微波萃取-气相色谱法测定血液中的可卡因及其代谢物爱冈宁甲基酯

建立了血液中可卡因(cocaine, COC)及其代谢物爱冈宁甲基酯(ecgonine methyl ester, EME)的气相色谱-质谱(CG-MS)和气相色谱-氢火焰离子化检测(GC-FID)方法。该方法采用微波萃取提取血液中的COC和EME,优化并确定了最佳提取条件: 以氯仿-异丙醇(体积比为9:1)混合溶液为提取溶剂,用0.05 mol/L Na2CO3-NaHCO3缓冲溶液调节样品溶液的pH至10.0,在40 ℃下微波萃取6 min;采用GC-MS对萃取液中的COC和EME进行定性,采用GC-FID进行定量检测。COC和EME的平均回收率分别为79.91%～99.85%,相对标准偏差(RSD)均小于3.10%,检出限(S/N=3)分别为60 mg/L和40 mg/L。该方法无需衍生化,快速、准确、灵敏,可同时检测血液中的COC和EME。     

Concentration profiles of cocaine, pyrolytic methyl ecgonidine and thirteen metabolites in human blood and urine: determination by gas chromatography-mass spectrometry

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.     

Electrospray Ionization Mass Spectra of Amino Acid Methyl Ester 5 ＇-Phosphoramidates of 2＇, 3＇-Isopropylideneuridine

Amino acid methyl ester phosphates were synthesized and determined by using positive-ion mode dectrospmy ionization mass spectrometry（ESIMS） in combination with multistage tandem mass spectrometry. The fragmentation pathways were investigated, and it was observed that most fragment ions contained the phosphoryl group. It was interesting to observe that the fragmentation pathways of the protonated molecule show some differences when compared with those of the sodium ion adduct. The methoxy group of amino acid methyl ester can migrate from the carbonyl group to the phosphoryl group in the sodium ion adduct.     

A novel rearrangement in electrospray ionization multistage tandem mass spectrometry of amino acid ester cyclohexyl phosphoramidates of AZT

Several amino acid ester cyclohexyl phosphoramidates of AZT as anti-HIV prodrugs were synthesized and investigated by electrospray ionization tandem mass spectrometry (ESI-MS(n)). A novel methoxy group migration from the carbonyl group to the phosphoryl group was observed in ESI-MS2. This migration is believed to be a general pathway for ions with a methyl ester moiety at the gamma-position to a phosphoric acid moiety, which is assisted with metal ions such as Li(+), Na(+) and K(+). Coordination between metal ions with both the carbonyl oxygen and phosphoryl oxygen might be a key factor responsible for this migration.     

EPA甲酯和DHA甲酯标准样品的制备和结构解析

从鱼油中分离制备得到二十碳五烯酸（EPA）甲酯和二十二碳六烯酸（DHA）甲酯,采用红外光谱、质谱、核磁共振波谱等手段对EPA甲酯和DHA甲酯的结构进行表征,提供了不饱和脂肪酸甲酯标准物质研制基础。样品的红外光谱（IR）、质谱（MS）、核磁共振谱图（1HNMR、13CNMR）所给出的结构信息与EPA甲酯和DHA甲酯的化学结构式相符,并通过DEPT谱和I3C-1H COSY化学位移相关谱（HMBC）对各共振峰进行了指认,样品的谱图数据与文献报道基本一致。     

Methyl group transfer upon gas phase decomposition of protonated methyl benzoate and similar compounds

Gas phase decompositions of protonated methyl benzoate and its conjugates have been studied by using electrospray ionization‐collision induced dissociation‐tandem mass spectrometry. Loss of CO₂ molecule, thus transfer of methyl group, has been observed. In order to better understand this process, the theoretical calculations have been performed. For methyl benzoate conjugates, it has been found that position of substituent affects the loss of CO₂ molecule, not the electron donor/withdrawing properties of the substituent. Therefore, electrospray ionization‐mass spectrometry in positive ion mode may be useful for differentiation of isomers of methyl benzoate conjugates.     

