The Analysis, Determination Of Polar Component With No Ultraviolet Activity And Its Application In Pharmaceutical Analysis

There are many components with no chromophore, which usually called no ultraviolet activity components, among so many drug ingredients except the components with conjugated chromophore; they have no absorption in ultraviolet-visible spectral region, such as bisphosphonates, polyalcoho and alkaloid. They have always been the research focus as their strong physiological activity. However, it is difficult to adopt conventional analytical methods for the analysis of no ultraviolet activity components due to the lack of chromophores. For the polar, low volatile ingredients, it is not only difficult to retain on non-polar stationary phase, but hard to be analyzed with GC. Till now, the reported methods for the analysis of the polar component with no ultraviolet activity are mostly ion-exchange chromatography coupled with refractive index detection and MS detection.The aim of the project is to adopt modern analytical means, such as evaporative light scattering detection (ELSD), flow injection analysis system coupled with chemiluminescence detector (FIA-CL) and capillary electrophoresis (CE), to establish the following new methods for the determination of bisphosphonate, polyalcoho and alkaloid. The developed approaches provide new means for the quality control of pharmaceutical products contained such components. (1) A simple method has been developed for analysis of bisphosphonates by reversed phase-high performance liquid chromatography by combining ELSD, volatile n-amylamine as the mobile phase additive. (2) A simple and rapid CE-UV detection method has been established for the determination of bisphosphonate with end absorption. (3) It is developed a new and specific RPLC-indirect UV detection method for the analysis of amino bisphosphonate based on AQC as the derivatizing reagent. The derivatisation procedure is very simple, the derivatives are very stable and the excess reagent is hydrolyzed to 6-aminoquinoline (AMQ) in less than 2 min, avoiding the special treatment. (4) In additional, it is found that the CL intensity of luminol-hydrogen peroxide-copper (Ⅱ) system was increased in the presence of bisphosphonates. Based on this phenomenon, a novel and specific FIA-CL approach is developed to assay bisphosphonates. The results indicate that no interference is observed among adjuvant, bisphosphonate and its related substance (phosphate and phosphite). The sensitivity is 200 times higher than the ELSD method and the same as molybdenum blue spectrophotometric method. (5) The routine means for the determination of polyalcoho is refractive index detection, but the sensitivity of the method is low, a HPLC-ELSD method in our research is developed for the analysis of inositol. (6) Previously reported methods for the determination of alkaloid usually applied UV detector, but the sensitivity and specificity were low. The HPLC-ELSD method we established for oxymatrine is simple, rapid and specific.PART 1 The quality study and control of bisphosphonates (1) Determination of Incadronate and Its Related Substances by HPLC-ELSDObjective: To establish a simple, rapid HPLC-ELSD method for the assay of incadronate and its related substances.Methods: The separations were performed on an Inertsil C8 column (150 mm×4.6 mm, 5μm) at 30℃, with the mobile phase of 0.0175 mol·L-1 n-amylamine aqueous solution (adjusted to pH 7.3 with acetic acid)-acetonitrile (90:10), at a flow rate of 1.0 mL·min-1.Results: The calibration curve was linear in the range of 31.5~1.01×103μg·mL-1 (r=0.999 7), the LODs of incadronate and its related substance (phosphate and phosphite) were 15.8, 3.13, 3.17μg·mL-1, respectively. The average recovery of incadronate for injection was 100% with RSD of 0.9%.Conclusion: A specific HPLC-ELSD method with volatile n-amylamine as the mobile phase additive was established for the simultaneous analysis incadronate and its related substance (phosphate and phosphite). Not only can the problems of their retention and detection be solved but also the requests of ELSD can be satisfied. The developed RP-HPLC method provides an approach for the routine analysis and quality control of incadronate bulk materials and preparations.