Capillary electrophoretic separation of protease inhibitors used in human immunodeficiency virus therapy

A new capillary electrophoretic method for trace analysis of gamma-cyclodextrin, gamma-CD, in a sample of beta-CD has been developed, building on our recent work in which the tetraphenylborate ion, Ph4B-, was found to bind to gamma-CD three orders of magnitude more strongly than to beta-CD. The method involves measurement of the change of net electrophoretic mobility of Ph4B- and its CD complexes in a background electrolyte containing a fixed concentration of beta-CD. Good linearity was found between 1/deltamu and 1/Cgamma where deltamu is the difference in the mobility of Ph4B- in the beta-CD solution at a given and at zero concentration of gamma-CD, and Cgamma the gamma-CD concentration. The limit of detection for gamma-CD in a beta-CD sample was found to be 0.020% (w/w), and high precision and accuracy were obtained.     

Secondary ion mass spectrometry of nucleic acid components: Pyrimidines,purines, nucleosides and nucleotides

Biologically important involatile organic compounds including nucleotides, nucleosides, purines and pyrimidines have been analysed by secondary ion mass spectrometry. For the emission of molecule-like secondary ions small amounts of the substances were deposited as thin layers on silver foils and bombarded with 3 keV [AR]⁺ ions. All the compounds investigated yielded intense molecular ions of the general composition [M±H]^± and [M + Ag]⁺, but only a few characteristic fragment ions due to simple bond cleavages. Similarities and differences as compared with spectra obtained by other mass spectrometric ionization techniques are illustrated and discussed.     

Determination of nucleotides and nucleosides in milks and pediatric formulas: a review

Nucleotides and nucleosides play important roles as structural units in nucleic acids, as coenzymes in biochemical pathways, and as sources of chemical energy. Milk contains a complex mixture of nucleotides, nucleosides, and nucleobases, and because of the reported differences in their relative levels in bovine and human milks, pediatric formulas are increasingly supplemented with nucleotides. Liquid chromatography is the dominant analytical technique used for the quantitation of nucleospecies and is commonly applied using either ion-exchange, reversed-phase, or ion-pair reversed-phase modes. Robust methods that incorporate minimal sample preparation and rapid chromatographic separations have been developed for routine product compliance analysis. This review summarizes the analytical techniques used to date in the analysis of nucleospecies in bovine and human milks and infant formulas.     

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.     

PROSIT: pseudo-rotational online service and interactive tool, applied to a conformational survey of nucleosides and nucleotides

A Pseudo-Rotational Online Service and Interactive Tool (PROSIT) designed to perform complete pseudorotational analysis of nucleosides and nucleotides is described. This service is freely available at http://cactus.nci.nih.gov/prosit/. Files containing nucleosides/nucleotides or DNA/RNA segments, isolated or bound to other molecules (e.g., a protein) can be uploaded to be processed by PROSIT. The service outputs the pseudorotational phase angle P, puckering amplitude numax, and other related information for each nucleoside/nucleotide detected. The service was implemented using the chemoinformatics toolkit CACTVS. PROSIT was used for a survey of nucleosides contained in the Cambridge Structural Database and nucleotides in high-resolution crystal structures from the Nucleic Acid Database. Special cases discussed include nucleosides having constrained sugar moieties with extreme puckering amplitudes, and several specific DNA/RNA helices and protein-bound DNA oligonucleotides (Dickerson-Drew dodecamer, RNA/DNA hybrid viral polypurine tract, Z-DNA enantiomers, B-DNA containing (L)-alpha-threofuranosyl nucleotides, TATA-box binding protein/TATA-box complex, and DNA (cytosine C5)-methyltransferase complexed with an oligodeoxyribonucleotide containing transition state analogue 5,6-dihydro-5-azacytosine). When the puckering amplitude decreases to a small value, the sugar becomes increasingly planar, thus reducing the significance of the phase angle P. We introduce the term "central conformation" to describe this part of the pseudorotational hyperspace in contrast to the conventional north and south conformations.     

