Determination of sparfloxacin in plasma and urine by a simple and rapid liquid chromatographic method.

A simple, specific, sensitive, and rapid method has been developed and validated for the determination of sparfloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with ultraviolet detection. Plasma proteins were efficiently removed by precipitation with perchloric acid after the addition of grepafloxacin as an internal standard. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a C18 reversed-phase column. The quantification limit was 0.025 mg/L in plasma and 0.5 mg/L in urine. The coefficients of variation (CV) were less than 10% for intra-day and inter-day analyses. The recovery of sparfloxacin added to plasma and urine ranged from 96.7% to 97.9%. The method has been successfully applied to pharmacokinetic studies.     

An isocratic high-performance liquid chromatographic system for simultaneous determination of theophylline and its major metabolites in human urine

A simple and highly selective isocratic high-performance liquid chromatography method is presented for the simultaneous determination of theophylline and its major metabolites in human urine using β-hydroxyethyl theophylline as an internal standard. The method utilizes direct injection of diluted urine samples followed by separation and quantitation by reversed-phase isocratic elution and ultraviolet detection. The assay is accurate and reproducible with a sensitivity of 1 μg ml⁻¹ for theophylline and 0.5 μg ml⁻¹ for its metabolites. The assay was employed for the analysis of theophylline and its major metabolites in urine following the oral administration of theophylline to four healthy volunteers.     

Simultaneous determination of 2,4-D and MCPA in canine plasma and urine by HPLC with fluorescence detection using 9-anthryldiazomethane (ADAM)

A method for the simultaneous determination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) in canine plasma and urine has been developed. This method used derivatization of extracted samples with 9-anthrylmethane (ADAM) for analysis by reversed-phase high-performance liquid chromatography with fluorescence detection. Precision and accuracy were within the accepted limits of 15% and 85-115%, respectively, for both analytes in plasma and urine. Calibration curves for 2,4-D and MCPA in plasma were linear (r2 > 0.99) between 0.50 and 5.0 mg/L and 5.0 and 100 mg/L. Calibration curves for 2,4-D and MCPA in urine were linear (r2 > 0.99) between 5.0 and 70.0 mg 2,4-D/L and 10.0 and 70.0 mg MCPA/L. The lower limit of detection was 62.5 ng/mL for both 2,4-D and MCPA.     

Simultaneous determination of metoprolol and alpha-hydroxymetoprolol in human plasma and urine by liquid chromatography with a preliminary observation on metoprolol oxidation in Japanese subjects

A simple, sensitive, and highly reliable liquid chromatographic method using fluorescence detection is described for the simultaneous determination of metoprolol and alpha-hydroxymetoprolol in plasma and urine. This method involves a single extraction of the compounds with the internal standard pindolol from alkalinized plasma or urine into dichloromethane. A reconstituted aliquot with a mobile phase is injected onto a reversed-phase, Zorbax ODS column, and the detection is achieved by the excitation and emission wavelengths at 230 and 300 nm, respectively. The assay is reproducible and precise for metoprolol and alpha-hydroxymetoprolol in both plasma and urine samples, as judged by a coefficient of variation of less than 9.2% at all concentrations examined. The standard curves for metoprolol and alpha-hydroxymetoprolol are linear over 10-200 ng/ml in plasma and over 0.5-10 micrograms/ml in urine. The lower detection limit is 2 ng/ml for each of the compounds in plasma using a 0.5-ml sample. Preliminary data on the oxidation polymorphism of metoprolol in Japanese subjects are reported using the current assay method. In 183 Japanese subjects no poor metabolizer of metoprolol has been identified so far.     

Preliminary evaluation of the Abbott TDx for benzoylecgonine and opiate screening in whole blood

A method for the detection of benzoylecgonine (cocaine metabolite) and opiates in whole blood is described. This method employs the Abbott TDx fluorescence polarization immunoassay technique, which was designed for urine analysis. Drug-free whole blood was spiked with varying concentrations of benzoylecgonine, morphine, and codeine. Samples were prepared for analysis by adding 300 microL of 10% trichloroacetic acid to 300 microL of blood. Specimens were vortexed and centrifuged with 50 microL of supernatant required per assay. Precision studies of six replicate samples spiked with benzoylecgonine at 0.5 mg/L gave a within-run CV of 4.7% and a between-run CV of 4.7% with a detection limit of 0.1 mg/L. Within-run CVs for morphine at 0.5 mg/L and 0.1 mg/L were 1.7% and 7.9% respectively. The detection limits for morphine and codeine were 0.05 mg/L. Correlation coefficients for spiked whole blood calibration curves of benzoylecgonine, morphine, and codeine were 0.984, 0.999, and 0.997 respectively. This preliminary evaluation demonstrates a potential application of the TDx fluorescence polarization immunoassay technique to the analysis of drugs in whole blood.     

