Evaluation of an ODS column modified with zwitterionic/nonionic mixed surfactants and its application to direct injection determination of inorganic anions.

An octadecylsilica (ODS) column modified with zwitterionic/nonionic mixed surfactants was evaluated for the direct injection determination of inorganic anions in biological fluids by ion chromatography. A zwitterionic surfactant (sulfobetaine-type) and a nonionic surfactant (polyoxyethylene-type) were used for a stationary-phase modification. When aqueous electrolyte solutions with concentrations of sub-mM to several mM were used as a mobile phase, the zwitterionic surfactant coated on the ODS surface exhibited unique separation selectivity for ionic species, while the nonionic surfactant coated on the ODS might have formed a hydrophilic network over the ODS surface and restricted matrix proteins from adsorbing on the stationary phase. Consequently, the mixed surfactant-modified column system allowed an efficient ion chromatographic separation of inorganic anions as well as a size-exclusive removal of column-fouling proteins. This separation system was applied to the direct injection determination of UV-absorbing anions in human saliva. The detection limits for nitrite, nitrate, iodide and thiocyanate were 3.1, 2.7, 4.5 and 25 microM, respectively, with UV detection at 210 nm (injection volume; 20 microl), and their relative standard deviations for 5 replicate measurements of saliva samples spiked with 100 microM each of those anions were 1.4, 0.9, 2.2 and 5.5%, respectively.     

Methylcellulose-immobilized reversed-phase precolumn for direct analysis of drugs in plasma by HPLC.

We evaluated a new restricted access media (RAM) precolumn for direct analysis of drugs in plasma using a column switching HPLC system. The new RAM material was prepared by the modification of the external surface of porous silica with hydrophilic methylcellulose (MC), followed by modification of the internal surface with octadecylsilane (ODS). The external surface of the MC-immobilized ODS silica material (MC-ODS) suppressed the adsorption of proteins, while the internal surface of MC-ODS retained various types of drugs, such as ketoprofen, propranolol, caffeine and atenolol in plasma samples. In addition, MC-ODS allowed direct analysis of drugs in a 1000-microL plasma sample to monitor trace amounts of analytes contained. Reduced efficiency and clogging of the MC-ODS precolumn and/or the analytical column were not observed even after the repetitive injection of plasma sample up to 40 mL. Our results indicated that the MC-ODS precolumn could be used in pharmacodynamic and clinical studies.     

Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4 degrees C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.     

Synthesis of mixed-functional-phase silica supports for liquid chromatography and their applications to assays of drugs in serum

Mixed-functional-phase (MFP) silica supports were designed for the direct injection determination of drugs in serum. The MFP silicas were synthesized from porous silica materials in three or four steps: introduction of 3-glycidoxypropyl phases, introduction of phenyl, butyl or octyl phases and hydrolysis of the oxirane ring to diol phases, or these three steps plus further introduction of glycerylpropyl (i.e., diol) phases. Although the further introduction of glycerylpropyl phases resulted in a reduction in the column efficiency, serum proteins were completely recovered in the first injection of serum samples. The prepared MFP packing materials can be used for the direct injection determination of hydrophobic and hydrophilic drugs in serum.     

Zwitterionic ion chromatography using a dynamically coated column and mobile phase recycling

An investigation into the use of zwitterionic ion chromatography for the determination of inorganic anions in water samples was carried out. When using an ODS stationary phase precoated with Zwittergent 3-14 and a pure water mobile phase, the stability of the adsorbed coating was insufficient for quantitative work. Recycling of the water mobile phase was used to stabilise the zwitterionic coating, and resulted in improved retention time precision (15.2% RSD down to 2.4% RSD for nitrate). Post-detection cation- and anion-exchange columns in acid and hydroxide form removed sample ions from the recycling mobile phase, with the desorbed Zwittergent 3-14 passing through unretained and passing back through the pump to the analytical column. A 200-ml volume of mobile phase was recycled over a 3-week period with retention times for sulphate, chloride and nitrate standards injected at the start and end of the period varying less than 2.5%. The same system was then used with a mobile phase containing 2 mM Zwittergent 3-14. This resulted in further improvements in retention time (0.2-0.5% RSD, n = 10) and peak area precision (2.6-6.0% RSD, 1 mM standards) and improved peak efficiencies (2421-4047 N). The developed method was applied to water samples, and results compared to those obtained using anion-exchange chromatography. All sample cations were exchanged to sodium using an off-line cation-exchange procedure prior to injection.     

