Determination of nitrate in forages by using selective ion electrode: collaborative study

Each of 10 collaborating laboratories analyzed 4 blind duplicate pairs of forage samples for nitrate, by using a potentiometric method. Two forage controls and a 100 000 mg KNO3/L standard were also provided. Nitrate was extracted into an aqueous Al2(SO4)3 solution containing 70 mg KNO3/L and quantitated with a nitrate-selective electrode. Standards were prepared using extracting solution as diluent. Nitrate concentrations in forage samples ranged from less than 0.50 to 4.35% KNO3. Repeatability coefficients of variation (CVo) ranged from 1.74 to 3.61%, and reproducibility coefficients of variation (CVx) ranged from 6.92 to 7.66%. Mean recovery of a 0.55% KNO3 spike was 94.5%. The method has been adopted official first action.     

Performance characteristics of methods of analysis used for regulatory purposes. I. Drug dosage forms. D. High pressure liquid chromatographic methods

Precision parameters of high pressure liquid chromatographic methods approved by AOAC for the analysis of drug dosage forms were recalculated on a consistent statistical basis, using the computer program "FDACHEMIST." Eleven collaborative studies of 12 compounds in 66 dosage forms analyzed by an average of 9 laboratories per study, with a total of 1150 determinations, were reviewed. For the approved methods and methods awaiting approval (9 studies, 11 compounds, 54 dosage forms, and 959 determinations), the average repeatability relative standard deviation (within-laboratory; RSDo) was 1.0%; reproducibility relative standard deviation (among-laboratories, including within-; RSDx) was 2.5%; the ratio RSDo/RSDx was an unusually low 0.40, with an average outlier rate of 0.6% of the reported values. The line of best fit for RSDx plotted against - log concentration increases with decreasing concentration, extending approximately from RSDx = 2% at 100% concentration to RSDx = 3.6% at 0.01% concentration, a change in RSDx of about 0.4% for each 10-fold decrease in concentration, independent of analyte and matrix.     

Performance characteristics of methods of analysis used for regulatory purposes. I. Drug dosage forms. C. Automated methods

For analysis of drug dosage forms, precision measures of AOAC approved automated methods, usually containing a spectrophotometric or fluorometric measurement step, were recalculated on a consistent statistical basis, using a computer program "FDACHEMIST." Ten collaborative studies of 14 compounds in 38 materials, consisting of various dosage forms, usually in 10 replications by an average of 7 laboratories, with a total of 2461 determinations, were reviewed. The average relative standard deviations within-laboratory (RSDo) and among-laboratories (RSDx) were 1.1 and 1.9%, respectively, and the ratio of RSDo/RSDx was 0.57, with an average outlier rate of 0.57% of the reported values. The line of best fit for RSDx plotted against - log concentration increases slightly with decreasing concentration, extending from an RSDx of about 1.6% at 100% concentration to an RSDx of 2.2% at 0.1% concentration, a change in RSDx of about 0.2% for a 10-fold decrease in concentration, independent of analyte and matrix.     

Performance characteristics of methods of analysis used for regulatory purposes. I. Drug dosage forms. E. miscellaneous methods

Precision parameters of miscellaneous methods for the analysis of drug dosage forms approved by AOAC since 1972, and not previously reviewed in this series, were recalculated on a consistent statistical basis by using the computer program FDACHEMIST. Seventeen published collaborative studies were reviewed; the studies encompassed 19 analytes in 80 different materials (dosage forms), 102 collaborative assays, approximately 10 laboratories per study, and principally direct spectrophotometric, polarographic, and spectroscopic methods, for a total of 1451 determinations. The average repeatability relative standard deviation (within-laboratories, RSDo) for the instrumental methods was 1.5%; the reproducibility relative standard deviation (among-laboratories, including within-, RSDx) was 2.6%; the ratio RSDo/RSDx of the averages was 0.57, with an average outlier rate of 2.7% of the reported determinations. The line of best fit of RSDx for the instrumental methods plotted against the negative logarithm of the concentration increases slightly with decreasing concentration, extending from an RSDx of approximately 2.0% at 100% concentration to an RSDx of 3.4% at 0.001% (10 ppm) concentration; this represents an RSDx change of approximately 0.3% (absolute) for each 10-fold decrease in concentration, independent of analyte, matrix, and method. A method for determining precipitated allergenic protein by the micro-Kjeldahl technique appeared to be outside this general relation, showing an RSDx of about 13% at a concentration of 0.015% (150 ppm) nitrogen.     

