HPLC semi‐preparative separation of diclazuril enantiomers and racemization in solution

Diclazuril has been widely used in poultry feed for prevention and treatment of coccidiosis, and its chiral separation is rarely reported. Herein, semi‐preparative separation method of diclazuril enantiomers has been developed through normal‐phase high‐performance liquid chromatography. Effects of chiral stationary phases, alcoholic modifiers, and column temperature on separation of diclazuril were discussed in detail. Both the single‐urea‐bound 4‐chlorophenylcarbamoylated β‐cyclodextrin and amylose tris(3,5‐dimethylphenylcarbamate)‐coated chiral stationary phases showed strong ability in separation of diclazuril by using n‐hexane–trifluoroacetic acid–ethanol. Then, semi‐preparative separation of diclazuril was carried out through stacked injection, and the "enantiomeric excess" purities of two fractions were over 98%. Next, the electronic circular dichroism profiles of these two fractions in ethanol solution displayed the mirror image of each other in the range 360–200 nm. Moreover, effects of acidic/basic additive, time, and temperature on racemization of diclazuril enantiomers in ethanol solution have been studied in detail through normal‐phase high‐performance liquid chromatography. Racemization of diclazuril enantiomers was remarkably accelerated through adding triethylamine at high temperature. We envision that this systematic investigation of diclazuril at an enantiomeric level would provide valuable information in future studies involving enantioselective bioactive, metabolic, and toxicological activities.     

HPLC enantioseparation of racemic bupropion,baclofen and etodolac: modification of conventional ligand exchange approach by pre‐column formation of chiral ligand exchange complexes

Separation of racemic mixture of (RS)‐bupropion, (RS)‐baclofen and (RS)‐etodolac, commonly marketed racemic drugs, has been achieved by modifying the conventional ligand exchange approach. The Cu(II) complexes were first prepared with a few l ‐amino acids, namely, l ‐proline, l ‐histidine, l ‐phenylalanine and l ‐tryptophan, and to these was introduced a mixture of the enantiomer pair of (RS)‐bupropion, or (RS)‐baclofen or (RS)‐etodolac. As a result, formation of a pair of diastereomeric complexes occurred by ‘chiral ligand exchange’ via the competition between the chelating l ‐amino acid and each of the two enantiomers from a given pair. The diastereomeric mixture formed in the pre‐column process was loaded onto HPLC column. Thus, both the phases during chromatographic separation process were achiral (i.e. neither the stationary phase had any chiral structural feature of its own nor did the mobile phase have any chiral additive). Separation of diastereomers was successful using a C₁₈ column and a binary mixture of MeCN and TEAP buffer of pH 4.0 (60:40, v/v) as mobile phase at a flow rate of 1 mL/min and UV detection at 230 nm for (RS)‐Bup, 220 nm for (RS)‐Bac and 223 nm for (RS)‐Etd. Baseline separation of the two enantiomers was obtained with a resolution of 6.63 in <15 min. Copyright © 2016 John Wiley & Sons, Ltd.     

Liquid chromatographic enantioseparation of (RS)‐etodolac using (S)‐levofloxacin and determination of absolute configuration of the diastereomeric derivatives

(RS)‐Etodolac was isolated from commercial tablets and was purified and characterized to be used as racemic standard. A pair of diastereomeric derivatives was synthesized using (S)‐levofloxacin as a chiral derivatizing reagent. The derivatization reaction was carried out under conditions of stirring at room temperature (30°C for 1.5 h) as well as under microwave irradiation; the derivatives obtained by the two methods were compared. Reaction conditions for derivatization were optimized with respect to mole ratio of chiral derivatizing reagent and (RS)‐etodolac. No racemization was observed throughout the study. Separation of diastereomeric derivatives was successful using C₁₈ column and a binary mixture of methanol and triethyl ammonium phosphate buffer of pH 4.5 (80:20, v/v) as mobile phase at a flow rate of 1 mL min^?1 and UV detection at 223 nm. An efficient approach for recognizing chirality and determining the absolute configuration of the diastereomeric derivatives of (RS)‐etodolac is described, which in turn is a measure of the enantiomeric purity of (RS)‐etodolac since the diastereomeric derivatives were separated and isolated using preparative thin‐layer chromatography.     

