Improved HPLC Method Research Articles

Cefoperazone rapidly and sensitive quantitative assessment via a validated RP-HPLC method for different dosage forms, in-use stability, and antimicrobial activities

Cefoperazone (Cfz) is a member of the third generation of parenteral cephalosporin antibiotics. It is used on a wide scale in prescribed antibiotic drugs as anti-infection, especially for Gram-negative and also against Gram-positive microorganisms. The current study aimed to find a rapid RP-HPLC method of Cfz analysis with high linearity, repeatability, sensitivity, selectivity, and inexpensive. In our developed method, there is no need to use special chemical reagents, a high percentage of organic solvent, a high flow rate, further guard column. The chromatographic system comprises an ODS column (150 mm × 4.6 mm × 5 μm). The mobile phase was prepared by mixing KH2PO4 solution: acetonitrile (80:20) with a flow rate of 1.0 mL/min at detection wavelength 230 nm, at room temperature using injection volume 20 μL. The method manifested a satisfied linearity regression R2 (0.9993) with a good repeatability range (0.34–0.92%) with LOD and LOQ; 4.04 μg/mL and 12.24 μg/mL respectively. The method proved its efficiency via system suitability achievement in the robustness and ruggedness conduction according to the validation guidelines. The shorter analysis time makes the method very valuable in quality control to quantify the commercial Cfz in pharmaceutical preparations. This improved HPLC method has been successfully applied for Cfz analysis for Peracef and Peractam vials in our routine finished and stability studies testing laboratories. Additionally, the detection limit of Cfz has been tested in our quality control lab to detect the smallest amount of traces that may be present after the cleaning process of the production machines for cephalosporins preparations. In a precedent for the first time, we were able to use the current analysis method to determine the minimum inhibitory concentration (MIC) and minimum bacteriostatic concentration (MBC). The conventional broth micro-dilution tube method was used to determine MIC at 250 µg/mL and MBC at 125 µg/mL of Cfz against the standard strain of Burkholderia cepacia (B. cepacia) ATCC 25416 as Gram-negative bacteria in vitro.

Rapidly, sensitive quantitative assessment of thiopental via forced stability indicating validated RP-HPLC method and its in-use stability activities

Thiopental sodium (Tho) is an intravenous anesthetic. The current study aimed to find a rapid RP-HPLC method of Tho analysis with high linearity, repeatability, sensitivity, selectivity, and inexpensive. In our developed method, there is no need to use special chemical reagents, a high percentage of organic solvent, a high flow rate, or a further guard column. The chromatographic system consists of an ODS column (150 mm × 4.6 mm × 5 μm). The mobile phase was prepared by mixing KH2PO4 solution: methanol (40:60) with a flow rate of 1.2 mL/min at a detection wavelength of 230 nm, at room temperature using an injection volume of 10 μL. The method manifested a satisfied linearity regression R2 (0.9997) with a good repeatability precision range (0.16–0.47%) with LOD and LOQ; 14.4 μg/mL and 43.6 μg/mL respectively. Additionally, the method proved its efficiency via system suitability achievement in robustness and ruggedness, according to the validation guidelines. The shorter analysis time makes the method very valuable in quality control to quantify the commercial Tho in pharmaceutical preparations. This improved HPLC method has been successfully applied for Tho analysis for Thiopental UP Pharma 500 mg vials and Thiopental Eipico 1.0 g vials in our routine finished and stability studies testing laboratories. Additionally, the detection limit of Tho has been tested in our quality control lab to detect the smallest amount of traces that may be present after the cleaning process of the production machines for cephalosporins preparations. The method has shown positive results for Tho in low-level raw materials and pharmaceutical formulations.

