Collision‐induced dissociation study of poly(2‐ethyl‐2‐oxazoline) using survival yields and breakdown curves

Poly(2‐ethyl‐2‐oxazoline), a synthetic polymer was analysed by mass spectrometry using different ion sources. Two distributions could be identified in the mass spectra which related to two different polymer series (one with hydrogen and hydroxyl end‐groups and the other with methyl and hydroxyl end‐groups). The fragmentation behaviour of the protonated oligomers was studied in a quadrupole time‐of‐flight mass spectrometer (MS) with electrospray, atmospheric pressure chemical ionization and direct analysis in real time soft ionization techniques. Three product ion series were identified in the MS/MS spectra independently of the ion source used. Based on the results, a mechanism was proposed for the dissociation by means of the accurate mass of the product ions, pseudo MS³ experiments and the energy dependence of the product ion intensity, i.e. breakdown curves. The survival yield method was used to highlight the correlation between the size of the oligomers and the laboratory frame collision energy. Copyright © 2012 John Wiley & Sons, Ltd.     

Negative charge induced dissociation: fragmentation of deprotonated N‐benzylidene‐2‐hydroxylanilines in electrospray ionization mass spectrometry

Unimolecular reactivities of different N‐benzylidene‐2‐hydroxylaniline anions were investigated in gas phase by electrospray ionization tandem mass spectrometry. All the collision‐induced dissociation spectra of N‐benzylidene‐2‐hydroxylaniline anions show similar ions at phenyl anions, neutral loss of benzonitrile and benzoxazole anions, respectively. The possible fragmentation pathway was probed through deuterium labeling and various group substituents experiments. Computational results were applied to shed light on the mechanism of fragmentation patterns. The proton in the CH=N is reactive in the formation of the concerned ions. Its direct transfer to the oxygen results in 2‐hydroxyphenyl anion. Proton abstraction between benzoxazole and phenyl anion leads to the formation of benzene and benzoxazole anion. Copyright © 2014 John Wiley & Sons, Ltd.     

Low‐energy collision‐induced dissociation (low‐energy CID), collision‐induced dissociation (CID), and higher energy collision dissociation (HCD) mass spectrometry for structural elucidation of saccharides and clarification of their dissolution mechanism in DMAc/LiCl

The dissolution mechanism of oligosaccharides in N,N‐dimethylacetamide/lithium chloride (DMAc/LiCl), a solvent used for cellulose dissolution, and the capabilities of low‐energy collision‐induced dissociation (low‐energy CID), collision‐induced dissociation (CID), and higher energy collision dissociation (HCD) for structural analysis of carbohydrates were investigated. Comparing the spectra obtained using 3 techniques shows that, generally, when working with monolithiated sugars, CID spectra provide more structurally informative fragments, and glycosidic bond cleavage is the main pathway. However, when working with dilithiated sugars, HCD spectra can be more informative providing predominately cross‐ring cleavage fragments. This is because HCD is a nonresonant activation technique, and it allows a higher amount of energy to be deposited in a short time, giving access to more endothermic decomposition pathways as well as consecutive fragmentations. The difference in preferred dissociation pathways of monolithiated and dilithiated sugars indicates that the presence of the second lithium strongly influences the relative rate constants for cross‐ring cleavages vs glycosidic bond cleavages, and disfavors the latter. Regarding the dissolution mechanism of sugars in DMAc/LiCl, CID and HCD experiments on dilithiated and trilithiated sugars reveal that intensities of product ions containing 2 Li⁺ or 3 Li⁺, respectively, are higher than those bearing only 1 Li⁺. In addition, comparing the fragmentation spectra (both HCD and CID) of LiCl‐adducted lithiated sugar and NaCl‐adducted sodiated sugar shows that while, in the latter case, loss of NaCl is dominant, in the former case, loss of HCl occurs preferentially. The compiled evidence implies that there is a strong and direct interaction between lithium and the saccharide during the dissolution process in the DMAc/LiCl solvent system.     

