Removal of ceftriaxone and cefotaxime antibiotics from industrial wastewater via complexation with Cu(II).

A high-performance liquid chromatographic method (HPLC) with UV detection is described for determination of ceftriaxone sodium (CFX) and cefotaxime sodium (CFM) content in pharmaceutical industrial wastewater. These methods are based on the detection of these antibiotics via the formation of chelate complexes with Cu(II). The developed Liquid Chromatographic method offers symmetric peak shape, good resolution and reasonable retention time for both drugs. The removal percentage reached about 100 and 92.1% at pH 7.2 for CFX and CFM, respectively. In UV detection, the removal of the chelating antibiotics were based on forming of chelate complexes with Cu(II) which detected at λmax = 253 and 244 nm for CFX and CFM, respectively. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5.99-59.86 µg mL-1 for CFX and 14.33-71.63 µg mL-1 for CFM. The proposed method can be used for the quality control of industrial wastewater containing CFX and CFM.

Optimization of a Solid-Phase Extraction Coupled with a High-Performance Liquid Chromatography and Diode Array Ultraviolet Detection Method for Monitoring of Different Antibiotic Class Residues in Water Samples.

BACKGROUND: The increased use of cephalosporin antibiotics in the last few years as well as the detection of their residues in wastewater treatment plants and hospital wastewater poses a risk for infiltration of their residues into environmental water samples. OBJECTIVE: A simplified, sensitive, and convenient solid-phase extraction (SPE) procedure coupled with either HPLC or fast HPLC methods with diode array detection was developed and validated to screen the residues of six different cephalosporin antibiotics: cefoperazone, cefipime, ceftazedime, ceftriaxone, cefdinir, and cefotaxime, along with amoxicillin, levofloxacin, and ciprofloxacin in water samples. METHODS: An HPLC-diode array detector (HPLC-DAD) method and a fast HPLC method, based on a core-shell stationary phase, were developed for the fast screening of the antibiotic compounds. In addition, the SPE step was optimized to enable the extraction of the studied drugs with high accuracy of the recovered amounts of residues. RESULTS: The method sensitivity was enhanced by the coupling of SPE with HPLC-DAD and fast HPLC to achieve low LODs; from 0.2 to 3.8 ng/mL and from 0.65 to 12.2 ng/mL, respectively. The developed methods were augmented by LC-MS/MS determination for confirmation of identity and quantity of any positively identified sample. The method was applied to the analysis of water samples collected from a rural site. In Addition, an example application of cleaning validation of cefotaxime-contaminated stainless-steel surfaces was provided. CONCLUSION: The method's simplicity and high sensitivity encourage its application in monitoring of antibiotic residues in different types of water samples such as environmental samples and samples from cleaning validation activities. HIGHLIGHTS: HPLC-DAD and fast HPLC methods were developed for separation of nine different antibiotics. The combination with the SPE procedure achieved low detection limits; from 0.2 to 3.8 ng/mL for SPE-HPLC-DAD and from 0.65 to 12.2 ng/mL for SPE-fast HPLC.

Development of a comprehensive two-dimensional liquid chromatographic mass spectrometric method for the non-targeted identification of poly- and perfluoroalkyl substances in aqueous film-forming foams.

In this research, we developed an online comprehensive two-dimensional liquid chromatographic (LC × LC) method hyphenated with high-resolution mass spectrometry (HRMS) for the non-targeted identification of poly- and perfluorinated compounds (PFASs) in fire-fighting aqueous-film forming foams (AFFFs). The method exploited the combination of mixed-mode weak anion exchange-reversed phase with a octadecyl stationary phase, separating PFASs according to ionic classes and chain length. To develop and optimize the LC × LC method we used a reference training set of twenty-four anionic PFASs, representing the main classes of compounds occurring in AFFFs and covering a wide range of physicochemical properties. In particular, we investigated different modulation approaches to reduce injection band broadening and breakthrough in the second dimension separation. Active solvent and stationary phase assisted modulations were compared, with the best results obtained with the last approach. In the optimal conditions, the predicted peak capacity corrected for undersampling was higher than three-hundred in a separation space of about 60 min. Subsequently, the developed method was applied to the non-targeted analysis of two AFFF samples for the identification of homologous series of PFASs, in which it was possible to identify up to thirty-nine potential compounds of interest utilizing Kendrick mass defect analysis. Even within the samples, the features considered potential PFAS by mass defect analysis elute in the chromatographic regions discriminating for the ionic group and/or the chain length, thus confirming the applicability of the method presented for the analysis of AFFF mixtures and, to a further extent, of environmental matrices affected by the AFFF.

