Percent Relative Standard Deviation Research Articles

RP-HPLC Method Validation for Estimation of Tenofovir Disoproxil Fumarate in Pharmaceutical Oral Dosage Form

A simple, efficient and reproducible Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for the estimation of Tenofovir Disoproxil Fumarate in Pharmaceutical formulation has been validated. The Separation was carried out on C-18 column 4.6 mm x 250 cm x 5 µm column at ambient temperature using Buffer: Acetonitrile in the ratio of 60: 40 as a mobile phase. The flow rate was 1 ml/min and effluent was detected at 260nm. The retention times was found to be 3 mts. The percentage recovery was within the range between 99.23% to 101.44 %. The method was linear in the range of 50%-150% for Tenofovir Disoproxil Fumarate where r2 = 0.999. The percentage relative standard deviation for accuracy and precision was found to be less than 2%. The method was validated according to ICH guidelines and the acceptance criteria for specificity, linearity, accuracy, precision, ruggedness and robustness were met in all cases in detecting Tenofovir Disoproxil Fumarate. Hence the method could be successfully applied for routine analysis of Tenofovir Disoproxil Fumarate in the solid dosage form.

Development of Micro-Column Preconcentration Method Using a Restricted-Access Poly(protoporphyrin-co-vinyl pyridine) Adsorbent for Copper Determination in Water and Milk Samples by FIA-FAAS

For years, researchers have focused on the determination of metal ions at trace levels in environmental and food samples using analytical methods that employ techniques with low cost acquisition and maintenance and without microwave-assisted acid digestion procedures or aggressive reagents. Therefore, the present study deals with the synthesis and application of a novel, restricted-access poly(protoporphyrin-co-vinyl pyridine) adsorbent to preconcentrate copper in water samples and bovine milk that have only been subjected to pH adjusting (pH 6.0) and filtration using posterior on-line determination by FAAS. Regarding macromolecules, the restricted-access property of the adsorbent was achieved using the hydrophilic compound 2-hydroxyethyl methacrylate (HEMA). This method is based on the preconcentration of Cu2+ ions using a flow-injection system which is buffered with 0.05 mol L−1 of Britton–Robinson (BR) at a pH of 6.0 and has a flow rate of 14.0 mL min−1 through a mini-column packed with 50.0 mg of adsorbent. The elution was carried out using 0.40 mol L−1 of HCl toward the FAAS detector. The developed method provided a preconcentration factor of 44.7-fold, low limits of detection (LOD) (0.90 µg L−1) and quantification (LOQ) (2.90 µg L−1), tolerance to interfering ions (95.0 and 103.0%), and intra-day and inter-day precision assessed as the RSD (percentage of relative standard deviation), which ranged from 3.08 to 4.80%. The restricted-access poly(protoporphyrin-co-vinyl pyridine) adsorbent demonstrated outstanding features to exclude macromolecules, bovine serum albumin (BSA), and humic acid (HA) from an aqueous medium. Lake water and bovine milk samples were analyzed by the proposed preconcentration method with minimal sample pretreatment (which was based mainly on pH adjusting and filtration using an analytical curve with external calibration), yielding recovery values from addition and recovery tests ranging from 91.7 to 101.9%. The developed method shows great advantages over previously published methods, avoiding the time-consuming use of concentrated acids in a microwave-assisted acid digestion procedure.

Validated high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in fruits of Terminalia chebula, Phyllanthus emblica, and Quercus infectoria.

A simple and rapid high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in dried fruits of Terminalia chebula, Phyllanthus emblica, and Quercus infectoria has been developed. The chromatographic development was carried out on precoated silica gel 60 F254 plates in a mixture of toluene:ethyl acetate:chloroform:formic acid (4:8:1:3 v/v/v/v). The plate was scanned densitometrically at a wavelength of 280nm. The retention factor value of gallic acid and ellagic acid was found to be 0.63±0.2 and 0.53±0.1, respectively. The developed method was validated in terms of linearity, precision, accuracy, sensitivity, robustness, specificity and stability as per the international conference of harmonization guidelines. The method showed good linear relationship over a range of 100-600ng/band (gallic acid) and 100-500ng/band (ellagic acid) with a regression coefficient (r2 ) of 0.997 (gallic acid) and 0.996 (ellagic acid). The method showed high accuracy (99.65%-100.85%). The percentage relative standard deviation of intra-day and inter-day precision studies was not more than 2%. The method is highly robust and has displayed high specificity. The developed method is new, simple, and accurate and can be successfully employed in routine analysis of raw materials and formulations containing gallic acid and ellagic acid.

