LC-MS/MS Analysis Elucidates a Daily Rhythm in Orexin A Concentration in the Rat Vitreous Body

Orexins are two neuropeptides synthesised mainly in the brain lateral hypothalamic area. The orexinergic system provides arousal-dependent cues for a plethora of brain centres, playing a vital role in feeding behaviour, regulation of the sleep–wake cycle and circadian rhythms. Recently, orexins were found to be produced in the retina of an eye; however, their content in the vitreous body and possible daily pattern of expression have not yet been explored. In this manuscript, we describe the development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method designed for quantitative bioanalysis of orexin in the rat vitreous body. Orexin was extracted from vitreous body samples with a water:acetonitrile:formic acid (80:20:0.1; v/v/v) mixture followed by vortexing and centrifuging. Separation was performed on a reverse-phase HPLC column under gradient conditions. Orexin was analysed via multiple-reaction monitoring (MRM) in the positive electrospray mode. The total analysis time for each sample was less than 5.0 min. Once the method was fully optimised, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. The calibration curves for orexin (1–500 ng/mL) were constructed using a linear regression with a 1/x2 weighting. The lower limit of quantitation for orexin was 1.0 pg/mL for the vitreous body. Intra-day and inter-day estimates of accuracy and precision were within 10% of their nominal values, indicating that the method is reliable for quantitation of orexin in the rat vitreous body. From the physiological perspective, our results are the first to show daily rhythm of orexin synthesis by the retina with possible implications on the circadian regulation of vision.

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Development and Validation of an LC-MS/MS Assay to Quantitate 2′,4′,6′-Trihydroxyacetophenone in Rat and Dog Plasma and its Application to a Pharmacokinetic Study

Molecules ◽

10.3390/molecules25194373 ◽

2020 ◽

Vol 25 (19) ◽

pp. 4373

Author(s):

Hee Jo Yoo ◽

Se-Jung Hwang ◽

Jeong-Hun Lee ◽

Wang-Seob Shim ◽

Yun-Woong Choi ◽

...

Keyword(s):

Pharmacokinetic Study ◽

Ammonium Acetate ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Dog Plasma ◽

Limit Of Quantitation ◽

Regulatory Guidelines ◽

One Step ◽

Standard Calibration ◽

Development And Validation

In the present study, a simple, rapid, and reliable bioanalytical method was developed using liquid chromatography with tandem-mass spectrometry (LC-MS/MS) to quantify 2′,4′,6′-trihydroxyacetophenone (THAP) in rat and dog plasma with 2′,4′,6′-trihydroxybenzaldehyde as an internal standard (IS). The LC-MS/MS instrument was operated in the multiple reaction monitoring (MRM) mode to detect THAP at m/z transition 166.89 > 82.8 and IS at 152.89 > 82.8, respectively. A simple, one-step protein precipitation (PP) method was employed with acetonitrile for sample preparation. Utilizing a Gemini C18 column, THAP and IS were separated with an isocratic mobile phase consisting of 10 mM ammonium acetate and methanol (10:90, v/v) at a flow rate of 0.2 mL/min. Total chromatographic run time was 2.5 min per sample injection. The standard calibration curve for THAP was linear (r2 ≥ 0.9987) over the concentration range of 0.1 to 100 µg/mL with the lower limit of quantitation (LLOQ) of 0.1 µg/mL (S/N ratio > 10). According to the regulatory guidelines from the U.S. Food and Drug Administration (FDA) and the Korea Ministry of Food and Drug Safety (MFDS), our newly developed biomedical analytical method was fully and adequately validated in terms of selectivity, sensitivity, linearity, intra- and inter-day precision and accuracy, recovery, matrix effect, stability, and dilution integrity. Our validated assay was successfully utilized in a nonclinical pharmacokinetic study of THAP in rats and dogs.

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Quantitative LCMS for ceramides/dihydroceramides: pregnancy baseline biomarkers and potential metabolic messengers

10.1101/2020.02.24.963462 ◽

2020 ◽

Author(s):

Qianyang Huang ◽

Shiying Hao ◽

Xiaoming Yao ◽

Jin You ◽

Xiao Li ◽

...

