Reversed-phase High-performance Liquid Chromatographic Technique Research Articles

Green analytical quality by design RP-HPLC technique with fluorimetric detection for simultaneous assay of cinnarizine and dompridone.

A green and sensitive factorial design-assisted reversed-phase high-performance liquid chromatography (RP-HPLC) approach with fluorimetric detection has been developed for simultaneous determination of cinnarizine and domperidone in pure materials, commercial single and combined tablets. Both drugs are co-formulated in one dosage form and are commonly used for the effective treatment of motion sickness in the ratio of 4 : 3 for cinnarizine and domperidone, respectively. To achieve the developed method's best performance and reliability, a 23 full factorial design was applied during the optimization of chromatographic conditions. Subsequently, isocratic elution on a C18 column with a mobile phase mixture of methanol and 0.02 M potassium dihydrogen phosphate buffer (85 : 15 v/v, pH 3.0) was performed to achieve the best separation. Moreover, the flow rate of 0.8 ml min-1 was applied during the analysis with fluorescence detection at λex 283 nm/λem 324 nm. The elution process was time effective; it consumed less than 6 min for a complete run as retention time for cinnarizine and domperidone, was 4.5 and 3.5, respectively. Good linearity of the proposed method was achieved within the concentration ranges of 0.2-4.5 and 0.4-5.0 µg ml-1 for domperidone and cinnarizine, respectively. The suggested approach was carefully validated according to the International Council for Harmonization (ICH) guidelines. Moreover, green analytical procedure index (GAPI), national environmental methods index (NEMI), analytical greenness (AGREE) and analytical eco-scale methods were applied to assess and confirm method greenness.

Development and validation of a green RP-HPLC method for detection and quantitation of meropenem trihydrate in the bulk: A comparison with HPTLC method

This research introduces an eco-friendly green Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) method for detecting and quantifying meropenem trihydrate. Traditional methods use buffered solutions, gradient mobile phases, and longer retention times, but this method offers a rapid RP-HPLC technique with a C8 column and isocratic mobile phase (60% methanol, 40% ultra-pure water), eliminating the need for buffers and acetonitrile. It features a short 2.1-minute retention time and a high r² value of 0.9995. It delivers accuracy (101.1-102.3%) and precision (RSD ≤ 2%), coupled with low LOD and LOQ values of 1.72 and 5.20 µg/ml. Aqueous dilution simplifies sample preparation, reducing degradation and interference. The method is compared with an HPTLC method, showing an extended linear range (6.25-200 µg/ml for HPLC, 7.81-62.5 µg/ml for TLC) and high sensitivity, making it significant for meropenem trihydrate quality control in bulk and dosage forms.

Selective Stability Indicating Liquid Chromatographic Method Based on Quality by Design Framework and In Silico Toxicity Assessment for Infigratinib and Its Degradation Products.

Infigratinib, a protein kinase inhibitor employed in the therapeutic management of cholangiocarcinoma, was subjected to various stress conditions, including hydrolytic (acidic and alkaline), oxidative, photolytic, and thermal stress, in accordance with the rules established by the International Council for Harmonization. A cumulative count of five degradation products was observed. The application of the Quality by Design principle was utilized in the development of a rapid and specific separation method for Infigratinib and its degradation products. The methodology employed in this study was derived from an experimental design approach, which was utilized to examine the critical process parameters associated with chromatographic systems. The reversed-phase high-performance liquid chromatography technique, employing a C18 column and a mobile phase composed of a gradient mixture of 25 mM ammonium acetate buffer at pH 6.0 and acetonitrile, successfully facilitated the chromatographic separation. The methodology was expanded to include the utilization of UPLC-quadrupole tandem mass spectrometry in order to conduct a comprehensive analysis of the structural properties and characterize the degradation products. Overall, five degradation products were found in different stress conditions. The method was verified at certain working points, wherein a linearity range (5.0-200.0 µg/mL) was developed and other parameters such as accuracy, repeatability, selectivity, and system suitability were evaluated. Finally, the toxicity and mutagenicity of Infigratinib and its degradation products were predicted using in silico software, namely DEREK Nexus® (version 6.2.1) and SARAH Nexus® (version 3.2.1). Various toxicity endpoints, including chromosomal damage, were predicted. Additionally, two degradation products were also predicted to be mutagenic.

