METHOD DEVELOPMENT AND VALIDATION OF THE CHROMATOGRAPHIC ANALYSIS OF FLUTICASONE PROPIONATE AND SALMETEROL XINAFOATE COMBINATION IN SOLUTIONS AND HUMAN PLASMA USING HPLC WITH UV DETECTION

Objective: A simple, Rapid, and sensitive HPLC method utilizing UV detection was developed and validated for the simultaneous estimation of Fluticasone propionate (FP) and Salmeterol xinafoate (SX) in solutions and in vitro human plasma. Methods: Chromatographic analysis was done on SUPELCO® RP-C18 column (150 x 4.6 mm, 5 μm particle size) with an isocratic mobile phase composed of methanol, acetonitrile, and water (50:20:30, v/v) mixture while flow rate was set to 1 ml/min. Detection with UV at maximum absorbance wavelength (ʎmax) values of 236 and 252 for FP and SX, respectively. Spiked plasma samples were liquid-liquid extracted by diethyl ether and reconstituted using methanol. Results: Method was accurate and precise over a linear (R2>0.995) range of (0.067-100 µg/ml) and (0.0333-50 µg/ml) for FP and SX, respectively. LOD/lOQ values were 0.13/0.6 and 0.06/0.3 µg/ml for FP and SX, respectively. The developed method was successfully applied for the analysis of FP and SX in spiked human plasma samples. The method is considered to be accurate and precise over a linear (R2>0.9969) range of (6.67-66.67 µg/ml) and (3.33-33.3 µg/ml) for FP and SX, respectively. Extraction efficiency was approved by recovery values of (94.98–102.46 %) and (96.54–102.62 %) for FP and SX, respectively. Conclusion: This validated method revealed simple and cheap extraction procedures and detectors, non-buffered mobile phase, and short retention times with excellent resolution.

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Validation of a simple HPLC method to quantify methotrexate concentrations in human plasma

MedPharmRes ◽

10.32895/ump.mpr.6.1.5 ◽

2021 ◽

Vol 6 (1) ◽

pp. 27-32

Author(s):

Thuan Thi Minh Nguyen ◽

Minh Hue Nguyen

Keyword(s):

Human Plasma ◽

Chromatographic Analysis ◽

Mobile Phase ◽

High Performance ◽

Internal Standard ◽

Immunosuppressive Agent ◽

Hplc Method ◽

Plasma Samples ◽

Injection Volume ◽

Oasis Hlb

Methotrexate (MTX) is a chemotherapy and immunosuppressive agent widely used to treat cancer, autoimmune diseases in children and adult patients, and ectopic pregnancy. However, MTX is highly toxic to the liver, kidney, and nervous system. This study aimed to quantify the concentration of MTX in human plasma using high-performance liquid chromatography (HPLC). MTX and its internal standard (para aminoacetophenone-PAPA) in plasma samples were extracted simultaneously with methanol. Sample purity was performed using the 1 cc OASIS HLB cartridges. Sample injection volume of 10 µL was analyzed on a Lichrocart Supersil 125-4 column C18 maintained at 40 °C on a Waters 2695 XE equipped with a PDA detector set at 303 nm. The mobile phase contained phosphate buffer (pH 6.0) and methanol at a ratio of 80:20 (v/v) and was maintained at a flow rate of 1 ml/min. The results showed that the total time of chromatographic analysis was 15 min. MTX and PAAP were found in the chromatograms at retention times of 2.3 and 5.2 min, respectively. The linear range of the MTX from 0.5 to 25 µg/mL. Intra-day and inter-day imprecision for MTX ranged from 3.42 to 8.128%. LLOQ of MTX was 0.5 µg/mL and the extraction effects were above 77%. In conclusion, we developed and validated a simple HPLC method to determine the MTX concentrations in human plasma.

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A NEW BIOANALYTICAL METHOD DEVELOPMENT & VALIDATION FOR SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND SITAGLIPTIN PHOSPHATE MONOHYDRATE IN HUMAN PLASMA BY USING RP-HPLC

INDIAN DRUGS ◽

10.53879/id.51.12.10224 ◽

2014 ◽

Vol 51 (12) ◽

pp. 18-25

Author(s):

S. A Kumar ◽

◽

M Debnath ◽

J. V. L. N. S Rao ◽

G Sankar

Keyword(s):

