Determination of fosfomycin in chicken plasma samples by gas chromatography: application to pharmacokinetic studies

Summary A sensitive gas chromatographic method is described for the analysis of fosfomycin (FOS) in chicken plasma using phenylphosphonic acid (PPA) as the internal standard. Plasma samples were ultrafiltered, and the ultrafiltrate was then evaporated to dryness. The residue was reconstituted with a silylation reagent for the derivatization of FOS and PPA. The methodology involves the use of a HP-5 capillary column and a flame ionisation detector (FID). The retention times of FOS and PPA were 4.63 and 8.68 minutes, respectively. Response was linear in the range of 1–150 μ mL⁻¹. The detection and quantitation limits were 1 and 2.1 μ mL⁻¹, respectively. Recovery was determined as 109% for FOS. The method was applied to the determination of FOS in chicken plasma samples collected during pharmacokinetic studies.     

HPLC Determination of DCJW in Rat Plasma and Its Application to Pharmacokinetics Studies

A high-performance liquid chromatography–UV method for determining DCJW concentration in rat plasma was developed. The method described was applied to a pharmacokinetics study of intramuscular injection in rats. The plasma samples were deproteinized with acetonitrile in a one-step extraction. The HPLC assay was carried out using a VP-ODS column and the mobile phase consisting of acetonitrile–water (80:20, v/v) was used at a flow rate of 1.0 mL min⁻¹ for the effective eluting DCJW. The detection of the analyte peak area was achieved by setting a UV detector at 314 nm with no interfering plasma peak. The method was fully validated with the following validation parameters: linearity range 0.06–10 μg mL⁻¹ (r > 0.999); absolute recoveries of DCJW were 97.44–103.46% from rat plasma; limit of quantification, 0.06 μg mL⁻¹ and limit of detection, 0.02 μg mL⁻¹. The method was further used to determine the concentration–time profiles of DCJW in the rat plasma following intramuscular injection of DCJW solution at a dose of 1.2 mg kg⁻¹. Maximum plasma concentration (C _max) and area under the plasma concentration–time curve (AUC) for DCJW were 140.20 ng mL⁻¹ and 2405.28 ng h mL⁻¹.     

Simultaneous Determination of Lidocaine, Proline and Lomefloxacin in Human Urine by CE with Electrochemiluminescence Detection

A new method was developed for the simultaneous determination of lidocaine, proline and lomefloxacin in human urine by capillary electrophoresis-electrochemiluminescence detection with Ru(bpy)₃ ²⁺. Conditions of the separation and detection were investigated and optimized. It was proved that 20 mM phosphate buffer at pH 6.7 could achieve the most favorable resolution, and the high sensitivity of detection was obtained by using the detection potential at 1.15 V and 5 mM Ru(bpy)₃ ²⁺–60 mM phosphate buffer at pH 7.6 in the detection reservoir. The detection limits were 0.02 μg mL⁻¹ for lidocaine, 0.03 μg mL⁻¹ for proline and 0.06 μg mL⁻¹ for lomefloxacin. Relative standard deviations of the ECL intensity and the migration time were 3.5 and 1.1% for 6 μg mL⁻¹ lidocaine, 3.2 and 1.0% for 6 μg mL⁻¹ proline and 3.7 and 1.2% for 6 μg mL⁻¹ lomefloxacin, respectively. A baseline separation for lidocaine, proline and lomefloxacin was achieved within 360 s. The developed method was successfully applied to determine the amounts of lidocaine, proline and lomefloxacin in human urine. The recovery and RSD were in the range of 93.3–97.2 and 3.8–4.9%, respectively.     

Determination of phenols in waters using micro-pumped multicommutation and spectrophotometric detection: an automated alternative to the standard procedure

An automated and greener spectrophotometric procedure has been developed for the determination of phenol in water at 700 nm. The method uses the reaction between phenol, sodium nitroprusside, and hydroxylamine hydrochloride in a buffered medium at pH 12.3. The flow manifold comprises four solenoid micro-pumps employed for sample and reagent introduction into the reaction coil and to transport the colored product formed to the detector. The linear dynamic range was 50–3,500 ng mL⁻¹ (R = 0.99997; n = 6) and the method provided a limit of detection (3σ) of 13 ng mL⁻¹. The sampling throughput was estimated to be 65 measurements per hour and the coefficient of variation was 0.5% (n = 10) for a 1.0 μg mL⁻¹ phenol concentration. Recoveries of 92–105% were obtained for phenol determination in spiked water samples at concentration levels from 50 to 5,000 ng mL⁻¹. The use of multicommutation reduced the reagent consumption 25-fold, the sample consumption 225-fold, and the waste generation 30-fold compared with the batch procedure. The proposed method is an environmentally friendly alternative to the official 4-aminoantipyrine method since it avoids the use of chloroform.     

