HPLC-ELSD法测定清开灵栓中胆酸和猪去氧胆酸的含量

目的:建立以高效液相色谱-蒸发光散射(HPLC-ELSD)检测法测定清开灵栓中胆酸和猪去氧胆酸含量的方法。方法:色谱柱为Diamonsil C18(250mm×4.6mm,5μm),流动相为0.1%甲酸-乙腈(梯度洗脱),ELSD检测器漂移管温度为110℃,氮气流速为2.0L·min-1。结果:胆酸和猪去氧胆酸的进样量分别在0.59～7.40μg(r=0.9996)、0.38～4.8μg(r=0.9997)范围内与各自峰面积积分值呈良好线性关系;二者平均回收率分别为99.20%和98.57%,RSD分别为1.4%(n=6)、1.8%(n=6)。结论:本方法简便、准确、分离效果好,可用于清开灵栓的质量控制。     

Optimization of extraction technique and validation of developed RP-HPLC-ELSD method for determination of terpene trilactones in Ginkgo biloba leaves

Terpene trilactones are potent and selective antagonists of platelet activating factor. The present study deals with standardization of efficient extraction method and validation of newly developed simple, sensitive and rapid reversed phase high performance liquid chromatographic method with evaporative light scattering detection (RP-HPLC-ELSD) method for the quantitative determination of ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), ginkgolide J (GJ) and bilobalide (BB) within 8 min in Ginkgo biloba leaf extract. The analysis was conducted on a Zorbax RP-C₁₈ column with gradient elution of methanol–water–tetrahydrofuran. The method was validated for accuracy, precession, limit of detection and quantification. The drift tube temperature of evaporative light scattering detector was set to 90 °C and nitrogen flow rate was 1.5 standard liter/min (SLM).     

应用HPLC／ELSD法测定西洋参中拟人参皂苷F11的含量

目的：测定西洋参中拟人参皂苷Ｆ１１的含量。方法：应用高效液相色谱法－蒸发光散射检测器（ＨＰＬＣ／ＥＬＳＤ）,ＰｌａｔｉｎｕｍＣ１８色谱柱,乙腈－水（３０∶７０）为流动相,并优化选择了蒸发光散射检测器参数,测定了西洋参中拟人参皂苷Ｆ１１的含量。结果：拟人参皂苷Ｆ１１在２１９２～１０９６μｇ范围内呈良好线性（ｒ＝０９９９２,ｎ＝５）,回收率为（１０２６±４１）％。结论：本方法精密度、重现性良好,适用于人参皂苷类成分的分析测定。     

Simultaneous analysis of bioactive metabolites from Ziziphus jujuba by HPLC–DAD–ELSD–MS/MS

A high-performance liquid chromatography (HPLC) with diode array detector (DAD), evaporative light scattering detector (ELSD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of nine representative metabolites of the alkaloid, flavonoid and triterpenoid classes from Ziziphus jujuba var. spinosa. The optimal chromatographic conditions were obtained on an ODS column and the mobile phase was composed of water and methanol with 0.1% formic acid using a gradient elution system. Using the developed methods, all of the validation parameters were successfully obtained. In addition, effectiveness of diverse extraction methods was compared to each other for the development of standard analytic method. The verified method was successfully applied to the quantitative determination of representative metabolites in commercial samples of Z. jujuba and Z. mauritiana from different markets in Korea, China and Myanmar. The analytical results showed that the contents of the nine analytes vary significantly with sources and species, thus demonstrating its potential for the detection of this plant.     