Human Plasma ◽

Mobile Phase ◽

Method Development ◽

Hplc Method ◽

Precipitation Method ◽

Metformin Hydrochloride ◽

Simultaneous Estimation ◽

Dihydrogen Phosphate ◽

Rp Hplc

A new RP-HPLC method for the quantitative determination of metformin and sitagliptin in human plasma was developed and validated as per US-FDA guidelines. The drug was spiked in the plasma and extracted with mobile phase by precipitation method. The extracted analyte was injected into Symmetry C18 (4.6 x 150 mm, 3.5μm, Make: XTerra) or equivalent, maintained at ambient temperature and effluent was monitored at 254 nm. The mobile phase consisting of potassium dihydrogen phosphate [pH 5.8]: acetonitrile [HPLC Grade] (65:35 v/v). The flow rate was maintained at 0.9 mL/min. The calibration curve for metformin and sitagliptin was linear from 10.0 to 35.0 µg/mL (r2= 0.999) and 1.0 to 3.5 µg/mL (r2= 0.998) respectively. The inter-day and intra-day precision was found to be within limits. The lower limit of quantification (LLOQ) for metformin and sitagliptin were found to be 0.026 and 0.70 μg/mL respectively. The average % recovery for metformin and sitagliptin were found to be 98.82-100.03 & 99.76-100.89 % respectively and reproducibility was found to be satisfactory. This RP-HPLC method is suiTable for determining the concentration of metformin and sitagliptin in human plasma and it can applied for routine analysis for determination of the metformin and sitagliptin from dosage form during pharmacokinetic study.

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ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

PLoS ONE ◽

10.1371/journal.pone.0250265 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0250265

Author(s):

Hubert Hayden ◽

Nahla Ibrahim ◽

Johannes Klopf ◽

Branislav Zagrapan ◽

Lisa-Marie Mauracher ◽

...

Keyword(s):

Human Plasma ◽

Dynamic Range ◽

Neutrophil Extracellular Traps ◽

Plasma Samples ◽

Dna Complexes ◽

High Background ◽

Isotype Control ◽

Extracellular Traps

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

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Validation of developed method by RP-HPLC for simultaneous estimation of famotidine and ibuprofen in human plasma and studying the stability of the drugs in plasma

Kathmandu University Journal of Science Engineering and Technology ◽

10.3126/kuset.v12i1.21564 ◽

2018 ◽

Vol 12 (1) ◽

pp. 34-48

Author(s):

K. S Ashutosh ◽

D. Manidipa ◽

R. J. V. L. N. Seshagiri ◽

S. D. Gowri

Keyword(s):

Human Plasma ◽

Mobile Phase ◽

Simultaneous Estimation ◽

Hplc Separation ◽

Reverse Phase ◽

Rp Hplc ◽

The Stability ◽

Sodium Dihydrogen ◽

Isocratic Mode

The RP-HPLC separation was carried out by reverse phase chromatography on a Symmetry C18 (4.6 x 150 mm, 3.5 μm, make: XTerra) with a mobile phase composed of sodium dihydrogen ortho phosphate [pH 2.5] and acetonitrile in the ratio of 30:70 v/v in an isocratic mode at a flow rate of 1.2 mL/min. The run time was maintained for 8.0 min. The detection was monitored at 236 nm. The accuracy was calculated in human plasma and the % recovery was found 99.80 - 99.85 for famotidine and 99.56 -99.85.5 for ibuprofen and reproducibility was found to be satisfactory. The calibration curve for famotidine in human plasma was linear over 3.32 to 6.65 μg/mL and 100- 200 μg/mL for ibuprofen in human plasma respectively. The inter-day and intra-day precision in human plasma was found within limits. The proposed method has adequate sensitivity, reproducibility, and specificity for the determination of famotidine and ibuprofen in plasma. The LLOQ obtained by the proposed method in human plasma were 1.24 and 5.0 μg/mL for famotidine and ibuprofen respectively. The proposed method is simple, fast, accurate, and precise for the quantification of famotidine and ibuprofen in plasma as per the ICH guidelines.Kathmandu University Journal of Science, Engineering and TechnologyVol. 12, No. I, June, 2016, Page: 34-48

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ANALYTICAL METHOD DEVELOPMENT AND ITS VALIDATION FOR EVALUATION OF POLYHERBAL FORMULATION

INDIAN DRUGS ◽

10.53879/id.55.10.10920 ◽

2018 ◽

Vol 55 (10) ◽

pp. 66-68

Author(s):

◽

A. P Jadhav

Keyword(s):

