Study On Qualitative And Quantitative Methodes Of Pristinamycins

The times of antibiotics were coming when penicillin was found and applied to the clinic,and it started a new epoch to treat diseases by antibiotics. Antibiotics have made a significant contribution to human health.With the extensive application of antibiotics,the problem of bacterial drug resistance has become serious year by year. The new antibiotics which have effect to the infection of drug resistant strain have become emergency request. The research and development of new antibiotics has been an urgent task.The discovery of pristinamycins has been provided new solution to those problems. Pristinamycins is a naturally occurring antibiotic of the Streptogramin class, which was first separated from Streptomyces pristinaespiralis by Mancy in 1962. It has strong bactericidal activity against gram-positive bacteria,which can be mainly used for the treatment of gram-positive bacteria infection including the drug-resistance bacteria infection.Pristinamycins is composed of pristinamycinⅠ(PNⅠ), a kind of cyclic depsipeptides, and pristinamycinⅡ(PNⅡ), a kind of polyunsaturated macrolactone.The antibacterial activity of the combination of two kind in vitro is at least 10 times than that of a single and the combination also expanded the antibacterial spectrum. At the same time the synergistic action has a strong bactericidal action while a single one only has a effect of bacteriostasis. Pristinamycins,which is different from other antibiotics,has notable characteristics of synergistic effect. It is hard to cause antibiotic resistance due to the two compoents have totally different chemcial constitutions. Moreover it is the only one which can be taken orally in the anti-drug-resistant antibiotic family. The research and development of pristinamycins will be significant to solve the problem of drug-resistance bacteria infections and will great contribut to produce the self-made pristinamycins for there is no relevant study in our country.Objective: To qualitative and quantitative analysis of pristinamycins, three new methodes were established , i.e. the HPLC method, the HPCE method and the LC-MS method.Method: 1. HPLC method. (1) Optimization chromatographic condition: the best separation condition was chosen by optimizing different columns, solvent proportion of mobile phase ,flow rate and column temperature. (2) System suitablity test: On the optimized chromatographic separation condition, the resolution of pristinamycinⅠA and pristinamycinⅡA, the number of theoretical plates of pristinamycinⅠA and pristinamycinⅡA was determinated. (3) Specificity test: By treat with heating, base, acid, hydrogen peroxide (H2O2), strong light and high moisture, the samples of pristinamycins were analyzed. (4) Precision test: The sample solution was analyzed for six times; the peak area of pristinamycins was determinaed, and relative standard deviation was calculated. (5) Stability test: By determinaed sample solutions at different time on the room temperature, the stability of the sample solution was determined. (6) Linearity and range of calibration curve: Prepared a series of the reference solutions and determined peak area, then calibration curve was obtained by the content of pristinamycins and the peak area. (7) Limit of detection test: Dilute the reference solution until the ratio of signal and noise ( S/N )was not less than 3.The limit of detection was determined. (8) Sample analysis: Determine the content of three batchs of pristinamycins. 2. HPCE method. (1) By optimizing factors which affect the separation, such as the pH value , concentration of buffers, the supplied voltage and temperature, the optimum conditions for separation were selected. (2) System suitablity test: On the optimized chromatographic separation condition, the number of theoretical plates of pristinamycinⅠAand pristinamycinⅡA was determinated. (3) Specificity test: By treat with base, acid and hydrogen peroxide (H2O2), the samples of pristinamycins were analyzed.(4)Precision test : The sample solution was analyzed for six times; the peak area of pristinamycins was determined, and relative standard deviation was calculated. (5)Limit of detection (LOD) test: Dilute the reference solution until the ratio of signal and noise ( S/N )was not less than 3. The limit of detection was determinaed. (6) Sample analysis: Determine the content of three batchs of pristinamycins. 