An automated procedure for the determination of rat adrenal ascorbic acid used in the U.S.P. XVII bioassay of corticotropin is described. Ascorbic acid analyses of manually filtered, adrenal gland homogenates are performed with buffered 2,6-dichloro-indophenol sodium. A sample-rinse ratio of 1 : 2 is used and sample carryover is not detectable. AutoAnalyzer Sampler 11, proportioning pump, colorimeter, and recorder are used. The coefficient of variation for the automatic procedure is approximately 1 per cent at an effective sampling rate of 6o/hr. An IBM 1620 computer is used for calculations of ascorbic acid determinations and for the bioassay of corticotropin using a 3 X 3 statistical design. Assay results using automated and manual ascorbic acid values compare favorably.
HE DEPLETION of ascorbic acid of rat adrenal
Tglands is used as the measure of response in the bioassay of corticotropin as directed b y U.S.P. XVII (1). For the subcutaneous assay, hypophysectomized rats are injected in random order with varying doses of standard or test preparations using a 3 X 3 design. Three hours after injection, the paired adrenals are removed, freed of excess tissue, weighed, and homogenized in 2.5% metaphosphoric acid. The ascorbic acid content of each pair of adrenal glands is then determined using buffered 2,6-dichloro-indophenol sodium. Statistical analyses are applied to ascorbic acid values to determine potency, precision, and validity of t h e assay. This report describes a n automated procedure for the determination of r a t adrenal ascorbic acid used in t h e bioassay of corticotropin. The automated method, based on the U.S.P. XVII procedure, requires manual filtration of adrenal gland homogenates. Automatic sampler,* proportioning pump, colorimeter, and recorder are used, permitting the analysis of 60 samples per hour, including wash between samples.
MATERIALS AND METHODS
Instruments-Automatic
sampler, proportioning pump, colorimeter with 524 mp filters and 1.5-cm. flow cell, recorder, and chart reader.
Reagents-(a) 2,G-Dichloro-indophenol sodium, 48.0 mg./1000 ml. Filter and store in a brown bottle under refrigeration. ( b ) Sodium acetate trihydrate, 45.3 Gm./lOOO ml. Adjust to pH 7 with acetic acid. Store in a brown bottle under refrigeration. ( c ) Color reagent, equal parts of 2,