Silver (Ι)‐assisted enantiomeric analysis of ginsenosides using electrospray ionization tandem mass spectrometry

For identification of ginsenoside enantiomers, electrospray ionization mass spectrometry (ESI‐MS) was used to generate silver complexes of the type [ginsenoside + Ag]⁺. Collision induced dissociation of the silver‐ginsenoside complexes produced fragment ions by dehydration, allowing differentiation of ginsenoside enantiomers by the intensity of [M + Ag ? H₂O]⁺ ion. In the meanwhile, an approach based on the distinct profiles of enantiomer‐selective fragment ion intensity varied with collision energy was introduced to refine the identification and quantitation of ginsenoside enantiomers. Five pairs of enantiomeric ginsenosides were distinguished and quantified on the basis of the distribution of fragment ion [M + Ag ? H₂O]⁺. This method was also extended to the identification of other type of ginsenoside isomers such as ginsenoside Rb2 and Rb3. For demonstrating the practicability of this novel approach, it was utilized to analyze the molar ratio of 20‐(S) and 20‐(R) type enantiomeric ginsenosides in enantiomer mixture in red ginseng extract. The generation of characteristic fragment ion [M + Ag ? H₂O]⁺ likely results from the reduction of potential energy barrier of dehydration because of the catalysis of silver ion. The mechanism of enantiomer identification of ginsenosides was discussed from the aspects of computational modeling and internal energy. Copyright © 2012 John Wiley & Sons, Ltd.     

液相色谱-大气压化学电离质谱法分析人参中的人参皂甙

研究了用反相高效液相色谱-大气压化学电离质谱(HPLC/APCI-MS)分析人参皂甙的方法。液相色谱采用乙腈-水流动相进行梯度洗脱,质谱采用正负离子同时扫描并结合二级质谱进行定性,用选择反应离子模式(SRM)测定检测限。实验发现虽然人参皂甙是热不稳定物质,但在大气压化学电离质谱的高温汽化过程中仍能检测到很强的负离子分子离子峰,而且随着汽化温度的升高,人参皂甙的负离子分子离子峰的强度增加。该方法对人参皂甙Rb1和Rg1的检测限分别为1.2×10-13 g和3.0×10-14 g,并检测出白参中包括丙二酰人参皂甙在内的29种人参皂甙。该法灵敏度高,重复性好,结果准确,能有效地对药材提取物中的多种人参皂甙进行检测和结构分析。     

Mass spectrometric behavior of anabolic androgenic steroids using gas chromatography coupled to atmospheric pressure chemical ionization source. Part I: Ionization

The detection of anabolic androgenic steroids (AAS) is one of the most important topics in doping control analysis. Gas chromatography coupled to (tandem) mass spectrometry (GC–MS(/MS)) with electron ionization and liquid chromatography coupled to tandem mass spectrometry have been traditionally applied for this purpose. However, both approaches still have important limitations, and, therefore, detection of all AAS is currently afforded by the combination of these strategies. Alternative ionization techniques can minimize these drawbacks and help in the implementation of a single method for the detection of AAS. In the present work, a new atmospheric pressure chemical ionization (APCI) source commercialized for gas chromatography coupled to a quadrupole time‐of‐flight analyzer has been tested to evaluate the ionization of 60 model AAS. Underivatized and trimethylsylil (TMS)‐derivatized compounds have been investigated. The use of GC–APCI–MS allowed for the ionization of all AAS assayed irrespective of their structure. The presence of water in the source as modifier promoted the formation of protonated molecules ([M+H]⁺), becoming the base peak of the spectrum for the majority of studied compounds. Under these conditions, [M+H]⁺, [M+H‐H₂O]⁺ and [M+H‐2·H₂O]⁺ for underivatized AAS and [M+H]⁺, [M+H‐TMSOH]⁺ and [M+H‐2·TMSOH]⁺ for TMS‐derivatized AAS were observed as main ions in the spectra. The formed ions preserve the intact steroid skeleton, and, therefore, they might be used as specific precursors in MS/MS‐based methods. Additionally, a relationship between the relative abundance of these ions and the AAS structure has been established. This relationship might be useful in the structural elucidation of unknown metabolites. Copyright © 2014 John Wiley & Sons, Ltd.     

