Search results for: isocratic

Authors: Sofiqul Islam, V. Murugan, Prema Kumari, Hari

Abstract:

Gentamicin is a broad spectrum water-soluble aminoglycoside antibiotics produced by the fermentation process of microorganism known as Micromonospora purpurea. It is widely used for the treatment of infection caused by both gram positive and gram negative bacteria. Gentamicin consists of a mixture of aminoglycoside components like C1, C1a, C2a, and C2. The molecular structure of Gentamicin and its related substances showed that it has lack of presence of chromophore group in the molecule due to which the detection of such components were quite critical and challenging. In this study, a simple Reversed Phase-High Performance Liquid Chromatographic (RP-HPLC) method using ultraviolet (UV) detector was developed and validated for quantification of the related substances present in Gentamicin drug substances. The method was achieved by using Thermo Scientific Hypersil Gold analytical column (150 x 4.6 mm, 5 µm particle size) with isocratic elution composed of methanol: water: glacial acetic acid: sodium hexane sulfonate in the ratio 70:25:5:3 % v/v/v/w as a mobile phase at a flow rate of 0.5 mL/min, column temperature was maintained at 30 °C and detection wavelength of 330 nm. The four components of Gentamicin namely Gentamicin C1, C1a, C2a, and C2 were well separated along with the related substance present in Gentamicin. The Limit of Quantification (LOQ) values were found to be at 0.0075 mg/mL. The accuracy of the method was quite satisfactory in which the % recovery was resulted between 95-105% for the related substances. The correlation coefficient (≥ 0.995) shows the linearity response against concentration over the range of Limit of Quantification (LOQ). Precision studies showed the % Relative Standard Deviation (RSD) values less than 5% for its related substance. The method was validated in accordance with the International Conference of Harmonization (ICH) guideline with various parameters like system suitability, specificity, precision, linearity, accuracy, limit of quantification, and robustness. This proposed method was easy and suitable for use for the quantification of related substances in routine analysis of Gentamicin formulations.

Keywords: reversed phase-high performance liquid chromatographic (RP-HPLC), high performance liquid chromatography, gentamicin, isocratic, ultraviolet

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Authors: Anil P. Dewani, Alok S. Tripathi, Anil V. Chandewar

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Present manuscript reports the development and validation of a quantitative high performance liquid chromatography method for the pharmacokinetic evaluation of Glimepiride (GLM) and Ilaprazole (ILA) in rat plasma. The plasma samples were involved with Solid phase extraction process (SPE). The analytes were resolved on a Phenomenex C18 column (4.6 mm× 250 mm; 5 µm particle size) using a isocratic elution mode comprising methanol:water (80:20 % v/v) with pH of water modified to 3 using Formic acid, the total run time was 10 min at 225 nm as common wavelength, the flow rate throughout was 1ml/min. The method was validated over the concentration range from 10 to 600 ng/mL for GLM and ILA, in rat plasma. Metformin (MET) was used as Internal Standard. Validation data demonstrated the method to be selective, sensitive, accurate and precise. The limit of detection was 1.54 and 4.08 and limit of quantification was 5.15 and 13.62 for GLM and ILA respectively, the method demonstrated excellent linearity with correlation coefficients (r2) 0.999. The intra and inter-day precision (RSD%) values were < 2.0% for both ILA and GLM. The method was successfully applied in pharmacokinetic studies followed by oral administration in rats.

Keywords: pharmacokinetics, glimepiride, ilaprazole, HPLC, SPE

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Authors: Hassan M. Albishri, Abdullah Almalawi, Deia Abd El-Hady

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A simple and ecofriendly cyclodextrine-surfactant modified UHPLC (CDS-UPLC) method for rapid and sensitive simultaneous determination of multi water-soluble vitamins such as ascorbic acid, pyridoxine hydrochloride and thiamine hydrochloride in commercial pharmaceuticals and milk samples have been firstly developed. Several chromatographic effective parameters have been changed in a systematic way. Adequate results have been achieved by a mixture of β-cyclodextrine (β-CD) and cationic surfactant under acidic conditions as an eco-friendly isocratic mobile phase at 0.02 mL/min flow rate. The proposed CDS- UHPLC method has been validated for the quantitative determination of multivitamins within 8 min in food and pharmaceutical samples. The method showed excellent linearity for analytes in a wide range of 10-1000 ng/µL. The repeatability and reproducibility of data were about 2.14 and 4.69 RSD%, respectively. The limits of detection (LODs) of analytes ranged between 0.86 and 5.6 ng/µL with a range of 81.8 -115.8% recoveries in tablets and milk samples. The current first CDS- UHPLC method could have vast applications for the precise analysis of multivitamins in complicated matrices.

