Susceptibility to macrolide antibiotics of Bordetella pertussis and Bordetella parapertussis strains isolated from whooping cough patients in 1968 and in 1997-99]

The activity of erythromycin, azithromycin, clarithromycin, dirithromycin, oleandomycin, roxithromycin, spiramycin and josamycin against 21 and 34 B. pertussis strains and against 6 and 8 B. parapertussis strains isolated respectively in the years 1968 and 1997-99 was examined. The antibiotic agar dilution method was used. The minimum concentration of macrolides which inhibited growth of B. pertussis and B. parapertussis was calculated for 50% (MIC50) and 90% (MIC90) of isolates. The susceptibility to macrolides of B. pertussis and B. parapertussis strains isolated in the years 1968 and 1997-99 did not differ significantly. The MIC90 values of erythromycin were the same for B. pertussis (MIC90 = 0.125 mg/l) and B. parapertussis strains (MIC90 = 0.25 mg/l) recovered in 1968 as for those recovered in the years 1997-99. The most active antibiotic against all strains was azithromycin (MIC90 = 0.06 mg/l). The least active antibiotics were oleandomycin (MIC90 = 2-4 mg/l) and spiramycin (MIC90 = 8 mg/l). The study showed that erythromycin remains the antibiotic of choice for treatment of whooping cough and in case of emergence of B. pertussis and/or B. parapertussis strains erythromycin resistant, can be replaced by azithromycin.     

Simple and rapid analysis of lamotrigine, a novel antiepileptic, in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A simple and rapid method for the quantitation of concentrations of lamotrigine, a novel antiepileptic, in human serum was developed with high-performance liquid chromatography, using a solid-phase extraction technique. The mobile phase was composed of acetonitrile-10 mM phosphate buffer (pH 3.5) containing 5 mM sodium octanesulphonate (27:73, v/v), and components were detected at 265 nm. Retention times of acetanilide as an internal standard and lamotrigine were 3.4 and 10.3 min, respectively. The coefficients of variation were 3.1–4.5% and 4.4–9.8% for the within-day and between-day precision estimates, respectively. The extraction recovery of lamotrigine added to blank serum was 86–107%. The quantitation limit of lamotrigine was ca.0.2 μg/ml in 100 μl of serum. These results suggest that the method employed in this study is useful for the routine monitoring of sereum concentrations of lamotrigine in epileptic patients.     

Determination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection

In this study, a high-performance liquid chromatographic method was developed for the quantitative determination of erythromycin (EM), roxithromycin (RXM), and azithromycin (AZM) in rat plasma with amperometric detection under a standardized common condition using clarithromycin (CAM) as an internal standard. This method was also proved to be applicable for the determination of CAM by employing RXM as an internal standard. Each drug was extracted from 150 μl of plasma sample spiked with internal standard under an alkaline condition with tert.-butyl methyl ether. The detector cell potential for the oxidation of the drugs was set at +950 mV. The linearity of the calibration curves were preserved over the concentration ranges of 0.1–10 μg/ml for EM and RXM, and 0.03–3.0 μg/ml for CAM and AZM. Coefficients of variation and relative error were less than 9% and ±7%, respectively. The analytical method presented here was proved to be useful for the investigation of the pharmacokinetic characteristics of EM, CAM, RXM, and AZM in rats.     

Analysis of Ecteinascidin 743, a new potent marine-derived anticancer drug, in human plasma by high-performance liquid chromatography in combination with solid-phase extraction

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the quantification of the novel anticancer drug Ecteinascidin 743 in human plasma. The sample pretreatment of the plasma samples involved a solid-phase extraction (SPE) on cyano columns. Propyl-p-hydroxybenzoate was added after the sample pretreatment to correct for variability in injection volumes. The separation was performed on a Zorbax SB-C₁₈ column (75×4.6 mm I.D., particle size 3.5 μm) with acetonitrile–25 mM phosphate buffer, pH 5.0 (70:30, v/v) as the mobile phase. The flow-rate was 1.0 ml/min and the eluent was monitored at 210 nm. The accuracies and precisions of the assay fall within ±15% for all quality control samples and within ±20% for the lower limit of quantitation, which was 1.0 ng/ml using 500 μl of plasma. The overall recovery of the sample pretreatment procedure for Ecteinascidin 743 was 87.0±5.9%. The drug was found to be stable in human plasma at −30°C for at least 2 months. At room temperature Ecteinascidin 743 was stable in human plasma for 5 h at most.     

