Sucrose rinse modulates the salivary behavior of carbonic anhydrase VI and its buffering capacity: a longitudinal study in 4 to 6.5-year-old children.

Background: Carbonic anhydrase VI (CA VI) is crucial in regulating oral pH and predicting susceptibility to dental caries. The hypothesis posits that caries activity may alter the CA VI function, diminishing its capacity to regulate pH effectively and potentially exacerbating cariogenic challenges. This 1-year cohort study sought to investigate the enzymatic activity of salivary CA VI and buffering capacity following a 20% sucrose rinse in 4 to 6.5-year-old children. Method: This research involved 46 volunteers categorized into three groups based on their caries status after follow-up: caries-free (CFee), arrested caries (CArrested), and caries active (CActive). Children underwent visible biofilm examination and saliva collection for salivary flow rate, buffering capacity, and CA VI analyses before and after a 20% sucrose rinse. Results: A reduction in the buffering capacity was observed after sucrose rinse in all groups. The CA VI activity decreased significantly in CFee and CArrested groups after sucrose rinse, although it did not change in the CActive group. An improvement in the buffering capacity and salivary flow rate was found at follow-up when compared with the baseline. After 1-year follow-up, buffering capacity and salivary flow rate increased in all groups, whilst the CA VI activity reduced only in CFree and CArrested children. Conclusion: Sucrose rinse universally reduces the salivary buffering capacity, while caries activity may disrupt CA VI activity response during a cariogenic challenge. After a year, increased salivary flow enhances buffering capacity but not CA VI activity in caries-active children.

Solubility vs Dissolution in Physiological Bicarbonate Buffer.

BACKGROUND: Phosphate buffer is often used as a replacement for the physiological bicarbonate buffer in pharmaceutical dissolution testing, although there are some discrepancies in their properties making it complicated to extrapolate dissolution results in phosphate to the in vivo situation. This study aims to characterize these discrepancies regarding solubility and dissolution behavior of ionizable compounds. METHODS: The dissolution of an ibuprofen powder with a known particle size distribution was simulated in silico and verified experimentally in vitro at two different doses and in two different buffers (5 mM pH 6.8 bicarbonate and phosphate). RESULTS: The results showed that there is a solubility vs. dissolution mismatch in the two buffers. This was accurately predicted by the in-house simulations based on the reversible non-equilibrium (RNE) and the Mooney models. CONCLUSIONS: The results can be explained by the existence of a relatively large gap between the initial surface pH of the drug and the bulk pH at saturation in bicarbonate but not in phosphate, which is caused by not all the interfacial reactions reaching equilibrium in bicarbonate prior to bulk saturation. This means that slurry pH measurements, while providing surface pH estimates for buffers like phosphate, are poor indicators of surface pH in the intestinal bicarbonate buffer. In addition, it showcases the importance of accounting for the H2CO3-CO2 interconversion kinetics to achieve good predictions of intestinal drug dissolution.

Drug Crystal Precipitation in Biorelevant Bicarbonate Buffer: A Well-Controlled Comparative Study with Phosphate Buffer.

The purpose of the present study was to clarify whether the precipitation profile of a drug in bicarbonate buffer (BCB) may differ from that in phosphate buffer (PPB) by a well-controlled comparative study. The precipitation profiles of structurally diverse poorly soluble drugs in BCB and PPB were evaluated by a pH-shift precipitation test or a solvent-shift precipitation test (seven weak acid drugs (pKa: 4.2 to 7.5), six weak base drugs (pKa: 4.8 to 8.4), one unionizable drug, and one zwitterionic drug). To focus on crystal precipitation processes, each ionizable drug was first completely dissolved in an HCl (pH 3.0) or NaOH (pH 11.0) aqueous solution (450 mL, 50 rpm, 37 °C). A 10-fold concentrated buffer solution (50 mL) was then added to shift the pH value to 6.5 to initiate precipitation (final volume: 500 mL, buffer capacity (ß): 4.4 mM/ΔpH (BCB: 10 mM or PPB: 8 mM), ionic strength (I): 0.14 M (adjusted by NaCl)). The pH, ß, and I values were set to be relevant to the physiology of the small intestine. For an unionizable drug, a solvent-shift method was used (1/100 dilution). To maintain the pH value of BCB, a floating lid was used to avoid the loss of CO2. The floating lid was applied also to PPB to precisely align the experimental conditions between BCB and PPB. The solid form of the precipitants was identified by powder X-ray diffraction and differential scanning microscopy. The precipitation of weak acids (pKa ≤ 5.1) and weak bases (pKa ≥ 7.3) was found to be slower in BCB than in PPB. In contrast, the precipitation profiles in BCB and PPB were similar for less ionizable or nonionizable drugs at pH 6.5. The final pH values of the bulk phase were pH 6.5 ± 0.1 after the precipitation tests in all cases. All precipitates were in their respective free forms. The precipitation of ionizable weak acids and bases was slower in BCB than in PPB. The surface pH of precipitating particles may have differed between BCB and PPB due to the slow hydration process of CO2 specific to BCB. Since BCB is a physiological buffer in the small intestine, it should be considered as an option for precipitation studies of ionizable weak acids and bases.