(2) Determination of zoledronic acid and its preparations by Capillary Zone ElectrophoresisObjective: To estebalish a simple and rapid capillary zone electrophoresis (CZE) method for the analysis of zoledronic acid and its related substances.Methods: An uncoated fused-silica capillary column of 50 cm×50μm was used. The separation was achieved using 3 mmol·L-1 phosphate buffer solution (pH 7.9) as background electrolyte solution with the electrokinetic injection of 2 kV (5 s) and a running voltage of 25 kV. The detection wavelength was 210 nm.Results: The calibration curve was linear in the range of 31.2~998μg·ml-1 (r=0.999 7), the average recoveries of zoledronic acid for injection and zoledronic acid injection were 99.6% and 100%, respectively and with RSD of 1.1% and 1.0%, respectively. The LOD was 10.4μg·mL-1, the active ingredient-zoledronic acid was successfully separated from its related substance.Conclusion: A simple and rapid CZE method was developed for the simultaneous analysis of zoledronic and its related substance (imidazolylacetic acid). It is more simple and rapid than the reported methods such as ion-pair chromatography and ion-exchange chromatography, the migration time of zoledronic acid and imidazolylacetic acid is 4.2 min and 2.4 min, respectively. The method is accurate and reliable; it provides a new reliable means for the quality control of zoledronic acid.(3) Simultaneous analysis of alendronate and its related subatance (γ-aminobutyric acid) by HPLC with AccQ-Fluor as the derivatizing reagentObjective: To establish a new derivatization method for the simultaneous determination of alendronate and its related substance (γ-aminobutyric acid).Methods: The analysis was performed on a Diamonsil C18 column (150 mm×4.6 mm, 5μm particles) with acetonitrile-35 mmol·L-1 sodium acetate buffer (containing 1% triethylamine, adjusted to pH 6.8 with acetic acid) (13.5:86.5, v/v) as a mobile phase in isocratic mode at 35℃and detected at 254 nm using a UV detector. The flow-rate was 0.7 mL·min-1.Results: The linearity of response was examined for alendronate andγ-aminobutyric acid using solutions in the range of 15.5–990μg·mL-1, 0.307–960μg·mL-1, respectively. The correlation coefficients of the linear regression of the standard curves were greater than 0.999 5 and the LOD of alendronate andγ-aminobutyric acid was 7.75 and 0.154μg·mL-1, respectively.Conclusion: A new specific and sensitive RPHPLC-UV method has been established for the analysis of alendronate and its related substance (γ-aminobutyric acid), based on AQC as the derivatizing reagent. The results indicate that no interference is observed among adjuvant, bisphosphonate and its related substance (phosphate and phosphite). The developed method seems to be an advantageous alternative to traditional methods with OPA or FMOC as the derivatizing reagent, because of simplicity, high sensitivity, and good stability in the simultaneous determination of alendronate and its related substance (γ-aminobutyric acid). The derivatisation procedure is very simple and the derivatives are stable at least one week. The excess reagent is hydrolyzed to 6-aminoquinoline (AMQ) in less than 2 min, avoiding the laborious analytical isolation. Not only could it be applied to the quality control of alendronate bulk material and its preparations, but to the dissolution test of alendronate tablet. (4) Novel, rapid method for determination of ibandronate using flow injection analysis with chemiluminescence detectorObjective: To estiblish a novel, rapid method for the determination of ibandronate by combining the flow injection with chemiluminescence (FIA-CL).Methods: The solution of ibandronate was diluted with Cu (II) solution (1×10-4 mol·L-1). One pump was used to carry the ibandronate solution and luminol solution (1×10-6 mol·L-1) with two channels and the other pump was used to deliver carrier solution (1% H2O2 solution). The mixed solution was injected into flow cell by a six-way injection valve where it met with the carrier solution. The two pumps speeds was 1.2 mL·min-1 and 2.9 mL·min-1, respectively. The same procedure was carried out to measure the blank signal. The optimised method was validated for specificity, precision, linearity and accuracy.Results: Under the optimum conditions, the net enhancement of CL intensity (ΔI) is proportional to the concentration of ibandronate in solution. The linear relationship was obtained in the range of 1.56 to 99.9μg·mL-1. The limit of detection (S/N=3) is 0.53μg·mL-1 with the relative standard derivation (R.S.D., n=11) of 1.8% for ibandronate and the average recovery was approximately 100%. The results indicate that no interference is observed among adjuvant, bisphosphonate and its related substance (phosphate, phosphite and 3-methylpentylaminopropionic acid).Conclusion: A novel, rapid and specific FIA-CL method has been established for the analysis of ibandronate. It takes a serial of advantages over the reported methods: the analysis cycle is only 40 s; the sensitivity is 200 times higher than the ELSD method and equal to the molybdenum blue spectrophotometric method; no interference is observed among adjuvant, ibandronate and its related substance (phosphate, phosphite and 3-methylpentylaminopropionic acid). The proposed method can be used for the quality control and also can realize automation for scene analysis of ibandronate.