Octadecylsilane Column Research Articles

Polypyrrole as Adsorbent in Magnetic Solid Phase Extraction for Progesterone Determination from Human Plasma.

A straightforward and effective chromatographic method has been created for the analysis of progesterone from human plasma using a composite based on polypyrrole/magnetic nanoparticles in the sample preparation procedure. The quantification of progesterone is necessary due to its importance in human development and fertility. The employed conditions used acetonitrile:ultrapure water (70:30, v/v) as the mobile phase at 1.0 mL min-1 and an octadecyl silane column (Phenomenex Gemini, 250 mm × 4.6 mm, 5 μm) at a wavelength of 235 nm. The composite and its precursors were synthesized and properly characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy/energy dispersive spectroscopy, thermogravimetric analysis, and point of zero charge. The main factors affecting the extraction recovery of progesterone from pool human plasma samples employing magnetic solid phase extraction have been studied, such as sample pH (without adjustment), sample volume (1000 μL), washing solvent (ultrapure water), eluent (acetonitrile), eluent volume (1000 μL), and amount of adsorbent (10 mg). The extraction recoveries ranged from 98% to 102%, and linearity ranged from 5 to 3000 ng mL-1. The correlation coefficient (r) was ≥0.99, and acceptable relative standard deviation (precision), relative error (accuracy), and p-values (robustness) were observed. Lastly, the plasma samples from pregnant women were successfully analyzed by the validated method.

Development and Validation of HPLC-FLD Analysis of Perampanel in MEPS-Processed Rat Plasma Sample

Perampanel, a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥ 12 years) with refractory partial-onset seizures. A simple high-performance liquid chromatographic method fluorescence detection (HPLC-FLD) was developed to analyze perampanel in rats’ plasma and validated for bioanalytical purposes. Rats’ plasma (50 µL) was processed by microextraction packed sorbent (MEPS). The analytes were separated using a Hypersil Gold octadecyl silane column (250 × 4.6 mm internal diameter, 5 μm particle size) with isocratic elution. A mobile phase consisting of acetonitrile–methanol–water (275:275:450, v/v/v; containing 50 µL triethylamine and pH adjusted to 3.25 with orthophosphoric acid) was used in this analysis. The flow rate was 1.25 mL/min. Analytes were monitored at an excitation wavelength of 285 nm and an emission wavelength of 430 nm. The linearity range for this validated method was from 3.75 to 300 ng/mL. No endogenous peaks were found in the elution of analytes in drug-free rats’ plasma. Intra- and inter-batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria. The results indicate that the present method is simple, selective, reproducible, and suitable for the analysis of perampanel in small volume samples. The robustness of the method was accessed using MODDE® design of experiments software version 12.5.

DEGRADATION DETERMINATION OF TINOSORB-S IN SUNSCREEN PREPARATIONS

Objectives: The main objective was to determine the Tinosorb-S degradation in the sunscreen preparations with a simple economic linear and specific analytical method. Methods: The RP-HPLC was achieved with 100% methyl alcohol as the mobile phase, a flow rate of 2.5 mL/min., and an octadecylsilane column (300 × 3.9 cm, 10 µ) at 254 nm. Results: The developed analytical method of Tinosorb-S was statistically validated for accuracy and linearity of 70–130 µg/mL. The correlation coefficient was 0.996, and the % RSD of precision was 0.957. The recovery percentage was 100.01–105.04, and the excellent thermal and photolytic stability of Tinosorb-S was confirmed by the thermal, dry, and wet chromatograms compared with the standard; no such degradation was developed, and the recovery was 99.52%, 99.44%, and 99.45%, respectively. Acid, oxidative, and alkaline decomposition of Tinosorb-S were established by screening the decomposition peaks, and the percentage recovery was 41.31%, 40.38%, and 40.44%, respectively. Conclusions: The stress degradation study of Tinosorb-S is important in stability to evaluate the maximum protecting power of skin from harmful UV rays. Conditions like photolytic degradation, exposure to thermal conditions, acid hydrolysis, oxidation, and alkali hydrolysis were studied with a cost-effective, specific, linear, high-performance liquid chromatographic method with a photodiode array detector.

