High-pressure liquid chromatography: I. Quantitative separation of purine and pyrimidine nucleosides and bases

A high-pressure liquid column chromatographic method has been developed for the separation and identification of ribonucleosides, deoxyribonucleosides, and bases. This method is capable of detecting these components at 1.0 to 5.0 ng applied; is reproducible under conditions of constant pressure, temperature, and pH; and is rapid, requiring about 30 min for a complete chromatographic separation of ribonucleosides from deoxyribonucleosides and from bases. These separations were carried out under different pH values and buffers, namely, phosphate buffer containing 2.5% methanol at pH 6.9 and 3.0 or 50 mm sodium borate buffer, pH 9.0. These different conditions were utilized to obtain more definitive identification and quantitation of normal metabolites and their counterparts, the antimetabolites. The advantage of this method is that the 20 naturally occurring components are separated from each other by an isocratic elution method, alleviating the need for a gradient elution system, which produces a drift in the baseline with increasing concentration of the eluting buffer, especially when the instrument is operating at maximum sensitivity, thus hindering the quantitation of the separated components. The potential application of this method for the quantitation of plasma metabolites and antimetabolites such as Ara-C², Ara-U, and F-pyrimidine is describe.     

Single-run high-performance liquid chromatography of nucleotides, nucleosides, and major purine bases and its application to different tissue extracts

A high-performance liquid chromatography (HPLC) method is described for the separation and quantitation of nucleotides, nucleosides, purine bases, and related compounds in one single run. The separation of a standard mixture of at least 24 components is achieved within 35 min on glass columns (30 cm, 3-mm i.d.) with C-18 reversed-phase particles of 5 micron, and ammonium dihydrogen phosphate (0.15 M, pH 6.00) and a slow linear gradient of methanol/acetonitrile (to 15%) as eluting solvent. The method has been applied to microsamples of different cells and tissues. Samples (2.5 mg dry wt) were cooled in liquid nitrogen, lyophilized, and extracted with 0.6 N perchloric acid. After neutralization with potassium bicarbonate, the extract (20 microliter) was directly injected into the column. To illustrate the wide applicability of the method, representative chromatograms are shown of extracts of biopsies from heart tissue, skeletal muscle, and brain and liver and from hepatocytes, erythrocytes, and yeast cells, under different conditions, known to induce changes in purine metabolism.     

High-performance liquid chromatographic method with electrochemical detection for the analysis of O6-methylguanine

An improved system consisting of a combination of high-performance liquid chromatographic methods with electrochemical detection for the separation and analysis of the DNA adduct O⁶-methylguanine (O6MG) has been developed. This adduct is produced by the interaction of methylating agents with DNA and induces mispairing in the DNA of the target cells. A good separation of modified from unmodified bases is first achieved with an HPLC system using a Partisil 10 SCX column and a salt gradient. A second HPLC step with electrochemical detection and a C18 column is used for farther separation and quantitation of O⁶-methylguanine. This method shows a linear response up to 15 pg of O6MG tested. The lowest amount detected was 0.5 pg of O6MG and is highly reproducible. This method is useful to study DNA damage as a product of cellular metabolism and its effects on the process of carcinogenesis.     

Two-step high-performance liquid chromatography method for the determination of myo-inositol and sorbitol

A simple two-step HPLC method for the separation and quantitation of myo-inositol and sorbitol in extracts of glomeruli from rat kidneys is described. The limit of detection is 2 ng. The procedure involves fractionation of the sugar alcohols on a Waters Sugar Pak column, preparation of the p-nitrobenzoate derivatives, and further purification with quantitation by absorbance at 254 nm using a Waters mu Porasil column. The applicability of the procedure to determination of sorbitol and myo-inositol in biological samples was demonstrated by the finding of marked alterations in sorbitol and myo-inositol content of glomeruli isolated from diabetic compared to that from normal rat kidneys.     

Highly sensitive analysis of the antifolate pemetrexed sodium, a new cancer agent, in human plasma and urine by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of pemetrexed (LY231514, ALIMTA) in human urine and plasma. Plasma samples were spiked with the internal standard lometrexol and extracted using Certify II columns. Pemetrexed was assayed in diluted urine by an external calibration method. A C₈ column was used for the separation of analytes with a mobile phase composed of sodium formate buffer and acetonitrile. Between- and within-day precision and accuracy were acceptable down to the limit of quantitation of 5 ng/ml in plasma. This method was used successfully for an investigation of the disposition of pemetrexed in patients receiving 500 mg/m² as a 10-min infusion.     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Rapid and selective high-performance liquid chromatographic method for the determination of metronidazole and its active metabolite in human plasma, saliva and gastric juice

A rapid and selective HPLC method has been developed for the separation and quantitation of metronidazole and its hydroxylated metabolite in human plasma, saliva and gastric juice. The assay requires a simple protein precipitation step prior to analysis and is selective, sensitive and reproducible. The limits of quantitation (0/5-ml sample) were at least 0.25 μg/ml for metronidazole and 0.20 μg/ml for its hydroxy metabolite. A Hypersil ODS 5 μm (150×4.6 mm I.D.) column was used with a mobile phase of acetonitrile-aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90) delivered at a flow-rate of 1.0 ml/min.     

Separation and quantitation of phospholipids and their ether analogues by high-performance liquid chromatography.

