Proportion Of Acetonitrile Research Articles

Developing an improved UHPLC method for impurity profile analysis of ceftriaxone using analytical quality by design.

In this study, we developed a new reversed-phase ultra-high-performance liquid chromatography (UHPLC) method for comprehensively measuring impurities in ceftriaxone. The method was developed based on the Chinese Pharmacopoeia (ChP) HPLC method, which is limited by the lack of selectivity to potential impurities and a long running time. Screening experiments showed that octylamine concentration, mobile phase pH, and organic phase ratio were critical method parameters. Further optimisation and Monte-Carlo simulations were performed to map out the design space. The selected working conditions resulted in a complete separation of the impurity profile in approximately 10 min. A multivariate approach confirmed that the method was robust, and the proportion of acetonitrile should be carefully controlled. Additionally, the developed UHPLC method could be transferred back to HPLC in a single step using a Columns Calculator, providing a new approach for the rapid and effective development of the HPLC method. Our findings could serve as a reference for developing the next version of the ChP.

Silica-Embedded Polyurea Microspheres with Rough Surface for Matte Leather Finishing

A type of polyurea microspheres with rough surfaces and different sizes (1-12 μm), which was used for waterborne polyurethane matte leather coating, were prepared by precipitation polymerization in H 2 O/acetonitrile system, using isophorone diisocyanate as polymerized monomer, 2,4-diaminobenzenesulfonic acid sodium salt as hydrophilic monomer and nano-SiO 2 as surface roughener. The effect of the loading of isophorone diisocyanate and 2,4-diaminobenzenesulfonic acid sodium salt, the volume ratio of H 2 O/acetonitrile and the addition stage of nano-SiO 2 on the particle size and surface roughness were investigated so as to achieve the excellent matte effect of the coating. The results indicated that the adding of hydrophilic monomer can reduce the particle size of microspheres and improve its re-dispersibility in water, while the increase of the proportion of acetonitrile in the mixed solvent and the loading of polymerized monomer can enlarge the size of the polyurea microspheres. As surface roughener, with the increase of nano-SiO 2 loading, the surface roughness and the particle size of microspheres increase. When nano-SiO 2 is added at the stage of the reaction system getting turbid, the maximum surface roughness of polyurea microspheres can be achieved. Polyurea microspheres with particle size (1-2 μm) and high surface roughness have better matting effect, and the gloss of the prepared waterborne polyurethane coating can be adjusted to less than 1.6° (60°-incident Angle).

Stable isotope labelling-flow injection analysis-mass spectrometry for rapid and high throughput quantitative analysis of 5-hydroxymethylfurfural in drinks

In this work, we reported a stable isotope labelling-flow injection analysis-mass spectrometry (SIL-FIA-MS) method for high throughput analysis of 5-hydroxymethylfurfural (HMF) content in drinks. 3-Nitrophenylhydrazine (3-NPH) and 13 C 6 -3-Nitrophenylhydrazine hydrochloride ( 13 C 6 -3-NPH) were used as pared labelling reagents to selectively label HMF. With a simple centrifugal operation, a certain volume of sample was directly mixed with 3-NPH solution. After adding a small amount of 13 C 6 -3-NPH labelled HMF as internal standard, the sample can be directly transferred for the subsequent MS analysis owing to the contamination-free feature of excess 3-NPH. By using a multi-well thermostatic oscillator to carry out bath derivatization, the average time cost for each sample pretreatment was less than 1 min. Coupled with a triple-quadrupole mass spectrometer, FIA-MS with multiple reaction monitoring (MRM) was used for the rapid and high throughput detection of the labelled HMF in negative ion mode. The time cost for MS detection including injection process for each sample was about 1.5 min. Namely, the whole procedure including sample pretreatment and MS detection can be completed within 3 min per sample, which was very suitable for routine analysis for its high throughput and simplification. By labelling HMF with 3-NPH, the characteristic fragment ion after labelling and detection in interference-lesser negative ion mode enabled high detection specificity even without chromatographic separation. By using 13 C 6 -3-NPH labelled HMF as internal standard to counteract the matrix effect during MS analysis, the detection accuracy and precision were guaranteed. In order to achieve the best conditions for the derivatization of HMF, several derivatization conditions including the acid concentration, the proportion of acetonitrile (ACN), the derivatization reagent concentration, reaction temperature and reaction time were investigated in detail. Under the optimized derivatization, quantitative analysis method was validated in ranges of 0.050–5.0 μg/mL. The relative standard deviations (RSDs) of inter-day and intra-day precision were less than 5.42% and 4.89%, respectively, showing satisfied reproducibility. The proposed method has been successfully applied to the analysis of HMF in sugar-sweetened drinks for feasibility verification in real samples. • SIL-FIA-MS was realized by using MS response-free and contamination-free 3-NPH and 13 C 6 -3-NPH for labelling. • Rapid and high throughput MS analysis for HMF was realized. • The method also offers competitive advantages in accuracy, specificity, simplification, automatability and LODs. • The method is promising to determine other aldehydes in foods with proper modifications.

