Cetyl-alcohol-reinforced hollow fiber solid/liquid-phase microextraction and HPLC-DAD analysis of ezetimibe and simvastatin in human plasma and urine.

A new cetyl-alcohol-reinforced hollow fiber solid/liquid-phase microextraction (CA-HF-SLPME) followed by high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed for simultaneous determination of ezetimibe and simvastatin in human plasma and urine samples. To prepare the CA-HF-SLPME device, the cetyl-alcohol was immobilized into the pores of a 2.5 cm hollow fiber micro-tube and the lumen of the micro-tube was filled with 1-octanol with the two ends sealed. Afterwards, the prepared device was introduced into 10 mL of the sample solution containing the analytes with agitation. Under optimized conditions, calibration curves plotted in spiked plasma and urine samples were linear in the ranges of 0.363-25/0.49-25 µg L-1 for ezetimibe/simvastatin and 0.193-25/0.312-25 µg L-1 for ezetimibe/simvastatin in plasma and urine samples, respectively. The limit of detection was 0.109/0.174 µg L-1 for ezetimibe/simvastatin in plasma and 0.058/0.093 µg L-1 for ezetimibe/simvastatin in urine. As a potential application, the proposed method was applied to determine the concentration of selected analytes in patient plasma and urine samples after medication and satisfactory results were achieved. In comparison with reference methods, the CA-HF-SLPME-HPLC-DAD method demonstrates considerable potential in the biopharmaceutical analysis of selected drugs.

Matrix Effect Compensation in Small-Molecule Profiling for an LC-TOF Platform Using Multicomponent Postcolumn Infusion.

The possible presence of matrix effect is one of the main concerns in liquid chromatography-mass spectrometry (LC-MS)-driven bioanalysis due to its impact on the reliability of the obtained quantitative results. Here we propose an approach to correct for the matrix effect in LC-MS with electrospray ionization using postcolumn infusion of eight internal standards (PCI-IS). We applied this approach to a generic ultraperformance liquid chromatography-time-of-flight (UHPLC-TOF) platform developed for small-molecule profiling with a main focus on drugs. Different urine samples were spiked with 19 drugs with different physicochemical properties and analyzed in order to study matrix effect (in absolute and relative terms). Furthermore, calibration curves for each analyte were constructed and quality control samples at different concentration levels were analyzed to check the applicability of this approach in quantitative analysis. The matrix effect profiles of the PCI-ISs were different: this confirms that the matrix effect is compound-dependent, and therefore the most suitable PCI-IS has to be chosen for each analyte. Chromatograms were reconstructed using analyte and PCI-IS responses, which were used to develop an optimized method which compensates for variation in ionization efficiency. The approach presented here improved the results in terms of matrix effect dramatically. Furthermore, calibration curves of higher quality are obtained, dynamic range is enhanced, and accuracy and precision of QC samples is increased. The use of PCI-ISs is a very promising step toward an analytical platform free of matrix effect, which can make LC-MS analysis even more successful, adding a higher reliability in quantification to its intrinsic high sensitivity and selectivity.

A dispersive liquid-liquid microextraction method based on the solidification of a floating organic drop combined with HPLC for the determination of lovastatin and simvastatin in rat urine.

A dispersive liquid-liquid microextraction method based on solidification of floating organic drop combined with HPLC was developed for the determination of lovastatin and simvastatin in rat urine for the first time. 1-Dodecanol and methanol were used as the extraction and disperser solvents, respectively. Several important parameters influencing the micro-extraction efficiency were studied and systematically optimized, including the type and volume of extraction solvent and disperser solvent, extraction time, pH and salt concentration. The analytes were separated on a Kromasil C18 column at 30°C with a mobile phase of methanol and 0.2% acetic acid in water (83:17, v/v) and detected at 238 nm. Under the optimal conditions, the maximum number of enrichment factors for both analytes was 27. The linear ranges were 20.08-1004 and 20.00-1000 µg/L with the correlation coefficients ranging from 0.9990 to 0.9994 for lovastatin and simvastatin, respectively. The volume of organic solvent consumed in extraction was <0.3 mL, and the extraction time was 10 min. The newly developed environment-friendly sample pretreatment method will be a good alternative to conventional techniques, such as solid-phase extraction, liquid-liquid extraction and protein precipitation, for the HPLC determination of lovastatin and simvastatin in biological samples.

Determination of simvastatin-derived HMG-CoA reductase inhibitors in biomatrices using an automated enzyme inhibition assay with radioactivity detection.