The use of linear ion trap for qualitative analysis of phytochemicals in Korean ginseng tea

A new approach to qualitative analysis of ginsenosides in challenging matrices was developed on the basis of high‐performance liquid chromatography/tandem mass spectrometry. Using the extracts from samples of ginseng tea, the approach was validated. Analysis of extracts was carried out using a reversed‐phase chromatography with SB‐C₁₈ sorbent. For compound identification, electrospray ionization and a quadrupole/linear ion trap mass‐spectrometer in different modes were used. A meticulous study of the fragmentation of ginsenosides in the linear ion trap and its application for analysis of these compounds was performed in this work. The accuracy of the identification was proven with standards of ginsenosides Rb1, Rg1, Re, Rf, Rd, Rb2, Rb3 and Rc. Copyright © 2012 John Wiley & Sons, Ltd.     

Fast liquid chromatography/multiple‐stage mass spectrometry of coccidiostats

Drugs that are used as medicines and also as growth promoters in veterinary care are considered as emerging environmental contaminants and in recent years concern about their potential risk to ecosystems and human health has risen. In this paper we used a method based on liquid chromatography/electrospray tandem mass spectrometry to analyze eight coccidiostatic compounds: diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, lasalocid, monensin, salinomycin, maduramicin and nasarin. Multiple‐stage mass spectrometry (MSⁿ) based on the precursor ions [M+Na]⁺ (polyether ionophores), [M+H]⁺ (robenidine) and [M–H]^? (diclazuril and dinitrocarbanilide) was used to study the fragmentation of these compounds. MSⁿ data and genealogical relationships were used to propose a tentative assignment of the different fragment ions. Loss of water, decarboxylations, ketone β‐cleavages and rearrangement of cyclic ethers and amide groups were some of the fragmentations observed for these compounds. Liquid chromatography with a sub‐2 µm particle size column was coupled to tandem mass spectrometry (LC/MS/MS) allowing the separation of these compounds in less than 7 min. Method detection limits ranging from 11 to 71 ng L^?1 and run‐to‐run values in terms of relative standard deviation (RSD) (up to 12%) were obtained. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS

A high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry( HPLC-ESI-MS/MS) method was developed for the analysis and identification of ginsenosides in the extracts of raw Panax ginseng(RPG) and steamed Panax ginseng at high temperatures(SPGHT). A total of 25 ginsenosides were extracted include of which 10 low-polar ginsenosides, such as ginsenosides F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3 and so on, were identified according to their HPLC retention time and MS/MS data. The results indicated that the low polar ginsenosides were seldom found in RPG. For the exploration of the transformation pattern of the ginsenosides in steam processing, the standards of ginsenosides Re, Rg1, Rb1, Rc, Rb2, Rb3 and Rd were selected and hydrolyzed at a temperature of 120 ℃. The results show that these polar ginsenosides can be converted to low-polar ginsenosides such as Rg2, Rg6, F4, Rk3 and Rg5 by hydrolyzing the sugar chains.     

Study of adduct ions of meso-phenyl-substituted tetrabenzoporphyrins by fast-atom bombardment mass spectrometry

Mass spectra of meso-phenyl-substituted tetrabenzoporphyrins were investigated by fast-atom bombardment mass spectrometry and tandem mass spectrometry. A cluster of adduct ions with mass-to-charge ratio values higher than the corresponding molecular ions of the porphyrins has been observed. The mass number differences among the series of cluster ions are constant depending on the para-phenyl substituents. Under certain conditions, dimers or trimers of molecular ions with low abundances have been detected. To trace the origin of the adduct ions, a series of experiments based on mass spectrometry have been carried out. The mass spectrum of tetrabenzoporphyrin showed no adduct ions with mass number differences of 90 even with the addition of phenylacetic acid. The mass spectrum of meso-tetraphenylte-trabenzoporphyrin ¹³C-labeled at the meso carbons showed adduct ions with mass number differences of 91. Product spectra of [2M + H]⁺ or [3M + H]⁺ of porphyrins exhibited adduct ions. All these results suggest that fragmentations of [2M + H]⁺ or [3M + H]⁺ may be one of the many possible routes to form the adduct ions, and the mass number differences among the series of these cluster ions should correspond to the benzyl group from the meso positions of meso-phenyl-substituted tetrabenzoporphyrins.     