Equivalent Method Development and Cross Validation of Pharmacopoeial Method of Montelukast Sodium

The purpose of this exploration research work article is to develop equivalent method and evaluate its equivalency (Cross validation) against pharmacopoeial method of Montelukast sodium for the evaluation and assessment of process related impurities i.e. Methyl MLK impurity in Montelukast sodium by HPLC method and its principles. The method mentioned in European Pharmacopoeia (EP) and United States Pharmacopoeia (USP) does not sufficiently separates impurity C and impurity D , as these impurities elutes under the main peak and the pharmacopoeial methods were also not able to detect the Methyl MLK impurity which is not listed in USP monograph. So our prime design of experiment is to develop of new high-performance liquid chromatographic (HPLC) method which eliminates the drawback of two pharmacopoeial methods and this proposed created strategy is fit for recognition and detection of Methyl MLK impurity and separation of all process related impurities of Montelukast sodium mentioned in EP and USP monographs. An efficient strategy screening and scouting in which various C-18 columns were tried and tested. LUNA C-18 column utilized in RP HPLC mode ended up being the phenomenal decision for the technique streamlining. The proportion of acetonitrile and trifluoroacetic acid (TFA) in water and in the mobile phase, column temperature, flow rate and diluents were considered as basic strategy boundary. The method developed equivalency was checked in terms of Specificity, LOD, Quantitation (LOQ), Precision, Linearity, Relative response factor (RRF), and Accuracy.

QuEChERS and 96-well plate solid phase extraction for determination of vancomycin and norvancomycin in fish meat by UPLC–MS/MS

Vancomycin and norvancomycin are glycopeptide antibiotics for gram-positive bacteria infection, but indiscriminately used in aquaculture. In this study, a QuEChERS (quick, easy, cheap, effective, rugged, and safe)/96-well solid-phase extraction (SPE) plate method was used to extract vancomycin and norvancomycin in fish meat samples, and the drugs were further analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). The parameters, such as the sorbent of cation exchange resin, the proportion of acetonitrile (15%) in extractant, the mobile phase of water (0.1% formic acid)/acetonitrile, were optimized. The method was validated in terms of linearity (0.9990–0.9994), LOD (0.51 μg·kg−1), LOQ (1.73 μg·kg−1), intra-dayprecision (<5.19%), inter-day precision (<6.30%), and recovery (86.7–98.6%). Finally, the method was successfully applied to contaminated and randomly collected samples. The results indicated that the proposed method meet the daily monitoring requirements for vancomycin and norvancomycin.