Association between sleep pattern, salivary cariogenic bacteria and fungi populations, pH and buffering capacity in children: A comparative study.

BACKGROUND: Sleep quality has a significant impact on a child's health and is linked to oral and systemic diseases. It affects the circadian rhythm, which plays a crucial role in regulating the balance of the endocrine and hormonal systems. Current research has focused on exploring its role in the development of caries, which is influenced by inherent oral factors such as the composition of the oral microbiome and pH levels. OBJECTIVES: This study aimed to investigate the relationship between bacterial population, pH, and buffering properties of saliva and sleep patterns in 8- to 12-year-old children. MATERIAL AND METHODS: This cross-sectional study was conducted on 85 elementary school children aged 8-12 years. After obtaining written consent, non-stimulating saliva samples were collected using the spitting method. The participants' sleep pattern information was obtained with the use of the Persian version of the Children's Sleep Habits Questionnaire (CSHQ). Based on the results of the CSHQ, the participants were divided into 2 groups: those with appropriate sleep patterns; and those with inappropriate sleep patterns. The study compared the bacterial population of Streptococcus mutans, Lactobacillus spp. and Candida albicans, as well as the buffering capacity and pH of the saliva between the 2 groups. The statistical analysis employed the χ2 test, the independent samples t-test and Spearman's correlation. RESULTS: The group with inappropriate sleep patterns had significantly lower pH and buffering capacity (p < 0.001) and significantly higher colony counts of Lactobacillus and S. mutans (p < 0.001 and p = 0.012, respectively). There was no association between C. albicans and sleep patterns (p = 0.121). CONCLUSIONS: Inappropriate sleep patterns increase the population of caries-causing bacteria and reduce salivary pH and buffering capacity. This can be a significant factor in the development of dental caries in children aged 8-12 years.

Dissolution Profiles of Immediate Release Products of Various Drugs in Biorelevant Bicarbonate Buffer: Comparison with Compendial Phosphate Buffer.

PURPOSE: The purpose of this study was to clarify the extent to which the dissolution profiles of immediate release (IR) products of various drugs differ between biorelevant bicarbonate buffer (BCB) and compendial phosphate buffer (PPB). METHODS: The dissolution profiles of the IR products of fifteen poorly soluble ionizable drugs were measured in BCB and PPB. BCB was set to be relevant to the small intestine (pH 6.8, 10 mM). The pH was maintained using the floating lid method. The Japanese pharmacopeia second fluid (JP2, 25 mM phosphate buffer, nominal pH 6.8) was used as compendial PPB. The compendial paddle apparatus was used for the dissolution tests (500 mL, 50 rpm, 37°C). RESULTS: In 11/15 cases, a difference in dissolved% (< 0.8 or > 1.25-fold) was observed at a time point. In 4/15 cases, the ratio of the area under the dissolution curve was not equivalent (< 0.8 or > 1.25-fold). In the cases of free-form drugs, the dissolution rate tended to be slower in BCB than in JP2. In the case of salt-form drugs, a marked difference was observed for the cases that showed supersaturation. However, no trend was observed in the differences. CONCLUSIONS: Many IR products showed differences in the dissolution profiles between biorelevant BCB and compendial PPB. With the floating lid method, BCB is as simple and easy to use as PPB. Biorelevant BCB is recommended for dissolution testing.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Exploiting Principal Component Analysis (PCA) to reveal temperature, buffer and metal ions' role in neuromelanin (NM) synthesis by dopamine (DA) oxidative polymerization.

