Characterization of the precolumn bio trap 500 C18 for direct injection of plasma samples in a column-switching system

Summary A new restricted access media (RAM) type of precolumn, Bio Trap 500 C₁₈, for direct injection of plasma samples in column-switching systems was evaluated with respect to the elution of plasma proteins in different mobile phases, the loading capacity of plasma samples, the chromatographic behavior during plasma injections and protein contamination of the packing and sealings. More than 95% of plasma proteins could be excluded from the precolumn within three minutes for all selected mobile phases. Quantitative analyte recoveries could be obtained by injecting plasma samples ranging from 5 to 500 μL with the analyte mass>150 ng onto a BioTrap 500 C₁₈ column (20×4 mm I.D.). One precolumn tolerated about 15 mL of plasma injection without out noticeable change in retention and pressure. Clogging of the precolumn was encountered (≥45 mL of plasma) due mainly to the adsorption of proteins on the packing. The performance of the analytical column (Kromasil C₁₈) was also examined. The column efficiency decreased by 60% after processing 45 mL plasma in total.     

Simple high-performance liquid chromatographic method for the determination of a new phytochemical drug, fellavine, and its metabolites in human and rat plasma and urine.

The use of a small precolumn instead of an injection loop for the determination of a new phytochemical drug, fellavine, and its metabolites is described. The method combines the direct injection of plasma and urine into the reversed-phase precolumn with separation on a Spheri-5 RP-18 analytical column. Different sorbents in the precolumn were compared. A recovery of fellavine and its metabolites from biological fluids except rat plasma of almost 100% was achieved on Chrompack RP (30-40 microns) and LiChrosorb RP-18 (7 microns). For rat plasma only the last sorbent gave 80% fellavine recovery. The influence of the protein binding on the fellavine recovery was examined. The limit of detection was equal to 0.05 micrograms/ml fellavine for plasma and 0.02 micrograms/ml for urine. To enhance the limit of detection longer precolumns were perferred.     

Direct HPLC analysis of ketoprofen in horse plasma applying an ADS-restricted access-phase.

Making up part of the unique family of restricted access materials (RAM) the Lichrospher ADS (alkyl-diol silica) sorbents have been developed as special packing materials for precolumns used for LC-integrated sample processing of biofluids. The advantage of such phases consists of direct injection of untreated biological fluids without sample clean-up and elimination of the protein matrix together with an on-column enrichment. The plasma samples, with internal standard phenacetin added (not essential), were brought onto the precolumn (C-18 ADS, 25 micron, 25 x 4 mm i.d.) using a phosphate buffer, 0.1 M, pH 7.0. After washing with the buffer, the ADS column was backflushed with the mobile phase phosphate buffer 0. 05 M pH 7.0: acetonitrile (80:20), thus transporting the analytes onto a reversed-phase column Ecocart 125-3 HPLC cartridge with a LiChrocart 4-4 guard column, both packed with LiChrospher 5 micron 100 RP-18; after separation detection was performed in UV at 260 nm. Essential features of the method include the novel precolumn packing, the absence of sample pretreatment, a quantitave recovery, good precision and accuracy, as well as a considerable reduction of analysis time compared to conventional manual methods applied in bioavailability studies.     

A Miniaturized Precolumn for On-Line Trace Enrichment and Direct Plasma Injection in Narrow-Bore Liquid Chromatography

Abstract

A precolumn for on-line trace enrichment in narrow-bore liquid chromatography has been designed and some critical parameters like precolumn length, inlet capillary I.D. and flow-rate have been studied. The precolumn allows direct injection of plasma and serum samples without any previous clean-up. Due to the trace enrichment, concentration sensitivity is improved 50-fold when 100 μl samples are applied as compared to standard micro-loop injections of 0.5 μl. The applicability of the system has been demonstrated by the analysis of the anti-cancer drug etoposide (VP-16) in human plasma with comparable or even better results than those obtained with conventional-size systems.     

Application of the restricted-access precolumn packing material alkyl-diol silica in a column-switching system for the determination of ketoprofen enantiomers in horse plasma

The group of LiChrospher ADS (alkyl-diol silica) sorbents that make part of a unique family of restricted-access materials, have been developed as special packings for precolumns used in the LC-integrated sample processing of biofluids. The advantage of these sorbents lies in the direct injection of untreated biological fluids, that is without sample clean-up, the elimination of the protein matrix with a quantitative recovery together with an on-column enrichment. The present method is based on previous work applying UV detection at 260 nm for ketoprofen determinations. Plasma samples introduced to the ADS precolumn using a 0.1 M phosphate buffer, pH 7.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.01 M phosphate buffer-6% (v/v) 2-propanol-5 mM octanoic acid at a pH of 5.5, thus transporting the analytes to the chiral-HSA (human serum albumin) (100x4.0 mm) column where the separation of the ketoprofen enantiomers was achieved with a resolution factor of 1.4. The developed column-switching method was fully applicable to plasma injections.     

