Comparison of HPLC and CE methods for the determination of cetirizine dihydrochloride in human plasma samples

Two methods, capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC), for analysis of cetirizine dihydrochloride in small sample volumes of human plasma were compared. The CE and HPLC assays were developed and validated by analyzing a series of plasma samples containing cetirizine dihydrochloride in different concentrations using these two methods. The extraction procedure is simple and no complicated purification steps or derivatization are required. The analysis time in the HPLC method was shorter than that in the CE method, but solvent consumption was considerably lower in the CE method. The calibration curve was linear to at least 10-1000 ng/mL both for CE and HPLC with r(2) = 0.9993 and r(2) = 0.9994, respectively. The detection limits for cetirizine dihydrochloride were 3 and 5 ng/mL with CE and HPLC (a UV detector was applied in the both cases), respectively. Both methods were selective, robust and specific, allowing reliable quantification of cetirizine dihydrochloride, and could be useful for clinical and biomedical investigations.     

Spectrofluorimetric determination of paroxetine hydrochloride in its formulations and human plasma

A sensitive spectrofluorimetric procedure for the determination of paroxetine-HCl in pharmaceutical formulations and human plasma has been described. The native fluorescence of the drug has been studied under different conditions. Maximum fluorescence intensity was obtained in methanol at 340 nm using 290 nm for excitation. Different surfactants showed negative effect on the fluorescence intensity of paroxetine-HCl. Regression analysis of Beer's plot showed good correlation (r=0.9999) between fluorescence intensity and concentration over the range of 0.05-0.40 microg ml-1 with lower limit of detection (LOD) of 0.015 microg ml-1. The drug was successfully determined in its tablets with average % recovery of 98.00+/-0.99% which was in accordance with those given by a compendial method. The method was also applied to the determination of paroxetine-HCl in spiked human plasma with average recovery of 77.70+/-1.06%.     

Determination of sertraline in rat plasma by HPLC and fluorescence detection and its application to in vivo pharmacokinetic studies

A simple, rapid, and sensitive HPLC method based on 9H‐fluoren‐9‐ylmethyl chloroformate derivatization for the quantification of sertraline in rat plasma has been developed, requiring a plasma sample of only 0.1 mL, which was deproteinized and derivatized for 5 min in two single steps. The obtained derivative was stable at room temperature and was determined by HPLC using a fluorescence detector. The analytical column was a C(18) column and the mobile phase was acetonitrile and water (80:20, v/v). Calibration curves were linear in the range of 10–500 ng/mL. The limit of detection was approximately 3 ng/mL, and the lower limit of quantification was established at 10 ng/mL. The bias of the method was lower than 10%, and the within day as well as between day, relative standard deviations were lower than 12%. This analytical method was successfully applied to characterize sertraline pharmacokinetics in rats following intravenous (t_1/2 = 213 ± 48 min, Cl = 43.1 ± 8.7 mL/min, V_d = 11560 ± 1861 mL) and oral (C_max = 156 ± 76 ng/mL, t_max = 63.8 ± 16.3 min) administration of 2 and 5 mg, respectively.     

Spectrofluorimetric determination of moxifloxacin in tablets, human urine and serum

A spectrofluorimetric method to determine the antibiotic moxifloxacin is proposed and was applied to pharmaceuticals, human urine and serum. The fluorimetric method allows the determination of 30-300 ng mL-1 moxifloxacin in aqueous solution containing phosphoric acid-phosphate buffer (pH 8.3) with lambda exc = 287 nm and lambda em = 465 nm. Detection and quantification limits were 10 and 30 ng mL-1, respectively, with a relative standard deviation (n = 10) of 2%. This method was applied to the determination of moxifloxacin in three Spanish commercial pharmaceutical formulations. Another variant of the method in micellar medium allows the direct measurement of moxifloxacin in human serum and urine by standard additions. The enhanced fluorescence of moxifloxacin in 8 mM sodium dodecyl sulfate (SDS) solution at pH 4.0 (acetic acid-acetate buffer) for lambda exc = 294 nm and lambda em = 503 nm shows the same linear range as the aqueous method with a 25% lower slope (with detection and quantification limits of 15 and 60 ng mL-1, respectively, and a relative standard deviation of 1.3%), but permits the background fluorescence for urine and serum blanks to be minimized. Hence, sufficient sensitivity is reached to determine therapeutic concentrations of the drug in urine (average recovery 102 +/- 2%) and serum (average recovery 105 +/- 2%) samples.     

