Determination of fat-soluble vitamins in a pharmaceutical dosage form by solid-phase extraction and reversed-phase liquid chromatography

A rapid and precise method for the determination of fat-soluble vitamins from a water-based multivitamin mixture was developed utilizing solid-phase extraction and reversed-phase high-performance liquid chromatography (HPLC). Cholecalciferol, alpha-tocopherol acetate, and retinol palmitate were extracted in a single stage from the matrix using Bond Elut C18 columns. The vitamins were then chromatographed on a Hypersil 5-microns C18 column, using a water:methanol gradient, and quantified simultaneously using individual UV absorption maxima. The recovery of added analytes varied from 92.6 to 100.6%. The assay coefficient of variation was 1.7-2.7%. The sample preparation method and HPLC assay described here are practical for pharmaceutical quality control purposes.     

RP-HPLC法测定盐酸尼非卡兰粉针的含量

目的 建立反相高效色谱法测定盐酸尼非卡兰粉针的含量.方法 采用Hypersil ODS柱(4.6mm×250mm,5μm),检测波长269nm.流动相为甲醇-0.02mol·L-1醋酸铵(60∶40);流速:1.0mL·min-1.结果 盐酸尼非卡兰在9.35～95.7μg.mL-1浓度范围内线性关系良好(r=0.99998).平均回收率为101.29%,RSD=0.28%.结论 本法快速,简便,准确,重复性好.     

反相高效液相色谱法检测小鼠血清中丹皮酚浓度

目的:建立反相高效液相色谱法(RP-HPLC)测定血清中丹皮酚(paeonol,Pae)浓度.方法:血清样品用氯仿单步萃取,萃取物以氮气流吹干后,用甲醇溶解用于色谱分析.色谱条件:采用Lichrospher C18柱(4.6 mm×250 mm,5μm);以甲醇-乙腈-水(30:40:30)为流动相;流速为0.8 mL·min-1;检测波长为274 nm;柱温为25℃;进样量为20 μL.结果:Pae最低检测浓度为0.01 mg·L-1;血清样品浓度在0.02～12.80 mg·L-1范围内呈良好线性关系,回归方程为Y=2712.1836X-5.7137,r=0.9999,n=5;相对回收率为(101.2±1.2)%,平均萃取回收率为(79.7±3.0)%;日内RSD与日间RSD均小于10.0%(n=5).结论:该方法灵敏、简便、快速、重复性好,适用于血药浓度测定及药动学研究.     

反相高效液相色谱法测定罗哌卡因血药浓度

目的:建立准确、快速测定罗哌卡因血药浓度的反相高效液相色谱法。方法:血样用乙醚和正己烷提取,布比卡因作内标,氮气吹干,流动相溶解进样。色谱条件:采用AgilentC_(18)色谱柱(4.6 mm×150 mm,5μm),流动相:0.045 mol/L磷酸二氢钾(pH3.0)-甲醇(55:45),流速:1.0 mL/min,检测波长:210 nm,柱温:30℃。结果:罗哌卡因在0.250 9～5.018μg/mL范围内线性关系良好(r=0.999 6),平均回收率102.0%,日内、日间精密度RSD均<15%,符合方法学要求。结论:本法快速、稳定,可用于罗哌卡因血药浓度监测。     

改良RP-HPLC法测定血浆中罗哌卡因的含量

目的:采用反相高效液相色谱法测定犬血浆中罗哌卡因的浓度。方法:待测样品经蛋白沉淀处理后,外标法进行高效液相紫外检测,选用乙腈作为蛋白沉淀剂,色谱柱采用Intertsil C18柱(4.6 mm×250 mm,5μm),流动相为甲醇-水相,体积比为50 50,水相为0.005 nmol.ml-1己烷基磺酸钠,冰醋酸调节pH至3.5,检测波长为215 nm,流速为:1.4ml.min-1;柱温为室温;柱压为2 800 PSI,进样体积为10μl,灵敏度为0.02AUFS。结果:在选定的色谱条件下,罗哌卡因与血浆中的杂质分离良好,罗哌卡因的保留时间为9.8 min。罗哌卡因的检测浓度线性范围为0.1~25μg.ml-1(r=0.9992),最低检测浓度为0.05μg.ml-1,回归方程为Y=16212.66X+37016.53,相关系数r=0.9992,回收率为91.2%~93.6%,RSD为2.10%~3.40%。结论:本法用于罗哌卡因类药物给药后血药浓度的检测,操作简便、可靠、快捷、准确,重现性好。     

Determination of polythiazide in pharmaceutical dosage forms by high-pressure liquid chromatography.

A method is presented for the quantitative analysis of polythiazide tablets by high-pressure liquid chromatography. The powdered tablets are extracted with methanol, containing quinoline as the internal standard, and assayed by comparison of peak heights after liquid chromatography. The chromatographic conditions employed may be adapted for the determination of many other thiazide and nonthiazide diuretics.     

