Ammonium Formate Concentration Research Articles

A Robust Chromatographic Method for Drug Release profiling of liposomal doxorubicin HCl

Liposomes are excellent drug delivery vehicles for chemotherapeutics as they may change the pharmacokinetics of therapeutic compounds, resulting in altered tissues distribution, and in some cases, reduced cytotoxicity and enhanced distribution and efficacy of the active pharmaceutical ingredient (API) at target tissues. Drug release profiles of liposomal formulations are crucial to support equivalence evaluation and quality control in pre- and post-approval stages. We developed an automated chromatographic method for quantifying the drug release profile of liposomal formulations containing doxorubicin to overcome the shortcomings of currently available methods. The newly developed method employs nanoparticle exclusion chromatography (nPEC), using a monolithic silica column coated with polyvinylpyrrolidone to separate the released drug from liposomal encapsulated drug. We evaluated the effects of pH, temperature, and ammonium formate concentration on the drug release rate. The optimized release buffer consisting of 5 % sucrose, 20 mM l-histidine, and 200 mM ammonium formate was selected for the drug release profiling of five liposomal formulations at 47 °C. The drug release profiles of five liposomal doxorubicin formulations were similar. Our automated method requires very small amounts of the sample and provides release profiles with high sensitivity and accuracy. In addition, this method can be applied to other liposomal products to allow for simple, fast, and accurate analysis of in vitro drug release profiling.

Assessment of growth rate of <i>Yarrowiali polytica</i> and <i>Pichia guilliermondii</i> species in an ammonium formate solution of rubber wastewater

The growth of microorganisms is influenced by several physico-chemical parameters that need to be controlled before startinga biological treatment plant for better process efficiency. The influence of temperature (25-40 °C), and ammonium formateconcentration (1.59-7.94 mM) on the growth of two yeast strains (Y.lipolytica and P.guilliermondii )was examined in a batchprocess. Temperature has a direct impact on the kinetic growth parameters with an activation energy (Ea) of 14.3 kcal/mol andR2 0.95 for Y.lipolytica and 12.5 kcal/mol and R2 0.97 for P.guilliermondii, indicating a dominant biological regime.

Leaching Behavior of Rare Earth Elements and Aluminum from Weathered Crust Elution-Deposited Rare Earth Ore with Ammonium Formate Inhibitor

In situ leaching of weathered crust elution-deposited rare earth ore usually uses ammonium sulfate as the leaching agent, which poses challenges such as low mass transfer efficiency, high consumption of the leaching agent and long leaching periods. In order to intensify the leaching process of rare earth elements and reduce the impurity of leaching accompanying rare earth, ammonium sulfate and ammonium formate were mixed as a novel compound leaching agent to treat weathered crust elution-deposited rare earth ore. The effects of ammonium formate concentration, liquid/solid ratio, leaching agent pH and leaching temperature on the leaching process of rare earth (RE) and aluminum (Al) were investigated and evaluated using the chromatographic plate theory. Results showed that ammonium formate could effectively enhance the mass transfer efficiency of rare earth and significantly inhibit the mass transfer efficiency of aluminum. Leaching agent pH has a greater impact on the mass transfer efficiency of aluminum. A higher leaching temperature could enhance the mass transfer efficiency of rare earth and aluminum by providing a driving force to overcome the resistance of diffusion. The optimum conditions for leaching rare earth and aluminum are 0.1 mol/L ammonium sulfate compounded with 0.032 mol/L ammonium formate, pH 4–8 of the leaching agent, 0.8:1 liquid:solid (mL/g) ratio and room temperature. Under this condition, the mass transfer efficiency of rare earth was improved, and the mass transfer efficiency of aluminum was significantly inhibited.

