scholarly journals A Novel HPLC Method for the Concurrent Analysis and Quantitation of Seven Water-Soluble Vitamins in Biological Fluids (Plasma and Urine): A Validation Study and Application

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Margherita Grotzkyj Giorgi ◽  
Kevin Howland ◽  
Colin Martin ◽  
Adrian B. Bonner
Keyword(s):  
Biological Fluids ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Water Soluble ◽  
Linear Gradient ◽  
Plasma And Urine Samples ◽  
Urine Separation ◽  
Concurrent Analysis ◽  
Alcohol Dependent

An HPLC method was developed and validated for the concurrent detection and quantitation of seven water-soluble vitamins (C, B1, B2, B5, B6, B9, B12) in biological matrices (plasma and urine). Separation was achieved at 30°C on a reversed-phase C18-A column using combined isocratic and linear gradient elution with a mobile phase consisting of 0.01% TFA aqueous and 100% methanol. Total run time was 35 minutes. Detection was performed with diode array set at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples (24 plasma and urine samples from abstinent alcohol-dependent males). Interday and intraday precision were <4% and <7%, respectively, for all vitamins. Recovery percentages ranged from 93% to 100%.

Reversed-Phase High Performance Liquid Chromatographic Analysis of Three First Line Anti-Tuberculosis Drugs

2019 ◽  
Vol 69 (12) ◽  
pp. 3590-3592
Author(s):  
Nela Bibire ◽  
Romeo Iulian Olariu ◽  
Luminita Agoroaei ◽  
Madalina Vieriu ◽  
Alina Diana Panainte ◽  
...  
Keyword(s):  
High Performance ◽  
Biological Fluids ◽  
Gradient Elution ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Assay Method ◽  
Active Pharmaceutical Ingredients ◽  
First Line ◽  
Pharmaceutical Ingredients ◽  
Liquid Chromatographic

Active pharmaceutical ingredients such as isoniazid, pyrazinamide and rifampicin are among the most important first-line anti-tuberculosis drugs. A simple, rapid and sensitive reversed phase-high performance liquid chromatographic assay method for the simultaneous determination of isoniazid, pyrazinamide and rifampicin has been developed. Separation of the interest compounds was achieved in a 10 min chromatographic run in gradient elution mode on a Zorbax SB-C18 stainless steel column (150 � 4 mm, 5 mm) using a guard column containing the same stationary phase. The gradient elution was carried out with a mobile phase of 10% CH3CN aqueous solution for channel A and 50% CH3CN in pH = 6.8 phosphate buffer (20 mM), to which 1.5 mL triethylamine were added for channel B. Quantification of the analyzed substances was carried out spectrophotometrically at 269 nm. Detection limits of 0.48 mg/L for isoniazid, 0.52 mg/L for pyrazinamide and 0.48 mg/L for rifampicin were established for the developed assay method. The present work showed that the proposed analysis method was advantageous for simple and rapid analysis of the active pharmaceutical ingredients in pharmaceuticals and biological fluids.

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.

High Performance Liquid Chromatography Coupled with Radioactivity Detection: A Powerful Tool for Determining Drug Metabolite Profiles in Biological Fluids

1986 ◽  
Vol 41 (1-2) ◽  
pp. 115-125 ◽  
Author(s):  
K.-O. Vollmer ◽  
W. Klemisch ◽  
A. von Hodenberg
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Biological Fluids ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Isolation And Purification ◽  
Carbon 14 ◽  
Tracer Techniques ◽  
Metabolite Profiles

Abstract High performance liquid chromatography coupled with continuous radioactivity detection rep­resents an advancement in drug metabolism research. Using radioactive substances labelled in biologically stable positions, all metabolites can be specifically detected by radioactivity measure­ment. Thus no clean-up of biological fluids is required prior to HPLC. This can prevent artefact formation from unstable metabolites, reduces recovery problems and facilitates quantitation. Separation of highly polar and unpolar metabolites is possible in a single chromatographic run using gradient elution and reversed phase materials. This technique is also well-suited for prepara­tive isolation and purification of metabolites for subsequent structure elucidation. Various metabolite profiles of drugs labelled with carbon-14 or tritium are shown. Metabolites of the following drugs are presented: norfenefrine, etozolin, thymoxamine, naloxone, and levobunolol. We review the general methodology and report our experience with this technique. In principle, this technique may be useful for all biological systems in which tracer techniques are applied.