(5) Rapid determination of alendronate using flow injection analysis with chemiluminescence detectionObjective: To estiblish a rapid method for the determination of alendronate by combining the flow injection with chemiluminescence (FIA-CL).Methods: The solution of alendronate was diluted with Cu (II) solution (1×10-4 mol·L-1). One pump was used to carry the alendronate solution and luminol solution (4×10-7 mol·L-1) with two channels and the other pump was used to deliver carrier solution (0.1% H2O2 solution). The two pumps speeds was 1.2 mL·min-1 and 2.9 mL·min-1, respectively. The mixed solution was injected into flow cell by a six-way injection valve where it met with the carrier solution. The same procedure was carried out to measure the blank signal. The optimised method was validated for specificity, precision, linearity and accuracy.Results: Under the optimum conditions, the net enhancement of CL intensity (ΔI) is proportional to the concentration of alendronate in solution. Alendronate can be determinated in the range of 1.24 to 49.6μg·mL-1 with the limit of detection (S/N=3) of 0.41μg·mL-1 and the relative standard derivation (R.S.D., n=11) of 1.3%. The average recovery was 99.8%. The results indicate that no interference is observed among adjuvant, alendronate and its related substance (phosphate and phosphite).Conclusion: The FIA-CL method established for the analysis of alendronate is rapid, sensitive and specific. The analysis cycle of the proposed method is only 40 s. However, the sensitivity is 200 times higher than the ELSD method and equal to molybdenum blue spectrophotometric method. The method can not only provide a new means for the quality control of alendronate, but can be used for dissolution test of alendronate tablets. It is suitable to the scene analysis of alendronate.PART 2 The analysis, determination of other polar components with no ultraviolet activity and its application in pharmaceutical analysis(1) Determination of Inositol in Lysine, Inositol and Vitamin B12 Oral Solution by HPLC-ELSDObjective: To establish a simple and rapid HPLC-ELSD method for the analysis of inositol of compound preparation.Methods: The separations were performed on a Kromasil NH2 column, with the mobil phase of methanol-water (60:40), at a flow rate of 0.8 mL·min-1.Results: The calibration curve was linear in the range of 157~2.51×103μg·mL-1 (r=0.999 6), the LOD was 39.3μg·mL-1 and the average recovery of inositol was 99.6% with RSD of 0.8%. The active ingredient inositol was successfully separated from the other substances.Conclusion: A new, simple and rapid HPLC-ELSD method for the analysis of inositol of compound preparation has been established without complicated pretreatment. It avoids the complication, time-consuming derivazation step and instability of the derivatives. The method is more sensitive than the traditional refractive index detection and it provides a new reliable approach for the determination of inositol of food and drugs.(2) Determination of Oxymatrine in matrine capsule by HPLC-ELSDObjective: To establish a simple, rapid HPLC-ELSD method for the assay of oxymatrine.Methods: The separation was performed on a Diamonsil C18 column, with the mobile phase of acetonitrile-aqueous solution (0.5% triethylamine, adjusted to pH 5.5 with acetic acid) (90:10), at a flow rate of 1.0 mL·min-1.Results: The calibration curve was linear in the range of 16.0~513μg·mL-1 (r=0.999 9), the LOD was 4.00μg·mL-1, the average recovery of was 100% with RSD of 0.8%.Conclusion: Under the optimization chromatographic conditions, satisfactory separation of oxymatrine and its related substance (N-oxysophocarpine), which may be the most possible present among other alkaloid during purification process, was achieved on a C18 column with good peak shapes. The developed HPLC-ELSD method is simpler and faster than the ion-suppression chromatography, ion-pair chromatography and amino-column-RP-HPLC. It provides a new means for the determination of drugs containing oxymatrine.