High-performance liquid chromatographic quantification of allysine as bis-p-cresol derivative in elastin

Summary. The first step in normal cross-linking in elastin is the formation of α-aminoadipic-δ-semialdehyde, allysine, through oxidative deamination of specific peptidyl lysine by the enzyme lysyl oxidase (EC 1.4.3.13). For the analysis of allysine, allysine was derivatized with p-cresol. The derivatization was carried out by acid hydrolysis (6N HCl containing 5% (w/v) p-cresol at 110°C for 48 h) accompanied with the hydrolysis of elastin. A bis-p-cresol derivative of allysine was isolated from bovine ligamentum nuchae elastin hydrolysates, and was characterized by UV, FAB-MS and NMR. This derivative was identified as 2-amino-6,6-bis(2-hydroxy-5-methylphenyl)hexanoic acid. A rapid, sensitive reverse-phase high-performance liquid chromatographic method with UV detection was developed for the quantitative determination of allysine as its bis-p-cresol derivative. The lower limit of detection of the bis-p-cresol derivative was 58 pmol in the standard sample with a 20-μl injection at a signal-to-noise ratio of 3. This method was applied to the determination of allysine in bovine ligamentum nuchae, aorta, lung, and rat aorta elastin. The allysine content in rat aorta elastin dramatically increased from 1 week to 2 weeks of age. Received June 30, 2000 Accepted September 22, 2000     

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.     

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.     

Preparative high-performance liquid chromatographic separation of proteins with HyperD ion-exchange supports

HyperD ion-exchange media combine the mechanical strength of a rigid polystyrene-mineral composite skeleton with the high protein-binding capacity of a three-dimensional soft gel located inside the skeleton. The skeleton solid matrix is completely filled with functionalized, highly hydrophilic, chemically stable ion-exchange hydrogels. These materials gave very efficient columns for protein separation with superior dynamic capacity, high resolving power and excellent protein recovery. Various protein mixtures were used to study the chromatographic performance of these new stationary phases. Comparisons between different particle size packing materials demonstrated the potential of this ion-exchange material for use on a large scale.     

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.     

Rapid high-performance liquid chromatographic assay for atovaquone

A rapid high-performance liquid chromatography assay has been developed for the drug atovaquone, which is currently being used to treat Pneumocystis carinii pneumonia and Taxoplasma gondii encephalitis associated with the acquired immunodeficiency syndrome (AIDS). Protein is precipitated from plasma with acetonitrile-aqueous 1% acetic acid (85:15). The supernatant is assayed on a C₆ column using methanol-10 mM triethylamine in aqueous 0.2% trifluoroacetic acid (76:24) with detection at 254 nm. The working assay range was 0.5 to 50 μg/ml. Recovery was 97% and the between-day coefficients of variation were 2.1% at 50 μg/ml and 10.3% at 1 μg/ml. A number of drugs commonly used to treat AIDS and its complications did not interfere with the assay.     

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.     

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.     

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.     

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.     

Unstable proteins: how to subject them to chromatographic separations for purification procedures

The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.     

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

A simple and practical high-performance liquid chromatographic analysis has been developed for measuring teniposide (VM26) in human plasma. The present analytical method has improved extraction efficiency from human plasma, therefore allowing determination of VM26 in a clinical setting using ultraviolet detection alone. Furthermore, sample preparation was simplified and shortened through use of a one-step extraction procedure. VM26 and internal standard (ibuprofen) were extracted from human plasma (0.5 ml) with ethyl acetate. A phenyl μBondapak column eluted with a mobile phase, consisting of acetonitrile–distilled water–acetic acid (30:68:2, v/v/v) was used for separation, and quantitation was achieved with a UV monitor set at 240 nm. Average extraction efficiency was 96.8±6.6% for VM26 between 1 and 25 μg/ml, and 91.4±4.3% for internal standard, with both intra- and inter-day coefficients of variation being less than 10%. The detection limit with a 100-μl injection was estimated at 0.2 μg/ml with a signal-to-noise ratio of 3 for VM26 in human plasma. The stability data of VM26 in plasma, standard and stock solutions were also obtained. The present method was found to be an alternative to the previously reported method with an electrochemical detection, and can be easily applied to routine clinical pharmacokinetic studies of VM26.     

Paired-ion high-performance liquid chromatographic assay for plasma choline

Choline was isolated from deproteinized plasma by cation-exchange chromatography. Isolated choline was directly converted to the 3,5-dinitrobenzoate derivative and was analyzed by paired-ion high-performance liquid chromatography with UV detection at 254 nm. An internal standard, 3-hydroxy-N,N,N-trimethylpropanaminium iodide was used for quantitation of plasma choline.Linearity was achieved from 1–500 nmole/ml with a reproducibility of ± 6%. Plasma choline concentrations below 1 nmole/ml could not be accurately measured while plasma choline concentrations in the μmole/ml range deviated from linearity.     

Improved high-performance liquid chromatographic method in the analysis of adenovirus particles

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.     

Rapid and sensitive high-performance liquid chromatographic analysis of halogenopyrimidines in plasma

Recent studies have stressed the need for individual adjustment of 5-fluorouracil (5-FU) dosage. Most of the techniques previously reported are not well adapted to routine application. We describe a sensitive, selective and simple HPLC technique under isocratic conditions for the quantitation of 5-FU and other halogenopyrimidines. The proportion of reagents and internal standard were optimised to allow the use of minitubes, particularly adapted to large series of plasma assays. High extraction yield, 75% for 5-FU and 90% for 5-bromouracil and 5-chlorouracil, was obtained using 1.2 ml isopropanol–ethyl acetate (15:85, v/v) following precipitation of plasma proteins with 300 mg ammonium sulfate. The mobile phase was 0.01 M phosphate buffer (pH 3.0). Uracil and 5-fluorouracil were fully resolved with Spherisorb ODS2 column. The limits of quantitation and detection in human plasma were 6 ng ml⁻¹ and 3 ng ml⁻¹, respectively, for all compounds studied. The total analysis time required for each run was 25 min. Final results could be given within 90 min of blood sampling. At least 50 plasma samples could be analysed per day. This method has been successfully used for monitoring 5-FU-based treatments.     

Separation and analysis of haematoporphyrin derivative components by high-performance liquid chromatography

High-performance liquid chromatography has been used to separate and analyse the components of haematoporphyrin derivative, a material used in cancer phototherapy. Both haematoporphyrin derivative in the solid form and the solution derived from it have been quantitatively analysed on reversed-phase columns. The factors (low pH, presence of ion-pairing reagent and solvent) that are of importance in optimising these separations are discussed.     

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from a C₁₈ reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96-70%. The limit of quantification was 0.1 μg/ml. The assay was reproducible, the within-day coefficient of variation (n=6) was <8% for serum, lungs and liver. The between-day variability (n=6) was <7.7% for serum, liver and lungs at 1 μg/ml or 1 μg/g tissue concentration. The assay was linear within the range 1–40 μg/ml (r²=0.999).