Helen Archontaki

Development and optimization of a reversed-phase high-performance liquid chromatographic method for the determination of acetaminophen and its major metabolites in rabbit plasma and urine after a toxic dose

A reversed-phase high-performance liquid chromatographic method with detection at 242 nm was developed, optimized and validated for the determination of acetaminophen (A) and its major metabolites glucuronide (AG) and sulfate (AS) conjugates in rabbit plasma and urine after a toxic dose. m-Aminophenol was used as internal standard (IS). A Hypersil BDS RP-C18 column (250 x 4.6 mm), 5 microm particle size, was equilibrated with a mobile phase composed of aqueous buffer solution of KH2PO4 0.05 M containing 1% CH3COOH (pH 6.5) and methanol (95:5, v/v). Its flow rate was 1.5 ml/min. Calibration curves of A, AG and AS were linear in the concentration ranges of 0.5-250, 1-200, 0.5-100 microg/ml in plasma and 1-200, 0.5-150, 0.5-100 microg/ml in urine matrix, respectively. Limits of detection and quantitation were calculated in all cases and extensive recovery studies were also performed. Intra-day relative standard deviation (R.S.D.) for A, AG and AS in plasma was less than 5, 4, 2% and in urine less than 4, 7, 4%, respectively, while the corresponding inter-day values were 7, 6, 4% and 5, 8, 6%, respectively.

Determination of intralumenal individual bile acids by HPLC with charged aerosol detection.

An isocratic HPLC charged aerosol detector (CAD) method was developed, validated, and applied for the determination of individual bile acids in human gastric and duodenal aspirates. The method requires a low volume of aspirates (50–100 μl) and minimal sample pretreatment. A Hypersil BDS RP-C18 column (250 × 4.6 mm, 5 μm particle size) was equilibrated with a mobile phase composed of methanol-[ammoniun formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 v/v. Its flow rate was 1 ml/min. The elution times for taurocholate, glycocholate, taurochenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate, cholate, and glycodeoxycholate were approximately 9.9, 16.2, 18.2, 21.3, 31.6, 34.5, and 38.5 min, respectively. Calibration curves in the mobile phase were constructed in the concentration range of 0.5–500 μM. Limits of detection and quantification were in the range of 0.07–0.60 μM and 0.20–1.80 μM, respectively. This method was applied first, in gastric aspirates collected in the fasted state, in which bile acid presence is minimal and, second, in duodenal aspirates collected in the fed state, in which a large number of potentially interfering compounds exists. Intra-day relative standard deviation in fasted gastric aspirates and in fed duodenal aspirates was less than 2.2% and 6.0%, respectively.

Acidic hydrolysis of bromazepam studied by high performance liquid chromatography. Isolation and identification of its degradation products

A kinetic study on the acidic hydrolysis of bromazepam was carried out in 0.01 M hydrochloric acid solution at 25 and 95 degrees C. A reversed-phase HPLC method was developed and validated for the determination of bromazepam and its degradation products. Bromazepam degraded by a consecutive reaction with a reversible first step. Two degradation products were isolated and identified by infrared, 1H and 13C nuclear magnetic resonance and mass spectroscopy. Spectroscopic data indicated that N-(4-bromo-2-(2-pyridylcarbonyl)phenyl)-2-aminoacetamide was the intermediate degradation product of this acid hydrolysis, whereas 2-amino-5-bromophenyl-2-pyridylmethanone was the final one. Therefore, the mechanism of this acid-catalysed hydrolysis involved initial cleavage of the 4,5-azomethine bond, followed by slow breakage of the 1,2-amide bond. Statistical evaluation of the HPLC method revealed its good linearity and reproducibility. Detection limits were 3.8 x 10(-7) M for bromazepam, 6.25 x 10(-7) M for the intermediate and 8.16 x 10(-7) M for the benzophenone derivative.

Investigation of the interactions of silibinin with 2-hydroxypropyl-β-cyclodextrin through biophysical techniques and computational methods.