Development and Validation of a New Method for Simultaneous Estimation of Ramipril and Carvedilol by HPLC

The main target of this current research work was intended to construct and make affirmation of a facile, economical, very sensitive with high precision and accuracy, reversed-phase high performance liquid chromatographic method for simultaneous estimation of two drugs Ramipril and Carvedilol. The validation parameters were verified based on the standard requirements of International Council for Harmonization (ICH), U.S. Food and Drug Administration (FDA) and United States Pharmacopoeia (USP) by the determination of linearity, accuracy and precision. To develop the method, a C18 column (with a dimension of 250 × 4.6 mm, 5 μ, SUFELCOSILTM LC-18) was used. The mobile phase was comprised of aqueous KH2PO4 buffer solution and acetonitrile at the ratio of 55:45 (V/V) and flow rate was 1 mL per minute. 220 nm wavelength in the ultra-violet region was used to monitor the effluents and the retention times were found at 5.1±0.1 and 8.1±0.1 minutes for Ramipril and Carvedilol, respectively. Percent recovery for both drugs was above 98%, which demonstrated that the accuracy protocol was maintained. The linearity responses of the method for both Ramipril and Carvedilol were higher than 0.995 and the percentage of Relative Standard Deviation (RSD) (precision) for both of these two drugs were lower than the highest permissible limit or less than 2% (according to FDA). Therefore, it is easily perceptible that the corresponding RP-HPLC method was highly accurate, effective, rapid and precise which can offer huge potential for the application of simultaneous assay of Ramipril and Carvedilol in pure forms. Bangladesh Pharmaceutical Journal 26(1): 15-19, 2023 (January)

Validation of digital droplet PCR in detection and quantification of FLT3-TKD mutations in acute myeloid leukaemia

Background: Oncogenic mutation in the tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is a known recurrent mutation in AML.1 Com mon techniques used in its detection and/or characterisation include Sanger sequencing and nextgeneration sequencing, with recognised sensitivity limitations.2 Aim/Objective: To validate the use of digital droplet PCR (ddPCR) in the detection and quantification of common FLT3-TKD mutations (D835Y, D835H, D835V). Patients/Method: Samples from patients (seven D835Y, four D835H, two D835V), one external QAP and a commercial D835Y positive control were tested using a commercial Bio-rad ddPCR assay. Results: All samples with known FLT3-TKD mutations were correctly detected (15/15) with no false positives in the normal control. The assay showed cross-reactivity against FLT3-TKD mutations they were not-specific for, which persisted despite annealing temperature and digestion enzyme augmentation. The cross reactive events were reliably distinguishable from true positive events due to lower fluorescence amplitude. For the D835Y assay, repeatability studies showed a percentage relative standard deviation of 0.78% and a limit of detection of 0.01%. Conclusion: ddPCR is suitable for the detection and quantification of FLT3-TKD mutation in the diagnostic setting. Methodology optimisation may further improve upon on the sensitivity allowing for its use in minimal residual disease detection3.

Optimization of HPLC-MS/MS method for determination of antimalarial adulterants in herbal products.

