反相高效液相色谱法测定动物体液和组织中的HMBA

介绍了用甲醇沉淀萃取法、反相高效液相色谱法分离测定生物样品中的抗癌药物六亚甲基二乙酰胺（ＨＭＢＡ）的新方法。药物的回收率在９３．３％～１０４％之间。对鼠和兔的血、尿及８种组织１５３个样品中的ＨＭＢＡ进行了测定,得到了满意的结果。     

Gas Chromatographic Determination of Hexamethylene Bisacetamide in Plasma and Urine

Abstract

A rapid, simple and specific gas chromatographic method has been developed to measure the differentiating agent hexamethylene bisacetamide (HMBA) in biological samples. Addition of a homolog as an internal standard, ultrafiltration and then direct packed column GC-FID analysis of the ultrafiltrate gives a detection limit of less than 50 μg/ml (0.25 mM) for HMBA in plasma or urine. Ultrafiltration is quantitative and assay precision is better than 4.3% for the 1–5 mM range. This method has been applied to determine the bolus dose pharmacokinetics and disposition of HMBA in a single small animal such as a rat. The developed assay should be suitable for therapeutic monitoring of human patients undergoing HMBA treatment.     

Determination of ritodrine in biological fluids of the pregnant sheep by fused-silica capillary gas chromatography using electron-capture detection

A sensitive and selective gas chromatographic assay method employing splitless injection, fused-silica capillary columns and electron-capture detection is reported for the quantitation of the tocolytic drug, ritodrine, in a variety of biological fluids obtained from the pregnant ewe and fetus. This method has improved sensitivity and selectivity over previously published assay procedures. A 25 m x 0.31 mm I.D., cross-linked 5% phenylmethylsilicone, fused-silica capillary column was employed for all analyses. Linearity of response was observed over the range 2.5-75 ng of ritodrine base per 0.05-0.5 ml of biological fluid, representing approximately 1-75 pg at the detector. The coefficient of variation was less than 10% over the range 2.5-75 ng of added ritodrine. The minimum quantifiable amount is approximately 2.5 ng from a 0.5-ml biological fluid sample. Applicability of this method to biological fluids, obtained from ovine subjects, is demonstrated by the analysis of samples obtained during the course of ritodrine placental transfer studies.     

Liquid chromatographic analysis of doxazosin in human serum with manual and robotic sample preparation

A specific method for the determination of the antihypertensive drug doxazosin in human serum is described. The method utilizes the related drug prazosin as an internal standard and is based on a simple extraction scheme followed by analysis by reversed-phase ion suppression high-performance liquid chromatography (HPLC) on an alumina-based column with fluorescence detection. The method is completely automated with a flexible robotic system for the analysis of drugs in biological fluids. The robotic automation of the method allows a 20% increase in the sample throughput and the savings of about 7 man-hours a day. Both the manual and robotic procedures yield precise quantitative results over the therapeutically relevant concentration range of 0.5 to 20 ng/mL of serum.     

Biological Sample Injection for Open Tubular Liquid Chromatography

Abstract

Two injection procedures for open tubular liquid chromatography have been evaluated for their ability to directly sample biological components in minute volumes of body fluids. The techniques provided R.S.D.s in peak heights of less than 12% for injections of low nL volumes of human serum spiked with a test compound. A dynamically modified open tubular column was employed to separate the anti-tumor drug doxorubicin hydrochloride and two of its metabolites.     

On-line coupling of sequential injection extraction with restricted-access materials for sample clean-up and analysis of drugs in biological matrix

In this contribution, the on-line coupling of solid phase extraction (SPE), based on a restricted-access material (RAM), with sequential injection technique (SIA) for the analysis of biological samples, is described. The SIA-RAM system was tested with a new potential antileucotrienic drug (VUFB-19363 (Quinlukast)) for serum analysis. The method is based on SPE with the novel internal-surface reversed-phase column packing material-alkyl-diol silica (ADS). The supports tolerate direct and repetitive injection of proteinaceous fluids (plasma, serum) and allow reversed-phase partitioning at the internal surface. A column packed with a 25 microm C18 alkyl-diol support was used for direct serum injection. Using a 6-port selection valve and the system of three mobile phases, the polar matrix compounds and metabolites are removed by sequentially aspirated mobile phases with lower content of the organic part (methanol-water (2:98) and following acetonitrile-water (20:80)) to the waste, and then, the analyte enriched on the column is eluted by a strong mobile phase (acetonitrile-methanol-water (40:20:40)) to the UV detector without transfer loss. With the fully automated SIA system, a total analysis time of less than 10 min was achieved. The only off-line sample pre-treatment step required to remove particulate matter was centrifugation. The studies showed a range of linearity (2-40 microg ml(-1)) and a high recovery (93.6-96.8%) of drug from the biological matrix with coefficients of variation (RSD) less than 5.0% (n = 6). This paper introduces a new, simple and robust analytical technique suitable for screening determination and direct analysis of drugs in biological materials.     