Keyword(s):

High Throughput ◽

Multiple Reaction Monitoring ◽

Cellular Membrane ◽

Mass Spectrometric ◽

Precipitation Method ◽

Enhanced Sensitivity ◽

Limit Of Quantitation ◽

Liquid Chromatography Tandem Mass ◽

Tandem Mass Spectrometric ◽

Development And Validation

AbstractCeramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 mins by using stable isotope-labeled ceramides as internal standards (ISs). This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring (MRM) for mass spectrometric detection. To qualify for clinical applications, our assay was validated against the FDA guidelines: the Lower Limit of Quantitation (LLOQ as low as 1 nM), linearity (R2>0.99), precision (Coefficient of Variation<15%), accuracy (Percent Error<15%), extraction recovery (>90%), stability (>85%), and carryover (<0.1%). With enhanced sensitivity and specificity from this method, we have, for the first time, determined the serological levels of ceramides and dihydroceramides to reveal unique temporal gestational patterns. Our approach could have value in providing insights into disorders of pregnancy.

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Development and Validation of HPLC/UV-Spectrophotometric Procedures for Metronidazole Quantitative Determination

International Journal of Pharmaceutical Quality Assurance ◽

10.25258/ijpqa.v9i3.13663 ◽

2018 ◽

Vol 9 (3) ◽

Author(s):

Klimenko Lina Yu ◽

Shkarlat Galyna L ◽

Shovkova Zoia V ◽

Yaremenko Vitaliy D ◽

Shpychak Oleg S

Keyword(s):

Quantitative Determination ◽

Sodium Hydroxide Solution ◽

Gradient Elution ◽

Calibration Model ◽

Column Temperature ◽

Medical Laboratories ◽

Accuracy And Precision ◽

Development And Validation ◽

Gradient Elution Mode ◽

Potential Object

Metronidazole is the most popular representative of antiprotozoal medicines from the group of 5-nitroimidazoles. Metronidazole blocks the enzymes of alcohol dehydrogenase and acetaldehyde dehydrogenase, therefore when its joint taking with alcohol it is observed the strong intoxication syndrome and fatal poisonings too. Therefore metronidazole can be a potential object of chemical toxicological investigations. The purpose of our paper is to develop HPLC/UV-procedure of metronidazole quantification with application of the system of HPLC-analyzer MiLiChrome® A-0230 implemented in practice of forensic medical laboratories in Russia and Ukraine and carry out step-by-step validation of the developed procedure. Chromatographic conditions: Eluent A (0.2 M LiClO4 – 0.005 M HClO4) and Eluent B (acetonitrile) wereused as the mobile phase components; HPLC microcolumn Ø2×75 mm and ProntoSIL 120-5-C18 AQ, 5 μm were used as an analytical column; temperature was 40°С; flow rate was 100 μl/min; gradient elution mode was from 5% to 100% Eluent B for 40 min, then 100% Eluent B for 3 min; detection was performed at 277 nm. Retention time for metronidazole is 5.95 min. Since metronidazole is easy soluble and stable enough in the solutions of diluted alkalis 0.001 M sodium hydroxide solution has been proposed for preparation of the solutions in developing HPLC/UV-procedure of metronidazole quantification. Validation of the procedure has been carried out in the variants of the method of calibration curve and method of standard by such parameters as in process stability, linearity/calibration model, accuracy and precision within 3 different analytical runs using different batches of reagents and different glassware; experiments have been performed by three different analysts. New procedure of metronidazole quantitative determination by the method of HPLC/UV has been developed. Its validation has been carried out and acceptability for application has been shown.

