Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography-tandem mass spectrometry.

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography-tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1'-carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H]+ signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M-CO2 +H]+ and [M-ΙmCO2 +H]+ as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti-Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples.

Serum and tissue pharmacokinetics of silibinin after per os and i.v. administration to mice as a HP-ß-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-ß-CD) lyophilized product. 60 C57Bl/6J mice were divided into groups of 5, each group representing one sampling time point. SLB-HP-ß-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-ß-CD product was 10 times higher than after administration of pure SLB.

Investigation of the interactions of silibinin with 2-hydroxypropyl-ß-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-ß-cyclodextrin (HP-ß-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-ß-CD in identical manner. Molecular dynamics calculations have been performed to examine the thermodynamic characteristics associated with the silibinin-HP-ß-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-ß-CD complex comparatively to silibinin in MCF-7 human cancer cells, MTT assays have been performed.

Gas chromatographic-mass spectrometric quantitation of busulfan in human plasma for therapeutic drug monitoring: a new on-line derivatization procedure for the conversion of busulfan to 1,4-diiodobutane.

A simplified gas chromatographic-mass spectrometric (GC-MS) analytical method, involving a novel derivatization procedure was developed for monitoring busulfan (Bu) plasma concentrations in populations undergoing bone marrow transplantation. Plasma samples (500 µL) containing Bu-d8 as internal standard were extracted with ethyl acetate (2 mL) followed by centrifugation (1800 rpm, 5 min) and evaporation of the organic layer under nitrogen flow (50 °C). The dry residue was reconstituted with 100 µL iodine solution in acetonitrile (0.25%, w/v) and 3 µL were injected into the GC-MS system at 250 °C. Conversion of Bu to 1,4-diiodobutane was accomplished on-line without the need of an extra derivatization step. MS was operated at selected ion monitoring mode at m/z 183 and 191 corresponding to Bu and Bu-d8 derivatives. Total analysis time was 11.5 min. Calibration curves were linear (mean r=0.9996) over a concentration range of 25-3651 ng/mL using a (1/x)-weighted scheme. Limit of detection and lower limit of quantitation were 10.6 and 25 ng/mL, respectively. Overall accuracy Er (%) was ranging from -5.10% to 10.5%. Within- and between-run RSD (%) were lower 4.51% and 2.15%, respectively. Overall recovery of Bu was equal to 69.3±4.56% (RSD (%)). The present method is sensitive and specific, requiring a simple sample preparation procedure and short analysis time, advantages crucial for therapeutic drug monitoring of Bu in clinical practice and application in pharmacokinetic studies.

Development and validation of a liquid chromatographic method for the simultaneous determination of aniracetam and its related substances in the bulk drug and a tablet formulation.

Simultaneous determination of aniracetam and its related impurities (2-pyrrolidinone, p-anisic acid, 4-p-anisamidobutyric acid and (p-anisoyl)-4-methyl-2-pyrrolidinone) was accomplished in the bulk drug and in a tablet formulation using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS-CN column (150 mm × 4.0 mm, 5 µm) using a gradient elution program with solvent A composed of phosphate buffer (pH 4.0; 0.010 M) and solvent B of acetonitrile-phosphate buffer (pH 4.0; 0.010 M) (90:10, v/v). The flow rate of the mobile phase was 1.0 mL min(-1) and the total elution time, including the column re-equilibration, was approximately 20 min. The UV detection wavelength was varied appropriately among 210, 250 and 280 nm. Injection volume was 20 µL and experiments were conducted at ambient temperature. The developed method was validated in terms of system suitability, selectivity, linearity, range, precision, accuracy, limits of detection and quantification for the impurities, short term and long term stability of the analytes in the prepared solutions and robustness, following the ICH guidelines. Therefore, the proposed method was suitable for the simultaneous determination of aniracetam and its studied related impurities.

Robust and sensitive high-performance liquid chromatographic-UV detection technique for the determination of tigecycline in rabbit plasma.