Aspects of trapping efficiency and matrix effects in the development of a restricted‐access‐media‐based trap‐and‐elute liquid chromatography with mass spectrometry method

Online restricted access media with liquid chromatography and tandem mass spectrometry for the direct analysis of small molecules in biological fluids represents an interesting alternative to time‐demanding traditional sample preparation techniques. In this study, important considerations concerning the development of a restricted access media with liquid chromatography and tandem mass spectrometry method for the analysis of dansylated estrogens in biological matrix are presented. Parameters influencing peak tailing and trapping efficiency were evaluated. The key factors included the ion strength of the mobile phase, a loading flow rate of the sample onto the trap column, and selection of a proper stationary phase of the trap column for a given set of analytes. These parameters have proven to be essential for minimizing any unwanted chromatographic peak tailing. The bulk derivatization of the analytes in the biological fluids and its relationship to the observed matrix effects was evaluated as well.     

Liquid Chromatographic Assay for Amodiaquine in Tablets and Biological Fluids

Abstract

A high performance liquid chromatographic assay for quantitating amodiaquine (A) in tablets, urine, plasma, bile and saliva is described. The method involved acid extraction of the drug from tablets and chloroform extraction of its base from the biological fluids after alkalinization with ammonia. Quinidine was used as the internal standard. A μ-Bondapak phenyl column was used for separation together with a mobile phase made of methanol, water and glacial acetic acid (pH 2.3). Good chromatograms with efficient separation of drug and internal standard peaks were observed. Retention times of 5.2 and 7.1 min. were obtained for the drug and the internal standard respectively. Correlation between areas under the curve and drug concentration was high. The mean percentage recovery of A from tablets was 102.03%, while from the biological fluids, it ranged from 85.2 to 104.61%. Urine and saliva obtained from volunteers and bile obtained from animals administering amodiaquine showed chromatograms similar to those obtained for blank samples spiked with A. Interference from table't excipients of biological fluids was undetectable or negligible. The method was found to be precise and simple.     

High-performance liquid chromatographic separation of the enantiomers of hydroxychloroquine and its major metabolites in biological fluids using an alpha 1-acid glycoprotein stationary phase.

An enantioselective two-stage off-line assay has been developed for the analysis of hydroxychloroquine and its three major metabolites in biological fluids. The first non-stereoselective stage of the assay (PRP-1 column) separates and quantitates parent drug and metabolites. Fractions containing hydroxychloroquine and each of the metabolites are collected manually, evaporated, reconstituted in mobile phase and re-injected onto an alpha 1-acid glycoprotein column to separate and determine proportions of individual enantiomers. Preliminary results from patients samples indicate that the disposition of hydroxychloroquine and its major metabolites is enantioselective. p6     

Liquid-solid extraction and HPLC determination of amiodarone metabolites in biological fluids

Summary An improved liquid-solid extraction procedure for the purification and determination of amiodarone and desethylamiodarone in biological fluids is proposed. The sample is passed through a silica-C₁₈ column, and after washing, the analytes are eluted with methanol. The determination is then accomplished by RP HPLC using an octadecyl silica column and a mobile phase of methanol containing 0.0015% of ammonium hydroxide. The effect of ammonia concentration on the capacity factors of the analytes has been used for estimating the acid dissociation constants of the investigated secondary and tertiary amines in methanol.     