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.     

UV spectrophotometric determination of piperine in pepper preparations: collaborative study

Eight collaborating laboratories performed replicate analyses for piperine on 5 samples representing pepper raw spice, oleoresins, and soluble seasonings. Piperine is extracted into ethylene dichloride and measured at maximal absorbance 342-345 nm with a UV light source. Piperine content is calculated using an absorbance factor derived from piperine. Intralaboratory coefficients of variation (CVo) ranged from 0.5 to 3.1%; interlaboratory coefficients of variation (CVx) ranged from 3.0 to 5.8%. The method has been adopted as an official method of the American Spice Trade Association and as an official first action method by AOAC.     

Atomic absorption spectrophotometric determination of tin in canned foods, using nitric acid-hydrochloric acid digestion and nitrous oxide-acetylene flame: collaborative study

Twenty-six collaborators participated in a study to evaluate an atomic absorption spectrophotometric (AAS) method for the determination of tin in canned foods. The 5 foods evaluated were meat, pineapple juice, tomato paste, evaporated milk, and green beans, each spiked at 2 levels. The concentration range of tin in the samples was 10-450 micrograms/g, and each level was sent as a blind duplicate. Statistical treatment of results revealed no laboratory outliers and 6 individual or replicate-total outliers, accounting for 3.3% of the data. Repeatability (within-laboratory) coefficient of variation (CVo) ranged from 2.2 to 48%, depending on the tin level and food evaluated. For samples containing greater than or equal to 80 micrograms/g of tin, repeatability CV averaged 5.6% including outliers and 3.7% after their rejection. Overall among-laboratories coefficient of variation (CVx) varied from 3.3 to 58%; at levels greater than or equal to 80 micrograms/g, it averaged 7.3% with outliers and 5.3% after their rejection. Recovery of tin, based on spiking levels, ranged from 100.0 to 112.8% and averaged 105.4%. Detection limit range is 2-10 micrograms/g, and lower quantitation limit is 40 micrograms/g. This method has been adopted official first action.     

Atomic absorption spectrophotometric determination of liver copper: collaborative study

Eleven collaborating laboratories conducted replicate analyses on 4 blind duplicate pairs of bovine liver samples that either had naturally acquired copper levels or were spiked with one of 3 copper levels. A National Bureau of Standards Bovine Liver sample (SRM 1577, 193 +/- 10 mg copper/kg) and a 1000 mg copper/L standard were also submitted to the collaborators. The method requires the tissue to be digested with concentrated HNO3 at 60 degrees C, diluted to volume with water, and analyzed by atomic absorption spectrophotometry. The intralaboratory coefficients of variation (CVo) ranged from 5.6 to 19%; the interlaboratory CVx values ranged from 7.1 to 21%. The lower limit of detection was estimated to be 1 mg copper/kg tissue. The method has been adopted official first action.     

Multiresidue gas chromatographic method for determining organochlorine pesticides in poultry fat: collaborative study

A gas chromatographic electron capture detector method is described for the quantitative determination of organochlorine pesticide residues in poultry fat. The samples are rendered and cleaned up using automated gel permeation chromatography. The collaborative samples consisted of 10 fortified samples and one incurred residue sample, all in duplicate. Fortification levels ranged from 0.15 to 1.0 ppm for alpha-BHC, lindane, cis- and trans-chlordane, octachlor epoxide, o,p' and p,p'-DDT, p,p'-DDE, p,p'-TDE, hexachlorobenzene, heptachlor epoxide, dieldrin, endrin, methoxychlor, mirex, and toxaphene. The average recovery was 91.9% with a range of 81-102%. The ranges of coefficients of variation were: CVo = 3.39-14.79%; CVL = 0-16.6%; and CVx = 5.82-19.0%. The results indicate accuracy and precision comparable to other official methodology. The method has been adopted official first action.     