Validation of an HPLC method for the determination of fleroxacin and its photo-degradation products in pharmaceutical forms

HPLC determination of fleroxacin in dosage forms was carried out using either reversed-phase column YMC pack ODS-AQ or Supelco LC Hisep shielded hydrophobic phase column, with UV detection at 280 nm. The mobile phase for ODS column consisted of 50:50:0.5 v/v/v and for Hisep column 15:85:0.5 v/v/v acetonitrile-water-triethylamine. The pH of the mobile phase was adjusted to 6.30 for ODS column and to 6.85 for Hisep column, with H3PO4. Linear response was obtained in the concentration range of fleroxacin between 0.01 and 1.30 micrograms/mL. Detection limit was 4.8 ng/mL. Recovery test in the determination of fleroxacin in "Quinodis" tablets (Hoffmann La Roche, nominal mass 400 or 200 mg) was 98-101% for both columns. The effect of the composition and pH of the mobile phase on spectra, retention time and dissociation constants of fleroxacin was discussed. The proposed method could be also used for separation of the photo-degradation products of fleroxacin. Ten degradation products were separated on the ODS-AQ column, thus confirming the suitability of the proposed method for stability study of fleroxacin in pharmaceuticals.     

Serum Injection of the HPLC Column for Carbamazepine Assay

Abstract

Serum was injected directly on an HPLC column packed with C₁, 6.5 μm particle size, 300 Å pore-packing material for carbamazepine determination. The use of a wide-pore column with low hydrophobicity eliminated excessive pressure buildup in the column from protein precipitation which is usually caused by the high concentration of organic solvents in the mobile phase. This simple approach can be utilized for the determination of other drugs and endogenous compounds in serum.     

Determination of Danazol in Plasma by High Performance Liquid Chromatography

Abstract

An HPLC method has been developed for the determination of danazol in human plasma. the chromatographic conditions consisted of an ODS column 80 × 7.0 mm, 3 μm; a mobile phase of 71:29, methanol:20 mM potassium dihydrogen phosphate. A UV-Visible detector was set at 288 nm to monitor the danazol peak. Danazol was extracted from plasma with acetonitrile which was salted out with potassium carbonate. Prior to salting out, cadmium sulfate was mixed with the acetonitrile-plasma mixture to remove any interfering constituents. the extracted acetonitrile layer was evaporated and the residue was reconstituted in the mobile phase before injection. the method was found to be reproducible with relative standard deviation (%rsd) from 2.3 to 7.2%. A number of clinically important drugs did not interfere with danazol determination.     

Speciation of mercury in salmon egg cell cytoplasm in relation with metallomics research