Use of liquid chromatography/tandem mass spectrometry and online databases for identification of phosphocholines and lysophosphatidylcholines in human red blood cells

In this work a systematic strategy integrating liquid chromatography/tandem mass spectrometry (LC/MS/MS) and online databases was developed to identify phosphocholines (PC) and lysophosphatidylcholines (LPC) in human red blood cells (RBCs). First of all, the neutral loss scan of 59 and the precursor ion scan of m/z 184 were performed to find out the possible lipids with phosphocholine head‐group structure in RBCs. The acquired [M+H]⁺ and [M+Na]⁺ adduct ions were then identified online using the Human Metabolome Database (HMDB) and the LIPID MAPS, which were then further confirmed by their MS/MS fragmentation. Based on the comparison of chemical structures of the detected PC and LPC with their corresponding MS/MS fragmentation pathways, several new diagnostic fragments or fragmentation pathway were found. These include, (1) the neutral losses of 183 could be used as a diagnostic fragmentation to discriminate PC and LPC; (2) product ions at m/z 104 could be used to distinguish LPC and their sn‐2 isomers; (3) fragment ions at m/z 184 are characteristic fragmentation that could be used for discrimination of sodiated ions [M+Na]⁺ and protonated ions [M+H]⁺, especially for co‐eluted PC or LPC with a molecular weight difference of 22. The structures of the above‐mentioned fragment ions were confirmed by quadrupole time‐of‐flight (Q‐TOF) MS. Furthermore, a PC and LPC focused LC/MS semi‐quantification approach was also developed and validated. This approach could be useful for future lipidomic study. Copyright © 2009 John Wiley & Sons, Ltd.     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Tandem mass spectrometry of lithium-attachment ions from polyglycols

A detailed study has been carried out of the fast atom bombardment tandem mass spectrometry (MS/MS) behavior of lithium-attachment ions from three glycol polymers: linear poly(ethylene glycol), linear poly(propylene glycol), and an ethoxylated fatty alcohol. Collisional activation was carried out in the “collision octapole” of a BEoQ hybrid mass spectrometer at a translational energy of 50 eV, with collision gas air. It was found that [M + Li]⁺ ions provide a number of advantages as precursors for practical MS/MS analysis as compared to the use of [M + H]⁺ or [M + Na]⁺ ions. First, [M + Li]⁺ ions are much more intense than the corresponding [M + H]⁺ ions. Second, [M + Li]⁺ ions dissociate to lithiated organic fragments with reasonable efficiency, which is not the case with [M + Na]⁺ precursors. Third, product ions are generally formed over the entire mass range for low molecular weight polyglycols. The most intense product ions are lithiated, linear polyglycol oligomers. These ions are formed via internal hydrogen transfer reactions which are facilitated by lithium (charge-induced). Two series of less intense product ions are formed via charge-remote fragmentations involving l,4-hydrogen elimination. A fourth product ion series consists of lithiated radical cations; these form via homolytic bond cleavages near chain ends. Overall, MS/MS analysis of [M + Li]⁺ polyglycol ions proved to be quite useful for chemical structure elucidation.     

纳升电喷雾萃取电离质谱快速测定人参皂苷

以自行研制的纳升电喷雾萃取电离源（NanoEESI）为基础,建立了在无需色谱分离条件下快速测定人参皂苷的质谱分析方法,用NanoEESI串联质谱分析鉴定了人参中的多种人参皂苷,并将该方法用于人参、桔梗和商陆的快速鉴定。实验表明,人参中的皂苷易于在电离过程中结合钠离子,并形成[M+Na]+正离子从而被检测分析,而桔梗和商陆等样品中不含人参皂苷成份,在串联质谱中也没有相应的特征碎片离子,从而达到鉴别中药材真伪的目的。本方法简便、快速、灵敏、特征性强、重现性好,对道地中药材快速鉴定提供了思路,具有很好的实用价值。     

Differentiation of Regioisomeric Esters of Sucrose by Ionspray Tandem Mass Spectrometry

Abstract

Structural characterization and differentiation of distinct regioisomenc esters of sucrose were obtained using lonspray ionization and low energy tandem mass spectrometry. Low energy CAD MS/MS analyses of the protonated molecules [M + H]⁺ provided characteristic fingerprint patterns, and permitted differentiation of the various regioisomers. MS/MS analyses of selected intermediate fragment ions formed during the ionization process provided additional structural data, and established the fragmentation routes of their [M + H]⁺ precursors.     

