New Assay Methods Established And Application For BPs In Pharmaceutical

BPs are new category of bone resorption inhibitor drugs developing in the recent 20 years, which had been widely used in the management of skeletal disorder, including malignant hypercalcemia, postmenopausal osteoporosis, Paget's disease and so on.The assays for BPs presented some difficulties, since the flowing characters: strong polar, ionic, low bioavailability and so on. Molybdenum blue spectrophotometry method is still used in routine quality control analysis of BPs, in which BPs were oxidized to orthophosphate ions firstly, so it was inevitable that trace phosphate, phosphite and excipients interfere with analysis of BPs, thus the precision and accuracy were low relatively. Although ion-pair HPLC-ELSD method for identification and routine determination of BPs was established, the sensitivity was so low that was not suitable for trace quantity biological specimen. Many indirect methods based on derivatization have been reported for the chromatographic separation and determination of BPs, which contain primary or secondary amine groups, for example alendronate and pamidronate, but the indirect methods were tedious, time-consuming and can be used for only analysis a few of BPs, as majority of BPs without derivative group, therefore there is still a need for specific methods for assay of BPs.The aim of our research is to introduce specific, simple and sensitive methods for routine analysis and quality control of BPs, which is helpful for developing BPs quality. Here, we present specific, simple and sensitive methods for assay of BPs according to the flowing the two parts:PART 1 Formation of Novel OPM and Analytical Application in Pharmaceuticalâ… . Studies on the Mechanism of Novel OPM Formation and Characters of OPMObjective: To study the formation mechanism of novel OPM and some characters of OPM, such as stoichiometric ratio and molar absorptivity.Methods: OPM were formed directly when mixing BPs with MoO in acidic media; their characters were studied by UV spectrophotometry method. Job's method of consecutively variation and continuous variation method were applied for definition of stoichiometric ratio of BPs to Mo in OPM, according to the strong absorbance of OPM in wave range of 220～300nm.Results: The novel OPM are obtained, which are stable and have strong absorbance at 254nm. The stoichiometric ratio of BPs to Mo in OPM was determined to be 1:5. The molar absorptivity of OPM are 8.61×10³,9.58×10³,6.89×10³, 7.53×10³,8.08×10³,8.80×10³ (L·mol^-1·cm^-1) of Alen, Pami, Zole, Eti, Inca and Iban respectively. The forming of the OPM does not depend on the presence of N-substituendum in BPs.Conclusion: Novel OPM are obtained by reacting of BPs with MoO in acidic media, which are stable and have strong absorbance. The organo-phosphorus is coordination center in the OPM molecular structure.â…¡. Assay of BPs in Pharmaceutical by Spectrophotometry Based on the Novel OPMObjective: To establish a novel spectrophotometric method for quantitative analysis of BPs and for dissolution test of low dose Iban tablet with high accuracy and precision.Methods: Basing on OPM formed directly mixing BPs with MoO in acidic media, UV spectrophotometry was developed with a detection wavelength of 254nm.Results: Good linear relationship between absorbance and the concentrations of Alen, Pami, Zole, Eti, Inca and Iban were established in the range of 5.00～50.0, 4.92～49.2, 5.26～52.6, 5.00～50.0, 5.16～51.6 and 4.92～49.2μg·ml-1 (r≥0.9989), respectively. The LOD of Alen, Pami, Zole, Eti, Inca and Iban were 0.677, 0.916, 0.710, 0.607, 0.810 and 0.781μg·mL^-1 and LOQ were 0.203, 0.275, 0.213, 0.182, 0.243 and 0.234μg·mL^-1 respectively. The average recovery were 96.8～102.6% (n=3), RSD were less than 1.5%. The dissolution was quite fast: 86% of Iban was dissolved within 8min and the drug dissolution was complete dissolved about 10min.Conclusions: A specific and sensitive method was developed to assay of BPs in Pharmaceutical, and it can be conveniently adopted for dissolution test of low dose Iban tablets. Compared with molybdenum blue method, it could not be interferenced byphosphate, phosphite or common excipients, what's more, it is so simple and rapid without any pretreatment.PART 2 Study of Chromatographic Method for Assay of BPsâ… . Assay of Eti and Iban by HPCE with Indirect UV Detection Objective: To establish HPCE with indirect UV detection for assay of Eti and Iban.Method: An uncoated fused-silica capillary column was used, and its electrolyte system contained sodium benzoate (7mmol·L^-1)-KH2PO4 (7mmol·L^-1) (pH 8.0), applied voltage 20kV and detection wavelength 224nm.Results: Good linear relations of Eti and Iban were obtained in the range of 62.60～1002μg·mL^-1 and 61.00～975μg·mL^-1 (r>0.999), with the detection limit of 7.825μg·mL^-1 and 7.744μg·mL^-1 respectively. The average recoveries of Eti and Iban during 99.5%～100.3%, RSD were less than 1.5% (n=3). Conclusion: The method is simple, rapid and economic, which provides a new reliable means for quality control of Eti and Iban.â…¡. Analysis of Iban by Ion Pair RP-HPLC-ELSDObjective: To establish a sensitive and specific RP-HPLC–ELSD method for analysis of Iban and its preparation.Method: Separations were performed on an UltimateTM XB-C18 (4.6×250mm, 5μm) column an isocratic mobile phase: acetonitrile-methanol-20mM triethylamine (pH 8.5, adjusted by acetic acid)-( 90:2:8). The mobile phase flow rate was 1.0ml·L^-1, at room temperature. Analyses were detected by ELSD.Results: In quantitative analysis, the method showed that the calibration curve was linear in the range of 30.70～980μg·mL^-1, regression equation is lgA=1.960 lgC+2.047 (r=0.999), with the detection limit of 7.675μg·mL^-1. The average recoveries of Iban tablets were 98.8%, 99.2% and 100.5% and RSD were less than 1.1%.Conclusion: A sensitive and specific RP-HPLC-ELSD method was developed for analysis of Iban and its preparation. It is more sensitive than reported RP-HPLC-ELSD method since micro Mol. Wt and stronger volatile ion pair diethylamide was selected. It provides a new reliable means for quality control of Iban. â…¢. Determination of Zole Dosage Formulation by Ion Pair RP-HPLC-UV with Post-column Photochemical DerivatizationObjective: To develop a sensitive and specific ion pair RP-HPLC-UV method with post-column photochemical derivatization for the determination of Zola dosage formulation and its related substance.Methods: They were separated by Phenomenon C18 column and eluant of triethylamine (20mM adjusted to pH 7.0 with acetic acid)-methanol (99:1) at flow rate of 1.0ml·min^-1 at room temperature. The sensitive detection of Zole was based on its oxidation to orthophosphate by the on-line peroxydisulfate- assisted photolysis flow by post-column reaction with molybdate and Vitamin C to yield phosphomolybdate, then measured by UV detection.Results: Zole was successfully separated from its related substance (phosphate and phosphite). A good linear relation was obtained in the range of 20.00～500.0μg·mL^-1 (r=0.999), with the detection limit of 1μg·mL^-1. The average recoveries of Zole for injection and Zole injection were 98.7～101.0% and 99.6～100.4% respectively with RSD less than 2.1%.Conclusion: The ion pair RP-HPLC-UV method is simple, accurate and specific,and tedious sample pretreatment is unnecessary. The ion pair RP-HPLC-UV method provides an approach for the routine analysis and quality control of Zole in pharmaceutical.