Simultaneous Quantification of Neomycin and Bacitracin by LC-ELSD

The first simultaneous quantification of neomycin and bacitracin using liquid chromatography evaporative light scattering detection as an alternative to MS detection and pre-/post-column derivatisation, respectively, was the aim of this study. The developed method was validated for two strength of neomycin and one strength of bacitracin in sterile pharmaceutical formulation and is a fast and efficient tool for content uniformity tests in quality control. With this method the separation of neomycin from sulfate and the base line separation of the four major components of bacitracin (bacitracin A, B1, B2 and B3) was achieved. These four components are responsible for 96% of the microbiological activity. A Phenomenex Synergi POLAR analytical column (250 mm × 4.6 mm, 4 μm I.D.) in combination with 0.5% perfluoropropionic acid and acetonitrile in gradient mode, the peaks of interest could be separated with high efficiency within 14 min. The calibration was performed using a second order regression with an R ² = 0.9999 for neomycin (B and C) sulfate and R ² = 0.9996 for bacitracin A, B1, B2 and B3. The results of the accuracy evaluation were 99.2 and 99.7%, respectively, for neomycin and 100.8% for bacitracin. Injection precision results are 0.4–1.5 RSD% recorded for six injections. The established method has a high potential for routine high-throughput analyses in the pharmaceutical industry.

     

Simultaneous Quantification of Neomycin and Bacitracin by LC-ELSD

The first simultaneous quantification of neomycin and bacitracin using liquid chromatography evaporative light scattering detection as an alternative to MS detection and pre-/post-column derivatisation, respectively, was the aim of this study. The developed method was validated for two strength of neomycin and one strength of bacitracin in sterile pharmaceutical formulation and is a fast and efficient tool for content uniformity tests in quality control. With this method the separation of neomycin from sulfate and the base line separation of the four major components of bacitracin (bacitracin A, B1, B2 and B3) was achieved. These four components are responsible for 96% of the microbiological activity. A Phenomenex Synergi POLAR analytical column (250 mm × 4.6 mm, 4 μm I.D.) in combination with 0.5% perfluoropropionic acid and acetonitrile in gradient mode, the peaks of interest could be separated with high efficiency within 14 min. The calibration was performed using a second order regression with an R ² = 0.9999 for neomycin (B and C) sulfate and R ² = 0.9996 for bacitracin A, B1, B2 and B3. The results of the accuracy evaluation were 99.2 and 99.7%, respectively, for neomycin and 100.8% for bacitracin. Injection precision results are 0.4–1.5 RSD% recorded for six injections. The established method has a high potential for routine high-throughput analyses in the pharmaceutical industry.     

高效液相色谱法分离测定牦牛乳中的8种蛋白质

建立了同时分离和测定牦牛乳中4种酪蛋白和4种乳清蛋白的反相高效液相色谱方法。脱脂牦牛乳经分散剂处理后,采用C4色谱柱(250 mm×4.6 mm,300,5μm i.d.)进行分离,以0.1%的三氟乙酸水溶液和0.1%的三氟乙酸乙腈溶液为流动相,流速为0.8 mL/min,梯度洗脱,二极管阵列检测器(DAD)在220nm波长下检测,外标法定量。结果表明,牦牛乳中8种主要蛋白质在40 min内完全分离,在各自的线性范围内呈良好线性,除α-乳白蛋白外,其余7种蛋白的相关系数均大于0.99。8种蛋白质的回收率为86%～103%,相对标准偏差(RSDs)为1.7%～8.7%;检出限(LODs)为10.7～39.2 mg/L,定量下限(LOQs)为35.7～130.7 mg/L。该方法的准确度和精密度均较高,能够满足实际检测的要求。     

An Improved Narrow-Bore LC Method for Quantification of Alfuzosin in Pharmaceutical Formulations

