Determination of caffeic acid in rabbit plasma by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method involving UV detection was developed for determination of caffeic acid in rabbit plasma. A Lichrosphere CN column (250 mm × 4 mm I.D., 5 μm) was used as the stationary phase and the mobile phase consisted of 2% acetic acid solution at a flow-rate of 1.0 ml/min. The UV absorbance was monitored at 320 nm. The plasma sample was acidified by the addition of 0.01 parts of concentrated phosphoric acid (85%) to maintain caffeic acid stability. After a simple clean-up procedure, the limit of quantitation achieved was 0.1 μg/ml, and the standard curve was found to be linear over the concentration ranges of 0.1–2.0 μg/ml and 0.1–40 μg/ml. The coefficient of variation for within- and between-run precision and accuracy was less than 10%, and the recovery was 82.3%.     

Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin,SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).     

Bioanalysis of diclofenac as its fluorescent carbazole acetic acid derivative by a post-column photoderivatization high-performance liquid chromatographic method

A sensitive and selective bioanalytical liquid chromatographic method for diclofenac is described. The drug was detected as a fluorescent derivative, which was demonstrated by ¹H NMR and mass spectrometric studies to be carbazole acetic acid. Diclofenac was derivatized by UV irradiation of the substance performed as a post-column photoreaction. The reactor was a PTFE capillary wound around a 254-nm UV lamp. Diclofenac was isolated from the plasma samples by precipitation of the proteins with acetonitrile. A 50-μl volume of the supernatant was injected onto a Nucleosil C₁₈ column. The mobile phase was 32% acetonitrile in pH 6.6 buffer. Carbazole acetic acid was detected by a fluorescence detector using an excitation wavelength of 288 nm and an emission wavelength of 360 nm. The recovery was 92%, the standard curve was linear in the range 10–5500 ng diclofenac per ml plasma, and the relative standard deviation at 10 and 5000 ng of diclofenac per ml plasma was 9.0% and 3.3%, respectively. The limit of detection was 6 ng/ml at an injection volume of 50 μl. Chromatograms of human and rat plasma containing diclofenac are shown.     

High-performance liquid chromatographic method for the simultaneous determination of HIV-1 protease inhibitors indinavir,saquinavir and ritonavir in plasma of patients during highly active antiretroviral therapy

A new high-performance liquid chromatographic method for the simultaneous determination of indinavir, saquinavir and ritonavir in human plasma is described. Quantitative recovery following liquid–liquid extraction with diethyl ether from 500 μl of human plasma was achieved. Subsequently, the assay was performed with a linear gradient starting at 67 mM potassium dihydrogenphosphate–acetonitrile (65:35 to 40:60, v/v) as a mobile phase, a Phenomenex C₁₈ column and UV detection at 240 and 258 nm, respectively. Linear standard curves were obtained for concentrations ranging from 75 to 20 000 ng/ml for indinavir, from 10 to 6000 ng/ml for saquinavir, and from 45 to 30 000 ng/ml for ritonavir. The calculated intra- and inter-day coefficients of variation were below 6%.     

High performance liquid chromatographic method for the determination of cetirizine and ambroxol in human plasma and urine--a boxcar approach

