scholarly journals Development, Validation, and Application of the LC-MS/MS Method for Determination of 4-Acetamidobenzoic Acid in Pharmacokinetic Pilot Studies in Pigs

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4437
Author(s):  
Paulina Markowska ◽  
Zbigniew Procajło ◽  
Joanna Wolska ◽  
Jerzy Jan Jaroszewski ◽  
Hubert Ziółkowski
Keyword(s):  
Sample Preparation ◽  
Analytical Method ◽  
Half Life ◽  
High Performance ◽  
Internal Standard ◽  
Inosine Pranobex ◽  
Pilot Studies ◽  
Pharmacokinetics Study ◽  
Limit Of Quantitation ◽  
One Step

Each drug has pharmacokinetics that must be defined for the substance to be used in humans and animals. Currently, one of the basic analytical tools for pharmacokinetics studies is high-performance liquid chromatography coupled with mass spectrometry. For this analytical method to be fully reliable, it must be properly validated. Therefore, the aims of this study were to develop and validate a novel analytical method for 4-acetamidobenzoic acid, a component of the antiviral and immunostimulatory drug Inosine Pranobex, and to apply the method in the first pharmacokinetics study of 4-acetamidobenzoic acid in pigs after oral administration. Inosine Pranobex was administered under farm conditions to pigs via drinking water 2 h after morning feeding at doses of 20, 40, and 80 mg/kg. For sample preparation, we used liquid–liquid extraction with only one step—protein precipitation with 1 mL of acetonitrile. As an internal standard, we used deuterium labeled 4-acetamidobenzoic acid. The results indicate that the described method is replicable, linear (r2 ≥ 0.99), precise (2.11% to 13.81%), accurate (89% to 98.57%), selective, and sensitive (limit of quantitation = 10 ng/mL). As sample preparation requires only one step, the method is simple, effective, cheap, and rapid. The results of the pilot pharmacokinetics study indicate that the compound is quickly eliminated (elimination half-life from 0.85 to 1.42 h) and rapidly absorbed (absorption half-life from 0.36 to 2.57 h), and that its absorption increases exponentially as the dose is increased.

scholarly journals METHOD DEVELOPMENT AND VALIDATION FOR ANALYSIS OF DEOXYARBUTININ ANHYDROUS EMULSION SYSTEM USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

2019 ◽  
pp. 172-175 ◽  
Author(s):  
MUCHTARIDI MUCHTARIDI ◽  
IDA MUSFIROH ◽  
AHMAD FAUZI
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Analytical Method ◽  
High Performance ◽  
Method Development ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Emulsion System ◽  
Relative Standard ◽  
Limit Of Quantitation

Objective: The aim of this study is to develop a simple, precise and accurate analytical method of deoxyarbutin in anhydrous emulsion system preparation. Methods: The analysis was conducted using high-performance liquid chromatography (HPLC). Chromatographic analysis was carried out using a reversed phase-C18 column. The mobile consists of two phases methanol and water (60: 40 v/v) at a flow rate of 1.0 ml/min. The determinations were performed using UV detector set at 225 nm. All validation procedures were added with hydroquinone as an internal standard. Results: The method showed coefficient correlation is 0.9978, relative standard deviation (RSD) smaller than 2%, Limit of Detection (LOD) and Limit of Quantitation (LOQ) are 0.599 µg/ml and 1.817 µg/ml respectively. The total amount deoxyarbutin in anhydrous emulsion preparation is 1.964+0.02 % with 98% recovery percentage. Conclusion: The developed HPLC analytical method meets the validation criteria made by International Conference on Harmonisation (ICH).

scholarly journals DEVELOPMENT AND VALIDATION OF BIOANALYTICAL HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF CILNIDIPINE AND NEBIVOLOL IN HUMAN PLASMA

2017 ◽  
Vol 9 (10) ◽  
pp. 253
Author(s):  
Aruna G. ◽  
Bharathi K ◽  
Kvsrg Prasad
Keyword(s):  
Human Plasma ◽  
Analytical Method ◽  
Internal Standard ◽  
Hplc Method ◽  
Pharmacokinetic Parameters ◽  
Simultaneous Estimation ◽  
Time Data ◽  
Bioanalytical Method Validation ◽  
Limit Of Quantitation ◽  
The Mean