Determination of idebenone in plasma by HPLC with post-column fluorescence derivatization using 2-cyanoacetamide

Idebenone is a benzoquinone analog that is used in the treatment of several neurological disorders including Friedreich's ataxia. It was found that the reaction of idebenone with 2-cyanoacetamide under alkaline conditions generates fluorescent products, and the reaction was considered to proceed via Craven's reaction. The reaction mixture from idebenone gave fluorescence with excitation and emission maximum wavelengths at 358 nm and 409 nm, respectively. It was adopted for HPLC with post-column fluorescence derivatization of idebenone. Idebenone in the plasma showed a linear response in the range of 0.5-32 ng (25-1600 ng/mL), and the quantitation limit (S/N=10) was 12.5 ng/mL. The detection limit (S/N=3) of the standard solution of idebenone was 0.1 ng. The HPLC system was applied to the human blood plasma obtained by finger prick. The plasma sample obtained by finger prick gave a similar chromatogram to that of venous blood obtained by venipuncture.     

Pharmacokinetics of clenbuterol in the ostrich.

The aim of this study was to investigate the pharmacokinetics of clenbuterol in the ostrich as no such data is available. Clenbuterol (2 mg) was given as a single oral dose to nine ostriches. Blood samples were collected over a period of 96 h after administration and urine for a period of 5 d. Plasma and urine samples were frozen at -20 degrees C pending analysis. Clenbuterol was quantified using a gas chromatograph-mass selective detector. The method for quantification of clenbuterol in plasma was validated by analysing spiked quality control samples at different concentrations. The limit of quantification was determined to be 0.75 ng ml-1 with an absolute recovery of more than 80%. The geometric mean maximum plasma clenbuterol concentration was 4.40 ng ml-1 with 3.0 h as the median time for maximum concentration. The plasma elimination half-life was 19.7 h. The clenbuterol concentration was above 0.75 ng ml-1 in plasma for 48 h and above 1.0 ng ml-1 in urine for 5 d. These data can be useful in residue analysis for clenbuterol in ostriches.     

Fluorescence detection with an immersed flow cell in capillary electrophoresis

An immersed flowcell has been developed for fluorescence detection in capillary electrophoresis (CE) by modifying an ordinary HPLC spectrofluorimetric detector flowcell. The minimum detectability obtained for riboflavine was about 4 fmol. The use of the flowcell to measure a fluorescence spectrum by the stopped-flow method of shutting down the DC high voltage during a wavelength scan, has also been achieved successfully.     

HPLC-electrospray mass spectrometry for the analysis of temoporfin-poly(ethylene glycol) conjugates

The photodynamic therapeutic agent temoporfin, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) conjugated with poly(ethylene glycol) 2000 (PEG), has been analysed by high performance liquid chromatography (HPLC), linked to electrospray ionisation mass spectrometry (ESI-MS). Sufficient separation of m-THPC-PEG 2000 oligomers was achieved, enabling determination of molecular mass. The use of ESI-MS alone could not achieve this, because of too great a complexity in the mass spectrum, resulting from the presence of four PEG 2000 side chains with a wide molecular mass distribution. The technique is applicable to similar PEG conjugated compounds.     