Keywords: ecofriendly, cyclodextrine-surfactant, multivitamins, UHPLC

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Authors: Ian Marc G. Cabugsa, Kim Ryan A. Won

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Fast, reliable and simultaneous HPLC analysis of theobromine and caffeine in cacao and cocoa products was optimized in this study. The samples tested were raw, fermented, and roasted cacao beans as well as commercially available cocoa products. The HPLC analysis was carried out using step gradient solvent system with acetonitrile and water buffered with H3PO4 as the mobile phase. The HPLC system was optimized using 273 nm wavelength at 35 °C for the column temperature with a flow rate of 1.0 mL/min. Using this method, the theobromine percent recovery mean, Limit of Detection (LOD) and Limit of Quantification (LOQ) is 118.68(±3.38)%, 0.727 and 1.05 respectively. The percent recovery mean, LOD and LOQ for caffeine is 105.53(±3.25)%, 2.42 and 3.50 respectively. The inter-day and intra-day precision for theobromine is 4.31% and 4.48% respectively, while 7.02% and 7.03% was for caffeine respectively. Compared to the standard method in AOAC using methanol in isocratic solvent system, the results of the study produced lesser chromatogram noise with emphasis on theobromine and caffeine. The method is readily usable for cacao and cocoa substances analyses using HPLC with step gradient capability.

Keywords: cacao, caffeine, HPLC, step gradient solvent system, theobromine

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Authors: Muzaffar Iqbal, Essam Ezzeldin, Khalid A. Al-Rashood

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Pomalidomide is a second generation oral immunomodulatory agent, being used for the treatment of multiple myeloma in patients with disease refractory to lenalidomide and bortezomib. In this study, a sensitive UPLC-MS/MS assay was developed and validated for high-throughput determination of pomalidomide in rat plasma using celecoxib as an internal standard (IS). Liquid liquid extraction using dichloromethane as extracting agent was employed to extract pomalidomide and IS from 200 µL of plasma. Chromatographic separation was carried on Acquity BEHTM C18 column (50 × 2.1 mm, 1.7 µm) using an isocratic mobile phase of acetonitrile:10 mM ammonium acetate (80:20, v/v), at a flow rate of 0.250 mL/min. Both pomalidomide and IS were eluted at 0.66 ± 0.03 and 0.80 ± 0.03 min, respectively with a total run time of 1.5 min only. Detection was performed on a triple quadrupole tandem mass spectrometer using electrospray ionization in negative mode. The precursor to product ion transitions of m/z 272.01 → 160.89 for pomalidomide and m/z 380.08 → 316.01 for IS were used to quantify them respectively, using multiple reaction monitoring mode. The developed method was validated according to regulatory guideline for bioanalytical method validation. The linearity in plasma sample was achieved in the concentration range of 0.47–400 ng/mL (r2 ≥ 0.997). The intra and inter-day precision values were ≤ 11.1% (RSD, %) whereas accuracy values ranged from - 6.8 – 8.5% (RE, %). In addition, other validation results were within the acceptance criteria and the method was successfully applied in a pharmacokinetic study of pomalidomide in rats.

Keywords: pomalidomide, pharmacokinetics, LC-MS/MS, celecoxib

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Authors: Atifa Mushtaq, Tanweer Khaliq, Hafiz Alam Sher, Asia Farid, Anila Kanwal, Maliha Sarfraz

