A preformulation study on the kinetics of HI-6 in concentrated solution

The degradation kinetics of HI-6 have been investigated at various temperatures and at different pHs at a concentration of 200 mg/ml. Both aqueous and other hydrophilic solvents (glycols and glycerol-water mixtures) have been used. The degradation of HI-6 follows pseudo-first-order kinetics with respect to HI-6. The observed rate seems to depend on a hydroxyl ion-catalyzed reaction (k_OH) and an un/water catalyzed reaction (k₀) which show influence at pH below 3. The pH profile shows a positive slope less than one, which seems to depend on an error in the determination of the rate constants at higher pH. k_OH was determined to be 1 · 10⁹ andk₀ to be 0 · 011 h⁻¹ at 60° C. The observed rate seems to be independent of the dielectric constant of the solvent. The activation energy k₀ has been determined to be 82.0 kJ mol⁻¹. Based upon these data the predicted shelf life (t_90%) at pH 0 will not be long than 13 days at 25 ° C and 9 months at 0°C. Thus, the systems studied seem to be unsuitable to formulate a stable intramuscular formulation of HI-6.     

Molecular docking and pharmacogenomics of vinca alkaloids and their monomeric precursors, vindoline and catharanthine

Vinblastine and vincristine are dimeric indole alkaloids derived from Catharanthus roseus (formerly: Vinca rosea). Their monomeric precursor molecules are vindoline and catharanthine. While vinblastine and vincristine are well-known mitotic spindle poisons, not much is known about vindoline and catharanthine. Vindoline and catharanthine showed weak cytotoxicity, while vinblastine, vincristine, and the semisynthetic vindesine and vinorelbine revealed high cytotoxicity towards cancer cells. This may reflect a general biological principle of poisonous plants. Highly toxic compounds are not only active towards predators, but also towards plant tissues. Hence, plants need mechanisms to protect themselves from their own poisons. One evolutionary strategy to solve this problem is to generate less toxic precursors, which are dimerized to toxic end products when needed. As shown by in silico molecular docking and biochemical approaches, vinblastine, vincristine and vinorelbine bound with high affinity to α/β-tubulin and inhibited tubulin polymerization, whereas the effects of vindoline and catharanthine were weak. Similarly, vinblastine produced high fractions of mono- and multipolar mitotic spindles, while vindoline and catharanthine did only weakly affect bipolar mitotic spindle formation. Here, we show that vinblastine contributes to cell death by interference with spindle polarity. P-glycoprotein-overexpressing multidrug-resistant CEM/VCR1000 cells were highly resistant towards vincristine and cross-resistant to vinblastine, vindesine, and vinorelbine, but not or only weakly cross-resistant to vindoline and catharanthine. In addition to tubulin as primary target, microarray-based mRNA signatures of responsiveness of these compounds have been identified by COMPARE and signaling pathway profiling.     

Flow cytometric evaluation of the effect of various doses of vindesine sulphate on mouse spermatogenesis

Spermatogenesis, a rapidly proliferating cell system, is highly susceptible to damage by radiotherapy and/or chemotherapy. Vindesine, a semisynthetic vinca alkaloid, was given as a single injection to adult male Swiss albino mice to study its effects on testicular weight and male germ cell turnover pattern using flow cytometry. Testicular weight declined significantly at Day 7 to 14 and from Day 14 to 35 after administration of 1 and 2 mg/kg b wt vindesine, respectively. Flow cytometric evaluation of various testicular cell types after the administration of 2 mg/kg b wt vindesine revealed a significant increase in the relative percentage of spermatogonial cells at Day 21 and 35 posttreatment. In contrast, the relative percentage of primary spermatocytes declined significantly at Day 7 and 14 posttreatment. Similarly, a significant reduction in the relative percentage of round spermatids was observed from Day 7 to 35 posttreatment. The relative percentage of elongated spermatids declined significantly at day 35 post-treatment. These changes are reflected in the transformation ratios. While the 4C:2C ratio did not exhibit any significant change below 1 mg/kg vindesine, it declined significantly after 1 mg/kg (Day 14) and 2 mg/kg (Day 7 to 35, except Day 28 posttreatment) vindesine treatment. Treatment of male mice with 2 mg/kg vindesine resulted in a significant decline in 1C:2C ratio from 7 to 35 d post-treatment. The 4C:S-phase ratio decreased significantly at Day 7 and 14 posttreatment for all the drug doses above 0.05 mg/kg. A significant reduction in the 1C:4C ratio was observed at day 21 to 35 posttreatment as a result of 2 mg/kg vindesine administration.     