A Study of Importance of Health Benefits in Curcumin

Abstract: The present study represents the importance of curcumin which is present in turmeric. Curcumin is belonging to the group of curcuminoids which are natural phenols responsible for turmeric yellow colour. The main constituent of this curcuminoid is it contains enormous number of therapeutic properties such as antioxidant, anti-inflammatory, radio protective, anti-cancer and neuro protective. Curcuminoid is used as dietary supplements, food additives, medical treatment and cosmetics. An average human should consume 2500 mg of curcumin per day to avoid the carcinogenic and cardiovascular defects. Various samples were analysed using improved HPLC method.

Determination of monosaccharide composition in human serum by an improved HPLC method and its application as candidate biomarkers for endometrial cancer

Altered glycan levels in serum have been associated with increased risk of cancer. In this study, we have developed and validated a HPLC-based method to analyze monosaccharide composition (D-mannose, Glucosamine, Galactosamine, Glucuronic acid, D-glucose, D-galactose, D-xylose, L-fucose) in human serum, with L-rhamnose, being used as internal standard. Monosaccharides obtained from hydrolyzed serum samples were derivatized by 1-Phenyl-3-methyl-5-pyrazolone. A ZORBAX XDB-C18 column(150×4.6mm) was used for chromatographic separation with 100 mM ammonium acetate buffer (NH4Ac-HAc, PH=5.5, solvent A), acetonitrile (ACN, solvent B) as a mobile phase. The calibration standard curves for the eight monosaccharides showed good linearity over the range of 2.5-500μg/mL with R2 > 0.995. The relative standard deviation values for intra-day and inter-day precision were ≤ 5.49%. Recovery was 69.01-108.96%. We observed that this column exhibited high specificity and selectivity to separate monosaccharides from serum. This method was then applied to quantitatively analyze the serum monosaccharide levels in 30 patients with endometrial cancer and 30 matched healthy controls. Statistical analysis indicated that the serum monosaccharide levels were significantly higher in patients compared with healthy controls (P value< 0.0001). Overall, we report here a simple, reliable, low-cost, and reproducible HPLC method for the separation and quantification monosaccharides in the human serum, which has potential value to serve as a screening marker for endometrial cancer.

Improved HPLC method for the determination of ribavirin concentration in red blood cells and its application in patients with COVID-19.

Ribavirin is a synthetic, broad‐spectrum antiviral drug. Ribavirin is recommended as an antiviral drug in the Interim Guidance for Diagnosis and Treatment (the seventh edition) of COVID‐19. The ribavirin levels in red blood cells may be closely related to both its efficacy and adverse drug reactions. In this study, a simple and fast HPLC–UV method was established to determine the concentrations of total ribavirin in the red blood cells of 13 patients with COVID‐19. Phosphorylated ribavirin was dephosphorylated by phosphatase incubation to obtain the total amount of ribavirin in red blood cells. The chromatographic column was an Atlantis C18. The recoveries were 85.45–89.05% at three levels. A good linear response was from 1 to 200 μg/ml, with a correlation coefficient of r 2 = 0.9991. The concentration of total ribavirin in the red blood cells of the patients ranged from 30.83 to 133.34 μg/ml. The same samples without phosphatase incubation ranged from 4.07 to 20.84 μg/ml. About 85% of ribavirin was phosphorylated in red blood cells. In addition, we observed changes in these patients' hematological parameters and found that the erythrocyte, hemoglobin and hematocrit declined to the lowest levels on the fifth day after discontinuation of ribavirin (p < 0.05).

A simple alternative and improved HPLC method for the estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in solid oral dosage form.

