极性非紫外活性成分的分析、测定及其在药物分析中的应用
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摘要
在众多的药物成分中,除了具有共轭生色团结构的成分外,还有许多成分无生色团,其在紫外-可见光区无吸收,通常称之为“非紫外活性成分”,如双膦酸盐(该类药物中大部分无紫外吸收基团)、多元醇、生物碱等,这些物质一般都具有很强的生理活性,一直是人们研究的热点。但是由于这些成分无紫外吸收,采用常规的分析方法困难,特别是某些不但无紫外吸收而且极性大、不易挥发的成分,在非极性色谱柱上难保留,同时很难采用气相色谱法进行分析测定。目前,文献对该类成分的分析多采用离子交换色谱配以示差折光检测器或质谱检测器。
本课题采用现代的分析手段如蒸发光检测器(ELSD)、流动注射-化学发光、毛细管电泳等,建立了双膦酸盐、多元醇、生物碱的新的检测方法,如下所示的这些方法可为含有此类成分的药物的质量控制提供有效可靠的分析手段。(1)本课题采用挥发性的正戊胺作为流动相添加剂,结合蒸发光检测器建立了双膦酸盐的反相液相色谱分析方法;(2)对于具有末端紫外吸收的双膦酸盐,建立了毛细管电泳-紫外检测的分析方法。(3)对于氨基双膦酸盐建立了以AQC为柱前衍生试剂的新的、特异的反相液相色谱-间接紫外检测的方法,该衍生反应步骤简便,衍生产物稳定,过量的衍生试剂不需要经过特殊处理,其可在2分钟内水解成AMQ,且水解产物不干扰测定。(4)此外,试验发现双膦酸盐对鲁米诺-H2O2-Cu(Ⅱ)化学发光体系有增敏作用,据此建立了高效、特异的流动注射-化学发法测定双膦酸盐,试验结果表明药物中的辅料及其有关物质(磷酸盐和亚磷酸)均不干扰测定,其检测灵敏度可达蒸发光检测的100倍,灵敏度与传统的钼蓝法相当。(5)多元醇类成分常规的分析方法为示差折光检测法,但其灵敏度低,本课题采用蒸发光检测器建立了肌醇的分析方法。(6)生物碱类无紫外吸收,文献多采用其末端紫外吸收进行检测,但方法的灵敏度低、干扰多,本课题同样采用蒸发光检测器建立了一种简便、快速、特异性好的HPLC- ELSD法分析测定氧化苦参碱。
一、双膦酸类药物的分析与测定
(一)英卡膦酸二钠及有关物质的HPLC-ELSD法测定
目的:建立简便、特异的HPLC-ELSD法测定英卡膦酸二钠及其有关物质。
方法:采用Inertsil C8色谱柱(150 mm×4.6 mm, 5μm),流动相为添加了0.0175 mol·L-1正戊胺的水溶液(乙酸调节pH至7.3)-乙腈(90:10);流速1.0 mL·min-1;柱温30℃。
结果:英卡膦酸二钠在31.5~1.01×103μg·mL-1的浓度范围内线性关系良好(r=0.999 7),英卡膦酸二钠及其有关物质(磷酸盐和亚磷酸)的最低检测限分别为15.8、3.13、3.17μg·mL-1。注射用英卡膦酸二钠的平均回收率为100%,RSD为0.9%。
结论:本文采用可挥发性的正戊胺作为流动相添加剂,建立了同时分析英卡膦酸二钠及其有关物质(磷酸盐和亚磷酸)的特异的HPLC-ELSD法,即解决了它们的保留与检测问题,又同时满足了ELSD的使用要求。所建立的反相液相色谱法为英卡膦酸二钠原料药及其制剂的常规分析及质量控制提供了有效可靠的手段。
(二)毛细管区带电泳测定唑来膦酸及其制剂的含量
目的:建立简便、快速分析唑来膦酸及与其有关物质的毛细管区带电泳方法。
方法:采用非涂渍石英毛细管(50 cm×50μm)为分离通道,3 mmol·L-1磷酸盐缓冲液(pH 7.9)为运行缓冲体系;工作电压25 kV;进样电压2 kV,进样时间5 s;紫外检测波长为210 nm。
结果:唑来膦酸在31.2~998μg·mL-1的范围内线性关系良好(r=0.999 7);注射用唑来膦酸和唑来膦酸注射剂的平均回收率分别为99.6%、100%,RSD分别为1.1%、1.0%;最低检测限为15.6μg·mL-1,该条件下唑来膦酸与其有关物质分离良好。
结论:本文建立了一种简单、快速且可以同时分析唑来膦酸及其有关物质咪唑乙酸的毛细管区带电泳法,该法较文献报道的离子对色谱法、离子交换色谱法操作过程简便,分析速度快(唑来膦酸与咪唑乙酸的迁移时间分别为4.2 min、2.4 min),结果准确可靠,为唑来膦酸生产及制剂过程中的质量控制提供了一种新的可靠的分析手段。
(三)以AQC为衍生试剂的高效液相色谱法同时分析阿仑膦酸钠及其有关物质(γ-氨基丁酸)
