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.     

Development and validation of an UPLC method for rapid determination of ibuprofen and diphenhydramine citrate in the presence of impurities in combined dosage form

A novel, stability-indicating gradient reverse-phase ultra-performance liquid chromatographic method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in the presence of degradation products and process related impurities in combined dosage form. The method was developed using C18 column with mobile phase containing a gradient mixture of solvent A and B. The eluted compounds were monitored at 220 nm. Ibuprofen and diphenhydramine citrate were subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Major unknown impurity formed under oxidative degradation was identified using LC-MS-MS study. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantitation, accuracy, precision and robustness. The described method was linear over the range of 0.20-6.00 μg/mL (r>0.998) for Ibuprofen and 0.084-1.14 μg/mL for diphenhydramine citrate (r>0.998). The limit of detection results were ranged from 0.200-0.320 μg/mL for ibuprofen impurities and 0.084-0.099 μg/mL for diphenhydramine citrate impurities. The limit of quantitation results were ranged from 0.440 to 0.880 μg/mL for ibuprofen impurities and 0.258 to 0.372 μg/mL for diphenhydramine citrate impurities. The recovery of ibuprofen impurities were ranged from 98.1% to 100.5% and the recovery of diphenhydramine citrate impurities were ranged from 97.5% to 102.1%. This method is also suitable for the simultaneous assay determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms.     

New gradient high-performance liquid chromatography method for determination of donepezil hydrochloride assay and impurities content in oral pharmaceutical formulation

A new gradient HPLC method has been developed and validated for the determination of both assay and related substances of donepezil hydrochloride in oral pharmaceutical formulation. Different kinds of columns and gradient elution programs were tested in order to achieve satisfactory separation between the active substance, four impurities and an interfering excipient used in the formulation. The best results were obtained using an Uptisphere ODB C-18 column 250 mm x 4.6 mm, 5 microm, UV detection at 270 nm and a gradient elution of phosphate buffer (0.005 M, pH 3.67) and methanol as the mobile phase. The method was validated with respect to linearity, precision, accuracy, specificity and robustness. It was also found to be stability indicating, and therefore suitable for the routine analysis of donepezil hydrochloride and related substances in the pharmaceutical formulation.     

High throughput screening of active pharmaceutical ingredients by UPLC

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.     

Isolation and characterization of process related impurities of olanzapine using HPLC and ESI-MS/MS

An RP-HPLC method for the simultaneous separation and determination of olanzapine (OLZ) and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on Inertsil ODS 3V (4.6 mm x 250 mm; particle size 5 microm) column using 0.2 M ammonium acetate (pH = 4.50) and ACN as mobile phase in gradient elution mode. The analytes were monitored by a photo diode array (PDA) detector set at 254 nm and the flow rate was kept at 1.0 mL/min. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. Four unknown process impurities observed consistently during the analysis of different batches of OLZ were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR. The proposed RP-HPLC method was successfully applied to the analysis of commercial formulations. The method could be of use not only for rapid and routine evaluation of the quality of OLZ in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations.     

Simultaneous separation and determination of process-related substances and degradation products of venlafaxine by reversed-phase HPLC

A simple and rapid gradient RP HPLC method for simultaneous separation and determination of venlafaxine and its related substances in bulk drugs and pharmaceutical formulations has been developed. As many as four process impurities and one degradation product of venlafaxine have been separated on a Kromasil KR100-5C18 (4.6 mm x 250 mm; particle size 5 microm) column with gradient elution using 0.3% diethylamine buffer (pH 3.0) and ACN/methanol (90:10 v/v) as a mobile phase. The column was maintained at 40 degrees C and the eluents were monitored with photo diode array detection at 225 nm. The chromatographic behaviour of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. The inter- and intraday assay precision was < 4.02% (%RSD) and the recoveries were in the range of 96.19-101.14% with %RSD < 1.15%. The correlation coefficients (r2) for calibration curves of venlafaxine as well as impurities were in the range of 0.9942-0.9999. The proposed RP-LC method was successfully applied to the analysis of commercial formulations and the recoveries of venlafaxine were in the range of 99.32-100.67 with %RSD <0.58%. The method could be of use not only for rapid and routine evaluation of the quality of venlafaxine in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Forced degradation of venlafaxine was carried out under thermal, photo, acidic, basic and peroxide conditions and the acid degradation products were characterized by ESI-MS/MS, 1H NMR and FT-IR spectral data.     