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The study was designed to assess the various pharmacokinetic parameters of a commercially available clarithromycin Tablet (Klaricid® 250 mg Abbot, Pakistan) in plasma sample of healthy adult female volunteers by applying a rapid, sensitive and accurate HPLC-UV analytical method. The human plasma samples were evaluated by using an isocratic High Performance Liquid Chromatography (HPLC) system of Sykam consisted of a pump with a column C18 column (250×4.6mn, 5µm) UV-detector. The mobile phase comprises of potassium dihydrogen phosphate (50 mM, pH 6.8, contained 0.7% triethylamine), methanol and acetonitrile (30:25:45, v/v/v) was delivered with injection volume of 20µL at flow rate of 1 mL/min. The detection was performed at λmax 275 nm. By applying this method, important pharmacokinetic parameters Cmax, Tmax, Area under curve (AUC), half-life (t1/2), , Volume of distribution (Vd) and Clearance (Cl) were measured. The parameters of pharmacokinetics of clarithromycin were calculated by software (APO) pharmacological analysis. Maximum plasma concentrations Cmax 2.78 ±0.33 µg/ml, time to reach maximum concentration tmax 2.82 ± 0.11 h and Area under curve AUC was 20.14 h.µg/ml. The mean ± SD values obtained for the pharmacokinetic parameters showed a significant difference in pharmacokinetic parameters observed in previous literature which emphasizes the need for dose adjustment of clarithromycin in Pakistani population.

Keywords: Pharmacokinetc, Clarothromycin, HPLC, Pakistan

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Authors: S. Jain, R. Savalia, V. Saini

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A simple, accurate, precise, sensitive and specific RP-HPLC method was developed and validated for simultaneous estimation of Metoprolol Succinate and Olmesartan Medoxomil in bulk and tablet dosage form. The RP-HPLC method has shown adequate separation for Metoprolol Succinate and Olmesartan Medoxomil from its degradation products. The separation was achieved on a Phenomenex luna ODS C18 (250mm X 4.6mm i.d., 5μm particle size) with an isocratic mixture of acetonitrile: 50mM phosphate buffer pH 4.0 adjusted with glacial acetic acid in the ratio of 55:45 v/v. The mobile phase at a flow rate of 1.0ml/min, Injection volume 20μl and wavelength of detection was kept at 225nm. The retention time for Metoprolol Succinate and Olmesartan Medoxomil was 2.451±0.1min and 6.167±0.1min, respectively. The linearity of the proposed method was investigated in the range of 5-50μg/ml and 2-20μg/ml for Metoprolol Succinate and Olmesartan Medoxomil, respectively. Correlation coefficient was 0.999 and 0.9996 for Metoprolol Succinate and Olmesartan Medoxomil, respectively. The limit of detection was 0.2847μg/ml and 0.1251μg/ml for Metoprolol Succinate and Olmesartan Medoxomil, respectively and the limit of quantification was 0.8630μg/ml and 0.3793μg/ml for Metoprolol and Olmesartan, respectively. Proposed methods were validated as per ICH guidelines for linearity, accuracy, precision, specificity and robustness for estimation of Metoprolol Succinate and Olmesartan Medoxomil in commercially available tablet dosage form and results were found to be satisfactory. Thus the developed and validated stability indicating method can be used successfully for marketed formulations.

Keywords: metoprolol succinate, olmesartan medoxomil, RP-HPLC method, validation, ICH

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Authors: D. S. Mohale, A. P. Dewani, A. S.tripathi, A. V. Chandewar

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Present work demonstrates the applicability of high-performance liquid chromatography (HPLC) with UV-Vis detection for the quantification of malondialdehyde as malondialdehyde-thiobarbituric acid complex (MDA-TBA) in-vivo in rats. The HPLC method for MDA-TBA was achieved by isocratic mode on a reverse-phase C18 column (250mm×4.6mm) at a flow rate of 1.0mLmin−1 followed by detection at 532 nm. The chromatographic conditions were optimized by varying the concentration and pH of water followed by changes in percentage of organic phase optimal mobile phase consisted of mixture of water (0.2% triethylamine pH adjusted to 2.3 by ortho-phosphoric acid) and acetonitrile in ratio (80:20v/v). The retention time of MDA-TBA complex was 3.7 min. The developed method was sensitive as limit of detection and quantification (LOD and LOQ) for MDA-TBA complex were (standard deviation and slope of calibration curve) 110 ng/ml and 363 ng/ml respectively. Calibration studies were done by spiking MDA into rat plasma at concentrations ranging from 500 to 1000 ng/ml. The precision of developed method measured in terms of relative standard deviations for intra-day and inter-day studies was 1.6–5.0% and 1.9–3.6% respectively. The HPLC method was applied for monitoring MDA levels in rats subjected to chronic treatment of levofloxacin (LEV) (5mg/kg/day) for 21 days. Results were compared by findings in control group rats. Mean peak areas of both study groups was subjected for statistical treatment to unpaired student t-test to find p-values. The p value was <0.001 indicating significant results and suggesting increased MDA levels in rats subjected to chronic treatment of LEV of 21 days.