Testing of the anabolic stanozolol in human hair by gas chromatography–negative ion chemical ionization mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of stanozolol in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride and the sonication in methanol of 100 mg of powdered hair for 2 h. After elimination of the solvent, the hair sample was solubilized in 1 ml 1 M NaOH, 15 min at 95°C, in the presence of 10 ng stanozolol-d₃ used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C₁₈) and a liquid–liquid (pentane) extraction. After evaporation of the final organic phase, the dry extract was derivatized using 40 μl MBHFA–TMSI (1000:20, v/v), incubated for 5 min at 80°C, followed by 10 μl of MBHFBA, incubated for 30 min at 80°C. The derivatized extract was analyzed by a Hewlett-Packard GC–MS system with a 5989 B Engine operating in the negative chemical ionization mode of detection. Linearity of the detector response was observed for stanozolol concentrations ranging from 5 to 200 pg/mg with a correlation coefficient of 0.998. The assay was capable of detecting 2 pg of stanozolol per mg of hair when approximately 100 mg hair material was processed, with a quantification limit set at 5 pg/mg. Intra-day precision was 5.9% at 50 pg/mg and 7.8% at 25 pg/mg with extraction recoveries of 79.8 and 75.1%, respectively. The analysis of a 3-cm long hair strand, obtained from a young bodybuilder (27 year old) assuming to be a regular user of Winstrol (stanozolol, 2 mg), revealed the presence of stanozolol at the concentration of 15 pg/mg.     

Determination of a chemoprotective agent, 2-(allylthio)pyrazine, in plasma, urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemoprotective agent, 2-(allylthio)pyrazine (I), in human plasma and urine, and in rat blood and tissue homogenate using diazepam as an internal standard. The sample preparation was simple; 2.5 volumes of acetonitrile were added to the biological sample to deproteinize it. A 50–100 μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column. The mobile phase employed was acetonitrile–water (55:45, v/v), and it was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector at 330 nm. The retention times for I and the internal standard were 4.0 and 5.1 min, respectively. The detection limits of I in human plasma and urine, and in rat tissue homogenate (including blood) were 20, 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 6.1%) in a concentration range from 0.02 to 10 μg/ml for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.     

Thermal biology of the fossorial rodent Ctenomys fulvus from the Atacama desert, northern Chile

The Andean tuco-tuco, Ctenomys fulvus (Rodentia: Ctenomyidae) inhabits one of the most arid regions of the world, the Salar de Atacama, Northeast of Antofagasta, Chile (23°17′06″S, 68°05′43″W; 2.240 m.a.s.l). We found that a stable microclimate in burrows, a low evaporative water loss (EWL), and a diet of roots (59% water content) are the main factors that permit the survival of this fossorial species in harsh desert conditions. Large circadian variation in T_a was observed above ground. Daily ΔT_a (T_a max − T_a min) = 37.9±0.2°C in summer and in winter. In contrast, circadian variation of T_a inside the burrows was only 5.8±0.5°C in the same seasons. Relative humidity (RH) was 1.9–3.1% during the day, increasing to maximum values of 27% at night and early morning. Inside the burrows RH was higher and quite stable, ranging between 53.1 and 65%, independent of the time of day and season. EWL, measured between 10 and 25°C, was low (1.26 mg/g h), and a moderate increase of 13–20% was observed at higher temperatures. The low EWL may prevent dehydration. However, because of the low heat loss capability, animals became hyperthermic (0.8–1.6°C) in dry air at T_a=30–35°C. As T_a during afternoon normally exceeded 35°C, the microclimate of burrows provided the only way to avoid the lethal effects of hyperthermia.     

