Sil C18 Research Articles

Analytical Method Development and Validation for the Simultaneous Estimation of Sulfadiazine and Pyrimethamine by RP-HPLC Method

Background Sulfadiazine and Pyrimethamine is a combination drug of choice to treat malaria and used in those living in or will be travelling to an area where there is chance of getting malaria. Objective The main objective of the study was to validate the simultaneous estimation and the method established with resisting small changes in flow rate and ratio of organic compounds and proposed method ensures its use in routine quality control analysis of pharmaceutical formulation. MethodologyA HPLC AllianceWater 2695 with UVVIS DetectorPDA detector UV lab India UV 3000 series and Zodiac C18 250 mm times 4.6 mm times 5 microm column were used. A new method was established for simultaneous estimation of Sulfadiazine and Pyrimethamine by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Sulfadiazine and Pyrimethamine by using Zodiac sil C18 column 4.6times150 mm 5micro flow rate was 1mLmin mobile phase ratio was 7030 vv methanolphosphate buffer KH2 PO4 and K2 HPO4 phosphate pH 3 pH was adjusted with orthophosphoric acid detection wavelength was 240 nm. Results The results were in good agreement with those obtained with official HPLC with maximum absorption of 240 nm by preparing mobile phase 7030 methanolphosphate buffer with flow rate 1 mLmin and was run for 10 minutes by selecting column Zodiac silica RP C18 4.5times100 mm 3.0 microm of ambient temperature. All the results were obtained with good precision accuracy and robustness as per ICH guidelines. Conclusion It can be concluded that the proposed RPHPLC method was accurate precise sensitive specific robust and reproducible for the simultaneous analysis of Sulfadiazine and Pyrimethamine with less tailing factor and also economical. Zodiac sil C18 column 4.6times150mm5micro flow rate was 1mLmin mobile phase ratio was 7030 vv methanol phosphate buffer KH2 PO4 and K2 HPO4 phosphate pH 3 pH was adjusted with orthophosphoric acid detection wavelength was 240 nm.

DEVELOPMENT AND VALIDATION OF REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ASSAY METHOD FOR ESTIMATION OF FENOFIBRATE IN TABLET DOSAGE FORM PREPARED USING CRYSTALLO-CO-AGGLOMERATES OF THE DRUG

The aim of the present study was to develop a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method and validate for the determination of fenofibrate in tablet dosage forms. RP-HPLC method was developed using Hi Q Sil C18 (250 cm × 4.6 mm, 5 μm) and mobile phase comprising 1 mM ammonium acetate buffer: Acetonitrile (10:90 v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 290 nm. The retention time was found to be 6.15 ± 0.03 min. Validation of the method was performed for precision, accuracy, linearity, robustness, specificity and sensitivity to conform to the International Conference on Harmonization (ICH) guidelines. The data of linear regression analysis indicated a good linear response in the concentration range of 5 μg/mL–30 μg/mL with correlation co-efficient (R2) of 0.997. The developed method was found to be simple, sensitive, accurate and repeatable for assay of tablets of fenofibrate prepared using crystallo-co-agglomerates of the drug.

STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF PIOGLITAZONE, AMLODIPINE, ATORVASTATIN AND CLOPIDOGREL

A new simple, rapid, isocratic and stability indicating reverse phase high performance liquid chromatography (HPLC) method has been developed for simultaneous estimation of pioglitazone (P), amlodipine (AM), atorvastatin (AT) and clopidogrel (CP). Isocratic separation was achieved on Millennium M Sil C18 (125 mm ´4.6 mm) column, with mobile phase comprising of methanol: 0.02 M KH2PO4 (55:45 V/V), pH adjusted 4.0 with 0.01% ortho phosphoric acid. The flow rate was maintained 1mL/min and analytes were screened with U.V. detector. The method was validated according to ICH guidelines with respect to linearity, accuracy, precision and specificity. The drugs were exposed to various stress conditions like acidic and basic hydrolysis, oxidation, exposure to UV light and temperature (dry heat) and the stressed sample were analysed by the proposed method. No co-eluting, interfering peaks from excipients, impurities were observed during stress condition and all the degraded peaks are well resolved from parent peaks.

