A high-performance thin-layer chromatography densitometric method for the separation of isomeric ceftriaxone in powder for injection formulation.

The aim of this study was to develop and validate a High-Performance Thin Layer Chromatographic (HPTLC) method for simultaneous determination of ceftriaxone and ceftriaxone e-isomer in powder for injection formulation. Ceftriaxone sodium injection is an antibiotic that used globally. It has Z/E geometrical conformation, in which ceftriaxone sodium and 3 ene-isomer have Z- conformation while (E)-isomer has E- conformation and the potential toxicity of ceftriaxone (E)-isomer has been reported. Thus, to safeguard the public health, a simple and easy to use, rapid and reliable method was developed for qualitative and quantitative determination of ceftriaxone sodium and its (E)-isomer. Samples were applied on HPTLC glass plates precoated with silica gel 60F254 by using Linomat semi-auto sampler. Separation was carried out using acetone, triethyl amine, water, chloroform and ethyl acetate as a mobile phase in different ratios. The Rf values of separated compounds were 0.51 ± 0.01 and 0.62 ± 0.01 for ceftriaxone sodium and ceftriaxone (E)-isomer respectively. The method was validated by studying Specificity, Linearity, Accuracy, Precision, Robustness, Limit of Detection (LOD) and Limit of Quantification (LOQ) and Solution stability. The developed method was successfully, sensitive, simple, precise, accurate, robust and applicable for the simultaneous determination of ceftriaxone sodium and ceftriaxone (E)-isomer in powder for injection formulation.

Removal of ceftriaxone and cefotaxime antibiotics from industrial wastewater via complexation with Cu(II).

A high-performance liquid chromatographic method (HPLC) with UV detection is described for determination of ceftriaxone sodium (CFX) and cefotaxime sodium (CFM) content in pharmaceutical industrial wastewater. These methods are based on the detection of these antibiotics via the formation of chelate complexes with Cu(II). The developed Liquid Chromatographic method offers symmetric peak shape, good resolution and reasonable retention time for both drugs. The removal percentage reached about 100 and 92.1% at pH 7.2 for CFX and CFM, respectively. In UV detection, the removal of the chelating antibiotics were based on forming of chelate complexes with Cu(II) which detected at λmax = 253 and 244 nm for CFX and CFM, respectively. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5.99-59.86 µg mL-1 for CFX and 14.33-71.63 µg mL-1 for CFM. The proposed method can be used for the quality control of industrial wastewater containing CFX and CFM.

The comparison of the quality of selected brands of antibiotics in Tanzania sourced from different geographical regions.

OBJECTIVES: The quality of amoxicillin capsules, ceftriaxone for injection, and ciprofloxacin tablets was evaluated to determine whether there is any difference in quality when comparing the country of origin. This was undertaken because it has been claimed that antibiotics manufactured in Europe are of superior quality to those originating from Africa or Asia. METHODS: Samples of amoxicillin capsules, ceftriaxone for injection, and ciprofloxacin tablets were collected from three randomly selected wholesale pharmacies in each city, namely Arusha, Dar es Salaam and Mwanza, Tanzania. The collected samples of collected brands were subjected to quality control testing as per their respective pharmacopoeial monographs. Amoxil 250 mg capsules (Glaxo Wellcome, Mayenne, France), Rocephin (Roche, Switzerland) and Cipro-Denk 500 (Allphamed Pharbil Arzneimittel GmbH, Gottingen, Germany) were used as reference brands for the other generic brands of amoxicillin, ceftriaxone and ciprofloxacin, respectively. RESULTS: A total of 31 brands (10 different brands of amoxicillin capsules, 9 of ceftriaxone sodium injections, and 12 of ciprofloxacin tablets) were collected from the targeted regions and subjected to quality control testing. All samples of collected brands complied with the requirements of their respective pharmacopoeial monographs. CONCLUSIONS: There was no significant difference in quality between brands of amoxicillin capsules, ceftriaxone for injection, and ciprofloxacin tablets manufactured in Africa and Asia against those manufactured in Europe in terms of compliance with the respective pharmacopoeial monographs.

