Junko Wakai

Chiral separation of propranolol and its ester derivatives on an ovomucoid-bonded silica : influence of pH, ionic strength and organic modifier on retention, enantioselectivity and enantiomeric elution order

SummaryThe retention, enantioselectivity and enantiomeric elution order of racemic propranolol (PP) and its ester derivatives (O-acetyl,-propyl,-butyl and-valeryl PP) on an ovomucoid-bonded silica column have been investigated with respect to pH, ionic strength and organic modifier. For these cationic solutes, an increase in the organic modifier content and/or a decrease in the pH result in a decreased retention of both enantiomers. Enantioselectivity of the ester derivatives was higher than of underivated PP. The enantiomeric elution order was (S)/(R) for PP and (R)/(S) for the four ester derivatives, when ethanol or 2-propanol was used as the organic modifier. When methanol or acetonitrile was used as the organic modifier, inversion of the enantiomeric elution order was observed for O-valeryl PP with the use of methanol and for PP and O-propyl PP with acetonitrile. These results suggest that at least two chiral binding- or recognitionsites are present in a protein molecule and/or conformational changes occur in the chiral binding- or recognition-site(s) of the protein molecule bonded to a silica matrix.

High-performance liquid chromatographic assay of ampicillin, amoxicillin and ciclacillin in serum and urine using a pre-column reaction with 1,2,4-triazole and mercury(II) chloride

High-performance liquid chromatographic methods have been developed for the determination of ampicillin (ABPC), amoxicillin (AMPC) and ciclacillin (ACPC) in serum and urine. The methods involve acetylation of these aminopenicillins with acetic anhydride in aqueous solutions (pH 9.0) at ambient temperature for 3 min followed by reaction with 2 M 1,2,4-triazole and 10–3M mercury(II) chloride in solution (pH 9.0) at 60 °C for 10 min. The resulting products were separated on a C18 column following stabilisation in an eluent containing sodium thiosulphate. They were detected at 328 nm for ABPC and AMPC and 327 nm for ACPC.The methods have been applied to assays of these aminopenicillins in serum and urine. The procedures, which permit the accurate determination of aminopenicillin concentrations in serum down to 0.05 µg ml–1 for AMPC and 0.1 µg ml–1 for ABPC and ACPC, are specific to intact penicillins without interference from the corresponding penicilloates. At an aminopenicillin concentration of 1 µg ml–1 in serum, the within- and between-run precisions were 0.97–3.51% and 2.07–3.55%, respectively.

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.

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.

Synthesis of a mixed-functional silica support for direct injection analysis of drugs in serum by liquid chromatography

SummaryA new mixed-functional silica material has been developed for direct injection analysis of drugs in serum. The mixed-functional material is synthesized from porous silica by three steps; introduction of 3-glycidoxypropyl groups, introduction of phenyl groups, and hydrolysis of the oxirane ring to diol groups. The prepared column packing can be used for direct injection analysis of hydrophobic and hydrophilic drugs in serum over the eluent pH range employed for ordinary siloxane-bonded silica. The recovery of drugs from serum was almost 100%, regardless of the difference in their protein bindings.

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.

Ion-exclusion chromatography of carboxylic acids with conductivity detection Peak enhancement using a cation-exchange hollow-fibre membrane and an alkaline solution

Abstract A sensitive method for the determination of carboxylic acids is described. They were separated using a dilute aqueous solution of sulphuric acid as an eluent on a strong cation-exchange column, passed through a cation-exchange hollow-fibre membrane surrounded by an alkaline solution and detected by electrical conductivity. They gave positive and/or negative chromatographic peaks, depending on the concentration of the alkaline solution used. For most monocarboxylic acids, the response was enhanced 16 to 93 times as both the positive and negative peaks, while the latter gave 1.8 to 3 times higher responses compared with the former. The quantification of carboxylic acids in a wine sample as their negative peaks 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.

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 × 10−3 M mercuric chloride, and 1 × 10−2 M ethylenediaminetetraacetic acid disodium salt in solution. The limits of accurate determination were 0.5 μg mL−1 for I, 2.0 μg mL−1 for II and III, and 1.0 μg mL−1 for IV in neat urine samples with a 10 μL injection. At concentrations of compounds I‐IV of 5 μg 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.