Monitoring the reactions of cisplatin with nucleotides and methionine by reversed-phase high-performance liquid chromatography using cationic and anionic pairing ions

Methodology, based on reversed-phase high-performance liquid chromatography, is described for monitoring the reactions of cisplatin with DNA, nucleotides, and methionine. Cisplatin was determined in DNA ultrafiltrates on solvent-generated anion exchangers which were prepared by coating the surface of a reversed-phase column with hexadecyltrimethylammonium bromide. These systems were also applicable to studies on the reactions of cisplatin with nucleotides. The retention of the nucleotides studied (5'-AMP, 5'-GMP, 5'-CMP, and 5'-TMP) was described by means of an ion-exchange model and was manipulated by controlling the phosphate concentration in the mobile phase and its pH. The results indicate that cisplatin interacts predominantly with adenosine and guanosine groups on the DNA molecule and that binding is limited by the rate of conversion to an aquated intermediate. Whereas reversed-phase HPLC systems employing cationic pairing ions were applicable to the analysis of mixtures containing cisplatin and anionic solutes, systems employing alkyl sulfonates were required to monitor the reaction of cisplatin with methionine which produces cationic products. Retention, in this latter system, was optimized by the addition of acetonitrile to the mobile phase and by controlling the concentration and chain length of alkylsulfonate in the mobile phase. Although an octadecylsilylsilica, reversed-phase column was preferred for the analytical separation of the methionine-platinum complexes, a polystyrene-divinylbenzene colume was preferred for preparative work.     

Analysis of hydroxyproline in urine by high-performance liquid chromatography after dabsyl-chloride derivatization

Summary In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l.     

Analysis of clozapine and norclozapine by high-performance liquid chromatography

A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C₁₈ SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C₁₈ reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210–365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 and 8.0% and the inter-assay C.V.s were from 1.1. to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring.     

Analysis of thiamine and its phosphate esters by high-performance liquid chromatography.

High-performance liquid chromatography was used to separate thiamine and its phosphate esters after conversion to corresponding highly fluorescent thiochrome derivatives by alkaline oxidation. These compounds were absorbed on LiChrosorb-NH₂, eluted with acetonitrile-90 mm potassium phosphate buffer (pH 8.4), and determined spectrofluorometrically. A complete, rapid, and quantitative separation of thiochrome and its phosphate derivatives was made and the minimum amount detected was 1 pmol for each of these compounds.     

Analysis of minocycline by high-performance liquid chromatography in tissue and serum

A sensitive and rapid reversed-phase high-performance liquid chromatography assay can be used to accurately determine serum and tissue minocycline concentrations. Minocycline is a broad spectrum tetracycline derivative with many applications. Tissue and serum samples were obtained from guinea pigs that had received either topical or intravenous minocycline. Samples were extracted using a Sep-Pak C₁₈ cartridge and were injected into a μBondapak C₁₈ column with an isocratic methanol mobile phase. Samples were analyzed using UV detection and produced sharp peaks with a retention time of 2.5 min. The lower limit of detection was 100 ng and drug recovery was 61%. This method greatly facilitated the analysis of minocycline while allowing for sensitivity.     

Analysis of sialyllactoses in blood and urine by high-performance liquid chromatography

A sensitive and highly selective high-performance liquid chromatography (HPLC)-based method has been developed for the analysis of oligosaccharides in biological fluids. In this method, a sample of biological fluid, such as blood serum or urine, is filtered through a 10,000 molecular weight cutoff filter cartridge to remove large molecules such as proteins and lipids. The carbohydrates in the filtrate are then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) as described previously [Anal. Biochem. 180, 351-357, (1989)]. The derivatized carbohydrates are separated by reverse-phase HPLC and monitored by UV absorbance at 245 nm. Quantitative analysis of the carbohydrates can be achieved based on their integration values relative to a standard calibration curve. Since neutral and acidic carbohydrates can be separated by using Dowex 1-X8 anion exchange resin, this method can be used specifically to analyze neutral, acidic, and total carbohydrates in the biological fluids. Because PMP specifically reacts with reducing aldoses, interference from noncarbohydrate components present in the biological fluids is essentially eliminated. This method has proven to be highly sensitive, requiring as little as 5 pmol of analyte for reliable analysis. It has also been used successfully for pharmacokinetic analysis of carbohydrate drugs in human blood and urine samples.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E. coli

Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products. Received: 10 May 1997 / Accepted: 19 June 1997     

Analysis of dipalmitoyl phosphatidylcholine in amniotic fluid by high-performance liquid chromatography

A novel method for the determination of dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid by high-performance liquid chromatography (HPLC) is described. Aliquots of 50 μl of amniotic fluid were hydrolyzed with phospholipase C from Bacillus cereus and the resulting dipalmitoylglycerol analyzed by HPLC. Run-to-run and day-to-day precision for DPPC analysis were 4.2 and 6.1%, respectively, and analysis time was 10 min. Recoveries for DPPC ranged between 92 and 98%. In summarizing, this method provides a high precision and fast turnaround time means for the analysis of DPPC in amniotic fluid.     

Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion-exchange high-performance liquid chromatography with suppressed conductivity detection

Phosphatidylinositol 4,5-biphosphate (PIP(2)) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP(2) levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP(2) are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP(2) amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 micromol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP(2), whereas overexpression of Type II PI4-kinase can increase both PIP and PIP(2). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and the D3 isomers of PIP(2) are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP(2) remains low; exogenous PIP(2) is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.     

Simultaneous determination of three naphthoquinones in the leaves of Impatiens balsamina L. by reversed-phase high-performance liquid chromatography

Introduction – Naphthoquinones; lawsone ( 1 ), lawsone methyl ether ( 2 ) and methylene‐3,3′‐bilawsone ( 3 ) are the main active compounds of Impatiens balsamina leaves. Objective – To develop and validate an HPLC method for simultaneous quantitative determination of 1 – 3 in I. balsamina leaf extracts. Methodology – The method utilised a Supelco® C₁₈ column (5 µm, 4.6 × 150 mm) at 25°C with the mixture of 2% aqueous acetic acid : methanol (gradient elution as follows: 0–10 min, 25 : 75; 10–20 min, 32 : 68; 20–35 min, 55 : 45) as the mobile phase at a flow‐rate of 1 mL/min, and UV detection at 280 nm. The parameters of linearity, repeatability, reproducibility, accuracy specificity and sensitivity of the method were evaluated. Results – The recovery of the method was 96–101% and linearity (r² ≥ 0.9995) was obtained for all naphthoquinones. A high degree of specificity, as well as repeatability and reproducibility (RSD less than 5%), were also achieved. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative study of glycosyltransferase activities in Caco-2 cells before and after enterocytic differentiation using lectin-affinity high-performance liquid chromatography.

Human colonic adenocarcinoma Caco-2 cells differentiate into enterocytes by induction with sodium butyrate after confluence. Our previous studies have shown that there are high levels of H type 1 blood group antigen and core 2 structure present in O-glycans of the glycoproteins from these differentiated cells and these O-glycans appear to be indispensable for the process of differentiation of the cells (J. Amano and M. Oshima, 1999, J. Biol. Chem. 274, 21209-21216). Here, we have determined the glycosyltransferase activities using lectin-affinity HPLC because the method enabled easy separation and identification of mixtures of isomeric oligosaccharide structures due to the high resolution and reproducibility. The activities of beta 3-galactosyltransferase, alpha 2-fucosyltransferase, which are responsible for H type 1 antigen biosynthesis, and core 2 beta 6-N-acetylglucosaminyltransferase in differentiated Caco-2 cells were higher than those in undifferentiated cells. These results demonstrate that an increase in specific glycosyltransferase activities brought on a change of the O-glycan structures during differentiation.     

Carbohydrate and carbon metabolite accumulation responses in leaves of ozone tolerant and ozone susceptible spinach plants after acute ozone exposure

The objective of this study was to determine whether exposure of plants to ozone (O₃) increased the foliar levels of glucose, glucose sources, e.g., sucrose and starch, and glucose-6-phosphate (G6P), because in leaf cells, glucose is the precursor of the antioxidant, L-ascorbate, and glucose-6-phosphate is a source of NADPH needed to support antioxidant capacity. A further objective was to establish whether the response of increased levels of glucose, sucrose, starch and G6P in leaves could be correlated with a greater degree of plant tolerance to O₃. Four commercially available Spinacia oleracea varieties were screened for tolerance or susceptibility to detrimental effects of O₃ employing one 6.5 hour acute exposure to 25O nL O₃ L^-1 air during the light. One day after the termination of ozonation (29 d post emergence), leaves of the plants were monitored both for damage and for gas exchange characteristics. Cultivar Winter Bloomsdale (cv Winter) leaves were least damaged on a quantitative grading scale. The leaves of cv Nordic, the most susceptible, were approximately 2.5 times more damaged. Photosynthesis (Pn) rates in the ozonated mature leaves of cv Winter were 48.9% less, and in cv Nordic, 66.2% less than in comparable leaves of their non-ozonated controls. Stomatal conductance of leaves of ozonated plants was found not to be a factor in the lower Pn rates in the ozonated plants. At some time points in the light, leaves of ozonated cv Winter plants had significantly higher levels of glucose, sucrose, starch, G6P, G1P, pyruvate and malate than did leaves of ozonated cv Nordic plants. It was concluded that leaves of cv Winter displayed a higher tolerance to ozone mediated stress than those of cv Nordic, in part because they had higher levels of glucose and G6P that could be mobilized during diminished photosynthesis to generate antioxidants (e.g., ascorbate) and reductants (e.g., NADPH). Elevated levels of both pyruvate and malate in the leaves of ozonated cv Winter suggested an increased availability of respiratory substrates to support higher respiratory capacity needed for repair, growth, and maintenance.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - ASC L-ascorbic acid - APX ascorbate peroxidase - Ce CO₂ concentration in air in the measuring cuvette during photosynthesis measurements - Ci CO₂ concentration in the leaf intercellular spaces during photosynthesis measurement - Chl chlorophyll - DHA dehydroascorbic acid - DHA reductase dehydroascorbate reductase - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - Gluc glucose - GR glutathione reductase - G_sw stomatal conductance with units as mmol H₂O m^-2 s^-1 - GSSG oxidized glutathione - GSH reduced glutathione - G1P glucose-1-phosphate - G6P glucose-6-phosphate - G6P dehydrogenase glucose-6-phosphate dehydrogenase - 6PG 6-phosphogluconate - 6PG dehydrogenase 6-phosphogluconate dehydrogenase - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - MAL malate - MDHA reductase monodehydroascorbate reductase - PE post-emergence - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - PYR pyruvate - Pn net CO₂ photoas-similation in leaves - PPFD photosynthetic photon flux density with units of mol photons m^-2 s^-1 - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf weight - TCA cycle tricarboxylic acid cycle - Triose-P DHAP+GAP     

