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Simultaneous multiresponse optimization of an HPLC method to separate seven cephalosporins in plasma and amniotic fluid: Application to validation and quantification of cefepime, cefixime and cefoperazone

An HPLC method for the separation of seven cephalosporins [Cefepime (CEP), ceftazidime (CTA), ceftizaxime (CTI), ceftriaxone (CTR), cefotaxime (COT), cefixime (CIX) and cefoperazone (COP)] in human plasma and amniotic fluid has been developed. Optimization of the chromatographic method was performed in three steps: a series of initial experiments followed by two sets of experiments based on different experimental designs. The initial experiments were performed to decide the basic analytical requirements of the method. Then screening experiment fractional factorial design was used in order to decrease the number of parameters by eliminating parameters which having insignificant effect on responses. The parameters having significant effect were further optimized through a full factorial design. Having studied two responses (retention times and resolutions), a desirability function that assess the responses together, was used to find experimental conditions where the system generated desirable results. The desirable results were obtained with XTerra C18 (250 mm x 4.6mm, 5 microm i.d.) column, 40 mM phosphate buffer, pH 3.2, 18% MeOH, 0.85 mL min(-1) flow rate and 32 degrees C column temperature. Gradient elution with MeOH was applied. A simple and efficient solid-phase extraction was applied for the preparation of plasma and amniotic fluid samples. The validation parameters of the method were evaluated in accordance with ICH guideline. The method validated was applied to the analysis of CEP and COP in maternal venous, fetal venous and fetal arterial plasma, and to the analysis of CIX in maternal venous plasma and amniotic fluid.

Method development and validation for the analysis of meloxicam in tablets by CZE.

A capillary zone electrophoresis assay for the analysis of meloxicam has been developed and validated. The influence of buffer concentration, buffer pH, methanol as organic modifier, capillary temperature, applied voltage and injection time was systemically investigated in a fused silica capillary (i.d. 50 microm, total length 44 cm and effective length 35.5 cm). Optimum results were obtained with a 100 mM borate buffer (pH 8.5) containing 5% methanol, capillary temperature 25 degrees C, applied voltage 20 kV and injection time 3 s hydrodynamic injection. The detection wavelength was set to 205 nm. Diflunisal was used as internal standard. The method showed good selectivity, accuracy, precision, linearity and sensitivity according to the evaluation of the validation parameters. The method was applied to the determination of six pharmaceutical preparations including two dosage forms. The relative standard deviation of 7 replicate analyses for each sample was less than 0.66%. The results were compared with a spectrophotometric method reported in literature and no significant difference was found statistically.

Polarographic behaviour of meloxicam and its determination in tablet preparations and spiked plasma.

Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID), which has a higher activity cyclooxygenase-2 (COX-2) than against cyclooxygenase-1 (COX-1), with potentially high anti-inflammatory and analgesic action. The voltammetric behaviour of meloxicam was studied using direct current (DC), differential pulse polarography (DPP) and cyclic voltammetry (CV). The influence of several variables (including nature of the buffer, pH, concentration, modulation amplitude, scan rate, drop size, etc.) was examined in DPP method for meloxicam. The best DPP response was obtained in acetate buffer pH 4.88. The peak currents were measured with a static mercury drop electrode at -1.49 V versus Ag/AgCl. Calibration curve for meloxicam was linear at a concentration range from 0.38 to 15.0 microg ml(-1). The method was validated and applied to the determination of meloxicam in tablets, which were in two different dosage forms. A spectrophotometric method reported in the literature was utilized as a comparison method. There were no significant differences between the results obtained by two methods. DPP method is also available and applicable for the determination of mentioned substance in plasma. Mean recovery was 99.20+/-0.37%. It was concluded that the developed method was accurate, sensitive, precise, reproducible and useful for the quality control of meloxicam in pharmaceuticals and spiked plasma.

