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Liquid chromatographic determination of vitamin K1 in infant formulas and milk.

Vitamin K1 in infant formulas and milk products is determined by reversed-phase liquid chromatography (LC) with UV detection. The sample is hydrolyzed enzymatically, and the vitamin is extracted with hexane. Fractionation by normal-phase semi-preparative LC is followed by analytical LC, with quantitation by the internal standard technique. Recovery of the analyte was 97.4 +/- 2.8%. Linearity was established between 0.05 and 4.0 micrograms/mL. The limit of quantitation is 0.5 microgram/100 g for milk powder, which allows the method to quantitate endogenous levels of vitamin K1.

Animals↗

Matrix-matched quantitative analysis of trace-elements in calcium carbonate shells by laser-ablation ICP-MS: application to the determination of daily scale profiles in scallop shell (Pecten maximus).

A micro-scale method has been developed for analysis of trace-element concentration profiles in the calcium carbonate shell of the Great Scallop (Pecten maximus). UV laser ablation at 266-nm coupled with ICP-MS detection was used to analyse daily calcite striae of shell samples to obtain high temporal resolution of trace element incorporation. Analysis of scallop shells was carefully examined to determine the quality of calcium carbonate ablation and calibration. An accurate external calibration method based on matrix matching was developed. Twelve sodium-free enriched calcium carbonate standards containing up to twenty-four elements were prepared, by co-precipitation with aqueous ammonia and NH(4)HCO(3), and subsequently back-calibrated in the laboratory. These CaCO(3) standards were found to be homogenous and their use enabled sensitive quantitative analysis (detection limits of a few ng g(-1)) over a wide range of concentrations (0.1 to 500 microg g(-1)). Use of these CaCO(3) standards was also evaluated by analysis of three calcium-rich certified reference materials. Because calibration was consistent with the certified results, this analytical method is a sensitive tool for analysis of environmental calcium carbonate matrices. Repeated analysis of scallop shell samples collected simultaneously at the same location showed that the trace elements are homogeneously distributed along a stria. The reliability of such in-situ records of biogenic calcium carbonate (scallop shells) is apparent from the inter-individual and inter-annual reproducibility of the trace element profiles.

Animals↗

Quantitative analysis of steroid hormone receptors and their messenger ribonucleic acids.

Ligand binding will remain the basic technique for receptor determinations in many laboratories. It can easily be set up and it is inexpensive. It has, however, the disadvantage of requiring relatively large amounts of tissue or cultured cells, if the proper multipoint measurements for Scatchard plots are to be done. Furthermore, each of the bound/free separation techniques mentioned above has its own caveat that has to be respected if analytically correct results are to be obtained. The great advantage of receptor immunoassays is their technical simplicity and the possibility of measuring a single dose of a receptor sample (in contrast to the Scatchard plot approach in ligand-binding assays). The possibility of using a single dose (even if assayed in duplicate) is a very valuable feature in all instances when only small tissue samples are available for assay. It would be very attractive for many laboratories to set up their own receptor immunoassays. The availability of suitable antibodies may not be a major obstacle. However, the necessity of possessing a supply of a highly purified receptor standard preparation may pose a problem. This is why commercial kits seem to be used so frequently. The analysis of receptor mRNA is a complement of or alternative to receptor quantitation. It must be realized, however, that special skills, as well as a molecular biology laboratory environment and equipment, are required for successful analytic work in this area. Solution hybridization is to be preferred as an approach to obtain results of a quantitative character. However, the specificity of hybridization should be checked by Northern blots. The same is true for dot/slot hybridization, which is a suitable method for semiquantitative assessments of a series of samples. Last but not least, the in situ hybridization provides invaluable information on the tissue and cell distribution of the mRNA analyzed.

Animals↗

Method development and validation for zotarolimus concentration determination in stented swine arteries by liquid chromatography/tandem mass spectrometry detection.