目的:建立一种新的衍生方法同时测定阿仑膦酸钠及其有关物质(γ-氨基丁酸)。
方法:采用Diamonsil C18色谱柱(150 mm×4.6 mm, 5μm),以乙腈-35 mmol·L-1乙酸钠缓冲液(内含1%三乙胺,乙酸调pH6.8)(13.5:86.5)为流动相进行等度洗脱。柱温35℃;紫外检测波长254 nm;流速0.7 mL·min-1.。
结果:阿仑膦酸钠及γ-氨基丁酸在15.5~990μg·mL-1、0.307~960μg·mL-1的范围内线性关系良好(相关系数均不低于0.999 5)。二者的最低检测限分别为7.75、0.154μg·mL-1。在该条件下,阿仑膦酸钠、γ-氨基丁酸以及衍生副产物AMQ之间分离良好。
结论:本文建立了一种以AQC为衍生试剂的反相液相色谱-紫外检测法同时分析阿仑膦酸钠及其有关物质(γ-氨基丁酸),方法特异性好、灵敏度高,有关物质及制剂中的辅料不干扰阿仑膦酸钠的测定。本试验所建立的衍生方法较文献采用的OPA以及FMOC衍生方法优点多:衍生过程简便;衍生产物稳定(至少一周);过量的衍生试剂不需经过特殊处理,2分钟内即可迅速水解,水解产物AMQ不干扰测定。本方法不仅可用于阿仑膦酸钠原料药及其制剂的质量控制,还可用于阿仑膦酸钠片剂的溶出度试验。
(四)一种新颖的、快速的流动注射-化学发光法测定伊班膦酸钠
目的:通过联用流动注射与化学发光技术,建立一种新颖的、快速、特异的测定伊班膦酸钠的方法。
方法:将伊班膦酸钠溶液用1×10-4 mol·L-1的Cu (II)溶液稀释。一个具有两个通路的蠕动泵用来分别传送伊班膦酸钠溶液及1×10-6 mol·L-1的鲁米诺溶液,另一蠕动泵用来输送载液(1% H2O2溶液),泵速分别为1.2、2.9 mL·min-1,溶液经六通阀混合后注入流通池。用同样的程序测定空白信号,并对所建立的方法进行方法学验证。
结果:在优化的条件下,发光信号的增加值(ΔI)正比于伊班膦酸钠溶液的浓度。伊班膦酸钠在1.56~99.9μg·mL-1的浓度范围内线性关系良好,最低检测限为0.53μg·mL-1,RSD 1.8%,平均回收率约为100%。伊班膦酸钠注射剂中的辅料及有关物质(磷酸盐、亚磷酸以及3-甲基戊胺基甲酸)均不干扰测定。
结论:本文建立了一种新颖的、快速、特异的流动注射-化学发光法分析伊班膦酸钠。该方法较文献报道的方法分析优点多:仅40 s即为一个分析周期;检测灵敏度可达蒸发光检测的100倍,与传统的钼蓝法相当;其有关物质及制剂中的辅料均不干扰伊班膦酸钠测定。该方法可用于伊班膦酸钠的质量控制,并可实现自动化、用于生产过程中的现场分析。
(五)一种快速的流动注射-化学发光法测定阿仑膦酸钠
目的:通过联用流动注射与化学发光技术,建立一种快速测定阿仑膦酸钠的方法。
方法:将阿仑膦酸钠溶液用1×10-4 mol·L-1的Cu (II)溶液稀释。一个具有两个通路的蠕动泵用来分别传送这一阿仑膦酸钠溶液及4×10-7 mol·L-1的鲁米诺溶液,另一蠕动泵用来输送载液(0.1% H2O2溶液),泵速分别为1.2、2.9 mL·min-1,溶液经六通阀混合后注入流通池。用同样的程序测定空白信号,并对所建立的方法进行方法学验证。
结果:在优化的条件下,发光信号的增加值(ΔI)正比于阿仑膦酸钠的浓度。阿仑膦酸钠在1.24~49.6μg·mL-1的浓度范围内线性关系良好,最低检测限为0.41μg·mL-1,RSD 1.3%,平均回收率为99.8%。阿仑膦酸钠片剂中的辅料及有关物质(磷酸盐、亚磷酸)均不干扰测定。
结论:本文所建立的流动注射-化学发光法分析速度快、灵敏度高、特异性好:40 s即为一个分析周期;检测灵敏度可达蒸发光检测的100倍,与传统的钼蓝法相当;其有关物质及制剂中的辅料均不干扰测定。该方法可为阿仑膦酸钠的质量控制提供新的分析手段同时可用于阿仑膦酸钠片剂的溶出度试验,并可实现自动化,用于生产过程中的现场分析。
第二部分其他极性非紫外活性成分的分析、测定及其在药物分析中的应用
(一)高效液相色谱-蒸发光散射检测法测定赖氨肌醇维B12口服液中肌醇含量
目的:建立简便、快速的HPLC-ELSD法测定复方制剂中肌醇含量。
方法:采用Kromasil NH2色谱柱,流动相为甲醇-水(60:40),流速0.8 mL·min-1,柱温室温。
结果:肌醇在157~2.51×103μg·mL-1浓度范围内线性关系良好(r=0.999 6),最低检测限为39.3μg·mL-1,平均回收率99.6%,RSD为0.8%。在该条件下,样品中辅料及其他成分不干扰肌醇测定。
