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

MedPharmRes ◽  
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.

Improved liquid-chromatographic assay of labetalol in plasma.

1987 ◽  
Vol 33 (8) ◽  
pp. 1450-1452 ◽  
Author(s):  
D R Luke ◽  
G R Matzke ◽  
J T Clarkson ◽  
W M Awni
Keyword(s):  
High Performance Liquid Chromatography ◽  
Mobile Phase ◽  
High Performance ◽  
Internal Standard ◽  
Excitation Wavelength ◽  
Plasma Samples ◽  
Standard Curve ◽  
Low Concentrations ◽  
Styrene Divinylbenzene ◽  
Liquid Chromatographic

Abstract This is an assay for labetalol in plasma by "high-performance" liquid chromatography, with 5-(2-[4-(4-chlorophenyl)ethyl]) salicylamide hemihydrate as the internal standard. Plasma samples (500 microL) are extracted with acetonitrile, evaporated under nitrogen, reconstituted in the mobile phase, and injected onto a PRP-1 (Hamilton) column packed with particles of poly(styrene-divinylbenzene) copolymer. Fluorescence, enhanced by post-column introduction of NH4OH, was measured in the effluent (excitation wavelength 340 nm, emission wavelength 418 nm). Retention times for labetalol and the internal standard were 1.99 and 3.32 min, respectively. Inter- and intraday CVs for high and low concentrations of the drug were less than 7.5%. The assay standard curve is linear from 1 to 250 micrograms/L. Some commonly co-administered drugs were tested and did not interfere.

Liquid chromatographic analysis of disopyramide and its mono-N-dealkylated metabolite.

1979 ◽  
Vol 25 (3) ◽  
pp. 405-408 ◽  
Author(s):  
J J Lima
Keyword(s):  
Chromatographic Analysis ◽  
Mobile Phase ◽  
High Performance ◽  
Antiarrhythmic Drugs ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Coefficients Of Variation ◽  
Liquid Chromatographic Analysis ◽  
Liquid Chromatographic ◽  
Lower Limits

Abstract We describe a rapid, sensitive, and specific "high performance" liquid chromatographic analysis for disopyramide and its mono-N-dealkylated metabolite in serum, urine, and saliva. We used a mu-Bondapak CN column and an acetate buffer mobile phase containing methanol. Retention times for the two compounds and the internal standard, p-chlorodisopyramide, were 3.4, 4.1, and 6.3 min, respectively. The lower limits of sensitivity for drug and metabolite were 50 and 80 micrograms/L, respectively, with maximum coefficients of variation of 4.6 and 12%, respectively. Currently used antiarrhythmic drugs did not interfere with the analysis of disopyramide, and the pharmacokinetics of the drug, obtained from studies of one subject, agree well with reported values.

scholarly journals Determination of Carbamazepine and Its Impurities Iminostilbene and Iminodibenzyl in Solid Dosage Form by Column High-Performance Liquid Chromatography

2010 ◽  
Vol 93 (4) ◽  
pp. 1059-1068 ◽  
Author(s):  
Predrag Lj Džodić ◽  
Ljiljana J ivanovi ◽  
Ana D Proti ◽  
Mira L Zeevi ◽  
Biljana M Joci
Keyword(s):  
Mobile Phase ◽  
High Performance ◽  
Dosage Form ◽  
Internal Standard ◽  
Hplc Method ◽  
Solid Dosage ◽  
Rp Hplc ◽  
Good Repeatability ◽  
Chemometric Approach

Abstract An accurate and precise RP-HPLC method was developed and validated for the determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in a tablet formulation with fluphenazine as an internal standard. Buffermethanol (50 + 50, v/v) was used as the mobile phase. During validation, specificity, linearity, precision, accuracy, LOD, LOQ, and robustness of the method were tested. The method was proven to be specific against placebo interference. Linearity was evaluated over the concentration range of 100500, 0.050.25, and 0.10.5 g/mL, and the r values were 0.9994, 0.9997, and 0.9979 for carbamazepine, iminostilbene, and iminodibenzyl, respectively. Intraday precision of the method was good, and RSD was below 2 for all analytes. The accuracy of the method ranged from 100.69 to 102.10, 99.76 to 102.66, and 99.26 to 100.08 for carbamazepine, iminostilbene, and iminodibenzyl, respectively. LOD was 0.0125, 0.025, and 0.05 g/mL and LOQ was 0.05, 0.05, and 0.1 g/mL for carbamazepine, iminostilbene, and iminodibenzyl, respectively. Robustness of the method was proven by using a chemometric approach. The method was successfully applied to the analysis of commercially available carbamazepine tablets and showed good repeatability, with RSD below 2.

