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The optimization of HPLC method involves several variables whose influence has been widely studied. However, inmost of the cases, only process variables are taken into account. In this work, the influence of mixture compositionon peak quality parameters of Pitavastatin calcium in bulk and tablet dosage form has been studied using a mixturesimplex design. A simplex centroid design with axial points in a pseudo-component representation was generated fromthe pure mixture components. Twelve ternary mixture mobile phases corresponding to augmented design points weretested to separate the drug in sample. The statistical analysis was performed to generate the polynomial equation foreach response. The desirability approach was used to determine the optimal mobile phase composition. Furthermore,the method was validated as per the ICH guidelines using specificity, linearity, accuracy, precision, sensitivity, systemsuitability, and robustness. The results of experimental design were statistically tested for full and in portion to getbest fitted model which accurately describe changes in the proportion of these solvents in the mobile phase close to theregion of optimal peak quality. The method demonstrated optimum chromatographic separation with isocratic elutionof the mobile phase containing a mixture of acetonitrile-water (pH 3.0)-tetrahydrofuran (43:55:02, v/v/v) with a flowrate at 1.0 ml/minute. Design of experiment optimization strategy is a powerful tool to acquire the maximum qualitydata while performing minimum number of experiments. The mobile phase composition was successfully optimizedusing simplex centroid mixture design with desirability approach. Additionally, developed method can be appliedfor routine quantitative analysis of Pitavastatin calcium in bulk and tablet dosage form as it was found to be simple,sensitive, and robust.
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Chromatography can be described as a mass transfer process involving adsorption using a nonpolar stationary phase and a mobile polar phase titrating through the column. The active component of the column, the sorbent or the stationary phase , is typically a granular material made of solid particles (e.g. silica, polymers, etc.,). The component of the sample mixture are separated from each other by means of mobile phase and different degrees of interaction with the sorbent particles based on their relative polarity. In the present study we have extracted piperine from grounded pepper using different chemicals such as petroleum ether, acetone and methanol. Petroleum ether extraction showed higher piperine content of 9.12% than methanol and acetone 3.15% and 3.37% respectively.
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OBJECTIVE： To establish a method to determine the contents of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla. METHODS： The separation degree of ionic liquid 1-butyl-3-methylimidazolium chloride （C4mimCl） as mobile phase additive was compared with that of mobile phase without additives and with traditional additive triethylamine （which damaged the chromatographic column）. The optimum concentration of C4mimCl was screened and the contents of rhynchophylline and isorhynchophylline in U. rhynchophylla from 4 habitats in Jiangxi province were determined by the newly established method. The determination was performed on Dikmatech Diamonsil Plus C18 column， the mobil phase was acetonitrile-buffer （0.1％ phosphoric acid+3.0 mmol/L C4mimCl）， gradient elution. UV detection wavelength was set at 245 nm and the flow rate was 1 mL/min. Sample size was 10 μL. RESULTS： When mobile phase had no additives or 3.0 mmol/L triethylamine and 3.0 mmol/L C4mimCl were added as additives， the separation of rhynchophylline from the front peak was 1.02， 1.23 and 1.72， and the separation from the back peak was 1.06， 6.00 and 4.25， respectively. The symmetry factors were 0.81， 0.86 and 1.13， respectively. The separation of isorhynchophylline from the front peak was 0.96， 3.89 and 4.05， and the separation from the back peak was 1.02， 2.34 and 2.36， respectively. The symmetry factors were 0.88， 0.81 and 0.96， respectively. The linear range of rhynchophylline and isorhynchophylline were 4.93-157.76 （r＝0.999 9） and 4.98-159.50 μg/mL （r＝1.000）， respectively. The quantitative limits were 0.486 4， 0.793 6 μg/mL， respectively. RSDs of precision， repeatability， stability and durability tests were all less than 5％ （n＝6）. The recovery rates were 102.9％-107.8％ （RSD＝1.7％，n＝6） and 95.4％-106.3％ （RSD＝3.9％，n＝6）， respectively. The content of rhynchophylline and isorhynchophylline in U. rhynchophylla from 4 habitats were 0.758-1.343 and 1.511-1.823 mg/g， respectively. CONCLUSIONS： Addition of C4mimCl into mobile phase can enhance its separation. Established HPLC method is rapid， accurate and reproducible， which can be used for content determination of rhynchophylline and isorhynchophylline in U. rhynchophylla.
