Therapeutic Drug Monitoring 83752570 1986 Raven Press, New York Influence of Tube Type, Storage Time, and Temperature on the Total and Free Concentration of Valproic Acid Charles G. Tarasidis, *William R. Garnett, +Berry J. Kline, and John M. Pellock Radford Community Hospital, Radford, *Department of Pharmacy and Pharmaceutics, School of Pharmacy, 4Department of Neurology, Medical College of VirginialVirginia Commonwealth University, Richmond, Virginia, and *Squibb Pharmaceutical Company, New Brunswick, New Jersey, U.S.A. ‘Summary: The influence of storage conditions on the total and free concentra- tion of valproic acid (VPA) was studied in six normal male subjects who it gested 750 mg of VPA (3 x 250 mg Depakene capsules; Abbott Laboratories). Blood samples were collected in various types of Vacutainer tubes (red top, no additives; green top, sodium heparin; blue top, sodium citrate; and purple top, EDTA) 2h post administration of VPA. Either these samples were centrifuged immediately or stored for various periods of time at room temperature or re- frigerated, or the supernate was frozen prior to analysis. Free VPA samples were obtained utilizing the Amicon ultrafiltration system. All VPA samples were analyzed by gas-liquid chromatography. Total VPA concentrations ob- tained from plasma collected with sodium citrate were lower (p < 0.05) than either serum or plasma collected with other anticoagulants, There were no differences (p > 0.05) in total or free VPA concentrations between samples. collected in serum or in plasma collected with heparin or EDTA. Storing samples for 96 h at room temperature did not alter the total VPA concentra- tions but was found to increase the free fraction of VPA (p < 0.05). The refrig- eration or freezing of the supernate from the blood samples for 7 days did not alter (p > 0.05) the total or the free fraction of VPA. The results of this study demonstrate that total and/or free VPA may be collected from either serum or plasma, provided sodium citrate is not used to collect plasma. If the free frac- tion of VPA is to be determined and samples cannot be assayed immediately, the samples should be refrigerated for not longer than 24 h or the supernate frozen until analysis. Key Words: Valproic acid —Time— Temperature—Con- centrations Valproic acid (VPA) is an antiepileptic drug (AED) that has been approved for the treatment of, absence seizures and has been proven useful in a variety of other generalized seizure types (1). The upper limit of the therapeutic range remains unde- fined. It has been suggested that the free concen- Address correspondence and reprint requests to Dr. W. R. Garnett at Department of Pharmacy and Pharmaceutics, School of Pharmacy, Medical College of Virginia/Virginia Common wealth University, Box 581, MCV Station, Richmond, VA 23298-0001, U.S.A. 373 tration of VPA may be a better therapeutic guide than the total concentration (2). This may be espe- cially true at higher concentrations because the per- centage of drug bound to piasma proteins de- creases. The use of free levels has been enhanced by the validation of rapid ultrafiltration devices for separation of free from bound drug (3) ‘The reliability and validity of reported total and free drug concentrations must be established. EDTA plasma has been shown to be unsuitable for analysis of VPA by EMIT. Also, citrate causes a negative interference in the concentration of VPA374 C. G. TARASIDIS ET AL. when measured by enzyme multiplied immuno- assay and gas-liquid chromatography (4). Heparin decreases the binding characteristics of several drugs in vivo (5), although serum as well as heparin or EDTA plasma has been shown to give compa- rable values in samples analyzed within 2 h by gas chromatography (6). Environmental factors have also been shown to alter free concentration deter- mination (7) A review of several hospitals revealed that VPA samples were collected in a variety of tube types (no additives, sodium citrate, EDTA, and heparin) and stored at variable conditions prior to assay. Therefore, it is the purpose of this study to (a) com- pare serum and plasma VPA concentrations, (b) compare plasma VPA collected in Vacutainer tubes, containing sodium citrate, EDTA, and heparin, and (©) determine