A novel method was developed for the simultaneous estimation of the doravirine, lamivudine, and tenofovir disoproxil fumarate in the pharmaceutical dosage form. The chromatogram was run through an Ascentis C18 column (150 × 4.6 mm, 2.7 μm), with the mobile phase consisting of a phosphate buffer and acetonitrile in the ratio of 50:50 (v/v). The mobile phase was pumped through the column at a flow rate of 1 mL/min. The column temperature was maintained at 30°C. The optimized wavelength for doravirine, lamivudine, and tenofovir disoproxil fumarate was 230.0 nm. The retention times for doravirine, lamivudine, and tenofovir disoproxil were 2.222, 2.764, and 3.403 min, respectively; the relative standard deviation (%) values of method precision for doravirine, lamivudine, and tenofovir disoproxil were 0.6, 0.6, and 0.1, respectively. The % recovery was 100.20%, 100.15%, and 100.36% for doravirine, lamivudine, and tenofovir disoproxil fumarate, respectively. The limit of detection and limit of quantification values were obtained from regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate, and were 0.24 and 0.73 ppm, 0.53 and 1.60 ppm, and 0.47 and 1.43 ppm, respectively. The regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate were y = 17,541x + 117,303, y = 15,555x + 10,791, and y = 15,250x + 31,663, respectively. The method developed was accurate, simple, precise, sensitive, and economical. Hence, it could be adopted for regular quality control for estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in pharmaceutical industries.

Improved HPLC method for the quantification of free fatty acids in parenteral lipid emulsions

Rationale: Total free fatty acid (FFA) concentration of oil-in-water (o/w) emulsions is a critical quality parameter and thus necessary to quantify. Here we present an improved method for the FFA measurement by HPLC with fluorescence detection that can distinguish between different fatty acid (FA) species, which will further enhance our understanding of underlying degradation mechanisms and thereby can foster the development of innovative lipid emulsions.

Improved HPLC Conditions to Determine Eumelanin and Pheomelanin Contents in Biological Samples Using an Ion Pair Reagent.

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility.

Determination of Active Vitamin B12 (Cobalamin) in Dietary Supplements and Ingredients by Reversed-phase Liquid Chromatography (P13-040-19)

ObjectivesVitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. A high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed and validated, for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. MethodsA Plackett-Burman factorial study was used to identify factors that contributed to the extraction of cobalamins. Significant factors were selected to produce the improved HPLC method for cobalamin separation. This method was then subjected to a single-laboratory validation according to the AOAC International guidelines for linearity, suitability, detection limits, accuracy, and precision. ResultsThe method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005% w/w - 85% w/w), high precision (repeatability relative standard deviations ranged from 1.08% to 3.06%), and high accuracy (>96% spike recovery rate). The method detection and quantification limits are less than 0.16 and 0.52 μg/mL, respectively. ConclusionsTo our best knowledge, the method is simpler, less time-consuming, and more economical than other published methods for its intended uses. Funding Sources1Eurofins Supplement Analysis Center, Eurofins Scientific, Inc.2MilliporeSigma Supporting Tables, Images and/or Graphs▪

In vivo anti-hyperuricemic and xanthine oxidase inhibitory properties of tuna protein hydrolysates and its isolated fractions

This study follows recent attempts to discover natural xanthine oxidase (XO) inhibitors from foods, focusing herein on under-researched fish proteins. The anti-hyperuricemic function of tuna flesh hydrolysate (TPH) produced using Alcalase 2.4L was confirmed in potassium oxonate-induced hyperuricemic rats. TPH was separated using 80 wt% aqueous ethanol. The ethanol-soluble fraction (ESF) abundant in small peptides (<1000 Da) afforded the highest XO inhibition. Separation of ESF by Sephadex G-15 and UPLC/MS/MS revealed 13 di-/tri-peptides (12 are newly identified XO inhibitors). Their XO inhibitory activities were assessed using corresponding synthetic peptides via an improved HPLC method. Results indicate that Phe-containing peptides were more potent XO inhibitors than Trp-containing peptides, with Phe-His having the highest XO inhibitory activity (IC50 = 25.7 mM). Molecular docking studies revealed the importance of two hydrogen bonds and one π-π stacking interaction with Phe-914 in XO for XO-peptide inhibitor binding. Phe-containing di-/tri-peptides could be potent XO inhibitors against hyperuricemia.

Erratum to: HPLC Determination of Bioactive Sulfur Compounds, Amino Acids and Biogenic Amines in Biological Specimens.

