A Quantitative Estimation of Piperacillin and Tazobactam in Generic and Branded Pharmaceutical Formulations by Reverse Phase High Performance Liquid Chromatography

Purpose: To develop a sensitive and rapid reverse phase high\nperformance liquid chromatography (HPLC) method for the measurement of\nthe levels of zidovudine (ZVD) and nevirapine (NVP) in human plasma. \nMethods: Standard stock solutions for HPLC analysis were prepared by\ndissolving ZVD and NVP in methanol. In the HPLC measurement, sample\ndetection was carried out at 246 nm using an ultraviolet (UV)-photo\ndiode array (PDA) detector. Plasma sample pretreatment consisted of\nprotein precipitation extraction with methanol. The compounds were\nseparated using a mobile phase consisting of a pH 3.0 solution\n(obtained by adjusting the pH of water with orthophosphoric acid):\nacetonitrile (73:27 v/v) on a Phenomenex LUNA C18, column (250×4.6\nmm i.d., 5µm) at a flow rate of 0.9 mL min. The total run time for\nthe assay was 10.2 min-1 . The method was validated over the range of\n300-9600 ng mL-1 and 200-6400 ng mL-1 for ZVD and NVP, respectively. \nResults: The lowest limits of quantification (LLOQ) and of detection\n(LOD) were 300 and 63 ng mL-1 for ZVD and 200 and 17 ng mLfor NVP,\nrespectively. The method was found to be accurate, with accuracy\nranging from -10.92 to +9.57 % and precise, with intra-day, inter-day\nas well as analyst to analyst precision of 0.68 to 9.38 %. Extraction\nrecoveries of the drugs from plasma were 91.39, 95.01, 89.51 % for ZVD\nand 90.93, 93.26, 92.13 % for NVP, for LQC (low quality control), MQC\n(medium quality control) and HQC (high quality control) samples,\nrespectively. Stability data revealed that the drugs were stable in\nplasma under various test conditions. Conclusion: This assay can be\nsuitably used for the determination of zidovudine (ZVD) and nevirapine\n(NVP) in human plasma and should be useful in HIV clinical trials and\nclinical therapeutic drug monitoring (TDM) programs. It would also be\npotentially useful in the determination of pharmacokinetic profiles and\nin bioequivalence studies in HIV research.

Flavonoids are significant secondary metabolites of vascular plants. They have been proven to possess numerous types of anti-bacterial, anti-tumor, anti-oxidant and anti-inflammatory bioactivities etc. Thereby, there are many flavonoids extracted and purified from plants and have been used for biological tests. Nevertheless, the traditional extraction methods require a large amount of initial sample, tedious and professional techniques and low yield of target compound. Until present, the advanced development of genetic engineerings and recombinant protein as platform allow synthesis of high amount of different types of flavoid in short time. Escherichia coli is one of the most popular host for whole-cell biotransformation of flavonoids and their derivatives due to its simple genetical, physiological system and fast growth. In this paper, we described the biosynthetic method of morin-3-O-rhamnopyranoside from a genetically engineered E. coli using morin as substrate. In particular, the E. coli harboring biosynthetic gene cluster of activated TDP-L-rhamnose and gene encoding for glycosyltransferase was used as host for biotransformation. Morin was fed into the culture broth of recombinant E. coli for rhamnosylation. The formation of morin-3-O-rhamnopyranoside was then quantitied and qualified using Thin Layer Chromatography (TLC), Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) assays. The highest yield of product (56.5 µM) was achieved after 48h, 32 oC and pH = 7.2-8.0. This result showed that morin-3-O-rhamnopyranoside was succesfully synthesized in the genetically engineered E. coli. Furthermore, metabolic engineering of intra-cellular system for improving absorption of substrate as well as excretion of product or enhancement of glycosyltransferase activity may increase the final yield of biotransformation process.

Eleven optical brighteners have been qualitatively and quantitatively determined using C18, C8, or C2 reverse phase high performance liquid chromatography (HPLC) columns. Comparable results also were obtained using radially compressed reverse phase HPLC cartridges. Mobile phases consist of acetonitrile and methanol in water with 0.05 M phosphate buffer. Quaternary ammonium salts also are added to the mobile phase for ion pair formation. Ultraviolet (UV) detection at 340 nm gives more than adequate sensitivity for brighteners formulated between 0.05 and 2.0%. Sample preparation is simple and all components can be eluted within 15 min. Powdered and liquid detergents, fabric softeners, and bleach boosters have been routinely analyzed and results closely agree with those obtained using thin layer chromatography and densitometry.

