Determination of memantine in plasma and vitreous humour by HPLC with precolumn derivatization and fluorescence detection

A new HPLC procedure with precolumn derivatization and rimantadine as the internal standard for determining memantine, a candidate agent for the treatment of glaucoma in plasma and vitreous humour, has been developed and validated. Precolumn derivatization was performed with 9-fluorenylmethyl-chloroformate-chloride (FMOC-Cl) as the derivatization reagent and followed by a liquid-liquid extraction with n-hexane. Optimal conditions for derivatization were an FMOC-Cl concentration of 1.5 mM, a reaction time of 20 min, the temperature at 30°C, the borate buffer pH 8.5, and a borate buffer-acetonitrile ratio of 1:1. The derivatives were analyzed by isocratic HPLC with the fluorescence detector λex 260 nm λem 315 nm on a Novapack C(18) reversed-phase column with a mobile phase of acetonitrile-water (73:27, v/v), 40°C, and a flow rate of 1.2 mL/min. The linear range was 10-1000 ng/mL with a quantification limit of ～ 10 ng/mL for both types of samples. This analytical method may be suitable for using in ocular availability studies.     

高效液相色谱法测定南瓜粉中的4-氨基丁酸

 采用强阳离子交换柱分离 ,pH梯度洗脱 ,邻苯二甲醛 (OPA)柱后衍生 ,荧光λex=338nm ,λem=42 5nm检测的高效液相色谱法测定了南瓜粉中的 4 氨基丁酸 (GABA)。若以GABA的峰高Y(μV)对进样质量X(μg)进行线性回归 ,则线性方程为Y =45 6 6X +1396 ,r =0 9998;GABA的平均回收率 (n =3)为 99%。方法稳定、快速、灵敏、准确。     

Analysis of coptisine, berberine and palmatine in adulterated Chinese medicine by capillary electrophoresis-electrospray ion trap mass spectrometry

A rapid and simple reversed-phase liquid chromatographic method that did not require the derivatization of 4-amino-3-hydroxybutyric acid (GABOB) was developed and validated. The method proved to be suitable for the determination of GABOB concentrations in finished pharmaceutical product (tablets). The method was developed using a RP-18 column, UV detection at 210 nm and 0.01 M sodium heptasulphonate solution, at pH 2.4, as the mobile phase. Different validation parameters were included and satisfactorily evaluated. The specificity of the method was demonstrated. Linearity was established in the range 0.40–0.60 mg/ml (r=0.997). The method showed excellent accuracy (100.1%). Precision (repeatability) gave a relative standard deviation value of 0.68%, while the intermediate precision was 1.70%. A robustness test showing the influence of different pH values and counter-ion concentrations was also performed.     

Development and Validation of a Liquid Chromatographic Method Useful for the Determination of Amino Acids in New and Commercial Alimentary Supplements

In this study, a new liquid chromatographic method with pre-column derivatization was developed and validated for the simultaneous quantification of acetylcarnitine taurinate, asparagine, potassium aspartate, asparagine and carnosine in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde in placebo solutions (DPD). Experimental parameters affecting the derivatization and chromatographic separation were investigated. The reaction was carried out at room temperature for 10 min. The adducts were separated on a Synergi Hydro-RP 80 Å column using a mobile phase consisting of 11 mM aqueous tetrapropylammonium bromide (pH 3.5)/methanol by gradient elution conditions at a flow rate of 0.8 mL/min. UV absorbance detection was set at λ = 320 nm. The validation parameters such as linearity, sensitivity, accuracy, precision, specificity and ruggedness were highly satisfactory. Linear responses were obtained by placebo solutions (determination coefficient ≤ 0.9994). Intra-day precision (RSD) was ≤1.06 % for corrected peak area and ≤1.14 % for retention times (t _R) without significant differences between intra- and inter-day data. Recovery studies showed good results for all the examined compounds (from 97.72 to 101.5 %) with RSD ≤1.30 %.     