Pre-concentration of vitamin K1 (phylloquinone) at carbon paste electrodes and its determination in plasma by adsorptive stripping voltammetry

Linear sweep voltammetry was used to study the accumulation behaviour of vitamin K1 at carbon paste electrodes prepared with different types of graphite and pasting agents. The vitamin was found to undergo strong accumulation, but this depended on the type of graphite and pasting agent used. A carbon paste electrode containing Nujol - Ultra Carbon Ultra Superior Purity graphite (25 + 75 m/m) gave the highest sensitivity with adsorptive stripping voltammetry; the optimum accumulation time was 15 min at an open circuit. A variety of procedures were investigated in order to separate vitamin K1 from plasma prior to adsorptive stripping analysis. These procedures were evaluated for plasma levels of the vitamin that are likely to be encountered in pharmacokinetic studies. A solvent extraction method using hexane and ethanol gave the best recovery (91%) and detection limits [180 ng ml-1 in the supporting electrolyte (450 ng ml-1 in plasma)]. However, the analysis time could be reduced by 50% (with some loss of sensitivity) by using ethanol to deproteinate the plasma with the measurement being made directly on the resulting supernatant. As the calibration graphs are linear, quantification can be performed by the method of single standard additions; therefore, relatively short analysis times are feasible.     

Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ～ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.     

Simultaneous quantitative analysis of ketanserin and ketanserinol in plasma by RP-HPLC with fluorescence detection

A sensitive and selective high-performance liquid chromatographic assay for the quantification of ketanserin and ketanserinol in human plasma was developed and validated. The procedure involves extraction of ketanserin and ketanserinol from plasma using an Extrelut NT-1 solid-phase extraction column. The chromatograph was equipped with a Hypersil BDS column (100 x 4.5 mm, 3 micro m particle size). Separation was performed with a mixture of acetate buffer 0.01 M, pH 4.9-methanol-acetonitrile (52:40:8, v/v/v). Detection was performed with fluorescence detection (lambda(ex) = 332 nm and lambda(em) = 410 nm). Calibration curves were linear (r(2) = 0.999) in the range 0-400 ng/mL for both ketanserin and ketanserinol. The repeatability coefficient for ketanserin and ketanserinol was 3.1 and 3.0%, respectively. The reproducibility coefficient for ketanserin and ketanserinol was 10.5 and 9.1%, respectively. The limit of quantification for both ketanserin and ketanserinol was 2.0 ng/mL. The mean recovery yield for both ketanserin and ketanserinol was 60%. In an 8 h work day approximately 60 samples, including calibration and reference standards, could be processed.     

Determination of warfarin at trace-levels in water by solid-phase spectrofluorimetry

A new selective and sensitive spectrofluorimetric method for the determination of warfarin at trace levels (0.2-50.0 ng/ml) in water is proposed. Warfarin is fixed on Sephadex QAE A-25 gel (at pH = 7.0) and its fluorescence is measured directly in the solid-phase using a 1-mm silica cell at 312/385 nm with a detection/quantification limit of 0.06/0.2 ng/ml, a relative standard deviation of 2.3% and recoveries between 95 and 105%. The method is applied to the determination of warfarin residues in water.     

Determination of warfarin at trace-levels in water by solid-phase spectrofluorimetry

A new selective and sensitive spectrofluorimetric method for the determination of warfarin at trace levels (0.2–50.0 ng/ml) in water is proposed. Warfarin is fixed on Sephadex QAE A-25 gel (at pH = 7.0) and its fluorescence is measured directly in the solid-phase using a 1-mm silica cell at 312/385 nm with a detection/quantification limit of 0.06/0.2 ng/ml, a relative standard deviation of 2.3% and recoveries between 95 and 105%. The method is applied to the determination of warfarin residues in water.     

An improved HPLC method for rapid quantitation of diazepam and its major metabolites in human plasma

A rapid and specific HPLC method was developed and validated for simultaneous determination of diazepam and its main active metabolites, desmethyldiazepam, oxazepam and temazepam in human plasma. Plasma samples were extracted using toluene. HPLC system included a Chromolith Performance RP-18e 100 mm x 4.6mm column, using 10mM phosphate buffer (pH 2.5)-methanol-acetonitrile (63:10:27, v/v) as mobile phase running at 2 mL min(-1). UV detector (lambda=230 nm) was used. The calibration curves were linear in the concentration range of 2-800 ng mL(-1) for diazepam and 2-200 ng mL(-1) for the three metabolites (r(2)>0.99). The lower limit of quantification was 2 ng mL(-1) for all analytes. Within and between-day precisions in the measurement of QC samples were in the range of 1.8-18.0% for all analytes. The developed procedure was used to assess the pharmacokinetics of diazepam and its main metabolites following single dose administration of 10mg diazepam orally to healthy subjects.     

