Homocysteine and other thiols in plasma and urine: automated determination and sample stability

1993 ◽  
Vol 39 (2) ◽  
pp. 263-271 ◽  
Author(s):  
T Fiskerstrand ◽  
H Refsum ◽  
G Kvalheim ◽  
P M Ueland
Keyword(s):  
Column Switching ◽  
Fluorescence Yield ◽  
Reversed Phase ◽  
Hplc Method ◽  
Analytical Recovery ◽  
Single Column ◽  
Successful Separation ◽  
Heparin Plasma ◽  
Sampling Procedures ◽  
High Degree

Abstract We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sample output is approximately 70 samples in 24 h. Serum and heparin plasma can also be analyzed. Hemolysis up to approximately 2.0 g/L of hemoglobin did not interfere with the analytical recovery of homocysteine or cysteine. Collection of blood, separation of plasma from whole blood, and acid precipitation must be standardized to obtain reproducible thiol results. Our modifications and the standardization of blood-sampling procedures have substantially improved the method and broadened its applications.

Liquid-chromatographic quantification compared with gas-chromatographic-mass-spectrometric determination of verapamil and norverapamil in plasma.

1988 ◽  
Vol 34 (12) ◽  
pp. 2502-2503 ◽  
Author(s):  
P A Hynning ◽  
P Anderson ◽  
U Bondesson ◽  
L O Boréus
Keyword(s):  
Phosphate Buffer ◽  
High Performance ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Hplc Method ◽  
Gas Chromatography Mass Spectrometry ◽  
Analytical Recovery ◽  
Liquid Chromatographic ◽  
Chromatographic Mass

Abstract A high-performance liquid chromatographic (HPLC) method for determining verapamil and norverapamil in plasma is presented and compared with gas chromatography/mass spectrometry (GC-MS). The plasma samples were extracted at alkaline pH with hexane containing 2-butanol (20 mL/L) and then back-extracted into phosphate buffer (0.1 mol/L, pH 3.0). For chromatography we used a reversed-phase column (Supelcosil LC-18 DB) with a mobile phase of the phosphate buffer and acetonitrile (70/30 by vol). Fluorescence detection was used (excitation at 203 nm, emission at 320 nm). Overall analytical recovery was 85%. Standard curves were linear from 1 to 1000 micrograms/L. The detection limit was 1 microgram/L. The assays are accurate and precise. We found no interferences by those substances tested. Results by HPLC and GC-MS agreed well (r = 0.99) for both verapamil and norverapamil determinations.

scholarly journals Simultaneous estimation of ledipasvir and sofosbuvir in bulk and its dosage forms by stability indicating RP-HPLC method

2019 ◽  
Vol 10 (3) ◽  
pp. 1631-1639
Author(s):  
Ismail Y ◽  
Vijaya Vara Prasad M
Keyword(s):  
Reversed Phase ◽  
Dosage Forms ◽  
Hplc Method ◽  
Fixed Dose ◽  
Simultaneous Estimation ◽  
Isocratic Elution ◽  
Hepatitis C Infection ◽  
Rp Hplc ◽  
High Degree ◽  
Degradation Evaluation

The fixed-dose ledipasvir-sofosbuvir combination offers an effective and well-tolerated pill for the treatment of chronic hepatitis C infection. Only a few analytical works were carried out to estimate the Ledipasvir and Sofosbuvir drug combination in the various dosage forms. This work aimed to simplify the estimation process using RP-HPLC methodology. The method was developed on a reversed phase Agilent C18 (4.6 x 150 mm, 5 µm) column. The isocratic elution process was performed using a mobile phase ratio of Methanol (70% v/v): Water (30 % v/v) with 0.6 ml/min flow rate. Elute was scanned using the PDA detector at the wavelength of 235 nm. The results of the elution process showed that the Ledipasvir and Sofosbuvir elute the peak at a concentration of 9 μg/ml and 40 μg/ml with retention times of 7.745 min and 2.345 min respectively. The percentage purity of Ledipasvir and Sofosbuvir was found to be 99.40 % w/v and 98.20 % w/v. The proposed method was found to be a high degree of precision and reproducibility. The percentage recovery was found to be 99.92 % for Ledipasvir and 99.82 % for Sofosbuvir. The LOD and LOQ were measured, and the results were within limits. The developed validation method can be applied for degradation evaluation of Ledipasvir and Sofosbuvir for the various dosage forms.