Simultaneous determination of rocuronium and its eight impurities in pharmaceutical preparation using high-performance liquid chromatography with amperometric detection

A sensitive and selective HPLC method with amperometric detection (HPLC-ED) for the determination of rocuronium bromide and its eight impurities has been developed. The analysis was performed on Hypersil 100 Silica column 5 microm (250 mm x 4.6 mm; Thermo Electron). The mobile phase consisting of 4.53 g l(-1) solution of tetramethylammonium hydroxide adjusted to pH 7.4 with 85% phosphoric acid:acetonitrile (1:9), was found the best for the separation and determination of the studied compounds. The chromatograms were recorded over 10 min using the amperometric detection at a potential +0.9 V of the glassy carbon electrode versus the reference electrode Ag/AgCl. The limit of quantitation was 45 ng ml(-1) for rocuronium and from 25 to 750 ng ml(-1) for the examined impurities. The proposed HPLC-ED method was successfully applied to the analysis of rocuronium and its impurities in Esmeron solution for injection.     

Considerations on the possibilities and limitations of comprehensive normal phase-reversed phase liquid chromatography (NPLC x RPLC)

A comprehensive normal phase system LC-reversed phase LC (NPLC x RPLC) was evaluated for the separation of a pharmaceutical mixture and citrus oil extracts. NPLC was performed on a 25 cm x 1 mm ID x 5 microm dp diol phase. In the second dimension, an RP 18 monolithic column (10 cm L x 4.6 mm ID x 2 microm macropore size) and an octadecyl silicagel-packed column (5 cm L x 4.6 mm ID x 3.5 microm dp) were applied for the analyses of the pharmaceutical sample and the citrus oil extracts, respectively. A two-position/ten-port switching valve was used as interface. Under optimised LC conditions, the high degree of orthogonality between NP and RP resulted in peak capacities of 300 for the pharmaceutical sample and of 450 for the citrus oil extract composed of lemon and orange oil. Despite the features of NPLC x RPLC, several shortcomings related with the solvent incompatibility between the two LC modes were identified and the practical consequences were discussed.     

A novel validated LC method for quantitation of lopinavir in bulk drug and pharmaceutical formulation in the presence of its potential impurities and degradation products

A simple isocratic liquid chromatographic method was developed for determination of lopinavir from its related impurities and assay for the first time. This method involves the use of a C(8) (Symmetry Shield RP8, 150 x 4.6 mm, 5 microm) column. The method was validated over the range of limit of quantitation (LOQ) to 120% of impurity specification limit and LOQ to 150% of working concentration for assay. The mobile phase consisted of a mixture of 50 mM of potassium phosphate buffer, acetonitrile and methanol in the ratio of 40:50:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 210 nm. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The developed method was validated for linearity, range, precision, accuracy and specificity. This method was successfully applied for content determination of lopinavir in pharmaceutical formulations. The method can be conveniently used in a quality control laboratory for routine analysis for assay and related substances as well for the evaluation of stability samples of bulk drugs and pharmaceutical formulations.     

A comparison of performance of various analytical columns in pharmaceutical analysis: conventional C18 and high throughput C18 zorbax columns

New improved types of analytical columns Zorbax Eclipse XDB-C18 (75 mm x 4.6 mm i.d., 3.5 microm) and Zorbax Eclipse XDB-C18 (50 mm x 4.6 mm i.d., 1.8 microm) have been tested for determination of estradiol (active substance), methylparaben, propylparaben (preservatives) and estrone (degradation product) and compared with the conventional C18 columns (250 mm x 3.0 mm i.d., 5.0 microm). The Zorbax columns differ with their particle size, column length and ODS (octadecylsilica) type as well. Higher flow-rates (up to about 2.5 ml min(-1)) could be applied regardless to back-pressure. The analysis - previously done at 40 degrees C - could be performed even at ambient temperature. Analytical run was shortened to 3.5 min (from 12 min used for the conventional C18 column) with the same or better retention characteristics. System suitability data for all Zorbax columns show the advantages of these columns for the practical use in routine quality control of pharmaceuticals, particularly from the point of view of speed of analysis and solvent consumption.     

