Internal Standard Solution Research Articles

Active TBC in Butadiene by HPLC

Abstract A high performance liquid chromatographic method has been developed for the determination of active 4-tert-butyl-catechol (TBC) In 1,3-butadiene. Following evaporation of the butadiene from an aqueous m-nitrophenol internal standard solution, a 20-microliter aliquot is injected onto a reversed-phase liquid chromatographic column. Recovery op TBC was found to be quantitative over a 17-242 ppm range with a relative standard deviation of 3.8%.

Determination of Minoxidil in Bulk Drug and Pharmaceutical Formulations by Ion-Pairing High-Performance Liquid Chromatography

An ion-pairing liquid chromatographic method with UV detection is described for the determination of minoxidil in bulk drug, compressed tablet, and topical solution formulations. The chromatographic system consists of a microparticulate octadecylsilica column and a mobile phase composed of sodium dioctylsulfosuccinate in aqueous methanol (pH 3). The bulk drug and the topical solution samples are prepared by the dissolution of the drug in internal standard solution. Sample preparation for the compressed tablet formulation involves dissolving the drug from an aliquot of pulverized sample and centrifuging to remove insoluble excipients. Quantitative recovery of minoxidil from formulation excipients was demonstrated; assay precision was <1% CV.

Performance Characteristics of Methods of Analysis Used for Regulatory Purposes. I. Drug Dosage Forms. B. Gas Chromatographic Methods

Gas chromatographic methods for the analysis of drug dosage forms consist of a simple extraction, dilution with an internal standard solution, and injection, or, even simpler, dilution with the internal standard solution and injection. These methods were used in 7 collaborative studies of the determination of 12 pharmaceuticals, published in the Journal of the AOAC during 1973-1983. A total of 43 individual materials consisting of various dosage forms were each analyzed, usually in duplicate, by an average of 8 laboratories, with a total of 582 reported determinations. The average within-laboratory coefficient of variation (CVo) was 1.25% and the average among-laboratories coefficient of variation (CVx) was 2.41%, for a CVo/CVx ratio of 0.52, at an average outlier rate of 1.4% of the reported values. The line of best fit for CVx plotted against concentration increases with decreasing concentration, extending from a CVx of approximately 1.8% at 100% concentration to a CVx of approximately 3.2% at 1% concentration. The change in CVx for a 10-fold decrease in concentration is approximately 0.7% CVx, independent of analyte and matrix.

Gas Chromatographic Determination of Nordihydroguaiaretic Acid in Larrea Divaricata

Abstract A glc analysis method utilizing a flame ionization detector was developed to determine nordihydroguaiaretic acid (NDGA). Lyophilized samples of an aqueous extract from leaves of L. divaricata were reconstituted in water at a concentration of 50 mg/ml. A 100 mcl aliquot of this solution had 50 mcl of the internal standard solution, p,p'-biphenol (0.4 mg/ml in methanol) added followed by extraction with benzene:isopropyl alcohol (98:2), evaporation, and trimethylsilylation with N,O-bis-(trimethylsilyl)-acetamide. Glc analysis was accomplished using a 10% OV-101 on 80/100 Gas Chrom Q column. Accuracy and precision for the glc method was demonstrated over the range of anticipated concentration of NDGA in the L. divaricata leaves. The content of NDGA present in the leaves was found to be 1.6%.

Liquid Chromatography of Liquid Formulations Containing 2,4-Dichlorophenoxyacetic Acid, Dicamba, and 2-(2-Methyl-4-chlorophenoxy)propionic Acid as Their Salts

Abstract A high pressure liquid chromatographic method has been developed for liquid herbicide combinations that contain different combinations of 3 active ingredients including 2,4-dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy)propionic acid (MCPP), and dicamba. A reverse phase column in the ion suppression mode and a binary solvent system separate all 3 herbicides quantitatively on a single chromatogram. The internal standard solution may contain 2 internal standards, salicylic acid and butyrophenone, for use with certain combinations of the herbicides. The solvent system resolves the compounds of interest from all significant impurities.

Thiopental monitoring by gas-chromatography.

A gaschromatographic method is presented for the determination of thiopental (and of its metabolite pentobarbital) in cases of severe head injuries treated by induction of therapeutic barbiturate coma. To 1 ml serum the internal standard and saturated ammonium sulphate solution are added. The mixture is extracted by chloroform and the concentrated organic phase is injected into the gaschromatograph (stationary phase: SP 2510 DA). Imprecision from day to day: Coefficient of variation 7.7%; recovery 97%. The specificity was checked by comparison with the retention time of more than 80 drugs. One determination is accomplished within 1 hour.

