Reversed-phase HPLC sample preparation

Chassaigne H, and Lobinski R (1998) Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS. Analyst 123 2125- 2130. Chemosphere (1999) Special issue - Sources of error in methylmercury determination during sample preparation, derivatisation and detection. Chemosphere 39 1037-1224. 

The sample extracts that show either toxicity or no dose response on initial testing should be fractionated. An aliquot of the extract is solvent exchanged to acetonitrile, and an initial analytical scale separation is made to assess the distribution of constituents in the sample. This separation is accomplished by using a Qg reversed-phase system eluted for 45 min with a linear gradient of 0-100% acetonitrile in water. If >75% of the sample elutes after the solvent composition of 80% and 20% acetonitrile, then the fractions are isolated by preparative reversed-phase HPLC. Fraction A is eluted with 100% water fraction B is eluted with a linear mobile-phase gradient from 100% to 75% water and 25% acetonitrile fractions C, D, and E are eluted with gradients with final compositions of 50%, 75%, and 100% acetonitrile. 

HPLC Separations. SAMPLE PREPARATION FOR INJECTION. Isolated residue organics were dissolved in water/acetonitrile solvent mixtures for reverse-phase HPLC separations as follows sample was dissolved in a minimum volume of acetonitrile and diluted with water until... 

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

Inject extract into the normal- or reversed-phase HPLC system (see Basic Protocol). SAMPLE PREPARATION FROM MEATS... 

As the levels of tocopherols and tocotrienols in meat samples are usually lower than in oil and fat samples, a larger sample size is needed in the sample preparation. The meat sample is homogenized to weaken the sample matrix. As in Basic Protocol 2, saponification, heating, and liquid/liquid extraction are used to increase the recovery and remove interference compounds. Satisfactory results can be achieved using a reversed-phase HPLC method. 

This method is used to simplify a chromatogram by reducing the number of compounds in a sample to the six aglycons. This protocol describes the dilution, preparation (including solid phase extraction and acid hydrolysis), filtration, and reversed-phase HPLC analysis of the sample. 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

Reversed-phase HPLC is used for the analysis of the different groups of phenols, phenolic acids, hydroxycinnamic acids, flavonoids, and procyanidins in grapes and wines (22,46,47,77-80). However, due to the presence of a large quantities of various compounds, wine analysis is difficult. Thus, different sample preparation procedures, including fractionation and extraction, are often applied when various groups of phenolic compounds are studied together. 

A reversed-phase HPLC procedure was proposed for the determination of seven phenolic acids in green coffee samples (144). The sample preparation technique involved extraction, alkaline hydrolysis, and liquid/liquid extraction. The chromatographic separation was achieved us-... 

Sample Preparation. SFC grade C02 (< 5 ppm O2) was purchased from Scott and pyrene (99 %) was obtained from Aldrich. The pyrene purity was checked by reversed phase HPLC (C18) and all reagents were used as received. Stock solutions of pyrene were prepared in absolute ethanol. 

The most common analytical technique for the analysis of FFAs and their breakdown products has been chromatography. HPLC has been used for the analysis of FFAs (Christie, 1997 Lues et ah, 1998 Zeppa et ah, 2001). Analysis of short-chain fatty acids (C2-C4) may be relatively simple (Zeppa et ah, 2001). However, the analysis of long-chain fatty acids (>C6) may require derivatization. They are extracted using solvents, converted to bromophenacyl esters, and analyzed by reverse-phase HPLC. GC (with sample preparation and derivatization) has been the method of choice for analysis of fatty acids. An ideal but difficult procedure is to extract FFAs from the aqueous phase and organic phase and combine them (IDF, 1991). The challenge is to overcome the effects of partitioning and extraction of compounds that interfere with the analysis. ISO and IDF have detailed some of the extraction methods for lipids and liposoluble compounds in milk products (ISO, 2001b). Several other methods, which are mainly different in the extraction and derivatization steps, were reviewed by Collins et ah (2004). 

Building on published literature and based on empirical trials, an efficient reverse-phase HPLC system and sample preparation scheme has been developed for vitamin D3 in... 

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... 

Minimal sample preparation (dilution in HPLC mobile phase) is necessary. A standard reversed-phase HPLC method is used for all the samples associated with a drug candidate to reduce time-consuming method development/method refinement procedures. Standard reversed-phase methods typically involve a 20-30 min cycle time and provide information on a wide range of compounds. The incorporation of a standard method strategy allows the use of autosampling procedures and standard system software for data analysis. 

Normal phase (NP) separations are comparatively rarely used in environmental analysis. Again, the reasons lie in the range of analytes amenable to this mode of separation, and in the limited compatibility of typical normal phase HPLC (NP-HPLC) mobile phases with mass spectrometric detection (this also applies to IC). Not only for this reason has interest recently grown in hydrophilic-lipophilic interaction chromatography (HILIC), which represents a viable alternative to the separation of very polar compounds with mobile phases that have a much better compatibility with MS detection, for example, acetonitrile/water with a low water content, typically below 10%, 32 Nonetheless, NP chromato-graphy retains its important role in sample preparation, particularly for the cleanup of complex environmental samples. In the off-line approach, fractions are collected and the relevant one is injected into the reversed phase HPLC (RP-HPLC) system, often after solvent exchange. 

A sensitive reverse-phase HPLC method has been developed for the analysis of etodolac in tablet formulation [22]. The chromatographic separation was achieved using a reverse-phase Cu column, having dimensions of 3.3 cm x 0.46 cm i.d. (3 pm particles) and which was maintained at 30°C. The mobile phase consisted of pH 6.0 phosphate buffer / methanol (60 40 v/v), and was eluted at 1 mL/min. Analyte detection was effected on the basis of UV detection at 230 nm. Diazepam was used as an internal standard. The sample preparation entailed grinding the etodolac tablets, followed by extraction with methanol (using sonication). A retention time of 1.46 min was obtained for etodolac under these conditions, and the method was found to be linear, precise, and accurate over the concentration range of 0.01 to 0.1 mg/mL. 

The sample preparation methodology for the determination of TCDD at these low levels is an active area of development as indicated by the improved procedure reported here and used to analyze the milk for the NIEHS study. It uses reagent modified adsorbants, a higher efficiency basic alumina column than previously (19) and a new degree of separation, reverse phase HPLC as an integral part of the procedure. All of these are directed towards improving the specificity of the sample preparation for 2,3,7,8-TCDD. Increased specificity is needed since the lowest detection limit achievable in all previous studies of TCDD in environmental samples has been dictated by the presence of other substances in the samples which have not been removed by the sample preparation. 

A method using HPLC has also been proposed (Schulte, 1994) whereby an oil solution is first eluted through a silica gel column to separate the steradienes from the more polar lipids. The eluate is then concentrated and analysed on a reverse phase HPLC column using UV detection at 235 nm. This method involves a little more sample preparation than the LC-GC method proposed by Grob et al. (1992) but the equipment is more widely available. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

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