Validation and application of sub-2 µm core-shell UHPLC-UV-ESI-Orbitrap MS for identification and quantification of ß-carotene and selected cleavage products with preceding solid-phase extraction.

A validated ultrahigh-performance liquid chromatography method using 1.7 µm core-shell particles is presented for the identification and quantification of ß-carotene (BC) and related cleavage products (CPs) in primary cell culture media. Besides BC, apo-4'-, apo-8'-, apo-10'-, and apo-12'-carotenals, as well as 5,6-epoxy-ß-carotene, were selected as target analytes. Detection was performed via an 80-Hz diode array detector and an electrospray ionization-linear quadrupole ion trap-Orbitrap XL mass spectrometer, both hyphenated in series. Total analysis time was below 6 min with peak widths <12 s. Addition of trifluoroacetic acid and tetrahydrofuran to the mobile phase allowed for the mass spectrometric detection of BC and related CPs and reduced peak tailing due to improved solubility of hydrophobic analytes. Intra-day and inter-day precision for UV and mass spectrometric detection were ≤1.5 % for retention times and ≤5.1 % for peak areas. Instrumental linearity was confirmed by Mandel's fitting test between 0.25 (or 1.00 µg/mL) and 5.00 µg/mL for UV detection. The higher sensitivity of mass spectrometric detection allowed for the coverage of three concentration domains between 0.025 and 5.00 µg/mL in linearity testing. Homoscedasticity was confirmed between 0.10 and 5.00 µg/mL for Orbitrap XL MS. The limits of quantification were between 52.6 and 889.4 ng/mL for UV detection and between 19.3 and 102.4 ng/L for mass spectrometric detection. Offline solid-phase extraction from culture media fortified with BC and CPs provided intra- and inter-day recoveries between 65.8 and 102.4 % with coefficients of variation ≤6.2 %. Primary rat hepatocyte cultures treated with BC and subjected to different oxidative stress conditions contained 5,6-epoxy-BC and apo-4'-carotenal besides residual BC. Apparently, 5,6-epoxy-BC was formed in the medium via autoxidation of BC by ambient oxygen.

Solid-phase extraction and GC-MS analysis of potentially genotoxic cleavage products of ß-carotene in primary cell cultures.

A validated method for the simultaneous determination of prominent volatile cleavage products (CPs) of ß-carotene in cell culture media has been developed. Target CPs comprised ß-ionone (ß-IO), cyclocitral (CC), dihydroactinidiolide (DHA), and 1,1,6-trimethyltetraline (TMT). CPs were extracted by solid-phase extraction applying a phenyl adsorbent, eluted with 10% (v/v) tetrahydrofuran in n-hexane, and identified and quantified by gas chromatography-mass spectrometry with electron impact ionization. Method validation addressed linearity confirmation over two application ranges and homoscedasticity testing. Recoveries from culture media were between 71.7% and 95.7% at 1.0 µg/ml. Precision of recoveries determined in intra-day (N = 5) and inter-day (N = 15) assays were <2.0% and <4.8%, respectively. Limit of detection and limit of quantification of the analysis method were <18.0 and <53.0 ng/ml for ß-IO, CC, and TMT, whereas 156 and 474 ng/ml were determined for DHA, respectively. Although extractions of blank matrix proved the absence of interfering peaks, statistical comparison between slopes determined for instrumental and total method linearity revealed significant differences. The method was successfully applied in selecting an appropriate solvent for the fortification of culture media with volatile CPs, including the determination of their availability over the incubation period. For the first time, quantification of volatile CPs in treatment solutions and culture media for primary cells becomes accessible by this validated method.

Profiling preparations of recombinant birch pollen allergen Bet v 1a with capillary zone electrophoresis in pentamine modified fused-silica capillaries.

Three preparation batches of the recombinant birch pollen allergen Bet v 1a have been analyzed by capillary zone electrophoresis (CZE) using a separation electrolyte consisting of 100 mmol L(-1) phosphate at pH 6.50 with 2.0 mmol L(-1) tetraethylenepentamine (TEPA) added. TEPA improved the resolution by wall shielding and selective attachment to allergens, but reduced migration repeatability at concentrations >2.0 mmol L(-1). Heterogeneity of preparations determined by CZE and electrospray ionization-quadrupole-time-of flight-MS were in accordance and revealed chemically modified (carbamylated) allergens in one of the preparations. The method was validated according to the ICH-guidelines. Repeatability of effective electrophoretic mobility (mu(eff)) was <0.55% R.S.D. (n = 5). Migration time corrected peak areas were used for quantification. Limit of quantification (LOQ) was 25 microg mL(-1) for the major isoform Bet v 1a, based on a signal-to-noise ratio of 10, and detector response was linear between LOQ and 0.90 mg mL(-1). Purity of the different rBet v 1a preparations was determined to be between 40 and 92% depending on the manufacturing protocol.