Immune Response ◽

Ethyl Acetate ◽

Analytical Method ◽

Mobile Phase ◽

Method Development ◽

Simultaneous Estimation ◽

Active Constituents ◽

Polyherbal Formulation ◽

Marker Compounds ◽

Hptlc Method

Talekt capsule is a branded polyherbal formulation used for enhancing the immune response to dermal infections and also for treating skin disorders. Curcumin and embelin, which are the active constituents of Haridra and Vidanga, respectively were used as marker compounds for developing a simple, accurate, precise and robust HPTLC method for simultaneous estimation. The mobile phase of toluene: ethyl acetate: formic acid (6.5: 3: 0.2 v/v/v) was used for separation. 381nm was used as wavelength for analysis. The Rf value obtained for curcumin, and embelin was found to be 0.51 ± 0.2 and 0.33 ± 0.2, respectively. The developed method was validated on the basis of ICH Q2 (R1) guidelines.

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RP-HPLC Method Development and Its Validation for Quantitative Determination of Rimonabant in Human Plasma

Journal of Analytical Methods in Chemistry ◽

10.1155/2012/625979 ◽

2012 ◽

Vol 2012 ◽

pp. 1-4 ◽

Cited By ~ 2

Author(s):

Shravan Bankey ◽

Ganesh Tapadiya ◽

Jasvant Lamale ◽

Deepti Jain ◽

Shweta Saboo ◽

...

Keyword(s):

Human Plasma ◽

Mobile Phase ◽

Method Development ◽

Bioequivalence Study ◽

Hplc Method ◽

Liquid Extraction ◽

Liquid Liquid Extraction ◽

Rp Hplc ◽

Hplc Method Development

A simple, accurate, and precise HPLC method was developed and validated for determination of rimonabant in human plasma. Following liquid-liquid extraction, chromatographic separation was accomplished using C18 column with mobile phase consisting of acetonitrile : water (90 : 10, v/v), drug was detected at 260 nm using UVdetector. The LOD and LOQ were 3.0 and 10.0 μg/L, respectively. The method is linear in the interval 50.0–1000.0 μg/L. The average extraction recovery of drug from plasma was found to be 92.2%. The percent CV of the method was found to be less than 10.8%, and accuracy was found between 94.5 and 106.7%. The assay may be applied to a pharmacokinetic and bioequivalence study of rimonabant.

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Simultaneous Estimation of Pantoprazole and Domperidone in Pure Powder and a Pharmaceutical Formulation by High-Perfomance Liquid Chromatography and High-Performance Thin-Layer Chromatography Methods

Journal of AOAC International ◽

10.1093/jaoac/90.1.142 ◽

2007 ◽

Vol 90 (1) ◽

pp. 142-146 ◽

Cited By ~ 11

Author(s):

Bhavesh H Patel ◽

Bhanubhai N Suhagia ◽

Madhabhai M Patel ◽

Jignesh R Patel

Keyword(s):

Liquid Chromatography ◽

Thin Layer ◽

Thin Layer Chromatography ◽

Mobile Phase ◽

High Performance ◽

Hplc Method ◽

Uv Detection ◽

Simultaneous Estimation ◽

Phase Quantification ◽

Layer Chromatography

Abstract This paper describes validated high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) methods for the simultaneous estimation of pantoprazole (PANT) and domperidone (DOM) in pure powder and capsule formulations. The HPLC separation was achieved on a Phenomenex C18 column (250 mm id, 4.6 mm, 5 μm) using 0.01 M, 6.5 pH ammonium acetate buffer-methanol-acetonitrile (30 + 40 + 30, v/v/v, pH 7.20) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetatemethanol (60 + 40, v/v) as the mobile phase. Quantification was achieved with ultraviolet (UV) detection at 287 nm over the concentration range 400-4000 and 300-3000 ng/mL with mean recovery of 99.35 ± 0.80 and 99.08 ± 0.57% for PANT and DOM, respectively (HPLC method). Quantification was achieved with UV detection at 287 nm over the concentration range 80-240 and 60-180 ng/spot with mean recovery of 98.40 ± 0.67 and 98.75 ± 0.71% for PANT and DOM, respectively (HPTLC method). These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of PANT and DOM in pure powder and capsule formulations.

