Analysis of fat-soluble vitamins. XXIII. High performance liquid chromatographic assay for vitamin D in vitamin D3 and multivitamin preparations.

Vitamin D is determined in preparations containing other fat-soluble vitamins by high performance liquid chromatography (HPLC). The unsaponifiable residue is extracted and separated from interferences by reverse phase chromatography; the fraction corresponding to vitamin D3 is collected and quantitated using normal phase chromatography (amylalcohol-n-hexane as mobile phase) by measuring the vitamin D3 and pre-vitamin D3 peaks at 254 nm. Previtamin D3 content is calculated as vitamin D3 with a conversion factor (determined on the equipment used). Application of the method to vitamin AD3 mixtures in oils gives 98-102% recovery. The reproducibility, using an external standard, is 2-3%, calculated as the coefficient of variation; with an internal standard, the coefficient of variation is 1-1.5%. The method measures potential vitamin D3 content in preparations containing greater than or equal to 200 IU/g in the presence of all known vitamin D3 isomers, vitamin A, and vitamin E.     

Reverse phase liquid chromatographic determination of some food additives

Liquid chromatographic methods are described for the separation and determination of non-nutritive sweeteners, namely, acesulfame, aspartame, saccharin, and dulcin; preservatives such as benzoic acid and p-hydroxybenzoic acid; and caffeine and vanillin in ready-to-serve beverages, ice candy, ice cream, squash beverage, tomato sauce, and dry beverage mix samples. These additives are separated on a muBondapak C18 column using methanol-acetic acid-water (20 + 5 + 75) as mobile phase and detected by UV absorption at 254 nm. Caffeine, vanillin, dulcin, and benzoic acid can be analyzed quickly by using a mobile phase of methanol-acetic acid-water (35 + 5 + 60). Aspartame can be separated in the presence of caffeine and vanillin by using the mobile phase pH 3 acetate buffer-methanol (95 + 5). Retention factors and minimum detectable limits are described. The percentage error and the percent relative standard deviation for 6 replicate samples ranged from 0.3 to 2.8 and from 1.64 to 3.60, respectively. Recovery of additives added to the foods named and analyzed by the direct method and by extraction ranged from 98.0 to 100.6% and from 91.6 to 101.8%, respectively. The proposed LC techniques are simple, rapid, and advantageous because all the additives can be detected in a single step, which makes it useful for the routine analysis of various food products.     

Reverse phase liquid chromatographic determination of bisacodyl in dosage forms

A method is described for the determination of bisacodyl in enteric-coated tablets and suppositories by liquid chromatography (LC). The method will also determine the hydrolysis degradation products monoacetylbisacodyl and desacetylbisacodyl. The sample is dissolved in 2-propanol, and the extract is diluted with the mobile phase and injected into a liquid chromatograph fitted with a mu Bondapak C18 column and an ultraviolet detector set at 254 nm. The column is eluted with methanol-acetonitrile-0.01M citric acid (25 + 25 + 50). The pooled mean recovery value for bisacodyl from commercial enteric-coated tablets and suppositories was 99.7% with a pooled coefficient of variation (CV) of 0.72%. For content uniformity assays, the CVs were 0.7 and 1.0% for groups of 10 individual commercial suppositories and tablets, respectively. Differences between assay values by the LC and USP XX methods were 0.2% of declared for enteric-coated tablets (n = 5) and 1.0% of declared for suppositories (n = 2). The LC method can determine as little as 0.015 microgram of the monoacetyl or desacetyl degradation product.     

Spectrofluorometric assay of thiamine hydrochloride in injections and multivitamin preparations

A spectrofluorometric method is described for the determination of thiamine hydrochloride. The method is based on formation of a fluorescent product by oxidation of thiamine HCl with 2,3,5,6-tetrabromo-1,4-benzoquinone in aqueous acetonitrile solution. The reaction product is stable for at least 6 h and shows excitation and emission maxima around 355 and 420 nm, respectively. The method is highly selective for thiamine HCl in the presence of other B vitamins. Thiamine HCl can be determined at concentrations as low as 20-160 ng/mL of the final solutions. When the method was applied to the determination of thiamine HCl added to some commercially available multivitamin preparations, recoveries were 98.78-99.98%.     