The quantification of paracetamol, paracetamol glucuronide and paracetamol sulphate in plasma and urine using a single high-performance liquid chromatography assay

A range of analytical methods exist for the determination of paracetamol in biological fluids. However, to understand the fate of paracetamol and the effect of other drugs on its disposition in vivo, the major metabolites require quantification in urine and plasma. A method to simultaneously quantify paracetamol, paracetamol glucuronide (PG) and paracetamol sulphate (PS) in plasma and urine with superior sensitivity is therefore desired, especially if the volume of plasma available is low. A simple isocratic reverse phase high-performance liquid chromatography (HPLC) assay with spectrophotometric detection has been developed. The method, requiring only 100 microl of plasma and 50 microl of urine, utilizes a reversed-phase C18 column, a wavelength of 254 nm for detection and a mobile phase composed of potassium dihydrogen orthophosphate (0.1 M)-isopropanol-tetrahydrofuran (THF) (100:1.5:0.1, v/v/v) adjusted to pH 3.7 with phosphoric acid. The method is sensitive and linear in plasma within a concentration range from 0.4 to 200 microM for paracetamol, PG and PS. For PG and PS in urine, the method is sensitive and linear within a concentration range from 100 to 20,000 microM. Over these ranges, accuracy and precision were less than 12%. The assay has been used to measure concentrations of paracetamol and the two metabolites in plasma collected by finger-prick sampling and of the metabolites in urine from healthy volunteers administered a single oral dose of 1000 mg of paracetamol.     

同时分析酒石酸美托洛尔和富马酸比索洛尔与阿替洛尔的气相色谱-质谱联用法研究

目的建立同时分析酒石酸美托洛尔、富马酸比索洛尔和阿替洛尔的气相色谱-质谱联用法。方法样品经N,O-双（三甲基硅烷）三氟乙酰胺衍生化后,用气相色谱-质谱联用仪进行分离与分析。结果3种心脏病常用药物酒石酸美托洛尔、富马酸比索洛尔和阿替洛尔的检出限分别为16．73、33．58、21．46mg／L,对应的线性范围均为0．05—1．00g／L,回收率为91．2％、98．8％、88．4％。结论建立了同时分离分析3种心脏病药物的方法,该方法样品处理简便,色谱分离完全,结果准确可靠,为后期心脏病患者尿液中的药物代谢组学研究提供了基础。     

Simultaneous plasma level analysis of clomipramine, N-desmethylclomipramine, and fluvoxamine by reversed-phase liquid chromatography

A simple reversed-phase liquid chromatographic method enabling the simultaneous analysis in plasma of the tricyclic antidepressant clomipramine, its demethylated metabolite, and the selective serotonin reuptake inhibitor fluvoxamine, was developed. The drugs and dibenzepine, the internal standard, were extracted from 1 mL plasma through an automated solid-phase procedure, eluted in a total chromatographic time of approximately 14 min and detected by means of an ultraviolet spectrophotometer preset at 254 nm. An assay sensitivity of 10 microg/L was observed for all analytes. Recoveries for these drugs and their metabolites ranged between 65% and 98% and their coefficient of variation (within-day and day-to-day) between 1.9% and 2.9%. In spiked plasma, within-day and day-to-day imprecision data were less than 5%. The simultaneous determination of clomipramine, N-desmethylclomipramine, and fluvoxamine with adequate sensitivity and accuracy may be useful for the monitoring of drug treatment in depression and obsessive-compulsive disorder, where combinations of such drugs are employed.     