scholarly journals High-Performance Liquid Chromatographic Determination of Proquazone and Its m-Hydroxy Metabolite in Spiked Human Plasma and Urine

2007 ◽  
Vol 90 (4) ◽  
pp. 971-976
Author(s):  
Ekram M Hassan ◽  
Azza A Gazy ◽  
Mohamed H Abdel-Hay ◽  
Tarek S Belal
Keyword(s):  
Human Plasma ◽  
Mobile Phase ◽  
High Performance ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Urine Samples ◽  
Spiked Human Plasma ◽  
Liquid Chromatographic ◽  
Hydroxy Metabolite

Abstract A simple and rapid high-performance liquid chromatographic method for the determination of proquazone (PQZ) and its major metabolite, m-hydroxyproquazone, in spiked human plasma and urine was developed. Plasma samples were purified using acetonitrile as a protein precipitant, while urine samples were diluted only with the mobile phase and filtered prior to injection. Samples containing the parent compounds and glafenine (internal standard) were eluted from a reversed-phase C8 column using acetonitrile-0.025 M sodium acetate (60 + 40) adjusted to pH 5 as the mobile phase and detected at 234 nm. Peak area ratios of the analytes versus internal standard were used for calibration. The mean recoveries from plasma and urine samples spiked with PQZ and its m-hydroxy metabolite ranged from 97.87 to 103.88%. The relative standard deviation for the within- and between-day analyses were <4%. The proposed method was applied for the assay of PQZ in laboratory-made tablets.

scholarly journals Development of RP-HPLC Method for the Estimation of Rabeprazole in Pure and Tablet Dosage Form

2008 ◽  
Vol 5 (s2) ◽  
pp. 1149-1153 ◽  
Author(s):  
A. Lakshmana Rao ◽  
B. N. V. Ravi Kumar ◽  
G. G. Sankar
Keyword(s):  
Mobile Phase ◽  
High Performance ◽  
Dosage Form ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Hplc Method ◽  
Assay Method ◽  
Control Analysis ◽  
Tablet Dosage ◽  
Liquid Chromatographic

A simple, rapid, sensitive and precise High Performance Liquid Chromatographic (HPLC) method has been developed for the estimation of rabeprazole in bulk and tablet dosage form. In this method RP-C18column (150 mm x 4.6 mm I.D, 5 µ m particle size) with mobile phase consisting of methanol and water in the ratio of 65:35 v/v in isocratic mode was used. The detection wavelength is 284 nm and the flow rate is 0.8 mL/min. Tinidazole is used as internal standard. In the range of 0.25-20 µ g/mL, the linearity of rabeprazole shows a correlation coefficient of 0.9999. The drug and internal standard were eluted at 4.41± 0.05 and 2.16± 0.04 min. respectively. The intra- and inter-day variation was found to be less than 1% showing high precision of the assay method. The detection limit was found to be 100 ng/mL. The mobile phase selected for the proposed method is simple, fast, accurate and precise and hence can be applied for routine quality control analysis of rabeprazole in bluk and its tablet dosage from.

scholarly journals DEVELOPMENT AND VALIDATION OF A SIMPLE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–UV METHOD FOR THE DETERMINATION OF VINORELBINE - A CHEMOTHERAPEUTIC DRUG IN SPIKED HUMAN PLASMA