Reversed-Phase High-Performance Liquid Chromatography Quantification, Gas Chromatography-Mass Spectrometry Study and In Vitro Antioxidant Potential of Clerodendrum serratum Linn. Roots

The present study was aimed to identify and to quantify the phytoconstituents by gas chromatography-mass spectrometry, reversed-phase high-performance liquid chromatography techniques and further to evaluate their in vitro antioxidant potential. In this study, the roots of Clerodendrum serratum Linn. was subjected to soxhlet extraction using n-hexane, chloroform, ethyl acetate and ethanol solvents and the obtained extracts (CSRH, CSRC, CSREA and CSRA) were analyzed by gas chromatography-mass spectrometry technique. The caffeic acid and beta-sitosterol were quantified from the extracts using reversed-phase high-performance liquid chromatography technique. Total phenolic and flavonoid content were determined using established methods viz. Folin-Ciocalteu and aluminium chloride colorimetric assay. Antioxidant potential was assessed using in vitro methods such as 2,2-diphenyl-1-picrylhydrazyl scavenging activity; ferric reducing ability of plasma assay and total antioxidant activity. The gas chromatography-mass spectrometry analysis revealed the presence of 48 compounds, out of which 41 compounds were identified and among them caffeic acid and beta-sitosterol were quantified using reversed-phase high-performance liquid chromatography technique. The abundance of phenolic and flavonoid content was found in CSREA and CSRA fractions. The half maximal inhibitory concentration values of 2,2-diphenyl-1-picrylhydrazyl assay was found to be 1.24, 3.36, 10.61 μg/ml for the CSRA, CSREA and CSRC fractions respectively and the total antioxidant activity was found to be 960±0.25, 766±0.14 and 816.6±0.28 μg/mg for the CSRC, CSREA and CSRA fractions respectively showing strong antioxidant activity. The ferric reducing ability of plasma assay showed strongest reducing power for CSRA and CSREA fractions. The results showed that the CSRA and CSREA fractions had strong antioxidant potential. It may be concluded from the results that the Clerodendrum serratum Linn. roots possesses significant antioxidant potential may be due to presence of potent phenolic and flavonoid compounds and these finding supports the traditional use of plant to treat the oxidative stress and related disorders.

Ibuprofen And Diphenhydramine Citrate Related Substances In Tablet Dosage Form May Be Determined Simultaneously Using A Reversed-Phase High-Performance Liquid Chromatographic Technique

Ibuprofen and diphenhydramine citrate have been combined into one dosage form, and a novel graded reverse-phase elevated HPLC technology has been created for the simultaneous detection of related chemicals in this dosage form. This technique is also capable of detecting degradation products and impurities associated with the manufacturing process. The approach involved the utilization of a C18 column and a mobile phase composed of a gradient mixture of solvent A and B. Moreover, this method is well-suited for the determination of the assay of citrate versions of ibuprofen and diphenhydramine from pharmaceutical sources. Furthermore, the technique is stability-indicating, indicating its suitability for evaluating the shelf-life of pharmaceutical products.

Innovated formulation of oseltamivir powder for suspension with stability study after reconstitution using a developed ion-pair reversed phase high-performance liquid chromatography method.

ABSTRACTOseltamivir is an antiviral neuraminidase inhibitor used for the treatment and prophylaxis of influenza infection with viruses A and B. The mechanism of oseltamivir antiviral activity is by inhibiting the activity of the viral neuraminidase enzyme present on the surface of the virus, which stops viral replication and infectivity. Oral suspensions of oseltamivir phosphate are dispensed orally capsules and suspension. However, the use of oral suspension for pediatric administration is preferable and is prepared as a powder for suspension. The reconstituted suspension degrades rapidly within a few days. The objective of this work is to establish a stable formulation of oseltamivir phosphate as a suspension and to assure the stability conditions for prolonged use after reconstitution in aqueous form. In addition, this required formulation should maintain a high rate of dissolution, which subsequently leads to higher bioavailability. In this study, oseltamivir forms an inclusion complex with the natural and safe polymer hydroxypropyl beta-cyclodextrin, which resembles a host because its structural cavity carries the oseltamivir molecule in the aqueous preparation and provides a protective property against environmental challengers. In addition, a high-performance liquid chromatography (HPLC) stability-indicating method of analysis has been developed using an ion-pair reversed-phase HPLC technique that is validated for precision, accuracy, reproducibility, and specificity for the determination of oseltamivir in suspension. The results of this work show the relatively long shelf life of the reconstituted oseltamivir oral powder for suspension in the new pediatric formulation, and the developed HPLC method was precisely suitable for stability study.

STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF ANTIDIABETIC DRUGS, DAPAGLIFLOZIN AND SAXAGLIPTIN

For simultaneous measurement of Dapagliflozin (DAPA) and Saxagliptin (SAXA) in combination, as well as quantification of their major degradation products, a simple reversed-phase high-performance liquid chromatographic technique was devised and validated. The separation was accomplished using an ODS C18 column (250 mm 4.6 mm, i.d.5 m) with isocratic elution at room temperature. The optimum mobile phase consists of an aqueous phase (sodium acetate buffer, pH 4) and an organic phase (acetonitrile: methanol, 40:40) at a ratio of (20:80). The flow rate was set to 1.0 ml min-1, and effluents were measured using a 228 nm diode array detector. This method showed good linearity over a range of 2.0-12 μg ml-1 and 1-6 μg ml-1 for DAPA and SAXA respectively. The retention times of samples were 2.31 and 2.90 minutes for DAPA and SAXA respectively. According to the International Conference Harmonization (ICH) criteria, the drugs in combination were submitted to different stress degradation studies. The suggested approach is appropriate for stability investigations, according to the results of the stress degradation experiments. The proposed method was simple, rapid and precise for the study of a new formulation that has been approved for the cure of diabetes mellitus.

STABILITY INDICATING HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND DAPAGLIFLOZIN IN API AND PHARMACEUTICAL DOSAGE FORM

A simple and specific stability indicating reversed-phase high-performance liquid chromatography technique has been developed and validated for the concurrent estimation of metformin hydrochloride and dapagliflozin in bulk and pharmaceutical dosage form. The ideal conditions were established for the study or analysis of the drug such as chromatographic separation was carried out on THERMO fisher ODS C18 column containing mobile phase of water and acetonitrile 65:35 % v/v of pH 6.8 adjusted with 0.1 % ortho phosphoric acid at a flow rate of 1 ml/minutes detected wavelength at 240 nm. The retention time was found to be 2.13 minutes and 5.41 minutes for metformin hydrochloride (MET) and dapagliflozin (DAPA) respectively. The proposed method was found to be linear in the concentration range of 100-600 ug/ml for MET (R2=0.9999) and 1-6 ug/ml for DAP (R2=0.9996), respectively. Method was validated according to ICH guidelines. Co-relation coefficients for both the drugs were found to be less than one. The mean % recoveries obtained were found to be 99.06-100.32% for metformin and 99.1-100.18% for dapagliflozin respectively. Stress testing is carried out for both drugs in acid, base, peroxide, photolytic and thermal degradation. The developed method can be effectively applied for routine analysis in simultaneous determination of metformin hydrochloride and dapagliflozin in bulk and combined tablet dosage form.

Solid-phase synthetic method for N-alkyl-4-alkylamino-6-arylthieno[3,2-d]pyrimidine-2-carboxamide derivatives

Abstract Herein, we describe a solid-phase synthetic method for synthesizing N-alkyl-4-alkylamino-6-arylthieno[3,2-d]pyrimidine-2-carboxamide derivatives. The derivatives consist of the biologically active 6-phenylthieno[3,2-d]pyrimidine scaffold. The template mediated synthetic strategy involving Suzuki coupling reactions between methyl 3-amino-5-bromothiophene-2-carboxylate and arylboronic acids afforded methyl 3-amino-5-arylthiophene-2-carboxylates. Cyclocondensation reactions involving methyl 3-amino-5-arylthiophene-2-carboxylates and methyl cyanoformate afforded esters, that when subjected to hydrolysis reactions yielded 6-aryl-4-oxo-3,4-dihydrothieno[3,2-d]pyrimidine-2-carboxylic acids (template compounds). These carboxylic acid templates were coupled with primary alkylamine-loaded acid-sensitive methoxybenzaldehyde (AMEBA) resins. The amide coupling reactions were followed by direct amination reactions mediated by benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP). The compounds were subsequently cleaved from the solid support, purified using the reverse phase-high performance liquid chromatography technique (RP-HPLC), and passed through a strong anion exchange (SAX) resin pretreated with water to yield the N-alkyl-4-alkylamino-6-arylthieno[3,2-d]pyrimidine-2-carboxamide derivatives. The reaction conditions for the solid-phase transformations were optimized using a solution-phase model using 2,4-dimethoxybenzyl-protected isobutylamine as a reactant. 2,4-Dimethoxybenzyl-protected isobutylamine, and not AMEBA resin-loaded isobutylamine was used during the process. Substituent variation experiments were performed using 6-aryl-4-oxo-3,4-dihydrothieno[3,2-d]pyrimidine-2-carboxylic acids and a variety of primary and secondary amine building blocks. Additionally, we could include the Suzuki coupling step in a modified solid-phase synthetic sequence.