A comparative study of the repeat dose toxicity of grepafloxacin and a number of other fluoroquinolones in rats

Grepafloxacin is a new oral fluoroquinolone with potent activity against community acquired respiratory pathogens, including Streptococcuspneumoniae, and pharmacokinetic properties which allow once daily dosing. As part of its safety evaluation a study of 4 weeks duration was performed to compare the toxicity of grepafloxacin with that of a number of commercially available quinolones in the rat. Groups of eight male Sprague-Dawley rats received either control material or grepafloxacin, enoxacin, lomefloxacin, ofloxacin or ciprofloxacin at an oral dosage of 300 mg/kg/day for 4 consecutive weeks. Effects related to the antibacterial activity of the drugs were seen as increased caecal weight, decreased urinary excretion of sodium, increased water consumption, decreased urine volume, increased urine osmolality, soft stools and suppressed body weight gain. It is well documented that fluoroquinolones can cause lesions in the cartilage of the major diarthrodial joints, and blister formation or erosion on the joint surface was observed in all quinolone-treated groups other than the grepafloxacin group. Some quinolones, have been found to cause crystalluria, which is often associated with secondary nephropathy in laboratory animals due to the poor solubility of quinolones under the alkaline conditions of the urine. In the present study, needle-like crystals in the urinary sediment were observed in enoxacin and ciprofloxacin treated groups only. In conclusion, grepafloxacin was well tolerated and showed a low potential for joint toxicity and crystalluria compared to other quinolones.     

Effects of sparfloxacin, grepafloxacin, moxifloxacin, and ciprofloxacin on cardiac action potential duration

Fluoroquinolone antibiotics have been associated with QT prolongation following administration to humans. This study compares the effects of four fluoroquinolones, sparfloxacin, grepafloxacin, moxifloxacin and ciprofloxacin on action potential duration recorded from canine isolated cardiac Purkinje fibres. Left and right ventricular Purkinje fibres were isolated from canine hearts and continuously superfused with physiological salt solution. Action potential duration at 90% repolarization was recorded via intracellular microelectrodes. Sparfloxacin, grepafloxacin, moxifloxacin and ciprofloxacin prolonged action potential duration in a concentration dependent manner. Mean concentrations causing a 15% prolongation of action potential duration recorded at a stimulation frequency of 1 Hz were: sparfloxacin 4.2+/-0.7 microg/ml; grepafloxacin 9.3+/-0.9 microg/ml; moxifloxacin 9.9+/-1.6 microg/ml and ciprofloxacin 72.8+/-26.4 microg/ml. Prolongation was inverse frequency dependent with larger increases in action potential duration occurring when the stimulation frequency was reduced to 0.5 Hz. These results indicate that effects on action potential duration vary within this class of compound. Rank order of potency was sparfloxacin > grepafloxacin = moxifloxacin > ciprofloxacin.     

Activity of moxifloxacin and other quinolones against pneumococci resistant to first-line agents, or with high-level ciprofloxacin resistance

The activity of moxifloxacin and other quinolones was assessed against 288 epidemiologically diverse isolates of Streptococcus pneumoniae, many of them resistant to one or more first-line agents and/or with increased ciprofloxacin resistance (minimum inhibitory concentrations, MICs 16- > 64 mg/l compared with 1-2 mg/l for most isolates). Moxifloxacin and grepafloxacin were the most active quinolone analogues, inhibiting about 90% of the isolates at < or = 1 mg/l, whereas levofloxacin inhibited 64% of isolates at < = 1 mg/l and ciprofloxacin inhibited 42%. Moxifloxacin also was the most active agent against isolates with elevated ciprofloxacin resistance (MIC 16- > 64 mg/l): moxifloxacin MICs of around 4 mg/l were seen for most such isolates, compared with 16-32 mg for levofloxacin and grepafloxacin. The activity of moxifloxacin against pneumococci resistant to one or more first-line agent suggests it will have a useful therapeutic role, although its activity against highly ciprofloxacin resistant isolates seems marginal.     

A simple fluorescence high-performance liquid chromatographic assay for dihydroquinidine in serum

A simple and rapid reversed-phase C-18 high-performance liquid chromatography (HPLC) assay without a prior extraction step for dihydroquinidine (HQD) in serum is described. The method is selective and useful for monitoring HQD and its presumed metabolites, quinidine (QD), N-oxide QD, 3-hydroxy QD (3-OHQD), and 10-11-diol QD. The detection limit for HQD was 0.2 +/- 0.1 mg/L, and the peak heights--concentration curve was linear between 0.5 and 10.0 mg/L. The coefficients of variation within and between runs were identical, 5.0 +/- 1.5%. A 100 +/- 2.5% recovery was obtained by direct injection into the chromatograph of the supernatant following acetonitrile protein precipitation. The total assay time was less than 15 min.     

