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.     

The effect of age and acetylator phenotype on the pharmacokinetics of sulfasalazine in patients with rheumatoid arthritis.

The pharmacokinetic disposition of sulfasalazine and its metabolites was studied in 8 young and 12 elderly patients with active rheumatoid arthritis. Equal numbers of slow and fast acetylators were included in each age group. Patients received enteric-coated sulfasalazine 2g daily for 21 days; specimens of serum and urine were collected for 96 h after administration on days 1 and 21. The elimination half-life of sulfasalazine was greater in the elderly patients. Many disposition parameters of sulfapyridine differed in slow and fast acetylators; of greatest significance were the increased values of steady-state serum concentration in the slow acetylators. There was no effect of age on any sulfapyridine disposition parameters. Values for the steady-state serum concentrations of N-acetyl-5-acetylsalicylic acid were greater in elderly than in young patients. The metabolism of sulfapyridine was markedly affected by acetylator phenotype and this was reflected in the composition of sulfapyridine-related material in the urine. Thus, age is a determinant of the steady-state concentrations of salicylate moieties but acetylator phenotype plays a greater role in determining the serum concentration of sulfapyridine, which has greater therapeutic implications in rheumatology.     

Acetylator phenotype and serum levels of sulfapyridine in patients with inflammatory bowel disease

Summary Twenty-eight outpatients receiving sulfasalazine for inflammatory bowel disease were monitored. Assessment of acetylator phenotype according to the percentage of acetylated sulfapyridine in serum provided a clear distinction between rapid and slow acetylators. In comparison, the percentage of acetylated sulfapyridine in saliva or urine was a less precise index of phenotype. Determination of saliva concentrations of sulfapyridine and N⁴-acetylsulfapyridine did not provide a reliable estimate of serum levels. Slow acetylators had significantly higher serum concentrations of sulfapyridine (21.9±14.0 [SD] µg/ml) than rapid acetylators (8.8±4.3 µg/ml) and had a higher incidence of toxicity (not statistically significant,p>0.05). Serum concentrations of sulfapyridine were significantly higher in patients with symptoms of toxicity (23.2±15.9 µg/ml) than those without (13.9±9.5 µg/ml) (p<0.05). However, serum concentrations of total sulfapyridine (sulfapyridine plus N⁴-acetylsulfapyridine) were not significantly different in patients with (32.9±21.2 µg/ml) or without (22.8±12.0 µg/ml) toxicity (p>0.05). For all patients serum concentrations of sulfapyridine (total sulfapyridine) ranged from 3.5 to 73.1 (5.7 to 95.1) µg/ml in patients with controlled disease and 6.3 to 38.0 (14.0 to 54.7) µg/ml in patients with active disease. A significant correlation between clinical status of disease and serum drug concentrations was only apparent for rapid acetylators (p<0.05). The daily sulfasalazine dosage (mg/kg of body weight, log value) and serum drug concentrations (log values) were highly correlated (p<0.05). For clinical evaluation of inflammatory bowel disease patients determination of serum sulfapyridine concentrations appears to be more important for monitoring toxicity than therapeutic efficacy of sulfasalazine. Assessment of acetylator status appears to be useful for predicting serum sulfapyridine levels in patients receiving sulfasalazine therapy.     

导数光谱法测定生物样品中柳氮磺吡啶及其代谢产物

目的建立一种同时测定生物样品中柳氮磺吡啶,5-氨基水杨酸和磺胺吡啶含量的方法。方法选用蛋白沉淀法,以丙酮或乙醇作为沉淀剂对生物样品进行前处理,并采用一级导数光谱法消除三者紫外吸收的干扰。结果实验的各项指标均达到国家规定的生物药物分析标准,生物样品萃取回收率均>79.1%,方法回收率在70.80%~115.6%,日内日间精密度(n=5)均<10%。结论该方法简便、快速、可靠,适用于上述3种药物的生物样品分析。     

N-Acetyltransferase 2 Genotype-Related Efficacy of Sulfasalazine in Patients with Rheumatoid Arthritis