Application of comprehensive two-dimensional gas chromatography to drugs analysis in doping control

Comprehensive two-dimensional gas chromatography (GC×GC) now occupies a niche within the GC technology regime. The technique is undeniably unique in the manner in which the experiment is conducted, the way results are presented and the interpretive opportunities offered. For the 1000th volume of this journal it is appropriate to expand upon these features, and review the progress made in GC×GC to date. Firstly, brief general comment is made on multidimensional procedures, and to review key aspects of GC×GC. The use of the targeted multidimensional GC method allows absolute retentions in the second dimension of a GC×GC experiment to be estimated, and also offers a novel way to obtain enhanced response for resolved solutes. Then, to illustrate the utility of the technique, the application of GC×GC to the screening of drugs and their metabolites in biological fluids is described using prolintane metabolites in canine urine as an example, with samples taken at four time intervals after administration. This example illustrates the first application of GC×GC in the field of forensic toxicology, an area traditionally dominated by GC–MS. Most drug compounds were found to be retained on the 0.8-m second column for a greater time than the modulation period (3 s) used for initial analysis, under the conditions described. Hence a 0.4-m D2 BPX50 (50% phenyl methyl polysilphenylene) column was then used throughout, with most compounds retained less than 4 s. For the standard drug mixture, three overlapping drugs on the first dimension column (BPX5) were subsequently baseline resolved on the BPX50 column. For prolintane administration samples, the parent drug and metabolites could be effectively resolved from background matrix peaks. Likewise a 23-drug spike standard in horse urine blank gave acceptable resolution of the drugs from matrix peaks.     

The role of nitric oxide in cancer improved methods for measurement of nitrite and nitrate by high-performance ion chromatography

The short lifetime of nitric oxide (NO) in vivo impedes its quantitation directly; however, the determination of nitrite and nitrate ions as the end-products of NO oxidation has proven a more practical approach. High-performance ion chromatographic analysis of nitrite in biological fluids is hampered by the large amount of chloride ion (up to 100mmol/l) which results in insufficient peak resolution when utilizing conductimetric detection. Analysis of both anions in small sample volumes is also constrained by the need to minimise sample handling to avoid contamination by environmental nitrate. We report a means to remove Cl⁻ ions from small sample volumes using Ag⁺ resin which facilitates quantitation of either nitrite and nitrate anions in biological samples, using silica or polymer based ion-exchange resins with conductimetric or electrochemical and spectrophotometric detection. Including a reversed-phase guard column before the anion-exchange guard and analytical column also greatly extends column lifetime.     

Determination of melagatran in rabbit plasma by high-performance liquid chromatography with automated column switching

Melagatran is an active thrombin inhibitor showing oral and parenteral bioavailability for antithrombotic therapy. A simple and convenient liquid chromatographic method has been developed and applied to the analysis of melagatran in rabbit plasma. The clean-up and separation of the sample solutions were performed by automated on-line column switching HPLC. The method validation shows the suitability of the column switching liquid chromatographic system for the quantitation of melagatran in biological fluids.     

Microextraction techniques in antibiotic monitoring in body fluids: Recent trends and future

The analysis of drugs in biomedical discipline targets a broad range of aims such as therapeutic drug monitoring, pharmacokinetic study to investigate the drug bioavailability, bioequivalence tests to evaluate the effect of formulation parameters, toxicology, and forensic science. Because of the low levels of typical antibiotics in plasma, blood, urine, exhalation samples, and other biological fluids as well as complex matrix of biological media, adequate sample preparation methods should be implemented for quantification of antibiotics. In this review, developments in well-established microextraction techniques for the clinical analysis of biological samples will be reviewed and discussed. This article presents an overview of microextraction methods for biological samples, focusing especially on antibiotics.     

Large-bore coated columns for sampling and concentration of organic volatiles in air, headspace and water analysis

Large-bore coated (LBC) columns were used as sampling and concentrating traps in analyses for traces of organic volatiles in air and water. This simple technique utilizes long metal columns thinly coated with SE-30 for direct trapping of the organics. The sample is simply passed through the LBC column; the trapped organics are then thermally desorbed onto a conventional porous polymer pre-column or onto a second LBC column. If desired, this can be shorter or narrower bore than the initial LBC sampling column. The sample is finally desorbed onto the gas chromatographic column for analysis. Multiple transfers between LBC columns are possible, with increased concentration at each transfer, resulting in a "concentration pump" effect. The technique offers the advantages of great simplicity, efficiency and ease of sample transfer. Samples are obtained with low back-pressure and minimal interfering artifacts. The system shows almost complete imperturbability to moisture. Indifference to moisture and the low back-pressure enable direct sampling of very large volumes of air and even breath. Direct sampling of aqueous systems was also possible. The latter area was not fully investigated but offers potential for water pollution analysis and in direct examination of biological fluids and aqueous flavor extracts where heat sensitivity is a problem. With LBC columns the sampling and concentration sequence exposes the substances sought to no more drastic conditions than those they will be subjected to in the process of gas chromatographic analysis.     

