Synthesis of mixed-functional-phase silica supports for liquid chromatography and their applications to assays of drugs in serum.

Mixed-functional-phase (MFP) silica supports were designed for the direct injection determination of drugs in serum. The MFP silicas were synthesized from porous silica materials in three or four steps: introduction of 3-glycidoxypropyl phases, introduction of phenyl, butyl or octyl phases and hydrolysis of the oxirane ring to diol phases, or these three steps plus further introduction of glycerylpropyl (i.e., diol) phases. Although the further introduction of glycerylpropyl phases resulted in a reduction in the column efficiency, serum proteins were completely recovered in the first injection of serum samples. The prepared MFP packing materials can be used for the direct injection determination of hydrophobic and hydrophilic drugs in serum.

Characterization of an internal-surface reversed-phase silica support for liquid chromatography and its application to assays of drugs in serum.

Internal-surface reversed-phase (ISRP) silica supports having N-octanoylaminopropyl phases bound to the internal surfaces of the porous silica and N-(2,3-dihydroxypropyl)aminopropyl phases bound to the external surfaces were synthesized from silica particles differing in nominal pore diameters and specific surface areas. These ISRP supports were characterized with regard to physical and chromatographic properties. The support with an N-octanoylaminopropyl phase coverage of 485 mumol/g and an average pore diameter of 65 A was the most suitable for the direct-injection determination of hydrophilic or hydrophobic drugs in serum or plasma. Non-steroidal anti-inflammatory (acetylsalicylic acid and salicylic acid) and tricyclic antidepressant drugs (desipramine and nortriptyline) in serum were successfully determined with this support and an acidic eluent.

Beta-cyclodextrin bonded silica for direct injection analysis of drug enantiomers in serum by liquid chromatography.

A new beta-cyclodextrin (CD) bonded silica has been designed for direct injection analysis of drug enantiomers in serum for liquid chromatography. The new beta-CD bonded silica is synthesized by three steps: introduction of a 3-glycidoxypropyl phase, introduction of a beta-CD carbamate-bonded phase, and hydrolysis of the oxirane ring to a diol phase. Although the recovery of serum proteins from only a beta-CD bonded silica (having no diol phase) was poor, they were completely recovered from a mixed functional silica having beta-CD and diol phases. Direct injection analysis of drug enantiomers in serum can be attained with the mixed functional silica support over the eluent pH range of 3-7. The recovery of racemic drugs from serum was almost 100%.

Internal-surface reversed-phase silica support for direct injection determination of drugs in biological fluids by liquid chromatography.

A new internal-surface reversed-phase (ISRP) silica support has been designed for direct injection analysis of drugs in biological fluids by liquid chromatography. The support, prepared by using a new enzyme, polymyxin acylase, has N-octanoylaminopropyl phases, bound only to the internal surfaces of the porous silica, and N-(2,3-dihydroxypropyl)-aminopropyl phases on the external surfaces in order to be nonadsorptive to proteins. The average pore diameter of the prepared ISRP silica support was 50 A, which is small enough to exclude macromolecules such as serum proteins from the pores. The new ISRP support can be used for the direct injection analysis of hydrophilic and hydrophobic drugs in serum or plasma without destructive accumulation of proteins over the eluent pH range of 3 to 7. The recovery of drugs from serum was almost 100%, regardless of the difference in their protein bindings.

Determination of cyclodextrins and branched cyclodextrins by reversed-phase chromatography with pulsed amperometric detection and a membrane reactor.

A high-performance liquid chromatographic method has been developed for the determination of cyclodextrins (CDs) and branched CDs. The method involves their separation on a reversed-phase column using a mixture of water and acetonitrile as an eluant, eluant pH modification with a cation-exchange membrane reactor surrounded by 1.5 M sodium hydroxide solutions, and pulsed amperometric detection with a gold working electrode. The calibration graphs constructed by peak height versus injected amount were linear over the ranges 50-1000 pmol. The detection limits for CDs and branched CDs were about 1-5 pmol at a signal-to-noise ratio of 3. The method was successfully applied to the assay of beta-CD in serum samples.

Direct serum injection in micellar liquid chromatography. Recovery of serum proteins and assay of hydrophilic drugs.

The recovery of serum proteins from reversed-phase and internal-surface reversed-phase (ISRP) silica supports following direct serum injection was investigated using an eluent containing a micellar solution of sodium dodecyl sulphate (SDS). The results indicated that the recoveries of serum proteins were 98-103% for both supports. On the basis of the above findings, the separation and recovery of hydrophilic drugs (cephalosporins and salicylic acid) from human serum were investigated using acidic eluents including micellar solutions of SDS. They were completely separated from the components of serum, and the recoveries were 94-98% despite protein binding. Although the recommended eluent pH range is 6.0-7.5 for the ISRP support, eluents of pH 2-8 can be used with the micellar chromatographic system.

