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Irreproducibility during initial development of a high performance liquid chromatography method is seldom investigated in a thorough and careful manner when working in an industrial setting. For a drug project in the early developmental stages, the LC method is often changed, sometimes drastically, if one encounters irreproducibility in the method. Too often a method is deemed irreproducible due to column lot variability, diluent effects, or pH effects with little or no experiments taking place for justification and little thought given to why the irreproducibility may have occurred in the first case. In this paper, a case study is presented in which a systematic approach was carried out in order to determine the exact reason why method irreproducibility was occurring. The findings of the investigation were then drawn onto change a method with poor reproducibility into one that is rugged without the need to start from the beginning and develop a new method.

The fruits of Silybum marianum L. (milk thistle) has been used for the treatment of hepatic and biliary diseases since ancient times. As separation techniques improved several isomers of silybin—the first determined active compound of milk thistle—have been described. Moreover, a new variety, the white-flowered S. marianum was discovered which contains deoxy-derivatives of some of the flavanolignans found in the purple-flowered variety. Based on this observation Nyiredy presumed that the deoxy-derivatives of all known flavanolignans of the purple-flowered variety are present in the white-flowered one. To prove this prediction several separations have been developed by the use of “PRISMA” principle.

This paper describes a rapid micellar electrokinetic capillary chromatographic (MEKC) method for the mapping and identification of biopharmaceutical grade cell culture media. The method has been validated and we currently use it as an identity test for raw material release testing of cell culture media. The MEKC method is capable of resolving components of cell culture medium with specificity, matching the sample profile with the expected component profile, and precision. The precision of the assay was shown by comparison of peak migration times and relative peak areas of three injections of test media on each of three days. The intra-day and inter-day peak migration times and UV214 nm responses (% total peaks areas) of the assay were within acceptable ranges. The intermediate precision (% Relative Standard Deviation) of peak migration times was less than 3%. The intermediate precision (% Relative Standard Deviation) of relative percent peak areas was less than 10% for major peaks present. A linear relationship between response (% total peaks areas) and the concentration of the cell culture medium was observed, with the average correlation coefficient of >0.96 for major peaks (each accounting for greater than 2% of total peaks areas). Based on the identification by chromatographic profile, linearity, and precision, the MEKC method for the mapping of cell culture media components is suitable for the intended use.

The improvements in separation and analysis of complex mixtures by LC-NMR during the last decade have shifted its emphasis from data acquisition to data analysis. For correct data analysis, not only high quality datasets are necessary, but adequate software and adequate databases for semi (or fully)-automated assignments of complex molecules are needed. Only by using NMR, when necessary in combination with MS, the identification of molecules, as present for example in natural products, can be achieved. Here we report on the ongoing efforts required for the construction of an NMR database of flavonoids, implemented for automated assignments of flavonoids. The procedure is demonstrated for a series of flavonoids.

Comprehensive gas chromatography is an excellent technique for separating complex mixtures. If, however high-boiling compounds such as polychlorinated biphenyls have to be analysed, proper adjustiment of the temperature of the modulator turns out to be very important in maintaining the performance of the system. It is shown that miniaturisation of the column-to-modulator coupling, adequate selection of the modulating conditions and, for halogenated compound mixtures, the use of a micro electron-capture detector, can solve experimental problems previously encountered with high boilers.

For better identification of chemical compounds in natural resins HPLC with UV-visible detection has been complemented with spectrofluorimetric detection. The fluorimetric response of many standards has been studied and typical patterns were obtained for several resins which are also found in ancient samples.

The dual separation mechanism exhibited by capillary electrochromatography is demonstrated by the simultaneous separation of sulfonamides as charged and neutral species over a pH range of 2.5 to 6.9. The neutral sulfonamides were separated according to the difference in their hydrophobicities while charged components were separated by the differences in their electrophoretic mobilities. The limitation of capillary electrochromatography for acidic components was also examined.

“Tokaji Aszu”. is the most famous Hungarian wine type with a very delicious, unique aroma. Tokaji Aszu wines are produced from noble rotten grapes by a special vinification technology. In the last few years certain wineries have made an effort to introduce some changes into this traditional process. The present work examines the effect of different vinification technologies on some aroma components in one year old Aszu wines. The volatile aroma components were determined by gas chromatography-mass spectrometry using a Supelcowax 10 60m×0.32 mm capillary column. Wine samples were extracted with Freon 11 prior to the analysis. The chromatograms contained 150–190 peaks. About 40 aroma components were used for comparison of the wines. The results show some limited changes in the aroma composition of the wines due to vinification technological changes. Higher levels of several hydroxy-, oxo-, dicarboxylic acid esters and lactones were found in Aszu wines produced in the traditional way. Attempts have been made to compare instrumental data to sensory properties of the wines.

Surface-enhanced Raman spectra (SERS) ofp-dimethylaminobenzylidenerhodanine have been recorded on silica gel 60 F254 and Si60 F254 Raman TLC plates. Spectra were enhanced by use of a silver sol prepared according to the modified Lee-Meisel procedure. The standard deviations of the intensities and the band ratios for the seven most intense peaks were calculated for 30 parallel measurements. Although the Raman plate gives more reproducible results, several experimental difficulties are encountered in the development of chromatograms.

Univ Estadual Paulista, Inst Quim Araraquara, Dept Quim Analit, BR-14800900 Araraquara, SP, Brazil

A few samples of polychrome Chinese archaeological objects of art have undergone analytical pyrolysis coupled with GC-MS to evaluated the possibility of contemporary characterization of the pigments as well as the organic media used. The red pigment cinnabar (HgS) is easily media used. The red pigment cinnabar (HgS) is easily detected, whereas in the case of organic components positive results are only found where the matrices are resistant to archaeological environmental conditions.