Recent application of acidic 1,3-azolium salts as promoters in the solution-phase synthesis of nucleosides and nucleotides

Acidic 1,3-azolium salts are prepared from Brønsted acids and 1,3-azoles such as imidazole, thiazole, and oxazole. Acidic imidazolium salts are frequently employed as promoters for the synthesis of nucleotides using the phosphoramidite method in a solution phase. Recently, it was revealed that thiazolium and oxazolium salts catalyzed Vorbrüggen-type N-glycosylation reactions to give nucleosides. These reactivities are attributed to the stronger Brønsted acidities of the thiazolium and oxazolium salts relative to those of the imidazolium salts. This digest focuses on recent progress in the applicability of acidic 1,3-azolium salts as promoters in the solution-phase synthesis of nucleosides and nucleotides.     

High-performance liquid chromatographic determination of 2',3'-didehydro-3'-deoxythymidine, a new anti-human immunodeficiency virus agent, in human plasma and urine.

Sensitive and selective high-performance liquid chromatographic techniques have been developed for the determination of 2'-3'-didehydro-3'-deoxythymidine, d4T (BMY-27857), in human plasma and urine. The methods had linear standard curves over the concentration ranges 0.025-25.0 and 0.5-100 micrograms/ml for the plasma and urine matrices, respectively. Both methods used solid-phase extraction for isolating d4T and the internal standard, thymidine oxetane, from the biological matrix. In addition, the analytical column, mobile phase, instrumentation and chromatographic conditions used for both methods were identical. The ultraviolet absorbance of the column effluent was monitored at 266 nm. Results of analysis of quality control samples indicated that the intra-assay precision values, as measured by percent relative standard deviation, were within 12 and 3%, and accuracy samples deviated less than 10 and 5% from nominal values for the plasma and urine assays, respectively.     

The use of tat and env sequences from human immunodeficiency virus 1 in phylogenetic epidemiological studies

Using nucleotide sequences from the first exon of the tat gene of the human immunodeficiency virus 1 (HIV-1), we tested the hypothesis that a Florida dentist (a common source) infected five of his patients in the course of dental procedures against the null hypothesis that the dentist and each individual of the dental group independently acquired the virus within the local community. This novel approach of analyzing the tat gene region was used because it may, in some circumstances, be more informative for phylogenetic epidemiology than the more commonly used C2-V3 envelope gene region. The first exon of the tat gene was polymerase chain reaction (PCR)-amplified and directly sequenced from uncultured peripheral blood mononuclear cells. Patient's sequences were compared with sequences from six HIV-1 infected heterosexual couples unrelated to the dentist or the five patients, but from the same general geographic area. In addition, a sixth infected dental patient, previously inferred to have acquired HIV-1 from a source other than the dentist, was included. Multiple phylogenetic analyses demonstrated that the sequences of the five patients were significantly more closely related to each other than to sequences of the controls. Our results using tat sequences, combined with envelope sequence data, strongly support a common phylogenetic epidemiological relationship among these five patients, and the HIV-1 infected dentist who treated them. Correct recovery of known epidemiological relationships among couples included in the analysis further strengthens this conclusion.     

Analysis and validation of the phosphorylated metabolites of two anti-human immunodeficiency virus nucleotides (stavudine and didanosine) by pressure-assisted CE-ESI-MS/MS in cell extracts: sensitivity enhancement by the use of perfluorinated acids and alcohols as coaxial sheath-liquid make-up constituents