柱前手性衍生化反相高效液相色谱法分离拉贝洛尔对映体

以乙酰葡萄糖异硫氰酸酯（GITC）作柱前手性衍生化试剂,用反相高效液相色谱法成功地分离了拉贝洛尔的两对对映异构体,并以荧光检测和紫外检测作对照,确认了4个衍生物的色谱峰。     

Simple Determination of 1-Deoxynojirimycin in a New Dietary Supplement by Liquid Chromatography

A simple and fast chromatographic method using ultraviolet diode-array detector (UV-DAD) was developed for the high performance liquid chromatography determination of the content of 1-deoxynojirimycin (DNJ) in a new dietary supplement in the form of granules for oral solution preparation. The derivatization reaction was carried out at room temperature for 15 min at pH 7. The reaction reached completeness at a reagent to analyte molar ratio of about 60. The chromatographic separations were performed on a C18 Phenomenex Synergi Fusion stainless steel column (250 mm?×?4.6 mm; 4 µm) with detection at λ?=?254 nm. The mobile phase consisted of triethylammonium (TEA) phosphate buffer (0.05 M; pH 3) and acetonitrile under gradient conditions at a flow-rate ramping from 1 to 1.2 mL/min. The validation parameters (linearity, sensitivity, accuracy, precision, specificity and stability) were satisfactory. Intra-day precision (relative standard deviation, RSD) was ≤?2.23% for peak area and retention time without significant differences between intra- and inter-day data. Recovery studies gave good results (93.59%; n?=?15) with a RSD of 2.64%. The developed method is suitable for the quality control of DNJ in raw material and industrial products. The method can be applied in any analytical laboratory and does not require sophisticated instrumentation.     

Development and validation of a stability-indicating column high-performance liquid chromatographic assay method for determination of nebivolol in tablet formulation

A simple, precise, and accurate isocratic reversed-phase (RP) stability-indicating column high-performance liquid chromatographic (HPLC) assay method was developed and validated for determination of nebivolol in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on a Phenomenex Luna C8 (2) column (250 mm x 4.6 mm id, 5 microm particle size) using mobile phase composed of acetonitrile-pH 3.5 phosphate buffer (35 + 65, v/v) at a flow rate of 1.0 mL/min, and detection was performed at 280 nm using a photodiode array detector. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The method was linear in the drug concentration range of 40-160 microg/mL with a correlation coefficient of 0.9999. The repeatability relative standard deviation (RSD) for 6 samples was 0.69%, and the intermediate precision (RSD) for 6 samples was 1.39%. The accuracy (recovery) was between 98.57 and 99.55%. Degradation products produced as a result of stress studies did not interfere with detection of nebivolol, and the assay can thus be considered stability-indicating.     

Reversed phase-HPLC for rapid determination of polyphenols in flowers of rose species

A rapid, simple, sensitive, robust, and improved HPLC method was developed and validated for determination of 10 polyphenols, namely gallic acid, catechin, epicatechin, rutin, m-coumaric acid, quercitrin, myricetin, quercetin, apigenin, and kaempferol in fresh flowers of Rosa bourboniana and R. brunonii and in both fresh flowers and marc (left after industrial distillation of rose oil) of R. damascena. Six polyphenols, gallic acid, rutin, quercitrin, myricetin, quercetin, and kaempferol, were detected and quantified in all extracts. The chromatographic separation of 10 polyphenols was achieved in less than 16 min by RP-HPLC (Phenomenex, Luna C18 (2) column, 5 microm, 250 mm x 4.6 mm) using linear gradient elution of water and acetonitrile (0.02% trifluroacetic acid) with a flow rate of 1 mL/min at lambda 280 nm. Standard calibration curves were linear in the range of 0.39-500 microg/mL. Good results were achieved with respect to repeatability (RSD <3%) and recovery (98.6-100.8%). The method was validated for linearity, accuracy, repeatability, LOD, and LOQ.     

药用植物铃兰中吖丁啶-2-羧酸的测定

使用强阳离子交换柱分离、pH梯度洗脱、柱后衍生、荧光λex＝338nm和λem＝425nm检测的高效液相色谱法,成功地分析了药用植物铃兰中的吖丁啶－2－羧酸,方法回收率为96．4％。     

荧光衍生中性糖的高效液相色谱分析

在HypersilODS2色谱柱上,利用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-乙基肼基甲酸酯(BCEC)作柱前衍生化试剂,采用梯度洗脱对5种中性糖荧光衍生物进行了优化分离.65℃下在乙腈溶剂中以冰乙酸作催化剂,衍生反应6.5h后获得稳定的荧光产物,衍生反应完全.激发和发射波长分别为λex=333nm,λem=390nm.线性回归系数均在0.999以上,检测限为24.3～62.1fmol.     