the effect of time and temperature on the VPA (total and free) concentrations. METHODS ‘To most closely mimic the clinical setting, blood samples were obtained from six normal male sub- jects 2 h after administration of drug. All subjects gave informed consent prior to initiation of the study. Each subject received a single 750-mg dose of VPA (3.x 250 mg Depakene capsules: Abbott Laboratories, lot number 36-368-AF-21) on an empty stomach, and blood was collected in 24 Vac- utainer tubes 2 h after drug ingestion. There were six red tops (no additives; Becton Dickinson, 2F714), six green tops (heparin sodium; Becton Dickinson, 6483-2F097), six blue tops (sodium ci- trate; Becton Dickinson, 6419-2E073), and six purple tops [ethylenediaminetetraacetic acid (EDTA); Becton Dickinson, 6451-2B642VC]; each containing 5 ml of blood. ‘Once the blood had been drawn into each of the 24 Vacutainer tubes, the tubes were handled as follows. Four of the 24 tubes (one red top, one green, one blue, and one purple) were centrifuged immediately for 10 min at 2,500 rpm and the super- nate used for immediate analysis. Four more samples (one from each group of tube types) were stored at room temperature, ~22°C, for 24 h prior to centrifuging for analysis. Four more samples (one from each group) were stored at room temper- ature for 96 h prior to collecting the supernate for analysis. Eight more samples (two from each group) were refrigerated at 57°C for 24 or 96 h be- fore the supernate was obtained for analysis. The Therapeutic Drug Monitoring, Vol. 8, No, 3, 1986 remaining four samples were centrifuged immedi- ately after the blood had been drawn, and the su- pernate was frozen for 7 days before analysis. All frozen samples were allowed to thaw at room tem- perature prior to analysis, ~2 h. ‘Once the supernate was obtained for analysis, total and free fraction of VPA was measured. Free (unbound) VPA was obtained utilizing the Ami- con ultrafiltration system, The membrane used had a molecular weight cutoff of 25,000 (type CF 25). Membrane ultrafiltration was accomplished through centrifugation at 2,500 rpm for 40. min Once ultrafiltration was completed, total as well as free VPA levels were measured using gas-liquid chromatography by the method of Wood et al. (8). The coefficient of variation was 5.2% for the total VPA (bound plus unbound) levels and 3.9% for the free VPA (unbound) levels. The sensitivity limit of the assay was 2.5 ug/ml. ‘Two-way mixed-model analysis of variance was used to determine statistical differences in the total and free fractions of VPA. Duncan's multiple range test was the test statistic used (9), and a signifi- cance level was set at p < 0.05. RESULTS ‘The total concentration, free concentration, and free fraction of VPA for each subject with respect to tube type and storage condition are summarized in Table 1. The immediate concentrations represent the total concentration and free concentration 2h post administration of a single 750-mg dose of VPA. The concentrations at various times represent the influence of storage time and storage temperature. Influence of Time and Temperature on Total VPA Concentrations ‘Samples collected in tubes containing sodium ci- trate were found to yield significantly less total VPA at all times and storage temperatures than samples collected in tubes containing no additives, heparin, or EDTA. There were no differences within the other tube types. Within the same tube type there was no change in total VPA over time and storage temperatures (Fig. 1). Influence of Time and Temperature on Free Fraction of VPA Concentrations Significant differences were found for the sam- ples collected in tubes containing sodium citrateEFFECTS OF STORAGE ON VPA CONCENTRATIONS 375 TABLE 1. Valproic acid concentration (ugiml) “Tube ype Time Red Greea Blue Purple Immediate . Total 769 = 11.03 18.35 = 11.82 ree 5.06 = Lid 43510 Fre 65 = 055 60 = 089 24 hat room temperature “Tol 756= 1037 Fre 588 = 106 eFree 766 = 052 S945 = 2.08 7362 = 1341 SIS = 10. 