Optimization and validation of RP-HPLC method for simultaneous estimation of palbociclib and letrozole

A simple, rapid, and robust RP-HPLC method have been developed and validated to measure palbociclib (PB) and letrozole (LT) at single wavelength (254?nm). A isocratic elution of samples performed on Intersil C₈ (4.6?mm?×?250?mm particle size 5?μm) column with mobile phase consisting 0.02 M sodium dihydrogen phosphate buffer (pH 5.5): acetonitrile: methanol (80:10:10 v/v/v) delivered at flow rate 1.0?mL?min^?1. A good linear response was achieved over the range of 5–50?μg?mL^?1. The LODs for PB and LT were found to be 0.098 and 0.0821 µg?mL^?1, while the LOQs for PB and LT were 0.381–0.315 µg?mL^?1, respectively. The method was quantitatively evaluated in terms of system suitability test, linearity, precision, accuracy (recovery) and robustness as per standard guidelines. The method is simple, convenient and suitable for the analysis of PB and LT in bulk drug.     

RP-HPLC-UV method development and validation for simultaneous determination of terbutaline sulphate,ambroxol HCl and guaifenesin in pure and dosage forms

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Surface modification of PLGA nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel

Background

Poly lactic-co-glycolic acid (PLGA) based nanoparticles are considered to be a promising drug carrier in tumor targeting but suffer from the high level of opsonization by reticuloendothelial system due to their hydrophobic structure. As a result surface modification of these nanoparticles has been widely studied as an essential step in their development. Among various surface modifications, human serum albumin (HSA) possesses advantages including small size, hydrophilic surface and accumulation in leaky vasculature of tumors through passive targeting and a probable active transport into tumor tissues.

Methods

PLGA nanoparticles of docetaxel were prepared by emulsification evaporation method and were surface conjugated with human serum albumin. Fourier transform infrared spectrum was used to confirm the conjugation reaction where nuclear magnetic resonance was utilized for conjugation ratio determination. In addition, transmission electron microscopy showed two different contrast media in conjugated nanoparticles. Furthermore, cytotoxicity of free docetaxel, unconjugated and conjugated PLGA nanoparticles was studied in HepG2 cells.

Results

Size, zeta potential and drug loading of PLGA nanoparticles were about 199 nm, −11.07 mV, and 4%, respectively where size, zeta potential and drug loading of conjugated nanoparticles were found to be 204 nm, −5.6 mV and 3.6% respectively. Conjugated nanoparticles represented a three-phasic release pattern with a 20% burst effect for docetaxel on the first day. Cytotoxicity experiment showed that the IC50 of HSA conjugated PLGA nanoparticles (5.4 μg) was significantly lower than both free docetaxel (20.2 μg) and unconjugated PLGA nanoparticles (6.2 μg).

Conclusion

In conclusion surface modification of PLGA nanoparticles through HSA conjugation results in more cytotoxicity against tumor cell lines compared with free docetaxel and unconjugated PLGA nanoparticles. Albumin conjugated PLGA nanoparticles may represent a promising drug delivery system in cancer therapy.     

HPLC法同时测定Beagle犬血浆中烟酸和烟尿酸的浓度

目的建立比格犬血浆中烟酸及其代谢物烟尿酸的RP-HPLC测定方法。方法血浆样品用甲醇沉淀蛋白,上清液氮气流吹干,残留物用流动相复溶后分析,以富马酸为内标。色谱柱:Diamon-sil C18柱(4.6 mm×200 mm,5μm),流动相:甲醇-0.04 mol.L-1四丁基溴化铵溶液(体积比为30∶70),流速:1.0 mL.min-1,柱温:20℃,检测波长:262 nm。结果烟酸在0.20~80.0 mg.L-1质量浓度内线性关系良好,高、中、低3种质量浓度QC样品的日内、日间RSD均小于8.0%,提取回收率在90.0%~97.8%之间;烟尿酸在0.10~5.0 mg.L-1浓度范围内线性关系良好,高、中、低3种浓度QC样品的日内、日间RSD均小于9.1%,提取回收率在95.9%~99.5%之间。结论该方法适用于血浆中烟酸和烟尿酸浓度的测定和药动学研究。     

