Integrated on-line sample clean-up using cation exchange restricted access sorbent for the LC determination of atropine in human plasma coupled to UV detection.

A new, simple and fully automated liquid chromatographic (LC) method with UV detection has been developed for the direct determination of atropine in plasma. Sample clean-up was based on the use of cation exchange restricted access material (RAM) in a pre-column, coupled to LC by means of a column switching system. After direct injection of a 200 microl-volume of plasma sample, the biological matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate adjusted to pH 3.0 and methanol (97:3; v/v). By rotation of the switching valve, atropine was then eluted in the back-flush mode for 2 min and transferred to the analytical column packed with octadecyl silica by the LC mobile phase constituted of a mixture of acetonitrile and potassium phosphate buffer (pH 3.0; 50 mM) containing 2 mM sodium heptanesulfonate (16:84; v/v). The UV detection was performed at 220 nm. The method was validated according to a new approach based on accuracy profile over a concentration range from 25 ng/ml, corresponding to the limit of quantitation, to 1000 ng/ml. The method was then applied for the determination of atropine in plasma after intravenous administration to hospitalised patients.

Development and validation of a fully automated LC method for the determination of cloxacillin in human plasma using anion exchange restricted access material for sample clean-up.

In the framework of a preliminary investigation on the plasma profile of cloxacillin after oral administration, a simple and rapid LC method was developed for the direct determination of this compound in human plasma. The on-line sample clean-up was carried out using a weak anion exchanger (diethylaminoethyl groups) as restricted access material (RAM). The effects of the washing liquid pH, the ionic strength and the addition of organic modifier to the washing liquid were studied in order to obtain an efficient sample clean-up and a high recovery of cloxacillin. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of phosphate buffer (pH 4.0; 25 mM) and acetonitrile (72:28, v/v). The UV detection was performed at 215 nm. The most appropriate regression model of the response function as well as the limit of quantitation (LOQ) were first selected during the pre-validation step. These criteria were then assessed during the formal validation step. The LOQ was 50 ng/ml. The method was also validated with respect to analyte recovery, precision, trueness, accuracy and linearity. Finally, it was successfully applied for the analysis of the first plasma samples obtained from patients having taken an oral dose of 500 mg cloxacillin.

Use of a novel cation-exchange restricted-access material for automated sample clean-up prior to the determination of basic drugs in plasma by liquid chromatography.

A new kind of silica-based restricted-access material (RAM) has been tested in pre-columns for the on-line solid-phase extraction (SPE) of basic drugs from directly injected plasma samples before their quantitative analysis by reversed-phase liquid chromatography (LC), using the column switching technique. The outer surface of the porous RAM particlescontains hydrophilic diol groups while sulphonic acid groups are bound to the internal surface, which gives the sorbent the properties of a strong cation exchanger towards low molecular mass compounds. Macromolecules such as proteins have no access to the internal surface of the pre-column due to their exclusion from the pores and are then flushed directly out. The retention capability of this novel packing material has been tested for some hydrophilic basic drugs, such as atropine, fenoterol, ipratropium, procaine, sotalol and terbutaline, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The elution profiles of the different compounds and the plasma matrix as well as the time needed for the transfer of the analytes from the pre-column to the analytical column were determined in order to deduce the most suitable conditions for the clean-up step and develop on-line methods for the LC determination of these compounds in plasma. The cationic exchange sorbent was also compared to another RAM, namely RP-18 ADS (alkyl diol silica) sorbent with respect to retention capability towards basic analytes.

Evaluation of a novel anion-exchange restricted-access sorbent for on-line sample clean-up prior to the determination of acidic compounds in plasma by liquid chromatography.

A new kind of silica-based restricted-access material (RAM) with anionic properties has been tested in pre-columns for on-line solid-phase extraction of acidic compounds from directly injected plasma samples prior to their determination by reversed-phase liquid chromatography (LC), using the column-switching technique. The outer surface of the porous RAM particles contains hydrophilic diol groups while diethylaminoethyl (DEAE) groups are bound to the internal surface which gives the sorbent the properties of a weak anion exchanger towards low-molecular-mass compounds. Due to an appropriate pore diameter (about 6 nm), macromolecules, such as proteins, are physically excluded from the pores and flushed directly out during the sample clean-up process, while small compounds have access to the inner surface and can be retained mainly by electrostatic interactions. The retention capability of this novel packing material has been tested for some hydrophilic acidic compounds such as aspartic acid, glutamic acid, ascorbic acid and acetylcysteine as well as for some more hydrophobic drugs such as naproxen, ibuprofen and diclofenac, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The efficiency of the sorbent to clean-up complex matrices was also tested using human plasma and urine samples. A generic washing liquid composition was then selected in order to obtain efficient and selective sample clean-up as well as a high recovery of the acidic analytes.

Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for on-line sample clean-up.

A new automated method for the quantitative analysis of cyproterone acetate (CPA) in human plasma has been developed using on-line solid phase extraction (SPE) prior to the LC-MS/MS determination. The method was based on the use of a pre-column packed with internal-surface reversed-phase material (LiChrospher RP-4 ADS, 25 mm x 2 mm) for sample clean-up coupled to LC separation on an octadecyl silica stationary phase by means of a column switching system. A 30 microl plasma sample volume was injected directly onto the pre-column using a mixture of water, acetonitrile and formic acid (90:10:0.1 (v/v/v)) adjusted to pH 4.0 with diluted ammonia as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase consisting of water, methanol and formic acid (10:90:0.1 (v/v/v)). The dispensing flow rates of the washing liquid and the LC mobile phase were 300 microl min(-1). Medroxyprogesterone acetate (MPA) was used as internal standard. The MS ionization of the analytes was achieved using electrospray (ESI) in the positive ion mode. The pseudomolecular ionic species of CPA and MPA (417.4 and 387.5) were selected to generate daughter ions at 357.4 and 327.5, respectively. Finally, the developed method was validated according to a new approach using accuracy profiles as a decision tool. Very good results with respect to accuracy, detectability, repeatability, intermediate precision and selectivity were obtained. The LOQ of cyproterone acetate was 300 pg ml(-1).

Fully automated method for the liquid chromatographic determination of cyproterone acetate in plasma using restricted access material for sample pre-treatment.

A new fully automated method for the quantitative analysis of an antiandrogenic substance, cyproterone acetate (CPA), in plasma samples has been developed using on-line solid-phase extraction (SPE) prior to the determination by reversed-phase liquid chromatography (LC). The automated method was based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher RP-4 ADS) for sample clean-up coupled to LC analysis on an octadecyl stationary phase using a column-switching system. A 200-microL volume of plasma sample was injected directly on the precolumn packed with restricted access material using a mixture of water-acetonitrile (90:10, v/v) as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase which consisted of a mixture of phosphate buffer, pH 7.0-acetonitrile (54:46, v/v). The elution profiles of CPA and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were determined. Different compositions of washing liquid and mobile phase were tested to reduce the interference of plasma endogenous components. UV detection was achieved at 280 nm. Finally, the developed method was validated using a new approach, namely the application of the accuracy profile based on the interval confidence at 90% of the total measurement error (bias+standard deviation). The limit of quantification of cyproterone acetate in plasma was determined at 15 ng mL(-1). The validated method should be applicable to the determination of CPA in patients treated by at least 50 mg day(-1).

Fully automated LC method for the determination of sotalol in human plasma using restricted access material with cation exchange properties for sample clean-up.

A simple and rapid fully automated bio-analytical method for the liquid chromatographic (LC) determination of sotalol in human plasma has been described. The method is based on the use of a new kind of porous silica restricted access material (RAM) with cation exchange properties for sample clean-up. 100 microl of plasma samples were directly injected into the precolumn coupled on-line to a reversed-phase column (RP-Select B) by means of column switching system. The plasma matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate and methanol (97:3; v/v). By rotation of the switching valve, the analytes were then eluted in back-flush mode for 2 min and transferred to the analytical column by the LC mobile phase constituted of a mixture of methanol and 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM 1-octanesulphonic acid sodium salt (20:80; v/v). The flow-rate was 1.0 ml/min and sotalol was detected using fluorescence detection at 235 and 300 nm as excitation and emission wavelengths, respectively. The method was then validated using a new approach based on accuracy profile over a concentration range from 5 to 500 ng/ml. The limit of quantitation (LOQ) was 5 ng/ml and the total analysis time was 19 min.