As opposed to inorganic composition, the characterization of organic content in patinas of bronze monuments has been neglected. Analytical pyrolysis coupled with gaschromatographyάss-spectrometry (Py/GC-MS) has been shown to be a valid and rapid technique to identify organic materials in corrosion patina of bronze monuments. In this paper six renaissance Italian outdoor bronze monuments were analyzed in order to determine organic composition of the patina. Results show the presence of both environmental organic compounds and chemicals coming from protective treatment.

Improvements to an established HPLC method are introduced. The modified method is more efficient for separation and detection of the toxins responsible for paralytic shellfish poisoning (PSP). The PSP toxin content of two strains of Alexandrium tamarense and approximately forty shellfish samples collected from different locations in China have been analyzed with this HPLC method. Only one shellfish sample, collected from Lianyungang, China, contained PSP toxins.

The thermal degradation products of the 7α- and 7β-cholesteryl 3β-acetate hydroperoxides obtained by thermal oxidation of cholesteryl 3β-acetate are reported and discussed. The hydroperoxides were degraded in both the vapour and condensed phases. The main components identified were 7-ketocholesteryl 3β-acetate, 7α- and 7β-hydroxycholesteryl 3β-acetates and the products generated from the loss of the hydroperoxide group and/or of acetic acid. Remarkable is the finding of the epoxy-hydroxy derivatives of cholesteryl acetate among the degradation products.

The present paper describes the development of a method for the differentiation of hydroxycinnamic acid derivatives by HPLC with photodiode-array detection. Furthermore spectral data of the compounds under investigation are given. Whereas p-coumaric acid derivatives are distinguishable from caffeic and ferulic acid derivatives by the positions of their spectrum maxima and their convexity interval value, it is not possible to distinguish between caffeic and ferulic acid derivatives because of overlapping spectrum maxima and convexity interval values., The authors acknowledge the financial support of the Spanish "Comision Interministerial de Ciencia y Tecnologia" (proyect ALI92-0928).

A crude chloroform extract of the roots ofPodophyllum emoddii var. chinesis Sprague was studied by micellar electrokinetic chromatography. Four peaks belonging to podophyllotoxin, 4′-desmethyl-podophyllotoxin, kaempferol and quercetin were identified by known standards. The four substances are valuable pharmaceutical ingredients of the plant.

The separation of fullerenes with a monomeric octadecylsilica bonded phase using n-hexane or toluene/methanol mobile phase systems is described. Analytical and preparative separations, incorporating on-line UV/VIS spectral measurements, confirmed the existence of large fullerenes such as C76, C78 and C84. However, isomers of C78 and C84 were not conclusively found.

A complete assignment of the polychlorodibenz-p-dioxins (CLxDD) and polychlorodibenzofurans (ClxDF) (x=4–8) separated on the new polar polysiloxane phase which contains 44% methyl, 28% phenyl, 20% cyanopropyl and 8% polyoxyethylene (Carbowax) groups (DB DIOXIN) is presented.

A computer-assisted prediction system for small peptide sequencing has been constructed based on the retention prediction approach in reversed-phase liquid chromatography. The system has the basic function to predict retention of small peptides at any chromatographic conditions and this function can be useful for the prediction of sequencing. The outline of the system construction and the performance is discussed.

Urinary steroid metabolites were measured by capillary gas chromatography in 25 women (aged 33.3±12.0 years) with effluvium and 16 control healthy laboratory workers women (aged 34.6±8.4 years). There was significant elevation in the level of a number of steroids and steroid metabolites. The elevation of most steroid metabolites refers to the increased steroid secretion of the adrenal and the high cortisol metabolites levels may be do to stress in some of the patients with effluvium.

A knowledge base for determining residual solvents in pharmaceuticals has been established using static headspace gas chromatography (HS-GC). Fifty-one solvents which belong to four different classes according to the guideline for residual solvents regulated by the International Conference on Harmonization were studied. Two columns with opposite polarity, SPB-1 and HP-FFAP, were used and the relative retention time was the qualitative parameter in the knowledge base. With the aid of the knowledge base, unknown residual solvents in pharmaceuticals can be identified promptly and suitable methods for determining residual solvents can be developed rapidly.

A gas chromatographic (GC) method for the routine analysis of fully halogenated chlorofluorocarbons (CFCs) in aerosol cans is described. The identification of CFCs by GC was found to be in full agreement with those by GC-mass-spectrometery. The method has been applied to the analysis of CFCs in 448 aerosol products. The most commonly used fully halogenated CFC propellants in aerosol cans were found to be CFC11, CFC12 and CFC114.

The retention behaviour of steroids was investigated using normal and reversed-phase high-performance TLG systems. Homologous series of esters and n-alcohols were applied as the mobile phases. The electrostatic interactions operating in these chromatographic systems were analysed by studing the relationship between the log (1/Rf) values and the molecular polarizabilities of the solvents.

This paper describes a test (“DMD-test”) to determine chromatographic properties of C8- or C18-bonded phases. The test was developed to select HPLC columns, for clinical and forensic toxicological screening in particular. The three compounds for the DMD-test are: diphenhydramine, 5-(p-methylphenyl)-5-phenylhydantoin (=MPPH) and diazepam. The isocratic mobile phase (acetonitrile/buffer pH2.3) is suitable for analysis of biological samples. 24 commercially available columns were tested. From our work this test enables the selection of commercial HPLC columns with reproducible properties. In this way a reference LC retention list can be developed to identify analytical results on an inter- or intralaboratory basis.