Metabolomics profiling for diagnosis of acute renal failure after cardiopulmonary bypass.

RATIONALE: Acute renal failure (ARF) is one of the most serious complications of cardiopulmonary bypass (CPB) surgery. Serum creatinine level is a key compound examined to understand whether renal function is normal. However, its level may vary based on age, gender, race, muscle mass, nutrition, and drugs taken by an individual. In addition, it may not be detected without a 50% reduction in renal function and may lead to delays in treatment. New markers are needed for early diagnosis of ARF. They were determined for early diagnosis of ARF after CPB. Metabolic differences in plasma samples of individuals who developed and did not develop ARF after cardiopulmonary bypass were determined. METHODS: This study was the first to perform an untargeted metabolomics analysis for early diagnosis of ARF after CPB surgery. Plasma samples were taken from 105 patients (9 ARF patients) at five time points to identify the time at which a more accurate ARF diagnosis can be made. A total of 687 samples, including quality control samples, were analyzed. RESULTS: Two hundred twenty-six metabolites were identified using retention index libraries. Based on the statistical evaluations, tryptophan, threonine, and methionine were found in lower concentrations in patients with ARF compared to the control group at all time points. Whereas gluconic acid, hypoxanthine, and lactic acid showed a decreasing trend over time, longitudinal analysis showed that cysteine, hippuric acid, and uric acid levels increased over time in the ARF group. CONCLUSIONS: These metabolites are candidate biomarkers for early diagnosis of ARF as well as biomarkers for tracking the recovery of ARF patients.

Development of molecular imprinted nanosensor for determination of tobramycin in pharmaceuticals and foods.

In this study, we developed quartz crystal microbalance (QCM) nanosensor for the real-time detection of tobramycin (TOB). Firstly, the modification of gold surface of QCM chip was performed by self-assembling monolayer formation of allyl mercaptane to introduce polymerizable double bonds on the chip surface. Then, TOB imprinted poly(2-hydroxyethyl methacrylate-methacryloylamidoglutamic acid) [p(HEMA-MAGA)] film was generated on the gold surface. The nonmodified and TOB-imprinted p(HEMA-MAGA) surfaces were characterized by using atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, ellipsometry and contact angle measurements. The proposed method was validated according to the ICH guideline. The linearity range and the detection limit (S/N=3) were obtained as 1.7×10(-11)-1.5×10(-10) M and 5.7×10(-12) M, respectively. The developed method was applied to pharmaceuticals, and food samples such as chicken egg white and milk extract for the determination of TOB. In addition, association kinetics analysis and isotherm models were applied to the data to explain the adsorption process that took place.

Voltammetric determination of candesartan cilexetil in its Cu(II) complex and application to pharmaceutical formulations.

The aim of this study was to develop a new, simple, rapid, and sensitive square-wave adsorptive stripping voltammetric method for the determination of candesartan cilexetil in bulk form and pharmaceutical formulations by complex formation with Cu(II). The experimental and instrumental parameters affecting the response of the candesartan cilexetil-Cu(II) complex were investigated and optimized. This method was based on electrochemical reduction of candesartan cilexetil-Cu(II) complex at a hanging mercury drop electrode in phosphate buffer at pH 3.0 containing CuCl2 x 2H2O. A well-defined reduction peak was observed at -325 mV with 30 s accumulation time and -150 mV accumulation potential versus an Ag/AgCl reference electrode in the proposed method. The developed voltammetric method was validated in terms of linearity, sensitivity, precision, accuracy, recovery, selectivity, robustness, and ruggedness. The linear calibration range was 0.50-1.77 microg/mL (r = 0.9972). The LOD and LOQ of this method were 0.10 and 0.50 microg/mL (n = 7), respectively. The developed method was fully validated and applied to the pharmaceutical formulations, including candesartan cilexetil.

Simultaneous determination of ezetimibe and simvastatin in pharmaceutical preparations by MEKC.

A micellar electrokinetic capillary chromatography method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical preparations. The influence of buffer concentration, buffer pH, sodium dodecyl sulphate (SDS) concentration, organic modifier, capillary temperature, applied voltage, and injection time was investigated, and the method validation studies were performed. The optimum separation for these analytes was achieved in less than 10 min at 30 degrees C with a fused-silica capillary column (56 cm x 50 microm i.d.) and a 25mM borate buffer at pH 9.0 containing 25mM SDS and 10% (v/v) acetonitrile. The samples were injected hydrodynamically for 3 s at 50 mbar, and the applied voltage was +30.0 kV. Detection wavelength was set at 238 nm. Diflunisal was used as internal standard. The method was suitably validated with respect to stability, specificity, linearity, limits of detection and quantification, accuracy, precision, and robustness. The limits of detection and quantification were 1.0 and 2.0 microg/mL for both ezetimibe and simvastatin, respectively. The method developed was successfully applied to the simultaneous determination of ezetimibe and simvastatin in pharmaceutical preparations.