Characterization of multiple fragmentation pathways initiated by collision‐induced dissociation of multifunctional anions formed by deprotonation of 2‐nitrobenzenesulfonylglycine

The correlation of anion structure with the fragmentation behavior of deprotonated nitrobenzenesulfonylamino acids was investigated using tandem mass spectrometry, isotopic labeling and computational methods. Four distinct fragmentation pathways resulting from the collision‐induced dissociation (CID) of deprotonated 2‐nitrobenzenesulfonylglycine (NsGly) were characterized. The unusual loss of the aryl nitro substituent as HONO was the lowest energy process. Subsequent successive losses of CO, HCN and SO₂ indicated that an ortho cyclization reaction had accompanied loss of HONO. Other pathways involving rearrangement of the ionized sulfonamide group, dual bond cleavage and intramolecular nucleophilic displacement were proposed to account for the formation of phenoxide, arylsulfinate and arylsulfonamide product ions at higher collision energies. The four distinct fragmentation pathways were consistent with precursor–product relationships established by CID experiments, isotopic labeling results and the formation of analogous product ions from 2,4‐dinitrobenzenesulfonylglycine and the Ns derivatives of alanine and 2‐aminoisobutyric acid. The computations confirmed a low barrier for ortho cyclization with loss of HONO and feasible energetics for each reaction step in the four pathways. Computations also indicated that three of the fragmentation pathways started from NsGly ionized at the carboxyl group. Overall, the pathways identified for the fragmentation of the NsGly anion differed from processes reported for anions containing a single functional group, demonstrating the importance of functional group interactions in the fragmentation pathways of multifunctional anions. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of an exception to the ‘even‐electron rule’ upon low‐energy collision induced decomposition in negative ion electrospray tandem mass spectrometry

Electrospray‐generated precursor ions usually follow the ‘even‐electron rule’ and yield ‘closed shell’ fragment ions. We characterize an exception to the ‘even‐electron rule.’ In negative ion electrospray mass spectrometry (ES‐MS), 2‐(ethoxymethoxy)‐3‐hydroxyphenol (2‐hydroxyl protected pyrogallol) easily formed a deprotonated molecular ion (M‐H)^? at m/z 183. Upon low‐energy collision induced decomposition (CID), the m/z 183 precursor yielded a radical ion at m/z 124 as the base peak. The radical anion at m/z 124 was still the major fragment at all tested collision energies between 0 and 50 eV (E_lab). Supported by computational studies, the appearance of the radical anion at m/z 124 as the major product ion can be attributed to the combination of a low reverse activation barrier and resonance stabilization of the product ions. Furthermore, our data lead to the proposal of a novel alternative radical formation pathway in the protection group removal of pyrogallol. Copyright © 2009 John Wiley & Sons, Ltd.     

Glutathionylated γG and γA subunits of hemoglobin F: a novel post‐translational modification found in extremely premature infants by LC‐MS and nanoLC‐MS/MS

Oxidative stress plays an important role in the development of various disease processes and is a putative mechanism in the development of bronchopulmonary dysplasia, the most common complication of extreme preterm birth. Glutathione, a major endogenous antioxidant and redox buffer, also mediates cellular functions through protein thiolation. We sought to determine if post‐translational thiol modification of hemoglobin F occurs in neonates by examining erythrocyte samples obtained during the first month of life from premature infants, born at 23 0/7 – 28 6/7 weeks gestational age, who were enrolled at our center in the Prematurity and Respiratory Outcomes Program (PROP). Using liquid chromatography‐mass spectrometry (LC‐MS), we report the novel finding of in vivo and in vitro glutathionylation of γG and γA subunits of Hgb F. Through tandem mass spectrometry (nanoLC‐MS/MS), we confirmed the adduction site as the Cys‐γ94 residue and through high‐resolution mass spectrometry determined that the modification occurs in both γ subunits. We also identified glutathionylation of the β subunit of Hgb A in our patient samples; we did not find modified α subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of γG and γA of Hgb F occurs in premature infants. Additional studies of this post‐translational modification are needed to determine its physiologic impact on Hgb F function and if sG‐Hgb is a biomarker for clinical morbidities associated with oxidative stress in premature infants. Copyright © 2014 John Wiley & Sons, Ltd.     