Reverse phase liquid chromatographic determination of aspartame in beverages and beverage mixes

A method is described for determining the artificial sweetener aspartame in beverages and beverage mixes by liquid chromatography. Aspartame is separated on a microC18 column, using a mobile phase of acetic acid, water, and isopropyl alcohol at pH 3.0 and UV detection at 254 nm. Beverages are filtered through 0.45 micron filters and injected directly into the chromatograph. Aspartame is eluted in approximately 7 min. Detection of aspartame is confirmed by a UV scan of the trapped peak. Aspartame is quantitated in the presence of other beverage additives such as saccharin, caffeine, sodium benzoate, artificial colors, and artificial flavors. Results are presented for spiked soda beverages, beverages from fruit-flavored mixes, instant tea, reconstituted presweetened drink mixes, and a powdered tabletop sweetener.     

Reverse phase liquid chromatographic determination and confirmation of aflatoxin M1 in cheese

A systematic method is proposed for determination and confirmation of aflatoxin M1 in cheese by liquid chromatography (LC). A sample of cheese is extracted with chloroform, cleaned up on 2 silica gel columns followed by a Sep-Pak C18 cartridge, and chromatographed on a 5 microns octadecyl silica column with fluorometric detection. The sample extract or standard is treated with n-hexane-trifluoroacetic acid (TFA) (4 + 1) for 30 min at 40 degrees C. Analysis by LC with TFA-treatment of the extract provides quantitative data. Multiple assays of 5 samples of Gouda cheese spiked with aflatoxin M1 at levels of 0.5, 0.1, and 0.05 ng/g showed average recoveries of 93.2, 91.6, and 92.4%, with coefficients of variation of 2.63, 3.97, and 4.52%, respectively. Assay of 5 naturally contaminated cheeses resulted in 0.051-0.448 ng/g of aflatoxin M1. Limit of quantitation is about 0.01 ng/g. The identity of aflatoxin M1 is confirmed by treating aflatoxin M1 or the M2a derivative with TFA-methanol (or ethanol) (3 + 1). The TFA-methanol reaction products of M2a could be detected quantitatively.     

Reverse phase high pressure liquid chromatographic determination of vitamin K1 in infant formulas

A reverse phase high pressure liquid chromatographic (HPLC) method for quantitating vitamin K1 in enzymatic hydrolysates of infant formula is described. The vitamin is extracted with n-pentane before determination by isocratic and isothermal reverse phase HPLC. Recovery of vitamin K1 added to 5 infant formulas ranged from 84 to 103%.     

Reverse phase ion pair high pressure liquid chromatographic determination of ethylenediaminetetraacetic acid in crabmeat and mayonnaise.

A method is described for the determination of ethylenediaminetetraacetic acid (EDTA) in crabmeat and mayonnaise. EDTA is extracted from the food sample with water and converted to its copper chelate, which is then quantitated by reverse phase ion pair high pressure liquid chromatography with ultraviolet detection. Maximum sensitivity is obtained with detection at about 254 nm; higher wavelengths may be used for enhanced specificity. Cleanup procedures for crabmeat and mayonnaise were improved by using a radiotracer method. Analyses of crabmeat and mayonnaise samples spiked at 3 different levels showed greater than 90% recovery of EDTA.     

Liquid chromatographic determination of pindolol and related compounds in raw materials

A liquid chromatographic method for the assay of pindolol and related compounds in the bulk drug has been developed. The method resolves 6 known and several unknown impurities from the drug and each other by using a nitrile column, an acetonitrile-sodium acetate buffer (35 + 65), and a UV detector set at 219 nm. Minimum quantifiable amounts of impurities are 0.02% or less relative to the drug. Ten lots of pindolol raw material were evaluated for purity and drug content. Total levels of impurities in these samples, quantitated against pindolol, ranged from about 0.03 to 0.24%. Assay results were within the range of 98.5 to 101.5%.     

