Capillary electrophoresis for regulatory analysis: is it ready? Problems encountered during an interlaboratory study utilizing capillary electrophoresis.

Recently, the Northeast Regional Laboratory of the Food and Drug Administration conducted a collaborative study on the method to determine histamine in tuna by capillary electrophoresis. This study, under the guidance of the AOAC International, was the first of its kind involving the use of CE and was undertaken to establish official monograph status. The results of the collaborative study, however, were highly variable. Percent recovery data obtained by 11 collaborators ranged from 54 to 281%, with an average of 111%. In this paper, problems with reproducibility, and factors such as low sensitivity, noisy baselines, and variable migration times encountered by collaborators using various commercial capillary electrophoresis units are discussed.

Capillary zone electrophoretic determination of histamine in fish.

Histamine, the principal causative agent in scombroid food poisoning, was analyzed in seafood by a new, rapid, and sensitive method using capillary zone electrophoresis (CZE) with UV detection at 210 nm. Incurred histamine in methanolic fish extracts migrated within 4 min in a fused silica capillary filled with 0.02 M citrate buffer, pH 2.5, under an applied potential of 375 V/cm. The analytical response was linear from 0.5 to 100 ppm histamine (correlation coefficient, r = 0.999). The coefficients of variation for migration time and peak area response were < 1 and < 3%, respectively. Recovery of histamine in fortified fish composites was satisfactory. CZE was considered for alternative application in seafood speciation.

Identification and separation of five cephalosporins by micellar electrokinetic capillary chromatography.

The cephalosporins are a group of structurally related, broad spectrum beta-lactam antibiotics isolated from the mold Cephalosporium. Methods of analysis of cephalosporin antibiotics include microbiological, titrimetric and chromatographic assays. Chromatographic techniques, including high-performance liquid chromatography, have been extensively utilized for specific and sensitive assays of beta-lactam antibiotics in a variety of matrices, i.e. clinical and pharmaceutical. Several of the drawbacks of HPLC in the analysis of cephalosporins in food and biological samples include matrix interferences and low resolution due to column adsorption. Recently, the applicability of capillary electrophoresis to the resolution of beta-lactam antibiotics has been demonstrated in the literature. In this paper we employed sodium borate and an anionic surfactant, sodium dodecyl sulfate (SDS), in a separation technique called micellar electrokinetic capillary chromatography with UV detection, to resolve a mixture of five cephalosporins--cefuroxime, cephalexin, cephapirin, cefamandole nafate and cephalothin. The presence of SDS in the running buffer above the critical micelle concentration, creates a pseudostationary phase enabling high-efficiency chromatographic separations. The effect of the ion-pairing reagent, pentanesulfonic acid sodium salt, on the resolution of the cephalosporin mixture in conjunction with SDS was also examined.

Liquid chromatographic determination of penicillin V potassium in tablets: collaborative study.

A liquid chromatographic method for the determination of penicillin V potassium in tablets was collaboratively studied by 7 laboratories. The method uses an octadecyl silane reverse-phase column, a mobile phase of acetonitrile-methanol-0.01 M potassium phosphate monobasic (21 + 4 + 75 v/v/v), photometric detection at 225 nm, and sulfadimethoxine as an internal standard. Each collaborator received 6 samples: powdered composites of 2 commercial tablet preparations and 1 synthetic tablet powder mixture, each with blind duplicates. The mean repeatability and reproducibility relative standard deviations for commercial samples were, respectively, 1.10 and 1.46% (250 mg dosage), and 0.84 and 2.82% (500 mg dosage). The average standard recovery from the synthetic formulation was 99.1%, with repeatability and reproducibility relative standard deviations of 1.30 and 3.66%, respectively. The method has been adopted official first action.

UV spectrophotometric determination of hydralazine hydrochloride in tablets: collaborative study.

A UV spectrophotometric method for the determination of hydralazine hydrochloride in tablets was collaboratively studied by 5 laboratories. The method is based on conversion of hydralazine to a tetrazolo [5,1-alpha]phthalazine derivative which shows an absorption maximum at about 274 nm. Each collaborator received blind duplicate samples of 2 commercial powdered composites from 10 and 100 mg tablets, and 1 synthetic tablet formulation. Each collaborator also received a set of 10 tablets for determination of content uniformity. The pooled mean recovery of hydralazine hydrochloride from the synthetic formulation was 101.2 +/- 0.94%. The mean assay values for 10 and 100 mg tablets were 95.6 +/- 0.98 and 101.0 +/- 0.73% of the declared amounts, respectively, with corresponding CV values of 1.02 and 0.73%. The pooled mean for individual tablet assay was 99.8 +/- 3.26% of the declared value, with a CV of 3.29%. The method has been adopted official first action.

Ultraviolet spectrophotometric determination of hydralazine hydrochloride in tablets following derivatization with nitrite.

Routine use of the USP XXI spectrophotometric method for the content uniformity determination of hydralazine hydrochloride tablets has shown that tablet excipients can significantly alter the spectral characteristics of the drug and thus cause inaccurate assay values to be obtained. Because of this problem, a simple and reliable alternative spectrophotometric assay method, based on the conversion of hydralazine to tetrazolo [5,1-alpha]phthalazine with nitrite ions under acidic conditions, was developed. The derivative showed an absorption maximum at about 274 nm and obeyed Beer's law over the concentration range 4-40 micrograms/mL. Mean recoveries of hydralazine hydrochloride added to commercial coated and uncoated tablets were 101.0% (n = 10) and 100.8% (n = 8), respectively. The proposed method was found suitable for the assay not only of individual tablets but also of tablet composites.

Liquid chromatographic determination of penicillin V potassium in tablets and powders for oral solution.

A reverse-phase liquid chromatographic method is described for the assay of penicillin V potassium in tablets and powders for oral solution. Under isocratic conditions, the combined use of an octadecylsilane column, with a mobile phase composed of acetonitrile-methanol-0.01M monobasic potassium phosphate (21 + 4 + 75, v/v), and photometric detection at 225 nm, separated penicillin V potassium from excipients, related compounds, and degradation products. Sulfadimethoxine was used as an internal standard. Detector responses were linearly related to concentrations of penicillin V over the range 25-225 micrograms/mL (r = 0.99997). Standard addition recoveries ranged from 98.8 to 99.9% (mean 99.5%, n = 8) for tablets and from 97.9 to 101.6% (mean 99.8%, n = 8) for powders for oral solution. The liquid chromatographic assay results were compared with those obtained by the official iodometric titration method. The proposed method is simple, selective, stability-indicating, and free from interference by excipients and degradation products.