Performance characteristics of methods of analysis used for regulatory purposes. I. Drug dosage forms. F. Gravimetric and titrimetric methods

The original gravimetric and titrimetric methods approved by AOAC for the analysis of pharmaceutical preparations, particularly during the period 1915-1950, show precision, recovery, and outlier parameters approximately the same as those exhibited by the previously reviewed instrumental methods that are currently used. Fifty-nine published collaborative studies utilized gravimetric methods and 85 used titrimetric. The studies of the gravimetric methods encompassed 47 analytes, 95 dosage forms, and 136 assays; the corresponding figures for the titrimetric studies are 72, 112, and 152. An average of approximately 7 laboratories participated per study. The line of best fit of the relative standard deviation between-laboratories (RSDR) plotted against the negative logarithm of the fractional concentration, C, extends from 1.2 and 1.0% for the gravimetric and titrimetric methods, respectively, at 100% concentration to 2.2 and 2.8% at 1.0% concentration. Below this concentration the precision of the titrimetric methods degenerates faster than that of the gravimetric methods. Above about 0.1% concentration the gravimetric and titrimetric methods are somewhat more precise than the instrumental methods in current use for drug analysis. The difference, however, is not statistically significant and the general equation, RSDR = 2 exp(1-0.5 log C), is also applicable to gravimetric and titrimetric methods above a concentration level of about C = 0.001 (0.1%).     

Multiresidue gas chromatographic method for determining organochlorine pesticides in meats: validation study for swine and beef fats

A validation of a previously studied method for determining organochlorine residues in poultry fat was conducted to extend the usefulness of the method to beef and swine fats. The validation samples consisted of 16 materials all analyzed in duplicate. Fortification levels ranged from 0.02 to 1.2 ppm for alpha-BHC, lindane, cis- and trans-chlordane, octachlor epoxide, o,p'- and p,p'-DDT, p,p'-DDE, p,p'-TDE, hexachlorobenzene, heptachlor epoxide, dieldrin, endrin, methoxychlor, mirex, and toxaphene. The average recovery was 101% with a range of 79-113%, not including toxaphene. The ranges of coefficients of variation were CVo = 0-23.37% and CVx = 3.74-26.19%. The results were comparable to the previous collaborative study of the same method for poultry fat. The extended method has been adopted official first action.     

Liquid chromatographic determination of methocarbamol in injection and tablet dosage forms: collaborative study

A reverse phase liquid chromatographic method was developed for determining methocarbamol in injection and tablet dosage forms. The injections require dilution only; the tablets require a filtration step before introduction into the chromatograph. Response for methocarbamol was linear over the range 0-18 micrograms, using an ultraviolet detector at 274 nm. Recoveries by the author ranged from 96.1 to 101.9% for authentic injection formulations and 98.0 to 101.0% for authentic tablet formulations. A collaborative study of the method by 6 laboratories resulted in standard deviations of 1.70 and 2.22 for injection and tablet dosage forms, respectively. The method has been adopted official first action.     

Determination of amphetamine by Schiff base formation and quantitative gas-liquid chromatography.

A procedure was developed to determine amphetamine salts in solid dosage forms. Amphetamine was extracted from the solid matrix with dilute hydrochloric acid and reacted with cyclohexanone in a strongly basic aqueous methanolic solution. The Schiff base reaction product was extracted with hexane for gas chromatographic determination. Reaction time and optimum conditions were studied. Phenethylamine, similarly treated, was used as an internal standard. Results compared favorably with those obtained by using USP methods.     

Liquid chromatographic determination of morphine sulfate and some contaminants in injections and bulk drug material

A liquid chromatographic (LC) procedure is described for the assay of morphine sulfate in bulk drug material and injection solutions. The bulk drug and injection samples are prepared by direct dilution with LC mobile solvent. The average bulk drug purity (5 manufacturers) determined by the LC method was 99.9% with a difference of 0.1% from the average purity (anhydrous) found by the official USP XX procedure. The average LC recovery (19 studies) of morphine sulfate added to injection samples was 99.4% with a coefficient of variation (CV) of 1.14%. Morphine sulfate content was determined in triplicate for 53 injection samples (1-15 mg morphine sulfate/mL) formulated by 6 manufacturers, using the proposed LC procedure. Individual sample CV (n = 3) averaged 1.14%. The LC method is simple and specific for morphine sulfate. Major degradation products, preservatives, and some contaminants and related compounds are separated during LC.     