A column switching high performance liquid chromatographic method with estimable sensitivity and accuracy was developed for the determination of cetirizine and ambroxol in human plasma using nebivolol as the internal standard. Plasma samples were prepared by liquid-liquid extraction in methylene chloride and a mixture of diethylether (80:20, v/v). The extracted samples were injected into a multifunctional clean-up column Supelcosil LCABZ (50 mm × 4.6 mm, 5 μm particle size) using mobile phase 1 comprising acetonitrile-phosphate buffer (pH 3.5; 20 mM) (20:80, v/v). The eluate of cetirizine and ambroxol were separated to an analytical Kromasil C(8) micro bore column (50 mm × 0.3 mm, 5 μm particle size) via a column switching device. A Kromasil C(18) analytical column (250 mm × 2.1 mm, 5 μm particle size) was used as a separation column. Mobile phase 2 consisting acetonitrile-triethylamine (0.5%) in phosphate buffer (pH 3.5; 20mM) (55:45, v/v) was used for the compound elution. The eluents were detected at 230 nm with photodiode array detector. An aliquot of 150 μl of plasma sample was introduced into the pretreatment column via the auto sampler using mobile phase 1 at a flow rate of 0.5 ml/min, column switching valve being positioned at A. The pretreatment column retained cetirizine, ambroxol and nebivolol (IS) in the column leaving the residual proteins of plasma eluted in void volume and drained out. The switching valve was shifted to position B at 7.5 min. Cetirizine, ambroxol and IS were eluted from the pretreatment column between 7. 5 and 11.5 min and introduced to the concentration column. Finally, cetirizine, ambroxol and IS were introduced to the separation column by switching valve using mobile phase 2 at a flow rate of 0.4 ml/min. During the analysis the pretreatment column was washed for the next analysis and resume to the position A. The total run time was 25 min for a sample. The procedure was repeated for urine analysis also. The method was linear from 2 to 450 ng/ml and 7-300 ng/ml for cetirizine and ambroxol respectively in plasma and 1-500 ng/ml and 5-400 ng/ml, respectively for cetirizine and ambroxol in urine. Intra-day and inter-day precision of cetirizine and ambroxol was below 15% in terms of coefficient of variation and accuracy of cetirizine and ambroxol was ranged from 94 to 101.6% and 91.1 to 100.2%, respectively. The method demonstrated high sensitivity and selectivity and therefore, applied to evaluate pharmacokinetics of cetirizine and ambroxol in healthy human volunteer after a single oral administration. Urine samples obtained from healthy human volunteers and clinical subjects with renal impairment have also been analyzed by the method to compare the elimination pattern. The method was precise and accurate for the estimation of cetirizine and ambroxol both in blood and in urine.     

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.     

Simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, respectively.     

HPLC同时测定伤科黄水中6个生物碱的含量

建立HPLC同时测定伤科黄水中6个生物碱的方法。采用XBridge C₁₈色谱柱(3. 5μm,2. 1 mm×100 mm),柱温35℃,测定波长280 nm,以0. 1%磷酸溶液(每100 mL加0. 3 g十二烷基苯磺酸钠)(A)-乙腈-水-磷酸-十二烷基苯磺酸钠(90∶10∶0. 1∶0. 3)(B)为流动相,进行梯度洗脱(0～30 min,B%:35～70; 30～31 min,B%:70～35; 31～40min,B%:35)。经方法学验证,黄柏碱、药根碱、表小檗碱、黄连碱、巴马汀、小檗碱等共6个生物碱分离情况良好,在测定时间段内无明显干扰峰;加样回收率均在95%～115%之间,RSD%均小于5%;精密度RSD%均小于5%;在测定浓度范围内(1～50μg/mL)线性关系良好,相关系数(R^2)大于0. 999。3个不同批次供试品的测定结果较一致。本研究建立的HPLC分析方法可用于同时测定伤科黄水中6个生物碱的含量。     

Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C₁₈ reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 μl of whole blood or plasma sample. Calibration curves were linear (r²=0.99) over a 1–10 μg/ml concentration range and intra- and inter-day precision were between 4–11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Determination of the antihypertensive drug cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine by solid-phase extraction and high-performance liquid chromatography with photometric detection

A liquid chromatographic method with photometric detection for the determination of cilazapril and its active metabolite and degradation product cilazaprilat in urine and pharmaceuticals has been developed. The chromatographic method consisted of a μBondapak C₁₈ column maintained at 30±0.2°C, using a mixture of methanol-10 mM phosphoric acid (50:50 v/v) as mobile phase at a flow-rate of 1.0 ml/min. Enalapril maleate was used as internal standard. The detection was performed at a wavelength of 206 nm. A study of the retention of cilazapril and cilazaprilat using solid–liquid extraction has been carried out in order to optimise the clean-up procedure for urine samples, which consisted of a solid–liquid extraction using C₈ cartridges. Recoveries greater than 85% are obtained for both compounds. The method was sensitive, precise and accurate enough to be applied to the determination of urine samples obtained from three hypertensive patients up to 24 h after intake of a therapeutic dose (detection limit of 70 ng/ml for cilazapril and cilazaprilat in urine). A comparison of the method developed using photometric and amperometric detection has been carried out.     