Micellar electrokinetic and high-performance liquid chromatographic determination of potential manufacturing impurities in pholcodine

Quantitative high-performance liquid chromatographic (HPLC) and micellar electrokinetic chromatographic (MEKC) methods have been developed for the determination of four structurally related potential manufacturing impurities, including morphine, of the opiate derivative pholcodine. Pholcodine and the four impurities were separated by MEKC in less than 14 min using a 70 cm x 75 microm I.D. uncoated fused-silica capillary (25 kV at 30 degrees C) and a running buffer consisting of 10% acetonitrile (v/v) in 20 mM borate-phosphate buffer pH 8.0 containing 40 mM sodium dodecyl sulphate (SDS). The MEKC method was compared to a HPLC method using a 5 microm Luna phenyl-hexyl column (150 x 4.6 mm I.D.) eluted with a mobile phase consisting of a mixture of 10% (v/v) acetonitrile, 7% (v/v) tetrahydrofuran in 20 mM phosphate buffer pH 8.0. Both methods were fully validated and a comparison was made regarding selectivity, linearity, precision, robustness and limits of detection and quantitation. The presence of the impurities in different samples of pholcodine drug substance was investigated using both methods.     

Simultaneous determination of paracetamol, chlorzoxazone, and related impurities 4-aminophenol, 4'-chloroacetanilide, and p-chlorophenol in pharmaceutical preparations by high-performance liquid chromatography

This paper presents a simple, specific, and precise high-performance liquid chromatographic method for the simultaneous determination of paracetamol (PCM), chlorzoxazone (CXZ), and their related impurities in bulk raw materials and solid dosage forms. The mobile phase consisted of water-methanol-glacial acetic acid (60 + 40 + 2, v/v/v). A column containing octadecylsilane chemically bonded to porous silica particles (Spherisorb ODS 1, 25 cm x 4.6 mm, 5 microm) was used as stationary phase. Detection was performed using a variable wavelength ultraviolet-visible detector set at 272 nm for all compounds. Solutions were injected into the chromatograph under isocratic condition at a constant flow rate of 1.2 mL/min. The method was validated according to International Conference on Harmonization requirements and demonstrates good accuracy and precision and a wide linearity range. The method separates PCM, CXZ, and 3 major impurities [4-aminophenol (4AP), 4'-chloracetanilide (4CA), and p-chlorophenol (PCP)] with fair resolution in less than 15 min. The developed method is rapid and sensitive (limit of detection for 4AP, 4CA, and PCP established at 31.25, 39.06, and 65.16 ng/mL, respectively) and, therefore, suitable for quality control and stability studies of these compounds in dosage forms.     

Simultaneous liquid chromatographic determination of ezetimibe and simvastatin in pharmaceutical products

A reversed-phase liquid chromatographic (LC) method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical dosage forms. The LC method was carried out on a Synergi fusion C18 column (150 mm x 4.6 mm id) maintained at 45 degrees C. The mobile phase consisted of phosphate buffer 0.03 M, pH 4.5-acetonitrile (35 + 65, v/v) run at a flow rate of 0.6 mL/min, and detection was made using a photodiode array detector at 234 nm. The chromatographic separation was obtained within 15.0 min, and calibration graphs were linear in the concentration range of 0.5-200 microg/mL. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range for both compounds. Moreover, the proposed method was successfully applied for the routine quality control analysis of pharmaceutical products.     

超高效液相色谱法同时测定布洛芬注射液中布洛芬和精氨酸的含量

建立了一种同时测定布洛芬注射液中布洛芬和精氨酸含量的超高效液相色谱方法。精氨酸与衍生化试剂2,4-二硝基氟苯(DNFB)反应后,与布洛芬同时在超高效液相色谱-二极管阵列检测器(UPLC-PDA)上检测。采用BEH C18色谱柱(50 mm×2.1 mm, 1.7 μm),以乙腈-0.05 mol/L磷酸二氢钾缓冲液(pH 2.5)为流动相进行梯度洗脱,流速为0.4 mL/min,柱温为30 ℃,检测波长分别为357 nm(精氨酸衍生物)和220 nm(布洛芬)。结果表明,布洛芬与精氨酸分别在2.0～100.5 mg/L和1.7～84.5 mg/L范围内呈良好的线性关系,相关系数(r)均为0.9997;平均回收率分别为99.8%和99.6%,相对标准偏差(RSDs)分别为0.37%和0.25%;定量限(信噪比（S/N）=10)分别为0.1 ng和0.2 ng;检出限(S/N=3)分别为0.03 ng和0.05 ng。本方法快速、准确,重复性好,可较全面地评价布洛芬注射液的质量。     

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man.