Distinguishing of 2‐MAPB and 6‐MAPB: Solution of the problem

Differentiation of new psychoactive substance (NPS), 6‐(2‐methylaminopropyl)benzofuran (6‐MAPB), and its positional isomer, 2‐(2‐methylaminopropyl)benzofuran (2‐MAPB), by means of gas chromatography/mass spectrometry (GC/MS) with quadrupole detection is ambiguous. Reliable distinguishing of the two isomers could be achieved by MS/MS spectra recorded after collision‐induced dissociation (CID) of precursor ions. Both electron ionization (EI) and electrospray ionization (ESI) methods could be used for these purposes.     

离子相互作用模型在HCl-LiCl-MgCl2-H2O体系20˚C时溶解度预测中的应用

Component solubility in HCl-LiCl-MgCl2-H2O system of high ionic strength at 20℃ was predicted by using the Pitzer＇s ion-interaction model. The results indicated that the model supplied a very good prediction of the component solubility of the system mentioned above. The values of parameters of β^0, β^1 and C^＊ of HCl, LiCl and MgCl2 were obtained from optimization of literature data, while those of θMN and ψMNX were calculated from a least-squares optimization procedure to couple activity coefficient with solubility data. According to the ion-interaction model, no additional parameters need to be determined for more complex systems. The study provided theoretical basis for the manufacture process, which was proposed by Gao and employed to extract LiCl and MgCl2·6H2O from salt lake brine.     

Synthesis of polyethylene‐octene elastomer/SiO2‐TiO2 nanocomposites via in situ polymerization: Properties and characterization of the hybrid

In this study, a silicic acid and tetra isopropyl ortho titanate ceramic precursor and a metallocene polyethylene‐octene elastomer (POE) or acrylic acid grafted metallocene polyethylene‐octene elastomer (POE‐g‐AA) were used in the preparation of hybrids (POE/SiO₂? TiO₂ and POE‐g‐AA/SiO₂? TiO₂) using an in situ sol‐gel process, with a view to identifying a hybrid with improved thermal and mechanical properties. Hybrids were characterized using Fourier transform infrared spectroscopy, ²⁹Si solid‐state nuclear magnetic resonance (NMR), X‐ray diffraction, differential scanning calorimetry, thermogravimetry analysis, dynamic mechanical thermal analysis, and Instron mechanical testing. Properties of the POE‐g‐AA/SiO₂? TiO₂ hybrid were superior to those of the POE/SiO₂? TiO₂ hybrid. This was because the carboxylic acid groups of acrylic acid acted as coordination sites for the silica‐titania phase to allow the formation of stronger chemical bonds. ²⁹Si solid‐state NMR showed that Si atoms coordinated around SiO₄ units were predominantly Q₃ and Q₄. The 10 wt % SiO₂? TiO₂ hybrids gave the maximum values of tensile strength and glass transition temperature in both POE/SiO₂? TiO₂ and POE‐g‐AA/SiO₂? TiO₂. It is proposed that above this wt %, excess SiO₂? TiO₂ particles caused separation between the organic and inorganic phases. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1690–1701, 2005     

Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and specific high‐performance liquid chromatography mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of β‐hydroxy‐β‐methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water–acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C₈ (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30–4600 ng/mL (r > 0.998) for HMB. The intra‐ and inter‐day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench‐top, autosampler freeze–thaw cycles and long‐term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

1H, 13C and 15N NMR spectroscopic characterization of unexpected degradation products of the nifedipine analogue bis(2‐cyanoethyl) 2,6‐dimethyl‐4‐(2‐nitrophenyl)‐1,4‐dihydro‐3,5‐pyridinedicarboxylate

In the course of saponification experiments with bis(2‐cyanoethyl) 2,6‐dimethyl‐4‐(2‐nitrophenyl)‐1,4‐dihydro‐3,5‐pyridinedicarboxylate ( 1 ), an analogue of the calcium channel blocker nifedipine, three unexpected degradation products were isolated. The compounds were identified as 3‐(2‐acetamido‐1‐carboxy‐1‐propenyl)‐1‐hydroxy‐2‐indolecarboxylic acid ( 3 ), 9‐hydroxy‐1,3‐dimethyl‐β‐carboline‐4‐carboxylic acid ( 4 ) and 6‐hydroxy‐2,4‐dimethyl‐5‐oxo‐5,6‐dihydrobenzo[c][2,7]naphthyridine‐1‐carboxylic acid ( 6 ). The structures of these compounds were deduced from one‐ and two‐dimensional ¹H, ¹³C and natural abundance ¹⁵N NMR experiments (¹H,¹H‐COSY, gs‐HSQC, gs‐HMBC, ¹⁵N gs‐HMBC), and corroborated by comparison of their NMR data with the respective data for structurally similar compounds. Copyright © 2002 John Wiley & Sons, Ltd.     