Quantification of small molecules in plasma with direct analysis in real time tandem mass spectrometry, without sample preparation and liquid chromatographic separation

Recently, a new ion source, Direct Analysis in Real Time (DART), has been introduced which allows direct biological sample introduction into a mass spectrometry (MS) system. The elimination of conventionally required sample preparation and separation by high-performance liquid chromatography (HPLC) prior to MS analysis represents a remarkable opportunity to reduce assay turn-around time, environmental impact and capital/manpower investment. This new technology initially was used in various qualitative applications to directly detect chemicals on solid surfaces, in liquids and gases. In this study, a DART source operating under ambient pressure with ground potential was installed onto a Sciex 4000 tandem mass spectrometer and employed in the sample analysis of plasma based on direct introduction into the DART-MS/MS system. Reasonable precision and accuracy (%CV and %Error, both <10%) were achieved of a significant number of compounds tested in biological fluids. In addition, the limit of detection for 80% of the tested compounds reached 5 ng/mL or lower which is sufficient for pharmaceutical drug discovery support. Finally, experimental conditions that significantly impacted assay performance were investigated with respect to optimization and limitation. Because of its simplicity, fast data acquisition (3-5 s) and low cost, DART has the potential to significantly impact quantitative pharmaceutical analysis in biological matrices.     

HPLC柱切换技术在临床药物分析中的应用

综述了近十年来高效液相色谱柱切换技术在药物分析中的应用情况，主要介绍了柱切换技术在生物样品纯化，富集和手性分离方面的实际应用，该技术可用于直接进样分析，特别适用于临床药物分析，可和于药代动力学，生物利用度等研究。     

Reverse Phase HPLC Determination of Azq in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method employing a simple sample preparation procedure and utilizing an internal standard was developed to measure the new antitumor agent AZQ in biological fluids. A single chloroform extraction gave drug recoveries of greater than 88% from plasma, urine and CSF in the range of expected physiological concentrations (20–800 ng/ml). Isocratic reverse phase HPLC with UV detection at 340 nm resulted in a limit of quantisation of 5 ng/ml although smaller amounts of the drug could be detected. This assay was successfully applied to determine the single dose plasma pharmacokinetics of AZQ in rats. The potential of this method for determining AZQ disposition and pharmacokinetics in human subjects was demonstrated by analysis of patient CSF.     

Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic-coated capillaries

In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6 min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4 x 10? plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.     

Bio-compatible in-tube solid-phase microextraction capillary for the direct extraction and high-performance liquid chromatographic determination of drugs in human serum

A restricted access material (RAM), alkyl-diol-silica (ADS), was used to prepare a highly bio-compatible solid-phase microextraction (SPME) capillary for the automated and direct in-tube extraction of several benzodiazepines from human serum. The bifunctionality of the ADS extraction phase prevented fouling of the capillary by protein adsorption while simultaneously trapping the analytes in the hydrophobic porous interior. This the first report of a restricted access material utilized as an extraction phase for in-tube SPME. The approach simplified the required apparatus in comparison to existing RAM column switching procedures, and more importantly eliminated the excessive use of extraction solvents. The biocompatibility of the ADS material also overcame the existing problems with in-tube SPME that requires an ultrafiltration or other deproteinization step prior to handling biological samples, therefore further minimizing the sample preparation requirements. The calculated oxazepam, temazepam, nordazepam and diazepam detection limits were 26, 29, 22 and 24 ng/ml in serum, respectively. The method was linear over the range of 50-50 000 ng/ml with an average linear coefficient (R2) value of 0.9998. The injection repeatability and intra-assay precision of the method were evaluated with five injections of a 10-microg/ml serum sample (spiked with all compounds), resulting in an average RSD<7%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.