(RS)‐Propranolol: enantioseparation by HPLC using newly synthesized (S)‐levofloxacin‐based reagent,absolute configuration of diastereomers and recovery of native enantiomers by detagging

Diastereomers of (RS)‐propranolol were synthesized using (S)‐levofloxacin‐based new chiral derivatizing reagents (CDRs). Levofloxacin was chosen as the pure (S)‐enantiomer for its high molar absorptivity (ε_o ～ 24000) and availability at a low price. Its ‐COOH group had N‐hydroxysuccinimide and N‐hydroxybenzotriazole, which acted as good leaving groups during nucleophilic substitution by the amino group of the racemic (RS)‐propranolol; the CDRs were characterized by UV, IR, ¹H‐NMR, high resolution mass spectrometry (HRMS) and carbon, hydrogen, nitrogen, and sulphur fundamental elemental components analyser (CHNS). Diastereomers were separated quantitatively using open column chromatography; absolute configuration of the diastereomers was established and the reagent moiety was detagged under microwave‐assisted acidic conditions. (S)‐ and (R)‐propranolol as pure enantiomers and (S)‐levofloxacin were separated, isolated and characterized. Optimized lowest‐energy structures of the diastereomers were developed using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6‐31G* basis set (based on density functional theory) for explanation of elution order and configuration. In addition, RP HPLC conditions for separation of diastereomers were optimized with respect to pH, concentration of buffer, flow rate of mobile phase and nature of organic modifier. HPLC separation method was validated as per International Conference on Harmonization guidelines. With the systematic application of various analytical techniques, absolute configuration of the diastereomers (and the native enantiomers) of (RS)‐propranolol was established. Copyright © 2016 John Wiley & Sons, Ltd.     

A novel approach for enantioseparation as applied to (RS)‐etodolac from pharmaceutical formulations: LC MS and density functional theory support for confirmation of diastereomers so separated

In the present studies formation of diastereomers of (RS)‐etodolac was confirmed using LC‐MS when [M + H]⁺ or [M]⁺ were recorded for the diastereomers. The lowest energy optimized structures of two diastereomers were drawn, which confirmed the three‐dimensional geometry of the diastereomers. This supports the optimized analytical separation conditions. In addition, separation of diastereomers was successful using a C₁₈ column and a binary mixture of methanol and triethyl ammonium phosphate buffer of pH 4.5 (80:20, v/v) as mobile phase at a flow rate of 1 mL min^?1 and UV detection at 223 nm. The separation method was validated as per International Conference on Harmonization guidelines. (RS)‐Etodolac was isolated from commercial tablets and purified and characterized to be used as racemic standard. Three pairs of diastereomers were synthesized using enantiomerically pure amines, namely, (R)‐(+)‐α‐methyl benzyl amine, (S)‐(?)‐α,4‐dimethylbenzylamine and (R)‐(?)‐1‐cyclohexylethylamine. Derivatization reactions were carried out under conditions of stirring at room temperature (30 °C for 2 h) as well as under microwave irradiation (MWI), and the two types of diastereomers were compared. Reaction conditions for derivatization were optimized with respect to mole ratio of chiral derivatizing agent and (RS)‐etodolac and MWI time. No racemization was observed throughout the study. Copyright © 2015 John Wiley & Sons, Ltd.     

Methods and approaches for determination and enantioseparation of (RS)‐propranolol

Propranolol, a β‐adrenergic receptor antagonist, is a chiral compound that is marketed as a racemate, but only the (S)‐(?)‐enantiomer is responsible for the β‐adrenoceptor blocking activity. Different chromatographic methods have been applied for separation and determination of enantiomers of (RS)‐propranolol. In this article a review is presented on different liquid chromatographic methods used for enantioseparation of (RS)‐propranolol, using both HPLC and TLC. In addition, some aspects of enantioseparation under achiral phases of liquid chromatography have been briefly mentioned.     