The common mobile phase hexane/isopropanol/water used for separation of phospholipids on high-performance liquid chromatography silica columns poses several problems, such as incomplete separation and rapid column deterioration. By inclusion of 5 mM ammonium sulfate in the aqueous phase, we were able to substantially improve the chromatographic resolution and obtain complete separation of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, and sphingomyelin. In addition, ammonium sulfate prevented column degeneration and greatly improved reproducibility. A new quantitation method for alkenylacyl, alkylacyl, and diacyl forms of phospholipids was also developed based on derivatization with [(3)H]acetic anhydride. Separation and quantitation of the radioactive acetyl diradylglycerols were performed by straight-phase HPLC coupled to a radioactive flow detector and enabled detection of the various ether analogues at the picomole level with high reproducibility. The described methods are mild and nondestructive and can therefore be easily combined with analysis of either molecular species or fatty acid and aldehyde composition of the individual phospholipids.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

Determination of epsilon (gamma-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation

A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the epsilon (gamma-glutamyl)lysine isopeptide bond, which is usually formed by protein-crosslinking transglutaminases between polypeptide chains. The procedure is based on the separation and quantitation of epsilon (gamma-glutamyl)lysine isodipeptide following exhaustive proteolytic digestion of the crosslinked peptide. It involves preliminary separation steps on a cation exchanger resin and a silica HPLC column, precolumn derivatization with phenylisothiocyanate, and reversed-phase high-pressure liquid chromatographic separation on a C18 column. The derivatized isodipeptide gave a linear concentration-response relationship, with a detection limit of 10 pmol/mg of protein. The combination of the preliminary separation steps and the sensitive detection system permits the determination of the epsilon (gamma-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.     

Simultaneous microdetermination of theophylline, caffeine and phenobarbital in blood collected on paper

A sensitive and selective gas chromatographic method has been developed for the simultaneous determination of theophylline, caffeine and phenobarbital. Blood collection is performed by dropping 30 μl of blood onto a disc of a special paper. Vinbarbital is used for quantitation by the internal standard method. The chromatographic separation is performed on a 3% OV-17 column, after pentylation of the methylxanthine and internal standard, and the compounds are detected with a nitrogen-sensitive detector.The sensitivity of the method allows the monitoring of theophylline therapy in premature newborns by the differential determination of caffeine and theophylline. The sampling method does not affect the accuracy and precision and is very suitable for the collection of small blood samples.     

An ultraperformance liquid chromatography method for the normal-phase separation of lipids

An ultraperformance liquid chromatography method using normal-phase solvents, a silica column, and evaporative light-scattering detection is presented. The method is based on a quaternary gradient profile and is capable of resolving the major neutral and polar lipids present in plasma and animal tissue in under 5 min, with a total cycle time of 11 min. Limits of quantitation for 7 different lipid classes were on the order of 200 ng of material on column which enables an accurate analysis from as little as 20 μL of plasma or 50 mg of tissue for typical samples. Intraday and interday precision for the determination of the major lipid classes in human plasma ranged from 3.6 to 10.5% CV with a variability in retention time of less than 6%. The utility of the method is demonstrated through the separation and quantitation of lipids in mouse plasma, liver, and heart tissue.     

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separted and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of < 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.     

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.     

Development and validation of a liquid chromatography method for simultaneous determination of three process-related impurities: yeastolates, triton X-100 and methotrexate

Yeastolates, triton X-100 (TX-100) and methotrexate (MTX) are common process-related impurities (PRI) in cell-based bioproduction of many active biopharmaceuticals. In this study, a reverse phase high performance liquid chromatography (RP-HPLC) method coupled with ultraviolet (UV) detection was developed for simultaneous determination and quantitation of these impurities. The chromatographic separation was achieved using a Jupiter C4 column and analyses of yeastolates, TX-100 and MTX were monitored at 257, 280 and 302 nm, respectively. The method was further validated with respect to selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. The limits of quantitation for yeastolates, TX-100 and MTX were determined to be 27 ppm, 10 ppm and 41 ppb, respectively. Finally, the suitability of the method for analyses of recombinant human hyaluronidase (rHuPH20) in-process (viral inactivation, QFF, PS, APB and CHT filtered, final viral filtrate) and final manufacturing materials was demonstrated, and trace levels of yeastolates, TX-100 and MTX were reliably measured except for three matrices early in the purification process in which TX-100 was not accurately determined due to interfering effects.     

Determination of mirtazapine in human plasma by liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of mirtazapine in human plasma is presented. The method is based on a liquid-liquid extraction and reversed-phase chromatography with fluorimetric detection. The separation was performed on a Luna microm C(18)(2) 50 x 4.6 mm I.D. column using an isocratic elution. Zolpidem hemitartrate was used as the internal standard. The between-day precision expressed by relative standard deviation was less than 5% and inaccuracy does not exceed 6%. A low limit of quantitation (1.5 ng/ml) and a short time of analysis (4 min) makes this assay suitable for pharmacokinetic studies.     

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.     

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.     

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.     

Separation of oxidatively damaged DNA nucleobases and nucleosides on packed and monolith C18 columns by HPLC-UV-EC

This study involves the incorporation of a commercially available Phenomenex Onyx C18 monolith column into the separation and detection of oxidative DNA damage. It includes thorough investigation of monolith performance and a comparison of the performance of monolith columns with a commercially available packed Restek reverse phase Ultra C18 column for the separation of DNA bases and nucleosides. The performance of the monolith was examined using efficiency, resolution, plate height, asymmetry and retention times, and each case showed improved or at least comparable results in the separation of a mix of DNA bases and nucleosides. A 90% reduction, from just under 40min to just under 4min, was obtained in the elution time of this separation. To the best of our knowledge, this is the first report of a fast monolith column separation successfully coupled to both a UV-vis and EC detector, which is especially useful for the analysis of oxidative DNA damage. The determination of 8-oxoG and 8-OH-dG, oxidation products of guanine and 2'-deoxyguanosine, respectively, may be compromised by their ease of oxidation and therefore the fast separation, selective and sensitive detection, with no artifactual oxidation, detailed in this report, is ideal.