Response surface modeling of ceftriaxone removal from hospital wastewater.

In recent decades, an emerging concern of widespread antimicrobial resistance has been raised due to the existence of pharmaceutical samples such as antibiotics in an aqueous medium. Herein, antibiotic ceftriaxone (CTX) removal from hospital wastewater employing a hybrid process of electrocoagulation (EC) and adsorption (AD) was investigated. The response surface methodology (RSM) was employed to study the influences of main operating variables, including initial CTX concentration, pH, current density, reaction time, and chitosan dosage, on the removal efficiency of the treatment process. Under the optimum condition of the employed EC/AD hybrid treatment process, where initial CTX concentration, pH solution, the current density, adsorbent dosage, and reaction time were set at 20.0 mg L-1, 7.5, 6.0 mA cm-2, 0.75 g L-1, and 12.5 min, respectively, the removal efficiency of 100% was achieved. Analysis of variance (ANOVA) confirmed that the developed quadratic treatment model is highly significant. The applied EC/AD hybrid treatment process revealed the electrical energy consumption of 0.84 kWh m-3 and 0.2168 kWh (g Al)-1 per cubic meter of hospital wastewater and gram of consumed aluminum electrode, respectively. The second-order kinetic model with R2 of 0.9514 and the Langmuir isotherm model with R2 of 0.973 best fit the developed EC/AD hybrid treatment process, and qm was found to be 111.1 mg g-1. The obtained experimental results confirmed that the CTX concentration of the hospital wastewater was reduced to zero after applying the EC/AD hybrid process.

Highly sensitive detection of Ceftriaxone in water, food, pharmaceutical and biological samples based on gold nanoparticles in aqueous and micellar media.

A colorimetric assay with excellent sensitivity is reported to detect Ceftriaxone in aqueous and micellar solutions. Ceftriaxone could induce the aggregation of gold nanoparticles through hydrogen-bonding interaction and electrostatic attraction. As a result of aggregation, the surface plasmon resonance band around 520 nm decreases and a new band appears at 620 nm. The effect of surfactants was investigated on the aggregation. The band around 620 nm is shifted to around 685 nm in Triton X-100 micellar media and that is seen color conversion from red to deep blue which is clearly detectable by the naked eye. The results were improved in Triton X-100 micellar media as compared to aqueous media so that the lowest measured concentration and detection limit in micellar media have decreased 10 and 8 times, respectively. Triton X-100 showed strong effect on the stabilization of the solutions. The method has been successfully applied for the analysis of various real samples.

A Critical Review of Analytical Methods for Determination of Ceftriaxone Sodium.

Ceftriaxone sodium is a third-generation semi-synthetic antibiotic belonging to the class of cephalosporins. Is administered only by parenteral route and has the ability to cross the blood-brain barrier. It has bactericidal action; its main activity is related to the Gram-negative bacteria, being also able to act against Gram-negative bacilli resistant to the first- and second-generation cephalosporins. The present study presents a survey of the characteristics, properties and analytical methods used for the determination of ceftriaxone sodium, for the gathering of data searches were carried out in scientific articles in the world literature, as well as in the official compendia. It is necessary to create awareness about the importance of developing effective and reliable analytical methods for quality control and consequently for conducting pharmacokinetic, bioavailability, bioequivalence studies as well as for the therapeutic monitoring of this drug. Most of the methods found use high-performance liquid chromatography, but also methods that use absorption spectroscopy ultraviolet, infrared spectroscopy, spectrofluorimetry and microbiological methods have been presented. A discussion was presented highlighting the need to develop new ecological methods using less toxic solvents, rapid analysis and miniaturization of the samples.

Enhanced antimicrobial de-escalation for pneumonia in mechanically ventilated patients: a cross-over study.