Synthesis of a heterodinuclear ruthenium(II)–platinum(II) complex linked by l-cysteine methyl ester

A potential anticancer heterodinuclear ruthenium(II)–platinum(II) complex, [ruthenium(II)(4,4′-dimethyl-2,2′-bipyridine)₂(5-(l-cysteine-methyl ester)-1,10-phenanthroline)-trans-chlorodiammineplatinum(II)] chloride, [Ru(Me₂bipy)₂(5-(l-cysteine-Me)-phen)-trans-Pt(NH₃)₂Cl]Cl₃, was synthesised. l-Cysteine methyl ester was used to link the two metal centres, as more conventional straight chain diaminoalkanes and 2-mercaptoethylamine failed to couple to the phenanthroline ligand. From the precursor mononuclear ruthenium(II) complexes, which were separated into their Δ- and Λ-isomers by column chromatography, the dinuclear complex was synthesised and characterised by ¹H and ¹³C NMR, UV–Vis, circular dichroism, fluorescence and electrospray ionisation mass spectrometry.     

Determination, purity assessment and chiral separation of levodopa methyl ester in bulk and formulation pharmaceuticals

A sensitive, reliable and reproducible HPLC method with electrochemical detection (HPLC-ECD) has been developed for the separation and quantification of levodopa methyl ester (LDME) and its impurities such as levodopa (l-DOPA), 3-methoxytyrosine (MTS) and l-tyrosine (TS) in bulk drug and pharmaceutical dosage form. The separation was performed on an LC18 column by isocratic elution with methanol-acetonitrile-50 mm potassium dihydrogen phosphate (8:2:90, v/v/v) containing 5 mm sodium 1-hexanesulfonate, 5 mm EDTA and 5 mm sodium chloride, adjusted with phosphoric acid to a pH of 3.2 as mobile phase. The correlation coefficients of linear regression for LDME, L-DOPA, MTS and TS were more than 0.999. The detection limits for L-DOPA, MTS and TS were 3.15, 2.04 and 2.88 ng/mL, respectively. The precision was checked in terms of F-test variance ratio using potentiometric titration as reference. The separation of dopa methyl ester enantiomers by chiral chromatography is also described. This method is capable of separating the two enantiomers with a selection of 1.4 and a resolution of 8.4. Both methods were found to be stable and useful in the quality control of the bulk material and formulations.     

Synthesis and properties of new luminescent hole transporting materials of triarylamine with dehydroabietic acid methyl ester moieties

A series of triarylamines based on dehydroabietic acid methyl ester moieties (6a–h) were synthesized for possible application as hole transporting materials for organic electroluminescent devices. The target compounds were characterized by elemental analysis, FT-IR, NMR, and mass spectrometry. Their optical, electrochemical, and thermal properties were investigated using UV–vis, PL spectroscopy, cyclic voltammetry (CV), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), respectively. CV measurements show that the compounds present suitable HOMO values (in a range of −4.63 to −5.11 eV) for hole injection, which is confirmed by theoretical calculations. All compounds were thermally stable. Organic light-emitting diode devices having 6f, 6g, and 6h as a hole transporting layer showed better performance of maximum brightness, turn-on voltage, and maximum luminous efficiency than a comparable device NPB. These compounds could be excellent candidates for applications in OLED devices.     

A novel methyl migration to the phosphoryl group with the formation of cyclic aminoacylphosphoramidates in electrospray ionization tandem mass spectra of amino acid ester phosphoramidates of antiviral nucleosides

The mass spectral behavior of amino acid methyl ester phosphoramidate derivatives have been investigated using electrospray ionization multistage mass spectrometry (ESI-MS(n)) and moderate theoretical calculations at the B3LYP/6-31G(d) level. A novel methyl group migration to the phosphoryl group with the formation of the intermediate cyclic aminoacylphosphoramidate was found. The proposed structures of the rearrangement ions were confirmed by high-resolution tandem mass spectrometry. A possible mechanism involving the pentacoordinate phosphoric-carboxylic phosphate anhydride was proposed, in which the metal ion coordination with the phosphoryl and carbonyl groups and the intrinsic properties of phosphoryl group might be the key factors responsible for this novel migration.     