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Measurement of Native and Degraded Forms of Plasminogen in Human Plasma

10.1055/s-0039-1687224 ◽

1979 ◽

Author(s):

M.F. Scully ◽

V.V. Kakkar

Keyword(s):

Human Plasma ◽

Final Concentration ◽

Hexanoic Acid ◽

Chromogenic Substrate ◽

Plasma Samples ◽

In Vitro Degradation ◽

Presence And Absence ◽

Rate Of Activation

A method has been devised to measure the levels of native and degraded forms of plasminogen either alone or in mixtures using the known differences in the rate of activation as measured using the chromogenic substrate, S2251. Preliminary studies using the purified zymogens ascertained the conditions under which maximum differences in the rate of activation are observed. Blood is taken into citrate and trasylol (final concentration 1000K IU/ml of blood to prevent in-vitro degradation of plasminogen) and plasma prepared. Plasminogen is precipitated at 13% Na2SO4 and washed 5 times with 17% NA2SO4 to remove trasylol and antiplasmins. The fractions are reconstituted in saline and the rate of activation by urokinase measured in the presence and absence of 6-amino-hexanoic acid (final concentration 2.5mM), the ratio of these activities being linearly related to the percentage of degraded plasminogen in the mixture. The observed rate is not dependent an the concentration of plasminogen in the mixture. The behaviour of degraded and native plasminogen by this procedure was shown to be the same as that of the purified zymogens by the assay of plasma samples in which the original plasminogen was replaced by purified degraded and native plasminogen although the recovery of native plasminogen was less than the degraded forms. Various patient plasma samples have been assayed by this method

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Simultaneous Quantification of Dexpanthenol and Resorcinol from Hair Care Formulation Using Liquid Chromatography: Method Development and Validation

Scientifica ◽

10.1155/2016/1537952 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8

Author(s):

Amit Kumar De ◽

Partha Pratim Chowdhury ◽

Shyamaprasad Chattapadhyay

Keyword(s):

Mobile Phase ◽

Method Development ◽

Seborrheic Dermatitis ◽

Simultaneous Estimation ◽

Chromatographic Technique ◽

Personal Care ◽

Chromatography Method ◽

Mobile Phase Flow Rate ◽

Simultaneous Quantification ◽

Hair Care

The current study presents the simultaneous quantification of dexpanthenol and resorcinol from marketed hair care formulation. Dexpanthenol is often present as an active ingredient in personal care products for its beautifying and invigorating properties and restorative and smoothing properties. On the other hand resorcinol is mainly prescribed for the treatment of seborrheic dermatitis of scalp. The toxic side effects of resorcinol limit its use in dermatological preparations. Therefore an accurate quantification technique for the simultaneous estimation of these two components can be helpful for the formulation industries for the accurate analysis of their product quality. In the current study a high performance liquid chromatographic technique has been developed using a C18 column and a mobile phase consisting of phosphate buffer of pH = 2.8 following a gradient elution. The mobile phase flow rate was 0.6 mL per minute and the detection wavelength was 210 nm for dexpanthenol and 280 nm for resorcinol. The linearity study was carried out using five solutions having concentrations ranging between 10.34 μg·mL−1and 82.69 μg·mL−1(r2=0.999) for resorcinol and 10.44 μg·mL−1and 83.50 μg·mL−1(r2=0.998) for dexpanthenol. The method has been validated as per ICH Q2(R1) guidelines. The ease of single step sample preparation, accuracy, and precision (intraday and interday) study presents the method suitable for the simultaneous quantification of dexpanthenol and resorcinol from any personal care product and dermatological preparations containing these two ingredients.

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AN INSULIN ASSAY BASED ON THE INCORPORATION OF LABELLED GLYCINE INTO PROTEIN OF ISOLATED RAT DIAPHRAGM

Journal of Endocrinology ◽

10.1677/joe.0.0190259 ◽

1959 ◽

Vol 19 (3) ◽

pp. 259-262 ◽

Cited By ~ 11

Author(s):

K. L. MANCHESTER ◽

P. J. RANDLE ◽

F. G. YOUNG

Keyword(s):

Human Plasma ◽

Plasma Samples ◽

Rat Diaphragm ◽

Normal Human Plasma ◽

Advantages And Disadvantages ◽

Straight Line ◽

Normal Human ◽

Isolated Rat

SUMMARY Insulin increases the incorporation of [14C]glycine into the protein of isolated rat diaphragm in vitro. This effect of the hormone can be used as the basis of a straight-line assay for insulin. The advantages and disadvantages of such a method of assay are discussed. Normal human plasma also stimulates incorporation of glycine into diaphragm protein, and the activities of two plasma samples were equivalent to 2 and 10 mu. insulin/ml. plasma, respectively.

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