Neuromelanin (NM) plays a well-established role in neurological disorders pathogenesis; the mechanism of action is still discussed and the investigations in this field are limited by NM's complex and heterogeneous composition, insolubility, and low availability from human brains. An alternative can be offered by synthetic NM obtained from dopamine (DA) oxidative polymerization; however, a deep knowledge of the influence of both physicochemical parameters (T, pH, ionic strength) and other compounds in the reaction media (buffer, metal ions, other catecholamines) on DA oxidation process and, consequently, on synthetic NM features is mandatory to develop reliable NM preparation methodologies. To partially fulfill this aim, the present work focuses on defining the role of temperature, buffer and metal ions on both DA oxidation rate and DA oligomer size. DA oxidation in the specific conditions is monitored by UV-Vis spectroscopy and Principal Component Analysis (PCA) is run either on the raw spectra to model the background absorption increase, related to small DA oligomers formation, or on their first derivative to rationalize DA consumption. After having studied three case studies, 3-Way PCA is applied to directly evaluate the effect of temperature and buffer type on DA oxidation in the presence of different metal ions. Despite the proof-of-concept nature of the work and the number of compounds still to be included in the investigation, the preliminary results and the possibility to further expand the chemometric approach represent an interesting contribution to the field of in vitro simulation of NM synthesis.

Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

PURPOSE/STUDY QUESTION: Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? METHODS: Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). RESULTS: Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). CONCLUSION: The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing. Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

Assessing the Efficacy of Buffered Versus Nonbuffered Lidocaine in Dental Extractions: A Double-Blinded Randomized Controlled Trial.

BACKGROUND: Injections using buffered lidocaine may decrease discomfort, have a quicker onset, and be a more efficacious local anesthetic. Previous studies have been inconclusive in the oral context. PURPOSE: To address if bicarbonate buffered 2% lidocaine can decrease pain from the use of local anesthesia, has a quicker onset time, and is more efficacious. STUDY DESIGN: The design was a single-center double-blinded randomized control trial, set in an outpatient oral and maxillofacial clinic housed in the University of Cincinnati Medical Center. Inclusion criteria for the study were patients requiring a single tooth extraction due either to caries or periodontal disease. PREDICTOR VARIABLE: The predictor variable was the local anesthetic used either nonbuffered 2% lidocaine with 1:100,000 epinephrine (control) or bicarbonate buffered 2% lidocaine with 1:100,000 epinephrine (study) was randomly assigned. MAIN OUTCOME VARIABLES: Primary outcome variables were injection pain score, and postoperative pain, time to anesthetic onset, and the number of rounds of injections required to achieve adequate anesthesia. COVARIATES: The covariates were jaw involved, age, sex, and race, American Society of Anesthesiologists score, body mass index, current tobacco use, history of psychiatric illness, chronic pain, and preoperative pain score. ANALYSES: Test statistics were calculated using Wilcoxon rank-sum test, Kruskal-Wallis test, Spearman rank correlation test, χ2 test for bivariate analyses, and Fisher's exact test. P values ≤ .05 were considered statistically significant. RESULTS: The final sample was 114 subjects. The mean age of the sample was 42.97 years, standard deviation ±13.43 years. The sample was 39.47% male. The racial demographics were Caucasian (62.28%) and African American (33.33%). Buffered lidocaine did not have a statistically significant relationship with any of the outcomes. The jaw involved had a statistically significant association to the injection pain score (P value = .006), and the number of rounds of anesthetic required (P value = .047). Age showed a statistically significant association to injection pain score (P value = .032), and the number of rounds of anesthetic required (P value = .027). Finally, preoperative pain had a statistically significant relationship with injection pain score (P value = < .001). CONCLUSION AND RELEVANCE: In this study, bicarbonate buffered lidocaine did not exhibit any discernible advantages over nonbuffered lidocaine for any study outcomes.

Evaluation of acute intravenous toxicity of HEPES: Is Good's buffer good and safe enough for clinical utilization in nuclear medicine?

OBJECTIVE: Good's buffer or HEPES has advantages over other buffers commonly used in radiopharmaceutical preparation as it exhibits significantly lower complexation tendency with metal ions. However, use of HEPES buffer for radiolabeling reactions, meant for clinical applications, has been underrated due to the non-availability of sufficient toxicity data. The objective of the present study is to find the evidences towards safety of intravenous administration of HEPES through systemic toxicological studies in small animal model to support its safe application for clinical exploitation. EXPERIMENTAL: A pilot study was performed to investigate the lethal dose of HEPES in female Sprague Dawley rats by administering seven different doses of HEPES solution (150 to 2000 mg/kg), through intravenous pathway. Similarly, for determining maximum tolerated dose (MTD), gradually increasing doses of HEPES (50 to 950 mg/kg) were administered in the same species via similar pathway. Various hematological and clinical pathological investigations were carried out in order to find out the safe administration dose of HEPES in rats. RESULTS: No mortality was observed up to 2000 mg/kg doses of HEPES. The doses beyond 300 mg/kg resulted few temporary adverse effects, though these were found to disappear within 4-5 days of dosing. CONCLUSION: The amount of HEPES to be administered during clinical intervention is usually much lower (typically 1-2.5 mg per kg of body weight of healthy adult) than the MTD determined in rat model during present report. Hence, the utilization of this buffer for preparation of radiolabeled drugs for human investigation may be safe. However, further detailed investigations may be warranted for supporting the candidature of Good's buffer for regular clinical exploitation.