Direct GC determination of methylmercury chloride on HBr-methanol-treated capillary columns

Summary Gas chromatography with electron capture detection (GC-ECD) for the analysis of methylmercury choloride (MMC) using a packed column and a capillary column has been investigated. The columns were 2% silicone OV-227 Uniport HP glass column and a DB-17 capillary column, each pretreated by about ten injections of HBr-methanol solution. MMC was separated as a sharp peak by the HBr-teated column and determined directly by ECD without derivatisation. The mass spectrum of MMC indicated that halide exchange from chloride to bromide proceeded during separation. The minimum detectable concentrations were approximately 5 ng mL⁻¹ on the packed column, and 2 ng mL⁻¹ on the capillary. Calibration curves showed good linearity between 5–200 ng mL⁻¹ for the packed column, and between 2–200 ng mL⁻¹ for the capillary. Relative standard deviations of peak areas were 0.95% for the packed column and 0.43% for the capillary at the level of 100 ng mL⁻¹ in both cases. The column treatment technique was applicable to determination of methylmercury in fish samples.     

MEKC Determination and Pharmacokinetic Study of Danshensu in Rabbits after Intragastric Administration of the Aqueous Extract from Danshen

A micelle eletrokinetic capillary chromatography (MEKC) method was used to determine Danshensu in rabbit blood plasma and tissues (liver, lung, kidney, brain, heart and spleen). The separation was achieved with a buffer consisting of 30 mmol L⁻¹ borax and 50 mmol L⁻¹ SDS (pH 9.0), and with an applied voltage of 7.0 kV. Validation of the method showed good sensitivity, reproducibility and precision. The calibration curve for Danshensu was linear over the concentration range of 0.4–400 μg mL⁻¹. The limit of detection (LOD) was 0.08 μg mL⁻¹ (S/N = 3). The validated method has been successfully applied for the pharmacokinetic and the tissue distribution studies of Danshensu after intragastric administration of the aqueous extract from traditional Chinese medicine Danshen.     

Development and validation of an HPLC method for the simultaneous determination of clozapine and desmethylclozapine in plasma of schizophrenic patients

Summary A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of levels of clozapine (CLZ) and its active metabolite N-desmethylclozapine (DMC) in human plasma. The analysis was performed on a 5 μm C8 reversed phase column (150×4.6 mm i.d.), with acetonitrile-phosphate buffer (pH 3.5), as the mobile phase. The detection voltage was +800 mV and the cell and column temperature were 50°C. Linear responses were obtained between 2 ng mL⁻¹ and 100 ng mL⁻¹. Absolute recovery for both clozapine and desmethylclozapine exceeded 88% and the detection limit was 1 ng mL⁻¹. Repeatability, intermediate precision and accuracy were satisfactory. The method, which is rapid, sensitive and selective, has been applied to therapeutic drug monitoring in schizophrenic patients following administration of Leponex^? tablets. In 21 patients in steady state at a mean daily clozapine dosage of 358 mg (ranging from 150 to 500 mg day⁻¹), clozapine levels averaged 379 ng mL⁻¹ (ranging from 102 to 818 ng mL⁻¹) and DMC levels averaged 233 ng mL⁻¹ (ranging from 70 to 540 ng mL⁻¹). The method requires only a very small amount of plasma (100 μL), and thus it is suitable for pharmacokinetic studies, as well as for therapeutic drug monitoring.     

Determination of eight penicillins in serum from cattle and pigs by generic HPLC method

Summary An HPLC method was developed for determination of amoxicillin, penicillin G, penicillin V, ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in serum from pigs and cattle. Serum was cleaned up by solid-phase extraction (SPE), ultra-filtered and derivatised. The method was linear in the range tested up to 2000 ng mL⁻¹ of individual penicillins in serum. Limits of detection were 11–14 ng mL⁻¹. Mean recoveries were 90–103% in the range 20–2000 ng mL⁻¹. The relative repeatability, standard deviation was <10% at 20 ng mL⁻¹ level and <6% in the range 100–2000 ng mL⁻¹.     