Application of experimental design in HPLC method optimisation for the simultaneous determination of multiple bioactive cannabinoids

The scientific interest in Cannabis sativa L. analysis has been rapidly increasing in recent years, especially for what concerns cannabinoids, plant secondary metabolites which are well known for having many biological properties. High-performance liquid chromatography (HPLC) is frequently used for both the qualitative and quantitative analysis of cannabinoids in plant extracts from C. sativa and its derived products. Many studies have been focused on the main cannabinoids, such as ∆9-tetrahydrocannabinolic acid (∆9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA) and their decarboxylated derivatives, such as ∆9-tetrahydrocannabinol (∆9-THC), cannabidiol (CBD) and cannabigerol (CBG). In addition to the abovementioned compounds, the plant produces other metabolites of the same chemical class, and some of them have shown interesting biological activities.In the light of this, it is important to have efficient analytical methods for the simultaneous separation of cannabinoids, which is quite complex since they present similar chemical-physical characteristics. The present work is focused on the use of the Design of Experiments technique (DoE) to develop and optimise an HPLC method for the simultaneous separation of 14 cannabinoids. Experimental design optimisation was applied by using a Central Composite Face-Centered design to achieve the best resolution with minimum experimental trials. Five significant variables affecting the chromatographic separation, including ammonium formate concentration, gradient elution, run time and flow rate, were studied. A multivariate strategy, based on Principal Component Analysis (PCA) and Partial Least Squared (PLS) regression, was used to define the best operative conditions. The developed method allowed for the separation of 12 out of 14 cannabinoids. Due to co-elution phenomena, HPLC coupled with a triple quadrupole mass analyser (HPLC-ESI-MS/MS) was applied, monitoring the specific transitions of each compound in the multiple reaction monitoring (MRM) mode. Finally, the optimised method was applied to C. sativa extracts having a different cannabinoid profile to demonstrate its efficiency to real samples.The methodology applied in this study can be useful for the separation of other cannabinoid mixtures, by means of appropriate optimisation of the experimental conditions.

Diquat and Paraquat Analysis in Rice using a Zwitterionic Hydrophilic Interaction Chromatography – tandem Mass Spectrometry

The analysis of polar herbicides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is challenging due to their complicated interactions. Diquat and paraquat, the bipyridilium group of polar herbicides, are widely used worldwide. This study aimed to optimize analytical conditions of diquat and paraquat in rice matrix using a hydrophilic interaction liquid chromatography column (Obelisc R; 2.1 x 150 mm, 5 µm). The mixtures of acetonitrile and ammonium formate buffer in water were used as mobile phase. All buffer solutions were acidified to a pH of 2.5 using concentrated formic acid. The results showed that 10 mM buffer concentration of ammonium formate was optimal compared to others, including 5, 20, and 50 mM. A mixture of methanol and water (6:4, v/v) containing 1% formic acid was used as an extractable solvent for sample preparation. Method quantitation limit (MQL) of diquat and paraquat in rice samples was 10 µg kg-1. Recoveries at different concentrations (i.e., 20, 50, and 100 µg kg-1) were higher than 70% for diquat, and 80% for paraquat with RSD < 20%, respectively. Diquat and paraquat in rice samples purchased from the local market were lower than MQL, with good recoveries for standard spiked samples.

Leaching Process of Weathered Crust Elution-Deposited Rare Earth Ore With Formate Salts.

To strengthen the rare earth leaching process and weaken the hydration of clay minerals for preventing landslides, it is of great importance to adopt a green and sustainable leaching agent in the industry. In this work, the leaching process of weathered crust elution-deposited rare earth ores with formate salts (ammonium formate, potassium formate, and sodium formate) was investigated. The effects of formate salts on the linear swelling ratio and zeta potential of the clay minerals were studied. The experimental results showed that ammonium formate could effectively recover the rare earth elements from weathered crust elution-deposited rare earth as well as inhibit the leaching of impurity aluminum. At room temperature, when the ammonium formate concentration was 1% wt, the leaching efficiencies of rare earth and aluminum were 87 and 37%, respectively. Compared with traditional inorganic ammonium salts, the inhibition effect of impurity aluminum was obvious. In addition, the results of the linear swelling ratio in the clay minerals showed that the inhibit ability of formate salts on the hydration of clay minerals enhanced with the increase of the formate concentration, and the order of the inhabitation on the clay minerals followed: 1% ammonium formate > 1.5% potassium formate > 2.5% sodium formate > distilled water. Based on the double layer theory, ammonium formate and potassium formate could effectively compress clay mineral particles to avoid water intake, which could increase the interaction between clay mineral particles and greatly reduce the electronegative property of the clay minerals, so as to effectively reduce the surface hydration of clay minerals to decrease the swelling of rare earth ore. The results of this experiment have important and practical significance in guiding the prevention of landslides, promoting the in-situ leaching technology, and effectively protecting the ecological environment in mining areas.