BACKGROUND: Antibiotics are commonly administered to hospitalized patients with infiltrates for possible bacterial pneumonia, often leading to unnecessary treatment and increasing the risk for resistance emergence. Therefore, we performed a study to determine if an enhanced antibiotic de-escalation practice could improve antibiotic utilization in mechanically ventilated patients with suspected pneumonia cared for in an academic closed intensive care unit (ICU). METHODS: This was a prospective cross-over trial comparing routine antibiotic management (RAM) and enhanced antimicrobial de-escalation (EAD) performed within two medical ICUs (total 34 beds) at Barnes-Jewish Hospital, an academic referral center. Patients in the EAD group had their antibiotic orders and microbiology results reviewed daily by a dedicated team comprised of a second-year critical care fellow, an ICU attending physician and an ICU pharmacist. Antibiotic de-escalation recommendations were made when appropriate based on microbiologic test results and clinical response to therapy. RESULTS: There were 283 patients evaluable, with suspected pneumonia requiring mechanical ventilation: 139 (49.1%) patients in the RAM group and 144 (50.9%) in the EAD group. Early treatment failure based on clinical deterioration occurred in 33 (23.7%) and 40 (27.8%) patients, respectively (P = 0.438). In the remaining patients, antimicrobial de-escalation occurred in 70 (66.0%) and 70 (67.3%), respectively (P = 0.845). There was no difference between groups in total antibiotic days ((median (interquartile range)) 7.0 days (4.0, 9.0) versus 7.0 days (4.0, 8.8) (P = 0.616)); hospital mortality (25.2% versus 35.4% (P = 0.061)); or hospital duration (12.0 days (6.0, 20.0) versus 11.0 days (6.0, 22.0) (P = 0.918). CONCLUSIONS: The addition of an EAD program to a high-intensity daytime staffing model already practicing a high-level of antibiotic stewardship in an academic ICU was not associated with greater antibiotic de-escalation or a reduction in the overall duration of antibiotic therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02685930 . Registered on 26 January 2016.

An ultrasensitive and selective method for the determination of Ceftriaxone using cysteine capped cadmium sulfide fluorescence quenched quantum dots as fluorescence probes.

In this paper, l-cysteine (Cys) coated CdS quantum dots (QDs) have been prepared, which have excellent water-solubility and are highly stable in aqueous solution. These QDs is proposed as sensitizers for the determination of Ceftriaxone. The quantum dot nanoparticles were structurally and optically characterized by Ultra Violet-Visible absorption Spectroscopy (UV-vis absorption spectroscopy), Fourier transform infrared spectroscopy (FT-IR spectra) and photoluminescence (PL) emission spectroscopy. High resolution transmission electron microscopy (HRTEM) confirms that the Cys-CdS QDs have a spherical structure with good crystallinity. Therefore, a new simple and selective PL analysis system was developed for the determination of Ceftriaxone (CFX). Under the optimum conditions, The response of l-Cys capped CdS QDs as the probe was linearly proportional to the concentration of Ceftriaxone ions in the range of 1.6×10(-9)-1.1×10(-3)M with a correlation coefficient (R2) of 0.9902. The limit of detection of this system was found to be 1.3nM. This method is simple, sensitive and low cost.

Identification of a new isomer from a reversible isomerization of ceftriaxone in aqueous solution.

A reversible isomerization of ceftriaxone in aqueous solution was observed, and the structure of the isomer was determined by mass spectrometry and various 1D and 2D NMR techniques. The mechanism of isomerization was also discussed. Finally, molecular docking simulations were performed and the antimicrobial activities of the isomers were measured. This showed that the biological activity of ceftriaxone was stronger than that of its isomer. The results reported in this article may be important to quality control requirements and to the stability of ceftriaxone products.

Potassium permanganate-acridine yellow chemiluminescence system for the determination of fluvoxamine, isoniazid and ceftriaxone.

Based on the oxidation of acridine yellow by permanganate in basic medium, a new chemiluminescence system was developed for the sensitive determination of some important drugs. The remarkable inhibiting effect of fluvoxamine, ceftriaxone and isoniazid on this reaction was applied to their detection. A possible mechanism was proposed for this system based on chemiluminescence emission wavelengths and experimental observations. Under optimum conditions, calibration graphs were obtained for 1 × 10(-9) to 1 × 10(-6) mol/L of fluvoxamine; 2 × 10(-8) to 8 × 10(-6) mol/L of ceftriaxone and 5 × 10(-8) to 4 × 10(-5) mol/L of isoniazid. This proposed method was satisfactorily used in the determination of these drugs in pharmaceutical samples and human urine and serum.