Cyclodextrins (CDs) are a well-known class of supermolecules that have been widely used to protect drugs against conjugation and metabolic inactivation as well as to enhance the aqueous solubility and hence to ameliorate the oral bioavailability of sparingly soluble drug molecules. The hepatoprotectant drug silibinin can be incorporated into CDs, and here we elucidate the interaction between the drug and the host at the molecular level. The complexation product of silibinin with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) is characterized by Differential Scanning Calorimetry, mass spectrometry, solid and liquid high-resolution NMR spectroscopy. The chemical shift changes using (13)C CP/MAS on the complexing of the guest with the host provided significant information on the molecular interactions, and they were in agreement with the 2D NOESY results. These results point out that in both solid and liquid forms, the drug is engulfed and interacts with HP-β-CD in identical manner. Molecular dynamics calculations have been performed to examine the thermodynamic characteristics associated with the silibinin-HP-β-CD interactions and to study the stability of the complex. To approximate the physiological conditions, the aqueous solubility and dissolution characteristics of the complex at pH states simulating those of the upper gastrointestinal tract have been applied. To evaluate the antiproliferative activity of silibinin-HP-β-CD complex comparatively to silibinin in MCF-7 human cancer cells, MTT assays have been performed.

Effect of pH and water-soluble polymers on the aqueous solubility of nimesulide in the absence and presence of β-cyclodextrin derivatives

The aqueous solubility of nimesulide in the absence and presence of β‐cyclodextrin (β‐CD) and its alkyl derivatives hydroxypropyl‐β‐CD and methyl‐β‐CD was studied. We also investigated the effect of water‐soluble polymers, hydroxypropylmethyl‐cellulose, sodium‐carboxymethyl‐cellulose, polyvinyl‐ pyrrolidone and polyethyleneglycol on the solubilization efficacy and complexation ability of cyclodextrins with nimesulide. The solubility of nimesulide in the absence and presence of cyclodextrins and polymers was studied using a phase solubility technique combined with a spectrophotometric method. The study was carried out at 25°C and pH values of 6.0 and 7.0. Conditions in terms of polymer concentration and polymer heating with and without sonication were optimized. Values of the solubility enhancement factor of nimesulide in the presence of each cyclodextrin and in the absence and presence of each polymer were determined and the formation constants, K, of the inclusion complexes formed calculated. β‐CDs increased the aqueous solubility of nimesulide in the following order: methyl‐β‐CD > β‐CD > hydroxypropyl‐β‐CD. Addition of hydroxypropylmethyl‐cellulose at a concentration of 0.1% (w/v) had the greatest influence on complexation of all three β‐CDs with nimesulide, while preheating of the polymer at 70°C under sonication resulted in an additional two‐fold increase in the aqueous solubility of the drug. Sodium‐ carboxymethyl‐cellulose, polyvinylpyrrolidone and polyethyleneglycol had minor effects on the aqueous solubility of nimesulide. Thus β‐CD, hydroxypropyl‐β‐CD and methyl‐β‐CD are proposed as good solubilizing agents for nimesulide in the presence and absence of hydroxypropylmethyl‐ cellulose in order to enhance its oral bioavailability.

Kinetic study on the degradation of prazepam in acidic aqueous solutions by high-performance liquid chromatography and fourth-order derivative ultraviolet spectrophotometry

A reversed-phase HPLC method was developed for the kinetic investigation of the acidic hydrolysis of prazepam which was carried out in hydrochloric acid solutions of 0.01, 0.1 and 1.0 M. In addition, a fourth-order derivative method for monitoring the parent compound itself was proposed and evaluated. One intermediate was observed by HPLC, which should be formed from breakage of the azomethine linkage. Further slow hydrolysis of the amide bond led to the benzophenone product that was isolated and identified. The mechanism of hydrolysis was biphasic, showing a consecutive reaction with a reversible step. Relative standard deviation was less than 2% for HPLC and less than 5% for the derivative method. Detection limits were 1.2 x 10(-7) M for the former method and 6.7 x 10(-7)M for the latter. Accelerated studies at higher temperatures were employed. Results of HPLC and fourth-order derivative methods were statistically the same.

Serum and tissue pharmacokinetics of silibinin after per os and i.v. administration to mice as a HP-β-CD lyophilized product.