清开灵系列品种中胆酸和猪去氧胆酸的通用检测定方法研究

目的:建立一个通用检测方法测定清开灵系列品种中胆酸和猪去氧胆酸的含量。方法:采用高效液相色谱-蒸发光散射检测法。色谱柱为Agilent Zorbax SB-C18(250mm×4.6mm,5μm),流动相为乙腈-0.5%乙酸溶液(40:60,V/V),流速为0.8mL·min-1,蒸发光散射检测器的漂移管温度为110℃,N2流速为2.0mL·min-1,进样量为5～25μL。结果:胆酸和猪去氧胆酸的进样量均在0.5～3.5μg(r=0.9999或0.9998)范围内与各自峰面积积分值呈良好的对数线性关系;不同剂型清开灵系列品种中胆酸的平均加样回收率在93.8%～99.5%之间,RSD在0.8%～4.6%之间(n=6);猪去氧胆酸的平均加样回收率在95.9%～105.6%之间,RSD在0.9%～4.9%之间(n=6)。结论:该方法简便、准确、分离效果好、专属性强,可作为清开灵系列品种的通用检测方法。     

Comparison of electrospray ionization mass spectrometry and evaporative light scattering detections for the determination of Poloxamer 188 in itraconazole injectable formulation

A high performance liquid chromatography (HPLC) method for Poloxamer 188 using size-exclusion chromatography (SEC) was developed and two different detection mechanisms, evaporative light scattering (ELSD) and electrospray ionization mass spectrometry (ESI-MS), were compared for their quantification capabilities in itraconazole formulation. Both detection techniques coupled with SEC separation were highly effective for the determination of Poloxamer 188, which is difficult to analyze by other common HPLC methods. As expected, ESI-MS detection provided sensitivity and selectivity superior to ELSD. But since the analyte is an excipient in the formulation, high sensitivity was not required and ELSD's simplicity and ruggedness made it more appropriate for routine analysis of this formulation.     

指纹图谱和化学模式识别研究黄芪的不同基原和生长方式

目的：基于化学组分信息探究不同基原、生长方式的黄芪药材的质量差异。方法：采用高效液相色谱-蒸发光散射检测技术（HPLC-ELSD）建立指纹图谱分析方法,对收集的28批不同基原、不同生长方式的黄芪样品进行相似度评价和化学模式识别分析。结果：建立的黄芪HPLC指纹图谱共鉴定出11个共有峰,聚类分析将28批样品分为蒙古黄芪和膜荚黄芪两类,其中蒙古黄芪明显分为野生/仿野生黄芪和移栽黄芪两类。正交偏最小二乘法判别分析（OPLS-DA）筛选出5个野生/仿野生黄芪与移栽黄芪的差异性成分。结论：本研究建立的黄芪HPLC-ELSD指纹图谱结合化学模式识别方法,较为全面地反映出黄芪药材化学成分的总体质量特征,可区分不同基原和不同生长方式的黄芪药材,可为黄芪药材质量控制提供参考。     

偏振荧光光度法同时测定氨基酸注射液及动、植物浸出液中酪氨酸、色氨酸的研究

酪氨酸、色氨酸和苯丙氨酸具有荧光,本文使用互相垂直的偏振片有效地消除了苯丙氨酸的干扰和溶剂散射光的影响;附加滤光片,进一步提高了酪氨酸与色氨酸荧光测定的选择性。线性范围分别为0.02～12.0mg·L^-1(酪氨酸)与0.01～2.5mg·L^-1(色氨酸)。此法可直接用于氨基酸注射液和动、植物浸出液中游离酪氨酸、色氨酸的分析,结果满意。     

Determination of steroidal saponins in Tribulus terrestris by reversed-phase high-performance liquid chromatography and evaporative light scattering detection.

This paper describes the first analytical method suitable for the determination of steroidal saponins in Tribulus terrestris. A separation by high performance liquid chromatography (HPLC) was achieved by using a reversed-phase (RP-18) column, evaporative light scattering (ELS) detection, and a water/acetonitrile gradient as the mobile phase. The marker compound, protodioscin, was detected at a concentration as low as 10.0 microg/mL. Several different samples of plant material were successfully analyzed, and depending on origin and plant part used for extraction, significant differences in the composition of the saponins were observed. The analysis of market products showed considerable variations of 0.17 to 6.49% in the protodioscin content.     