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE ESTIMATION OF LEDIPASVIR IN BULK AND TABLET DOSAGE FORM

Objective: The objective of this study was to develop a stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the estimation of ledipasvir (LDP) in bulk and tablet formulation.
 Methods: Stability-indicating RP-HPLC method was developed and validated for the estimation of LDP in bulk and tablet formulation. RP-HPLC was carried out on HiQ SiL C18 columns (250 mm × 4.6 mm, 5 μ particle size) using mobile phase acetonitrile:1 mM ammonium acetate buffer in the ratio of 90:10 v/v at a flow rate of 1 ml/min. The analytes were monitored using MD 2010 PDA detector at 333 nm.
 Results: The retention time was found to be 3.843 min. The proposed method was found to be having linearity in the concentration range of 5–30 μg/ml. The number of theoretical plates obtained was 4236.50 which indicate the efficient performance of the column. The limit of detection was 0.305 μg/ml and limit of quantification was 0.923 μg/ml, which indicate the sensitivity of the method; the high percentage recovery indicates that the proposed method is highly accurate. The developed method has been validated according to the ICH guidelines and found to be simple, specific, precise, and accurate.
 Conclusion: The proposed method is precise, accurate, and stability indicating. Therefore, the proposed method can be used for routine quality control and analysis of LDP during stability studies in bulk samples and tablet dosage forms.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF IMATINIB MESYLATE AND ITS DIMER IMPURITY IN PHARMACEUTICAL FORMULATION BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The present study is to develop a simple, specific, and validated reverse-phase high-performance liquid chromatography (HPLC) method for the determination of imatinib mesylate and its dimer impurity in pharmaceutical dosage form.Methods: A HPLC instrument incorporated with column HiQ Sil C18 (250 mm × 4.6 mm, 5 μm), mobile phase as methanol and acetate buffer pH 3.5 in the ratio of 80:20 v/v was used for the determination of the imatinib mesylate and its dimer impurity. The detection wavelength was set at 273 nm. The flow rate of the mobile phase was 1.0 mL/min.Results: The retention time for imatinib mesylate was 8.060, and for dimer impurity, it was 11.398. The calibration plot was linear (R2=0.9971) and the % mean recoveries for imatinib mesylate were in the range of 99.83–101.57, and for dimer impurity, it was in the range of 98.16–99.18. The limit of detection concentration was found to be 0.570 μg/ml for imatinib mesylate and 0.033 μg/ml dimer impurity and limit of quantification concentration was 1.728 μg/ml for imatinib mesylate and 0.099 μg/ml dimer impurity.Conclusion: The projected method was validated and successfully functional for the estimation of imatinib mesylate and dimer impurity in formulations. It can be adopted apparently for routine quality control and research tests.

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF LIDOCAINE AND PRILOCAINE IN TOPICAL FORMULATION

Objective: A simple, specific, accurate, and precise method, namely, reverse phase high-performance liquid chromatography was to develop for simultaneous estimation of Lidocaine (LDC) and prilocaine (PLC) in a topical local anesthetic cream.Method: The mixture of PLC and LDC was separated on Hi Q Sil C18 HS column, (250 mm × 4.6 mm, 5 μm), column temperature ambient and flow rate 1.2 mL/minutes. The mobile phase was acetonitrile: 0.01 M diethylamine solution (pH adjusts to 6.8 with orthophosphoric acid) (60:40) with detection at 225 nm.Results: The retention time was found to be 6.075±0.12 minutes for PLC and 8.642±0.15 minutes for LDC, respectively. Linearity was observed in the concentration range of 1-6 μg/mL for both LDC and PLC, respectively. The method was validated according to International Conference on Harmonization guideline and values of linearity, precision, robustness, limit of detection, limit of quantitation, selectivity, and recovery were found to be in good accordance with the prescribed value.Conclusion: The proposed method can be useful in the quality control of LDC and PLC in their topical formulation.

DETERMINATION THE CONCENTRATION OF ASIATICOSIDE IN CENTELLA ASIATICA (L.) URB. – APIACEAE AT QUANG DIEN DISTRICT, THUA THIEN HUE PROVINCE

Background: Centella asiatica (L.) Urb. - Apiaceae has been used in traditional medicine to cure many diseases such as cholera dysentery, pimples, discharge, cough, sore throat, hemorrhoids and detoxification. The objective is to determine the amount of active ingredient asiaticoside in Centella asiatica (L.) Urb at Quang Tho 2 cooperatives, Quang Dien District, Thua Thien Hue province. Materials and method: C.asiatica after being harvested, washed and drained was dried in sunlight or in dryer with different temperature. Each 20g of C.asiatica was extracted using various methods with dissolving appropriate solvents. The extracted solution was diluted by extracted solvent. This diluted solution was filtered through 0.45μm membrane filter for the purpose of chromatography. Results: The chromatography condition: mobile phase consisting of methanol: Trifluoracetic acid 1% (60: 40); Column: HiQ Sil C18 (4.6 x 250 mmm) 3μm; flow rate at 0.7 ml/min; The maximum detection wavelength: 215 nm; retention time of asiaticosid (tR): 18.7 minutes. Conclusion: The content of asiaticoside in sample is from 0.09% to 1.35% depending on the initial sample handling and sample extraction method. Keywords: Centella asiatica (L.) Urb. - Apiaceae, asiaticoside, HPLC.