Enantioseparation of (RS)‐fexofenadine and enhanced detection as the diastereomeric amide and anhydride derivatives using liquid chromatography‐mass spectrometry

Enantioselective analysis of (RS)‐fexofenadine was carried out by achiral HPLC via a derivatization approach using N‐hydroxy‐benzotriazolyl‐(S)‐naproxen ester (synthesized for this purpose) and three chirally pure amines as chiral derivatizing reagents. There occurred formation of amide and anhydride types of diastereomeric derivatives. These were separated and isolated by HPLC (analytical and preparative). The structures and configurations were verified via recording full‐scan product ion mass spectra using LC‐MS, ¹HNMR spectra, Chem3D Pro 12.0 software and the software Gaussian 09 Rev.A.02 program and hybrid density functional B3LYP with 6‐31G basis set supplemented with polarimetry. Experimental conditions for synthesis and separations were optimized and the elution order was established. Analytical separation was performed on a C₁₈ analytical column with different ratios of MeCN–TEAP buffer and MeOH–TEAP buffer (v/v) adjusted to pH 7.5 as mobile phase at a flow rate of 0.7 mL min^‐1. Detection was performed via UV absorbance at 225 nm. The method was validated in accordance with International Conference on Harmonization guidelines. The detection limits were 6.25 and 7.87 ng mL^‐1 for first and second eluting diastereomeric derivatives, respectively.     

Sensitive enantioseparation and determination of isoprenaline in human plasma and pharmaceutical formulations

A simple, sensitive and fast RPHPLC method was developed and validated for the enantioselective determination of (RS)‐isoprenaline (Ipn) in human plasma. The enantiomers were converted to diastereomeric derivatives using s‐triazine (cyanuric chloride) based chiral derivatizing reagents. l ‐isoleucine and l ‐methionine were introduced as chiral auxiliary in s‐triazine and two new monochloro‐s‐triazine reagents were synthesized. These reagents were characterized and used for synthesis of diastereomeric derivatives of (RS)‐Ipn spiked in human plasma. (RS)‐Ipn was isolated (purified and characterized) from a commercial pharmaceutical formulation and was used as the standard racemic sample. Structures of the two diastereomeric derivatives were optimized for lowest energy using the Gaussian 09 Rev A. 02 program and hybrid density functional B3LYP with 6‐31G* basis set which showed the spatial orientation of hydrophobic groups on stereogenic centers in the diastereomeric derivatives. The results were correlated with the mechanism of separation and elution order. Limit of detection values were found to be 24.6 and 26.8 ng mL^?1 for the first and second eluting diastereomeric derivatives, respectively.     

Ultrafiltration‐LC–MS combined with semi‐preparative HPLC for the simultaneous screening and isolation of lactate dehydrogenase inhibitors from Belamcanda chinensis

Stroke represents the fourth leading cause of death in the USA and the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke and natural products are considered a promising source of novel lactate dehydrogenase inhibitors. In this study, we used PC12 cells to determine the protective effect of extracts from the herb Belamcanda chinensis following toxic challenge. Using ultrafiltration high‐performance liquid chromatography coupled with photo‐diode array detection and electrospray ionization mass spectrometry, we screened and identified isoflavonoids from Belamcanda chinensis extracts. Semi‐preparative high‐performance liquid chromatography was then applied to separate and isolate the active constituents. Using these methods, we identified six major compounds in Belamcanda chinensis as lactate dehydrogenase inhibitors: tectoridin, iristectorin A, iridin, tectorigenin, irigenin, and irisflorentin, which were then isolated to >92% purity. This is the first report that Belamcanda chinensis extracts contain potent lactate dehydrogenase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from Belamcanda chinensis guided by ultrafiltration high‐performance liquid chromatography coupled with photo‐diode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