Detection and identification of volatile organic compounds in blood by headspace gas chromatography as an aid to the diagnosis of solvent abuse

A gas chromatographic method has been developed for the detection and identification of some volatile organic compounds in whole blood, plasma or serum. After incubation of the sample (200 μl) together with the internal standard solution in a sealed vial, a portion of the headspace in analysed using a 2-m glass column packed with 0.3% (w/w) Carbowax 20M on Carbopack C, 80–100 mesh. The column oven, after a 2-min isothermal period, is programmed from 35 to 175°C at 5°/min and held for 8 min. the effluent is monitored by both flame-ionisation and electron-capture detection, and peak assignment is by means of retention time and relative detector response. The method has proved applicable to the detection of bromchlorodifluoromethane, n-butane, carbon tetrachloride, chlorobutanol, cryofluorane (Halon 114), dichlorodifluoromethane (Halon 12), ethyl acetate, halothane, isobutane, isopropanol, isopropyl nitrate, methyl ethyl ketone, propane, tetrachloroethylene, toluene, 1,1,1-trichlorothane, 2,2,2-trichlorethanol, trichloroethylene and trichlorofluoromethane (Halon 11) in blood specimens obtained from patients suspected of abusing these agents.

High-performance Liquid Chromatographic Determination of Suprofen in Drug Substance and Capsules

A rapid, simple, stability-indicating assay procedure for suprofen, a new analgesic agent, in suprofen drug substance and in capsules was developed using high-performance liquid chromatography. Suprofen was extracted from the sample matrix with methanol and diluted with internal standard solution, and an aliquot was chromato-graphed on a reversed-phase column using acetonitrile-pH 3.0 buffer solution as the mobile phase. The selectivity of the chromatographic system for intact suprofen was demonstrated by resolving suprofen from synthetic intermediates, potential impurities, and reaction products resulting from accelerated stress conditions. The method is linear, quantitative, and reproducible. Either peak height or peak area ratios can be used for quantitation.

Rapid high-performance liquid chromatographic method for the measurement of verapamil and norverapamil in blood plasma or serum

A simple high-performance liquid chromatographic method for the simultaneous measurement of plasma verapamil and norverapamil concentrations has been developed. The sample (100 μl) is vortex-mixed for 30 sec with 4 M sodium hydroxide solution, pH 13 (50 μl), internal standard solution (aqueous 5,6-benzoquinoline, 0.20 mg/l) (50 μl) and methyl tert.-butyl ether (200 μl). After centrifugation at 9950 × g for 2 min, a portion (100 μl) of the resulting extract is analysed on a microparticulate (5 μm) silica column using a methanolic solution of potassium bromide (3.0 mM) and perchloric acid (0.37 mM) as the mobile phase, and the column effluent is monitored by fluorescence detection using an excitation wavelength of 203 nm. A specimen, together with a quality control sample, can be analysed, in duplicate, within 30 min. The limit of accurate measurement of the assay is 2 μg/l, and no potential sources of interference have been identified. The method has advantages of speed, small sample requirement and complete resolution of the three major metabolites of verapamil.

High Pressure Liquid Chromatographic Analysis of Chlorpyrifos Containing Insecticidal Formulations: Collaborative Study

Abstract A high pressure liquid chromatographic method for determining chlorpyrifos was collaboratively studied. Three liquid and 2 solid formulations were analyzed by 12 collaborators. Samples were weighed and diluted with acetonitrile containing the internal standard, 1,4-dibromonaphthalene. Chlorpyriphos is separated on a Zorbax ODS column with acetonitrile- water-acetic acid (82 ± 17.5 ± 0.5) at 2 mL/min and monitored at 300 nm. Clay samples are extracted with the internal standard solution by agitation for 5 min. Coefficients of variation ranged from 1.28 to 2.40%. Results of all 12 collaborators fell within the 5% two-tail limits. The method has been adopted official first action.

Rapid micro-method for the measurement of paracetamol in blood plasma or serum using gas-liquid chromatography with flame-ionisation detection

A simple method for the measurement of plasma paracetamol concentrations using a novel extraction/acetylation procedure prior to gas-liquid chromatographic analysis has been developed. The sample (100 μl) is vortex-mixed for 30 sec with 0.067 mole/l phosphate buffer, pH 7.4 (50 μl), internal standard solution (N-butyryl- p-aminophenol (200 mg/l) in chloroform) (50 μl) and “acetylation reagent” (acetic anhydride-N-methylimidazole (catalyst)-chloroform, 5:1:30) (20 μl). After centrifugation at 9950 g for 3 min, a portion (5 μl) of the resulting extract is analysed on a 1.5 m × 4 mm I.D. glass column packed with 3% (w/w) C 87 hydrocarbon (Apolane-87) on Chromosorb W HP, 100–120 mesh, maintained at 235°C. A specimen together with a quality control sample can be analysed, in duplicate, within 20 min. The limit of accurate measurement of the assay is 10 mg/l, and few potential sources of interference have been identified. The method has advantages of speed and reproducibility over other gas-liquid chromatographic procedures and, in addition, of selectivity over spectrophotometric techniques. The procedure provides a useful alternative to liquid chromatographic methods for emergency paracetamol measurements.