High performance liquid chromatography characteristic spectrum of artificial musk.

To determine the high performance liquid chromatography(HPLC)characteristic spectrum of artificial musk. HPLC was performed on a HiQ Sil C18 analytical column(4.6 mm×250 mm)with the mobile phase of methanol/water(90:10 v/v);the detection wavelength was 254 nm and the column temperature was maintained at 25 ℃. The similarities among 10 batches of artificial musk samples were anlysised in accordance with the System for Evaluating the Similarities among the Chromatographic Fingerprints of Traditional Chinese Drugs,which is recommended by Chinese Pharmacopoeia Commission. The similarities among these 10 batches of artificial musk ranged between 0.999 and 1.000. HPLC is a convenient,accurate and reliable technique for establishing the specific spectum of artificial musk and therefore can be used for the quality control of this product.

QUANTITATIVE DETERMINATION OF MILNACIPRAN HCL IN RABBIT PLASMA BY HPLC AND ITS APPLICATION TO PHARMACOKINETICS STUDY

A simple, sensitive, and specific reverse phase high performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of Milnacipran HCl in rabbit plasma. Milnacipran HCl and internal standard (IS, Venlafaxine HCl) were extracted by protein precipitation method with chloroform. The separation was performed on a HiQ sil C18 column (250 mm × 4.6 mm i.d., 5 µm). The wavelength was set at 220 nm. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (0.0125 M) and acetonitrile (72:28%, v/v) with 0.20% triethylamine at a flow rate of 1.0 mL/min. The pH of the solution was adjusted to 3.65 with 0.1 M orthophosphoric acid. The calibration curve was linear over the concentration range 0.1–25 µg/mL. The intra-day and inter-day precision was ranged from 3.9 to 7.3% and 5.2 to 10.8%, respectively. Finally, this proposed method was successfully applied to rabbit pharmacokinetics study and yielded the most comprehensive data on systemic exposure of Milnacipran HCl to date.

Synthesis and characterization of norverapamil and quantification of verapamil and nor-verapamil in plasma

Verapamil is a calcium channel blocking agent which has found widespread use in the management of supraventricular tachyarrhythmias, angina pectoris, hypertrophic cardiomyopathy and hypertension. It is converted to its biologically active metabolite nor-verapamil in liver by cytochrome P450. In present communication, synthesis and characterization of nor-verapamil and development of reverse phase high performance liquid chromatographic method for the quantification of nor-verapamil along with verapamil in plasma has been carried out. The characterization of nor-verapamil was carried out using GC-MS, FT-IR and NMR spectroscopy. The separation was carried out with an isocratic JASCO RP-HPLC system using 5 μm KYA TECH HiQ Sil C18 HS column (250 mm × 4.6 mm internal diameter) as a stationary phase and methanol: water: 0.01 M orthophosphoric acid: triethylamine [70: 30: 2: 0.5, v/v/v/v] as mobile phase. The flow rate was maintained at 1.0 mL/min and UV detection at 222 nm. The calibration for verapamil and nor-verapamil were found to be linear over concentration range of 50–300 ng/mL with correlation coefficient (n = 6) of 0.9995 and 0.9997, respectively. This method was validated according to USFDA guidelines. The method was found to be simple, accuare, precise sensitive and selective for the determination of verapamil and nor-verapamil in plasma and thus useful in bioequivalence studies of verapamil.