Rapid monitoring of benzodiazepines in clinical samples by using on-line column switching HPLC

1991 ◽  
Vol 37 (6) ◽  
pp. 804-808 ◽  
Author(s):  
C M Moore ◽  
K Sato ◽  
Y Katsumata
Keyword(s):  
Signal To Noise Ratio ◽  
Column Switching ◽  
Reversed Phase ◽  
Clinical Samples ◽  
Analytical Column ◽  
Drug Concentrations ◽  
Analytical Recovery ◽  
Rapid Monitoring ◽  
On Line ◽  
Line Sample

Abstract We have determined routinely administered benzodiazepines and some of their metabolites in clinical samples by using on-line column switching for sample clean-up and reversed-phase high-pressure liquid chromatography (HPLC). The plasma sample is directly injected onto a cyanopropyl (CN) pre-column, washed with buffer, and eluted onto an analytical column (C18, octadecylsilane). Analytical recovery from drug-supplemented plasma samples was greater than 85% for all the benzodiazepines tested. The response of the analytical column varied linearly with drug concentrations over the range 0.01-1 mg/L in plasma, with CVs ranging from 0.3% to 1.8% within-day and 5.5% to 15.7% between days. The detection limit (signal-to-noise ratio greater than 3) was 0.01 mg/L (1 ng on column) for all of the benzodiazepines and their metabolites. The advantages of the technique include the extreme cleanliness of the traces, no requirement for off-line sample preparation, and ease of automation.

Isocratic reversed-phase HPLC method to measure pyrimethamine extracted from plasma of infants treated for toxoplasmosis

1991 ◽  
Vol 37 (7) ◽  
pp. 1281-1283 ◽  
Author(s):  
T H Zytkovicz ◽  
J Salter ◽  
L Hennigan ◽  
R Timperi ◽  
J Maguire ◽  
...  
Keyword(s):  
Reversed Phase ◽  
Hplc Method ◽  
Plasma Samples ◽  
Reversed Phase Hplc ◽  
Organic Extraction ◽  
Analytical Recovery ◽  
Increased Sensitivity ◽  
Isocratic Hplc

Abstract An isocratic HPLC method for measuring pyrimethamine extracted from infant plasma is reported. The method is an improvement over previously published methods by requiring lower volumes of plasma (100 microL) and having increased sensitivity to pyrimethamine at 210 nm. The procedure, which entails a basic organic extraction and subsequent HPLC chromatography of the reconstituted extract, can detect 1.4 ng and quantify 4.0 ng of pyrimethamine per 40-microL injection, with two analyses per 100-microL sample. Analytical recovery of pyrimethamine added to plasma at 10, 50, and 125 ng/100 microL averaged 80%, 92%, and 101%, respectively (n = 20). Within- and between-day CVs were less than 7%. Studies of various plasma samples from adults and infants (n = 15) revealed no interference from other plasma peaks with the analyte of interest.

scholarly journals Rapid RP-HPLC method for simultaneous estimation of metformin, pioglitazone, and glimepiride in human plasma

2020 ◽  
Vol 32 (1) ◽  
pp. 16-21 ◽  
Author(s):  
Mahmoud M. Sebaiy ◽  
Sobhy M. El-Adl ◽  
Mohamed M. Baraka ◽  
Amira A. Hassan
Keyword(s):  
Human Plasma ◽  
High Performance ◽  
Reversed Phase ◽  
Hplc Method ◽  
Simultaneous Estimation ◽  
Simulation Studies ◽  
Rp Hplc ◽  
Short Analysis Time ◽  
High Degree

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed for rapid and simultaneous separation and estimation of 3 antidiabetic drugs, namely, metformin, pioglitazone, and glimepiride, in human plasma within 3 min. Separation was carried out on a MAGELLEN 5U C18 (5 μm, 150 mm × 4.60 mm) using a mobile phase of MeOH–0.025 M KH2PO4 adjusted to pH 3.20 using ortho-phosphoric acid (85:15, v/v) at ambient temperature. The flow rate was 1 mL/min, and the maximum absorption was measured at 235 nm. The retention time of metformin, pioglitazone, and glimepiride was noted to be 1.24, 2.32, and 2.77 min, respectively, indicating a very short analysis time compared to that of other reported methods. Also, limits of detection were reported to be 0.05, 0.26, and 0.10 μg/mL for metformin, pioglitazone, and glimepiride, respectively, showing a high degree of method sensitivity. The method was then validated according to the FDA guidelines for the determination of the three drugs clinically in human plasma, in particular, regarding pharmacokinetic and bioequivalence simulation studies.

