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A gas-liquid chromatographic (GLC) method, using the butyryl esters of sterols, has been developed for the measurement of cholesterol, stigmasterol, sitosterol, and campesterol in foods. An immobile phase of 1 % SE-30 coated on 100–120 mesh Gas-Chrom Q packed in a 6′ × 4 mm id glass column operated at 255°C was the most satisfactory of 7 column packings evaluated. Extraction with chloroform-methanol gave 98.7 % recovery with a coefficient of variation of 1.8% for cholesterol added to a variety of foods. When cholesteryl palmitate was added to vegetable oil and the butyryl derivative was prepared, followed by GLC analysis, the recovery was 99.3% with a coefficient of variation of 0.9%. Amounts as low as 1 mg/100 g food can be detected with a precision of 2.5%. The results of the analysis of a variety of foods for cholesterol, campesterol, sitosterol, and stigmasterol are given.

A collaborative study was conducted to compare the USP XVII (Emmerie-Engel) method and three GLC methods for quantitatively determining a-tocopherol, a-tocopheryl acetate, and a-tocopheryl succinate in pharmaceutical products. Nine collaborating laboratories analyzed seven pharmaceuticals encompassing tablets, capsules, and liquids. The mean coefficients of variation were: USP method, 28.6%; GLC method A, 10.2%; GLC method B, 10.8%; GLC method C, 18.0%. GLC method A was the method of choice of those evaluated. The vitamin E analogs are extracted with n-hexane from ground tablets and capsules; liquid preparations are diluted with n-hexane or injected directly onto the GLC column. The compounds are chromatographed on an 8′ × 4 mm id glass column paeked with 5% SE-30 on 100-120 mesh silanized Gas Chrom P, operated between 270-285°C, and the carrier gas flow rate adjusted so that ∝-tocopheryl acetate appears within 23-27 min. Acetic anhydride-pyridine reaction is utilized to determine whether ∝-tocopherol or ∝-tocopheryl succinate is present in a sample, since these two analogs have similar retention times. It is recommended that GLC method A be adopted as official first action.

A collaborative study was conducted to compare the NF XII method and a GLC method for quantitatively determining menadione in pharmaceutical products. Ten collaborating laboratories analyzed 6 Pharmaceuticals encompassing tablets, capsules, and a liquid. Menadione is extracted with diethyl ether from ground tablets; slip capsules are opened and the contents are emptied into the ether along with the capsule parts. After filtration and removal of the ether, the residue is redissolved in n-hexane. The menadione sodium bisulfite injectable is transferred to a separatory funnel and extracted with chloroform and sodium hydroxide, evaporated to dryness, and redissolved in n-hexane. Menadione is chromatographed on a 6’ × 3mm id glass column packed with 1% neopentyl glycol succinate on 100–120 mesh Gas Chrom Q and operated at 135°C. The carrier gas flow is adjusted so that menadione appears within 5.5–6.5 mhi. Because of the coefficients of variation, additional study is recommended.

A collaborative study for the analysis of vitamin E in multivitamin tablets was conducted and the method was subsequently approved as official first action by the AOAC. Additional data were obtained by an extensive collaborative study sponsored by the Pharmaceutical Manufacturers Association, which verified the usefulness of GLC for the assay of α-tocopherol, α-tocopheryl acetate, and α-tocopheryI acid succinate. The mean coefficient of variation obtained from the 8 participating laboratories for duplicate samples, duplicate injections, was ±2.04, 1.75, and 2.71%, respectively. Further collaborative studies were conducted on samples of mixed tocopherol concentrate, multivitamin tablets, and multivitamin soft gelatin capsules. The mean coefficient of variation obtained from 13 collaborators was in the range of ±5%. The accumulated data demonstrate the utility of this technique for improved specificity and reliability of the analysis of vitamin E.

A chemical method for the determination of calcium pantothenate in pharmaceutical preparations is presented. The method involves a number of modifications of a method of Zappala and Simpson to make it applicable to a wide variety of materials. The pantothenate molecule is cleaved with acid, and the resultant β-alanine is treated with a chlorinating solution and then with potassium iodide. The free iodine is measured spectrophotometrically at 358 mμ. Interfering color, which cannot be eliminated by washing off the outer coating of tablets, is taken into account by the use of a sample blank. The results, obtained from many types of pharmaceutical samples, are in good agreement with the official microbiological assay. Beer's law is followed over a suitable working range, 0.25–1.50 mg.