A CE method utilizing triple quadrupole electrospray (ES) MS (MS/MS) detection was developed and validated for the simultaneous measurement of nucleoside 5'-triphosphate and 5'-monophosphate anabolites of the anti-HIV (human immunodeficiency virus) didanosine (ddAMP, ddATP) and stavudine (d4TMP, d4TTP), among a pool of 14 endogenous 5'-mono-, di-, and triphosphate nucleosides. These compounds were spiked and extracted from peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. An acetic acid/ammonia buffer (pH 10, ionic strength of 40 mM) was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of +30 kV and an overimposed pressure of 28 mbar (0.4 psi). The application of pressure assistance was needed to provide stable ES conditions for successful coupling. The coupling was carried out with a modified sheath-flow interface, with one uninterrupted capillary (80 cmx 50 microm id; 192 microm od) in a dimension that fits into the ESI needle to get a stable ion spray. Some CE-MS parameters such as overimposed pressure, sheath-liquid composition, sheath-liquid and sheath-gas flow rates, ES voltage, and the CE capillary position were optimized in order to obtain an optimal sensitivity. The use of perfluorinated alcohols and acids in the coaxial sheath-liquid make-up (2,2,2-trifluoroethanol + 0.2 mM tridecafluoroheptanoic acid) appeared to provide the best MS sensitivity and improve the stability of spray. The linearity of the CE-MS and CE-MS/MS methods was checked under these conditions. Validation parameters such as accuracy, intraday and interday precision, and LOQs were determined in CE-MS/MS mode. Finally, the quantitation of d4T-TP and ddA-TP was validated in this CE-MS/MS system.     

Simultaneous determination of paracetamol and dextropropoxyphene in human plasma by liquid chromatography/tandem mass spectrometry: application to clinical bioequivalence studies

A liquid chromatography/mass spectrometry method for simultaneous determination of paracetamol and dextropropoxyphene in human plasma is described. Paracetamol and dextropropoxyphene, together with their internal standards (tolbutamide and pyrroliphene), were extracted from 0.5 mL of plasma using solid-phase extraction. The chromatography was performed using a Thermo Hypersil APS-2 Amino column (250 mm x 4.6 mm, 5 microm) with a mobile phase consisting of acetonitrile and 0.4% glacial acetic acid in water (20:80). The total run time was 6 min for each sample. The triple-quadrupole mass spectrometer was operated in both positive (for detection of dextropropoxyphene and its IS pyrroliphene) and negative (for detection of paracetamol and its IS tolbutamide) modes using a polarity-switching technique. Multiple reaction monitoring was used for quantification. The method was linear over the concentration range of 0.1-20 microg/mL for paracetamol and 0.5-80 ng/mL for dextropropoxyphene. The intra- and inter-day precision were less than 10%, and the accuracy ranged from 92.2-110.9%. The lower limits of quantification were 0.1 microg/mL for paracetamol and 0.5 ng/mL for dextropropoxyphene. The present method provides a robust, fast and sensitive analytical tool for both paracetamol and dextropropoxyphene, and has been successfully applied to a clinical bioequivalence study in 14 subjects.     

Capillary electrophoresis hyphenated to inductively coupled plasma-mass spectrometry: a novel approach for the analysis of anticancer metallodrugs in human serum and plasma

The development of metal-based chemotherapeutics lacks methods which are capable of providing early indication on the potential of new metal complexes as future anticancer drugs. Since most of these compounds are administered intravenously, serum proteins are the first available biological binding partners in the bloodstream. For platinum-based anticancer drugs the interaction with serum proteins is regarded as an important contribution to the side effects accompanying chemotherapy. In contrast, newly developed ruthenium compounds are thought to be transported into the tumor in a protein-bound form. In here, the application of CE hyphenated to inductively coupled plasma (ICP)-MS, applying Polybrene-coated capillaries, is demonstrated for studying the interaction of indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) with HSA and transferrin, which are important transport proteins. Furthermore, the applicability of the method to human serum and plasma and, more importantly, to real-world patient samples was proven. KP1019 was found to bind to a high degree to HSA both in serum, plasma and the patient samples. Only minor fractions of ruthenium were found attached to other proteins.     

