Achiral liquid chromatography with circular dichroism detection for the determination of carnitine enantiomers in dietary supplements and pharmaceutical formulations

A simple and enantioselective method for the separation and determination of carnitine enantiomers in dietary supplements and pharmaceutical formulation samples is proposed. This method is based on achiral liquid chromatographic separation of carnitine enantiomers from interferences and direct circular dichroism (CD) detection. The calibration curve of the anisotropy factor (g) versus the enantiomeric excess was linear, with a correlation coefficient (R²) of 0.996. The precision evaluated by UV peak area and CD peak area was suitable (RSD <5% in all cases). The usefulness of the proposed method was demonstrated by analysing natural dietary supplements and pharmaceutical formulation samples. This method has the advantages of being rapid and precise, without using an expensive chiral column. The method was suitable for the simultaneous determination of both enantiomers and for assessing the chemical purity of carnitine.     

HPLC法测定L-(+)-扁桃酸的光学纯度

目的采用HPLC法测定L-(+)-扁桃酸的光学纯度。方法采用Chirlcel OJ-H手性色谱柱,以正己烷-乙醇-三氟乙酸(96∶4∶0.3)为流动相,检测波长220 nm,流速1.0 m L·min-1,柱温30℃。结果 L-(+)-扁桃酸与D-(-)-扁桃酸的分离良好,L-(+)-扁桃酸0.2~1.2×103μg·m L-1与峰面积的线性关系良好(r=0.9998),检测限为0.4 ng(S/N=3),定量限为1.6 ng(S/N=10)。结论所用方法快速、准确、灵敏,适于L-(+)-扁桃酸光学纯度的测定。     

Determination of the antidepressant fluoxetine in human plasma by LC with UV detection

A selective and sensitive isocratic high-performance liquid chromatographic (HPLC) method was developed for the quantitative analysis of low concentrations of fluoxetine (FLX) in human plasma, with ultra-violet detection at 226 nm. A reversed-phase column, LiChrospher® 60 RP-Select B (125×3 mm i.d., 5 μm) (Merck), was used to resolve FLX and diazepam (DZP) (internal standard) from endogenous matrix interferences. FLX was isolated from plasma by liquid-liquid extraction. Two identical HPLC systems were used, both validated under the same study conditions. Each chromatographic separation was completed in 30 min and the results showed a mean relative recovery of 101 and 99.3% and an overall precision (RSD%) of 4.78 and 6.09 for each HPLC system. The standard curve was linear for FLX concentrations over the range of 5.00–50.0 ng ml⁻¹ (R=0.997 and 0.998).The limit of quantitation of FLX was 5 ng ml⁻¹ for both HPLC systems. The method described was applied to the analysis of plasma samples obtained from healthy subjects treated with one single oral dose of 40 mg of fluoxetine.     

乳糖醇的HPLC—蒸发光散射检测器测定

建立了HPLC－蒸发光散射检测器（ELSD）法测定乳糖醇含量的方法,采用Rezex柱,水为流动相,在0．05－1．0mg范围内乳糖醇进样量的自然对数与其峰面积的自然对数呈良好的线性关系（r=0.9994),方法平均回收率为99．73％（RSD＝0．3％）,最低检测限为1ug。本法灵敏,准确,稳定性好。     

高效液相色谱-蒸发光散射检测法测定三乙醇胺乳膏的含量

目的:建立高效液相色谱-蒸发光散射检测法(high performance liquid chromatography-evaporative light-scatting detector,HPLC-ELSD)测定三乙醇胺乳膏含量的方法。方法:采用Lichrospher CN色谱柱(250 mm×4.6 mm,5μm);流动相为甲醇-水(含0.1%甲酸)(70∶30),流速1.0 mL·min-1;蒸发光散射检测器参数:漂移管温度为50℃,雾化室温度为70℃,载气流速为1.2 L·min-1。结果:三乙醇胺的色谱峰与辅料峰能够较好的分离,三乙醇胺峰理论塔板数均>2000;软膏80%,100%和120%3个浓度水平加样回收率分别为99.83%(RSD=0.8%,n=3),100.07%(RSD=1.19%,n=3)和98.97%(RSD=1.81%,n=3);三乙醇胺的线性方程为:y=1.404 7x+2.311 6(r=0.999 5),浓度范围25～300 mg·L-1,线性关系良好;三乙醇胺的检测限为2 mg·L-1。结论:本法简便、快速、结果准确、可靠、重现性好,可用于三乙醇胺乳剂含量测定,能够对药品质量进行控制。     