The use of herbal products is booming all over the world because of being believed as safer than conventional drugs and free of side effects. However, there are untrustworthy manufacturers who adulterate herbal products by adding conventional drugs which might eventually lead to microbial resistance and herb-to-drug interactions. There is a need to develop methods for detecting adulterants in herbal products. A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous identification and determination of conventional antimalarials (chloroquine, quinine, sulfadoxine, pyrimethamine, mefloquine, lumefantrine, amodiaquine, artemisinin, dihydroartemisinin, artesunate and artemether) in herbal products was developed. Stable isotopically labelled compounds (artemether-d3, quindine-d3, and sulfadoxine-d3) were used as internal standards (ISs) for quantitative analysis. Extraction of analytes was performed using methanol: water: formic acid (90:10:0.1, v/v) and chromatographic separation was done in a gradient mode using mobile phase A: Ultrapure water containing 0.1% formic acid and 1mM ammonium formate and mobile phase B: Acetonitrile/methanol (50:50) containing 0.1% formic acid and 1mM ammonium formate. The calibration curves were linear (r2 ≥ 0.991) over the range of 0.001-0.3µgmL-1 for all compounds. The limit of detection (LOD) ranged from 0.002 to 0.02μgmL-1 while the limit of quantification (LOQ) ranged from 0.006 to 0.08μgmL-1. Accuracy, expressed as recovery of spiked herbal products ranged from 52 to 128%. The precision, expressed as percent relative standard deviation (%RSD) at two concentration levels, ranged from 1.0 to 13.8%. The matrix effect expressed as the matrix factor (MF) ranged from 0.77 to 0.97. The developed method was used to identify and quantify conventional antimalarials in herbal product samples from Tanzania. Ten out of 50 herbal products were found to contain amodiaquine, sulfadoxine, pyrimethamine, mefloquine, dihydroartemisinin, artemether and lumefantrine. The developed method is considered a valuable tool for getting a better understanding of the adulteration of conventional antimalarials in herbal products.

Simultaneous Determination of Vitamin E and γ-Oryzanol in Rice Bran Oil via HPSEC-PDA without Sample Pretreatment.

Vitamin E (tocopherols and tocotrienols) and γ-oryzanol are two minor constituents of rice bran oil (RBO) and are known to be potential bioactive compounds. The content of γ-oryzanol, a unique antioxidant found only in RBO, is a key factor in determining the retail price of the oil. Limitations of conventional HPLC columns for vitamin E and γ-oryzanol analysis are the alteration of these components and the time-consuming need for pretreatment of samples by saponification. High-performance size exclusion chromatography (HPSEC) equipped with a universal evaporative light scattering detector (ELSD) is a versatile tool for screening optimum mobile phase conditions because components of the sample can be separated and detected in the same run. In this work, the RBO components (triacylglycerol, tocopherols, tocotrienols, and γ-oryzanol) assessed on a single 100-Å Phenogel column using ethyl acetate/isooctane/acetic acid (30:70:0.1, v/v/v) as the mobile phase provided baseline separations (R s >1.5) with a total run time of 20 min. The HPSEC condition was then transferred to determine the content of tocopherols, tocotrienols, and γ-oryzanol in RBO products using a selective PDA detector. The limit of detection (LOD) and limit of quantification (LOQ) of α-tocopherol, α-tocotrienol, and γ-oryzanol were 0.34 and 1.03 μg/mL, 0.26 and 0.79 μg/mL and 2.04 and 6.17 μg/mL, respectively. This method was precise and accurate, with a percentage of relative standard deviation (%RSD) of the retention time of less than 0.21%. The intra-day and inter-day variations were 0.15-5.05% and 0.98-4.29% for vitamin E and γ-oryzanol, respectively. The recoveries of tocopherols, tocotrienols, and γ-oryzanol ranged between 90.75% and 107.98%. Thus, the developed HPSEC-ELSD-PDA method is a powerful analytical tool for determining the vitamin E and γ-oryzanol present in oil samples without requiring any sample pretreatment.

LEFLUNOMIDE TABLET FORMULATION: DEVELOPMENT AND VALIDATION OF AN RP-HPLC TECHNIQUE

Objective: To develop a Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for Leflunomide using rapid, cheap, economical, and less composition of the mobile phase. To validate the method's specificity, linearity, precision, accuracy, robustness, and ruggedness were all validated as per regulatory requirements ICH Q2 [R1] guidelines. Methods: The method employed solving of Development and validation based on the measurement of absorbance at one wavelength, 251 nm, λ max, Inertsil-ODS C18 analytical column (250 x 4.6 mm, 5μ). 1.0 ml/min of a mobile phase consisting of water and methanol (40:60v/v) for Leflunomide tablet formulation. Results: The method showed excellent linear response with correlation coefficient (R2) values of 0.999 for a Leflunomide. The percent recoveries for a drug were found within the acceptance limit of (99.93%–100.34%). Intra-and inter-day precision studies of the new method were less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0. It can be concluded from the results that the present method for validation determination of Leflunomide in tablets is specific, rapid, and simple with good sensitivity. Conclusion: This analytical method is also applicable in ordinary laboratories and also technique may be used to measure the drug and assess the uniformity and purity of the dosage formulation as well as for quality control of commercial Leflunomide tablets.