Characterization of Retention Mechanisms in Mixed-Mode HPLC with a Bimodal Reversed-Phase/Cation-Exchange Stationary Phase

Mixed-mode HPLC (MM-HPLC), combining different interactions or retention modes in a single column, can be an interesting alternative to reversed-phase HPLC, notably to achieve the combined retention of polar and non-polar species. In the present fundamental study, we have selected one bimodal stationary phase allowing for both reversed-phase and weak cation-exchange retention modes (Acclaim mixed-mode WCX-1 LC). First, the mobile phase buffer composition (buffer pH ranging from 5 to 7 and concentration ranging from 20 to 100 mM) was explored with a small set of probe compounds (15 acids, bases and neutrals) to ensure adequate retention and peak shapes for the target compounds, and to evaluate the relative contributions of reversed-phase and ion-exchange mechanisms. Second, retention values measured for 63 probe compounds with various proportions of acetonitrile (ranging from 30 to 80%) served to establish linear solvation energy relationships based on (a) the usual and (b) a modified version of the solvation parameter model comprising additional descriptors to take account of interactions with ionizable species to bring some insights into the retention mechanisms. Finally, temperature effects at the low (30%) and high (60%) proportions of acetonitrile were observed between 20 and 40 °C (with 5 °C increments) and Van’t Hoff plots were drawn to measure the changes in interactions energies when the mobile phase composition changed.

Preparation, characterization and usage of molecularly imprinted polymer for the isolation of quercetin from hydrolyzed nettle extract.

Quercetin (3,3',4',5,7-pentahydroxyflavone, QC) is a health-beneficial flavonoid, widely occurring in leaves, fruits, and flowers of various plants. In this work aiming isolation, purification and pre-concentration of QC, QC imprinted polymers (QC-MIPs) in different molar ratios {template:monomer:cross-linker (1:4:20, 1:5:30, 1:8:40, 1:10:50)} were prepared thermally through bulk polymerization by using QC as the template molecule, 4-vinylpyridine (4-VP), methacrylic acid (MAA), acrylamide (AA) as the functional monomers, ethylene glycol dimethacrylate (EDMA) as the cross-linker and 2,2'-azobisisobutyronitrile (AIBN) as initiator in the porogens of acetone and tetrahydrofuran. Their recognition and selectivity properties were investigated in solutions containing QC and other similar-structure phenolics by equilibrium binding experiments using different proportions of acetonitrile (ACN)-dimethylsulfoxide (DMSO) mixtures and methanol (MeOH) as solvents. The MIP with 1:4:20 molar ratio of QC:4-VP:EDMA was established as the most suitable for recognition of QC. Sorption parameters of the MIP and the NIP (non-imprinted polymer) were calculated by using Freundlich and Langmuir isotherms with QC solutions in ACN:DMSO (98:2, v/v). The mentioned MIP was found to be highly selective for quercetin over other phenolic compounds (rutin, catechin, etc.). Thus, molecularly imprinted solid-phase extraction (MISPE) procedures were applied for selective pre-concentration and purification of QC from synthetic mixtures of phenolic compounds and nettle extract, known as a source of official and folk medicine. The results demonstrated the possibility of direct extraction of certain pharmacophoric constituents such as QC and QC derivatives from nettle by MIP separation.

Development and Validation of a Reverse Phase Ultra Performance Liquid Chromatographic Method for Simultaneous Estimation of Nebivolol and Valsartan in Pharmaceutical Capsule Formulation

The present study describes the development and validation of sensitive, novel Ultra Performance Liquid Chromatographic technique to simultaneously evaluate Nebivolol and Valsartan in commercial pharmaceutical capsule formulation. The technique was performed utilizing thermo C18 (4.6 mm×50 mm, 1.9 μm) column having a mobile phase comprising of 10 mM ammonium dihydrogen phosphate pH adjusted to 3.00 ± 0.02 with dilute orthophosphoric acid as buffer, with a ratio of buffer: acetonitrile 60:40 (v/v) and with a flow rate of 0.4 mL min-1. The eluted components were monitored out at 220 nm utilizing a photo diode array detector. The retention times for NBL and VST were 0.48 and 0.83 min respectively. The developed Ultra Performance Liquid Chromatographic method was validated according to ICH guidelines to confirm specificity, linearity, accuracy and precision. The homoscedasticity of the variances and lack of fit in the evaluation of linearity was established using the Cochran’s C test and F-test respectively. The repeatability variances and time different intermediate variances were assessed simultaneously. The time different variances expressed as percentage relative standard deviations (%RSD) and accuracy were well within the limits as prescribed by ICH. In order to designate suitability in the experimental design approach, a robustness test was carried out. To assess robustness 3 aspects were taken into consideration, namely, proportion of flow rate, proportion of acetonitrile in mobile phase and pH; all the three factors have no significant effect on response (assay). Experimental design based robustness with the aid of Full Factorial design (FFD) provided an effective way to simultaneously asses Nebivolol and Valsartan. This method was successfully used to analyze fixed dose capsule samples of Nebivolol as well as Valsartan and can be utilized for regular lab investigation of Nebivolol and Valsartan in capsules.