Determination of Arsenic Species in Edible Insects by LC-ICP-MS.

Edible insects may contain arsenic. Analysis of arsenic species is necessary in order to accurately assess arsenic exposure. An analytical method was validated and used to determine and quantitate arsenic species in edible insects. Arsenic species were extracted from edible insects by heating at 100 °C in 0.3 mol/L nitric acid. The concentration of arsenic species was then determined by liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) using an octadecylsilane (ODS) column with a mobile phase containing an ion-pair reagent. The limit of detection (0.007-0.012 mg/kg), limit of quantitation (0.021-0.038 mg/kg), repeatability (1.2-3.2%), intermediate precision (2.8-4.5%), and trueness (recoveries 97-102% based on spiked samples) of the proposed method were satisfactory for inorganic arsenic, dimethylarsinic acid, and arsenobetaine in edible insects. Total arsenic was detected in all samples obtained in Japan (Asian forest scorpion, diving beetles, giant water bug, grasshoppers, June beetles, mole crickets, male rhino beetle, female rhino beetle, sago worms, and silkworm pupae) and consisted of mostly inorganic arsenic. Beetles in particular showed relatively high levels. Arsenic content varies among edible insect species. Feed control is important, as arsenic concentrations in edible insects may be feed dependent. Arsenic species in edible insects were analyzed by LC-ICP-MS using an ODS column with a mobile phase containing an ion-pair reagent. Inorganic arsenic was detected in most samples, with concentrations ranging from <0.04 to 29.3 mg/kg.

Optimization of Extraction and Separation Process of Notoginsenoside Fc from Panax notoginseng Leaves.

Response surface methodology (RSM) was used to determine the optimal conditions for ultrasound-assisted extraction (UAE) of Notoginsenoside Fc (Fc) from panax notoginseng leaves. The experiment utilized a Box-Behnken design (BBD) and separation conditions were optimized. The optimum extraction conditions were as follows: extraction time = 1.5 h, ethanol concentration = 86%, liquid-to-solid ratio = 19:1. The experimentally obtained values were in accordance with the values predicted by the RSM model. We determined that the RSM model was able to successfully simulate the optimal extraction of Fc from the leaves. Further, Fc was enriched from Panax notoginseng through nine macroporous resins, and HPD-100 macroporous resins were selected for preliminary enrichment of Fc due to its economic costs and benefits. Subsequently, octadecyl silane (ODS) column chromatography was used to improve the purity of Fc to over 90% after separation by ODS column chromatography. Fc with a purity greater than 95% can be obtained by recrystallization. This is the first study that has focused on the extraction and enrichment of Fc from Panax notoginseng leaves using macroporous resin combined with ODS column chromatography, which provides the possibility for further application of Fc.

SIMULTANEOUS REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (RP-HPLC) ESTIMATION OF AZELASTINE HYDROCHLORIDE, FLUTICASONE PROPIONATE, AND PHENYL ETHYL ALCOHOL IN DYMISTA (MEDA) NASAL SPRAY

This work reported a reverse phase-high performance liquid chromatography method for quantitative estimation of Azelastine Hydrochloride, Fluticasone Propionate, and Phenylethyl Alcohol in Nasal Spray formulation. The assay involved an isocratic elution of these components by using Intersil Octadecylsilane (C18) column (25 cm x 4.6 mm x 5 μm) using mobile phase composition of Buffer: Solvent Mixture (40:60 %v/v) and pH adjusted to 6.5 with dilute orthophosphoric acid. The flow rate was 1.4 mLmin-1 and the analytes are monitored at 254 nm. Separation was completed within 20 minutes. Calibration curves were linear with correlation coefficient more than 0.99 over a concentration range of 50%, 75%, 100%, 125%, and 150% for Azelastine Hydrochloride, Fluticasone Propionate and Phenylethyl Alcohol. The method was proven to be accurate between 50 to 150% with 98 to 102 % recovery of the actives from a spiked placebo. The method was shown to be precise yielding acceptable results for the system reproducibility and method repeatability. All the validation parameters were within the acceptance range according to International Conference on Harmonization norms. The developed method is simple and rapid which could be applied for routine estimation of the formulation.