Determination of vitamin D3 in liquid multivitamin preparation, using reverse phase, solid phase extraction liquid chromatography

A method is described for the determination of vitamin D3 in a liquid multivitamin preparation by liquid chromatography. Samples are purified on a disposable reverse phase extraction (SPE) column with a mobile phase of methanol-2-propanol (97 + 3) and are analyzed on a Zorbax ODS (5 micron) column with an acetonitrile-2-propanol-water (90 + 8 + 2) solvent system. Vitamin D3 is completely resolved from other interfering compounds within approximately 21 min and is detected with a UV detector at 254 nm. A mean of 98.5% of theory with a coefficient of variation of 3.8% was found for determination of vitamin D3 in a commercial preparation.     

Analysis of pharmaceutical dosage forms for oxfendazole: I. Reverse phase liquid chromatographic determination of oxfendazole in swine premix

A reverse phase liquid chromatographic (LC) procedure is described for quantitating oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] in swine premix. Sample preparation consists of extracting oxfendazole with an acetone-methanol mixture. An aliquot of the extract is then centrifuged to separate undissolved premix excipients. Internal standard is added to the supernate and the sample is further diluted with water-acetonitrile-phosphoric acid (80 + 20 + 1). Oxfendazole is quantitatively determined using a Partisil-5-ODS-3 column with acetonitrile-0.01 M phosphate buffer (pH 6.0) as the mobile phase. The method is stability specific and yields a mean recovery of 101.1 +/- 0.4% for the 1.35% premix formulation. The dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.     

Validation of liquid chromatographic method for assay of chlorthalidone in tablet formulations

A stability-indicating, reverse phase liquid chromatographic (LC) method was developed for assay of chlorthalidone in tablet formulations. The chromatographic system separates the parent compound from its potential hydrolysis product (4'-chloro-3'-sulfamoyl-2-benzophenone carboxylic acid), which can be quantitated at low levels, and another degradation product (2-chloro-5-(1-methoxy-3-oxo-1-isoindolinyl)benzenesulfonamide), which was found during the experimental work. The procedure can also be used for content uniformity determinations. The general utility of the method was demonstrated by the assay of several product brands. The validated procedure was shown to be accurate, precise, reproducible, and specific.     

Nonaqueous reverse phase liquid chromatographic analysis for cholesterol in milk chocolate

A method using liquid chromatography was developed for the analysis of cholesterol in milk chocolate products. The method involves saponification of the sample with methanolic KOH followed by extraction with ether. Potentially interfering components are eliminated through the use of a silica Sep-Pak cleanup step before injection. The nonaqueous reverse phase LC system consists of a C18 column and an isopropanol-hexane mobile phase with direct detection at 205 nm. Recoveries of 1, 3, and 5 mg cholesterol added to 1 g sample of milk chocolate were 88.6, 102.8, and 110.1%, respectively. Studies conducted with [4-14C]-cholesterol were undertaken to further document the accuracy of the method.     

Reverse phase high pressure liquid chromatographic determination of some iodoamino acid contaminants in sodium liothyronine or sodium levothyroxine.

Some iodoamino acids have been separated and determined by high pressure liquid chromatography on octadecylsilane reverse phase packing with a mobile phase consisting of methanol, potassium phosphate monobasic, and orthophosphoric acid at 44 degrees C. The method separates and quantitates mixtures of 3,5-diiodothyronine, liothyronine, isoliothyronine, and levothyroxine. The procedure provides an accurate, sensitive, and rapid estimation of decomposition and/or impurities in standards in approximately 30 min, at the less than 75 pmole level, using an ultraviolet detector at 254 nm.     