RP-HPLC Estimation of Ramipril and Telmisartan in Tablets

A rapid high performance liquid chromatographic method has been developed and validated for the estimation of ramipril and telmisartan simultaneously in combined dosage form. A Genesis C18 column having dimensions of 4.6×250 mm and particle size of 5 μm in isocratic mode, with mobile phase containing a mixture of 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid): methanol:acetonitrile (15:15:70 v/v/v) was used. The mobile phase was pumped at a flow rate of 1.0 ml/min and the eluents were monitored at 210 nm. The selected chromatographic conditions were found to effectively separate ramipril (R_t: 3.68 min) and telmisartan (R_t: 4.98 min) having a resolution of 3.84. The method was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantitation. Linearity for ramipril and telmisartan were found in the range of 3.5-6.5 μg/ml and 28.0-52.0 μg/ml, respectively. The percentage recoveries for ramipril and telmisartan ranged from 99.09-101.64% and 99.45-100.99%, respectively. The limit of detection and the limit of quantitation for ramipril was found to be 0.5 μg/ml and 1.5 μg/ml respectively and for telmisartan was found to be 1.5 μg/ml and 3.0 μg/ml, respectively. The method was found to be robust and can be successfully used to determine the drug content of marketed formulations.     

RP-HPLC法测定降血压肽/聚乳酸-羟基乙酸微球中主药含量及包封率

目的:建立测定降血压肽(AHP)/聚乳酸-羟基乙酸(PLGA)微球中AHP含量和包封率的反相高效液相色谱(RP-HPLC)法。方法:样品经二氯甲烷破坏并用水萃取后进行测定。反相色谱柱为Eclipse XDB-C18,流动相为乙腈-水(含0.05%三氟乙峰酸)均=得1到7∶很83好,流的速分为离1,.0A mHPL.检m测in浓-1,度柱线温性为范30围℃为,2检5～测6波25长μ为g.1m9L9-n(1mr,=进0样.99量9为9),2平0μ均L回。收结率果为:在9此8.7色8%谱,条日件内下和A日H间P与精辅密料度及分溶别为剂0.53%、0.86%(n=5)。结论:所建立的方法操作简单、快速,结果准确、可靠,可用于AHP/PLGA微球中主药含量及包封率的测定。     

Development and validation of RP-HPLC method for sildenafil citrate in rat plasma-application to pharmacokinetic studies

Sildenafil citrate (SIL) is used in the treatment of erectile dysfunction and other chronic disorders. For the pharmacokinetic investigation of SIL we developed a simple and sensitive method for the estimation of SIL in rat plasma by reverse phase high-performance liquid chromatography (RP-HPLC). The drug samples were extracted by liquid–liquid extraction with 300 μl of acetonitrile and 5 ml of diethyl ether. Chromatographic separation was achieved on C₁₈ column using methanol:water (85:15 v/v) as mobile phase at a flow rate of 1 ml/min and UV detection at 230 nm. The retention time of SIL was found to be 4.0 min having a separation time less than 5 min. The developed method was validated for accuracy, precision, linearity and recovery. Linearity studies were found to be acceptable over the range of 0.1–6 μg/ml. The method was successfully applied for the analysis of rat plasma sample for the application in pharmacokinetic study, drug interaction, bioavailability and bioequivalence.     

RP-HPLC法测定盐酸青藤碱PLGA纳米粒的包封率及载药量

目的：建立盐酸青藤碱PLGA纳米粒的包封率及载药量的测定方法。方法：SHIM-PACK VP-ODS柱,流动相为甲醇-10mol·L-1 NaH2PO4溶液（38∶62）,柱温为30℃,检测波长为262nm,流速1mL·min-1。结果：盐酸青藤碱在0.5～50μg·mL-1范围内线性关系良好,R2=0.9999,溶剂和辅料对其无明显干扰,平均回收率为95.31%,日内和日间RSD分别为1.31%和1.59%（n=5）。结论：该方法简便快速,准确可靠,可用于盐酸青藤碱PLGA纳米粒包封率及载药量的测定。     

Simultaneous determination of emtricitabine,tenofovir alafenamide fumarate and dolutegravir sodium by validated stability-indicating RP-HPLC-DAD method

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Development and validation of indirect RP-HPLC method for enantiomeric purity determination of D-cycloserine drug substance