Liquid chromatographic determination of penicillins by postcolumn degradation with sodium hypochlorite using an hollow-fibre membrane reactor.

A sensitive, high-performance liquid chromatographic method involving postcolumn degradation with sodium hypochlorite and using a hollow-fibre membrane as a reactor is described for the determination of penicillins. Penicillins were separated on a C18 column followed by postcolumn reaction with sodium hypochlorite and sodium hydroxide using aminated and sulphonated hollow-fibre membrane reactors immersed in each solution, and detected at 270-280 nm based on the UV absorbances of the degradation products. At penicillin concentrations of 2 micrograms/ml, the precisions (relative standard deviation) were 2.28-4.78%. The detection limits of the proposed method were 2.5-25 ng for each penicillin at a signal-to-noise ratio of 3. Ampicillin and its metabolites [(5R,6R)-ampicilloic acid, the (5S,6R)-epimer and (2R)-pierazine-2',5'-dione] in human serum and urine were simultaneously determined by this method.

Liquid chromatographic determination of amino acids using a hollow-fiber membrane reactor.

A high-performance liquid chromatographic method using a passive hollow-fiber membrane reactor (HFMR) has been developed for the determination of amino acids. This method involves gradient elution of 18 common amino acids on a C18 column using sodium octanesulfonate as an ion-pairing agent, postcolumn derivatization with o-phthalaldehyde and 2-mercaptoethanol (combined with hypochlorite oxidation for the imino acid proline) introduced into the main flow stream using sulfonated and aminated HFMRs immersed in the reagent solutions, and fluorometric detection of derivatives (lambda ex = 340 nm, lambda em = 450 nm). The detection limits of the proposed method were 0.4 to 20 pmol for each amino acid at a signal-to-noise ratio of 3. For 18 amino acids in amounts of 250-2500 pmol, the precisions were on the order of 1.5-4.8% (relative standard deviation, n = 10). The assay of amino acids in a protein hydrolysate sample by the proposed method is also described.

Liquid chromatographic determination of penicillins by postcolumn alkaline degradation using a hollow-fiber membrane reactor.

A high-performance liquid chromatographic method using a hollow-fiber membrane reactor is described for the determination of penicillins. This method involves separation of penicillins on a C18 column, postcolumn reaction with sodium hydroxide and mercury (II) chloride introduced into the main flow stream using sulfonated hollow-fiber membrane reactors immersed in each solution (4 M sodium hydroxide and 3 X 10(-2) M mercury (II) chloride plus 10(-2) M nitric acid), and detection at 290 nm based on the uv absorbance of the degradation products. At penicillin concentrations of 5 micrograms/ml, within- and between-run precisions (relative standard deviation) were 0.24-2.39 and 1.19-4.13%, respectively. The detection limits of the proposed method were 1-5 ng at a signal-to-noise ratio of 3. The method was applied to assays of ampicillin and its metabolites in human serum and urine.

High-performance liquid chromatographic determination of ampicillin and its metabolites in rat plasma, bile and urine by post-column degradation with sodium hypochlorite.

A high-performance liquid chromatographic method has been developed for the determination of ampicillin (1) and its metabolites [(5R,6R)-ampicilloic acid (2), the (5S,6R)-epimer (3) and the corresponding (2R)-piperazine-2',5'-dione (4)] in rat plasma, bile and urine. The method involves the separation of 1-4 from the background components of the biological fluids on a reversed-phase C18 column, using sodium heptanesulphonate as an ion-pairing agent and methanol in the mobile phase, followed by post-column degradation with 1.5 M sodium hydroxide-0.02% sodium hypochlorite solution at ambient temperature, and detection of the degradation product(s) of each compound at 270 nm. The detection limits were about 25 ng for each compound at a signal-to-noise ratio of 3. At concentrations of 2-5 micrograms/ml of each compound, the within- and between-run precisions (relative standard deviation) were 0.77-7.15% and 1.76-5.96%, respectively. The plasma, biliary and urinary levels of 1 and its metabolites were determined by the proposed method after intravenous administration of 1 to rats.

Fluorimetric determination of amino acids by high-performance liquid chromatography using a hollow-fibre membrane reactor.

A high-performance liquid chromatographic method has been developed for the determination of primary amino acids. The method involves separation of primary amino acids on a C18 column using sodium heptanesulphonate as an ion-pairing agent, post-column derivatization with o-phthalaldehyde and 2-mercaptoethanol introduced into the main flow stream using a sulphonated hollow-fibre membrane reactor immersed in their solutions and fluorimetric detection of derivatives (lambda ex = 340 nm, lambda em = 450 nm). A higher or similar sensitivity was obtained compared with the conventional post-column derivatization method. The detection limits were 0.2-2.3 pmol at a signal-to-noise ratio of 3. For amounts of 48-110 pmol, the precision was of the order of 1.1-4.0% (relative standard deviation, n = 20).