Speciation of mercury in salmon egg cell cytoplasm was investigated by surfactant-mediated high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS), where an ODS (octadecylsilica) column coated with a bile acid derivative, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), was used for species separation. Prior to the speciation analysis, total Hg in the cell cytoplasm was determined by ICP-MS at m/z 202 in a flow injection mode. For the precise measurement, salmon egg cell cytoplasm was diluted five-fold with 0.1 M Tris (Tris(hydroxymethyl)aminomethane)-HNO₃ buffer solution, and the standard addition method was employed. Thus, the total concentration of Hg in cell cytoplasm was estimated to be 12.4 ng g⁻¹ on the wet weight basis. Next, the cell cytoplasm diluted five-fold with 0.1 M Tris-HNO₃ buffer solution was analyzed by surfactant-mediated HPLC with the dual detection system of a UV absorption detector and an ICP-MS instrument. Two peaks corresponding to some proteins and small molecules were mainly observed in those chromatograms. When salmon egg cell cytoplasm was diluted five-fold with 0.01 M Tris buffer solution or pure water, some precipitates appeared probably because of precipitation of hydrophobic proteins in cytoplasm. After the precipitates were eliminated with a membrane filter, the filtrate was subjected to the analysis by surfactant-mediated HPLC/UV/ICP-MS. As a result, the peaks for small molecular species of Hg were clearly observed at the retention time near 4.0 min (corresponding to low-molecular weight zone) in the chromatograms with UV absorption detection as well as with Hg- and S-specific ICP-MS detections. The small molecule bound with Hg was identified as cysteine through the cysteine-spiked experiment. In addition, the protein fraction on the chromatogram obtained by using the CHAPS-coated ODS column was further analyzed by SEC (size exclusion chromatography). Consequently, several protein peaks with molecular weight of 300, 50 and 12 kDa were observed in all the detections of UV absorption, Hg and Se, although two peaks among them were coincident in the case of S. These results indicate that Hg in salmon egg cell cytoplasm binds with proteins containing selenocysteine and/or cysteine residues in proteins.     

Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C₁₈ columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C₁₈ RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.     

Development and validation of an HPLC method for the determination of verapamil residues in supports of cleaning procedure

Analytical method validation, determining the recovery rate from the equipment surface, and stability of a potential contaminant are important steps of a cleaning validation process. An HPLC method for the determination of the verapamil residues on stainless steel surfaces of the equipment employed in drug manufacture is described. The cleaning validation sample impurities as well as excipients of the commercial sample did not interfere in the analysis which proved the selectivity of the method. The validation of the method demonstrated acceptable levels of the linearity, precision and accuracy. Cotton swabs, moistened with methanol were used to remove any residues of drugs from stainless steel surfaces, and give recoveries of above 78.59% for three diferent concentration levels. The precision of the results, reported as the relative standard deviation (RSD, %), were below 1.58%. Low quantities of the drug residues were determined by HPLC using a Hypersil ODS column (125 × 4.0 mm, 5 μm) at 25°C with the mobile phase metanol-water-triethylamine (70: 30: 0.2, v/v/v) at a flow rate of 0.6 mL/min, injection volume of 50 μL and detection at 278 nm.     

Application of ion-pair high performance liquid chromatography for analysis of hyoscyamine and scopolamine in solanaceous crude drugs

A new ion-pair high-performance liquid chromatographic method for the analysis of hyoscyamine and scopolamine in various kinds of crude drugs derived from solanaceous plants was evaluated using sodium dodecyl sulfate as a counter ion. A reversed-phase chromatographic system consisting of a chemically bonded ODS silica gel column with phosphate buffer (pH 2.5)-acetonitrile (65:35) containing 17.5 mM sodium dodecyl sulfate as the mobile phase was used. Hyoscyamine and scopolamine in crude drugs derived from Scopolia, Atropa, Hyoscyamus and Datura species were separated from other compounds in the crude drugs and determined within 20 min after direct injection of the solution extracted with the mobile phase. The results for various kinds of samples are presented.     

厚朴酚键合硅胶高效液相色谱固定相分离嘌呤、嘧啶、蝶呤及黄酮

将新制备的厚朴酚键合硅胶固定相(MSP)用于嘌呤、嘧啶、蝶呤及黄酮类化合物的液相色谱分离分析。选取了4种嘌呤、8种嘧啶、4种蝶呤及5种黄酮类药物作为极性化合物的代表,以商品反相碳十八烷基键合硅胶柱(ODS)作参照,研究了新固定相对碱性化合物的选择性和相关分离机理。实验发现,在简单流动相条件下,厚朴酚键合硅胶固定相对上述药物表现出较高的选择性及分离效果。尽管MSP没有进行封尾处理,但含氮类极性化合物(嘌呤、嘧啶、蝶呤)仍表现出基本对称的色谱峰形。多数药物在两柱上的洗脱顺序大致相同,说明疏水作用始终存在,这说明新固定相具有反相色谱性能。比较研究还发现,MSP在分离上述极性药物时能够提供除疏水性作用之外的其他作用位点。例如,在分离嘌呤、嘧啶及蝶呤时,氢键和偶极作用明显存在;同时MSP与溶质结构中的芳环(硫唑嘌呤、紫花牡荆素)之间有较强的π-π电子相互作用等,使得含氮类极性化合物和黄酮的保留一般比ODS强,分离度也有一定的改善。多种作用可以合理地解释MSP柱对极性溶质有更强的分离能力,厚朴酚键合硅胶固定相可在一定程度上弥补ODS单一疏水作用的不足,有利于分类碱性化合物。     