结论:本文建立了一种新的、简便、快速的HPLC-ELSD法测定复方制剂中的肌醇含量,样品不需预处理,避免了衍生等步骤给分析带来的不便,较通常采用的折光检测法灵敏度高,该方法的建立为食品、药品中该成分的测定提供了一种新的可靠的分析手段。
(二)苦参素胶囊中氧化苦参碱的HPLC-ELSD测定目的:建立HPLC-ELSD法测定苦参素胶囊中的氧化苦参碱。
方法:采用Diamonsil C18色谱柱,以乙腈-水(含0.5%三乙胺,醋酸调pH 5.5)(10:90)为流动相,流速1.0 mL·min-1,柱温室温。
结果:氧化苦参碱在16.0~513μg·mL-1浓度范围内线性关系良好(r=0.999 9),最低检测限为4.00μg·mL-1,平均回收率为100%,RSD为0.8%。
结论:本文采用普通的C18色谱柱,在优化的色谱体系下即可使氧化苦参碱得到很好的保留且峰形好,并可与其在纯化制备过程中最有可能混入的其他生物碱(氧化槐果碱)达到基线分离。本文所建立的HPLC-ELSD法较文献采用的离子抑制色谱、离子对色谱、氨基柱-RP-HPLC法简便、快速,为含有该成分药物的测定提供了一种新的分析手段。
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-ELSD
Objective: 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 Electrophoresis
Objective: 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 reagent
Objective: 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 detector
Objective: 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 detection
Objective: 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-ELSD
Objective: 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-ELSD
Objective: 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.
本课题采用现代的分析手段如蒸发光检测器(ELSD)、流动注射-化学发光、毛细管电泳等,建立了双膦酸盐、多元醇、生物碱的新的检测方法,如下所示的这些方法可为含有此类成分的药物的质量控制提供有效可靠的分析手段。(1)本课题采用挥发性的正戊胺作为流动相添加剂,结合蒸发光检测器建立了双膦酸盐的反相液相色谱分析方法;(2)对于具有末端紫外吸收的双膦酸盐,建立了毛细管电泳-紫外检测的分析方法。(3)对于氨基双膦酸盐建立了以AQC为柱前衍生试剂的新的、特异的反相液相色谱-间接紫外检测的方法,该衍生反应步骤简便,衍生产物稳定,过量的衍生试剂不需要经过特殊处理,其可在2分钟内水解成AMQ,且水解产物不干扰测定。(4)此外,试验发现双膦酸盐对鲁米诺-H2O2-Cu(Ⅱ)化学发光体系有增敏作用,据此建立了高效、特异的流动注射-化学发法测定双膦酸盐,试验结果表明药物中的辅料及其有关物质(磷酸盐和亚磷酸)均不干扰测定,其检测灵敏度可达蒸发光检测的100倍,灵敏度与传统的钼蓝法相当。(5)多元醇类成分常规的分析方法为示差折光检测法,但其灵敏度低,本课题采用蒸发光检测器建立了肌醇的分析方法。(6)生物碱类无紫外吸收,文献多采用其末端紫外吸收进行检测,但方法的灵敏度低、干扰多,本课题同样采用蒸发光检测器建立了一种简便、快速、特异性好的HPLC- ELSD法分析测定氧化苦参碱。
一、双膦酸类药物的分析与测定
(一)英卡膦酸二钠及有关物质的HPLC-ELSD法测定
目的:建立简便、特异的HPLC-ELSD法测定英卡膦酸二钠及其有关物质。
方法:采用Inertsil C8色谱柱(150 mm×4.6 mm, 5μm),流动相为添加了0.0175 mol·L-1正戊胺的水溶液(乙酸调节pH至7.3)-乙腈(90:10);流速1.0 mL·min-1;柱温30℃。