Flow-Injection Fluorimetric Determination of Thiamine in Pharmaceutical Preparations

 A new fluorimetric procedure for the determination of thiamine using flow injection analysis is proposed. The method is based on the derivatization reaction of the primary amine group with o-phthalaldehyde in the presence of 2-mercaptoethanol using fluorimetric detection. The calibration graph based on peak area was linear in the range 0.2–6 ng mL⁻¹. The detection limit was close to 0.1 ng mL⁻¹. The method was applied to the determination of the vitamin in commercial pharmaceutical preparations. Received March 31, 1999. Revision October 15, 1999.     

Development and Validation of Improved Reversed Phase-HPLC Method for Simultaneous Determination of Curcumin, Demethoxycurcumin and Bis-Demethoxycurcumin

Isocratic reversed phase high performance liquid chromatographic (HPLC) method using RP C18 column was developed for simultaneous determination of the curcuminoids. Mobile phase consisted of acetonitrile:0.1% trifluro-acetic acid (50:50) and flow rate was 1.5 mL min⁻¹ and elution was monitored at 420 nm. Validation in selected conditions showed that the chosen method is sensitive, selective, precise and reproducible with linear response of detector for the simultaneous determination of curcumin (C), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC). The limits of detection were 27.99, 31.91 and 21.81 ng mL⁻¹ for C, DMC and BDMC, respectively. Limits of quantitation for C, DMC and BDMC, were 84.84, 96.72 and 66.10 ng mL⁻¹, respectively. Linear range was form 100 to 600 ng mL⁻¹. The mean ± SD percent recoveries of curcuminoids were 99.87 ± 0.34, 100.09 ± 0.48 and 100.10 ± 0.60% of C, DMC and BDMC, respectively. Further, the method was used for quantitation of curcuminoids from turmeric rhizome.     

Development of a Validated Stability-Indicating LC Method for Nitazoxanide in Pharmaceutical Formulations

A reversed-phase liquid chromatographic (LC) method was developed for the assay of nitazoxanide (NTZ) in solid dosage formulations. An isocratic LC separation was performed on a Phenomenex Synergi Fusion C₁₈ column (250 mm × 4.6 mm, i.d., 4 μm particle size) using a mobile phase of 0.1% o-phosphoric acid solution, pH 6.0: acetonitrile (45:55, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was achieved with a photodiode array detector at 240 nm. The detector response for NTZ was linear over the concentration range from 2 to 100 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method were proved using stress conditions. The RSD values for intra-day precision were less than 1.0% for tablets and powder for oral suspension. The RSD values for inter-day precision were 0.6 and 0.7% for tablets and powder for oral suspension. The accuracy was 100.4% (RSD = 1.8%) for tablets and 100.9% (RSD = 0.3%) for powder for oral suspension. The limits of quantitation and detection were 0.4 and 0.1 μg mL⁻¹. There was no interference of the excipients on the determination of the active pharmaceutical ingredient. The proposed method was precise, accurate, specific, and sensitive. It can be applied to the quantitative determination of drug in tablets and powder for oral suspension.     

Rapid HPLC method for acetyl starch determination in blood

Summary Colloidal plasma substitutes of chemically modified starch are used in surgery and in emergency medicine. Acetyl starch (ACS) is a new plasma substitute based on an amylopectin acetic ester. Metabolic cleavage of the ACS ester substituents leads to improved degradation and elimination of infused polymer. To determine the metabolic fate of ACS a rapid LC-method for ACS quantitiation in blood samples was needed. For this purpose a size-exclusion chromatography (SEC) system with improved sensitivity is outlined using a refractive index detector. The limit of detection is 0.005 mg mL⁻¹. From 0.10–5.00 mg mL⁻¹ a linear relationship (correlation coefficient R=0,9999) between the RI signal and ACS concentration is obtained. Recoveries of ACS from blood plasma range 102.3–107.7% for ACS 200/0.5 (range 0.20–7.94 mg mL⁻¹) and 103.0–111.4% for ACS 200/0.7 (range 0.19–9.33 mg mL⁻¹). Only small differences between runs are obtained. In the inter assay test coeficients of variation of 1.8% and of 2.6% respectively are obtained for ACS 200/0.5 and ACS 200/0.7.     