Methods in protein sequence analysis constitute important fields in rapid progress. We have experienced a continuous increase in analytical sensitivity coupled with decreases in time necessary for pur

The chlorogenic acid, rosmarinic acid, and caffeic acid contents in 100 selected plants were determined using reversed phase high performance liquid chromatography equipped with diode array detector. The optimum condition was 0.2% phosphoric acid in water (solvent A) and methanol (solvent B) as the mobile phase, which was set at 45% B for 20 minutes at a flow rate of 1.2 mL/min. The column temperature was maintained at 30 oC and the detection wavelength was 325 nm. Among 100 selected plants, 39.64% contained all 3 compounds, 40.54% contained 2 compounds, 14.41% contained only 1 compound, and 5.41% could not detect any of the 3 compounds. The highest contents of chlorogenic acid, rosmarinic acid, and caffeic acid were found in Lonicera japonica flowering buds, Melissa officinalis leaves, and Coffea canephora seeds at the concentration of 9.900 ± 0.004, 19.908 ± 0.171, and 1.233 ± 0.003 g/100 g of dried plant, respectively.

The present work describes the development and subsequent validation of a novel, simple, selective and stability indicating gradient reversed phase high performance liquid chromatography method for the quantitative determination of related substances, assay of cabozantinib and nivolumab and its application to dissolution studies. The chromatographic method was optimized using the impurity-spiked solution. A good resolution between the peaks was achieved under selected chromatographic conditions. The separation was accomplished on an X-Bridge C18, 150×4.6 mm, 3.5 μ column connected to a photo diode array detector using 0.1 % orthophosphoric acid in water as mobile phase A and acetonitrile as mobile phase B, under gradient elution. The mobile phase flow rate was maintained at 1.0 ml/min. The detection of the constituents was done at 216 nm using a ultra-violet detector. Recovery studies were satisfactory and the correlation coefficient for two active pharmaceutical ingredients and their related substances, 0.999 indicates the linearity of the method within the limits. Limit of detection and limit of quantification for all impurities and cabozantinib and nivolumab were established with respect to the test concentration. Specificity, accuracy, precision, ruggedness and robustness were determined as part of the method validation. The performance of the method was validated according to the current International Council for Harmonisation requirements. Moreover, the dissolution study was performed on active pharmaceutical ingredients to estimate the recovery using the same method. Validation of the developed reversed phase high performance liquid chromatography procedure revealed that all the degradation products formed during stress conditions and related impurities were well separated from their active pharmaceutical ingredients and peaks were well resolved from each other with appropriate retention time. The method was characterized by good linearity, specificity, low values of limit of detection and quantisation, accuracy, precision, ruggedness and robustness. All statistical results were within the acceptance criteria and the proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine dissolution analysis and employed for quality control of drug samples during stability studies.

A simple, economical, specific, accurate, precise and validated Reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for the simultaneous estimation of Cetrizine hydrochloride and Phenylephrine hydrochloride in synthetic mixture and its combined dosage form. The chromatographic separation was achieved on C18 column (Intersil, 220 mm 4.6 mm i.d., 10 ?m particle size) at ambient temperature using disodium hydrogen phosphate (Na 2 HPO 4 ) buffer; pH was adjusted to 6.50 using ortho-phosphoric acid and a mobile phase of Methanol: Acetonitrile: Disodium hydrogen phosphate (80: 4: 19, v/v) at flow rate 2.0 ml/min. Quantification was achieved with UV detector at wavelength 270 nm. The retention time was found to be 1.2 0.04 min and 3.1 0.06 min for cetirizine HCl and phenylephrine HCl respectively. The calibration curves were linear with correlation coefficient 0.998 and 0.999 in the range of 10-50 ?g/ml for Cetrizine hydrochloride and Phenylephrine hydrochloride. The methods were validated in terms of linearity, precision, accuracy, LOD, LOQ and ruggedness according to ICH guideline.

Th accurate rapid, simple and selective reversed phase high performance liquid chromatography (RP-HPLC) has been established and validated for the determination of captopril (CAP). Chromatographic separation was accomplished using prepacked ODSI C18 column (250 mm × 4.6 mm with 5 μm particle size) in isocratic mode, with mobile phase consisting of water: acetonitrile (60:40 v/v), pH adjusted to 2.5 by using 85% orthophosphoric acid at a flow rate of 1 mL/min and UV detection was performed at 203 nm. RP-HPLC method used for the analysis of CAP in mobile phase and rabbit plasma was established and validated as per ICH-guidelines. It was carried out on a well-defined chromatographic peak of CAP was established with a retention time of 4.9 min and tailing factor of 1.871. The liquid–liquid extraction method was used for extraction of CAP from the plasma. Excellent linearity (R2 = 0.999) was shown over range 3.125–100 µg/mL with mean percentage recoveries ranges from 97 to 100.6%. Parameters of precision and accuracy of the developed method meet the established criteria. Intra and inter-day precision (% relative standard deviation) study was also performed which was less than 2% which indicate good reproducibility of the method. The limit of detection (LOD) and quantification for the CAP in plasma were 3.10 and 9.13 ng/mL respectively. The method was suitably validated and successfully applied to the determination of CAP in rabbit plasma samples.