Establishment of an Agrobacterium tumefaciens-mediated transformation system for Tilletia foetida.

Tilletia foetida causes wheat common smut disease with severe loss of yield production and seed quality. In this study, a low-cost, rapid, and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for T. foetida mutagenesis was constructed: Transformants were screened with hygromycin B at 100 µg/ml, cefotaxime sodium concentrations with 200 µg/ml, Acetosyringone (AS) concentration at 200 µmol/l, 1 × 106 T. foetida hypha cells/ml, co-cultivation at 22 °C with 24 h and culture was incubated at 16 °C up to day 7. Fourteen transformants were randomly selected and confirmed using the specific primers to amplify the fragment of hygromycin phosphotransferase gene. At the same time, PCR analysis was performed to detect Agrobacterium tumefaciens Vir gene to eliminate false positives. The transformants were cultivated up to 8 generations on hygromycine B-containing complete medium (CM) and confirmed by PCR. The results indicated that 80% of T. foetida transformants were hygromycine B resistant. In conclusion, our analyses identified an efficient T-DNA insertion system for T. foetida and the results will be useful for further understanding the pathogenic mechanism via generation of the insertional mutants.

The first isolation of Clostridium difficile RT078/ST11 from pigs in China.

We investigated the molecular characteristics and antimicrobial susceptibility of Clostridium difficile isolated from animals in China. We obtained 538 rectal swabs from pigs, chickens and ducks in 5 provinces during 2015 and 2016. C. difficile isolates were characterized by detection of toxin genes, multilocus sequence typing and ribotyping. And antimicrobial susceptibility testing was performed using the agar dilution method. Out of 538 samples, 44 (8.2%) were C. difficile positive with high prevalence in pigs (n = 31). Among these, 39 (88.6%) were toxigenic including 14 (31.8%) that were A+B+CDT+ and 13 (29.5%) A+B+. The remaining 12 (27.3%) were A-B+. We identified 7 ST types and 6 PCR ribotypes. The most predominant type was ST11/RT078 with toxin profile A+B+CDT+ and all were isolated from piglets with diarrhea. ST109 isolates possessed two different toxigenic profiles (A-B-CDT- and A-B+CDT-) and although it was not the most prevalent sequence type, but it was widely distributed between chickens, ducks and pigs in the 5 provinces. All C. difficile isolates were fully susceptible to vancomycin, metronidazole, fidaxomicin, amoxicillin/clavulanate and meropenem but retained resistance to 4 or 5 of the remaining antibiotics, especially cefotaxime, tetracycline, ciprofloxacin, cefoxitin. The RT078/ST11 isolates were simultaneously resistant to cefotaxime, tetracycline, cefoxitin, ciprofloxacin and imipenem. This is the first report of the molecular epidemiology of C. difficile isolated from food animals in China. We identified the epidemic strain RT078/ST11 as the predominate isolate among the animals we screened in our study.

Determination of cefotaxime, cefoperazone, ceftazidime and cefadroxil using surface plasmon resonance band of silver nanoparticles

The aim of this work is to evaluate simple, sensitive, effective and validated procedures for the determination of cefotaxime, cefoperazone, ceftazidime and cefadroxil. In this study, the methods based on the ability of the cited drugs to reduce Ag+ ions to silver nanoparticles (Ag-NPs) in the presence of Polyvinyl Pyrrolidone (PVP) as a stabilizing agent producing very intense surface plasmon resonance peak of Ag-NPs (λmax. = 410-430 nm). The plasmon absorbance of the Ag-NPs allows the quantitative spectrophotometric determination of the cited drugs. The calibration curves are linear with concentration ranges of 0.4-3.2, 1-8, 0.5-4.0 and 1.5-9.0 µg/mL for cefotaxime, cefoperazone, ceftazidime and cefadroxil, respectively. Apparent molar absorptivity, detection and quantitative limits are calculated. Applications of the proposed methods to representative pharmaceutical formulations are successfully presented. The extracellular synthesis of nanoparticles is fast, and the method doesn't require various elaborate treatments and tedious extraction procedures.