Development of a method for the determination of danofloxacin in plasma by HPLC with fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of danofloxacin (DAN) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. DAN and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column, and eluted with aqueous solution-acetonitrile (80:20 v/v). The fluorescence of the column effluent was monitored at lambda(ex) = 338 and lambda(em) = 425 nm. The retention times were 2.80 and 4. 40 min for DAN and SAR, respectively. The method was shown to be linear from 1 to 1500 ng/mL (r(2) = 0.999). The detection and quantitation limit were 1 and 5 ng/mL, respectively. Mean recovery was determined as 80% by the analysis of plasma standards containing 150, 750 and 1500 ng/mL. Inter- and intra-assay precisions were 4.0% and 2.4%, respectively.     

Comparative Evaluation of CE and HPLC for Determination of Cotinine in Human Urine

Two rapid and popular methods—capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) have been compared for analysis of cotinine in human urine. Cotinine was analyzed in less than 7 min, with detection limits of 5 and 3.2 ng mL⁻¹ for CE and HPLC, respectively. The performance of the methods was evaluated in terms of sensitivity, specificity, precision, accuracy, and limits of detection and quantification. Calibration plots were linear in the range 50–4,000 ng mL⁻¹, at least, and mean recoveries were satisfactory for both techniques. The methods were successfully used for quantification of cotinine in urine.     

Spectrofluorimetric determination of piroxicam in the presence and absence of beta-cyclodextrin

The complexation between beta-cyclodextrin (beta-CD) and piroxicam (PX) was investigated by both fluorescence and absorption spectrometry. A 1:2 guest:host stoichiometry for the complex was established, and its association constant was calculated by applying a non-linear regression method to the changes brought about by the presence of beta-CD in both the fluorescence and absorbance spectra of PX. During the study of the influence of the pH on the fluorescence emission of the complex, an efficient enhancement of the signals at acidic pH was observed. This suggests that the protonated form of PX is included more effectively than the ionized form in the beta-CD cavity. Based on the results obtained, spectrofluorimetric methods for the determination of PX were developed. The best limits of detection and quantification were obtained using beta-CD at an acidic pH. The dynamic range in this latter case was 0.02-1 microgram ml-1. The method was applied satisfactorily to the determination of piroxicam in a pharmaceutical preparation.     

Comparing Electrochemical,Fluorescence, and Ultraviolet Detectors for HPLC Analysis of the Decapeptide,Nafarelin

Abstract

This report describes a reverse-phase HPLC technique to determine the concentration of nafarelin (a decapeptide luteinizing hormone-releasing hormone analog) in aqueous solutions for intranasal administration. Pursuant to the method development we evaluated three different detectors with respect to sensitivity, linearity, specificity and reliability. The three detector types investigated were: spectrophotometric (225 nm), electrochemical (at +1.2 v), and fluorescence (excitation = 282 nm, emission = 332 nm). All three detectors gave satisfactorily linear response, and gave equivalent results for nafarelin samples assayed in parallel. The lower detection limits for the three detectors were: ultraviolet = 1.5 ng, electrochemical = 2.0 ng, and fluorescence = 0.6 ng. Thus, the three detector types are nearly equally sensitive for nafarelin analysis. For routine determinations the ultraviolet detector is superior to the electrochemical and fluorescence detectors with respect to convenience of operation.     

Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection.

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C18 reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C18 ODS Hypersil analytical column (5 microns particle size; 250 mm x 4.6 mm I.D.) using an acetonitrile-water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N',N'-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75-400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.     

HPLC‐ESI‐MS/MS analysis and pharmacokinetics of luteoloside,a potential anticarcinogenic component isolated from Lonicera japonica,in beagle dogs

Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC‐ESI‐MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol–acetonitrile (1:1, v/v). A Phenomenex Gemini C₁₈ column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0–2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra‐day and inter‐day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC‐ESI‐MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.