高效液相色谱直接进样法测定血浆中头孢布烯的含量

目的:直接进样高效液相色谱法测定人血浆中的头孢布烯的含量.方法:选用浸透限制固定相,紫外检测器,使头孢布烯在流动相pH 7.0条件下与内标及血浆中的蛋白质得到良好的分离.结果:制作了头孢布烯的标准曲线,测定方法的变异系数在4.1%～7.3%,回收率近100%.结论:浸透限制固定相色谱法测定血浆中的头孢布烯,省去了前处理,快速简单,更适用于临床治疗药物监测.     

Rapid hydrophilic interaction chromatography determination of lysine in pharmaceutical preparations with fluorescence detection after postcolumn derivatization with o-phtaldialdehyde

A rapid procedure for the determination of lysine based on hydrophilic interaction chromatography (HILIC) separation of arginine and lysine with fluorescence detection has been developed. The separation conditions and parameters of lysine postcolumn derivatization with o-phtaldialdehyde (OPA)/2-mercaptoethanol were studied. The various HILIC columns were employed using isocratic elution. Fluorescence detection was performed at excitation and emission wavelength of 345 nm and 450 nm, respectively. An advantage of the reported method is a simple sample pre-treatment and a quick and very sensitive HPLC method. The developed method was successfully applied for analysis of commercial samples of Ibalgin Fast tablets (Zentiva, Czech Republic).     

Determination of metformin and its prodrugs in human and rat blood by hydrophilic interaction liquid chromatography

Simple and specific hydrophilic interaction liquid chromatography (HILIC) method with ultraviolet (UV) detection was developed for the simultaneous determination of highly water-soluble metformin and its more lipophilic prodrugs in human and rat blood samples. The sample preparation was accomplished by precipitating proteins with acetonitrile, which enabled the direct injection of supernatants to the HPLC. Chromatographic separation was performed on an analytical normal phase silica column using a mixture of 0.01 M ammonium acetate pH 5.0 and acetonitrile (40:60, v/v) as a mobile phase at flow rate of 1 ml/min and at the wavelength of 235 nm. The method was validated in terms of specificity, linearity, accuracy, precision, recovery, and analyte stability. The UV-HILIC method was suitable for detecting both metformin and one of its more lipophilic prodrugs simultaneously in human and rat blood samples.     

Determination of inorganic pharmaceutical counterions using hydrophilic interaction chromatography coupled with a Corona CAD detector

A simple generic approach was investigated for the determination of inorganic pharmaceutical counterions in drug substances using conventional high performance liquid chromatographic (HPLC) instruments. An intuitive approach combined Corona^® charged aerosol detection (CAD) with a polymer-based zwitterionic stationary phase in the hydrophilic interaction chromatography (HILIC) mode. Two generic methods based on this HILIC/CAD technique were developed to quantitate counterions such as Cl⁻, Br⁻, SO₄²⁻, K⁺, Ca²⁺ and Mg²⁺ in different pharmaceutical compounds. The development and capability of this HILIC/CAD technique analysis were examined.HILIC/CAD was compared to ion chromatography (IC), the most commonly used methodology for pharmaceutical counterion analysis. HILIC/CAD was found to have significant advantages in terms of: (1) being able to quantitate both anions and cations simultaneously without a need to change column/eluent or detection mode; (2) imposing much less restriction on the allowable organic percentage of the eluents than IC, and therefore being more appropriate for analysis of counterions of poorly water-soluble drugs; (3) requiring minimal training of the operating analysts. The precision and accuracy of counterion analysis using HILIC/CAD was not compromised. A typical precision of <2.0% was observed for all tested inorganic counterions; the determinations were within 2.0% relative to the theoretical counterion amount in the drug substance. Additionally, better accuracy was shown for Cl⁻ in several drug substances as compared to IC. The main drawback of HILIC/CAD is its unsuitability for many of the current silica-based HILIC columns, because slight dissolution of silica leads to high baseline noise in the CAD detector.As a result of the universal detection characteristics of Corona^® CAD and the unique separation capabilities of a zwitterionic stationary phase, an intuitive and robust HPLC method was developed for the generic determination of various counterions in different drug substances. HILIC/CAD technique is a useful alternative methodology, particularly for determination of counterions in low-solubility drugs.     