An isocratic HPLC method with detection at 248 nm was developed and fully validated for the determination of tigecycline in rabbit plasma. Minocycline was used as an internal standard. A Hypersil BDS RP-C18 column (250 x 4.6 mm, 5 microm particle size) was used with the mobile phase phosphate buffer (pH 7.10, 0.070 M)-acetonitrile (76 + 24, v/v) at a flow rate of 1.0 mL/min. The elution time of tigecycline and minocycline was approximately 8.1 and 9.9 min, respectively. Calibration curves of tigecycline were linear in the concentration range of 0.021-3.15 microg/mL in plasma. The LOD and LOQ in plasma were estimated as 7 and 21 ng/mL, respectively. The intraday and interday precision values of the method were in the range of 5.0-7.1 and 5.6-9.1%, while the corresponding accuracy values were in the ranges of 92.8-111.1 and 97.6-102.3%, respectively. At the LOQ, the intraday precision was 18.7%, while intraday and interday accuracy values were 97.3 and 98.0%, respectively. Robustness of the proposed method was studied using a Plackett-Burman experimental design. A pharmacokinetic profile is presented for confirmation of the applicability of the method to pharmacokinetic studies.

Effect of cyclodextrin complexation on the aqueous solubility and solubility/dose ratio of praziquantel.

Praziquantel (PZQ), the primary drug of choice in the treatment of schistosomiasis, is a highly lipophilic drug that possesses high permeability and low aqueous solubility and is, therefore, classified as a Class II drug according to the Biopharmaceutics Classification System (BCS). In this work, beta-cyclodextrin (beta-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) were used in order to determine whether increasing the aqueous solubility of a drug by complexation with CDs, a BCS-Class II compound like PZQ could behave as BCS-Class I (highly soluble/highly permeable) drug. Phase solubility and the kneading and lyophilization techniques were used for inclusion complex preparation; solubility was determined by UV spectroscopy. The ability of the water soluble polymer polyvinylpyrolidone (PVP) to increase the complexation and solubilization efficiency of beta-CD and HP-beta-CD for PZQ was examined. Results showed significant improvement of PZQ solubility in the presence of both cyclodextrins but no additional effect in the presence of PVP. The solubility/dose ratios values of PZQ-cyclodextrin complexes calculated considering the low (150 mg) and the high dose (600 mg) of PZQ, used in practice, indicate that PZQ complexation with CDs may result in drug dosage forms that would behave as a BCS-Class I depending on the administered dose.

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

The aqueous solubility of nimesulide in the absence and presence of beta-cyclodextrin (beta-CD) and its alkyl derivatives hydroxypropyl-beta-CD and methyl-beta-CD was studied. We also investigated the effect of water-soluble polymers, hydroxypropylmethyl-cellulose, sodium-carboxymethyl-cellulose, polyvinylpyrrolidone 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 degrees 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. beta-CDs increased the aqueous solubility of nimesulide in the following order: methyl-beta-CD > beta-CD > hydroxypropyl-beta-CD. Addition of hydroxypropylmethyl-cellulose at a concentration of 0.1% (w/v) had the greatest influence on complexation of all three beta-CDs with nimesulide, while preheating of the polymer at 70 degrees 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 beta-CD, hydroxypropyl-beta-CD and methyl-beta-CD are proposed as good solubilizing agents for nimesulide in the presence and absence of hydroxypropylmethyl-cellulose in order to enhance its oral bioavailability.

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.

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 microl) and minimal sample pretreatment. A Hypersil BDS RP-C(18) column (250 x 4.6 mm, 5 microm 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 microM. Limits of detection and quantification were in the range of 0.07-0.60 microM and 0.20-1.80 microM, 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.

Estimation of chromatographic parameters on two silica-based columns connected in series under nonaqueous reversed phase liquid chromatographic conditions.

Empirical equations were produced to relate important chromatographic parameters on two silica-based columns serially linked, in isocratic nonaqueous RP HPLC, to retention times and peak widths of the separated compounds on the individual columns. These equations were derived because the experimental data seemed to deviate from the values expected, applying basic chromatographic theoretical equations. The chromatographic parameters studied were retention time, peak width, resolution, number of theoretical plates, capacity factor, and separation factor. In addition, empirical linear relationships were produced for the estimation of the above mentioned parameters of the serial systems, in direct and reverse order, relating them to those obtained on each column, separately. The experimentally obtained values were in good agreement with those estimated by the derived equations.

C(18) columns for the simultaneous determination of oxytetracycline and its related substances by reversed-phase high performance liquid chromatography and UV detection.

