Quantification and Metabolite Identification of Sulfasalazine in Mouse Brain and Plasma Using Quadrupole-Time-of-Flight Mass Spectrometry.

Sulfasalazine (SAS), an anti-inflammatory drug with potent cysteine/glutamate antiporter system xc-(SXC) inhibition has recently shown beneficial effects in brain-related diseases. Despite many reports related to central nervous system (CNS) effect of SAS, pharmacokinetics (PK) and metabolite identification studies in the brain for SAS were quite limited. The aim of this study was to investigate the pharmacokinetics and metabolite identification of SAS and their distributions in mouse brain. Using in vivo brain exposure studies (neuro PK), the PK parameters of SAS was calculated for plasma as well as brain following intravenous and oral administration at 10 mg/kg and 50 mg/kg in mouse, respectively. In addition, in vivo metabolite identification (MetID) studies of SAS in plasma and brain were also conducted. The concentration of SAS in brain was much lower than that in plasma and only 1.26% of SAS was detected in mouse brain when compared to the SAS concentration in plasma (brain to plasma ratio (%): 1.26). In the MetID study, sulfapyridine (SP), hydroxy-sulfapyridine (SP-OH), and N-acetyl sulfapyridine (Ac-SP) were identified in plasma, whereas only SP and Ac-SP were identified as significant metabolites in brain. As a conclusion, our results suggest that the metabolites of SAS such as SP and Ac-SP might be responsible for the pharmacological effect in brain, not the SAS itself.

Highly luminescent nitrogen-doped carbon dots for simultaneous determination of chlortetracycline and sulfasalazine.

Here, we have presented a green and facile strategy to fabricate nitrogen-doped carbon dots (N-CDs) and their applications for determination of chlortetracycline (CTC) and sulfasalazine (SSZ). The fluorescent N-CDs, prepared by one-step hydrothermal reaction of citric acid and l-arginine, manifested numerous excellent features containing strong blue fluorescence, good water-solubility, narrow size distribution, and a high fluorescence quantum yield (QY) of 38.8%. Based on the fluorescence quenching effects, the as-synthesized N-CDs as a fluorescent nanosensor exhibited superior analytical performances for quantifying CTC and SSZ. The linear range for CTC was calculated to be from 0.85 to 20.38 µg ml-1 with a low detection limit of 0.078 µg ml-1 . Meanwhile, the linear range for SSZ was estimated to be from 0.34 to 6.76 µg ml-1 with a low detection limit of 0.032 µg ml-1 . Therefore, the N-CDs hold admirable application potential for constructing a fluorescent sensor for pharmaceutical analysis.

A solid-phase luminescence sensor based on molecularly imprinted polymer-CdSeS/ZnS quantum dots for selective extraction and detection of sulfasalazine in biological samples.

A new solid phase luminescence sensor (SPLS) based on molecularly imprinted polymer (MIP) as recognition element and semi-conductor CdSeS/ZnS alloyed quantum dots (QDs) as fluorophore has been synthesized and developed for the determination of sulfasalazine. The surface modified quantum dot was attached to the surface of the glass slide. MIP and Non-imprinted polymer (NIP) with the recognition site for Sulfasalazine (SSZ) were synthesized and attached to the immobilized QDs. A trace amount of sulfasalazine was determined using fluorescence spectroscopy method based on quenching effect of sulfasalazine on the QDs luminescence with a fluorescence resonance energy transfer (FRET) mechanism. The MIP-QDs based sensor showed high recognition capability of sulfasalazine in comparison with the NIP-QDs based sensor. Various experimental parameters affecting fluorescence intensity such as the reactions time and pH were investigated and optimized. Under optimal conditions, the MIP-QDs sensor showed good linearity for sulfasalazine in the range of 0.02-1.5 µmol L-1 with a determination coefficient (R2) of 0.9986. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were found to be 0.0071 and 0.024 µmol L-1, respectively. The intra-day and inter-day RSDs were 2.6-5.1% and 3.5-6.9%, respectively. The MIP-QDs based sensor was successfully used to the determination of sulfasalazine in human plasma and urine with the recoveries in the range of 90-107%, offering reliability and applicability of the sensor to the complex matrices.

Ultrasonic preparation of near-infrared emission cluster-based YbIII and NdIII coordination materials: Ratiometric temperature sensing, selective antibiotics detection and "turn-on" discrimination of l-arginine.