Effects of incorporated drugs on degradation of novel 2,2'-bis(2-oxazoline) linked poly(lactic acid) films

Earlier studies have indicated that the degradation rate of poly(lactic acid) (PDLLA) can be modified by using 2,2′-bis(2-oxazoline) as a chain extender in polymer synthesis to form a lactic acid-based poly(ester-amide) (PEA). In the present study, the effect of an incorporated drug on the degradation rate of the PEA was evaluated. The model drugs, neutral guaifenesin, acidic sodium salicylate (pK_a 3.0) and basic timolol (pK_a 9.2), were incorporated into solvent cast PDLLA and PEA films. The drug content in the films was 2% (w/w). The degradation studies were carried out in PBS (pH 7.4, 37 °C); the resulting decrease in molecular weight of polymers was determined by size exclusion chromatography and the weight loss of films was measured. In addition, the drug release from the films in PBS (pH 7.4, 37 °C) was studied. The model drugs were released from the PDLLA and PEA films in a biphasic or triphasic manner. The final fast release phase of the drugs from both PDLLA and PEA films started when the molecular weight (M_n) of the polymer had decreased close to 15,000 g/mol. The degradation rate of the PDLLA films was clearly enhanced by incorporated sodium salicylate or timolol. Whereas, the degradation rate of the PEA film was not enhanced by the incorporated drugs. The present results indicate that when compared to the PDLLA film, degradation rate of the PEA film in the presence of the drug is more predictable.     

A study of toxicity and comparative therapeutic efficacy of vindesine-prednisone vs. vincristine-prednisone in children with acute lymphoblastic leukemia in relapse

Summary Vindesine (des-acetyl Vinblastine) is a synthetic derivative of vinblastine, and was produced with the hope that it would have less neurotoxicity and hematopoietic toxicity than other vinca alkaloids.Phase I and II studies also demonstrated significant activity in lymphoid malignancies, especially Acute Lymphoblastic Leukemia (ALL). The present study was designed to compare therapeutic effectiveness of twice weekly vindesine (2 mg/M²/dose) plus Prednisone (60 mg/M²/dose) (Treatment 1) to weekly Vincristine (2 mg/M²/dose) plus Prednisone (60 mg/M²/day) (Treatment 2). All patients were less than 21 years of age, and had documented bone marrow relapse (blast count>25%). In 39 patients presumed sensitive to vincristine, there were 11 complete responses out of 20 patients (55%) randomized to receive vindesine/ prednisone and 7 complete responses out of 19 patients (37%) treated with Vincristine/Prednisone. In 37 patients resistant to vincristine, there were 7 complete responses (19%). Vindesine was more toxic than Vincristine. Major toxicities of vindesine included paraesthesias, peripheral neuropathy and ileus. Vindesine hematological toxicity appeared greater, but such toxicity is hard to assess in patients with bone marrow disease. In this study, vindesine and vincristine had similar efficacy, but vindesine use was associated with more toxicity.     

The ICH guidance in practice: stress degradation studies on indinavir sulphate and development of a validated specific stability-indicating HPTLC assay method

