HPLC methods requirements

Traditionally HPLC methods were used for isoflavone analysis from foods [Wang et al., 1990], and gas chromatography/mass spectrometry (GC/MS) to determine isoflavones and their metabolites in human biological fluids including urine [Adlercreutz et al., 1991 Kelly et al., 1993], plasma [Adlercreutz et al., 1993], and feces [Adlercreutz et al., 1995 Kurzer et al., 1995]. HPLC with photodiode array (PDA) detection was introduced in 1994 to measure these analytes in human urine [Franke and Custer, 1994 Xu et al., 1994]. Compared to GC/MS, HPLC methods require fewer steps for sample preparation and analysis and demand less technician time and less expensive instrumentation. 

Detectors Many compendial HPLC methods require the use of spectrophotometric detectors. Such a detector consists of a flow-through cell mounted at the end of the column. A beam of ultraviolet radiation passes through the flow cell and into the detector. As compounds elute from the column, they pass through the cell and absorb the radiation, resulting in measurable energy level changes. 

Developing an HPLC method requires a clear specification of the goals of the separation. The primary objective could be (1) resolution, detection and characterisation or quantitation of one or a few substances in a product, so that it is important to separate only a few sample components and complete separation of the sample is not necessary (2) complete resolution, characterisation and quantitation of all sample components (3) isolation of purified sample components for spectral identification or for other assays. Further points that should be considered include the required sensitivity (especially for trace analysis), accuracy, precision, character of sample matrices (which determines sample dissolution, extraction or pretreatment necessary for possible concentration of sample analytes or for removing interference), expected frequency of analyses and the HPLC equipment available. 

Fluorescence detection at 284/310 nm (extinction/ emission wavelengths) leads to a detection limit of 1.3 mmol/L (0.14 mg/mL for / -cresol). Identification of phenol and /7-cresol may be confirmed by liquid chroma- tography/mass spectrometry. Because HPLC methods require only simple extraction, e.g., by ethyl acetate, and do not require further steps such as derivatization, they j are simple and rapid compared with gas chromatography or gas chromatography/mass spectrometry. Such methods I are useful for monitoring serum phenols in dialyzed patients as an index of hemodialysis adequacy. How- ever, the separation of the three isomers of cresol can only be performed by adding 3-cyclodextrin to the c liquid phase. q... 

Various aspects of the chromatography of vitamin B 2 and related corrinoids have been reviewed (59). A high performance Hquid chromatographic (hplc) method is reported to require a sample containing 20—100 p.g cyanocobalamin and is suitable for premixes, raw material, and pharmaceutical products (60). 

Separation of enantiomers by physical or chemical methods requires the use of a chiral material, reagent, or catalyst. Both natural materials, such as polysaccharides and proteins, and solids that have been synthetically modified to incorporate chiral structures have been developed for use in separation of enantiomers by HPLC. The use of a chiral stationary phase makes the interactions between the two enantiomers with the adsorbent nonidentical and thus establishes a different rate of elution through the column. The interactions typically include hydrogen bonding, dipolar interactions, and n-n interactions. These attractive interactions may be disturbed by steric repulsions, and frequently the basis of enantioselectivity is a better steric fit for one of the two enantiomers. ... 

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

You require to develop an HPLC method for the determination of a high-molecular-weight aliphatic alcohol that has no UV absorption. Unfortunately, you only have a UV detector available. How would you attempt the analysis ... 

By utilizing the HPLC method, it is possible to determine the level of each individual toxin in sample solutions. This provides a "toxin profile" that can be very useful in PSP toxin research studies. The ability to examine relative changes in toxin concentration and profile has greatly facilitated studies relating to toxin production by dinoflagellates, metabolism of toxins in shellfish, and movement of toxins up the food chain. Since the HPLC method is easily automated and requires only very small sample sizes (< 1 g tissue), it has clear advantages over other analytical procedures for the toxins in many research situations. Two examples of the utilization of HPLC for the study of the PSP toxins follow. 

HPLC has also been utilized in more complex food chain transfers. It has been known for some time that the toxins can be responsible for fish kills in the Bay of Fundy 18). The vectors for these fish kills are zooplankton that feed on toxic dinoflagellates. In two related studies (79 Sullivan, unpublished), HPLC was utilized to investigate the transport of toxins from dinoflagellates to zooplankton and then to fish. The HPLC method is ideally suited for this since only very small sample sizes (ca. 100,000 dinoflagellate cells) are required. 

However, since fluorometric methods require sophisticated instrumentation, their applicabihty is limited because of cost. In conclusion, spectroscopic methods usually enable crude estimates of chlorophylls in an extract, but in most cases accurate and detailed analysis of a specific composition requires separation of the mixture into individual compounds using methods such as HPLC. 

Traditionally, Irganox B blends (Irganox 1010/1076 and Irgafos 168) are analysed by HPLC methods, which require a long sequence of preliminary actions milling,... 

The near-IR technique has been used very successfully for moisture determination, whole tablet assay, and blending validation [23]. These methods are typically easy to develop and validate, and far easier to run than more traditional assay methods. Using the overtone and combination bands of water, it was possible to develop near-IR methods whose accuracy was equivalent to that obtained using Karl-Fischer titration. The distinction among tablets of differing potencies could be performed very easily and, unlike HPLC methods, did not require destruction of the analyte materials to obtain a result. 

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