Quantitative analysis of ezetimibe in human plasma by gas chromatography-mass spectrometry.

A new, specific and sensitive GC-MS method with electron impact ionization technique was developed for quantitative analysis of ezetimibe (EZE) in human plasma. Prior to GC analysis, EZE was derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), which is a trimethyl silylating reagent. The derivatization reaction was optimized and parameters such as catalyst, derivatization time, temperature, solvent and the volume of silylating reagent were investigated. Trimethylsilyl ether derivative of EZE was determined in selected ion monitoring (SIM, mass-to-charge ratio (m/z): 326) mode. The method was validated with respect to LOD and LOQ, precision, accuracy, linearity, specificity, stability, and recovery. The LOQ and LOD were found as 15 and 10 ng/mL, respectively. The linearity of the method ranged from 15 to 250 ng/mL. The correlation coefficient of the calibration curve was 0.9977 +/- 0.0004 (+/- S.E.M.). The intra- and inter-day precisions (RSD) were less than 6% and accuracies (bias) for intra- and inter-day accuracy were found between -4.04 and 9.71% at four different concentration levels (15, 40, 100, 250 ng/mL). The proposed method was successfully applied to real human plasma samples for determination of total EZE.

Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography.

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20mM pH 9.0 borate buffer containing 30mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DIF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50microm internal diameter, 44cm total length, 35.5cm effective length) at 25 degrees C with the application of 3s of hydrodynamic injection at 50mbar pressure and a potential of 29kV. Detection wavelength was 200nm. Under these conditions, the migration times were found to be 5.174min for PAR, 5.513min for CAF, 7.195min for DIF, and 9.366min for PRO. Linearity ranges for the method were determined as 2-200microgmL(-1) for PAR and CAF and 3-200microgmL(-1) for PRO. Limit of detections were found as 0.6microgmL(-1) for PAR and CAF and 0.8microgmL(-1) for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.

Simultaneous determination of rosiglitazone and metformin in plasma by gradient liquid chromatography with UV detection.

A novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in human plasma. The analysis was performed on a phenyl column (250mmx4.6mm i.d., 5mum) using a gradient method starting with mobile phase composed of acetonitrile:5mM acetate buffer pH 5.5 (75:25, v/v). The flow rate was 1mLmin(-1). UV detection was performed at 245nm and verapamil was used as internal standard. The total run time was less than 10min. Sample preparation included a simple protein precipitation step with acetonitrile. Validation experiments were performed to demonstrate stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 100ngmL(-1) for rosiglitazone and 250ngmL(-1) for metformin. The extraction recoveries were 100.02-105.0% for rosiglitazone and 105.64-103.88% for metformin. The method was applied with success to plasma samples obtained from diabetic patients undergoing treatment with rosiglitazone and metformin.

Determination of magnesium, calcium, sodium, and potassium in blood plasma samples by capillary zone electrophoresis.

A capillary zone electrophoretic assay has been developed and validated for analysis of magnesium, calcium, sodium, and potassium in blood plasma samples. Optimum results were obtained with 20 mmol L(-1) imidazole (pH 2.8) and 0.5 mmol L(-1) oxalic acid containing 5% methanol, capillary temperature 25 degrees C, applied voltage 30 kV, hydrodynamic injection time 3 s, and a poly(vinyl alcohol)-coated capillary (i.d. 50 microm, total length 64.5 cm and effective length 56 cm). Indirect detection was performed at 214 nm. Cadmium was used as internal standard. The migration times of magnesium, calcium, sodium, and potassium were 4.25, 3.79, 3.96, and 2.79 min, respectively. The method was applied to the determination of magnesium, calcium, sodium, and potassium in blood plasma samples. The results were compared with those from atomic absorption spectrophotometry and no statistically significant difference was found (P > 0.05).

Determination of piribedil in pharmaceutical formulations by micellar electrokinetic capillary chromatography.