Stability indicating LC-method for estimation of paracetamol and lornoxicam in combined dosage form

A simple, specific and stability indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of paracetamol and lornoxicam in tablet dosage form. A Brownlee C-18, 5 μm column having 250×4.6 mm i.d. in isocratic mode, with mobile phase containing 0.05 M potassium dihydrogen phosphate:methanol (40:60, v/v) was used. The flow rate was 1.0 ml/min and effluents were monitored at 266 nm. The retention times of paracetamol and lornoxicam were 2.7 min and 5.1 min, respectively. The linearity for paracetamol and lornoxicam were in the range of 5-200 μg/ml and 0.08-20 μg/ml, respectively. Paracetamol and lornoxicam stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation and dry heat degradation. The proposed method was validated and successfully applied to the estimation of paracetamol and lornoxicam in combined tablet dosage form.     

Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Phenoxyethanol,Methylparaben, Propylparaben,Mometasone Furoate,and Tazarotene in Topical Pharmaceutical Dosage Formulation

A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of phenoxyethanol (PE), methylparaben (MP), propylparaben (PP), mometasone furoate (MF), and tazarotene (TA) in topical pharmaceutical dosage formulation. The desired chromatographic separation was achieved on the Waters X-Bridge™ C18 (50×4.6mm, 3.5μ) column using gradient elution at 256 nm detection wavelength. The optimized mobile phase consisted of 0.1%v/v orthophosphoric acid in water as solvent-A and acetonitrile as solvent-B. The method showed linearity over the range of 5.88–61.76 μg/mL, 0.18–62.36 μg/mL, 0.17–6.26 μg/mL, 0.47–31.22 μg/mL, and 0.44–30.45 μg/mL for PE, MP, PP, MF, and TA, respectively. The recovery for all of the components was in the range of 98–102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples, in which the spectral purity of PE, MP, PP, MF, and TA along with the separation of degradation products from the analyte peaks was achieved. The proposed method was successfully applied for the quantitative determination of PE, MP, PP, MF, and TA in a cream sample.     

Determination of ibuprofen in ointments by reversed-phase liquid chromatography

A selective reversed-phase HPLC method for the determination of ibuprofen in different ointment bases is described. Following a simple dilution step, the drug was separated from interfering compounds on an octadecylsilica column protected by a precolumn. A mobile phase of aqueous tetrahydrofuran buffered by phosphate was used. Detection was monitored at 219 nm. After each 10 injections of lipophilic samples, the columns were washed with tetrahydrofuran and n-hexane. Recoveries of ibuprofen from a new pilot preparation spiked to contain 4, 5, and 6% of the drug were 100.4, 100.3, and 99.7%, respectively. The precision at each concentration was less than 0.8%. The analysis of ibuprofen degeneration products showed that the method also provides an indication of the stability of the drug.     

Spectrophotometric determination of glutathione and of its oxidation product in pharmaceutical dosage forms.

A simple and sensitive spectrophotometric method suitable for the stability control of pharmaceutical dosage forms containing glutathione (gamma-glutamyl-cysteinyl-glycine), GSH, is described. Besides GSH, the method quantitatively determines its oxidation product, GSSG. The colour reactions of GSH and GSSG with ammonium tetrachloropalladate have been investigated and the optimum reaction conditions, spectral characteristics and composition of the yellow water-soluble complexes have been established. The assay results of pharmaceutical formulations showed good accuracy and precision over the concentration range of 5 x 10(-5)-6 x 10(-4) M GSH.     

Rapid simultaneous determination of telmisartan, amlodipine besylate and hydrochlorothiazide in a combined poly pill dosage form by stability-indicating ultra performance liquid chromatography

A simple, precise and rapid stability-indicating ultra-performance liquid chromatography (UPLC) method is developed for the simultaneous quantitative determination of Telmisartan, Amlodipine besylate and Hydrochlorothiazide from their innovative poly pill combination drug product in the presence of degradation products. It involves a 100 mm x 2.1 mm, 1.7 μm C-18 column. The separation is achieved on a simple gradient method. The mobile phase A contains a mixture of sodium perchlorate buffer pH 3.2 (0.053M): acetonitrile in the ratio 90:10, v/v, and mobile B contains a mixture of sodium perchlorate buffer pH 3.2 (0.053M): acetonitrile in the ratio 20:80, v/v. The flow rate is 0.6 mL min(-1) and the column temperature is maintained at 55°C.The gradient program (T/%B) is set as 0/5, 1.2/5, 1.6/40, 4/40, 4.1/5 and 4.5/5. The detector wavelength is 271 nm for Hydrochlorothiazide and Telmisartan and 237 nm for Amlodipine. The retention times of Telmisartan, Amlodipine, and Hydrochlorothiazide are 3.6 minutes, 3.2 minutes and 0.9 minutes; respectively. The total runtime for the separation of the three active compounds and their degradation products is 4.5 minutes. The described method is validated with respect to system suitability, specificity, linearity, precision and accuracy. The precision of the assay method is evaluated by carrying out six independent assays of T, A and H (0.032 mg mL(-1) of T, 0.004 mg mL(-1) of A, 0.01 mg mL(-1) of H). The accuracy of the method is evaluated in triplicate at three concentration levels, i.e. 50%, 100% and 150% of target test concentration (0.64 mg mL(-1) of T, 0.08 mg mL(-1) of A, 0.2 mg mL(-1) of H). The described method is linear over the range, 16 to 48 μg mL(-1) for T, 2 to 6 μg mL(-1)A and 5 to 15 μg mL(-1) for H. The method is fast and suitable for high-throughput analysis allowing the analysis of about 250 samples per working day.     