支原体对抗生素的体外敏感性研究

目的研究非淋菌性尿道(宫颈)炎(NGU)患者支原体对12种抗生素敏感性,指导临床治疗。方法男性标本取尿道拭子．女性标本取宫颈拭子,采用支原体培养鉴定及药敏试剂盒进行体外药敏测定。结果126株解脲支原体(Uu)对12种抗生素敏感性从高至低依次为交沙霉素、强力霉素、美满霉素、克拉霉素、四环素、阿奇霉素、罗红霉素、司帕沙星、甲砜霉素、红霉素、可乐必妥和壮观霉素。12株人型支原体(Mh)对12种抗生素敏感性从高至低依次为交沙霉素、强力霉素、美满霉素、司帕沙星、四环素、壮观霉素、可乐必妥、甲砜霉素、红霉素、阿奇霉素、克拉霉素和罗红霉素。结论支原体耐药情况相当普遍。Uu、Mh对相同抗生素具有不同的敏感性。治疗时可依据药敏结果选择用药。     

On-line microdialysis coupled with microbore liquid chromatography for the determination of unbound chloramphenicol and its glucuronide in rat blood

On-line microdialysis coupled with microbore liquid chromatography was used to investigate the pharmacokinetics of chloramphenicol and its glucuronide in rat blood. A microdialysis probe was inserted into a jugular vein of male Sprague–Dawley rats. Chloramphenicol succinate (20 mg/kg, intravenously) was then administered via a femoral vein. Dialysates were automatically injected onto a LC system, via an on-line injector. Samples were eluted with a mobile phase containing acetonitrile-10 mM monochloroacetic acid (30:70, v/v, pH 3.0). The UV detector wavelength was set at 278 nm. The limit of quantitation for chloramphenicol was 10 ng/ml. The in vitro recoveries of chloramphenicol and chloramphenicol glucuronide at 500 ng/ml were 32.2±0.3% and 11.4±0.7%, respectively (n=6). Intra- and inter-assay accuracy and precision of the analyses were ≤10% in the range of 0.01 to 5.0 μg/ml.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

Determination of sparfloxacin in serum and urine by high-performance liquid chromatography

A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 × 4.0 mm I.D., 5 μm particle size) protected by a guard column (Perisorb RP-18, 30 × 4.0 mm I.D., 30–40 μm particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5–100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) − 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0–97.8%; method comparison c(bioassay) = 1.092c(HPLC) − 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.     

Simplified method for determination of clarithromycin in human plasma using protein precipitation in a 96-well format and liquid chromatography-tandem mass spectrometry

A simplified method to determine clarithromycin concentrations in human plasma using protein precipitation in a 96-well plate and liquid chromatography-tandem mass spectrometry was developed and validated. Plasma proteins were precipitated with acetonitrile and roxithromycin was used as the internal standard. After vortex mixing and centrifugation, the supernatants were directly injected onto a Phenomenex Luna Phenyl-Hexyl column (50 mm x 2.0 mm ID, 3 microm). The mobile phase consisted of water and methanol (30:70, v/v) containing 0.1% formic acid and 5mM ammonium acetate. The flow rate was 0.22 mL/min and the total run time (injection to injection) was less than 3 min. Detection of the analytes was achieved using positive ion electrospray tandem mass spectrometry in selected reaction monitoring (SRM) mode. The linear standard curve ranged from 100 to 5000 ng/mL and the precision and accuracy (inter- and intra-run) were within 7.9% and 4.9%, respectively. The method was successfully used to determine clarithromycin concentrations in human plasma samples obtained from healthy subjects who were given clarithromycin 500 mg for 3 days. The method is rapid, simple, precise and directly applicable to clarithromycin pharmacokinetic studies.     

