There is not yet a suitable assay for male fern extract. Variation in proportions of phloroglucinol derivatives are not reflected by the gravimetric determination for filicin (B.P. 1968). Neither the paper chromatographic method of Klevstrand (1961) nor the thin-layer method of von Schantz, Ivars & others (1962) are satisfactory. The present paper describes a two-dimensional thin-layer method for the separation of the two major constituents in Dryopteris jilix-mas (L) Schott., namely flavaspidic acid and filicic acid, followed by their ultraviolet spectrophotometric determination. ExperimentalSilica gel G (Merck): washed with purified water, twice with dry ethanol and twice with dry chloroform, filtered off and dried in a current of air between washings. After final drying the material was powdered and then stored in an airtight container.Method. The two-dimensional method of Fish & Kirk (1968) was used. Plates were prepared with the pre-treated silica gel (25 g) mixed with ascorbic acid (150 mg) in buffer solution, pH 6.0. Before spreading, the plates were soaked overnight in detergent solution, throughly rinsed and dried.Commercial extract of male fern B.P. was dissolved in ether (100 mg/ml) and, by means of a Hamilton syringe fitted with a PB600 dispenser, 10 pl of the solution was spotted near one corner of a plate, using a spotting template (Brain & Hardman, 1968). Alongside each test plate, a control (blank) plate was developed twodimensionally, then both plates were dried in the dark at 25" to remove all traces of solvent (about 2-3 h).The male fern extract (1 g) was treated by the official assay process (B.P. 1968) and 10 pl of the chloroform solution of filicin obtained (75 ml) was spotted on to plates and chromatographed as above.Samples of flavaspidic acid and filicic acid were obtained as described previously (Fish & Kirk, 1968) and fixed volumes (10 pl) of ethereal solutions containing known amounts of either of these compounds were also chromatographed. From the results calibration curves were constructed ; they were linear over the range 0.001-Elution and determination. The test plates were examined in ultraviolet light of 366 nm and the dark purple, fluorescence-quenched areas corresponding to flavaspidic acid and filicic acid were marked. The adsorbent from those areas, and from areas corresponding exactly in size and position on the blank plates, were separately removed using the glass transfer tool described by Bird, Brickley & others (1963).Each portion of adsorbent was extracted repeatedly by mixing with dry chloroform (12 x 0.4 ml), the solutions being bulked and made up to volume (5 ml).
30 u ml-1) to 5% dextrose solution (Baxter Labs.), which was kept sterile at ,-Onstant temperature of 15O, 2 5 ' and 3 5 ' and sampled at time intervals. Table 1. Loss and recovery of heparin anticoagulant activity in dextrose unstable (Okuno & Nelson, 1975); its apparently erratic behaviour given solutions Solution in which heparin was dissolved (30 u ml-1) within 5 h, % activity+ , h Fall in activity+ Time for recovery of Dextrose 5% 65 24 -48 'I + NaCl 0.9% 40 5 -7 I' + NaCl 2% 0 -*calculated as percentage of zero-time anticoagulant activity.The decline in activity frequently occurred by 1 h. Addition of 0.9% NaCl to the dextrose solution clearly modified the pattern of activity change which was abolished altogether by addition of 2% NaC1. The eventual recovery of full anticoagulant activity showed that heparin had not been destroyed. Also, the acceleration of recovery when 0.9% NaCl had been added together with abolition of the effect when 2% NaCl was present, suggest that a reversible salt-sensitive rearrangement or interaction of the heparin molecule took place in presence of a certain dextrose concentration. 4.5% dextrose solution, the only change observed wasa 15% variation in activity over 5 h; lower concentrations of dextrose caused no change. The pattern of activity change in 5% dextrose was independent of temperature for the temperatures used, supporting the view that degradative change was not occurring.Concurrent chemical determination of heparin using toluidine blue, which measures macroanion activity (McIntosh, 1941) showed no variation in heparin concentration throughout 72 h, indicating no change in available ester sulphate and confirming belief about the integrity of the heparin molecule in such conditions. Whether, in use, the temporary modification of anticoagulant activity in 5% dextrose solution is rapidly restored on injection into the completely different -in vivo environment has still to be determined. Meanwhile, doubt about the complete suitability of 5% dextrose solution as a vehicle for heparin infusion persists and the need for vigilance and improvement in the laboratory control of heparin therapy is underlined. Jacobs,
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