Relationship between the color stability and impurity profile of cefotaxime sodium.

The color grade, mainly introduced in the processes of semisynthesis and storage, is an important index used to evaluate the quality of cefotaxime sodium. Because the drug itself is prone to degradation under susceptible conditions, including those involving moisture, heat, ultraviolet light, acids, alkalis, and oxidants, and a series of degradation products as impurities are generated. In this study, the factors affecting color grade stability and the degradation mechanisms of cefotaxime sodium were investigated by designing different accelerated stability tests under the aforementioned conditions. The degradation extent was studied by using analytical methods, such as a solution color comparison method, ultraviolet spectrophotometry, and HPLC. The relationship between the color grade stability of cefotaxime sodium and its impurity profile has been explored, and a reasonable degradation mechanism has been proposed. The manufacturing conditions of inspection have been optimized, and a scientific basis for drug packaging, storage, and transportation conditions has been established. The results show that the color grade stability of cefotaxime sodium is related to the impurity profile to some degree, and the difference between the actual color and the standard color can reflect the levels of impurities to some extent.

A Critical Review of Analytical Methods for Quantification of Cefotaxime.

Bacterial resistance to antibiotics is a growing phenomenon in the world. Considering the relevance of antimicrobials for population and the reduction in the registration of new antimicrobials by regulatory, proper quality control is required in order to minimize the spread of bacterial resistance and ensure the effectiveness of a treatment, as well as safety for the patient. Among the antimicrobials is cefotaxime, a drug belonging to third-generation cephalosporins, which is highly active against Gram-negative bactéria and is used to treat central nervous system infections such as meningitis and septicemia. Due to the critical importance of quality control in regard to drugs and pharmaceutical products, combined with bacterial resistance to antibiotics, this study aims to conduct a detailed review of analytical methods for cefotaxime. Using a critical review of literature, this paper describes the analytical methods published to quantify cefotaxime in different matrices; a large number of methods by HPLC and spectrophotometry were observed. Despite the advantages of the techniques, most methods reported have large environment and occupational impact, which enfatizes the need to adopt green procedures in quantifying cefotaxime.

Application of acetone acetals as water scavengers and derivatization agents prior to the gas chromatographic analysis of polar residual solvents in aqueous samples.

The sensitivity of gas chromatography (GC) combined with the full evaporation technique (FET) for the analysis of aqueous samples is limited due to the maximum tolerable sample volume in a headspace vial. Using an acetone acetal as water scavenger prior to FET-GC analysis proved to be a useful and versatile tool for the analysis of high boiling analytes in aqueous samples. 2,2-Dimethoxypropane (DMP) was used in this case resulting in methanol and acetone as reaction products with water. These solvents are relatively volatile and were easily removed by evaporation enabling sample enrichment leading to 10-fold improvement in sensitivity compared to the standard 10µL FET sample volumes for a selection of typical high boiling polar residual solvents in water. This could be improved even further if more sample is used. The method was applied for the determination of residual NMP in an aqueous solution of a cefotaxime analogue and proved to be considerably better than conventional static headspace (sHS) and the standard FET approach. The methodology was also applied to determine trace amounts of ethylene glycol (EG) in aqueous samples like contact lens fluids, where scavenging of the water would avoid laborious extraction prior to derivatization. During this experiment it was revealed that DMP reacts quantitatively with EG to form 2,2-dimethyl-1,3-dioxolane (2,2-DD) under the proposed reaction conditions. The relatively high volatility (bp 93°C) of 2,2-DD makes it possible to perform analysis of EG using the sHS methodology making additional derivatization reactions superfluous.

Spectrophotometric complexation of cephalosporins with palladium (II) chloride in aqueous and non-aqueous solvents.

The complexation reaction of cephalosporins namely cefotaxime (CTX), cefuroxime (CRX), and cefazolin (CEFAZ) with palladium (II) ions have been studied in water and DMF in 25 °C by the spectrophotometric methods. The method is based on the formation of yellow to yellowish brown complex between palladium (II) chloride and the investigated cephalosporins in the presence of sodium lauryl sulfate (SLS) as surfactant. The complexation process was optimized in terms of pH, temperature and contact time. The stoichiometry of all the complexes was found to be 2:1 (metal ion/ligand) for CTX, CRX, and 1:2 for CEFAZ. The stoichiometry of palladium (II)-cephalosporins was estimated by mole ratio and continuous variation methods and emphasized by the KINFIT program. These drugs could be determined by measuring the absorbance of each complex at its specific λmax. The results obtained are in good agreement with those obtained using the official methods. The proposed method was successfully applied for the determination of these compounds in their dosage forms.