Simultaneous liquid chromatographic determination of chloramphenicol and antiepileptic drugs (phenobarbital, phenytoin, carbamazepine, and primidone) in plasma

A method for the simultaneous analysis of chloramphenicol and four antiepileptic drugs (phenobarbital, phenytoin, carbamazepine, and primidone) in plasma by high-performance liquid chromatography (HPLC) is described. The method involves a preliminary extraction of 0.1 ml of plasma with diethyl ether containing phenacetin as an internal standard, chromatography with a reversed-phase column with a methanol-water mobile phase, and detection by measuring ultraviolet absorbance at 210 nm. The method demonstrated sufficient precision, sensitivity, and specificity: the recoveries of the drugs were greater than 95% with the exclusion of primidone (80.3%); the maximum within-day and day-to-day coefficients of variation for all drugs were less than 5%; the lower detection limits were 0.5 microgram/ml or less for all drugs analyzed; and six other antibiotics, phenylethylmalondiamide, carbamazepine-10,11-epoxide, and chloramphenicol esters did not interfere with the analysis. The HPLC method was tested for clinical applicability by analyzing plasma samples from a volunteer who received concurrent single doses of chloramphenicol, phenobarbital, and phenytoin. This method can be used for studying drug interactions between chloramphenicol and antiepileptic drugs and for monitoring the concentrations of these drugs in plasma when administered concurrently, to prevent concentration-related side effect(s) of each drug.     

Determination of a new isoquinolinedione derivative, 7-anilino-5,8-isoquinolinedione,in plasma,urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new isoquinolinedione derivative, 7-anilino-5,8-isoquinolinedione (IQO4), in rat plasma, urine, blood and tissue homogenates using diazepam as an internal standard. A 2 volume of acetonitrile was added to deproteinize the biological sample. A 50 microl aliquot of the supernatant was injected onto a C(18) reversed-phase column. The mobile phase, 0.05 M acetate buffer (pH 3):acetonitrile:methanol (40:40:20, v/v/v), was run at a flow rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 298 nm. The retention times for IQO4 and the internal standard were approximately 5 and 7 min, respectively. The detection limits of IQO4 in rat plasma, urine and tissue homogenates (including blood) were 0.05, 0.1 and 0.1 microg/ml, respectively. The coefficients of variation of the assay were below 9.4% for rat plasma, urine and tissue homogenates. No interferences from endogenous substances were found.     

Determination of oxprenolol and its glucuronide metabolite in plasma and urine using high-performance liquid chromatography

A HPLC assay is presented for the determination of oxprenolol (1) and its glucuronic acid conjugate (2) in human plasma and urine. The procedure employs a selective re-extraction using alprenolol (3) as the internal standard, followed by reversed-phase chromatography and UV-detection. The minimal detectable concentration is 10 ng/ml in plasma and 50 ng/ml in urine, using 1.0 and 0.5 ml of plasma and urine, respectively. Within-run and day-to-day variations are below 10% at all concentrations examined. Plasma and urine samples of either healthy volunteers or patients with renal failure are free of interferences from endogenous compounds and drugs frequently used in these patients. The glucuronic acid conjugate of oxprenolol is determined as the parent compound after hydrolytic cleavage with beta-glucuronidase/arylsulfatase. The specificity and selectivity of this cleavage are also demonstrated.     

Concentrations of moxifloxacin in plasma and urine, and penetration into prostatic fluid and ejaculate, following single oral administration of 400 mg to healthy volunteers

The spectrum of chronic bacterial prostatitis (CBP) comprises Gram-negative, Gram-positive and atypical pathogens. Because of its broad spectrum of activity, moxifloxacin might be a suitable antibiotic for the treatment of CBP. In this pharmacokinetic study, plasma concentrations and the penetration of moxifloxacin into prostatic fluid and ejaculate were investigated. Twelve healthy male volunteers received a single oral dose of 400mg moxifloxacin and at the same time received 3.24 g of iohexol intravenously to assess urinary contamination of prostatic fluid and ejaculate. Plasma concentrations were determined at 0, 0.5, 1, 2, 3 and 4h and prostatic fluid and ejaculate (mean+/-standard deviation (S.D.)) were determined at 3.5+/-0.4h and 3.6+/-0.4h, respectively, following administration of drugs. Urinary concentrations were determined in the urine collected from 0-4.5h. Concentrations of moxifloxacin and iohexol in plasma, secretions and urine were determined by high-performance liquid chromatography. The mean+/-S.D. peak plasma concentration of moxifloxacin was 2.8+/-0.5 mg/L observed after 1.6+/-0.9h. In prostatic fluid, the concentration of moxifloxacin was 3.8+/-1.2 mg/L and the prostatic fluid/plasma ratio was 1.6+/-0.5. In ejaculate, the concentration was 2.5+/-0.7 mg/L and the ejaculate/plasma ratio was 1.0+/-0.2. Moxifloxacin concentrations in prostatic fluid were ca. 60% (P<0.05) higher than in plasma and concentrations in ejaculate were approximately the same as in plasma. Therefore, moxifloxacin might be a good alternative for the treatment of CBP, but further studies are warranted to establish this indication.     