Drug-eluting stents have attracted significant attention in the medical community and pharmaceutical industry due to their proven success in significantly reducing restenosis. Abbott Laboratories is developing a drug-eluting stent coated with zotarolimus and swine was recently used as an animal model for the pre-clinical study of stent implantation. In this article, we present a detailed experimental design and results for the validation and sample analysis of zotarolimus drug concentration in stented swine artery samples. Introduction of tissue quality control (QC) samples allows evaluation of the entire analytical process as well as the stability of the drug in both original tissue and homogenized tissue samples. In addition, a novel approach using 100% swine blood as the homogenization solution was developed for the consistency of the liquid-liquid extraction recovery and stability of the zotarolimus in tissue homogenates. Standards were prepared by spiking zotarolimus working solution in swine blood and tissue QC samples were used along with the artery samples during the sample analysis. The linear dynamic range of blood standard samples is from 0.61 to 333.20 ng/mL to accommodate the predicted artery homogenate concentrations. Overall tissue QC %CV during the method validation was from 4.4% to 8.6%. The overall %bias of tissue QC samples during the method validation was from -7.3% to 16.6%. The method was successfully applied for the analysis of swine artery samples. A similar approach for method validation and sample analysis has been successfully applied for the analysis of swine myocardium, kidney and liver tissue samples.

Animals↗

Closed-form confidence intervals on measures of precision for an interlaboratory study.

Within the pharmaceutical industry it is common practice to transfer analytical methods from one laboratory to other laboratories. An experiment or interlaboratory study is performed to estimate the repeatability, the intermediate precision, and the reproducibility of the analytical method. These measures of precision are quantified by appropriate sums of variance components from an analysis of variance model describing the structure of the data. In the literature, several methods have been described for calculating approximate (closed-form) confidence intervals on sums of variance components, i.e., Welch, Satterthwaite, and modified large-sample (MLS). Comparisons between these methods have been performed for one-way and two-way classification analysis of variance models only. Interlaboratory studies though often need higher order classifications. Therefore, these methods for constructing confidence intervals are compared on the measures of precision from a specfic three-way classification analysis of variance model that is frequently used for method transfer studies. Using a simulation study, the coverage probability for these methods is evaluated in situations where variance components may be estimated negatively with the standard moment estimates and where either the standard moment estimates are adjusted to zero or remain unadjusted. The MLS method is superior to the other two methods in case the standard moment estimates are used. If the adjusted moment estimates are used, then the method of Satterthwaite performs similar to the MLS method for many settings of the variance components and sample sizes but much better for some particular settings. The method of Satterthwaite performs better than the method of Welch for all the selected settings of variance components and sample sizes, irrespective of the standard or adjusted moment estimates.

Analysis of Variance↗

[Comparison of chemical and radiochemical methods in determination of steroid hormones].

While in some cases steroids can be measured directly in serum or plasma by radioimmunoassay (RIA), in other cases, especially when analyses are carried out in urine, the samples must be processed before RIA can be performed. The operations involved in the preparation of urinary or blood extracts suitable for the RIA of steroid hormones are examined and compared in terms of practicability with the analytical procedures currently used for the chemical determination of the same steroids or their metabolites.

17-Hydroxycorticosteroids↗

A multiwell format assay for heparanase.

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

Blood Platelets↗

Immunochemical analytical methods for the determination of peanut proteins in foods.

Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.

Allergens↗

Use of pyrolysis molecular beam mass spectrometry (py-MBMS) to characterize forest soil carbon: method and preliminary results.