Developing a versatile gradient elution LC/ELSD method for analyzing cellulose derivatives in pharmaceutical formulations

Liquid chromatography combined with evaporative light scattering detection is a powerful tool for analyzing polymeric excipients used in pharmaceutical formulations. A versatile, gradient elution liquid chromatographic method utilizing evaporative light scattering detection (ELSD) has been developed for analyzing several types of cellulose ether and ester derivatives in pharmaceutical formulations. This single method was proven to be capable of differentiating six types of cellulose ether and ester derivatives. The influence of ELSD instrument parameters on the detector response and sensitivity has been studied by a statistical design of experiments. It was found that lowering gas flow rate increased peak area response significantly. Increasing nebulizer temperature also increased peak area response. In contrast, evaporator temperature has very minor impact on peak area response, but had a significant impact on noise level. Thus, signal to noise ratio was significantly lower for low evaporator temperature setting. Despite the logarithmic relationship between peak area responses versus concentrations, sufficient selectivity, precision and accuracy were achieved. The method has been validated for assaying hypromellose acetate succinate (HPMCAS) polymer in a pharmaceutical formulation.     

Simultaneous determination of jujuboside A, B and betulinic acid in semen Ziziphi spinosae by high performance liquid chromatography-evaporative light scattering detection

A reverse phase high performance liquid chromatographic (HPLC) method with evaporative light scattering detection (ELSD) was developed for simultaneous determination of jujuboside A, B and betulinic acid in semen Ziziphi spinosae. The analysis was performed by gradient elution, using an aqueous mobile phase (containing 0.1% acetic acid) modified by acetonitrile. The evaporator tube temperature of ELSD was set at 45 °C, and with the nebulizing gas flow-rate of 1.8 l/min. The method was validated for accuracy, reproducibility, precision and limits of detection and quantification. Quantification of the three active compounds in semen Ziziphi spinosae from different locations was performed by this method, which provides a new tool for quality assessment of semen Ziziphi spinosae.     

Simultaneous determination of 13 bioactive compounds in Herba Artemisiae Scopariae (Yin Chen) from different harvest seasons by HPLC-DAD

Herba Artemisiae Scopariae is a Chinese herbal medicine widely used for the remedy of liver diseases. A high performance liquid chromatography method coupled with diode array detection was developed to simultaneously determine 13 different bioactive compounds in Herba Artemisiae Scopariae (Yin Chen) including chlorogenic acid (1), 6,7-dihydroxycoumarin (2), caffeic acid (3), 4-hydroxyacetophenone (4), scopoletin (5), rutin (6), hyperoside (7), isoquercitrin (8), scoparone (11), 7-methoxycoumarine (12) and quercetin (13). By using four different wavelengths in the HPLC analysis, the developed method was able to determine the bioactive compounds with excellent resolution, precision and recovery. The method was applied to determine the amounts of the bioactive compounds in nine samples from different cultivated regions and harvest seasons in China, and significant variations were revealed. Chlorogenic acid was the most abundant among the analyzed compounds. The samples harvested in the spring contained higher contents of chlorogenic acid than those collected in other seasons. Other phenolic acids as caffeic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and 4-hydroxyacetophenone accumulated at much higher amounts in about May to July. The samples analyzed contained a much lower level of the amount of other flavonoids and coumarins as rutin, hyperoside, isoquercitrin and scoparone.     

Simultaneous qualitative and quantitative analysis of triterpenic acids, saponins and flavonoids in the leaves of two Ziziphus species by HPLC-PDA-MS/ELSD