A high-performance liquid chromatography method for simultaneous quantification of metformin and ferulic acid in solid dosage forms.

Metformin is one of the most commonly used drugs in the world for the treatment of type 2 diabetes, while ferulic acid is a molecule that stands out for its antioxidant potential. Recent studies demonstrate hypoglycemic synergy between these molecules. The objective of this study is to develop and validate an analytical methodology by high-performance liquid chromatography for the simultaneous quantification of these drugs in pharmaceutical formulations. The method used an octadecylsilane column and a mobile phase composed of 6mM sodium lauryl sulfate in 15mM phosphate buffer:ACN (65:35). Ferulic acid and metformin were monitored at 232nm, with a mobile phase flow rate of 1ml/min and oven temperature at 40°C. The method was linear in the range of 5-25μg/ml for both molecules. In the presence of degradation products, satisfactory selectivity was achieved. Accuracy values were close to 100% and standard deviations in precision were less than 2%. In the robustness evaluation, the proposed variations did not interfere with the quantification. Therefore, it is concluded that the present method can be safely applied to the quality control of ferulic acid and metformin raw materials, as well as when they are combined in pharmaceutical formulations.

칠면초로부터 고순도 Rutin 및 Narcissin의 동시 분리 및 정제

Suaeda japonica is a useful candidate food material for the manufacture of blended tea due to its red color and excellent antioxidant activity. Rutin and narcissin are the main antioxidative flavonoids in the aerial parts of S.japonica, and these compounds have been reported to have antioxidant, anti-inflammatory, antibacterial, and anticancer activities. However, comparatively little is known about the biological activities of narcissin, which is present in small amounts in several plants. In this study, we developed an isolation method to obtain rutin and narcissin simultaneously in high purity from the aerial parts of S. japonica. The rutin and narcissin contents in the hot water extract of the aerial parts of S. japonica were 59.2 ± 1.3 mg/100 g and 171.3 ± 2.1 mg/100 g, respectively, as determined by ODS-HPLC. Briefly, the dried aerial parts (50 g) of S. japonica were extracted with hot water, and the filtrate obtained was extracted with water-saturated n-butanol (BuOH). The BuOH fraction was purified by medium-performance liquid chromatography using an octadecylsilane column. The purities of the rutin (27.6 mg) and narcissin (79.9 mg) isolated both exceeded 99% by LC-MS and 1 H-NMR analysis, and their total recoveries were estimated to be &gt; 91%.

Quantification of Pitolisant: A RP - HPLC study

Objective An attempt was made to develop a simple, isocratic and robust reverse phase high performance liquid chromatographic method for Pitolisant. Material and methods An Inertsil octa decyl silane column and photo diode array detector were employed for the quantification of Pitolisant at 268 nm using a mobile phase consisting of acetonitrile and 0.1 % v/v formic acid (90:10 % v/v) at a flow rate of 1.0 mL/min. for the efficient elution of Pitolisant. Results The developed method was validated for various parameters and the linearity range was observed at 25-150 µg/mL. The correlation coefficient, limit of detection and limit of quantification calculated from the calibration curve were 0.9998, 0.3 µg/mL and 1.0 µg/mL respectively. The mean percentage recovery was 99.9 %. The formulation was subjected to stress at various conditions resulting in minor degradants at different retention times. Conclusion An isocratic, fast, robust reverse phase – high performance liquid chromatographic method was developed which can be exploited for the estimation of Pitolisant in bulk and tablets. Key words: Pitolisant, High performance liquid chromatography, Photo diode array detector, Limit of detection, Degradation studies

A cytotoxic triterpenoid from a Periplaneta americana-derived, Gordonia hongkongensis WA12-1-1.

