HPLC-PDA

Zanatta, C.R et ah. Determination of anthocyanins from camu-camu (Myrciaria dubia) by HPLC-PDA, HPLC-MS, and NMR, J. Agric. Food Chem., 53, 9531, 2005. 

The simplest and cheapest procedure to obtain standards is based on selective extraction followed by crystallization. A method developed to obtain lycopene from tomato residue using factorial experimental design consisted of a preliminary water removal with ethanol, followed by extraction with EtOAc and two successive crys-talhzation processes using dichloromethane and ethanol (1 4), producing lycopene crystals with 98% purity, measured by HPLC-PDA. Using this approach, bixin was extracted with EtOAc from annatto seeds that were previously washed with... 

Schieber, A., Detection of isorhamnetin glycosides in extracts of apples (Malus domestica cv. Brettacher ) by HPLC-PDA and HPLC-APCIMS/MS, Phytochem. Anal., 13, 87, 2002. 

HPLC determination can be carried out for most of the neonicotinoids. The average recoveries of imidacloprid in the various crops by the HPLC/PDA method were 88-94% at a fortification level of 0.25 mg and 96-99% at a fortification level of 0.05 mg kg . The overall average recovery for 30 samples was 95% with an RSD of 4.7%.12... 

The final step of method development is validation of the HPLC method. Optimisation of chromatographic selectivity [110], performance verification testing of HPLC equipment [591], validation of computerised LC systems [592] and validation of analysis results using HPLC-PDA [34] were reported. The feasibility of automated validation of HPLC methods has been demonstrated [593]. Interlaboratory transfer of HPLC methods has been described [594]. 

Aromatic amines formed from the reduction of azo colorants in toy products were analysed by means of HPLC-PDA [703], Drews et al. [704] have applied HPLC/ELSD and UV/VIS detection for quantifying SFE and ASE extracts of butyl stearate finish on various commercial yarns. From the calibrated ELSD response the total extract (finish and polyester trimer) is obtained and from the UV/VIS response the trimer only. Representative SFE-ELSD/UV finish analysis data compare satisfactorily to their corresponding SFE gravimetric weight recovery results. GC, HPLC and SEC are also used for characterisation of low-MW compounds (e.g. curing agents, plasticisers, by-products of curing reactions) in epoxy resin adhesives. 

HPLC-PDA-MS) are already being used. Although HPLC-NMR-MS provides a very powerful approach for compositional and structural analysis, it by no means represents the limit of what is possible in terms of hyphenation. On-line extraction and the attachment of multiple detectors (e.g. IR, F) make the technique even more powerful. Other analytical laboratories such as TG-DTA-DSC-FTIR, TD-CT/Py/GC-MS/FTIR and HPLC-UV/NMR/IR/MS have been put to work, but do not represent practical solutions for routine polymer/additive analysis. 

HPLC is a universal separation technique that is capable of separating both volatiles and non-volatiles without the need for derivatization. We are developing methods that employ both on-line photodiode array (PDA) detection and mass selective detection, HPLC/PDA/MS. This approach also utilizes an ion-trap mass... 

Figure 3.5 Three-dimensional display of the photodiode array absorbance data obtained by HPLC/PDA/MS for a M. truncatula extract. The first dimension is HPLC retention time, second is wavelength, and third is absorbance. The data can be rapidly previewed for specific absorbance regions characteristic of functional groups.

HPLC/PDA/MS analysis of an alfalfa root extract. The HPLC retention time, the UV absorbance spectrum, and the mass spectrum readily identify the peak eluting at 45 minutes as medicarpin, a known phytoalexin in alfalfa. 

HPLC/PDA/MS has also been used to compare the saponin profiles in multiple cultivars of alfalfa and M. truncatula. Comparative profiles are provided (Fig.3.8). It is interesting that these closely related legumes yielded different saponin profiles. The saponin profile of M. truncatula is more complex than alfalfa and may provide a richer source for mining putative pharmaceuticals. 

Figure 3.8 Comparative saponin profiles for two cultivars of alfalfa and one cultivar of M. truncatula obtained by reverse-phase HPLC/PDA/MS using electrospray ionization and an ion trap mass spectrometer. The profiles illustrate the increased complexity of saponins in M. truncatula and offer a richer source for bio-prospecting of natural products.

HPLC/PDA/MS/MS characterization of saponins. We then applied this approach to... 