The components of soil organic matter (SOM) and their degradation dynamics in forest soils are difficult to study and thus poorly understood, due to time-consuming sample collection, preparation, and difficulty of analyzing and identifying major components. As a result, changes in soil organic matter chemical composition as a function of age, forest type, or disturbance have not been examined. We applied pyrolysis molecular beam mass spectrometry (py-MBMS), which provides rapid characterization of SOM of whole soil samples. to the Tionesta soil samples described by Hoover, C.M., Magrini, K.A., Evans, R.J., 2002. Soil carbon content and character in an old growth forest in northwestern Pennsylvania: a case study introducing molecular beam mass spectrometry (PY-MBMS). Environmental Pollution 116 (Supp. 1), S269-S278. Our goals in this work were to: (1) develop and demonstrate an advanced, rapid analytical method for characterizing SOM components in whole soils, and (2) provide data-based models to predict soil carbon content and residence time from py-MBMS analysis. Using py-MBMS and pattern recognition techniques we were able to statistically distinguish among four Tionesta sites and show an increase in pyrolysis products of more highly decomposed plant materials at increasing sample depth. For example, all four sites showed increasing amounts of older carbon (phenolic and aromatic species) at deeper depths and higher amounts of more recent carbon (carbohydrates and lignin products) at shallower depths. These results indicate that this type of analysis could be used to rapidly characterize SOM for the purpose of developing a model, which could be used in monitoring the effect of forest management practices on carbon uptake and storage.

Carbon↗

Capillary electrophoresis determinative and LC-MS confirmatory method for screening selected imidazolinone herbicides from soil.

Residues of imazapyr, imazamox, imazapic, imazethapyr, imazaquin, and imazamethabenz (meta and para) are extracted from soil with 0.5 N sodium hydroxide. The pH is adjusted to 2.0-2.2, and the resulting precipitate is filtered. Compounds are trapped onto a tC18 solid-phase extraction (SPE) cartridge, then eluted from the cartridge and passed through a strong anion exchange (SAX) SPE cartridge onto a benzenesulfonic acid strong cation exchange (SCX) cartridge using ethyl acetate. After eluting the analytes from the SCX cartridge using saturated potassium chloride in methanol, the solution is evaporated and redissolved in 1% formic acid in water. The sample is then desalted using a tC18 SPE cartridge and eluted with methanol. After evaporating the methanol to dryness, the compounds are partitioned from acidic solution (pH 3.5) into methylene chloride. The methylene chloride is evaporated to dryness and the residues are then dissolved in Milli-Q water (Millipore, Bedford, MA, U.S.A.) in preparation for analysis by capillary electrophoresis. Results are calculated by direct comparison of the sample peak heights to the peak heights of bracketing standards. The validated sensitivity of the method (LOQ, limit of quantitation) is 2.0 ppb for each compound. Confirmation for individual residues greater than 2.0 ppb is provided by liquid chromatography-electrospray ionization mass spectrometry (LC-ESMS) of the final extract.

Chromatography, Liquid↗

Ultrarapid capillary electrophoresis method for the determination of reduced and oxidized glutathione in red blood cells.

We describe a very rapid high-performance capillary electrophoresis method for the separation and quantification of reduced (GSH) and oxidized (GSSG) glutathione in red blood cells. Two procedures for sample preparation have been compared, Microcon-10 membrane filtration and acid precipitation. The separation is obtained in an uncoated capillary using a high ionic strength borate buffer at pH 7.8. The intra-assay coefficients of variation (CVs%) are 1.53 and 1.66 for GSH and GSSG, respectively. The run is shorter than 90 s and the migration time is highly reproducible both for GSH (CV% 0.22) and GSSG (CV% 0.17). When the filtration step is used only GSH is found, whereas both GSH and GSSG are detectable after acid precipitation, suggesting that GSSG revealed after acid treatment may be an artefact due to GSH oxidation. Because of its good analytical performance this method could be used for routine red blood cell glutathione measurement in healthy or pathological conditions.

Electrophoresis, Capillary↗

Assessment of commercial laboratories performing hair mineral analysis.