Liquid chromatographic determination of barbiturates using a hollow-fibre membrane for postcolumn pH modification.

A sensitive, high-performance liquid chromatographic method is described for the determination of barbiturates by postcolumn pH modification. The barbiturates (barbital, phenobarbital, hexobarbital and amobarbital) were separated on a C18 column using a mixture of methanol and water as an eluent. Then the pH of the eluent was raised to 10 by introducing ammonia or ammonium ion through a sulphonated hollow-fibre membrane inserted between the column and the detector. The detection was based on the primary ionized barbiturates at 240 nm. At barbiturate concentrations of 2.0 micrograms/ml, the within- and between-experiment precision (relative standard deviation) was 0.65-3.28 and 0.76-1.90%, respectively. The limits of detection were about 0.5-2.5 ng at a signal-to-noise ratio of 3. The method was applied to the determination of amobarbital in saliva.

Liquid chromatographic determination of amoxicillin and its metabolites in human urine by postcolumn degradation with sodium hypochlorite.

A high-performance liquid chromatographic method has been developed for the determination of amoxicillin and its metabolites [(5R,6R)-amoxicilloic acid, the (5S,6R) epimer, and the (2R)-piperazine-2',5'-dione] in human urine. They were separated from the background components of urine on a reversed-phase C18 column using sodium heptylsulphonate as an ion-pairing agent and methanol as an organic mobile phase modifier. The eluent was led to the postcolumn degradation with 1.5 M sodium hydroxide plus 0.02% sodium hypochlorite solution at ambient temperature. The degradation product(s) of each compound was detected at 270 nm. The proposed method permits detection of I, II, III, and IV down to 1 microgram/ml in neat urine samples. At a concentration of 5 micrograms/ml of each compound, within- and between-run precisions (relative standard deviation) were 1.12-5.79 and 0.80-2.70%, respectively. The urinary levels of I and its metabolites were determined by the proposed method after administration of I to humans.

Liquid chromatographic determination of ampicillin and its metabolites in human urine by postcolumn alkaline degradation.

A high-performance liquid chromatographic method has been developed for the determination of ampicillin (I) and its metabolites [5R,6R)-penicilloate (II), the (5S,6R)-epimer (III), and piperazine-2,5-dione (IV)) in human urine. The assay was based on the measurement of the absorbance at 300 nm following the postcolumn alkaline degradation with 0.75 M sodium hydroxide, 2 X 10(-3) M mercuric chloride, and 1 X 10(-2) M ethylenediaminetetraacetic acid disodium salt in solution. The limits of accurate determination were 0.5 microgram mL-1 for I, 2.0 microgram mL-1 for II and III, and 1.0 microgram mL-1 for IV in neat urine samples with a 10 microL injection. At concentrations of compounds I-IV of 5 micrograms mL-1, within- and between-run precisions were 1.10-4.03% and 0.93-2.34%, respectively. The urinary levels of I and its metabolites were quantified by the proposed method.

High-performance liquid chromatographic determination of 6-aminopenicillanic acid by postcolumn alkaline degradation.

A high-performance liquid chromatographic method has been developed for the determination of 6-aminopenicillanic acid in amino acid mixtures and human serum. The separation of 6-aminopenicillanic acid was carried out on a C18 column using sodium heptylsulfonate or tetrabutylammonium bromide as an ion-pairing agent and methanol as an organic mobile phase modifier. Detection was based on a postcolumn reaction with sodium hydroxide, mercury(II) chloride, and ethylenediaminetetraacetic acid disodium salt followed by measurement of ultraviolet absorbance (at 300 nm) of the reaction product(s). The method is quantitative for 6-aminopenicillanic acid concentrations down to 0.1 microgram/ml in human serum samples with a 20-microliter injection. At a concentration of 2 micrograms/ml, the within- and between-run precisions (relative standard deviation) were 1.29-3.91% and 2.30%, respectively.

A sensitive high performance liquid chromatographic assay of a new metabolite of ampicillin in man.

A sensitive, high-performance liquid chromatographic method has been developed for the determination of piperazine-2,5-dione, a new metabolite of ampicillin, in human urine. Piperazine-2,5-dione was separated from human urine on a C18-column using phosphate buffer-methanol (pH 3.5) as eluent. Subsequently, the effluent underwent a postcolumn reaction with mercurous chloride and was then detected at 305 nm. It was found that piperazine-2,5-dione was also excreted in the urine of a volunteer dosed with ampicillin.