scholarly journals HPLC Determination of Erythrocyte Methotrexate Polyglutamates after Low-Dose Methotrexate Therapy in Patients with Rheumatoid Arthritis

2003 ◽  
Vol 49 (10) ◽  
pp. 1632-1641 ◽  
Author(s):  
Thierry Dervieux ◽  
Diana Orentas Lein ◽  
John Marcelletti ◽  
Ken Pischel ◽  
Katie Smith ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Low Dose ◽  
Direct Injection ◽  
Reversed Phase ◽  
Hplc Method ◽  
Stability Study ◽  
Limit Of Quantification ◽  
Methotrexate Polyglutamates ◽  
Routine Monitoring ◽  
High Concentrations

Abstract Background: Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential γ-linked glutamic residues to MTX. We sought to develop an innovative HPLC method for the quantification of these metabolites in erythrocytes. Methods: Two alternative approaches were developed. In the first approach, MTXPGs from 50 μL of packed erythrocytes were converted to MTX in the presence of plasma γ-glutamyl hydrolase and mercaptoethanol at 37 °C. In the second approach, MTXPG species (up to the hepta order of glutamation) from 100 μL packed erythrocytes were directly quantified in a single run. In both methods, the MTXPGs were extracted from the biological matrix by a simple perchloric acid deproteinization step with direct injection of the extract into the HPLC. The chromatography used a C18 reversed-phase column, an ammonium acetate/acetonitrile buffer, and postcolumn photo-oxidation of MTXPGs to fluorescent analytes. Results: Intra- and interday imprecision (CVs) were <10% at low and high concentrations of analytes for both methods. The limit of quantification was 5 nmol/L. In 70 patients with rheumatoid arthritis receiving weekly low-dose MTX, the mean (SD) total MTXPG concentration measured after conversion of MTXPGs to MTX was similar to the total MTXPG concentration calculated from the sum of individual MTXPG species [117 (56) vs 120 (59) nmol/L; r = 0.97; slope = 1.0]. The triglutamate predominated over all other MTXPG species (36% of total), the pentaglutamate was the highest order of glutamation detected, and a stability study revealed no change in the polyglutamation pattern in erythrocytes 48 h after phlebotomy when the specimen was stored at 2–8 °C. Conclusion: The proposed method for quantification of erythrocyte MTXPGs is rapid, sensitive, and accurate and can be applied to the routine monitoring of MTX therapy.

DEVELOPMENT OF VALIDATED RP-HPLC METHOD FOR THE QUANTITATIVE ANALYSIS OF CHROMIUM PICOLINATE III IN A DIET VINEGAR FORMULATION

INDIAN DRUGS ◽  
2013 ◽  
Vol 50 (05) ◽  
pp. 48-52
Author(s):  
A Lodhi ◽  
◽  
A Jain ◽  
B. Biswal
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Hplc Method ◽  
Chromium Picolinate ◽  
Limit Of Quantification ◽  
Pharmaceutical Dosage Forms ◽  
Liquid Chromatographic Method ◽  
Liquid Chromatographic

A validated high performance liquid chromatographic method was developed for the determination of chromium picolinate in pharmaceutical dosage forms. The analysis was performed at room temperature using a reversed-phase ODS, 5µm (250×4.6) mm column. The mobile phase consisted of acetonitrile: buffer (60:40 V/V) at a flow rate of 0.5 mL/min. The PDA-detector was set at 264 nm. The developed method showed a good linear relationship in the concentration range from 1.5 – 12.5 µg/mL with a correlation coefficient from 0.999. The limit of detection and limit of quantification were 0.0540513 and 0.1637919 µg/mL respectively.

scholarly journals HPLC determination of ketamine, norketamine, and dehydronorketamine in plasma with a high-purity reversed-phase sorbent