Routine quality evaluation of benzodiazepine drugs to USP-NF specifications

A GLC procedure was developed for the evaluation of diazepam, chlordiazepoxide, and flurazepam formulations to USP-NF specifications for drug content, content uniformity, impurities, and identity by retention times and peak areas. The polyimide column, instrument zone temperatures, gas flows, internal standard solution, extraction solvent, and auxiliary equipment were the same for each drug. No derivatization of the samples was required. The GLC assay values (mean of 10 individual dosage units) for diazepam and flurazepam products were in good agreement with the results obtained by the pharmacopeial composite assays. With chlordiazepoxide capsules, when the levels of the two pharmacopeial impurities determined by GLC were added to the GLC assay results (mean of 10), the aggregate values were consistent with the drug content results found by the nonspecific USP method. The procedure can be made sensitive to impurity levels of ∼0.01% for 2-amino-5-chlorobenzophenone and to ∼0.2% for 7-chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one 4-oxide. With the equipment used, the estimated potential outputs in lots per working day for a complete quality profile (drug content, content uniformity, purity, and identity) were seven for chlordiazepoxide if no impurity test was required, five if such a test was required, eight for diazepam, and seven for flurazepam.

Determination of acetazolamide in biological fluids by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) assay for acetazolamide is presented. A 100-microliter sample is mixed with an aliquot of the internal standard solution and the mixture, buffered at pH 4.5, is extracted with ethyl acetate. The extract is evaporated to dryness and the residue is analyzed by HPLC, using a reverse-phase octadecylsilane column. The wavelength of the detection is 254 nm. The coefficient of variation (CV) in the within-day analysis of replicate 10-microgram/ml acetazolamide samples in human blood plasma was 6.5%, while the between-day CV was 7.1%. The procedures was developed for the 1-25 microgram/ml acetazolamide concentration range. The internal standard used is similar in chemical structure to acetazolamide and can be readily prepared in one step from a commercially available precursor. In addition to blood serum or plasma, the assay can also use aqueous and vitreous humor samples. Theophylline and acetaminophen interfere in the assay. The technique was used to determine the concentration of acetazolamide in the blood serum of human volunteers after an oral dose of the drug, and in the aqueous and vitreous humors of rabbits after an intravenous dose of acetazolamide.

A rapid and simple microassay for lidocaine in human blood plasma using gas-liquid chromatography with nitrogen detection.

Lidocaine is widely used in antiarrhythmic therapy, and the need to monitor the drug concentration in the blood of patients receiving lidocaine is well established. We have developed a new assay for lidocaine designed for the routine clinical therapeutic drug monitoring laboratory. A 100-microliter aliquot of blood plasma is treated with 50 microliters of internal standard solution and the mixture alkalinized. The drug and internal standard are extracted with toluene and analyzed by nitrogen-detection gas-liquid chromatography. A single extraction is used, and no solvent evaporation is required, resulting in a simple and rapid procedure. Using the single-point standard, the within-day coefficient of variation (CV) was 6.4%, while the between-day CV was 7.4%. Monoethylglycinexylidide, the major plasma metabolite of lidocaine, and several drugs potentially present in patients receiving lidocaine were shown not to interfere. The procedure has significant advantages over previously published similar methods.

Determination of phenol in injectable biological products by gas chromatography

A gas chromatographic method for the determination of phenol in a wide variety of biological products has been developed by extending a USP procedure. The procedure and its extension were validated for preparations of cholera vaccine, typhoid vaccine, tuberculin purified protein derivative, pneumococcal polysaccharide vaccine, horse serum-derived products such as antivenins, several types of aqueous, glycerinated and alum precipitated allergenic extracts and others. The average recovery of phenol at the 0·3–0·5% phenol level was 99·6%. The average precision was measured as a relative standard deviation of 0·43%. Glycerin contained in glycerinated allergenic extracts and tuberculin purified protein derivative did not interfere with quantitation. Horse serum-derived antitoxins, antivenins and antirabies serum produced a precipitate upon the addition of the internal standard solution, benzyl alcohol in 100% methanol. This precipitate was eliminated by the use of 5% (or 0%) methanol in water as diluent in the internal standard solution. Two aqueous allergenic extracts were encountered that produced peaks that were not adequately resolved from the phenol and benzyl alcohol peaks. Lowering the injection temperature to 185 °C and the column temperature to 125 °C produced satisfactory separations. Gas chromatography/mass spectrometry was used to determine the purity of peaks representing benzyl alcohol and phenol in tuberculin, cholera vaccine, typhoid vaccine and several allergenic extracts.