The secondary metabolites produced by microorganisms are a source of novel compounds with antitumor activities. In this study, we isolated biologically active secondary metabolites produced by microorganisms in the intestinal tract of Periplaneta americana. Based on the 16S rRNA gene sequencing, Gordonia hongkongensis WA12-1-1 was identified as the main microorganisms in the intestinal tract of P. americana. The obtained sequence was deposited in the National Center for Biotechnology Information (NCBI) database under the accession number MZ348554. The isolated secondary metabolites were separated and purified by thin layer chromatography, silica gel column chromatography, Sephadex column chromatography, open octadecyl silane column chromatography, high-performance liquid chromatography (HPLC), and semipreparative HPLC. Next, the structure of individual compounds was determined by ultraviolet spectroscopy, nuclear magnetic resonance, and mass spectrometry. A total of 20 compounds were isolated from the secondary metabolites produced by G. hongkongensis WA12-1-1. A total of 12 compounds were obtained from the crude ethyl acetate extract of the culture supernatant and eight from the cellular fraction. Compound 1 was identified as a triterpenoid named gordonterpene and showed cytotoxicity against A549 and HepG2 cell lines. These findings form a basis for further studies on the bioactivity of gordonterpene to tumor cells.

Isolationof diterpenes fromAraucariaspBrazilian brown propolisand development of a validated high-performance liquid chromatography method for its analysis.

Propolis comprises a complex resinous product composed of plant's parts or exudates, pollen, bee wax, and enzymes. Brazilian brown propolis from Araucaria sp displays several biological activities. Considering the lack of validated analytical methods for its analysis, we are reporting the development of a validated high-performance liquid chromatography with photodiode array detector method to analyze Araucaria brown propolis. The crude propolis were extracted and chromatographed, furnishing six main diterpenes. The isolated standards were used to draw the analytical curves, allowing the studies of selectivity, precision, accuracy, recovery, robustness, the determination of limits of detection and limits of quantification. The mobile phase consisted of 0.1% acetic acid in water and acetonitrile, using an octadecylsilane column, 1mL/min flow rate and detection at 200 or 241nm. Relative standard deviation values obtained for intra-day and inter-day precision were lower than 4% for all diterpenes. From the five parameters for robustness, wavelength detection and flow rate were the critical ones. Limits of detection and quantification ranged from 0.808 to 10.359 μg/mL and from 2.448 to 31.392 μg/mL, respectively. The recoveries were between 105.03 and 108.13%, with relative standard deviation values around 5.0%. The developed method is precise, sensitive, and reliable for analyzing Araucaria brown propolis.

Phenylpropanoids, lignan glucosides, feruloyltyramines, and flavonoid glycosides, as antioxidative compounds from aerial parts of Suaeda japonica

Sixteen antioxidative compounds were purified and isolated from the aerial parts of Suaeda japonica by column chromatography using Amberlite XAD-2 and octadecylsilane columns and high-performance liquid chromatography. These compounds, esculin (1), citrusin D (2), scopolin (3), cichoriin (4), isorhamnetin 3-O-rutinoside-7-O-β- D-glucopyranoside (5), 3'-methoxytaxifolin 7-O-β-D-glucopyranoside (6), (+)-lyoniresinol 3a-O-β-D-glucopyranoside (7), (2R,3R,4S)-isolariciresinol 3a-O-β-D-glucopyranoside (8), N-cis-feruloyltyramine 4-O-β-D-glucopyranoside (9), N-cis-feruloylmethoxytyramine 4-O-β-D-glucopyranoside (10), trans-icariside H1 (11), cis-icariside H1 (12), N-trans- feruloyltyramine 4-O-β-D-glucopyranoside (13), rutin (14), kaempferol-3-O-rutinoside (15), and narcissin (16), were identified for the firtst time in S. japonica. The structures of these compounds were determined by mass spectrometry and nuclear magnetic resonance spectroscopic analyses. Among the compounds, 14 showed the highest 1,1-diphenyl- 2-picrylhydrazyl free radical-scavenging activity and 8 exhibited the highest reactive oxygen species-scavenging activity in Huh7 cells.