PURPOSE: For the individual optimization of drug therapy with sulfasalazine (SASP), we studied the influence of the N-acetyltransferase 2 (NAT2) genotype on the pharmacokinetics, efficacy, and incidence of adverse reactions of SASP in patients. METHODS: Ninety-six rheumatoid arthritis (RA) patients were treated or had been treated with 0.5 and/or 1.0 g/day of SASP. The wild-type allele (NAT2*4) and three variant alleles (NAT2*5B, *6A, and *7B) of NAT2 were determined by the polymerase chain reaction-restriction fragment length polymorphism method. Plasma concentrations of SASP and its two metabolites, sulfapyridine (SP) and N-acetylsulfapyridine (AcSP), were estimated by HPLC. Therapeutic efficacy and incidence of adverse reactions were also monitored as recommended by the American College of Rheumatology. RESULTS: Patients were classified into three groups by NAT2 genotyping: Rapid Type (homozygote for NAT2*4), Intermediate Type (heterozygote for NAT2*4 and variant alleles), and Slow Type (homozygote for variant alleles). There was no clear difference in the genotype frequencies between RA patients and healthy subjects. NAT2 genotypes significantly affected both the plasma concentration ratios of SP to AcSP (SP/AcSP) and the efficacy of SASP (p < 0.05). Adverse reactions to SASP were found in 26 (27.1%) out of 96 patients, and there was no difference among the three genotype groups. CONCLUSIONS: NAT2 gene polymorphism is related to the plasma SP/AcSP ratio and the efficacy of SASP.     

Systematic analysis of diuretic doping agents by HPLC screening and GC/MS confirmation

The simultaneous analysis of diuretic agents by reversed-phase liquid chromatography with a diode-array detector (DAD) was performed by using a gradient elution with acetonitrile and phosphate buffer on a Hypersil-ODS column. For the spiked urine the extraction recovery of solid-phase extraction (SPE) using Sep-Pak C18 cartridge was compared with that of liquid-liquid extraction (LLE) with diethyl ether at various pH. The standard calibration curves were linear from 0.20-20.0 micrograms/mL for all diuretic agents except amiloride, 1.0-20.0 micrograms/mL, and the detection limit was about 0.2 micrograms/mL for 3 mL of urine, except that of amiloride, which was 1.0 micrograms/mL. The confirmation analysis was performed by gas chromatography/mass spectrometry (GC/MS) following methylation. The characteristic mass fragment ions obtained by electron-impact (EI) ionization (70 eV) provided identification of each diuretic agent.     

N,N'-diethyl-m-toluamide (m-DET): analysis of an insect repellent in human urine and serum by high-performance liquid chromatography.

A procedure for monitoring m-DET in human urine and serum is described. m-DET is removed from the urine specimen by partitioning into diethyl ether, but solid-phase extraction is used to remove it from human serum. The urine and serum m-DET values are determined by HPLC with a UV detector. The limit of detection was 0.09 micrograms/mL in urine and 0.09 micrograms/g for serum. The percent of m-DET recovered from human urine spiked between 0.50 and 8.00 micrograms/mL was 90 +/- 5.4%. For human serum spiked between 0.25 and 10.00, the percent recovered was 96 +/- 5.9%. The pooled relative standard deviations (RSD) for spiked matrices were 0.06 for urine and 0.06 for serum.     

Inhibition of cytotoxicity by sulfasalazine. I. Sulfasalazine inhibits spontaneous cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from control and inflammatory bowel disease patients

We have studied the effects of sulfasalazine and its metabolites on cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from both control and inflammatory bowel disease (IBD) patients. Sulfasalazine and sulfapyridine, as well as hydrocortisone and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD peripheral blood cells. Sulfasalazine and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD intestinal mononuclear cells cultured for 72 h in media alone. In contrast, 5-aminosalicylate, indomethacin and benzylimidazole had no effect on cytotoxicity by any cell population. Lectin-induced, antibody-dependent and interleukin-2-induced cell-mediated cytotoxicity, as well as lymphokine-activated killing were not inhibited by the drugs: inhibitory effects in these assays were primarily upon the underlying spontaneous cell-mediated cytotoxicity. The inhibition induced by sulfasalazine, sulfapyridine and nordihydroguaiaretic acid could not be reversed by adding the lipoxygenase metabolites leukotriene B4 or 12-hydroxyeicosatetraenoic acid. These findings demonstrate that spontaneous cell-mediated cytotoxicity by control and IBD mononuclear cells can be inhibited by sulfasalazine.     

Analysis of corticosteroids in urine by HPLC and thermospray LC/MS.