Currently near-infrared (NIR) luminescence of lanthanide ions has received great attention because of their unique emissions in the near-infrared region (800-1700 nm). These NIR luminescent materials behave excellent applications in many fields such as sensors and probes in optical amplification, laser systems, biological systems and organic light-emitting diodes. In this work, two new near-infrared (NIR) emission three-dimensional (3D) YbIII and NdIII cluster-based coordination materials, namely {[Yb2(L)2(DMF)(H2O)4]·(DMF)2 (H2O)}n (NIR-MOF 1) and [Nd(L)(DMF)2]n (NIR-MOF 2) (H3L = terphenyl-3,4″,5-tricarboxylic acid) have been synthesized through the facile sono-chemical preparation methods. Both the near-infrared materials 1 and 2 have been characterized by single crystal X-ray diffraction, powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM). Further the mixed-lanthanide near-infrared emission material Nd0.35Yb0.65L (NIR-MOF 3) can also be prepared under the sono-chemical conditions. NIR-MOF 3 can be successfully applied as the ratiometric NIR-MOF-based thermometer, which should origin from the emission intensity ratio between Yb3+ (976 nm) and Nd3+ (1056 nm) in the temperature range of 308-348 K. Besides these, the micro-morphologies of NIR-MOF 1 can be deliberately tuned through different sono-chemical reaction factors (reaction time, reaction temperature and sono-chemical powers). These tuned nano-sized materials NIR-MOF 1 (100 W, 80 min) can be utilized as the fluorescent sensing material to distinguish furazolidone and sulfasalazine from other antibiotics. At the same time, NIR-MOF 2 can be applied as the first example of MOFs-based sensors for discriminating l-arginine from other amino acids through the "turn-on" mode in the near-infrared emission region.

NiO nanoparticles modified carbon paste electrode as a novel sulfasalazine sensor.

The sol-gel synthesized NiO nanoparticles were characterized and used for the modification of a carbon paste electrode (CPE) in electrocatalytic voltammetric determination of sulfasalazine. Calcination temperature of NiO had an important role on the peak current of the modified NiO-CPE and calcined NiO at 200 °C had the best and maximum peak current for the corresponding electrode. Jahn-Teller and a Z-out effects for the singlet electrons in their 3dz2 orbital for calcined NiO at 200 °C relatively instable such electrons and hence they can easily participate in the electro-oxidation process. The electrochemical impedance spectroscopy (EIS) results confirmed that the modified CPE by calcined NiO at 200 °C has lower charge transfer resistance. In interaction effects study between the experimental variables by using the response surface methodology (RSM) approach, the optimized run included the 17.5% of NiO modifier, 0.5 M NaOH, amplitude of 295 mV, step potential of 7 mV and frequency of 15 Hz. A calibration plot in the range of 0.009-1.6 µM with limits of detection (LOD) and quantification (LOQ) of 0.002 and 0.006 µM was obtained, respectively. The modified electrode showed good selectivity and applicability for SSZ determination in complex matrixes.

Feasibility of amlodipine besylate, chloroquine phosphate, dapsone, phenytoin, pyridoxine hydrochloride, sulfadiazine, sulfasalazine, tetracycline hydrochloride, trimethoprim and zonisamide in SyrSpend(®) SF PH4 oral suspensions.

The objective of this study was to evaluate the feasibility of 10 commonly used active pharmaceutical ingredients (APIs) compounded in oral suspensions using an internationally used suspending vehicle (SyrSpend(®) SF PH4 liquid): (i) amlodipine, (as besylate) 1.0mg/mL; (ii) chloroquine phosphate,15.0 mg/mL; (iii) dapsone, 2.0 mg/mL; (iv) phenytoin, 15.0 mg/mL; (v) pyridoxine hydrochloride, 50.0 mg/mL; (vi) sulfadiazine, 100.0 mg/mL; (vii) sulfasalazine, 100.0 mg/mL; (viii) tetracycline hydrochloride, 25.0 mg/mL; (ix) trimethoprim, 10.0 mg/mL; and (x) zonisamide, 10.0 mg/mL. All suspensions were stored both at controlled refrigeration (2-8 °C) and controlled room temperature (20-25 °C). Feasibility was assessed by measuring the percent recovery at varying time points throughout a 90-day period. API quantification was performed by high-performance liquid chromatography (HPLC-UV), via a stability-indicating method. Given the percentage of recovery of the APIs within the suspensions, the expiration date of the final products (API+vehicle) was at least 90 days for all suspensions with regard to both the controlled temperatures. This suggests that the vehicle is stable for compounding APIs from different pharmacological classes.