Objective: To develop and validate a modified isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of cilnidipine and nebivolol in human plasma to be used for pharmacokinetic studies.Methods: The drug was extracted from plasma samples by direct protein precipitation technique using acetonitrile. Amlodipine was used as internal standard (IS). Samples were analyzed on BDS C18 column (250 x 4.6 mm, 5 µm), applying ortho phosphoric acid (0.1%): Acetonitrile, at a ratio of 45:55 v/v in isocratic mode as a mobile phase at a flow rate of 1 ml/min to attain adequate resolution. Separations were performed at room temperature and monitored at a wavelength of 260 nm after injection of 50μl samples into the HPLC system. The analytical method was validated according to FDA bioanalytical method validation guidance. The method was applied for pharmacokinetic study of cilnidipine and nebivolol tablets-10 mg and 5 mg were administered as a single dose to 6 healthy male rabbits under fasting condition. Twelve blood samples were withdrawn from each rabbit over 24 h periods. From the plasma concentration-time data of each individual, the pharmacokinetic parameters; Cmax, Tmax, AUC0-t and AUC0-∞ were calculated.Results: A peak area was obtained for cilnidipine and nebivolol at 3.943 and 4.719 min retention time respectively. Linearity was established at a concentration range of 0.20-20 μg/ml (r2=0.999, n=8) for cilnidipine and 0.02-2 μg/ml (r2=0.999, n=8) for nebivolol. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 0.2μg/ml for cilnidipine and 0.02 μg/ml for nebivolol. The coefficients of variation (%cv) of the intra-day and inter-day precision of cilnidipine at 600, 1000 and 1600ng/ml levels were found to be 6.90%, 6.19%, 5.22%; and 7.74%, 6.54%, 5.77%, respectively, which are lower than the accepted criteria limits (15-20 %). The mean recovery (%) cilnidipine at 600, 1000, and 1600ng/ml was found to be 101.03%, 99.27% and 104.87%, and for nebivolol 60, 100, and 160 ng/ml was found to be 106.13%, 107.03% and 98.06% respectively. Stability at different conditions and in autosampler was also established. The mean pharmacokinetic parameters; Cmax, Tmax, AUC0-t and AUC0-∞ were 6 ng/ml, 2 hr, 96.76 mg. hr/ml, 63.45 mg. hr/ml for cilnidipine and 5.8ng/ml, 2hr, 74.78 mg. hr/ml, 100.25 mg. hr/ml for nebivolol respectively.Conclusion: The present analytical method was found to be specific, sensitive, accurate and precise for quantification of cilnidipine and nebivolol in human plasma. It can be successively applied for pharmacokinetics, bioavailability and bioequivalence studies.

scholarly journals A sensitive and reliable RP-HPLC method for the detection of cimetidine – a H2 receptor antagonist in human plasma

2019 ◽  
Vol 8 (3) ◽  
pp. 671-674
Keyword(s):  
Human Plasma ◽  
High Performance ◽  
Internal Standard ◽  
Bioequivalence Study ◽  
High Concentration ◽  
H2 Receptor Antagonist ◽  
Chromatography Method ◽  
Thaw Cycle ◽  
Limit Of Quantitation ◽  
The Mean

Bioanalytical methods for bioequivalence studies require high sensibility and rapidity due to the large number of samples and the low plasma concentration of drugs. The present study aimed to develop and validate a high-performance liquid chromatography method to quantify cimetidine (CMT) in human plasma and to apply it in a bioequivalence study. Spiked plasma of 500 µl (l, m and h concentration) was used for the assay. The HPLC injection volume was 20μl of the reconstitute sample where, 2 ml of ethyl acetate used for extraction purposes. Cimetidine was prepared separately for low (80 ng/ml), medium (2000 ng/ml) and high (3600 ng/ml) concentrations and internal standard (ranitidine) concentration was 3000 ng/ml. Freeze thawing and long terms stability were conducted at -25º c. The individual calibration curve for spiked standards was linear with R2= 0.99. The inaccuracy values for QC samples were within 15% of the actual value and not more than 20% for the LOQ. The limit of quantitation (LOQ) was 40 ng/ml, which was also the lowest concentration of cimetidine that was quantitated with the variability of 5.9%. The within day precision and between day precision for LOQ were 10.8 and 5.9 respectively. The retention time for the analyte was 4.1-4.5 minutes during the within a day and between day results. The mean % inaccuracy values for low, medium and high concentration were 6.8, 5.6 and 7.8 respectively for within day and 2.4, 6.1 and 7.9 respectively for between days. The within day and between day % inaccuracy for LOQ concentration was 12.4 and 5.5 respectively. The mean recoveries for low, medium and high concentration of cimetidine were 80.2, 70.9 and 74.2. The overall mean recovery for cimetidine was 75.1%. The maximum inaccuracy for freeze thaw cycle and long term stability samples for low, medium and high was found with CV less than 15% for all concentrations, indicating that cimetidine is stable. The developed method was precise and accurate and was suitable to be applied for the bioequivalence study of cimetidine.