Keywords: malondialdehyde-thiobarbituric acid complex, levofloxacin, HPLC, oxidative stress

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Authors: Anil P. Dewani, Ravindra L. Bakal, Anil V. Chandewar

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Present work demonstrates the applicability of high-performance liquid chromatography (HPLC) with UV detection for the determination of malondialdehyde as malondialdehyde-thiobarbituric acid complex (MDA-TBA) in-vivo in rats. The HPLC-UV method for MDA-TBA was achieved by isocratic mode on a reverse-phase C18 column (250mm×4.6mm) at a flow rate of 1.0mLmin−1 followed by UV detection at 278 nm. The chromatographic conditions were optimized by varying the concentration and pH followed by changes in percentage of organic phase optimal mobile phase consisted of mixture of water (0.2% Triethylamine pH adjusted to 2.3 by ortho-phosphoric acid) and acetonitrile in ratio (80:20 % v/v). The retention time of MDA-TBA complex was 3.7 min. The developed method was sensitive as limit of detection and quantification (LOD and LOQ) for MDA-TBA complex were (standard deviation and slope of calibration curve) 110 ng/ml and 363 ng/ml respectively. The method was linear for MDA spiked in plasma and subjected to derivatization at concentrations ranging from 100 to 1000 ng/ml. The precision of developed method measured in terms of relative standard deviations for intra-day and inter-day studies was 1.6–5.0% and 1.9–3.6% respectively. The HPLC method was applied for monitoring MDA levels in rats subjected to chronic treatment of ciprofloxacin (CFL) (5mg/kg/day) for 21 days. Results were compared by findings in control group rats. Mean peak areas of both study groups was subjected for statistical treatment to unpaired student t-test to find p-values. The p value was < 0.001 indicating significant results and suggesting increased MDA levels in rats subjected to chronic treatment of CFL of 21 days.

Keywords: MDA, TBA, ciprofloxacin, HPLC-UV

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Authors: Thidarat Duangyod, Chanida Palanuvej, Nijsiri Ruangrungsi

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Pentace burmanica Kurz., belonging to the Malvaceae family, is commonly used for anti-diarrhea in Thai traditional medicine. A method for quantification of (+)-catechin and (-)-epicatechin in P. burmanica stem bark from 12 different Thailand markets by reverse-phase high performance liquid chromatography (HPLC) was investigated and validated. The analysis was performed by a Shimadzu DGU-20A3 HPLC equipped with a Shimadzu SPD-M20A photo diode array detector. The separation was accomplished with an Inersil ODS-3 column (5 µm x 4.6 x 250 mm) using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) as mobile phase at the flow rate of 1 ml/min. The isocratic was set at 20% B for 15 min and the column temperature was maintained at 40 ºC. The detection was at the wavelength of 280 nm. Both (+)-catechin and (-)-epicatechin existed in the ethanolic extract of P. burmanica stem bark. The content of (-)-epicatechin was found as 59.74 ± 1.69 µg/mg of crude extract. In contrast, the quantitation of (+)-catechin content was omitted because of its small amount. The method was linear over a range of 5-200 µg/ml with good coefficients (r2 > 0.99) for (+)-catechin and (-)-epicatechin. Limit of detection values were found to be 4.80 µg/ml for (+)-catechin and 5.14 µg/ml for (-)-epicatechin. Limit of quantitation of (+)-catechin and (-)-epicatechin were of 14.54 µg/ml and 15.57 µg/ml respectively. Good repeatability and intermediate precision (%RSD < 3) were found in this study. The average recoveries of both (+)-catechin and (-)-epicatechin were obtained with good recovery in the range of 91.11 – 97.02% and 88.53 – 93.78%, respectively, with the %RSD less than 2. The peak purity indices of catechins were more than 0.99. The results suggested that HPLC method proved to be precise and accurate and the method can be conveniently used for (+)-catechin and (-)-epicatechin determination in ethanolic extract of P. burmanica stem bark. Moreover, the stem bark of P. burmanica was found to be a rich source of (-)-epicatechin.

