High-performance liquid chromatographic determination of zinc protoporphyrin and porphyrin carboxylic acids in urine

A method for the reversed-phase high-performance liquid chromatographic separation of zinc protoporphyrin and porphyrin carboxylic acids with fluorescence detection and its application are described. A mu Bondapak C18 column was employed for all the experiments in this study. The method required a pretreatment of the column with a two-component mobile phase containing 0.1 M NaH2PO4 in acetonitrile (28:15, v/v, pH 5.3) for 10 min prior to sample injection. Separation was achieved isocratically by increasing the concentration of acetonitrile in the mobile phase (0.1 M NaH2PO4:acetonitrile, 18:130, v/v, pH 5.3) 4 min after injection to complete the elution. The flow rates and the period of pretreatment of the column were studied to optimize the separation. The method was applied to determining zinc protoporphyrin and porphyrin carboxylic acids of heme biosynthesis in urine.     

Optimization of a solid-phase extraction method for determination of indapamide in biological fluids using high-performance liquid chromatography

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C(8) column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0-100.0 ng/ml for serum, and 50.0-500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.     

Determination of amphotericin B in human serum by liquid chromatography

A rapid reversed-phase high-performance liquid chromatographic method with a 30-mm long column is described for assaying amphotericin B in serum. After deproteinization of serum samples with methanol, the supernatant was injected onto a reversed-phase C₁₈ column, using 2.5 mM Na₂EDTA-acetonitrile (70:30, v/v) as the mobile phase. Amphotericin B was eluted at 1.5 min. Calibration plot of the peak area against concentration was linear from 0.05 to 25 μg/ml (C.V. of 3%). Within-day and day-to-day imprecision (C.V.) ranged between 1.33% and 3.61%. The application was evaluated in 55 serum samples from patients treated with amphotericin B.     

Influence of specific albumin ligand markers used as modifiers on the separation of benzodiazepine enantiomers by chiral liquid chromatography on a human serum albumin column

Specific ligand markers for the various binding sites of human serum albumin (HSA) have been described in the literature. Some of these markers (medium chain fatty acids, warfarin, digoxin, and bilirubin) were used as mobile phase modifiers. Using a high performance liquid chromatographic (HPLC) column containing HSA as stationary phase, their influence was investigated on the separation in this phase of the enantiomers of three benzodiazepines (temazepam, oxazepam, and lorazepam). Displacement effects were observed with medium chain fatty acids. This influence was proportional to the chain length and to the concentration of acid. Allosteric cooperative effects were noted with digoxin for the three benzodiazepines. Both displacement and cooperative effects were observed with warfarin. Stereoselectivity was decreased for temazepam and oxazepam and increased for lorazepam. © 1993 Wiley-Liss, Inc.     

Reversed-phase HPLC separation of human serum employing a novel saw-tooth gradient: toward multidimensional proteome analysis

A novel reversed-phase (RP-) HPLC gradient profile applicable to multidimensional separations of complex protein mixtures is reported. This gradient profile elutes small numbers of proteins from the RP-HPLC column in discrete intervals while minimizing the amount of band broadening between elution intervals and maintaining constant flow through the HPLC column. Eluting the proteins in discrete intervals eases the instrumental requirements necessary for performing multidimensional separations and can be used to aid in the collection of well-defined fractions. The saw-tooth gradient was applied to the successful isolation of albumin from less abundant proteins in whole human serum and provides adequate separation of proteins in a low-molecular weight (LMW) fraction of human serum with resolution comparable to that achieved using a typical linear gradient profile.     

Chemochromatography: its use for the separation of nikkomycins X and Z

A novel mode of reversed-phase high-performance liquid chromatography in which the mobile phase reacts chemically with the compounds to be separated was developed. Nikkomycin X and nikkomycin Z, two natural isomeric nucleoside peptide antibiotics, move as a single peak on a C18 reversed-phase column using an aqueous trifluoroacetic acid mobile phase. Addition of sodium bisulfite (1.0%) to the mobile phase results in the formation of a polar bisulfite addition product with nikkomycin X, but not with nikkomycin Z, inside the HPLC column. This type of reactive chromatography, or chemochromatography, led to the analytical and preparative separation of nikkomycins X and Z which are normally very intractable to separation by conventional chromatographic techniques.     