Use of direct injection precolumn techniques for the high-performance liquid chromatographic determination of the retinoids acitretin and 13-cis-acitretin in plasma.

A previously developed highly sensitive high-performance liquid chromatographic method for the determination of retinoids, using direct injection of large plasma volumes, on-line solid-phase extraction and ultraviolet detection, was improved and fully validated for the determination of acitretin and 13-cis-acitretin in plasma samples. The addition of acetonitrile to improve the recovery was performed on-line by a T-piece, avoiding any cis-trans isomerization which could occur when acetonitrile was added prior to storage in the autosampler. About 30 injections could be made onto one precolumn despite the large injection volume (1 ml of plasma containing the internal standard). Full automation was attained by the use of automated precolumn replacement. In addition, forward- and back-flush purging of the precolumn enhanced the longevity of the analytical column. This consisted of three coupled C18 columns of 125 mm length each. The quantification limit was 0.3 ng/ml, using ultraviolet detection at 360 nm, and the mean inter-assay precision was 3.8% for the two compounds.     

Alkyl-Diol Silica (ADS): restricted access precolumn packings for direct injection and coupled-column chromatography of biofluids

The direct and repetitive injection of untreated biological fluids (e.g., hemolyzed blood, plasma, serum, cell culture and tissue homogenates) onto an HPLC-system and the subsequent analysis of low-molecular weight compounds (e.g. drugs, xenobiotics, metabolites) is rendered possible by a coupled-column configuration and special precolumn packings. For this purpose a new family of chemically and enzymatically tailored reversed-phase packing materials have been prepared. The LC-integrated sample clean-up with these restricted access (bimodal) phases is based on the complete nonadsorptive size exclusion of macromolecules (e.g. proteins) and on the simultaneous dynamic partitioning of the target molecules. The bonded phase which exclusively covers the internal pore surface of a glyceryl-modified silica is a butyryl-(C-4), capryloyl-(C-8) or stearoyl-(C-18) moiety. These ligands allow a classical reversed-phase or ion-pair chromatography during the sample work-up step. The capacity of the n-alkyl phase is comparable with conventional silica based RP-materials. The broad hydrophobic retentive capability of these packings allows the extraction of a wide variety of compounds of biomedical interest. The electroneutral and hydrophilic particle exterior (glyceryl-residues) was generated using either soluble or immobilized enzymes (lipase, esterase) which cleave the fatty acid esters exclusively at the outer surface. Unwanted macromolecular components of a sample (e.g. proteins) are quantitatively eluted in the void volume due to the restricted access given by the pore size (6 nm) and the nonadsorptive external diol coverage. The lifetime of a precolumn (25 × 4 mm I.D.) packed with these novel bimodal, i.e. RP-SEC phases exceeds more than 200 injections of 500 l plasma. In addition to the synthesis, this paper describes an application of each of these Alkyl-Diol Silica (ADS) precolumn packings in fully automated coupled-column HPLC systems for the analysis of drugs and endogenous compounds in different biological matrices.Dedicated to Professor Dr. Dr. h.c. mult. J.F.K. Huber on the occasion of his 70th birthday     

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.     

The use of precolumns for solvent focusing in capillary column gas chromatography

In trace analysis by capillary GC it is often desirable to use larger than normal injection volumes to obtain sufficient sensitivity. This, however, results in a wider solvent peak and tailing, and may reduce column efficiency. This paper describes the use of a short length of a capillary precolumn coated with a stationary phase of polarity similar to that of the sample solvent and a film thickness greater than that of the analytical column; provided the right combination of polarities of injection solvent and liquid phases are used, the precolumn focuses the solvent band, thereby enabling the maximum injection volume to be increased without measurably reducing efficiency. Typical precolumn dimensions are 1 m length, 0.32 mm i.d., and 0.5 μm stationary phase film thickness. Using a precolumn increases the maximum injection volume up to 8 or 10 μl, or three times that appropriate for a conventional analytical column, with little or no loss in efficiency.     

Sample preparation for gas chromatographic determination of halogenated volatile organic compounds in environmental and biological samples

In this review, the wide spectrum of the techniques of isolation and/or preconcentration and final determination of halogenated volatile organic compounds (HVOCs) in water, air, soil, sediment and biological fluids are presented and discussed. The techniques discussed are solvent microextraction, solid phase extraction, gas extraction (static and dynamic techniques), membrane processes and passive sampling. Also, direct techniques, such as direct aqueous injection into gas chromatography (GC) column and membrane inlet mass spectrometry, are presented. Main attention is paid to the practical application of these techniques during all HVOCs determination.     

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.     