The objective of this investigation was to create and apply a reliable reverse phase high performance liquid chromatography (RP-HPLC) technique for the concurrent measurement of donepezil (DPZ) and quercetin (QT) in rat blood samples for pharmacokinetic research. This is the first publication that introduces a method for DPZ and QT simultaneous determination in rat plasma by high-performance liquid chromatography (HPLC). Using a mobile phase of methanol and HPLC grade water (pH 2.8; adjusted with 0.05% v/v orthophosphoric acid) 45:55 v/v with 2-3 drops of triethylamine (TEA) in an isocratic elution mode at flow rate of 1.0ml/min, DPZ and QT were successfully separated chromatographically on a Hypersil gold C-18 column (250 mm × 4.6 mm, 5μm). The retention time for DPZ and QT was observed to be 6.3 and 12.3 minutes and was detected at an isobestic wavelength of 273 nm using a UV detector. The method was shown to be precise (%RSD < 2%), accurate (96–100%), and specific for the simultaneous detection of DPZ and QT. Several freeze-thaw cycles of the treated plasma samples did not significantly affect the analyte’s stability. The method’s applicability was subsequently confirmed through an oral pharmacokinetic investigation in rats. Since the results were deemed trustworthy, the validated RP-HPLC method may be used to simultaneously detect and quantify both drugs. The method worked well for evaluating the pharmacokinetic characteristics in wistar rats following a single oral dose of 5 mg/kg of QT and 10 mg/kg of DPZ. It is well acknowledged that the chromatographic process is straightforward, robust, accurate, exact, and repeatable.

A reversed phase high performance liquid chromatography (RP-HPLC) method was described for simultaneous determination of seven effective constituents (orientin, isoorientin, vitexin, isovitexin, chlorogenic acid, caffeic acid and ferulic acid) in Bamboo leaf from nine cultivars of Phyllostachys pubescens. The seven compounds were simultaneously determined by HPLC with Sunfire C18 ODS column (4.6 × 250 mm, 5 μm) by gradient elution using acetonitrile (A) and 0.8% acetic acid (B) as the mobile phase. The flow rate was 1.0 mL·min-1; the detection wavelength was 335 nm with column temperature at 25°C. Finally, these effective components were separated clearly and respectively in 28 min, the linear ranges of orientin, isoorientin, vitexin, isovitexin,chlorogenic acid, caffeic acid and ferulic acid were 0.093 to 0.623 μg (r i¼ 0.9994), 0.058 to 0.385 μg (r i¼ 0.9994), 0.045 to 0.301 μg (r i¼ 0.9987), 0.041 to 0.272 μg (r i¼ 0.9992), 0.059 to 0.396 μg (r i¼ 0.9997), 0.046 to 0.312 μg (r i¼ 0.9992), 0.103 to 0.684 μg (r i¼ 0.9983), respectively; The average recoveries (n = 3) were 98.75, 98.34, 98.32, 99.70, 98.61, 98.20 and 99.31%, respectively. The validated method is simple, accurate and can be widely applied to quantification of the four flavonoids and three phenolic acid compounds in 9 different cultivars of P. pubescens. Key words: Reversed phase high performance liquid chromatography (RP-HPLC), orientin, isoorientin, vitexin, isovitexin, chlorogenic acid, caffeic acid, ferulic acid.

Objective To develop a simple,sensitive,and accurate method for the determination of shionone in rat plasma after administration of shionone.Methods The separation was developed by HPLC on a Waters shieldTM RP18 column (150mm × 3.9mm,5 μm) with a mobile phase composed of acetonitrile-0.05％ phosphoric acid water (98:2) at a flow rate of 1.0 mL/min.UV Detector was set at 200nm.Results The linear range of the standard curves was 0.043-1.720 μg/ml with the correlation coefficient of 0.995.The intra-and inter-day precisions were all below 10％.Conclusion The developed method can be successfully applied to the pharmacokinetic study. Key words: Shionone; Reversed phase high performance liquid chromatography; Pharmacokinetics