The leaves of Ziziphus jujuba and Z. jujuba var. spinosa have been utilized as crude drugs for their health benefits in China for thousands of years. To control their quality, a reliable method based on high-performance liquid chromatography coupled with photo diode array and electrospray ionization tandem mass spectrometry detection (HPLC-PDA-ESI-MS/MS) was developed for exploration of the chemical profiles of these jujube leaves. As the results, fourteen constituents including three flavonoids, two saponins and nine triterpenic acids were identified or tentatively characterized. Then, twelve of them such as quercetin-3-O-rutinoside, zizyphus saponins I and II, ceanothic acid, alphitolic acid, maslinic acid, 2α-hydroxyursolic acid, zizyberanalic acid, epiceanothic acid, ceanothenic acid, betulinic acid, and oleanolic acid were selected as the chemical markers and were determined using an HPLC coupled with evaporative light scattering detection (ELSD) method. The separation was carried out on a Waters Sunfire C₁₈ column with 0.2% acetic acid and acetonitrile as the mobile phase under gradient elution. The operating conditions of ELSD were set as 80 °C for drift tube temperature and 2.7 l/min for nitrogen flow rate. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of eight batches of Z. jujuba and Z. jujuba var. spinosa leaves from different collections.     

Comparative study on the analytical performance of different detectors for the liquid chromatographic analysis of tobramycin

Pulsed electrochemical detection (PED) with a triple step potential–time waveform has been used successfully for the direct detection of several non-chromophoric aminoglycosides at alkaline pH following ion-pair liquid chromatography (LC). This direct detection method has helped to overcome the various flaws observed in the analysis of aminoglycosides using time consuming derivatization procedures. However, this technique also suffers from some disadvantages. Hence, improvements of PED as well as alternative detection techniques would be welcome for the detection of drug molecules lacking UV absorbing chromophores. In this study a comparison was made between the analytical performance of different brands of PED detectors and two different evaporative light scattering detectors (ELSD) from the same manufacturer. Different PED waveforms (triple, quadruple and six potential–time waveforms) were also examined for the detection of tobramycin, a representative example of the group of non-chromophoric aminoglycosides. As starting point, the LC–PED method of the European Pharmacopoeia (Ph. Eur.) was taken. As the Ph. Eur. method prescribes the use of a non-volatile mobile phase, an alternative one had to be used in combination with ELSD. The analytical performance was compared with regards to sensitivity, linearity and long-term stability.     

HPLC detection of miltefosine using an evaporative light scattering detector

Miltefosine has recently been introduced as leishmanicidal drug for oral administration (Impavido). Previous communications report about the use of liquid chromatography coupled with mass spectrometry detection to detect miltefosine in pharmaceutical preparations and biological fluids. We report about a new method to detect miltefosine using an evaporative light scattering detector (ELSD). The absolute recovery of the analyte was greater than 98.0%. The limit of quantification for miltefosine in plasma at a signal-to-noise ratio of 7.3 was 0.34 microg/ml. The precision of the assay yielded coefficients of variation ranging from 1.8 to 4.5% and an accuracy of 97-107%. Our method advances the qualitative and quantitative detection of miltefosine by combining rapid and efficient solid phase extraction and analysis with an evaporative light-scattering detector.     

HPLC法同时测定清开灵注射液中黄芩苷和千层纸素A-7-O-β-d-葡糖醛酸苷的含量

目的:建立同时测定清开灵注射液中黄芩苷和千层纸素A-7-O-β-d-葡糖醛酸苷含量的方法,并比较不同厂家、批号产品中黄芩苷和千层纸素A-7-O-β-d-葡糖醛酸苷的含量差异。方法:采用高效液相色谱法。色谱柱为Agilent ZORBAX SB-C18,流动相为甲醇-0.1%磷酸溶液(梯度洗脱),流速为1.0 ml/min,检测波长为274 nm,柱温为30℃,进样量为10μl。结果:黄芩苷、千层纸素A-7-O-β-d-葡糖醛酸苷检测质量浓度分别在10.1²52.0、1.0252.0、1.0⁶.0μg/ml范围内与各自峰面积积分值呈良好的线性关系(r=0.999 9);精密度、稳定性、重复性试验的RSD≤1.6%;平均加样回收率分别为99.64%、99.03%,RSD分别为2.7%、0.9%(n=6)。不同厂家、批号产品中黄芩苷、千层纸素A-7-O-β-d-葡糖醛酸苷的质量浓度范围分别为4.1516.0μg/ml范围内与各自峰面积积分值呈良好的线性关系(r=0.999 9);精密度、稳定性、重复性试验的RSD≤1.6%;平均加样回收率分别为99.64%、99.03%,RSD分别为2.7%、0.9%(n=6)。不同厂家、批号产品中黄芩苷、千层纸素A-7-O-β-d-葡糖醛酸苷的质量浓度范围分别为4.151⁴.849、0.0974.849、0.097⁰.157 mg/ml。结论:该方法简单、准确、可靠,适用于清开灵注射液的质量控制;不同厂家、批号清开灵注射液中黄芩苷和千层纸素A-7-O-β-d-葡糖醛酸苷的含量存在较大差异。     