Keywords: pentace burmanica, (+)-catechin, (-)-epicatechin, high performance liquid chromatography

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Authors: S. G. Vasantharaju, Viswanath Guptha, Raghavendra Shetty

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Introduction: Aminophylline is a methylxanthine derivative belonging to the class bronchodilator. From the literature survey, reported methods reveals the solid phase extraction and liquid liquid extraction which is highly variable, time consuming, costly and laborious analysis. Present work aims to develop a simple, highly sensitive, precise and accurate high-performance liquid chromatography method for the quantification of Aminophylline in rat plasma samples which can be utilized for preclinical studies. Method: Reverse Phase high-performance liquid chromatography method. Results: Selectivity: Aminophylline and the internal standard were well separated from the co-eluted components and there was no interference from the endogenous material at the retention time of analyte and the internal standard. The LLOQ measurable with acceptable accuracy and precision for the analyte was 0.5 µg/mL. Linearity: The developed and validated method is linear over the range of 0.5-40.0 µg/mL. The coefficient of determination was found to be greater than 0.9967, indicating the linearity of this method. Accuracy and precision: The accuracy and precision values for intra and inter day studies at low, medium and high quality control samples concentrations of aminophylline in the plasma were within the acceptable limits Extraction recovery: The method produced consistent extraction recovery at all 3 QC levels. The mean extraction recovery of aminophylline was 93.57 ± 1.28% while that of internal standard was 90.70 ± 1.30%. Stability: The results show that aminophylline is stable in rat plasma under the studied stability conditions and that it is also stable for about 30 days when stored at -80˚C. Pharmacokinetic studies: The method was successfully applied to the quantitative estimation of aminophylline rat plasma following its oral administration to rats. Discussion: Preclinical studies require a rapid and sensitive method for estimating the drug concentration in the rat plasma. The method described in our article includes a simple protein precipitation extraction technique with ultraviolet detection for quantification. The present method is simple and robust for fast high-throughput sample analysis with less analysis cost for analyzing aminophylline in biological samples. In this proposed method, no interfering peaks were observed at the elution times of aminophylline and the internal standard. The method also had sufficient selectivity, specificity, precision and accuracy over the concentration range of 0.5 - 40.0 µg/mL. An isocratic separation technique was used underlining the simplicity of the presented method.

Keywords: Aminophyllin, preclinical pharmacokinetics, rat plasma, RPHPLC

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Authors: Jignasha Derasari, Patel Krishna M, Modi Jignasa G.

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Chemical stability of pharmaceutical molecules is a matter of great concern as it affects the safety and efficacy of the drug product.Stability testing data provides the basis to understand how the quality of a drug substance and drug product changes with time under the influence of various environmental factors. Besides this, it also helps in selecting proper formulation and package as well as providing proper storage conditions and shelf life, which is essential for regulatory documentation. The ICH guideline states that stress testing is intended to identify the likely degradation products which further help in determination of the intrinsic stability of the molecule and establishing degradation pathways, and to validate the stability indicating procedures. A simple, accurate and precise stability indicating RP- HPLC method was developed and validated for simultaneous estimation of Amiloride Hydrochloride and Furosemide in tablet dosage form. Separation was achieved on an Phenomenexluna ODS C18 (250 mm × 4.6 mm i.d., 5 µm particle size) by using a mobile phase consisting of Ortho phosphoric acid: Acetonitrile (50:50 %v/v) at a flow rate of 1.0 ml/min (pH 3.5 adjusted with 0.1 % TEA in Water) isocratic pump mode, Injection volume 20 µl and wavelength of detection was kept at 283 nm. Retention time for Amiloride Hydrochloride and Furosemide was 1.810 min and 4.269 min respectively. Linearity of the proposed method was obtained in the range of 40-60 µg/ml and 320-480 µg/ml and Correlation coefficient was 0.999 and 0.998 for Amiloride hydrochloride and Furosemide, respectively. Forced degradation study was carried out on combined dosage form with various stress conditions like hydrolysis (acid and base hydrolysis), oxidative and thermal conditions as per ICH guideline Q2 (R1). The RP- HPLC method has shown an adequate separation for Amiloride hydrochloride and Furosemide from its degradation products. Proposed method was validated as per ICH guidelines for specificity, linearity, accuracy; precision and robustness for estimation of Amiloride hydrochloride and Furosemide in commercially available tablet dosage form and results were found to be satisfactory and significant. The developed and validated stability indicating RP-HPLC method can be used successfully for marketed formulations. Forced degradation studies help in generating degradants in much shorter span of time, mostly a few weeks can be used to develop the stability indicating method which can be applied later for the analysis of samples generated from accelerated and long term stability studies. Further, kinetic study was also performed for different forced degradation parameters of the same combination, which help in determining order of reaction.