2019 ◽  
Vol 12 (1) ◽  
pp. 369
Author(s):  
Useni Reddy Mallu ◽  
Venkateswara Rao Anna ◽  
Bikshal Babu Kasimala
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Internal Standard ◽  
Hplc Method ◽  
Pharmacokinetic Studies ◽  
Chemotherapeutic Drug ◽  
Spiked Human Plasma ◽  
Rp Hplc

Objective: Vinorelbine (VNRB) is a chemotherapeutic drug used to treat non-small cell lung cancer and breast cancer. Literature survey reveals that there are no reverse-phase high-performance liquid chromatography (RP-HPLC) methods reported for the estimation of VNRB in spiked human plasma. Hence, the present work aimed to develop a simple and efficient RP-HPLC method for the estimation of VNRB in human plasma.Methods: Specimen preparation for the measurement of VNRB was performed through liquid-liquid extraction using methanol as extracting solvent and reconstructed with mobile phase. Paclitaxel (PCTX) was used as internal standard. HPLC method was optimized and validated as per the US FDA bioanalytical guidelines. VNRB and internal standard were separated on Kromasil® C18 (250×4.6 mm; id 5 μ) using acetate buffer (pH=5.9) and methanol in the ratio of 85:15 (v/v) at 1 ml/min flow rate. Eluted compounds were recorded using UV detector at 235 nm.Results: The retention time of PCTX and internal standard was found to be 4.3 and 9.0 min, respectively. The analytical measuring ranges were found to be 5–750 ng/ml (r2>0.9998). The method was found to be simple, accurate, precise, and stable and there is no interference of plasma matric components.Conclusion: The described HPLC method allows for the measurement of total and free PCTX in both plasma and cord blood and can utilize for the estimation of drug in pharmacokinetic studies.

scholarly journals Development and validation of a simple bio-analytical HPLC-UV method for estimation of irbesartan in human plasma

2020 ◽  
Vol 11 (3) ◽  
pp. 3846-3849
Author(s):  
Ashok P ◽  
Narenderan S T ◽  
Meyyanathan S N ◽  
Babu B ◽  
Jawahar N
Keyword(s):  
Human Plasma ◽  
Mobile Phase ◽  
High Performance ◽  
Internal Standard ◽  
Precipitation Method ◽  
Quantification Limit ◽  
Us Fda ◽  
Development And Validation ◽  
Analytical Hplc

The present study was aimed to develop and validate a simple, sensitive and economical bio-analytical high-performance liquid chromatographicultraviolet method for the determination of irbesartan in human plasma. The method involves the use of simple precipitation method for the determination of irbesartan, using methanol as precipitating agent and losartan as internal standard. The separation was achieved using Zorbax C18 column (150 x 4.6 mm, 5µm), mobile phase consists of methanol and 0.2% formic acid in water at the ratio 85:15, v/v using detection wavelength of 237 nm. Further, the developed method was validated as per US-FDA guidelines for accuracy, precision, linearity, stability, detection and quantification limit. The method developed was found to be linear over the concentration ranging from 5 to 500 ng/ml with a correlation coefficient of 0.9987. The LOD and LLOQ of the method were found to be 1 ng/ml and 5 ng/ml, respectively.

Rapid Determination of Metronidazole and 2-Hydroxymetronidazole in Murine Blood Plasma

2021 ◽  
Author(s):  
Nina Zemanová ◽  
Pavel Anzenbacher ◽  
Tomáš Hudcovic ◽  
Eva Anzenbacherová
Keyword(s):  
Plasma Proteins ◽  
Mobile Phase ◽  
High Performance ◽  
Rapid Determination ◽  
Internal Standard ◽  
Hplc Method ◽  
Primary Metabolite ◽  
Relative Standard ◽  
Protozoan Infections