Catalytic Transfer Hydrogenation of Low-erucic-acid Rapeseed Oil over a Ni-Ag0.15/SBA15 Catalyst.

The kinetics of catalytic transfer hydrogenation (CTH) of low-erucic-acid rapeseed oil using ammonium formate as a hydrogen donor over a Ni-Ag0.15/SBA15 catalyst were studied. Then, a kinetic model for the hydrogenation of low-erucic-acid rapeseed oil was established, and it was found that the reaction rate constants of hydrogenations of 9c-18:1 and 12c-18:1 oleic acid were 0.1262 and 0.0148, and the catalytic selectivity of linoleic acid was 2.04. For the catalyst loading of 0.23%, the hydrogenation temperature was 80°C, the ammonium formate concentration was 0.32 mol/50 mL, and the low-erucic-acid rapeseed oil was hydrogenated in 90 min; it was also found that the iodine value of low-erucic-acid rapeseed oil was 80 g I2/100 g, the oleic acid content was 65%, and the trans fatty acids (TFAs) content was only 6.7%. Therefore, CTH may be widely used in the modification of oils and fats.

Salt and solvent effects in the microscale chromatographic separation of heparan sulfate disaccharides

The analysis of heparan sulfate disaccharides poses a real challenge both from chromatographic and mass spectrometric point of view. This necessitates the constant improvement of their analytical methodology. In the present study, the chromatographic effects of solvent composition, salt concentration, and salt type were systematically investigated in isocratic HILIC-WAX separations of heparan sulfate disaccharides. The combined use of 75% acetonitrile with ammonium formate had overall benefits regarding intensity, detection limits, and peak shape for all salt concentrations investigated. Results obtained with the isocratic measurements suggested the potential use of a salt gradient method in order to maximize separation efficiency. A 3-step gradient from 14 mM to 65 mM ammonium formate concentration proved to be ideal for separation and quantitation. The LOD of the resulting method was 0.8–1.5 fmol for the individual disaccharides and the LOQ was between 2.5–5 fmol. Outstanding linearity could be observed up to 2 pmol. This novel combination provided sufficient sensitivity for disaccharide analysis, which was demonstrated by the analysis of heparan sulfate samples from porcine and bovine origin.

Effect of a novel compound on leaching process of weathered crust elution-deposited rare earth ore

The leaching process of weathered crust elution-deposited rare earth ore is a typical noncatalytic heterogeneous reaction in liquid–solid systems. In order to intensify the leaching process of rare earth and reduce the impurity leaching accompanying with rare earth, ammonium sulfate and ammonium formate were mixed as a novel compound leaching agent, in which ammonium formate was used as an inhibitor for aluminum, as well as an aid agent to speed up rare earth leaching. Effects of ammonium formate concentration, liquid/solid ratio, leaching agent pH and leaching temperature on the leaching process of rare earth and aluminum were investigated, and then were systematically evaluated by the leaching kinetic theory. It is found that ammonium formate could effectively enhance the leaching rate of rare earth and significantly inhibit the leaching efficiency of aluminum. Leaching agent pH has a greater impact on the leaching efficiency of aluminum, and no effects on that of rare earth. A higher leaching temperature in the tested range could enhance the leaching rate of rare earth and aluminum, but it will largely increase the production cost. The optimum conditions of leaching rare earth and aluminum are 0.1 mol/L ammonium sulfate compounded with 0.032 mol/L ammonium formate, 1:1 liquid:solid (mL/g), pH 5.0 ∼ 8.0 and room temperature. At these conditions, the leaching efficiency of rare earth and aluminum are 92.97% and 37.79% respectively. Moreover, the leaching equilibrium time for rare earth was 320 min shorter than the traditional leaching process by the ammonium sulfate, confirming an acceleration from this novel compound on the leaching of rare earth, especially for the difficult-to-infiltrate weathered crust elution-deposited rare earth ore. The leaching results are well fitted with the shrinking core model with apparent activation energy of 15.83 kJ/mol for RE and 9.60 kJ/mol for Al in the temperature range of 283 ∼ 323 K, indicating a diffusion controlled reaction, and the reaction orders are 1.06 for RE and −1.77 for Al.