scholarly journals Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatography procedure

1998 ◽  
Vol 44 (7) ◽  
pp. 1481-1488 ◽  
Author(s):  
Maria Shipkova ◽  
Paul Dieter Niedmann ◽  
Victor William Armstrong ◽  
Ekkehard Schütz ◽  
Eberhard Wieland ◽  
...  
Keyword(s):  
Mycophenolic Acid ◽  
High Performance ◽  
Internal Standard ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Free Concentration ◽  
Elution Chromatography ◽  
Glucuronide Metabolite

Abstract We describe a reversed-phase HPLC method for determination of total mycophenolic acid (MPA), its free concentration (MPAf), and the glucuronide metabolite (MPAG), based on simple sample preparation and gradient elution chromatography. The compounds were quantified in parallel by absorbance at 254 nm and 215 nm in the internal standard mode. Linearity was verified up to 50 mg/L for MPA and up to 500 mg/L for MPAG (r &gt;0.999). Detection limits at 215 and 254 nm were, respectively, 0.01 and 0.03 mg/L for MPA, and 0.03 and 0.1 mg/L for MPAG. The recovery of MPA was 95–106%;recovery of MPAG was 96–106%. The imprecision (CV) for MPA (0.2–25 mg/L) was &lt;8.4% (254 nm) and &lt;4.4% (215 nm) within day (n = 12) and &lt;9.2% (254 nm) and &lt;6.2% (215 nm) between days (n = 12). The imprecision for MPAG (10–250 mg/L) was &lt;4.9% (254 nm) and &lt;3.4% (215 nm) within day, and &lt;6.1% (254 nm) and &lt;5.9% (215 nm) between days. For quantification of MPAf, 100 μL of ultrafiltrate was applied directly to the column. The detection limit was 0.005 mg/L at 215 nm and 0.015 mg/L at 254 nm. In the range between 18–210 μg/L, the within-day CVs were &lt;11.8% (n = 12) and the between-day CVs were &lt;15.8% (n = 12).

scholarly journals A Simplified, Specific HPLC Method of Assaying Thiamine and Riboflavin in Mushrooms

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Mohammad F. Hossain ◽  
Mamoon Rashid ◽  
Rajjit Sidhu ◽  
Randy Mullins ◽  
Susan L. Mayhew
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Extraction Process ◽  
Reversed Phase ◽  
Hplc Method ◽  
Water Soluble ◽  
Qualitative And Quantitative Analysis ◽  
Food Industries ◽  
Qualitative And Quantitative ◽  
Rp Hplc

Mushrooms have been used as part of the average diet and as a nutraceutical for thousands of years due to their immense health benefits. The purpose of this study was to develop a simple, fast, accurate, specific, reproducible, and robust chromatographic method to identify and quantify two water-soluble vitamins: thiamine (B1) and riboflavin (B2) in mushrooms. The method employed for qualitative and quantitative analysis of these vitamins was Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) equipped with Ultraviolet–Visible (UV-Vis) Detector. The extraction process involved acid hydrolysis followed by enzymatic dephosphorylation with takadiastase enzyme. Chromatographic separation was achieved with a Shimadzu prominence HPLC system using isocratic elution mode on a Waters Xterra® MS C-18 column (4.6mm × 150mm, 5 μm) integrated with a XBridge® BEH C-18 Guard column (2.1mm × 5 mm, 5 μm). The mobile phase of this study consisted of buffer and methanol in the ratio of 80:20, where the buffer contained sodium-1-hexanesulfonate, glacial acetic acid, methanol, and pH adjusted to 3.0 with diethylamine. Vitamins were detected simultaneously at their lambda max wavelengths B1: 245nm and B2: 268nm using dual-wavelength UV detection technique to get their highest response. The proposed method was found to be specific, linear R>1.0, accurate, precise (% recovery ± SD; B1:104.45±4.5 and B2: 104.88±2.04), sensitive, (limit of detection for B1 and B2 was 0.043 and 0.029 μg/mL, respectively), and robust for mushrooms analysis. No coeluting peaks were observed at the retention time of the vitamins and all the peaks were spectrally homogenous. The standard and sample solutions were found to remain stable at cold temperature for 72 hours. In summary, our data suggest that the proposed method could be used in food industries to monitor the product quality during routine quality control purposes.