Keywords: amiloride hydrochloride, furosemide, kinetic study, stability indicating RP-HPLC method validation

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Authors: Indu Gaur, Neha Bhadauria, Shilpi Shilpi, Susmita Goswami, Prem D. Sharma, Prabir K. Paul

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The concept of organic agriculture has been accepted as novelty in Indian society, but there is no data available on the human pathogens colonizing plant parts due to such practices. Also, the pattern and mechanism of their colonization need to be understood in order to devise possible strategies for their prevention. In the present study, human pathogenic bacteria were isolated from organically grown tomato plants and five of them were identified as Klebsiella pneumoniae, Enterobacter ludwigii, Serratia fonticola, Stenotrophomonas maltophilia and Chryseobacterium jejuense. Tomato plants were grown in controlled aseptic conditions with 25±1˚C, 70% humidity and 12 hour L/D photoperiod. Six weeks old plants were divided into 6 groups of 25 plants each and treated as follows: Group 1: K. pneumonia, Group 2: E. ludwigii, Group 3: S. fonticola, Group 4: S. maltophilia, Group 5: C. jejuense, Group 6: Sterile distilled water (control). The inoculums for all treatments were prepared by overnight growth with uniform concentration of 108 cells/ml. Leaf samples from above groups were collected at 0.5, 2, 4, 6 and 24 hours post inoculation for the colony forming unit counts (CFU/cm2 of leaf area) of individual pathogens using leaf impression method. These CFU counts were used for the in vivo colonization assay and adherence assay of individual pathogens. Also, resistance of these pathogens to at least 12 antibiotics was studied. Based on these findings S. fonticola was found to be most prominently colonizing the phylloplane of tomato and was further studied. Tomato plants grown in controlled aseptic conditions same as mentioned above were divided into 2 groups of 25 plants each and treated as follows: Group 1: S. fonticola, Group 2: Sterile distilled water (control). Leaf samples from above groups were collected at 0, 24, 48, 72 and 96 hours post inoculation and homogenized in suitable buffers for surface and cell wall protein isolation. Protein samples thus obtained were subjected to isocratic SDS-gel electrophoresis and analyzed. It was observed that presence of S. fonticola could induce the expression of at least 3 additional cell wall proteins at different time intervals. Surface proteins also showed variation in the expression pattern at different sampling intervals. Further identification of these proteins by MALDI-MS and bioinformatics tools revealed the gene(s) involved in the interaction of S. fonticola with tomato phylloplane.

Keywords: cell wall proteins, human pathogenic bacteria, phylloplane, solanum lycopersicum

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Authors: L. García, N. Cobas, M. López