P–006 Simultaneous determination of bisphenol A and S in the samples of human seminal fluid

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Fialková ◽  
T Král ◽  
J Kohoutek ◽  
K Franzová ◽  
M Ješeta ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Bisphenol A ◽  
Endocrine Disruptors ◽  
Analytical Method ◽  
Seminal Fluid ◽  
Limit Of Detection ◽  
Semen Parameters ◽  
Ms Detection ◽  
Validation Criteria ◽  
Limit Of Quantitation

Abstract Study question Can we quantitatively determine concentrations of endocrine disruptors namely bisphenol A and S in seminal fluid? Summary answer We developed selective analytical method to simultaneously screen for the presence of bisphenol A (BPA) and S (BPS). What is known already The male reproductive system involves processes, which may be influenced by the disruption of the endocrine system by chemicals called endocrine disruptors (EDs). There is a growing evidence that EDs such as bisphenol A and S may be responsible for the decline in male reproductive health. To date, the claimed adverse effects on male fertility are largely based on the results from studies assessing the relationship between urinary BPA and BPS concentration and semen parameters. The best evidence of an adverse effect of BPA and BPS directly on spermatozoa could be provided by measuring bisphenols concentration directly in seminal fluid. Study design, size, duration To selectively and quantitatively analyzed bisphenols in any biological matrix advanced analytical tools and selective sample preparation protocols must be employed. In this study we developed targeted analytical method based on liquid chromatography tandem mass (LC-MS/MS) detection to measure bisphenol A and S in seminal fluid samples obtained from IVF clinic. A total of 140 samples were analysed. Participants/materials, setting, methods BPA and BPS was extracted from 140 seminal fluid samples using solvent extraction followed by preconcentration step. Samples were analyzed on Agilent 6495 Triple Quadrupole (Agilent Technologies, Santa Clara, CA) operating in the ESI-negative mode. Two MS/MS transitions were used for quantitative LC-MS/MS analyses. Chromatographic separation was achieved on Waters™ ACQUITY™ UPLC™BEH C18 (100 × 2.1 mm, 1.7 µm) column using gradient elution with a mixture of 0.1mM ammonium fluoride and methanol as mobile phases. Main results and the role of chance We developed selective sample preparation method for detection of BPA and BPS in seminal fluid followed by LC-MS/MS detection. The method validation was performed based on FDA guidelines. Validation criteria included limit of detection (LOD), limit of quantitation (LOQ), accuracy and precision. Due to the lack of the certified reference material the validation criteria of the method were assessed in pool of spiked seminal samples. The accuracy of the LC-MS/MS method was evaluated as a percent recovery of the amount of target analyte added into the sample. Recovery rates were above 80% for both analytes. LOD was 0.04 ng/mL for BPA and 0.01 ng/mL for BPS. LOQ was 0.14 ng/mL and 0.02 ng/mL for BPS. Measured BPA concentration ranged from 0.04 ng/mL to 1.62 ng/mL. For BPS, the concentration ranged from 0.01 ng/mL to 0.47 ng/mL. BPA and BPS were detected in 64% and 81% of samples, respectively. Interestingly, BPA showed lower detection frequency compared to BPS. These results are consistent with other studies performed on urine samples. Limitations, reasons for caution The limitation of the developed method is the time-consuming sample preparation and analysis cost. Wider implications of the findings: These results document for the first time the presence of BPS in seminal fluid. Knowing the concentration of BPA and BPS in seminal fluid is crucial for mitigating the associated health risks and initiating intervention and prevention strategies. Our future work will evaluate the influence of BPS concentration on spermatozoa. Trial registration number AZV NV18–01–00544; CZ.02.2.69/0.0/0.0/19_074/0012727

scholarly journals Determination of Tianeptine in Tablets by High-Performance Liquid Chromatography with Fluorescence Detection

