A robust high resolution reversed-phase HPLC strategy to investigate various metabolic species in different biological models

Recent advancements in the field of omics sciences have paved the way for further expansion of metabolomics. Originally tied to NMR spectroscopy, metabolomic disciplines are constantly and growingly involving HPLC and mass spectrometry (MS)-based analytical strategies and, in this context, we hereby propose a robust and efficient extraction protocol for metabolites from four different biological sources which are subsequently analysed, identified and quantified through high resolution reversed-phase fast HPLC and mass spectrometry. To this end, we demonstrate the elevated intra- and inter-day technical reproducibility, ease of an MRM-based MS method, allowing simultaneous detection of up to 10 distinct features, and robustness of multiple metabolite detection and quantification in four different biological samples. This strategy might become routinely applicable to various samples/biological matrices, especially for low-availability ones. In parallel, we compare the present strategy for targeted detection of a representative metabolite, L-glutamic acid, with our previously-proposed chemical-derivatization through dansyl chloride. A direct comparison of the present method against spectrophotometric assays is proposed as well. An application of the proposed method is also introduced, using the SAOS-2 cell line, either induced or non-induced to express the TAp63 isoform of the p63 gene, as a model for determination of variations of glutamate concentrations.     

Determination of polyanionic macromolecules by size-exclusion chromatography

This report presents an extension of a method developed for determination of dextran sulfate in rat serum. The drug is a negatively charged polysaccharide with a molecular mass of 8000. It is fractionated by molecular size and separated from serum components by high-performance size-exclusion chromatography. Sensitive detection is achieved by the post-column complexation of the analyte with 1,9-dimethylmethylene blue (DMMB). A metachromatic complex is formed; the absorbance maximum of the complex is shifted from that of the free dye. Various glycosaminoglycans and other macromolecular polyanions interact with DMMB. Several can be determined using the chromatographic conditions developed for dextran sulfate. The method provides a simple procedure for quantitation of these compounds. Compared to spectrophotometric assays, less sample preparation is required, selectivity is enhanced, and molecular mass information is provided. With modification of eluent composition, dye concentration, and detection wavelength, the method can be validated for determination of additional compounds.     

Finasteride Concentrations and Prostate Cancer Risk: Results from the Prostate Cancer Prevention Trial

ObjectiveIn the Prostate Cancer Prevention Trial (PCPT), finasteride reduced the risk of prostate cancer by 25%, even though high-grade prostate cancer was more common in the finasteride group. However, it remains to be determined whether finasteride concentrations may affect prostate cancer risk. In this study, we examined the association between serum finasteride concentrations and the risk of prostate cancer in the treatment arm of the PCPT and determined factors involved in modifying drug concentrations.MethodsData for this nested case-control study are from the PCPT. Cases were drawn from men with biopsy-proven prostate cancer and matched controls. Finasteride concentrations were measured using a liquid chromatography-mass spectrometry validated assay. The association of serum finasteride concentrations with prostate cancer risk was determined by logistic regression. We also examine whether polymorphisms in the enzyme target and metabolism genes of finasteride are related to drug concentrations using linear regression.

Results and Conclusions

Among men with detectable finasteride concentrations, there was no association between finasteride concentrations and prostate cancer risk, low-grade or high-grade, when finasteride concentration was analyzed as a continuous variable or categorized by cutoff points. Since there was no concentration-dependent effect on prostate cancer, any exposure to finasteride intake may reduce prostate cancer risk. Of the twenty-seven SNPs assessed in the enzyme target and metabolism pathway, five SNPs in two genes, CYP3A4 (rs2242480; rs4646437; rs4986910), and CYP3A5 (rs15524; rs776746) were significantly associated with modifying finasteride concentrations. These results suggest that finasteride exposure may reduce prostate cancer risk and finasteride concentrations are affected by genetic variations in genes responsible for altering its metabolism pathway.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00288106","term_id":"NCT00288106"}}NCT00288106     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