Simultaneous determination of oxytetracycline, 4-epioxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline on C(18) columns has been accomplished using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS RP-C(18) column (250 mm x 4.6 mm) and on a Waters C(18) Symmetry column (150 mm x 3.9mm), 5 microm particle size each. These columns were equilibrated with mobile phases consisted of methanol-acetonitrile-0.1M phosphate buffer pH 8.0 (12.5:12.5:75, v/v/v) and (15:15:70, v/v/v), respectively. The flow rate was 1.0 ml/min and the total elution time was 15 and 5 min, respectively. Both methods were applied to oxytetracycline raw material, human and veterinary formulations, where the excipients did not interfere. External standard calibration curves were linear for 4-epioxytetracycline, oxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline in the concentration range of 0.27-200 microM, 0.05-200 microM, 0.03-200 microM, 0.35-200 microM and 0.20-200 microM on column A and 0.08-200 microM, 0.15-200 microM, 0.09-200 microM, 0.25-200 microM and 0.47-200 microM on column B, respectively. Day-to-day relative standard deviation of the determination for every component was less than 3%. Concerning the first column, limits of detection and quantification of the above compounds were in the concentration ranges of 10-106 nM and 30-352 nM, respectively, whereas on the second column these ranges became 27-144 nM and 81-475 nM, respectively. Recovery of the separated compounds was 95-105%.

Solubilization and quantification of lycopene in aqueous media in the form of cyclodextrin binary systems.

An optimized kneading method for the preparation of lycopene-cyclodextrin binary systems was developed leading to solubilization of lycopene in water and 5% (w/v) dextrose solution. Lycopene quantification in the prepared binary systems was performed by a developed spectrometric method that followed a successful single-step extraction with dichloromethane. Storage stability characteristics of the binary systems were studied at 4 degrees C in solution and at -20 degrees C in the lyophilized products. Lycopene content was monitored at lambda(max)=482 nm, the limit of detection was 0.41 microg/ml and relative standard deviation was less than 3.1%. The results obtained with the spectrometric method were confirmed by a HPLC method. In the presence of cyclodextrins, lycopene concentration in water was 8.0+/-1.0, 27.1+/-3.2 and 16.0+/-2.2 microg/ml for beta-CD, HP-beta-CD and Me-beta-CD, respectively. In 5% (w/v) aqueous dextrose solutions the corresponding values were 16.0+/-1.8, 48.0+/-5.1 and 4.0+/-0.5 microg/ml, respectively. At 4 degrees C, storage stability of lycopene-cyclodextrin binary systems in water or 5% (w/v) aqueous dextrose solutions, was limited (t(1/2)=1-4 days). Addition of the antioxidant sodium metabisulfite increased the stability of lycopene-HP-beta-CD binary system in water. At -20 degrees C, the lyophilized lycopene-cyclodextrin binary systems were stable for at least 2 weeks.

Development and validation of a non-aqueous reversed-phase high-performance liquid chromatographic method for the determination of four chemical UV filters in suncare formulations.

A non-aqueous reversed-phase high performance liquid chromatographic method (RP-HPLC) with UV detection at 313 nm was developed and validated for simultaneous determination of methylene bis-benzotriazolyl tetramethylphenol (Tinosorb M) along with three other chemical UV filters, octocrylene (Eusolex OCR), octyl methoxycinnamate (Eusolex 2292) and octyl salicylate (Eusolex OS) in suncare products. An isocratic elution was performed on a Hypersil BDS RP-C18 column (250 mm x 4.6 mm), 5 microm particle size, using a mobile phase consisted of methanol-acetonitrile (90:10, v/v) with a flow-rate of 1.5 ml/min. The determination of the four UV filters was not interfered by the excipients in the products. The method of external standard, as well as the standard addition method was used for the determination. The external standard calibration curves were linear for Eusolex OCR, Eusolex 2292, Eusolex OS, and Tinosorb M in the concentration ranges of 0.5-100 microM, 0.5-100 microM, 0.5-200 microM, and 0.2-100 microM, respectively. Day-to-day relative standard deviation of the determination was within 3%. Limits of detection and quantitation of the above compounds were found equal to 36 and 110 nM, 220 and 660 nM, 170 and 520 nM, 44 and 130 nM, respectively. The recovery of these four chemical UV filters from the spiked samples was 96-103%.