A new chiral purity method was developed for D-cycloserine (D-cys) by reverse phase HPLC and validated. Chiral derivatizing reagents, viz., o-phthalaldehyde and N-acetyl-L-cysteine were utilized in this method. The resultant diastereomers were resolved using Zorbax SB Phenyl HPLC column under isocratic elution. A mobile phase of 95:05 (v/v), 20mM Na(2)HPO(4) (pH 7), and acetonitrile, respectively, was used with the flow rate of 1.0 mL/min and UV detection at 335 nm. The method development with different chiral stationary phases and chiral derivatization reagents were also investigated. The stability of diastereomer derivative and influence of organic modifier and pH of the mobile phase were studied and optimized. The stability-indicating capability of the method was established by performing stress studies under acidic, basic, oxidation, light, humidity and thermal conditions. The detection and quantitation limit of L-cycloserine (L-cys) were 0.015 and 0.05% (w/w), respectively. A linear range from 0.05 to 0.30% (w/w) was obtained with the coefficient of determination (r(2)) 0.998. The recovery obtained for L-cys was between 92.9 and 100.2%. This method was applied successfully in pharmaceutical analysis to determine the content of L-cys in D-cys bulk drug.     

Development and validation of a reverse phase liquid chromatography method for the quantification of rasagiline mesylate in biodegradable PLGA microspheres

In the present study, a reverse phase high performance liquid chromatographic method was developed and validated for the determination of rasagiline mesylate in biodegradable microspheres. Chromatographic separation was carried out on a RP-18 column using a mobile phase consisting of acetonitrile:water (5:95, v/v) adjusted at pH 3.1. Flow rate was 1.0 ml min⁻¹ and UV detection at 290 nm. Acyclovir was used as the internal standard. The calibration curve was linear over the range 0.5–20.0 μg ml⁻¹. R.S.D. for precision was <1.8%. Accuracy ranged between 99.01% and 102.55% with a R.S.D. lower than 1.3%. LOD and LOQ were 0.07 μg ml⁻¹ and 0.23 μg ml⁻¹, respectively. The method was simple, rapid, and easy to apply, making it very suitable for routine analysis of rasagiline mesylate in biodegradable PLGA microspheres. It could be also used with reliability for the determination of the drug in other pharmaceutical dosage forms.     

Development and validation of an ion-pair RP-HPLC method for the determination of oligopeptide-20 in cosmeceuticals

Oligopeptide-20 is a growth-factor mimicking peptide used in cosmeceuticals. This article describes the development and validation of an ion-pair reversed-phase liquid chromatography method that allows, after liquid-liquid extraction, the quantification of oligopeptide-20 in cosmetic creams. Chromatographic separation was achieved on a cyanopropyl Hypersil analytical column (100 mm × 2.1 mm i.d., 5 μm particle size), using a mobile phase of acetonitrile-heptafluorobutyric acid (pH = 2.5, 9.0 mM) (70:30, v/v) containing 0.045% diethylamine at a flow rate of 0.50 mL min⁻¹. Ultraviolet (UV) spectrophotometric detection at 225 nm was used. The method had linear calibration curve over the range 1.35-4.95 μg mL⁻¹ for oligopeptide-20. The intra- and inter-day RSD values were less than 3.3%, while the relative percentage error, %E_r, was less than 1.9. The developed method was applied successfully to the quality control of a cosmetic cream containing 0.003% (w/w) oligopeptide-20.     

Development and validation of a stability indicating RP-HPLC method for the simultaneous determination of related substances of albuterol sulfate and ipratropium bromide in nasal solution

A simple, sensitive and specific RP-HPLC method was developed for the quantification of related impurities of albuterol sulfate (AS) and ipratropium bromide (IB) in liquid pharmaceutical dosage form. The chromatographic separation employs gradient elution using an inertsil C8-3, 250 mm × 4.6 mm, 5 μm columns. Mobile phase consisting of solvent A (solution containing 2.5 g of potassium dihydrogen phosphate and 2.87 g of heptane-1-sulfonic acid sodium salt per liter of water, adjusted to pH 4 with orthophosphoric acid) and solvent B (acetonitrile) delivered at a flow rate of 1.0 ml min⁻¹. The analytes were detected and quantified at 210 nm using photodiode array (PDA) detector. The method was validated as per ICH guidelines, demonstrating to be accurate and precise (repeatability and intermediate precision level) within the corresponding linear range of known impurities of AS and IB. The specificity of the method was investigated under different stress conditions including hydrolytic, oxidative, photolytic and thermal as recommended by ICH guidelines. Relevant degradation was found to take place under hydrolytic and oxidative conditions. Robustness against small modification in pH, column oven temperature, flow rate and percentage of the mobile phase composition was ascertained. Lower limit of quantification and detection were also determined. The peak purity indices (purity angle < purity threshold) obtained with the aid of PDA detection and satisfactory resolution between related impurities established the specificity of the determination. All these results provide the stability indicating capability of the method.     

