Modulation of mobile phase composition in flow-injection/sequential-injection chromatography exploiting multisyringe flow analysis

In this paper, a time-based multicommutated flow system is proposed for appropriate selection and modulation of mobile phase composition in flow-injection (FI)/sequential-injection (SI) chromatography. The novel flow assembly involves the on-line coupling of a short monolithic reversed-phase chromatographic column with a multisyringe flow injection set-up furnished with a set of solenoid valves. The proposed hyphenated technique was applied to the simultaneous spectrophotometric determination of thiamine (B₁), pyridoxine (B₆) and cyanocobalamin (B₁₂) which were taken as model analytes. The separation method capitalizes on a dual isocratic elution protocol involving the use of a single forward stroke of the multisyringe pump for initial delivery of 50 mmol L⁻¹ ammonium acetate (pH 7.0) for 2.4 min followed by 50 mmol L⁻¹ ammonium acetate–methanol (80:20, v/v) for 6.4 min at 0.5 mL min⁻¹ and room temperature. Detection was performed at the maximum wavelength for each target vitamin—280 nm for B₁, 325 nm for B₆, and 360 nm for B₁₂. A first-order, two-level full-factorial design was utilized to ascertain the significant variables influencing the chromatographic separation and the magnitude of the interaction effects. The experimental design method revealed that resolution of the target vitamins is highly dependent on the pH, percentage of organic modifier, and their second-order interaction. The multisyringe flow-injection-based monolithic column separation method, which should be viewed as an expeditious and cost-effective alternative to the high-performance liquid chromatography counterpart, was applied to the separation and determination of B₁, B₆, and B₁₂ in different pharmaceutical dosage forms in less than 9 min. Statistical comparison of the results from the proposed procedure with those from the HPLC method endorsed by the US Pharmacopeia revealed there were no significant differences at the 95 % confidence level.     

Application of chemometric approach for development and validation of high performance liquid chromatography method for estimation of ropinirole hydrochloride

A systematic Quality by Design approach was employed for developing an isocratic reversed‐phase liquid chromatographic technique for the estimation of ropinirole hydrochloride in bulk drug and pharmaceutical formulations. LiChrospher RP 18‐5 Endcapped column (25 cm × 4.6 mm id) at ambient temperature (25 ± 2°C) was used for the chromatographic separation of the drug. The screening of factors influencing chromatographic separation of the active pharmaceutical ingredient was performed employing fractional factorial design to identify the influential factors. Optimization of the selected factors was carried out using central composite design for selecting the optimum chomatographic conditions. The mobile phase employed was constituted of Solvent A/Solvent B (65:35 v/v) (Solvent A [methanol/0.05 M ammonium acetate buffer, pH 7, 80:20 v/v] and Solvent B [high performance liquid chromatography grade water]) and used at 0.6 mL/min flow rate, while UV detection was performed at 250 nm. Linearity was achieved in the drug concentration range 5–100 µg/mL (R² = 0.9998) with limits of detection and quantification of 1.02 and 3.09 µg/mL, respectively. Method validation was performed as per ICH guidelines followed by forced degradation studies, which indicated good specificity of the developed method for detecting ropinirole hydrochloride and its possible degradation products in the bulk drug and pharmaceutical formulations.     

Chromatographic analysis of some drugs employed in erectile dysfunction therapy: Qualitative and quantitative studies using calixarene stationary phase

In this study, the effect of change in chromatographic process variables on the retention behavior of four drugs employed in erectile dysfunction therapy on a calixarene stationary phase is described. Three of these drugs are known to treat erectile dysfunction, namely, sildenafil citrate, tadalafil, and apomorphine hydrochloride, and one drug that is used as opioid analgesic, tramadol hydrochloride, which is quiet widely misused to treat premature ejaculation. The results indicate the importance of considering the structure and pK_a values of drugs to be separated along with mobile phase composition. A new optimized, rapid, and accurate liquid chromatography method is also established for simultaneous determination of sildenafil citrate, tadalafil, and apomorphine hydrochloride in pharmaceutical preparations and bulk powders. The chromatographic separation of the three pharmaceuticals was achieved on a calixarene column in less than 10 min using a binary mobile phase of 35% acetonitrile and 65% 50 mM sodium perchlorate pH2.5 at 1 mL/min flow rate. The method was validated for system efficiency, linearity, accuracy, precision, limits of detection and quantitation, specificity, stability, and robustness. Statistical analysis proved that the method enabled reproducible and selective quantification of all three analytes in bulk drugs and in pharmaceutical preparations.     