A mechanism for the loss of 60 u from peptides containing an arginine residue at the C-terminus

The loss of 60 u from protonated peptide ions containing an arginine residue at the C-terminus has been investigated by means of low energy tandem mass spectrometry. The lowest energy conformation of singly charged bradykinin is thought to involve a salt-bridge structure, which may lead to the formation of two isomeric forms. It is thought that one isomer retains the ionizing proton at the C-terminal end of the peptide, leading to the formation of the [b _n?1 + H + OH]⁺ fragment ion, and the other isomer retains the charge at the N-terminus, leading to the formation of the [M + H ? 60]⁺ fragment ion. It was found that the formation of the [M + H ? 60]⁺ ion occurs only from singly charged precursor ions. In addition, the loss of 60 u occurs from peptides in which the charge is localized at the N-terminus. These results indicate that the mechanism of formation of the [M + H ? 60]⁺ ion may be driven by a charge-remote process.     

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.     

Rate and extent of gas-phase hydrogen/deuterium exchange of bradykinins: evidence for peptide zwitterions in the gas phase

The gas-phase H/D exchange of bradykinin [M + H]⁺, [M + Na]⁺, [M + 2H]²⁺, and [M + H + Na]²⁺ ions; des-Arg¹-bradykinin, des-Arg⁹-bradykinin, and bradykinin fragment 2-7 [M + H]⁺ ions; and O-methylbradykinin [M + H]⁺ and [M + 2H]²⁺ ions with D₂O have been examined by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at 9.4 T. The different peptides vary widely in exchange rate and extent of deuterium incorporation. H/D exchange was slowest and deuterium incorporation was least for bradykinin [M + H]⁺, [M + H + Na]²⁺ and bradykinin methyl ester [M + 2H]²⁺ ions. In contrast, H/D exchange and extent of deuteration are higher for des-Arg¹-bradykinin, des-Arg⁹-bradykinin, and bradykinin fragment 2-7 [M + H]⁺ ions; and highest for bradykinin [M + Na]⁺ and [M + 2H]²⁺, and O-methylbradykinin [M + H]⁺. Because the most likely site of protonation is the guanidino group of arginine, the above reactivity pattern strongly supports a zwitterion form for protonated gas-phase bradykinin.     

The observation of broad metastable ion peaks in the surface-induced dissociation mass spectra of protonated and cationated peptides

The product ion mass spectra of protonated and cationated peptides of relative molecular mass (RMM) 555–574 Da have been obtained by surface-induced dissociation of MH⁺ and [M + Cat]⁻ ions in a four-sector tandem mass spectrometer equipped with a specially designed collision cell. A linked scan of the electric and magnetic sector field strengths of the second mass spectrometer was used to transmit the fragment ions arising from collisions with a stainless steel surface. The resulting mass spectra contained broad metastable ion peaks produced by the dissociation of MH⁺ and [M + Cat]⁺ ions before the second magnetic sector, in the fourth field-free region of the instrument.     

Mechanism of formation of alkali metal cation adducts in plasma desorption mass spectrometry of biomolecules

Experimental data on correlations between the directions of the incident primary ion and the ejected protonated [M + H]⁺ and alkali metal adduct [M + Cs]⁺ molecules for three peptide samples with an incident beam of 72.3 MeV ¹²⁷I¹⁴⁺ ions are reported. Measurements were carried out in a linear time-of-flight mass spectrometer by monitoring the secondary ion yield as a function of electrostatic deflection in a direction perpendicular to the spectrometer axis. No difference was observed in the direction in which [M + H]⁺ and [M + Cs]⁺ ionic species are preferentially desorbed. The results obtained suggest that alkali metal cation attachment to biomolecules in plasma desorption mass spectrometry is realized in a close spatial location and time interval with protonation. Formation of ion-molecule complexes occurs at an early stage of the desorption event and precedes their ejection into the gas phase.     