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Development and Validation of 2-Azaspiro [4,5] Decan-3-One (Impurity A) in Gabapentin Determination Method Using qNMR Spectroscopy

Molecules ◽

10.3390/molecules26061656 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1656

Author(s):

Nataliya E. Kuz’mina ◽

Sergey V. Moiseev ◽

Mikhail D. Khorolskiy ◽

Anna I. Lutceva

Keyword(s):

Test Procedure ◽

Active Pharmaceutical Ingredient ◽

Test Methods ◽

Intermediate Precision ◽

Coefficients Of Variation ◽

Validation Parameters ◽

Limit Of Quantitation ◽

Student’S T ◽

Development And Validation ◽

Student’S T Test

The authors developed a 1H qNMR test procedure for identification and quantification of impurity A present in gabapentin active pharmaceutical ingredient (API) and gabapentin products. The validation studies helped to determine the limit of quantitation and assess linearity, accuracy, repeatability, intermediate precision, specificity, and robustness of the procedure. Spike-and-recovery assays were used to calculate standard deviations, coefficients of variation, confidence intervals, bias, Fisher’s F test, and Student’s t-test for assay results. The obtained statistical values satisfy the acceptance criteria for the validation parameters. The authors compared the results of impurity A quantification in gabapentin APIs and capsules by using the 1H qNMR and HPLC test methods.

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Development and Validation of a Rapid RP-HPLC Method for the Estimation of Esmolol Hydrochloride in Bulk and Pharmaceutical Dosage Forms

E-Journal of Chemistry ◽

10.1155/2010/470785 ◽

2010 ◽

Vol 7 (3) ◽

pp. 807-812 ◽

Cited By ~ 2

Author(s):

Vanita Somasekhar ◽

D. Gowri Sankar

Keyword(s):

Limit Of Detection ◽

Hplc Method ◽

Detector Response ◽

Reverse Phase Hplc ◽

Limit Of Quantification ◽

Pharmaceutical Dosage Forms ◽

Rp Hplc ◽

Accuracy And Precision ◽

Development And Validation

A reverse phase HPLC method is described for the determination of esmolol hydrochloride in bulk and injections. Chromatography was carried on a C18column using a mixture of acetonitrile, 0.05 M sodium acetate buffer and glacial acetic acid (35:65:3 v/v/v) as the mobile phase at a flow rate of 1 mL/min with detection at 275 nm. The retention time of the drug was 4.76 min. The detector response was linear in the concentration of 1-50 μg/mL. The limit of detection and limit of quantification was 0.614 and 1.86 μg/mL respectively. The method was validated by determining its sensitivity, linearity, accuracy and precision. The proposed method is simple, economical, fast, accurate and precise and hence can be applied for routine quality control of esmolol hydrochloride in bulk and injections.

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Development and Validation of UV Spectrophotometric Method for the Determination of Cefuroxime Axetil in Bulk and Pharmaceutical Formulation

Journal of Scientific Research ◽

10.3329/jsr.v6i1.14879 ◽

2013 ◽

Vol 6 (1) ◽

pp. 133-141 ◽

Cited By ~ 2

Author(s):

S. Binte Amir ◽

M. A. Hossain ◽

M. A. Mazid

Keyword(s):

Spectrophotometric Method ◽

Cost Effective ◽

Cefuroxime Axetil ◽

Pharmaceutical Formulation ◽

Uv Spectrum ◽

Accuracy And Precision ◽

Relative Standard ◽

Label Claim ◽

Development And Validation

The present study was undertaken to develop and validate a simple, sensitive, accurate, precise and reproducible UV spectrophotometric method for cefuroxime axetil using methanol as solvent. In this method the simple UV spectrum of cefuroxime axetil in methanol was obtained which exhibits absorption maxima (?max) at 278 nm. The quantitative determination of the drug was carried out at 278 nm and Beers law was obeyed in the range of (0.80-3.60) µg/ml. The proposed method was applied to pharmaceutical formulation and percent amount of drug estimated (95.6% and 96%) was found in good agreement with the label claim. The developed method was successfully validated with respect to linearity, specificity, accuracy and precision. The method was shown linear in the mentioned concentrations having line equation y = 0.05x + 0.048 with correlation coefficient of 0.995. The recovery values for cefuroxime axetil ranged from 99.85-100.05. The relative standard deviation of six replicates of assay was less than 2%. The percent relative standard deviations of inter-day precision ranged between 1.45-1.92% and intra-day precision of cefuroxime axetil was 0.96-1.51%. Hence, proposed method was precise, accurate and cost effective. Keywords: UV-Vis spectrophotometer; Method validation; Cefuroxime axetil; Recovery studies. © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v6i1.14879 J. Sci. Res. 6 (1), 133-141 (2013)