STABILITY INDICATING HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF METOPROLOL SUCCINATE AND TELMISARTAN

A simple, rapid, accurate, precise, selective, and reproducible stability‐indicating HPLC method has been developed for simultaneous estimation of metoprolol succinate and telmisartan using a mobile phase consisting mixture of Methanol: 10 mM potassium dihydrogen phosphate buffer: 10 mM hexane sulphonic acid (80:10:10,v/v/v) at the flow rate of 1 mL/min and detection wavelength at 223.0 nm. HiQ Sil C18 (250 × 4.6 mm, 5 µm) column was used as stationary phase. The retention time for metoprolol succinate and telmisartan were 3.067 min and 5.653 min, respectively. Linearity was observed in the concentration range of 5–80 µg/mL for metoprolol succinate and 5–60 µg/mL for telmisartan. The coefficient of correlation for metoprolol succinate and telmisartan was found to be 0.9990 and 0.9980, respectively. The results of analysis have been validated statistically and by recovery studies. Both the drugs were subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat, and photolytic degradation. The degradation products of telmisartan and metoprolol succinate were well resolved from the pure drugs with significant differences in the retention time values. This method can be successfully employed for simultaneous quantitative analysis of metoprolol succinate and telmisartan in bulk drugs and formulations.

Synthesis, Characterization and Quantification of Simvastatin Metabolites and Impurities

Simvastatin is used in treatment of hypercholesterolemia because it regulates cholesterol synthesis as a result of its β-hydroxy acid acting as an inhibitor of 3-hydroxy-methylglutaryl coenzyme A (HMG-CoA). The present communication deals with synthesis, characterization and development of accurate, precise and sensitive Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for simultaneous estimation of simvastatin and its synthetic impurities. The impurities methyl ether and β-hydroxy acid of simvastatin were synthesized in the laboratory and characterized by MS, NMR and FT-IR spectroscopy. The separation of simvastatin and its impurities was carried out on an isocratic JASCO RP-HPLC system using KYA TECH HIQ SIL C18 column (150 × 4.6 mm internal diameter, particle size 5 μm) operating at ambient temperature using acetonitrile:water (80:20 v/v) with 0.1% orthophosphoric acid as mobile phase. The method developed for HPLC analysis of three impurities along with simvastatin was validated using ICH Q2B (R1) guidelines and it complied with these guidelines. The results of analysis were found to be in the range of 98.14% to 101.89% for all analytes with acceptable accuracy and precision. The method can be used for detection and quantification of synthetic impurities in bulk or formulations of simvastatin.

Determination and quantification of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography–tandem mass spectrometry

A novel method using liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS) has been optimized and established for the qualitative and quantitative analysis of ten active phenolic compounds originating from the pigeon pea leaves and a medicinal product thereof (Tongluo Shenggu capsules). In the present study, the chromatographic separation was achieved by means of a HiQ Sil C18 V reversed-phase column with a mobile phase consisting of methanol and 0.1% formic acid aqueous solution. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the selected reaction monitoring (SRM) analysis was employed for the detection of ten analytes which included six flavonoids, two isoflavonoids and two stilbenes. All calibration curves showed excellent coefficients of determination ( r 2 ≥ 0.9937) within the range of tested concentrations. The intra- and inter-day variations were below 5.36% in terms of relative standard deviation (RSD). The recoveries were 95.08–104.98% with RSDs of 2.06–4.26% for spiked samples of pigeon pea leaves. The method developed was a rapid, efficient and accurate LC–MS/MS method for the detection of phenolic compounds, which can be applied for quality control of pigeon pea leaves and related medicinal products.

Spectrophotometric and reversed-phase high-performance liquid chromatographic methods for simultaneous determination of escitalopram oxalate and clonazepam in combined tablet dosage form.

Simple, accurate, precise, and sensitive ultraviolet spectrophotometric and reversed-phase high-performance liquid chromatographic (RP-HPLC) methods for simultaneous estimation of escitalopram oxalate (ESC) and clonazepam (CLO) in combined tablet dosage form have been developed and validated. The spectroscopic method employs an absorbance correction method using 238.6 and 308 nm as 2 wavelengths for estimation with methanol and water as solvents. Beer's law is obeyed in the concentration range of 10.0-50.0 and 0.5-3.0 micro/mL for ESC and CLO, respectively. The RP-HPLC method uses a Jasco HPLC system with HiQ SiL C18 column (250 x 4.6 mm id) acetonitrile-0.005 M tetrabutylammonium hydrogen sulfate (55 + 45, v/v) as the mobile phase, and satranidazole as an internal standard. The detection was carried out using an ultraviolet detector set at 287 nm. For the HPLC method, Beer's law is obeyed in the concentration range of 10.0-60.0 and 0.5-3.0 microg/mL for ESC and CLO, respectively. Both methods have been successfully applied for the analysis of the drugs in a pharmaceutical formulation. Results of analysis were validated statistically and by recovery studies.