A sensitive, selective, precise and stability-indicating high-performance thin layer chromatography (HPTLC) method for analysis of indinavir sulphate both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon tetrachloride/chloroform/methanol/10% v/v ammonia (4:4.5:1.5:0.05, v/v/v/v). Densitometric analysis of indinavir sulphate was carried out in the absorbance mode at 260 nm. This system was found to give compact spots for indinavir sulphate (Rf value of 0.43 +/- 0.02, for six replicates). Indinavir sulphate was subjected to acid and alkali hydrolysis, oxidation, dry and wet heat treatment, and photo degradation. The drug undergoes degradation under acidic and basic conditions, oxidation, dry and wet heat treatment, and photo degradation. Also the degraded products were well resolved from the pure drug with significantly different Rf values. The method was validated for linearity, precision, robustness, limit of detection (LOD), limit of quantitation (LOQ), specificity and accuracy. Linearity was found to be in the range of 100-6000 ng/spot with significantly high value of correlation coefficient r2 = 0.997 +/- 0.64. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.999 +/- 0.002 in the working concentration range of 1000-6000 ng/spot. The LOD and LOQ were 40 and 120 ng/spot, respectively. Statistical analysis proves that the method is repeatable and specific for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of acid degradation process. Arrhenius plot was constructed and activation energy was calculated.     

Formulation and in vitro/in vivo evaluation of terbutaline sulphate incorporated in PLGA (25/75) and L-PLA microspheres

Terbutaline sulphate (TBS) is widely used in the treatment of bronchial asthma, chronic bronchitis and emphysema. Because of its short biological half life and dosing schedule, a long acting TBS formulation is required to improve patient compliance. The objective of this study was to develop a TBS containing biodegradable microsphere formulation. Poly(D, L-lactide-co-glyco-lide) (PLGA) and poly(L-lactic acid) (L-PLA) were chosen as matrix materials. A solvent evaporation method was used for preparation of microspheres. Surface morphology, particle size distribution and encapsulation efficiency were investigated. In vitro release studies were performed in pH 7.4 phosphate buffer. In vivo distribution of microspheres were studied in the Swiss albino male mice. All microspheres were spherical in shape and had a porous surface with mean diameters of 9–21 μm. The encapsulation efficiency was influenced by the polymer type, but not the molecular weight. About 90% of the initial amount was trapped in PLGA microspheres, and the remainder was on the surface. In the case of L-PLA, 50% of the total drug was associated with the surface of microspheres. The in vitro release pattern was biphasic characterized by an initial burst phase followed by a slower phase. The L-PLA microspheres released ～92% of the initial payload in 72 h. On the other hand, TBS release was increased with an increase in the molecular weight of PLGA. Biodistribution of L-PLA microspheres was characterized by an initially high uptake (35%) by the lungs. All these results suggest that L-PLA and PLGA microspheres have the potential to be used for passive lung targeting.     

Stability Studies of Gabapentin in Aqueous Solutions

Gabapentin is a -aminobutyric acid analogue, which has been shown to be an effective antiepileptic. The solution stability of gabapentin in buffered systems was studied in order to facilitate the formulation of a liquid product. The degradation of the drug was followed as a function of pH, buffer concentration, ionic strength, and temperature. The results indicated that the rate of degradation was proportional to the buffer concentration and temperature. The pH–rate profile of gabapentin degradation showed that the rate of degradation was minimum at an approximate pH of 6.0. Further, the data suggested a slower solvent-catalyzed degradation rate for the zwitterionic species compared to the cationic or anionic species in the pH range of 4.5 to 7.0. There was no influence of ionic strength on the rate of degradation. Arrhenius plots of the data indicated that a shelf life of 2 years or more at room temperature may be obtained in an aqueous solution at a pH value of 6.0.     

Structure-activity relationships of dimeric Catharanthus alkaloids. 2. Experimental antitumor activities of N-substituted deacetylvinblastine amide (vindesine) sulfates.

While structure-activity relationships for vinblastine (VLB), vincristine, deacetyl-VLB, and deacetyl-VLB amide (vindesine, VDS) in several tumor and leukemia models have been reported previously, the present study explores these relationships for a series of N-substituted vindesine analogues. These compounds were prepared from the reaction of deacetyl-VLB acid azide with the appropriate amines and were characterized by mass spectral analysis, 1H and 13C NMR spectra, electrometric titration, and infrared spectra. N-Alkylvindesines have reduced activity compared to that of VDS against the Gardner lymphosarcoma (GLS). N-beta-Hydroxyethyl-VDS surpasses vindesine in its activity against the Ridgway osteogenic sarcoma and the GLS, whereas against the B16 melanoma it is less active than VDS. N-beta-(4-Hydroxyphenethyl)-VDS, envisaged as a substrate for the enzyme tryosinase, was shown to be more active than VDS against the B16 melanoma but has only marginal activity against the GLS. In terms of collective antitumor activity against the model systems used, vindesine emerges as the congener with optimum qualities. Bis(N-ethylidenevindesine) disulfide, the first example of a bridged bisvindesine and comparable to VDS in its antitumor profile, shows evidence of activity against a P388/VCR leukemia strain known to be resistant to maytansine as well as to vincristine.     