A robust, automated enzyme inhibition assay method was developed and validated for the determination of HMG-CoA reductase inhibitory activities in plasma and urine samples following simvastatin (SV) administration. The assay was performed on Tecan Genesis 150 and 200 systems equipped with 8-probe and 96-well plates. Plasma samples containing HMG-CoA reductase inhibitors were treated with acetonitrile for protein precipitation before being incubated with HMG-CoA reductase, [14C]-HMG-CoA, and NADPH for a fixed length of time at a fixed temperature. The product, [14C]-mevalonic acid, was lactonized and separated from excess substrate via a small ion exchange resin column, and radioactivity was counted on a scintillation counter. HMG-CoA reductase inhibitors were measured before and after base hydrolysis. The two values obtained for each sample are referred to as 'active' and 'total' HMG-CoA reductase inhibitor concentrations. Simvastatin acid (SVA), the beta-hydroxy acid of SV, was used as a standard to generate a calibration curve of HMG-CoA reductase activity versus SVA concentration (ng/ml). Three calibration ranges, 0.4-20, 2-50, and 50, 100 ng/ml, in human and animal plasma and urine were validated. The assay precision was less than 8.5%, CV in plasma and less than 10.4% in urine. The assay accuracy was 93.6-103.0 and 98.1-103.9% for the 0.4 20 and 2-50 ng/ml calibration ranges, respectively, in human plasma, and was 97.3-105.1, 94.4- 105.2, and 90.2-95.7%, for calibration range 5-100 ng/ml in rat plasma, dog plasma and human urine, respectively.

Effect of the traditional Chinese medicine tongxinluo on endothelial dysfunction rats studied by using urinary metabonomics based on liquid chromatography-mass spectrometry.

A urinary metabonomic method based on ultra-fast liquid chromatography coupled with ion trap-time of flight mass spectrometry (UFLC/MS-IT-TOF) was employed to study the preventive efficacy and the metabolic changes caused by simavastatin and the traditional Chinese medicine tongxinluo in endothelial dysfunction rats. Principal component analysis (PCA) was applied to study metabolic patterns of endothelial dysfunction rats and healthy control rats. 1-Methyladenosine, indoxyl sulfate, hippuric acid, riboflavin, coproporphyrin, and p-cresol glucuronide were identified as potential biomarkers, indicating that pathways of adenine, tryptophan, phenylalanine, riboflavin and porphyrin metabolism were disturbed in endothelial dysfunction rats. Applications of simvastatin and tongxinluo to endothelial dysfunction rats improved endothelial function according to the results of histopathology and measurements of endothelin-1 and nitric oxide. Metabonomic studies suggested that tongxinluo prevents endothelial dysfunction by regulating multiple metabolic pathways to their normal state, whereas simvastatin only altered selected metabolic pathways. This research demonstrated that metabonomics is a powerful and promising tool for disease investigation and the efficacy evaluation of complex traditional Chinese medicines.

Glucuronidation of statins in animals and humans: a novel mechanism of statin lactonization.

The active forms of all marketed hydroxymethylglutaryl (HMG)-CoA reductase inhibitors share a common dihydroxy heptanoic or heptenoic acid side chain. In this study, we present evidence for the formation of acyl glucuronide conjugates of the hydroxy acid forms of simvastatin (SVA), atorvastatin (AVA), and cerivastatin (CVA) in rat, dog, and human liver preparations in vitro and for the excretion of the acyl glucuronide of SVA in dog bile and urine. Upon incubation of each statin (SVA, CVA or AVA) with liver microsomal preparations supplemented with UDP-glucuronic acid, two major products were detected. Based on analysis by high-pressure liquid chromatography, UV spectroscopy, and/or liquid chromatography (LC)-mass spectrometry analysis, these metabolites were identified as a glucuronide conjugate of the hydroxy acid form of the statin and the corresponding delta-lactone. By means of an LC-NMR technique, the glucuronide structure was established to be a 1-O-acyl-beta-D-glucuronide conjugate of the statin acid. The formation of statin glucuronide and statin lactone in human liver microsomes exhibited modest intersubject variability (3- to 6-fold; n = 10). Studies with expressed UDP glucuronosyltransferases (UGTs) revealed that both UGT1A1 and UGT1A3 were capable of forming the glucuronide conjugates and the corresponding lactones for all three statins. Kinetic studies of statin glucuronidation and lactonization in liver microsomes revealed marked species differences in intrinsic clearance (CL(int)) values for SVA (but not for AVA or CVA), with the highest CL(int) observed in dogs, followed by rats and humans. Of the statins studied, SVA underwent glucuronidation and lactonization in human liver microsomes, with the lowest CL(int) (0.4 microl/min/mg of protein for SVA versus approximately 3 microl/min/mg of protein for AVA and CVA). Consistent with the present in vitro findings, substantial levels of the glucuronide conjugate (approximately 20% of dose) and the lactone form of SVA [simvastatin (SV); approximately 10% of dose] were detected in bile following i.v. administration of [(14)C]SVA to dogs. The acyl glucuronide conjugate of SVA, upon isolation from an in vitro incubation, underwent spontaneous cyclization to SV. Since the rate of this lactonization was high under conditions of physiological pH, the present results suggest that the statin lactones detected previously in bile and/or plasma following administration of SVA to animals or of AVA or CVA to animals and humans, might originate, at least in part, from the corresponding acyl glucuronide conjugates. Thus, acyl glucuronide formation, which seems to be a common metabolic pathway for the hydroxy acid forms of statins, may play an important, albeit previously unrecognized, role in the conversion of active HMG-CoA reductase inhibitors to their latent delta-lactone forms.