Abstract Metronidazole is a drug used to treat bacterial and protozoan infections. Nowadays, it is one of the most frequently prescribed drugs worldwide. The main aim of this paper is to present a rapid, reliable and simple high-performance liquid chromatography (HPLC) method to determine metronidazole along with its primary metabolite, 2-hydroxymetronidazole, in plasma or serum using paracetamol as an internal standard. A total of 100% methanol was used to denature plasma proteins. After centrifugation, the supernatant was evaporated under nitrogen flow. The samples were dissolved in the mobile phase and injected into a Li-Chrospher RP-18 column. A total of 10 mmol/L NaH2PO4: acetonitrile (90:10, v/v) solution with a flow rate of 1 mL/min was used as the mobile phase. Metronidazole and 2-hydroxymetronidazole were detected at two different wavelengths at 320 nm and 311 nm, respectively. The method is characterized by high precision (relative standard deviation % < 6). The method was used for the determination of metronidazole and 2-hydroxymetronidazole in murine blood using small amounts of plasma (≤100 μL).

scholarly journals High Performace Liquid Chromtographic Determination of Nicardipine Hydrochloride in Human Plasma

2004 ◽  
Vol 1 (1) ◽  
pp. 38-42
Author(s):  
Y. S. R. Krishnaiah ◽  
V. Satyanarayana ◽  
P. Bhaskar
Keyword(s):  
Ethyl Acetate ◽  
Human Plasma ◽  
High Performance ◽  
Potassium Dihydrogen Phosphate ◽  
Internal Standard ◽  
Immediate Release ◽  
Hplc Method ◽  
Single Step ◽  
Dihydrogen Phosphate ◽  
Precision And Accuracy

A sensitive high-performance liquid chromatographic method was developed for the estimation of nicardipine hydrochloride in human plasma. Varying amount of nicardipine hydrochloride (2.5 to 150 ng/0.5 mL) and fixed quantity (100 ng/0.5 mL) of nifedipine (internal standard) was added to blank human plasma, and a single step extraction was carried out with ethyl acetate. The mixture was centrifuged, ethyl acetate layer separated, dried and reconstituted with 100 μL of acetonitrile. Twenty microliters of this solution was injected into a reverse phase C-18 column using a mobile phase consisting of acetonitrile: 0.02 M potassium dihydrogen phosphate (pH 4.0) in the ratio of 60:40 v/v and the eluents were monitored at 239 nm. The method was validated for its linearity, precision and accuracy. The calibration curve was linear in the range of 5-150 ng/0.5 mL of plasma and the lower detection limit was 2.5 ng/0.5 mL of plasma. The intra- and inter-day variation was found to be less than 2.5% indicating that the method is highly precise. The mean recovery of nicardipine hydrochloride from plasma samples was 89.6±2.60%. The proposed HPLC method was applied for the estimation of nicardipine hydrochloride in human plasma after oral administration of an immediate release nicardipine hydrochloride capsule (dose 30 mg) to 6 adult male volunteers. There was no interference of either the drug metabolites or other plasma components with the proposed HPLC method for the estimation of nicardipine hydrochloride in human plasma. Due to its simplicity, sensitivity, high precision and accuracy, the proposed HPLC method may be used for biopharmaceutical and pharmacokinetic evaluation of nicardipine hydrochloride and its formulations in humans

Development and Application of HPLC Method for Clinical Pharmacokinetic Study of Domperidone

2016 ◽  
Vol 9 (6) ◽  
pp. 3531-3535
Author(s):  
Prasad Neerati ◽  
Bhargavi Latha A ◽  
Y. Shravan Kumar ◽  
Y Madhusudan Rao
Keyword(s):  
Pharmacokinetic Study ◽  
Mobile Phase ◽  
High Performance ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Hplc Method ◽  
Assay Method ◽  
Uv Detector ◽  
Liquid Chromatographic Method ◽  
Liquid Chromatographic

A simple and sensitive high performance liquid chromatographic method for quantification of domperidone in human serum was developed and validated. Domperi-done and internal standard (IS) propranolol hydrochloride were extracted into acetonitrile and separated using an isocratic mobile phase on a Phenomenx C18 column. The eluent was monitored by UV detector at 270 nm at a flow rate of 1.0 mL min–1. The linearity range of proposed  method was 2.5–1000 ng/ml. The intra-day and inter-day coefficient of variation and percent error values of the assay method were less than 15% and mean recovery was more than 97 and 96% for domperidone and IS, respectively. The method was found to be precise, accurate and specific during the study. The method was successfully applied for pharmacokinetic study of domperidone in humans.  

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