Comparison of polysaccharide-based and protein-based chiral liquid chromatography columns for enantioseparation of drugs

Two different columns—Lux Cellulose-1 and Chiralpak CBH—were evaluated for their chiral recognition abilities for eight drugs comprising three β-blockers, one antacid, and four cathinones in polar-organic elution mode and reversed-phase elution mode, respectively. The factors that affected the enantioseparation were tested and optimized to develop a suitable chiral separation method whose LC conditions are compatible with MS detection. In polar-organic elution mode with the Lux Cellulose-1 column, methanol and acetonitrile were tested as the main components of the mobile phase. In addition, the effects of adding isopropanol as organic modifier, acidic additives (formic acid), and basic additives (diethylamine) were evaluated. In reversed-phase elution mode with the Chiralpak CBH column, the effect of type and concentration of organic modifier (isopropanol, acetonitrile, and methanol), the mobile phase pH (6.4 and 5.0), and buffer concentration (1mM-20mM ammonium acetate) were evaluated. The best enantioseparation was achieved with the Chiralpak CBH column with a mobile phase composed of 5mM ammonium acetate aqueous (pH = 6.4)/methanol (95/5, v/v) at a flow rate of 0.1 mL/min and a temperature of 30°C. Under these conditions, six of eight chiral drugs were baseline separated.     

Simple high-performance liquid chromatography method for the simultaneous determination of serum caffeine and paraxanthine following rapid sample preparation

A simple reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of caffeine and paraxanthine in human serum is described. Serum proteins are precipitated with perchloric acid and the resulting supernatant neutralized for direct injection onto an HPLC column. The method uses a phosphate–methanol mobile phase (85:15, v/v) at pH 4.9 with a flow-rate of 1.75 ml/min and quantitation is by UV absorbance at 274 nm. Elution times are approximately 18 min for caffeine and 8 min for paraxanthine. Theobromine and theophylline have elution times of 5.4 and 9.4 min and do not interfere in the assay. The intra-assay and between-assay means for precision and accuracy for both drugs are: 4.5% C.V. and 3.3% deviation. The sensitivity of the method is 50 ng/ml for each drug.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching : I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17x4.6 mm I.D.), filled with C₁₈ Corasil 37–53 μm. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-1000 ng/ml was between 1.0 and 4.7% for all compounds.     

Comparison of ion-pair and amide-based column reversed-phase liquid chromatography for the separation of thiamine-related compounds

Two reversed-phase chromatographic methods for the separation of thiamine and related compounds are compared. The first procedure is based on the ion-pair technique using an octadecylsilica column, while the second uses a new amide-based stationary phase, which avoids the need to form ion-pairs, leading to narrower peaks and a simpler mobile phase. Analyses were performed by gradient elution and a photo-diode array was used for detection. Specificity was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity index with commercial standards. The procedures were applied to the determination of thiamine-related compounds in pharmaceutical preparations and urine. No preliminary sample treatment was required.     

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.     

Enantioselective and diastereoselective separation of synthetic pyrethroid insecticides on a novel chiral stationary phase by high-performance liquid chromatography

A novel chiral packing material for high-performance liquid chromatography (HPLC) was prepared by connecting (R)-1-phenyl-2-(4-methylphenyl) ethylamine (PTE) amide derivative of (S)-isoleucine to aminopropyl silica gel through 2-amino-3,5-dinitro-1-carboxamido-benzene unit. This chiral stationary phase was applied to the enantioselective and diastereoselective separation of five pyrethroid insecticides by HPLC under normal phase condition. To achieve satisfactory baseline separation an optimization of the variables of mobile phase composition was required. The two enantiomers of fenpropathrin and four stereoisomers of fenvalerate were baseline separated using hexane-1,2-dichloroethane-2-propanol as mobile phase. The results show that the enantioselectivity of CSP is better than Pirkle type 1-A column for these compounds. Only partial separations for the cypermethrin and cyfluthrin stereoisomers were observed. Seven peaks and eight peaks were observed for cypermethrin and cyfluthrin, respectively. The elution orders were assigned by using different stereoisomer-enriched products.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

Resolution of human mercapt- and nonmercaptalbumin by high-performance liquid chromatography

Gel-exclusion high-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on PGP 2000 column (0.10 M sodium phosphate buffer, 0.30 M NaCl, pH 6.86) showed at least two peaks, the principal component corresponding to human mercaptalbumin (HMA) and the second one to human nonmercaptalbumin (HNA). Mechanism for the separation of HMA and HNA might be due to weak resin-HSA interaction. HPLC analysis of bovine plasma albumin (BPA) showed a single peak on PGP 2000 column. The elution volume of HSA was larger than that of BPA, resulting in a clear resolution of HSA and BPA.     

Optical resolution of some biologically active compounds by chiral liquid chromatography on BSA-silica (resolvosil) columns

Optical resolution on the analytical scale of a number of racemic pharmaceuticals and some other biologically active compounds has been studied using immobilized bovine serum albumin (BSA) as the stationary phase. For some of the compounds the elution order was determined by the use of optically enriched fractions obtained from a preceding passage of a sample through a preparative column containing microcrystalline triacetylcellulose (MCTA). The reversal in the sign of optical rotation shown in the polarimetric elution profile from the latter, combined with the integrated peak area ratio obtained on resolution on the analytical column, gave directly the order of elution. For one of the benzothiadiazines studied (bendroflumethiazide), increasing the pH of the mobile phase produced opposite effects on the retention of the two enantiomers, leading to a large effect on the separation factor. For many of the compounds studied, high separation factors (α > 2) could be achieved.     