1998 ◽  
Vol 44 (3) ◽  
pp. 560-564 ◽  
Author(s):  
Sébastien Bolze ◽  
Roselyne Boulieu
Keyword(s):  
Low Dose ◽  
Reversed Phase ◽  
Hplc Method ◽  
Pharmacokinetic Studies ◽  
Liquid Liquid Extraction ◽  
Extraction Separation ◽  
Acetate Mixture ◽  
New Stationary Phase ◽  
High Column

Abstract We developed an isocratic, selective, and very sensitive HPLC method for the determination of ketamine and its two main metabolites in plasma. The compounds were extracted from plasma by a liquid–liquid extraction with a dichloromethane:ethyl acetate mixture followed by an acidic back-extraction. Separation was achieved on a new stationary phase, Purospher RP-18 end-capped, with a mobile phase containing acetonitrile:0.03 mol/L phosphate buffer (23:77 by vol) adjusted to pH 7.2. Because of the high column efficiency and the significant improvement of peak symmetry, the quantification limit could be down to 5 μg/L for ketamine and norketamine (NK). The intraday and interday CVs ranged from 1.7% to 5.8% and 3.1% to 10.2% for all compounds respectively. The method is sensitive enough for monitoring ketamine, NK, and dehydroketamine in plasma during pharmacokinetic studies after an intravenous bolus of a low dose of ketamine.

scholarly journals Vitamin B1 Status Assessed by Direct Measurement of Thiamin Pyrophosphate in Erythrocytes or Whole Blood by HPLC: Comparison with Erythrocyte Transketolase Activation Assay

2000 ◽  
Vol 46 (5) ◽  
pp. 704-710 ◽  
Author(s):  
Dinesh Talwar ◽  
Helen Davidson ◽  
Josephine Cooney ◽  
Denis St. JO’Reilly
Keyword(s):  
At Risk ◽  
Whole Blood ◽  
Reference Intervals ◽  
Reversed Phase ◽  
Hplc Method ◽  
Healthy Population ◽  
Limit Of Quantification ◽  
Thiamin Deficiency ◽  
Activation Test ◽  
Activation Assay

Abstract Background: The concentration of thiamin diphosphate (TDP) in erythrocytes is a useful index of thiamin status. We describe an HPLC method for TDP and its results in patients at risk of thiamin deficiency. Methods: We used reversed-phase HPLC with postcolumn derivatization with alkaline potassium ferricyanide and fluorescence detection. Samples were deproteinized and injected directly onto a C18 column. TDP concentrations in erythrocytes were compared with those in whole blood. Reference intervals for erythrocyte TDP (n = 147; 79 males and 68 females; mean age, 54 years) and whole blood TDP (n = 124; 68 males and 56 females; mean age, 54 years) were determined in an apparently healthy population. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency. Results: The method was linear to at least 200 μg/L. The between-run CV was <8%. The lower limit of quantification for both whole blood and packed erythrocytes was 300 pg on column with a detection limit of 130 pg on column. Recovery of TDP from blood samples was >90%. TDP in erythrocytes correlated strongly with that in whole blood (r = 0.97). Reference intervals for erythrocyte and whole blood TDP were 280–590 ng/g hemoglobin and 275–675 ng/g hemoglobin, respectively. Of the 63 patients suspected of thiamin deficiency, 46 were normal by both TDP and activation tests, 13 were deficient by both tests, 1 was deficient by the activation test but had normal erythrocyte TDP concentrations, and 4 were normal by the activation test but had low TDP. Conclusions: The HPLC method is precise and yields results similar to the erythrocyte activation assay.

scholarly journals Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood

2008 ◽  
Vol 54 (5) ◽  
pp. 901-906 ◽  
Author(s):  
Jun Lu ◽  
Elizabeth L Frank
Keyword(s):  
Whole Blood ◽  
Clinical Laboratory ◽  
Vitamin B1 ◽  
Current Method ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Phosphate Esters ◽  
Healthy Population ◽  
Limit Of Quantification