Direct injection analysis of oxypurinol and metformin in wastewater by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry

The increasing global prevalence of gout and diabetes has led to a rise in the use of their respective medications, allopurinol and metformin. These are excreted via urine as oxypurinol and metformin and are discharged into wastewater and the environment. Current environmental monitoring of those two polar chemicals requires labour intensive and potentially inefficient sample pre-treatments, such as using solid-phase extraction or freeze-drying. This study validated a sensitive and simple method using direct-injection LC–MS/MS for the simultaneous measurement of oxypurinol and metformin in wastewater. The final method utilised a hydrophilic interaction liquid chromatography together with simple filtration through 0.2 μm regenerated cellulose filter followed by dilution in acetonitrile with a dilution factor of 10. The developed method was validated with the limit of quantifications (LOQ) of 0.11 and 0.34 μg/L for metformin and oxypurinol, respectively. The new method was applied to 42 influent wastewater samples and 6 effluent samples collected from 6 Australian wastewater treatment plants. Both compounds were detected well above the LOQ at concentrations 29–214 μg/L in influent and 2–53 μg/L in effluent for metformin, and 24–248 μg/L in influent and 4–81 μg/L in effluent for oxypurinol, demonstrating its high applicability.     

Diastereomeric and enantiomeric high-performance liquid chromatographic separation of synthetic anisodamine

In order to investigate the enantiomeric pharmacokinetics and biotransformation of synthetic anisodamine (654-2), a cholinoceptor antagonist widely used in clinic in China, it has been preparatively separated into two racemates (I and II) by using ZORBAX Eclipse XDB-C18 column. The diastereo- and/or enantioseparations of 654-2, I and II were carried out by HPLC using CHIRALPAK AD-H as chiral stationary phase (CSP) and acetonitrile-2-propanol-DEA 97:3:0.1 (v/v/v) as mobile phase. The methods were optimized by studying mobile phase modifiers, concentration of modifier and column temperature. The HPLC method for the simultaneous separation of two pairs of enantiomers of 654-2 has been validated.     

亲水作用色谱法（HILIC）的方法开发策略及其在铂类抗癌药分析中的应用

亲水作用色谱法解决了大多数极性化合物的色谱分离问题,其已成为了包括铂类抗癌药在内的许多极性化合物的色谱分离的首选。亲水作用色谱法在极性化合物的分离与检测、药代动力学研究等的应用均日益广泛。如何高效的完成亲水作用色谱的方法开发是药物分析科学家和药代动力学科学家都面临的重要问题,然而目前还没有专门的文献对此进行系统化的整理和研究。本文以此为切入点,围绕亲水作用色谱法方法开发中的固定相选择、流动相筛选、pH值与洗脱的优化等方面,综述了亲水作用色谱（HILIC）的方法开发策略,以及其在铂类抗癌药分析中的应用。     

色谱手性固定相在手性药物拆分中的应用

色谱手性固定相（chiral stationary phases，CSPs）在手性药物分析和制备拆分中具有很重要的作用。本研究对5种带有CSPs的手性色谱技术以及在高效液相色谱（high performance liquid chromatography，HPLC）和超临界流体色谱（supereritical fluid chromatography，SFC）中CSPs的4种流动相模式和6种类型进行了综述，提出了CSPs色谱柱的筛选策略，并讨论了手性药物拆分从分析到制备的转变。     

Determination of new retention indices for quick identification of essential oils compounds

The classical methods of chromatographic identification of compounds were based on calculation of retention indices by using different stationary phases. The aim of the work was to differentiate essential oils extracted from different plant species by identification of some of their major compounds. The method of identification was based on the calculation of new retention indices of essential oils compounds fractionated on a polar chromatographic column with temperature programming system. Similar chromatograms have been obtained on the same column for one plant family with two different temperature gradients allowing the rapid identification of essential oils of different species, sub-species or chemotypes of Citrus, Mentha and Thymus.     

Stability indicating ion chromatography method for the simultaneous determination of ibandronate sodium drug substance and its impurities

A simple and sensitive ion chromatography method has been developed for the simultaneous assay of ibandronate sodium drug substance and the determination of its impurities. The separation was achieved on Allsep™ anion column 150 mm × 4.6 mm, 7 μm particle diameter. The mobile phase consisted of 1% (v/v) aqueous formic acid and acetone 98:2% (v/v); flow rate 1.0 ml min⁻¹ at ambient temperature. The analytes were monitored by conductometric detector. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolytic, thermal and humidity degradation. Considerable degradation was achieved only under oxidative conditions. Mass balance was demonstrated in all stress conditions. The method was validated for specificity, precision, linearity, solution stability and accuracy. The limits of detection (LOD) and limits of quantification (LOQ) for impurities were in the range of 0.36–0.80 μg ml⁻¹ and 1.00–2.40 μg ml⁻¹, respectively. For ibandronate LOD was 38 μg ml⁻¹ and LOQ was 113 μg ml⁻¹. The average recoveries for impurities and ibandronate were in the range of 99.0–103.1% and the method can be successfully applied for the routine analysis of ibandronate sodium drug substance.     