Development of spectrophotometric method for simultaneous estimation of dexketoprofen and dicyclomine

Objectives: The objective of this work was to develop a simple, fast, and reliable ultraviolet (UV) spectrophotometric method for simultaneous estimation of dexketoprofen and dicyclomine in tablet dosage forms. Materials and Methods: Dexketoprofen and dicyclomine solutions were prepared separately in the range of 2 µg/mL–10 µg/mL. The solutions were scanned over a range of 200–400 nm. The isoabsorptive point for both drugs was determined. The developed method was evaluated for accuracy, precision, and linearity. In linearity correlation coefficient was found. Results: A simple, fast, and reliable UV spectrophotometric method was developed for quantitative estimation of dexketoprofen and dicyclomine. The precision study showed that the percent relative standard deviation was within the range of acceptable limits, and the percent recovery was found to be in the range of 0.3–1.5 (intraday) and 0.23–1.7 (interday). In the accuracy study, the percent recovery was found to be 97 (50%), 96 (100%), and 95 (150%). Conclusion: The developed spectrophotometric method in this study was simple, accurate, precise, specific, sensitive, and reproducible and can be directly and easily applied to pharmaceutical dosage forms.

Smartphone digital image colorimetry for quantification of serum proteins.

Colorimetric determination of proteins in serum is proposed based on the biuret method and replacing ultraviolet-visible spectrometric (UV-Vis) detection with a simple and affordable smartphone digital image colorimetric (SDIC) method. Optimum SDIC conditions were found as a detection wavelength of 555 nm, a region of interest of 1600 px2, and 9.0 cm between the detection camera and sample solution. Under the optimum conditions, the coefficient of determination was 0.9982 within a linear dynamic range of 0.022 to 0.35 g dL-1. The precision of the method based on the percent relative standard deviation was below 5%. The limit of detection and limit of quantitation were found to be 0.007 and 0.022 g dL-1 respectively, which were sufficient for the quantification of the total protein, albumin, and globulin in serum. The method was validated with an independent experiment using a UV-Vis method and both methods showed good statistical agreement, indicating the accuracy of the proposed SDIC method.

White sustainable luminescent determination of nifuroxazide using nitrogen-sulphur co-doped carbon quantum dots nanosensor in bulk and various pharmaceutical matrices.

Nifuroxazide (NFX) is an antimicrobial agent that is frequently used as an intestinal antiseptic and recently was proven to have anticancer properties. This work employs the use of nitrogen and sulphur co-doped carbon quantum dots (NSC-dots) luminescent nanoparticles to propose a highly sensitive, sustainable, white and green spectrofluorometric method for NFX detection in bulk and pharmaceutical dosage forms. l-Cysteine and citric acid were the precursors to synthesize water soluble NSC-dots by a quick and environmentally-friendly hydrothermal process. NSC-dots' native fluorescence was measured at λem = 416 nm following excitation at 345 nm. Addition of NFX resulted in quantitative quenching of NSC-dots' luminescence, which represents the principle over which this luminescent method was based. Additionally, the mechanism of fluorescence quenching was studied and discussed. The analytical procedure was validated according to the ICH-guidelines. Linear response for NFX was obtained in the dynamic range 0.04-15 μg mL-1. The estimated NFX detection and quantification limits were 0.005 and 0.015 μg mL-1, respectively. The proposed method was employed for NFX quantification into two commercial pharmaceutical dosage forms. The calculated percentage recoveries (R%), percentage relative standard deviations (RSD%), and percentage error (Er%) were satisfactory. Comparison with other reported methods showed that the proposed method is superior in several aspects. Evaluation of the whiteness of the proposed method using the RGB 12 algorithm combined with the most widely used greenness evaluation tools, the Analytical Eco-Scale and AGREE, demonstrated its superiority and sustainability over other previously published spectrofluorimetric methods for the assay of NFX in various dosage forms.