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Introduction: High-performance liquid chromatography is one of the powerful qualitative and quantitative tech-niques, generally used for the analysis of pharmaceutical preparations. HPLC’s virtue lies in its versatility. We can use it to separate compounds of molecular weights from 54-450000Daltons. Amounts of material to be detected can vary from pictograms and nanograms to micrograms and milligrams to grams depends on which kind of detectors used for chromatographically separations. This article was prepared with an aim to review different aspects of HPLC, such as principle, types, and its applications. Keywords: High performance liquid chromatography, applications, mobile phase, stationary phase
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Objective To establish a method for the chiral separation and determination of S-isomer in epinephrine hydrochloride injection by HPLC with chiral mobile phase additives. Methods Column of Purospher? STAR RP-18 (4. 6 mm×250 mm, 5 μm) was used. The mobile phase was acetonitrile-10 mmol·L-1 sodium dihydrogen phosphate buffer containing 10 mmol·L-1 sulfobutylether-b-cyclodextrin (pH adjusted to 3. 0 with phosphoric acid) (98. 5:1. 5), detection wavelength was 280 nm, the flow rate was 0. 8 mL·min-1 , and the column temperature was 30 ℃ . Results Good linear relationship was established between the peak area and the concentration of S-isomer over the range of 5. 02-1501. 50 μg·mL-1 (R2 =0. 999 7). The detection limit was 0. 05 μg·mL-1 . Conclusion The proposed method shows high repeatability and durability. It can be employed for the quality control of S-isomer in epinephrine hydrochloride injection.
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A simple, specific, accurate, precise and sensitive Reverse Phase High Performance Liquid Chromatographic method has been developed for the quantitation of Tinidazole in both pure and pharmaceutical dosage forms. . Chromatographic separation achieved isocratically on a ODS2 reverse phase column [Use Symmetry C18, 250 X 4.6mm, 5μ] utilizing a mobile phase of Methanol. The flow rate was 0.5 ml/min and the effluents were monitored at 310 nm. The retention time was 4.610min. The linearity was in the range of 5-25μg / ml. This method was validated for linearity, precision and accuracy. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug.
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AIM@#Variation in structure-related components in plant products prompted the trend to establish methods, using multiple or total analog analysis, for their effective quality control. However, the general use of routine quality control is restricted by the limited availability of reference substances. Using an easily available single marker as a reference standard to determine multiple or total analogs should be a practical option.@*METHOD@#In this study, the Ultra-HPLC method was used for the baseline separation of the main components in ginseng extracts. Using a plant chemical component database, ginsenosides in ginseng extracts were identified by Ultra-HPLC-MS analysis. The charged aerosol detection (CAD) system with post-column compensation of the gradient generates a similar response for identical amounts of different analytes, and thus, the content of each ginsenoside in ginseng extracts was determined by comparing the analyte peak area with the reference standard (determination of total analogs by single marker, DTSM). The total ginsenoside content was determined by the summation of reference standard and other ginsenoside components.@*RESULTS@#The results showed that DTSM approaches were available for the determination of total ginsenosides in a high purity ginseng extract because of the removal of impurities. In contrast, DTSM approaches might be suitable for determination of multiple ginsenosides without interference from impurities in the crude ginseng extract.@*CONCLUSION@#Future practical studies similar to the present study should be conducted to verify that DTSM approaches based on CAD with post-column inverse gradient for uniform response are ideal for the quality control of plant products.
Subject(s)
Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Ginsenosides , Mass Spectrometry , Panax , Chemistry , Reference StandardsABSTRACT
Objective:To improve the determination method for penicillin V potassium tablets. Methods:An Inertis ODS-SP C18 column (150 mm × 4. 6 mm, 5 μm) was used. The mobile phase consisted of A and B(60∶40) [ A: pH 3. 5 phosphate buffer(0. 5 mol·L-1 potassium dihydrogen phosphate solution, adjusting pH to 3. 5 with phosphoric acid)-acetonitrile-water (10∶30∶60);B:pH 3. 5 phosphate buffer-acetonitrile-water (10∶55∶35)]. The flow rate was 1. 0 ml·min-1, the detection wavelength was 268nm, the column temperature was room temperature and the injection volume was 10 μl. Results:The linear range of penicillin V potassium was 0. 032 9-0. 821 6 mg·ml-1(r=0. 999 9, and the average recovery was 99. 9%( RSD=0. 6%, n=9). Conclusion:The method is reproducible and accurate, and can be used in the content determination of penicillin V potassium tablets and capsules.
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Objective To establish a self-made chiral column for enantiomeric separation of salbutamol. Methods We used different concentrations of acid,alkali additives in the polar phase flow for enantiomers separation of salbutamol by using 3, 4-dichlorophenyl isocyanate vancomycin chiral column, and discussed the chiral recognition mechanism. Results The ratio of acid to alkali additive in the mobile phase was 0.01%:0.01% (V/V), the flow rate was 1ml/min, the column temperature was 25℃, and the best separation of enantiomers of salbutamol was obtained,the selective factor was 1.16, the separation degree reached 1.41. Conclusion Self-made 3, 4-two chlorophenyl isocyanate vancomycin chiral column is effective for salbutamol separation, and it can be as a reference for developing other similar chiral stationary phase.