Sequential injection chromatographic determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations

A new separation method based on a novel reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations in this contribution.The coupling of short monolith with SIA system results in an implementation of separation step to until no-separation low-pressure method.A Chromolith^® Flash RP-18e, 25-4.6 mm column (Merck, Germany) and a FIAlab^® 3000 system (USA) with a six-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-water (20:90, v/v), pH 2.5 adjusted with 98% phosphoric acid, flow rate 0.48 ml min⁻¹, UV detection was at 213 nm.The validation parameters have shown good results: linearity of determination for both compounds including internal standard (ethylparaben) >0.999; repeatability of determination (R.S.D.) in the range 0.5-5.4% at three different concentration levels, detection limits in the range 0.5-2.0 μg ml⁻¹, and recovery from the pharmaceutical preparation in the range 99.3-99.9%. The chromatographic resolution between peak compounds was >5.0 and analysis time was <9 min under the optimal conditions. The method was found to be applicable for routine analysis of the active compounds ambroxol hydrochloride and doxycycline in various pharmaceutical preparations.     

Separation of structurally related primary aliphatic amines using hydrophilic interaction chromatography with fluorescence detection after postcolumn derivatization with o‐phthaldialdehyde/mercaptoethanol

The retention behavior of primary aliphatic amines (homologous series of aliphatic alkyl amines and cycloalkyl amines) and positional isomers of alkylamines in the hydrophilic interaction chromatography mode was studied. The study was carried out on a TSKgel Amide‐80 column followed by postcolumn derivatization with fluorescence detection to describe the retention mechanism of tested compounds. The effect of chromatographic conditions including column temperature, acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.8), and salt concentration in the mobile phase was investigated. The final mobile phase consisted of acetonitrile and solution of 20 mM potassium formate pH 3.5 in ratio 80:20 v/v. The analyses were carried out at mobile phase flow rate of 1.0 mL/min and the column temperature of 20°C. The developed method was fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy, and precision according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The proposed new methods were proved to be highly sensitive, simple, and rapid, and were successfully applied to the determinations of isopropylamine, cyclohexylamine, and cyclopropylamine in relevant active pharmaceutical ingredients.     

Dual separation mode for simultaneous determination of antihypertensive drug combinations by high-performance liquid chromatography

A simple, reproducible and efficient dual separation mode high performance liquid chromatographic (HPLC) method was developed for simultaneous determination of antihypertensive drug combinations including; hydrochlorothiazide (HCTZ), valsartan (VAL), amiloride (AML) and captopril (CAP). The newly developed Platinum™ column, which provides a dual-mode separation with its polar and non-polar sites, was used for rapid separation of these co-administered drugs. Good resolution was obtained when Platinum™ column was used compared with C₁₈ column. Additionally, simple isocratic mode with mobile phase containing methanol and 0.02 mole L⁻¹ phosphate buffer adjusted to pH 3.0 (45:55, v/v) was used for separation. The flow rate was 0.5 mL min⁻¹ and effluent was monitored at 270 nm. All the investigated drugs were completely separated within less than 6 min. The linearity range obtained for the developed HPLC method was 0.5-100 μg mL⁻¹ with detection limits of 0.13-1.2 μg mL⁻¹ for all the studied drugs. The method was validated in accordance with the requirements of ICH guidelines and shown to be suitable for intended applications. The method was successfully used for determination of the studied drugs in pure form and pharmaceutical dosage forms without prior need for separation. The method is valuable for quality control laboratories for simultaneous determination of these co-administered antihypertensive drugs in binary, ternary and quaternary mixtures.     

Direct separation of the enantiomers of ramosetron on a chlorinated cellulose‐based chiral stationary phase in hydrophilic interaction liquid chromatography mode

Ramosetron is an enantiopure active pharmaceutical ingredient marketed in Japan since 1996 and later in a few Southeast Asian countries predominantly as an antiemetic for patients receiving chemotherapy. In this study, a simple and rapid high‐performance liquid chromoatography method for the separation of ramosetron and its related enantiomeric impurity by using hydrophilic interaction liquid chromatography mode is presented. Chiral resolution was performed on an analytical column (100 mm × 4.6 mm id) packed with 3 μm particles of cellulose‐based Chiralpak IC‐3 chiral stationary phase. Using a mobile phase containing acetonitrile–water–diethylamine (100:10:0.1, v/v/v) and setting the column temperature at 35°C, the resolution value was 7.35. At a flow rate of 1 mL/min, the enantioseparation was completed within 5 min. The proposed method was partially validated and it has proven to be sensitive with limit of detection and limit of quantitation of the (S)‐enantiomer impurity of 44.5 and 133.6 ng/mL.     