Development and validation of a spectrofluorimetric method for the quantification of ceftriaxone in pharmaceutical formulations and plasma.

We describe the development and validation of a new, simple, sensitive and cost-effective method for the determination of ceftriaxone in commercial formulations and spiked human plasma. The method proposes the conversion of ceftriaxone into a fluorescent product by reacting with ortho-phthalaldehyde (OPA) in the presence of sulfite at room temperature. The reaction medium is buffered to pH 10 using borate buffer. The derivatized reaction product is highly fluorescent and exhibits maximum fluorescence intensity at λ(em) = 386 nm after excitation at λ(ex) = 324 nm. The experimental parameters affecting progress of the derivatization reaction were carefully studied and optimized. Under optimum experimental conditions, the method has an excellent correlation coefficient of 0.9984 with a broad linear range of 0.4-20 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 1.30 × 10(-3) and 3.90 × 10(-3) µg/mL, respectively. The interference effects of common excipients on the quantification of drug were investigated and no interference effect was observed. The proposed method has been successfully applied to the determination of ceftriaxone in pharmaceutical formulations and spiked human plasma samples. The method has been validated statistically through percent recovery studies using standard addition and by comparison with a reference HPLC method. The developed method exhibits excellent inter- and intraday precision.

A fully validated microbiological assay to evaluate the potency of ceftriaxone sodium

Ceftriaxone (CFTX) sodium is a third-generation, broad-spectrum cephalosporin that is resistant to beta-lactamases. An alternative bioassay for the assessment of the potency of this drug in pharmaceutical formulations has not been previously reported. Thus, this paper reports the development and full validation of a 3 x 3 agar diffusion bioassay using a cylinder-plate method to quantify CFTX sodium in pharmaceutical samples. The strain Staphylococcus aureus ATCC 6538P was used as the test microorganism, and the results of the proposed bioassay displayed high linearity, precision, accuracy, specificity and robustness. All potency results were statistically analyzed using an analysis of variance (ANOVA) and were found to be linear (r=0.99999) in the range of 16-64 µg/mL, accurate (100.5%), and precise [repeatability: relative standard deviation (RSD)=1.4%; intermediate precision: between-day RSD=2.1% and between-analyst RSD=2.5%]. The specificity of the bioassay was determined by evaluating a degraded sample (50 ºC) at 0, 24 and 48 hours as compared against the results from the pharmacopeial liquid chromatography method for CFTX. The results validated the proposed microbiological assay, which allows reliable quantitation of CFTX in pharmaceutical samples. Moreover, it is a useful, simple and low-cost alternative method for monitoring the quality of this medicine.

A ceftriaxona sódica é uma cefalosporina de terceira geração de uso parenteral, com amplo espectro de atividade e resistente a b-lactamases. Este estudo apresenta o desenvolvimento e validação de um bioensaio por difusão em ágar usando o método de cilindros em placas para determinação da potência deste antibiótico. A validação desenvolvida apresentou bons resultados em termos de linearidade, precisão, exatidão, especificidade e robustez. Empregou-se o Staphylococcus aureus ATCC 6538P como micro-organismo teste. Os resultados dos ensaios foram tratados estatisticamente utilizando-se análise de variância (ANOVA). O método apresentou linearidade (r=0,99999) na faixa de doses selecionada (16-64 µg/mL), precisão (repetibilidade: DPR=1,4%; precisão intermediária: inter-dias DPR=2,1% e inter-analistas: DPR=2,5%) e exatidão de 100,5%. A especificidade do bioensaio foi avaliada através da análise comparativa, por cromatografia líquida de alta eficiência, de amostras degradadas a 50 ºC nos tempos zero, 24 e 48 h. Os resultados encontrados demonstraram a validade do bioensaio proposto, o qual permite a quantificação confiável de ceftriaxona sódica em produtos farmacêuticos comerciais. Por ser metodologia simples e de baixo custo constitui-se em alternativa para a análise de rotina do controle de qualidade de medicamentos.