2007 ◽  
Vol 90 (3) ◽  
pp. 720-724
Author(s):  
Sevgi Tatar Ulu
Keyword(s):  
Lower Limit ◽  
High Performance ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Linear Calibration ◽  
Fluorescence Detector ◽  
Relative Standard ◽  
Validation Parameters ◽  
Limit Of Quantitation

Abstract A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-Cl). A mobile phase consisting of acetonitrile10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were <0.46 and <0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.

scholarly journals DEVELOPMENT OF A DIRECT METHOD OF ANALYZING TRANEXAMIC ACID LEVELS IN WHITENING CREAM USING REVERSED PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

2020 ◽  
pp. 88-92
Author(s):  
BAITHA PALANGGATAN MAGGADANI ◽  
JIHAN YASMINA ◽  
HARMITA HARMITA
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Tranexamic Acid ◽  
Analytical Method ◽  
High Performance ◽  
Limit Of Detection ◽  
Direct Method ◽  
Pharmaceutical Products ◽  
Optimal Method ◽  
Limit Of Quantitation

Objective: Whitening cream is a cosmetic that contains ingredients that can alleviate hyperpigmentation. Tranexamic acid (TA) is one of the potentialanti-pigmentation agents that work through inhibiting plasmin. TA is used in cosmetic formulations at a concentration of 2.5% as a whitening andmoisturizing agent. To date, research on TA in both cosmetics and other pharmaceutical products using high-performance liquid chromatography(HPLC) has not been done directly (without derivatization). Therefore, this study aimed to develop a simple and rapid analytical method for TA(without derivatization) in cosmetic cream samples using reverse-phase HPLC and water as a solvent.Methods: Optimization was conducted by evaluating several parameters that affect sample extraction, as well as composition and mobile phasetypes. The optimal method must fulfill suitability and validation requirements. The optimal method should be able to detect and quantify TA in creamsamples without derivatization.Results: The optimal analysis condition used a ultraviolet detector at a wavelength of 210 nm, acetonitrile: double-distilled water: phosphoric acid(64:34:2) as the mobile phase and a flow rate of 0.8 mL/min. The retention time of the analyte occurred in the 2nd min.Conclusion: The analytical method that met the validation requirements was characterized using parameters such as accuracy, precision, linearity,selectivity, limit, of detection, and limit of quantitation. This method is applicable for analyzing TA content in samples with a concentration of 1.02%.

scholarly journals ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF AMLODIPINE IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY/MASS SPECTROMETRY

2018 ◽  
Vol 11 (7) ◽  
pp. 393
Author(s):  
Akiful Haque M ◽  
Shanthi Priya D K ◽  
Dibyalochan Mohanty ◽  
Vasudha Bakshi ◽  
Narender Boggula
Keyword(s):  
Mass Spectrometry ◽  
Human Plasma ◽  
Analytical Method ◽  
High Performance ◽  
Method Development ◽  
Solid Phase ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Economic Method ◽  
Positive Ion

Objective: The objective of the present investigation was to develop a novel, simple, and economic method for the estimation of amlodipine in positive ion mode in human plasma using amlodipine maleate d4 as an internal standard.Methods: The chromatographic separation was performed on Zorbax SB, C18, 50 mm*4.6 mm, and 3.5 mm. The mobile phase was prepared with a mixture of 5 mm ammonium acetate in 0.1% formic acid: High performance liquid chromatographic (HPLC) grade methanol:HPLC grade acetonitrile (40:30:30) that run isocratically at the flow rate of 0.700 ml/min and run time at 2.50 min.Results: The analytical method is valid for the estimation of amlodipine, in human plasma over a range of 0.100 ng/ml–9.990 ng/ml with the detection of amlodipine m/z - 409.10 (parent) and 238.00 (product), and internal standard Amlodipine Maleate d4 m/z - 413.20 (parent), and 238.00 (product) in positive ion mode. The results of carryover test, matrix effect, linearity, precision and accuracy, stabilities, dilution integrity, and run size evaluation test presented in this report are within the acceptance range.Conclusion: A sensitive method for the separation and determination of amlodipine in plasma has been developed based on solid-phase extraction with disposable extraction cartridges in combination with LC and mass spectrophotometers (MS/MS).

