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

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.

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

Clinical Chemistry ◽

10.1093/clinchem/46.5.704 ◽

2000 ◽

Vol 46 (5) ◽

pp. 704-710 ◽

Cited By ~ 125

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.

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Development and Validation of an HPLC Method for Simultaneous Quantification of Clopidogrel Bisulfate, Its Carboxylic Acid Metabolite, and Atorvastatin in Human Plasma: Application to a Pharmacokinetic Study

Journal of Analytical Methods in Chemistry ◽

10.1155/2015/892470 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 7

Author(s):

Octavian Croitoru ◽

Adela-Maria Spiridon ◽

Ionela Belu ◽

Adina Turcu-Ştiolică ◽

Johny Neamţu

Keyword(s):

Carboxylic Acid ◽

Internal Standard ◽

Gradient Elution ◽

Reversed Phase ◽

Hplc Method ◽

Acid Metabolite ◽

Buffer Solution ◽

Solution Ph ◽

Limit Of Quantification ◽

Simultaneous Quantification

A simple, sensitive, and specific reversed phase liquid chromatographic method was developed and validated for simultaneous quantification of clopidogrel, its carboxylic acid metabolite, and atorvastatin in human serum. Plasma samples were deproteinized with acetonitrile and ibuprofen was chosen as internal standard. Chromatographic separation was performed on an BDS HypersilC18column (250 × 4.6 mm; 5 μm) via gradient elution with mobile phase consisting of 10 mM phosphoric acid (sodium) buffer solution (pH = 2.6 adjusted with 85% orthophosphoric acid) : acetonitrile : methanol with flow rate of 1 mL·min−1. Detection was achieved with PDA detector at 220 nm. The method was validated in terms of linearity, sensitivity, precision, accuracy, limit of quantification, and stability tests. Calibration curves of the analytes were found to be linear in the range of 0.008–2 μg·mL−1for clopidogrel, 0.01–4 μg·mL−1for its carboxylic acid metabolite, and 0.005–2.5 μg·mL−1for atorvastatin. The results of accuracy (as recovery) with ibuprofen as internal standard were in the range of 96–98% for clopidogrel, 94–98% for its carboxylic acid metabolite, and 90–99% for atorvastatin, respectively.

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Determination of the Designer Drugs 3,4-Methylenedioxymethamphetamine, 3,4-Methylenedioxyethylamphetamine, and 3,4-Methylenedioxyamphetamine with HPLC and Fluorescence Detection in Whole Blood, Serum, Vitreous Humor, and Urine

Clinical Chemistry ◽

10.1093/clinchem/46.12.1968 ◽

2000 ◽

Vol 46 (12) ◽

pp. 1968-1977 ◽

Cited By ~ 43

Author(s):

Karine M Clauwaert ◽

Jan F Van Bocxlaer ◽

Els A De Letter ◽

Serge Van Calenbergh ◽

Willy E Lambert ◽

...

Keyword(s):

Blood Serum ◽

Whole Blood ◽

Limit Of Detection ◽

Gradient Elution ◽

Vitreous Humor ◽

Hplc Method ◽

Designer Drugs ◽

Fluorometric Detection ◽

Limit Of Quantification

Abstract Background: The popular designer drugs 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) can be determined in serum, whole blood, and urine, but also in vitreous humor. The latter matrix is interesting when dealing with decomposed bodies in a toxicological setting. Methods: After extraction, chromatographic separation was achieved on a narrow-bore C18 column by gradient elution with fluorometric detection; results were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: The method was linear over the range of 2–1000 μg/L for whole blood, serum, and vitreous humor, and 0.1–5 mg/L for urine. Extraction recoveries were >70%, imprecision (CV) was 2.5–19%, and analytical recoveries were 95.5–104.4%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.8 and 2 μg/L, respectively, for whole blood, serum, and vitreous humor, and 2.5 μg/L and 0.1 mg/L, respectively, for urine. Excellent correlations between the quantitative LC-fluorescence and LC-MS/MS results were obtained. We found the following concentrations in a thanatochemical distribution study in rabbits: in serum, 5.3–685 μg/L for MDMA and from the LOQ to 14.5 μg/L for 3,4-methylenedioxyamphetamine (MDA); in whole blood, 19.7–710 μg/L for MDMA and from the LOQ to 17.8 μg/L for MDA; in vitreous humor, 12.1–97.8 μg/L for MDMA and from the LOQ to 3.86 μg/L for MDA. In routine toxicological urine samples, concentrations ranged from LOQ to 14.62 mg/L for MDA, from LOQ to 157 mg/L for MDMA, and from LOQ to 32.54 mg/L for MDEA. Conclusions: The HPLC method described is sensitive, specific, and suitable for the determination of MDMA, MDEA, and MDA in whole blood, serum, vitreous humor, and urine.