[Level of evidence for therapeutic drug monitoring of ceftriaxone]. / Niveau de preuve du suivi thérapeutique pharmacologique de la ceftriaxone.

Ceftriaxone is a third generation cephalosporin with an original pharmacokinetics based on a long elimination half-life among cephalosporins, a high protein binding and a dual renal and biliary elimination. Also the pharmacokinetic parameters of ceftriaxone are highly variable in clinical situations such as severe renal insufficiency, liver and renal insufficiency, the elderly, the neonates less than 1 week of age and critically ill patients. In these clinical situations associated or not with high minimal inhibitory concentration (MIC) level, the relationship concentration-clinical outcome based on the ratio between trough plasma concentration and MIC can allow a dose adjustment. Consequently, therapeutic drug monitoring (TDM) of ceftriaxone could be possibly useful in these situations, whereas the necessity of TDM has still to be demonstrated to monitor toxicity.

Comparison between original and generic versions of ceftriaxone sodium preparation for injection: compatibility with calcium-containing product.

The purpose of this study was to compare the compatibility of ROCEPHINE® Intravenous, the original manufacturer's ceftriaxone sodium preparation for injection, and seven generic versions thereof, with various calcium chloride injection 2%. The influence of calcium ion concentration, storage time and shaking strength on the appearance and quantity of insoluble microparticles in mixed solutions was examined using a light obscuration particle counter. In all products, the observed number of insoluble microparticles was proportional to the calcium ion concentration, storage time and shaking strength after the addition of calcium chloride solution. In several of the generic products, the number of insoluble microparticles was significantly higher than those of the original product, while in others it was lower. We evaluated the quality of the original and 7 generic preparations, measured the content of impurity and pH of the various ceftriaxone solutions, as impurity content and pH of solution are possible factor affecting compatibility. Three impurities were found in all products. The impurity content of several generic products, as estimated from their peak area on high performance liquid chromatography (HPLC), was significantly lower than that of the original product. pH of solution was difference between products. Although it was difficult that impurity and pH of solution verify critical factor affecting compatibility. The results show that there are differences in the appearance of insoluble microparticles between the original product and seven generic products, when calcium chloride injection 2% solution is added to the product.

Validation of an HPLC-UV method for the determination of ceftriaxone sodium residues on stainless steel surface of pharmaceutical manufacturing equipments.

In pharmaceutical industry, an important step consists in the removal of possible drug residues from the involved equipments and areas. The cleaning procedures must be validated and methods to determine trace amounts of drugs have, therefore, to be considered with special attention. An HPLC-UV method for the determination of ceftriaxone sodium residues on stainless steel surface was developed and validated in order to control a cleaning procedure. Cotton swabs, moistened with extraction solution (50% water and 50% mobile phase), were used to remove any residues of drugs from stainless steel surfaces, and give recoveries of 91.12, 93.8 and 98.7% for three concentration levels. The precision of the results, reported as the relative standard deviation (RSD), were below 1.5%. The method was validated over a concentration range of 1.15-6.92 µg ml(-1). Low quantities of drug residues were determined by HPLC-UV using a Hypersil ODS 5 µm (250×4.6 mm) at 50 °C with an acetonitrile:water:pH 7:pH 5 (39-55-5.5-0.5) mobile phase at flow rate of 1.5 ml min(-1), an injection volume of 20 µl and were detected at 254 nm. A simple, selective and sensitive HPLC-UV assay for the determination of ceftriaxone sodium residues on stainless steel surfaces was developed, validated and applied.

[Rapid determination of ceftriaxone in mixed saliva of practically healthy subjects and patients with infectious and somatic pathologies].

A method for spectroscopic determination of ceftriaxone in saliva of healthy subjects and patients with upper respiratory tract infections was developed. The concentration range is 1-50 mcg/ml. The method is express and simple, and can be used to determine ceftriaxone in clinical tests and pharmacokinetic studies. Possible determination of ceftriaxone in aqueous solutions was demonstrated which is useful in analysis of drugs.

