Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagent

Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60 degrees C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human alpha(1)-acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein.     

Sensitive determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, in a rat biological sample by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

4-(4-Chlorophenyl)-4-hydroxypiperidine (CPHP), one of the metabolites of haloperidol, is considered to exhibit brain toxicity. CPHP concentrations in plasma and tissue homogenates (each 200 microL) from rats were analyzed by HPLC fluorescence detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, the derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was carried through the procedure as an internal standard. The regression equation for CPHP showed a good linearity in the range of 0.03-1 microg/mL with a detection limit of 0.008 microg/mL. The coefficient of variation was less than 11.6%. Plasma concentration-time courses of CPHP after intraperitoneal or per oral administration of CPHP, haloperidol or reduced haloperidol were examined, and the pharmacokinetic parameters were estimated. Additionally, CPHP levels in various tissues at 8 h after intraperitoneal administration of these compounds were compared. The method was simple and sensitive, useful for determination of CPHP in rat biological samples using as little as 200 microL of sample volume and could be applied for pharmacokinetic study.     

Protein-based chiral stationary phases for high-performance liquid chromatography enantioseparations

The enantioseparations of various compounds using proteins as the chiral selectors in high-performance liquid chromatography (HPLC) are considered in this review. The proteins used include albumins such as bovine serum albumin and human serum albumin, glycoproteins such as alpha1-acid glycoprotein, ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as trypsin, alpha-chymotrypsin, cellobiohydrolase I, lysozyme, pepsin and amyloglucosidase, and other proteins such as ovotransferrin and beta-lactoglobulin. This review deals with the properties of HPLC chiral stationary phases based on proteins, and the enantioselective properties and chiral recognition mechanisms of these stationary phases.     

Comparison of SSI with APCI as an interface of HPLC-mass spectrometry for analysis of a drug and its metabolites

Sonic spray ionization (SSI) was compared with atmospheric pressure chemical ionization (APCI) as an interface of high-performance liquid chromatography (HPLC)-mass spectrometry (MS) for sensitive analyses of a neuroleptic drug, haloperidol and its two metabolites, such as reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), in biological samples. For both SSI and APCI interfaces, HPLC-MS-MS gave higher sensitivity than HPLC-MS. The sensitivities by HPLC-SSI-MS-MS for haloperidol and reduced haloperidol were 100 and 30 times higher, respectively, than those by HPLC-APCI-MS-MS; no spectrum with recognizable peaks was obtained for CPHP with the APCI interface. Therefore, detection limits and regression equations were examined by the HPLC-SSI-MS-MS for human plasma and urine samples spiked with the above drug and its metabolites. Haloperidol, reduced haloperidol, and CPHP showed good linearity in the ranges of 5-800, 10-800, and 100-800 ng/mL, respectively, for both human plasma and urine; their detection limits were 2.5, 5, and 75 ng/mL, respectively, using a new polymer HPLC column which enabled direct application of biological samples.     

High-performance liquid chromatographic method for the simultaneous determination of R-(-)- and S-(+)-hexobarbital in rat plasma

The enantiospecific determination of R- and S-hexobarbital in rat plasma is described. The method involves liquid-liquid extraction of racemic hexobarbital from plasma, separation of the underivatized enantiomers by high-performance liquid chromatography on an alpha 1-acid glycoprotein column and ultraviolet detection. The mobile phase consists of a phosphate buffer (pH 5.4) containing 0.4% 2-propanol as organic modifier. An alpha 1-acid glycoprotein guard column is used to increase the lifetime of the analytical column. Heptabarbital is the achiral internal standard. With detection limits of ca. 0.05 microgram/ml for both R- and S-hexobarbital, the assay is suitable for pharmacokinetic studies of the enantiomers in rats.     

Alpha 1-acid glycoprotein high-performance liquid chromatography column (EnantioPAC) as a screening tool for protein binding

An attempt was made to correlate retention behavior on a high-performance liquid chromatographic (HPLC) system employing an immobilized alpha 1-acid glycoprotein (AAG) column with AAG binding behavior for various compounds. Protein binding was assessed by propranolol displacement studies in an equilibrium dialysis system using isolated AAG. HPLC retention behavior poorly correlated with propranolol displacement from AAG. This system is not suitable for use as a screening tool for AAG affinity.     

Tandem electric field gradient focusing system for isolation and concentration of target proteins

Two electric field gradient focusing (EFGF) systems, one based on a hollow dialysis fiber and the other based on a shaped ionically conductive polymer were serially integrated to trap and concentrate selected proteins while simultaneously desalting and removing other unwanted proteins from the sample. A series of experiments were performed to test the EFGF systems individually and after integration. Online concentration of amyloglucosidase indicated a concentration limit of detection of approximately 20 ng mL(-1) (200 pM) from a sample volume of 100 microL. Concentration of human alpha1-acid glycoprotein with simultaneous removal of human serum albumin was also demonstrated. Elimination of small buffer components while concentrating trypsin inhibitor, and selective concentration and separation of myoglobin from a mixture were performed using the integrated EFGF system.     