Developing a High-performance Liquid Chromatography Method for Simultaneous Determination of Loratadine and its Metabolite Desloratadine in Human Plasma

2020 ◽  
Vol 20 (13) ◽  
pp. 1053-1059
Author(s):  
Mahmoud M. Sebaiy ◽  
Noha I. Ziedan
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Allergic Diseases ◽  
Reversed Phase ◽  
Hplc Method ◽  
H1 Receptor ◽  
Chromatography Method

Background: Allergic diseases are considered as the major burden on public health with increased prevalence globally. Histamine H1-receptor antagonists are the foremost commonly used drugs in the treatment of allergic disorders. The target drug in this study, loratadine, belongs to this class of drugs and its biometabolite desloratadine which is also a non-sedating H1 receptor antagonist with anti-histaminic activity being 2.5 to 4 times greater than loratadine. This study aimed to develop and validate a novel isocratic Reversed-phase High-Performance Liquid Chromatography (RP-HPLC) method for rapid and simultaneous separation and determination of loratadine and its metabolite, desloratadine in human plasma. Methods: The drug extraction method from plasma was based on protein precipitation technique. The separation was carried out on a Thermo Scientific BDS Hypersil C18 column (5μm, 250 x 4.60 mm) in a mobile phase of MeOH: 0.025M KH2PO4 adjusted to pH 3.50 using orthophosphoric acid (85: 15, v/v) at an ambient temperature. The flow rate was maintained at 1 mL/min and maximum absorption was measured using the PDA detector at 248 nm. Results: The retention times of loratadine and desloratadine in plasma samples were recorded to be 4.10 and 5.08 minutes, respectively, indicating a short analysis time. Limits of detection were found to be 1.80 and 1.97 ng/mL for loratadine and desloratadine, respectively, showing a high degree of sensitivity of the method. The method was then validated according to FDA guidelines for the determination of the two analytes in human plasma. Conclusion: The results obtained indicate that the proposed method is rapid, sensitive in the nanogram range, accurate, selective, robust and reproducible compared to other reported methods.

Development and Validation of a Novel Reversed Phase High Performance Liquid Chromatography with Refractive Index Detector Method for Assay of Polyvinyl Alcohol in an Ophthalmic Solution

2020 ◽  
Vol 16 (7) ◽  
pp. 867-871
Author(s):  
Harun Ergen ◽  
Muge Guleli ◽  
Cigdem Sener ◽  
Cem Caliskan ◽  
Sercan Semiz ◽  
...  
Keyword(s):  
Refractive Index ◽  
Liquid Chromatography ◽  
Polyvinyl Alcohol ◽  
High Performance ◽  
Ophthalmic Solution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Solution Stability ◽  
Technical Requirements ◽  
Refractive Index Detector

Introduction: Polyvinyl alcohol (PVA), a polymer, is in demand due to its usage in different applications such as pharmaceutical, biomedical and textile, paper, food industries. Methods: A new sensitive reversed phased high-pressure liquid chromatography (RP-HPLC) method with refractive index detector (RID) was developed for determination of PVA in an ophthalmic solution containing dexpanthenol and PVA as active substances and it was validated according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline. Results: Chromatographic separation was achieved on a Chiral-AGP (150 mm × 4.0 mm, 5 μm) column kept at 30°C with an isocratic flow at a flow rate of 1.0 ml/min. The detector temperature was 30°C, the retention time of PVA was around 1.0 min and the total run time was 5 minutes. Conclusion: The proposed method showed linearity, accuracy, precision, specificity, robustness, solution stability, and system suitability results within the acceptance criteria.

scholarly journals Development and Validation of Stability-Indicating RP-HPLC Method for the Estimation of Lenalidomide and its Impurities in Oral Solid Dosage Form

2019 ◽  
Vol 35 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Somana Siva Prasad ◽  
G. V. Krishna Mohan ◽  
A. Naga Babu
Keyword(s):  
Limit Of Detection ◽  
Degradation Products ◽  
Pharmaceutical Formulations ◽  
Hplc Method ◽  
Stability Robustness ◽  
Rp Hplc ◽  
Mobile Phases ◽  
Limit Of Quantitation ◽  
Successful Separation ◽  
Quality By Design Approach

