ABSTRACT
Objective:To explore the quality of inventory samples of a biobank stored in a deep freezer from 0 to over 10 years in Shanghai Ruijin Hospital. Methods:We extracted 24 pairs of stocked gastric cancer samples between 2003 and 2014. We used 1%aga-rose gel electrophoresis to analyze DNA and RNA purity and integrity while adding the RNA integrity number (RIN) for precise analysis. Bicinchonininc acid (BCA) assay was used for protein concentration evaluation. Coomassie brilliant blue method was used for protein integrity assay. Results: The samples were divided into four groups according to cryopreservation period (9 years). No significant difference in DNA integrity was found between the groups (P>0.05);however, DNA degradation in normal gastric mucosa was faster than that in gastric cancer tissue (P=0.023). The RIN significantly declined when the storage period was 6 years or longer (P=0.018). No significant difference in protein concentration was observed between different groups. Using Coo-massie brilliant blue method, we found significant differences in preserved proteins with different molecular weights. Proteins with varying molecular weights were detected in the groups with the following cryopreservation periods:>9 years, a small number of low-molecular-weight (average 36.5 KD) proteins;6-8 years, medium-molecular-weight (average 65.63KD) proteins;3-5 years, high-molecu-lar-weight (average 127.5 KD) proteins;<2 years, high-molecular-weight (average 160 KD) proteins. Conclusion:Cryopreservation does not exert an obvious effect on DNA. If the cryopreservation period is more than 5 years, serious degradation of RNA should occur;like-wise, degradation of proteins with higher molecular weight should occur.
ABSTRACT
Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a widely used technique in the quantitative analysis of small molecules, part of peptides and proteins in biological matrix. When compared with LC, the LC-MS/MS posesses many advantages, such as high sample throughput, high sensitivity and resolutions, simple method development and simultaneous quantification of multiple components. At the stage of method development, the main practices for analysts are to validate analytical methods according to the regulated requirements. However, due to the quantitative mechanisms of LC-MS/MS, even though the used method is fully validated, analytical inaccuracy problem, which is the so called bioanalytical risk (pitfalls), may still exist, if we do not pay attentions to some details. In the present paper, the factors which may cause inaccuracy in quantitative analysis are summarized, especially those could not be found during routine method validation. Those factors include back transformation of unstable drug metabolites to analyte in the handling of biosamples, matrix effects, in-source conversion, and ion interference caused by ions with similar or same mass-to-charge ratio, etc. In addition to QA/QC oversight and strict method validation, the preventing strategies may contain the follows: selecting biosample processing method with high proficiency in eliminating matrix interferences, enhancing chromatographic separation, selection of suitable precursor-product ions and validation with incurred samples, etc.
ABSTRACT
OBJECTIVE: To develop an HPCE method of tetramethylpyrazine hydrochloride in rat plasma using pyramide as the internal standard. METHODS: The separation was performed on a fused-silica uncoated capillary with an inner diameter (ID) of 75 μm, an effective length of 50 cm and a total length of 60 cm. The optimum HPCE conditions were as follows: 100 mmol·L-1 SDS-30 mmol·L-1 sodium tetraborate solution (40:60) was used as the background electrolyt, the samples were injected at the anodic end at 0.5 psi for 6 s, separation voltage was 20 kV at positive power, capillary temperature was 20°C, and detection wavelength was 295 nm. RESULTS: The linear range was 1.12-286.72 mg·L-1(r=0.9996), and the inter-day and intra-day precisions expressed as RSDs were less than 4.21% and 4.74%, respectively. The average recoveries were 91.22%-97.48%, and the limit of detection was 0.16 mg·L-1. CONCLUSION: This method is rapid and accurate to quantify tetramethylpyrazine hydrochloride in rat plasma.