Comparison of sample preparation methods for the high-performance liquid chromatographic analysis of cell culture extracts for triphosphate ribonucleosides and deoxyribonucleosides

We compared four different procedures for the purification and concentration of nucleoside triphosphates in cell extracts prior to HPLC analysis. Two methods involved precipitation, with either acetonitrile or calcium fluoride. The acetonitrile procedure yielded reasonable recovery and sufficient purity for the subsequent HPLC analysis. The calcium fluoride coprecipitation procedure gave both good recovery and purity; but the recovery was shown to be dependent on the concentration of the nucleoside triphosphates. The other two methods involved small Sep-Pak cartridges. The silica cartridge procedure yielded unfavorable recoveries in periodate-treated cell extracts, apparently due to poor solubility of nucleoside triphosphates in the requisite solvents. The strong anion exchange cartridge procedure yielded both good recovery and purity. This procedure was found to be fast, efficient, and reliable for purifying and concentrating nucleotides in cell extracts.     

Automation of high-performance liquid chromatographic sample clean-up for mass fragmentographic assays

A previously described high-performance liquid chromatographic (HPLC) sample clean-up procedure has been automated by attaching a (DuPont) auto-sampler and a time-controlled fraction collector to the HPLC equipment. To obtain the required reliability for unattended operation, the sample intake was controlled by volume rather than by time, and the system was protected against sample loss due to non- or improper operation of the injection valve. The capacity of the system depends on the HPLC run time per sample but varies from 45 to 135 samples per 24 h. The recovery and reproducibility are comparable to the manually operated system, while carry-over to subsequent samples is prevented by intermittent injection of the HPLC solvent system as flush fluid.     

Determination of diprivan in urine by a supported liquid membrane technique and liquid chromatography-electrochemical detection

A supported liquid membrane technique was used for the extraction and enrichment of propofol in a spiked sample of urine. An acidic solution of propofol and thymol as an internal standard was passed over the membrane and after enrichment the acceptor solution was analyzed by LC with an electrochemical detector. The acceptor and donor pH, flow-rate, and volume of donor and different membrane solvents were varied to optimize the extraction efficiency. The detection limit for 100 ml of a spiked urine sample was 10 ppt of propofol.     

Development of a high-performance liquid chromatographic method for the analysis of staurosporine

Staurosporine (Stsp), a protein kinase inhibitor, has been found to have a differential effect on the proliferation of normal and transformed cells in vitro. Hence, Stsp might be used in cancer therapy to arrest normal proliferating cells in G1, while permitting tumor cells to continue proliferation. The patient could then be treated with a therapeutic agent of maximum toxicity for actively proliferating tumor cells. To facilitate investigations of Stsp in vivo, we have developed an HPLC method for measuring the levels of Stsp in blood. Using a rat model, plasma containing Stsp is treated with acetone to precipitate proteins and extract the Stsp. The acetone extract is then subjected to reversed-phase HPLC on a μBondapak C₁₈ column. Using a linear elution gradient of acetonitrile containing trifluoroacetic acid, Stsp elutes as a sharp peak at ca. 35 min which can be detected by UV absorption at 292 nm. No blood or reagent components interfere with its quantification. The calibration curve, ranging from 0.1 to 2.0 μg Stsp, demonstrated a linear response to Stsp concentration having a correlation coefficient (r²) of 0.990. Precision analysis demonstrated that the method will yield results that are ±11.6% from the mean 95% (two standard deviations) of the time. This method was used to measure Stsp levels in plasma after administering an injection of 0.2 mg Stsp into the jugular vein of rats. No Stsp could be detected in the plasma 5 min after injection, even though enough Stsp was administered to be easily detectable if it was simply contained in the plasma. Thus, it is concluded that some compartment other than the plasma must adsorb the Stsp from the plasma and sequester it in vivo.     