Optimization of titanium dioxide enrichment of phosphopeptides and application in the &lt;I&gt;Thermoanaerobacter tengcongensis&lt;/I&gt; phosphoproteome analysis

Using titanium dioxide is a very good strategy for the phosphopeptide enrichment. There are many other factors can affect the enrichment efficiency, and the optimization of parameters was needed for better enrichment results. In this study, the peptide mixtures of six standard proteins were used as the model samples to evaluate and optimize the parameters such as the proportion of acetonitrile and trifluoroacetic acid in loading buffer and the TiO2-to-peptide ratio. The results showed that 80% (v/v) acetonitrile, 1% (v/v) trifluoroacetic acid and 40 : 1 (m/m) TiO2-to-peptide ratio were the optimum parameters to obtain the best enrichment selectivity and maximum phosphopeptides identification. For the first time, the optimum enrichment conditions were applied for the phosphoproteome analysis of the Thermoanaerobacter tengcongensis, an anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengchong, China, and 25 phosphorylated proteins were identified in the preliminary experiment. The results provided a reference for further study on this organism survived under extreme environment.

GRP-127 On-Line Quality Control of Cytotoxic Drugs: Ultra-Fast Chromatographic Separation of Vinca Alkaloids

BackgroundDue to repetitive and tedious handling tasks, production of anticancer drugs for infusion is associated with a high risk of non-conformity. Thus, on-line quality control is necessary to improve the...

Systematic evaluation of retention behavior of carbohydrates in hydrophilic interaction liquid chromatography

A systematic evaluation of retention behavior of carbohydrates in hydrophilic interaction liquid chromatography (HILIC) was performed. The influences of mobile phase, stationary phase and buffer salt on the retention of carbohydrates were investigated. According to the results, the retention time of carbohydrates decreased as the proportion of acetonitrile in mobile phase decreased. Increased time of carbohydrates was observed as the concentration of buffer salt in mobile phase increased. The retention behavior of carbohydrates was also affected by organic solvent and HILIC stationary phase. Furthermore, an appropriate retention equation was used in HILIC mode. The retention equation lnk = a + blnC(B) + cC(B) could quantitatively describe the retention factors of carbohydrates of plant origin with good accuracy: the relative error of the predicted time to actual time was less than 0.3%. The evaluation results could provide guidance for carbohydrates to optimize the experimental conditions in HILIC method development especially for carbohydrate separation

DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF CHLORHEXIDINE AND P-CHLOROANILINE IN VARIOUS PHARMACEUTICAL FORMULATIONS

□ A simple, rapid and sensitive isocratic reversed-phase (RP) high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of chlorhexidine (CHX) and p-chloroaniline (CAL) in various pharmaceutical formulations. Compound separation was achieved in less than 10 min with an XBridge C18 column that was maintained at 40°C and a mobile phase consisting of 32:68 (v/v) of acetonitrile and a pH 3.0 phosphate buffer solution (a 0.05 M monobasic sodium phosphate solution containing 0.2% of triethylamine). Analyses were performed at a flow rate of 2 mL min−1 and at a detection wavelength of 239 nm. The method was shown to be selective, linear, accurate, and precise in intra-day and inter-day analyses. The robustness of the method was shown by slightly changing the flow rate, column oven temperature, and proportion of acetonitrile in the mobile phase. The method was found, however, to be very sensitive to the pH of the mobile phase buffer. The method was successfully validated following the guidelines of the International Conference on Harmonization (ICH). This validation proved that the method was suitable for the determination of CHX and CAL in toothpaste, mouthwash, wound cleanser, and skin and hand disinfectants.