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-S?rensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.     

Reversed-phase ultra-performance liquid chromatographic method development and validation for determination of impurities related to torsemide tablets

A simple RP-ultra-performance LC method was developed and validated for determination of impurities related to torsemide tablets. The rapid method provided adequate separation of all known related impurities and degradation products. Separation was achieved on a Zorbax SB-C18 column (50 x 4.6 mm id, 1.8 microm particle size) with binary gradient elution, and detection was performed at 288 nm. The drug product was subjected to oxidative, hydrolytic, photolytic, and thermal stress conditions to prove the specificity of the proposed method. The linearity and recovery were investigated for known impurities in the range of 0.025 to 1.0%, with respect to the drug concentration in the prepared sample. The linearity of the calibration curve for each of the impurities and torsemide was found to be very good (r2 > 0.999). Relative response factors for each of the known impurities were established by the slope ratio method from the linearity study.     

Characterization of impurities in spiramycin by liquid chromatography/ion trap mass spectrometry

A reversed-phase liquid chromatography/tandem mass spectrometry method is described for the investigation of spiramycin and related substances. The method uses an XTerra C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of acetonitrile, methanol, water and ammonium acetate solution, pH 6.5. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) operated in the positive ion mode. Using this method, the fragmentation behavior of spiramycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total 17 compounds were identified, among which three reported as specified impurities in the European Pharmacopoeia. The other impurities showed mainly a modification in the forosamine sugar or in the substituent at C-3 and C-6 positions. In one impurity, the mycarose sugar is absent.     

High-performance liquid chromatographic determination of pantoprazole and its main impurities in pharmaceuticals

An RP-HPLC method for simultaneous separation and quantification of pantoprazole and its five main impurities in pharmaceutical formulations was developed and validated. The separation was accomplished on a Zorbax Eclipse XDB C18 column (5 microm particle size, 150 x 4.6 mm id) using a gradient with mobile phase A [buffer-acetonitrile (70 + 30, v/v)], and mobile phase B [buffer-acetonitrile (30 + 70, v/v)]. The buffer was 0.01 M ammonium acetate solution with addition of 1 mL triethylamine/L of the solution, adjusted to pH 4.5 with orthophosphoric acid. The eluent flow rate was 1 mL/min, the temperature of the column was 30 degrees C, and the eluate was monitored at 290 nm. Linearity (r = 0.999), recovery (97.6-105.8%), RSD (0.55-1.90%), and LOQ (0.099-1.48 microg/mL) were evaluated and found to be satisfactory. The proposed method can be used for simultaneous identification and quantification of the analyzed compounds in pharmaceutical formulations.     

Evaluation of an International Pharmacopoeia method for the analysis of nelfinavir mesilate by liquid chromatography

A gradient LC method for the determination of related substances in nelfinavir mesilate (NFVM) has been recently published in the International Pharmacopoeia. The method uses a base deactivated reversed phase C18 column (25 cm x 4.6 mm I.D.), 5 microm kept at a temperature of 35 degrees C. The mobile phases consist of acetonitrile, methanol, phosphate buffer pH 3.4 and water. The flow rate is 1.0 ml/min. UV detection is performed at 225 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards NFVM components was investigated on 18 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm I.D.), 5 microm. A two level fractional factorial design was applied to examine the robustness of the method. The method shows good selectivity, precision, linearity and sensitivity. Seven commercial samples were examined using this method.     

A validated method for analysis of chromium picolinate in nutraceuticals by reversed phase high performance liquid chromatography

A validated high performance liquid chromatographic method was developed for the determination of chromium picolinate in pharmaceutical dosage forms. The analysis was performed at room temperature using a reversed-phase Supelcosil LC-18 (250 x 4.6 mm, 5 microm) column. The mobile phase consisted of acetonitrile:water (40:60 v/v) at a fl ow rate of 0.8 mL/min. The UV-detector was set at 264 nm. The developed method showed a good linear relationship in the concentration range from 0.125 to 12.5 microg/mL with a correlation coefficient from 0.999. The limit of detection and limit of quanti fi cation were 0.091 and 0.181 microg/mL, respectively.