Copper(I) complex of 1,3‐DimethylBarbituric acid modified SBA‐15 and its catalytic role for the synthesis of 2,3‐Dihydroquinazolin‐4(1H)‐ones and Imidazoles

An efficient one‐pot method for synthesis of 2,3‐dihydroquinazolin‐4(1H)‐ones and tri/tetra substituted‐1H‐imidazoles has been accomplished in the presence of catalytic amounts of Cu(I)‐1,3‐dimethylbarbituric acid modified SBA‐15 as heterogeneous catalyst with good to excellent yields. The catalyst is reusable and can be applied several times without any decrease in product yield. The synthesized catalyst was characterized by scanning electron microscopy (SEM), X‐ray diffraction (XRD), energy dispersive X‐ray spectroscopy (EDS), thermal gravimetric analysis (TGA), N₂ adsorption/desorption isotherms (BET), Fourier transform infrared spectroscopy (FT‐IR) and atomic absorption spectroscopy (AAS).     

The first automated 470.59 MHz 19F NMR‐controlled titration: dissociation constants and ion‐specific chemical shifts of 2‐amino‐4‐fluoro 2‐methylpent‐4‐enoic acid

2‐Amino‐4‐fluoro‐2‐methylpent‐4‐enoic acid, obtained as a 1 : 1 salt with trifluoro‐acetic acid, was characterized by ¹H and ¹⁹F high‐resolution NMR spectroscopy. High‐precision potentiometry led to the dissociation constants pK = 1.879 and pK = 9.054. The first automated 470.59 MHz ¹⁹F NMR‐controlled titration yielded the dynamic chemical shift 〈δ_F〉 as a function of pcH or τ and the ion‐specific chemical shifts: δ_F(H₂L⁺) = ?94.81 ppm, δ_F(HL) = ?94.21 ppm, δ_F(L^?) = ?92.45 ppm. The deprotonation gradients were found to be Δ₁ = ?0.60 ppm and Δ₂ = ?1.76 ppm. Copyright © 2002 John Wiley & Sons, Ltd.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

SPE–HPLC–MS/MS method for the trace analysis of tobacco‐specific N‐nitrosamines and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol in rabbit plasma using tetraazacalix[2]arene[2]triazine‐modified silica as a sorbent

Tobacco‐specific N‐nitrosamines (TSNAs), including N′‐nitrosonornicotine, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone, N′‐nitrosoanatabine, and N′‐nitrosoanabasine, have been implicated as a source of carcinogenicity in tobacco and cigarette smoke. We present a rapid and effective method comprising SPE based on tetraazacalix[2]arene[2]triazine‐modified silica as sorbent and analysis with HPLC–MS/MS for the determination of TSNAs and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), a metabolite of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone, in rabbit plasma. The linear dynamic ranges were 10–2000 pg/mL for NNAL and 4–2000 pg/mL for the four TSNAs with good correlation coefficients (>0.9965). The LODs were in the range of 0.9–3.7 pg/mL, and the LOQs were between 2.9 and 12.3 pg/mL. The accuracies of the method were also evaluated and found to be in the range of 90.1–113.3%. This method is promising to be applied to the preconcentration and determination of TSNAs and NNAL in smoke and human body fluids.     

Electrochemically Initiated Catalytic Oxidation of 5‐Thio‐2‐Nitrobenzoic Acid (TNBA) in the Presence of Thiols at a Boron Doped Diamond Electrode: Implications for Total Thiol Detection

The electrochemical response of 5,5‐dithiobis(2‐nitrobenzoic acid) (DTNB) to increasing additions of thiol species has been examined at a boron doped diamond electrode. A reaction has been shown to occur with a range of biologically relevant thiols and proceeds via a CECC' process. A total thiol detection methodology has been developed showing that the sensitivities of the standard addition plots are independent of the individual thiol species added to the solution. The analytical utility of the reaction process has been assessed using chronoamperometry with the corresponding data producing detection limits of 5.7 μM, 4.4 μM and 5.8 μM for the detection of cysteine, homocysteine and glutathione respectively.     