High-performance liquid chromatographic assay of fleroxacin in human serum using fluorescence detection

A HPLC method has been developed for the direct assay of fleroxacin in serum, without previous extraction. Serum samples, after the addition of sodium dodecylsulfate (0.5%), were injected directly into an LC Hisep column. The mobile phase consisted of acetonitrile, water and triethylamine in a per cent volume ratio 18:80:2. The pH of the mobile phase was adjusted to 6.50 with the addition of phosphoric acid. The drug was detected fluorometrically at lambda (ex )=280 nm and lambda (em )=450 nm . The linear concentration range of fleroxacin was between 0.01 and 2.0mg/l with a detection limit of 1ng/ml.     

Determination of primary biogenic amines in various food products using isocratic HPLC with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde as a new precolumn fluorogenic reagent

A simple HPLC approach has been successfully established for the sensitive determination of six biogenic amines (BAs) in food samples. The method involves derivatization with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde newly synthesized as a new fluorogenic reagent followed by LC isocratic elution mode. The optimization of both derivatization and separation conditions is carefully studied. Related analyses of the eluted compounds, in the presence of MeOH/THF/H(2)O (78:2.5:19.5 v/v/v) as a mobile phase containing 8 mM, pH 6.0 phosphate buffer solution, have been carried out on a C(18) column. The LOD (S/N = 3) of 2.5 nM, RSD value from 1.0 to 5.1% in peak areas, and good response linearity (R(2) >0.9936) are provided with fluorescence detection at lambda(ex)/lambda(em) = 480/545 nm. Obviously, recovery ranging from 95 to 107% in this method demonstrates its accuracy for determination of histamine, tyramine, 2-phenylethylamine, putrescine, cadaverine, and spermidine in the storage fish sample. Thus, the present method could be developed to monitor BAs in fish, cucumber, and spinach samples.     

Development and validation of a high-performance liquid chromatographic method for determination of eprosartan in bulk drug and tablets

A simple, precise, and accurate isocratic RP-HPLC method was developed and validated for determination of eprosartan in bulk drug and tablets. Isocratic RP-HPLC separation was achieved on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase 0.5% formic acid-methanol-acetonitrile (80 + 25 + 20, v/v/v, pH 2.80) at a flow rate of 1.0 mL/min. The retention time of eprosartan was 7.64 +/- 0.05 min. The detection was performed at 232 nm. The method was validated for linearity, precision, accuracy, robustness, solution stability, and specificity. The method was linear in the concentration range of 10-400 microg/mL with a correlation coefficient of 0.9999. The repeatability for six samples was 0.253% RSD; the intraday and interday precision were 0.21-0.57 and 0.33-0.71% RSD, respectively. The accuracy (recovery) was found to be in the range of 99.86-100.92%. The drug was subjected to the stress conditions hydrolysis, oxidation, photolysis, and heat. Degradation products produced as a result of the stress conditions did not interfere with detection of eprosartan; therefore, the proposed method can be considered stability-indicating.     

Development and Validation of a Liquid Chromatographic Method Useful for the Determination of Amino Acids in New and Commercial Alimentary Supplements

In this study, a new liquid chromatographic method with pre-column derivatization was developed and validated for the simultaneous quantification of acetylcarnitine taurinate, asparagine, potassium aspartate, asparagine and carnosine in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde in placebo solutions (DPD). Experimental parameters affecting the derivatization and chromatographic separation were investigated. The reaction was carried out at room temperature for 10 min. The adducts were separated on a Synergi Hydro-RP 80 Å column using a mobile phase consisting of 11 mM aqueous tetrapropylammonium bromide (pH 3.5)/methanol by gradient elution conditions at a flow rate of 0.8 mL/min. UV absorbance detection was set at λ = 320 nm. The validation parameters such as linearity, sensitivity, accuracy, precision, specificity and ruggedness were highly satisfactory. Linear responses were obtained by placebo solutions (determination coefficient ≤ 0.9994). Intra-day precision (RSD) was ≤1.06 % for corrected peak area and ≤1.14 % for retention times (t _R) without significant differences between intra- and inter-day data. Recovery studies showed good results for all the examined compounds (from 97.72 to 101.5 %) with RSD ≤1.30 %.

     

Development and Validation of a Micellar Liquid Chromatographic Method with UV Detection for Determination of Azithromycin in Tablets and Capsules

A rapid and simple micellar liquid chromatographic method that does not require use of specific chromatographic columns has been developed and validated for azithromycin determination. The method uses a Hypersil C₁₈ column at 60 °C, 1-butanol–pH 6.86 phosphate buffer solution–water, 15:25:60 (v/v), containing 0.10 M sodium dodecyl sulfate, as mobile phase, and UV-detection at 215 nm. Different characteristics of the method were validated satisfactorily. The specificity, accuracy, linearity, precision (repeatability), and robustness of the method were demonstrated. The method proved suitable for determination of the azithromycin content of capsules and uncoated tablets.Revised: 5 February and 11 March 2004     

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).     