Abstract Background: Thiamine (vitamin B1) deficiency is associated with severe diseases such as beriberi and Wernicke encephalopathy. Although most Americans have sufficient dietary intake, thiamine deficiency is observed in the alcohol-dependent and elderly populations. Measurement of thiamine concentration in whole blood provides an assessment of vitamin B1 status in at-risk individuals. Method: We used TCA to precipitate proteins in whole blood. Thiamine and its phosphate esters were derivatized using potassium ferricyanide to thiochromes, which were separated by gradient elution on a reversed-phase HPLC column and detected by fluorescence. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Results obtained with this method were compared with those produced by the method currently used in our clinical laboratory. Reference values of thiamine and its phosphate esters were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements. To shorten analysis time, our method used whole blood rather than washed erythrocytes, did not require lengthy enzymatic dephosphorylation, and had a simple mobile phase. Results: The method was linear to 4000 nmol/L. The lower limit of quantification was 3 nmol/L. The within-run CV was <3.5% and total CV was <9.4%. This method correlated with our current method (r = 0.97). Approximately 90% of the total thiamine content in whole blood was present as thiamine diphosphate (TDP). The means (ranges) for an apparently healthy population were 114 (70–179) nmol/L for TDP and 125 (75–194) nmol/L for total thiamine. Results for separation and measurement of free thiamine and thiamine phosphate esters in whole blood were obtained within 5.5 min. Conclusion: We developed an HPLC method that allows separation and measurement of free thiamine and thiamine phosphate esters in whole blood and provides more rapid results than other methods.

scholarly journals Development and Validation of an RP-HPLC Method for CB13 Evaluation in Several PLGA Nanoparticle Systems

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
J. Álvarez-Fuentes ◽  
L. Martín-Banderas ◽  
I. Muñoz-Rubio ◽  
M. A. Holgado ◽  
M. Fernández-Arévalo
Keyword(s):  
Size Distribution ◽  
High Performance ◽  
Limit Of Detection ◽  
High Performance Liquid Chromatographic ◽  
Plga Nanoparticles ◽  
Reversed Phase ◽  
Hplc Method ◽  
Array Detector ◽  
Limit Of Quantification ◽  
Rp Hplc

A simple, fast, and reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for determining of a cannabinoid derivate, which displays potent antihyperalgesic activity, 1-naphthalenyl[4-(pentyloxy)-1-naphthalenyl]methanone (CB13) into PLGA nanoparticles. Separation was achieved in a C18 column using a mobile phase consisting of two solvents: solvent A, consisting of acetonitrile : water : acetic acid (75 : 23.7 : 1.3 v/v), and solvent B, consisting of acetonitrile. An isocratic method (70 : 30 v/v), with a flow rate of 1.000 mL/min, and a diode array detector were used. The developed method was precise, accurate, and linear over the concentration range of analysis with a limit of detection and a limit of quantification of 0.5 and 1.25 μg/mL, respectively. The developed method was applied to the analysis of CB13 in nanoparticles samples obtained by three different procedures (SEV, FF, and NPP) in terms of encapsulation efficiency and drug release. Nanoparticles size and size distribution were also evaluated founding that NPP method presented the most lowest particle sizes with narrow-size distribution (≈320 nm) and slightly negative zeta potential (≈−25 mV) which presumes a suitable procedure for the synthesis of PLGA-CB13 nanoparticles for oral administration.

A high-speed liquid-chromatographic method for measuring urinary porphyrins.