Fast HPLC for quality control of Harpagophytum procumbens by using a monolithic silica column: method transfer from conventional particle-based silica column

The applicability of a monolithic C18-bonded silica column for the rapid HPLC separation of ingredients in medicinal plants and their phytopharmaceutical preparations has been evaluated in the author's laboratory. In this presentation, an existing method for the determination of the iridoid glycoside harpagoside in Harpagophytum procumbens (Devil's Claw) was successfully transferred from a conventional particle-based C18 silica column to a monolithic silica column. The very high porosity of the stationary phase allows chromatography with a much lower backpressure than on conventional columns. Therefore, the flow rate could be easily increased from 0.8 mL/min (particle-based column) to 5 mL/min (monolithic column) and the run-time reduced from 30 to 5 min (that is a reduction about 85% !), without losing any chromatographic resolution of the compound of interest. The amount of harpagoside was measured with the original method on a conventional particle-based silica column and on the adapted method on a monolithic silica column. The statistical mean t-test showed no significant differences of the variances and the means indicating that the fast HPLC method is an acceptable alternative. The shorter analysis time makes the method very valuable for commercial quality control of Harpagophytum extracts and its pharmaceutical preparations.     

High‐performance liquid chromatographic column packings with different particle sizes: Chromatographic behavior for the quality analysis of HuanglianShangqing pill

The chromatographic separation of traditional Chinese medicines is still a highly challenging task in analytical science with respect to its hundreds and thousands of chemical compounds, while increase of separation efficiency can greatly improve the separation power of chromatographic column for traditional Chinese medicine. In this study, 13 bioactive components in HuanglianShangqing pill were selected as an index to optimize the separation conditions and evaluate the system suitability of three commercially available columns packed with 1.8, 3.5, and 5.0 μm particles. The chromatographic separations were obtained by the most appropriate Eclipse Plus C₁₈ column (100 × 2.1 mm, 3.5 μm) within 45 min using gradient elution with aqueous‐ammonium acetate (10 mmol/L, pH 5.0) and acetonitrile, at a flow rate of 0.3 mL/min and an operating temperature of 30°C. The quality of HuanglianShangqing pill was assessed through combining simultaneous quantification of 13 compounds with fingerprint analysis. For the qualitative analysis, mass spectrometry was used to confirm the 13 compounds. All the validation data conformed to the acceptable requirements. For the fingerprint analysis, 32 peaks were selected as the common peaks at 254 nm to evaluate the similarities among HuanglianShangqing pills obtained from ten manufacturers.     

Development and validation of a generic high‐performance liquid chromatography for the simultaneous separation and determination of six cough ingredients: Robustness study on core–shell particles

A generally applicable high‐performance liquid chromatographic method for the qualitative and quantitative determination of pharmaceutical preparations containing phenylephrine hydrochloride, paracetamol, ephedrine hydrochloride, guaifenesin, doxylamine succinate, and dextromethorphan hydrobromide is developed. Optimization of chromatographic conditions was performed for the gradient elution using different buffer pH values, flow rates and two C₁₈ stationary phases. The method was developed using a Kinetex® C₁₈ column as a core–shell stationary phase with a gradient profile using buffer pH 5.0 and acetonitrile at 2.0 mL/min flow rate. Detection was carried out at 220 nm and linear calibrations were obtained for all components within the studied ranges. The method was fully validated in agreement with ICH guidelines. The proposed method is specific, accurate and precise (RSD% < 3%). Limits of detection are lower than 2.0 μg/mL. Qualitative and quantitative responses were evaluated using experimental design to assist the method robustness. The method was proved to be highly robust against 10% change in buffer pH and flow rate (RSD% < 10%), however, the flow rate may significantly influence the quantitative responses of phenylephrine, paracetamol, and doxylamine (RSD% > 10%). Satisfactory results were obtained for commercial combinations analyses. Statistical comparison between the proposed chromatographic and official methods revealed no significant difference.     