结果:英卡膦酸二钠在31.5~1.01×103μg·mL-1的浓度范围内线性关系良好(r=0.999 7),英卡膦酸二钠及其有关物质(磷酸盐和亚磷酸)的最低检测限分别为15.8、3.13、3.17μg·mL-1。注射用英卡膦酸二钠的平均回收率为100%,RSD为0.9%。
结论:本文采用可挥发性的正戊胺作为流动相添加剂,建立了同时分析英卡膦酸二钠及其有关物质(磷酸盐和亚磷酸)的特异的HPLC-ELSD法,即解决了它们的保留与检测问题,又同时满足了ELSD的使用要求。所建立的反相液相色谱法为英卡膦酸二钠原料药及其制剂的常规分析及质量控制提供了有效可靠的手段。
(二)毛细管区带电泳测定唑来膦酸及其制剂的含量
目的:建立简便、快速分析唑来膦酸及与其有关物质的毛细管区带电泳方法。
方法:采用非涂渍石英毛细管(50 cm×50μm)为分离通道,3 mmol·L-1磷酸盐缓冲液(pH 7.9)为运行缓冲体系;工作电压25 kV;进样电压2 kV,进样时间5 s;紫外检测波长为210 nm。
结果:唑来膦酸在31.2~998μg·mL-1的范围内线性关系良好(r=0.999 7);注射用唑来膦酸和唑来膦酸注射剂的平均回收率分别为99.6%、100%,RSD分别为1.1%、1.0%;最低检测限为15.6μg·mL-1,该条件下唑来膦酸与其有关物质分离良好。
结论:本文建立了一种简单、快速且可以同时分析唑来膦酸及其有关物质咪唑乙酸的毛细管区带电泳法,该法较文献报道的离子对色谱法、离子交换色谱法操作过程简便,分析速度快(唑来膦酸与咪唑乙酸的迁移时间分别为4.2 min、2.4 min),结果准确可靠,为唑来膦酸生产及制剂过程中的质量控制提供了一种新的可靠的分析手段。
(三)以AQC为衍生试剂的高效液相色谱法同时分析阿仑膦酸钠及其有关物质(γ-氨基丁酸)
目的:建立一种新的衍生方法同时测定阿仑膦酸钠及其有关物质(γ-氨基丁酸)。
方法:采用Diamonsil C18色谱柱(150 mm×4.6 mm, 5μm),以乙腈-35 mmol·L-1乙酸钠缓冲液(内含1%三乙胺,乙酸调pH6.8)(13.5:86.5)为流动相进行等度洗脱。柱温35℃;紫外检测波长254 nm;流速0.7 mL·min-1.。
结果:阿仑膦酸钠及γ-氨基丁酸在15.5~990μg·mL-1、0.307~960μg·mL-1的范围内线性关系良好(相关系数均不低于0.999 5)。二者的最低检测限分别为7.75、0.154μg·mL-1。在该条件下,阿仑膦酸钠、γ-氨基丁酸以及衍生副产物AMQ之间分离良好。
结论:本文建立了一种以AQC为衍生试剂的反相液相色谱-紫外检测法同时分析阿仑膦酸钠及其有关物质(γ-氨基丁酸),方法特异性好、灵敏度高,有关物质及制剂中的辅料不干扰阿仑膦酸钠的测定。本试验所建立的衍生方法较文献采用的OPA以及FMOC衍生方法优点多:衍生过程简便;衍生产物稳定(至少一周);过量的衍生试剂不需经过特殊处理,2分钟内即可迅速水解,水解产物AMQ不干扰测定。本方法不仅可用于阿仑膦酸钠原料药及其制剂的质量控制,还可用于阿仑膦酸钠片剂的溶出度试验。
(四)一种新颖的、快速的流动注射-化学发光法测定伊班膦酸钠
目的:通过联用流动注射与化学发光技术,建立一种新颖的、快速、特异的测定伊班膦酸钠的方法。
方法:将伊班膦酸钠溶液用1×10-4 mol·L-1的Cu (II)溶液稀释。一个具有两个通路的蠕动泵用来分别传送伊班膦酸钠溶液及1×10-6 mol·L-1的鲁米诺溶液,另一蠕动泵用来输送载液(1% H2O2溶液),泵速分别为1.2、2.9 mL·min-1,溶液经六通阀混合后注入流通池。用同样的程序测定空白信号,并对所建立的方法进行方法学验证。
结果:在优化的条件下,发光信号的增加值(ΔI)正比于伊班膦酸钠溶液的浓度。伊班膦酸钠在1.56~99.9μg·mL-1的浓度范围内线性关系良好,最低检测限为0.53μg·mL-1,RSD 1.8%,平均回收率约为100%。伊班膦酸钠注射剂中的辅料及有关物质(磷酸盐、亚磷酸以及3-甲基戊胺基甲酸)均不干扰测定。
结论:本文建立了一种新颖的、快速、特异的流动注射-化学发光法分析伊班膦酸钠。该方法较文献报道的方法分析优点多:仅40 s即为一个分析周期;检测灵敏度可达蒸发光检测的100倍,与传统的钼蓝法相当;其有关物质及制剂中的辅料均不干扰伊班膦酸钠测定。该方法可用于伊班膦酸钠的质量控制,并可实现自动化、用于生产过程中的现场分析。