Simultaneous determination of active ingredients in Erigeron breviscapus (Vant.) Hand-Mazz. by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis (CE) with electrochemical detection (ED) method was developed for the determination of the pharmacologically active ingredients in Erigeron breviscapus (Vant.) Hand-Mazz. and its extract phytopharmaceuticals in this work. Under the optimum conditions, nine analytes, baicalein, naringenin, scopoletin, kaempferol, apigenin, scutellarin, luteolin, caffeic acid and protocatechuic acid were separated within 24 min in a borax buffer (pH 8.7). Notably, excellent linearity was obtained over two orders of magnitude with detection limits (S/N=3) ranged from 1.0 x 10(-7) g/mL to 5.6 x 10(-7) g/mL for all nine analytes. This method was successfully used in the analysis of E. breviscapus (Vant.) Hand-Mazz. and its phytopharmaceuticals with a relatively simple extraction procedure, and the assay results were satisfactory.     

Comparison of liquid chromatography and capillary electrophoresis methods for quantification of sodium residuals

Liquid chromatography (LC) and capillary electrophoresis (CE) methods were developed to perform the determination of residual sodium in mother liquors and successive washes of an active pharmaceutical ingredient (API). The addition of sodium chloride to the product solution results in rapid and complete crystallization of the API. The LC method was coupled to evaporative light scattering detection (ELSD) while the CE approach was based on indirect UV detection. Both methods were fully validated. Selectivity, response function, trueness, precision, accuracy, linearity and limits of detection (LOD) and quantification (LOQ) were the criteria investigated. The LC-ELSD method was found to be more sensitive than the CE/indirect UV approach. The methods were found to be valid over concentration ranges of 62-500 and 235-1500 ppm for the LC and the CE methods, respectively. Both methods were compared and used for the determination of actual samples coming from different batches of the same API chemical synthesis.     

中药复方制剂中黄芩苷含量的HPLC检测法的建立

目的建立中药复方制剂中黄芩苷含量的HPLC检测法。方法采用高效液相色谱法检测以清开灵颗粒为代表的中药复方制剂中黄芩苷的含量,流动相为0.5%磷酸水溶液-甲醇(58︰42),检测波长280nm,流速为1.0mL/min,采用外标峰面积法计算清开灵颗粒中黄芩苷的含量。结果黄芩苷与有关物质完全分离,在0.1019～1.019μg范围内峰面积与浓度呈现良好的线性关系(R2=0.9999,n=5),平均回收率为99.44%(RSD=0.86%,n=5)。结论本实验方法简单、迅速、专一性强,适用于中药复方制剂中黄芩苷的含量检测。     

Determination of 24(R)-pseudoginsenoside F(11) in North American ginseng using high performance liquid chromatography with evaporative light scattering detection

A gradient liquid chromatographic method with evaporative light scattering detection (ELSD) for the determination of 24(R)-pseudoginsenoside F(11) in North American ginseng is described. Samples are analyzed by means of a reverse-phase column (Waters Spherisorb ODS-2, C(18)) using acetonitrile and water under gradient conditions as the mobile phase over 20 min. The evaporative light scattering detector (ELSD) used, was set at an evaporating temperature of 35 degrees C and nitrogen gas pressure of 3.4 bar. The detection limit (S/N>5) of 24(R)-pseudoginsenoside F(11) is 53 ng on column.