Isomer Specific Assay of Ester and Salt Formulations of 2-Methyl-4-chlorophenoxyacetic Acid and 2,4,5-Trichlorophenoxyacetic Acid by High Pressure Liquid Chromatography: Collaborative Study

Abstract HPLC methodology for ester and salt formulations of 2 methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-TA) has been substantiated in 2 collaborative studies. The methods closely follow the AOAC method for 2,4-dichorophenoxyacetic acid (2,4-DA), including preparation of samples by addition of a saponification internal standard solution to all samples. A microparticulate column in the reverse phase ion suppression mode separates all known impurities from the internal standard and MCPA, or 2,4,5-TA. The only differences in essential parameters between the 2 methods concern the internal standard, strength of solvent (acetonitrile), and pH of the acidic buffered eluant. One of the test samples is a combination of 2,4,5-TA and 2,4-DA as an ester formulation and each herbicide is quantitated on the same chromatogram. In respective acid equivalent, the coefficient of variation ranged from 1.67% for the 45% 2,4,5-TA ester formulation to 2.36% for the 22% 2,4,5-TA component in the combination ester formulation of 2,4-DA and 2,4,5-TA. There were 14 collaborators from the United States and Canada including 6 from industry and 8 from government agencies. The methods have been adopted as official first action.

Mannitol estimation in biological fluids by gas-liquid chromatography of trimethylsilyl derivatives.

We describe a procedure for estimating mannitol concentrations in biological fluids. Samples are mixed with internal standard solution (alpha-methylglucose), deproteinized if necessary, desalted, and dried. Specimens are then derivatized by adding pyridine/bis(trimethylsilyl)acetamide/trimethylchlorosilane and heating at 60 degrees C for 30 min. Samples are chromatographed on a 275-cm column of 10% OV-17, operated at 190 degrees C, and quantitated by peak-height measurement. The technique is linear, accurate, precise, sensitive, and free from interference. It has been used to measure mannitol in plasma, urine, and bile.

Mannitol estimation in biological fluids by gas-liquid chromatography of trimethylsilyl derivatives.

Abstract We describe a procedure for estimating mannitol concentrations in biological fluids. Samples are mixed with internal standard solution (alpha-methylglucose), deproteinized if necessary, desalted, and dried. Specimens are then derivatized by adding pyridine/bis(trimethylsilyl)acetamide/trimethylchlorosilane and heating at 60 degrees C for 30 min. Samples are chromatographed on a 275-cm column of 10% OV-17, operated at 190 degrees C, and quantitated by peak-height measurement. The technique is linear, accurate, precise, sensitive, and free from interference. It has been used to measure mannitol in plasma, urine, and bile.

Gas-Liquid Chromatographic Method for Determining Bolstar Insecticide in Formulations: Collaborative Study

Abstract A gas-liquid chromatographic (GLC) method for determining Bolstar insecticide in liquid technical material and emulsifiable concentrate formulations was collaboratively studied using Youden’s matched pair scheme. Three matched sample pairs were analyzed by 21 laboratories using integrator area measurements and/or peak height measurements. Samples of a technical material containing about 78% active ingredient, a commercial 6 lb/ gal. emulsifiable concentrate containing about 64% active ingredient, and a synthetic emulsifiable concentrate containing about 64% active ingredient were dissolved in a measured amount of internal standard solution, tetracosane in toluene, and were subjected to gas-liquid chromatography on a column of 1.5% SE-30 + 1.5% OV-210. Bolstar was detected using flame ionization. The mean coefficient of variation by integrator area measurement for the 6 samples was 1.22%, and the mean coefficient of variation by peak height measurement for the 6 samples was 1.65%. The method was adopted as official first action.

Determination of chlorinated phenols in surface-treated lumber by HPLC.

A solution of internal standard in acetonitrile is used to extract samples of wood from lumber which had been commercially treated with chlorinated phenols. The tetrachlorophenol(TCP) and pentachlorophenol( PCP) were analytically separated from each other and from the other wood extractive compounds using a high performance liquid chromatograph (HPLC). The ultraviolet (UV) absorptions of the TCP, PCP, and internal standard were automatically measured as they eluted. The UV absorption peaks were integrated, and the amounts of TCP and PCP present were calculated with a dedicated microcomputer. Compared with the method previously used, this method is faster (up to 144 samples per 48 hours), has the same accuracy and precision, and it is linear over the concentration range used.