(五)一种快速的流动注射-化学发光法测定阿仑膦酸钠
目的:通过联用流动注射与化学发光技术,建立一种快速测定阿仑膦酸钠的方法。
方法:将阿仑膦酸钠溶液用1×10-4 mol·L-1的Cu (II)溶液稀释。一个具有两个通路的蠕动泵用来分别传送这一阿仑膦酸钠溶液及4×10-7 mol·L-1的鲁米诺溶液,另一蠕动泵用来输送载液(0.1% H2O2溶液),泵速分别为1.2、2.9 mL·min-1,溶液经六通阀混合后注入流通池。用同样的程序测定空白信号,并对所建立的方法进行方法学验证。
结果:在优化的条件下,发光信号的增加值(ΔI)正比于阿仑膦酸钠的浓度。阿仑膦酸钠在1.24~49.6μg·mL-1的浓度范围内线性关系良好,最低检测限为0.41μg·mL-1,RSD 1.3%,平均回收率为99.8%。阿仑膦酸钠片剂中的辅料及有关物质(磷酸盐、亚磷酸)均不干扰测定。
结论:本文所建立的流动注射-化学发光法分析速度快、灵敏度高、特异性好:40 s即为一个分析周期;检测灵敏度可达蒸发光检测的100倍,与传统的钼蓝法相当;其有关物质及制剂中的辅料均不干扰测定。该方法可为阿仑膦酸钠的质量控制提供新的分析手段同时可用于阿仑膦酸钠片剂的溶出度试验,并可实现自动化,用于生产过程中的现场分析。
第二部分其他极性非紫外活性成分的分析、测定及其在药物分析中的应用
(一)高效液相色谱-蒸发光散射检测法测定赖氨肌醇维B12口服液中肌醇含量
目的:建立简便、快速的HPLC-ELSD法测定复方制剂中肌醇含量。
方法:采用Kromasil NH2色谱柱,流动相为甲醇-水(60:40),流速0.8 mL·min-1,柱温室温。
结果:肌醇在157~2.51×103μg·mL-1浓度范围内线性关系良好(r=0.999 6),最低检测限为39.3μg·mL-1,平均回收率99.6%,RSD为0.8%。在该条件下,样品中辅料及其他成分不干扰肌醇测定。
结论:本文建立了一种新的、简便、快速的HPLC-ELSD法测定复方制剂中的肌醇含量,样品不需预处理,避免了衍生等步骤给分析带来的不便,较通常采用的折光检测法灵敏度高,该方法的建立为食品、药品中该成分的测定提供了一种新的可靠的分析手段。
(二)苦参素胶囊中氧化苦参碱的HPLC-ELSD测定目的:建立HPLC-ELSD法测定苦参素胶囊中的氧化苦参碱。
方法:采用Diamonsil C18色谱柱,以乙腈-水(含0.5%三乙胺,醋酸调pH 5.5)(10:90)为流动相,流速1.0 mL·min-1,柱温室温。
结果:氧化苦参碱在16.0~513μg·mL-1浓度范围内线性关系良好(r=0.999 9),最低检测限为4.00μg·mL-1,平均回收率为100%,RSD为0.8%。
结论:本文采用普通的C18色谱柱,在优化的色谱体系下即可使氧化苦参碱得到很好的保留且峰形好,并可与其在纯化制备过程中最有可能混入的其他生物碱(氧化槐果碱)达到基线分离。本文所建立的HPLC-ELSD法较文献采用的离子抑制色谱、离子对色谱、氨基柱-RP-HPLC法简便、快速,为含有该成分药物的测定提供了一种新的分析手段。
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-ELSD
Objective: 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 Electrophoresis
Objective: 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 reagent
Objective: 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 detector
Objective: 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 detection
Objective: 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-ELSD
Objective: 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-ELSD
Objective: 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.