The effect of ferrous sulphate and sucralfate on the bioavailability of oral gemifloxacin in healthy volunteers

Sucralfate is a cytoprotectant with antacid properties and ferrous sulphate is commonly prescribed for iron-deficiency anaemia. This open, randomized, single-dose, five-way crossover study investigated the effect of sucralfate and ferrous sulphate on the bioavailability of gemifloxacin, a novel fluoroquinolone antimicrobial. Twenty-seven healthy male volunteers received gemifloxacin, 320 mg p.o., alone, 3 h after sucralfate (2 g) or ferrous sulphate (325 mg), or 2 h before sucralfate or ferrous sulphate. Each subject received all five dosing regimens in random order with at least 6 days between regimens. Plasma samples collected up to 48 h after dosing with gemifloxacin, were assayed for gemifloxacin to determine pharmacokinetic parameters. Administration of gemifloxacin 3 h after sucralfate produced a marked decrease of 53% in the area under the plasma concentration–time curve from time zero extrapolated to infinity (AUC_0–∞), and a decrease of 69% in the maximal plasma concentration (C_max). Administration of gemifloxacin 3 h after ferrous sulphate resulted in only a modest reduction of 11% in AUC_0–∞ and of 20% in C_max, which was not considered to be clinically significant. In contrast, at the doses used neither sucralfate nor ferrous sulphate altered gemifloxacin bioavailability when it was administered 2 h before either of these agents. Gemifloxacin was well tolerated in all the regimens. The results of this study support the dosing recommendation that gemifloxacin can be safely administered at least 2 h before sucralfate or ferrous sulphate, or at least 3 h after ferrous sulphate.     

Voltammetric determination of vitamins in a pharmaceutical formulation

Direct current polarography and differential pulse polarographic methods have been developed for the qualitative as well as quantitative analysis of vitamin B₁, B₂ and B₆. Thiamin (Vitamin B₁) produced a well-defined wave in 0.1 M KCl at pH 5.2 with E_1/2=−1.2 V and E_p=−1.22 V versus saturated calomal electrode (SCE). Riboflavin (Vitamin B₂) gave two distinct waves in Britton Robinson buffer at pH 1.8 with E_1/2 VALUES=−0.13 and −0.34 V versus SCE and at pH 6.5 with E_1/2=−1.10 V and E_p=−1.2 V versus SCE. Pyridoxin (Vitamin B₆) produced a well-defined wave in Britton Robinson buffer at pH 6.5 with E_1/2=−1.7 V and E_p=−1.68 V versus SCE. All the three Vitamins under study are reversibly reduced at the electrode surface. The number of electrons involved in the electrode process for vitamin B₁ and B₆ is one in each case where as for the two waves of B₂ it is one and two, respectively. This has been confirmed by the measurement of E_3/4–E_1/4 values and also from the log plot slopes for the reduction waves. The wave height of polarogram was found to be proportional to the vitamin concentration. The developed methods have been standardised for the determination of these compounds in pharmaceutical formulation. The concentration of Vitamin B₁, B₂ and B₆ are found to be 9.96, 9.92 and 3.01 mg, respectively in 240 mg of capsule powder of a standard company (name has not been disclosed due to secrecy purpose). The results have been found to be in excellent agreement to that claimed by the manufacturer. The observed data has been subjected to statistical analysis, which revealed high reliability and precision.     

Toxicology of vindesine (desacetyl vinblastine amide) in mice, rats, and dogs.