A simple rapid and stability-indicating LC method using a narrow-bore column has been developed, fully validated, and applied to the quantification of alfuzosin in pharmaceutical formulations. Chromatography was achieved isocratically on a narrow-bore, 5-μm particle size, C₈ analytical column. The mobile phase was a 35:65 (v/v) 0.0125 m ammonium formate–acetonitrile at a flow rate of 0.35 mL min^?1. Detection was by UV absorption at 245 nm. Evaluation over the range 200–800 ng mL^?1 revealed linearity was good. Limits of detection and quantification for alfuzosin were 22.9 and 69.5 ng mL^?1, respectively. Intra-day and inter-day RSD were less than 6.4%, and the relative percentage error was less than ?1.7% (n = 5). Accelerated degradation performed under different stress conditions including oxidation, hydrolysis, and heat, proved the selectivity of the procedure. The method was successfully used for quality-control and content-uniformity testing of commercial tablets.     

Simultaneous Quantification of Two Major Classes of Constituents in Erigeron breviscapus and Its Extract Injection by RP-HPLC

The objective of this research was to establish a simple, practical and efficient method for routine quantitative analysis of Erigeron breviscapus and its extract injection to control their qualities. The reversed phase high performance liquid chromatographic method was adopted to determine simultaneously the contents of two major classes of constituents namely phenolic acids and flavonoids, which were usually ignored in previous studies of E. breviscapus. Under the optimum conditions, three flavonoids including scutellarin, scutellarein and apigenin and four phenolic acids including caffeic acid, chlorogenic acid, 3,4-O-dicaffeoylquinic acid and 3,5-O-dicaffeoylquinic acid were successfully separated on a Zorbax SB-C₁₈ column (250 × 4.6 mm I.D., 5.0 μm particle size) at 25 °C. Of the three flavonoids, scutellatin is a flavone glucuronide. The mobile phase was a mixture of acetonitrile and 1.0% (v/v) aqueous acetic acid employing gradient elution at a flow rate of 1.0 mL min^?1 and the detection wavelength was set at 330 nm. Regression equations revealed good linear relationship between the peak areas of the analytes and their concentrations (r ² > 0.9990). The relative standard deviations of retention time and peak area were less than 0.33 and 1.45%. The intra- and inter-day precisions as determined from sample solutions were below 1.66 and 2.35%. And the recoveries ranged from 96.5 to 101.8%. The proposed method has been successfully applied to the simultaneous quantification of two major classes of constituents in E. breviscapus and its extract injection for the first time.     

Two-Stage,One-Dimensional Thin Layer Chromatographic Method for Separation of Lipid Classes

Abstract

A thin-layer chromatographic (TLC) technique was developed for routine analysis of lipid classes of blood, milk, tissue and egg yolk. This procedure provided rapid and reproducible separations suitable for in situ quantitation by densitometry

The spotted TLC plate was subjected to two developments in one dimension to separate, in ascending order from the origin: phospholipids, monoglycerides, free fatty acids, cholesterol, 1,2-diglycerides, 1,3-diglycerides, triglycerides, and cholesterol esters. Development 1: chloroform: methanol: acetic acid (98:2:1) to 17.0 cm. Development 2: hexane: ethyl ether: acetic acid (94:6:0.2) to the top of the plate. After air drying, the plate was dipped into a solution of 3% cupric acetate in 8% phosphoric acid for 3 seconds, and heated at 130°C for 30 minutes to char the separated lipid classes. The chromatograms were scanned at 350 nm.

Overloading of TLC plates with sample using the spotter resulted in the appearance of distorted kidney-shaped spots. A mathematical model and general explanation for the shape of these distorted spots was described.     

A Validated LC Method for Separation and Quantification of Voriconazole and Its Enantiomer

A simple liquid chromatographic method was developed for the separation and quantification of voriconazole and its enantiomer in drug substance. The separation was achieved on Chiral cel-OD (250 mm × 4.6 mm × 10 μm) using mobile phase consisting of n-hexane and ethanol in the ratio 9:1 (v/v) with a flow rate of 1.0 mL min⁻¹, at 27 °C column temperature and detection at 254 nm with an injection volume of 20 μL. Ethanol was used as diluent. The method is capable of detecting the (2S, 3R) enantiomer down to 0.0075% and can quantify down to 0.021% with respect to sample concentration. The method is rapid and the resolution achieved was about 3.0. This method can be employed for the quantification of (2S, 3R) enantiomer in voriconazole drug substance.     