引文
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18 USP28-NF23, 63
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24 Sanders JM., Ghosh S, Chan JM, et al. Quantitative Structure-Activity Relationships for γδT Cell Activation by Bisphosphonates. J. Med. Chem, 2004, 47(2):375~384
25 Kunzmann V, Bauer E, Feurle J, et al. Stimulation of γδT cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood, 2000, 96(2):384~392
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1 Bezzi M, Hasmim M, Bieler G, et al. Zoledronate Sensitizes Endothelial Cells to Tumor Necrosis Factor-induced Programmed Cell Death. J. Biol. Chem, 2003, 278(44):43603~43614
2 Coxon JP, Oades GM, Kirby RS, et al. Zoledronic acid induces apoptosis and inhibits adhesion to mineralized matrix in prostate cancer cells via inhibition of protein prenylation. BJU Int, 2004, 94(1):164~170
3 郑国钢, 方滢芝. 高效液相色谱法测定注射用唑来膦酸的含量. 药学实践杂志, 2003, 21(6):343~345
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1 Russell RG, Rogers MJ. Bisphosphonates: from the laboratory to the clinic and back again. Bone, 1999, 25(1):97~106
2 Davas I, Altintas A, Yoldemir T, et al. Effect of daily hormone therapy and alendronate use on bone mineral density in postmenopausal women. Fertility and Sterility, 2003, 80(3):536~540
3 Yieng-Hau RH, Qin XZ. Determination of alendronate sodium by ion chromatography with refractive index detection J. Chromatogr. A, 1996, 719(2):345~352
4 Eric WT, Dominic PI, Marvin AB. Determination of alendronate in pharmaceutical dosage formulations by ion chromatography with conductivity detection. J. Chromatogr. A, 1992, 596(2):217~224
5 Eric WT, Steven DC, Richard JF, et al. Determination of bisphosphonate drugs in pharmaceutical dosage formulations by ion chromatography with indirect UV detection. J. Pharm.Biomed. Anal, 1994, 12(8):983~991
6 Isabela T, Luigi S, Simona RS, et al. Development and application of a high-performance liquid chromatography-mass spectrometry method to determine alendronate in human urine. J. Chromatogr. A, 2007, 1160(1-2):21~33
7 Xie Z, Jiang Y, Zhang DQ. Simple analysis of four bisphosphonates simultaneously by reverse phase liquid chromatography using n-amylamine as volatile ion-pairing agent. J. Chromatogr. A, 2006, 1104(1-2):173~178
8 Tsai EW, Singh MM, Lu HH, et al. Application of capillary electrophoresis to pharmaceutical analysis : Determination of alendronate in dosage forms. J. Chromatogr. A, 1992, 626(2):245~250
9 Jadranka K, Ivana J, Jovan N, et al. Spectrophotometric determination of alendronate in pharmaceutical formulations via complex formation with Fe(III) ions. J. Pharm. Biomed. Anal, 2002, 28(6):1215~1220
10 Paraskevas DT, Constantinos KZ, Georgios AT, et al. Normal spectrophotometric and stopped-flow spectrofluorimetric sequential injection methods for the determination of alendronic acid, an anti-osteoporosis amino-bisphosphonate drug, in pharmaceuticals. Anal. Chim. Acta, 2005, 547(1):98~103
11 Sparidans RW, Hartigh JD, Vermeij P. High-performance ion-exchange chromatography with in-line complexation ofbisphosphonates and their quality control in pharmaceutical preparations. J. Pharm. Biomed. Anal, 1995, 13(12):1545~1550
12 Sami KAD, Imad IH, Samer MAN. Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine. Talanta, 2004, 64(3):695~702
13 Kline WF, Matuszewski BK. Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection. J. Chromatogr, 1992, 583(2):183~193
14 Yun MH, Kwon KI. High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: Application to a pharmacokinetic study in humans. J. Pharm. Biomed. Anal, 2006, 40(1):168~172
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1 Ralph M, Robert RR. Bisphosphonate action on bone structure and strength: Preclinical and clinical evidence for ibandronate. Bone, 2007, 41(5):S16~S23