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Simultaneous Determination of Four Herbicide and Pesticide Residues in Chinese Green Tea Using QuEChERS Purification Procedure and UPLC-MS/MS Analysis

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.634-638.1586 ◽

2013 ◽

Vol 634-638 ◽

pp. 1586-1590

Author(s):

Su Fang Wang ◽

Shou Jie Zhang ◽

Chun Hong Dong ◽

Guo Qing Wang ◽

Jun Feng Guo ◽

...

Keyword(s):

Formic Acid ◽

Simultaneous Determination ◽

Green Tea ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Graphitized Carbon Black ◽

Ms Analysis ◽

Relative Standard ◽

Limit Of Quantitation

A method for simultaneous determination of residuals of four herbicides and pesticides, simazine, carboxin, diflubenzuron and rotenone, in Chinese green tea was developed. In the proposed method, the tea powder was placed in a centrifuge tube with a plug, extracted in saturated aqueous sodium chloride solution and acetonitrile, agitated using vortex oscillator, and then centrifuged 5 min at 4000 rpm. The supernatant solution was purified by primary secondary amine (PSA) sorbent, C18 power, and graphitized carbon black powder, respectively. Then the purified extracts were dissolved with acetonitrile:0.1% formic acid aqueous solution (40:60, V/V) and agitated, filtered using a syringe with 0.22 μm nylon filter prior to UPLC-MS/MS analysis. The UPLC analysis was performed on an ACQUITY UPLC® HSS T3 column (2.1 mm×100 mm, 1.8 µm), using acetonitrile-0.1% formic acid as mobile phase with the flow rate as 0.3 mL•min-1. Injection volume was 10 µL. Positive ionization mode was applied, and the ions were monitored in the multiple reaction monitoring (MRM) mode with curtain gas 0.069 MPa, collision gas 0.052 MPa, ESI ion spray voltage 5000 V, temperature 550 °C, nebulizer gas 0.24 MPa, and turbo gas 0.28 MPa. The limit of detection (LOD) and limit of quantitation (LOQ) of the proposed method are 1 μg•kg-1and 5 μg•kg-1, respectively. The average recoveries of the four pesticides at 10, 20, and 50 µg•kg-1spiking levels range from 77.4% to 95.3%. TheSupersSuperscript textcript textrelative standard deviation (RSD) (n=6) range form 11.83% to 4.52%.

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Spectrophotometric Determination of Olmutinib in Bulk by Area under Curve and First Order Derivative Methods and its Validation as per ICH guidelines

Journal of Drug Delivery and Therapeutics ◽

10.22270/jddt.v9i4-a.3466 ◽

2019 ◽

Vol 9 (4-A) ◽

pp. 349-354

Author(s):

BALU KHANDARE ◽

Atish C. Musle ◽

Sanket S. Arole ◽

Pravin V. Popalghat

Keyword(s):

Area Under Curve ◽

Limit Of Detection ◽

Order Derivative ◽

Spectrometric Method ◽

First Order ◽

Accuracy And Precision ◽

Ich Guidelines ◽

Validation Parameters ◽

Limit Of Quantitation

Abstract: A simple, precise and economical UV-spectrophotometric method has been developed for the estimation of Olmutinib from bulk. Two methods were developed First method (A) applied was area under curve (AUC) in which the area was integrated in wavelength from 262-272nm. Second method (B) was first order derivative spectrometric method. In this method absorbance at λmin=256.57nm, λmax=282.83nm and zero cross=267.68nm was measured. Calibration curves were plotted for the method by using instrumental response at selected wavelength and concentration of analyte in the solution. In both the methods, linearity was observed in the concentration range of 2-12µg/ml at the λmax=267.68nm. Accuracy and precision studies were carried out and results were satisfactorily obtained. The drug at each of the 80 %, 100 % and 120 % levels showed good recoveries that is in the range of 98.00 to 99.00% for both methods, hence it could be said that the method was accurate. Limit of detection (LOD) and limit of quantitation (LOQ) were determined for the method. The method was validated as per International Conference on Harmonization. All validation parameters were within the acceptable limit. The developed method was successfully applied to estimate the amount of Olmutinib in pharmaceutical formulation.

