Isocratic reversed-phase HPLC method

FIGURE 21 (a) An isocratic reversed-phase HPLC method developed for assay, with which 15%... 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

A list of the 64 analytes and their method performance is shown in Table 6.6. The increased number of analytes is possible because of improvements to the collision region of the MS/MS system that provide increased sensitivity and reduced memory effects. In addition, robotic systems for sample handling and on-line solid-phase extraction (SPE) of plasma samples were integrated with the LC/MS/MS system (Figure 6.22). An isocratic reversed-phase HPLC method provided a cycle time of 4.5 min per sample. The on-line sample preparation and short analysis resulted in an increased sample throughput that required less time from the scientist. The... 

Toukairin-Oda reported an isocratic, reversed-phase HPLC method for determination of all six nutritionally active Bg vitamers in food (96). PLP fluorescence was enhanced by precolumn potassium cyanide treatment to convert PLP to the highly fluorescent 4-pyridoxic acid-5 -phosphate. However, potassium cyanide causes oxidation of PL to 4-pyridoxic acid lactone, which shows little fluorescence at the acid pH (pH = 3.5) of the mobile phase used (Table 2). This problem is circumvented by duplicate analysis (1) without prior potassium cyanide treatment to determine all the B vitamers except PLP, and (2) after potassium cyanide treatment to determine PLP as 4-pyridoxic acid-5-phosphate. This method has been applied to fruit juices, wheat flour, cream cheese, eggs, and baker s yeast. 

Sultana et al. [88] developed a reversed-phase HPLC method for the simultaneous determination of omeprazole in Risek capsules. Omeprazole and the internal standard, diazepam, were separated by Shim-pack CLC-ODS (0.4 x 25 cm, 5 m) column. The mobile phase was methanol-water (80 20), pumped isocratically at ambient temperature. Analysis was run at a flow-rate of 1 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 3.5 ng/ml at a signal-to-noise ratio of 4 1. The limit of quantification was set at 6.25 ng/ml. The calibration curve was linear over a concentration range of 6.25—1280 ng/ml. Precision and accuracy, demonstrated by within-day, between-day assay, and interoperator assays were lower than 10%. 

Air drawn through a cartridge containing silica gel coated with acidified DNPH derivative eluted with acetonitrile and determined using isocratic reverse phase HPLC with UV detection at 360 nm (U.S. EPA 1988, Method TOll) recommended flow rate 1 L/min sample volume 100 L. 

Perucka and Oleszek have reported extraction and determination of capsaicinoids in fresh fruit of hot pepper using spectrophotometry and HPLC techniques [79]. Cooper et al. developed a reversed-phase HPLC method utilizing a conventional Cjg column to separate capsaicin, dihydrocapsaicin, and nordihydrocapsaicin present in hot peppers [121]. The isocratic mobile phase (60 40 [v/v] methanol/water at a flow rate of 1.5 mL/min) was employed and achieved the separation of these three capsaicinoids in 28 min [121]. Krajewska and Power developed a reversed-phase HPLC... 

The above HPLC methods are used for the analysis of limonin and/or nomilin in citrus juices. Herman et al. (1989) used a reverse phase HPLC method to quantify limonoids in fruits, seeds, leaves, stems and seedlings of Citrus ichangensis. The extraction procedure of Hasegawa et al. (1984) as described in Section 2.1.1 was followed. The dried extracts were dissolved in 50% methanol prior to injection. A reverse phase C-18 column (4.6 X 250 mm) was used. The column was eluted isocratically with water-methanol-acetonitrile (49 41 10) at a flow rate of 1 ml/min. Limonoids were detected by UV absorption at 210 nm. This... 

Kutnink et al. (39) reported an isocratic reversed-phase HPLC-UV method for the determination of MNA in human urine. A method for urinary MNA and 2-Py using isocratic reversed-phase HPLC-UV detection after a simple anion exchange cleanup procedure has been described by Carter (40). However, UV detection suffers from sensitivity. In 1987, Shibata (41) developed a highly sensitive HPLC method with fluorescence detection. MNA was reacted with acetophenone in a strongly alkaline medium at 0°C in the presence of a large amount of isonicotinamide. After 10 min, formic acid was added and the mixture was kept at 0°C for another 15 min. The mixture was heated in a boiling water bath for 5 min. 

Isocratic reverse phase HPLC On-line method... 

Usually, mobile phases of acetonitrile and acetone have been used in the analysis of TG from milk fat, most often in isocratic elution (114,115) and in gradient eiution, and they provide a resolution of 50 chromatographic peaks (Numela). One of the main difficulties in the analysis of TG is the identification of the chromatographic peaks, because of the small number of mixed TGs in a pure state. Bornaz et al. (115) and Dotson et al. (114) identified butterfat chromatographic peaks from the relationship between the retention time and the theoretical carbon number according to the model proposed by El-Hamdy and Perkins (87). An alternative method is the fractionation of total TG in milk fat by reversed-phase HPLC and analysis of the fatty acids in each fraction (116,117). 

In one method, the Dopa formed during the reaction was partially purified by ion-exchange and aluminum oxide chromatography and the amount present quantified by reversed-phase HPLC (ODS column). The mobile phase consisted of 0.1 M potassium phosphate buffer at pH 3.5. The column was eluted isocratically and the eluent monitored by means of an electrochemical detector. 

The performance of the SEC method was compared to that of an isocratic reversed phase (RP)-HPLC method (UV detection). 

Measurement of labelling yield and subsequent radiochemical purity requires a suitable analytical technique, and the method of choice for radio-labelled peptides is reversed phase HPLC with on-line UV and radiometric detection. It is important to use as stringent a separation method as possible with isocratic or slow mobile phase composition gradients over the peptide peak. Ideally, more than one mobile phase system should be used (e.g. a phosphate buffer-methanol system in addition to the standard water-acetonitrile system), since these may show the presence of new impurities. It is important to recognize that HPLC analyses only measure those components that elute from the column. Insoluble, highly lipophilic or positively charged species may bind to the solid phase. It is very important to verify the absence of these species by a complimentary technique such as thin layer chromatography (TLC) and to ensure that the two techniques produce similar results. 

In a similar manner to the lightpipe in GC-IR, a flow cell interface can be used where the eluent flows into a transmission flow cell with IR windows at each end and the IR beam passes through the cell perpendicular to the flow and continuously records IR spectra. This only works if the solvents do not absorb so strongly in the IR region of interest and mask the analyte transmission/absorption, or if the mobile phase absorption spectrum is subtracted (which only works well when the LC method is isocratic). In fact, the solvents used in normal phase HPLC are more compatible with IR than the more polar ones used in reversed phase HPLC. 

We will now proceed to discuss method development strategies. First, we will contemplate the choice of isocratic or gradient methods. Then we will develop an efficient method development strategy. The focus of the method development is the analysis of low-molecular-weight ionizable compounds by reversed-phase HPLC. 

After initial purification and group fractionation of the leukotrienes, the different fractions are usually subjected to reverse phase HPLC, using either gradient or isocratic elution systems. However, although HPLC methods usually have a high... 

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