There is an increasing interest for analytical methods aimed to detect biological sulfur-containing amines, because of their involvement in human diseases and metabolic disorders. This work describes an improved HPLC method for the determination of sulfur containing amino acids and amines from different biological matrices. We optimized a pre-column derivatization procedure using dabsyl chloride, in which dabsylated products can be monitored spectrophotometrically at 460 nm. This method allows the simultaneous analysis of biogenic amines, amino acids and sulfo-amino compounds including carnosine, dopamine, epinephrine, glutathione, cysteine, taurine, lanthionine, and cystathionine in brain specimens, urines, plasma, and cell lysates. Moreover, the method is suitable for the study of physiological and non-physiological derivatives of taurine and glutathione such as hypotaurine, homotaurine, homocysteic acid and S-acetylglutathione. The present method displays good efficiency of derivatization, having the advantage to give rise to stable products compared to other derivatizing agents such as o-phthalaldehyde and dansyl chloride.

HPLC Determination of Bioactive Sulfur Compounds, Amino Acids and Biogenic Amines in Biological Specimens.

There is an increasing interest for analytical methods aimed to detect biological sulfur-containing amines, because of their involvement in human diseases and metabolic disorders. This work describes an improved HPLC method for the determination of sulfur containing amino acids and amines from different biological matrices. We optimized a pre-column derivatization procedure using dabsyl chloride, in which dabsylated products can be monitored spectrophotometrically at 460 nm. This method allows the simultaneous analysis of biogenic amines, amino acids and sulfo-amino compounds including carnosine, dopamine, epinephrine, glutathione, cysteine, taurine, lanthionine, and cystathionine in brain specimens, urines, plasma, and cell lysates. Moreover, the method is suitable for the study of physiological and non-physiological derivatives of taurine and glutathione such as hypotaurine, homotaurine, homocysteic acid and S-acetylglutathione. The present method displays good efficiency of derivatization, having the advantage to give rise to stable products compared to other derivatizing agents such as o-phthalaldehyde and dansyl chloride.With this method, we provide a tool to study sulfur cycle from a metabolic point of view in relation to the pattern of biological amino-compounds, allowing researchers to get a complete scenario of organic sulfur and amino metabolism in tissues and cells.

Improved HPLC method for rooibos phenolics targeting changes due to fermentation

The phenolic composition of rooibos (Aspalathus linearis) herbal tea is inherently linked to its health-promoting properties. “Fermentation” (oxidation) develops the characteristic flavour and colour of the herbal tea product. Changes to the phenolic composition during fermentation include conversion of aspalathin, a dihydrochalcone unique to rooibos and the major bioactive compound, to eriodictyol-glucopyranoside isomers. These compounds have not been quantified in rooibos plant material to date, due to separation challenges. In this study an improved HPLC method using a core shell column was developed to provide effective separation of a phenolic precursor (enolic phenylpyruvic acid-2-O-glucopyranoside, PPAG) and 15 rooibos tea phenolics (aspalathin, four eriodictyol-glucopyranoside isomers and 11 other major phenolic compounds) in a reasonable time. Compounds were tentatively identified using authentic reference standards and/or tandem mass spectrometry. The HPLC method utilising diode-array detection for quantitation was validated. After evaluation of different extraction solvents, a 40% aqueous acetonitrile solution was deemed optimal for extraction of phenolic compounds from rooibos plant material. Green, semi-fermented and fermented plant material from 10 sub-divided rooibos bushes were extracted and analysed. Water extracts, representing food ingredient extracts, were also analysed. The largest decreases with fermentation were observed for the dihydrochalcones, with moderate to small decreases for flavonols. Eriodictyol-glucopyranoside isomer concentrations increased following fermentation.