1988 ◽  
Vol 34 (1) ◽  
pp. 103-105 ◽  
Author(s):  
P M Johnson ◽  
S L Perkins ◽  
S W Kennedy
Keyword(s):  
High Speed ◽  
High Performance ◽  
Porphyria Cutanea Tarda ◽  
Clinical Chemistry ◽  
Direct Injection ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Hplc Method ◽  
Chemistry Laboratory ◽  
Liquid Chromatographic

Abstract We describe a rapid quantitative and qualitative "high-performance" liquid-chromatographic (HPLC) method for measuring porphyrins in urine. Direct injection of acidified, filtered urine onto a 3-micron (particle size) 3-cm-long reversed-phase column fully resolves uroporphyrin, hepta-, hexa-, and pentacarboxylic acid porphyrins, and coproporphyrin. Instrument response is linearly related to concentration over the range 25 to 300 nmol/L. The method provides data essential for the differential diagnosis of porphyric states, including porphyria variegata and porphyria cutanea tarda. This relatively inexpensive method requires a run time of only 8 min per sample, making it particularly suitable for routine use in the clinical chemistry laboratory.

scholarly journals STABILITY INDICATING VALIDATED HPLC METHOD FOR THE DETERMINATION OF ZANUBRUTINIB IN BULK AND PHARMACEUTICAL DOSAGE FORM

2020 ◽  
pp. 159-162
Author(s):  
M. VIJAYA KUMARI ◽  
CH. BALASEKHAR REDDY
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Pharmaceutical Preparations ◽  
Reversed Phase ◽  
Hplc Method ◽  
Limit Of Quantification ◽  
Intermediate Precision ◽  
Pharmaceutical Dosage Form ◽  
Acceptable Limit ◽  
Relative Standard

Objective: An accurate, rapid economical and straight forward, reliable assay technique was evolved and showed for the evaluation of zanubrutinib using reversed-phase high-performance liquid chromatography. Methods: In the proposed method, efficient chromatographic separation was achieved applying acetonitrile and 0.1% orthophosphoric acid (50:50 v/v) as a mobile phase with a flow of 1 ml/min and the wavelength was observed at 220 nm. Chromatography was administered isocratically at ambient temperature and run time was approximately 6 min and the retention time (Rt) was observed as 4.358 min. Results: The method was justified as per ICH guidelines. System suitability parameters were studied by injecting the quality six fold and results were well under acceptance criteria. Linearity study was administered between 10% and 150% levels, regression coefficient value was observed as 0.999. Limit of detection and limit of quantification were observed as 0.02 μg/ml and 0.2 μg/ml, respectively. Precision was found to be 0.74 for repeatability and 0.68 for intermediate precision. Recovery of the drug was found to be 98–102%, indicates that the recovery is in the acceptable limit. Validation results were found to be satisfactory and the method applicable for bulk and formulation analysis. Hence, it was evident that the proposed method was said to be suitable for regular analysis and quality control of pharmaceutical preparations. Conclusion: The validation results were in good agreement with the acceptable limit. Relative standard deviation values which are <2.0% indicating the accuracy and precision of this method. Assay of retail formulation was administered and found to be 100.24% was present using the above method. Stress conditions of degradation in acidic, alkaline, peroxide, and thermal were studied. This developed method showed reliable, precise, accurate results under optimized conditions.

scholarly journals HPLC Determination of Thiopurine Nucleosides and Nucleotides in Vivo in Lymphoblasts following Mercaptopurine Therapy

2002 ◽  
Vol 48 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Thierry Dervieux ◽  
Yaqin Chu ◽  
Yi Su ◽  
Ching-Hon Pui ◽  
William E Evans ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Lymphoblastic Leukemia ◽  
Direct Injection ◽  
Ammonium Phosphate ◽  
Reversed Phase ◽  
Target Tissue ◽  
Chromatographic System ◽  
Water Detection ◽  
High Concentrations