Fluorescence derivatisation of urinary corticosteroids for high-performance liquid chromatographic analysis

Corticosteroids containing a C₂₁ primary hydroxyl group were derivatised with 9-anthroyl cyanide. The reagent was prepared as a solution in acetonitrile, containing 0.1% triethylamine, at a concentration of 2 mg/ml. Approximately 1 μg of corticosteroid was reacted with 100 μl of this reagent, at 45°C for 2 h. The fluorescent derivatives were separated by HPLC on a silica column, 250×4.6 mm I.D., by stepwise elution, with a mobile phase of 2-propanol–hexane (2:98) for 20 min, followed by 2-propanol–hexane (7:93) from 20 to 40 min. The fluorescence detector was set to 370-nm excitation and 470-nm emission. The relatively low temperature for derivatisation avoided reaction with secondary hydroxyl groups and also prevented thermal degradation of the corticosteroids.     

Anion-exchange high-performance liquid chromatographic analysis of inositol phosphates

The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.     

Dual ultraviolet wavelength high-performance liquid chromatographic method for the forensic or clinical analysis of seventeen antidepressants and some selected metabolites

A sensitive method suitable for the determination of tricyclic and other antidepressants in postmortem and clinical specimens is presented. The procedure, which utilizes reversed-phase HPLC combined with dual ultraviolet wavelength detection, enables the separation of 17 commonly prescribed antidepressants and some selected metabolites in a single extraction. Peak purity was confirmed using absorbance ratios at 220 nm and 254 nm wavelengths and revealed little interference from other eluting analytes. The blood detection limit for most antidepressants was 50 ng/ml. The most commonly observed antidepressants in 281 forensic cases analysed over a two-year period with the described method were dothiepin, amitriptyline, nortriptyline and doxepin.     

Sensitive high-performance liquid chromatographic determination of nifedipine in dog plasma using an automated sample preparation system with laboratory robot

Nifedipine, a calcium-channel blocking drug was analysed in dog plasma after oral dosing with two different formulations. Sample preparation was automated with a laboratory robot. Quantitative determination of the drug was performed on a reversed-phase HPLC system with electrochemical detection (ED) using an internal standard. Validation of the analytical method showed that the system is well suited for pharmacokinetic studies on dogs. The assay was linear in the range 1–50 ng/ml. Inter-day and intra-day variability were between 6.43–18.15% C.V. and 1.57–5.53% C.V., respectively.     

Analysis of some tryptophan and phenylalanine metabolites in urine by a straight-phase high-performance liquid chromatographic technique

A high-performance liquid column chromatographic technique is reported for the analysis of some tryptophan and phenylalanine acid metabolites in the urine. An acidified and NaCl-saturated urine sample is loaded on to a C₁₅-bonded silica microcolumn. After washing the microcolumn with clean and deionized water, the metabolites of interest are selectively extracted by successive elutions with organic solvents of variable polarity. Acids are eluted first and the neutral compounds with the next fraction. Basic compounds and other neutral substances of higher polarities were eliminated during the washing procedure.The chromatography was performed in the straight-phase isocratic elution mode utilizing 5-μm silica-gel columns loaded with a triethanolammonium perchlorate—perchloric acid aqueous solution. The separations achieved have permitted the application of the chromatographic technique to the analysis of urinary metabolites with acceptable accuracy.     

Derivatization and high-performance liquid chromatographic analysis of pentaazapentacosane pentahydrochloride

A rapid high-performance liquid chromatographic method for determination of the dansyl derivative of pentaazapentacosane (PAPC) pentahydrochloride has been developed. The chromatographic system uses a reversed-phase C₈ column, a mobile phase of acetic acid buffer and acetonitrile and UV detection. The dansylation conditions were optimized with a pH of 11.0 and a 20-fold dansyl chloride excess. The yield of dansyl PAPC increased 10-fold as the reaction pH was changed from 9.5 to 10.5. Under derivatization conditions of pH 8.5–11.0 and 1–30-fold excess dansyl chloride only perdansyl PAPC was found.     

Extractive acidogenic fermentation by a supported liquid membrane

An on-line extractive fermentation system using a supported liquid membrane, recently termed pertractive fermentation, was investigated in order to overcome end-product inhibition and to enhance the productivity of microbial acidogenesis. It was observed that the pH of the fermentation broth was the key parameter for the successful operation of this pertractive fermentation system. At pH 4.8, 300 ml of broth and 80 sq·cm of extracting area, microbial activity in the system was prolonged and the productivity was enhanced about 5 fold compared to conventional acidogenic fermentation without extraction.     