3.LC-MS method. To identify the main components and related substance of pristinamycins, a high performance liquid chromatography–mass spectrometric method (HPLC-MS) was established by optimizing the chromatographic condition and the mass spectrometric factors. Mass spectrometric detection was operated on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source in positive mode. The main components and related substances were identified by HPLC-MS and relationships was established between the two metheds.Results: 1. HPLC method. (1)The HPLC separation was performed by gradient elution on a Agilent ZorbaxC18 analytical column , with a mobile phase consisting of acetonitrile and 0.03mol·L-1 phosphate buffer(pH 2.3)at the flow rate of 1.0 ml/min. The detection wavelength was 210 nm. The column temperature was set at 30℃. Injection volume was 10μl. (2)System suitablitily test: On the optimized chromatographic separation condition, the resolution bewteen pristinamycinⅠA and pristinamycinⅡA was 2.1,and the number of theoretical plates of pristinamycinⅠA and pristinamycinⅡA was 6811, 11811 respectively. (3) Specificity test: By analyzed accelerate samples,the specificity was proved. (4) Precision test: The Precision at six times was good and the RSD of the peak area of pristinamycinⅠA and pristinamycinⅡA was 0.43% and 0.32% respectively. (5) Stability test: The RSD of the peak area of PNⅠA and PNⅡA was 1.93% and 1.89% respectively.The test solution was stable in 12 hours. (6) Linearity and range of calibration curve:The linear range for PNⅠA was 0.01~0.51mg/ml. The calibration curve was Y=2.856×107X+5.381×104,(r=0.9998). The linear range for PNⅡA was 0.03~1.38mg/ml. The calibration curve was Y=2.700×107X+ 3.011×105,(r=0.9997). (7) Limit of detection test: The detection limit of pristinamycinⅠA and pristinamycinⅡA was 0.023ng and 0.025ng respectively. (8) Sample analysis: The content of pristinamycinⅠA of three batchs was 26.18%, 26.23% and 26.21% respectively, The content of pristinamycinⅡA of three batchs was 69.81%,69.30% and 69.36% respectively. 2 HPCE method. (1)Using 55mmol·L-1 phosphate buffer(pH 2.5), the base-line separation of the two components of pristinamycins was achieved and the new method was validated. (2)System suitablitily test: On the optimized separation condition, the number of theoretical plates of pristinamycinⅠA was 222400 and the number of theoretical plates of pristinamycinⅡA was 145760. (3) Specificity test: By analyzed accelerate samples,the specificity of the method was proved. (4) Precision test: The Precision of two components at six times was good and the RSD of the peak area of pristinamycinⅠA and pristinamycinⅡA was 1.40%and 2.56% respectively. (5) Limit of detection (LOD) test: The limit of detection of pristinamycinⅠA and pristinamycinⅡA was 0.02% and 0.01% respectively. (6) Sample analysis: The content of pristinamycinⅠA for three batchs was 25.9%,26.2% and 26.1% respectively and that of pristinamycinⅡA was 69.7%,69.6% and 69.0% respectively. 3.LC-MS method. (1)By using electrospray ionzation and positive ion monitoring, the main components of pristinamycins were analyzed. The temperature of the desolvation and the source block were set at 250℃and 115℃respectively. The flow rate of cone gas(N2) was 280L/h. The capillary voltage and cone voltage were 3.0KV and 30KV respectively. The separation was performed by gradient elution with a mobile phase consisting of 15mmol·L-1 ammonium acetate(pH 3.0)and acetonitrile at the flow rate of 1.0 ml/min. In the program of gradient elution, acetonitrile was 25% in 0-15min increased to 32% within 15-22min then to 80% within 22-40min. (2)In the LC-MS method, two main components, pristinamycinⅡA(Rt 17.60min) and pristinamycinⅠA(Rt 25.79min) were identified. (3) The main related substances were identified as pristinamycinⅠB, pristinamycinⅠC, pristinamycinⅡB and the homologous compound of pristinamycinsⅡA. (4) The peak of main components and related substances which identifed by LC-MS were found in HPLC chromatogram. The relationship was established between the two metheds.Conclusion: Three new methodes were established, i.e. the HPLC method, the HPCE method and the LC-MS method. By validation, the three new methods were proved to be specific,accurate and sensitive. They may be used to qualitative and quantitative analysis of pristinamycins in the new drug development for quality control and stability study.