Preparative isolation and purification of 12 main antioxidants from the roots of Polygonum multiflorum Thunb. using high‐speed countercurrent chromatography and preparative HPLC guided by 1,1′‐diphenyl‐2‐picrylhydrazyl‐HPLC

A hyphenated strategy by off‐line coupling of 1,1′‐diphenyl‐2‐picrylhydrazyl‐high‐performance liquid chromatography, high‐speed countercurrent chromatography, and preparative high‐performance liquid chromatography was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the targeted guidance of 1,1′‐diphenyl‐2‐picrylhydrazyl‐high‐performance liquid chromatography experiment, 12 compounds were identified as potential antioxidants and readily isolated by high‐speed counter‐current chromatography and preparative high‐performance liquid chromatography. Ultraviolet spectroscopy, mass spectrometry, and ¹H NMR spectroscopy were employed to identify their structures, which were assigned as gallic acid ( 1 , 6.2 mg, 98.28%), catechin ( 2 , 8.8 mg, 90.69%), epicatechin ( 3 , 4.1 mg, 96.71%), polydatin ( 4 , 5.3 mg, 94.91%), 2,3,5,4′‐tetrahydroxy stilbene‐2‐Ο‐β‐D‐glucoside ( 5 , 20.2 mg, 95.23%), piceatannol ( 6 , 5.3 mg, 96.85%), rutin ( 7 , 5.4 mg, 97.92%), resveratrol ( 8 , 5.2 mg, 96.94%), isorhapontigenin ( 9 , 11.4 mg, 94.81%), hyperoside ( 10 , 9.7 mg, 98.52%), rhein ( 11 , 4.9 mg, 97.46%), and emodin ( 12 , 8.2 mg, 95.74%). Notably, compounds 6 and 9 were isolated from Polygonum multiflorum for the first time. In addition, antioxidant activity of compounds 1–12 were evaluated, and compounds 1–8 and 10 exhibited stronger antioxidant activity than ascorbic acid (positive control). These results indicated that the proposed method is a highly efficient strategy to screen and isolate antioxidants from complex natural products.     

High‐performance liquid chromatographic enantioseparation of (RS)‐bupropion using isothiocyanate‐based chiral derivatizing reagents

A high‐performance liquid chromatographic (HPLC) method for enantioseparation of bupropion was developed using two isothiocyanate‐based chiral derivatizing reagents, (S)‐1‐(1‐naphthyl) ethyl isothiocyanate, (S)‐NEIT, and (R)‐α‐methyl benzyl isothiocyanate, (R)‐MBIT. The diastereomers synthesized with (S)‐NEIT were enantioseparated by reversed‐phase HPLC using gradient elution with mobile phase containing water and acetonitrile, whereas diastereomers synthesized with (R)‐MBIT were enantioseparated using triethyl amine phosphate buffer and methanol. Derivatization conditions were optimized and the method was validated for accuracy, precision and limit of detection. The limit of detection was found to be 0.040–0.043 µg/mL for each of the diastereomers prepared with (S)‐NEIT. Copyright © 2013 John Wiley & Sons, Ltd.     

Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration,enzyme‐immobilized magnetic beads,semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

Nonsteroidal anti‐inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase‐2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase‐2 inhibitors can also act through cyclooxygenase‐independent mechanisms. In this study, using ultrafiltration, enzyme‐immobilized magnetic beads, high‐performance liquid chromatography, and electrospray‐ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase‐2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase‐2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography, and high‐speed counter‐current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

The chiral bioconversion and preclinical pharmacokinetic analysis of (R)‐(+)‐rabeprazole in beagle dogs by HPLC and HPLC‐MS/MS