The method for simultaneous determination and confirmation of nine corticosteroids in urine by high-performance liquid chromatography with a diode array detector and thermospray liquid chromatography-mass spectrometry was studied. HPLC was performed on a C18 column with a gradient mobile phase system of water and acetonitrile. The calibration curve was linear from 20 ng/mL to 1.0 micrograms/mL for each corticosteroid, and the detection limit was 10 ng/mL in 5 mL of urine. The extraction recovery of each corticosteroid from the spiked urine was equal to or greater than 85% by liquid-liquid extraction (LLE) at pH 9 using diethyl ether and approximately 79% or more by solid-phase extraction (SPE) with a Sep-Pak C18 cartridge. The mass spectra obtained with the positive ion mode showed protonated molecular species [M + H]+, ammonium adduct ion [M + NH4]+, [MH-60]+, [MH-30]+, and [MH-18]+. The LC/MS detection limits ranged from 10 to 50 ng in the scan mode and from 1 to 5 ng in the selected ion monitoring (SIM) mode.     

Relative systemic availability of sulfapyridine from commercial enteric-coated and uncoated sulfasalazine tablets.

The absorption of sulfapyridine after a single 2.0-Gm oral dose of sulfasalazine, the drug of choice in the treatment of inflammatory bowel disease, as commercial uncoated and enteric-coated and uncoated tablets was evaluated in four healthy male adults. The peak plasma concentration of sulfapyridine after the enteric-coated tablets occurred at 20 hours on the average (compared to 14 hours for the uncoated tablets) and was only 50% of that attained from the uncoated tablets (P less than 0.05). The low relative extent of systemic availability of sulfapyridine from the enteric-coated tablets (65.5 +/- 6.3 per cent, mean +/- S.E.) compared to uncoated tablets may be due to absorption rate-dependent presystemic metabolism, since the relative extent of sulfapyridine absorption was 92.7 +/- 6.2 per cent compared to uncoated tablets. These findings suggest that enteric-coated and uncoated tablets of sulfasalazine are not bioequivalent. It remains to be determined whether the clinical efficacy of sulfasalazine from enteric-coated tablets is affected.     

Validated RP-HPLC Method for Simultaneous Quantitation of Losartan Potassium and Metolazone in Bulk Drug and Formulation

A HPLC method has been described for simultaneous determination of Losartan potassium and Metolazone in formulation. This method is based on a HPLC separation of the two drugs on the Thermo Hypersil BDS-C(18) (250 mm × 4.6 mm, 5.0 μm) with isocratic conditions and a simple mobile phase containing acetonitrile:water (60:40) at a flow rate of 0.8 mL/min using UV detection at 237 nm. This method has been applied to a marketed formulation without interference of excipients. The linear regression analysis data for the calibration plots showed a good linear relationship over the concentration range of 2-12 μg/mL for Losartan potassium and 0.2-1.2 μg/mL for Metolazone, respectively. The method was validated for precision, robustness and recovery. Statistical analysis showed that the method is repeatable and selective for the estimation of Losartan potassium and Metolazone.     

High-performance liquid chromatographic assay of the aldose reductase inhibitor spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567) in plasma and urine and its pharmacokinetics in humans

Two selective high-performance liquid chromatographic (HPLC) methods have been developed for the quantitative determination of spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567; 1) in plasma and urine, with an assay sensitivity of 0.25 micrograms/mL for plasma and 0.13 micrograms/mL for urine. The plasma assay procedure involved precipitation of proteins with acetonitrile followed by dilution with water. The diluted supernatant was analyzed on an ODS column eluting with acetonitrile:0.5% phosphoric acid (30:70) adjusted to pH 7.2 with concentrated ammonium hydroxide. The urine assay procedure involved extraction of 1 with 10% n-butanol in hexane, followed by back extraction with 0.05 M sodium hydroxide. The basic extract was neutralized and analyzed on a phenyl column eluting with acetonitrile:10 mM potassium phosphate (30:70; monobasic, pH 5.6). The pharmacokinetics of 1 was investigated in humans following single and multiple oral doses. The elimination half-life from 12 normal subjects following single 100-400-mg oral doses was independent of dose, and the overall mean half-life was 66 +/- 9 h. The overall mean oral clearance (assuming a bioavailability of 100%) was 11 +/- 3 mL/min, and the mean apparent volume of distribution was 59 +/- 13 L. The mean urinary recovery of intact drug during the first 24 h after dosing was 1.2 +/- 0.4% of the administered dose. During once daily 100-mg oral dosing of 1 to five subjects for 21 d, plasma concentrations of 1 reached apparent steady-state by 7 d.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new method for the determination in blood and urine of a novel triazolobenzodiazepine (estazolam) by HPLC

