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

2020 ◽  
Vol 21 (14) ◽  
pp. 5134 ◽  
Author(s):  
Shosuke Ito ◽  
Sandra Del Bino ◽  
Tomohisa Hirobe ◽  
Kazumasa Wakamatsu
Keyword(s):  
Dicarboxylic Acid ◽  
Biological Samples ◽  
Potassium Phosphate ◽  
Structural Diversity ◽  
Tricarboxylic Acid ◽  
Reversed Phase ◽  
Hplc Method ◽  
Original Method ◽  
Ion Pair ◽  
Improved Method

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.

Isomeric separation and identification of tetra-, tri-, and di-β-sulphonic phthalocyanine zinc complexes

2011 ◽  
Vol 15 (02) ◽  
pp. 140-148 ◽  
Author(s):  
Zhou Jiang ◽  
Wenyi He ◽  
Huisheng Yao ◽  
Jian Wang ◽  
Naisheng Chen ◽  
...  
Keyword(s):  
High Performance ◽  
Condensation Reaction ◽  
Reversed Phase ◽  
Hplc Method ◽  
Ion Pair ◽  
Zinc Complexes ◽  
Separation Technology ◽  
Isomeric Structure ◽  
Rp Hplc ◽  
Hplc Peak

The synthesis, isomeric separation, and identification of β-sulphonic phthalocyanine zinc complexes were reported. While the sulphonic phthalocyanines have been studied extensively, the development of separation technology may enable deeper insights into their isomeric constitution. An ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) method was developed to separate the sulphonic phthalocyanine isomers. The results showed that the product of the condensation reaction is a mixture of all possible isomers with statistical distribution. Several isomers were obtained and structural determination was undertaken by NMR. Based on the IP-RP-HPLC elution sequence of these well-identified isomers, a relationship between the structure and efficiency was deduced: closely spaced intervals of sulphonic groups lead to higher hydrophobicity and shorter retention times on HPLC. Based on this relationship, each HPLC peak was assigned to the corresponding isomeric structure.

Reversed-phase Ion-pair HPLC Method for the Direct Analysis of 1-OH Midazolam Glucuronide in Human Serum

1999 ◽  
Vol 21 (4) ◽  
pp. 416-420 ◽  
Author(s):  
Katja M. van Rij ◽  
Dirk Compas ◽  
Eleonora L. Swart ◽  
Pieter N. F. C. de Goede ◽  
Daniël J. Touw
Keyword(s):  
Human Serum ◽  
Direct Analysis ◽  
Reversed Phase ◽  
Hplc Method ◽  
Ion Pair

Development and Validation of a Reversed-Phase Column Liquid Chromatographic Method for the Determination of Five Cephalosporins in Pharmaceutical Preparations

2011 ◽  
Vol 94 (5) ◽  
pp. 1440-1446 ◽  
Author(s):  
Ehab F Elkady ◽  
Samah S Abbas
Keyword(s):  
Binary Mixture ◽  
Sodium Benzoate ◽  
Potassium Phosphate ◽  
Pharmaceutical Preparations ◽  
Reversed Phase ◽  
Hplc Method ◽  
Column Liquid Chromatographic ◽  
Development And Validation ◽  
Liquid Chromatographic

Abstract A new, simple, rapid, and precise RP-HPLC method has been developed and validated for the determination of five cephalosporins, namely, cefalexin, cefoperazone, ceftriaxone, ceftazidime, and cefepime. The method has been applied successfully for simultaneous determination of cefalexin in a binary mixture with sodium benzoate in a suspension, and cefoperazone in a binary mixture with sulbactam in vials. Chromatographic separation was achieved on a Waters μBondapak® C18 column (250 × 4.6 mm id, 10 μm particle size) using the mobile phase monobasic potassium phosphate (50 mM, pH 4.6)–acetonitrile (80 + 20, v/v) with UV detection. A flow rate of 1 mL/min was applied. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 30–300, 3–30, and 15–120 μg/mL for the studied cephalosporins, sodium benzoate, and sulbactam, respectively. The optimized method proved to be specific, robust, and accurate for QC of the cited drugs in their pharmaceutical preparations.

Ion-pair reversed-phase HPLC method for the identification and determination of phosphothiamine in cocarboxylase hydrochloride

1997 ◽  
Vol 31 (5) ◽  
pp. 274-275
Author(s):  
S. V. Kuz'min ◽  
E. E. Ishina ◽  
M. L. Shishova ◽  
V. A. Kozlov
Keyword(s):  
Reversed Phase ◽  
Hplc Method ◽  
Ion Pair ◽  
Reversed Phase Hplc

Reversed-phase liquid-chromatographic determination of purine compounds in serum applied to studies of hypouricemia.

1986 ◽  
Vol 32 (2) ◽  
pp. 287-290 ◽  
Author(s):  
T Kojima ◽  
T Nishina ◽  
M Kitamura ◽  
N Kamatani ◽  
K Nishioka
Keyword(s):  
Uric Acid ◽  
High Performance ◽  
Potassium Phosphate ◽  
Chromatographic Determination ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Hplc Method ◽  
Uric Acid Concentration ◽  
Liquid Chromatographic Determination ◽  
Liquid Chromatographic

Abstract With this rapid (20 min), sensitive (0.1 mumol/L), and reproducible reversed-phase "high-performance" liquid-chromatographic (HPLC) method hypoxanthine, xanthine, uric acid, allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), and oxypurinol(4,6-dihydroxypyrazolo[3,4-d]pyrimidine) concentrations can be determined simultaneously in de-proteinized serum. We use an Amicon MPS-1 centrifugal ultrafiltration device, then inject the de-proteinized ultrafiltrate into a Toyosoda ODS-120A HPLC column and elute with a mobile phase consisting of potassium phosphate buffer (40 mmol/L, pH 2.2) containing 20 mL of methanol per liter. We used this method to investigate purine metabolism in 10 cases of hypouricemia (uric acid concentration in serum less than 60 mumol/L).

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