Reverse phase liquid chromatographic determination of dexamethasone acetate and cortisone acetate in bulk drug substances and dosage forms: method development

The determination of the steroid acetates was evaluated for ruggedness of the method by using an octyldecylsilane column, 254 nm detection, and acetonitrile-water as mobile phase. Mobile phase pH, oven temperature, and columns from various manufacturers had no dramatic effect on the chromatography. The method was then optimized for dexamethasone acetate and cortisone acetate bulk drug and dosage forms. For dexamethasone acetate, the bulk drug substance should be dried at 105 degrees C before use, and the sample should be dissolved in 50% acetonitrile-buffer pH 6 for stability. Cortisone acetate, on the other hand, was found to be nonhygroscopic and hence could be used as received. For stability, the sample should be stored in 50% acetonitrile-buffer pH 4.     

Validation of liquid chromatographic method for assay of calcitriol and alfacalcidol in capsule formulations

The validation of a liquid chromatographic procedure suitable for the determination of calcitriol and alfacalcidol in their respective formulations labeled to contain at least 0.25 micrograms drug per unit is described. The capsule content is diluted and chromatographed in 15-20 min on silica columns (5 micron) with a mobile phase of hexane-tetrahydrofuran-methylene dichloride-isopropanol (72 + 12 + 12 + 4, v/v) with detection at 254 nm. The calibration curve is linear. Recoveries of "spikes" averaged 101% with a standard deviation of 2%. Precision was better than 1.5%.     

Reverse-phase liquid chromatographic determination of clioquinol in cream and ointment preparations: collaborative study

Seven laboratories participated in a collaborative study to analyze, in duplicate, 2 synthetic formulations and 2 commercial preparations, labeled to contain 3% clioquinol. Clioquinol is determined as its nickel (II) complex by reverse-phase liquid chromatography on a phenyl-bonded column with a mobile phase of acetonitrile-methanol-water, containing ammonium acetate and nickel chloride. Detection is at 273 nm and diphenylamine is added as an internal standard. Mean recoveries were 99.1 and 101.1%, respectively, for the ointment and cream synthetic preparations and 96.7 and 99.7%, respectively, for the commercial ointment and cream. All results are consistent with the variability of other methods at this concentration range. The method has been approved interim official first action.     

Nonaqueous reverse phase liquid chromatographic system for separation and quantitation of provitamins A

A reverse phase liquid chromatographic (LC) system has been developed for separating the main naturally occurring carotenoids that have provitamin A activity. The system produces baseline separation of beta-carotene, alpha-carotene, and beta-cryptoxanthin (beta, beta-carotene-3-ol, 472-70-8) from biologically inactive zeinoxanthin (beta, epsilon-carotene-3-ol, 24480-38-4) and from a pigment believed to be alpha-cryptoxanthin (beta, epsilon-carotene-3'-ol). Some cis-isomers are also separated. These separations are obtained on a C-18 column, isocratically, with methanol-chloroform eluant. For quantitation, peak areas from detection at 475 nm are compared with that of an internal standard, 1-(phenylazo)-2-naphthalenol (842-07-9), which elutes prior to the provitamins. Provitamin amounts are calculated from absorbance ratios. Prior to LC, esters are saponified, and interfering pigments are removed from ester-free extracts by adsorption on magnesia.     

Ion-pair reverse phase liquid chromatographic determination of sodium acifluorfen in feed

Ion-pair reverse phase liquid chromatography (LC) and UV detection at 280 nm have been used to determine sodium acifluorfen (sodium-5-[2-chloro-4-(trifluoromethyl)-phenoxy]-2-nitrobenzoate), an experimental diphenyl ether herbicide, in dog feed. Sodium-5-(2,4-dichlorophenoxy)-2-nitrobenzoate is used as the internal standard. The feed is homogenized in 0.01N HCl, followed by ethyl acetate extraction, and centrifugation. The organic layer is removed and evaporated and the residue is reconstituted in methanol and filtered before LC analysis (mobile phase methanol-water (58 + 42), 0.005M in tetrabutylammonium phosphate and 0.045M in (NH4)2HPO4, at pH 7.4). The ion-pair technique offers a high degree of control over the retention characteristics of the herbicide and internal standard. The use of the internal standard permits precise and accurate quantitation and substantially reduces analysis time compared with the external standard method.