HPLC-ELSD法测定异帕米星含量

目的　探讨利用HPLC 蒸发光散射检测器 (ELSD)测定异帕米星含量的方法。方法　色谱条件为 :ZorbaxSB C18色谱柱 (15 0mm× 4 6mm ,5 μm ) ,流动相 :0 5 %五氟丙酸水溶液—甲醇 (4 2∶2 2 ,V/V) ,流速 :0 64ml/min ;漂移管温度 110℃ ,载气流速 2 65L/min。利用阿米卡星为对照绘制随行标准曲线 ,根据测定的异帕米星标准品的峰面积值确定含量 ;并与经典的微生物检定法对测定结果的准确性进行验证。结果　该方法的重复性、灵敏度均较理想 ;HPLC 蒸发光散射检测器的测定结果 (69 7% )与微生物检定法的测定结果(70 7% )非常接近。结论　在没有异帕米星对照品的情况下 ,以结构相似含量已知的阿米卡星为对照 ,采用蒸发光散射检测器测定异帕米星的含量是可行的     

Study of the interaction between mercury (II) and bovine serum albumin by spectroscopic methods

Mercury is a significant environmental pollutant that originates from industry. Mercury will bind with albumin and destroy biological functions in humans if it enters the blood. In this paper, the interaction between mercury (II) and bovine serum albumin (BSA) was investigated in vitro by fluorescence, UV–Vis absorption and circular dichroism (CD) under simulated physiological conditions. This study proves that the probable quenching mechanism of BSA by mercury (II) was mainly static quenching due to the formation of a mercury (II)–BSA complex. The quenching constant K_a and the corresponding thermodynamic parameters (ΔH, ΔS and ΔG) at four different temperatures were calculated by a modified Stern–Volmer equation and the van’t Hoff equation, respectively. The results revealed that the interaction between mercury (II) and BSA was mainly enthalpy-driven and that hydrogen bonding and van der Waals forces played a major role in the reaction. The obtained data for binding sites of n approximately equal to 1 indicated that there was a single class of binding site for the BSA with mercury (II). The value of the distance r (3.55 nm), determined by Föster's non-radioactive energy transfer theory, suggested that the energy transfer from BSA to mercury (II) occurred with a high probability. The conformational investigation from synchronous fluorescence, CD spectroscopy and three-dimensional fluorescence showed that the presence of mercury (II) resulted in micro-environmental and conformational changes of the BSA molecules, which may be responsible for the toxicity of mercury (II) in vivo.     

Assay and purity control of oxytetracycline and doxycycline by thin-layer chromatography — a comparison with liquid chromatography

A thin-layer chromatographic (TLC) method using densitometry is described for the assay and purity control of oxytetracycline and doxycycline. With a mobile phase of dichloromethane—methanol—water (59:35:6, v/v/v) and a silica gel thin-layer, previously sprayed with 10% sodium edetate solution adjusted to pH 9.0, all the potential impurities of oxytetracycline or doxycycline are well separated from the main components and from each other. Results obtained with TLC are compared with those obtained by previously established liquid chromatography (LC) methods using poly(styrene-divinylbenzene) stationary phases. A good correlation was obtained (r > 0.9999). For TLC the relative standard deviation (RSD) for the assay of the main component was <2%, for LC the RSD was <1%.     

Identification and control of impurities in streptomycin sulfate by high-performance liquid chromatography coupled with mass detection and corona charged-aerosol detection

For the control of impurities in streptomycin sulfate a reversed phase ion-pair high performance liquid chromatography (HPLC) method using charged aerosol detection (CAD) was developed. With this method, 21 impurities could be separated and tentatively identified using a combination of exact mass measurement by TOF-MS and MS/MS experiments with a triple quadrupole MS. For three impurities the suggested structures could be confirmed by in situ formation. The CAD detector response was found to be linear over 2 orders of magnitude allowing a straightforward quantification of all impurities. A limit of quantification of 0.09% for streptomycin sulfate and of 0.008% for streptidine sulfate (referred to the concentration of the 5 mg/ml test solution) could be achieved. The HPLC method was applied to the purity testing of 12 samples of commercially available streptomycin sulfate from different manufacturers. Impurity levels between 4.6% and 16.0% were found. The current European Pharmacopoeia monograph for streptomycin sulfate only limits streptomycin B by a TLC test to 3.0%. Therefore, the results of this study underline the importance of introducing a state-of-the-art test for the control of impurities in the monograph. The new HPLC-CAD method is considered suitable for this purpose.     