Large Volume Injection of Biological Samples Dissolved in a Non-Eluting Solvent: A Way to Increase Sensitivity and a Means of Automating Drug Determination Using Hplc

Abstract

The injected volume of a sample dissolved in the mobile phase of an HPLC system must be maintained as small as possible so as to minimize the loss in efficiency. Generally this requirement limits the sensitivity of HPLC methods devoted to trace quantity determinations of drugs in biological fluids. In order to avoid this limitation and to increase the effective sensitivity of HPLC methods for determination of drugs such as antrafenine, nifuroxazide and cipropride, the samples were dissolved in a non-eluting solvent and a large volume (> 100 μl) was injected on to the chromatographic column.

The above-mentioned compounds and their internal standards were dissolved in a series of eluting and non-eluting solvents and increasing volumes (5 to 1000 μl) were injected. Peaks corresponding to injections made in an eluting solvent showed retention times independent of the injection volume but their variances increased with the volume injected. In contrast, peaks corresponding to injections made in a non-eluting solvent, similar to the mobile phase, had a variance independent of the injection volume but their retention times increased linearly with the injection volume. The repeated injection of such non-eluting solvents had no influence on chromatographic behaviour. Peaks corresponding to compounds injected in a non-eluting solvent made with components different from those of the mobile phase had a variance independent of the injection volume but their retention times varied both with the injection volume and with the interval between injection.

The application of non-eluting solvents has been defined theoretically and it has been demonstrated that solutions composed of 25% of the mobile phase diluted with the least eluting of its components act as non-eluting solvents and can be injected in large volume without loss in efficiency. This feature could be used to inject all the samples volume or only part of it, manually or automatically, since any automatic injector can be used with large volumes.

Thus, using the relatively simple procedure of making injections with a non-eluting solvent it is possible to increase both sensitivity and the rate of sample analysis.     

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was 相似文献   

高效迎头分析法测定药物-人血清白蛋白混合液中游离药物浓度

 用高效迎头分析法 (HPFA)测定了药物 人血清白蛋白 (HSA)混合液中游离药物的浓度。样品溶液不经任何处理直接进样到装有内表面反相固定相的色谱柱中 ,用 67mmol/L磷酸盐缓冲液 ( pH 7 4 ,I =0 17mol/L)作流动相。当进样体积足够大时 ,游离药物以平顶峰的形式被洗脱出来 ,平顶峰区域洗脱液中的药物浓度等于样品溶液中游离药物的浓度。收集平顶峰区域的洗脱液 ,然后将一定体积的洗脱液注入到反相色谱柱中 ,测定游离药物的浓度。用该法测定酮基布洛芬 HSA和头孢哌酮 HSA两种混合液中游离药物的浓度。     

Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection

A simple, sensitive and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of amino acids in human serum. The method involves precipitation of the serum proteins with methanol followed by pre-column derivatization of amino acids with o-phthalaldehyde-2-mercaptoethanol or o-phthalaldehyde-sodium sulfite. HPLC separation of the derivatives was performed using an ODS column with an isocratic mobile phase system and electrochemical detection (+0.75 V). The response was linear over the range 5-300 microM for all amino acids. The method allows quantitative determination of glutamic acid, asparagine, serine, glutamine, histidine, taurine, alanine, arginine, methionine, isoleucine, ornithine, leucine, phenylalanine, lysine and tryptophan at concentrations as low as 0.5-5.0 pmol (signal-to-noise ratio=2). Using this method, the levels of amino acids in serum from healthy donors and patients with ischemic stroke were determined.     