High-performance liquid chromatographic determination of liposomal nystatin in plasma and tissues for pharmacokinetic and tissue distribution studies

A reliable reversed-phase high-performance liquid chromatographic method was developed for the determination of liposomal nystatin in plasma. Nystatin is extracted by 1:2 (v/v) liquid–liquid extraction with methanol. Separation is achieved by HPLC after direct injection on a μBondapak™ C₁₈ analytical column with a mobile phase composed of 10 mM sodium phosphate, 1 mM EDTA, 30% methanol and 30% acetonitrile adjusted to pH 6. Detection is by ultraviolet absorbance at 305 nm. Quantitation is based on the sum of the peak area concentration of the two major isomers of nystatin, which elute at 7.5–8.5 and 9.5–10.5 min. The assay was linear over the concentration range of 0.05 to 50 μg/ml. The lower limit of quantitation was 0.05 μg/ml, sufficient for investigating the plasma pharmacokinetics of liposomal nystatin in preclinical studies. Accuracies and intra- and inter-day precision showed good reproducibility. With minor modifications, this method also was used for assaying nystatin in various non-plasma body fluids and tissues.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

ELISA法检测乙型脑炎减毒活疫苗中卡那霉素和庆大霉素的残留量

用ELISA试剂盒检测乙脑减毒活疫苗中卡那霉素和庆大霉素的残留量。以间接竞争ELISA法检测线性范围内加入高、中、低3种浓度的卡那霉素和庆大霉素,测定其在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率,以及在10倍稀释乙脑减毒活疫苗中的回收率。高、中、低浓度的卡那霉素在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率为101.40%～124.80%之间;原倍疫苗稳定剂对庆大霉素的测定有明显干扰,在原倍疫苗稳定剂中低浓度和中浓度的庆大霉素的回收率高达2280%和575%,但其它测定条件下回收率在90%～125%之间。在10倍稀释的乙脑疫苗中加入一定量的卡那霉素和庆大霉素,回收率分别为124%和103.25%。用10倍稀释法测定1人份规格的乙脑减毒活疫苗17批、5人份规格的乙脑减毒活疫苗19批。乙脑减毒活疫苗10倍稀释后,可用ELISA试剂盒检测卡那霉素和庆大霉素残留量。     

Simple,rapid and sensitive method for the determination of indomethacin in plasma by high-performance liquid chromatography with ultraviolet detection

A micro method for determination of indomethacin in plasma was developed. Following deproteinization of plasma with acetonitrile containing internal standard (mefenamic acid), the separation of indomethacin and internal standard was achieved by high-performance liquid chromatography using a 7 μm LiChrosorb-RP18 column (250×4 mm I.D.) at 50°C. The mobile phase was 6 mM phosphoric acid–acetonitrile (50:50). The flow-rate was kept at 2.0 ml/min and the column effluent was monitored at 205 nm. The coefficients of variation of the method estimated at 0.2 and 1.0 μg/ml were 4.2 and 2.3%, and the detection limit of the drug was about 0.05 μg/ml (S/N=5). The method requires minimum pretreatment of the plasma with a small sample volume (25 μl), and is very suitable for therapeutic drug monitoring of indomethacin in premature infants with symptomatic patent ductus arteriosus.     