甲磺酸罗哌卡因中2,6-二甲基苯胺的HPLC-电化学法测定

建立了测定甲磺酸罗哌卡因中 2 ,6 -二甲基苯胺的高效液相色谱 -电化学检测法。采用 C1 8柱 ,流动相为含乙二胺四乙酸二钠和磷酸氢四丁铵的 0 .5 mol/ L磷酸盐缓冲液 -乙腈 (6 0∶ 4 0 ) ,流速 1.0 m l/ min,电极电压为 0 .90 V。2 ,6 -二甲基苯胺在 5× 10 - 3～ 6× 10 - 2 μg/ ml的范围内线性良好 (r=0 .9980 ) ,RSD为 2 .7%。     

高效液相色谱-电雾式检测器测定氨溴特罗口服液中羟乙基纤维素含量

目的：建立测定氨溴特罗口服液中羟乙基纤维素的高效液相色谱-电雾式雾式检测器(HPLC-CAD)方法。方法：采用TSKgtl G2000SW (60cm×7.5mm,10 μm),以10 mmol/L乙酸铵(pH4.5):乙腈=95:5为流动相,柱温：30 ℃,流速：1.0 mL/min,电雾式检测器的蒸发温度为50 ℃。结果：该方法对羟乙基纤维素专属性良好,精密度、平行性和回收率均满足分析要求,峰面积线性关系良好(r＞0.995)。结论：新建方法专属性好、操作简便,可以满足氨溴特罗口服液中羟乙基纤维素分析的要求。     

HPLC-ELSD法同时测定新氢化可的松乳膏中硫酸新霉素和氢化可的松的含量

目的建立HPLC-蒸发光散射检测器(ELSD)法,同时测定新氢化可的松乳膏中硫酸新霉素和氢化可的松的含量。方法色谱条件:C18色谱柱(150 mm×4.6 mm,5μm);流动相:乙腈-0.1 mol/L三氟乙酸溶液(梯度洗脱);漂移管温度:110℃;载气流速:2.6 L/min。用硫酸新霉素和氢化可的松对照品分别测定新氢化可的松乳膏中硫酸新霉素和氢化可的松的含量。结果硫酸新霉素进样量在0.7～4.3μg范围内(r=0.999 7),氢化可的松进样量在1.0～2.9μg范围内(r=0.999 9)。结论该方法快速、简便、重现性好,灵敏度高。     

高效液相色谱法测定洋参雪哈口服液中三组分含量

目的建立测定洋参雪哈口服液含量的高效液相色谱(HPLC)法。方法色谱柱为Agilent HC-C18柱(250mm×4.6mm,5μm),以乙腈(A)与水(B)为流动相进行梯度洗脱,检测波长为203nm。结果人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1进样量分别在0.31～1.56μg,0.82～4.13μg,0.96～8.01μg范围内与峰面积线性关系良好,平均加样回收率为98.71%,RSD=0.81%(n=6)。结论HPLC法重现性好,结果准确,可用于洋参雪哈口服液的质量控制。     

HPLC-示差折光检测器测定米格列醇片的含量及有关物质

目的建立高效液相色谱-示差折光检测法测定米格列醇片的含量和有关物质。方法用磺酸基阳离子交换键合硅胶(250×4.6mm,5μm)为填充剂;以乙腈-三氟乙酸溶液(4.5→1000mL)(20∶80)为流动相;示差折光检测器检测;检测器温度为40℃。理论板数以米格列醇峰计算,应不低于2000。结果空白辅料不干扰测定,米格列醇主峰和杂质峰分离良好,定量限浓度约为10μg·mL^-1。米格列醇浓度在26.28~210.2μg·mL^-1范围内,与峰面积线性关系良好,线性方程A=2611.5C-2802,r=0.9988;平均回收率100.9%(RSD=1.0%;n=9);溶液在8h内稳定。结论本法专属性强、灵敏度高、准确度和稳定性好,可作为米格列醇片有关物质和含量测定的检测方法。     

Determination of the optical purity of (R)-terbutaline by H-NMR and RP-LC using chiral derivatizing agent, (S)-(−)-α-methylbenzyl isocyanate

A simple, convenient and precise ¹H-NMR and indirect HPLC methods were used for the determination of (S)-terbutaline in (R)-terbutaline. The enantiomers were converted to diastereomeric derivatives using (S)-(−)-α-methylbenzyl isocyanate and were successfully separated on an ODS column within 40 min with R_S=1.41 and α=1.09. Interaction between chiral solutes by the formation of the diastereomeric complexes also led to differentiations of the ¹H-NMR spectra of enantiomers and optical purities were determined on the basis of the peak area of the enantiomeric amine proton resonance. The effect of various experimental parameter, such as reaction time, reaction temperature and concentration of chiral derivatizing agent on the derivatization reaction and composition of mobile phase on the ODS column is discussed. Validation data such as recovery, linearity and detection limit are also presented. The results from ¹H-NMR and RP-HPLC methods were compared with those from chiral HPLC method and no racemization was found during the experiments. NMR results had agreed with those obtained by indirect HPLC method and two methods could be used as a quality control method for the enantiomeric purity determination of (R)-terbutaline.     