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

INDIAN DRUGS ◽

10.53879/id.50.05.p0048 ◽

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.

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

Clinical Chemistry ◽

10.1093/clinchem/44.7.1481 ◽

1998 ◽

Vol 44 (7) ◽

pp. 1481-1488 ◽

Cited By ~ 90

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 >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 <8.4% (254 nm) and <4.4% (215 nm) within day (n = 12) and <9.2% (254 nm) and <6.2% (215 nm) between days (n = 12). The imprecision for MPAG (10–250 mg/L) was <4.9% (254 nm) and <3.4% (215 nm) within day, and <6.1% (254 nm) and <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 <11.8% (n = 12) and the between-day CVs were <15.8% (n = 12).

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Clinical measurement of serum amiodarone and desethylamiodarone by using solid-phase extraction followed by HPLC with a high-carbon reversed-phase column

Clinical Chemistry ◽

10.1093/clinchem/39.3.496 ◽

1993 ◽

Vol 39 (3) ◽

pp. 496-500 ◽

Cited By ~ 12

Author(s):

M A Jandreski ◽

W E Vanderslice

Keyword(s):

Serum Proteins ◽

Clinical Laboratory ◽

Limit Of Detection ◽

Solid Phase ◽

Reversed Phase ◽

Hplc Method ◽

Extraction Column ◽

Assay Precision ◽

Phase Column ◽

Reversed Phase Column

Abstract We describe a rapid, simple HPLC method routinely used in our clinical laboratory for determining amiodarone and its metabolite desethylamiodarone. These compounds are released from serum proteins by pretreatment with an acidic solution and then extracted onto a C2 reversed-phase clean-up column. After elution from the extraction column, the compounds are separated and quantified by HPLC with a C18 reversed-phase column and spectrophotometric detection. The standard curves for the drug and metabolite are linear up to 20.0 mg/L, with a lower limit of detection of 0.16 mg/L. The CVs for intra-assay precision were 5.0% at 0.58 mg/L and 2.9% at 5.96 mg/L; for inter-assay precision, they were 9.6% at 0.52 mg/L and 6.1% at 2.09 mg/L. Lipemia, hemoglobin, and bilirubin up to 300 mg/L do not interfere with this assay. None of > 550 cardiac patients' samples tested contained a compound that interferes with this assay.

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Routine clinical monitoring of sirolimus (rapamycin) whole-blood concentrations by HPLC with ultraviolet detection

Clinical Chemistry ◽

10.1093/clinchem/42.12.1943 ◽

1996 ◽

Vol 42 (12) ◽

pp. 1943-1948 ◽

Cited By ~ 72

Author(s):

K L Napoli ◽

B D Kahan

Keyword(s):

Therapeutic Drug Monitoring ◽

Drug Monitoring ◽

Whole Blood ◽

Clinical Status ◽

Reversed Phase ◽

Therapeutic Drug ◽

Full Time ◽

Limit Of Quantification ◽

Blood Concentrations ◽

Ultraviolet Detection

Abstract During phase I/II clinical trials of sirolimus (rapamycin; SRL), therapeutic drug monitoring was performed with a multistep liquid-liquid extraction of 1-mL aliquots of whole blood followed by reversed-phase HPLC with ultraviolet detection. Blood was sampled according to a standardized protocol and clinical status. SRL concentrations were interpolated from calibration curves with a linear range of 0-50 micrograms/L and 1 microgram/L lower limit of quantification. Quality control was monitored over 68 consecutive analytical runs by using frozen aliquots of SRL-supplemented pooled whole blood at 4, 12, and 32 micrograms/L. These samples showed mean concentrations of 3.7 +/- 0.6, 10.9 +/- 1.1, and 29.6 +/- 2.6 micrograms/L, respectively. This method for therapeutic drug monitoring of SRL permits one full-time technician to analyze 100 clinical specimens per week with a 24-h turnaround time. With this method, a strong linear relation (r2 = 0.946, Sy/x = 0.41, n = 115) between the average SRL concentration over a 24-h period and the SRL concentration at the 24th h was revealed.

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

Journal of AOAC International ◽

10.1093/jaoac/93.5.1503 ◽

2010 ◽

Vol 93 (5) ◽

pp. 1503-1514 ◽

Cited By ~ 9

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.

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

The Scientific World JOURNAL ◽

10.1100/2012/737526 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 3

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.

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A New Validated Stability indicating RP-HPLC Method for Simultaneous Quantification of Impurities of Fluticasone Propionate and Salmeterol Xenafoate in Metered Dose Inhalation Aerosol

Asian Journal of Chemistry ◽

10.14233/ajchem.2021.23101 ◽

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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