In order to accurately investigate the preclinical pharmacokinetics of (R)‐(+)‐rabeprazole sodium injection, a reliable high‐performance liquid chromatography (HPLC) method was developed using a Chiral‐AGP column to prove that there is no chiral bioconversion of (R)‐(+)‐rabeprazole to (S)‐(?)‐rabeprazole in beagle dogs after single intravenous administration of (R)‐(+)‐rabeprazole sodium injection. An HPLC–tandem mass spectrometry (HPLC‐MS/MS) method for analysis of (R)‐(+)‐rabeprazole was developed and validated, and used to acquire the pharmacokinetic parameters in beagle dogs. (R)‐(+)‐Rabeprazole and internal standard omeprazole were extracted from plasma samples by protein precipitation and separated on a C₁₈ column using methanol–5 mm ammonium acetate as mobile phase. Detection was performed using a turbo‐spray ionization source and mass spectrometric positive multi‐reaction monitoring mode. The linear relationship was achieved in the range from 2.5 to 5000 ng/mL. The method also afforded satisfactory results in terms of sensitivity, specificity, precision, accuracy and recovery as well as the stability of the analyte under various conditions, and was successfully applied to a preclinical pharmacokinetic study in beagle dogs after single intravenous administrations of (R)‐(+)‐rabeprazole sodium injection at 0.33, 2 and 6 mg/kg. Copyright © 2013 John Wiley & Sons, Ltd.     

Indirect reversed‐phase high‐performance liquid chromatographic and direct thin‐layer chromatographic enantioresolution of (R,S)‐Cinacalcet

Enantioresolution of the calcimimetic drug (R,S)‐Cinacalcet was achieved using both indirect and direct approaches. Six chiral variants of Marfey's reagent having l ‐Ala‐NH₂, l ‐Phe‐NH₂, l ‐Val‐NH₂, l ‐Leu‐NH₂, l ‐Met‐NH₂ and d ‐Phg‐NH₂ as chiral auxiliaries were used as derivatizing reagents under microwave irradiation. Derivatization conditions were optimized. Reversed‐phase high‐performance liquid chromatography was successful using binary mixtures of aqueous trifluoroacetic acid and acetonitrile for separation of diastereomeric pairs with detection at 340 nm. Thin silica gel layers impregnated with optically pure l ‐histidine and l ‐arginine were used for direct resolution of enantiomers. The limit of detection was found to be 60 pmol in HPLC while in TLC it was found to be in the range of 0.26–0.28 µg for each enantiomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Indirect enantioresolution of (R,S)‐mexiletine by reversed‐phase high‐performance liquid chromatography via diastereomerization with [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride], (S)‐naproxen and nine chiral reagents synthesized as variants of Marfey's reagent

Eleven chiral derivatizing reagents (CDRs) were used for preparation of diastereomers of (R,S)‐mexiletine containing a primary amino group in close proximity to the stereogenic center. One anhydride, namely [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride] was synthesized and (S)‐naproxen was used as such as the chiral derivatizing reagent. The other nine CDRs were synthesized by substituting one of the fluorine atoms in 1,5‐difluoro‐2,4‐dinitrobenzene with six amino acid amides and three amino acids. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography. The method was validated for linearity, accuracy, limit of detection and limit of quantification. The limit of detection was found in the range of 10–30 pmol. Copyright © 2010 John Wiley & Sons, Ltd.     

Preparation of poly(vinylalcohol)/poly(acrylamide‐co‐vinyl imidazole)/γ‐Fe2O3 semi‐IPN nanocomposite and their application for removal of heavy metal ions from water

