Liquid chromatographic determination of clobetasone-17-butyrate in ointments

A liquid chromatographic (LC) method has been developed for determination of clobetasone-17-butyrate in ointment using clobetasone propionate as an internal standard. Separation was carried out on a C18 reverse-phase column using water-methanol as a mobile phase. Methylparaben and propylparaben (both sodium salt) used as preservatives did not interfere with separation. Compounds are detected photometrically at 235 nm. Mean assay results for 0.05% commercial ointments were 100.36% (n = 5). Mean recovery of clobetasone-17-butyrate added to commercial ointment was 99.89%.     

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.     

Determination of small quantities of atropine in commercial preparations by liquid chromatography with fluorescence detection

A method is presented for the determination of small quantities of atropine in commercial preparations by liquid chromatography (LC) with fluorescence detection. The sample is extracted with CHCl3 from basic suspension, the CHCl3 is evaporated on the steam bath, and the dry residue is dissolved in a small volume of CH3OH. A reverse phase column is used for the LC analysis; the eluting solvent is prepared by mixing 950 mL CH3OH with 50 mL water containing 1 g of the sodium salt of 1-pentanesulfonic acid. The fluorescence detector is set at an excitation wavelength of 255 nm and an emission wavelength of 285 nm. Several commercial tablets and injections containing atropine in combination with other ingredients and a commercial sample of belladonna extract were analyzed by the proposed method. Recoveries of atropine sulfate from aqueous solutions averaged 100.7% with a relative standard deviation (RSD) of 3.35% for atropine sulfate levels of 0.12 mg. Recoveries of atropine sulfate from synthetic injection formulations were 99.8 and 100.0% with RSDs of 2.03 and 2.35%, respectively; the atropine sulfate concentrations of commercial injections with the same formulations were found to be 97.0 and 100.0% of the labeled amounts with RSDs of 0.53 and 1.46%, respectively.     

Determination of fluoride in fluoride tablets and solutions by ion-selective electrode: collaborative study

A proposed method using the fluoride (F) ion-selective electrode has been developed for determining the fluoride ion concentration in tablets and solutions containing sodium fluoride. The method has been subjected to collaborative study. Eight samples consisting of 2 authentic fluoride solutions, 2 commercial fluoride solutions, and 4 commercial fluoride tablets were sent to each of 11 collaborators together with a copy of the method. Single assays on the authentic fluoride solutions known to contain 1 mg F/5 mL were performed with average recoveries of 99.5 and 99.6% and relative coefficients of variation (CV) of 2.11 and 1.91%, respectively. Single assays of 2 commercial fluoride solutions declared at 1 mg F/5 mL gave mean values of 0.994 and 0.990 mg and relative CV values of 1.88 and 2.36%, respectively. Single assays of 2 commercial fluoride tablet preparations declared at 0.5 mg F gave mean values of 0.485 and 0.478 mg and relative CV values of 3.12 and 3.71%, respectively. Single assays of 2 commercial fluoride tablet preparations declared at 1 mg F gave mean values of 0.991 and 0.981 mg and relative CV values of 2.99 and 2.85%, respectively. The method has been adopted official first action.     

Processing effects on the composition of sea buckthorn juice from Hippophae rhamnoides L. Cv. Indian Summer.

Sea buckthorn juice is one product that can be derived from the sea buckthorn berry, a new alternative crop for the Canadian western provinces. Fresh pressed juice separates into three phases when allowed to stand overnight in the refrigerator: an upper cream phase, juice in the middle portion, and a sediment at the bottom. Enzymatic hydrolysis with commercial, broad spectrum carbohydrate hydrolyzing enzyme preparations reduced the juice viscosity, assisted juice separation, and provided an opalescent juice. Soluble solids averaged 10.2 degrees Brix, pH averaged 3.13, ascorbic acid averaged 174.2 mg/100 mL, and titratable acidity averaged 1.97% as malic acid all determined on centrifuged (10 000 rpm, 15 min) juice. Soluble sugars included glucose, fructose, and an unidentified component that was not sucrose or other common soluble monomeric or dimeric sugar. Quinic acid was quantitatively most important, while malic was next, and oxalic, citric, and tartaric acids were minor components. Washing berries by dipping reduced soluble solids (degrees Brix) in juice suggesting uptake of wash water.     