A fast and simple micellar electrokinetic capillary chromatographic method was developed for the analysis of piribedil in pharmaceutical formulations. The effects of buffer concentration, buffer pH, sodium dodecyl sulphate (SDS) concentration, organic modifier, applied voltage and injection time were investigated. Optimum results were obtained with a 50 mM borate buffer at pH 8.0 containing 50 mM SDS by using a fused silica capillary (50 microm internal diameter, 72 cm effective length). The sample was injected hydrodynamically for 4 s at 50 mbar pressure and the applied voltage was +30 kV. The detection wavelength was set at 205 nm. Diflunisal was used as an internal standard. The analysis was performed at 25 degrees C and the total run time was 14 min. The method was suitably validated with respect to linearity range, limit of detection and quantification, precision, accuracy, specificity and robustness. The linear calibration range was 5-100 microg mL(-1) and the limit of detection was determined as 1 microg mL(-1). The method developed was successfully applied to the determination of piribedil in pharmaceutical formulations. The results were compared with a spectrophotometric method reported in the literature and no significant difference was found statistically.

Determination of rofecoxib, in the presence of its photodegradation product, in pharmaceutical preparations by micellar electrokinetic capillary chromatography.

A simple and rapid micellar electrokinetic capillary chromatographic (MEKC) method for analysis of rofecoxib (ROF) and its photodegradation product (PDP) in pharmaceutical preparations has been developed and validated. Analyses were conducted in a fused silica capillary (72 cm effective length, 50 microm i.d.) with a background electrolyte consisting of 25 mmol L(-1) borate buffer at pH 7.0 containing 15 mmol L(-1) sodium dodecyl sulfate (SDS) and 10% acetonitrile (ACN). The separation was performed by voltage-controlled system, applying 30 kV at 30 degrees C, detecting at 225 nm; injection was hydrodynamic at 50 mbar for 2 s. Nifedipine was used as internal standard (IS). Under the optimum conditions ROF, PDP, and IS were well separated with in 10 min. The method was validated with regard to linearity, limit of detection and quantitation, precision, accuracy, specificity, and robustness. The detection limit of the method was low, 0.8 microg mL(-1), and the linearity range was wide, 2.5 to 125 microg mL(-1). The method was highly efficient-5x10(5) plates m(-1) for ROF. The method was applied to the tablet form of ROF-containing pharmaceutical preparations. The data were compared with those from the voltammetric method described in literature. No statistically significant difference was found.

Electrochemical studies and square-wave voltammetric determination of fenofibrate in pharmaceutical formulations.

The electrochemical reduction of fenofibrate at a hanging mercury drop electrode (HMDE) was investigated by cyclic voltammetry, square-wave voltammetry, and chronoamperometry. Different buffer solutions were used over a wide pH range (3.0-10.0). The best definition of the analytical signals was found in borate buffer (pH 9.0)-tetrabutylammonium iodide mixture containing 12.5% (v/v) methanol at -1.2 V (versus Ag/AgCl). According to cyclic voltammetric studies, the reduction was irreversible and diffusion controlled. The diffusion coefficient was 2.38x10(-6) cm2 s(-1) as determined by chronoamperometry. Under optimized conditions of square-wave voltammetry, a linear relationship was obtained between 0.146-4.96 microg mL(-1) of fenofibrate with a limit of detection of 0.025 microg mL(-1). Validation parameters such as sensitivity, accuracy, precision, and recovery were evaluated. The proposed method was applied to the determination of fenofibrate in pharmaceutical formulations. The results were compared with those obtained by a published high-performance liquid chromatography method. No difference was found statistically.

Determination of nifedipine in human plasma by square wave adsorptive stripping voltammetry.

A simple, sensitive and selective square-wave adsorptive stripping voltammetric method has been developed and validated for the determination of nifedipine (NIF) in plasma. The assay was performed after single extraction of NIF from alkalinised plasma into organic phase. The adsorption behaviour of NIF on a hanging mercury drop electrode (HMDE) was explored by square-wave and cyclic voltammetry. The drug was accumulated at HMDE and a well-defined peak was obtained at -730 mV versus Ag/AgCl in borate buffer of pH 9.0 including 0.01 M KCl. The linear concentration range was 2.89 x 10(-9) M-3.61 x 10(-7) M (1.00-125.01 ng ml(-1)) when using 30 s accumulation time at -300 mV. Limit of detection and limit of quantification were 1.21 x 10(-9) M (0.42 ng ml(-1)) and 2.89 x 10(-9) M (1.00 ng ml(-1)) respectively. The intra-day relative standard deviation (RSD) ranged from 1.93 to 4.12% at three concentrations and the inter-day RSDs varied from 2.53 to 6.68%. The method was applied, to the plasma of pregnant women suffering from pregnancy induced hypertension, for the determination of NIF. The percentage recoveries varied from 96.26 to 99.49%. It has been shown that NIF could be determined in the presence of its main metabolite (dehydronifedipine) by the developed method.