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Resolution of Antibiotic Mixtures in Serum Samples by High-Voltage Electrophoresis

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Resolution of Antibiotic Mixtures in Serum Samples by High-Voltage Electrophoresis J Clin Pathol: first published as 10.1136/jcp.28.6.435 on 1 June 1975. Downloaded from J. clin. Path., 1975, 28, 435-442 Resolution of antibiotic mixtures in serum samples by high-voltage electrophoresis D. S. REEVES AND H. A. HOLT From the Department of Medical Microbiology, Division ofPathology, Southmead Hospital, Bristol BSJO SNB SYNOPSIS The use of high-voltage electrophoresis and bioautography for the separation mixtures of antibiotics received in clinical serum samples is described. The electrophoretic characteristics of 39 antibiotics are given as is the accuracy of the method for quantitating three antibiotics. In screening 189 consecutive serum samples submitted for routine antibiotic assay, undisclosed anti- biotics were revealed by the technique in 35. The treatment of infection by administering more Methods than one antibiotic simultaneously to patients in hospital is common practice. Increasingly labora- MATERIALS tories are being asked to assay sera from such The sources of the antibiotics used are shown in patients, and although it is frequently possible to table I. They were prepared for electrophoresis as measure at least one element in a mixture by the use solutions in distilled water or pooled human serum. of a selectively resistant organism or by inactivating Samples from patients examined for their anti- a component, such procedures are not always biotic content were those which had been received available. A further problem is that, in our ex- for assay, mostly of gentamicin, in controlling http://jcp.bmj.com/ perience, sera for assay sometimes contain anti- therapy. They were stored at -20°C for examination biotics ofwhich the laboratoryis not made aware and in batches. which can adversely affect assay results. The clinician requesting the investigation may not always know, SEPARATION OF ANTIBIOTICS or just simply forgets, about antibiotics which a The method used was adapted from that described patient is receiving or has recently received. There- by Lightbown and de Rossi (1965). The apparatus fore a technique is needed to detect the presence of (Stogate Instruments Ltd) (fig 1) used consisted of a on October 3, 2021 by guest. Protected copyright. extraneous antibiotics and to identify them. If the carefully levelled, water-cooled bed on which the technique could be made quantitative then this glass platten carrying the agar was placed. The water would be an advantage. supply to the bed was from a mixer-tap, allowing the With these criteria in mind we adapted the well- temperature of the platten to be varied. At each end known technique of high-voltage electrophoresis to of the platten there were two buffer boxes, the distal resolving antibiotic mixtures. To our knowledge its ones containing the platinum electrodes (25 cm of adaptation for use with the type of concentrations 0-38 mm diameter wire). The platten and boxes were found in the sera of patients on treatment has not all contained in a Perspex safety-cabinet with a lid previously been described. Using this method we which operated micro-switches controlling the determined the relative mobility of 39 antimicrobial output of the power pack. The power pack (Stogate agents used in clinical practice and examined the Instruments Ltd) was capable of delivering a conditions for their optimal detection and also the maximum of 200 mA and 2500 volts. limits of detection. We also determined the accuracy Electrophoresis was performed in 0-9 % agarose of quantitative assay of three antibiotics. Finally, a (BDH) in pH 5-6 buffer.' Seventy millilitres of 1 8 % retrospective survey was made of sera submitted for agarose in water was melted, and an equal volume of antibiotic assay from patients to determine the buffer was added. A rectangular sheet of glass was frequency of extraneous antimicrobial agents. 'Tris-(hydroxy-methyl)-aminomethane 27-21 g Maleic acid 26-10 g Received for publication 7 January 1975. Distilled water to adjust to pH 56 with SN NaOH. 435 J Clin Pathol: first published as 10.1136/jcp.28.6.435 on 1 June 1975. Downloaded from 436 D. S. Reeves and H. A. Holt Drug Source depth of 1-3 mm. The frame was removed when the agarose was set. Sample volumes of 10 ,ul were Penicillin G (3B271B), cephaloridine Glaxo Laboratories Ltd (805 02A 0573), lividomycin placed in wells cut with a 4 mm diameter cork borer. (AP003H), streptomycin (H82202 The buffer boxes each contained 600 ml of half- 1271) strength buffer and were connected to the agar and Ampicillin (Ref. Std 29), cloxacillin Beecham Research (F-511/1), flucloxacillin (Std No. 4), Laboratories Ltd each other with presoaked lint wicks running the carbeniciffin (Ref. Std No. 8), full width of the plate. After closing the lid of the carfecillin (Ref. Std 2), ticarcillin (Ref. Std) safety box the electrophoresis