Determination of finasteride in human plasma by liquid–liquid extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of finasteride in human plasma is presented. The method is based on liquid–liquid extraction with hexane–isoamylalcohol (98:2, v/v) and reversed-phase chromatography with spectrophotometric detection at 210 nm. The mobile phase consists of acetonitrile–15 mM potassium dihydrogenphosphate (40:60, v/v). Clobazam is used as the internal standard. The limit of quantitation is 4 ng/ml and the calibration curve is linear up to 300 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Adaptation of the Griess reaction for detection of nitrite in human plasma

The determination of nitrite in human plasma or serum has been most frequently used as a marker of nitric oxide (NO) production. In addition, it has recently been suggested that nitrite could act as a vasodilating agent at physiological concentrations by NO delivery. Therefore, nitrite determination in biological fluids is becoming increasingly important. The most frequently used method to measure nitrite is based on the spectrophotometric analysis of the azo dye obtained after reaction with the Griess reagent. This method has some limitations regarding detection limit and sensitivity, thus resulting unsuitable for nitrite detection in plasma. We have identified some drawbacks and modified the original procedure to overcome these problems. By the use of the newly developed method, we measured 221±72 nM nitrite in human plasma from healthy donors.     

Development and validation of a new gas flame ionization detector method for the determination of finasteride in tablets

A simple, rapid, and sensitive method based on gas chromatography with flame ionization detection is described for the determination of finasteride in tablets. The method is based on the derivatization of finasteride with N,O-bis(trimethylsilyl)trifluoroacetamide-1% trimethylchlorosilane at 60 degrees C for 30 min. The method was validated for specificity, linearity, precision, accuracy, robustness, and limit of quantification. The degree of linearity of the calibration curves, the percentage recoveries of finasteride, and the limit of detection (LOD) and limit of quantification (LOQ) for the gas chromatographic method were determined. The assay was linear over the concentration range of 10 to 50 microg ml(-1) (R approximately 0.999). LOQ and LOD (signal/noise ratio = 10) were found to be 10 and 2 microg ml(-1), respectively. The method was found to be simple, specific, precise, accurate, and reproducible. All of the validation parameters were within the acceptance range. The developed method was applied successfully to estimate the amount of finasteride in tablets. The results were compared statistically with those obtained by the official method using t and F tests. There was no significant difference between the two methods with respect to mean values and standard deviations at the 95% confidence level.     

High-performance liquid chromatographic determination of creatinine in serum, and a correlation of the results with those of the Jaffé and enzymic methods

A high-performance liquid chromatographic method for the determination of creatinine in control sera is reported, based on its separation from deproteinated serum components on the ion-exchange material HEMA Bio 1000 SB and ultraviolet detection at 230 nm. Groups of eleven to fourteen samples of human serum and several control materials were simultaneously analysed by the Jaffé, enzymic ultraviolet and enzymic peroxidase aminophenazone methods. Another group (52–115 sera) was analysed for correlations with spectrophotometric methods. The precision of the chromatographic method ranges between 2.0 and 1.0% (relative standard deviation) for serum creatinine concentrations of 115.1 to 471 μmol/l, respectively. A very good accuracy was found in analyses of reference materials Kontrollogen-L and -LP. Some results of analyses of the other control sera were higher and the other lower than those obtained by the Jaffé and enzymic methods, because both interferences and enzyme inhibitors were encountered. Correlations between the chromatographic and spectrophotometric methods were good.     

Application of the method of measurement of fluorescence intensity in vivo in biological tissues for pharmacokinetic studies of different chlorin-based photosensitizers

A precise and reproducible method of quantitative determination of the photosensitizer (PS) Photolon in liver samples of laboratory animals by means of a spectrophotometric assay with preliminary extraction has been developed. Conditions of the PS extraction have been optimized and validated for the quantitative determination of the PS Photolon by the spectrophotometric assay and the main analytical characteristics have been investigated. Using the method of quantitative determination of the PS Photolon in liver tissue samples a quantitative estimation of in vivo fluorescence of the liver tissue was performed after PS administration to animals. There was a high correlation (R = 0.99) between results obtained by spectrophotometry ex vivo and spectrofluorimetry in vivo. The method of fluorescence detection in vivo is applicable for studies of the pharmacokinetics of different photosensitizers.     