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OBJECTIVE: To performe chiral separation of three pharmaceutical racemates collected in Ch. P 2010 containing N-alkyl groups, including verapamil hydrochloride, chlorphenamine maleate, and chloroquine diphosphate by using SBE-β-CD as a mobile-phase additive in an HPLC system. METHODS: The analysis was carried out on an Alltima C18 column. The mobile phase consisted of acetonitrile and a phosphate solution added with SBE-β-CD and NaH2PO4, whose pH value was adjusted to 2.5 by phosphoric acid. The flow rate was set at 0.8 mL · min-1, and the column temperature was 25°C. The UV detection was carried out at 332, 264 and 278 nm, respectively. The effects of different factors on the separation effect were investigated, including CD types (β-CD, HP-β-CD), SBE-β-CD concentration, pH value, oven temperature, and buffer concentration. RESULTS: The established method was able to separate the three racemates and the mechanism had been discussed. CONCLUSION: Three racemates can be separated on C18 column using SBE-β-CD as a chiral mobile phase additive. This method might be applied to separate various types of biologically important enantiomers which have N-alkyl groups in the chemical structure.
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A new, simple, accurate, and precise high-performance thin-layer chromatographic (HPTLC) method has been established for quantitative analysis of valsartan in tablet formulations. Standard and sample solutions of valsartan were applied to precoated silica gel G 60 F254 HPTLC plates and the plates were developed with chloroform: acetonitrile: toluene: glacial acetic acid, in the ratio 1:8:1:0.1 (v/v) (v/v), as mobile phase. UV detection was performed at 254 nm. The retention factors of valsartan was 0.65. The calibration plot for Valsartan standard was linear with r =0.9999, slope = 5.328 and intercept=356.9. The limit of detection and limit of quantitation of Valsartan were found to be 5and 16 ng per spot respectively. The percentage recovery was found to be 99.37% for Valsartan. The method showed good repeatability and recovery with relative standard deviation less than 2. Method was validated in accordance with the requirements of ICH guidelines and was shown to be suitable for purpose. The method is selective and specific can be used for determination of the routine analysis of valsartan in tablets. Tablet excipients did not interfere with the chromatography.
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The status of the use of carcinogenic or strong virose organic solvents as mobile phase in the tests of partial drugs of Chinese medicine is summarized and analyzed based on the Pharmacopeia of the People's Republic of China (2005 ed) Volume 1. A few new testing methods that were used as the mobile phase with non-benzene and its homologue in the national standards published by SFDA are used for reference. Finally, the proposals to investigate the reasonable methods of safe mobile phase fleetly and limit the use of carcinogenic or strong virose organic solvents as the mobile phase in the test of drugs strictly are presented.
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BACKGROUND: Optimal use of tricyclic antidepressants (TCAs) requires serum monitoring to determine if the appropriate therapeutic range has been attained and to assess possible side effects. This study was to evaluate the resolution capacity of the following four mobile phases which were previously reported; mobile phase I (methanol, acetonitrile and 5 mmol/L Na2HPO4: 41/15/44 by volume), II (methanol, acetonitrile and 5 mmol/L Na2HPO4,: 15/60/25 by volume), III (acetonitrile and 2-propanol: 95/5 by volume) and IV (methanol and n-butylamine: 99.5/0.5 by volume). METHODS: Amitriptyline (AT), nortriptyline (NT), imipramine (IMI) and doxepin (DOX) were used for the selection of appropriate mobile phase in high performance liquid chromatographic (HPLC) determination. TCAs were extracted from serum with hexane, isoamyl alcohol (99:1). The drug was re-extracted into 0.1 N HCl and an aliquot was injected into the HPLC. The analytical column was C-18 reversed phase column (3.9 mm x 30 cm; Waters, USA) with the flow rate of 1.5 mL/min. The UV detector signal was monitored at 254 nm. RESULTS: Mobile phase I disclosed 9.8-15.8 retention time (min), 5.1-8.8 capacity ratio and 1.0-2.2 resolution factors for the above four TCAs. Precision studies using this mobile phase demonstrated a coefficient variation of 2.4-4.7% in the concentration range of 500-125 ng/mL. Analytical recovery of AT and IMI was 85-90% at a concentration of 125 ng/mL and 250 ng/mL. CONCLUSIONS: Mobile phase I provided a reliable and excellent resolution of TCAs in the use of HPLC with the C-18 reversed phase column and UV absorbance detector.