Flow-injection enhanced chemiluminescence method for determination of ciprofloxacin in pharmaceutical preparations and biological fluids

A novel rapid and sensitive analytical method, enhanced chemiluminescence with flow-injection sampling, is described for determination of ciprofloxacin. The method is based on the chemiluminescence reaction of the potassium permanganate–sodium thiosulfate–ciprofloxacin system. An enhanced chemiluminescence reaction was developed, and optimum conditions for CL emission were investigated. The chemiluminescence intensity was linearly dependent on ciprofloxacin concentration in the range 1.0×10⁻⁸–1.0×10⁻⁵ g mL⁻¹. The detection limit was 4×10⁻⁹ g mL⁻¹. The relative standard deviation was 1.8% for eleven measurements of 2.0×10⁻⁷ g mL⁻¹ ciprofloxacin standard solution. The new method enables simple, sensitive, and rapid determination of ciprofloxacin and has been successfully used for determination of ciprofloxacin in biological fluids and in ciprofloxacin hydrochloride tablet and injection.     

A novel kinetic-spectrophotometric method for determination of nitrites in water

A simple, selective and sensitive kinetic method for the determination of nitrite in water was developed. The method is based on the catalytic effect of nitrite on the oxidation of methylene blue (MB) with bromate in a sulfuric acid medium. During the oxidation process, absorbance of the reaction mixture decreases with the increasing time, inversely proportional to the nitrite concentration. The reaction rate was monitored spectrophotometrically at λ = 666 nm within 30 s of mixing. Linear calibration graph was obtained in the range of 0.005–0.5 μg mL⁻¹ with a relative standard deviation of 2.09 % for six measurements at 0.5 μg mL⁻¹. The detection limit was found to be 0.0015 μg mL⁻¹. The effect of different factors such as acidity, time, bromate concentration, MB concentration, ionic strength, and order of reactants additions is reported. Interference of the most common foreign ions was also investigated. The optimum experimental conditions were: 0.38 mol L⁻¹ H₂SO₄, 5 × 10.4 mol L⁻¹ KBrO₃, 1.25 × 10.5 mol L⁻¹ MB, 0.3 mol L⁻¹ sodium nitrate, and 25°C. The proposed method was conveniently applied for the determination of nitrite in spiked drinking water samples.     

Simultaneous Determination of Baicalin, Baicalein, Wogonin, Oxysophocarpine, Oxymatrine and Matrine in the Chinese Herbal Preparation of Sanwu-Huangqin-Tang by Ion-Paired HPLC

A sensitive and reliable ion-paired high-performance liquid chromatographic method has been established for the simultaneous quantification of six major active ingredients, namely baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the Chinese herbal preparation, Sanwu-Huangqin-Tang. HPLC analyses were performed on a Phenomenex luna C₁₈ column with mobile phase of methanol–acetonitrile–aqueous phosphoric acid at a flow rate of 0.9 mL min⁻¹. The complete separation was achieved within 35 min for the six target constituents. A good linear regression relationship between peak-areas and concentrations was obtained over the range of 12.10–242.0 μg*mL⁻¹ for baicalin, 5.05–101.0 μg*mL⁻¹ for baicalein, 0.95–19.0 μg*mL⁻¹ for wogonin, 2.75–55.0 μg*mL⁻¹ for oxysophocarpin, 2.75–55.0 μg*mL⁻¹ for oxymatrine and 4.90–98.0 μg*mL⁻¹ for matrine, respectively. The repeatability was evaluated by intra- and inter-day assays with relative standard deviation (RSD) being less than 5.1%. The recoveries, measured at three concentration levels, varied from 93.8 to 102.1%. The assay was successfully applied for determination of six bioactive compounds in Sanwu-Huangqin-Tang. The interaction of chemical constituents was observed when the herbs were used in compatibility. The results indicated that the developed assay method was rapid, accurate and could be readily utilized as a quality control method for Sanwu-Huangqin-Tang.     