ABSTRACTSofosbuvir (SOF) and ledipasvir (LED) are recently approved and coformulated as directly acting antiviral agents used for treatment of hepatitis C virus (HCV). A reversed phase high performance liquid chromatography - diode array detector (RP-HPLC/DAD) method was developed and validated for the first time for the analysis of newly formulated anti-HCV combination, in pure form, pharmaceutical formulation and in human plasma. In the developed method, separation was performed on Zorbax® Eclipse C18 column using a gradient mixture of acetonitrile–water as a mobile phase and scanning was performed at 260 nm (for SOF) and 330 nm (for LED). The two drugs were completely separated from each other and from plasma, where plasma peak appeared at 2.76 ± 0.05 min, SOF at 4.25 ± 0.05, and LED at 7.35 ± 0.05. The developed method showed high sensitivity, the drugs showed linearity in the range of 1–45 µg/mL for both pure form and spiked human plasma. Three freeze–thaw cycles were performed separately at two different temperatures, −8 and −20°C. No significant loss of the studied drugs were observed during repeated thawing and freezing. Validation parameters such as accuracy, precision, robustness, and ruggedness were tested in compliance with USP recommendations, where acceptable results were obtained. Applying to pharmaceutical formulation showed no interference from tablet excipients.

Full analysis of triglycerides of natural fats and oils has been investigated by the combination of argentation high performance liquid chromatography (HPLC) with nonaqueous reversed phase (NARP) HPLC. An infrared detector was used in argentation HPLC, because it indicated molar responsibility to all triglycerides. After peak trapping with argentation HPLC, each triglyceride fraction was analyzed with NARP chromatography using the glyceride‐selective post‐column reactor detector. The results of the analyses of triglycerides of palm oil and cocoa butter by the proposed method agreed well with those reported earlier.

Objective: An accurate, precise, rapid and economical reverse phase High Performance Liquid Chromatography (HPLC) method has been developed and validated for the estimation of etravirine in pharmaceutical dosage forms, using PDA detector. Method: Elution was carried out using a mobile phase-A and B consisting of HPLC grade and flow rate was set on 1 ml/minute at 310 nm wave length. The retention time for Etravirine (ETR), impurity-1 and impuriey-2 was found to be 15.813, 12.043 and 17.704 respectively minutes. Result: Analytical method was developed using HPLC Shimadzu (with power stream) gradient chromatographic technique. Data were passed through the spinchrom software. Separation was achieved on Xselect HSS T3 (150 × 4.6 mm, 3.5 μm) column and using mobile phase A (Buffer) was used at pH 4.0 and mobile phase B (Methanol: Acetonitrile: Water (90:5:5 v/v)) by gradient programme. Flow rate was maintained at 1 ml/min with 310 nm PDA detection. The Retention Time (RT) obtained for Etravirine (ETR), impurity 1 and impurity 2 was at 15.813 min, 12.043 min and 17.704 min respectively with injection volume 10 μL and the detection was made at 310 nm. The % recovery of impurity-1 and impurity-2 observed was above 90% from LOQ level to 150%. The correlation coefficient r2 was 0.9993 for impurity-1 and 0.9997 for impurity-2. The method was found unaffected by change in method variance during the robustness study. During the stress study with acid, base, peroxide and temperature, maximum degradation was observed with peroxide indicating the sensitivity of the molecule toward oxidative stress. Conclusion: The developed method is precise, accurate, robust and linear and hence can be routinely used for the related substance analysis of metformin hydrochloride and teneligliptin hydrobromide hydrate tablet in the quality control laboratory at manufacturing site during the commercial manufacturing. Results of all validation parameter were within the limits as per International Conference on Harmonization (ICH) guideline. Keywords Reverse Phase High Performance Liquid Chromatography (RP-HPLC); Etravirine (ETR); Method development and validation

Haplamine, a pyranoquinoline alkaloid, was isolated from the genus Haplophyllum. The inter-species variability of haplamine metabolism was determined by reversed phase high performance liquid chromatography (HPLC) with UV detection. Microsomes from the liver of rats, mice, rabbits, guinea-pigs and humans were incubated with haplamine. After incubation, samples were extracted with a mixture of ethyl acetate and isopropyl alcohol (90 : 10; v/v). Haplamine and its metabolites were separated by HPLC using Nucleosil C18 Nautilus (5 microm) connected with a precolumn of the same type. The HPLC mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85 : 15; v/v) (B) used in a gradient mode (17 to 27 % B for 10 min, 27 to 90 % B for 37 min, 90 to 17 % B for 3 min, and finally 17 % B for 3 min) at 1 mL/min. Quantitative and qualitative results showed significant inter-species differences in haplamine metabolism. Qualitative similarities were found between guinea-pigs, rabbits, and humans. The metabolites were isolated by HPLC and identified by GC/MS after silylation. The phase I metabolites identified in human liver microsomes were TRANS/CIS-3,4-dihydroxy-9-O-desmethylhaplamine, TRANS/CIS-3,4-dihydroxyhaplamine and 9-O-desmethylhaplamine.