High-performance liquid chromatographic determination of diadenosine 5′,5‴-p1,p4-tetraphosphate with precolumn fluorescence derivatization and its application to metabolism study in whole blood

Diadenosine 5′,5‴-p¹,p⁴-tetraphosphate (Ap4A) was converted with chloroacetaldehyde to the fluorescent di-1,N⁶-ethenoadenosine derivative within 60 min at 80°C. It was separated by reversed-phase HPLC and detected fluorimetrically (excitation and emission wavelengths of 275 and 410 nm, respectively). The detection limit of Ap4A was ca. 0.2 μg/ml in plasma when 10 μl of the sample was applied to the column. The rate of degradation of Ap4A added to whole blood (5 μg/ml) was examined using this method. Half-lives (means ± S.E., n = 3) were 0.88 ± 0.30 min (in rat blood), 13.7 ± 3.6 min (in dog blood and 17.2 ± 1.4 min (in human blood). A marked species difference in the degradation rate of Ap4A in blood was observed.     

Determination of urinary thiamine by high-pressure liquid chromatography utilizing the thiochrome fluorescent method

A sensitive, reproducible, and specific method for the determination of urinary thiamine has been established. Unique to this method is the use of high-pressure liquid chromatography (HPLC) to separate the fluorescent thiamine derivative from interfering fluorescent compounds. Urine samples were passed through a Decalso cation-exchange column, washed with 0.5 M KCl to remove some interfering compounds, and eluted with 3.4 M KCl. The eluted thiamine was converted to the fluorescent derivative, thiochrome, by reaction with alkaline potassium ferricyanide. The reaction mixture was extracted with isobutanol and subjected to HPLC monitored by a fluorescent detector.Within-day and day-to-day coefficients of variation proved to be 2.5% and 1.2%, respectively. Recovery of added thiamine (range 0.04 to 2.0 μg/ml) averaged 99.9 ± 5.3%. The sensitivity of this method was 0.03 μg/ml.     

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 μg/ml. Recoveries varied from 91 to 10% with coefficients of variation ranging from 0.387 to 7.95%.     

Simple,rapid and micro high-pressure liquid chromatographic method for the simultaneous determination of tolbutamide and carboxy tolbutamide in plasma

A rapid high-pressure liquid chromatographic (HPLC) assay is described for the quantitative analysis of tolbutamide and its major metabolite, carboxy tolbutamide, in plasma. An aliquot (25–100 μl) of plasma was prepared for chromatography by deproteinization as follows. One volume of plasma and 2.5 volumes of acetonitrile were vortex mixed for a few seconds and then centrifuged for approx. 1 min. A 50-μl sample of the clear supernatant was injected into the chromatograph. A μBondapak C₁₂ reversed-phase column was used with a mobile phase of acetonitrile-0.05% phosphoric acid (45:55) at a flow-rate of 1.5 ml/min. The column effluent was monitored by a variable-wavelength UV detector set at 200 nm. Tolbutamide and its metabolite had retention times of 5.75 and 3.25 min, respectively. The procedure yields reproducible results with sensitivity adequate for routine clinical monitoring of plasma levels or for single-dose pharmacokinetic studies. A number of commonly used drugs do not interfere with the method. A single plasma sample can be analyzed in approx. 9 or 10 min.     

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from a C₁₈ reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96-70%. The limit of quantification was 0.1 μg/ml. The assay was reproducible, the within-day coefficient of variation (n=6) was <8% for serum, lungs and liver. The between-day variability (n=6) was <7.7% for serum, liver and lungs at 1 μg/ml or 1 μg/g tissue concentration. The assay was linear within the range 1–40 μg/ml (r²=0.999).     

Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 microl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 microm I.D.x 30 mm 10 microm Kromasil C(18) pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 microm I.D.x 100 mm 3.5 microm Kromasil C(18) analytical column. Loading flow rates up to 100 microl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M-H](-) at m/z 217.08. The method was validated over the concentration range 0.2-40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4-7.3 and 7.0-8.1%, respectively, and the recoveries were in the range 96.2-97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.