Development and validation of a HPLC-UV method for the simultaneous detection and quantification of paclitaxel and sulforaphane in lipid based self-microemulsifying formulation

2019 ◽  
Vol 57 (10) ◽  
pp. 931-938 ◽  
Author(s):  
Mohammad M Kamal ◽  
Sami Nazzal
Keyword(s):  
Analytical Method ◽  
Mobile Phase ◽  
High Performance ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Reversed Phase ◽  
Limit Of Quantitation ◽  
Development And Validation ◽  
Simultaneous Delivery ◽  
Detection And Quantification

Abstract Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH 5.0) as the mobile phase. PTX and SFN peaks were detected at 202 nm with high resolution without interference from excipients. This method showed linearity within 2.5–100 μg/mL range with r2 > 0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964 μg/mL for PTX and 0.4419 and 1.3389 μg/mL for SFN, respectively. A total of 98–101% of the injected samples was recovered with RSD of 0.06–0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.

scholarly journals Development and validation of a SPE-UHPLC-fluorescence method for the analysis of ochratoxin-a in certain Turkish wines

2019 ◽  
Vol 38 (2) ◽  
pp. 161
Author(s):  
Elif Mine Oncu Kaya
Keyword(s):  
Ochratoxin A ◽  
Mobile Phase ◽  
High Performance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Internal Standard ◽  
Fluorescence Method ◽  
Limit Of Quantitation ◽  
Development And Validation

A sensitive Ultra-High Performance Liquid Chromatography (UHPLC)-fluorescence method was developed and validated for the determination of ochratoxin-A (OTA) in Turkish wine samples. Naphthalene was used as an internal standard in this study. OTA was separated on a C18 (3.0 mm × 100 mm × 1.8 µm) column and analyses were run under isocratic conditions, with a mobile phase consisting of water/acetonitrile/acetic acid (50:50:1, v/v/v). The flow rate and injection volume were 0.5 ml min−1 and 10 μl, respectively. The excitation and emission wavelengths were 330 nm and 460 nm for OTA, respectively, and 220 nm and 325 nm for internal standard, respectively. A solid-phase extraction (SPE) clean-up procedure on a C18 cartridge was used prior to the analysis of the wine samples by UHPLC. The developed method was validated with respect to linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), stability and robustness. The method presented good RSD (< 4 %) and recovery (102.6–105.2 %) values. The LOD and LOQ values were 0.01 ng ml–1 and 0.05 ng ml–1, respectively. All other parameters were acceptable. OTA amounts were found in the range of 2.72‒7.40 µg kg‒1 in the Turkish wine samples.

scholarly journals Targeted Clinical Metabolomics Platform for the Stratification of Diabetic Patients

2019 ◽  
Author(s):  
Linda Ahonen ◽  
Sirkku Jäntti ◽  
Tommi Suvitaival ◽  
Simone Theilade ◽  
Claudia Risz ◽  
...  
Keyword(s):  
Analytical Method ◽  
High Performance ◽  
Clinical Laboratory ◽  
Plasma Metabolites ◽  
Diabetic Patients ◽  
Diabetes Clinic ◽  
High Throughput Analysis ◽  
Limit Of Quantitation ◽  
Liquid Chromatography Tandem Mass

AbstractBackgroundSeveral small molecule biomarkers have been reported in the literature for prediction and diagnosis of (pre)diabetes, its co-morbidities and complications. Here, we report the development and validation of a novel, quantitative, analytical method for use in the diabetes clinic. This method enables the determination of a selected panel of 36 metabolite biomarkers from human plasma.MethodsBased on a review of the literature and our own data, we selected a panel of metabolites indicative of various clinically-relevant pathogenic stages of diabetes. We combined these candidate biomarkers into a single ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and optimized it, prioritizing simplicity of sample preparation and time needed for analysis, enabling high-throughput analysis in clinical laboratory settings.ResultsWe validated the method in terms of limit of (a) detection (LOD), (b) limit of quantitation (LOQ), (c) linearity (R2), (d) linear range, and (e) intra- and inter-day repeatability of each metabolite. The method’s performance was demonstrated in the analysis of selected samples from a diabetes cohort study. Metabolite levels were associated with clinical measurements and kidney complications in type 1 diabetes (T1D) patients. Specifically, both amino acids and amino acid-related analytes were associated with macro-albuminuria. Additionally, specific bile acids were associated with kidney function, anti-hypertensive medication, statin medication and clinical lipid measurements.ConclusionsThe developed analytical method is suitable for robust determination of selected plasma metabolites in the diabetes clinic.

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