Rapid method for monitoring N-nitrosodimethylamine in drinking water at the ng/L level without pre-concentration using high-performance liquid chromatography-chemiluminescence detection

As a contaminant in drinking water, N-nitrosodimethylamine (NDMA) is of great concern because of its carcinogenicity; it has been limited to levels of ng/L by regulatory bodies worldwide. Consequently, a rapid and sensitive method for monitoring NDMA in drinking water is urgently required. In this study, we report an improvement of our previously proposed HPLC-based system for NDMA determination. The approach consists of the HPLC separation of NDMA, followed by NDMA photolysis to form peroxynitrite and detection with a luminol chemiluminescence reaction. The detection limit for the improved HPLC method was 0.2ng/L, which is 10 times more sensitive than our previously reported system. For tap water measurements, only the addition of an ascorbic acid solution to eliminate residual chlorine and passage through an Oasis MAX solid-phase extraction cartridge are needed. The proposed NDMA determination method requires a sample volume of less than 2mL and a complete analysis time of less than 15min per sample. The method was utilized for the long-term monitoring of NDMA in tap water. The NDMA level measured in the municipal water survey was 4.9ng/L, and a seasonal change of the NDMA concentration in tap water was confirmed. The proposed method should constitute a useful NDMA monitoring method for protecting drinking water quality.

An improved HPLC method for single-run analysis of the spectrum of hop bittering compounds usually encountered in beers

A single-run reverse phase-high performance liquid chromatography method for the quantification of humulinones, α-acids, iso-α-acids and reduced iso-α-acids (where present) in commercial beer samples is presented. The method utilizes a binary solvent system consisting of (A) 1% v/v acetic acid and (B) 0.1% v/v orthophosphoric acid in acetonitrile. Separation was achieved on a Purospher® star RP-18 column (250 × 46 mm, 3 µm) with a flow rate of 0.5 mL/min. The compounds of interest eluted within 32 min. The method was fully validated according to International Conference on Harmonization guidelines and subsequently applied to monitor degradation of hop acids in a storage trial where four lager beers were aged at 28 and 38 °C for 70 and 60 days, respectively. Results confirmed the widely reported degradation through storage of trans-iso-α-acids whilst demonstrating that the HLPC method was sufficiently sensitive to monitor and model this degradation. One beer exhibited a significantly lower (P < 0.05) rate of trans-iso-α-acid degradation than the other conventionally hopped beers in the study, which might have been linked to its higher pH (4.71 vs 4.36). The relative stability of reduced iso-α-acids during ageing was also confirmed.

An improved high performance liquid chromatography method for the separation of carotenoids extracted from Phaffia rhodozyma

Separation of analytes is an important factor affecting the effectiveness of carotenoids analysis from a complex extraction. In this study, a high performance liquid chromatography procedure with improved separation was developed to determine carotenoids extracted from P. rhodozyma. Mixture design experiment was employed to optimize the organic solvent constitution, which showed that the optimal organic phase was composed (v/v) of 61% of methanol, 32% of tetrahydrofuran, and 7% of acetonitrile. Furthermore, the gradient elution procedure optimization demonstrated that the gradient elution started at the optimal organic phase concentration of 60% lead to the best separation effect. Meanwhile, 18 compounds in total 25 detected peaks were separated. Based on their absorption spectra, astaxanthin, s-carotene, zeaxanthin, β-cryptoxanthin and lycopene were identified by matching their retention times with standards. Interestingly, our improved HPLC method was effective to classify P. rhodozyma strains and monitor the degradation of astaxanthin in accordance with their carotenoids composition.

Curcuminoids in Turmeric Roots and Supplements: Method Optimization and Validation

Curcuma longa L. rhizomes are used extensively as a spice in food preparations and dietary supplements for their anti-inflammatory and antioxidant properties. An expert review panel (ERP) evaluated analytical methods for the quantitation of individual curcuminoids for the purpose of identifying a method for official method status. It was requested that several modifications be undertaken to improve method performance prior to subjecting the chosen method to a single-laboratory validation. Two separate Plackett-Burman factorial studies were used to identify factors that contributed to the chromatographic separation and extraction of curcuminoids. Significant factors were further optimized to produce the improved HPLC method for curcuminoid separation. This method was then subjected to a single-laboratory validation according to the AOAC International guidelines for linearity, detection limits, precision, and accuracy. The two most significant factors impacting the quantitation of curcuminoids were column temperature and extraction solvent, which were optimized to 55 °C and 100 % methanol, respectively. The validation was performed on 12 raw materials and finished products containing turmeric roots. The method precision was reported using HorRat values which were within recommended ranges of the AOAC guidelines. Overall accuracy of the method was accessed at three separate levels for each analyte and ranged from 99.3–100.9 %. The validated method is suitable for quantitation of individual curcuminoids in turmeric raw materials and finished products and is recommended for consideration as an official method by the AOAC International.