High-throughput simultaneous analysis of buprenorphine, methadone, cocaine, opiates, nicotine, and metabolites in oral fluid by liquid chromatography tandem mass spectrometry

A method for simultaneous determination of buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), anhydroecgonine methyl ester (AEME), morphine, codeine, 6-acetylmorphine (6AM), heroin, 6-acetylcodeine (6AC), nicotine, cotinine, and trans-3′-hydroxycotinine (OH-cotinine) by liquid chromatography tandem mass spectrometry in oral fluid (OF) was developed and extensively validated. Acetonitrile (800 μL) and OF (250 μL) were added to a 96-well Isolute-PPT+protein precipitation plate. Reverse-phase separation was achieved in 16 min and quantification was performed by multiple reaction monitoring. The assay was linear from 0.5 or 1 to 500 μg/L. Intraday, interday, and total imprecision were less than 13% (n?=?20), analytical recovery was 92–114% (n?=?20), extraction efficiencies were more than 77% (n?=?5), and process efficiencies were more than 45% (n?=?5). Although ion suppression was detected for EME, cocaine, morphine, 6AC, and heroin (less than 56%) and enhancement was detected for BE and nicotine (less than 316%), deuterated internal standards compensated for these effects. The method was sensitive (limit of detection 0.2–0.8 μg/L) and specific (no interferences) except that 3-hydroxy-4-methoxyamphetamine interfered with AEME. No carryover was detected, and all analytes were stable for 24 h at 22 °C, for 72 h at 4 °C, and after three freeze–thaw cycles, except cocaine, 6AC, and heroin (22–97% loss). The method was applied to 41 OF specimens collected throughout pregnancy with a Salivette® OF collection device from an opioid-dependent BUP-maintained pregnant woman. BUP ranged from 0 to 7,400 μg/L, NBUP from 0 to 71 μg/L, methadone from 0 to 3 μg/L, nicotine from 32 to 5,020 μg/L, cotinine from 125 to 508 μg/L, OH-cotinine from 11 to 51 μg/L, cocaine from 0 to 419 μg/L, BE from 0 to 351 μg/L, EME from 0 to 286 μg/L, AEME from 0 to 7 μg/L, morphine from 0 to 22 μg/L, codeine from 0 to 1 μg/L, 6AM from 0 to 4 μg/L, and heroin from 0 to 2 μg/L. All specimens tested negative for EDDP and 6AC. This method permits a fast and simultaneous quantification of 16 drugs and metabolites in OF, with good selectivity and sensitivity.     

Chlorination of N-tritylproline methyl ester

The reaction of N-tritylproline methyl ester with tert-butyl hypochlorite gave 2,4-dichloro-2-methoxycarbonyl-5-pyrroline, which is readily converted to 2-methoxycarbonyl-4-chloropyrrole. The intermediate products were identified by Chromatographic mass spectrometry when the reaction was carried out at low temperatures.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 7, pp. 894–896, July, 1988.     

Mechanism of the formation of a dehydrated ion by an unusual loss of oxygen at the 4'-carbonyl group of abscisic acid methyl ester in electron ionization mass spectrometry

Methyl ester of abscisic acid (ABA), a plant hormone, gives a dehydrated ion at m/z 260 in electron ionization mass spectrometry (EI-MS). This dehydrated ion had been considered to be derived only from the elimination of the tertiary hydroxyl group at C-1'. We found that 34% of the dehydrated ion was formed by elimination of the oxygen atom at the 4'-carbonyl group, and the remaining 66% by elimination of the 1'-hydroxyl group. This unusual elimination of the carbonyl oxygen was shown with [4'-(18)O]ABA methyl ester. Involvement of the 4'-carbonyl oxygen in dehydration was observed in methyl ester of phaseic acid (PA), a natural metabolite of ABA, but not in 1'-deoxy-ABA methyl ester or isophorone. This suggested that the 1'-hydroxyl group was necessary for the elimination of the 4'-carbonyl oxygen. ABA methyl esters labeled with stable isotopes showed that hydrogen atoms at the 1'-hydroxyl group and at C-4 or -5 or -3' or - 5' or -7' were eliminated with the 4'-carbonyl oxygen. These results allow us to propose a formation mechanism of the dehydrated ion derived from the elimination of 4'-carbonyl oxygen and hydrogen atoms at C-4 and 1'-oxygen in ABA methyl ester as follows: first, ionization at the 1'-hydroxyl group occurs to give an ion radical, and the proton at the 1'-oxygen migrates to the 4'-carbonyl oxygen after the bond fission between C-1'-C-6'; second, migration of the proton at C-4 to the 1'-oxygen is followed by migration of the protons at C-5 and C-7' to C-4 and C-5, respectively; finally, the proton at the 1'-oxygen migrates to the 4'-hydroxyl group, and H(2)O at C-4' is eliminated to give the dehydrated ion. Our findings point out that a dehydrated ion is not always derived from the elimination of a hydroxyl group.     