Phase diagram for the hexane-[Y(NO3)3(TBP)3]-acetonitrile liquid ternary

A phase diagram for the hexane-[Y(NO3)3(TBP)3]-acetonitrile (1-2-3) liquid ternary at T = 298.15 K was studied. This ternary consists of two pairs of incompletely miscible liquids (hexane-acetonitrile and [Y(NO3)3(TBP)3]-hexane). It contains two homogeneous liquid fields and one two-phase liquid field. One phase is depleted of acetonitrile and contains variable proportions of [Y(NO3)3(TBP)3] and hexane; the other contains variable proportions of acetonitrile and [Y(NO3)3(TBP)3] and small proportions of hexane. Intermolecular interaction parameters and excess Gibbs energies g E were calculated for binaries and the liquid ternary along binodal curves proceeding from miscibility data for the [Y(NO3)3(TBP)3]-hexane and hexane-acetonitrile binaries and the liquid ternary and using equations of the NTRL model. For the liquid ternary, g E > 0.g E decreases in the following order of pairs of liquids: (1, 3), (1, 2), (2, 3). A computer algorithm is given for calculating binodal curves and tie-lines for the liquid ternary with the use of equations of the NTRL model proceeding from the intermolecular interaction parameters.

Determination of Tanshinones in Salvia miltiorrhiza Bunge by High-Performance Liquid Chromatography with a Coulometric Electrode Array System

A method was developed for the determination of tanshiones (Cryptotanshinone, tanshinone I, and tanshinone IIA) in Salvia miltiorrhiza Bunge (S. miltiorrhiza Bunge) using high performance liquid chromatography with the coulometric electrode array system (HPLC-CEAD). The analysis was carried out on a column of Hypersil C18 (250 mm × 4.6 mm, 5 µm) with a mobile phase of sodium acetate (pH 2.8, 50 mM) and acetonitrile in the proportion of 45:55. Detection potentials of four electrodes in series were set at − 600, − 400, 100, and 500 mV, respectively. The pH of the mobile phase and the proportion of acetonitrile were optimized in this work. Calibration curves showed good linearity with correlation coefficients of more than 0.9979, the average recoveries of tanshiones were more than 94%, and the relative standard deviations were less than 5.3%. The HPLC-CEAD method was stable, sensitive, and reproducible for the determination of tanshiones S. miltiorrhiza Bunge.

Determination of four water-soluble compounds in Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to determine the four water-soluble components-Danshensu (I), protocatechuic acid (II), protocatechuic aldehyde (III) and salvianolic acid B (IV) in Chinese medicine plant Salvia miltiorrhiza Bunge using high-performance liquid chromatography with a coulometric electrode array detection (HPLC-CEAD) system. Heat reflux extraction was used to pretreat the sample. This analysis was carried on a column of Hypersil C18 (250 mm x 4.6 mm, 5 microm) with a mobile phase of sodium acetate (pH 2.5, 50 mM) and acetonitrile in gradient mode. An ESA electrochemical detector monitored the four compounds. Potentials of four electrodes in series were set at 100, 150, 200 and 250 mV, respectively. Optimization of the pH of mobile phase and the proportion of acetonitrile were also performed. Calibration curve showed good linearity with correlation coefficients (r) more than 0.9937. Average recoveries of the four compounds were more than 92% and relative standard deviations were less than 6.6%. This method appeared to be stable, sensitive and reproducible for determination of the four water-soluble compounds in Chinese medicine plant S. miltiorrhiza Bunge.