A rapid and simple ultra high performance liquid chromatography method for the simultaneous determination of methoxyphenol derivatives involved in the eugenol catabolic pathway

An efficient ultra high performance liquid chromatography method of separation was developed for the analysis of six important methoxyphenol derivatives involved in the eugenol catabolic pathway. In the present study, an Acquity UPLC BEH C₁₈ column was used for the chromatographic separation of the industrially important phenolic compounds such as vanillin, vanillic acid, ferulic acid, coniferyl alcohol, and coniferyl aldehyde obtained during microbial transformation of eugenol. Eluted components were identified using the dual wavelength (254 and 310 nm) UV detector. A gradient method of elution using mobile phase of aqueous 1 mM trifluoroacetic acid (Solvent A) and methanol (Solvent B) at a flow rate of 0.3 mL/min separated all the five intermediate methoxyphenol derivatives along with their precursor eugenol within 15 min with stable baseline resolution. Method validation was performed for the accurate quantification of vanillin, coniferyl aldehyde, and eugenol using the parameters of linearity, specificity, precision, limit of detection, limit of quantification, and robustness. The developed method would be helpful for clear separation and identification of the five most important intermediate metabolites of the eugenol catabolism pathway.     

Establishing the chromatographic fingerprint of traditional Chinese medicine standard decoction based on quality by design approach: A case study of Licorice

A novel analytical quality by design approach for developing a chromatographic fingerprint was established for analyzing complex traditional Chinese medicine, using a licorice standard decoction as an example. Considering the characteristics of integrity and ambiguity, the resolution of eight common peaks, total peak number, capacity factor distributions, and peak purity were selected as potential critical method attributes for assessing the quality of the chromatographic fingerprint. A central composite design was used to evaluate the relationship between critical method attributes and critical method parameters, including column temperature, wavelength, flow rate, formic‐acid concentration, and gradient parameters. A standard probability method was employed to calculate the design space of the fingerprint analysis parameters and evaluate the robustness of the methodology. The optimized high‐performance liquid chromatography fingerprint conditions were acetonitrile and 0.1% formic acid water gradient elution (0‐5 min, 5–19% A; 5–10 min, 19% A; 10–50 min, 19–42% A; 50–54 min, 42–100% A; 54–60 min, 100% A), column temperature 25±5°C, detection wavelength 265 nm. The design space of fingerprint analytical method based on the analytical quality by design approach not only met the requirements of the fingerprint analysis, but also improved the robustness and applicability of the fingerprint method.     

Fast analysis of isoflavones by high-performance liquid chromatography using a column packed with fused-core particles

The recent development of fused-core technology in HPLC columns is enabling faster and highly efficient separations. This technology was evaluated for the development of an fast analysis method for the most relevant soy isoflavones. A step-by-step strategy was used to optimize temperature (25-50 °C), flow rate (1.2-2.7 mL/min), mobile phase composition and equilibration time (1-5 min). Optimized conditions provided a method for the separation of all isoflavones in less than 5.8 min and total analysis time (sample-to-sample) of 11.5 min. Evaluation of chromatographic performance revealed excellent reproducibility, resolution, selectivity, peak symmetry and low limits of detection and quantification levels. The use of a fused-core column allows highly efficient, sensitive, accurate and reproducible determination of isoflavones with an outstanding sample throughout and resolution. The developed method was validated with different soy samples with a total isoflavone concentration ranging from 1941.53 to 2460.84 μg g⁻¹ with the predominant isoflavones being isoflavone glucosides and malonyl derivatives.     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Comparison between micellar liquid chromatography and capillary zone electrophoresis for the determination of hydrophobic basic drugs in pharmaceutical preparations

The determination of highly hydrophobic basic compounds by means of conventional reversed-phase liquid chromatographic methods has several drawbacks. Owing to the characteristics of micellar liquid chromatography (MLC) and capillary electrophoresis (CE), these techniques could be advantageous alternatives to reversed-phase chromatographic methods for the determination of these kinds of compounds. The objective of this study was to develop and compare MLC and CE methods for the determination of antipsychotic basic drugs (amitryptiline, haloperidol, perphenazine and thioridazine) in pharmaceutical preparations. The chromatographic determination of the analytes was performed on a Kromasil C(18) analytical column; the mobile phase was 0.04 m cetyltrimethylammonium bromide (CTAB), at pH 3, containing 5% 1-butanol, at a flow rate of 1 mL/min. The CE separation was performed in a fused-silica capillary with a 50 mm tris-(hydroxymethyl)-aminomethane buffer, pH 7, at an applied voltage of 20 kV, using barbital as internal stardard. The proposed methods are suitable for a reliable quantitation of these compounds in the commercial tablets and drops in terms of accuracy and precision and require a very simple pre-treatment of the samples. By comparing the performance characteristics and experimental details of the MLC and CE methods we conclude that CE seems to be slightly better than MLC in the determination of highly hydrophobic compounds in pharmaceuticals in terms of resolution and economy, taking into account that the limits of detection are not a handicap in pharmaceutical samples.     