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4 Sami ADK, Imad IH, Samer ANM. Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine. Talanta, 2004, 64(3):695~702
5 Ptá?ek P, K??ma J, Macek J. Determination of alendronate in human urine as 9-fluorenylmethyl derivative by high-performance liquid chromatography. J. Chromatogr. B, 2002, 767(1):111~116
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12 Xie Z, Jiang Y, Zhang DQ. Simple analysis of four bisphosphonates simultaneously by reverse phase liquid chromatography using n-amylamine as volatile ion-pairing agent. J. Chromatogr. A, 2006, 1104(1-2):173~178
13 Li BX, He YZ, Xu CL. Simultaneous determination of three organophosphorus pesticides residues in vegetables using continuous-flow chemiluminescence with artificial neural network calibration. Talanta, 2007, 72(1):223~230
14 Wang L, Yang P, Li YX, et al. A flow-injection chemiluminescence method for the determination of some estrogens by enhancement of luminol–hydrogen peroxide–tetrasulfonated manganese phthalocyanine reaction. Talanta, 2006, 70(1):219~224
15 Ramos-Ferńandez JM, Bosque-Sendra JM, Gar??a-Camp?na AM, et al. Chemiluminescence determination of amikacin based on the inhibition of the luminol reaction catalyzed by copper. J. Pharm. Biomed. Anal, 2005, 36(5):969~974
16 Anthony FL, Peter WG. Flow injection analysis of imidacloprid in natural waters and agricultural matrixes byphotochemical dissociation, chemical reduction, and nitric oxide chemiluminescence detection. Anal. Chim. Acta, 2007, 590(2):151~158
17 Hu YF, Zhang ZJ, Yang CY. The determination of hydrogen peroxide generated from cigarette smoke with an ultrasensitive and highly selective chemiluminescence method. Anal. Chim. Acta, 2007, 601(1):95~100
18 Anastasios E, Paraskevas D T, Maria N, et al. Determination of methimazole and carbimazole by flow-injection with chemiluminescence detection based on the inhibition of the Cu(II)-catalysed luminol–hydrogen peroxide reaction. Anal. Chim. Acta, 2004, 505(1):129~133
19 Marques KL, Santos JLM, Lima JFC. A catalytic multi-pumping flow system for the chemiluminometric determination of metformin. Anal Bioanal Chem, 2005, 382(2):452~457
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1 Russell RG, Rogers MJ. Bisphosphonates: from the laboratory to the clinic and back again. Bone, 1999, 25(1):97~106
2 Davas I, Altintas A, Yoldemir T, et al. Effect of daily hormone therapy and alendronate use on bone mineral density in postmenopausal women. Fertility and Sterility, 2003, 80(3):536~540
3 Yieng-Hau RH, Qin XZ. Determination of alendronatesodium by ion chromatography with refractive index detection. J. Chromatogr. A, 1996, 719(2):345~352
4 Eric WT, Dominic PI, Marvin AB. Determination of alendronate in pharmaceutical dosage formulations by ion chromatography with conductivity detection. J. Chromatogr. A, 1992, 596(2):217~224
5 Eric WT, Steven DC, Richard JF, et al. Determination of bisphosphonate drugs in pharmaceutical dosage formulations by ion chromatography with indirect UV detection. J. Pharm. Biomed. Anal, 1994, 12(8):983~991
6 Isabela T, Luigi S, Simona RS, et al. Development and application of a high-performance liquid chromatography-mass spectrometry method to determine alendronate in human urine. J. Chromatogr. A, 2007, 1160(1-2):21~33
7 Xie Z, Jiang Y, Zhang DQ. Simple analysis of four bisphosphonates simultaneously by reverse phase liquid chromatography using n-amylamine as volatile ion-pairing agent. J. Chromatogr. A, 2006, 1104(1-2):173~178
8 Tsai EW, Singh MM, Lu HH, et al. Application of capillary electrophoresis to pharmaceutical analysis: Determination of alendronate in dosage forms. J. Chromatogr. A, 1992, 626(2):245~250
9 Reed DG, Martin GP, Konieczny JM, et al. The determination of alendronate sodium in tablets by inductively coupled plasma (ICP). J. Pharm. Biomed. Anal, 1995,13(8):1055~1058