Isomer separation and analysis of amphiphilic polysialogangliosides using reversed-phase liquid chromatography-mass spectrometry.

Gangliosides are amphiphilic, acidic glycosphingolipids possessing one or more sialic acid residues and several isobaric structural isomers with different abundances and bioactivities. Therefore, the distinction between these isomers is crucial for their proper profiling. Although liquid chromatography-mass spectrometry has been successfully employed for this purpose, the distinction process can still be improved, particularly regarding liquid chromatography. Recently, a reversed-phase liquid chromatography method that could separate disialoganglioside isomers was reported; however, the distinction of trisialoganglioside isomers using reversed-phase liquid chromatography has not been demonstrated. Here, we investigated the practicality of a reversed-phase liquid chromatography with an octadecylsilane column for separating polysialoganglioside isomers and successfully achieved the isomer separation of disialogangliosides and trisialogangliosides for the first time. We also confirmed several crucial factors in the mobile-phase composition, which affect the differential retention and mass spectral response of the isomers. First, an organic modifier, acetonitrile, exhibited superior selectivity against polysialogangliosides over methanol. Second, ammonium bicarbonate was the best ammonium salt additive among those tested, in terms of the separation efficiency and mass spectral response. Third, as the ammonium salt concentration increased, the negative electrospray ionization response was extensively suppressed, and the retention of gangliosides increased.

Single quad mass analyzer coupled UPLC method for impurity profile of Brimonidine tartrate and Timolol maleate: Application in their binary mixture ophthalmic formulation

The main objective of the study is to develop a suitable and rapid UPLC/PDA method by coupling online to Quadrupole Dalton analyzer (QDa), a mass detector for the identification and impurity profiling of Brimonidine Tartrate (BRIM)/Timolol maleate (TIMO) in the ophthalmic formulation. Chromatographic separation was achieved on ethylene bridged hybrid octadecylsilane column having 1.7μm particle size in gradient mode using high pure heptafluorobutyric acid as a buffer (A) and water, methanol, and acetonitrile (B) as mobile phase with a flow rate of 0.3mlmin-1. Based on the spectral maxima, BRIM and its impurities were monitored at 248nm, and TIMO and its impurities were monitored at 295nm. During evaluation of stress conditions and stability data unknown degradants are observed and identified as m/z 218.01 (DP1) and m/z 390.03 (DP2) using QDa-ESI+ scanning mode technique. The performance of the method was systematically validated according to ICH Q2 (R1) guidelines and the method shown very good sensitivity (≥0.5μg.mL-1) and linearity (r2≥0.999) with consistent recoveries and less than 5% RSD for all compounds. Hence, the proposed UPLC/PDA/QDa method is a simple, sensitive and comprehensive technique where identification and quantification can be done. It gives for complete impurity profile evaluation of BRIM/TIMO in the ophthalmic formulation during quality control in the pharmaceutical industry.

Antifungal Activity of Cyclic Tetrapeptide from Bacillus velezensis CE 100 against Plant Pathogen Colletotrichum gloeosporioides.

The aim of this study was to investigate the antifungal activity of a cyclic tetrapeptide from Bacillus velezensis CE 100 against anthracnose-causing fungal pathogen Colletotrichum gloeosporioides. Antifungal compound produced by B. velezensis CE 100 was isolated and purified from ethyl acetate extract of B. velezensis CE 100 culture broth using octadecylsilane column chromatography. The purified compound was identified as cyclo-(prolyl-valyl-alanyl-isoleucyl) based on mass spectrometer and nuclear magnetic resonance analyses. This is the first report of the isolation of a cyclic tetrapeptide from B. velezensis CE 100 culture filtrate. Cyclic tetrapeptide displayed strong antifungal activity at concentration of 1000 µg/mL against C. gloeosporioides mycelial growth and spore germination. Our results demonstrate that the antifungal cyclic tetrapeptide from B. velezensis CE 100 has potential in bioprotection against anthracnose disease of plants caused by C. gloeosporioides.