Simultaneous estimation of four proton pump inhibitors—lansoprazole,omeprazole, pantoprazole and rabeprazole: development of a novel generic HPLC‐UV method and its application to clinical pharmacokinetic study

A highly selective, sensitive and accurate HPLC method has been developed and validated for the estimation of four proton‐pump inhibitors (PPI), lansoprazole (LPZ), omeprazole (OPZ), pantoprazole (PPZ) and rabeprazole (RPZ), with 500 µL human plasma using zonisamide as an internal standard (IS). The sample preparation involved simple liquid–liquid extraction of LPZ, OPZ, PPZ and RPZ and IS from human plasma with ethyl acetate. The baseline separation of all the peaks was achieved with 0.1% triethylamine (pH 6.0):acetonitrile (72:28, v/v) at a flow rate of 1 mL/min on a Zorbax C₈ column. The total chromatographic run time was 11.0 min and the simultaneous elution of IS, OPZ, RPZ, PPZ and LPZ occurred at approximately 2.42, 4.45, 5.02 and 9.37 min, respectively. The method was proved to be accurate and precise at linearity range of 20.61–1999.79 ng/mL with a correlation coefficient (r) of ≥0.999. The limit of quantitation for each of the PPI studied was 20.61 ng/mL. The intra‐ and inter‐day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous Determination of Rosiglitazone and Metformin in Pharmaceutical Preparations by LC

In the present study, a novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in pharmaceutical preparations. The separation was achieved on a phenyl column (250 × 4.6 mm i.d., 5 μm) using a mobile phase composed of acetonitrile:10.0 mM phosphate buffer pH 5.5 (70:30, v/v). The flow rate was 1 mL min⁻¹. UV detection was performed at 245 nm and verapamil was used as internal standard. The developed method was validated in terms of stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 0.02 μg mL⁻¹ for both drugs. The method developed was successfully applied to the simultaneous determination of rosiglitazone and metformin in pharmaceutical preparations. The results were compared to two methods reported in the literature and no significant difference was found statistically.     

Valuation of environmental influence of recently invented high-performance liquid chromatographic method for hypoglycemic mixtures of gliflozins and metformin in the presence of melamine impurities: Application of molecular modeling simulation approach

Molecular modeling is the science of representing molecular structures numerically and simulating their behavior with the equations of quantum and classical physics. Coupling molecular modeling and simulation with chromatographic resolution for pharmaceutical products constitutes a new technique in pharmaceutical analysis. An innovative high-performance liquid chromatographic (HPLC) methodology was developed for the quantification of metformin hydrochloride (MET), empagliflozin (EMP), and canagliflozin (CAN) in bulk, laboratory-developed combinations, pharmaceutical tablets, and in the presence of melamine. Chromatographic separation was accomplished using a Symmetry column with 0.03 M potassium dihydrogen phosphate buffer and 0.02 M heptane sulphonic acid: acetonitrile as the mobile phase. Molecular modeling using molecular operating environment software was applied to properly select the stationary phase suitable for the developed HPLC method. Additionally, molecular modeling estimates and validates binding between the studied analytes and the stationary phase to clarify and explain the chromatographic separation and elution order. In accordance with the International Conference of Harmonization recommendations, the method was validated in terms of linearity, accuracy, precision, and selectivity. The linearity ranges (μg/ml) were 200–1500 (MET), 2–15 (EMP), and 20–150 (CAN) and the limit of detection values were in the ranges of 0.17–54.58 μg/ml. Analysis of pharmaceutical tablets using the suggested approach yielded satisfactory outcomes. As a result, it might be used in quality control laboratories to analyze the aforementioned medications.     

一种从蛇毒中纯化神经生长因子的新工艺

为了能从中华眼镜蛇蛇毒中简单快速地分离纯化神经生长因子（一种治疗各种神经性损伤和神经退行疾病的药物,简称NGF）,采用不同的色谱柱联用的方式对NGF的纯化工艺进行了研究。结果表明,采用DEAE Sepharose F.F.和Sephadex G 50二步柱色谱工艺可以从蛇毒中快速分离得到神经生长因子。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳和反相高效液相色谱分析, 证明所得到的NGF为单一组分,相对分子质量约为 29 000。对8　d龄鸡胚背根神经节采用体外培养法,结果证明,所得NGF具有明显的促进神经纤维