Analytical method development and validation studies for the estimation of H2 receptor antagonist drug (Ranitidine)

<p class="abstract"><strong>Background:</strong> An analytical science in the development, finding a new molecules and procreation of pharmaceuticals has been an extensive approached. From the evaluation over small quantities of complex biological substances to the quality monitoring of the finished dosage form, the use on analytical technology know- how covers an ample thoroughness over techniques or disciplines.</p><p class="abstract"><strong>Methods:</strong> Reversed phase high performance liquid chromatography (RP-HPLC) approach has been promoted for the discernment over Ranitidine (RAN) within pharmaceutical dosage form split of RAN was accomplished inside a unaccompanied chromatographic run of an Phenomenix column size 5 μm 4.6x250 mm along with UV analysis at 227 nm wavelength, below isocratic conditions, using Ammonium acetate and Methanol (pH 6.0) in 80:20 ratio. Validation parameters had been observed in accordance with exhibit linearity, accuracy, precision, Limit of Quantitation and Limit of detection in conformity in imitation of ICH guidelines. </p><p class="abstract"><strong>Results:</strong> The contemporary approached demonstrates significant linearity upon the range concerning 50-202.5 μg/ml for RAN followed by intra-day and inter-day precision, expressed so the relative standard deviation (RSD), on replicates is <2.0 and accuracy among the range over 98-102%.</p><p class="abstract"><strong>Conclusions:</strong> The flourished RP-HPLC technique was once innovative, suitable for detecting RAN in pure form and in pharmaceutical preparation.</p>

STABILITY-INDICATING METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF ROSUVASTATIN CALCIUM IN PHARMACEUTICAL DOSAGE FORM BY REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The present work was focused on the development and validation of the reversed-phase high-performance liquid chromatography (RP-HPLC) method, which is simple, rapid, precise, accurate, sensitive, economical, and stability-indicating for the quantification of rosuvastatin calcium in bulk and tablet formulation.
 Methods: The separation was attained on reversed-phase Princeton (C18) column with dimensions (250 mm × 4.6 mm, 5μ) employing buffer, which is a mixture of water (pH 3.0, adjusted with orthophosphoric acid) and methanol in the ratio (20:80) v/v as mobile phase, at flow rate 1.0 ml/min. and detection was carried out at wavelength 240 nm. The retention time under the optimized condition of rosuvastatin calcium was found to be 2.844 min.
 Results: The linearity of the method was demonstrated in the concentration range of 6-16 µg/ml for rosuvastatin calcium with a correlation coefficient (r2) of 0.9999. The percentage relative standard deviation was ˂2% and percentage recovery was found to be 100.12-101.37% for rosuvastatin calcium. The assay of marketed tablet formulations was found to be 98.99%.
 Conclusion: The developed RP-HPLC technique was found to be simple, specific, sensitive, rapid, linear, accurate, precise, and economical and will be used for regular quality control of rosuvastatin calcium in bulk and tablet formulations.

Quantitative assessments of adenosine triphosphatase hydrolytic activity by ultrafiltration-coupled ion-pair reversed-phase high-performance liquid chromatography.

Adenosine triphosphate is a universal energy currency that can directly provide energy required for a multitude of biochemical reactions and biophysical actions through adenosine triphosphatase catalyzed hydrolysis. Adenosine triphosphatase activity is thus one important feature for the characterization of protein function and cell activity. Herein, we optimized ion-pair reversed-phase high-performance liquid chromatography technique for highly efficient separation of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, and the method demonstrated good linearity. Moreover, by coupling a protein-removable ultrafiltration, we developed a sensitive and robust approach for quantification of adenosine triphosphatase hydrolytic activity. By this assay, we demonstrated that RecA filaments-catalyzed adenosine triphosphate hydrolysis approached a second-order reaction, and its rate constant was estimated as 0.057mM-1 min-1 . In addition, we explored the effects of DNA length on this reaction and revealed that the increase of the length of single-stranded DNA can promote the adenosine triphosphatase hydrolytic activity of RecA filaments. All these results confirm the feasibility of this new method in quantification of adenosine triphosphatase hydrolytic activity assays. Compared with previous complicated enzyme-coupled or homogeneous colorimetric measurements, the developed approach with high resolution separation allows a simple reaction system for adenosine triphosphatase assay and a sensitive detection free of interference from background noise.