Development and characterization of hyaluronic acid decorated PLGA nanoparticles for delivery of 5-fluorouracil

The present investigation was aimed to develop and explore the prospective of engineered PLGA nanoparticles as vehicles for targeted delivery of 5-fluorouracil (5-FU). Nanoparticles of 5-FU-loaded hyaluronic acid-poly(ethylene glycol)-poly(lactide-co-glycolide) (HA-PEG-PLGA-FU) copolymer were prepared and characterized by FTIR, NMR, transmission electron microscopy, particle size analysis, DSC, and X-ray diffractometer measurement studies. The nanoparticulate formulation was evaluated for in vitro release, hemolytic toxicity, and hematological toxicity. Cytotoxicity studies were performed on Ehrlich ascites tumor (EAT) cell lines using MTT cell proliferation assay. Biodistribution studies of ^99mTc labeled formulation were conducted on EAT-bearing mice. The in vivo tumor inhibition study was also performed after i.v. administration of HA-PEG-PLGA-FU nanoparticles. The HA conjugated formulation was found to be less hemolytic but more cytotoxic as compared to free drug. The hematological data suggested that HA-PEG-PLGA-FU formulation was less immunogenic compared to plain drug. The tissue distribution studies displayed that HA-PEG-PLGA-FU were able to deliver a higher concentration of 5-FU in the tumor mass. In addition, the HA-PEG-PLGA-FU nanoparticles reduced tumor volume significantly in comparison with 5-FU. Thus, it was concluded that the conjugation of HA imparts targetability to the formulation, and enhanced permeation and retention effect ruled out its access to the non-tumor tissues, at the same time favored selective entry in tumors, thereby reducing the side-effects both in vitro and in vivo.     

Analytical method development and validation of simultaneous estimation of rabeprazole,pantoprazole, and itopride by reverse-phase high-performance liquid chromatography

A simple, selective, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of rabeprazole (RP), pantoprazole (PP), and itopride (IP) has been developed. The compounds were well separated on a Phenomenex C₁₈ (Luna) column (250 mm × 4.6 mm, dp = 5 μm) with C₁₈ guard column (4 mm × 3 mm × 5 μm) with a mobile phase consisting of buffer containing 10 mM potassium dihydrogen orthophosphate (adjusted to pH 6.8): acetonitrile (70:30 v/v) at a flow rate of 1.0 mL/min and ultraviolet detection at 288 nm. The retention time of RP, PP, and IP were 5.35, 7.92, and 11.16 minutes, respectively. Validation of the proposed method was carried out according to International Conference on Harmonisation (ICH) guidelines. Linearity range was obtained for RP, PP, and IP over the concentration range of 2.5–25, 1–30, and 3–35 μg/mL and the r² values were 0.994, 0.978, and 0.991, respectively. The calculated limit of detection (LOD) values were 1, 0.3, and 1 μg/mL and limit of quantitation (LOQ) values were 2.5, 1, and 3 μg/mL for RP, PP, and IP correspondingly. Thus, the current study showed that the developed reverse-phase liquid chromatography method is sensitive and selective for the estimation of RP, PP, and IP in combined dosage form.     

Sensitive method for rapid estimation of Lornoxicam in bulk and its dosage form by RP-HPLC

Lornoxicam (6-chloro-4-hydroxy-2-methyl-N-2-pyridyl-2H-thieno-[2,3-e]-1,2-thiazine-3-carboxamide-1,1-dioxide) is persist as a non-steroidal anti-inflammatory drug of the oxicam class with analgesic, anti-inflammatory and antipyretic properties. A fast, accurate and sensitive chromatographic method for estimation of Lornoxicam was developed as no official method available for detection. The chromatographic separation employs isocratic elution by utilizing an inertsil ODS-C 18 , 250 mm×4.6 mm, 5 μm columns. Mobile phase consisting of solvent (40 mL acetonitrile and 60 mL 0.1 M phosphate buffer (pH 6.8 was adjusted with triethylamine)) endowed at a flow rate of 1.0 mL/min. The analyte was detected and quantified at 290 nm using UV detector. The method was validated according to ICH guidelines, illustrating to be accurate (recovery 99.08%-101.13%) and precise (intraday (0.27-1.32) and interday (0.59-1.59))within the corresponding linear range (10-60 μg/mL) with r 2 0.9992.     