In this study the authors report a new method to determine estazolam in blood and urine by high pressure liquid chromatography (HPLC). This compound is a triazolobenzodiazepine and shows an interesting psychopharmacological activity. The extraction from biological fluids was carried out using a mixture of ethylenechloride, methylenechloride, and ethylacetate (1:1:8). The HPLC analysis was carried out with a C18 column, using methanol:phosphate buffer (pH 7.5, 0.011M):acetonitrile (65:33:2) as mobile phase. The detector was set at lambda = 240 nm. The method shows good repeatability and a linear response in the range of 0.6 to 10 micrograms estazolam/mL both in serum and urine. A case of non-lethal acute intoxication in which the method was applied is also reported.     

Determination of trazodone and its metabolite, m-CPP, in serum and urine by HPLC

A sensitive and rapid method for the analysis of trazodone (TZD) and its metabolite, 1-(m-chlorophenyl)piperazine (m-CPP), in the serum and urine of rabbits treated with the drug was developed. The assay requires extraction from the biological fluids with adequately buffered organic solvents followed by HPLC. The assay allows good reproducibility, fair recovery, and excellent linearity in the range of 0.6 to 10 micrograms/mL for TZD and 1.2 to 20 micrograms/mL for m-CPP.     

Simultaneous determination of pseudoephedrine hydrochloride, chlorpheniramine maleate, and dextromethorphan hydrobromide by second-derivative photodiode array spectroscopy

The simultaneous determination of the active ingredients in multicomponent pharmaceutical products normally requires the use of a separation technique, such as HPLC or GC, followed by quantitation. Presented here is a rapid, validated, analytical method that does not require prior separation for the simultaneous determination of three drugs, pseudoephedrine hydrochloride, chlorpheniramine maleate, and dextromethorphan hydrobromide, in a tablet formulation. A diode array spectrophotometer, capable of multicomponent analysis, was used for the quantitation. The utility of this method was demonstrated in two ways: the analysis of a chewable pediatric tablet (formulation CP) containing 7.5 mg of pseudoephedrine hydrochloride, 0.5 mg of chlorpheniramine maleate, and 2.5 mg of dextromethorphan hydrobromide, and the dissolution analysis of a hydroxypropyl methylcellulose-based sustained-release tablet (formulation SR) containing 120 mg of pseudoephedrine hydrochloride, 8 mg of chlorpheniramine maleate, and 60 mg of dextromethorphan hydrobromide. The sensitivity of this assay is 7.5 micrograms/mL for pseudoephedrine hydrochloride, 1.0 micrograms/mL for chlorpheniramine maleate, and 5.0 micrograms/mL for dextromethorphan hydrobromide, using the second-derivative spectra of the absorbance with respect to wavelength. Determinations were made in 0.1 M sodium acetate buffer at pH 5.0 using a 1-cm quartz cell. Absorbance spectra, and their first and second derivatives, from 240 to 300 nm were used for the determination. The results obtained by this method compared favorably with the results obtained by a validated HPLC method.     

Liquid chromatographic procedure for the quantitative analysis of carboplatin in beagle dog plasma ultrafiltrate

A specific and reproducible high-performance liquid chromatographic (HPLC) procedure was developed for the quantitative analysis of carboplatin (JM-8) in dog plasma ultrafiltrate. Plasma ultrafiltrate samples were generated using Amicon Centrifree micropartition systems or Amicon Centriflo cones, and injected onto a microBondapak NH2 column. The mobile phase consisted of acetonitrile:methanol:0.005 M sodium perchlorate, pH 2.4 (77-75:13-15:10, v/v/v); the flow rate was 1.5 mL/min. Detection was performed by monitoring UV absorbance of the column effluent at 229 nm. Carboplatin eluted between 9.5 and 11.0 min. The internal standard, JM-10, eluted between 11.0 and 13.0 min. The peak height ratio of carboplatin:internal standard versus carboplatin concentration was linear over a range of 0.2 to 20.0 micrograms/mL. The limit of quantitation was 0.2 microgram/mL. The intra-assay precision of this method, as measured by percent relative standard deviation (%RSD), was within 12% for the theoretical concentrations 0.5, 5.0, and 50.0 micrograms/mL. Accuracies were within 11%. The results of the validation procedures indicated that this procedure was accurate and specific.     