477 = 4103 So6 = 082 6.5 = 08 24h post refrigeration ‘Total 75.8 = 10.0 Fre 523 = 116 SeFree 683 = 117 217 ox 07 9 hat room temperature "Total 78.06 = 13.9 Free 683 = 0.98 ce Free #82 17 wos 60 0.0 no 087 oss 9h post refrigeration “Tol 7665 = 132 03 6037 160 Free S56 = 138 Lin "83 94 Free 716 = 078 1238 108 Post freezing “Tol 7565 = 10.75 74.76 = 9.45 60.27 wars Fre S38 = 12 126 “S03 098 % 10 = 083 10 833 Los Red, no additives; blue, sodium citrate: green, heparin: purple, EDTA. and samples collected in tubes containing no addi- tives, heparin, or EDTA. There were no differences in the other tube types. Within the same tube type, storing samples for 96 h at room temperature was found to significantly increase the free fraction of VPA. The refrigeration or freezing of the supernate from blood samples for 7 days did not alter the free fraction of VPA (Fig. meD 2681 24%REF 96tRT 96EREF FZN-70 FIG. 1. Storage time and temperature changes within tube types: total valproic acid (VPA) concentrations. 800 op Neeson cate Bele op TawreTa FREE FRACTION OF VPA (% 12s Top Tibe-Som crete ‘2 cue on aes Soaum earn Sire top rowseTa FIG. 2. Storage time and temperature changes within tube types: free valproic acid (VPA) concentrations. DISCUSSION The proper collection and storage of drug samples is essential to the proper interpretation of drug concentrations, Under clinical conditions, we demonstrated that maintaining the blood/drug samples for extended periods of time at room tem- perature significantly increases the free VPA level regardless which tube type is used. However, the total VPA concentration did not differ significantly with time or storage temperature in any of the tube types utilized. The coefficient of variation of the assay for the free drug levels was 3.9%. The average variation for the reported free drug levels was at least two times the coefficient of variation of the assay in all but two cases (each of these specific tube type at a specific time and tempera- ture). Therefore, when a difference was demon- strated, it is unlikely that this difference can be at- tributed to the coefficient of variation of the assay. The total concentration of VPA in the sodium ci- trate tubes was ~20-25% less than in any of the other three tube types used. This difference can be explained by the aqueous solution of sodium citrate used in these tubes. The sodium citrate solution ap- proximated a volume of | ml, which would cause a 17% dilution effect on the total VPA level when 5 ml of blood was collected. Vacutainer tubes that contain no additives, hep- arin sodium, or EDTA may be used to measure total and free VPA concentrations. Storage of these tubes for 24-96 h by refrigeration or freezing for | week does not significantly alter the free fraction of VPA relative to immediate analysis. The blue top Therapeutic Drug Monitoring, Vo. 8, No. 2, 1986376 C. G. TARASIDIS ET AL. tubes, which contain aqueous sodium citrate, should not be used because of the dilution factor previously discussed. Also, allowing the tubes to stand at room temperature for extended periods of time (24 h) will increase the free fraction of VPA over that which is present in vivo. Time periods <24 h at room temperature have not been evalu- ated, and therefore conclusions cannot be drawn. It is undetermined whether these statistical differ- ences in sample collection technique result in clini cally significant differences in interpretation of VPA concentrations. However, the clinical useful- ness of free and total VPA concentration should be enhanced by a valid and reliable sample collection We have established that samples may be col- lected in tubes containing no additives, heparin, or EDTA because no significant difference is observed between the measured total VPA or free VPA con- centrations. Hence, plasma levels of total or fr VPA are not different from those found in serum. Also, samples that are collected for kinetic studies and are to be batch assayed should be centrifuged immediately and the supernate frozen for the time period prior to analysis. Finally, samples that must be shipped to a distant laboratory for analysis should be refrigerated for no longer than 96 h, and Therapeutic Drug Monitoring, Vo. 8, No.3, 1986 if the transportation time exceeds this limit, the samples should be centrifuged and the supernate shipped frozen. REFERENCES 1. Loiseau P. Rational use of valproate: indications and drug regimen in epilepsy. 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