女性生殖道支原体感染与不孕症的关系及药敏分析

目的探讨女性生殖道支原体感染与不孕症之间的关系及对11种抗生素的敏感率,指导临床明确诊断,合理用药。方法随机选择2007年至2009年来长治市妇幼保健院就诊的女性不孕症患者360例,进行宫颈分泌物支原体培养和药敏试验。结果 360例宫颈分泌物标本中检出解脲支原体(Uu)阳性者161例、人型支原体(Mh)阳性者3例、解脲支原体(Uu)+人型支原体(Mh)混合阳性者29例;对阳性标本都做了11种抗生素的药敏试验,其中交沙霉素敏感率为93.47%、美满霉素敏感率为92.00%、强力霉素敏感率为90.21%、克拉霉素敏感率为83.69%、甲砜霉素敏感率为73.91%、环脂红霉素敏感率为61.95%和阿奇霉素敏感率为53.26%等。结论女性不孕症伴随高发的生殖道支原体感染,支原体感染与不孕症可能有关;长治地区生殖道支原体对交沙霉素、美满霉素、强力霉素、克拉霉素、甲砜霉素敏感性较高,环脂红霉素、阿奇霉素次之,对红霉素、罗红霉素敏感性较低,对环丙沙星、左氧氟沙星敏感性很低。     

Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

Human prepubertal testicular cells in culture: Steroidogenic capacity, paracrine and hormone control

The neonatal human Leydig cell undergoes a transient period of activation during the first months of life. The biological significance of this activation is unknown. Furthermore, little is known about the hormonal regulation of this biological process, even though it coincides with an elevation of LH levels in serum. In order to study the function of human prepubertal testicular culture cells, obtained during the neonatal period, a method for maintaining primary culture cells (isolated from testes collected at necropsy) in culture was developed. Within 24 h after death, testes were collected from 1–36-month-old subjects. Subjects were divided into two age groups, based on the presence or absence of fetal Leydig cells: 1–7-month-old infants (group 1) and 12–36-month-old children (group 2). Testes were digested with collagenase, and cells were seeded in multi-well dishes. Cells were grown in serum-free conditioned media supplemented with 5 mg/l vitamin C, 0.2 IU/l vitamin E and 10% fetal bovine serum for 2 days. Cells were then grown for an additional 4 days in serum-free media in the presence or absence of hLH (40 IU/l), hCG (135 IU/l), rh FSH (1.5 IU/l), rhGH (0.12 IU/l) or insulin (0.9 μmol/l). Concentrations of steroids in media were determined by RIA on day 6 of culture. In basal conditions cells of group 1 (n = 11) secreted more testosterone, androstendione, 17-hydroxyprogesterone, progesterone and dehydroepiandrosterone (mean ± SE: 6.76 ± 1.86, 7.37 ± 1.82, 61.9 ± 1.86, 5.75 ± 1.74 and 8.51 ± 3.23 pmol/10⁶ cells/24 h, respectively) than cells of group 2 (n = 5) (2.95 ± 1.15, 1.50 ± 2.75, 1.44 ± 2.75, 0.78 ± 1.74 and 3.23 ± 1.32, respectively). Under hLH stimulation, cells of group 1 increased testosterone, androstendione and 17-hydroxyprogesterone secretions (to 38.2 ± 0.89, 13.5 ± 1.17 and 51.7 ± 3.23), while progesterone secretion remained unchanged (2.82 ± 1.20). Cell response to rhFSH and rhGH was similar to that of hLH. On the other hand, medium collected from cultures of cells isolated from a Sertoli cell tumor was able to stimulate testosterone secretion in subcultures of control testicular cells in a way similar to that of hCG. In conclusion, (1) these prepubertal human testicular cells can be maintained in primary culture for several days keeping their in vivo steroidogenic potential; (2) cells isolated from young infants can respond to hLH in culture; (3) response to rhFSH is probably mediated by a paracrine factor; (4) response to rhGH is observed in the absence of gonadotropins. Therefore, the early postnatal activation of the human testis might be under multiple pituitary hormone control; and, finally, (5) Sertoli cell tumors can secrete paracrine factors that stimulate steroidogenesis.     

Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 μl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 μl of a solution of CPT (1 μg/ml) in acetonitrile–1 mM orthophosphoric acid (90:10); 200 μl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C₁₈ (5 μm, 250×4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)–acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1–10 000 ng/ml) is 5.17% (range 2.15–8.27%) and for SN-38 (0.5–400 ng/ml) is 4.33% (1.32–7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03–10.8%) and 4.94% (2.09–9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m² of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (V_ss) is 203 l/m², and the total body clearance (Cl) is 14.8 l/h·m². The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Identification of amidated forms of GLP-1 in rat tissues using a highly sensitive radioimmunoassay

The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7–36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7–36)amide and GLP-1(1–36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7–36)amide, GLP-1(1–36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7–36)amide could be identified in the hypothalamus, in amounts that represented 55–94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1–36)amide, representing 0.8–3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7–36)amide (0–0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7–36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27–73% of total GLI, and in five of six specimens only GLP-1(7–36)amide could be identified on HPLC, in amounts representing 13–17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1–36)amide (13% of total GLI) in addition to GLP-1(7–36)amide (31% of total GLI). Acid–ethanol extraction of peptide-free rat plasma with added GLP-1(7–36)amide gave recoveries of 91±SEM 2% in the range 20–200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1±1.1 pmol/l and rose to a maximum of 15.4±3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.     

High performance liquid chromatographic determination of azithromycin in serum using fluorescence detection and its application in human pharmacokinetic studies

A fast and sensitive high-performance liquid chromatographic method for determination of azithromycin in human serum using fluorescence detection was developed. The drug and an internal standard (clarithromycin) were extracted from serum using n-hexan and subjected to pre-column derivatization with 9-fluorenylmethyl chloroformate as labeling agent. Analysis was performed on a phenyl packing material column with sodium phosphate buffer containing 2 ml/l triethylamine (pH 5.9) and methanol (29:71, v/v) as the mobile phase. The standard curve was linear over the range of 10-500 ng/ml of azithromycin in human serum. The means between-days precision were from 13.3% (for 10 ng/ml) to 2% (500 ng/ml) and the within-day precision from 11.9 to 1.7% determined on spiked samples. The accuracy of the method was 100.7-107.2% (between days) and 100.3-107.8% (within day). The limit of quantification was 10 ng/ml. This method was applied in a bioequivalence study of four different azithromycin preparations in 12 healthy volunteers.     

Isocratic high-performance liquid chromatographic determination of thiacetazone by direct injection of plasma into an internal surface reversed-phase column

Thi report describes the determination of thiacetazone in human and rat plasma by direct-injection high-performance liquid chromatography (HPLC). Plasma filtrate (50 μl) was injected directly into the internal surface reversed-phase (ISRP) mixed-functional phenyl column (Capcell Pak, 50×4.6 mm, 5 μm) and eluted with an aqueous mobile phase containing 7.5% acetonitrile at a flow-rate of 1 ml/min. With UV detection at 322 nm, thiacetazone eluted at 11.0 min whereas endogenous interferences eluted before 5 min. The lower detection limit for a 50-μl sample at a signal-to-noise ratio of 5 was 63 ng/ml, which was several hundred fold lower than its cytotoxic concentrations determined from in vitro cell line studies. At a concentration range of 0.17 to 2.7 μg/ml, the recovery of thiacetazone was 98.0±4.4% (mean±S.D.). The intra- and inter-day coefficients of variation were 3.0±1.4% and 4.2±2.1%, respectively. This method was successfully applied to study the pharmacokinetics of thiacetazone in rats. The direct injection method is simple, requires small sample volume and does not require sample extraction, internal standard, or gradient elution.