Comparative acute intravenous toxicity studies of vinblastine sulfate (VLB), vincristine sulfate (VCR), and vindesine in mice and rats indicated that vindesine was more toxic than VLB and less toxic than VCR. Rats were able to tolerate larger repeated doses of vindesine than dogs. Rats given intravenous doses totaling 0.15 mg/kg-wk vindesine for 3 months developed no remarkable signs of toxicity. Doses of 0.3 mg/kg-wk or greater produced anorexia, depressed blood cell counts, atrophic intestinal mucosa, inhibition of spermatogenesis, extramedullary hematopoiesis, and infections. Dogs were given total weekly intravenous doses of 0.04, 0.08, 0.1, or 0.16 mg/kg vindesine for 3 months. The only observed effect in the two lower dose groups was inhibition of spermatogenesis. Groups receiving 0.1 or 0.16 mg/kg developed leukopenia, slight erythropenia, inhibition of spermatogenesis, focal skeletal muscle degeneration, elevated lactic dehydrogenase, and an increase in bone marrow myeloid: erythroid ratio. No evidence of functional or structural changes in neural tissues was found. The above effects are common to animals given VCR at lower doses and for a shorter test period. It is therefore concluded that vindesine is less toxic in animals than VCR.     

LC/MS/MS structure elucidation of reaction intermediates formed during the TiO2 photocatalysis of microcystin-LR

Microcystin-LR (MC-LR), a cyanotoxin and emerging drinking water contaminant, was treated with TiO₂ photocatalysts immobilized on stainless steel plates as an alternative to nanoparticles in slurry. The reaction intermediates of MC-LR were identified with mass spectrometry (MS) at pH of Milli-Q water (pH_sq=5.7). Eleven new [M+H]⁺ were observed in the liquid chromatography mass spectrometry (LC/MS) chromatogram with some of them giving multiple peaks. Most of these reaction intermediates have not been reported from previous studies employing TiO₂ nanoparticles at acidic conditions (pH=4.0). Investigating the effects of pH (for 3.02 photocatalytic films showed that acidic conditions are preferable for the degradation. Combined with the limited surface area of the films and the absence of additional oxidants (i.e., H₂O₂) the degradation was slower and more intermediate steps were identified. Possible structures of the intermediates (formed at neutral pH) after analyzing the corresponding MS/MS spectra are reported. The collision-induced dissociation of the [M+H]⁺ of MC-LR and the intermediates 1011.5 and 1029.5 are discussed and possible fragmentation pathways and mechanisms are also proposed. Analysis of the MS/MS spectra indicates that the fragmentation of some amino acids is less favorable because of internal interaction with free groups of adjacent amino acids. The MS/MS spectra assisted in determining hydroxylation sites, by the formation or alteration of specific product ions such as m/z 599.     

Polyvinylamine-based capsules: A mechanistic study of the formation using alginate and cellulose sulphate

Capsules based on sodium alginate (SA) and sodium cellulose sulphate (SCS), have been prepared using polyvinylamines (PVAm) of varying intrinsic viscosities. The resulting capsules are relatively dense in nature, revealing a bursting force which is four times that observed for the classical SA/SCS/polymethylene-co-guanidine chemistry. Molar mass cutoffs were typically in the 10–70?kDa range. A mechanistic study was carried out where the reaction time, ionic strength and pH of the reaction mixture, as well as the stoichiometry of the polyanion blend and the PVAm molar mass were varied. It is postulated that both the SA-PVAm and the SCS-PVAm binary interactions contribute to the mechanical properties and the permeability of the resulting capsules. The polyvinylamine-based chemistry offers interesting alternatives to the PMCG system in that it provides a means to produce capsules at low, or zero, ionic strengths. Subtle changes in the pH, or the SA:SCS ratio, can also be used to tune the bursting force quite sensitively. The most appropriate capsules, for transplantation, would likely be formed at polyanion levels of 1.2?wt% with a PVAm molar mass below 17?kDa.     