Quantitation of dimethyl sulfoxide in solutions and tissues by high-performance liquid chromatography.

We have developed a rapid and simple method to determine the level of dimethyl sulfoxide (Me2SO) in both solutions and tissue samples. For analysis of Me2SO in a cryopreservation medium, the solution is simply diluted in 10% (vol/vol) methanol and centrifuged. Then an aliquot of the supernatant is assayed by high-performance liquid chromatography. For tissue samples, the wet weight is measured and the intact sample is extracted with 10% (vol/vol) methanol (e.g., 10 ml/g wet wt) in a sealed vial. The extract is then diluted and centrifuged, and an aliquot of the supernatant is assayed. The dry weight of the tissue is measured after the methanol-extracted sample is placed into either for 2 h and air-dried overnight. The water content of the tissue is calculated as the difference between the wet and the dry weights. The concentration of Me2SO in the aqueous compartment of the tissue can then be calculated by taking into account the concentration of Me2SO in the extract and the dilution factor, based on the tissue water volume and the volume of methanol used to extract the Me2SO. The calculated values for porcine myocardium samples correlated 1:1 with the actual Me2SO concentrations in the solutions in which the tissue samples were equilibrated. Finally, we present results documenting the usefulness of this assay by following the time course of Me2SO penetration into core versus peripheral regions of 1-cm3 samples of porcine myocardium.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

Analysis of ellagic acid in pomegranate rinds by capillary electrophoresis and high-performance liquid chromatography

Two novel methods for the analysis of ellagic acid in pomegranate (Punica granatum) rinds are proposed. Capillary electrophoresis (CE) was performed in a bare fused-silica capillary using a buffer solution of tri(hydroxymethyl)aminomethane:potassium dihydrogen phosphate (pH 8.4) with an applied voltage of 20 kV and UV detection at 254 nm. HPLC analysis was performed with a Zobax SB C(18) column and a mobile phase consisting of methanol:ethyl acetate:potassium dihydrogen phosphate: phosphoric acid at a flow rate of 1.0 mL/min. Under optimised conditions, the HPLC retention and the CE migration times for ellagic acid were 10.32 and 12.23 min, respectively. Calibration curves of peak area vs. concentration gave correlation coefficients of 0.9999 for HPLC and 0.9990 for CE. The detection limits for HPLC and CE were 2.8 and 2.2 microg/mL, respectively. Average recoveries were 98.32 +/- 1.2% for HPLC and 96.52 +/- 2.8% for CE. Both methods were shown to be suitable for the determination of ellagic acid in pomegranate rinds extraction; however, the CE method required less solvent and gave better column efficiency, whilst the HPLC provided superior precision.     

Analysis of oxidized and reduced pyridine dinucleotides in rat liver by high-performance liquid chromatography

NADP+, NAD+, NADPH, and NADH were assayed by selective extraction and isocratic reversephase HPLC. Sample preparation involves freeze clamping and powdering liver under liquid nitrogen, extraction of dinucleotides with basic (reduced species) or acidic (oxidized species) cold ethanol, and injection onto the HPLC for quantitation at 340 nm (reduced) and 254 nm (oxidized). The mobile phase for the oxidized species is pH 5.25, 0.2 M ammonium phosphate/methanol, and for the reduced species is pH 6.0, 0.2 M ammonium phosphate/methanol/tributylamine. The method is linear over the range 0.016 to 2.0 nmol for the reduced species, and from 0.005 to 0.8 nmol for the oxidized pyridine dinucleotides. The recoveries were from 94.5% for NAD+ to 99.3% for NADPH, with standard deviations of approximately 2.5% for all species other than NADP+, which had a standard deviation of 10.4%. The coefficients of variation for repeated determinations of standards over 3 months were less than 4%.     

Analysis of isoxazolyl penicillins and their metabolites in body fluids by high-performance liquid chromatography

A high-performance liquid chromatographic assay method to quantitate the isoxazolyl penicillins, their active metabolites, and their penicilloic acids in serum or urine is described. Separation and analysis is performed using reversed-phase chromatography. Urine samples, after the appropriate dilution, can be assayed directly. Serum samples (0.1 ml) are either extracted with methylene chloride or treated with perchloric acid—methanol. Serum levels as low as 0.4 μg/ml (extraction procedure) can be assayed accurately.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.