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Acrylamide, a probable carcinogen, is formed in high-temperature processed food (>120ºC) when the free amino acid asparagine reacts with reducing sugars, mainly glucose and fructose. Cane juices' repeated heating would potentially form acrylamide during brown sugar production. This study aims to determine if using panela in yogurt cake preparation increases acrylamide formation. A secondary aim is to analyze the acrylamide concentration in four cake confections with different caloric sweetener ingredients: beet sugar (BS), cane sugar (CS), panela (P), and a panela and chocolate mix (PC). The doughs were obtained by combining ingredients in a planetary mixer. A model system made up of flour (25%), caloric sweeteners (25 %), eggs (23%), yogurt (15.7%), sunflower oil (9.4%), and brewer's yeast (2 %) was applied to BS, CS and P cakes. The ingredients of PC cakes varied: flour (21.5 %), panela chocolate (21.5 %), eggs (25.9 %), yogurt (18 %), sunflower oil (10.8 %), and brewer’s yeast (2.3 %). The preparations were baked for 45' at 180 ºC. Moisture was estimated by AOAC. Protein was determined by the Kjeldahl method. Ash percentage was calculated by weight loss after pyrolysis (≈ 600 °C). Fat content was measured using liquid-solid extraction in hydrolyzed raw ingredients and final confections. Carbohydrates were determined by difference and total sugars by the Luff-Schoorl method, based on the iodometric determination of copper ions. Finally, acrylamide content was determined by LC-MS by the isocratic system (phase A: 97.5 % water with 0.1% formic acid; phase B: 2.5 % methanol), using a standard internal procedure. Statistical analysis was performed using SPSS v.23. One-way variance analysis determined differences between acrylamide content and compositional analysis, with caloric sweeteners as fixed effect. Significance levels were determined by applying Duncan's t-test (p<0.05). P cakes showed a lower energy value than the other baked products; sugar content was similar to BS and CS, with 6.1 % mean crude protein. Acrylamide content in caloric sweeteners was similar to previously reported values. However, P and PC showed significantly higher concentrations, probably explained by the applied procedure. Acrylamide formation depends on both reducing sugars and asparagine concentration and availability. Beet sugar samples did not present acrylamide concentrations within the detection and quantification limit. However, the highest acrylamide content was measured in the BS. This may be due to the higher concentration of reducing sugars and asparagine in other raw ingredients. The cakes made with panela, cane sugar, or panela with chocolate did not differ in acrylamide content. The lack of asparagine measures constitutes a limitation. Cakes made with panela showed lower acrylamide formation than products elaborated with beet or cane sugar.

Keywords: beet sugar, cane sugar, panela, yogurt cake

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Authors: Mindaugas Liaudanskas, Darius Kviklys, Kristina Zymonė, Raimondas Raudonis, Jonas Viškelis, Norbertas Uselis, Pranas Viškelis, Valdimaras Janulis

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Apples are among the most commonly consumed fruits in the world. Based on data from the year 2014, approximately 84.63 million tons of apples are grown per annum. Apples are widely used in food industry to produce various products and drinks (juice, wine, and cider); they are also used unprocessed. Apples in human diet are an important source of different groups of biological active compounds that can positively contribute to the prevention of various diseases. They are a source of various biologically active substances – especially vitamins, organic acids, micro- and macro-elements, pectins, and phenolic, triterpenic, and other compounds. Triterpenic compounds, which are characterized by versatile biological activity, are the biologically active compounds found in apples that are among the most promising and most significant for human health. A specific analytical procedure including sample preparation and High Performance Liquid Chromatography (HPLC) analysis was developed, optimized, and validated for the detection of triterpenic compounds in the samples of different apples, their peels, and flesh from widespread apple cultivars 'Aldas', 'Auksis', 'Connel Red', 'Ligol', 'Lodel', and 'Rajka' grown in Lithuanian climatic conditions. The conditions for triterpenic compound extraction were optimized: the solvent of the extraction was 100% (v/v) acetone, and the extraction was performed in an ultrasound bath for 10 min. Isocratic elution (the eluents ratio being 88% (solvent A) and 12% (solvent B)) for a rapid separation of triterpenic compounds was performed. The validation of the methodology was performed on the basis of the ICH recommendations. The following characteristics of validation were evaluated: the selectivity of the method (specificity), precision, the detection and quantitation limits of the analytes, and linearity. The obtained parameters values confirm suitability of methodology to perform analysis of triterpenic compounds. Using the optimised and validated HPLC technique, four triterpenic compounds were separated and identified, and their specificity was confirmed. These compounds were corosolic acid, betulinic acid, oleanolic acid, and ursolic acid. Ursolic acid was the dominant compound in all the tested apple samples. The detected amount of betulinic acid was the lowest of all the identified triterpenic compounds. The greatest amounts of triterpenic compounds were detected in whole apple and apple peel samples of the 'Lodel' cultivar, and thus apples and apple extracts of this cultivar are potentially valuable for use in medical practice, for the prevention of various diseases, for adjunct therapy, for the isolation of individual compounds with a specific biological effect, and for the development and production of dietary supplements and functional food enriched in biologically active compounds. Acknowledgements. This work was supported by a grant from the Research Council of Lithuania, project No. MIP-17-8.

Keywords: apples, HPLC, triterpenic compounds, validation

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