Development and validation of an HPLC-UV method for determination of iohexol in human plasma

An HPLC-UV analytical method for estimation of iohexol in human plasma was developed and validated. Protein precipitation and iohexol extraction from plasma (100 μl) was carried out by adding 800 μl perchloric acid (5%, v/v in water) containing iohexol related compound B as the internal standard followed by vortex mixing and centrifugation. The supernatant (90 μl) was then injected onto a μBondapak C 18 column (150 mm × 3.9 mm, 10 μm) maintained at 30 °C. The mobile phase comprised of various proportions of acetonitrile and water with a total run time of 12 min and the wavelength of the UV detector was set at 254 nm. The extraction recovery of iohexol from plasma was >95% and the calibration curve was linear ( r 2 = 0.99) over iohexol concentrations ranging from 10 to 750 μg/ml ( n = 8). The method had an accuracy of >92% and intra- and inter-day CV of <3.7% and <3.6%, respectively. The method reported is simple, reliable, precise, accurate and has the capability of being used for determination of iohexol in clinical settings.

Determination of tetracyclines in ovine milk by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to analyze residual tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), methacycline (MTC), doxycycline (DC)) in ovine milk, using high-performance liquid chromatography (HPLC) with a coulometric electrode array system. The samples were pretreated, using liquid–liquid extraction based on hexane. The chromatography was performed, using a C 18 column (150 mm × 4 mm i.d. and 5 μm) with a mobile phase: sodium phosphate monobasic dihydrate (pH 2.2, 0.05 M)–acetonitrile (78:22, v/v). The flow rate of mobile phase was kept constantly at 1ml/min. The residues were monitored by an ESA electrochemical detector. Potentials of four electrodes in series were set at 400, 660, 680 and 700 mV, respectively. The first electrode was set to remove those interfering substances that may co-elute with TCs and the other three electrodes were used for quantification. The maximal potential of our detection was 700 mV. Calibration curve showed good linearity and the detection limit of TCs was 12.5, 20, 25, 10 and 25 ng/ml, respectively. Optimization of the pH of the mobile phase, the proportion of acetonitrile and the pH of the pretreatment were also performed. Recoveries of TCs from spiked samples were more than 88% and the relative standard deviations were less than 4.3%. This method was reliable, sensitive, economical and suited for routine monitoring of TC residues in ovine dairy milk.

Preparation and characterization of drug-loaded polymethacrylate microspheres by an emulsion solvent evaporation method

Microspheres containing the anti-hypertension drug, felodipine, were prepared by the emulsion solvent evaporation method (o/o) using acrylate methacrylate copolymers, Eudragit RL PO and Eudragit RS PO, as wall materials. In order to increase the encapsulation efficiency, a mixed solvent system comprising 1:1 proportions of acetonitrile and dichloromethane was used as a dispersed phase. The morphology of the microspheres was evaluated using a scanning electron microscope, which showed a spherical shape with smooth surface. The mean sphere diameter was between 9.5-13.2 #119 m and the microencapsulation efficiencies ranged from 51.4-80.4%. The release profiles and encapsulation efficiencies depended strongly on the structure of the polymers used as wall materials. The release rate of the Eudragit RS PO microspheres was much lower than that of Eudragit RL PO microspheres. Whereas Eudragit RL PO microspheres followed the Higuchi rule, Eudragit RS PO microspheres exhibited a triphasic release profile. It is concluded that drug release rate can be controlled by choice of polymer type.

Radioimmunoassay of regulatory peptides in the presence of acetonitrile: marked improvement of cholecystokinin assays