A series of magnetic semi‐interpenetrating polymer network (semi‐IPN) hydrogels was prepared in one‐stage strategy composed of linear poly(vinyl alcohol) (PVA) chains and magnetic γ‐Fe₂O₃ nanoparticles entrapped within the cross‐linked poly(acrylamide‐co‐vinylimidazole) (poly(AAm‐co‐VI)) network. The influence of PVA, weight ratio of AAm:VI, γ‐Fe₂O₃, and MBA on the swelling properties of the obtained nanocomposite hydrogels was evaluated. The prepared magnetic semi‐IPN hydrogels were fully characterized and used as absorbent for removal of Pb(II) and Cd(II) from water. Factors that influence the metal ion adsorption such as solution pH, contact time, initial metal ion concentration, and temperature were studied in details. The experimental results were reliably described by Langmuir adsorption isotherms. The adsorption capacity of semi‐IPN nanocomposite for Pb(II) and Cd(II) were175.80 and 149.76 mg g^?1, respectively. The kinetic experimental data indicated that the chemical sorption is the rate‐determining step. According to thermodynamic studies, Pb(II) and Cd(II) adsorption on the hydrogels was endothermic and also chemical in nature. The prepared magnetic PVA/poly(AAm‐co‐VI) semi‐IPN hydrogels could be employed as efficient and low‐cost adsorbents of heavy metal ions from water. Copyright © 2016 John Wiley & Sons, Ltd.     

Enantioseparation of (RS)-Bupropion and determination of configuration

Racemate of (RS)-Bupropion (Bup) has been separated by derivatization approach and thin layer chromatography (TLC). It was extracted from commercially available tablets, purified, and characterized. A new chiral derivatization reagent (CDR) was synthesized by introducing L-leucine amide and butoxy group in cyanuric chloride (CC) moiety. Derivatization reaction was carried out using a two-fold molar excess of CDR under microwave irradiation (MWI). The diastereomeric pair was separated by TLC and the same were recovered by the preparative method; these were purified and characterized. Absolute configuration of the Bup moiety in the diastereomeric derivatives was confirmed by recording their ¹HNMR spectra and thus elution order was also established.     

Selective Pre-concentration and Solid Phase Extraction of Mercury（Ⅱ） from Natural Water by Silica Gel-loaded （E）-N-（1-Thien-2＇-ylethylidene）-1,2-phenylenediamine Phase

Silica gel-loaded （E）-N-（1-thien-2＇-ylethylidene）-1,2-phenylenediamine （TEPDA） phase was synthesized based on physical adsorption approaches. The stability of a chemically modified TEPDA especially in concentrated hydrochloric acid that was then used as a recycling and preconcentration reagent allowed the further uses of silica gel-loaded immobilized TEPDA phase. The application of this silica gel-loaded phase to sorption of a series of metal ions was performed by using different controlling factors such as the pH of the metal ion solution and the equilibration shaking time by the static technique. This difference was interpreted on the basis of selectivity incorporated in these sulfur containing silica gel-loaded TEPDA phases. Hg（Ⅱ） was found to exhibit the highest affinity towards extraction by these silica gel-loaded TEPDA phases. The pronounced selectivity was also confirmed by the determined distribution coefficients （Kd） of all the metal ions, showing the highest value reported for mercury（Ⅱ） extraction by the silica gel immobilized TEPDA phase. The potential applications of the silica gel immobilized TEPDA phase to selective extraction of mercury（Ⅱ） from aqueous solution were successfully accomplished and preconcentration of low concentration of Hg（Ⅱ） （30 pg·mL^-1） from natural tap water with a preconcentration factor of 200 for Hg（Ⅱ） off-line analysis was conducted by cold vapor atomic absorption analysis.     

Study of the effect of poly(vinyl alcohol) concentration on the gelation point of poly(vinyl alcohol) poly(acrylic acid) semi‐IPN systems as determined by viscoelastic measurements

We report on the determination of the gelation point of semi‐interpenetrating polymer networks (semi‐IPNs) composed of poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAAc) formed by a sequential method. The evolution of the viscoelasticity during the gelation reaction of acrylic acid (AAc) in solutions of PVA has been monitored through the sol‐gel transition with dynamic mechanical experiments. The gelation time of the system increased with PVA concentration; however, the molecular structure of the gel, composed of swollen clusters, is rather independent of the presence of PVA. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 1944–1949, 2005