Application of the Empore solid-phase extraction membrane to the isolation of drugs from blood: II. Mexiletine and flecainide.

A stabilized therapeutic drug monitoring procedure incorporating the novel Empore solid-phase extraction membrane (SPEM) for isolation of the antiarrhythmic drugs mexiletine (MEX) and flecainide (FLEC) from serum is described. Routinely, serum (0.5 ml), adjusted to pH 4.5, is passed through an octyl (C8) SPEM to extract the drugs. A methanol:water wash follows to remove proteins and interferences. MEX and FLEC are eluted from the membrane with mobile phase and an aliquot is injected directly onto a Zorbax cyanopropyl (CN) high-performance liquid chromatographic column with detection at 214 nm. Evaporating/concentrating techniques that can adversely influence the stability of the volatile MEX are unnecessary. Recovery for both drugs exceeds 90% and the assay is linear from 0.05 mg/L up to at least 6.0 mg/L for MEX and from 0.05 mg/L up to at least 3.0 mg/L for FLEC. Precision (between-run) coefficients of variation range from 2.3 to 3.0% (0.49-1.97 mg/L) for MEX and 3.7 to 5.9% (0.240-0.992 mg/L) for FLEC. Interferences are minimal. When we compared performance of the Empore SPEM and large-particle solid-phase sorbents packed in cartridges, we observed greater capacity per gram of sorbent and smaller elution volume with the membrane. Most important, concentrating steps that adversely affect the stability of MEX are avoided with the SPEM.     

The comparative arthropathy of fluoroquinolones in dogs.

1. Fluoroquinolone antibiotics are generally only prescribed to paediatric patients on compassionate grounds. This is because they are known to cause lesions in the cartilage of the major diarthroidal joints in immature experimental animals. As dogs are considered to be the most sensitive species, a series of studies was performed to compare the potential for grepafloxacin (a new fluoroquinolone) to cause arthropathy to that of ofloxacin and ciprofloxacin in juvenile (3 month old) beagles. 2. Grepafloxacin was administered once daily to male juvenile dogs at dosages of up to 100 mg/kg/day (intravenously), 60 mg/kg/day (orally) or 30 mg/kg/day (subcutaneously) for 1 week. Blister formation was observed on the surface of the joints in one of the three animals treated with grepafloxacin intravenously at 100 mg/kg/day. No abnormalities were observed at lower dosages or when grepafloxacin was administered orally or subcutaneously, regardless of dose. In animals treated with ofloxacin or ciprofloxacin at dosages of 10-30 mg/kg/day, blister formation or erosion was observed on the surface of joints regardless of dose or route of administration. 3. Histopathological examination of the joint surfaces of affected animals revealed the loss of cartilaginous matrix and chondrocytes, cavitation within the intermediate zone of cartilage accompanied by cartilage fibrillation or chondrocyte clustering, or loss of the surface layer which covers the cavitation (or loss of outer wall of the cavity). These findings were not present in the absence of grossly observed lesions. 4. Absorption following oral administration of grepafloxacin was low. Examination of plasma concentrations of drug following intravenous administration showed that joint toxicity was seen with ofloxacin and ciprofloxacin at maximum concentrations as low as 3.80 and 4.24 mg/l, respectively, while plasma levels of grepafloxacin of up to 11.95 mg/l failed to cause such lesions. When the concentration of grepafloxacin was 18.69 mg/l a single joint lesion was seen. Following subcutaneous administration of grepafloxacin, systemic exposure (area under the curve) of approximately 1.5 times that seen in man was not associated with joint lesions. However, lesions were noted for ofloxacin and ciprofloxacin treated animals at exposures equal to or below those seen in man. Therefore grepafloxacin appeared to have a relatively low potential for joint toxicity; this was not due to lack of penetration into the synovial fluid.     

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.