In this study, a novel, simple and precise RP-HPLC method has been developed for the quantitative analysis of Lenalidomide (LLM) in pharmaceutical formulations using analytical quality by design approach. An X-bridge-C18 column (150 mm × 4.6 mm × 3.5 µ) with mobile phases containing a Potassium dihydrogen orthophosphate anhydrous buffer and methanol in the ratio of (90:10 v/v) and (35:65 v/v) are used for the estimation of LLM and its degradation products. The flow rate of 0.8 mL/min is maintained and all degradation studies are performed at 210 nm using photodiode array (PDA) detector. Method Validation is carried out according to International Council for Harmonisation (ICH) guidelines and the parameters namely; precision, accuracy, specificity, stability, robustness, linearity, limit of quantitation (LOQ) and limit of detection (LOD) are evaluated. The present developed RP-HPLC method shows the purity angle of peaks is less than their threshold angle, signifying that it to be suitable for stability studies. Hence, the developed method can be used for the successful separation of LLM and its impurities in the pharmaceutical dosage formulations.

scholarly journals Development and validation of RP-HPLC method for estimation of brexpiprazole in its bulk and tablet dosage form using Quality by Design approach

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Amol S. Jagdale ◽  
Nilesh S. Pendbhaje ◽  
Rupali V. Nirmal ◽  
Poonam M. Bachhav ◽  
Dayandeo B. Sumbre
Keyword(s):  
Flow Rate ◽  
High Performance ◽  
Reversed Phase ◽  
Hplc Method ◽  
Control Analysis ◽  
Uv Detector ◽  
Mobile Phase Flow Rate ◽  
Rp Hplc ◽  
Quality By Design Approach ◽  
Bulk Drug

Abstract Background A new, sensitive, suitable, clear, accurate, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of brexpiprazole in bulk drug and tablet formulation was developed and validated in this research. Surface methodology was used to optimize the data, with a three-level Box-Behnken design. Methanol concentration in the mobile phase, flow rate, and pH were chosen as the three variables. The separation was performed using an HPLC method with a UV detector and Openlab EZchrom program, as well as a Water spherisorb C18 column (100 mm × 4.6; 5m). Acetonitrile was pumped at a flow rate of 1.0 mL/min with a 10 mM phosphate buffer balanced to a pH of 2.50.05 by diluted OPA (65:35% v/v) and detected at 216 nm. Result The developed RP-HPLC method yielded a suitable retention time for brexpiprazole of 4.22 min, which was optimized using the Design Expert-12 software. The linearity of the established method was verified with a correlation coefficient (r2) of 0.999 over the concentration range of 5.05–75.75 g/mL. For API and formulation, the percent assay was 99.46% and 100.91%, respectively. The percentage RSD for the method’s precision was found to be less than 2.0%. The percentage recoveries were discovered to be between 99.38 and 101.07%. 0.64 μg/mL and 1.95 μg/mL were found to be the LOD and LOQ, respectively. Conclusion The developed and validated RP-HPLC system takes less time and can be used in the industry for routine quality control/analysis of bulk drug and marketed brexpiprazole products. Graphical abstract

Development of an HPLC method for measuring orally administered 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in biological fluids

1990 ◽  
Vol 36 (1) ◽  
pp. 5-8 ◽  
Author(s):  
J G Goddard ◽  
G J Kontoghiorghes
Keyword(s):  
High Performance ◽  
Biological Fluids ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Hplc Method ◽  
Iron Chelators ◽  
Serum Samples ◽  
Thalassemic Patient ◽  
Serum And Urine

Abstract "High-performance" liquid-chromatographic (HPLC) methods have been developed for identifying 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in serum and urine. Ion pairing with heptane- or octanesulfonic acid in pH 2.0-2.2 phosphate buffer and reversed-phase chromatography were required to separate these compounds from endogenous compounds in both biological fluids. In both the 2-methyl and 2-ethyl series of 1-substituted compounds (H, methyl, ethyl, or propyl) the elution times increased in accordance with the n-octanol/water partition coefficients (propyl greater than ethyl greater than H greater than methyl). Urine samples were filtered (0.4 microns pore size) and injected either undiluted or after dilution with elution buffer. After the addition of internal standard, the plasma or serum samples were deproteinized by treatment with HCIO4, 0.5 mol/L, centrifuged, and the supernates were injected directly onto the HPLC. Using these procedures, we could identify 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in the serum and urine of a thalassemic patient who had received a 3-g dose of the drug and in the urine of other patients who had received the same dose. One or more possible metabolites were also observed in the chromatograms of both urine and serum. The 24-h urinary output of L1 (0.22-2.37 g) and iron (10.6-71.5 mg) varied but there was no correlation between the two with respect to quantity or concentration. Instead, urinary iron output was higher in patients with a greater number of transfused units of erythrocytes. This is the first study in humans to show that L1 is absorbed from the gut, enters the circulation, and is excreted in the urine.

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