Simultaneous determination of oxytetracycline, tetracycline, and chlortetracycline in milk by liquid chromatography

A simple, rapid procedure is described for the simultaneous quantification of 3 tetracycline drugs in bovine milk. Samples are prepared by dilution with an EDTA/phosphate buffer solution and filtration through a molecular weight cutoff filter. Analytes are concentrated on-column using a reverse-phase gradient elution system of oxalic acid, acetonitrile, and methanol. The limits of quantitation are approximately 15-50 ng/mL and the limits of detection are 10-20 ng/mL, depending on the compound. For oxytetracycline, over the range 50-1200 ng/mL, the average recovery and intralaboratory coefficient of variation were 97% and 4.1%, respectively. Over the same range, these parameters were, respectively, 97% and 5.0% for tetracycline, and 89% and 6.4% for chlortetracycline. The applicability of this procedure is demonstrated by separation and detection of incurred tetracycline residues in milk from treated animals.     

Gas-liquid chromatographic determination of total cholesterol in multicomponent foods.

A method is described for the determination of total cholesterol in multicomponent foods and also other products such as nonfat dry milk, dried whole egg solids, and certain candy bars. The lipid is extracted from the sample by a mixed solvent and saponified. The unsaponifiable fraction which contains the cholesterol and other sterols is extracted with benzene. An aliquot is evaporated to dryness and the residue is dissolved in dimethylformamide. The sterols are derivatized to form trimethylsilyl (TMS) ethers. The TMS-cholesterol derivative is quantitatively determined by gas-liquid chromatography, using 5alpha-cholestane as an internal standard. Nine laboratories participated in a collaborative study of the determination of total cholesterol in deviled ham sandwich spread, vegetable beef stew, corned beef hash, frozen chicken pot pie, pizza pepperoni, fish sticks, breaded shrimp, chocolate-covered candy bars, dried whole egg solids, and nonfat dry milk and the results are reported here. The coefficient of variation ranged from 5.64 to 23.2%, with an average coefficient of variation of 14.8%.     

Reverse-phase liquid chromatographic determination of dexamethasone acetate and cortisone acetate in bulk drug substance and dosage forms: collaborative study

A reverse-phase liquid chromatographic method for determination of dexamethasone acetate and of cortisone acetate was subjected to an interlaboratory study by 8 collaborators for each steroid acetate. Bulk drug substance, suspensions, and tablets were assayed. Bulk drug or dosage form is dissolved in an acetonitrile-buffer mixture and analyzed by an external standard method. The steroid acetate is resolved from extraneous components by reverse-phase chromatography and detected at 254 nm. The sample solutions are stable for at least 72 h. For dexamethasone acetate, coefficients of variation were 0.9 and less than or equal to 3.1% for the bulk drug substance and the suspensions, respectively. For cortisone acetate, coefficients of variation were 0.7% for bulk material, less than or equal to 2.0% for suspensions, and less than or equal to 2.5% for tablets. All dosage forms were commercial formulations. The 2 methods have been adopted official first action.     

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.     

Gas chromatographic determination of ethylene dibromide in flour products

A method based on gas chromatography with electron capture detection was developed for the determination of ethylene dibromide (EDB) extracted from flour products. The procedure relies on the organic extraction of flour/water mixtures and uses an internal standard, 1-bromo-3-chloropropane. Recoveries of EDB at 10 and 100 ppb were 80.1 +/- 2.8% (SD) and 84.4 +/- 4.3%, respectively; recovery of the internal standard at the working concentration 500 ppb was 98.3 +/- 6.7%. Calibration curves were linear over the range 5-400 ppb, with a mean overall coefficient of variation of less than 5%. The reliability of the procedure was assessed by using gas chromatography combined with mass spectrometry. Results are shown for determination of EDB in locally milled flour products.     

Evaluation of analytical methods for carotenoid extraction from biofortified maize (Zea mays sp.)

Biofortification of maize with beta-carotene has the potential to improve vitamin A status in vitamin A deficient populations where maize is a staple crop. Accurate assessment of provitamin A carotenoids in maize must be performed to direct breeding efforts. The objective was to evaluate carotenoid extraction methods and determine essential steps for use in countries growing biofortified maize. The most reproducible method based on coefficient of variation and extraction efficiency was a modification of Kurilich and Juvik (1999). Heat and saponification are required to release carotenoids from biofortified maize and remove oils interfering with chromatographic analysis. For maize samples with high oil content, additional base may be added to ensure complete saponification without compromising results. Degradation of internal standard before carotenoids were released from the maize matrix required the addition of internal standard after heating to prevent overestimation of carotenoids. This modified method works well for lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, and beta-carotene.