Development of a capillary electrophoresis assay based on free sulfate determination for the direct monitoring of sulfoesterase activity.

A capillary electrophoresis assay of sulfoesterase activity was developed that overcomes the main drawbacks encountered with the usual methods for sulfate determination in complex biological medium. Conditions are described allowing direct measurement of inorganic sulfate that is enzymatically produced in the reaction mixture. The main features of this method are electrokinetic sample introduction, which allows selective extraction of sulfate from the matrix into the separation capillary, counter-electroosmotic flow migration mode, indirect absorbance detection and use of an internal standard for quantitative performances. Likewise, perfect linearity was obtained for concentrations of sulfate up to 40 ppm. The limits of detection and quantification were 0.2 and 0.6 ppm, respectively. The run-to-run and day-to-day precision are 1 and 4.5%, respectively, for sulfate concentrations varying from 35 ppm down to 1 ppm. The accuracy was established for the synthetic p-nitrocatechol sulfate substrate by comparison with the classical spectrophotometric assay. The method was applied to the kinetic monitoring of the activity of a sulfoesterase extracted from the marine mollusc Pecten maximus on fucoidan, a bioactive sulfated fucose-based polysaccharide derived from brown algae. For the first time, a sulfoesterase activity was shown to be effective on such sulfated polysaccharides.     

High-performance liquid chromatography with spectrophotometric and electrochemical detection of a series of manganese(III) cationic porphyrins

Recent studies have revealed potent pharmacological activities of manganese-containing cationic porphyrins. An analytical method employing high-performance liquid chromatography with spectrophotometric and electrochemical detection (HPLC-UV/EC) suitable for in vivo applications is described for a series of manganese(III) cationic porphyrins with good separation and resolution. In particular, this method resolved the four atropisomers of manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+ or AEOL-10113), verified by mass spectrometry. Electrochemical and spectrophotometric methods of detection were compared using manganese(III) meso-tetrakis(1,3-diethylimidazolium-2-yl)porphyrin (MnTDE-2-ImP5+ or AEOL-10150), the lead catalytic antioxidant of this series. Both methods of detection were quantitative, but electrochemical detection, although less specific for in vivo applications, appears to be considerably more sensitive than spectrophotometric detection.     

Determination of in-vivo cytoplasmic orthophosphate concentration in yeast

Summary To investigate in-vivo concentrations of cytoplasmic phosphate, especially during dynamic conditions, a method has been developed that enables reproducible determination of cytoplasmic phosphate from 5 M up to 30 M. The method involves fast sampling, spontaneous inactivation of cell metabolism and differential extraction procedure to gain porosity of the outer cell membrane exclusively. To determine very low cytoplasmic phosphate concentrations, an enzymatic assay was linked to a sensitive spectrophotometric cycling method to increase the detection limit.     

Techniques for boron determination and their application to the analysis of plant and soil samples