Analytical tools for the analysis of ß-carotene and its degradation products.

ß-Carotene, the precursor of vitamin A, possesses pronounced radical scavenging properties. This has centered the attention on ß-carotene dietary supplementation in healthcare as well as in the therapy of degenerative disorders and several cancer types. However, two intervention trials with ß-carotene have revealed adverse effects on two proband groups, that is, cigarette smokers and asbestos-exposed workers. Beside other causative reasons, the detrimental effects observed have been related to the oxidation products of ß-carotene. Their generation originates in the polyene structure of ß-carotene that is beneficial for radical scavenging, but is also prone to oxidation. Depending on the dominant degradation mechanism, bond cleavage might occur either randomly or at defined positions of the conjugated electron system, resulting in a diversity of cleavage products (CPs). Due to their instability and hydrophobicity, the handling of standards and real samples containing ß-carotene and related CPs requires preventive measures during specimen preparation, analyte extraction, and final analysis, to avoid artificial degradation and to preserve the initial analyte portfolio. This review critically discusses different preparation strategies of standards and treatment solutions, and also addresses their protection from oxidation. Additionally, in vitro oxidation strategies for the generation of oxidative model compounds are surveyed. Extraction methods are discussed for volatile and non-volatile CPs individually. Gas chromatography (GC), (ultra)high performance liquid chromatography (U)HPLC, and capillary electrochromatography (CEC) are reviewed as analytical tools for final analyte analysis. For identity confirmation of analytes, mass spectrometry (MS) is indispensable, and the appropriate ionization principles are comprehensively discussed. The final sections cover analysis of real samples and aspects of quality assurance, namely matrix effects and method validation.

GEOGRAPHIC DIFFERENTIATION IN ATRIPLEX CONFERTIFOLIA.

Atriplexconfertifolia (Chenopodiaceae) consists of ploidy races extending from diploid through decaploid and is dissected into many racial groups by cytological and flavonoid relationships. On the basis of morphology, the species can be divided into two major subdivisions, one centered in western Nevada and inhabiting chiefly the Great Basin, and one centered in the Colorado Plateau. Western Nevada plants are distinguished by smaller and narrower leaves, as well as by darker spines and other charactristics. Because western Nevada is situated in the lee of the Sierra Nevada Range, it received reduced amounts of rainfall during Pleistocene and Holocene times. These reduced leaf dimensions of A. confertifolia of the rain shadow zone may thus reflect an evolutionary response to aridity.

Determination of regularly distributed plant protectants in raw and drinking waters, using a multiresidue method with cyclodextrin-modified micellar electrokinetic chromatography.

Eighteen plant protectant compounds were separated and determined by cyclodextrin-modified micellar electrokinetic chromatography (MEKC) in a multiclass/multiresidue method. The pesticides included are those dispersed in the greatest amounts today over agricultural acreage, and they represent 8 different classes of compounds (azoles, benzoic acids, chloroacetanilides, phenoxy acids, phenylureas, sulfonylureas, thiocarbamates, and triazines) covering a wide range of chemical reactivities and physicochemical properties. A 500 mL sample of tap water is preconcentrated by solid-phase extraction (SPE) with 300 mg combined polystyrene-divinylbenzene and methacrylate macroporous resins. Trapped analytes are eluted collectively with diethyl ether. Concentration and solvent change yield 250 microL of an acetone "concentrate," which is further worked up and concentrated 1:10 to produce the MEKC injection solution containing 10 mmol/L sodium dodecyl sulfate (SDS) surfactant. For MEKC, 2 phosphate/SDS buffer systems were designed, each allowing complete separation of all pesticides in a single run. Sensitivity was enhanced by a self-etched bubble cell and an injection procedure which employs stacking at reversed polarity. The ability of MEKC to determine plant protectants in raw and drinking waters at the 0.1 microgram/L level, as demanded by the guidelines of the European Union, was demonstrated with spiked tap waters. Recoveries were between 75 and 110%, and limits of quantification, evaluated as method detection limits according to guidelines of the U.S. Environmental Protection Agency, ranged between 0.03 and 0.10 microgram/L. The precisions of the relative migration times were all below 0.5%.

Serum plasmalogens in ischemic cerebrovascular disease.

Plasmalogens, a subclass of glycerophospholipids are ubiquitous constituents of cellular membranes and serum lipoproteins. Comparing concentrations of plasmalogens in sera from patients suffering from ischemic cerebrovascular disease with serum levels in a normal population significantly lower values were found for patient sera.

Effect of temperature cycling on the production of aflatoxin by Asperfillus parasiticus.