Development of a validated RP-HPLC/DAD method for the quantitative determination of methyl jasmonate in an insect repellent semi-solid formulation

A simple and efficient reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the first time for the estimation of a mosquito repellent, methyl jasmonate in a cream formulation, and validated as per the International Conference on Harmonization guidelines. Acetonitrile and water (75:25 v/v) were used as the mobile phase and the flow-rate of the mobile phase was kept constant at 1.0 mL/min. The analysis was performed isocratically on a C18 analytical column (250 × 4.4 mm, 5 μm) using a Diode Array Detector for the detection of methyl jasmonate at 214 nm. The presence of excipients did not interfere with the quantification of methyl jasmonate. The calibration curve was linear in the concentration range of 25–300 μg/mL. The relative standard deviations for intra-day and inter-day precision, and repeatability were less than 2%. The recovery ranged from 88.5% to 90.7% with relative standard deviations not higher than 2%. The limit of detection and quantification were 9.4 μg/mL and 28.5 μg/mL, respectively. System suitability parameters were within the accepted range. The proposed method was also robust. Thus, the present report puts forward a novel analytical method for the estimation of an emerging mosquito repellent, methyl jasmonate by using the RP-HPLC technique.

Validated RP-HPLC/DAD Method for the Quantification of Insect Repellent Ethyl 2-Aminobenzoate in Membrane-Moderated Matrix Type Monolithic Polymeric Device.

A simple, accurate and sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the estimation of ethyl 2-aminobenzoate (EAB) in a matrix type monolithic polymeric device and validated as per the International Conference on Harmonization guidelines. The analysis was performed isocratically on a ZORBAX Eclipse plus C18 analytical column (250 × 4.4 mm, 5 μm) and a diode array detector (DAD) using acetonitrile and water (75:25 v/v) as the mobile phase by keeping the flow-rate constant at 1.0 mL/min. Determination of EAB was not interfered in the presence of excipients. Inter- and intra-day relative standard deviations were not higher than 2%. Mean recovery was between 98.7 and 101.3%. Calibration curve was linear in the concentration range of 0.5-10 µg/mL. Limits of detection and quantification were 0.19 and 0.60 µg/mL, respectively. Thus, the present report put forward a novel method for the estimation of EAB, an emerging insect repellent, by using RP-HPLC technique.

Degradation of carotenoids in pumpkin (Cucurbita maxima L.) slices as influenced by microwave vacuum drying

ABSTRACTThe aim of this study was to investigate the influence of microwave vacuum drying on carotenoids in pumpkin (Cucurbita maxima L.) slices. Carotenoids were measured using the reverse-phase high-performance liquid chromatography technique. It was shown that compared with hot air drying, microwave vacuum drying inhibited color changes and significantly (p < 0.05) improved total carotenoid retention (89.1%) in pumpkin slices. During the microwave vacuum drying process, microwave power had an important effect on total carotenoid and all-trans carotenoids. As microwave power increased, the total carotenoid content significantly decreased (p < 0.05), and the levels of individual carotenoids, including all-trans-α-carotene, all-trans-β-carotene, and all-trans-lutein, generally decreased. However, there was an overall upward trend for the levels of 13-cis-β-carotene, 15-cis-β-carotene, 9-cis-β-carotene, and 9-cis-α-carotene. The trans carotenoid quality of the finished products was improved within a certain range of vacuum levels. In addition to the degradation induced by microwave energy, isomerization was considered to be responsible for the loss of all-trans carotenoids. These results indicated that inappropriate drying methods and conditions might result in high losses of all-trans carotenoids in pumpkins.

Optimization of Extraction Techniques and RP-HPLC Analysis of Antidiabetic and Anticancer Drug Mangiferin from Roots of ‘Saptarangi’ (Salacia chinensis L.)

Saptarangi (Salacia chinensis L.) is a chief source of mangiferin, which has antidiabetic and anticancer properties. Extraction using continuous shaking extraction (CSE), ultrasonic extraction (UE), microwave assisted extraction (MAE), and steam bath assisted extraction (SBAE) were evaluated for extraction of mangiferin (MF) from roots of S. chinensis. The extracts were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) and Fourier transform-infra red spectroscopy (FT-IR) analysis. Operational parameters such as different solvents and exposure period were also evaluated. N,N-Dimethyl formamide (DMF 30%, v/v) extracted higher quantity of MF compared to other solvents. The results shows that the extraction of MF by SBAE was more efficient followed by MAE, CSE, and UE methods. Maximum quantity of MF (391.80 ppm) was obtained by SBAE technique and DMF (30%) as a solvent system. SBAE is the easiest and best method for screening of MF from S. chinensis. Furthermore, use of FT-IR validates the RP-HPLC technique for identification and quantification.