Electroanalytical Characterization of Verapamil and its Voltammetric Determination in Pharmaceuticals and Human Serum

Abstract The electrooxidative behavior and determination of Verapamil HCl, one of the class IV anti‐arrhythmic agent, on a glassy carbon disc electrode were investigated for the first time by using cyclic, differential pulse (DPV), and Osteryoung square wave voltammetry (OSWV). Verapamil showed an irreversible oxidation behavior at all pH values and buffers studied. From the electrochemical response, the main oxidation step was found to be related to the methoxy group on the phenyl ring. DPV and OSWV were used to generate peak current versus concentration curves for verapamil. A linear response was obtained in the range comprised between 8×10−7 and 1×10−4 M for both techniques with detection limit of 1.61×10−7 M for DPV and 1.33×10−7 M for OSWV. The repeatability and reproducibility of the methods for all investigated media (such as supporting electrolyte and serum samples) were determined. Precision and accuracy were also checked in all media. The methods were proposed for the determination of verapamil in dosage forms adopting both DPV and OSWV modes. The methods were extended to the in vitro determination of verapamil in spiked serum samples. No electroactive interferences from the endogenous substances were found in human plasma.

Analysis of diflunisal by electrochemical methods.

A new differential pulse polarographic (DPP) and differential pulse adsorptive stripping voltammetric (DPAdSV) methods for the electrochemical behavior and quantitative determination of diflunisal were described. In these voltammetric methods, the peak potential of diflunisal was found as -0.31 V (vs. Ag/AgCI) with selected Britton--Robinson buffer (BR, pH 7.8) as a supporting electrolyte. The variation of the peak current with the concentration of diflunisal were linear in the 9.0--40.0 and 4.0--30.0 microg ml(-1) concentration ranges for DPP and DPAdSV methods, respectively. The limits of detection (LOD) were found as 5.0 and 0.1 microg ml(-1) for DPP and DPAdSV methods, respectively. The developed methods were validated by evaluation of the validation parameters. The characteristics of the peak current of diflunisal were examined in detail and the results proved that the peak current has an adsorption characteristic. The developed methods were proposed for rapid determination of diflunisal in commercial tablets. The recovery studies showed that developed assays had a good accuracy and precision with mean recoveries 99.92 and 100.02% and mean variation coefficients 0.29 and 0.24% in DPP and DPAdSV methods, respectively.

Maternal blood and amniotic fluid levels of moxifloxacin, levofloxacin and cefixime

Aim:  Moxifloxacin and levofloxacin are wide spectrum quinolones and cefixime is a third‐generation cephalosporin with a wider spectrum of activity against gram‐positive and gram‐negative bacteria and anaerobics. Although they are widely used, little is known about the amniotic fluid levels of these antibiotics. The aim of the present investigation was to study and compare the maternal blood and amniotic fluid levels of these antibiotics in second trimester pregnancy.

Indirect atomic absorption spectrometric determination of alkaloids using an air-acetylene flame

An assay method has been developed which involves the extraction of ion pairs consisting of alkaloids and the inorganic complex Ni(SCN)4–2, followed by the determination of nickel in the organic phase by atomic absorption spectrometry. The optimum experimental conditions for pH, concentration of Ni(SCN)4–2, shaking time, aqueous to organic phase ratio and the number of extractions were determined. Atropine sulphate and pilocarpine hydrochloride were chosen as typical molecules and the method developed was applied to pharmaceutical preparations of atropine sulphate in eye drops, injection solutions and tablets and also eye drops containing pilocarpine hydrochloride.

Maternal and fetal blood levels of moxifloxacin, levofloxacin, cefepime and cefoperazone.

Wide-spectrum quinolones such as moxifloxacin and levofloxacin as well as high-order cephalosporins such as cefoperazone and cefepime have increased antimicrobial activity. However, little is known about their distribution in fetal blood. Therefore, the aim of this study was to measure and compare maternal and fetal blood levels of these agents. For the measurement of blood levels, 9 pregnant women received cefepime hydrochloride, 10 received cefoperazone, 10 received moxifloxacin and 12 received levofloxacin intravenously. Maternal and umbilical cord blood samples were drawn during delivery. Antibiotic levels were analysed by high-performance liquid chromatography. Mean transplacental passage rates of moxifloxacin, levofloxacin, cefepime and cefoperazone were 74.84%, 66.53%, 23.21% and 12.68%, respectively, and mean transfetal passage rates were 90.78%, 84.22%, 79.17% and 79.78%, respectively. The transplacental passage rate for either quinolone was significantly higher than that of either cephalosporin, and the transplacental passage rate of cefoperazone was lower than that of cefepime. In conclusion, both quinolones have high transplacental passage rates. Cefepime and cefoperazone have a lower transplacental passage rate and thus may be used as prophylaxis in situations where transplacental passage is undesirable.