Ultrasound assisted selective catalytic transfer hydrogenation of soybean oil using 5% Pd/C as catalyst under ambient conditions in water

Catalytic transfer hydrogenation (CTH) is an alternative approach that does not require the use of potentially dangerous hydrogen gas. Pd/C is the most favoured catalyst for the selective hydrogenation of soybean oil yielding lower extent of formation of stearic acid and trans-isomer, which have adverse health effects. The present work deals with intensification of catalytic transfer hydrogenation of soybean oil in the presence of 5wt.% Pd/C using ultrasound under ambient reaction conditions. The effect of important operating parameters such as ultrasound power, temperature, type of hydrogen donor, catalyst loading and donor concentration on the progress of reaction has been investigated. It was established that the maximum extent of hydrogenation as indicated by reduction in iodine value from 135 to 95 was observed under optimized conditions of irradiation power as 100W, 22kHz frequency, 90% duty cycle, ammonium formate concentration of 0.32mol/50ml water and 2% (w/w) Pd/C loading at ambient temperature and pressure in the presence of water as solvent. The approach also offered excellent selectivity with much lower trans-isomer formation as compared to the conventional approach of high pressure hydrogenation. Overall, the work has successfully demonstrated process intensification benefits due to the use of ultrasound for the Pd/C catalyzed transfer hydrogenation of soybean oil.

Formic acid–ammonium formate mixture: A new system with extremely high dehydrogenation activity and capacity

Formic acid (FA)–ammonium formate (AF) mixture is studied as a possible new system for dehydrogenation over a common Pd/C catalyst synthesized via a modified impregnation-NaBH4 reduction method. The composition and morphology of the Pd/C catalyst are characterized using X-ray diffraction, energy-dispersive X-ray spectroscopy, transmission electron microscopy, high-resolution transmission electron microscopy, and inductively coupled plasma–atomic emission spectrometry. Results show that the initial turn-over frequency of the FA–AF mixtures with total FA and AF concentration of 10 mol dm−3 and FA molar fraction of 0.2 can reach 7959 h−1 at 50 °C, which is the highest value reported for heterogeneous Pd-based catalysts in FA-based systems. FA in this mixture can completely decompose and deliver an electrochemical capacity of 565.87 mAh mL−1. The activity and stability of the Pd/C catalyst in this system are greatly improved in comparison with those of FA–sodium formate system. The excellent dehydrogenation performance of FA–AF mixture can be attributed to the catalytic activity of formate toward the decomposition of FA, the effect of ammonia adsorption on Pd/C catalyst, and the higher solubility of AF in FA solution.

Optimized ultra performance liquid chromatography tandem high resolution mass spectrometry method for the quantification of paraquat in plasma and urine.

A simple, sensitive and specific ultra performance liquid chromatography coupled to electrospray tandem high resolution mass spectrometry (UPLC-ESI-HRMS/MS) method has been developed and validated for quantification of paraquat in plasma and urine. The sample preparation was carried out by one-step protein precipitation with acetonitrile. The paraquat was separated with a HILIC column in 10min. Detection was performed using Q Exactive Orbitrap mass spectrometer by Targeted-MS/MS scan mode. Methodological parameters, such as ammonium formate concentration, formic acid concentration, spray voltage, capillary temperature, heater temperature and normalized collision energy were optimized to achieve the highest sensitivity. The calibration curve was linear over the concentration range of LOQ-1000ng/mL. LOD was 0.1 and 0.3ng/mL, LOQ was 0.3 and 0.8ng/mL for urine and plasma, respectively. The intra- and inter-day precisions were <7.97% and 4.78% for plasma and urine. The accuracies were within the range 93.51-100.90%. The plasma and urine matrices had negligible relative matrix effect in this study. This method was successfully applied to determine paraquat concentration in plasma samples with hemoperfusion from 5 suspected paraquat poisoning patients.