Abstract Background: Mercaptopurine is a prodrug requiring intracellular activation to thiopurine nucleotides to exert antileukemic effect. We developed a reversed-phase liquid chromatographic assay for the quantification of mercaptopurine, thioguanine, and methylmercaptopurine nucleoside and nucleotide concentrations in the target tissue, the leukemic lymphoblast. Methods: Leukemic blasts were isolated from peripheral blood and bone marrow by a standard Ficoll-hypaque procedure. Proteins were removed by ultrafiltration in the presence of dithiothreitol. Thiopurine ribonucleotides were converted into their respective ribonucleosides by treatment of ultrafiltrate with acid phosphatase. Thiopurine nucleosides and bases were measured by direct injection of ultrafiltrate into the chromatographic system. Thiopurine nucleotide concentrations were calculated by subtracting the thiopurine nucleoside concentrations measured after treatment with acid phosphatase from those measured after direct injection of ultrafiltrate in the chromatographic system. Analytes were separated on a C18 Supelco column with ammonium phosphate-methanol eluent coupled with ultraviolet detection. Results: CVs for intra- and interday precision were 1.1–14% (median, 4.9%), and recovery of added analyte was 89–126% (median, 105%) at low and high concentrations of analytes, except for mercaptopurine riboside. The median signal for each of the five metabolites in lymphoblast samples was 98% (range, 80–106%) of that in water. Detection limits for thiopurine bases and nucleosides ranged from 0.5 to 4.5 pmol/5 × 106 cells. Conclusions: This method is suitable for measurement of thiopurine metabolite concentrations in lymphoblasts in children with acute lymphoblastic leukemia following a single dose of intravenous mercaptopurine.

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE ESTIMATION OF LERCANIDIPINE IN BIOLOGICAL MATRICES

INDIAN DRUGS ◽  
2012 ◽  
Vol 49 (07) ◽  
pp. 49-53
Author(s):  
A Singla ◽  
◽  
Y. Paul ◽  
B. Singh
Keyword(s):  
Limit Of Detection ◽  
Quantitative Estimation ◽  
Reversed Phase ◽  
Hplc Method ◽  
Biological Matrices ◽  
Limit Of Quantification ◽  
Rp Hplc ◽  
Uv Visible ◽  
Development And Validation ◽  
Isocratic Mode

A simple, precise, accurate, reproducible and robust analytical method was developed using reversed phase HPLC for quantitative estimation of lercanidipine in biological matrices. The method was validated as per the ICH and FDA guidelines. Analysis of the drug was performed in an isocratic mode employing methanol and acetonitrile (74:26) as the mobile phase on a C18 column. UV-Visible detector at 219 nm was found to be suitable for drug estimation. Linearity was observed in the range of 5 and 500 ng/mL (p<0.001). Recovery was found to range between 96.26% and 100.67%. The % RSD values for method precision and system precision were found to be 0.437 and 1.58, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were observed to be 0.20 ng/mL and 0.68 ng/mL, respectively.

A Bioanalytical Method for Quantification of Telmisartan in rat Plasma; Development, Validation and Application to Pharmacokinetic Study

2021 ◽  
pp. 2139-2144
Author(s):  
Khater Ahmed Saeed AL-Japairai ◽  
Bappaditya Chatterjee ◽  
Syed Mahmood ◽  
Samah Hamed Almurisi
Keyword(s):  
Pharmacokinetic Study ◽  
Limit Of Detection ◽  
Plasma Concentrations ◽  
Rat Plasma ◽  
Reversed Phase ◽  
Hplc Method ◽  
Pharmacokinetic Parameters ◽  
Sprague Dawley ◽  
Linear Calibration ◽  
Limit Of Quantification

The telmisartan was determined in a rat plasma using developed and validated a reversed-phase high performance liquid chromatographic (HPLC). The pre-treatment of the plasma sample involving liquid-liquid extraction using ethanol as the extracting solvent. The HPLC method validation has been shown a linear calibration curve over a plasma concentrations range of 0.7 to 10µg/mL with a correlation coefficient of 0.9979, the limit of detection and the limit of quantification were determined to be 0.025µg/ml and 0.07µg/ml, respectively. The precision and accuracy were in an acceptable limit. The pharmacokinetic parameters of telmisartan were adequately evaluated following a single oral dose (4mg/kg) in Sprague-Dawley rats. The results observed conclude that the developed bioanalytical HPLC method is appropriate and applicable as an analytical tool in the pharmacokinetic study of telmisartan.

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