Fig. 29.1 HPLC-PDA of (a) MSPD extract of control bovine kidney, (b) MSPD extract of bovine kidney fortified at the 20 ppm level, and (c) synthetic mixture of standards at levels of 15 ng per component injected. Peaks 1, spectinomycin 2, hygromycin B 3, streptomycin 4, dihydrostreptomycin. (Reprinted from Ref. 19 with permission from Elsevier Science.)...

Fig. 29.5.2 HPLC-PDA chromatograms of (A) blank and (B) fortified (20 ppb) egg control samples and related UV-Vis spectra. Peaks Nf, nitrofurazone Fz, furazolidone Ft, furaltadone. (Reprinted from Ref. 166, with permission from Elsevier Science.)...

For phenolics in fruit by-products such as apple seed, peel, cortex, and pomace, an HPLC method was also utilized. Apple waste is considered a potential source of specialty chemicals (58,62), and its quantitative polyphenol profile may be useful in apple cultivars for classification and identification. Chlorogenic acid and coumaroylquinic acids and phloridzin are known to be major phenolics in apple juice (53). However, in contrast to apple polyphenolics, HPLC with a 70% aqueous acetone extract of apple seeds showed that phloridzin alone accounts for ca. 75% of the total apple seed polyphenolics (62). Besides phloridzin, 13 other phenolics were identified by gradient HPLC/PDA on LiChrospher 100 RP-18 from apple seed (62). The HPLC technique was also able to provide polyphenol profiles in the peel and cortex of the apple to be used to characterize apple cultivars by multivariate statistical techniques (63). Phenolic compounds in the epidermis zone, parenchyma zone, core zone, and seeds of French cider apple varieties are also determined by HPLC (56). Three successive solvent extractions (hexane, methanol, aqueous acetone), binary HPLC gradient using (a) aqueous acetic acid, 2.5%, v/v, and (b) acetonitrile fol-... 

Human serum, prostate Lycopene isomers, a/p-carotene Saponification with KOH, extraction with hexane, centrifugation C-30 MeOH-MTBE HPLC/PDA (450 nm)/MS/ APCI(+) 0.93 pmol for lycopene 109... 

Since the melanin formation assay was run against a cell viability assay, the activity peak maximum at fraction Dll was most likely due to cytotoxicity. The dereplication of another active peak located from fractions D7 to D2 is illustrated in Figure 9. Every active fraction was analyzed by HPLC/PDA/MS. There was a peak located at Rt=16.33 min in the LC/MS total ion chromatogram of active fractions. This peak showed the same pattern of increasing to decreasing intensity as the trend exhibited by the melanin inhibition activities of those fractions. 

Often, low levels of carotenoids in biological samples provide significant challenges in quantification by HPLC-PDA alone. Electrochemical detection (ECD) has been successful in quantifying low concentrations of carotenoids (MacCrehan and Schonberger, 1987 Finckh et ah, 1995 Yamashita and Yamamoto, 1997). More information about ECD can be found in Chapter 2. ECD has also been successful in quantifying carotenoid isomers in foods, plasma, prostate tissue, cervical tissue, and buccal mucosal cells (Ferruzzi et ah, 1998,2001 Allen et ah, 2003 Unlu et ah, 2007). Electrochemical array detection for all-irans - 3-carotene has been reported to be 10 fmol on column, which is approximately 100-1000 times more sensitive than UVA is detectors (Ferruzzi et ah, 1998). 

Often a mass spectrometer is interfaced with an HPLC-PDA system. This technique is especially useful because isobaric species can be chromatographically separated before entering the MS. Interestingly, there are a large number of isobaric species in the field of carotenoids, such as lycopene, [3-carotene, oc-carotene, and y-carotene which all have a parent mass of 536 mu, or (3-cryptoxanthin, oc-cryptoxanthin, zeinox-anthin, and rubixanthin which all have a parent mass of 552 mu. 

Techniques used to identify new carotenoids are also employed to identify carotenoid metabolites in various photosynthetic organisms, as well as animals and humans. A new metabolite might be identified in a food or biological extract by HPLC-PDA, with the observation of a new peak with a UVA is spectrum similar to a carotenoid, or which produces an MS fragment similar to other known carotenoids. Alternatively, the metabolism may be induced in vitro by creating ideal biological conditions for generating metabolites (with intestinal mucosa, for example dos Anjos Ferreira et ah, 2004). 

The data presented show a limitation of HPLC-PDA analysis for trace amonnts of CYL in enviromnental samples but also underline the potential of an inexpensive and fast analysis for various purposes. 

Welkera M., Bickel, H., and Eastner, J. 2002. Technical note HPLC-PDA detection of cylindrospermopsin—opportunities and limits. Water Research 36 4659 1663. 

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