Simultaneous determination of (R)- and (S)-naproxen and (R)- and (S)-6-O-desmethylnaproxen by high-performance liquid chromatography on a Chiral-AGP column.

A high-performance liquid chromatographic method for the simultaneous determination of both enantiomers of naproxen and its metabolite 6-O-desmethylnaproxen has been developed. The separation is performed on a column containing alpha 1-acid glycoprotein as the chiral selector. The method has been used for the determination of the enantiomeric purity of the drug substance and the metabolite, and for the simultaneous determination of all four compounds in biological fluids.     

Simultaneous analysis of azidothymidine and its mono-, di- and triphosphate derivatives in biological fluids, tissue and cultured cells by a rapid high-performance liquid chromatographic method.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro in the human MT-4 lymphocyte cell line did not require a prior extraction, and AZT and its phosphorylated derivatives could be accurately analysed in one HPLC run. However, plasma, bile, liver homogenate and urine samples could not be injected directly into the chromatograph. Therefore, a solid-phase extraction procedure was developed, using azidodideoxyinosine as internal standard. The extractions of the compounds of interest from all but urine samples were reproducible, with recoveries between 65% (AZT triphosphate from plasma) and 100% (AZT from plasma).     

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.     

固相萃取-高效液相色谱法同时测定食品塑料包装材料中9种光稳定剂

建立了同时测定食品塑料包装材料中9种紫外光稳定剂含量的高效液相色谱方法。样品用甲醇-乙酸乙酯混合溶剂超声提取,经固相萃取小柱净化后,以ZORBAX SB-C18柱(250 mm×4.6 mm, 5 μm)为分离色谱柱,甲醇和水为流动相,梯度洗脱,以310 nm为检测波长进行定性、定量分析。该方法前处理简单、易操作,9种紫外光稳定剂分离效果良好。9种紫外光稳定剂在0.2～10 mg/L范围内呈良好的线性关系,线性相关系数大于0.999;方法检出限为0.05～0.1 mg/L;实际样品中的加标回收率为70.2%～89.0%,相对标准偏差为0.4%～4.5%。该方法简单、准确,能够满足食品塑料包装材料中紫外光稳定剂的检测要求。     

Determination of polycyclic aromatic compounds by high-performance liquid chromatography with simultaneous mass spectrometry and ultraviolet diode array detection

A major problem in the determination of polycyclic aromatic compounds (PACs) in environmental samples is the extreme complexity of the extracts, even after extensive fractionation. The combination of high-performance liquid chromatography (HPLC) with simultaneous mass spectrometry (MS) and ultraviolet diode array detection (DAD) is a powerful tool for the identification and quantitation of such species with a high degree of confidence. HPLC allows the selective separation of a wide variety of PACs, including thermally labile and high molecular weight compounds. Electron ionization MS with the moving belt interface provides high sensitivity and selectivity, as well as structural information such as molecular weight, functional groups, and elemental composition. The diode array detector helps to differentiate isomeric structures and confirm compound identity.     

Immobilized artificial membrane chromatography coupled with atmospheric pressure ionization mass spectrometry

Liquid chromatographic separations on monolayers of cell membrane phospholipids covalently immobilized to silica particles at high molecular density is used for mimicking solute partitioning into biological membranes that generally correlates with membrane transport. This technique called immobilized artificial membrane chromatography usually employs ultraviolet (UV) detection where a single compound is analyzed in a chromatographic run limiting thereby its throughput for drug discovery applications. For coupling with atmospheric pressure ionization mass spectrometry, the phosphate-buffered saline mobile phase was replaced with one that used ammonium acetate as a volatile buffer. While atmospheric pressure chemical ionization accommodated a purely aqueous effluent, interfacing with electrospray ionization required effluent splitting and the addition of an organic modifier (5%, v/v, acetonitrile). Neuropeptide FF antagonists as early-phase drug candidates were used for the comparative evaluation of the methods. Whereas electrospray ionization produced essentially no fragment ions, several compounds involved in our study yielded low-abundance molecular ions with atmospheric pressure chemical ionization. The use of mass spectrometry yielded data that correlated well with those obtained by the method employing UV detection. Both atmospheric pressure ionization methods permitted the simultaneous determination of the k'(IAM), capacity factors and, therefore, an increased-throughput ranking of potential new leads emerged from the drug discovery process based on affinity to artificial membranes.     