Efficacy of 5′-nor-anhydrovinblastine (vinorelbine), against freshly explanted clonogenic human tumor cells in vitro

Summary Vinorelbine (5-nor-anhydrovinblastine) is a semisynthetic vinca alkaloid currently undergoing extensive clinical evaluation. We have studied the antitumor effect of vinorelbine (final concentrations: 8.4–1000.0 ng/ml) against freshly explanted clonogenic cells from 102 human tumors using a capillary soft agar cloning system and have compared the compound's activity with vinblastine, vincristine, vindesine, paclitaxel, docetaxel, and other clinically used anticancer agents. Four specimens were excluded from further analyses (3 bacterial or fungal contamination, 1 benign histology). Fifty-one of the remaining 98 (52%) specimens had adequate colony formation in control capillaries. Vinorelbine showed concentration-dependent antitumor activity against a variety of solid rumors. At clinically relevant concentrations (0.1 × peak plasma concentrations in humans) vinorelbine inhibited 21 of 49 specimens (43%) and was as active as vinblastine, vincristine, vindesine, bleomycin, doxorubicin, 5-fluorouracil, mitomycin-C, cisplatin, methotrexate, and etoposide. However, paclitaxel (71% inhibition, p = 0.006) and docetaxel (78% inhibition, p = 0.002) were significantly more active than vinorelbine. Moreover, vinorelbine showed antitumor activity against several tumor types and in particular against breast cancer but also in non-small cell lung cancer. We conclude that vinorelbine has a wide spectrum of in vitro activity against freshly explanted human tumors and that the clinical activity of this compound against breast cancer and non-small cell lung cancer is reflected in vitro.     

Degradation kinetics of (+/-)-4'-ethyl-2-methyl-3-(1-pyrrolidinyl)propiophenone hydrochloride (HY-770) and structure-stability relationship among its analogues in aqueous solution

The kinetics and pathways for degradation of (+/-)-4'-ethyl-2-methyl-3-(1-pyrrolidinyl)propiophenone hydrochloride (HY-770; 1), a newly developed muscle-relaxing agent, and its analogues were studied in aqueous solution at 50 degrees C, ionic strength 0.5 M, and pH 8.0-12.0. Compound 1 and its four analogues followed pseudo-first-order degradation kinetics at constant pH and temperature. From the analysis of the pH degradation-rate profiles, it is evident that specific hydroxide ion-catalyzed degradations of ionized and un-ionized species occur for 1 and its structural analogue, 3'-fluoro-2-methyl-3-(1-pyrrolidinyl)propiophenone hydrochloride (HN-961; 5). The hydroxide ion-catalyzed degradation of the ionized species was found to be 100 times faster than that of the un-ionized species and to be the major process at pH less than 9.0. On the contrary, 1 was extremely stable in 0.5 M HCl at 50 degrees C, suggesting that the hydronium ion-catalyzed degradation and the spontaneous degradation of the ionized species is negligible. The Arrhenius plot for the degradation of 1 at 35-50 degrees C and pH 9.0 showed that the apparent energy of activation was 22.0 kcal/mol. The degradation rates of the five structural analogues were significantly dependent on the electron withdrawing effect of the benzene substituents of the molecule.     

两种工艺制备的麻疹减毒活疫苗加速稳定性可比性研究

目的  通过强制降解试验,对细胞工厂与转瓶培养两种工艺制备的麻疹减毒活疫苗进行加速稳定性可比性研究。方法  取细胞工厂与转瓶培养工艺制备的麻疹减毒活疫苗单次收获液和原液各3批于25 ℃保存10 d,疫苗成品3批于37 ℃保存10 d。按照中国药典2015版三部和企业标准进行病毒滴度检定。利用Minitab 19比较两种工艺产品的病毒滴度降解曲线。结果  所有样品的降解曲线均符合麻疹病毒的热敏感性。2种工艺制备的麻疹减毒活疫苗单次收获液病毒滴度第1组分别下降1.51和1.56 lg细胞培养半数感染量（CCID₅₀）/ml,第2组均下降1.33 lg CCID₅₀/ml,第3组均下降1.54 lg,原液分别下降1.20和1.43 lg CCID₅₀/ml,成品分别下降1.33和1.30 lg CCID₅₀/ml。2种工艺产品的降解曲线相似。结论   细胞工厂与转瓶培养工艺生产的麻疹减毒活疫苗具有相似的加速稳定性。     