Poly(ethyleneglycol) column for the determination of acetaminophen,phenylephrine and chlorpheniramine in pharmaceutical formulations

New polar reversed-phase stationary phases in HPLC provide specific selectivities which can help to solve traditional chromatographic problems related to the development of chromatographic methods with widely different retention times for the sample components. One such case is the analysis of pharmaceutical formulations against the common cold. Acetaminophen, phenylephrine and chlorpheniramine, compounds with different polarities, are frequently associated in these drugs. An isocratic and rapid HPLC method for the simultaneous determination of the three compounds, acetaminophen, phenylephrine and chlorpheniramine, in capsules as pharmaceutical formulations, including the separation of impurities (4-aminophenol and 4-chloracetanilide) and excipients, has been developed and validated. The final chromatographic conditions employed a Supelco Discovery HS PEG column poly(ethyleneglycol) 15x0.46 cm, 5 microm. The mobile phase was 20 mM phosphate buffer, pH 7.0-acetonitrile (90:10, v/v) at a flow-rate of 1 ml/min. UV detection was performed at 215 nm for all the compounds except acetaminophen, which was measured at 310 nm. Validation parameters permit us to consider this method suitable.     

用高效液相色谱测定重组人尿激酶原制剂的蛋白含量

目的:用RP-HPLC方法对注射用重组人尿激酶原制剂蛋白含量进行定量分析。方法:用反相C18柱、0.1%TFA水溶液与0.1%乙腈进行梯度洗脱,280nm波长紫外检测器监测;以重组人尿激酶原同质标准品作为对照品,根据进样量和相应的峰面积建立标准曲线方程,将待测定样品的峰面积代入标准曲线方程,可测得蛋白含量。结果:按照方法学验证要求对此方法进行了专属性、检测限、定量限、线形、精密度(重复性、中间精密度)、准确度(回收率)考察,线性范围为9～27μg,回收率在97%以上,RSD2.0%,完全满足对制剂蛋白的定量需求。结论:本方法准确,适用于注射用重组人尿激酶原成品制剂蛋白定量测定。     

Displacement effect during HPLC preparative purification of human insulin

HPLC plays a key role in the preparative purification of human insulin. A21-desamidoinsulin is one of the impurities that possesses the chromatographic behavior similar to that of insulin and hence separation from this by-product is rather difficult at the process scale. During the optimization of insulin reversed-phase HPLC purification, when a column was sufficiently overloaded, the effect of displacement of A21-desamidoinsulin molecules from active groups of sorbent by insulin ones was observed. It was suggested that monocarboxylic acid and organic modifier in mobile phase are responsible for the esterification during which the formed ester promotes the displacement effect. This effect was studied in order to optimize the purification of human insulin at the process scale.     

Two-dimensional high-performance liquid chromatographic method for assaying S-adenosyl-l-methionine and its related metabolites in tissues

S-Adenosyl-l-methionine (SAM) is a methyl-donor compound which is actively involved in a variety of biochemical reactions. An assay has been developed permitting the quantitative measurement of SAM and its related metabolites (S-adenosylhomocysteine, decar☐ylated SAM, methylthioadenosine, adenosine and adenine) in liver and cell cultures. As gradient reversed-phase chromatographic or cation-exchange chromatographic methods often resulted in overlapping peaks, a two-dimensional high-performance liquid chromatographic (HPLC) procedure was developed involving gradient reversed-phase chromatographic separation followed by ion-exchange chromatography. After precipitating large molecules in the sample by perchloric acid, gel permeation was carried out on a Sephadex G 25 column to separate small water-soluble metabolites from proteins and membrane fragments. The freeze-dried sample was injected onto an ODS column and a 0–10% acetonitrile gradient in 10 m M ammonium formate buffer (pH 2.9) (20 min, linear) was applied. The relevant fractions were collected and injected onto a cation-exchange column (Partisil SCX, 10 μm, 250 mm × 4.6 mm I.D.). Elution and quantification were carried out using ammonium formate buffers of various concentration (15–400 m M), pH 2.9. The detector response (254 nm) as a function of concentration was linear over the concentration range 30–500 pmol. The detection limits of the compounds after the two-dimensional chromatographic procedure ranged from 10 to 60 pmol and the recovery was higher than 70%. The reproducibility of the results obtained from given samples was within 9–22% for rat liver and 6–24% for mast cells.