长链不饱和脂肪酸甲酯的臭氧化反应

长链不饱和脂肪酸甲酯的臭氧化反应;选择性     

Chiral separation of N-methyl-dl-aspartic acid in rat brain tissue as N-ethoxycarbonylated (S)-(+)-2-octyl ester derivatives by GC-MS

A selective and sensitive analytical method was developed for enantiomeric separation and determination of N‐methyl‐DL‐aspartic acid (NMA). The method involved the conversion of each enantiomer into N‐ethoxycarbonylated (S)‐(+)‐2‐octyl ester derivative for the direct separation by gas chromatography–mass spectrometry (GC‐MS). The diastereomeric derivatives showed characteristic mass spectral properties for analysis by selected ion monitoring mode (SIM) and enabling enantioseparation on an achiral capillary column. Two enantiomers were baseline separated, and the detection limits for N‐methyl‐L‐aspartic acid (NMLA) and N‐methyl‐D‐aspartic acid (NMDA) were 0.07 and 0.03 ng/g, respectively. When applied to rat brain tissues for absolute configuration of NMA, only NMDA was determined, while NMLA was monitored as lower than the limit of detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of electrospray ionization mass spectrometry for N-diisopropyloxyphosphoryl dipeptide methyl esters

A systematic study of the fragmentation pattern of N-diisopropyloxyphosphoryl (DIPP) dipeptide methyl esters in an electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was presented. A combination of accurate mass measurement and tandem mass spectrometry had been used to characterize the major fragment ions observed in the ESI mass spectrum. It was found that the alkali metal ions acted as a fixed charge site and expelled the DIPP group after transferring a proton to the amide nitrogen. For all the N-phosphoryl dipeptide methyl esters, under the activation of a metal ion, the rearrangement product ion at m/z 163 was observed and confirmed to be the sodium adduct of phosphoric acid mono-isopropyl esters (PAIE), via a specific five-membered penta-co-ordinated phosphorus intermediate. However, no rearrangement ion was observed when a beta-amino acid was at the N-terminal. This could be used to develop a novel method for differentiating isomeric compounds when either alpha- or beta-amino acid are at the N-terminus of peptides. From the [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters (DIPP Xaa1 Xaa2 OMe), the peaks corresponding to the [M+Na Xaa1 C3H6]+ were observed and explained. The [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters with Phe located in the C-terminal, such as DIPPValPheOMe, DIPPLeuPheOMe, DIPPIlePheOMe, DIPPAlaPheOMe and DIPPPhePheOMe, had characteristic fragmentation. Two unusual gas-phase intramolecular rearrangement mechanisms were first proposed for this fragmentation. These rearrangements were not observed in dipeptide methyl ester analogs which did not contain the DIPP at the N-terminal, suggesting that this moiety was critical for the rearrangement.     

Matrix isolation FT-IR spectroscopy and molecular orbital study of sarcosine methyl ester

N-methylglycine methyl ester (sarcosine-Me) has been studied by matrix isolation FT-IR spectroscopy and molecular orbital calculations undertaken at the DFT/B3LYP and MP2 levels of theory with the 6-311++G(d,p) and 6-31++G(d,p) basis set, respectively. Twelve different conformers were located in the potential energy surface of the studied compound, with the ASC conformer being the ground conformational state. This form is analogous to the dimethylglycine methyl ester most stable conformer and is characterized by a NH?O intramolecular hydrogen bond; in this form, the ester group assumes the cis configuration and the OC-C-N and Lp-N-C-C (where Lp is the nitrogen lone electron pair) dihedral angles are ca. −17.8 and 171.3°, respectively. The second most stable conformer (GSC) differs from the ASC conformer essentially in the conformation assumed by the methylamino group, which in this case is gauche (Lp-N-C-C dihedral angle equal to 79.4°). On the other hand, the third most stable conformer (AAC) differs from the most stable form in the conformation of the OC-C-N axis (151.4°). These three forms were predicted to differ in energy by less than ca. 5 kJ mol⁻¹ and represent ≈95% of the total conformational population at room temperature. FT-IR spectra were obtained for sarcosine-Me isolated in argon matrices revealing the presence in the matrices of the three lowest energy conformers predicted by the calculations. The matrices were prepared by deposition of the vapour of the compound using two different nozzle temperatures, 25 and 60 °C. The relative populations of the three conformers trapped in the matrices were found to be consistent with occurrence of conformational cooling during matrix deposition and with a stabilization of the most polar GSC and AAC conformers in the matrices compared to the gas phase. Indeed, like it was previously observed for the methyl ester of dimethylglycine [Phys. Chem. Chem. Phys. 5 (2003) 52] the different strength of the interactions between the conformers and the matrix environment seem to lead to a change in the relative order of stabilities of GSC and ASC upon going from the gas phase to the matrices, with the first conformer becoming the conformational ground state in the latter media. The assignment of the bands observed in the matrix spectra to the three experimentally observed conformers of sarcosine-Me is presented and discussed.     

