis(12)-hupyridone after intravenous administration of 5 mg/kg and intraperitoneal administration of 10 and 20 mg/kg in rats.     

Determination of cidofovir in human plasma after low dose drug administration using high-performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method for the determination of cidofovir (CDV) in human plasma using high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Varian^® SAX extraction cartridges prior to chromatography. The internal standard was ¹³C5-Folic acid (¹³C5-FA). Chromatography was performed using a Luna C8(2) analytical column, 5 μm, 150 mm × 3.0 mm, using an isocratic elution with a mobile phase consisting of 43% methanol in water containing 12 mM ammonium acetate, at a flow rate of 0.3 mL/min. The retention times of CDV and ¹³C5-FA were 2.1 min and 1.9 min, respectively, with a total run time of 5 min. The analytes were detected by a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 280.0 → 262.1 for CDV and m/z 447.0 → 294.8 for ¹³C5-FA (IS). The assay was linear over the range 20–1000 ng/mL. Accuracy (101.6–105.7%), intra-assay precision (4.1–5.4%), and inter-assay precision (5.6–6.8%) were within FDA limits. No significant variation in the concentration of CDV was observed with different sample storage conditions. This method is simple, adaptable to routine application, and allows easy and accurate measurement of CDV in human plasma.     

Plasma pharmacokinetics and tissue distribution study of cajaninstilbene acid in rats by liquid chromatography with tandem mass spectrometry

Cajaninstilbene acid (CSA; 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) is a major active constituent of pigeonpea leaves, has been proven to be effective in clinical treatment of diabetes, hepatitis, measles and dysentery. A rapid and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of CSA in rat plasma and various tissues (brain, heart, lung, liver, spleen, small intestine and kidney) of rat for the first time. Rat plasma and tissue distribution pre-treated by protein precipitation with acetoacetate was analyzed using LC–MS/MS with an electrospray ionization (ESI) interface, and isoliquiritigenin was used as an internal standard. Chromatographic separation was achieved on a HIQ Sil C₁₈ column with the mobile phase of water and methanol (9:91, v/v) containing 0.1% formic acid and resulted in a total run time of 10 min. The isocratic elution mode pumped at a flow rate of 1.0 mL/min. The lower limit of quantification (LLOQ) which was 10 ng/mL. The calibration curve was linear from 10 to 6000 ng/mL (R = 0.9967) for plasma samples and 10 to 6000 ng/mL (R ≥ 0.9974) for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 0.6% to 6.1% and 1.5% to 6.6%, respectively, and the intra- and inter-day assay accuracy was 93.5–104.6% and 93.3–107.5%, respectively. Recoveries in plasma and tissues ranged from 95.0% to 106.8%. The method was successfully applied in pharmacokinetic and tissue distribution studies of CSA after oral administration to rats. The pharmacokinetics of CSA showed rapid absorption and elimination (T_max, 10.7 ± 0.31 min; t_1/2, 51.40 ± 6.54 min). After oral administration in rats, CSA was rapidly and widely distributed in tissues. High concentrations were found in liver and kidney indicating that CSA was possibly absorbed by liver and eliminated by kidney.     

3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1β, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC₅₀ = 10 nM), mouse (IC₅₀ = 9 nM) and human (IC₅₀ = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1β and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([³H]A-804598; 8.1 Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [³H]A-804598 labeled a single class of high affinity binding sites (K_d = 2.4 nM and apparent B_max = 0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [³H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r = 0.95, P < 0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [³H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.     

Determination of anabolic steroids in human urine by automated in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry

A simple, rapid and sensitive method was developed for determining the presence of seven anabolic steroids (boldenone, nandrolone, testosterone, methyltestosterone, epiandrosterone, androsterone, and atnozolol) in human urine. Glucuronide-conjugates of these compounds were hydrolyzed with β-glucuronidase. The anabolic steroids were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–mass spectrometry (LC–MS). The steroids were separated within 14 min by high performance liquid chromatography using a Chromolith RP-18e column and 5 mM ammonium formate/methanol (35/65, v/v) as a mobile phase at a flow rate of 1.0 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for the MS detection of these compounds. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 μL using a Supel-Q PLOT capillary column for the extraction. The extracted compounds could be desorbed readily from the capillary column by flow of the mobile phase, and no carryover was observed. Using the in-tube SPME LC–MS with SIM mode detection, good linearity of the calibration curve (r > 0.995) was obtained in the concentration range of 0.5–20 ng/mL, except for stanozolol. The detection limits (S/N = 3) of anabolic steroids were in the range 9–182 pg/mL and the proposed method showed 20–33-fold higher sensitivity than the direct injection method. The within-day and between-day precisions were below 4.0% and 7.3% (n = 5), respectively. This method was applied successfully to the analysis of urine samples without the interference peaks. The recovery rates of anabolic steroids spiked into urine samples were above 85%. This method is useful to analyze the urinary levels of these compounds in anti-doping tests.     

Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma using HPLC coupled with tandem mass spectrometry: Application to bioequivalence studies

A sensitive, specific and selective liquid chromatography/tandem mass spectrometric method has been developed and validated for the simultaneous determination of irbesartan and hydrochlorothiazide in human plasma. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard losartan were separated on a reverse phase C₁₈ column (50 mm × 4 mm, 3 μm) using water with 2.5% formic acid, methanol and acetonitrile (40:45:15, v/v/v (%)) as a mobile phase (flow rate of 0.70 mL/min). Irbesartan and hydrochlorothiazide were ionized using ESI source in negative ion mode, prior to detection by multiple reaction monitoring (MRM) mode while monitoring at the following transitions: m/z 296 → 269 and m/z 296 → 205 for hydrochlorothiazide, 427 → 175 for irbesartan. Linearity was demonstrated over the concentration range 0.06–6.00 μg/mL for irbesartan and 1.00–112.00 ng/mL for hydrochlorothiazide. The developed and validated method was successfully applied to a bioequivalence study of irbesartan (300 mg) with hydrochlorothiazide (12.5 mg) tablet in healthy volunteers (N = 36).