Analytical Validation of a Reversed-Phase Ion Pairing HPLC-DAD Method for the Simultaneous Determination of Anthropogenic Pollutants

Caffeine, trigonelline, nicotinamide, and hippuric acid are potential organic markers of anthropogenic activities in the environment. In this work, a selective, precise, accurate, and robust reverse phase high performance liquid chromatography with a diode array detection method was developed for their simultaneous determination. Chromatographic analysis was achieved on GraceSmart RP C18 (150 mm x 4.6 mm, 5 μm) column. The mobile phase was 20 mM ammonium acetate buffer (pH 4.5) and methanol in gradient elution mode at a flow rate of 1.0 mL min-1, with 5 mM heptafluorobutyric acid as an ion pairing agent. Detection was performed at 275 nm for caffeine, 265 nm for trigonelline, 261 nm for nicotinamide, and 250 nm for hippuric acid. The method was validated as per the International Conference on Harmonization guidelines. Linear responses were found in the concentration range of 10-200 μg mL-1 with a correlation coefficient of at least 0.999. The percent relative standard deviation of intra-day precision, inter-day precision, and robustness was less than 2 %. The accuracy, expressed as percentage recovery ranged between 93.23 – 99.22%. Values for limits of detection and quantification were between 0.07-0.34 μg mL-1, and 0.21-1.04 μg mL-1, respectively. Lake samples from public parks were collected and subjected to analysis using the validated method

Designing of a unique bioreceptor and fabrication of an efficient genosensing platform for neonatal sepsis detection.

We report the results of studies related to the fabrication of a nanostructured graphene oxide (GO)-based electrochemical genosensor for neonatal sepsis detection. Initially, we selected the fimA gene of E. coli for nenonatal sepsis detection and further designed a 20-mer long amine-terminated oligonucleotide. This designed oligonucleotide will work as a bioreceptor for the detection of the virulent fimA gene. An electrochemical genosensor was further developed where GO was used as an immobilization matrix. For the formation of a thin film of GO on an indium tin oxide (ITO)-coated glass electrode, an optimized DC potential of 10 V for 90 s was applied via an electrophoretic deposition unit. Thereafter, the designed oligonucleotides were immobilized through EDC-NHS chemistry. The nanomaterial and fabricated electrodes were characterized via X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy and cyclic voltammetry techniques. The fabricated genosensor (BSA/pDNA/GO/ITO) has the ability to detect the target fimA gene with a linear detection range of 10-12 M to 10-6 M, a lower detection limit of 10-12 M and a sensitivity of 114.7 μA M-1 cm-2. We also investigated the biosensing ability of the developed genosensor in an artificial serum sample and the obtained electrochemical results were within the acceptable percentage relative standard deviation (% RSD), indicating that the fabricated genosensor can be used for the detection of neonatal sepsis by using a serum sample.

Method Development and Validation of Gallic Acid in Liquid Dosage Form by Using RP-HPLC Method.

A simple, rapid, precise, sensitive, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of gallic acid in the pharmaceutical dosage form. Chromatographic separation of gallic acid was achieved on Waters Alliance-e 2695, by using Waters X-Terra RP-18 (150×4.6mm, 3.5μ) column and the mobile phase containing 0.1% formic acid and ACN in the ratio of 70:30% v/v. The flow rate was 1.0mL/min; detection was carried out by absorption at 275 nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for gallic acid was NLT 2000 and should not be more than 2 respectively. Percentage relative standard deviation of peak areas of all measurements is always less than 2.0. The proposed method was validated according to ICH guidelines. The method was found to be simple, economical, suitable, precise, accurate, and robust method for quantitative analysis of gallic acid.