Method development and validation for the simultaneous determination of meloxicam and pridinol mesylate using RP-HPLC and its application in drug formulations

A simple and reliable reversed-phase high-perfomance liquid chromatographic method has been developed and validated for the simultaneous determination of meloxicam and pridinol mesylate in their synthetic mixtures and combined tablet formulations. Both drugs were separated on a 250 mm x 4.6mm C18 column packed with 5 microm particles. The mobile phase, optimized through an experimental design, was a 51:9:40 (v/v/v) mixture of methanol, isopropanol and 50mM potassium phosphate buffer (pH 5.9), pumped at a flow rate of 1.0 ml min(-1). UV detection was performed at 225 nm. The method was validated in the sample concentration ranges of 33.7-61.8 mg l(-1) for meloxicam and 8.8-16.8 mg l(-1) for pridinol mesylate, where it demonstrated good linearity with r=0.9989 and 0.9987 (n=15), respectively. The assay was shown to be repeatable at concentration levels of 70%, 100% and 130%, with relative standard deviation values of 1.09% and 0.82% for meloxicam and pridinol, respectively. For independent 100% level samples, the intra-day precision was 0.4% and 1.0% while the intermediate precision was 0.7% and 1.0% for the drugs. The method demonstrated to be robust, resisting to small deliberate changes in pH, flow rate and composition (organic:aqueous ratio) of the mobile phase. The LOD values were 0.22 and 0.20 mg l(-1), while the LOQ were 1.7 and 1.1 mg l(-1), for meloxicam and pridinol, respectively. The applicability of the method was demonstrated by determining the drug content of two commercial pharmaceutical formulations, where it exhibited good performance.     

Development and validation of a simple HPLC method for simultaneous in vitro determination of amoxicillin and metronidazole at single wavelength

A simple, rapid, sensitive and robust reversed phase-HPLC method was developed and validated to measure simultaneously the amount of amoxicillin and metronidazole at single wavelength (254 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new floating-sustained release tablet formulation and its subsequent stability studies. An isocratic elution of filtered sample was performed on C18 column with buffered mobile phase (pH 4.0) and UV detection at 254 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.15 and 600 microg/ml for amoxicillin and 0.13 and 300 microg/ml for metronidazole were established. Intra and inter-day precision were less than 2.5%. The limits of detection (LOD) and quantification were 0.05 and 0.15 microg/ml for amoxicillin and 0.10 and 0.13 microg/ml for metronidazole. The determination of the two active ingredients was not interfered by the excipients of the products. Samples were stable in the release media (37 degrees C) and the HPLC injector at least for 12 h.     

RP-HPLC method for simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet formulation

A simple, precise and stability-indicating HPLC method was developed and validated for the simultaneous determination of bisoprolol fumarate and hydrochlorothiazide in pharmaceutical dosage form. The method involves the use of easily available inexpensive laboratory reagents. The separation was achieved on an Inertsil ODS 3V (25 cm × 4.6 mm) 5 μm column with isocratic flow. The mobile phase at a flow rate of 1.0 mL min⁻¹, consisted of 0.1 M potassium dihydrogen phosphate buffer and acetonitrile (70:30, v/v). The UV detection was carried out at 228 nm. A linear response was observed over the concentration range 2.5–50 μg mL⁻¹ of bisoprolol fumarate and the concentration range 6.25–125 μg mL⁻¹ of hydrochlorothiazide. Limit of detection and limit of quantitation for bisoprolol fumarate were 0.01 and 0.03 μg mL⁻¹, respectively and for hydrochlorothiazide were 0.01 and 0.05 μg mL⁻¹, respectively. The method was successfully validated in accordance to ICH guidelines acceptance criteria for specificity, linearity, accuracy, precision, robustness, ruggedness and system suitability. Individual drugs (bisoprolol fumarate and hydrochlorothiazide), their combinations and the tablets were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions. The resultant stressed samples were analyzed by the proposed method. The method gave high resolution among the degradation products and the analytes. The peak purity of analyte peaks in the stressed samples was confirmed by photodiode array detector. The method was used for accelerated stability study on marketed and in-house formulations. The analysis concluded that the method was selective for simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide and was stability-indicating.