Unraveling the Microscopic Mechanism of Molecular Ion Interaction with Monoclonal Antibodies: Impact on Protein Aggregation.

Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.

Evaluation of the efficacy of buffered local anesthetic in extraction of infected teeth-randomized double-blind study.

PURPOSE: In the presence of infection, acidic pH of a lignocaine local anesthetic causes undesirable effects such as burning on injection, relatively slow onset, and lack of numbness. Buffered lignocaine will increase the pH of the solution and may resolve above problems. Thus, the objective of this study is to compare the efficacy of buffered lignocaine with that of commercial lignocaine. METHOD: Seventy patients with infected teeth were randomly divided into two equal groups. The study group received buffered lignocaine (8.4% sodium bicarbonate added to 2% lignocaine mixture) while the control received commercial lignocaine preparation (2% lignocaine with 1:80,000 adrenaline). Burning while injection, pain using VAS scale and onset of action with EPT (electric pulp tester) were recorded. RESULTS: In the study group, the VAS score after injection was 1.20 ± 0.68 and the control group was 2.57 ± 0.92 (p = 0.001). There was a statistically significant reduction in pain reduction in the study group. The time of onset was 3.97 ± 0.71 and 5.67 ± 1.15 min, respectively, and the difference was statically significant. Only one-third of the study group experienced burning on injection as compared to two-thirds in the control group. CONCLUSION: Buffered lignocaine is more effective as compared to commercial lignocaine in the extraction of infected teeth. CTRI NUMBER: CTRI/2022/01/039476.

Fluorinated Ethylamines as Electrospray-Compatible Neutral pH Buffers for Native Mass Spectrometry.

Native electrospray ionization mass spectrometry (ESI-MS) has emerged as a potent tool for examining the native-like structures of macromolecular complexes. Despite its utility, the predominant "buffer" used, ammonium acetate (AmAc) with pKa values of 4.75 for acetic acid and 9.25 for ammonium, provides very little buffering capacity within the physiological pH range of 7.0-7.4. ESI-induced redox reactions alter the pH of the liquid within the ESI capillary. This can result in protein unfolding or weakening of pH-sensitive interactions. Consequently, the discovery of volatile, ESI-compatible buffers, capable of effectively maintaining pH within a physiological range, is of high importance. Here, we demonstrate that 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) offer buffering capacity at physiological pH where AmAc falls short, with pKa values of 7.2 and 5.5 for the conjugate acids of DFEA and TFEA, respectively. Native ESI-MS experiments on model proteins cytochrome c and myoglobin electrosprayed with DFEA and TFEA demonstrated the preservation of noncovalent protein-ligand complexes in the gas phase. Protein stability assays and collision-induced unfolding experiments further showed that neither DFEA nor TFEA destabilized model proteins in solution or in the gas phase. Finally, we demonstrate that multisubunit protein complexes such as alcohol dehydrogenase and concanavalin A can be studied in the presence of DFEA or TFEA using native ESI-MS. Our findings establish DFEA and TFEA as new ESI-compatible neutral pH buffers that promise to bolster the use of native ESI-MS for the analysis of macromolecular complexes, particularly those sensitive to pH fluctuations.

Predicting Colloidal Stability of High-Concentration Monoclonal Antibody Formulations in Common Pharmaceutical Buffers Using Improved Polyethylene Glycol Induced Protein Precipitation Assay.

Colloidal stability is an important consideration when developing high concentration mAb formulations. PEG-induced protein precipitation is a commonly used assay to assess the colloidal stability of protein solutions. However, the practical usefulness and the current theoretical model for this assay have yet to be verified over a large formulation space across multiple mAbs and mAb-based modalities. In the present study, we used PEG-induced protein precipitation assays to evaluate colloidal stability of 3 mAbs in 24 common formulation buffers at 20 and 5 °C. These prediction assays were conducted at low protein concentration (1 mg/mL). We also directly characterized high concentration (100 mg/mL) formulations for cold-induced phase separation, turbidity, and concentratibility by ultrafiltration. This systematic study allowed analysis of the correlation between the results of low concentration assays and the high concentration attributes. The key findings of this study include the following: (1) verification of the usefulness of three different parameters (Cmid, µB, and Tcloud) from PEG-induced protein precipitation assays for ranking colloidal stability of high concentration mAb formulations; (2) a new method to implement PEG-induced protein precipitation assay suitable for high throughput screening with low sample consumption; (3) improvement in the theoretical model for calculating robust thermodynamic parameters of colloidal stability (µB and εB) that are independent of specific experimental settings; (4) systematic evaluation of the effects of pH and buffer salts on colloidal stability of mAbs in common formulation buffers. These findings provide improved theoretical and practical tools for assessing the colloidal stability of mAbs and mAb-based modalities during formulation development.