Simple method for simultaneous quantification of two new derivatives of betulinic and ursolic acids with antimalarial activity by RP-HPLC-UV-CAD

Endemic in more than 90 countries and territories, malaria is the most widely, populational, and geographically, parasitic disease in the world. Plasmodium sp. resistance to available drugs is one of the biggest problems for malaria eradication. In this study, we develop a method for the simultaneous determination of two new derivatives of betulinic and ursolic acids with antimalarial activity designated 3-O-butanoylbetulinic and 3-O-butanoylursolic acids. An analytical method was developed by high-performance liquid chromatography coupled, in series, to ultraviolet (UV) and charged aerosol (CAD) detectors. The chromatographic system, operated isocratically by reversed-phase, consisted in a mobile phase composed of acetonitrile: water pH 3.0 adjusted with formic acid (85:15, v/v), flow rate of 1.2 mL/min and a PhenoSphere Next octadecylsilane column (250 mm x 4.6 mm, 5 μm particle size). Chromatograms were recorded simultaneously in UV and CAD, at a concentration of 50 µg mL-1, an injection volume of 20 μL at a controlled temperature of 50 °C. The method was found to be selective, linear (r &gt; 0.99), precise (RSD &lt; 2.0%), accurate, and robust for both analytes, and considered statistically validated, and can be applied to the identification and quantification of these new drug candidates.

Determination of Inorganic Arsenic in Fish Oil and Fish Oil Capsules by LC-ICP-MS.

As inorganic arsenic is a highly toxic compound, its concentration in foods should be determined. Develop an analytical method for determining inorganic arsenic in fish oil and fish oil capsules. Inorganic arsenic was extracted from fish oil by heating at 80°C in 1.6% tetramethylammonium hydroxide-ethanol. The concentration of inorganic arsenic in fish oil was determined by liquid chromatography (LC) inductively coupled plasma (ICP) MS using an octadecylsilane (ODS) column with a mobile phase containing an ion-pair reagent. The LOD (0.005, 0.004 mg/kg), LOQ (0.016, 0.011 mg/kg), repeatability (4.2, 3.5%), intermediate precision (5.4, 3.5%), and trueness (recoveries 94-109% based on spiked samples) of the proposed method were satisfactory for inorganic arsenic in fish oil and fish oil capsules. A low level of inorganic arsenic was detected only in anchovy oil among all fish oil samples that were used in this study. Inorganic arsenic levels were below the quantitation limit in all fish oil capsules. Inorganic arsenic was determined after extraction from fish oil by heating at 80°C in 1.6% tetramethylammonium hydroxide-ethanol. The level of inorganic arsenic in all fish oil samples examined in this study was lower than 0.1 mg/kg of the maximum level defined in the Codex. Arsenic speciation in fish oil and fish oil capsules was analyzed by LC-ICP-MS using an ODS column with a mobile phase containing an ion-pair reagent. A low level of inorganic arsenic was detected only in anchovy oil. No inorganic arsenic was detected in fish oil capsules.

IMPURITY PROFILING IMPURITY PROFILING OF THIAMINE HYDROCHLORIDE INJECTION BY RP-HPLC AND CHARACTERIZATION OF DEGRADATION PRODUCT BY LC-MS/MS/QTOF