Aspergillus parasiticus (NRRL 2999) was grown under cycling temperature conditions on rice and nutmeat substrates. Under conditions of diurnal and nocturnal time-temperature sequencing, total heat input is an important factor of toxin production. When expressed in degree hours per day, thermal input becomes more definitive and provides a finite number, which can be related to observable changes in the culture such as sporulation and toxin biosynthesis. Three well-defined levels of response were observed in relation to heat input: no growth was detected at thermal inputs of less than 208 degree hours/day; mycelial growth as well as copious amounts of an orange pigment were observed at thermal inputs between 208 and 270 degree hours/day; sporulation and aflatoxin biosynthesis occurred above 270 degree hours/day. Between the optimum and minimum thermal input, cycling temperatures significantly reduced the period of the trophophase over cultures receiving equal heat input at a constant rate. Cycling temperatures at the low and high extremes of the temperature range had little or no effect upon the growth pattern of the culture. Regardless of how temperature was manipulated, these responses were consistent with the heat input received by the culture. A. parasiticus did not compete well when mixed with natural fungal isolates from nutmeats and was easily overgrown by the wild isolates even at relatively high thermal input and when present in superior numbers. This factor and heat input generally below that required for toxin biogenesis at harvest time appear to be two significant factors that limit occurrence of aflatoxin on nut crops of the Willamette Valley. These factors are likely to have significance for other crops grown and harvested under similar circumstances.

Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes.

We report on the determination of active enzyme components in pure and crude lipases, using fluorescent inhibitors for covalent modification and visualization of the enzymatically active proteins. Lipase-specific compounds are triacylglycerol analogs, namely 1,2(2, 3)-di-O-alkylglyceroalkylphosphonic acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcohols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible toward nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protein complexes are formed which can be analyzed on the basis of their fluorescent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the analysis of a commercial lipase preparation from Rhizomucor miehei. After incubation of the enzyme with the fluorescent lipid, a single fluorescence band was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtained between fluorescence intensity and the amount of enzyme. Using a combination of different inhibitors, we were able to discriminate between lipases and esterases.

Inequivalence of fluorescent choline and ethanolamine phospholipids in the erythrocyte membrane: fluorescence lifetime determination in the frequency and time domain.

The fluorescence decay of 1-palmitoyl-2-diphenyl-hexatrienyl-propionyl-sn-glycero-3-phosphocholine and the respective ethanolamine derivative were determined in human erythrocyte ghost membranes by phase and modulation fluorometry. The obtained bimodal distribution decay model could independently be confirmed by pulse fluorometry and data analysis by the exponential series method. Distributional widths of labeled choline and ethanolamine phospholipids were different in ghost membranes but identical in phospholipid vesicles. Thus, it is concluded that both lipid classes containing the same fluorophore experience environments of different heterogeneity in the biological system.

Male-specific DNA in the dioecious species Atriplex garrettii (Chenopodiaceae).

The mechanism of sex determination in dioecious species of the genus Atriplex (Chenopodiaceae) has not been determined. This paper reports the discovery of a male-specific DNA fragment in the diploid dioecious species A. garrettii. DNA samples extracted individually from ten male and ten female plants were bulked by sex. Random amplified polymorphic DNA (RAPD) fragments were generated in the two bulks in order to identify markers that were polymorphic between male and female plants. A total of 158 decamer primers were tested. A 2075 base-pair (bp) male-specific DNA fragment generated with the OPAF-14 primer was identified. The fragment was cloned and partially sequenced and 24-mer primers that exclusively amplified this fragment were constructed. When 124 male plants, 126 female plants, and one hermaphroditic plant were tested individually, the male-specific 2075-bp DNA fragment was present in the hermaphrodite and all but one of the male plants, and was absent in all female plants. A smaller DNA fragment (~1800 bp) that was homologous to the 2075-bp fragment was amplified from the single male plant that lacked the 2075-bp fragment. Cytogenetic analysis revealed no apparent heteromorphic sex chromosomes. These observations suggest that sex determination in A. garrettii is genetic, with no evidence of heteromorphic sex chromosomes.

New fluorogenic triacylglycerol analogs as substrates for the determination and chiral discrimination of lipase activities.