Stability-indicating HPTLC determination of imatinib mesylate in bulk drug and pharmaceutical dosage form

A simple, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis of imatinib mesylate both as a bulk drug and in formulations was developed and validated. The method employed HPTLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of chloroform:methanol (6:4, v/v). The system was found to give compact spot for imatinib mesylate (R(f) value of 0.53+/-0.02). Densitometric analysis of imatinib mesylate was carried out in the absorbance mode at 276 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r(2)=0.9966+/-0.0013 with respect to peak area in the concentration range 100-1000 ng per spot. The mean value+/-S.D. of slope and intercept were 164.85+/-0.72 and 1168.3+/-8.26 with respect to peak area. The method was validated for precision, recovery and robustness. The limits of detection and quantitation were 10 and 30 ng per spot, respectively. Imatinib mesylate was subjected to acid and alkali hydrolysis, oxidation and thermal degradation. The drug undergoes degradation under acidic, basic, oxidation and heat conditions. This indicates that the drug is susceptible to acid, base hydrolysis, oxidation and heat. Statistical analysis proves that the method is repeatable, selective and accurate for the estimation of said drug. The proposed developed HPTLC method can be applied for identification and quantitative determination of imatinib mesylate in bulk drug and dosage forms.     

Determination of flunixin in equine plasma by reversed-phase liquid chromatography

Flunixin is determined in equine plasma by liquid chromatography on LiChrosorb RP-18 with 70% methanol in phosphate buffer pH 3.1 as the eluent, with detection at 284 nm. Plasma is deproteinized with methanol and the supernatant is then injected directly into the system. With a short pre-column (5 × 3 mm i.d.), which is replaced after 25–40 injections of sample, 420 plasma samples could be analysed on one analytical column. The detection limit in plasma is 0.30 μmol/l (89 ng/ml) and the method can be used in pharmacokinetic studies.     

Determination of meprobamate in pharmaceutical dosage forms also containing carbromal by liquid chromatography and indirect photometric detection

In a pharmaceutical form also containing carbromal, meprobamate could not be quantified selectively by classical methods described in pharmacopoeias due to a significant interference from carbromal. Consequently, reversed-phase HPLC methods have been developed to separate the two active ingredients using indirect photometric detection to visualize and determine meprobamate which has very poor chromophoric properties. Different parameters influencing the sensitivity of the indirect response, such as the nature of the highly absorbing compound added to the mobile phase (the marker) as well as the methanol content and the pH of this phase, have been studied. Two chromatographic systems containing benzoic acid or cinnamic acid as the marker, have been optimized and validated. Good linearity and reproducibility have been obtained with both systems but the cinnamic acid method has the advantage that meprobamate and carbromal can be determined simultaneously at 273 nm.     

Determination of linezolid in plasma by reversed-phase high-performance liquid chromatography

An HPLC-UV method was developed for assay of linezolid in dog, rat, mouse, and rabbit plasma. Linezolid and the internal standard were extracted on a solid phase cartridge (SPE) and separated on a reversed-phase column (C8, 4.6x150 mm, 5 microm) with 20% acetonitrile in water as mobile phase. The SPE quantitatively recovered linezolid and the internal standard from plasma samples. The chromatographic peak height ratio or peak area ratio based on UV absorbency at 251 nm was used for quantitative analysis. The assay procedures were simple and the assay was specific and had adequate precision and accuracy. Calibration standards with concentrations over the range of 0.01 20 microg/ml were validated for routine sample analysis to support the pharmacokinetic and toxicology studies with linezolid in dog, rat, mouse, and rabbit. Analysis of quality control samples showed the coefficients of variation were usually <10% and the measured and theoretical concentrations differed by <10% in most assays. Linezolid in the plasma samples was stable when stored at below -20 degrees C for at least 63 days, at room temperature (22-23 degrees C) for up to 24 h, and after three freeze-thaw cycles. This HPLC method has been successfully used in multiple laboratories to assay plasma samples from pharmacokinetic and toxicology studies with linezolid in the animal species.     

Determination of indoprofen in physiological fluids by reversed-phase liquid chromatography

A rapid, sensitive, reversed-phase high-performance liquid chromatographic procedure was developed for the quantitative analysis of indoprofen in plasma and urine. Minimal sample preparation is required for the analysis of unconjugated urinary or plasma drug levels. The method provided quantitative results for indoprofen levels of 0.5-50 microgram/ml of plasma and 0.5-200 microgram/ml of urine and had a lower detection limit of 1 ng. Total urinary indoprofen levels required enzymatic hydrolysis of the conjugated drug prior to analysis. Results are presented for the plasma and urinary excretion levels of indoprofen for a patient receiving a single oral dose.