Objective: To propose a comprehensive, simple, and affordable RP-HPLC method for impurity profiling and characterization of unknown degradation products of thiamine hydrochloride injectable formulation.&#x0D; Methods: The chromatographic separation employs gradient mode using the octadecyl silane column using a mobile phase consisting of phosphate buffer with ion pair reagent, acetonitrile, and methanol delivered flow rate at 1.2 ml/min. The detection was carried out at 248 nm using empower software. LC-MS/MS/QTOF hyphenated technique was used for isolation and characterization of unknown degradation impurity. The performance of the method was systematically validated as per ICH Q2 (R1) guidelines.&#x0D; Results: Degradation product observed in accelerated stability was characterized by LC-MS/MS/QTOF hyphenated technique and found m/z value 351.1604 and postulated as an oxidative degradation product of thiamine due to excipient interaction. The validated method was sensitive, selective, and specific data proves the method is precise and accurate from LOQ to 150% level and results are within 95-108% and less than 4.5% RSD. The developed method is linear from 0.03-58.83 µg/ml with a correlation coefficient of more than 0.990 and LOD and LOQ value ranged from 0.03 to1.51 μg/ml.&#x0D; Conclusion: An efficient RP-HPLC method for impurity profiling of thiamine injectable formulation was successfully developed and unknown degradation product observed instability condition samples characterized by LC-MS/MS/QTOF technique. The validated method can be successfully employed for the impurity profiling of thiamine injectable in the quality control department.

Development and validation of bioanalytical method for the determination of hydrazinocurcumin in rat plasma and organs by HPLC-UV

Hydrazinocurcumin is a semi-synthetic analogue of curcumin with superior anticancer and anti-angiogenic activities. In the present work a simple and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method for quantitative evaluation of hydrazinocurcumin in plasma and various organs of rats including liver, kidneys, brain, heart, lungs and spleen was developed. Hydrazinocurcumin was separated using octadecylsilane (Inertsil-ODS-3V) column in an isocratic mode using mobile phase consisting of methanol-acetonitrile- water (36:27:37 v/v) with flow rate of 1.0 ml/min. Ultra violet (UV) detection of hydrazinocurcumin and internal standard was carried out in dual-wavelength mode at 332 nm and 380 nm, respectively. The linearity of hydrazinocurcumin was found in the range 0.05–5 µg/ml with a correlation coefficient of r2 > 0.999. The developed bioanalytical method shown higher inter-day accuracy (98.04–105.94%) and precision (0.89–10.24). The average recoveries of hydrazinocurcumin from rat plasma and various organs were in the range of 96–101.75% and 92.25–99.0%, respectively. The bioanalytical samples shows good stability of hydrazinocurcumin at different storage and handling conditions. In conclusion, this validated HPLC-UV method could be applied effectively for evaluation of hydazinocurcumin for the pharmacokinetic and organ distribution studies.

A Novel Validation of Analysis Method for Determining Nicotine Levels in Human Blood Plasma (In vitro) High-Pressure Liquid Chromatography Ultraviolet Detector

The necessity of nicotine analysis in blood plasma is increasing along with the increased number of smokers and nicotine poisoning cases. One of the analytical methods for nicotine is using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detector because it has been commonly owned by instance in Indonesia. To guarantee accuracy, an analytical method can be used, and it must be validated. This research was the purpose of finding out the validity of the nicotine analysis method in human blood plasma (in vitro) using HPLC with UV detection. Blood plasma samples remained treated with centrifugation procedure by protein denaturation method using acetonitrile. The compounds were analyzed using methanol and buffer acetate 0.01 M (pH 5) 85:15 v/v as a mobile phase on an octadecylsilane column 250 mm, with UV detection at 254 and 260 nm, and flow rate 0.6 mL/minute. Parameter of analytical methods that were validated includes selectivity, accuracy, precision, repeatability, linearity, limit of detection (LoD), limit of quantification (LoQ), and system suitability. According to the result, the selectivity was 2.479, repeatability expressed by its variation coefficient = 0.701%, linearity at range 5–22 μg/mL expressed by coefficient correlation (r) = 0.996. Based on the chromatogram’s area under a curve, the LoD value was found 2.021 μg/mL, LoQ value was 6.737 μg/mL, the accurate percentage was 112.49 to 114.12%, and precision (% CV) was 2.15 to 3.95%. The system suitability from retention time and chromatogram’s area under curve showed % CV 0.70 and 1.64%. According to the experiment result, all parameters meet the requirements of validation criteria.