A new type of fluorogenic and isomerically pure 1(3)-O-alkyl-2,3 (3,2)-diacyl glycerols was synthesized that can be used as substrate for the determination of lipase activities. These compounds contain a fluorescent pyrene acyl chain and, as a potent quencher of pyrene fluorescence, a trinitrophenylamino acyl residue. In their intact form, the fluorogens show only low fluorescence intensity. Upon lipase-induced or chemical hydrolysis of the substrates, however, the fluorophore and quencher separate from each other. This leads to a gradual increase in pyrene fluorescence, reflecting the time-dependent progress of lipolysis and, under substrate saturation conditions, lipase activity. This lipase assay is continuous and does not require separation of substrate and reaction products. Short- and long-chain homologues as well as optical isomers of the fluorogenic alkyldiacyl glycerols were hydrolyzed by pancreatic lipase, hepatic lipase, and lipo-protein lipase at highly different rates depending on the substrate or enzyme preparation and source (e.g., postheparin plasma or cultured cells). It is proposed that a useful set of enantiomeric and/or homologous substrates in combination with appropriate reaction media might be applied to the selective determination of a lipase in a mixture of lipases, e.g., hepatic and lipoprotein lipase in PHP, for medical diagnostics.

Transfer of phospholipase A-resistant pyrene-dialkyl-glycerophosphocholine to plasma lipoproteins: differences between Lp[a] and LDL.

1-O-Hexadecyl-2-O-pyrenedecanyl-sn-glycero-3-phosphocholine, a non-hydrolyzable fluorescent diether analog of phosphatidylcholine (PC), was synthesized as a probe for studying phospholipid transfer to different lipoprotein classes with potential phospholipase activities. After incubation of total human plasma with the new probe at 37 degrees C for 4.5 h, a characteristic partition between the main lipoprotein fractions was observed. The fluorescent lipid was not degraded under these conditions and, therefore, served as a measure for choline glycerophospholipid distribution between plasma lipoproteins. In low density lipoprotein (LDL) and high density lipoprotein-3 (HDL3) the fluorescent PC analog showed only monomer fluorescence, whereas in Lp[a] and HDL2 monomer and excimer fluorescence were observed, indicating that the fluorescent phosphatidylcholine analog was incorporated into the respective lipoproteins to a different extent. According to the increased pyrene excimer fluorescence in Lp[a] compared with LDL the labeled phosphatidylcholine must be enriched and/or clustered in Lp[a]. Data from phospholipid and total fluorescence analyses are compatible with the assumption of higher label concentration in Lp[a]. On the other hand, transfer rates for serum protein-catalyzed lipid transport into isolated Lp[a] were slower as compared to LDL. It is suggested that slower lipid transfer to Lp[a] under these conditions is due to the decreased lipid mobility in the Lp[a] surface, whereas the higher extent of label partition into Lp[a] as observed in total plasma might be due to the higher affinity of apolipoproteins for phosphatidylcholine in Lp[a] (Sommer, A., et al. 1992. J. Biol. Chem. 267: 24217-24222). The use of a fluorescent dialkyl- instead of diacyl-glycerophosphocholine for transfer studies was mandatory, as we found that lipoproteins contained phospholipase A2 activity toward long-chain phosphatidylcholine. The lipoprotein-associated phospholipase A2 was three times more active in Lp[a] than in LDL. The degradation products formed by the phospholipase, fatty acids, and lyso-PC may add to the high atherogenic potential of Lp[a].

Organization of phosphatidylcholine and sphingomyelin in the surface monolayer of low density lipoprotein and lipoprotein(a) as determined by time-resolved fluorometry.

Fluorescent analogs of phosphatidylcholine (PC) and sphingomyelin (SM) labeled with diphenylhexatrienylpropionic acid (DPH) were prepared and incorporated into the surface layer of human low density lipoprotein (LDL) and lipoprotein(a) (Lp(a)). Fluorescence anisotropy measurements of DPH-PC and DPH-SM in both lipoprotein classes were carried out at different temperatures ranging from 20 to 37 degrees C. DPH-PC as well as DPH-SM were shown to reside in more rigid domains in Lp(a) than in LDL according to higher anisotropy values in Lp(a). In both LDL and Lp(a), DPH-PC experienced a more rigid environment than DPH-SM, suggesting different environments of PC and SM in the surface shell of the lipoproteins. Fluorescence lifetimes of the labeled lipoproteins were determined by phase and modulation fluorometry. We found bimodal Lorentzian distributions for the decay times of DPH-PC and DPH-SM in LDL and Lp(a). Lifetime distribution centers for labeled lipids were very similar except for DPH-PC in Lp(a) which was shifted to longer lifetimes, suggesting a less polar environment of PC in Lp(a) than in LDL. The distributional width of DPH-PC in Lp(a) was broader than in LDL. Accordingly, phosphatidylcholine must be localized in a more homogeneous environment in LDL as compared with Lp(a). On the other hand, no difference in distributional widths was observed for DPH-SM in both lipoproteins, showing that SM organization in Lp(a) is unaffected by apo(a). From the obtained fluorescence data we propose that apoproteins discriminate between the choline phospholipids and preferentially associate with phosphatidylcholine. This effect is enhanced in Lp(a) due to the presence of apolipoprotein(a).