Biotic and abiotic degradation of four cephalosporin antibiotics in a lake surface water and sediment.

Cephalosporins are widely used veterinary and human antibiotics, but their environmental fate and impacts are still unclear. We studied degradation of four cephalosporins (cefradine, cefuroxime, ceftriaxone, and cefepime) from each generation in the surface water and sediment of Lake Xuanwu, China. The four cephalosporins degraded abiotically in the surface water in the dark with half-lives of 2.7-18.7d, which were almost the same as that in sterilized surface water. Under exposure to simulated sunlight, the half-lives of the cephalosporins decreased significantly to 2.2-5.0d, with the maximal decrease for ceftriaxone from 18.7d in the dark to 4.1d under the light exposure. Effects of dissolved organic matter (DOM) and nitrate on photodegradation of the cephalosporins were compound-specific. While DOM (5 mg L(-1)) stimulated the photodegradation of only cefradine (by 9%) and cefepime (by 34%), nitrate (10 microM) had effects only on cefepime (stimulation by 13%). Elimination rates of the cephalosporins in oxic sediment (half-lives of 0.8-3.1d) were higher than in anoxic sediment (half-lives of 1.1-4.1d), mainly attributed to biodegradation. The data indicate that abiotic hydrolysis (for cefradine, cefuroxime, and cefepime) and direct photolysis (for ceftriaxone) were the primary processes for elimination of the cephalosporins in the surface water of the lake, whereas biodegradation was responsible for the elimination of the cephalosporins in the sediment. Further studies are needed on chemical structure, toxicity, and persistence of transformation products of the cephalosporins in the environment.

Plasma pharmacokinetics and milk levels of ceftriaxone following single intravenous administration in healthy and endometritic cows.

Pharmacokinetics and milk levels of ceftriaxone were studied in healthy and endometritic cows following single intravenous administration. The drug was detected up to 8 h of dosing in plasma of healthy and endometritic cows and the drug disposition followed three-compartment open model. The values of Vd(area), AUC, t(1/2beta), Cl(B), MRT and P/C ratio were 0.50 +/- 0.19 L.kg(-1), 62.2 +/- 23.3 microg.ml(-1).h, 1.02 +/- 0.07 h, 0.30 +/- 0.09 L.kg(-1).h(-1), 1.55 +/- 0.25 h and 0.52 +/- 0.27, respectively, in healthy and 1.55 +/- 0.52 L.kg(-1), 37.0 +/- 17.1 microg.ml(-1).h, 1.56 +/- 0.25 h, 0.56 +/- 0.14 L.kg(-1).h(-1), 2.14 +/- 0.34 h and 1.44 +/- 0.60, respectively, in endometritic cows. The drug was detected in milk for 36 h after administration. For MIC(90) of 0.5 microg.ml(-1) the most appropriate dosage for ceftriaxone, would be 9.0 mg.kg(-1) repeated at 6 h intervals for the treatment of endometritis in cows.

A novel sensor for cephalosporins based on electrocatalytic oxidation by poly(o-anisidine)/SDS/Ni modified carbon paste electrode.

In this work for first time, the electrocatalytic oxidations of some cephalosporins were carried out by poly(o-anisidine)/SDS/Ni modified carbon paste electrode using cyclic voltammetry, chronoamperometry and chronocoulometry methods. At first, poly(o-anisidine) was formed by cyclic voltammetry in monomer solution containing sodium dodesyl sulfate (SDS), on carbon paste electrode surface. Then, Ni(II) ions were incorporated to electrode by immersion of the polymeric modified electrode having amine group in 0.1molL(-1) Ni(II) ion solution. A good redox behavior was observed for the Ni(OH)(2)/NiOOH couple on the surface of this electrode. Cephalosporins were successfully oxidized on the surface of this nickel ions dispersed poly(o-anisidine) modified carbon paste electrode. The electrocatalytic oxidation peak currents of cephalosporins were linearly dependent on their concentration. Electrode was successfully applied to determine cephalosporins in pharmaceutical preparations.

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.