Kendrick-analogous network visualisation of ion cyclotron resonance Fourier transform mass spectra: improved options for the assignment of elemental compositions and the classification of organic molecular complexity.

Here, we propose a novel computational and visual approach for the analysis of high field Fourier transform ion cyclotron resonance mass spectra (FTICR/MS) based on successive and multiple atomic and Kendrick analogous mass difference analyses. Compositional networks based on elemental compositions and functional networks based on selected functional groups equivalents enable improved assignment options of elemental composition and classification of organic complexity with tunable validation windows. The approach is demonstrated through the analysis of a 12T FTICR mass spectrum of an intricate water soluble extract of a secondary organic aerosol with a previously established abundance in CHNOS molecules.

Chemiluminescence determination of cefotaxime sodium with flow-injection analysis of cerium (IV)-rhodamine 6G system and its application to the binding study of cefotaxime sodium to protein with on-line microdialysis sampling.

A simple, sensitive and rapid flow-injection chemiluminescence (CL) method has been developed for the determination of cefotaxime sodium based on the chemiluminescence reaction of cefotaxime sodium with ceric sulfate and rhodamine 6G in nitric acid solution. The concentration of cefotaxime sodium was proportional with the CL intensity in the range of 4×10(-8)-8×10(-6) mol L(-1). The detection limit (signal-to-noise ratio=3) was 1×10(-8) mol L(-1). Coupled to the technique of on-line microdialysis sampling, this method was successfully applied to study cefotaxime sodium-protein interaction. The drug and protein were mixed in different molar ratios in Ringer's solution, pH 7.4, and incubated at 37°C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 µL min(-1) and the recovery of cefotaxime sodium under experimental condition was 16.2%. The data obtained by the present Microdialysis-Flow Injection Analysis-CL method was analyzed with the Scatchard analysis and Klotz plot. The estimated association constant (K) and the number of the binding sites (n) on one of BSA molecule were 5.94×10(4) M(-1) and 1.29 (Klotz equation), respectively.

Method for the identification of lipid classes based on referenced Kendrick mass analysis.

A rapid method for the determination of lipid classes with high sensitivity is described. The referenced Kendrick mass defect (RKMD) and RKMD plots are novel adaptations of the Kendrick mass defect analysis that allows for the rapid identification of members of a homologous series in addition to identifying the lipid class. Assignment of lipid classes by the RKMD method is accomplished by conversion of the lipid masses to the Kendrick mass scale and then referencing the converted masses to each lipid class. Referencing of the masses to a given lipid class is achieved by first subtracting the heteroatom and lipid backbone contributions to the mass defect, leaving behind the contribution to the mass by the fatty acid constituents. The final step in the referencing makes use of spacing differences in mass defects between members of the same Kendrick class to identify members of the lipid class being referenced. The end result of this is that a lipid belonging to the class being referenced will have an integer RKMD with the value of the integer being the degrees of unsaturation in the lipid. The RKMD method was able to successfully identify the lipids in an idealized data set consisting of 160 lipids drawn from the glyceride and phosphoglyceride classes. As a real world example the lipid extract from bovine milk was analyzed using both accurate mass measurements and the RKMD method.

Simultaneous multiresponse optimization of an HPLC method to separate seven cephalosporins in plasma and amniotic fluid: application to validation and quantification of cefepime, cefixime and cefoperazone.

