Reverse phase method development columns

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

Hultman et al. [130] developed a LC/MS/MS method for the quantitative determination of esomeprazole and its two main metabolites 5-hydro-xyesomeprazole and omeprazole sulfone in 25 /il human, rat, or dog plasma. The analytes and their internal standards were extracted from plasma into methyl ferf-butyl ether-dichloromethane (3 2). After evaporation and reconstitution of the organic extract, the analytes were separated on a reversed-phase liquid chromatography column and measured by atmospheric-pressure positive ionization mass spectrometry. 

Nakamura et al. [42] developed a simple and rapid semi-micro colunm HPLC method with UV detection for the simultaneous determination of lornoxicam and other oxicams in human blood samples. The drugs including isoxicam as an internal standard were extracted from buffered plasma samples (pH 3) with dichloromethane and the resulting extracts were subjected to HPLC analysis. The separation was performed with a Ci8 reversed-phase semi-micro column (25 cm x 1.5 mm, 5 pm) at 35 °C. The mobile phase used was a mixture of acetonitrile-0.1 M acetate buffer (pH 5)-methanol, and the detection wavelength was set at 365 nm. The drugs were separated within 30 min without interference by the blood components. The detection limits of lornoxicam were 6.4 ng/ml in serum and 9.3 ng/ml in plasma at a signal-to-noise ratio of 3. The method was applied to the determination of lornoxicam in the sera of the patients. 

The majority of reversed-phase methods have been developed on covalently modified silica gel and the most popular stationary phase is octade-cylsilyl silica (ODS, Cig). Polymeric supports, such as functionalized polystyrene-divinylbenzene copolymers (MacBlane et al., 1987), are particularly useful when mobile phases of higher pH are required because of their resistance to degradation in alkaline solutions. The main drawback of polymeric supports is their reduced column efficiencies and their lower mechanical resistance to high pressures compared with silica gel. 

Are there any other analytical protocols available from the analytical chemist The more information about a compound, the easier it will be to develop a purification procedure. For example, in addition to the reversed-phase method, are there thin-layer chromatography (TLC) methods available for this compound This could help in deciding whether a silica column may be the appropriate tool for this separation. 

Several reversed-phase methods were also developed which do not use a C18 column. A reversed-phase method using a C8 Spherisorb column has been reported (54) to quantitate diltiazem and two of its metabolites (N-monodemethyl diltiazem and desacetyl diltiazem). A 10 pm particle size PRP-1 column (55), mobile phase of 60% acetonitrile and 0.01 M aqueous KH2PO4, 40% 0.005M aqueous tetrabutylammonium hydroxide and UV absorbance detection at 254 nm was used to determine diltiazem present in plasma. Several HPLC methods have been developed which use a cyano-bonded column. One such method was developed for the determination of diltiazem and its metabolite desacetyl diltiazem in human plasma (56). The analytes are extracted from plasma made basic with 0.5M aqueous dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in hexane. The method uses a cyanopropylsilane column with a mobile phase of 45% acetonitrile and 55% 0.05M aqueous acetate buffer (pH 4.0). The minimum detectable limit was 2 ng/mL in plasma. A similar HPLC method was developed by Johnson and Pieper (57) for the determination of diltiazem and three of its metabolites. Also, an HPLC method was developed (58) for the analysis of diltiazem and desacetyl diltiazem in plasma using UV detection at 237 nm, a Zorbax CN 6 pm particle size column and a mobile phase of 45% methanol, 55% 0.05M aqueous ammonium dihydrogen phosphate and 0.25% triethylamine adjusted to pH 5. 

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

Despite their structural similarity, atropine ( hyoscyamine) and homatropine cannot be assayed in the same way as hyoscine (6,7-epoxyatropine) as the former compounds are very strongly retained on unmodified silica. Reversed-phase methods usually suffer from analyte peak tailing and, although some success with base deactivated columns has been achieved, it has not proved possible to develop... 

HPLC was proven to be the most important instrumental analytical method for determination of EAs. Separation on HPLC and downstream detection by fluorescence detector or tandem mass spectrometer was performed. In the early 1970s, when the semisynthetic lysergic acid diethylamide 28 (LSD) became available in the drug scene, there was the need of a suitable analytical method for the detection of LSD. Since then, many HPLC analyses have been developed for the detection of EAs. Initially, normal phase HPLC was used for the determination of 4 with subsequent fluorescence detection. Today, reversed phase with C18 column materials is used more frequently for analysis of EAs [63, 71-73]. As mentioned before, EAs differ often in the position of the double bond in ring D of the 2. Clavine-type EAs contain sometimes a double bond at C-8 and C-9, whereas 3 and 4 carry a double bond at C-9 and C-10 instead, which influences the chromophoric features and is therefore a key parameter for the choice of excitation and emission wavelength of fluorescence detection. Extract mixtures containing EAs with a double bond at different positions (C-8 and C-9 or C-9 and C-10) should be analyzed in two runs to ensure the detection and quantification of the complete EAs. Another possibility is to use two fluorescence detectors subsequently [74]. 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

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