An Improved Method for Product Separation in Metathetical Reactions and its Demonstration for the Synthesis of Anhydrous Cesium Salts

In conventional metathetical reactions, product separation is based on solubility product differences, and the resulting products are often impure and require purification by recrystallization. A new approach to product separation is described that relies on the formation of an unstable, volatile by‐product, such as NH₄⁺CH₃O^—. This method provides very pure and anhydrous products in high yield and was demonstrated successfully for the syntheses of anhydrous cesium salts.     

An Improved Technique for Extraction,Identification, and Quantification of Leukotrienes

Abstract

An improved method for extraction, separation and quantification of leukotrienes is described. Critical steps include extraction in thoroughly cleaned Sep-Pak C₁₈ mini columns, elution with 90 percent methanol in water, addition of 0.25 percent Na₄EDTA to the solvent system of methanol/water 60/40, pH 7.4, injection of the samples in 1 ml of the same system after lowering the pH to 3.0 with glacial acetic acid and utilization of a Z-module containing a 5 μm C₁₈ reverse phase cartridge. Recovery of the leukotrienes was 94 pm 8% mean pm SD for LTC₄, 89 pm 5% for LTD₄, 89 pm 3% for LTB₄ and 84 pm 6% for LTE₄. We had no problems with precipitation of Na₄EDTA and occlusion of the HPLC fittings. The method is simple, reproducible and easily adaptable to any research laboratory.     

HPLC Method for Chiral Separation and Quantification of Antidepressant Citalopram and Its Precursor Citadiol

HPLC method enabling chiral separation and determination of citalopram (CIT), a widely used antidepressant, and its synthetic precursor citadiol in one analysis was developed and validated. Moreover, supercritical fluid chromatography was also tested and was proved to be less effective for this separation purpose. The optimized HPLC system was composed of Chiralcel OD-H column and n-hexane/propane-2-ol/triethylamine 96/4/0.1 (v/v/v) as mobile phase, column temperature 25 °C, flow rate 1.0 mL min^?1, UV detection at 250 nm. The effects of amount of propane-2-ol, triethylamine addition, and temperature on enantioselectivity and resolution of the enantiomers were evaluated. The method was found to be suitable for determination of the enantiomeric purity of CIT in bulk drugs. Enantiomers of CIT were determined in two commercially available pharmaceuticals.     

An Improved Method for the Synthesis of N-Phenoxyacetylribonucleosides

N-phenoxyacetylribonucleosides were prepared efficiently from the reaction of ribonucleosides with phenoxyacetylchloride and 1,2,4-triazole(for adenosine and cytidine) or 1-hydroxybenzotriazole (for guanosine).     

An Improved Method for the Preparation of o-Quinodimethanes

The use of zinc/silver couple for preparation of the o-quinodimethane from 3, 6-dimethyl-1, 2-bis (bromomethyl) benzene, 1, is described.     

An Improved Ion-Pair Reversed Phase LC Method for Analysis of Major Impurities of Phosphorothioate Oligonucleotide Cantide

An improved ion-pair reversed phase LC (IP-RP-LC) method was developed using triethylammonium/hexafluoroisopropanol (TEA/HFIP) buffer as the mobile phase as an alternative to capillary gel electrophoresis (CGE) and ion-exchange chromatography (IEC) for routine quality control of the synthetic 20-mer phosphorothioate oligonucleotide (PS-ODN) Cantide. The most commonly observed impurities included an oligomer with a deletion at the 5′-end (5′n-1) and a monophosphodiester (P=S/O). The method described here was sufficiently selective to simultaneously separate and identify these impurities. To the best of our knowledge, this is the first report on the separation of P=S/O analogs from PS-ODN using IP-RP-LC. Relatively high TEA concentrations and higher pH values were recommended. The separation performance was maintained even in the presence of multiple P=S/O fragments containing different PO residues. The method was successfully applied to the analysis of failure sequences of authentic crude Cantide as well as the purified product. In addition to 5′n-1 and P=S/O analogs, some minor impurities such as 3′n-1 oligomers were also partially resolved.     