An HPLC method for the separation of seven cephalosporins [Cefepime (CEP), ceftazidime (CTA), ceftizaxime (CTI), ceftriaxone (CTR), cefotaxime (COT), cefixime (CIX) and cefoperazone (COP)] in human plasma and amniotic fluid has been developed. Optimization of the chromatographic method was performed in three steps: a series of initial experiments followed by two sets of experiments based on different experimental designs. The initial experiments were performed to decide the basic analytical requirements of the method. Then screening experiment fractional factorial design was used in order to decrease the number of parameters by eliminating parameters which having insignificant effect on responses. The parameters having significant effect were further optimized through a full factorial design. Having studied two responses (retention times and resolutions), a desirability function that assess the responses together, was used to find experimental conditions where the system generated desirable results. The desirable results were obtained with XTerra C18 (250 mm x 4.6mm, 5 microm i.d.) column, 40 mM phosphate buffer, pH 3.2, 18% MeOH, 0.85 mL min(-1) flow rate and 32 degrees C column temperature. Gradient elution with MeOH was applied. A simple and efficient solid-phase extraction was applied for the preparation of plasma and amniotic fluid samples. The validation parameters of the method were evaluated in accordance with ICH guideline. The method validated was applied to the analysis of CEP and COP in maternal venous, fetal venous and fetal arterial plasma, and to the analysis of CIX in maternal venous plasma and amniotic fluid.

Effects of Chai-Qin-Cheng-Qi Decoction on cefotaxime in rats with acute necrotizing pancreatitis.

AIM: To investigate the effect of Chai-Qin-Cheng-Qi Decoction (CQCQD) on cefotaxime (CTX) concentration in pancreas of rats with acute necrotizing pancreatitis (ANP). METHODS: Sixty healthy male Sprague-Dawley rats were divided randomly into an ANP group (ANP model + CTX, n = 20), treatment group (ANP model + CTX + CQCQD, n = 20) and control group (normal rats + CTX, n = 20). ANP models were induced by retrograde intraductal injection of 3.5% sodium taurocholate (1 mL/kg), and the control group was injected intraductally with normal saline. All rats were injected introperitoneally with 0.42 g/kg CTX (at 12-h intervals for a continuous 72 h) at 6 h after intraductal injection. Meanwhile, the treatment group received CQCQD (20 mL/kg) intragastrically at 8-h intervals, and the ANP and control group were treated intragastrically with normal saline. At 15 min after the last CTX injection, blood and pancreas samples were collected for the determination of CTX concentration using validated high-performance liquid chromatography. Pathological changes and wet-to-dry-weight (W/D) ratio of pancreatic tissue were examined. RESULTS: Serum CTX concentrations in three groups were not significantly different. Pancreatic CTX concentration and penetration ratio were lower in ANP group vs control group (4.4 +/- 0.6 microg/mL vs 18.6 +/- 1.7 microg/mL, P = 0.000; 5% vs 19%, P = 0.000), but significantly higher in treatment group vs ANP group (6.4 +/- 1.7 microg/mL vs 4.4 +/- 0.6 microg/mL, P = 0.020; 7% vs 5%, P = 0.048). The histological scores and W/D ratio were significantly decreased in treatment group vs ANP and control group. CONCLUSION: CQCQD might have a promotive effect on CTX concentration in pancreatic tissues of rats with ANP.

Separation and determination of cefotaxime enantiomers in injections by capillary zone electrophoresis.

Cefotaxime enantiomers have specific effects on Gram-negative bacteria. For quality control of cefotaxime it was necessary to establish a method for enantioseparation by capillary zone electrophoresis (CZE) using cyclodextrin (CD) as a chiral selector. The effects of various parameters on enantioseparation were studied. A fused silica capillary (40 cm effective length x 75 microm ID) was used. The cefotaxime enantiomers were separated on the baseline under conditions of 0.5 mmol/L CM-beta-CD, 75 mmol/L NaH2PO4 buffer at pH 7.0 using UV detection at 280 nm. Applied voltage and capillary temperature were 20 kV and 25 degrees C, respectively. Under these conditions for enantioseparation, linear calibration curves were obtained in the range 2 approximately 160 microg/mL. The limit of detection for both isomers was less than 0.5 microg/mL. The method was used for analysis of pharmaceutical preparations (dosage forms) of cefotaxime from various factories. A simple and specific CZE method was successfully demonstrated for the separation of cefotaxime enantiomers. The enantioseparation method should be established and this method should be used to control the quality of cefotaxime.

Trace analysis of cefotaxime at carbon paste electrode modified with novel Schiff base Zn(II) complex.