10 Razak OA, Belal SF, Bedair MM, et al. The utilization of copper (II) phosphate for the anodic stripping voltammetric assay of alendronate sodium, desferrioxamine mesylate and lisinopril. Talanta, 2003, 59(5):1061~1069
11 Tsai EW, Singh MM, Lu HH, et al. Application of capillary electrophoresis to pharmaceutical analysis: Determination of alendronate in dosage forms. J. Chromatogr. A, 1992, 626(2):245~250
12 Sparidans RW, Hartigh JD, Vermeij P. High-performance ion-exchange chromatography with in-line complexation of bisphosphonates and their quality control in pharmaceutical preparations. J. Pharm. Biomed. Anal, 1995, 13(12):1545~1550
13 Paraskevas DT, Constantinos KZ, Georgios AT, et al. Normal spectrophotometric and stopped-flow spectrofluorimetric sequential injection methods for the determination of alendronic acid, an anti-osteoporosis amino-bisphosphonate drug, in pharmaceuticals. Anal. Chim. Acta, 2005, 547(1):98~103
14 Jadranka K, Ivana J, Jovan N, et al. Spectrophotometric determination of alendronate in pharmaceutical formulations via complex formation with Fe(III) ions. J. Pharm. Biomed. Anal, 2002, 28(6):1215~1220
15 Kline WF, Matuszewski BK. Improved determination of the bisphosphonate alendronate in human plasma and urine byautomated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection. J. Chromatogr, 1992, 583(2):183~193
16 Sami ADK, Imad IH, Samer ANM. Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine. Talanta, 2004, 64(3):695~702
17 Ptá?ek P, K??ma J, Macek J. Determination of alendronate in human urine as 9-fluorenylmethyl derivative by high-performance liquid chromatography. J. Chromatogr. B, 2002, 767(1):111~116
18 USP28-NF23, 63
19 Yun MH, Kwon KI. High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: Application to a pharmacokinetic study in humans. J. Pharm. Biomed. Anal, 2006, 40(1):168~172
20 Ramos-Ferńandez JM, Bosque-Sendra JM, Gar??a-Camp?na AM, et al. Chemiluminescence determination of amikacin based on the inhibition of the luminol reaction catalyzed by copper. J. Pharm. Biomed. Anal, 2005, 36(5):969~974
21 Anastasios E, Paraskevas DT, Maria N, et al. Determination of methimazole and carbimazole by flow-injection with chemiluminescence detection based on the inhibition of the Cu(II)-catalysed luminol–hydrogen peroxide reaction. Anal. Chim. Acta, 2004, 505(1):129~133
22 Marques KL, Santos JM, Lima JFC. A catalytic multi-pumping flow system for the chemiluminometric determination of metformin. Anal Bioanal Chem, 2005, 382(2):452~457
23 Long ML, Huang HL, Yan F, et al. Determination of Alendronate Sodium in Compound Alendronate Sodium Tablets by theMolybdenum Blue Colorimetry. Pharm J Chin PLA, 2006, 22(6):434~436
24 Li BX, He YZ, Xu CL. Simultaneous determination of three organophosphorus pesticides residues in vegetables using continuous-flow chemiluminescence with artificial neural network calibration. Talanta, 2007, 72(1):223~230
25 Yao HC, Zheng LM, Xin XQ. Preparation, Structures and Properties of Copper ( Ⅱ ) Phosphonates. Ghin. J. Inorg. Chem, 2004, 20(6):625~631
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22 Tenta R, Sourla A, Lembessis P, et al. Bone microenviroment-related growth factors, zoledronic acid and dexamethasone differentially modulate PTHrP expression in PC-3 prostate cancer cells. Horm Metab Res, 2005, 37(10):593~601
23 Tanta R, Sourla A, Lembessis P, et al. Bone-related growth factors and zoledronic acid regulate the PTHrP/PTH.1 receptor bioregulation system in MG-63 human osteosarcoma cells. Anticancer Res, 2006, 26(1A):283~291
24 Sanders JM., Ghosh S, Chan JM, et al. Quantitative Structure-Activity Relationships for γδT Cell Activation by Bisphosphonates. J. Med. Chem, 2004, 47(2):375~384
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