Gradient high performance liquid chromatography method for simultaneous determination of ilaprazole and its related impurities in commercial tablets

A methodology (HPLC) proposed in this paper for simultaneously quantitative determination of ilaprazole and its related impurities in commercial tablets was developed and validated. The chromatographic separation was carried out by gradient elution using an Agilent C₈ column (4.6 mm × 250 mm, 5 μm) which was maintained at 25 °C. The mobile phase composed of solvent A (methanol) and solvent B (solution consisting 0.02 mmol/l monopotassium phosphate and 0.025 mmol/l sodium hydroxide) was at a flow rate of 1.0 ml/min. The samples were detected and quantified at 237 nm using an ultraviolet absorbance detector. Calibration curves of all analytes from 0.5 to 3.5 μg/ml were good linearity (r ≥ 0.9990) and recovery was greater than 99.5% for each analyte. The lower limit of detection (LLOD) and quantification (LOQ) of this analytical method were 10 ng/ml and 25 ng/ml for all impurities, respectively. The stress studies indicated that the degradation products could not interfere with the detection of ilaprazole and its related impurities and the assay can thus be considered stability-indicating. The method precisions were in the range of 0.41–1.21 while the instrument precisions were in the range of 0.38–0.95 in terms of peak area RSD% for all impurities, respectively. This method is considered stability-indicating and is applicable for accurate and simultaneous measuring of the ilaprazole and its related impurities in commercial enteric-coated tablets.     

Chiral stationary phases for the liquid chromatographic separation of pharmaceuticals

Biological systems exhibit remarkable enantioselectivity which is important in biosynthesis, metabolism, storage and transport processes. It is, therefore, not surprising that the chirality of, for example, pharmaceuticals, pesticides and agrochemicals, has become the focus of extensive research in many laboratories throughout the world. Chiral liquid chromatography is currently very popular for enantiomeric separations since the selectivities afforded by a wide range of commercially available chiral phases enable the separations of many classes of compounds to be routine. This paper discusses the most important types of chiral phases currently in use and reviews their applications to the analysis of compounds of pharmacological interest.     

正相高效液相色谱大粒径手性固定相拆分普萘洛尔对映体研究

目的:建立正相高效液相色谱拆分盐酸普萘洛尔对映异构体的方法。方法:采用自行合成的纤维素衍生物大粒径手性固定相,考察了拆分方法,以正己烷-异丙醇-三乙胺溶液(95∶5∶0.1%)为流动相,流速0.5mL·min-1,检测波长为276nm。结果:盐酸普萘洛尔两对映异构体的保留时间分别为8.67mim、14.36min,分离度良好;日内、日间精密度的RSD均小于3%。加样回收率在95.5%～101.4%之间,RSD为2.4%。结论:结果表明所建立的大粒径纤维素衍生物手性固定相高效液相色谱拆分盐酸普萘洛尔制剂中对映异构体的方法,简便、可靠、高效,可用于盐酸普萘洛尔制剂中对映异构体的测定。     

亲水作用色谱串联质谱法测定人血浆中的拉米夫定

目的:建立简便、快速的亲水作用色谱串联质谱法(HILIC-MS/MS)测定人血浆中的拉米夫定。方法:以13C1,15N2(拉米夫定为内标,血浆样品经乙腈沉淀蛋白后,采用Luna HILIC(100 mm×3.0 mm,3μm)柱分离。流动相为乙腈-5 mmol·L-1醋酸铵-甲酸(95∶5∶0.01,v/v/v),进样体积2μL,样品分析时间3 min。采用ESI源正离子模式、多反应监测(MRM),用于定量分析的离子反应分别为m/z230(112(拉米夫定)和m/z233(115(内标)。结果:测定人血浆中拉米夫定的线性范围为8.0～2000 ng·mL-1,定量下限为8.0 ng·mL-1,日内、日间精密度(RSD)均小于9.0%,准确度(RE)在-7.1%~2.7%之间。本法成功应用于健康受试者口服2种拉米夫定片后的生物等效性研究。结论:采用稳定同位素内标的HILIC-MS/MS法更为简便、快捷和准确,适用于人血浆样品中拉米夫定的测定。     