Copper(II) complexes of imino-bis(methyl phosphonic acid) with some bio-relevant ligands. Equilibrium studies and hydrolysis of glycine methyl ester through complex formation

Binary and ternary complexes of Cu(II) involving imino-bis(methyl phosphonic acid) (IdP) abbreviated as H₄A and some selected bio-ligands, amino acids, peptides and DNA constituents (L), were examined. Cu(II) forms CuA and CuAH complexes with IdP. Ternary complexes are formed in a stepwise mechanism whereby iminodiphosphonic acid binds to Cu(II), followed by coordination of amino acid, peptide or DNA. The concentration distribution of the various complex species has been evaluated. The kinetics of base hydrolysis of glycine methyl ester in the presence of Cu(II)-IdP was studied in aqueous solution at different temperatures, and in dioxane-water solutions of different compositions at 25°C. The activation parameters are evaluated and discussed.     

The effect of water solubility of the monomers on composition drift on methyl acrylate-vinyl ester combinations

It has been shown theoretically that composition drift mainly depends on reactivity ratios and water solubilities. Minimum composition drift can be obtained by lowering the monomer-to-water ratio in monomer systems where the more reactive monomer is also the more water-soluble one. Investigating the effect of water solubility on composition drift while keeping the reactivity ratios constant can elucidate the importance of the water solubility. The monomer combinations methyl acrylate-vinyl acetate (MA-VAc), methyl acrylate-vinyl 2,2-dimethylpropanoate (MA-VPV), and methyl acrylate-vinyl 2-ethylhexanoate (MA-V2EH) are ideal monomer combinations for studying the effect of water solubility on composition drift since the reactivity ratios for this series of monomer systems are approximately equal. Solution copolymerizations are performed to elucidate maximum composition drift at extremely high monomer-to-water ratios. From comparing theoretical predictions with experimental results it could be concluded that composition drift for the monomer combination MA-VAc could only be reduced since the difference in water solubility was not large enough to compensate the effects of the large difference in reactivity ratios. However, for the monomer combinations MA-VPV and MA-V2EH the difference in water solubility was large enough to make minimum composition drift possible for low monomer-to-water ratios even for monomer combinations with reactivity ratios as far apart as in the MA-vinyl ester case. © 1996 John Wiley & Sons, Inc.     

A validated positive chemical ionization GC/MS method for the identification and quantification of amphetamine, opiates, cocaine, and metabolites in human postmortem brain

A sensitive and specific method for the simultaneous detection and quantification of amphetamine, opiates, and cocaine and metabolites in human postmortem brain was developed and validated. Analytes of interest included amphetamine, morphine, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, and anhydroecgonine methyl ester. The method employed ultrasonic homogenization of brain tissue in pH 4.0 sodium acetate buffer and solid phase extraction. Extracts were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide and N,O-bis(trimethylsilyl) trifluoroacetamide. Separation and quantification were accomplished on a bench-top positive chemical ionization capillary gas chromatograph/mass spectrometer with selected ion monitoring. Eight deuterated analogs were used as internal standards. Limits of quantification were 50 ng/g of brain. Calibration curves were linear to 1000 ng/g for anhydroecgonine methyl ester and 6-acetylmorphine, and to 2000 ng/g for all other analytes. Accuracy across the linear range of the assay ranged from 90.2 to 112.2%, and precision, as percent relative standard deviation, was less than 16.6%. Quantification of drug concentrations in brain is a useful research tool in neurobiology and in forensic and postmortem toxicology, identifying the type, relative magnitude, and recency of abused drug exposure. This method will be employed to quantify drug concentrations in human postmortem brain in support of basic and clinical research on the physiologic, biochemical, and behavioral effects of drugs in humans.