Cefotaxime a third generation cephalosporin drug estimation in nanomolar concentration range is demonstrated for the first time in aqueous and human blood samples using novel Schiff base octahedral Zn(II) complex. The cefotaxime electrochemistry is studied over graphite paste and Zn(II) complex modified graphite paste capillary electrodes in H(2)SO(4) (pH 2.3) using cyclic voltammetry and differential pulse voltammetry. Cefotaxime enrichment is observed over Zn(II) complex modified graphite paste electrode probably due to interaction of functional groups of cefotaxime with Zn(II) complex. Possible interactions between metal complex and cefotaxime drug is examined by UV-vis and electrochemical quartz crystal microbalance (EQCM) techniques and further supported by voltammetric analysis. Differential pulse voltammetry (DPV) with modified electrode is applied for the determination of cefotaxime in acidified aqueous and blood samples. Cefotaxime estimation is successfully demonstrated in the range of 1-500 nM for aqueous samples and 0.1-100 microM in human blood samples. Reproducibility, accuracy and repeatability of the method are checked by triplicate reading for large number of samples. The variation in the measurements is obtained less than 10% without any interference of electrolyte or blood constituents.

A comparative study of capillary zone electrophoresis and pH-potentiometry for determination of dissociation constants.

Acidity constants of six cephalosporin antibiotics, cefalexin, cefaclor, cefadroxil, cefotaxim, cefoperazon and cefoxitin are determined using capillary zone electrophoresis (CZE) and pH-potentiometric titrations. Since CZE is a separation method, it is not necessary for the samples to be of high purity and known concentration because only mobilities are measured. The effect on determination of dissociation constants of different matrices (serum, 0.9% NaCl, fermentation matrix) was examined. The advantages of CZE can be utilized in those fields where potentiometry has limitations (sample quantity, solubility, purity, simultaneous determinations), although pK(a) values that are close to each other can be determined by potentiometry with more accuracy.

Study on the compatibility of cefotaxime with tinidazole in glucose injection.

An efficient HPLC method for the compatibility study of cefotaxime with tinidazole in glucose injection is described, which has been developed for the simultaneous determination of cefotaxime and tinidazole in glucose injection. The appearance and pH value of the mixed solution were investigated and the concentrations of cefotaxime and tinidazole were determined by RP-HPLC with an Agilent ZORBAX Eclipse XDB-C8 column, gradient elution and dual wavelength detection on diode-array-detector (DAD) at room temperature (20 degrees C) within 24 h. It was found that the resulting appearance and pH value of the mixed solution showed slight changes, on the other hand, the quantity of cefotaxime decreased significantly. The results show that the mixed solution of cefotaxime with tinidazole in glucose injection must be used within 8 h in clinical due to the possible degradation of cefotaxime in tinidazole glucose injection. This study provides a convenient method for rational use of compatible drugs in clinical practice.

Acidity constants of cefetamet, cefotaxime and ceftriaxone; the effect of the substituent at C3 position.

Ionization constants of three cephalosporin antibiotics, cefetamet (CEF), cefotaxime (CFX) and ceftriaxone (CFTR) are determined using pH-potentiometric titrations at I=0.1 M (NaCl) and t=25 degrees C. Cefetamet and cefotaxime have three ionization groups: carboxylic, amide and aminothiazole. Besides those three, ceftriaxone possesses an hydroxytriazinone group as new and additional ionization center. In acid medium two overlapping acid-base processes are occurring with acidity constants being: pK1 2.93 (COOH) and pK2 3.07 (aminothiazole) for cefetamet, and pK1 2.21 (COOH) and pK2 3.15 (aminothiazole) for cefotaxime. In the case of ceftriaxone the situation is even more complicated, three overlapping processes coexist with pK1 2.37 (COOH), pK2 3.03 (aminothiazole) and pK3 4.21 (hydroxytriazinone). Protolysis of amide group is happening in the alkaline medium as completely separated process from those in acid medium. The acidity constants which correspond to amide group are pK3 10.65 (CEF), pK3 10.87 (CFX) and pK4 10.74 (CFTR). The influence of the C3 substituent on the dissociation process of the neighboring ionization group, particularly carboxylic group, was considered. The differences in acidity of CEF, CFX and CFTR (pK1: 2.93, 2.21 and 2.37, respectively) are likely to be caused by the stereoelectronic properties of substituents in the beta-position to the carboxylic group due to the combined inductive, hyperconjugative and resonance effects.