Kinetics of degradation of methotrexate in aqueous solution

The chemical stability of methotrexate (I) in aqueous solution protected from light was studied utilizing an HPLC procedure. The investigation of the kinetics of degradation took place in aqueous buffer solutions at 85°C over the pH-range of 0–12. The pH-rate profile obtained from first-order kinetic plots showed maximum stability at about pH 7. An Arrhenius-type plot indicated a shelf-life at pK ~ 8.5 of about 4.5 years at 25 °C.At pH above 6.5 N¹⁰-methyl-folic acid (II) was the only degradation product while in acidic solution several compounds are formed.     

Accelerated stability studies on Rocephin by high-efficiency liquid chromatography

The stability of Rocephin in aqueous solutions was studied for different pH values in research into accelerated instability. The pH range studied was 2.5, 4.5, 5.5, 6.5, 7.4 and 8.0 adjusted to an ionic strength of μ = 0.5; each of these values was subjected to temperatures of 50, 60, 70 and 80 °C. From the experimental values found for the concentrations of undegraded Rocephin as a function of time it was seen that the degradation process of this cephalosporin follows first-order kinetics.The technique used for the determination of Rocephin was high-efficiency liquid chromatography, using a reverse phase (RP-18) as the stationary phase.Linear relationships were established for each pH studied, between the logarithms of the experimental degradation constants and the reciprocals of the absolute temperatures, obtaining correlation coefficients greater than 0.91.These values were used to calculate the values of the degradation constants, half-lives and expiry date for each pH at temperatures of 37, 20 and ?4°G Simultaneously, the activation energy and the logarithm of the frequency factor were determined, giving average values of 13.11 kcal/mol and 5.62, respectively.The values obtained for the expiry date at 20°C, greater than 7 h for all the pH values mean that in terms of pH-regulated degradation this antibiotic may be used in the normal perfusion liquids.     

Metabolomics of the Bio-Degradation Process of Aflatoxin B1 by Actinomycetes at an Initial pH of 6.0

Contamination of food and feed by Aflatoxin B1 (AFB1) is a cause of serious economic and health problems. Different processes have been used to degrade AFB1. In this study, biological degradation of AFB1 was carried out using three Actinomycete species, Rhodococcus erythropolis ATCC 4277, Streptomyces lividans TK 24, and S. aureofaciens ATCC 10762, in liquid cultures. Biodegradation of AFB1 was optimised under a range of temperatures from 25 to 40 °C and pH values of 4.0 to 8.0. An initial concentration of 20 µg/mL of AFB1 was used in this study. The amount of AFB1 remaining was measured against time by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), coupled with UV and mass spectrometry (LC-MS). All species were able to degrade the AFB1, and no significant difference was found between them. AFB1 remained in the liquid culture for R. erythropolis, S. lividans and S. aureofaciens were 0.81 µg/mL, 2.41 µg/mL and 2.78 µg/mL respectively, at the end of the first 24 h. Degradation occurred at all incubation temperatures and the pH with the optimal conditions for R. erythropolis was achieved at 30 °C and pH 6, whereas for S. lividans and S. aureofaciens the optimum conditions for degradation were 30 °C and pH 5. Analysis of the degradative route indicated that each microorganism has a different way of degrading AFB1. The metabolites produced by R. erythropolis were significantly different from the other two microorganisms. Products of degradation were identified through metabolomic studies by utilizing high-resolution mass spectral data. Mass spectrometric analysis indicated that the degradation of AFB1 was associated with the appearance of a range of lower molecular weight compounds. The pathway of degradation or chemical alteration of AFB1 was followed by means of high resolution Fourier transform mass spectrometry (HR-FTMS) analysis as well as through the MS² fragmentation to unravel the degradative pathway for AFB1. AFB1 bio-degradation was coupled with the accumulation of intermediates of fatty acid metabolism and glycolysis. A plausible mechanism of degradation of AFB1 by Rhodococcus was hypothesized.