A Novel Approach to Monitor the Concentration of Phosphate Buffers in the Range of 1 M to 0.1 M Using a Silicon-Based Impedance Sensor.

We present a novel and easy approach using a silicon-based impedance chip to determine the concentration of the given aqueous buffer solution. An accurate determination of the post-dilution concentration of the buffers is necessary for ensuring optimal buffer capacity, pH stability, and to assess solution reproducibility. In this study, we focused on phosphate buffer as the test liquid to achieve precise post-dilution concentration determinations. The impedance chip consisting of a top gold ring electrode, where a test volume of 20 µL to 30 µL of phosphate buffer was introduced for impedance measurements within the frequency range of 40 Hz to 1 MHz. For impedance investigation, we used phosphate buffers with three different pH values, and the impedance was measured after diluting the phosphate buffers to a concentration of 1.00 M, 0.75 M, 0.50 M, 0.25 M, 0.10 M, 0.05 M, and 0.01 M. In order to analyze the distinctive changes in the measured impedance, an equivalent circuit was proposed and modeled. From the impedance modeling, we report that the circuit parameter RAu/Si showed exponential dependence on the concentration of phosphate buffer and no dependence on the pH values of the phosphate buffer and on the added volume inside the ring electrode. The proposed silicon-based impedance chip is quick and uses reduced liquid volume for post-dilution concentration measurements of buffers and has perspective applications in the pharmaceutical and biological domains for regulating, monitoring, and quality control of the buffers.

Strategies to Stabilize Dalbavancin in Aqueous Solutions; Section-1: the Effects of Metal Ions and Buffers.

PURPOSE: The effect of monovalent (Na+ and K+) and divalent (Ca2+, Mg2+, and Zn2+) metal ions combined with citrate or acetate buffers (pH 4.5) on the stability of dalbavancin in aqueous solutions was investigated. METHOD: RP-HPLC and HP-SEC were used to evaluate the stability of aqueous solutions of dalbavancin in different combinations of buffers and metal ions after four weeks of storage at 5°C and 55°C. A long-term study of formulations with divalent metal ions was conducted over six months at 5°C., 25°C and 40°C using RP-HPLC. RESULTS: All formulations in citrate buffered solutions precipitated. Dalbavancin solutions in 10 mM acetate buffer at 55°C were more stable in 10 mM CaCl2, 5 mM ZnCl2 and 10 mM MgCl2 than those containing 2 mM NaCl or 5 mM KCl, although the MgCl2 formulations precipitated slightly. No significant effect was observed for any of the divalent metal ions at 40°C for six months. CONCLUSION: Dalbavancin's stability in solution was improved by a combination of acetate and divalent metal ions at 55°C for four weeks. No effect was observed with acetate or metal ions alone, and no effect was observed after six months at 40°C suggesting that acetate and divalent metal ions together interact with dalbavancin via a thermally activated step to inhibit hydrolysis of the drug.

Phosphate buffer interferes dissolution of prazosin hydrochloride in compendial dissolution testing.

The purpose of this study was to elucidate the lack of supersaturation behavior in the dissolution profile of prazosin hydrochloride (PRZ-HCl) in the compendial dissolution test. The equilibrium solubility was measured by a shake-flask method. Dissolution tests were performed by a compendial paddle method with a phosphate buffer solution (pH 6.8, 50 mM phosphate). The solid form of the residual particles was identified by Raman spectroscopy. In the pH range below 6.5, the equilibrium solubility in phosphate buffer was lower than that in the unbuffered solutions (pH adjusted by HCl and NaOH). Raman spectra showed that the residual solid was a phosphate salt of PRZ. In the pH range above 6.5, the pH-solubility profiles in the phosphate buffer solutions and the unbuffered solutions were the same. The residual solid was a PRZ freebase (PRZ-FB). In the dissolution test, PRZ-HCl particles first changed to a phosphate salt within 5 min, then gradually changed to PRZ-FB after several hours. Since the intestinal fluid is buffered by the bicarbonate system in vivo, the dissolution behavior in vivo may not be properly evaluated using a phosphate buffer solution. For drugs with a low phosphate solubility product, it is necessary to consider this aspect.

Investigating freezing-induced acidity changes in citrate buffers.

Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.