Characterization of interactions between polyphenolic compounds and human serum proteins by capillary electrophoresis

The interaction of ten natural polyphenolic compounds (chlorogenic acid, apigenin, catechin, epicatechin, flavanone, flavone, quercetin, rutin, vicenin-2 and vitexin) with human serum albumin and mixtures of human serum albumin and α₁-acid glycoprotein under near physiological conditions is studied by capillary electrophoresis–frontal analysis. Furthermore, the binding of these polyphenolic compounds to total plasmatic proteins is evaluated using ultrafiltration and capillary electrophoresis. In spite of the relatively small differences in the chemical structures of the compounds studied, large differences were observed in their binding behaviours to plasmatic proteins. The hydrophobicity, the presence/absence of some functional groups, steric hindrance and spatial arrangement seem to be key factors in the affinity of natural polyphenols towards plasmatic proteins.     

Immobilized artificial membrane chromatography with mass spectrometric detection: a rapid method for screening drug-membrane interactions.

A high-performance liquid chromatography (HPLC)/electrospray ionization mass spectrometry method for measuring drug-membrane interactions was developed using immobilized artificial membrane (IAM) fast-screening mini-columns. The HPLC mobile phase consisted of phosphate-buffered saline (i.e., 5.0 mM phosphate buffer at pH 7.4, 1.35 mM KCl, and 68.5 mM NaCl) and acetonitrile. This method facilitated the measurement of IAM retention time of over ten compounds in one experiment, significantly reducing analysis time compared with the earlier IAM-HPLC method. The particular electrospray source used demonstrated the ability to tolerate the high salt-containing nonvolatile buffer used for retention time measurement.     

Determination of a new anti-allergenic agent, 1-[4-[3-[4-[bis-(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone, and its active acidic metabolite in plasma by high-performance liquid chromatography

High-performance liquid chromatographic (HPLC) methods were developed for the analysis of two compounds in a series of new antiallergenic agents, 1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone and its active acidic metabolite in plasma. The methods utilize ultraviolet or fluorescence detection, liquid-liquid extraction or solid-phase extraction and reversed-phase HPLC. The drugs were quantitated in samples from bioavailability studies performed in dogs. Calibrations were in the ng/ml concentration range for both compounds in plasma.     

High-performance liquid chromatographic separation and detection methods for anabolic compounds.

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

Simultaneous analysis of flunixin, naproxen, ethacrynic acid, indomethacin, phenylbutazone, mefenamic acid and thiosalicylic acid in plasma and urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

Simple and reproducible high-performance liquid chromatographic (HPLC) and gas chromatographic-mass spectrometric (GC-MS) methods have been developed for the simultaneous analysis of several acidic drugs in horse plasma and urine. Although the capillary GC-MS column provided better separation of the drugs than the reversed-phase C8 (3 microns, 75 mm) HPLC column, the total analysis time with HPLC was shorter than the total analysis time with GC-MS. The HPLC system equipped with a diode-array detector provided simultaneous screening (limit of detection 100-500 ng/ml) and confirmation (limit 1.0 micrograms/ml) of the drugs. The HPLC system equipped with fixed-wavelength ultraviolet and fluorescence detectors provided a relatively sensitive screening [limit of detection 50-150 ng/ml for ultraviolet and 10 ng/ml for fluorescence (naproxen only) detectors] of the drugs. However, the positive samples had to be confirmed by using either the diode-array detector or the GC-MS system. The GC-MS system provided simultaneous screening and confirmation of the drugs at very low concentrations (20-50 ng/ml).     

Simultaneous determination of rifampicin and sulbactam in mouse plasma by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method with ultraviolet detection has been developed and validated for the simultaneous determination of rifampicin and sulbactam in mouse plasma. Plasma samples were deproteinized with acetonitrile and separated by HPLC on a RP-18 (125 x 4 mm, 5 microm) column and gradient elution with potassium dihydrogen phosphate solution (pH 4.5; 50 mm) and acetonitrile at a flow-rate of 1.0 mL/min. Rifampicin and sulbactam were monitored at 230 nm and confirmed by means of their UV spectra using a diode-array detector. The method was linear at plasma levels from 1 to 100 microg/mL for rifampicin and from 5 to 200 microg/mL for sulbactam. The limits of quantification were 0.6 microg/mL for rifampicin and 4.2 microg/mL for sulbactam. The intra- and inter-day precisions of the method (RSD) were lower than 5% for both compounds. Average recoveries of rifampicin and sulbactam from mice plasma were 98.2 and 89.3%, respectively. The developed method was successfully applied to the determination of the pharmacokinetic profile of both compounds in mice.