Amomum subalatum leaf extract derived silver nanoparticles for eco-friendly spectrophotometric detection of Hg (II) ions in water

Herein, biologically synthesized silver nanoparticles (AgNP) from amomum subalatum (AS) leaf extract (i.e. AgNP-AS) for spectrophotometric detection of Hg2+ ions. The formation of AgNP-AS has been confirmed by UV-Visible, X-ray diffraction (XRD), Fourier transform infrared (FTIR) and Transmission electron microscopy (TEM). The different characterization results showed that AgNP-AS exhibit a strong absorption band at 432 nm, crystallinity and spherical morphology with 4.9 nm average particle size. The AgNP-AS shows a good sensitivity towards Hg2+ ions by a decrease in its absorption intensity with a limit of detection (LOD) of 0.41 μM. Also besides, AgNP-AS shows a good selectivity towards Hg2+ ions over other heavy metal ions. The detection mechanism was thoroughly investigated with increasing Hg2+ ions concentration by observing the variation in the zeta potential. Moreover, to extend the applicability, the AgNP-AS was successfully applied to river and tap water samples treated with known concentrations of Hg2+ ions with a percentage recovery and relative standard deviation (RSD) values in the range of 96–101.1% and 1.6–6.2%, respectively. Therefore, the as-prepared AgNP-AS can be practically applied to samples of drinking water for the detection of Hg2+ ions.

Determination of vitamins B1 and B6 in infant formula and food supplement samples using magnetic layered double hydroxide nanoadsorbent before liquid chromatography-tandem mass spectrometry

A magnetic Mg/Fe layered double hydroxide (magnetic LDH) nanocomposite was successfully prepared and used as a suitable adsorbent to simultaneously extract vitamins B1 and B6 from complex infant formula and food supplements. The important parameters affecting the extraction were optimized and the final parameters are as follows; the amount of adsorbent (20 mg), extraction time (6 min), elution time (8 min), and elution solvent ethanol in 750 µL. As-prepared nanoadsorbent was successfully in magnetic solid phase extraction set-up with no hazardous solvents and extraction performed in 6 min. The structure of nanoadsorbnet is lamellar, providing a wide surface to extract the analytes. The quantification of the analytes was accomplished using the liquid chromatography-tandem mass (LC/MS-MS) technique. Under optimum extraction conditions, the linearity ranged from 4 to 1000 ng/mL (vitamin B1) and 20 to 1000 ng/mL (vitamin B6), and the correlation of coefficients (R2) was obtained better than 0.99. The intra-day (n = 3) and inter-day precisions (n = 3 working days) calculated in the form of percent relative standard deviations (%RSDs) were obtained below 10.20%. The proposed method was successfully practiced for analyzing vitamins B1 and B6 in several brands of infant formulas and food supplements.

Accurate and sensitive determination of Sb(III) in water samples using UV-VIS spectrophotometry after simultaneous complexation and preconcentration with deep eutectic solvent/DTZ probe-based liquid-liquid microextraction.

This study describes the determination of trace levels of antimony(III) by UV-Vis spectrophotometer after preconcentration by the deep eutectic solvent/dithizone probe-based liquid-liquid microextraction method. Ditizone was used as a ligand to form the coordinated antimony complex before extraction in the preconcentration process. In the microextraction method developed in the study, deep eutectic solvent was used to dissolve the complexing agent; thus, the complexation was performed at the same time as the extraction of antimony complex by deep eutectic solvent. All variables likely to affect the ligand-antimony(III) complex, extraction efficiency, and spectroscopic measurement were optimized to lower the detection limit. Under the determined optimum conditions, the detection limit for Sb was calculated as 1.6 × 10-3mg/L. The detection limit obtained with the method is much lower than the value obtained in the Uv-Vis spectrophotometer with the traditional method. In this study, the percent relative standard deviation for the lowest concentration was calculated as 3.12% (n = 8). This value indicates that the analysis performed has high precision. The applicability of the method was determined by performing spiked recovery tests on tap water taken from different regions. Satisfactory recovery results were obtained between 91 and 105% at three different concentrations.

Development and validation of a stability-indicating, single HPLC method for sacubitril-valsartan and their stereoisomers and identification of forced degradation products using LC-MS/MS.