制备型高效液相色谱法在药物杂质研究中的应用

制备型高效液相色谱法是一种使用高压、大流量液体输送系统在高分辨率、大内径、高载量分离柱上进行样品高纯度分离的液相色谱制备方法。应用该方法分离的产品在纯度、回收率、分离效率等方面远远优于传统的制备方法,因此在药物研究、生产领域得到广泛应用。介绍制备型高效液相色谱法的研究概况,特点,影响制备型高效液相色谱分离纯化的因素,以及在药物杂质研究中的实际应用,并对制备型高效液相色谱技术存在的问题与发展方向进行总结。     

Developing and optimizing a validated isocratic reversed-phase high-performance liquid chromatography separation of nimodipine and impurities in tablets using experimental design methodology

In the present study an isocratic reversed-phase high-performance liquid chromatography was investigated for the separation of nimodipine and impurities (A, B and C) using statistical experimental design. Initially, a full factorial design was used in order to screen five independent factors: type of the organic modifier – methanol or acetonitrile – and concentration, column temperature, mobile phase flow rate and pH. Except pH, the rest examined factors were identified as significant, using ANOVA analysis. The optimum conditions of separation (optimum values of significant factors) determined with the aid of central composite design were: (1) mobile phase: acetonitrile/H₂O (67.5/32.5, v/v), (2) column temperature 40 °C and (3) mobile phase flow rate 0.9 ml/min. The proposed method showed good prediction ability (observed–predicted correlation). The analysis was found to be linear, specific, precise, sensitive and accurate. The method was also studied for robustness and intermediate precision using experimental design methodology. Three commercially available nimodipine tablets were analyzed showing good % recovery and %RSD. No traceable amounts of impurities were found in all products.     

A new hydrophilic interaction liquid chromatographic (HILIC) procedure for the simultaneous determination of pseudoephedrine hydrochloride (PSH), diphenhydramine hydrochloride (DPH) and dextromethorphan hydrobromide (DXH) in cough-cold formulations

A new HILIC method has been developed for the simultaneous determination of pseudoephedrine hydrochloride (PSH), diphenhydramine hydrochloride (DPH) and dextromethorphan hydrobromide (DXH) in cough-cold syrup. Mobile phase consists of methanol:water (containing 6.0 g of ammonium acetate and 10 mL of triethylamine per liter, pH adjusted to 5.2 with orthophosphoric acid), 95:5 (v/v). Column containing porous silica particles (Supelcosil LC-Si, 25 cm x 4.6 mm, 5 microm) is used as stationary phase. Detection is carried out using a variable wavelength UV-vis detector at 254 nm for PSH and DPH, and at 280 nm for DXH. Solutions are injected into the chromatograph under isocratic condition at constant flow rate of 1.2 mL/min. Linearity range and percent recoveries for PSH, DPH and DXH were 150-600, 62.5-250, 75-300 microg/mL and 100.7%, 100.1% and 100.8%, respectively. Method is stability indicating and excipients like saccharin sodium, sodium citrate, flavour and sodium benzoate did not interfere in the analysis. Compounds elute in order of increasing ionization degree caused by cation-exchange mechanism in a run time of less than 15 min. Mobile phase pH is manipulated to regulate ionization and ion-exchange interaction and thereby retention of compounds.     

Development of liquid chromatographic enantiomer separation methods and validation for the estimation of (R)-enantiomer in eslicarbazepine acetate

Chiral separation method development was carried out for eslicarbazepine acetate and its (R)-enantiomer on diverse chiral stationary phases. Better chiral selectivity was observed on cellulose tris-(3,5-dichlorophenylcarbamate) immobilized column (Chiralpak IC-3). Under polar organic mode (POM), with 100% acetonitrile as mobile phase and 0.5 ml/min flow, a resolution close to three was achieved. With normal phase (NP) mobile phase consisting dichloromethane:ethanol (90:10, v/v) and 1.0 ml/min flow, a resolution close to six was achieved. Detection was done by UV at 220 and 240 nm respectively. Both the methods were found to be robust and were validated with respect to robustness, precision, linearity, limit of detection, limit of quantification and accuracy. The proposed methods are suitable for the accurate estimation of (R)-enantiomer in bulk drug samples up to 0.1% when a 1 mg/ml analyte test solution is chromatographed.