Extraction, pre-high-performance liquid chromatographic purification, and high-performance liquid chromatographic analysis of plant nucleotides

Problems encountered in obtaining reliable analytical data by HPLC for the free nucleotide constituents of plant tissues are considered and methods of overcoming them experimentally assessed. Major problems include suppression of residual phosphatase activity during extraction, and removal of pigments, phenolics, alkaloids, and other uv-absorbing nonnucleotides, prior to HPLC. An optimal combination of extraction and pre-HPLC purification techniques is discussed which, in combination with HPLC by anion exchange, yields quantitatively reliable data. The optimized procedure involves extraction with a monophasic mixture of methanol: chloroform:formic acid:water and purification of the nucleotide extract by a batch treatment with poly-N-vinylpyrrolidone, followed by ligand-exchange chromatography. The main HPLC separation uses mu Bondapak NH2 in a linear phosphate gradient and gives good resolution of all the commonly occurring plant nucleotides in a single chromatographic run.     

Glutathione monoethyl ester: high-performance liquid chromatographic analysis and direct preparation of the free base form

Glutathione monoethyl ester (L-gamma-glutamyl-L-cysteinylglycine ethyl ester) was shown by R. N. Puri and A. Meister (1983, Proc. Natl. Acad. Sci. USA 80, 5258-5260) to be taken up by several tissues and intracellularly hydrolyzed to GSH. Since GSH itself is not significantly taken up by tissues, glutathione monoesters provide the most direct and convenient means available for increasing the intracellular GSH concentration of many tissues and cell types. In previous studies glutathione esters were prepared by HCl- or H2SO4-catalyzed esterification, and the product esters were precipitated as acidic salts by addition of ether to the reaction mixtures. In the present studies, glutathione monoethyl ester was synthesized by H2SO4-catalyzed esterification in the presence of sodium sulfate as the dehydrating agent. When no GSH remained, alcohol-washed Dowex-1 resin (hydroxide form) was added to remove sulfate and neutralize the reaction mixture. After the resin was removed by filtration, glutathione monoethyl ester crystallized in the chilled filtrate. The product was free of sulfate, GSH, and glutathione diester; its solutions in water or saline were neutral. Preparations obtained to date are nontoxic when administered to mice in doses up to at least 10 mmol/kg. Progress of the esterification reaction and purity of the product were determined quantitatively by HPLC after derivatization of the thiols with monobromobimane. Elution times of GSH, glutathione diester, and glutathione monoesters involving either the glutamyl or the glycyl carboxylate groups are reported.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

Laccase assay by means of high-performance liquid chromatography

Spectrophotometric determination of laccase activity may be affected by the formation of quinoid chromophores arising from nonenzymatic oxidations interfering with enzymatic reactions. Km values for guaiacol obtained by spectrophotometric and HPLC methods confirm the above hypothesis. HPLC results are particularly useful for the assay of laccase activity on natural phenolic extracts.     

Improved high-performance liquid chromatographic analysis of terazosin in human plasma

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25–100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25–100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.     

Rocket-powered high-performance liquid chromatographic analysis of plant ascorbate and glutathione

We describe a robust procedure for the extraction and high-performance liquid chromatographic analysis of L-ascorbate (vitamin C), glutathione (gamma-glutamyl cysteinylglycine), and their respective oxidized forms from various plant tissues. Parameters such as the choice of extraction buffer, tissue disruption technique, sample stability, and separation conditions have all been optimized. In particular we found that the inclusion of the reducing agent dithiothreitol as a "stabilizer" in extracts with high phenolic content actually promoted oxidation of these antioxidants. Further, by using commercially available short "Rocket" HPLC columns in combination with high mobile-phase flow rates, analysis times were reduced to only 6min, making the method suitable for the high-resolution screening of large numbers of samples.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.