Radioimmunoassay has made it possible to measure the levels of many hormones. However, samples for some hormones, such as cholecystokinin (CCK), need to be purified by reverse phase chromatography before assay. Usually, samples are eluted from cartridges or HPLC columns in about 50% acetonitrile, dried on a vacuum centrifuge, and then reconstituted in buffer. Drying and reconstituting samples is time consuming and introduces additional sources of error and peptide loss. The present study investigated the effect of acetonitrile on radioimmunoassays for CCK to see if samples containing acetonitrile could be assayed directly. The non-specific binding of a radiolabeled peptide, the zero binding (B 0), and the fall in the presence of 2.5 fmol unlabeled CCK were determined in the presence of various proportions of acetonitrile with 0.1% TFA. Additionally, standard curves were compared in the presence and absence of 200μl of 50% acetonitrile, ( n=5). For assays using two separate CCK antisera, increasing amounts of acetonitrile gave progressively higher zero binding and fall, thereby increasing sensitivity and antibody titer. The use of 200μl 50% acetonitrile, chosen to represent typical sample conditions, increased antiserum titers by three to four-fold, as well as increasing sensitivity considerably. For one antiserum (CCK2), the IC 20 was 0.36±0.02 fmol CCK/tube in the presence of acetonitrile and 1.45±0.08 fmol/tube in its absence ( P<0.001). For the other antiserum (Dino 7), the IC 20 was 0.40±0.02 fmol CCK/tube in the presence of acetonitrile and 0.63±0.01 fmol/tube in its absence ( P<0.001). A similar increase in sensitivity was seen with a gastrin assay. However, no significant change in the gastrin antibody titer was evident. Assays for several other hormones were unaffected by 200 μl of 50% acetonitrile. At volumes encountered in samples following chromatography, acetonitrile did not adversely affect radioimmunoassays for a number of hormones, and the sensitivity and antibody titer of the CCK assays were improved. Measurement of CCK samples without drying and reconstitution increases assay efficiency and sensitivity.

The 4,4′-dimethoxytrityl carbenium ion by deamination of 4,4′-dimethoxytritylamine in acetonitrile–aqueous perchloric acid: kinetics, equilibria, deuterium isotope effects, and possible ion-pair formation

The rate of deamination of 4,4′-dimethoxytritylammonium cation, generated from the corresponding amine in aqueous perchloric acid containing a low proportion of acetonitrile at constant total perchlorate concentration, is first order in both dimethoxytritylammonium and hydronium ions. The specificity of the hydronium ion kinetic effect indicates acid catalysis with second-order rate constant, kH. There is also appreciable reactivity at [H3O+] = 0, i.e. an uncatalysed (solvent-induced) reaction channel from the substituted tritylammonium cation with first-order rate constant, ko. The deuterium solvent kinetic isotope effects upon uncatalysed and catalysed reaction channels have been measured [koH/koD = 1.51 (±0.03), kHH/kHD = 1.2 (��0.1), 25 °C and ionic strength = 1 mol dm–3]. The deuterium solvent isotope effect upon the subsequent equilibrium between 4,4′-dimethoxytrityl trityl cation and water (to give the substituted alcohol and hydronium ion) extrapolated to zero perchlorate concentration has also been determined, and the value (KR+)H/(KR+)D = 3.4 (±0.4) leads to a fractionation factor of ca. unity for the alcohol. The deamination is accelerated by increasing perchlorate concentrations at constant acidity; perchlorate also appears to have an appreciable effect upon the subsequent equilibrium involving the substituted trityl cation, water, hydronium ion, and the alcohol. Acetic acid, monochloroacetic acid, and trifluoroethanol also enhance the rate but not, apparently, by general acid catalysis, whereas the rate and final equilibrium are relatively insensitive to the proportion of the acetonitrile cosolvent. A mechanism for deamination is proposed which involves initial reversible heterolysis of the carbon–nitrogen bond of the tritylammonium cation followed by parallel diffusional separation and hydronium ion assisted separation. On the basis of the isotope effects, we conclude that the latter reaction channel involves either step-wise protonation of the ammonia molecule followed by rate-limiting separation of the cation–cation pair, or a strongly asynchronous (uncoupled) concerted process. Perchlorate may exert its appreciable kinetic and equilibrium effects simply by modifying the nature of the medium, i.e. by specific kinetic salt effects upon the various elementary steps in the overall reaction. Alternatively, the effect of perchlorate upon the final equilibrium may be explained by the reversible formation of specific ion-pairs between the extensively delocalised substituted trityl cation and the non-polarising perchlorate anion; we found no evidence of covalent dimethoxytrityl perchlorate. Correspondingly, the effect of perchlorate upon the observed pseudo first-order deamination rate constant could be due to its direct involvement in additional solvent-induced and hydronium ion-catalysed routes.