Silibinin, the main active component of Silybum marianum is a hepatoprotective and antioxidant agent with antitumor effect, exhibiting very low aqueous solubility and oral bioavailability limiting its use in therapeutics. We characterized serum and tissue pharmacokinetics of SLB, calculated its absolute bioavailability and developed an open loop physiologically based pharmacokinetic (PBPK) model, after oral (per os, p.o) and intravenous (i.v.) administration in mice as water-soluble silibinin-hydroxypropyl-beta-cyclodextrin (SLB-HP-β-CD) lyophilized product. 60 C57Bl/6J mice were divided into groups of 5, each group representing one sampling time point. SLB-HP-β-CD lyophilized product was administered orally (50mg/kg) and i.v. (20mg/kg) after reconstitution with water for injection. Blood and tissue samples were collected at selected time points after animal sacrificed, properly treated and analyzed with HPLC-PDA for non-metabolized and total SLB. NONMEM pharmacokinetic analysis revealed a 2-compartment PK model to describe serum SLB pharmacokinetics, with zero order absorption after oral administration and was applied as forcing function to an open loop PBPK model incorporating heart, liver, kidneys and lungs. Tissue/plasma Kp values were estimated using i.v. data and can be used to predict tissue SLB distribution after oral administration. Absolute oral bioavailability of SLB from the lyophilized SLB-HP-β-CD product was 10 times higher than after administration of pure SLB.

Development and optimization of a reversed-phase high-performance liquid chromatographic method for the determination of piperacillin and tazobactam in tazocin injectable powder.

A reversed phase high-performance liquid chromatographic method with detection at 220 nm was developed and validated for the determination of piperacillin, I, and tazobactam, II, in Tazocin injectable powder. Acetaminophen was used as internal standard. A Hypersil BDS RP-C(18) column (250 x 4.6 mm), 5 microm particle size, was equilibrated with a mobile phase composed of aqueous solution of sodium dihydrogenphosphate-dihydrate (20 mM)-acetonitrile-methanol (70:15:15, v/v/v) and pH 5.0. Its flow rate was 1.0 ml/min. Calibration curves were linear for I and II in the concentration ranges of 3.0 x 10(-7)-2.0 x 10(-4) M and 7.0 x 10(-7)-2.0 x 10(-4) M, respectively. Limits of detection and quantitation were 1 x 10(-7), 3 x 10(-7) M for I and 2 x 10(-7), 7 x 10(-7) M for II, respectively. Relative standard deviation, for I and II was less than 0.40 and 0.75%, respectively. Extensive recovery studies were also performed.

Kinetic study on the acidic hydrolysis of lorazepam by a zero-crossing first-order derivative UV-spectrophotometric technique.

A zero-crossing first-order derivative UV-spectrophotometric technique for monitoring the main degradation product, 6-chloro-4-(2-chlorophenyl)-2-quinazoline carboxaldehyde, was developed to study the acidic hydrolysis of lorazepam in hydrochloric acid solutions of 0.1 M. Due to the complete overlap of the spectral bands of the parent drug and the hydrolysis product (the range between their spectral maxima was only 3 nm), the graphical methods of derivative spectrophotometry were not efficient. The relative standard deviation of the proposed technique was less than 2.4% and the detection limit was 6.6x10(-8) M. Accelerated studies at higher temperatures have been employed that enable rapid prediction of the long-term stability of this drug. Pseudo-first order reaction kinetics was observed. Kinetic parameters, k(obs) and t(1/2), were calculated, which were similar to those estimated by an HPLC method developed in our laboratory.

Chromatographic behavior of zwitterionic enalapril—Exploring the conditions for lipophilicity assessment

The chromatographic behavior of enalapril was investigated under different stationary and mobile phase conditions in an effort to unravel interferences in the underlying retention mechanism, which would affect its relation to octanol-water partitioning. Extrapolated retention factors, logk(w), were used as relevant chromatographic indices. The retention/pH profile was established and the peak split phenomenon, associated with cis/trans interconversion, was also monitored as a function of pH. The pH at maximum retention and minimum peak split occurrence was chosen for further investigation, so that the presence of zwitterionic structure was guaranteed and any effect of cis/trans interconversion could be ignored. Retention of zwitterionic enalapril was found to be very sensitive to mobile phase conditions in regard to organic modifier as well to the aqueous component. The use of morpholine-propanesulfonic acid (MOPS) as buffer and the presence of n-octanol as mobile phase additive proved critical factors for maximum suppression of secondary interactions. Nevertheless, the corresponding extrapolated retention factor was considerably larger than octanol-water logD value at the isoelectric point. However, logk(w) could be successfully converted to logD by means of a calibration equation established for ionized acidic compounds.