Simultaneous Determination of Diclofenac Sodium and Rabeprazole Sodium in Bulk and Pharmaceutical Dosage Form by LC

A simple, selective, rapid, precise and accurate reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of diclofenac sodium and rabeprazole sodium from pharmaceutical formulations. The method was developed using a HiQ SiL C18 (250 mm × 4.6 mm i.d.) column with a mobile phase consisting of methanol:water, (80:20 v/v), at a flow rate of 1.25 mL min−1. Detection was carried out at 284 nm. Indapamide was used as an internal standard. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed method can be used for the estimation of these drugs in combined dosage forms.

Diversity of Translation Start Sites May Define Increased Complexity of the Human Short ORFeome

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048-2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5'-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5'-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome.

Determination of contents of 10-Hydroxycamptothecin inCamptotheca acuminata by high-performance liquid chromatogram

The determination method of 10-hydroxycamptothecin inCamptotheca acuminatu fruits by high-performance liquid chromatogram (HPLC) was studied. The HPLC analysis was performed on a HIQ sil C18(4.6×250 mm) column with mobile phase of acetonitrile-water (3:7, V:V), flow rate 1 mL·min−1 and UV detective wavelength 266 nm. Extracting 10-hydroxycamptothecin by ultrasonic method from fruits ofC. acuminata to prepare samples for analysis was systematically discussed. The optimal extraction condition was carried out by 60% alcohol solution at 60°C for 50 minutes.

Chromatographic properties of reversed phase stationary phases under pressure and electro driven conditions. Effect of buffer composition

Four different reversed-phase (RP) stationary phases (CEC Hypersil C18, Zorbax Rx SIL C18, Zorbax 300 Rx SIL C18 and Zorbax PSM1000/C18) were examined under high-performance liquid chromatographic (pressure-driven, HPLC), and capillary lectro- chromatographic (electro-driven, CEC) conditions using an acetonitrile mobile phase combined with twenty different buffer systems (different cations, anions, pH and/or ionic strengths). Chromatographic performance tests under HPLC and CEC conditions were carried out using acidic, basic and neutral polar/non-polar compounds. Parameters such as plate number, retention factor and asymmetry were used to describe the behavior of the RP-columns under both HPLC and CEC conditions. The buffer systems differently influence chromatographic characteristics of porous RP-phases under CEC and HPLC conditions. Thus the choice of an appropriate buffer can be critical for an application applied on an entire system.

Reversed phase planar chromatography of dansyl DL amino acids with bovine serum albumin in the mobile phase

The chromatographic behavior of twelve dansyl DL amino acids, one D isomer and eleven L isomers on RP18W/UV254, RP18W/F254s, and Sil C18−50 UV254 plates developed with aqueous—organic solutions containing bovine serum albumin (BSA) as chiral complexing agent has been extensively investigated. Enantiomeric resolution is highly dependent on mobile phase pH and ionic strength, and on the concentration of both BSA and organic modifier. All the racemates have been resolved within a development time of 1 h 30 min. The selectivity factors (α) for the dansyl amino acids have been compared with those from planar chromatography for the corresponding DNP, DNPy, and Fmoc amino acids, and with those of the same dansyl derivatives on a column prepared from BSA bound to silica gel.

Ion-pair high performance liquid chromatographic separation and determination of thiabendazole, diphenyl and o-phenylphenol in citrus fruits and banana.

An ion-pair high performance liquid chromatographic method was described for the simultaneous determination of thiabendazole (TBZ), diphenyl (DP) and o-phenylphenol (OPP) in citrus fruits and for the high sensitive determination of TBZ in banana. The use of ion-pair reagents such as 1-laurylsulfate for TBZ enabled the separation of three fungicides from interfering peaks. The fungicides were chromatographed on a Finepack SIL C18 column with acetonitrile-methanol-water (40 : 25 : 35) mixture adjusted pH to 2.5 containing 0.01 M sodium 1-laurylsulfate as mobile phase. The three fungicides in citrus fruits were detected by fluorescence detector set at excitation (Ex) 285 nm and emission (Em) 325 nm, and TBZ in banana was detected at Ex 305 nm and Em 350 nm. The recoveries of TBZ, DP and OPP added to citrus fruits were 94.1-98.0%, 90.1-93.0%, 92.0-95.0%, respectively, and the recovery of TBZ added to banana was 92.1-94.3%. The detection limits of DP, OPP and TBZ in citrus fruits were 0.5, 0.1 and 0.1 mg/kg, respectively, and the detection limit of TBZ in banana was 0.005 mg/kg.