Selectivity comparisons of monolithic silica capillary columns modified with poly(octadecyl methacrylate) and octadecyl moieties for halogenated compounds in reversed-phase liquid chromatography

Stationary phase selectivities for halogenated compounds in reversed-phase HPLC were compared using C18 monolithic silica capillary columns modified with poly(octadecyl methacrylate) (ODM) and octadecyl moieties (ODS). The preferential retention of halogenated benzenes on ODM was observed in methanol/water and acetonitrile/water mobile phases. In selectivity comparison of selected analytes on ODM and ODS, greater selectivities for halogenated compounds were obtained with respect to alkylbenzenes on an ODM column, while similar selectivities were observed with a homologous series of alkylbenzenes on ODM and ODS columns. These data can be explained by greater dispersive interactions by more densely packed octadecyl groups on the ODM polymer coated column together with the contribution of carbonyl groups in ODM side chains. For the positional isomeric separation of dihalogenated benzenes (ortho-, meta-, para-), the ODM column also provided better separation of these isomers for the adjacently eluted isomers that cannot be completely separated on an ODS column in the same mobile phase. These results imply that the ODM column can be used as a better alternative to the ODS column for the separation of other halogenated compounds.     

Phospholipid-Modified ODS Monolithic Column for Affinity Prediction of Hydrophobic Basic Drugs to Phospholipids

A phospholipid-modified octadecyl silica (ODS) monolithic column was prepared and its interaction with basic hydrophobic drugs was studied. These drugs are of interest in pharmaceutical research because of their strong interaction with biomembranes. The amount of dimyristoylphosphatidylcholine trapped on the ODS surface was reproducible, and the results of the trinitrobenzenesulfonic acid assay suggested the formation of a monolayer on the surface. Both hydrophobic and electrostatic interactions acted between the model drugs and the phosphatidylcholine. The column was stable for 10 days at least. The column was applied to the affinity screening of basic drugs to phospholipid. Good correlation was obtained between log k and log P for the basic drugs lidocaine, quinidine, propranolol, imipramine, and chlorpromazine. The monolithic silica column allowed highly hydrophobic basic drugs such as imipramine and chlorpromazine to be assayed; these are difficult to analyze by using a conventional particle-packed column. These drugs were clearly separated from acidic drugs naproxen and warfarin on the log k versus log P plots. The thermodynamic studies revealed that the retention of the drug was an enthalpy-driven process, and that the decrease in enthalpy for the phospholipid-modified ODS monolithic column was larger than those for immobilized artificial membrane columns. Our results suggest that the phospholipid-modified ODS monolithic column is applicable to affinity screening of drugs to phospholipids.     

Some characteristics of a protein-coated ODS column and its use for the determination of drugs by the direct injection analysis of plasma samples

Summary It was found that an ODS column of small pore (120?) which was coated with denatured plasma proteins (protein-coated ODS) no longer adsorbed plasma proteins from aqueous solution but retained the characteristics of native ODS for small hydrophobic molecules. Elemental analysis and nitrogen desorption (BET) analysis showed that the protein-coated ODS contained ca 25 mg proteing⁻¹ dry resin and that the pore diameter or pore volume was similar to that of native ODS. The coated denatured proteins, which seemed to be adsorbed on the external surface of the porous resin, were not eluted under usual reverse-phase elution conditions. Operating as either an analytical column or a pre-column, this protein-coated ODS column was used to analyse spiked-drugs in plasma. The recovery of all the spiked-drugs (such as doxorubicin, methotrexate) was almost quantitative (98–102%) with good reproducibilities (c.v., less than 2%). The present method was useful for the determination of total, that is, free + bound-to-plasma-proteins, hydrophobic drugs in plasma in view of its accuracy and simplicity. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984