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography

The separation of 12 model compounds chosen among purine and pyrimidine bases and nucleosides was studied by using hydrophilic interaction chromatography (HILIC). The compounds investigated were small molecules with relevant properties for biomedical and pharmaceutical studies. The mixture of pyrimidines and purines was applied on a ZIC-HILIC 150 x 2.1 mm, 5 microm, and two TSKgel Amide-80 150 x 2.0 mm, 5 microm and 3 microm particle size columns. The retention of the analytes was studied by varying ACN%, ammonium formate concentration, pH, and column temperature. The results obtained confirmed the elution order of nucleobases, nucleosides, and nucleotides based on their hydrophobicity. The retention mechanism of the columns was studied considering the models used for describing partitioning and surface adsorption. The influence on retention of chromatographic conditions (ACN%, salt concentration, pH, and temperature) was described and discussed for both columns. The optimization of the conditions studied allowed to assess a gradient method for the separation of the 12 analytes. The developed method is a valuable alternative to existing methods for the separation of the compounds concerned.

Determination of soil amino acids by high performance liquid chromatography-electro spray ionization-mass spectrometry derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

Determination of amino acids by mass spectrometry (MS) is an important technique to investigate soil nitrogen transformation and cycling as amino acids being the major nitrogen-containing compounds in soil organic matter. However, researchers have long faced a critical problem in coupling an efficient separation technique to a sensitive MS detection system simultaneously. In this context, we established a new method of liquid chromatography coupled to mass spectrometry based on the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization method for convenient and accurate quantification of amino acids in soil samples. Baseline separation of 17 amino acid AQC-derivatives was achieved on an XTerra R MS C 18 column using ammonium formate as a mobile phase modifier. The concentration of ammonium formate and the pH of the mobile phase were optimized in order to obtain sensitive MS signals. The response curves were linear over the range of 50–800 μmol L −1 amino acids. The detection limits were 0.20–0.60 pmol μL −1 on column and 0.07–0.24 μg g −1 soil under the optimized conditions. The method has been applied successfully for the first time to determine amino acids in 4 types of soil samples, in which 15 amino acids were quantified by MS detector but methionine and cystine were below the detection limits. Both the recovery and the precision were satisfactory. Hence, this proposed technique shows a potential for the identification of amino acids in soil as well as tracing the transformation of soil amino acids with isotope dilution technique in nitrogen cycling investigation.

Direct determination of dialkyl phosphates by strong anion‐exchange liquid chromatography/atmospheric pressure chemical ionization mass spectrometry using a quadrupole ion trap instrument

The direct determination of dialkyl phosphates (DAPs) in water by strong anion-exchange (SAX) liquid chromatography/atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated. The SAX high-performance liquid chromatography (HPLC) column was eluted with methanol/water gradients containing ammonium formate (AF) separating the DAPs which included six dimethyl- and diethyl-substituted phosphates, thiophosphates, and dithiophosphates. The high buffer concentrations required for separation were compatible with -ve APCI, but in +ve APCI the DAPs were unstable giving anomalous ions such as [M+15]+ and [M+29]+. These ions are believed to result from ion molecule reactions with CH3OH2+ in the plasma. DAPs are very stable in -ve APCI being detected as abundant [M-H](-) ions, even with 200 mM AF. At higher AF concentration formate clusters ([M+45](-) and [M+91](-)) were seen. Fragmentation by collision-activated dissociation (CAD) was more efficient for deprotonated ethyl-substituted DAPs which lost ethylene followed by ethanol. APCI instrument detection limits were in the low ng/mL range and the response was highly linear. Isotope dilution quantitation using d10-diethyl dithiophosphate (DEDTP) as an internal standard produced an instrument detection limit of 2 ng DEDTP/mL and method detection limit (MDL) of 9.3 ng/mL with accuracy of 99% (spike concentration, 25 ng/mL). DAP mixtures required storage in cold, dry conditions and alcohol solvents should be avoided because of solvolysis reactions.

‘LC‐electrolyte effects’ improve the bioanalytical performance of liquid chromatography/tandem mass spectrometric assays in supporting pharmacokinetic study for drug discovery

The development of rapid and sensitive bioanalytical methods in a short time frame with acceptable levels of precision and accuracy is imperative for successful drug discovery. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate or formic acid increased analyte electrospray ionization (ESI) response and controlled against matrix effects. We designated these favorable effects 'LC-electrolyte effects'. In order to support rapid pharmacokinetic (PK) studies for drug discovery, we applied LC-electrolyte effects to the development of generic procedures that can be used to quickly generate reliable PK data for compound candidates. We herein demonstrate our approach using four model tested compounds (Compd-A, -B, -C, and -D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast liquid chromatographic (LC) gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) for individual measurement of the tested compounds in 20-microL plasma samples. Good linearity over the concentration range of 1.6 or 8-25000 ng/mL (r(2) > 0.99), precision (RSD, 0.45-13.1%), and accuracy (91-112%) were achieved through the use of a low dose of formic acid (0.4 mM or 0.015 per thousand) in the methanol/water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ion suppression caused by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with i.v./p.o. dosing of rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies.

