Simultaneous determination of ten nonsteroidal anti-inflammatory drugs from drinking water, surface water and wastewater using micro UHPLC-MS/MS with on-line SPE system.

A simple, accurate and sensitive micro UHPLC-MS/MS method was developed and validated for the simultaneous determination of 10 nonsteroidal anti-inflammatory drugs (NSAIDs) from different environmental matrices. The micro LC ‒ on-line SPE method described in this study allowed to determine the selected drugs at ultra-trace levels without the most commonly used complex off-line SPE sample preparation procedures. The presented method is capable of reaching satisfactory low LOQ values with analysing the sample directly after being diluted with water. In order to attain high sensitivity, mass spectrometry was carefully optimized for the analysis of the drugs. Fenoprofen, flurbiprofen and naproxen were found to produce CO2 loss during ionization, forming intense [M-H-CO2]- ions instead of [M-H]-. All the other compounds were analyzed through their [M+H]+ and [M-H]- ions. Effect of mobile phase pH on ionization was also studied. Lower pH resulted in higher ion intensities. For this reason, a reversed phase chromatographic separation was applied at pH 3.1 with formic acid at concentration of 0.01%. Matrix effects have been evaluated during validation and sample dilution was optimized focusing on the lowest achievable LOQ values. Analytes were determined from drinking water directly, from surface water and wastewater following dilution with purified water by 2 : 8 (v/v) and 1 : 9 (v/v), respectively. Finally, the method was applied to real sample analysis.

Baicalin is a substrate of OATP2B1 and OATP1B3.

The use and significance of baicalin, the main bioactive component found in Radix Scutellaria, have been on the rise due to its interesting pharmacological properties. Baicalin, a low passive permeability compound, is directly absorbed from the upper intestine and its hepatic elimination is dominant. However, interaction but no transport studies have implicated organic anion­transporting polypeptides in its cellular uptake. By using mammalian cells stably expressing the uptake transporters of interest, we are showing that baicalin is a potent substrate of Organic anion­transporting polypeptide 2B1 (OATP2B1) and less potent substrate of OATP1B3. OATP2B1 and OATP1B3 transport baicalin and may play a role in the hepatic uptake of baicalin formed in the intestine.

Simultaneous determination of thirteen different steroid hormones using micro UHPLC-MS/MS with on-line SPE system.

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC-MS/MS system allowed to decrease LOQ without any complex sample preparation protocol. The presented method is capable of reaching satisfactory low LOQ values for analysis of thirteen different steroid molecules from human plasma without the most commonly used off-line SPE or compound derivatization. Steroids were determined by using two simple sample preparation methods, based on lower and higher plasma steroid concentrations. In the first method, higher analyte concentrations were directly determined after protein precipitation with methanol. The organic phase obtained from the precipitation was diluted with water and directly injected into the LC-MS system. In the second method, low steroid levels were determined by concentrating the organic phase after steroid extraction. In this case, analytes were extracted with ethyl acetate and reconstituted in 90/10 water/acetonitrile following evaporation to dryness. This step provided much lower LOQs, outperforming previously published values. The method has been validated and subsequently applied to clinical laboratory measurement.

Charged derivatization and on-line solid phase extraction to measure extremely low cortisol and cortisone levels in human saliva with liquid chromatography-tandem mass spectrometry.

The aim of this study was to develop a sensitive, reliable and high-throughput liquid chromatography - electrospray ionization - mass spectrometric (LC-ESI-MS/MS) method for the simultaneous quantitation of cortisol and cortisone in human saliva. Derivatization with 2-hydrazino-1-methylpyridine (HMP) was one of the most challenging aspects of the method development. The reagent was reacting with cortisol and cortisone at 60°C within 1h, giving mono- and bis-hydrazone derivatives. Investigation of derivatization reaction and sample preparation was detailed and discussed. Improvement of method sensitivity was achieved with charged derivatization and use of on-line solid phase extraction (on-line SPE). The lower limit of quantitation (LLOQ) was 5 and 10pg/ml for cortisol and cortisone, respectively. The developed method was subsequently applied to clinical laboratory measurement of cortisol and cortisone in human saliva.

Pushing quantitation limits in micro UHPLC-MS/MS analysis of steroid hormones by sample dilution using high volume injection.

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume. However, if analyte concentration is extremely low, it might be necessary to inject high sample volumes. This is particularly critical for strong sample solvents and weakly retained analytes, which are often the case when preparing biological samples (protein precipitation, sample extraction, etc.). In that case, high injection volumes may cause band broadening, peak distortion or even elution in dead volume. In this study, we evaluated possibilities of high volume injection onto microbore RP-LC columns, when sample solvent is diluted. The presented micro RP-LC-MS/MS method was optimized for the analysis of steroid hormones from human plasma after protein precipitation with organic solvents. A proper sample dilution procedure helps to increase the injection volume without compromising peak shapes. Finally, due to increased injection volume, the limit of quantitation can be decreased by a factor of 2-5, depending on the analytes and the experimental conditions.

Calcineurin-inhibition Results in Upregulation of Local Renin and Subsequent Vascular Endothelial Growth Factor Production in Renal Collecting Ducts.