High-speed liquid chromatographic analysis of sulfasalazine (salicylazosulfapyridine).

A high-speed liquid chromatographic method for analysis of sulfasalazine (salicylazosulfapyridine) in bulk powder and tablet dosage form is presented. Analysis is accomplished with a reverse-phase partition column and 10% 2-propanol in pH 7.7 phosphate buffer as the mobile phase. The method of analysis utilizes a simple, one-step, solubilization procedure with dimethylformamide, addition of an internal standard, and chromatography. The method is specific for sulfasalazine in the presence of starting materials, degradation products, or by-products from its manufacture.

A simple and rapid liquid chromatographic method for the determination of major metabolites of sulfasalazine in biological fluids.

A simple and rapid assay for quantitation of sulfasalazine metabolites in rat urine and plasma was developed using high-performance liquid chromatography (HPLC). The method involves dilution of urine or plasma samples (0.1 mL) with methanol for protein precipitation, followed by mixing and centrifugation at 10,000 x g. Chromatography was accomplished with a reversed-phase ODS C-18 column (5 mu; 4.6 x 250 mm). The mobile phase consisted of 20% methanol in 5.0 mM phosphate buffer (pH 6.0), with 0.5 mM tetrabutylammonium chloride as an ion-pairing agent. The flow rate was 1.7 mL/min. An injection volume of 30 microL was used and the metabolites were quantitated by an ultraviolet detector at 254 nm. Benzamide was used as the internal standard. This method is linear in the range of 0.5 to 25 micrograms/mL for 5-aminosalicylic acid (5-ASA), acetylsulfapyridine (Ac-SP), and acetyl-5-aminosalicylic acid (Ac-5-ASA), and from 0.25 to 25 micrograms/mL for sulfapyridine (SP). The percent relative standard deviation ranged from 1 to 7.9% for the metabolite standard curves and precision studies. The limit of detection for 5-ASA, Ac-SP, and Ac-5-ASA is 100 ng/mL, and for SP is 50 ng/mL, in both urine and plasma. This method is rapid, precise, and accurate, and has been used to determine sulfasalazine metabolites in individual rat plasma and urine samples following an oral dose of 60 mg/kg of sulfasalazine.

Characterization of impurities in sulfasalazine.

The chemical structures of four impurities isolated from sulfasalazine were determined. Three impurities are the by-products of the reaction process in drug synthesis, i.e., during diazotization of sulfapyridine and during coupling with salicylic acid. Only one contaminant was identified as a starting material, sulfapyridine, in the drug synthesis. The four impurities were characterized as 2-[[p-(2-pyridylsulfamoyl)-phenyl]azo]hydroxybenzene (I), 3-[[P-(2-pyridylsulfamoyl) phenyl]-azo]salicylic acid (II), 5-[[p-[4-(2-pyridylanilino)]-N-phenyl]azo]salicylic acid (III), and sulfapyridine (IV). Compounds I-III are novel molecules, and IV is the precursor of sulfasalazine. The isolation of the impurities was accomplished by TLC and liquid extraction procedures. The methods used to characterize the impurities were a combination of IR, UV, and NMR spectroscopy, mass spectrometry, and TLC. For I and III, comparisons also were made with the synthesized materials to supplement the evaluation.

Efficient biliary excretion of susalimod, probably via the bromosulphthalein carrier, studied in a chronic bile fistula model in dogs.

Susalimod is a structural analogue of sulphasalazine, known to be extensively excreted in the bile in various animal species and for inducing bile duct hyperplasia after long-term treatment of the dog with doses exceeding 25 mg kg(-1). In this study local concentrations of susalimod in the bile duct were determined after oral administration in dogs. A chronic bile fistula experimental model was designed to affect the bile duct as little as possible. The dogs received repeated oral doses of 25-150 mg kg(-1) day(-1) for 5 days; these doses had been used in previous toxicology studies. Extremely high biliary concentrations of unchanged susalimod (20,000-43,000 microM) were measured. Biliary excretion approached saturation at the higher doses, resulting in super-proportional increases in peripheral plasma concentrations as the dose was increased. The maximal bile/plasma concentration ratio was 4300. The high biliary clearance was indicative of almost complete first-pass elimination at doses below saturation of the elimination process. Interaction studies with the biliary excretion marker bromosulphthalein (BSP) demonstrated that susalimod and BSP probably share the same carrier transport system in biliary excretion. The elimination of BSP from plasma was prolonged 20 times and the biliary excretion rate was markedly reduced when susalimod was co-administered with BSP. These results show that susalimod is highly enriched in the bile, in a saturable manner, after oral administration. The compound interacts with the biliary excretion of BSP, suggesting that it shares the same carrier-mediated transport system.