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Development and Validation of UPLC-MS/MS Method for Determination of Enasidenib in Rat Plasma and Its Pharmacokinetic Application

International Journal of Analytical Chemistry ◽

10.1155/2020/5084127 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Shuang-long Li ◽

Yong-liang Zhu ◽

Yi Zhang ◽

Shu-han Liu ◽

Xiang-die Wang ◽

...

Keyword(s):

Mass Transfer ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Gradient Elution ◽

Rat Plasma ◽

Bioanalytical Method Validation ◽

Fda Approval ◽

Elution Method ◽

Acquity Uplc ◽

Development And Validation

In our research, a straightforward UPLC-MS/MS method, with diazepam as the internal standard (IS), was proposed and acknowledged to determine the concentrations of enasidenib in rat plasma. When preparing the sample, we used acetonitrile for protein precipitation. The gradient elution method was used, and the mobile phase was acetonitrile and 0.1% formic acid. Diazepam was used as the IS. We used the Acquity UPLC BEH C18 column to separate enasidenib and IS. Under the positive ion electrospray ionization (ESI) source conditions, the mass transfer pairs of enasidenib were monitored by multiple reaction monitoring (MRM) to be m/z 474.2 ⟶ 456.1 and m/z 474.2 ⟶ 267.0, and the IS mass transfer pairs were m/z 285.0 ⟶ 154.0. Enasidenib had good linearity (r2 = 0.9985) in the concentration range of 1.0–1000 ng/mL. Besides, the values of intraday and interday precision were 2.25–8.40% and 3.94–5.46%, respectively, and the range of the accuracy values varied from −1.44 to 2.34%. Matrix effect, extraction recovery, and stability were compliant with FDA approval guidelines in terms of bioanalytical method validation. We had established a new method that had been applied to the pharmacokinetic study of enasidenib in rats.

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DEVELOPMENT AND VALIDATION OF ULTRAVIOLET-SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF FEBUXOSTAT FOR CONDUCTING IN-VITRO QUALITY CONTROL TESTS IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2018.v11i9.25550 ◽

2018 ◽

Vol 11 (9) ◽

pp. 115

Author(s):

Jaspreet Kaur ◽

Daljit Kaur ◽

Sukhmeet Singh

Keyword(s):

Spectrophotometric Method ◽

Limit Of Detection ◽

Pharmaceutical Formulations ◽

Dosage Forms ◽

Pharmaceutical Dosage Forms ◽

Relative Standard ◽

Limit Of Quantitation ◽

Development And Validation

Objective: A simple, accurate, and selective ultraviolet-spectrophotometric method has been developed for the estimation of febuxostat in the bulk and pharmaceutical dosage forms.Method: The method was developed and validated according to International Conference on Harmonization (ICH Q2 R1) guidelines. The developed method was validated statistically with respect to linearity, range, precision, accuracy, ruggedness, limit of detection (LOD), limit of quantitation (LOQ), and recovery. Specificity of the method was demonstrated by applying different stressed conditions to drug samples such as acid hydrolysis, alkaline hydrolysis, oxidative, photolytic, and thermal degradation.Results: The study was conducted using phosphate buffer pH 6.8 and λmax was found to be 312 nm. Standard plot having a concentration range of 1–10 μg/ml showed a good linear relationship with R2=0.999. The LOD and LOQ were found to be 0.118 μg/ml and 0.595 μg/ml, respectively. Recovery and percentage relative standard deviations were found to be 100.157±0.332% and <2%, respectively.Conclusion: Proposed method was successfully applicable to the pharmaceutical formulations containing febuxostat. Thus, the developed method is found to be simple, sensitive, accurate, precise, reproducible, and economical for the determination of febuxostat in pharmaceutical dosage forms.

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