CONTEXT: Hair mineral analysis is being used by health care practitioners and promoted by laboratories as a clinical assessment tool and to identify toxic exposures, despite a 1985 study that found poor reliability for this test. OBJECTIVE: To assess whether the reliability of data from commercial laboratories advertising multimineral hair analyses for nutritional or toxicity assessment has improved since the 1985 study. DESIGN, SETTING, AND PARTICIPANTS: A split hair sample taken from near the scalp of a single healthy volunteer was submitted for analysis to 6 commercial US laboratories, which analyze 90% of samples submitted for mineral analysis in the United States. MAIN OUTCOME MEASURES: Agreement of test results for each analyte, laboratory reference ranges, laboratory characteristics, and interpretation of health implications. RESULTS: Laboratory differences in highest and lowest reported mineral concentrations for the split sample exceeded 10-fold for 12 minerals, and statistically significant (P<.05) extreme values were reported for 14 of the 31 minerals that were analyzed by 3 or more laboratories. Variations also were found in laboratory sample preparation methods and calibration standards. Laboratory designations of normal reference ranges varied greatly, resulting in conflicting classifications (high, normal, or low) of nearly all analyzed minerals. Laboratories also provided conflicting dietary and nutritional supplement recommendations based on their results. CONCLUSIONS: Hair mineral analysis from these laboratories was unreliable, and we recommend that health care practitioners refrain from using such analyses to assess individual nutritional status or suspected environmental exposures. Problems with the regulation and certification of these laboratories also should be addressed.

Biomarkers↗

Congener-specific determination of dioxins and related compounds by gas chromatography coupled to LRMS, HRMS, MS/MS and TOFMS.

Their characteristics as persistent organic pollutant and their toxicity (2,3,7,8-TCDD is named as a known human carcinogen) make the dioxins and related compounds a focus of interest in environmental analytical chemistry. In view of the widespread distribution of dioxins in the environment, these compounds must be monitored in several matrices, such as air, effluents, soil, sludge and biological samples. The analytical methodologies are especially difficult owing to the complexity of the mixtures of congeners (210 PCDD/Fs and 209 PCBs) and to the low detection limits required (ppb to ppq). Moreover, time-consuming sample preparation steps are needed owing to the presence of a large number of interfering compounds. The different toxicity of each congener requires the development of congener specific methods. This review of trace dioxin determination by mass spectrometry (MS) includes sample preparation and chromatographic separation. In this Special Feature, the use of different MS techniques such as low-resolution MS (LRMS) and high-resolution MS (HRMS) is discussed in terms of selectivity and sensitivity. The performances of other MS techniques, such as tandem MS (MS/MS) and time-of-flight MS (ToFMS), are compared. Quantification techniques, especially the isotopic dilution method, are also discussed. Conclusions and future perspectives are outlined.

Carcinogens↗

Mass spectrometry for detection of 4-hydroxy-trans-2-nonenal (HNE) adducts with peptides and proteins.

Despite the great technical advancement of mass spectrometry, this technique has contributed in a limited way to the discovery and quantitation of specific/precocious markers linked to free radical-mediated diseases. Unsaturated aldehydes generated by free radical-induced lipid peroxidation of polyunsaturated fatty acids, and in particular 4-hydroxy-trans-2 nonenal (HNE), are involved in the onset and progression of many pathologies such as cardiovascular (atherosclerosis, long-term complications of diabetes) and neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, and cerebral ischemia). Most of the biological effects of HNE are attributed to the capacity of HNE to react with the nucleophilic sites of proteins and peptides (other than nucleic acids), to form covalently modified biomolecules that can disrupt important cellular functions and induce mutations. By considering the emerging role of HNE in several human diseases, an unequivocal analytical approach as mass spectrometry to detect/elucidate the structure of protein-HNE adducts in biological matrices is strictly needed not only to understand the reaction mechanism of HNE, but also to gain a deeper insight into the pathological role of HNE. This with the aim to provide intermediate diagnostic biomarkers for human diseases. This review sheds focus on the "state-of-the-art" of mass spectrometric applications in the field of HNE-protein adducts characterization, starting from the fundamental early studies and discussing the different MS-based approaches that can provide detailed information on the mechanistic aspects of HNE-protein interaction. In the last decade, the increases in the accessible mass ranges of modern instruments and advances in ionization methods have made possible a fundamental improvement in the analysis of protein-HNE adducts by mass spectrometry, and in particular by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass spectrometry. The recent developments and uses of combined analytical approaches to detect and characterize the type/site of interaction have been highlighted, and several other aspects, including sample preparation methodologies, structure elucidation, and data analysis have also been considered.