Atomic absorption spectrophotometric determination of mercury in mercury-containing drugs: collaborative study

An atomic absorption spectrophotometric (AAS) method applicable to a wide variety of mercury-containing drugs has been developed and subjected to a collaborative study. Samples were digested with a water-HCl-HNO3 (4 + 3 + 1) mixture, and the mercury was determined in solution by AAS. High levels of mercury were measured with a conventional air-acetylene flame, whereas low levels were measured by the flameless technique. Each of 7 collaborators received duplicate synthetic samples of a tincture, an ophthalmic solution, and an antiseptic solution, and duplicate commercial samples of an ointment and an injectable. The overall mean value found by collaborators for mercury in these samples was 100.13%. The corresponding overall repeatability SD (CV, %) and reproducibility SD (CV, %) values were 2.18 (2.18) and 3.38 (3.38), respectively. The proposed AAS method has been adopted official first action.     

Resolution and analysis of enantiomers of amphetamines by liquid chromatography on a chiral stationary phase: collaborative study

A rapid, accurate method for separating and determining the enantiomeric composition of amphetamine bulk drug and commercial preparations was developed and subjected to collaborative study. Amide derivatives of the amphetamine enantiomers are formed by using achiral 2-naphthoyl chloride. The resulting enantiomeric amides are then chromatographed on a commercially available chiral stationary phase with hexane-isopropyl alcohol-acetonitrile (97 + 3 + 0.5) mobile phase, with detection at 254 nm. Seven collaborators received bulk drug and commercial samples of amphetamine. The collaborators and authors determined the mean percent l- and d-amphetamine from 2 injections of each sample. The method can detect the presence of as little as 0.5% of the l-enantiomer in d-amphetamine, with reproducibility between laboratories of +/- 71.3%. The method has been adopted official first action for determination of the enantiomeric composition of amphetamine bulk drug and preparations.     

Cleaning heavy metal contaminated soil with soluble humic substances instead of synthetic polycarboxylic acids

Abstract

Soils contaminated with heavy metals constitute a serious and widespread ecological problem but to clean such soils requires strong chemicals such as polycarboxylates; frequently ethylenediaminetetraacetic acid and nitrilotriacetic acid are used. However, these compounds are synthetic and toxic and their replacement by natural products such as soluble humic substances as washing agents for cleaning heavy metal polluted soils would be environmentally very attractive. In fact, such a replacement seems possible at least on cadmium and copper contaminated soil inasmuch as humic substances, depending on the concentration, were found to extract up to 45% and 54% of total cadmium and copper from a highly contaminated calcareous soil. Even though higher amounts of the two metals were extracted by ethylenediaminetetraacetic acid and nitrilotriacetic acid, the humic substances undoubtedly extracted the most reactive fractions. However, the humic substances extracted only 4% of total lead and 17% of total nickel, whereas the percentages for the synthetic polycarboxylates were about 30% for nickel and lead. Ethylenediaminetetraacetic acid and nitrilotriacetic acid may therefore be replaced by humic substances as washing agents for cadmium, copper and maybe nickel contaminated soils, whereas they seem unsuited for cleaning lead contaminated soils, at least if the soils are as calcareous as the soil tested.     

Collaborative study of a spectrofluorometric assay for Rauwolfia serpentina tablets and powdered root.

Reserpine-rescinnamine group alkaloids are extracted from Rauwolfia serpentina preparations into a dimethylsulfoxide (DMSO)-methanol mixture and diluted with 0.5N H2SO4. The chloroform extract of this solution is passed through a 0.1N NaOH-Celite column and then through a silica gel column. The weakly basic alkaloids trapped on the latter column are eluted with a methanol mixture; a portion of the eluate is treated with nitrous acid and the reserpine-rescinnamine content is determined by measuring the intensity of fluorescence of the oxidation product. The following means and standard deviations (11 collaborators) were obtained for the determination of reserpine-rescinnamine group alkaloids in 4 samples of Rauwolfia serpentina (NF reference powder, 100 mg and 50 mg commercial tablets, and a 45 mg synthetic tablet formulation) : 0.174% +/- 0.0112, 0.131% +/- 0.0047, 0.160% +/- 0.0100, and 0.153% +/- 0.0083, respectively.     

Spectrophotometric determination of aminacrine hydrochloride in creams, jellies, and suppositories: interlaboratory study

A previously reported visible spectrophotometric method for the analysis of aminacrine hydrochloride in creams, jellies, and suppositories was studied collaboratively by 8 laboratories. Aminacrine hydrochloride was extracted into acidic ethanol and its visible spectrum recorded. The amount present was calculated by determining the net absorbance between the absorbance maximum at about 402 nm and one-half the sum of the absorbance of the minima at about 389 and 412 nm. Each collaborator received 4 creams (0.2%), 1 jel (0.2%), 1 molded suppository (6 mg/3.198 g), and 2 gelatin-encapsulated suppository samples (12 mg/6.661 g and 14 mg/6.863 g). The cream samples included blind duplicates prepared to contain 0.212% aminacrine hydrochloride, 15% sulfanilamide, and 2% allantoin. Mean recovery for the authentic cream was 104.7% with a coefficient of variation (CV) of 9.22%. The commercial products contained these respective amounts (CVs): creams, 100.0% (2.48%) and 101.5% (2.16%); jel, 118.0% (9.58%); molded suppository, 102.7% (1.88%); and gelatin encapsulated suppositories, 93.1% (1.0%) and 94.3% (1.60%). Standard aminacrine hydrochloride provided for the study was 99.6% pure by nonaqueous titration. Thin layer chromatographic identification of aminacrine hydrochloride was also tested collaboratively. The method was not adopted by AOAC.     