was run normally for Cephalothin (SJL43B), cephalexin Eli Lilly & Co Ltd 40 minutes at a constant current of 190 mA, which (Lot No. 413-214-163), cephazolin a (767), tobramycin (849-AD-1 14-1), gave starting potential of 800 volts. The time vancomycin (7AK27A), cycloserine varied according to the degree ofseparation required. (92692) During electrophoresis cold water at 18-20°C from Pivampicillin (206), fusidic acid Leo Laboratories Ltd (69024LA), mecillinam (GA27180) the mains was run through the bed. Lincomycin (13403), clindamycin Upjohn Ltd (M6612), novobiocin (M6663) Trmethoprim (49036), neomycin Burroughs, Wellcome & Co DETECTION OF ANTIBIOTICS IN RUNNING (43515) LAYER Amoxycillin (Ref. Std No. 5) Bencard (Beecham) The positions and amounts of antibiotics in the Cephacetrile (02/261/1), rifampicin Ciba/Geigy Laboratories running layer were determined by adding a seeded (01/711/3) layer ofagar onto the agarose after the completion of Colistin (B5240), clomocycline Pharmax Ltd (75/83) the electrophoresis. The seed layer was usually 200 Oxytetracycline (5649-810D-3) Lederle Laboratories ml of Oxoid DST agar incorporated with Bacillus Erythromycin (E856-058) Abbott Laboratories Ltd Doxycycline (903-58745) Pfizer Ltd subtilis (NCTC 6633) or Staphylococcus aureus Chloramphenicol (LLG 404 RI) Parke-Davis & Co (NCTC 6571). This was prepared on a similar glass Nalidixic acid (Ref. Std Dev 483) Winthrop Laboratories Rifamidc (020) Lepetit Pharmaceuticals Ltd platten to the running layer during electrophoresis Bacitracin (122C-2810) Sigma Chemical Co and, when set, was slid carefully onto the running Gentamicin (PHD 1008/1) Nicholas Laboratories Ltd layer making sure that it was in good contact and Kanamycin (NA 301) Bristol Laboratories Ltd Nitrofurantoin (B663/3) Smith, Kline & French alignment with it. If quantitative results were Laboratories Ltd required the seed layer was poured directly onto the running layer to minimize the distortion inevitably Table I Sources ofantibiotics used (with batch no. if http://jcp.bmj.com/ known) encountered in the first process. In this case.only a spore-seeded agar could be used because of the higher pouring temperature required. The seed placed on the platten and warmed. A metal frame layers were adjusted to have a pH of either 7 9 or held by 'bulldog' clips was placed temporarily on 6-0 according to the optimal requirements for the glass to retain the molten agarose and was detection or estimation of the antibiotic. sealed to it with a little molten agarose. The rest of Other variations used were Escherichia coli the agarose was then poured to give a layer of even (NCTC 10418) as indicator organism when the drugs on October 3, 2021 by guest. Protected copyright. Fig 1 Electrophoresis bed and buffer boxes withplatten and wicks in place. Lid ofsafety cabinet is open. 90 cm J Clin Pathol: first published as 10.1136/jcp.28.6.435 on 1 June 1975. Downloaded from Resolution ofantibiotic mixtures in serum samples by high-voltage electrophoresis 437 detected were inactive against Gram-positive species, drug was taken as its mobility proportional to that and the use of Wellcome Wellcotest agar (pH 7-3) of penicillin (to the anode end; RM value 1 0) or for the seed layer when sulphonamides and trimetho- streptomycin (to the cathode end; RM value 1 -0), prim were investigated. and thus these drugs were put as markers on all It was found that some antibiotics, particularly plates. Distances were measured from the centre of the aminoglycosides, ran as 'streaks' in the agarose. the application well to the centre of the zone of The addition of sodium chloride to a final concen- inhibition. In the case of pear-shaped and streaked tration of I % to the running layer corrected this, the zones the point chosen arbitrarily was the centre of zones being round or pear-shaped under these the smallest radius furthest from the well. For quan- conditions. However, electrophoresis was prolonged titative estimations areas were calculated from meas- because the increased conductivity of the agar limited urements taken using a magnifying zone-reader the voltage available from the power source. (Leebrook Scientific Co Ltd). In the case of round Finally, after the application of the seed layer, the zones the area was taken to be Hlr2; for oval zones frame was replaced, the seed layer surface sterilized a b with ultraviolet light, and a metal lid added for the formula used was 17 , where a was the overnight incubation at 37'C. longest dimension and b the shortest; for pear-shaped SCREENING OF SERA FOR UNSUSPECTED zones the formula used was 4/3b(a + b) - (17/2b2) ANTIBIOTICS where a and b were as for an oval. The veracity of When samples from patients were screened, sera this last formula was checked by tracing around the were initially electrophoresed in duplicate on two magnified zones on the zone-reader using cleared, separate plates, and a seed layer of different pH was old x-ray film, cutting out the zones and weighing used on the two plates.
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