Seven methods were used to compare the efficiency of total lipid extraction from 13 samples of eight different food products. The methyl esters of fatty acids and the butyrate esters of sterols were prepared and analyzed by gas liquid chromatography. On the basis of total lipid recovered and amounts of fatty acids and sterols present, a chloroform: methanol procedure was selected as the most effective method.

The USP XVII chemical method for the determination of folic acid is not applicable to pharmaceutical preparations containing p-aminobenzoic acid (PABA) or procaine hydrochloride (added to intertables to reduce discomfort). PABA is often present in older folic acid injectables in which a breakdown of the folic acid has occurred on long standing. A modified method is described to eliminate interference from these compounds by diazotizing the interfering material and then converting it to the corresponding phenol. The folic acid is then cleaved to form PABA, diazotized, and coupled with the Bratton-Marshall reagent. In a comparison of the two methods using known amounts of folic acid in the presence of PABA, better recovery of folic acid was achieved with the modified method than with the USP method. The application of this modification to commercial preparations is also demonstrated.

The official GLC method of the Association of Official Analytical Chemists (AOAC) for determining vitamin E was modified and collaboratively studied for the National Formulary (NF). The internal standard hexadecyl hexadecanoate (cetyl palmitate) was substituted for the dotriacontane used in the AOAC method, and some other minor changes were made. Eleven samples, representing all types of NF formulations and NF bulk materials, were analyzed by 11 laboratories. The coefficients of variation of the reproducibility and repeatability were 4.5 and 2.4%, respectively, for all laboratories and samples. The values were 3.4 and 1.6%, respectively, when the one laboratory statistically determined to be an outlier was excluded. The coefficients of variation of reproducibility and repeatability for o-tocopheryl acid succinate were 2.1 and 1.5%, respectively. All of these values lie within the 5% limit required by the NF.

The application of gas liquid chromatography (GLC) as an analytical tool for the determination of the fat-soluble vitamins A, D, E and K has yet to be utilized to its full potential. A review of the published work of many researchers in this field is presented. GLC methods to measure the vitamin A isomers have not been developed to any appreciable practical extent. Liquid liquid chromatography might well be the technique of choice. In the field of vitamin D there are indications that a practical GLC analysis is feasible for pharmaceutical preparations. The GLC applications for vitamin E are diverse, well defined and generally widely accepted in research laboratory situations and for regulatory and quality control usage. Vitamins K1 and K2 have been measured with limited success in a few research laboratories, but the GLC methods have not developed on a practical basis. However GLC is used for measuring vitamin K3 (menadione and menadione sodium bisulfite) on a fairly routine basis in quality control laboratories.

Publisher Summary Various vitamin E analogs are resolved from each other on a molecular-weight basis on a relatively nonpolar SE-30 immobile phase. The only exception to the molecular-weight separation occurs with α-tocopheryl succinate that occasionally appears as a component in pharmaceutical products; its behavior is very similar to that of α-tocopherol. This chapter discusses the techniques for the separation of various vitamin E analogs. The separation of the various vitamin E analogs in their natural state has the advantage of reducing the errors of recovery from derivative preparation. During the preparation of derivatives, the analyst must be alert to the efficiency of reaction and to possible alterations to analogs other than the intended added moiety. It is best to keep the overall operation as simple and uncomplicated as possible and yet achieve the intended result; thus, the vitamin E analogs in this method are not converted to derivatives.

Gas-liquid chromatography has been used to quantitatively determine menadione (vitamin K3) in pharmaceuticals, using a 1% neopentyl glycol succinate column and a hydrogen flame detector. The gas chromatographic measurements compare favorably with results by the National Formulary methods on the same samples.

A comparative study was made of 2 GLC calibration methods for determining the menadione and menadione sodium bisulfite content of pharmaceuticals. One method utilizes an external standard and the second method makes use of butylated hydroxyanisole as an internal standard. Measurements were made on 8 pure solutions of known concentration of menadione and on 5 pharmaceuticals containing menadione or menadione sodium bisulfite. No significant difference was found between the results obtained by the 2 methods.