BACKGROUND: Tacrolimus (Tac) and Cyclosporine A (CyA) calcineurin inhibitors (CNIs) are 2 effective immunosuppressants which are essential to prevent allograft rejection. Calcineurin inhibitors are known to be nephrotoxic. However, the precise mechanism of nephrotoxicity is not fully understood. In this study, we investigated the in vivo effects of CNIs on the local renal renin-angiotensin system in the collecting duct (CD). METHODS: Three-week-old mice were treated with either vehicle, CyA (2 mg/kg per day), Tac (0.075 mg/kg per day), CyA + Aliskiren (25 mg/kg per day), or Tac + Aliskiren for 3 weeks. Serum creatinine was measured. Renin and vascular endothelial growth factor (VEGF) contents in CD were evaluated with flow cytometry and multiphoton microscopy. The diameter of vessels was assessed with multiphoton microscopy, and the amount of renal collagen was determined by real-time polymerase chain reaction and Masson staining. RESULTS: The elevated level of serum creatinine in CNI groups was abolished by Aliskiren. Flow cytometric analysis found elevated renin content in principal cells, which was prevented by Aliskiren. This result was further confirmed with multiphoton microscopy. The VEGF content in CD correlated with reduced capillary diameter and with the formation of fibrotic islands. CONCLUSIONS: Calcineurin inhibitors induce production of renin in the CD that may contribute to decreased renal blood flow. In turn, CD responds with increased VEGF production, resulting in disproportional vessel growth, further worsening the local hypoxia and striped fibrosis surrounding the CDs. Aliskiren, a direct renin inhibitor blocks these effects and improves CNI-induced nephropathy by decreasing renin production in the CDs. Our data suggest that Aliskiren may be used for the prevention of CNI nephrotoxicity.

Multiple ABC Transporters Efflux Baicalin.

Baicalein, the aglycone formed by hydrolysis of baicalin in the intestine, is well absorbed by passive diffusion but subjected to extensive intestinal glucuronidation. Efflux of baicalin, the low passive permeability glucuronide of baicalein from enterocytes, likely depends on a carrier-mediated transport. The present study was designed to explore potential drug-herb interaction by investigating the inhibitory effect of baicalin on the transport of reporter substrates by transporters and to identify the transporters responsible for the efflux of baicalin from enterocytes and hepatocytes. The interaction of baicalin with specific ABC transporters was studied using membranes from cells overexpressing human BCRP, MDR1, MRP2, MRP3 and MRP4. Baicalin was tested for its potential to inhibit vesicular transport by these transporters. The transport of baicalin by the selected transporters was also investigated. Transport by BCRP, MRP3 and MRP4 was inhibited by baicalin with an IC50 of 3.41 ± 1.83 µM, 14.01 ± 2.51 µM and 14.39 ± 5.69 µM respectively. Inhibition of MDR1 (IC50 = 94.84 ± 31.10 µM) and MRP2 (IC50 = 210.13 ± 110.49 µM) was less potent. MRP2 and BCRP are the apical transporters of baicalin that may mediate luminal efflux in enterocytes and biliary efflux in hepatocytes. The basolateral efflux of baicalin is likely mediated by MRP3 and MRP4 both in enterocytes and hepatocytes. Via inhibition of transport by ABC transporters, baicalin could interfere with the absorption and disposition of drugs.

Unexpected retention behavior of baicalin: Hydrophilic interaction like properties of a reversed-phase column.

The original aim of this study was to develop a method for the determination of baicalin from membrane vesicles. The unconventional chromatographic separation ("inverse gradient elution" on a reversed phase column) was due to a lucky chance, which is detailed and discussed in this study. The validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is proved to be sensitive, rapid and selective. Chromatographic separation was performed on a Zorbax SB-C8 column (250 mm × 4.6 mm, i.d.; 5 µm) with 0.1% formic acid in water and methanol by linear gradient elution. Quantification of baicalin was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The calibration curve was linear (r = 0.9987) over the concentration range from 1 to 1000 nM. The coefficient of variation and relative error of baicalin for intra- and inter-assay at three quality control (QC) levels were 2.0-10.2% and -6.1 to 6.7%, respectively. The lower limit of quantification (LLOQ) for baicalin was 1 nM (0.446 ng/ml), without preconcentration of the sample. This method was subsequently applied to vesicular transport assays of baicalin in membrane vesicles successfully. The developed method can open up new area of research in the chromatographic separation of flavonoids and their glucuronides.

A Modified Procedure for Estimating the Impact of the Uptake on the Overall Biliary Clearance in Sandwich Culture of Rat Hepatocytes.

Sandwich culture of hepatocytes is commonly applied for the prediction of in vivo biliary clearance (CLbil). In this paper, we present a modified procedure for the determination of in vitro CLbil in sandwich culture of rat hepatocytes, which allows the estimation of the impact of uptake processes on the overall CLbil. The main point of this modification is the separation of uptake and efflux processes. Ten drugs from four biopharmaceutics drug disposition classification system classes were chosen in order to demonstrate the advantages of this method: 1) the uptake is performed identically before the canaliculi are opened, thus the efflux starts at the same intracellular concentration of the drugs and the effect of Ca2+/Mg2+ depletion on the uptake is excluded; 2) exact intracellular concentrations can be measured at the start and at the end of the efflux; 3) the biliary clearance can be determined irrespective of the uptake; 4) the canalicular and the sinusoidal transport can be measured simultaneously; 5) drug-drug interactions concerning uptake and efflux transporters can be estimated independently. Depending on the degree of uptake, CLbil,app (calculated using the concentration of drugs in the medium) was significantly higher (sulfasalazine, fluvastatin, rosuvastatin, atorvastatin) or lower (pravastatin, procainamide) than CLbil,int (calculated using the intracellular concentration of drugs). When the uptake had no impact on the CLbil, the apparent and intrinsic CLbil did not differ significantly (lovastatin, rifampicin, quetiapine). Our results confirm that transporters may play a significant role in the uptake of drugs both with high and poor permeability and solubility.