scholarly journals Simultaneous Determination of Eight Synthetic Permitted and Five Commonly Encountered Nonpermitted Food Colors in Various Food Matrixes by High-Performance Liquid Chromatography

2010 ◽  
Vol 93 (5) ◽  
pp. 1503-1514 ◽  
Author(s):  
Sumita Dixit ◽  
Subhash K Khanna ◽  
Mukul Das
Keyword(s):  
Simultaneous Determination ◽  
High Performance ◽  
High Sensitivity ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Food Category ◽  
Food Commodities ◽  
Food Colors

Abstract A simple and sensitive HPLC method has been developed for the simultaneous determination of eight permitted food colors and five commonly encountered nonpermitted colors in various food commodities, including sugar-, fat-, and starch-based food matrixes. The method uses a specific food category-based cleanup/treatment procedure before color extraction to avoid the interference of food matrixes, and to obtain the optimal color extraction. Analysis was performed on a reversed-phase C18 -Bondapak column with ammonium acetate and acetonitrile gradient elution as the mobile phase; a programmable max-specific visible detection was used to monitor colors to obtain the higher sensitivity and expanded scope needed for multicolor blends having diverse absorption maxima. All colors showed good linearity, with regression coefficients of 0.99740.9999. The LOD and LOQ values ranged from 0.01 to 0.12 mg/L, and from 0.04 to 0.83 mg/L or mg/kg, respectively. The intraday and interday precision tests produced good RSD values, and the recoveries from different food matrixes ranged from 82 to 104%. The method offers high sensitivity for analysis of a wide variety of food matrixes containing a broad scope of multicolor blends. Two nonpermitted colors, orange II and metanil yellow, were found. Also, a number of samples contained permitted colors at levels two-to seven-fold higher than those prescribed.

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 &lt;3.5% and total CV was &lt;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 A New Validated Stability indicating RP-HPLC Method for Simultaneous Quantification of Impurities of Fluticasone Propionate and Salmeterol Xenafoate in Metered Dose Inhalation Aerosol

2021 ◽  
Vol 33 (4) ◽  
pp. 867-872
Author(s):  
Surya Prakash Mamillapalli ◽  
Shirisha Koyya ◽  
B. Venkata Subbaiah ◽  
N. Annapurna
Keyword(s):  
Fluticasone Propionate ◽  
Drug Product ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Hplc Method ◽  
Forced Degradation ◽  
Stability Indicating ◽  
Simultaneous Quantification ◽  
Metered Dose ◽  
Dose Inhalation

A simple, specific, precise, accurate and stability indicating reversed phase HPLC method for simultaneous quantification of total 12 impurities of fluticasone propionate and salmeterol xenafoate in metered dose inhalation aerosol has been developed in the present work. Chromatographic separation between impurities of both compounds were achieved on Altima C18 250 × 4.6 mm, 5 μ column using a step-gradient elution at a flow rate of 1.4 mL/min, 0.1% v/v orthophosphoric acid as buffer and acetonitrile as mobile phase constituents. Forced degradation studies for drug product were performed and revealed that Salmeterol is acid sensitive (about 21.3%), degrades to IMP-D and fluticasone is alkali sensitive (about 7.6%) and degrades to IMP-A. All degradant and process related impurities of both compounds were monitored at 214 nm and spectral purity along with % mass balance is assessed using PDA detector, which proved stability indicating capability of the method. The developed method is fully validated as per current ICH guidelines, where precision is achieved at % RSD of < 5, Correlation of < 0.999 for linearity, LOD-LOQ at < 0.02% and < 0.05%, along with satisfactory system suitability results under robustness conditions.

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