Direct analysis of crude plasma samples by turbulent flow chromatography/tandem mass spectrometry

Summary Turbulent flow chromatography coupled to tandem mass spectrometry (TFC-MS-MS) has recently emerged as a potentially fast, sensitive and specific technique for the direct analysis of pharmaceutical compounds from crude plasma. TFC-MS-MS removes the need for time-consuming sample preparation procedures such as solid-phase extraction (SPE) or liquid-liquid extraction (LLE). A relatively high flow rate combined with the use, of an HPLC column with large porous particles allows the on-line clean up and quantification of compounds in plasma samples. Until, now, the amount of plasma directly injected into TFC systems has rarely exceeded 30 μL in order to prevent rapid column degradation. Increasing the injection volume also induces high carry-over levels, particularly for drugs with basic and/or lipophilic properties. This paper describes the first genetic TFC-MS-MS method developed in a 96-well format, which allows the direct injection of 200 μL of 1∶1 diluted plasma (equivalent to 100 μL neat plasma). An average, of 390 injections was carried out with each extraction column. More than 2000 dog plasma samples were injected into the system without any sign of carryover. The method was fully validated over a 5–500 ng mL⁻¹ range for three basic compounds: doxazosin, CP122,288 and dofetilide. The imprecision was 1.2 to 8.3% for doxazosin, 1.5 to 4% for CP122,288 and 1.6 to 9.2% for dofetilide. The inaccuracy ranged from 6% to 7.9%. This generic methodology was then used to assay two structurally unrelated development compounds, showing that the method accuracy and sensitivity were adequate for the early pharmacokinetic (PK) studies in animals.     

浸透限制固定相及其应用

讨论了浸透限制固定相的特性及其在药物分析中的应用。已发展起来的浸透限制固定相主要用于高效液相色谱法对生物体液中药物的直接进样分析。这种固定相填料是在疏水层上覆盖了一层亲水层,而亲水层允许小分子物质如药物自由出入于固定相的疏水部分,从而立体地阻止了大分子物质如蛋白质与固定相的疏水部分相互作用,使小分子物质得到分离。     

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).     

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.     

A new type of two-dimensional packed+capillary column GC system. III. Instrumentation

Summary The constractional details of precolumns, its oven and connector have been given with two types of multiflow control modules. Two types of phases made in China, a silanized pellicular beads and a non silanized pellicular silica beads, with an optimum particle size of 20μm to 30μm were packed in the precolumn 20mm in length with the column efficiency of 4620 plates/meter at column temperature of 49°C. A 25m×0.22mm i.d. capillary column coated with bonded methylsilicone phase from Chrompack connected with the precolumn (250°C) has an efficiency of 5376 plates/meter for n-octane. But it decreased to 3149 plates/meter by decreasing the precolumn temperature to 130°C.     

Determination of the N-methyl- -aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl- -aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75×4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate–ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250×4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.     

Combining restricted access material (RAM) and turbulent flow for the rapid on-line extraction of the cyclooxygenase-2 inhibitor rofecoxib in plasma samples

Restricted access material (RAM) has been used in the packing of a solid-phase extraction (SPE) column for on-line extractions under turbulent flow conditions. The bio-compatible RAM material works by the principle of size exclusion in addition to conventional reversed-phase chromatography, thereby allowing the extraction and preconcentration of small analyte molecules from biological samples such as plasma. Using small column dimensions (0.76 mm x 50 mm) and a consequently high linear velocity, turbulent flow was achieved during online sample extractions. The improved mass-transfer rate characteristic of turbulent flow allows fast sample cleanup without decreased extraction efficiency. The novel use of the RAM column, connected upstream to a C18 monolithic column, allowed the direct injection, extraction, separation, and MS/MS detection of plasma samples spiked with rofecoxib in a span of 5 min. Calibration curves obtained using this RAM turbulent flow coupled column method showed good linearity (R2 > 0.99) and reproducibility (%RSD < or = 7%). The lower limit of quantitation of rofecoxib in plasma samples was found to be 40 ng/ml. The extraction method showed good recovery of rofecoxib from a plasma matrix with minimal signal loss and robustness after more than 200 plasma injections.     

The Use of Non-Splitting Injection Techniques for Trace Analysis in Narrow-Bore Capillary Gas Chromatography

The use of hot splitless, cold splitless, and on-column injections for trace analysis in narrow-bore capillary GC is evaluated. Despite the low flow rates for the columns used, the required splitless times for splitless injections can be surprisingly short if liners with a small inside diameter are used. On-column injection can be applied by using an appropriate normal-bore precolumn coupled to the narrow-bore analytical column using a specially designed low dead volume column connector. The effects of the experimental conditions such as sample volume, injection temperature, and initial oven temperature on peak focusing and the discrimination and degradation behavior of the analytes are discussed. The possibilities to obtain sensitive and fast separations are illustrated by various applications.     

Determination of the N-methyl-D-aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl-D-aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75x4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate-ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250x4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.