An Improved HPLC Method for Simultaneous Estimation of Isocurcumenol, Ar-Turmerone and &lt;I&gt;α&lt;/I&gt;, &lt;I&gt;β&lt;/I&gt;-Turmerone in Hexane Soluble Fraction of &lt;I&gt;Curcuma Longa&lt;/I&gt; and Its Formulations

An Improved HPLC Method for Simultaneous Estimation of Isocurcumenol, Ar-Turmerone and &lt;I&gt;α&lt;/I&gt;, &lt;I&gt;β&lt;/I&gt;-Turmerone in Hexane Soluble Fraction of &lt;I&gt;Curcuma Longa&lt;/I&gt; and Its Formulations

Simultaneous Determination of Impurities in Ropinirole Tablets by an Improved HPLC Method Coupled with Diode Array Detection

A gradient HPLC method coupled with diode array detection was developed and fully validated for the analysis of impurities in ropinirole using a Kromasil® C8 100 A (250 × 4.6 mm, 5 μm) column with a flow rate 1.0 mL min−1 and detection at 250 nm. The mobile phase component A consisted of a mixture of 19.6 mM aqueous potassium dihydrogen phosphate–acetonitrile (98:2 v/v), pH adjusted to 7.0 with triethylamine and the mobile phase component B consisted of acetonitrile. The method was validated in terms of linearity, sensitivity, precision, accuracy and stability. The calibration curves for ropinirole and its five impurities showed good linearity (r > 0.998) within the calibration ranges tested. The intra- and inter-day RSD values were <3.9 %, while the relative percentage error E r was <5.8 % for all compounds. Accelerated stability studies performed under various stress conditions including oxidation, hydrolysis and UV light irradiation at 254 nm proved the selectivity of the procedure. Long-term stability studies performed on blistered tablets and under various conditions of heat and humidity indicate the presence of four of the studied impurities in less than 0.07 %. The method was applied successfully to the detection and determination of ropinirole impurities in pharmaceutical formulations.

An Improved HPLC Method for Quantification of Metanephrine with Coulometric Detection

A rapid and straightforward analytical method, based on the use of RP-HPLC with coulometric detection, was developed and validated for the quantification of metanephrine, an O-methylated product in catechol-Omethyltransferase enzymatic assays. The isocratic separation was achieved on a reverse column with a mobile phase consisting of 0.1 M sodium dihydrogen phosphate, 0.024 M citric acid monohydrate, 0.5 mM sodium octyl sulphate and 9% acetonitrile (%v/v). The method was found to be linear between 0.25 and 15 nmol/mL with a determination coefficient of 0.9997 for metanephrine. Intra-and interday precision and accuracy were in conformity with the criteria accepted in bioanalytical method validation and the LOD and LLOQ were 0.25 nmol/mL. The main focus of the developed method is the lower LLOQ achieved that can have important implications in laboratory research for COMT activity determinations, in particular for the methionine 108/158 variant obtained either from native or recombinant extracts. Another major advantage of the present method is the shorter run times on automated chromatographic systems that allow the analysis of several samples in a short time. In addition, metanephrine was stable in the samples for at least 24 h at room temperature, for at least 24 h in HPLC system injector and for at least three freeze/thaw cycles. The developed method demonstrated higher sensitivity, precision, accuracy, stability, and linearity when compared with the methods previously described. Finally, a catechol-O-methyltransferase