高效液相色谱法测定复合维生素B片中4种组分的含量

目的:采用离子对高效液相色谱法测定复合维生素B片中烟酰胺、维生素B6、维生素B2、维生素B1等4种水溶性维生素的含量。方法:以C18为色谱柱,0.005mol.L-1庚烷磺酸钠溶液(含0.5%冰醋酸和0.05%三乙胺)-甲醇(72∶28,v/v)为流动相,流速为1mL.min-1,检测波长为280nm。结果:烟酰胺、维生素B6、维生素B2、维生素B1的线性范围分别为0.315 4~15.77μg,0.052 16~2.608μg,0.052 27~2.613 5μg,0.447 4~22.37μg;方法的回收率均在98.5%~101.4%,RSD均为0.6%~1.8%。结论:本法简便,快速,为复合维生素B片产品的质量控制及多维类药物中水溶性维生素含量的测定提供了可靠依据。     

高效液相色谱法测定阴泰洗液中替硝唑的含量

目的 :建立测定阴泰洗液中替硝唑含量的高效液相色谱方法。方法 :以Nova-PakC18 为色谱柱 ,甲醇 -水 -冰醋酸 (22∶78∶0.1)为流动相 ,甲硝唑为内标 ,检测波长为310nm。结果 :替硝唑在20～100μg/ml浓度范围内线性关系良好 (r=0 9999) ;平均回收率为101.2 % (n=5) ,RSD=1 22 %。结论 :本方法简便、快速 ,测定结果准确 ,重现性好。     

HPLC-CAD法测定硫酸卡那霉素及注射液含量及有关物质

目的建立测定硫酸卡那霉素原料及其注射液中有关物质与卡那霉素含量的高效液相色谱-电喷雾检测器(HPLCCAD)方法。方法采用Waters HSS T3色谱柱(4.6mm×250mm,5μm),以0.2 mol/L三氟乙酸水溶液为流动相,流速0.3m L/min,柱温30℃,电喷雾检测器的喷雾器温度为35℃。结果新建方法对卡那霉素与硫酸盐、有关物质分离良好,精密度、重复性、回收率均满足分析要求。卡那霉素浓度与峰面积线性关系良好(R2>0.99),检出限可达到6.1ng。采用该方法对单硫酸卡那霉素、硫酸卡那霉素以及硫酸卡那霉素注射液进行了测定,检出的有关物质个数及含量均高于现行标准的结果。结论新建方法灵敏度更高、分离度更好,能检出更多杂质,可以满足硫酸卡那霉素原料及注射液有关物质及含量测定分析要求。     

一测多评法测定天然辣椒碱中2种主要成分

目的建立以1种对照品同时测定天然辣椒碱中2种成分的质量评价方法。方法以辣椒辣素为对照,采用一测多评法,同时测定辣椒辣素和二氢辣椒辣素的含量。结果测得辣椒辣素与二氢辣椒辣素的相对校正因子为1.12,同时采用外标法和一测多评法测定样品中二氢辣椒辣素含量,采用t检验对二者测定值进行比较,二者之间无显著差异(P>0.05);以辣椒辣素的保留时间为1.00,计算得二氢辣椒辣素的相对保留时间为1.355,相对保留时间及保留时间差的RSD均小于5%。结论采用一测多评法,以辣椒辣素为对照,利用相对校正因子可实现同时测定辣椒辣素和二氢辣椒辣素的含量。     

柱前衍生化–高效液相色谱法测定蒙古黄芪多糖中单糖的组成

目的测定蒙古黄芪多糖中的单糖组成。方法蒙古黄芪多糖样品用三氟乙酸溶液水解成单糖,用1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化,采用HPLC法于245nm波长处检测各单糖。结果蒙古黄芪多糖由甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、阿拉伯糖组成,物质的量比分别为0.02∶0.05∶0.17∶1∶0.18。结论此方法简单、快速、重复性好,可用于蒙古黄芪多糖中单糖组成分析。