Comparison of pharmacokinetic profiles of moxifloxacin in Caesarean versus non-pregnant sectioned women by fully validated HPLC with fluorescence detection.

In this study, a simple, rapid, cost-effective, and sensitive reversed-phase high-performance liquid chromatographic method has been developed and validated for the analysis of moxifloxacin in plasma. The chromatographic separation was achieved on a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm i.d.) connected to a Phenomenex C(18) column (4 mm x 3.0 mm i.d.) using a mixture of acetonitrile: 15 mM citrate buffer (pH 3) (23:77, v/v) as the mobile phase with isocratic system at a flow rate of 1 mL/min. Fluorescence detection was employed with excitation at 290 nm and emission at 500 nm. Lomefloxacin was used as internal standard. Plasma samples were prepared with addition of acetonitrile only. The method was fully validated according to the International Conference on Harmonization (ICH) guidelines. The results of the validation parameters were: linearity range, 3-6000 ng/mL (R(2) = 0.9994); mean recovery, 100.48 %; limit of quantification, 5 ng/mL; limit of detection, 1 ng/mL; and intra- and inter-day precision less than 3.2% and 5.1%, respectively. The robustness of the method was evaluated and confirmed with fractional factorial design. After validation studies, the method was applied in order to conclude the effects of pregnancy on postoperative pharmacokinetic profiles of moxifloxacin. For this aim, moxifloxacin was given to non-pregnant women (n=9) and caesarean-sectioned women (n=6) as a single intravenous dose (400 mg Avelox(R) infusion). Plasma samples were analyzed in order to compare pharmacokinetic profiles of pregnants and non-pregnants. Peak serum concentrations of non-pregnant and caesarean-sectioned women at the arterial port after the infusion were 4.95 +/- 1.50 and 1.56 +/- 0.16 microg/mL, respectively. The mean elimination half-life, volume of distribution and calculated area under the concentration-time curve (AUC)(0-infinity) were 5.54 +/- 0.73 h, 65.58 +/- 6.30 L and 49.95 +/- 6.30 microg.h/mL for non-pregnant women and 3.50 +/- 0.37 h, 215.85 +/- 24.87 L and 10.53 +/- 0.66 microg.h/mL for caesarean-sectioned women, respectively. These results indicated that pregnancy has a significant effect on the pharmacokinetics of moxifloxacin.

Determination of Rivaroxaban in Human Plasma by Solid-Phase Extraction–High Performance Liquid Chromatography

In this study, a solid-phase extraction (SPE)-high performance liquid chromatography (HPLC)-ultra violet (UV) method was developed for the determination of rivaroxaban (RIV), an oral anticoagulant drug, in human plasma samples. The concentration of RIV in plasma samples was increased 7.5 times and the interference coming from matrix components was avoided by using SPE. The extracted samples of RIV were analyzed by using an HPLC-UV method. RIV was approved in 2008 and many studies have been published in recent years in order to investigate its pharmacokinetic profile in various groups. In light of this information, it is clear that the RIV pharmacokinetic profile should be investigated in further studies; the HPLC-UV method presented in this study might be an easy method to apply, as it is a cheap and rapid alternative to HPLC-MS-MS for this purpose. A Phenomenex Luna 5-µm C18 100 Å LC column (250 × 4.6 mm) was used for the separation of RIV and prednisolone (internal standard). The total analysis time was <6 min. The method was validated according to the FDA guidelines and can be proposed for pharmacokinetic studies of RIV.