正戊醛合成方法的改进

正戊醛合成方法的改进刘瑞蓝，南志祥，郭治安（西北大学化学系西安７１００６９）关键词正戊醛，一步氧化法，共沸蒸馏，精馏醛类化合物的使用相当广泛，尤其在香料工业中越来越受到人们重视，但由于易氧化与易聚合，在加热条件下合成难度大，产率低，我们在过去研究工作...     

Specific HPLC Method for the Separation of Verapamil and Four Major Metabolites After Oral Dosing

Abstract

We have developed a high performance liquid chromatographic (HPLC) method which resolves verapamil, norverapamil, D620, D617 and what we believe to be another verapamil metabolite which has been previously unreported. An alkyl-phenyl column is used with a mobile phase of 0.005% sulfuric acid in methanol. The extraction recoveries of verapamil, norverapamil and the internal standard (imipramine) from plasma ranged between 98% and 104%. The day-to-day, and within-day coefficients of variations for verapamil and norverapamil at plasma concentrations of 7.3 and 233 ng/ml ranged between 1.7 and 6.1%. The limit of sensitivity was slightly less than 1 ng for both verapamil and norverapamil. Chromatograms of extracts of serum and urine obtained from five normal subjects who took single oral verapamil doses, indicated the presence of verapamil, norverapamil, and two other known metabolites. Chromatograms of serum extracts also indicated an additional peak which is probably another verapamil metabolite.     

喹烯酮合成工艺的改进

以邻硝基苯胺为起始原料,经过氧化、Beirut反应、缩合反应3步合成产品喹烯酮,总收率73.4%.产品经HPLC分析,质量分数为98.5%.并通过m.P.、IR、1H NMR和13C NMR确证其结构.     

三氯苯达唑中间体合成方法的改进

以2,3-二氯苯酚与3,4-二氯-6-硝基苯胺为原料,在催化剂的存在下进行成醚反应,制备三氯苯达唑中间体。探讨和优化了合成条件,并采用液相色谱、红外光谱分析等手段确认了产物的结构。以经过改进后的中间体进行合成三氯苯达唑,最终产率达到了36.7%。     

An Improved Method for the Preparation of 4,5-Diaminochrysazin

4,5-Diaminochrysazin was prepared from chrysazin by ethylation and nitration followed by reaction with hydroiodic acid (HI). Treatment of 1,8-diethoxy-4,5-dinitroanthraquinone with HI resulted not only in O-deethylation, but also in reduction of nitro groups with more than 95% yield. This is a unique finding and is reported for the first time with a definite improvement over literature methods and is suitable for scale-up.     

Improved High Performance Liquid Chromatographic Method for the Quantification of 3-Methylhistidine in Serum and Urine

Abstract

We optimized an high-performance liquid chromatographic (HPLC) method for the determination of 3-methylhistidine (3-MeHis) in biological fluids. After pre-column derivatization with OPA, analytical separation was achieved on a reversed-phase C18 column by a simple gradient between sodium propionate buffer and acetonitrile. the method is accurate, reproducible and sensitive, and allows the determination of urinary 3-MeHis levels in about 55 min. Other additional 16 amino acids may be easily quantified while the 3-MeHis peak is well resolved from an unknown urinary compound potentially interfering.     

An Improved Method for the Purification of Hepatic Proliferation Inhibitor by Anion-Exchange HPLC

Abstract

An improved method for the purification of hepatic proliferation inhibitor from rat liver by means of anion-exchange HPLC has been developed. The inhibitor can be purified on an anion exchange HPLC column by using a linear sodium phosphate gradient. The HPLC method allows repeated use of one column and is both rapid and reproducible. The hepatic proliferation inhibitor isolated by this method retains all of its biological activity and is homogeneous as revealed by reverse-phase HPLC.