[Localization of salazopyrin in colonic mucosa patients with ulcerative colitis].

In this study SASP metabolite levels were measured in colonic mucosal specimens and plasma samples from 31 patients with ulcerative colitis (UC) under treatment with the drug. Colonic tissue specimens were obtained by endoscopically guided biopsy and plasma was isolated from peripheral blood. Measurements were performed by HPLC according to the procedure of Fischer et al. The levels of 5-ASA and SP in either of colonic tissue or plasma were significantly lower than those of Ac-5-ASA and Ac-SP, respectively. The tissue level of 5-ASA had a significant correlation to the dosage of 5-ASA. The tissue levels of 5-ASA and Ac-5-ASA were low in an active stage of UC than during a remission period and the difference observed with respect to the latter metabolite was significant. These findings suggest that acetylation of 5-ASA is inhibited in the colonic mucosa in an active stage of the disease.

Quantitative HPLC analysis of mebeverine, mesalazine, sulphasalazine and dispersible aspirin stored in a Venalink monitored dosage system with co-prescribed medicines.

An HPLC method for the quantitative analysis of mebeverine HCl, 5-aminosalicylic acid (5-ASA), sulphasalazine and dispersible aspirin has been developed and then applied to these specific medicines when stored, with other medications, in Venalink blister packs (monitored dosage system) for periods of up to 35 days. Chromatographic separation was achieved on a reversed-phase C(12) column with an isocratic mixture of methanol, water and acetic acid as the mobile phase. The method was validated regarding: accuracy, precision, detection limits, quantification limits, specificity and robustness.

Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe.

Breast cancer resistance protein (BCRP) is an efflux transporter expressed in tissues that act as barriers to drug entry. Given that single nucleotide polymorphisms (SNPs) in the ABCG2 gene encoding BCRP are common, the possibility exists that these genetic variants may be a determinant of interindividual variability in drug response. The objective of this study is to confirm the human BCRP-mediated transport of sulfasalazine in vitro, evaluate interindividual variation in BCRP expression in human intestine and to determine the role of ABCG2 SNPs to drug disposition in healthy patients using sulfasalazine as a novel in vivo probe. To evaluate these objectives, pinch biopsies were obtained from 18 patients undergoing esophagogastro-duodenoscopy or colonoscopy for determination of BCRP expression in relation to genotype. Wild-type and variant BCRP were expressed in a heterologous expression system to evaluate the effect of SNPs on cell-surface trafficking. A total of 17 healthy individuals participated in a clinical investigation to determine the effect of BCRP SNPs on sulfasalazine pharmacokinetics. In vitro, the cell surface protein expression of the common BCRP 421 C>A variant was reduced in comparison with the wild-type control. Intestinal biopsy samples revealed that BCRP protein and mRNA expression did not significantly differ between patients with 34GG/421CC versus patients with 34GG/421CA genotypes. Remarkably, in subjects with 34GG/421CA genotype, sulfasalazine area under the concentration-time curve was 2.4-fold greater compared with 34GG/421CC subjects (P<0.05). This study links commonly occurring SNPs in BCRP with significantly increased oral sulfasalazine plasma exposure in humans. Accordingly, sulfasalazine may prove to have utility as in vivo probe for assessing the clinical impact of BCRP for the disposition and efficacy of drugs.

Immunochemical method for sulfasalazine determination in human plasma.

Sulfasalazine is an antibiotic used in the treatment of inflammatory bowel diseases. For the assessment of sulfasalazine in several biological matrices, an Enzyme-Linked Immunosorbent Assay (ELISA) method based on polyclonal antibodies was developed and characterized. The immunoassay showed a high sensitivity (IC50=0.51 ng mL(-1)) and specificity, a detection limit of 0.02 ng mL(-1) and a dynamic range of 0.06-3.75 ng mL(-1) (80-20% inhibition). The immunoassay performed well when it was applied to spiked plasma samples (from 0.5 to 2.0 ng mL(-1)) previously cleaned up by protein precipitation with methanol. Recoveries ranged from 83 to 119%, with a mean value of 99% (CV=13%). Since sulfasalazine remaining of a treatment reaches the systemic circulation in unchanged form, the immunoassay can be applied to the determination of this pharmaceutical in human plasma in order to facilitate the control of the patients through the application of personal doses.