Spectrophotometric quantification of fluoxetine hydrochloride: Application to quality control and quality assurance processes

Simple and rapid spectrophotometric methods have been developed for the microdetermination of fluoxetine HCl. The proposed methods are based on the formation of ion-pair complexes between fluoxetine and bromophenol blue (BPB), bromothymol blue (BTB), bromocresol green (BCG), and bromocresol purple (BCP) which can be measured at optimum λ_max. Optimization of reaction conditions was investigated. Beerșs law was obeyed in the concentration ranges of 0.5–8.0 μg mL⁻¹, whereas optimum concentration as adopted from the Ringbom plots was 0.7–7.7 μg mL⁻¹. The molar absorptivity, Sandell sensitivity, and detection limit were also calculated. The most optimal and sensitive method was developed using BCG. The correlation coefficient was 0.9988 (n = 6) with a relative standard deviation of 1.25, for six determinations of 4.0 μg mL⁻¹. The proposed methods were successfully applied to the determination of fluoxetine hydrochloride in its dosage forms and in biological fluids (spiked plasma sample) using the standard addition technique.     

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometric detection (SPE–CE–MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE–CE–MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L⁻¹ ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 μL min⁻¹ was isopropanol–water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08–10 ng mL⁻¹. Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL⁻¹. Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.     

Determination of risperidone at picogram level in human urine by luminol—H2O2 chemiluminescence

A simple flow-injection chemiluminescence method with synergistic enhancement has been investigated for the rapid and sensitive determination of antipsychotic risperidone. The synergistic action was significant in the chemiluminescence system of luminol—hydrogen peroxide with risperidone as an enhancer. The increased chemiluminescence intensity was correlated with risperidone concentration within the range from 10 pg mL⁻¹ to 1.0 ng mL⁻¹ with relative standard deviations lower than 5.0 % and the detection limit of 4 pg mL⁻¹. At a flow rate of 2.0 mL min⁻¹, the flow-injection chemiluminescence method exhibited both a high sensitivity and excellent selectivity giving a throughput of 120 times per hour. The proposed method was successfully applied to determine the risperidone content in human urine without any pretreatment. It was found that the excretive amounts of risperidone reached their maximum after taking 2.0 mg of risperidone for 1 h, with a total excretive ratio of 17.37 % in 8.5 h.     

Chemiluminescence determination of chromium(III) and total chromium in water samples using the periodate-lucigenin reaction

A new chemiluminescence (CL) method combined with flow injection technique is described for the determination of Cr(III) and total Cr. It is found that a strong CL signal is generated from the reaction of Cr(III), lucigenin and KIO₄ in alkaline condition. The determination of total Cr is performed by pre-reduction of Cr(VI) to Cr(III) by using H₂SO₃. The CL intensity is linearly related to the concentration of Cr in the range 4.0 × 10⁻¹⁰–1.0 × 10⁻⁶ g mL⁻¹. The detection limit (3s _b) is 1 × 10⁻¹⁰ g mL⁻¹ Cr and the relative standard deviation is 1.9% (5.0 × 10⁻⁸ g mL⁻¹ of Cr(III) solution, n = 11). The method was applied to the determination of Cr(III) and total Cr in water samples and compared satisfactorily with the official method.     

Analysis of Follicle-Stimulating Hormone by CE with Chemiluminescence Detection Based on Competitive Immunoassay

A competitive immunoassay and capillary electrophoresis with enhanced chemiluminescence detection have been used for determination of follicle-stimulating hormone (FSH) in human serum. The method is based on the competitive immunochemical reaction of FSH and horseradish peroxidase (HRP)-labeled FSH with anti-FSH, CE separation of antibody-bound and free HRP-labeled FSH, then chemiluminescence detection. The detection limit depends on the stability of the immune complex, which depends on analysis time and detector design, and on the chemiluminescence enhancer used. A unique chemiluminescence detector without dead volume or diluent effects was therefore used, and sodium tetraphenylboron was selected as the optimum enhancer. As a result sensitivity was substantially improved. Free HRP-labeled FSH and the immune complex could be separated within 15 min in alkaline borate buffer by use of a potential of 15 kV. Under the optimum conditions a calibration plot for FSH was established in the concentration range 0–100 mIU mL⁻¹; the detection limit was 0.06 mIU mL⁻¹. The concentration sensitivity achieved was 30 times better than that of ELISA.