Proteomic analysis of differential protein expression in a human hepatoma revertant cell line by using an improved two-dimensional electrophoresis procedure combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The proteomic profiles of a human hepatoma revertant, CL1, and its original cell line, SMMC7721, were compared by using an improved two-dimensional electrophoresis (2-DE) procedure, with multi-IPGstrips gels (length 相似文献   

Capillary electrokinetic fractionation mass spectrometry (CEkF/MS): Technology setup and application to metabolite fractionation from complex samples coupled at‐line with ultrahigh‐resolution mass spectrometry

Capillary electrokinetic fractionation (CEkF) is investigated as a new, simple, and robust approach for semipreparative and analytical sample analysis based on pK_a‐dependant pH‐driven electrophoretic mobility. CEkF was optimized with contactless conductivity detection and conducted with 10 kV reverse voltage for 10 min, then coupled on/at‐line to ESI/MS. We propose a semi‐empirical model with 14 representative compounds based on the correlation between sample/medium pH regulating the partial charge, the electrokinetic loading of the capillary and intensity (I) of analytes. According to the model, an empirical function (I = f (pH)) could be derived to calculate the acid dissociation constant (pK_a) of various model compounds based on their pH‐dependant MS intensity profiles with the RSD < 4.05. Using the ultrahigh‐resolution of ion cyclotron resonance Fourier transform MS, the pK_a model was further illustrated in real samples into the structure prediction of important compounds in wine over two vintages. The established CEkF was successfully used to selectively fractionate sulfur compounds from the complex wine samples at pH 1.66. The proposed CEkF approach should allow in the future the simultaneous pK_a evaluation of multiple constituents without complicated separation out of a complex mixture in metabolomics or environmental chemistry.     

Determination of levetiracetam in human plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to bioequivalence studies

The first liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of levetiracetam, an antiepileptic drug, in human plasma is described. The plasma filtrate obtained after solid-phase extraction (SPE), using a polymer-based, hydrophilic-lipophilic balanced (HLB) cartridge, was submitted directly to a short column LC/MS/MS assay. There was no significant matrix effect on the analysis. For validation of the method, the recovery of the free analytes was compared to that from an optimized extraction method, and the analyte stability was examined under conditions mimicking sample storage, handling, and analytical procedures. The extraction procedure yielded extremely clean extracts with a recovery of 79.95% and 89.02% for levetiracetam and the internal standard (IS), respectively. The intra-assay and inter-assay precision for the samples at the lower limit of quantitation (LLOQ) were 6.33 and 6.82%, respectively. The calibration curves were linear for the dynamic range of 0.5 to 50 microg/mL with a correlation coefficient r >/= 0.9971. The intra-assay accuracy at LLOQ, LQC, MQC, and HQC levels ranged from 81.60 to 95.40, 93.00 to 103.47, 95.97 to 104.09, and 91.15 to 95.18%, respectively, while the inter-assay accuracy at LLOQ, LQC, MQC and HQC levels varied from 80.20 to 95.40, 88.53 to 107.53, 95.97 to 108.45, and 91.15 to 112.70%, respectively. The method is rugged and fast with a total instrumental run time of 2 min. The method was successfully applied for bioequivalence studies in human subject samples after oral administration of 1000 mg immediate release (IR) formulations.     

Development and application of a high-performance liquid chromatography-mass spectrometry method to determine alendronate in human urine

Despite the high potential offered by electrospray ionization on highly polar compounds like biphosphonates, few applications have been developed. High-performance liquid chromatography (HPLC) separation methods suitable for such molecules cannot be used in tandem with mass spectrometry (MS) due to high non-volatile salt content; at the same time the sample preparation, in biological fluids, is also a challenging problem. In the past ion-pair chromatography was mainly used in the case of HPLC-MS of biphosphonates, but no application to quantitative pharmacokinetic (PK) studies has been presented. In this study, after preliminary tests with ion-pair chromatography showing a poor sensitivity, a combined derivatization of the amino group and the biphosphonate has been developed and tested in a PK study. Using this analytical approach we were able to fully validate the quantitation of alendronate in the range of 6.667-4860.0 ng/ml in urine (sample volume 2.0 ml); each analytical run was 5.0 min long. The sensitivity achieved permitted a correct evaluation of the alendronate urinary excretion over the full period of urine collection. Sample preparation despite its complexity permitted to process and analyze up to 200 samples in a working day.