Malva sylvestris L. extract suppresses desferrioxamine‐induced PGE2 and PGD2 release in differentiated U937 cells: the development and validation of an LC‐MS/MS method for prostaglandin quantification

Malva sylvestris is a species used worldwide as an alternative to anti‐inflammatory therapies; however, its mechanism of action remains unknown. In this paper, the anti‐inflammatory effects of M. sylvestris alcoholic extracts were evaluated by measuring the pro‐inflammatory mediators PGE₂ and PGD₂ in desferrioxamine‐stimulated phorbol 12‐myristate 13‐acetate‐differentiated U937 cells. An HPLC‐DAD fingerprint of the M. sylvestris extract was performed and caffeic acid, ferulic acid and scopoletin were identified and quantified. An HPLC‐MS/MS method was developed and validated to separate and measure the prostaglandins. The lower limits of detection (~0.5 ng/mL for PGE₂ and PGD₂) and quantification (1.0 ng/mL for PGE₂ and PGD₂) indicated that the method is highly sensitive. The calibration curves showed excellent coefficients of correlation (r > 0.99) over the range of 1.0–500.0 ng/mL, and at different levels, the accuracy ranged from 96.4 to 106.4% with an RSD < 10.0% for the precision study. This method was successfully applied using U937‐d cells. A significant dose‐dependent reduction of PGE₂ and PGD₂ levels occurred using 10 µg/mL (10.74 ± 2.86 and 9.60 ± 6.89%) and 50 µg/mL of extract (48.37 ± 3.24 and 53.06 ± 6.15%), suggesting that the anti‐inflammatory mechanisms evoked by M. sylvestris may be related to modulation of these mediators. Copyright © 2014 John Wiley & Sons, Ltd.     

Supramolecular hydrogen‐bonding patterns in two cocrystals of the N(7)–H tautomeric form of N6‐benzoyladenine: N6‐benzoyladenine–3‐hydroxypyridinium‐2‐carboxylate (1/1) and N6‐benzoyladenine–DL‐tartaric acid (1/1)

Two novel cocrystals of the N(7)—H tautomeric form of N⁶‐benzoyladenine (BA), namely N⁶‐benzoyladenine–3‐hydroxypyridinium‐2‐carboxylate (3HPA) (1/1), C₁₂H₉N₅O·C₆H₅NO₃, (I), and N⁶‐benzoyladenine–DL‐tartaric acid (TA) (1/1), C₁₂H₉N₅O·C₄H₆O₆, (II), are reported. In both cocrystals, the N⁶‐benzoyladenine molecule exists as the N(7)—H tautomer, and this tautomeric form is stabilized by intramolecular N—H...O hydrogen bonding between the benzoyl C=O group and the N(7)—H hydrogen on the Hoogsteen site of the purine ring, forming an S(7) motif. The dihedral angle between the adenine and phenyl planes is 0.94 (8)° in (I) and 9.77 (8)° in (II). In (I), the Watson–Crick face of BA (N6—H and N1; purine numbering) interacts with the carboxylate and phenol groups of 3HPA through N—H...O and O—H...N hydrogen bonds, generating a ring‐motif heterosynthon [graph set R₂²(6)]. However, in (II), the Hoogsteen face of BA (benzoyl O atom and N7; purine numbering) interacts with TA (hydroxy and carbonyl O atoms) through N—H...O and O—H...O hydrogen bonds, generating a different heterosynthon [graph set R₂²(4)]. Both crystal structures are further stabilized by π–π stacking interactions.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid–liquid extraction, followed by derivatization of the extract and detection of β₂‐agonists trimethylsilyl‐derivatives by fast‐gas chromatography/electron impact–mass spectrometry (fast‐GC/EI‐MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra‐ and inter‐assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast‐GC/MS sequences, based on the use of short columns, high carrier‐gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS‐sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright © 2009 John Wiley & Sons, Ltd.