NMR Assignments of Six Asymmetrical N-Nitrosamine Isomers Determined in an Active Pharmaceutical Ingredient by DFT Calculations.

N-nitrosamines, which are well-known pro-mutagens, are found in drugs, pickled food and tobacco. Therefore, controlling their concentrations is very important. When an HPLC, GC or NMR analysis is conducted to investigate certain asymmetrical N-nitrosamines, two sets of signals attributed to the asymmetric N-nitrosamine isomers are usually observed. However, few reports on the NMR assignment of asymmetrical N-nitrosamine isomers have been published. In this study, we investigated the NMR assignments of the Z/E isomers of six asymmetrical N-nitrosamines by means of density functional theory (DFT) calculations. The configuration of the major isomer of asymmetrical N-nitrosamine 3 was the Z-configuration. The configuration of the major isomers of asymmetrical N-nitrosamines 4-7 was the E-configuration. Then, we determined the Z/E ratios of these asymmetrical N-nitrosamines by means of variable temperature (VT) and room temperature (RT) 1H-NMR experiments. The ratios of the Z/E isomer 3 quickly increased beyond 100% in the VT 1H NMR experiments. The ratios of Z/E isomers 4-7 were increased in the range of 10-60% in the VT 1H NMR experiments. The results of this study indicate that identifying the isomers of asymmetrical N-nitrosamine is necessary to control the quality of N-nitrosamines for active pharmaceutical ingredients (APIs).

[Research on HPLC fingerprint of Rheum palmatum from Gansu Province called "quan-shui-da-huang"].

OBJECTIVE: To establish an analysis method for HPLC fingerprint of Rheum palmatum collected from Gansu province (called "Quan-shui-da-huang"), and provide basis for quality control of Quan-shui-da-huang. METHODS: The Cosmosil 5C18-PAQ column (250 mm x 4.6 mm, 5 microm) was used with the mixture of 0.1% acetic acid and acetonitrile as mobile phase in a gradient elution mode. The flow rate was 1 mL/min, column temperature was 30 degrees C and detection wavelength was set at 280 nm. The similarity evaluation, principal component analysis (PCA) and cluster analysis of crude drugs collected from different habitats were carried out. RESULTS: The analysis method of HPLC fingerprint was set up. The characteristic fingerprint was obtained by chemometrics methods. CONCLUSION: Under the selected chromatographic conditions, constituents in Rheum palmatum can be separated well. This method can be used for the quality control of Quan-shui-da-huang.

[Prevalence of diabetic retinopathy among the elderly in rural southern Shuangcheng city, Heilongjiang province].

OBJECTIVE: The purpose of this study was to understand the awareness, prevalence of diabetic retinopathy and treatment status of people aged over 50 and living in the rural areas of Shuangcheng city, Heilongjiang province, China. METHODS: Cluster sampling was used in randomly selected 5504 survey for ophthalmic clinical examination, in patients with diabetic retinopathy. A questionnaire in the state of knowledge about prevention and treatment was developed. RESULTS: Among the 5504 persons entering in the project, 5053 were examined on their eyes (91.8%). In this selected population, 56 persons (112 eyes) were diagnosed as diabetic retinopathy (1.108%), with 95% confidence interval (CI) as: 0.819% to 1.397%. Of 56 patients, 49 cases were non-proliferative diabetic retinopathy, accounting for 87.50% of the total number of patients with diabetic retinopathy; proliferative diabetic retinopathy 7 cases, accounting for 12.50% of the 112 eyes, 6.25% (7/112) having vitreous hemorrhage, 8.04% (9/112) having macular edema. For diabetic retinopathy prevalence rates, there was no significant difference in males and females. Between the per differential 10-year-old division, the difference was significant. Among the 60 to 69 group, a significantly higher prevalence rate was seen. Of the 112 eyes with diabetic retinopathy, 34 eyes (30.4%) were low vision [visual acuity < 20/60 (0.3) to ≥ 20/400 (0.05)]; 6 eyes (5.4%) were blind [visual acuity < 20/400 (0.05) to NLP]. The rate in the patients with PDR and fasting blood glucose was above 11.1 mmol/L was higher than having NPDR and fasting blood glucose below 11.1 mmol/L. Having fasting blood glucose 11.1 mmol/L and above with the course over five years among patients with PDR, the proportion of fasting blood glucose was higher than those with less than 11.1 mmol/L and diabetic retinopathy duration of less than five years. Of 56 patients with diabetic retinopathy, 38 cases (67.9%) did not receive any treatment. Among 18 cases (32.1%) with insulin or oral drug therapy, regularly using insulin or other medication (14.3%), only 1 (1.8%) case was given the treatment for diabetic retinopathy. Results from our survey showed that patients with diabetic retinopathy had a poor understanding about prevention and treatment of the disease. CONCLUSION: Long duration and high blood glucose in patients with diabetic retinopathy seemed to be the important risk factor. Early systematic drug use for prevention and blood glucose control was the key to prevent diabetic retinopathy. Patients with diabetic retinopathy in China had poor understanding about the prevention measures of the disease and lack of knowledge.

[Analysis of mutation in KRT12 gene in a Chinese family with Meesmann's corneal dystrophy].

OBJECTIVE: To investigate the mutation of KRT12 gene in a large Chinese family with Meesmann's corneal dystrophy by molecular genetic study and linkage analysis. METHODS: Mode of inheritance was determined in a family with Meesmann's corneal dystrophy provided by Xingtai Eye Hospital. The blood samples were obtained from members of this family, including both affected and unaffected members. The genome-DNA was extracted from the samples. Linkage analysis was conducted in the selected markers (D17S800, D17S930, D12S390, D12S96) in the locus around KRT12 and KRT3 genes. The exons of linked genes were sequenced directly. All of the members in this family were examined with slit lamp biomicroscope and photographed. Blood samples were collected from 100 normal subjects and analyzed as the controls. RESULTS: The mode of inheritance of corneal dystrophy in this family was identified as autosomal dominant inheritance. The clinical diagnosis was Meesmann's corneal dystrophy. Linkage analysis revealed a lod score of 2.41 with theta = 0.00 at markers D17S800 and D17S930. Linkage was revealed between corneal dystrophy and KRT12 gene. Sequences of KRT12 gene in affected members showed the mutant in exon 1, T419A and L132H. All affected members in this family were heterozygotes of this mutation. No mutation of this type was identified in all unaffected member of this family and in the 100 normal controls. CONCLUSION: Mutation of KRT12 gene in exon 1, T419A and L132H is the molecular genetic change leading to the occurrence of Meesmann's corneal dystrophy in this Chinese family.