Determinação de ácido rosmarínico em Cordia verbenacea por cromatografia líquida: aplicabilidade em estudo sazonal

RESUMO Neste estudo, uma técnica de cromatografia líquida de alta resolução em fase reversa (CLAE-FR) para a determinação de ácido rosmarínico em Cordia verbenacea foi desenvolvida e validada. A análise de regressão foi avaliada, com observação de uma boa linearidade (r = 0,9997). Os valores obtidos para a precisão e exatidão estão de acordo com as diretrizes do ICH e com a legislação brasileira. Os valores de repetibilidade e precisão intermediária foram 2,79% e 4,76%, respectivamente. Os limites de detecção e de quantificação de ácido rosmarínico foram de 1,92 µg/mL e 5,81 µg/mL, respectivamente. Os resultados mostraram que o método desenvolvido é uma técnica por CLAE-FR de confiança para a determinação de ácido rosmarínico em tintura de C. verbenacea. Além disso, essa metodologia foi aplicada em estudo sazonal, que revela uma correlação positiva relativamente forte entre o período de chuvas e o teor de ácido rosmarínico.

Comparison of Chromatographic and Titrimetric Methods for the Determination of the a-amino Nitrogen in Standard Solution and Fish Protein Hydrolysates

&lt;p&gt;Two analytical methods, reverse phase high-performance liquid chromatography (RP-HPLC) and formol titration, were compared for their accuracy and precision for the determination of a-amino nitrogen (a-AN) concentration in fish protein hydrolysates. The result showed that, when tested with pure amino acid solutions, RP-HPLC method had a better standard deviation (S. D.), coefficient of variation (C. V.), and standard error (S. E.) but a poorer recovery rate compared with formol titration technique. The observed a-AN value by RP-HPLC has good coincidence with the actual a-AN value and their correlation coefficient (r) is 0.9981, which was close to the r value (0.9995) between the a-AN by formol titration and the actual a-AN. However, when tested with protein hydrolysates, the a-AN obtained from RP-HPLC technique were significantly (p&amp;lt;0.05) lower than that from formol titration for the same sample. Therefore, the formol titration method can be used as a quick screening method to assess the quality fish hydrolystaes.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Determination of Tryptophan in Raw Materials, Rat Brain and Human Plasma by RP-HPLC Technique

This paper describes tryptophan (TRP) estimation in raw human plasma and rat brain by reversed-phase high-performance liquid chromatography (RP-HPLC). Estimation was carried out on a Purospher STAR C18 column using water-acetonitrile (90:10 v/v, at pH 2.7) mixture at a rate of 1.5 mL/min as mobile phase. Eluents were monitored at 273 nm by an ultraviolet detector. The method was linear (R(2) > 0.999), precise (intra-day and inter-day precision <2%) in the range of 0.25-20 µg/mL. The detection and quantification limits were 0.0144 µg/mL and 0.0437 µg/mL, respectively. In human plasma, Day 1 and Day 2 precision were 0.054-2.29% and 1.66-3.7%; whereas precisions in rat brain were 1.23-2.3% and 0.677-4.2%, respectively. The method was applied to study TRP level in human smokers and in arthritic rat brain. An efficient RP-HPLC method was developed for TRP determination that worked for clinical and research purposes.

Physical Properties, Molecular Structures, and Protein Quality of Texturized Whey Protein Isolate: Effect of Extrusion Temperature

Although extrusion technology has contributed much to increasing the effective utilization of whey, the effect of extrusion conditions on the functional properties of the proteins is not well understood. In this work, the impact of extrusion temperature on the physical and chemical properties, molecular structures, and protein quality of texturized whey protein isolate (WPI) was investigated at a constant moisture content and compared with WPI treated with simple heat only. The Bradford assay methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high-performance liquid chromatography techniques were used to determine protein solubility and to analyze compositional changes in the two major whey proteins, α-lactalbumin and β-lactoglobulin. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic techniques were applied to study the secondary and tertiary structures of the proteins. This study demonstrated that extrusion temperature is a critical but not the sole determining factor in affecting the functional properties of extruded WPI.