Liquid chromatographic determination of coumarin anticoagulants in tablets: collaborative study

A liquid chromatographic method for the determination of coumarin anticoagulants in tablets was collaboratively studied by 7 laboratories. The method uses an octadecylsilane-bonded microparticulate column, tetrahydrofuran-methanol-water-acetic acid mobile phase, and photometric detection at 311 nm. Each collaborator received samples of warfarin sodium, phenprocoumon, and dicumarol as a synthetic composite and as commercial individual and composited tablets. Pooled average assay values for synthetic and commercial tablet samples of warfarin sodium were 101.6 and 99.5%, respectively, with a combined reproducibility SD of 2.38% (CV = 2.37%) and combined repeatability SD of 1.49% (CV = 1.49%). Pooled average (SD) assay values for dicumarol and phenprocoumon commercial samples were 98.0 (2.27) and 101.3% (4.00), respectively. The content uniformity determinations of 2 mg warfarin sodium and 25 mg dicumarol tablets indicated average tablet contents (range) of 99.5% (91.0-116.0) and 98.0% (89.8-108.8), respectively. The method has been approved interim official first action.     

Production of cod (Gadus morhua) muscle hydrolysates. Influence of combinations of commercial enzyme preparations on hydrolysate peptide size range

The complement of enzyme activities of a selection of commercial protease preparations were determined using fluorogenic substrates. Alcalase was used in combination with other commercial enzyme preparations to produce cod muscle (Gadus morhua) hydrolysates. Each muscle hydrolysate was characterized with respect to the percentage degree of hydrolysis (DH %), peptide molecular weight range, and free amino acid content. The enzyme preparations containing predominantly protease or endopeptidase activities achieved high DH % and produced significant amounts of peptides below a molecular weight of 3000. Alcalase combined with exopeptidase-rich preparations produced hydrolysates rich in low-molecular-weight peptides. Selecting combinations of enzyme preparations with complementary activity profiles could be used to manipulate the peptide molecular weight profile of hydrolysates.     

Solubilized wheat protein isolate: functional properties and potential food applications

Solubility, foaming capacity/stability, water holding and fat absorption capacities, and emulsifying capacity/stability of a solubilized wheat protein isolate (SWPI) were compared with those of commercial protein, that is, sodium caseinate (NaCAS), dried egg white (DEW), nonfat dry milk (NFDM), and soy protein isolate (SPI). SWPI was highly soluble at pH 6.5-8.5. Foaming capacity of SWPI was superior to those of SPI, NFDM, and DEW, and its foaming stability was similar to those of the commercial proteins. Foaming properties of SWPI were greatly improved in the presence of 0.5% (w/v) CaCl(2). Water holding capacity of SWPI was greater than that of NaCAS, NFDM, and DEW, whereas its fat absorption capacity was comparable to that of SPI, NaCAS, and DEW. SWPI exhibited emulsifying properties similar to those of SPI. SWPI was incorporated at 5, 10, 15, or 20% into ice cream, chocolate chip cookies, banana nut muffins, and hamburger patties. Products containing <5% SWPI were acceptable to consumers.     

Liquid chromatographic determination of levodopa and levodopa-carbidopa in solid dosage forms: collaborative study

A liquid chromatographic method for the determination of levodopa in tablets and capsules and levodopa-carbidopa in tablets was collaboratively studied by 6 laboratories. Collaborators were supplied with duplicate powdered composites of levodopa (1 synthetic formulation, 1 commercial tablet, and 1 commercial capsule) and levodopa-carbidopa (1 synthetic formulation and 2 commercial tablets), along with individual levodopa-carbidopa tablets for content uniformity determinations. The repeatability coefficient of variation (CVo) and reproducibility coefficient of variation (CVx) for levodopa single component were 0.48 and 0.87%; for levodopa in combination, 0.50 and 0.90%; and for carbidopa, 0.77 and 1.20%, respectively. Overall, the recovery values for levodopa and carbidopa from synthetic formulations simulating tablets were 100.4 and 99.5%, respectively. The pooled CVDo and CVDx values for the individual tablet assays were 2.07 and 2.30% for levodopa, and 1.80 and 2.24% for carbidopa, respectively. The method has been adopted official first action for determination of the active ingredients in levodopa tablets and capsules and in levodopa-carbidopa tablets and for content uniformity testing in the combination dosage form.     