脂肪胺荧光衍生物的高效液相色谱分离及质谱鉴定

采用新型荧光衍生试剂2-(9-吖啶酮)-乙酸(AAA)进行柱前衍生并经荧光检测对脂肪胺进行了高效液相色谱(HPLC)分离和在线质谱定性.衍生物荧光激发和发射波长为λex=404nm,λem=440nm.30℃下在乙腈溶剂中用N-乙基-N′-[(3-二甲氨基)丙基]碳二亚胺盐酸盐(EDC)做催化剂,衍生反应20min后获得稳定的荧光产物.在HypersilBDSC18(4.6mm×100mm,5μm)色谱柱上,采用梯度洗脱对12种脂肪胺衍生物进行了优化分离.采用大气压化学电离源(APCISource)正离子模式进行在线柱后质谱定性,实现了各种脂肪胺衍生物的快速、准确测定.该方法具有良好的重现性,多数脂肪胺的线性回归系数大于0.9996,检测限为12.09~25.52fmol.     

RP-HPLC拆分普萘洛尔对映体的洗脱顺序及其荧光响应研究

应用反相高效液相色谱法,以（Ｒ,Ｒ）－Ｏ,Ｏ－二乙酰基酒石酸酐（ＤＡＴＡＡＮ）为手性衍生化试剂,拆分了普萘洛尔（ｐｒｏｐｒａｎｏｌｏｌ）对映体。实验结果显示：普萘洛尔对映体经衍生化而生成的两个非对映异构体分子的空间结构有着明显的差异,即（Ｒ,Ｒ,Ｒ）－异构体中存在着分子内氢键,而（Ｒ,Ｒ,Ｓ）－异构体中却没有,这种结构上的区别不仅成为控制洗脱顺序的主要因素,而且对其荧光响应也产生重要的影响。     

Rapid separation and determination of process-related substances of paracetamol using reversed-phase HPLC with photo diode array as a detector.

A simple and rapid gradient reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of paracetamol and its related compounds in bulk drugs and pharmaceutical formulations has been developed. As many as nine process impurities and one degradation product of paracetamol have been separated on a Symmetry C18 column (4.6 x 250 mm i.d., particle size 5 microm) with gradient elution using 0.01 M potassium dihydrogen phosphate buffer (pH 3.0) and acetonitrile as mobile phase and photo diode array detection at 215 nm. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH values. The correlation coefficients for calibration curves for paracetamol as well as impurities were in the range of 0.9951 - 0.9994. The proposed RP-LC method was successfully applied to the analysis of commercial formulations; the recoveries of paracetamol were in the range of 99-101%. The method could be of use not only for rapid and routine evaluation of the quality of paracetamol in bulk drug manufacturing units but also for detection of its impurities in pharmaceutical formulations.     

High-performance liquid chromatographic determination of tramadol and its O-desmethylated metabolite in blood plasma. Application to a bioequivalence study in humans

Simultaneous HPLC determination of the analgetic agent tramadol, its major pharmacodynamically active metabolite (O-desmethyltramadol) in human plasma is described. Simple methods for the preparation of the standard of the above-mentioned tramadol metabolite and N1,N1-dimethylsulfanilamide (used as the internal standard) are also presented. The analytical procedure involved a simple liquid-liquid extraction of the analytes from the plasma under the conditions described previously. HPLC analysis was performed on a 250x4 mm chromatographic column with LiChrospher 60 RP-selectB 5-microm (Merck) and consists of an analytical period where the mobile phase acetonitrile-0.01 M phosphate buffer, pH 2.8 (3:7, v/v) was used, and of a subsequent wash-out period where the plasmatic ballast compounds were eluted from the column using acetonitrile-ultra-high-quality water (8:2, v/v). The whole analysis, including the equilibration preceding the initial analytical conditions lasted 19 min. Fluorescence detection (lambda(ex) 202 nm/lambda(em) 296 nm for tramadol and its metabolite, lambda(ex) 264 nm/lambda(em) 344 nm for N1,N1-dimethylsulfanilamide) was used. The validated analytical method was applied to pharmacokinetic studies of tramadol in human volunteers.