高效液相色谱-飞行时间质谱联用法分析人参及人参皂苷与山楂配伍过程中的水解行为

利用高效液相色谱-飞行时间质谱联用的方法,分别对人参配伍山楂前后人参皂苷的变化进行分析,同时对人参皂苷Re、Rg1、Rb1、Rd与山楂配伍的水解规律进行系统研究,并与单独煎煮液、仿山楂配伍pH值煎煮液的水解产物进行比较,结果发现人参与山楂配伍后人参皂苷Rg1、Rb1含量明显减少,而人参皂苷Re、Rd、Rg2、Rg3、F2、Rh1含量明显增加,其中人参皂苷Re与山楂配伍后水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2,仿山楂配伍pH值水解产物为人参皂苷20(R)-Rg2、20(S)-Rg2、Rg4、Rg6;人参皂苷Rg1与山楂配伍后水解产物为20(S)-Rh1、20(R)-Rh1,仿山楂pH值水解产物为20(S)-Rh1、20(R)-Rh1、Rh4、Rk3;人参皂苷Rb1与山楂配伍后水解产物为Rd、20(S)-Rg3,仿山楂pH值水解产物为F2、20(S)-Rg3;人参皂苷Rd与山楂配伍后水解产物为F2、20(S)-Rg3、20(R)-Rg3,仿山楂pH值水解产物为20(S)-Rg3、20(R)-Rg3。研究表明,不同人参皂苷和山楂配伍后与仿山楂pH值的水解产物并不相同,人参与山楂配伍改变了人参皂苷成分的种类及含量。本研究为临床方剂中人参与山楂配伍后成分的变化提供物质基础数据。     

Determination of ginsenosides (ginseng saponins) in dry root powder from Panax ginseng, Panax quinquefolius, and selected commercial products by liquid chromatography: interlaboratory study

Twelve collaborating laboratories assayed 4 products, namely, Panax ginseng, Panax quinquefolius, and 2 ginseng products, for 6 ginsenosides: Rb1, Rb2, Rc, Rd, Re, and Rg1. Collaborators also received a negative control for the recovery study. Pure ginsenosides were provided as reference standards for the liquid chromatography (LC) analysis and the system suitability tests. The LC analyses were performed on the methanol extract using UV detection at 203 nm. For P. ginseng, individual ginsenosides were consistent in their means; repeatability standard deviations (RSDr) ranged from 4.17 to 5.09% and reproducibility standard deviations (RSDR) ranged from 7.27 to 11.3%. For P. quinquefolius, the Rb1 and Rb2 ginsenosides were higher and lower in concentration than P. ginseng, with RSDr values of 3.44 and 6.60% and RSDR values of 5.91 and 12.6% respectively, and other analytes at intermediate precisions. For ginseng commercial products, RSDr values ranged from 3.39 to 8.12%, and RSDR values ranged from 7.65 to 16.5%. A recovery study was also conducted for 3 ginsenosides: Rg1, Re, and Rb1. The average recoveries were 99.9, 96.2, and 92.3%, respectively. The method is not applicable for the determination of Rg1 and Re in ginseng product at levels <300 mg/kg.     

Field desorption mass spectrometry of oligosaccharides in the presence of metallic salts

In the field desorption mass spectrometry of oligosaccharides, traces of contaminating salts in polar solvents decrease the detection sensitivity, and in some cases no ion could be detected in the molecular range. The present work shows that the addition of alkaline salts (NaI or KI) greatly increases the sensitivity, and cationized molecular ions are obtained. The influence of the molar ratio sugar/salt on the nature and relative intensity of the desorbed species has been studied on mixtures of α-D -trehalose and NaI. At the low salt ratio (100:1), high molecular weight clusters [X M + Na]⁺ are preferentially formed. At the high salt ratio (1:10), mixed clusters [M + NaI + Na]⁺, doubly charged ions [M + 2Na]²⁺ and monomeric cationized ions [M + Na]⁺ are observed along with salt clusters [NaI + Na]⁺ and [2NaI + Na]⁺. In the range of molar ratios sugar/salt of 10:1 to 1:1, the field desorption mass spectra exhibit a cationized ion [M + Na]⁺, which contributes more than 80% of Σ₁₀₀. This cationization technique has been applied to the field desorption mass spectrometry of several oligosaccharides. In all cases, the salt effect causes the replacement of the [M + H]⁺ ion by a [M + C]⁺ ion. (Note: the term [M + C]⁺ and similar ones mean an association between the whole molecule studied (M) and an alkaline cation [C]⁺ ([K)]⁺, [Na]⁺, [Li]⁺).) Thus, di, tri- and tetrasaccharides exhibit intense [M + Na]⁺ ions in the presence of NaI and only a few fragment peaks are observed in their field desorption mass spectra. This technique has been applied successfully to the detection of the hexasaccharide ajugose in a natural sample of pentasaccharide, and has also allowed the unambiguous determination of the composition of a mixture of partially methylated trehaloses. The salt effects are dicussed in terms of selective adsorption on the emitter, pre-existing soluble complexes sugar-salt, interactions between these species in the electric field and dissociative desorption of ionic complexes.