Inhibition of neutrophil degranulation and superoxide production by sulfasalazine: Comparison with 5-aminosalicylic acid,sulfapyridine and olsalazine

Sulfasalazine is a potent inhibitor of superoxide production and granule enzyme release by stimulated neutrophils, and modulation of these responses may contribute to its anti-inflammatory properties. It is a composite drug consisting of 5-aminosalicylic acid and sulfapyridine joined through an azo linkage. To investigate which functional groups on the molecule are active against neutrophil responses, 5-aminosalicylic acid, sulfapyridine and olsalazine were added to cells stimulated with fMet-Leu-Phe or immune complexes. The inhibitory effects of sulfasalazine on superoxide production, degranulation and neutrophil-mediated collagen degradation were closely mimicked by olsalazine, with the other two compounds having little effect on either function. Thus the azo link appears to be the important structural feature of sulfasalazine that affects neutrophil responses. This suggests that sulfasalazine could be anti-inflammatory in its own right rather than just acting as a source of 5-aminosalicylic acid. Our finclings are also a favourable indication for olsalazine (Dipentum), which is currently under trial as an anti-inflammatory agent.     

N-乙酰基转移酶2基因型多态性与柳氮磺胺吡啶治疗炎症性肠病所产生不良反应的关系

目的探讨N-乙酰基转移酶2(NAT2)基因型多态性对柳氮磺胺吡啶(SASP)治疗炎症性肠病(IBD)时所产生不良反应的影响。方法在110例IBD患者及120例健康对照者中,采用聚合酶链反应-限制性片断长度多态性方法,检测NAT2野生型等位基因(NAT2*4)和3种突变型等位基因(NAT2*5B、*6A和*7B)频率,并对服用SASP的78例IBD患者进行NAT2基因型与SASP不良反应的相关性分析。结果慢型乙酰化基因型的IBD患者服用SASP后不良反应的发生率高于快型和中间型乙酰化基因型患者,差异有显著性(55%vs24.1%,OR=3.841,95%CI=1.322~11.161,P=0.011)。与磺胺吡啶(SP)剂量相关的不良反应在慢型乙酰化患者中的发生率也高于快型和中间型乙酰化患者(55%vs20%,OR=4.889,95%CI=1.625~14.704,P=0.003)。结论IBD患者SASP不良反应,特别是与SP剂量相关的不良反应,与NAT2慢型乙酰化基因型相关。     

Rapid sample preparation and high performance liquid chromatographic determination of total and unbound serum disopyramide

A rapid sample preparation procedure, which requires no solvent extraction or concentration, for the high performance liquid chromatographic (HPLC) determination of disopyramide is described. The chromatography is performed on a C-18 radial compression mu-Bondapak column and detection by absorbance at 254 nm with a run time of 12 min. The mobile phase is 10 mM sodium acetate (pH 4.5)/acetonitrile (3:1 vol/vol). For the total drug assay, 50 microliter 30% (wt/vol) trichloroacetic acid is added to 500 microliter serum, which causes the precipitation of protein. Following centrifugation, 100 microliter of supernatant is mixed with 25 microliters of internal standard (25 micrograms/ml, ethyl p-aminobenzoate), and 50 microliters of this mixture is injected into the HPLC. Unbound disopyramide is separated from protein-bound drug by filtration with an Amicon Centrifree filter, which removes 99.6% of protein and does not retain disopyramide. To 100 microliters of this filtrate is added 25 microliters of internal standard, and 50 microliters is injected into the HPLC. The assay is linear to at least 20 micrograms/ml. The total drug assay shows an average recovery of 93.0% with an average coefficient of variation (CV) of 3.4%. The unbound drug assay shows an average CV of 4.1%. The percentage of free drug in a sample containing 4.85 g/dl protein varies from 68.0 to 83.5% at concentrations of 2.5-10 micrograms/ml, which illustrates the concentration-dependent nature of the protein binding, and the need to measure the unbound fraction of drug. Of 31 drugs tested for interference, none was found to interfere.