Production of aromatic d-amino acids from α-keto acids and ammonia by coupling of four enzyme reactions

A multi-enzyme system composed of glutamate racemase, thermostable d-amino acid aminotransferase, glutamate dehydrogenase and formate dehydrogenase was employed for the production of aromatic d-amino acids, d-phenylalanine and d-tyrosine, from the corresponding α-keto acids, phenylpyruvate and hydroxyphenylpyruvate, respectively. The optimal concentration of ammonium formate for the production of these d-amino acids was found in the range of 0.25–1.0 M. The optimal concentration of α-keto acid was determined to be 50 mM, above which the productivity greatly decreased. To keep the concentration of α-keto acid around this concentration, α-keto acid was intermittently fed into the multi-enzyme system during the production period. By running the multi-enzyme system for 35 h, 48 g l −1 of d-phenylalanine and 60 g l −1 of d-tyrosine were produced with 100% of optical purity from the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate, respectively. The production levels of both aromatic d-amino acids were demonstrated to be dependent on the stability of glutamate racemase.

High performance liquid chromatography mobile phase composition optimization for the quantitative determination of a carboxylic acid compound in human plasma by negative ion electrospray high performance liquid chromatography tandem mass spectrometry.

A systematic investigation was undertaken to study the effects of varying concentrations of additives in the acetonitrile/water high performance liquid chromatography mobile phase, especially formic acid and ammonium formate, on the negative ion electrospray response of a carboxylic acid compound. The study showed that the response progressively decreased with increase in the formic acid concentration. While such a decrease in the response could be qualitatively explained by the decrease in the concentration of the ionized form of the carboxylic acid compound due to the lower pH of the mobile phase, the change in response was not as large as expected from the change of the concentration of the ionized form. The response also progressively decreased with increase in the ammonium formate concentration but the decrease cannot be explained by the change in the pH of the mobile phase. Although the best negative ion electrospray response was obtained with a water/acetonitrile mobile phase that contained no additives at all, the retention time of the analyte was not found to be adequately reproducible on repeated injections. Thus, this mobile phase was deemed unacceptable for practical, routine use. Comparing formic acid against ammonium formate, the former was preferable since it caused a smaller attenuation of the negative ion response. Equally important was the fact that addition of formic acid had the desirable effect of maintaining a reasonably high capacity factor (k') for the analyte even at a relatively high acetonitrile concentration. A concentration of 1 mM formic acid in the mobile phase was large enough to achieve the reproducible elongated retention time for the analyte, with a loss in the analyte response of about 60% only. It should be noted that the sensitivity achieved with the 1 mM formic acid mobile phase, in which the carboxylic acid is expected to be about 10% in the ionized form, is about 9 times better than the sensitivity achieved in the 1 mM ammonium formate mobile phase, in which the carboxylic acid is expected to be about 99% in the ionized form.

Formation of Deoxyfructosazine and Its 6-Isomer by the Browning Reaction between Fructose and Ammonium Formate

2 - (d - arabino - 1′,2′,3′,4 - Tetrahydroxybutyl) - 5 - (d - erythro - 2″,3″,4″ - trihydroxybutyl)pyrazine (“Deoxyfructosazine”) and 2-(d-arabino-1′,2′,3′,4′-tetrahydroxybutyl)-6-(d-erythro-2″,3″,4″-trihydroxybutyl)pyrazine (6-isomer) have been isolated from the browning mixture of fructose and ammonium formate. Especially, deoxyfructosazine was obtained in a considerable high yield, but its 6-isomer was formed only in a very low yield. In the reaction of glucose and ammonium formate, on the contrary, the 6-isomer was predominantly formed. Formation of these compounds was also dependent on the concentration of ammonium formate, and under appropriate conditions the yields of these compounds amounted to 0.38-0.42 g per g of the original sugars.