This paper reviews techniques for determining B concentration and isotopic ratio and their application to soil and plant samples. Boron concentration has been determined utilising spectrophotometry, potentiometry, chromatography, flame atomic emission and absorption spectrometry, inductively coupled plasma (ICP) optical emission (OES) and mass spectrometry (MS), and neutron activation analysis using neutron radiography and prompt- activation analysis. Isotopic ratios of B have been measured by ICP–MS, thermal ionisation mass spectrometry (TIMS) and secondary ion mass spectrometry (SIMS). For isotopic measurements, TIMS and SIMS are more sensitive and provide higher degrees of accuracy and resolution than ICP–MS, however, extensive sample preparation and purification, and time-consuming measurements limit their usefulness for routine analyses.While the spectrophotometric technique using a colorimetric reaction of B with azomethine-H has been the most extensively applied B determination method for soil and plant samples, colorimetric methods, in general, suffer from numerous interferences and have poor sensitivity and precision. The prompt- method can determine B concentration in intact samples which enables this method to be especially useful for some applications in agriculture. Research involving B behaviour in plant and soil environments would benefit from this technology. In recent years, the use of ICP–OES and ICP–MS for B determination in plant and soil samples has grown tremendously. The application of ICP–OES brought a significant improvement in B analysis because of its simplicity, sensitivity and multielement detection capability. However, besides matrix interferences, the two most sensitive emission lines for B suffer strong spectral interference from Fe. The ICP–OES is not adequately sensitive for some nutritional work involving low B concentrations and B translocation studies using the isotope tracer technique.Plasma is one of the most effective analyte ionisers and MS is the most sensitive ion detector. Coupling of plasma with MS resulted in the development of plasma source MS technology (ICP–MS) which has outperformed all previous analytical methods for trace element determination. Boron determination by ICP–MS suffers no spectroscopic interferences, and is considered the most practical and convenient technique for B isotope determination. The ability of ICP–MS to measure isotopic ratios as well as B concentration enables: (1) B concentration determination by the isotope dilution method, (2) verification of B concentration by isotope fingerprinting in routine analysis and (3) determination of total B concentration as well as B isotope ratio in the same run for biological tracer studies. Therefore, ICP–MS is the method of choice among the present-day technologies for determining B concentration and a convenient method for B isotope determination. In recent years, new generations of plasma-source MS instruments have been developed using alternative plasma generation methods and high-resolution mass spectrometers. These instruments are expected to bring further improvements in accuracy, sensitivity and precision of B determination.     

Polymer membrane ion-selective electrodes as a convenient tool for lipases and esterases assays

We have proposed a novel assay for lipases and esterases activity determination based on potentiometry with ion-selective electrodes (ISEs). Enzyme preparations, obtained from the living cells, are complex mixtures of various proteins, short peptides, lipids, carbohydrates, and other compounds. The most commonly used quantitative methods in enzyme studies are based on spectrophotometric or spectroflourimetric protocols which has significant limitations. They are not valid for samples that are turbid or strongly colored. To overcome those drawbacks we have proposed an assay based on potentiometry with ISEs for lipases and esterases activity determination. This electrochemical methodology represents an attractive tool for enzyme analysis, because of its low detection limit, independence from sample volume and from sample turbidity. The usefulness of this assay has been proven by the determination of the activity of various raw enzymes “acetone powders” isolated from animal tissues. Moreover, activities of fractions obtained during purification of one of those raw biocatalysts were also determined that way. The reliability of determination enzyme activity with ISE assay was proven by comparison with a classical spectrophotometric method.     

Separation and quantitative analysis of rat testis histones by one-dimensional gel electrophoresis

A procedure is presented for direct separation and quantitative analysis of histones of rat testis by use of one-dimensional cylindrical gel electrophoresis in acid/urea/polyacrylamide gels at two concentrations of urea. After separation and staining with amido black the histone fractions are determined by extraction of bound dye and spectrophotometric analysis. This method provides a rapid and accurate procedure for determination of microgram amounts of the histone subfractions which are unique to the testis as well as those which are found in somatic cells, and it is particularly useful for determination of molar proportions of the histones.     

Interference of some detergent micelles with the titration of protein

We have examined the possible interferences with spectrophotometric protein determination of some detergents commonly used for the solubilization of membrane proteins. The results show that some mixtures of Triton X100 with SDS or DOC, at given concentrations, interfere with BSA determination by the method of Lowry.     