Determination of the absolute molecular weight averages and molecular weight distributions of alginates used as ice cream stabilizers by using multiangle laser light scattering measurements

High-performance size exclusion chromatography with multiangle laser light scattering detection (HPSEC-MALLS) was used for characterizing complete molecular weight distributions for a range of commercial alginates used as ice cream stabilizers. For the samples investigated, molecular weight averages were found to vary between 115 000 and 321 700 g/mol and polydispersity indexes varied from 1. 53 to 3.25. These samples displayed a high content of low molecular weights. Thus, the weight percentage of material below 100 000 g/mol ranged between 6.9 and 54.4%.     

Liquid chromatographic determination of diazepam in tablets: collaborative study

A stability indicating liquid chromatographic method for the determination of diazepam in tablets was collaboratively studied by 6 laboratories. The method uses a C18 reverse phase column, a methanol-water mobile phase, p-tolualdehyde as the internal standard, and photometric detection at 254 nm. The collaborators were supplied with a synthetic tablet powder and 3 commercial tablet samples. The mean recovery of diazepam from the synthetic tablet powder was 100.2%. For all samples analyzed, the coefficient of variation was less than 1.5%. The method has been adopted official first action.     

Sensory-directed identification of creaminess-enhancing volatiles and semivolatiles in full-fat cream

Aimed at defining the chemical nature of creaminess-related flavor compounds in dairy products on a molecular level, a full-fat cream was analyzed for sensorially active volatiles and semivolatiles by means of activity-guided screening techniques. Application of the aroma extract dilution analysis on an aroma distillate prepared from pasteurized cream (30% fat) revealed delta-decalactone, (Z)-6-dodeceno-gamma-lactone, gamma-dodecalactone, delta-dodecalactone, and 3-methylindole with by far the highest flavor dilution (FD) factors among the 34 odor-active volatiles identified. Using a complementary approach involving silica column chromatography and fractionated high-vacuum distillation combined with sensory experiments enabled the additional identification of delta-tetradecalactone, delta-hexadecalactone, gamma-tetradecalactone, gamma-hexadecalactone, and delta-octadecalactone as semivolatile flavor components in the cream fat. Although a series of lactones is present in dairy cream, spiking of cream samples with individual lactones revealed that only the delta-tetradecalactone is able to enhance the typical retronasal creamy flavor of the product when added in amounts above its threshold level of 66 micromol/kg. Rather than contributing to the retronasal aroma of cream, first evidence was found that, particularly, gamma- and delta-octadecalactones and gamma- and delta-eicosalactones are able to influence the melting behavior of cream in the oral cavity.     

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.     

Immobilization of angiotensin-converting enzyme on glyoxyl-agarose

The assay of angiotensin-converting enzyme (ACE) inhibition by food-derived peptides is usually carried out by using soluble ACE in a batch process. The purification of this enzyme from tissues is not an easy task, and the resulting preparation loses activity very fast. In addition, ACE commercial preparations are very expensive. In this work the immobilization of ACE, through lysine amino groups, to 4% beads cross-linked (4 BCL) glyoxyl-agarose is described. The amount of immobilized enzyme increased with increasing concentrations of enzyme and with incubation time until a saturation point was reached at 50 mg protein/mL gel and 3.5 hours, respectively. The IC50 values for a noncompetitive sunflower peptide inhibitor were similar for the soluble (30.56 microM) and immobilized (32.7 microM) enzymes. An immobilized derivative was obtained that was 60 times more stable than the soluble enzyme at 60 degrees C. This procedure yields a derivative that can be reused and has increased thermal stability compared to that of the soluble enzyme. Thus, ACE immobilization is a good alternative to using soluble freshly prepared or commercial preparations because of economical and practical reasons.     

High pressure liquid chromatographic determination of oxazepam dosage forms: collaborative study

A reverse phase high pressure liquid chromatographic method for the determination of oxazepam in tablets and capsules was collaboratively studied by 9 laboratories. Collaborators were supplied with 6 samples that included synthetic and commercial formulations. Tablet and capsule composites are diluted with methanol and filtered. Oxazepam is determined at 254 nm by using a C18 column. Mean recoveries of oxazepam from synthetic tablet and capsule formulations were 97.2 and 99.0%, respectively. Mean coefficients of variation for tablets and capsules ranged from 1.85 to 2.86%. The method has been adopted official first action.