Aldehydes↗

Solid-phase microextraction combined with surface-enhanced laser desorption/ionization introduction for ion mobility spectrometry and mass spectrometry using polypyrrole coatings.

The successful application of polypyrrole (PPY) solid-phase microextraction (SPME) coatings as both an extraction phase and a surface to enhance laser desorption/ionization (SELDI) of analytes is reported. This SPME/SELDI fiber integrates sample preparation and sample introduction on the tip of a coated optical fiber, as well as acting as the transmission medium for the UV laser light. Using ion mobility spectrometry (IMS) detection, the signal intensity was examined as a function of extraction surface area and concentration of analyte. The linear relationship between concentration and signal intensity shows potential applicability of this detection method for quantitative analysis. Extraction time profiles for the fiber, using tetraoctylammonium bromide as test analyte, illustrated that equilibrium can be reached in less than one minute. To investigate the performance of the PPY coating, the laser desorption profile was studied. The fiber was also tested using a quadrupole time-of-flight (Q-TOF) mass spectrometer with leucine enkephalin as test analyte. Since no matrix was used, mass spectra free from matrix background were obtained. This novel SPME/SELDI fiber is easy to manufacture, and is suitable for studying low-mass analytes because of the intrinsic low background. These findings suggest that other types of conductive polymers could also be used as an extraction phase and surface to enhance laser desorption/ionization in mass spectrometry.

Journal Article↗

Quantification of docetaxel and its main metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

Docetaxel is an antineoplastic agent widely used in therapeutics. The objective of this study was to develop and validate a routine assay, using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), for the simultaneous quantification of docetaxel and its main hydroxylated metabolites in human plasma. A structural analogue, paclitaxel, was used as the internal standard. Determination of docetaxel and four metabolites (M1, M2, M3 and M4) was achieved using only 100 microL of plasma. Liquid-liquid extraction was used for sample preparation, with extraction efficiency of at least 90% for all analytes. Detection used positive-mode electrospray ionization in selected reaction monitoring mode. The lower limit of quantification (LLOQ) was 0.5 ng/mL for all analytes. The assay was linear in the calibration curve range 0.5-1000 ng/mL and acceptable precision and accuracy (<15%) were obtained with concentrations above the LLOQ. This method was sufficiently selective and sensitive for quantification of metabolites in plasma from cancer patients receiving docetaxel chemotherapy, and is suitable for routine analyses during pharmacokinetic studies.

Antineoplastic Agents↗

Direct microtitre plate enzyme immunoassay of folic acid without heat denaturation of serum.

A new and simple method for enzyme immunoassay of folic acid (FA) has been developed, which does not require extraction or heat denaturation of serum. FA-free serum for standards was prepared by a new immunosorbent technique as conventional methods were unsuccessful. The detection limit of the assay is 0.05 ng/ml. Intra- and interassay variabilities ranged between 5-13.3%. Analytical recoveries obtained after spiking with different amounts of FA ranged between 93-110%. We eliminated the interference of endogenous folate binding protein--a major problem in direct FA assay by incubating serum samples (or standards) with FA-HRP conjugate in antibody coated plates at 50 degrees C. Comparison of our data with results obtained by microbiological assay and also by heating samples in alkaline buffer showed good correlation.

Carrier Proteins↗

Manual and automated determination of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in urine.

This paper describes the determination of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in urine. The method involves sample clean-up by liquid-liquid extraction with ethyl acetate followed by high-performance liquid chromatographic (HPLC) analysis. The sample preparation may be performed either manually or automatically using a Zymark Py-robotic system. The chloro analog of the parent compound, CV-araU, is used as the internal standard. As low as 0.1 microgram of analyte per ml of urine can be measured. This sensitivity is adequate for pharmacokinetic studies but could be improved quite easily if necessary.

Antiviral Agents↗