Esterases as a marker for growth of BY-2 tobacco cells and early somatic embryos of the Norway spruce

Growth is one of the basic properties of biological systems. The methods which are commonly used for the determination of growth are usually difficult and not very accurate. In the present work we decided to use esterase activity as a growth marker in tobacco suspension culture (BY-2 line) and in early somatic embryos of Norway spruce (clone 2/32) grown on a semi-solid medium. Esterase activity correlates well with the classical growth characteristics of BY-2 and spruce early somatic embryos. Determination of esterase activity is based on spectrophotometric and spectrofluorimetric detection of reaction products, which arise from the enzymatic hydrolysis of two substrates (p -nitrophenyl acetate and fluorescein diacetate) by esterase. The spectrophotometric method enabled us to detect approximately 10⁴ BY-2 cells and 25 spruce embryos whereas the more sensitive spectrofluorimetric method allowed us to detect approximately 800 BY-2 cells and 5 early somatic embryos of Norway spruce.     

一种测定蛹虫草中虫草酸含量的酶标仪微量反应方法

研究和建立一种基于酶标仪-96孔板高通量测定虫草酸含量的检测方法,并对该方法进行性能评价。以酶标仪为检测仪器,在96孔板内按照设定反应条件微量加入样品和试剂进行显色反应,利用酶标仪测定吸光度值并计算虫草酸含量。通过检测精密度、重复性、回收率,并与分光光度计法进行比较,综合评价该方法的准确度、精确度。结果表明,测定数据具有较高的精密度（样品CM1的RSD值0.829%;样品CM2的RSD值1.772%)、重复性（标准样品B40的RSD值2.061%;样品CM2 的RSD值1.599%)、回收率（平均回收率99.24%,RSD值3.666%）,测定结果与分光光度法检测结果无显著差异(P>0.05)。结果表明,酶标仪微量法测定准确、重复性好,并可大大减少样品和试剂的用量,该方法方便、快捷、高效,可以替代分光光度法用于虫草酸含量的测定。     

Identification and quantification of some elements in the hog roundworm, Ascaris lumbricoides suum, and certain tissues of its host

Atomic absorption spectrophotometric and fluorometric analyses were utilized for the determination of several elements in the whole bodies of both male and female Ascaris lumbricoides suum and from the muscle and kidney of the swine host. Concentrations of cadmium, calcium, copper, lead, magnesium, manganese, iron, selenium, potassium, and zinc in these tissues are reported. Statistical analysis (Tukey's procedure) of the data indicated no differences in metal concentrations between male and female ascarids. There were three instances in which the metal concentrations were statistically different in worm tissues and both hog tissues.     

Determination of serine/threonine protein phosphatase type 2B PP2B in lymphocytes by HPLC

Current cyclosporin (CsA) and tacrolimus (FK506) monitoring methods are based on blood concentrations determination, but the optimal sampling times are not clearly defined. An alternative pharmacodynamic approach has recently been introduced, based on assaying lymphocytes activity of calcineurin (PP2B), a phosphatase that is partially inhibited by CsA and FK506. However, the princeps method uses large amounts of radioactive [32P]substrate, raising a number of safety issues. Here we describe an alternative method for PP2B activity determination, based on HPLC coupled with spectrophotometric detection. A 19-amino-acid peptide is phosphorylated by a protein kinase, and further dephosphorylated by lymphocyte PP2B in the presence of okadaic acid. The two peptides are separated by using reverse-phase chromatography with a detection wavelength of 205 nm. After lymphocyte isolation by density-gradient centrifugation, PP2B activity is derived from the dephosphorylated peptide concentration measured during the first 6 min of the enzymatic reaction. This technique gives reproducible results and good analytical sensitivity with 10(6) lymphocytes. With an average isolation rate of 59.6%, only 7 ml of blood is required, making the method suitable for lymphopenic patients. Moreover, PP2B activity is stable in blood samples kept for 24h at room temperature and in isolated lymphocytes stored for 48 h at -20 degrees C. We validated the method by comparing median PP2B activity in 10 healthy volunteers (285.0+/-46.5 pmol/min/10(6)lymphocytes) and in 10 liver transplant patients (147.8+/-71.0 pmol/min/10(6)lymphocytes) (p<0.001). The relation between calcineurin activity and tacrolimus blood concentrations was also studied.