The aim of this research work was to develop and validate a stability-indicating, single reversed-phase HPLC method for the separation of five impurities, including enantiomers, diastereomers, and degradation products in sacubitril-valsartan tablets. The method was developed using a Chiralcel OJ-RH column (150 × 4.6mm, 5μm) at 45°C with a gradient program of (T/%B) 0.01/25, 10.0/25, 25/38, 37.0/45, 39.0/25, and 45.0/25 at a flow rate of 0.8ml/min. Mobile phase A consisted of 1ml of trifluoroacetic acid in 1000 ml of Milli-Q water. Mobile phase B consisted of 1ml of trifluoroacetic acid in a mixture of acetonitrile and methanol in the ratio of 950:50 (v/v). Sacubitril, valsartan, and their five impurities were monitored at 254 nm. Degradation was not observed when sacubitril-valsartan was subjected to heat, light, hydrolytic, and oxidation conditions. In acid degradation study (1N HCl/60°C/2h) impurity 1 (m/z 383.44) was formed, and in base degradation study (0.1N NaOH/40°C/1h) impurities 1 and 5 (m/z 265.35) were formed; both impurities were confirmed using LC-MS. The degradation products, enantiomers, and diastereomers were well separated from sacubitril and valsartan, proving the stability-indicating power of the method. The developed method was validated per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The inter- and intra-day percentage relative standard deviation for sacubitril, valsartan, and their five impurities was less than 5.2%, recovery of the five impurities was between 93 and 105%, and linearity was ≥0.999. The limit of detection was 0.030-0.048 μg/ml, and the limit of quantification was 0.100-0.160 μg/ml.

Simple and Rapid High-Performance Liquid Chromatography Method for Simultaneous Determination of Picloram and 2,4-D in Pesticide Formulations

Picloram and 2,4-D are systemic herbicides used to control a wide range of broad-leaved weeds and post-emergence annual and perennial broad-leaved weeds, respectively. The use of plant protection products containing Picloram and 2,4-D necessitates the development of analytical methods, useful for the laboratories focused on control, for monitoring. In this study, we designed and validated an analytical method for the rapid determination of picloram and 2,4-D by HPLC-DAD. The method involves the extraction of the substances by sonication of the sample with methanol, followed by dilution in acetonitrile, and direct injection on a liquid chromatography system, based on the use of a Gemini C18 column. We used an isocratic mobile elution consisting of acetonitrile and water acidified 1% with H3PO4 (50:50, v/v). We validated the proposed method, which demonstrated linearity within the concentration range of 0.01–0.028 mg/mL for picloram and 2,4-D, with a correlation coefficient (R2) of 0.9993 for picloram and 0.9999 for 2,4-D. We considered precision, repeatability and selectivity in the validation. The repeatability of the method expressed as percent of relative standard deviation (%RSD) was lower than 1% for both substances. The proposed method is suitable for the simultaneous determination of picloram and 2,4-D in pesticide formulations.

A new Validated UV-visible Spectrophotometric Method for the Estimation of Pregabalin in its pure and dosage form using Bromophenol blue

A new simple, accurate precise, cost effective, rapid and sensitive UV-Visible spectrophotometric method was developed for the estimation of Pregabalin in its pure and dosage form using Bromophenol blue. The developed method was validated as per ICH guideline in terms of linearity, accuracy, precision, ruggedness, LOD and LOQ. In this method the solvent used for the preparation of Pregabalin was distilled water. The method is based on the formation of brown colored ion-pair complex of Pregabalin with Bromophenol Blue reagent. The ion pair complex was produced in presence of Acid Phthalate Buffer of pH 3. The developed brown colour was stable at room temperature. The developed brown coloured complex showed maximum absorbance at 592nm. Linearity was found in the concentration range of 50, 60, 70, 80 and 90µg/ml with regression coefficient value 0.995. Precision studies for intra-day and inter-day shows that percentage relative standard deviation was within range of acceptable limit. The recovery studies were carried out at three percentage levels that is 50%, 100% and 150% levels. The results shows that the method was found to be accurate. The limit of detection (LOD) was found to be 10.91µg/ml and limit of quantification (LOQ) was found to be 33.06µg/ml. A ruggedness study was carried out using different analysts; assure that the method was rugged. Hence the method can be applied for routine quality control of the drug.